Discover CircRes

This month on Episode 38 of Discover CircRes, host Cynthia St. Hilaire highlights original research articles featured in the Jue 24th, July 8th and July 22nd issues of the journal. This episode also features an interview with the 2022 BCBS Outstanding Early Career Investigator Award finalists, Dr Hisayuki Hashimoto, Dr Matthew DeBerge and Dr Anja Karlstadt.

Article highlights:

Nguyen, et al. miR-223 in Atherosclerosis.

Choi, et al. Mechanism for Piezo1-Mediated Lymphatic Sprouting

Kamtchum-Tatuene, et al. Plasma Interleukin-6 and High-Risk Carotid Plaques

Li, et al. 3-MST Modulates BCAA Catabolism in HFrEF

Cindy St. Hilaire: Hi, and welcome to Discover CircRes, the podcast of the American Heart Association's journal, Circulation Research. I'm your host, Dr Cindy St. Hilaire, from the Vascular Medicine Institute at the University of Pittsburgh. And today I'm going to be highlighting articles from our June 24th, July 8th and July 22nd issues of Circulation Research. I'm also going to have a chat with the finalists for the 2022 BCBS Outstanding Early Career Investigator Award, Dr Hisayuki Hashimoto, Dr Matthew DeBerge and Dr Anja Karlstadt.

Cindy St. Hilaire: The first article I want to share is from our June 24th issue and is titled, miR-223 Exerts Translational Control of Proatherogenic Genes in Macrophages. The first authors are My-Anh Nguyen and Huy-Dung Hoang, and the corresponding author is Katey Rayner and they're from the University of Ottawa. A combination of cholesterol accumulation in the blood vessels and subsequent chronic inflammation that's derived from this accumulation drive the progression of atherosclerosis. Unfortunately, current standard medications tackle just one of these factors, the cholesterol. And this might explain why many patients on such drugs still have vascular plaques. In considering treatments that work on both aspects of the disease, meaning lipid accumulation and inflammation, this group investigated the micro RNA 223 or miR-223, which is a small regulatory RNA that has been shown to suppress expression of genes involved in both cholesterol uptake and inflammatory pathways in both liver and immune cells.

Cindy St. Hilaire: The team showed that mouse macrophages deficient in miR-223, exhibited increased expression of pro-inflammatory cytokines and reduced cholesterol efflux compared with control cells. Overexpression of miR-223 had the opposite effects. Furthermore, atherosclerosis prone mice, whose hematopoietic cells lacked miR-223, had worse atherosclerosis with larger plaques and higher levels of pro-inflammatory cytokines than to control animals with normal levels of miR-223. These findings highlight miR-223's dual prompt, antiatherogenic action, which could be leveraged for future therapies.

Cindy St. Hilaire: The second article I want to share is from our July 8th issue of Circulation Research and is titled, Piezo1-Regulated Mechanotransduction Controls Flow-Activated Lymph Expansion. The first author is Dongwon Choi and the corresponding author is Young-Kwon Hong, and they're from UCLA.

As well as being super highways for immune cells, lymph vessels are drainage channels that help maintain fluid homeostasis in the tissues. This network of branching tubes grows as fluids begin to flow in the developing embryo. This fluid flow induces calcium influx into the lymphatic endothelial cells, which in turn promotes proliferation and migration of these cells, leading to the sprouting of lymph tubules. But how do LECs, the lymphatic endothelial cells, detect fluid flow in the first place? Piezo1 is a flow and mechanosensing protein known for its role in blood vessel development and certain mutations in Piezo1 cause abnormal lymphatic growth in humans.

Cindy St. Hilaire: This script found that Piezo1 is expressed in the embryonic mouse LECs and that the suppression of Piezo1 inhibits both flow activated calcium entry via the channel ORAI1, as well as downstream target gene activation. Overexpression of Piezo1, by contrast, induced the target genes. The team went on to show that mice lacking either Piezo1 or ORAI1 had lymphatic sprouting defects and that pharmacological activation of Piezo1 in mice enhanced lymphogenesis and prevented edema after tail surgery. Together, the results confirmed Piezo1's role in flow dependent lymphatic growth and suggest it might be a target for treating lymphedema.

Cindy St. Hilaire: The third article I want to share is also from our July 8th issue and is titled, Interleukin-6 Predicts Carotid Plaque Severity, Vulnerability and Progression. The first and corresponding author of this study is Joseph Kamtchum-Tatuene from University of Alberta.

Excessive plasma cholesterol and systemic inflammation are contributing factors in atherosclerosis. While traditional remedies have been aimed at lowering patient's lipid levels, drugs that tackle inflammation are now under investigation, including those that suppress Interleukin-6, which is an inflammatory cytokine implicated in the disease. Focusing on carotid artery disease, this group conducted a prospective study to determine whether IL-6 levels correlated with disease severity. 4,334 individuals were enrolled in the cardiovascular health study cohort. They had their blood drawn and ultrasounds taken at the start of the study and five years later. This group found IL-6 was robustly correlated with and predicted plaque severity independent of other cardiovascular risk factors. This study also determined that an IL-6 blood plasma level of 2.0 picograms/mls, identified individuals with the highest likelihood of plaque, vulnerability and progression. This threshold value could be used to select patients who might benefit from novel IL-6 lowering medications.

Cindy St. Hilaire: The last article I want to share is from our July 22nd issue of Circulation Research and is titled, Mitochondrial H2S Regulates BCAA Catabolism in Heart Failure. The first author is Zhen Li, and the corresponding author is David Lefer from Louisiana State University. Hydrogen sulfide, or H2S, is a compound that exerts mitochondrial specific actions that include the preservation of oxidative phosphorylation, mitochondrial biogenesis and ATP synthesis, as well as inhibiting cell death. 3-mercaptopyruvate sulfurtransferase, or 3-MST, is a mitochondrial H2S producing enzyme, whose functions in cardiovascular disease are not fully understood.

Cindy St. Hilaire: This group investigated the global effects of 3-MST deficiency in the setting of pressure overload induced heart failure. They found that 3-MST was significantly reduced in the myocardium of patients with heart failure, compared with non failing controls. 3-MST knockout mice exhibited increased accumulation of branch chain amino acids in the myocardium, which was associated with reduced myocardial respiration and ATP synthesis, exacerbated cardiac and vascular dysfunction, and worsened exercise performance, following transverse aortic constriction. Restoring myocardial branched-chain amino acid catabolism, or administration of a potent H2S donor, ameliorated the detrimental effects of 3-MST deficiency and heart failure with reduced injection fraction. These data suggest that 3-MST derived mitochondrial H2S, may play a regulatory role in branch chain amino acid catabolism, and mediate critical cardiovascular protection in heart failure.

Cindy St. Hilaire: Today, I'm really excited to have our guests, who are the finalists for the BCVS Outstanding Early Career Investigator Awards. Welcome everyone.

Hisayuki Hashimoto: Thank you.

Anja Karlstaedt: Hi.

Hisayuki Hashimoto: Hi.

Matthew DeBerge: Hello. Thank you.

Cindy St. Hilaire: So the finalists who are with me today are Dr Hisayuki Hashimoto from Keio University School of Medicine in Tokyo, Japan, Dr Matthew Deberge from Northwestern University in Chicago and Dr Anja Karlstaedt from Cedar Sinai Medical Center in LA. Thank you again. Congratulations. And I'm really excited to talk about your science.

Hisayuki Hashimoto: Thank you. Yes. Thanks, first of all for this opportunity to join this really exciting group and to talk about myself and ourselves. I am Hisayuki Hashimoto, I'm from Tokyo, Japan. I actually learned my English... I went to an American school in a country called Zaire in Africa and also Paris, France because my father was a diplomat and I learned English there. After coming back to Japan, I went to medical school. During my first year of rotation, I was really interested in cardiology, so I decided to take a specialized course for cardiology. Then I got interested in basic science, so I took a PhD course, and that's what brought me to this cardiology cardiovascular research field.

Matthew DeBerge: So I'm currently a research assistant professor at Northwestern University. I'm actually from the Chicagoland area, so I'm really excited to welcome you all to my hometown for the BCVS meeting.

Cindy St. Hilaire: Oh, that's right. And AHA is also there too this year. So you'll see a lot of everybody.

Matthew DeBerge: I guess I get the home field advantage, so to speak. So, I grew up here, I did my undergrad here, and then went out in the east coast, Dartmouth College in New Hampshire for my PhD training. And actually, I was a viral immunologist by training, so I did T cells. When I was looking for a postdoctoral position, I was looking for a little bit of something different and came across Dr Edward Thorpe's lab at Northwestern university, where the interest and the focus is macrophages in tissue repair after MI. So, got into the macrophages in the heart and have really enjoyed the studies here and have arisen as a research assistant professor now within the Thorpe lab. Now we're looking to transition my own independent trajectory. Kind of now looking beyond just the heart and focusing how cardiovascular disease affects other organs, including the brain. That's kind of where I'm starting to go now. Next is looking at the cardiovascular crosstalk with brain and how this influences neuroinflammation.

Anja Karlstaedt: I am like Hisayuki, I'm also a medical doctor. I did my medical training and my PhD in Berlin at the Charité University Medicine in Berlin, which is a medical faculty from Humboldt University and Freie University. II got really interested in mathematical modeling of complex biological systems. And so I started doing my PhD around cardiac metabolism and that was a purely core and computationally based PhD. And while I was doing this, I got really hooked into metabolism. I wanted to do my own experiments to further advance the model, but also to study more in crosstalk cardiac metabolism. I joined Dr Heinrich Taegteyer lab at the University of Texas in the Texas Medical Center, and stayed there for a couple of years. And while I was discovering some of the very first interactions between leukemia cells and the heart, I decided I cannot stop. I cannot go back just after a year. I need to continue this project and need to get funding. And so after an AHA fellowship and NIHK99, I am now here at Cedars Sinai, an assistant professor in cardiology and also with a cross appointment at the cancer center and basically living the dream of doing translational research and working in cardio-oncology.

Cindy St. Hilaire: Great. So, Dr Hashimoto, the title of your submission is, Cardiac Reprogramming Inducer ZNF281 is Indispensable for Heart Development by Interacting with Key Cardiac Transcriptional Factors. This is obviously focused on reprogramming, but why do we care about cardiac reprogramming and what exactly did you find about this inducer ZNF281?

Hisayuki Hashimoto: Thank you for the question. So, I mean, as I said, I'm a cardiologist and I was always interested in working heart regeneration. At first, I was working with pluripotent stem cells derived cardiomyocyte, but then I changed my field during my postdoc into directly programming by making cardiomyocyte-like cells from fiberblast. But after working in that field, I kind of found that it was a very interesting field that we do artificially make a cardiomyocyte-like cell. But when I dissected the enhanced landscape, epigenetic analysis showed that there are very strong commonalities between cardiac reprogramming and heart development. So I thought that, hey, maybe we can use this as a tool to discover new networks of heart development. And the strength is that cardiac reprogramming in vitro assay hardly opens in vivo assay, so it's really time consuming. But using dark programming, we can save a lot of time and money to study the cardiac transitional networks. And we found this DNF281 from an unbiased screen, out of 1000 human open reading frames. And we found that this gene was a very strong cardiac reprogramming inducer, but there was no study reporting about any functioning heart development. We decided to study this gene in heart development, and we found out that it is an essential gene in heart development and we were kind of able to discover a new network in heart development.

Cindy St. Hilaire: And you actually used, I think it was three different CRE drivers? Was that correct to study?

Hisayuki Hashimoto: Ah, yes. Yeah.

Cindy St. Hilaire: How did you pick those different drivers and what, I guess, cell population or progenitor cell population did those drivers target?

Hisayuki Hashimoto: So I decided to use a mesodermal Cre-driver, which is a Mesp1Cre and a cardiac precursor Cre-driver, which is the Nkx2-5 Cre and the cardiomyocyte Cre, which is the Myh6-Cre. So three differentiation stages during heart development, and we found out that actually, DNF281 is an essential factor during mesodermal to cardiac precursor differentiation state. We're still trying to dig into the molecular mechanism, but at that stage, if the DNF281 is not there, we are not able to make up the heart.

Cindy St. Hilaire: That is so interesting. Did you look at any of the strains that survived anyway? Did you look at any phenotypes that might present in adulthood? Is there anything where the various strains might have survived, but then there's a kind of longer-term disease implicating phenotype that's observed.

Hisayuki Hashimoto: Well, thank you for the question. Actually, the mesodermal Cre-driver knocking out the DNF281 in that stage is embryonic lethal, and it does make different congenital heart disease. And they cannot survive until after embryonic day 14.5. The later stage Nkx2-5 Cre and Myh6-Cre, interestingly, they do survive after birth. And then in adult stage, I did also look into the tissues, but the heart is functioning normally. I haven't stressed them, but they develop and they're alive after one year. It looks like there's really no like phenotype at like the homeostatic status.

Cindy St. Hilaire: Interesting. So it's kind of like, once they get over that developmental hump, they're okay.

Hisayuki Hashimoto: Exactly. That might also give us an answer. What kind of network is important for cardiac reprogramming?

Cindy St. Hilaire: So what are you going to do next?

Hisayuki Hashimoto: Thank you. I'm actually trying to dig into the transitional network of what kind of cardiac transitional network the ZNF281 is interacting with, so that maybe I can find a new answer to any etiology of congenital heart disease, because even from a single gene, different mutation, different variants arise different phenotypes in congenital heart disease. Maybe if I find a new interaction with any key cardiac transitional factors, maybe I could find a new etiology of congenital heart disease phenotype.

Cindy St. Hilaire: That would be wonderful. Well, best of luck with that. Congratulations on an excellent study.

Hisayuki Hashimoto: Thank you.

Cindy St. Hilaire: Dr DeBerge, your study was titled, Unbiased Discovery of Allograft Inflammatory Factor-1 as a New and Critical Immuno Metabolic Regulatory Node During Cardiac Injury. Congrats on this very cool study. You were really kind of focused on macrophages in myocardial infarction. And macrophages, they're a Jeckel Hyde kind of cell, right? They're good. They're bad. They can be both, almost at the same time, sometimes it seems like. So why were you interested in macrophages particularly in myocardial infarction, and what did you discover about this allograft inflammatory factor-1, or AIF1 protein?

Matthew DeBerge: Thank you. That's the great question. You really kind of alluded to why we're interested in macrophages in the heart after tissue repair. I mean, they really are the central mediators at both pro-inflammatory and anti-inflammatory responses after myocardial infarction. Decades of research before this have shown that inflammation has increased acutely after MI and has also increased in heart failure patients, which really has led to the development of clinical efforts to target inflammatory mediators after MI. Now, unfortunately, the results to target inflammation after MI, thus far, have been modest or disappointing, I guess, at worst, in the respect that broadly targeting macrophage function, again, hasn't achieved results. Again, because these cells have both pro and anti-inflammatory functions and targeting specific mediators has been somewhat effective, but really hasn't achieved the results we want to see.

Matthew DeBerge: I think what we've learned is that the key, I guess, the targeting macrophage after MI, is really to target their specific function. And this led us to sort of pursue novel proteins that are mediating macrophage factor function after MI. To accomplish this, we similarly performed an unbiased screen collecting peri-infarct tissue from a patient that was undergoing heart transplantation for end stage heart failure and had suffered an MI years previously. And this led to the discovery of allograft inflammatory factor-1, or AIF1, specifically within cardiac macrophages compared to other cardiac cell clusters from our specimen. And following up with this with post-mortem specimens after acute MI to show that AIF1 was specifically increased in macrophages after MI and then subsequently then testing causality with both murine model of permanent inclusion MI, as well as in vitro studies using bone marrow drive macrophages to dig deeper mechanistically, we found that AIF1 was crucial in regulating inflammatory programing macrophages, which ultimately culminated in worse in cardiac repair after MI.

Cindy St. Hilaire: That's really interesting. And I love how you start with the human and then figure out what the heck it's doing in the human. And one of the things you ended up doing in the mouse was knocking out this protein AIF1, specifically in macrophage cells or cells that make the macrophage lineage. But is this factor in other cells? I was reading, it can be intracellular, it can be secreted. Are there perhaps other things that are also going on outside of the macrophage?

Matthew DeBerge: It's a great question. First, I guess in terms of specificity, within the hematopoietic compartment, previous studies, as well as publicly available databases, have shown that AIF1 is really predominantly expressed within macrophages. We were able to leverage bone marrow chimera mice to isolate this defect to the deficiency to macrophages. But you do bring up a great point that other studies have shown that AIF1 may be expressed in other radio-resistant cell populations. I mean, such as cardiomyocytes or other treatable cells within the heart. We can't completely rule out a role for AIF1 and other cell populations. I can tell you that we did do the whole body knockout complementary to our bone marrow hematopoetic deficient knockouts, and saw that deficiency of AIF1 within the whole animal, recapitulate the effects we saw within the AIF1 deficiency within hematopoietic department.

Matthew DeBerge: It was encouraging to us that, again, the overall role of AIF1 is pro-inflammatory after MI.

Cindy St. Hilaire: I mean, I know it's early days, but is there a hint of any translational potential of these findings or of this protein?

Matthew DeBerge: Yeah, I think so. To answer your question, we were fortunate enough to be able to partner with Ionis that develops these anti-sensible nucleotides so that we could specifically target AIF1 after the acute phase during MI. We saw that utilizing these anti-sensible nucleotides to deplete AIF1, again, within the whole mouse, that we were able to reduce inflammation, reduce in heart size and preserve stock function. I think there really is, hopefully a therapeutic opportunity here. And again, with it being, perhaps macrophage specific is, even much more important as we think about targeting the specific function of these cells within the heart.

Cindy St. Hilaire: Very cool stuff. Dr Karlstaedt, the title of your submission is, ATP Dependent Citrate Lyase Drives Metabolic Remodeling in the Heart During Cancer. So this I found was really interesting because you were talking about, the two major killers in the world, right? Cardiovascular disease and cancer, and you're just going to tackle both of them, which I love. So obviously this is built on a lot of prior observations about the effects of cancer on cardiac metabolic remodeling. Can you maybe just tell us a little bit about what is that link that was there and what was known before you started?

Anja Karlstaedt: Yeah. Happy to take that question. I think it's a very important one and I'm not sure if I will have a comprehensive answer to this, because like I mentioned at the beginning, cardio-oncology is a very new field. And the reason why we are starting to be more aware of cancer patients and their specific cardiovascular problems is because the cancer field has done such a great job of developing all these new therapeutics. And we have far more options of treating patients with various different types of cancers in particular, also leukemias, but also solid tumors. And what has that led to is an understanding that patients survive the tumors, but then 10, 20 years later, are dying of cardiovascular diseases. Those are particular cardiomyopathies and congestive heart failure patients. What we are trying, or what my lab is trying to do, is understanding what is driving this remodeling. And is there a way that we can develop therapies that can basically, at the beginning of the therapy, protect the heart so that this remodeling does not happen, or it is not as severe.

Anja Karlstaedt: Also, identifying patients that are at risk, because not every tumor is created equally and tumors are very heterogeneous, even within the same group. To get to your question, what we found is, in collaboration actually with a group at Baylor College of Medicine, Peggy Goodell's group, who is primarily working on myeloid malignancies, is that certain types of leukemias are associated with cardiomyopathies. And so when they were focusing on the understanding drivers of leukemia, they noticed that the hearts of these animals in their murine models are enlarged on and actually developing cardiomyopathies. And I joined this project just very early on during my postdoc, which was very fortunate and I feel very lucky of having met them. What my lab is now studying here at Cedars is how basically those physiological stress and mutations coming from the tumors are leading to metabolic dysregulation in the heart and then eventually disease.

Anja Karlstaedt: And we really think that metabolism is at the center of those disease progressions and also, because it's at the center, it should be part of the solution. We can use it as a way to identify patients that are at risk, but also potentially develop new therapies. And what was really striking for us is that when we knock down ACLY that in a willdtype heart where the mouse doesn't have any tumor disease, ACLY actually is critically important for energy substrate metabolism, which seems counterintuitive, because it's far away from the mitochondria, it's not part of directly ADP provision. It's not part of the Kreb cycle. But what we found is that when we knock it out using a CRISPR-Cas9 model, it leads to cardiomyopathy and critically disrupts energy substrate metabolism. And that is not necessarily the case when the mouse has leukemia or has a colorectal cancer, which upregulated in the beginning, this enzyme expression. And so we have now developed models that show us that this could be potentially also therapeutic target to disrupt the adverse remodeling by the tumor.

Cindy St. Hilaire: That is so interesting. So one of the things I was thinking about too is we know that, I mean, your study is showing that, the tumor itself is causing cardiac remodeling, but we also know therapies, right? Radiation, chemotherapy, probably some immune modulatory compounds. Those probably do similar, maybe not exactly similar, but they also cause, adverse cardiac remodeling. Do you have any insights as to what is same and what is different between tumor driven and therapy driven adverse remodeling?

Anja Karlstaedt: So we do not know a lot yet. It's still an open question about all the different types of chemotherapeutics, how they are leading to cardio toxicities. But what we know, at least from the classic anti-cyclic treatments, is right now at the core, the knowledge is that this is primarily disrupting cardiac mitochondrial function. And through that again, impairing energy provision and the interaction, again, with the immune system is fairly unknown, but we know through studies from Kathryn Moore and some very interesting work by Rimson is that myocardial infarction itself can lead to an increase in risk for tumor progression. And what they have shown as independent of each other, is that the activation of the immune system in itself can lead to an acceleration of both diseases, both the cardiac remodeling, and then also the tumor disease. We don't fully understand which drivers are involved, but we do know that a lot of the cardiomyopathies on cardiotoxicities that are chemotherapeutically driven, all have also metabolic component.

Cindy St. Hilaire: Nice. Thank you. When I prepare for these interviews, I obviously read the abstracts for the papers, but I found myself also Googling other things after I read each of your abstracts. It was a rabbit hole of science, which was really exciting.

I now want to transition to kind of a career angle. You all are obviously quite successful, scientifically, at the bench, right? But now you are pivoting to a kind of completely opposite slash new job, right? That of, independent researcher. I would love to hear from each of you, if there was any interesting challenge that you kind of overcame that you grew from, or if there was any bit of advice that you wish you knew ahead of time or anything like that, that some of our trainee listeners and actually frankly, faculty who can pass that information onto their trainees, can benefit from.

Anja Karlstaedt: I think the biggest challenge for me in transitioning was actually the pandemic. Because I don't know how it was for Hisa and Matt, but trying to establish a lab, but also applying for faculty position during a major global pandemic, is challenging is not quite something that I expected that would happen. And so I think saying that and looking more conceptually and philosophically at this as, you can prepare as much as you want, but then when life just kicks in and things happen, they do happen. And I think the best is to prepare as much as you can. And then simply go with the flow. Sometimes one of my mentors, Dave Nikon, mentioned that to me when I was applying for faculty positions, it's sometimes good to just go with the flow. And as a metabolism person, I absolutely agree. And there are some things that you can do as a junior investigator.

Anja Karlstaedt: We need to have a good network. So just very important to have good mentors. I was blessed with have those mentors, Peggy Goodell's one of them, Heinrich Taegtmeyer was another. And now with this study that we are publishing, Jim Martin and Dave Nikon were incredible. Without them, this study wouldn't have been possible and I would not be here at Cedars.

Anja Karlstaedt: You need to reach out to other people because those mentors have the experience. They have been through some of this before. Even if they have never had a major event, like COVID-19 in their life before, because none of us had before, they had other experiences and you can rely on them and they set you then up for overcoming these challenges. And the other thing I would say, is put yourself out there, go and talk to as many people as possible or set conferences, present a poster, not only talks. Don't be disappointed if you don't get a talk, posters are really great to build this network and find other people that you probably wouldn't have encountered and apply for funding. Just again, put yourself out there and try to get the funding for your research. Even if it's small foundations, it builds up over time and it is a good practice to then write those more competitive grants.

Cindy St. Hilaire: Dr Hashimoto, would you like to go next?

Hisayuki Hashimoto: Just my advice is that, could be like a culture of difference, but in east Asia, like in Japan, we were taught to, do not disturb people, don't interrupt people and help people. But I realized that I wasn't really good at asking for help. After I am still not like fully independent, but I do have my own group and I have to do grant writing. I still work at the bench and then have to teach grad students, doing everything myself. I just realized it's just impossible. I didn't have time. I need like 48 hours a day. Otherwise, you won't finish it. I just realized that I wasn't really good at asking for help. So my advice would be, don't hesitate to ask for help. It's not a shame. You can't do everything by just yourself. I think, even from the postdoc, even from grad school, I think, ask for help and then get used to that. And then of course, help others. And that is the way I think to probably not get overwhelmed and not stress yourself. Science should be something fun. And if you don't ask for help and if you don't help someone, I think you are losing the chance of getting some fun part from the science.

Cindy St. Hilaire: That's great advice. I really like that, especially because I find at least, I started my lab seven years ago now. And I remember the first couple months/year, it was extremely hard to let go, right? Like I taught my new people how to do the primary cell culture we needed, but I was terrified of them doing it wrong or wasting money or making too many mistakes. But you realize, you got to learn to trust people. Like you said, you got to learn to ask for help. And sometimes that help is letting them do it. And you doing, you're being paid now to write grants and papers. That's a big brain, you're not paid to do the smaller things. That's really great advice. I like that. Thank you. Dr DeBerge, how about you?

Matthew DeBerge: So I guess towards a bit of life advice, I think two obvious things is one, be kind, science is hard enough as it is. So I think we should try to lift each other up and not knock each other down. And along those lines as the others have alluded to as well, one of the mantras we sort of adapted on the lab, is a rising tide raises all ships, this idea that we can work together to elevate each other's science and really, again, collaborate.

Towards the career side of things I'll just touch on, because I guess one thing I'll add, there's more than one path, I guess, to achieving your goals. I've been fortunate enough to have an NIH post-doctoral fellowship and had an AHA career development award, but I'm not a K99 recipient. Oftentimes, I think this is the golden ticket to getting the faculty job, so I'm trying to, I guess, buck trend, I just submitted an RO1. So fingers crossed that leads to some opportunity.

Even beyond academia, I'm not certain how much everyone here is involved in science Twitter, it's really become a thing over the last couple years, but I think, kind of the elephant in the room is that academia, it's really hard on the trainees nowadays to have a living wage, to go through this. I mean, I'm really excited to see my, fellow finalists here are starting their own groups and stuff, but for many, that's not the reality for many, it's just not financially feasible. So I think, kind of keeping in mind that there's many, many alternative careers, whether it's industry, whether it's consulting, science writing, etcetera, going back to what Dr Hash says, find what you love and really pursue that with passion.

Cindy St. Hilaire: I think it's something only, I don't know, five to 10% of people go into or rather stay in academia. And that means, 90 to 95% of our trainees, we need to prepare them for other opportunities, which I think is exciting, because it means it can expand our network for those of us in academia.

Anja Karlstaedt: I think right now it's even worse because it's about 2% of old postdocs that are actually staying and becoming independent researchers, independent or tenure track or research track. And I think I second, as what Matt said, because I play cello. I do music as a hobby and people always ask me if I'm a musician. And at the beginning I felt like, no, of course not. I'm not like Yoyo Ma. I'm just playing, it's a hobby. And then I, that got me thinking. I was like, no, of course you are because there's so many different types. And what we need to understand is that scientists, like you are always a scientist. It doesn't matter if you are working at Pfizer or if you are working at a small undergrad institution and you're teaching those next generation scientists, you are still scientist and we all need those different types of scientists because otherwise, if everybody is just a soloist, you are never going to listen to symphony. You need those different people and what we need to normalize beyond having those different career paths, is also that people are staying in academia and becoming those really incredible resources for the institutions and labs, quite frankly, of being able to retain those technologies and techniques within an institution. And I think that's something to also look forward to, that even if you're not the PI necessarily, you're the one who is driving those projects. And I hope to pass this on at some point also to my trainees that they can be a scientist, even if they're not running a lab and they become an Institute director and that's also critically important.

Cindy St. Hilaire: There's lots of ways to do science. Thank you all so much for joining me today. Either waking up at 5:00 AM or staying up past midnight, I think it is now in Japan or close to it. So Matt and I kind of made it out okay. It's like 8:00 or 9:00 AM.

Matthew DeBerge: Thank you.

Hisayuki Hashimoto: My apologies for this time zone difference.

Cindy St. Hilaire: I'm very glad to make it work. Congratulations to all of you, your presentations. I forget which day of the week they are on at BCVS, but we are looking forward to the oral presentations of these and congratulations to all of you. You are amazing scientists and I know I'm really looking forward to seeing your future work so best of luck.

Matthew DeBerge: Thank you.

Hisayuki Hashimoto: Thank you.

Anja Karlstaedt: Thank you so much.

Cindy St. Hilaire: That's it for the highlights from the June 24th, July 8th and July 22nd issues of Circulation Research. Thank you for listening. Please check out the CircRes Facebook page and follow us on Twitter and Instagram with the handle at CircRes and hashtag Discover CircRes. Thank you to our guests. The BCVS Outstanding Early Career Investigator Award Finalists, Dr Hisayuki Hashimoto, Dr Matthew DeBerge and Dr Anja Karlstaedt. This podcast is produced by Ashara Ratnayaka, edited by Melissa Stoner and supported by the editorial team of Circulation Research. Some of the copy text for the highlighted articles is provided by Ruth Williams. I'm your host, Dr Cindy St. Hilaire. And this is Discover CircRes, you're on the go source for the most exciting discoveries in basic cardiovascular research. This program is copyright of the American Heart Association, 2022. The opinions expressed by speakers in this podcast are their own and not necessarily those of the editors or of the American Heart Association. For more information visit ahajournals.org.

This month on Episode 37 of Discover CircRes, host Cynthia St. Hilaire highlights two original research articles featured in the May 27th issue of the journal and also provides an overview of the Compendium on Basic Models of Cardiovascular Disease featured in the June 10 issue of Circulation Research. This episode also features an interview between Dr Nikki Purcell, Circulation Research Social Media Editor and Associate Professor at Huntington Medical Research Institute and Dr Mark Feinberg, Dr Rulin Zhuang, and Dr Jingshu Chen from Brigham and Women's Hospital in Harvard Medical School to discuss their study, Perivascular Fibrosis Is Mediated by a KLF10-IL-9 Signaling Access in CD4+ T-Cells.

Article highlights:

Liang, et al. Tenascin-X Inhibits EndMT and Atherosclerosis

Jin, et al. Lineage Tracing of Pericardial Cavity Macrophages

Rosenzweig, et al. Basic Models of Cardiovascular Disease

Cindy St. Hilaire: Hi, and welcome to Discover CircRes the podcast of the American Heart Association's Journal, Circulation Research. I'm your host, Dr Cindy St. Hilaire from the. Vascular Medicine Institute at the University of Pittsburgh, and today I'm going to be highlighting articles from our May 27th and June 10th issues of Circulation Research. Dr Nikki Purcell, an Associate Professor from the Huntington Medical Research Institute and my colleague on the CircRes Editorial Board, is going to interview Dr Mark Feinberg, Dr Rulin Zhuang and Dr Jingshu Chen from Brigham and Women's Hospital in Harvard Medical School and they're going to discuss their study, Perivascular Fibrosis Is Mediated by a KLF10-IL-9 Signaling Access in CD4+ T-Cells.

Cindy St. Hilaire: The first article I'm going to highlight is titled Tenascin-X Mediates Flow Induced Suppression of EndMT and Atherosclerosis. The first author is Guozheng Liang and the corresponding author is Stefan Offermanns and they're from the Max Planck Institute. Atherosclerotic plaques in arteries arise when blood flow is reduced or turbulent. These are commonly regions where the vessels are curved or branched. Disturbed flow in these regions can prompt the local endothelial cells to undergo a process called e-to-mesenchymal transition or EndMT, which in turn contributes to atherosclerotic plaque formation. Exactly why turbulent, but not laminar flow prompts EndMT is not known. Using in vitro and in vivo experiments, this group discovered a critical EndMT suppressor protein expressed at high levels in endothelial cells exposed to lamina flow, but not exposed to turbulent flow. Mice that lack the protein, which is called Tenascin-X or TN-X, exhibited signs of EndMT and inflammation throughout their aorta and they were more prone to atherosclerosis. TN-X binds and blocks the function of the cytokine TGF beta, which is a potent driver of EndMT. Inhibiting the activity of TGF beta, whether with an anti TGF beta antibody or by deletion of its receptors, prevented the EndMT promoting effects of TN-X loss. Together, the results suggest that bolstering or mimicking the effects of TN-X may be a novel strategy for preventing atherosclerosis progression.

Cindy St. Hilaire: The second article I want to share is titled, Genetic Lineage Tracing of Pericardial Cavity Macrophages in the Injured Heart. The first author is Hengwei Jin and the corresponding author is Bin Zhou and they're from the Chinese Academy of Science. If the heart suffers an infarction, monocytes and macrophages travel to the injury site via the bloodstream and locally from within the heart itself. A recent Immunity paper claims that macrophages in the surrounding pericardial cavity also can infiltrate the heart to aid in its repair. New work from Jin and colleagues, however, does not support these findings. The discrepancies seem in part to be related to the way that pericardial cavity macrophages were tracked. In the Immunity study, pericardial cavity macrophages were tracked by labeling with fluorescent beads or transplantation of trackable pericardial cavity macrophages into the recipient mice. Both of these strategies showed the cells entering the myocardium after infarction.

Cindy St. Hilaire: However, in the Circ Research study, mice were engineered to have trackable endogenous pericardial cavity macrophages. Using these animals, the team found that while pericardial cavity macrophages did migrate to the surface of the heart, they did not significantly penetrate the tissue. Further discrepancies between the studies were apparent in loss of function experiments. Where the initial study found pericardial cavity macrophage loss led to increased myocardial fibrosis and left ventricle stiffness, the new study did not. Because myocardial infiltration of pericardial cavity macrophages represents a paradigm shift in heart injury research, the authors say its existence requires rigorous testing and for now at least, it remains debatable.

Cindy St. Hilaire: The June 10th issue of Circ Research is our Compendium on Basic ModelsoOf Cardiovascular Diseases. The articles in this compendium are Illuminating the Mechanisms Underlying Sex Differences in Cardiovascular Disease by Carrie Weiss and Karen Rue, Animal Models to Study Cardiac Arrhythmias by Daniel Blackwell and Bjorn Knollmann, Animal Models of Exercise from Rodents to Pythons by Margaret Hastings, Anthony Rosenzweig and colleagues, Animal Models of Atherosclerosis Supportive Notes and Tricks of the Trade by Anton Gastera and Goran Hanson and colleagues, Heart Failure with Preserved Ejection Fraction, Heterogeneous Syndrome, Diverse Preclinical Models by Jason Roh and colleagues, Large and Small Animals of Heart Failure with Reduced Ejection Fraction by Patrick Pilz, Ronglih Liao and colleagues, CRISPR Modeling and Correction of Cardiovascular Disease by Ning Liu and Eric Olson, Animal Models of Cardiovascular Complications of Pregnancy by Zoltan Arany, Denise Hilfiker-Kleiner and S Ananth Karumanchi, Animal Models of Dysregulated Cardiac Metabolism by Heiko Bugger, Nicole Burn and Dale Abel, Biomedical Imaging and Experimental Models of Cardiovascular Disease by Marielle Scherrer-Crosbie and David Sosnovik, Zebrafish Models of Cardiac Disease from Fortuitous Mutants to Precision Medicine by Juan Gonzalez-Rosa and Cellular and Engineered Organoid Cardiovascular Models by Dilip Thomas and Joe Wu and colleagues.

Nikki Purcell: Hi, I'm Dr Nicole Purcell, Associate Professor in the Cardiovascular Division at Huntington Medical Research Institute and today Dr Mark Feinberg, Dr Rulin Zhuang and Dr Jingshu Chen from Brigham and Women's Hospital in Harvard Medical School are with me to discuss their study, Perivascular Fibrosis Is Mediated by a KLF10-IL-9 Signaling Access in CD4 T-Cells in our May 27th issue of Circulation Research. Thank you for joining me today.

Mark Feinberg: Thanks for having us. We're glad to be here.

Rulin Zhuang: Thank you.

Nikki Purcell: There were a lot of authors involved in this study, and while all could not join us today, I appreciate you taking the time to discuss your findings. So, your paper is dealing with vascular disease, often associated with elevated blood pressure or hypertension. A hallmark of advanced vascular diseases fibrosis is in the heart. When we talk about fibrosis, most investigators would think of interstitial fibrosis, but your paper focuses on perivascular. So Dr Feinberg, what is perivascular fibrosis and what led you to focus in hypertension?

Mark Feinberg: Thanks, it's a great question. So perivascular fibrosis is characterized by an increased accumulation of connective tissue around blood vessels. There are many cell types that contribute to this process. Fibroblasts, obviously, that produce a lot of extracellular matrix and a wide range of collagens, but other cell types, muscle cells, which are sensitive to humoral factors, Ang-2, endothelial and other cytokines and growth factors, and more recently getting more attention, our immune cells, including infiltrated T-cells, which are important mediators of crosstalk between fibroblasts and extra matrix that actively modulate this fibrotic response. Clinically, perivascular fibrosis is a hallmark of several age related conditions that we see in our patients, hypertension, diabetes, chronic kidney disease, really all are involved with extensive extracellular remodeling. Many of our patients with elderly patients with hypertension have left ventricular hypertrophy, stiff heart and pure diastolic dysfunction, as well as arterial stiffness, which can contribute to a range of diseases from heart failure, MI, stroke and organ damage and including kidney disease.

Mark Feinberg: So we actually started off wondering if there are any key transcription factors that may be involved in CD-4 T cell effector functions, given potential role of CD-4 T cells in this hypertensive response, and that perhaps may underlie the development of blood pressure and organ injury. With that idea, if you can understand a signaling pathway, perhaps it might impact the development of blood pressure, cardiovascular modeling, particularly with interstitial fibrosis and end organ injury. To be honest, we did not expect to find a factor that regulated perivascular fibrosis and end organ damage, but had no effect on blood pressure or interstitial fibrosis. This was a real surprise in the subject of the paper.

Nikki Purcell: Like you said, the importance of T cells and perivascular fibrosis falling hypertension with Ang 2 infusion has recently been demonstrated, but you know, you guys were really focusing on what was the mechanism, trying to understand that. So, Dr Feinberg, can you elaborate on why you chose to focus on the CD-4+ T cells and hypertension in particular, how it came about the transcription factor, Kruppel like factor 10 or KLF 10?

Mark Feinberg: Yeah. Great question. So we over accumulating studies many years now that CD-4 T cells play a particular important role, mediating hypertension, and a variety of preclinical models. For example, studies from David Harrison's or Steven Crowley's groups perform some classical experiments using immunocompromised mice. These are either RAG1 or SCID mice, which as, you know, have defective T or B cells. What's really interesting about those seminal papers was one that Ang2 mediated increase in blood pressure and the associated cardiac and kidney injury in these mice was severely blocked and two only when there was adoptive transfer of CD-4 T cells did that restore these deleterious effects in response to Ang2, indicating really for the first time that CD-4 T cells are key mediators of blood pressure and organ injury, and predominantly they focus on interstitial fibrosis and remodeling. However, the factors that mediate the CD-4 T cell effect on end organ damage or blood pressure really have been poorly defined over the years.

Mark Feinberg: And so work from our group and others have identified over several years a transcription factor called KLF10 or Kruppel like factor 10 belongs to a family about 17 total Kruppel like factors. This one is expressed highly in CD-4 T cell subsets, both factors and T regulatory cells and work from our group and actually those are others have, have shown that KLF10 can regulate T cell factors. They're more hyperactivated and also the T regulatory subsets don't exhibit what we call immunosuppressive or anti-inflammatory properties. We've shown this now in the context of athero, we've developed CD-4 specific health and knockout mice, and when placed on a high fat diet, those mice developed obesity and insulin resistance. However, the role for KLF10 in CD-4 T cells and hypertension really was unknown and really came into this thinking that this was going to play a role in development of blood pressure and interstitial fibrosis. So it was, again, a real surprise for us.

Nikki Purcell: I'm glad you mentioned that. Dr Zhuang, given the role of the CD-4T+ cells in controlling hypertension, you would've expected the blood pressure to be increased in your CDF4KLF10 knockout mice, but surprisingly, there was no difference. Can you tell us why you think this happened?

Rulin Zhuang: Yeah, that's true. When we found severe cardiovascular fibrosis and vascular remodeling in TKO mice, after Ang2 infusion, the first potential explanation comes to us that there should be a difference in blood pressure, but we didn't find any difference in after 28 days and even 42 days after Ang2 treatment. So previous literatures did indicate that hypertension and is related cardiovascular injury could result from other forms of mediators, which is independent of blood pressure. Which suggests that blood pressure alone perhaps is not sufficient to predict end organ damage in hypertension. You know, it is because of the lack of difference in blood pressure that allow us to explore another molecule insights into perivascular fibrosis.

Nikki Purcell: So Dr Chen, you found that the CD-4 positive KLF10 knockout mice had perivascular fibrosis in multiple organs, both the aorta, hearts and kidneys following Ang2 infusion. How did you identify that IL-9 was mediating these effects on perivascular fibrosis?

Jingshu Chen: Actually after we found the phenotype in Ang2 treated TKO mice, we start to find some possible mediators involved in these multiple organ perivascular fibrosis. Firstly, we detect the expression of angiotensin to receptors in CD-4 positive T Cells, but no difference was observed between TKO and our Cre mice. KLF10 also have another name, which is TIEG1, TGFbeta Inducible Early Gene-1. So next we checked the TGFbeta signaling. But we didn't find any difference in the rational level of TGFbeta one to three between Cre and knockout mice in the CD-4+ T cells that made it. Also, we didn't find any impact of calcium in TGFbeta signaling in vivo supported by our different RNA seek dataset for some pathway analysis. We performed calcium flux profiling from our Ang2 treated TKO and Cre mice. Although we do find some cytokines, slightly changed TKO mice to treatment, IL-9 nine was the only one significantly increase dramatically in both male and female TKO miceIL-9 nine was reported in the regulation of the immune responses and played a pro fibrotic role in lung fibrosis and liver fibrosis. We'll assume that perhaps IL-9 contributes to perivascular fibrosis. We gave the recombinant IL-9, our control mice, which have a less perivascular fibrosis after Ang2 treatment. After we giving them recombinant IL-9, we do found more perivascular fibrosis, which is efficiently phenocopy what we observed in our transgenic mice. Also, our further study found that calcium could bind into IL-9 promoter and interact with HTAC-1 to inhibit IL-9 activation. That is where we make a conclusion that we consider that calcium deficient C4 positive release more IL-9 that introduce perivascular fibrosis.

Mark Feinberg: I might add that, the discovery of this phenotype was almost missed. And I think it was interesting when Rulin described some of the initial H and E from the hearts of these mice and remember him saying there wasn't a lot of interstitial fibrosis, but there was lots of thickening outside the blood vessel wall. And what was interesting is that several of the aortas didn't show this, but it turned out those aortas Rulin had actually stripped for different reasons. And then when he repeated it without stripping and looking at all the organs, aorta, heart, kidney, you could tell easily who was the knockout, just blank, looking at the H and E slide. And he searched very hard for interstitial fibrosis. I actually had a colleague, Rick Mitchell who's a cardiac pathologist at the Brigham, review these slides in a blinded manner who, who verified that there was a lot of perivascular fibrosis in multiple organs, but no clear difference in interstitial fibrosis. That made us really excited about our new pathway to explore.

Nikki Purcell: Nice. That leads you nice that you were talking about the strip because Dr Zhuang and Chen, you had through your RNA seek data found that calcium signaling were dominantly upregulated in those nonstripped aorta. That's the perivascular adventitia tissue wasn't removed in those, in the CD-4 + KLF10 knockout mice after Ang2 treatment. To further investigate calcium signal involved in the fibroblast and myophytes differentiation, you had gone and nicely isolated primary fibroblasts from these blood vessels. And that can be quite tricky. So can you tell us about what method was used to purify these fibroblasts for this study?

Jingshu Chen: Yeah, it's actually very interesting progress for isolating those because when we search literatures, there's actually no well-established method to isolate from the aortas. We actually go from the aorta digestion to make sure we have a very good viability of the digestive cells. And then after that, Dr Zhuang and I kept discussing at lab, like why we just use the BES to isolate it's use the antibody, find the BES to isolate the fibroblasts. We tried a lot of methods and then finally we find optimize the protocol by use of magnetic BES these, but also to remove a lot of other cell types and to make sure we get the good purity of the fibroblasts. It's a very nice protocol and we actually published in the Atherosclerosis journal. It is already online. So I hope it can benefit the field. Anyone who can use it.

Nikki Purcell: Yeah. It's these beautiful pictures that you've got from that isolation in the paper. You used both the cells as well as the tissue for several RNA Seq overlapping data sets in this manuscript. You used those stripped and non-stripped aortas from the KD-4 + KLF10 knockout Cre mice exposed to Ang2. But then as you talked about Dr Zhuang, you also use from a group of Ang2 to treated mice that received that anti IL-9 monoclonal antibody. What are some of the main findings that you found from these dataset that you'd like others to know?

Jingshu Chen: Followed by the discussion, we are having the non-stripped and stripped aortas at very first, we sent off on the stripped aortas for sequencing, and we don't find a lot of genes are regulated actually only 200, 300 genes were regulated. It comes to the question actually, the perivascular fibrosis happened in adventitia. We sent out again for the nonstripped aorta, which has a perivascular fibrosis areas included. And luckily, we find like thousands of genes were regulated and a lot of the genes by perform the gene oncology pathway analysis, we found, calcium pathways and fibrosis are very enriched for the top 10 significant pathways.

Nikki Purcell: Nice. And so once you found that you were very interested and you were able to isolate these primary fibroblasts from your KLF10 knockout mice, you then performed single cell RNA sequencing on these. Were there specific fibroblasts populations of interest that were driving this perivascular fibrosis that you found?

Jingshu Chen: Yes. The single cell RNA Seq we perform as we digest the whole aorta, including the adventitia tissues and then make a cell suspension center for Seq. And the beauty and the privilege of the single cell is you actually can cluster different cell types. And then you can extract only the fibrous part from the whole aortic cells. And because we perform the single cell, including 50,000 cells, it's a very good population for us to look into. And by extract only the fibroblast clusters, we actually reclustered and find, there's actually nine clusters within this fibroblast clusters and followed by that we actually perform a very interesting analysis called RNA velocity. It is actually measuring or calculating the splices and unspliced mRNAs. And then by using this analysis can predict the potential future directions of this, the cells. And by utilizing that analysis, we actually found actually fibroblast original type can gradually progress to a more fibrosis or a mild fibrous type by looking at the splices and unspliced ratio. And which is the picture we showed in the manuscript.

Mark Feinberg: I might add that I was really impressed with the single cell seq. When you look at the vascular fibrosis, it looks like chaos. And the single cell seq provides a fair amount of order that I had not anticipated. We would be able to appreciate what I mean by that is Jingshu was able to characterize these nine subgroups and with the velocity analysis, prioritize them in terms of who are the major drivers. And this, for example, the subgroup eight was highly expressed in this collagen eight, A1 for example, that we think is a pathological fibroblasts whereas the canonical fibroblasts markers were in this earlier fibroblast subgroup one, and there's many other fibroblast cellular identities in between, including those that have endothelial mesenchymal markers and many others. We've learned quite a bit from it. And I have to say for others, it might be interested it's worth the investment, because it provides clarity.

Nikki Purcell: Thank you. So because of the heterogeneity, was there any sex differences between your male and female knockout mice on hypertension and this perivascular fibrosis that you saw?

Mark Feinberg: Phenotypically we did not notice any difference. The knockout females do not similar to the males they don't have increased blood pressure. They still develop accelerated perivascular fibrosis and many of the IL-9 signaling pathway. We didn't just because of cost issues, I think did not send out for the female single cell seek just because the phenotype was similar. But it's a great question. We, we probably should just to verify things.

Nikki Purcell: So what are the translational implications of your findings and how might you use this for treating vascular fibrotic diseases?

Mark Feinberg: We hope this study highlights the importance that hypertension is clearly a heterogeneous disease and multiple signaling pathways are likely responsible separately for different phenotypic manifestations of an organ disease. In particular, we show that perivascular fibrosis and interstitial fibrosis are distinct and are controlled by the former, by the CD-4, KLF10, IL-9 signaling pathway. And this may be leveraged for therapeutic benefit someday in patients. While early, these preclinical studies, we hope will inform the potential feasibility of considering IL-9 neutralizing antibodies, for example, in a proof of concept study in patients with uncontrolled or what we call refractory hypertension or chronic kidney disease that have a lot of perivascular fibrosis and that maybe that would improve or benefit the end organ damage that unfortunately occurs in those patients.

Nikki Purcell: What's on the horizon in the lab, what future studies are needed or have come from this work that you'd like to tell us about?

Mark Feinberg: There's actually a lot to do. We have some preliminary data that suggests that these CD-4 T cells probably there's a lot of crosstalk, not just with fibroblasts, but between muscle cells and endothelial cells. We show in the supplement of this paper that there's impairment of some muscle cell dependent, vaso relaxation and contraction in myograph studies. We have some preliminary data to suggest that endothelial dependent vaso relaxations also ongoing. The big question is how does KLF10 be an IL-9, maybe in a parakin manner impact the progression of disease in the intima. Where we actually spend most of the time in the lab thinking about the inner most lining of the blood vessel wall. This was a real treat to think about it from outside in. That's a real major focus in the lab is trying to understand the signaling pathway and maybe there's some crosstalk with IL-9 and other factors that control KLF10.

Jingshu Chen: For me, it's because we have seen this heterogeneities of the aortic cells using single cell. It's actually a bunch of, for us to do, like we can utilize the single cell technology to see how IL-9 is affecting the aortic walls for example, the fibroblasts, and also we can be more like to do a cell-cell talk like how endothelial cells, fibroblasts or other immune of key cells, especially like KLF10 is knocked out in T cells, how the cells talk and how we can observe this by using the data mining technique.

Rulin Zhuang: Actually in our study we've showed in the last figure is about the visualization of the IL-9 and we do found there it's reversed the angio to induce the fibrovascular fibrosis. The actually previous paper showed IL-9 can treat liver and heart and lung fibrosis. We assume that is probably IL-9 is a kind of profibrotic cytokine and can be evaluated further in the future to seeing other fibrosis like heart, interstitial maybe, I don't know, but it's a good way to detect.

Nikki Purcell: Great. Thank you so much for joining me today, Dr Feinberg, Zhuang and Chen, and for discussing your exciting findings and I look forward to seeing your future work. Thank you.

Mark Feinberg: Thanks so much for having us.

Cindy St. Hilaire: That's it for highlights from the May 27th and June 10th issues of Circulation Research. Thank you for listening. Please check out the CircRes Facebook page and follow us on Twitter and Instagram with the handle @CircRes and #DiscoverCircRes. Thank you to our guest host Dr Nikki Purcell and our interviewees, Dr Mark Feinberg, Dr Rulin Zhuang and Dr Jingshu Chen. This podcast is produced by Ishara Ratnayaka, edited by Melissa Stoner and supported by the editorial team of Circulation Research. Some of the copy text for the highlighted articles is provided by Ruth Williams. I'm your host, Dr Cindy St. Hilaire and this is Discover CircRes, your on the go source for the most exciting discoveries in basic cardiovascular research. This program is copyright of the American Heart Association, 2022. The opinions expressed by speakers in this podcast are their own and not necessarily those of the editors or of the American Heart Association. For more information visit ahajournals.org.

This month on Episode 36 of Discover CircRes, host Cynthia St. Hilaire highlights original research articles featured in the April 29 and May 13 issues of Circulation Research. This episode also features a conversation with Dr Patricia Nguyen and Jessica D'Addabbo from Stanford University about their study, Human Coronary Plaque T-cells are Clonal and Cross-React to Virus and Self.

Article highlights:

Zanoli, et al. COVID-19 and Vascular Aging

Wang, et al. JP2NT Gene Therapy in a Mouse Heart Failure Mode

Harraz, et al. Piezo1 Is a Mechanosensor in CNS Capillaries

Zhao, et al. BAT sEVs in Exercise Cardioprotection

Cindy St. Hilaire: Hi, and welcome to Discover CircRes, the podcast of the American Heart Association's journal, Circulation Research. I'm your host, Dr Cyndy St. Hilaire, from the Vascular Medicine Institute at the University of Pittsburgh. And today, I'll be highlighting the articles from our April 29th and May 13th issues of Circulation Research. I also will speak with Dr Patricia Nguyen and Jessica D'Addabbo from Stanford University about their study, Human Coronary Plaque T-cells are Clonal and Cross-React to Virus and Self.

Cindy St. Hilaire: The first article I want to share is titled Vascular Dysfunction of COVID 19 Is Partially Reverted in the Long-Term. The first author is Agostino Gaudio and the corresponding author is Luca Zanoli. And they're from the University of Catania. Cardiovascular complications, such as endothelial dysfunction, arterial stiffness, thrombosis and heart disease are common in COVID 19. But how quickly such issues resolve, once the acute phase of the illness has passed, remains unclear. To find out, this group examined aortic and brachial pulse wave velocity, and other measures of arterial stiffness in 90 people who, several months earlier, had been hospitalized with COVID 19. These measurements were compared with data from 180 controls, matched for age, sex, ethnicity and body mass index, whose arterial stiffness had been assessed prior to the pandemic. 41 of the COVID patients were also examined 27 weeks later to assess any changes in arterial stiffness over time. Together, the data showed arterial stiffness was higher in COVID patients than in controls. And though it improved over time, it tended to remain higher than normal for almost a year after COVID.

Cindy St. Hilaire: This finding could suggest residual structural damage to the arterial walls or possibly, persistent low-grade inflammation in COVID patients. Either way, since arterial stiffness is a predictor of cardiovascular health, its potential longterm effects in COVID patients deserves further longitudinal studies.

Cindy St. Hilaire: The second article I want to share is titled Gene Therapy with the N-Terminus of Junctophilin-2 Improves Heart Failure in Mice. The first author is Jinxi Wang and the corresponding author is Long-Sheng Song from the University of Iowa. Junctophilin-2 is a protein with a split personality. Normally, it forms part of the heart's excitation contraction coupling machinery. But when the heart is stressed, JP2 literally splits in two, and sends its N-terminal domain, JP2NT, to the nucleus, where it suppresses transcription of genes involved in fibrosis, hypertrophy, inflammation and other heart failure related processes. However, if this stress is severe or sustained, the protective action of JP2NT is insufficient to halt the progressive failure. This group asked. "What if this N-terminal domain could be ramped up using gene therapy to aid a failing mouse heart?"

Cindy St. Hilaire: To answer this question, they injected adenoviral vectors encoding JP2NT into mice either before or soon after transaortic constriction, or TAC, tack, which is a method of experimentally inducing heart failure. They found, in both cases, that the injected animals fared better than the controls. Animals injected before TAC showed less severe cardiac remodeling than control mice, while those treated soon after TAC exhibited slower loss of heart function with reduced ventricle dilation and fibrosis. These data suggest that supplementing JP2NT, via gene therapy or other means, could be a promising strategy for treating heart failure. And this data provides a basis for future translational studies.

Cindy St. Hilaire: The third article I want to share is titled Piezo1 Is a Mechanosensor Channel in Central Nervous System Capillaries. The first and corresponding author is Osama Harraz from the University of Vermont. Neurovascular coupling is the process whereby transient activation of neurons leads to an upsurge in local blood flow to accommodate the increased metabolic needs of the cell. It's known that agents released from active neurons trigger changes in local capillaries that prompt vasodilation, but how these hemodynamic changes are sensed and controlled is not entirely clear. This group suspected that the mechanosensory protein Piezo1, a calcium channel that regulates dilation and constriction of other blood vessels, may be involved. But whether Piezo1 is even found in the microcirculation of the CNS was unknown. This group shows that Piezo1 is present in cortical capillaries of the brain and the retina of the mouse, and that it responds to changes in blood pressure and flow.

Cindy St. Hilaire: Ex vivo preparations of mouse retina showed that experimentally induced changes in hemodynamics caused calcium transients and related currents within capillary endothelial cells, and that these were dependent on the presence of Piezo1. While it is not entirely clear how Piezo1 influences cerebral blood flow, its pressure induced activation of CNS capillary endothelial cells suggest a critical role in neurovascular coupling.

Cindy St. Hilaire: The last article I want to share is titled Small Extracellular Vesicles from Brown Adipose Tissue Mediate Exercise Cardioprotection. The first authors are Hang Zhao and Xiyao Chen. And the corresponding authors are Fuyang Zhang and Ling Tao from the Fourth Military Medical University. Regular aerobic exercise is good for the heart and it increases the body's proportion of brown adipose tissue relative to white adipose tissue. This link has led to the idea that brown fat, possibly via its endocrinal activity, might somehow contribute to exercise related cardioprotection. Zhao and colleagues now show that, indeed, brown fat produces extracellular vesicles that are key to preserving heart health. While mice subjected to four weeks of aerobic exercise were better protected against subsequent heart injury than their sedentary counterparts, blocking the production of EVs prior to exercise significantly impaired this protection. Furthermore, injection of brown fat derived EVs into the hearts of mice lessened the impact of subsequent cardiac injury.

Cindy St. Hilaire: The team went on to identify micro RNAs within the vesicles responsible for this protection, showing that the micro RNAs suppressed an apoptosis pathway in cardiomyocytes. In identifying mechanisms and molecules involved in exercise related cardio protection, the work will inform the development of exercise mimicking treatments for people at risk of heart disease or who are intolerant to exercise.

Cindy St. Hilaire: Lastly, I want to bring up that the April 29th issue of Circulation Research also contains a short Review Series on pulmonary hypertension, with articles on: The Latest in Animal Models of Pulmonary Hypertension and Right Ventricular Failure, by Olivier Boucherat; Harnessing Big Data to Advance Treatment and Understanding of Pulmonary Hypertension, by Christopher Rhodes and colleagues; New Mutations and Pathogenesis of Pulmonary Hypertension: Progress and Puzzles in Disease Pathogenesis, by Christophe Guignabert and colleagues; Group 3 Pulmonary Hypertension From Bench to Bedside, by Corey Ventetuolo and colleagues; and Novel Approaches to Imaging the Pulmonary Vasculature and Right Heart, by Sudarshan Rajagopal and colleagues; and Understanding the Pathobiology of Pulmonary Hypertension Due to Left Heart Disease, by Jessica Huston and colleagues.

Cindy St. Hilaire: Today, Dr Patricia Nguyen and Jessica D'Addabbo, from Sanford University, are with me to discuss their study, Human Coronary Plaque T-cells are Clonal and Cross-React to Virus and Self. And this article is in our May 13th issue of Circulation Research. So, Trisha and Jessica, thank you so much for joining me today.

Jessica D'Addabbo: Thank you for having us.

Patricia Nguyen: Yes. Thank you for inviting us to your podcast. We're very excited to be here.

Cindy St. Hilaire: Yeah. And I know there's lots of authors involved in this study, so unfortunately we can't have everyone join us, but I appreciate you all taking the time.

Patricia Nguyen: This is like a humongous effort by many people in the group, including Roshni Roy Chowdhury, and Xianxi Huang, as well as Charles Chan and Mark Davis. So, we thank you.

Cindy St. Hilaire: So atherosclerosis, it stems from lipid deposition in the vascular wall. And that lipid deposition causes a whole bunch of things to happen that lead to a chronic inflammatory state. And there's many cells that can be inflammatory. And this study, your study, is really focusing on the role of T-cells in the atherosclerotic plaque. So, before we get into the nitty gritty details of your study, can you share with us, what is it that a T-cell does normally and what is it doing in a plaque? Or rather, let me rephrase that as, what did we know a T-cell was doing in a plaque before your study?

Patricia Nguyen: So, T-cells, as you know, are members of the adaptive immune system. They are the master regulators of the entire immune system, secreting cytokines and other proteins to attract immune cells to a diseased portion of the body, for example. T-cells have been characterized in plaque previously, mainly with immunohisto chemistry. And their characterization has also been recently performed using single cell technologies. Those studies have been restricted to mainly mirroring studies, studies in mice in their aortic walls, in addition to human carotid arteries. So, it is well known that T cells are found in plaque and a lot of attention has been given to the macrophage subset as the innate immune D. But let's not forget the T-cell because they're actually composed about... 50% in the plaque are T-cells.

Patricia Nguyen: And we were particularly interested in the T-cell population because we have a strong collaboration with Dr Mark Davis, who's actually the pioneer of T-cell biology and was the first to describe the T-cell receptor alpha beta receptor in his lab in the 1970s. So, he has developed many techniques to interrogate T-cell biology. And our collaboration with him has allowed us and enabled us to perform many of these single cell technologies. In addition, his colleague, Dr Chen, also was pivotal in helping us with the interrogation and understanding of the T-cells in plaque.

Cindy St. Hilaire: And I think one of the really neat strengths of your study is that you used human coronary artery plaques. So, could you walk us through? What was that like? I collect a lot of human tissue in my lab. I get a lot of aortic valves from the clinic. And it's a lot of logistics. And a lot of times, we're just fixing them, but you are not just fixing them. So, can you walk us through? What was that experimental process from the patient to the Petri dish? And also, could you tell us a little bit about your patient population that you sampled from?

Jessica D'Addabbo: So, these were coronary arteries that we got from patients receiving a heart transplant. So, they were getting a heart transplant for various reasons, and we would receive their old heart, and someone would help us dissect out the coronary arteries from these. And then, we would process each of these coronary arteries separately. And this happened at whatever hour the hearts came out of the patient.

Jessica D'Addabbo: So sometime, I was coming in at 3:00 AM with Dr Nguyen and we would be working on these hearts then, because we wanted the samples to be as fresh as possible. So, we would get the arteries. We would digest out the tissue. And then, we would have certain staining profiles that we wanted to look at so that we could put the cells on fax to be able to sort the cells, and then do all the downstream sequencing from there.

Cindy St. Hilaire: So, in terms of, I don't know, the time when you get that phone call that a heart's coming in to actually getting those single cells that you can either send a fax or send a sequencing, how long did that take, on a good day? Let's talk only about good days.

Jessica D'Addabbo: Yeah. A lot of factors went into that, sometimes depending on availability of things. But usually, we were ready with all of the materials in advance. So, I'd say it could be anywhere from six to 12 hours, it would take, to get everything sorted. Then, everything after that would happen. But that was just that critical period of making sure we got the cells fresh.

Patricia Nguyen: So we have to credit the CT surgeons at Stanford for setting up the program or the structure, infrastructure, that enables us to obtain this precious tissue. That is Jack Boyd and Joseph Woo of CT surgery. So, they have enabled human research on hearts by making these tissues available. Because as you know, a transplant... They can say the transplant's happening at 12:00 AM, but it actually doesn't happen until 4:00 AM. And I think it's very difficult for a lab to make that happen all the time. And I think having their support in this paper was critical. And this has allowed us, enabled us, to interrogate kind of the spectrum of disease, especially focusing on T-cells, which are... They make a portion of the plaque, but the plaque itself has not like a million cells that are immune. A lot of them are not immune. So, enabling us to get the tissue in a timely fashion where they're not out of the body for more than 30 minutes enables us to interrogate these small populations of cells.

Cindy St. Hilaire: That's actually the perfect segue to my next question, which is, how many cells in a plaque were you able to investigate with the single cell analysis? And what was the percentage again of the T-cells in those plaques or in... I guess you looked at different phases of plaque. So, what was that spectrum for the percentage of T-cells?

Patricia Nguyen: So, for 10X, for example, you need a minimum of 10,000 captured cells. You could do less, but the utility of the 10X is maximized with 10,000. So, many times before the ability to multiplex these tissues, we were doing like capturing 5,000 for example. And the number of cells follows kind of the disease progression, in the sense that as a disease is more severe, you have more immune cells, in general. And it kind of decreases as it becomes more fibrotic and scarred, like calcified. So, it was a bit challenging to get very early just lipid-only cells. And a lot of those, we captured like 3000 or something like that. And efficiency is like 80% perhaps. So, you kind of capture…

Cindy St. Hilaire: And also, how many excised hearts are going to have early athero? So, it's...

Patricia Nguyen: Well, there are... nonischemics will have...

Cindy St. Hilaire: Oh, okay. Okay.

Patricia Nguyen: So, the range was nonischemic to ischemic.

Cindy St. Hilaire: Oh great.

Patricia Nguyen: So, about a portion... I would say one third of the total heart transplants were ischemic. And a lot of them were non ischemic. But as you know, the nonischemic can mix with ischemia. And so, they could have mild to moderate disease in the other arteries, for example, but not severe like 70%/90% obstruction.

Cindy St. Hilaire: Wow. That's so great. That's amazing. Amazing sample size you have. So T-cell, it's kind of an umbrella term, right? There's many different types of T-cells. And when you start to get in the nitty gritty, they really do have distinct functions. So, what types of T-cells did you see and did you focus on in this study?

Jessica D'Addabbo: So, the two main types of T-cells are CD4 positive T-cells and CD8 positive T-cells. And we looked at both of these T-cells from patients. We usually sorted multiple plates from each. And then, with 10X, we captured both. But our major finding was actually that the CD8 positive t-cell population was more clonally expanded than the CD4 population, which led us to believe that these cells were more important in the coronary artery disease progression and in the study that we were doing because for a cell to be clonally expanded, it means it was previously exposed to an antigen. And so, if we're finding these T-cells that are clonally expanded in our plaques, then we're hypothesizing that they were likely exposed to some sort of antigen, and then expanded, and then settled into the plaque.

Cindy St. Hilaire: And when you're saying expansion, are you talking about them being exposed to the antigen in the plaque and expanding there? Or do you think they're being triggered in the periphery and then honing in as a more clonal population?

Patricia Nguyen: So, that's a great question. And unfortunately, I don't have the answer to that. So basically-

Jessica D'Addabbo: Next paper, next paper.

Patricia Nguyen: Exactly. So, we... Interesting to expand on Jessica's answer. Predominantly what was found, as you said, was memory T-cells, so memory T-cells expressing specific markers, so memory versus naive. And these were effector T-cells. And memory meaning they were previously expanded by antigen engagement, and just happened to be in the plaque for whatever reason. We do not know why T-cells specifically are attracted to the plaque, but they are obviously there. And they're in a memory state, if you will. And some of them did display activation markers, which suggested that they clonally expanded to an antigen. What that antigen is, is the topic of another paper. But certainly, it is important to understand that these patients that we recruit, because they were transplant patients, they're not actively infected, right? That is a exclusionary criteria for transplants, right?

Patricia Nguyen: So, that means these T-cells were there for unclear reasons. Why they're there is unclear. Whether they are your resident T-cells also is unclear, because the definition of resident T-cell still remains controversial. And you actually have to do lineage tracking studies to find out, "Okay, where... Did they come from the bone marrow? Did they come from the periphery? How did they get there?" Versus, "Okay. They were already there and they just expanded, for whatever reason, inside the plaque."

Cindy St. Hilaire: So, your title... It was a great title, with this provocative statement, "T-cells are clonal and cross react to virus and self." So, tell us a little bit more about this react to virus and self bit. What did your data show?

Jessica D'Addabbo: So, because of the way we sequenced the T-cell receptor, we were able to have paired alpha and beta chains. And because we knew the HLA type of the patients, we were able to put the sequences that we got out after we sequenced these through an algorithm called GLIPH, which allows us to look at the CDR3 region of the T cell receptor, which is the epitope binding region. And there are certain peptide. They're about anywhere from three to four amino acids long. These are mapped to certain binding specificities to known peptides. And so, basically, we were able to look at which epitopes were most common in our plaques. And we found that after comparing these to other epitopes, that these were actually more binding to virus.

Patricia Nguyen: So let me add to what Jessica stated, and kind of emphasize the value of the data set, if you will. So, this is, I believe, the first study that provides the complete TCR repertoire of coronary plaque, and actually any plaque that I know of, which is special because we know that there is specificity of TCR binding. It's more complicated than the antibody that binds directly from B cells to the antigen, because the T-cells bind processed antigen. So, the antigens are processed by antigen presenting cells like Dendritic cells and macrophages. And they have a specific HLA MHC class that they need to present to. And they need both arms, the antigen epitope and the MHC, to activate the T-cell. So unfortunately, it's not very direct to find the antigen that is actually activating the T-cell because we're only given a piece of it. Right?

Patricia Nguyen: But we have provided a comprehensive map of all the TCRs that we find in the plaque. And these TCRs have a sequence, an immuno acid sequence. And luckily, in the literature, there is a database of all TCR specificities. Okay. So, armed with our TCR repertoire, we can then match our TCR repertoire with an existing database of known TCR specificities. Surprisingly, the matching TCRs are specific to virus, like flu, EBV and CMB. And also, because this was done in the era of COVID, we thought it would be important to look at the coronavirus database. We did find that there were matches to the coronavirus database. Even though our finding is not specific to SARS, it does lend to some potential mechanistic link there as well.

So, because this is all computational, it is important to validate. So, the importance of validation requires us to put the TCR alpha beta chain into a Jurkat cell, which is a T-cell line that does not have alpha beta chains on it, and then expose it to what we think is the cognate antigen epitote, with the corresponding HLA MHC APC. Because you don't have all those pieces, it will not work. Yes. So importantly, we did find that what we predicted to have the specificity of a flu peptide had specificity to a flu peptide.

Patricia Nguyen: So then, the important question was, "Okay, these patients aren't infected, right? Why are these things here? Is there a potential cross reactivity with self peptides?"

Patricia Nguyen: So luckily, our collaborator, Dr Charles Chan, was able to connect us with another computational algorithm that he was familiar with, whereby we were able to take the peptide sequences from the flu and match them with peptide sequencing from proteins that are self and ubiquitous. And we demonstrated, again, these T-cells were activated in vitro. That is why we concluded that there's a potential cross reactivity between self and virus that can potentially lead to thrombosis associated with viral infections. Of course, this all needs to be proved in vivo.

Cindy St. Hilaire: Sure, sure.

Patricia Nguyen: It's that first step for other things.

Cindy St. Hilaire: The other big immune cell that we know is in atherosclerotic plaques and that's macrophages. And they can help to present antigens and things like that. And they also help to chew up the necrotic bits. And so, do you think that this T-cell component is an earlier, maybe disease driving, process or an adaptive process that goes awry as a secondary event?

Patricia Nguyen: So, I'm a fan of the T-cell. So... I'm with team T cell. I would like to think that it is playing an active role in pathology in this case and not a reactive role, in the sense of just being there. I think that the T-cell is actively communicating with other cells within the plaque, and promoting pro fibrotic and pro inflammatory reactions, depending on the T-cell. So, a subset of this paper was looking at kind of the interactions between the T-cell and other cells within the plaque, like macrophages and smooth muscle cells. And as we know, T-cells are activated and they produce cytokines. Those cytokines then communicate to other cells. And we found that, computationally, when you look at the transcriptome, there is a pro-inflammatory signature of the T-cell that resides in the more complex stage. And then, there's an anti-inflammatory signature that kind of resides in the transition between lipid and fibro atheroma, if you will.

Cindy St. Hilaire: So, do you know, or is it known, how dynamic these populations are? Obviously, the hearts that you got, the samples you got, didn't have active infections. But do you know perhaps even how long ago they happened, or even how soon after there might be an infection or an antigen presented that you could get this expansion? And could that be a real driver of rupture or thrombosis?

Patricia Nguyen: So, in theory, you would suppose that T-cells expanding and dividing and producing more and more cytokines would then lead to more macrophages coming, more of their production of proteinases that destroy the plaque. Right? So yes, in theory, yes. I think it's very difficult to kind of map the progression of T cell clonality in the current model that we have, because we're just collecting tissues. However, in the future, as organoids become more in science and kind of a primary tissue, where we can... For example, Mark Davis is making organoids with spleen, and also introducing skin to that.

Patricia Nguyen: And certainly, we could think of an organoid involving the vasculature with immune cells introduced. And so, I think, in the next phase, project 2.0, we can investigate what... like over time, if you could model atherosclerosis and the immune system contribution, T-cells as well as macrophages and other immune cells, you can then kind of map how it happens in humans. Because obviously, mice are different. We know that mice... Actually, the models of transgenic mice do not rupture. It's very hard to make them rupture. Right?

Cindy St. Hilaire: Well, if you stop feeding them high fat diet, the plaque goes away.

Patricia Nguyen: For sure, for sure. So I think.. I mean, Mark Davis is a huge proponent of human based research, like research on human tissue. And as a physician scientist, obviously I'm more inclined to do human based research. And Jessica's going to be a physician someday soon. And I'm sure she's more inclined to do human based research. And certainly, the mouse model and in vitro models are great because you can manipulate them. But ultimately, we are trying to cure human diseases.

Cindy St. Hilaire: Mice are not little humans. That's what we say in my lab. I similarly do a lot of human based stuff and it's amazing how great mice are for certain things, but still how much is not there when we need to really fully recapitulate a disease model.

So, my last question is kind of regarding this autoimmune angle of your findings. And that is, women tend to have more autoimmune diseases than men, but due to the fact that you are getting heart transplants, you've got a whole lot more men in your study than women. I think it was like 31 men to four women. But, I mean, what can you do? It's the nature of heart transplants. But I'm wondering, did you happen to notice...Maybe the sample size perhaps is too small, but were there any differences in the populations of these cells between women and men? And do you think there could be any differences regarding this more prevalence of autoimmune like reactions in women?

Patricia Nguyen: So, that's an interesting question, but you hit it on the nose when you said "Your sample is defined mainly by men." And in addition, the samples that were women tend to have less disease. And they tend to be nonischemic in etiology. So, I think that kind of restricts our analysis. And perhaps, I guess, future studies could model using female tissues, for example, instead of only male. But the limitation of all human studies is sample availability. And perhaps, human organoid research can be less limited by that. And certainly, mouse research has become more evenly distributed of male and female mice.

Cindy St. Hilaire: Yeah. Suffice it to say, human research is hard, but you managed to do an amazing and really important study. It was really elegant and well done. Congratulations on what is an epic amount of time. 12-hour experiments are no joke, and really beautiful data. So, thank you so much for joining me today, Dr Nguyen and Miss almost Dr D'Addabbo. Congrats and I'm really looking forward to seeing your future work.

Jessica D'Addabbo: Thank you so much.

Patricia Nguyen: Thanks so much.

Jessica D'Addabbo: Thank you for having us. This is wonderful.

Cindy St. Hilaire: That's it for the highlights from the April 29th and May 13th issues of Circulation Research. Thank you so much for listening. Please check out the Circ Res Facebook page and follow us on Twitter and Instagram with the handle @Circres and #Discover CircRes. Thank you to our guests: Dr Patricia Nguyen, and soon to be Doctor, Jessica D'Addabbo, from Stanford University.

This podcast was produced by Ishara Ratnayaka, edited by Melissa Stoner, and supported by the editorial team of Circulation Research. Copy text for the highlighted articles was provided by Ruth Williams. I'm your host, Dr Cindy St. Haler. And this is Discover CircRes, you're on the go source for the most exciting discoveries in basic cardiovascular research. This program is copyright of the American Heart Association 2022. The opinions expressed by the speakers of this podcast are their own and not necessarily those of the editors or of the American Heart Association. For more information, visit aha journals.org.

This month on Episode 35 of Discover CircRes, host Cynthia St. Hilaire highlights two original research articles featured in the April 1 issue of Circulation Research, as well as highlights from the Stroke and Neurocognitive Impairment Compendium in the April 15th issue. This episode also features a conversation with Dr Shubing Chen and Dr Yuling Han from Weill Cornell Medical College to discuss their study, SARS-CoV-2 Infection Induces Ferroptosis of Sinoatrial Node Pacemaker Cells.

Article highlights:

Pabel, et al. Effects of Atrial Fibrillation on the Ventricle

Pattarabanjird, et al. P62-Mediated B1b Cell Atheroprotection

Iadecola, et al. Introduction to the Compendium on Stroke and Neurocognitive Impairment

Cindy St. Hilaire: Hi and welcome to Discover CircRes, the podcast of the American Heart Association's Journal, Circulation Research. I'm your host, Dr Cindy St. Hilaire, from the Vascular Medicine Institute at the University of Pittsburgh. And today I'm going to be highlighting articles from our April issues of Circulation Research.

I'll also speak with Dr Shubing Chen and Dr Yuling Han from Weill Cornell Medical College, and they're with me to discuss their study, SARS-CoV-2 infection induces ferroptosis of Sinoatrial node pacemaker cells.

Cindy St. Hilaire: The first article I want to share is titled, Effects of Atrial Fibrillation on the Human Ventricle. The first author is Steffen Pabel and the corresponding author is Samuel Sossalla and they're from Regensburg University. Atrial fibrillation, or AFib, is the most common form of heart arrhythmia. Patients with AFib may experience shortness of breath, dizziness and weakness. And they're also at risk for more life-threatening complications, such as clot-induced stroke and heart failure. Focusing on heart failure, this study investigated how disruptions to rhythm in the atria might lead to changes in the ventricular myocardium. The team studied ventricular muscle tissue from 24 patients with AFib and 31 without AFib. While the levels of fibrosis were equivalent in ventricular myocytes from both the AFib and the non AFib patients, other cellular features were distinct. For example, patients with AFib had reduced systolic calcium release, prolonged action potential duration and increased oxidative stress, compared with the non AFib patient controls. These differences were largely recapitulated in ventricular myocytes derived from human induced pluripotent stem cells that had been electrically stimulated to either mimic AFib or normal sinus rhythm. The results indicate that AFib affects the ventricles just as well as the atria and might therefore be best studied and treated with the whole heart in mind.

Cindy St. Hilaire: The second article I want to share is titled B-1b Cells Possess Unique bHLH-Driven P62-Dependent Self-Renewal and Atheroprotection. The first author is Tanyaporn Pattarabanjird and the corresponding author is Colleen McNamara, from the University of Virginia.

Atherosclerosis is a complex and dynamic chronic inflammatory condition. However, not all immune cells exacerbate this disease. Some immune cells are actively dampening the inflammation. B-1 cells are such cells that do this, and they produce IgM antibodies that bind cholesterol, preventing its uptake into macrophages and therefore limiting macrophage driven inflammatory responses. Increased number of B1 cells, therefore, might be atheroprotective. In mice, deletion of the transcription factor ID3 leads to a boost in B-1 cell IgM production.

Cindy St. Hilaire: In this work the authors investigated the molecular mechanism underlying this effect and found that upon deletion of ID3 in mice B-1b cells, the level of P62 protein was increased. B-1b cell proliferation was found to be dependent on P62 and over expression of P62 in mouse B-1b cells increased cell numbers, raised plasma IgM levels and importantly, ameliorated diet-induced atherosclerosis in animals. The team went on to show that people with an ID3 mutation had an unusually high level of serum IgM and B-1b cell P62. This suggests that results from mice may hold true for humans, and if so, could inform the development of immunomodulatory treatments for atherosclerosis.

Cindy St. Hilaire: So the April 15th issue of Circulation Research is our Stroke And Neurocognitive Impairment Compendium. The last Circulation Research Compendium on Stroke was published about five years ago. In this year Dr Costantino Iadecola, Dr Mark Fisher and Dr Ralph Sacco focused this update on advances made over the past five years, with a focus on topics that were not addressed in the previous compendium, that best reflect the leading edge of basic in clinical science related to cerebral vascular diseases. Seemant Chaturvedi, Brian Mac Grory and colleagues provide an overview of preventative strategies according to stroke mechanism, including stroke of unknown cause. And the challenges of stroke prevention with antithrombotic therapy and subjects with increased hemorrhage risk are also considered.

Cindy St. Hilaire: Stéphanie Debette and Hugh Markus provide an account of the most recent developments in the genetics of cerebrovascular diseases. The gut microbiota is another factor that has recently been linked to stroke risk and Pedram Honarpisheh, Louise McCullough and colleagues provide a comprehensive overview of the microbiology and the microbiota, and the influence that stroke risk factors exert on its composition and homeostatic relationship with mucosal surfaces. Karin Hochrainer and Wei Yang provide a systematic review of the large amount of data and stroke proteomic from animal models and human patients. Matthias Endres and colleagues cover the dramatic effect that innate and adaptive immunity exert on stroke risk and on acute brain damage and post stroke sequelae, such as post-stroke cognitive impairment and depression.

Cindy St. Hilaire: Manuela De Michele, Alexander Merkler and colleagues discuss the cerebral vascular diseases that have emerged as a frequent manifestation of the maladaptive immune response to severe SARS-CoV-2 infection. Jessica Magid-Bernstein and Lauren Sansing review the current concepts on epidemiology, risk factors in etiology, clinical features, as well as the medical and surgical interventions for cerebral hemorrhage. Yunyun Xiong and Marc Fisher cover the progress that has been achieved in the treatment of acute ischemic stroke and Natalie Rost and Martin Dichgans and colleagues address the long term impact of stroke on cognitive function, which is becoming a significant healthcare challenge in the world's aging population.

Cindy St. Hilaire: So today I have Dr Shubing Chen and Yuling Han from Weill Cornell Medical College. And they're with me to discuss their study SARS-CoV-2 infection induces ferroptosis of Sinoatrial node pacemaker cells. And this article is in our April 1st issue of Circulation Research. So thank you both for joining me today.

Shubing Chen: Thank you. It's really nice to join the program, and it's really a great honor.

Cindy St. Hilaire: It's a really great article. I'm so excited to talk about. So there's a lot of research happening regarding SARS-CoV-2 virus and the patients who are infected and have COVID-19. And this paper is focusing on the impact of viral infection on the heart and specifically on the sinoatrial node, which is the primary cardiac pacemaker that keeps our hearts beating. So I was wondering if you could tell us what led you to focus on this particular aspect of COVID-19 symptoms, and also how early in the pandemic did you start this?

Shubing Chen: Yeah, so we started working on SARS-CoV-2 through back to early 2020 when very unfortunately, New York City was a pandemic center and we had a lot of patients in the hospital unit, and also postdoc students working very hard in the lab. So that's the time we start working on SARS-CoV-2. And I was trained as a stem cell biologist. And what we're really interest is to set up a platform to basically understand which type of cells can be infected by SARS-CoV-2 and if they can, how they respond to SARS-CoV-2 infection. Not only for SARS-CoV-2, we sent it as like a viral infection platform, but SARS-CoV-2 is one of the virus we study now. And it's kind of very surprising. We have a pretty broad platform. We have a lung organoid, we have colon organoids, we have pancreas, we have cardiomyocytes, pacemaker cells. And as expected, we see lung can be infected like colon and because patient had GI tract, liver can be infected, but very surprisingly we see very high cardiomyocytes infection as well as pacemakers.

So as we'll know that still big controversy in the field, whether we can detect SARS-CoV-2 like viral protein or viral RA in the heart, in particular, cardiomyocytes. But I think now everyone agree that the cardiomyocytes really can be very well infected actually. Because it's very difficult to get the pacemaker tissue and the sinoatrial tissue from the COVID patient. So we collaborate with Dr Ben Andora's lab at NYU to get this hamster model. So we basically take SA tissue from hamster and then other colleagues basically did the section imaging, and we confirm that the hC4 polymerase cells can be infected by SARS-CoV-2. And at that time we start to learn a more clinical studies they report the COVID patient, they develop arrhythmia, or some other problem, not only with cardiomyocyte, as well as the conduction system. So at that time, that's the time that we say maybe we should do something on the pacemaker and focus on that. So that's how the project was developed.

Cindy St. Hilaire: That is so interesting. And so I know humans infected, like you just said with SARS-CoV-2, they can develop arrhythmias. What's that timeframe? Is there a common timeframe that this happens? Does it normally happen very close to the infection or only in later stage? What's that window of when these arrhythmias are happening?

Shubing Chen: At least based on the clinical study we show right now, actually the patient can develop acute arrhythmia. So it can be very soon after they developed symptom for COVID.

Cindy St. Hilaire: Wow. That's amazing. So you mentioned this, your study utilized a hamster model, which you actually don't see a lot of. Most studies use a lot of rats or most studies I'm familiar with, especially in Circulation Research, they use more rats or more mouse models. So what advantages does that hamster model have and why were you interested in using it?

Shubing Chen: Yeah, that's actually really specific for SARS-CoV-2. As SARS-CoV-2 mainly use ACE2 as a key entry factor to enter the cells. Of course, there's additional receptor, like neutrophils is one. Like all this enzyme involved, but human and mouse ACE2, they have very different structure. So the SARS-CoV-2 virus combine with human ACE2 very well but not mouse ACE2. So from the beginning, the rat and mouse was not used as a very good model to study SARS-CoV-2 infection. Of course there are other models, like knockin human ACE2 in the mouse and also like ACE2 transgenic mice. That's how different mouse model use. But hamster you don't need any modification, but they are very promising to SARS-CoV-2 infection. And so that's a reason we decide to use that as an animal model to basically run in parallel with our human stem cell model.

Cindy St. Hilaire: We joke in my lab, mice are not little humans, but it's really true in a lot of cases, they're beautiful models in so many ways, but then when they don't work, they really don't work.

Shubing Chen: Yeah. Before COVID every time when we try to talk about our human stem cell, derived cells, organoids as a disease model. People always ask, why do you want to work on human organoids? Right? It's that we have all these beautiful animal models like as you mentioned, mouse or rats, that's very broadly used. And we have to find different reasons. And now when we start working on SARS-CoV-2, which is very clear example, that mouse are not identical to human. Yeah.

Cindy St. Hilaire: Yeah. That's great. I love finding additional models to use that are the best one for the question. So in order to investigate, I guess kind of the mechanism of how this was happening in the SAN cells, the sinoatrial node cells, you had to develop a new differentiation protocol that took the human embryonic stem cells, I think it was the H9 line you used, and essentially differentiate that cell line into a sinoatrial node-like cell. So I was wondering if you could tell us a little bit about A) how did you figure out that protocol and B) how does it work?

Shubing Chen: So it's actually a long story to cell line.

Cindy St. Hilaire: We can condense it. Let's get-

Shubing Chen: At least based on the clinical study we show right now, actually the patient can. Let's condense it. But it's as you can imagine, we did not develop this cell line only for this particular project. Actually, we start working on this cell line back to maybe six, seven years ago. The first postdoc we have who basically knockin the mCherry, Myh6. Which basically label the atrial cardiomyocytes. And another postdoc, Zanir, he basically put a GFP in the SARS2 locus. So now we have this duel reporter line we can visualize the SA nodal cells. And we really spend a lot of time on that because we think that unfortunately in our hand, there is not really no good antibody for SARS2. We think it's very, very important that you can see these cells. So after developing these lines and because my lab run a lot of chemical screening, where we run Zanir, we run several chemical screening to develop the protocol.

And Jialing Zhu, another postdoc in the lab, also pick up the project to further develop the protocol. And there is several years' work. We do have this good protocol to make pretty efficiently to make the cells. And it's not only our work. I want to say that. For example, Dr Sean Wu from Stanford, they did this beautiful study on the single cell RNC mouse conduction system and Dr Gordon Keller and many other labs also basically published protocol in the field. We are very excited about this duel reporter line. I think they gave us a lot of new opportunity and we are very happy to share this line. Yeah. So if anyone in the field are interested in that, just contact us.

Cindy St. Hilaire: Yeah. Anyone listening. That's great. So were you surprised to find the entry factors that SARS-CoV-2 uses to get into a cell, were you surprised to find them on these sinoatrial node cells? And I guess in the context of comparing these particular cells to other cells in the heart, are those entry factors higher in the sinoatrial node cells?

Shubing Chen: So it can be either surprised or not surprised let's say this way. So because one, we see the cardiomyocytes that can be infected, we were kind of surprised. And then we find actually several type of cells in the heart can be infected, like endothelial cells. I will say that the ACE2 expression of like ACE2 aminophenol in pacemaker cell, it's not significantly higher than cardiomyocytes. So we are not really saying, or seeing that SA nodal cells are more permissive to SARS-CoV-2 infections compared to cardiomyocytes, even in the petri dish, but they can be infected.

Cindy St. Hilaire: So you found SARS-CoV-2 infection in these sinoatrial nodal cells induces a process called ferroptosis. So Yuling, I was wondering if you could tell us what is ferroptosis and what is it doing in these pacemaker cells?

Yuling Han: For the ferroptosis, they was surprised so far that its by the RA sequencing of the SARS-CoV-2 infection make our cells. And the first process is mainly caused by the-

Shubing Chen: Error in iron.

Yuling Han: Yes. So more intake of the iron error and induced the RA's pathway and caused the cell deaths. So by our RA sequencing, we found the key factor involved in ferroptosis pathway is the GPS score was checked after the SARS-CoV-2 infection. So we focused on the ferroptosis pathway and found other key factors or checked after the infection makes in the pacemaker cells.

Cindy St. Hilaire: What is the ferroptosis doing that disrupts the SNA cells?

Shubing Chen: Ferroptosis is a type of cell death mechanism. So eventually it will cause cell death. And we think something that is really surprising, but we think it's very interesting, is we only see ferroptosis in the SARS-CoV-2 infected general atrial cells. So SA cells, we actually, as Yuling mentioned, when we develop this platform, we see different type of cell can be affected. And we are very curious what happened. So we see that we run a sequence on each individual cells we can see infection and along, we can see cell death like apoptosis in cardiomyocytes. We see apoptosis and only in SA nodal cells, we actually see the ferroptosis pathway as we come up.

Cindy St. Hilaire: Why do you think that is in that cell type versus in another? Do you have any ideas about why?

Shubing Chen: No, we don't have any idea yet to be honest, but we are working on that. But at least I think that it gave us some clue that we really need to use different type of whole cells to study the whole cell response. Because traditionally when we study viral infection and when we see lung, we always say, oh, the cell died. It's fairly simple. But now if we really study the details and we think it's maybe over simplified way to think about how cells can respond to viral infection, not only to SARS-CoV-2 infection. So it gives us the motivation, very strong motivation to now really study how different host tissues response to viral infection.

Cindy St. Hilaire: I thought that was really interesting, not all cell death is the same.

Shubing Chen: Yeah. And another thing is kind of a little bit surprising is we actually did a very careful comparison between the SA nodal cells and the cardiomyocyte. We only see ferroptosis come up as SA nodal cell, but not cardiomyocyte. Again, we don't understand why as maybe some host factor that is specific, we're working on that.

Cindy St. Hilaire: So in addition to working out this mechanism of what is going wrong when these cells are infected with the virus, you also used this embryonic stem cell like tool for a drug screen. So can you walk us through that process in terms of what you did to do that? Did you focus in on one specific type of drugs or was it just kind of an unbiased screen?

Yuling Han: For the sinoatrial pacemaker cells, we focus on the antiviral drugs screening. And we also did several other projects, like lot of night or some neuron cells. For the [they did drug screening to find some drugs to inhibit the SARS-CoV-2 entry. And for the dominic neuron, we found SARS-CoV-2 infection can cause neuro cells synapses. So we focus on the synapses associated drug screening, but for the pacemaker cells, they only did the antiviral drug screen.

Cindy St. Hilaire: And you came up with two drugs that you wrote about in the paper, deferoxamine and imatinib. So what are the mechanisms of action of those drugs? Are they targeting the same thing or are they targeting slightly different things?

Yuling Han: For the imatinib, we also found this drug inhibit SARS-CoV-2 entry and we did several other screenings, like the lung organoids and neuro cells. We also found this drugs. And the six drug, the mechanism is kept and the spec protein of SARS-CoV-2. And this was found by several other groups and published some paper this year. And we found this in 2020 maybe. And we published this paper before and we found this mechanism. And for another drug, we checked the RA sequencing data of SARS-CoV-2 affect the peacemaker cells. And we did several run of RA sequencing. And we compared the key factors, involved in SARS-CoV-2 entry. Several key factors like CTSL and like TMPS2 and among several run of RA sequencing. We only found the drug can decrease the expression of CTSL. So we also did PTR immunostaining, and then we found the drug decrease the expression level of CTSL.

Shubing Chen: Yeah. So actually the other drug, it's also an antiferroptosis drug. So we did the mechanism study and it's very nice to see, we also identify the drug from an unbiased chemical screen. And for the chemical screening, we actually have a pretty large platform and we have around 1200 FDA approved drugs. We have like a 2000 anatrofin amino acid that signal pathway regulators for most of the SARS-CoV-2 screening, as you did mention, we have multiple screening platform. We focus on FDA approved drug. So it's more like for the drug repurposing and for other screening we also write larger skills.

Cindy St. Hilaire: So we got a mechanism, we got a super specific cell type and we now have some drugs. So what are the translational implications of these findings? And I guess I'm thinking about that in terms of the time course of when a patient gets infected, has symptoms, has arrhythmia, like where could you possibly target this ferroptosis pathway? Meaning if someone already is exhibiting AFib as a result of the infection, is that actually too late? Or can you start to treat it to reverse it or prevent it from getting worse? Like what do you see as a therapeutic potential for using these drugs?

Shubing Chen: That's a very good question. I will say this way, I think when we identify all these drugs, it's very, very exciting. But for antiviral drug development perspective, we definitely want a drug that show broader spectrum. So for COVID patient, of course we want to protect their heart, but we also want to protect their lungs.

Cindy St. Hilaire: Exactly. Protect everything.

Shubing Chen: Exactly. Exactly. So for the real drug that can clinical use, I think the lack of broad spectrum antiviral drug, I think that will be the way to go for drug development and for the cardioprotective respective. So if the patient do have very severe cardio symptom, particularly like arrhythmia symptom, I think that can be considered. But I don't want to really say this is the drug to treat the COVID patient. I don't think that's a way to go, particularly for ferroptosis is a cell type. This is a phenotype, very specific for the pacemaker. And I think for us, as a basic scientist, is very, very important that we understand the biology and we can identify these normal chemical tools that we can manipulate the system that can facilitate the future drug development.

Cindy St. Hilaire: So do you think your findings and I mean findings at multiple levels, that a viral infection can induce apoptosis in one cell, but ferroptosis in another cell, but also the findings of viral infection in general, sufficient enough to drives sinoatrial node cell dysfunction. Do you think this is specific to SARS-CoV-2 and corona viruses or do you think this is something that is more broad with other viruses that maybe we just haven't recognized possibly because we don't have the tools yet?

Shubing Chen: That's a great question. I will say some other type of virus can also infect heart, at least cardiomyocyte, like a Coxsackie virus, regular virus three. And there's actually a lot of study on the viral infection on the cardiomyocytes. And for us, the most exciting part is we really have now in serious, limited starting materials to get these pacemaker cells. Like I SA nodal cells. So we can use this as a platform to study how other virus infect, how the viral infection in general cause cell dysfunction. Because in the study we also do the calcium blocks assay, we can monitor their beating and then we can do RN-seq to monitor their transcription changes. Because this we have this still reporting system, we can purify cells, we can even run larger scale, like epigenetic level, how they change. So that's a very useful tool to study how cell responds to viral infection. I'm very excited about that.

Cindy St. Hilaire: That's great. Well, Dr Chen and Dr Han, thank you so much for joining me today. Congratulations on a beautiful story. And I look forward to hearing more out all these different organoid and cell models you have.

Shubing Chen: Cindy, thank you. Thank you for so much for having us.

Cindy St. Hilaire: That's it for the highlights from the April issues of Circulation Research. Thank you for listening. Please check out the CircRes Facebook page and follow us on Twitter and Instagram with the handle @CircRes and #DiscoverCircRes. Thank you to our guests, Dr Shubing Chen and Dr Yuling Han. This podcast was produced by Ishara Rantikac edited by Melissa Stoner and supported by the editorial team of Circulation Research. Some of the copy text for highlighted articles was provided by Ruth Williams. I'm your host, Dr Cindy St. Hilaire, and this is Discover CircRes, you're on the go source for the most exciting discoveries in basic cardiovascular research. This program is copyright of the American Heart Association, 2022. The opinions expressed by speakers in this podcast are their own and not necessarily those of the editors or of the American heart Association. For more our information visit ahajournals.org.

This month on Episode 34 of Discover CircRes, host Cynthia St. Hilaire highlights four original research articles featured in the March 4 and March 18th issues of Circulation Research. This episode also features a conversation with Dr Mireille Ouimet and Sabrina Robichaud from the University of Ottawa Heart Institute to discuss their study, Autophagy is Differentially Regulated in Leukocyte and Non-Leukocyte Foam Cells During Atherosclerosis.

Article highlights:

Pauza, et al. GLP1R in CB Suppress Chemoreflex-Mediated SNA

Lim, et al. IL11 in Marfan Syndrome

Hohl, et al. Renal Denervation Prevents Atrial Remodeling in CKD

Liu, et al. Smooth Muscle Cell YAP Promotes Arterial Stiffness

Cindy St. Hilaire: Hi and welcome to Discover CircRes, the podcast of the American Heart Association's journal, Circulation Research. I'm your host, Cindy St. Hilaire from the Vascular Medicine Institute at the University of Pittsburgh, and today I'm going to be highlighting articles from our March issues of Circulation Research. I'm also going to speak with Dr Mireille Ouimet and Sabrina Robichaud from the University of Ottawa Heart Institute, and they're with me to discuss their study, Autophagy is Differentially Regulated in Leukocyte and Non-Leukocyte Foam Cells During Atherosclerosis.

The first article I want to share is titled GLP1R Attenuates Sympathetic Response to High Glucose via Carotid Body Inhibition. The first author is Audrys Pauza, and the corresponding authors are Julian Paton and David Murphy at the University of Bristol.

Cindy St. Hilaire: Hypertension and diabetes are risk factors for cardiovascular disease. And yet, for many patients with these two conditions, lowering blood pressure and blood sugar is insufficient for eliminating the risk. The carotid body is a cluster of sensory cells in the carotid artery, and it regulates sympathetic nerve activity. Because hypertension and diabetes are linked to increased sympathetic nerve activation, this group investigated the role of the carotid body in these disease states. They performed a transcriptome analysis of crowded body tissue, from rats with and without spontaneous hypertension. And they found among many differentially-expressed genes that the transcript encoding glucagon-like peptide-1 receptor or GLP1R, was considerably less abundant in hypertensive animals.

Cindy St. Hilaire: This was of particular interest because the gut hormone GLP-1 promotes insulin secretion and tends to be suppressed in Type 2 diabetes. Moreover, GLP1R agonists are already used as diabetic treatments. This group showed that treating rat carotid body with GLP1R agonist suppresses sympathetic nerve activation and arterial blood pressure, suggesting that these drugs may provide benefits in more than one way. Perhaps the carotid body could be a novel target for lowering cardiovascular disease risk in metabolic syndrome.

Cindy St. Hilaire: The second article I want to share is titled Inhibition of IL11 Signaling Reduces Aortic Pathology in Murine Marfan syndrome. The first author is Wei-Wen Lim, and the corresponding author is Stuart Cook and they're from the National Heart Center in Singapore. People with the genetic connective tissue disorder Marfan syndrome, are typically tall and thin with long limbs and are prone to skeletal, eye and cardiovascular problems, including a life-threatening weakening of the aorta. While Marfan syndrome patients commonly take blood pressure-lowering treatments to minimize risk of aortic aneurysm and dissection, there's currently no cure for Marfan syndrome or targeted therapy.

Cindy St. Hilaire: The cytokine IL11 is strongly induced in vascular smooth muscle cells upon treatment with the growth factor TGF-beta, which is over activated in Marfan syndrome patients. And TGF-beta is also considered a key feature of the syndrome's molecular pathology. This study found that IL11 is strongly upregulated in the aortas of Marfan syndrome model mouse, and that genetically eliminating IL11 in these animals protected them against aortic dilation, fibrosis, inflammation, elastin degradation and loss of smooth muscle cells. Treating Marfan syndrome mice with anti-IL11 neutralizing antibodies exhibited the same beneficial effects. These results suggest that perhaps inhibiting IL11's activity could be a novel approach for protecting the aortas of Marfan syndrome patients.

Cindy St. Hilaire: The next article I want to mention is titled Renal Denervation Prevents Atrial Arrhythmogenic Substrate Development in Chronic Kidney Disease. The first authors are, Mathias Hohl, Simina-Ramona Selejan and Jan Wintrich, and the corresponding authors also Mathias Hohl, and they're from Saarland University. People with chronic kidney disease have a two to three fold higher risk than the general population of developing atrial fibrillation, which is a common form of arrhythmia that can be life-threatening. Chronic kidney disease is associated with activation of the sympathetic nervous system, which can be damaging to the heart. Thus, this group examined myocardial tissues from atrial fibrillation patients with and without chronic kidney disease to see how they differ. They found that atrial fibrosis was more pronounced in patients with both conditions than in patients with atrial fibrillation alone, suggesting that chronic kidney disease perhaps exacerbates or even drives arterial remodeling.

Cindy St. Hilaire: Sure enough, induction of chronic kidney disease in rats led to greater atrial fibrosis and incidence of atrial fibrillation than seen in the control animals. Renal denervation is a treatment in which the sympathetic nerves are ablated, and it's a medical procedure that's used for treating uncontrolled hypertension, and it has also been shown in animals to reduce atrial fibrillation. Performing renal denervation in the rats with chronic kidney disease reduced atrial fibrosis and atrial fibrillation susceptibility. This study not only shows that chronic kidney disease induces atrial fibrosis and in turn atrial fibrillation, but also suggests that renal denervation may be used in chronic kidney disease patients to break this pathological link and prevent potentially deadly arrhythmias.

Cindy St. Hilaire: The last article I want to highlight is titled YAP Targets the TGFβ Pathway to Mediate High-Fat/High-Sucrose Diet-Induced Arterial Stiffness. First author is Yanan Liu and the corresponding author is Ding Ai from Tianjin Medical University. Metabolic syndrome is characterized as a collection of conditions that increase the risk of cardiovascular diseases, such as obesity, hypertension and diabetes. Among the tissue pathologies associated with metabolic syndrome is arterial stiffness, which itself is a predictor of cardiovascular disease incidence and mortality. To specifically investigate how arterial stiffness develops in metabolic syndrome, this group fed mice a high-fat, high-sugar diet, which is known to induce metabolic syndrome and concomitant arterial stiffness.

Cindy St. Hilaire: After two weeks on the diet, the animals' aorta has exhibited significant upregulation of TGF-beta signaling, which is a pathway known for its role in tissue fibrosis, and the aorta has also exhibited increased levels of yes-associated protein, or YAP, which has previously been implicated in vascular remodeling, collagen deposition and inflammation. YAP gain and loss of function experiments in transgenic mice revealed that while knockdown of protein in the animals' smooth muscle cells attenuated arterial stiffness, increased expression exacerbated the condition.

Cindy St. Hilaire: The team went on to show that YAP interacted with and prevented the activation of PPM-1 B, which is a phosphatase that normally inhibits TGF-beta signaling and thus fibrosis. Together the results suggest that targeting the YAP, PPM-1 B pathway, could be a strategy for reducing arterial stiffness and associated cardiovascular disease risk in metabolic syndrome.

Cindy St. Hilaire: Today, Sabrina Robichaud and Dr Mireille Ouimet from University of Ottawa Heart Institute are with me to discuss their study Autophagy is Differentially Regulated in Leukocyte and Non-Leukocyte Foam Cells During Atherosclerosis, which is in our March 18 issue of Circulation Research. So thank you both for joining me today.

Sabrina Robichaud: Thank you so much for having us. It's a pleasure.

Mireille Ouimet: Thank you for having us.

Cindy St. Hilaire: Yeah, and congrats on the study. So we know that LDL particles contain cholesterol and fats, and these are the initiating factors in atherosclerosis. And it's also really now appreciated that inflammation in the vessel wall is a secondary consequence to this lipid accumulation. Macrophages are an immune cell that, in the context of the plaque, gobble up this cholesterol to the point that they become laden with lipids and exhibit this foamy appearance, which we now call foam cells. And these foam cells can exhibit atheroprotective properties, one of them called reverse cholesterol transport, and that's really one of the focuses of your paper. So before we dig into what your paper is all about, could you give us a little bit of background about what reverse cholesterol transport is in the context of the atherosclerotic plaque? And maybe introduce how it links to this cellular recycling program, autophagy, which is also a big feature of your study.

Mireille Ouimet: Yes, so the reverse cholesterol transport pathway is a pathway that's very highly anti-atherogenic. It's linked to HDL function and the HDL protective effects, in that HDL can serve as a cholesterol acceptor for any excess cholesterol from arterial cells or other cells of the body and return this excess cholesterol to the liver for excretion into the feces. There is also trans-intestinal cholesterol efflux that can help eliminate any excess bodily cholesterol.

Mireille Ouimet: So reverse cholesterol transport is a way that we can eliminate excess cholesterol from foam cells in the vascular wall, and that's why we're really interested in the process. But the rate-limiting step of cholesterol efflux out of foam cells in plaques is actually, they have to be mobilized in the form of free cholesterol to be pumped out of the cells through the action of the ATP-binding cassette transporters. And so the rate-limiting step of the process is the hydrolysis of the cholesterol esters and the lipid droplets, because that's where the excess cholesterol is stored in foam cells.

Mireille Ouimet: And so for years, people investigated the actions of cytosol like lipases in mobilizing free cholesterol from lipid droplets, although the identity of those lipases are not well-known and in macrophage themselves, but our recent work showed a role for autophagy in the catabolism of lipid droplets. And in fact, in macrophage foam cells, 50% of lipid droplet hydrolysis is attributable to autophagy while the other half is mediated by neutral lipases, which makes it really important to investigate the mechanisms of autophagy-mediated lipid droplet catabolism.

Cindy St. Hilaire: That is so interesting. I guess I didn't realize it was that significant a component in that kind of rate-limiting step. That's so cool. So really, a lot of the cholesterol efflux studies, and maybe this is just limited to my knowledge of a lot of these cholesterol efflux studies, but to my knowledge, it's been really focused on the foam cell itself, the macrophage foam cell. However, there's been a lot of recent work that has now implicated vascular smooth muscle cells in this process. So could you share some of the research specific to smooth muscle cells and smooth muscle-derived foam cells that led you to want to investigate the contributions of smooth muscle cell-derived foam cells in cholesterol efflux?

Mireille Ouimet: Yeah, so you're right in the sense that macrophages have always been the culprit foam cells in the atherosclerotic plaques but pioneering work from several groups, including Edward Fisher and Gordon Francis, they've shown that the smooth muscle cells can actually acquire a macrophage-like phenotype becoming lipid-loaded and foamy. And there's been work specifically looking at the ABC transporters, and their ability to efflux cholesterol from these vascular smooth muscle cell-derived foam cells, because as they trans-differentiate into macrophage-like cells, they acquire the expression of ABCA1, but this is to a lower extent, as compared to their macrophage counterparts.

Mireille Ouimet: And the efflux is defective because there's an impairment in liposomal cholesterol processing of the lipoproteins that's really important to activate a like cell, and the expression of the ABC transporters, so vascular smooth muscle cell-derived foam cells are very poor effluxes.

Sabrina Robichaud: There's very few studies that look at the vascular smooth muscle cell foam cells, and the very few that did look at it mostly focused on the ABCA1 transporters, and did show that they were poor effluxes. And as we all know, ABC1 is not the only cholesterol transporters that can transport cholesterol out of cells, there's also ABCG1 which is also one of our major findings in our paper.

Cindy St. Hilaire: Can you tell us a little bit about the models you chose in the study and why you picked them? And also maybe a step back in terms of, what are the pros and cons of using mouse models in atherosclerotic studies?

Sabrina Robichaud: So we chose to use the GFP-LC3 reporter mouse model because it allows us to track in lifestyle the movement of LC3, which is the main component of the autophagosome which is involved in pathology. So by using this reporter model, we could infer whether or not the cells had high autophagy or low autophagy. And to induce atherosclerosis in these mice, instead of backcrossing them to either an LDLR knockout or an ApoE knockout, we chose to do the adeno-associated virus that encode the gain of function PCSK9 instead to kind of minimize the time for breeding. It did have the effect that we needed in terms of raising plasma cholesterol to induce the atherosclerosis. So that was one of the models that we used in our paper.

Mireille Ouimet: There's not very many good mouse models to study autophagy flux in vivo and GFP-LC3 is kind of the main one currently. We're working on developing some other tools to track lipophagy in vivo, but these things take time to put in place. So in the future, we hope to have some better tools to track lipophagy in real-time in vivo.

Cindy St. Hilaire: How difficult is it to measure autophagy flux in vivo? I know there's certain part like LC3 or P62, a lot of people use a western blot and it's like, oh, it's high, it must be active, but it's a flux. So it's a little bit more... There's more subtleties to that, dynamic than that. So how difficult is it to really measure this flux in in vivo tissues?

Mireille Ouimet: Yes, so now there are more recent mouse models that have been developed more recently to replace kind of the GFP-LC3 is the Rosella LC3. So it has both a red and a green tag, and so two LC3, so when autophagosomes are fused to lysosomes and are degraded, then there's preferential quenching of the GFP first, and then you have the red appearance that predominates so we know that then it's kind of like it a live flux measurements. Because we use the GFP-LC3 mouse, Sabrina treated her cells ex vivo. When we dissected out the aortic arches, digested the cells then we divided those into two components and added bafilomycin so that we can inhibit lysosome acidification to see the changes in the flux. And that's really to get the differences in untreated versus bafilomycin-treated.

Mireille Ouimet: When we inhibit the lysosome, then we're sure that it is a functional flux or not. But it's kind of an indirect way of measuring it, and it reads very complex when we're talking about P62 and LC3 degradation with or without lysosome inhibition, but you really need that lysosomal inhibition, to show that if you block the degradation of the autophagosomes that fuse in with a lysosome, then you get an increase in the LC3 and the P62, and that's when you know that the flux is you intact.

Mireille Ouimet: Because you could get an increase in LC3, that's just related to a defect in the breakdown of the autophagosome. But in our study, we've used phosphorylated ATG16L1, which is a now better marker of active autophagy. And I would recommend researchers to begin to use that rather than the combination of P62 and LC3 together with or without a lysosome inhibitors such as-

Cindy St. Hilaire: Oh, interesting. So let's repeat that, phosphorylated ATG-

Mireille Ouimet: 16L1, yes. So there's been an antibody that was developed by a colleague at the University of Ottawa, Dr Ryan Russell, and it's commercially available through cell signaling now, and it really has been a great tool to track active autophagy.

Cindy St. Hilaire: That's great. I remember my lab was looking at that at one point, and I was trying to explain the flux as... I don't know if people are going to remember this, but there's this amazing, I Love Lucy skit, where her and Ethel are working on a chocolate factory conveyor belt, and it picks up speed. And because she can't get it all done quick, she starts stuffing them in her mouth. And it's like, if you just took a snapshot of that, you would not know whether it's going too fast, or not functioning properly. And so I equate the flux experiments to that. Which are probably aging myself a lot on so.

Cindy St. Hilaire: All right, so sticking to kind of the autophagy angle, what were the differences you found in autophagy in early and late atherosclerotic plaques? Because I know you looked at those two time points, but also, importantly, between the macrophage foam cells and the smooth muscle cell-derived foam cells?

Sabrina Robichaud: So surprisingly, there weren't that big of a difference between each time point when we were looking at the individual cell type by themselves. Surprisingly, we did find that the macrophages did have a functional autophagy flux, even at the later stages of atherosclerosis, which was kind of interesting in itself. But when we looked at the vascular smooth muscle cell foam cells, though, that was a whole other story, and we found that these were actually defective at a very early stage and stayed defective up until the very late stage of atherosclerosis.

Cindy St. Hilaire: And what is the very early stage like? What's that definition with the smooth muscle cell?

Sabrina Robichaud: So we did a six-week time points in terms of our atherosclerosis study, and then a 25-week time point. So there are far apart, which shows like the very early, early stage and what would be considered the most effective autophagy at that point with the necrotic core and everything. So surprisingly, the two phenotype were quite similar at early and both late stages for both cell types, but were functional in the macrophages but dysfunctional in the smooth muscle cells.

Cindy St. Hilaire: So you mentioned at one point in the discussion that you observed inconsistent lipid loading of the smooth muscle cells, and you mentioned that a lipase, which is excreted from the foam cells can then be internalized by, I assume kind of neighboring or in the vicinity, smooth muscle cells. And so the question I had it's kind of one of those chicken-and-egg question, and it's, is the smooth muscle cell-derived foam cell an independent process? Does it happen alone or de novo as a function of a smooth muscle-mediated process? Or is it really dependent first on this macrophage foam cell providing this lipid that is efflux that is then internalized by a smooth muscle cell that kind of goes on to become a foam cells. It's kind of a question of like the continuum of an atherosclerotic plaque and what do you think is happening, either based on your data or just kind of a hunch?

Mireille Ouimet: That's an excellent question. And there's no doubt that macrophages really drive the initiating events of atherosclerosis. So I don't think that without the macrophage there would ever be a vascular smooth muscle cell, or there would be minimal vascular smooth muscle cell-derived foam cells. Definitely the inconsistencies that we observed in our study, were if we added like aggregated LDL on its own to a primary mouse vascular smooth muscle cell, we would get poor lipid loading and a very low percentage of those cells that would become foamy, relative to treating them with cyclodextrin complex cholesterol, for instance.

Mireille Ouimet: So free cholesterol, that's cell permeable, will go into the vascular smooth muscle cell, no problem, and generate the foaminess and then allow that cell to acquire the macrophage-like phenotype. But aggregated LDL on its own in our hands, just gave very poor loading. And when we treated the vascular smooth muscle cells with aggregated LDL along with macrophage-derived condition media, we got some improvements, but it was still kind of inconsistent. But then we thought if we treat the vascular smooth muscle cells with aggregated LDL in the presence of conditioned media from macrophage foam cells that were preloaded with the aggregated LDL, would that promote their foaminess to a greater extent? And it did.

Mireille Ouimet: So, there have been studies from Gordon Francis's lab that showed that adding recombinant lysosomal acid lipase to vascular smooth muscle cells that contained aggregated LDL, promoted the lysosomal hydrolysis of the aggregated LDL and to generate the foamy macrophages and allow the lysosomal processing. So we know that that vascular smooth muscle cells take up lysosomal acid lipase, and we know that macrophages undergo lysosome exocytosis and they can secrete lysosome acid lipase and acidify the extracellular milieu.

Mireille Ouimet: So work from Fred Maxfield group has shown the presence of these cell surface connected compartments that are acidified, containing macrophage-derived lysosomal acid lipase, that even hydrolyze extra cellularly-aggregated LDL for macrophages. So we're not sure whether there's probably a local production of free cholesterol in the plaque by macrophages, this free cholesterol could be taken up by the vascular smooth muscle cell. And also the vascular smooth muscle cells do express some scavenger receptors, whether the expression of these scavenger receptors like LRP or CD36 even goes up when they've taken up a little bit of the free cholesterol. And then that allows the aggregated LDL to come in and then there would be some lysosomal acid lipase secreted by the macrophage foam cells that would promote the lysosomal processing of this aggregated LDL. All of those are very complex questions that will require some addressing in vivo models.

Cindy St. Hilaire: You also mentioned in the paper that studies... There's a handful of them now. Studies have shown that between 30% and 70% of the cells that are staining positively for macrophage markers, meaning they're foam cells, are of the smooth muscle cell lineage. And so I believe people have seen that in mouse plaques with lineage tracing, but they've also used newer techniques to really see this also in human atherosclerotic plaques. So we know it's not just from a mouse, we know that smooth muscle cells can turn into a macrophage-like foam cell, and it's 30% to 70%, which is a huge range.

Cindy St. Hilaire: So do we know the factors that dictate whether a specific plaque is going to have more or less smooth muscle cell derived foam cells? And I guess more important to what you found in your paper is, how important would it be to know whether a plaque is on the 30% end or on the 70% end in terms of therapeutic strategies?

Sabrina Robichaud: Yeah, most of these studies, the range can be attributed to the different time points at which these studies have been collected early on will be a little bit more macrophage understanding would be at a later time point. Now of course in terms of therapeutics, as we saw in our paper, metformin actually will positively increase cholesterol efflux in the vascular smooth muscle cell foam cells, but not in the macrophages. So obviously, being able to know at which point there's a majority of macrophages versus vascular smooth muscle cells, definitely going to determine which therapeutic we're going to be able to use.

Sabrina Robichaud: Ideally, we would be able to find a therapeutic that would work in both foam cell, but from what we've seen, the mechanistic behind the autophagy dysfunction between both cell types are so different, that I'm not entirely sure that that would be possible, we would need some sort of combination therapy. But again, we need to be a little bit more targeted depending on the percentage of the foam cells that are comprising the plaque at that particular moment in time.

Cindy St. Hilaire: Yeah, so you mentioned there's a function of time there. If you look earlier, there's more macrophage, if you look later, the percent of smooth muscle cell-derived foam cell increases. Is there a point in a very advanced atherosclerotic plaque where it's just mostly smooth muscle cells? Or do those macrophage foam cells stay, and it's just the increasing number of smooth muscle cell-derived foam cells? Do we know?

Mireille Ouimet: This is an excellent question, and I was going to bring up the topic of clonal expansion of the vascular smooth muscle cells. So it's a very heterogeneous population and understanding that might be some of the differences that we see in different studies. It could be the model has one type of a smooth muscle cell that's expanding more than another, what are the factors that govern that? Does one clone take over at the later stages versus the earlier stages? We don't know.

Mireille Ouimet: But we were surprised in our studies to see that the macrophages that are present at least on the lumen of the plaques were very active in autophagy. They had the highest staining for the phospho-ATG16L1 in that late stage. So we're not sure if it's newly-recruited macrophages that come in, that are more active and in autophagy, and then have good lysosomal capacity that keeps degrading the lipid present in the plaque and tries to ingest it, but also as a consequence keeps releasing some of the degraded cholesterol into the milieu where the smooth muscle cells that are proliferating are internalizing it and becoming more foamy. So these are really great open questions that need to be addressed in the field.

Cindy St. Hilaire: So drug-eluting stents are coated with rapamycin or the various chemical compositions that are derived from rapamycin. And rapamycin itself induces autophagy. So while the thought behind using this coating on stents was to prevent smooth muscle cell proliferation, and thus restenosis or ingrowing of the stent, your study suggests that this could also help to promote autophagy in the cells underlying the stent. So has anyone gone in and looked at plaques that have been stented and either failed or not, and investigated the foam cell content or markers for autophagy activity?

Mireille Ouimet: Not to my knowledge, and this has been something we've definitely... We think that this is what's happening. Some of the protective effects of these drug-eluting stents that have everolimus or sirolimus or the rapamycin or rapamycin analogs, we do believe that some of their protective effect can be attributed to autophagy activation, but this remains to be demonstrated. We think that autophagy activation locally would promote reverse cholesterol transport and would be one of the processes that prevents restenosis because we can promote the efflux of cholesterol out.

Cindy St. Hilaire: Great. So I guess stemming from my question on the stents, what are the other translational implications of the findings of your study? And what would you like to see come out of this?

Mireille Ouimet: So one of the things is, as Sabrina mentioned, would be to target both foam cell populations because it seems as though the vascular smooth muscle cell foam cells are very much defective in their autophagy capacity, and they're very poor effluxes, but we could potentially restore autophagy in the cell population to promote reverse cholesterol transport.

And looking at prevention of atherosclerosis is a bit different than looking at regression, because regression is at a later stage where the plaques are more advanced. And if they're mostly vascular smooth muscle cell-derived, maybe then those drugs that we're considering that protect against the development of atherosclerosis are effective on the macrophage themselves early on, but might not be mimicking what we would see in the clinic where the patients that present are older.

Cindy St. Hilaire: Yeah, it's kind of really reminiscent of like the CANTOS trial and like, where do we want to target the therapy? It's going to be very different if it's an early smaller plaque, versus a late-stage possibly pro close to rupturing type of plaque. Well, Sabrina Robichaud and Dr Ouimet, thank you so much for joining me today. Congratulations again on a wonderful study, and I'm really looking forward to hearing more about this from your group.

Sabrina Robichaud: Thank you.

Mireille Ouimet: Thank you very much. And we also want to thank all the co-authors on the study, specifically also Adil Rasheed, who is co-first author on the work and Katey Rayner's group for all the support and involvement in this study.

Cindy St. Hilaire: That's it for the highlights from the March issues of Circulation Research. Thank you for listening. Please check out the CircRes Facebook page and follow us on Twitter and Instagram with the handle @circres and #DiscoverCircRes. Thank you to our guests, Sabrina Robichaud and Dr Mireille Ouimet Sabrina. This podcast is produced by Ashara Ratnayaka, edited by Melissa Stoner and supported by the editorial team of Circulation Research. Some of the copy text for highlighted articles was provided by Ruth Williams. I'm your host, Dr Cindy St. Hilaire and this is Discover CircRes, you're on-the-go source for the most up-to-date and exciting discoveries in basic cardiovascular research. This program is copyright of the American Heart Association 2022, The opinions expressed by speakers in this podcast are their own and not necessarily those of the editors or of the American Heart Association. For more information, visit ahajournals.org.

This month on Episode 33 of Discover CircRes, host Cynthia St. Hilaire highlights two original research articles featured in the February 4 issue of Circulation Research. In addition, she previews Circulation Research's Compendium on Women and Cardiovascular Health, featured in the February 18th issue. This episode also features a conversation with Dr Alastair Poole and Dr Laura Corbin from the University of Bristol and Dr Stephen White from the Manchester Metropolitan University about their study, Epigenetic Regulation of F2RL3 Associates with Myocardial Infarction and Platelet Function.

Article highlights:

Samargandy, et al. Blood Pressure Trajectories and Menopause

Gilchrist, et al. Research Goes Red Registry

Cindy St. Hilaire: Hi, and welcome to Discover CircRes, the podcast of the American Heart Association's Journal, Circulation Research. I'm your host, Dr Cindy St. Hilaire from the Vascular Medicine Institute at the University of Pittsburgh and today I'm going to be highlighting articles from our February issues of Circulation Research. I'm also going to speak with Dr Alastair Poole and Dr Laura Corbin from the University of Bristol and Dr Stephen White from the Manchester Metropolitan University about their study, Epigenetic Regulation of F2RL3 Associates with Myocardial Infarction and Platelet Function.

Cindy St. Hilaire: The first article I want to share is titled Trajectories of Blood Pressure in Midlife Women: Does Menopause Matter? The first author is Saad Samargandy, and the corresponding author is Samar El Khoudary from the University of Pittsburgh. Blood pressure increases with age, but after midlife, the rate of increase for women generally exceeds that for men. This observation has led to debate over whether menopause might influence the blood pressure trajectory.

Cindy St. Hilaire: To find out, this group examined data on over 3300 women of diverse ethnicity enrolled in the Study of Women's Health Across the Nation, or SWAN study. The women began the study between 42 and 52 years old, and they had 17 follow-up visits at roughly one-year intervals. At these visits, blood pressure, hormone levels, weight and other health parameters were measured.

Cindy St. Hilaire: Analysis of the data revealed women fell largely into three blood pressure trajectory groups. Those with low blood pressure before menopause and accelerated blood pressure after menopause, those with a linear increase linked to age, and those with high blood pressure before and a slower ascent afterwards. White, Chinese and Japanese women were more likely to be in the low to accelerated group, as were those with early menopause, while Latino and Black women were more likely to have high blood pressure in general. Together, the results indicate that for many women, menopause itself does not accelerate age-related blood pressure increase, and that women of menopausal age should be advised of this risk and have their blood pressure monitored regularly.

Cindy St. Hilaire: The second article I want to highlight is titled Research Goes Red: Early Experience With a Participant-Centric Registry. The first author is Susan Gilchrist and the corresponding author is Jennifer Hall from the American Heart Association. Cardiovascular disease is a leading cause of death for men and women alike, but there are particular factors such as pregnancy and menopause that may specifically influence the genesis, presentation and management of the condition in women.

Cindy St. Hilaire: With that in mind, for the past two decades, the AHA's Go Red for Women campaign has been raising awareness of and driving research into women's cardiovascular health issues. The latest Go Red initiative, an online platform called Research Goes Red, was launched in 2019 with the aim of empowering women to contribute to health research by, among other things, taking part in health surveys. In the last two years, the platform has garnered 15,000 registered individuals between the ages of 30 and 60. It has deployed six targeted health surveys and prompted two AHA-funded research studies based on participant responses: one on perimenopausal weight gain, and one on the use of social media to engage young women in cardiovascular disease awareness. While Research Goes Red has successfully amassed middle aged participants, the authors say that future goals should include increasing the number and the diversity of the registrants and encouraging researchers to use the registry not just for data, but for identifying potential trial participants.

Cindy St. Hilaire: I want to now mention the 15 articles that are featured in our Compendium on Women and Cardiovascular Disease that is featured in our February 18th issue of Circulation Research. And this also happens to correspond with February being the American Heart Month. So Susan Cheng and colleagues present A Scientific Imperative As Seen Through a Sharpened Lens: Sex, Gender and the Cardiovascular Condition. Genetic, molecular and cellular determinants of sex-specific cardiovascular traits is discussed by Teemu Niiranen and colleagues. Bonnie Ky et al. describe sex-specific cardiovascular risks of cancer and its therapies. Sex differences and similarities in valvular heart disease is presented by Francis Delling and colleagues. Cecile Lahiri and colleagues wrote about the cardiovascular implications of immune disorders in women. Joshua Smith and colleagues discuss sex differences in cardiac rehabilitation outcomes.

Cindy St. Hilaire: Pregnancy and reproductive risk factors of cardiovascular disease in women is reviewed by Michael Honigberg and colleagues. The impact of sex and gender on stroke is presented by Kathryn Rexrode and colleagues. Ersilia DeFilippis and colleagues cover heart failure subtypes and cardiomyopathies in women. Demilade Adedinsewo and colleagues wrote about cardiovascular disease screening in women, leveraging artificial intelligence, and digital tools. Sexual dimorphism in cardiovascular biomarkers, clinical research implications, is discussed by Jennifer Ho and colleagues. Connie Hess et al. review sex differences in peripheral artery disease. Janet Wei and colleagues provide an update on coronary arterial function and disease in women with non-obstructive coronary arteries. Sex differences in myocardial and vascular aging is presented by Hongwei Ji and colleagues. And lastly, arrhythmias in female patients, incidence, presentation and management, is reviewed by Andrea Russo and colleagues.

Cindy St. Hilaire: Today I have with me Drs Alastair Poole and Laura Corbin from the University of Bristol and Dr Stephen White from the Manchester Metropolitan University. And they're here with me to discuss their study, Epigenetic Regulation of F2RL3 Associates with Myocardial Infarction and Platelet Function. And this article is in our February 4th issue of Circ Res. Well, Drs Corbin, Poole and White, thank you so much for joining me today.

Laura Corbin: Thank you very much.

Stephen White: Thank you.

Alastair Poole: Thanks.

Cindy St. Hilaire: So this is a really neat study. It's bringing in a couple different fields. It's investigating what I'm calling a Venn diagram of these intersecting topics all related to cardiovascular disease: cigarette smoking, epigenetic modification and platelet activation. So can you maybe give us a little bit of background on the status of the field and how these three topics intersected at the start of your study?

Laura Corbin: So yeah, our working hypothesis was based on existing literature and it was really to look at whether smoking-induced epigenetic DNA hypermethylation of F2RL3 could increase risk of myocardial infarction and whether the route to that could be through platelet function. So there's quite a lot of literature going back probably to around about 2015 that shown that there are changes to the methylome in response to smoking. And DNA methylation is a way of cells controlling gene expression, but without having to actually make changes in the DNA sequence itself. So this could be a really important way that we know that smoking increases the risk of a number of cardiovascular diseases, but we don't really know how that happens. And one way that that could happen is through changes to methylation.

Cindy St. Hilaire: What is known about how cigarette smoke impacts the status of DNA methylation? How has that switched or changed? Maybe when someone is actively smoking, when someone quits, is it dynamic? What is known about that relationship?

Laura Corbin: Okay. So yeah, going back to about 2015, there was a number of studies that looked at methylation across the whole genome. So in a hypothesis-free untargeted manner, developments and technology meant that we could look at many, many sites across the genome at the same time. And so studies were done to look at changes that were associated with smoking. And what was found was those changes, actually quite a lot of changes across the genome in a number of different genes, but not really anything much beyond that. So F2RL3 was one of the first sites to be identified as being associated, methylation at that site associated with smoking. And it was replicated in several studies.

Laura Corbin: And it was also showing that there was a dose-response relationship. So the more a person smoked, the less methylated that site appeared to be. And then there's been some work done already, but we also did it in our paper to show that those methylation marks actually hang around for quite a long time once somebody quits smoking. But also that there's a lot of variation within an individual, so even if you smoke, it doesn't necessarily mean that you'll definitely have low methylation, there's still variation. So there's other factors that are involved in that.

Cindy St. Hilaire: So you were looking at a specific population of patients, can you tell us a little bit about that group of patients you were looking at? And you mentioned the variability in the amount of smoke they were exposed to, do you know that information? And I guess one of the base questions I had is I'm in Pittsburgh, which back in the '80s and earlier was a steel mill town that had a lot of pollution. And so I'm wondering if you're able to clearly separate out a cigarette smoker from maybe someone who is a light cigarette smoker, but lives in a more polluted area?

Laura Corbin: Okay. So there's two parts of the study that were looking at this in a human context, so in a whole person context. One of those was using data from the Copenhagen City Heart Study, and that's the one where we looked at the relationship between smoking and methylation and then between methylation and myocardial infarction. So that study is great because it's been tracking people over time and so we're able to use the samples that were collected before they had their cardiovascular event and look at methylation at that point. So we know that the event occurs after that point, which is important. And so we were able to verify in that population that we did see an association between smoking and methylation. We were able to show that it was a dose-dependent relationship. So if we look at something like in that dataset, we had things like the intensity of which people smoke, so pack years is one of the things that we looked at. And it did appear to correspond in an approximately linear fashion.

Laura Corbin: So we don't really know, I don't think, at this point, what impact other environmental exposures would have on the methylome and how that would interact with the cigarette smoking. That's actually a really interesting point that we'll probably come onto later about whilst we were looking here at the smoking effect on this methylation site, in the second part of the work, we were able to show that even in non-smokers, there's variability in methylation at this site, and that can still have impacts on the biology downstream. So yeah, it's an interesting point.

Stephen White: Just to maybe just jump in, there's very good amounts of literature now that show quite a good correlation between changes in air quality and cardiovascular events. So smogs, wildfires and so on, clearly correlate with an increase in cardiovascular events. But actually the opposite's also been observed in the more recent COVID lockdowns, where reduction in air pollution also mirrored a reduction in the number of cardiovascular events. So I think you raise a really interesting point about is it cigarette smoke alone or does air quality in general play an effect? And clearly it does play an effect, although we didn't correlate that within this current dataset.

Cindy St. Hilaire: Your study looked at DNA methylation patterns at cytosine, phosphate, guanidine or CPG sites in the genome. Can you tell us a little bit more about what these islands are and how they change throughout maybe different cells in the body, but also maybe in the same cell, but throughout the course of life or the course of, in this case, cigarette exposure?

Stephen White: So if we just want to focus in on our study, what we showed was that exposure to cigarette smoke changes endothelial cell methylation. It also changes megakaryocyte methylation patterns in the same way. And I think one of the surprising things was that only 48 hours of exposure to cigarette smoke significantly changes the methylation pattern of the F2RL3 locus. So it's quite a dynamic event, but it does show that these can be quite rapidly regulated. And Laura's really nice work shows that the methylation on cessation of smoking, that pattern does actually go down, but it's a 20-year process. So it looks like it can be rapidly induced, but may actually remain as a methylation mark for a considerable length of time.

Stephen White: And I think one of the things we did in our study was actually to triangulate not only the observational data and the association data in patients, but actually start to look at a mechanism of how that might actually relate to changes in gene expression. So we showed that this particular CPG site is right next to a binding site for a transcription factor, and transcription factors are the cell's way of regulating how much of a particular gene is expressed. And we show that changes in methylation changed the binding of this transcription factor and therefore change the amount of this particular gene that was made.

Cindy St. Hilaire: Yeah. Actually, I want to start to talk about that locus you were interested in. So what was known about the F3RL2 locus? How big is it, but also what genes are there and what did you start to investigate with your in vitro modeling?

Stephen White: So I think when we started, we had the observation that a change in methylation at the F2RL3 locus was associated with the risk of cardiovascular events. And then it was a detective expedition into the gene using various in silico analyses that identified the methylation site that we are interested in, or most interested in, is right next to a transcription factor binding site.

Stephen White: So we then went on to show that binding of that transcription factor is sensitive to methylation, that if we would just excise that piece of DNA, we can show that that has the ability to regulate F2RL3 expression or the expression of a reporter gene. And then if you knocked out the transcription factor binding site, you lose that regulation. So it was a series of detective work and experimental steps that allowed us to put a mechanism behind the observation that changes in methylation might truly affect the level of gene expression of the F2RL3, otherwise known as PAR4 to platelet biologists. So get that in there.

Alastair Poole: I first came across it when another member of our team actually mentioned it to me over a casual conversation actually a few years ago that F2RL3 gene was regulated in this way. To me as a platelet biologist, F2RL3 didn't mean a lot, but when I was told then it was the gene that encodes PAR4, it meant everything. And so platelet biologists, we talk about PAR4, which is of course the protein product of the F2RL3 gene. And PAR4 is one of several really key receptors on a platelet surface that responds to, in this case, to changes in thrombin generation, thrombin activity, which is of course the major effectively end product of the coagulation cascade.

Alastair Poole: So it's what couples coagulation and platelet biology together, thrombin. And there are two major receptors on platelets that operate in response to changes in thrombin and that's PAR1 and PAR4. And they're both very important genes, but yeah, really interestingly, you have this rather selective effect on PAR4 and the paper actually shows it is indeed a selective effect on PAR4 as opposed to PAR1 in terms of epigenetic regulation of its responsiveness to PAR4 activation.

Cindy St. Hilaire: So I want to tap back onto something that Laura had mentioned briefly, and that is talking about your platelet assays where you isolated platelets from a specific subset of the patients. And I believe it was figure three, and you looked at patients who in adolescence had exhibited differences in the methylation pattern at the site in the F3RL2 locus. What do we know about that innate or early-age change? And then I would love to hear more about this actual experiment, how you looked at the patients earlier versus current and what the thinking was behind that.

Laura Corbin: So yeah, this part of the work was done in a birth cohort study called the Avon Longitudinal Study of Parents and Children, which is based at the University of Bristol. And this is a really great study, a great resource that we have, and in fact, it's open to all researchers so anyone could use it, where mothers were recruited during pregnancy, which was in around 1991 to 1992. And then those children that were born from those pregnancies have then been followed up ever since.

Laura Corbin: So that was the data that we were able to use for this part of the study. And what we wanted to do was to look at how this could work functionally, so look at the platelet function, but we really wanted at this point to step away from the smoking. Because obviously if you're going to look at platelet function in smokers versus non-smokers, it's incredibly difficult then to say that that's coming through a specific pathway, because we know that smoking induces lots and lots of changes in methylation, in proteins, all sorts of things going on. So we couldn't see a way of doing that part of the experiment with a comparison of smokers versus non-smokers. But what we know is that there's natural variation in methylation across all sites, including F2RL3.

Laura Corbin: So we had historic data from earlier time points, so two earlier time points from when the children were under 20. And we looked at those measures for F2RL3 and then just simply ranked people according to whether they had high or low methylation, and then used those two ranks together to then work out who had a consistently high versus consistently low level. And then we invited participants back into the clinic to have samples taken from those up and lower ends of the distribution. At that point, we were just really hoping that that methylation pattern would continue because this was then, I think they aged about 24 by the time we did this work, so it was some time after. And we restricted our selection just to people who were non-smokers, so never smokers based on the information they provided, but also asking them when they came in for that clinic just to verify that they were non-smokers.

Laura Corbin: And then we had a look at the methylation again. This time we looked across four sites in the region, which are the sites presented in the paper. And luckily for us, there was still that mean difference between the high group and the low group. But what we were able to then do is to compare people with high and low methylation, but without all of the trouble of isolating that pathway in amongst all the other smoking effects. And also not just the smoking effects, but the other confounding factors that come with smoking. So we know that smoking is correlated with a lot of other lifestyle factors. So if you ever do a smoking versus non-smoking comparison, it's really hard to work out exactly which bits are coming from smoking and which pathways it might be going down. So this was the idea behind this part of the study was to just really zoom in on F2RL3 methylation in the absence of all of the other noise in the other experimental designs.

Laura Corbin: So yeah, the natural variation we see in the non-smoking healthy participants in this part of the study is actually quite a lot less than we see when we look at smokers compared to non-smokers, but it was still enough to then go on and look at the platelet function. And then the differences we saw in the platelet's responses, there is nothing pathological there. It was just very subtle changes in the response when stimulated in the lab.

Alastair Poole: The only other thing I could add would be that platelets are very complicated cells. Every cell of the body is very complicated. Platelets are certainly very complicated. PAR4, F2RL3, is just one of very many components of the platelet that modulate its activity. So platelets are controlled by multiple forces sort of thing, at which F2RL3 and PAR4 is just one of them. So biology is very good at compensating for one level going up in one part of a pathway and going down compensatory wise in another part of a pathway. There isn't necessarily a direct relationship between one pathway enhancement and an overall effect because of the compensation.

Cindy St. Hilaire: Why would it be easy?

Alastair Poole: Yeah. Yeah. It's just very complex, the biology. So yeah, I completely get what you're saying, Laura, that we obviously don't want to frighten people that maybe they've got a propensity to enhance thrombosis based upon a single gene methylation difference because it will be much more complex than that.

Cindy St. Hilaire: Yeah. I think that's one of the beautiful things about your study is with the luck of having this sample population, you were able to ask these really precise questions that... You can't just start a study now and ask that sort of question. So it was really elegant in that sense.

Cindy St. Hilaire: Do we know the mechanism of how cigarette smoke induces these methylation changes, or maybe even the specific components of the smoke? And I guess I'm thinking that in terms of vaping that's becoming more and more popular, obviously the company selling those products want to advertise them as safer, but it comes down to is it all of the mixture of the cigarette smoke or is it one component that we know impacts the methyltransferases and demethyltransferases in this process?

Alastair Poole: Those are two follow-on routes of our study that I have to say that we discussed previously amongst ourselves and identified those as definitely very important follow-up areas. So do e-cigs have similar effects and that's a study that definitely needs to be done. We have done a little bit of work to try to investigate that initially, but I think that's a very important follow-on study. But yeah, you're also right that one of the key things that we want to understand and is, the missing piece in a way, is how is methylation at a molecular mechanistic level altered by smoking? Steve, I don't know whether you have any further details to add to that.

Stephen White: I think one of the key molecular pathways seems to be the antioxidant response. And so that's largely controlled by another transcription factor called NRF2. And so if you think about smoking or poor air quality, all of those things do combine through this particular pathway that senses free radical damage, free radical stress. So as Alastair said, it's an area we are going to carry on to look at and it's a big area of my own lab's investigations. But oxygen stress is probably the mediating factor, but the actual nuts and bolts about how the demethylase is targeted to this particular locus is still an area of active investigation.

Cindy St. Hilaire: All right, well, I will be on the lookout for those future studies because it's a really interesting topic, just the whole interplay of all of this. Are there any translational implications for these findings? Do you think potentially we could screen patients, say, to see their methylation status? I don't know if megakaryocytes are easy to isolate, but is it in a circulating cell, would this possibly be able to be turned into a screening tool or a diagnostic tool to predict thrombic events in patients?

Alastair Poole: It is possible. I think it would not be possible to isolate megakaryocytes very easily. There are a small number in the peripheral circulation, but the majority are not in the peripheral circulation. But we and others have used other blood cells as proxy measures. So actually, the gene methylation changes that we identified here come from other leukocytes, white blood cells, and those effectively are a cell that are exposed to smoke in the same way, or the smoke products in the same way. So we'd use a proxy cell for that.

Alastair Poole: Yes, I suppose it is possible. As you say, there's a natural variation in methylation status of that gene and there's, layered on top of that, a smoking induced. And I suppose that it would be an interesting further investigation to understand whether, effectively, your natural methylation status of that gene happened to give you an enhanced risk of a cardiovascular event. The work we've done seems to suggest that that may well be the case and therefore you could imagine possibly a personalized medicine approach that might include understanding the methylation status of F2RL3 as part of that.

Cindy St. Hilaire: Well, it was a beautiful study. I love these studies that bring in lots of different fields or specialties to ask interesting questions. So Dr Corbin and Dr Poole from the University of Bristol and Dr White from Manchester Metropolitan University, thank you so much joining me today.

Stephen White: Thank you. Our pleasure.

Alastair Poole: Thank you.

Laura Corbin: I'd also just like to acknowledge all of our co-authors as it really was a big team effort, especially the guys who are not represented on the call today, which is the folk from the Copenhagen City Heart Study, and also to all of the participants of that study and the Children of the '90s Study that contributed to the work. Thanks very much.

Cindy St. Hilaire: That's it for the highlights from our February issues of Circulation Research. Thank you so much for listening. Please check out the CircRes Facebook page and follow us on Twitter and Instagram with the handle @CircRes and the hashtag #DiscoverCircRes. Thank you to our guests, Drs Alastair Poole, Laura Corbin and Stephen White.

Cindy St. Hilaire: This podcast is produced by Ashara Ratnayaka, edited by Melissa Stoner, and supported by the editorial team of Circulation Research. Some of the copy text for highlighted articles is provided by Ruth Williams. I'm your host, Dr Cindy St. Hilaire, and this is Discoverer CircRes, your on-the-go source for the most exciting discoveries and basic cardiovascular research.

Cindy St. Hilaire: This program is copyright of the American Heart Association 2022. The opinions expressed by speakers of this podcast are their own and not necessarily those of the editors or of the American Heart Association. For more information, please visit ahajournals.org.

This month on Episode 32 of Discover CircRes, host Cynthia St. Hilaire highlights four original research articles featured in the January 7 and January 21 issues of Circulation Research. This episode also features a conversation with Ms Natalie Harris and Dr Kathleen Caron from the University of North Carolina Chapel Hill about their study, VE-Cadherin Is Required for Cardiac Lymphatic Maintenance and Signaling.

Article highlights:

Carlson, et al. AKAP18δ Controls CaMKIIδ Activity

Gan, et al. sEV and Adipocyte ER Stress Following MI/R

Khan, et al. Long-term Risk Prediction of Heart Failure

Awan, et al. Wnt5a Is Essential for Cholesterol Homeostasis

Cindy St. Hilaire: Hi, and welcome to Discover CircRes, the podcast of the American Heart Association's journal Circulation Research. I'm your host, Dr Cindy St. Hilaire from the Vascular Medicine Institute at the University of Pittsburgh. Today, I'm going to be highlighting the articles from our January issues of Circulation Research. I'm also going to speak with Ms Natalie Harris and Dr Kathleen Caron from the University of North Carolina Chapel Hill about their study, VE-Cadherin Is Required for Cardiac Lymphatic Maintenance and Signaling.

Cindy St. Hilaire: The first article I want to share is titled AKAP18δ Anchors and Regulates CaMKII Activity at Phospholamban-SERCA2 and Ryanodine Receptors. The first and corresponding author for this article is Cathrine Carlson, and the study was conducted at University of Ohio. In cardiac muscle cells, calcium is continuously released and taken up by the sarcoplasmic reticulum to drive alternating contractions and relaxations. The kinase, CaMKII, regulates this calcium signaling via phosphorylation of the sarcoplasmic reticulum proteins ryanodine receptors also called RYR.

Cindy St. Hilaire: These receptors promote calcium release, and phospholamban promotes calcium uptake via the transporter SERCA, but how CaMKII localizes to and associates with these sarcoplasmic reticulum factors was unclear. Because AKAP18 delta enables phosphorylation of phospholamban and calcium uptake into the sarcoplasmic reticulum, this group suspected it might be involved. The team's immuno precipitation and functional experiments in rodent cardiomyocytes show that AKAP18 delta associates with CaMKII and phospholamban SERCA2 as well as with CaMKII and ryanodine receptors, and that these interactions are linked to CaMKII activity.

Cindy St. Hilaire: The team identified two separate CaMKII binding domains within the AKAP18 delta protein, one that inhibits the kinase and one that actuates it, suggesting they may somehow serve to fine tune CaMKII activity. While such regulatory details remain to be resolved, the isolated domains may be utilized as tools for studying calcium handling in cardiomyocytes, and for developing therapeutic CaMKII regulating reagents for treating arrhythmia.

Cindy St. Hilaire: The second article I want to share is titled Ischemic Heart-Derived Small Extracellular Vesicles Impair Adipocyte Function. The first author is Lu Gan, and the corresponding authors are Yajing Wang and Yu Cao from Thomas Jefferson University. While diabetes and obesity increase a person's risk of myocardial infarction, suffering a myocardial infarction itself can lead to metabolic dysfunction. One of the main regulators of systemic metabolic homeostasis is the body's adipose tissue, but whether and how an injured heart communicates with adipocytes was unclear.

Cindy St. Hilaire: The infarcted heart is known to release microRNA containing extracellular vesicles, also called EVs, and so this group hypothesized that these EVs might constitute a heart-to-fat communication system. They isolated circulating EVs before and after myocardial infarction in mice, and incubated these vesicles with cultured adipocytes. After 24 hours, differences in adipocyte gene and protein expression were apparent. Notably, a key cardioprotective metabolic factor called adiponectin was downregulated in cells treated with the extracellular vesicles from myocardial infarcted mice, while genes involved in endoplasmic reticulum stress were increased.

Cindy St. Hilaire: Analysis of the myocardial infarction extracellular vesicle content showed an increased abundance of specific microRNAs, and the team went on to show that inhibiting production of these microRNAs or the EVs themselves, prevented adipocyte ER stress and adiponectin production in mice after myocardial infarction. Together, these data hints that such microRNA inhibition may be a clinical strategy that can be used to prevent infarction-associated metabolic dysfunction in humans.

Cindy St. Hilaire: The next article I want to share is titled Development and Validation of A Long-Term Incident Heart Failure Risk Model. The first and corresponding author of this study is Sadiya Khan from Northwestern University. Heart failure contributes to approximately 1.2 million hospitalizations, and 300,000 deaths in the U.S. annually. Heart failure also has an estimated healthcare cost of over $10 billion. With both the incident rates and costs expected to rise in the future, a method for predicting an individual's heart failure risk would enable preventative interventions such as diet and blood pressure treatments to be initiated early, thus prolonging the number of healthy years.

Cindy St. Hilaire: To develop such a prediction tool, this group studied decades of health data from over 24,000 individuals that was collected as part of five separate, long-running national heart, lung and blood institute studies. The individuals included in the model for development were at baseline aged between 20 and 59 years old, and had no cardiovascular disease diagnosis at that time. Analysis of their body mass indices, blood pressures, total cholesterol levels, high density lipoprotein levels, smoking statuses, diabetes diagnoses, and other cardiovascular health data over several decades enabled the team to develop an equation for predicting an individual's likelihood of developing heart failure in the next 30 years. The hope is such personalized risk assessments will help to guide patient-doctor discussions regarding cardiovascular health, lifestyle choices and medical interventions.

Cindy St. Hilaire: The last article I want to share is titled Wnt5a Promotes Lysosomal Cholesterol Egress and Protects Against Atherosclerosis. The first authors are Sarah Awan and Magalie Lambert, and the corresponding author is Philippe Boucher from the University of Strasbourg. The Wnt family of signaling proteins drives many developmental processes, such as cell fate determination, proliferation and migration. Recently, Wnt signaling has been implicated in lipid homeostasis. Mutations that impair Wnt signaling have been shown to cause hyperlipidemia in mice, and in humans, decreased Wnt signaling activity inversely correlates with atherosclerosis severity.

Cindy St. Hilaire: Because the protein Wnt5a in particular has been shown to inhibit cholesterol accumulation in cells, this group investigated the role of Wnt5a protein in mice and human cells. Mice whose vascular smooth muscle cells lacked Wnt5a developed more severe atherosclerosis compared to control animals, and human smooth muscle cells lacking Wnt5a accumulated far greater amounts of cholesterol in the lysosomes than did cells with normal levels of Wnt5a. The group then showed that Wnt5a normally associates with lysosomes, where it promotes the catabolism of lysosomal cholesterol via activating lysosomal lipase, and promoting cholesterol egress via the endoplasmic reticulum. In revealing how cholesterol efflux is trafficked by Wnt5a, these findings may help to inform future cholesterol regulating therapies.

Cindy St. Hilaire: Today, Natalie Harris and Dr Kathleen Caron from the University of North Carolina Chapel Hill are here with me to discuss their study, VE-Cadherin Is Required for Cardiac Lymphatic Maintenance and Signaling, which is featured in our January 7th issue of Circulation Research. Thank you both for joining me today.

Kathleen Caron: Thanks, Cindy, for having us. We're really honored and excited to talk with you.

Cindy St. Hilaire: I'm excited too, because I think this is my first lymphatic paper I'm talking about. That's where I'm going to start my questions. Your study is investigating cardiac lymphatics. But like I said, I haven't talked a lot about lymphatics here, so I was wondering if you could at least give a little bit of background about what the role is of the lymphatic system, especially because I feel like it's the unappreciated member of the circulation, and also give us a little bit of background on what cardiac lymphatics are.

Kathleen Caron: That's a really great question. We sometimes talk about lymphatic vessels as the third vascular system or the understudied vascular system. I'm hoping that that's not the case so much anymore, because the lymphatic field has really boomed in the past 15 years or so. I think where we are right now in the field is in early days, we and others had discovered key signaling molecules, and transcription factors, and growth factors that are important and specific to the lymphatic vasculature as compared to blood endothelial cells. Through those unique tools, now, the field has fast forwarded where we're starting to look into organ-specific functions of lymphatics.

Kathleen Caron: We're appreciating that perhaps a little unlike the blood vascular system, which has one main function of delivering blood, lymphatics actually have very different functions depending on the organ that they're in. Some of the more common ones that you'll read about in textbooks in about a paragraph in a medical textbook are that lymphatics are important for immune cell trafficking through the lymph nodes, so they're the major route of trafficking for immune cells and for their maturation. Lymphatics are also important for draining interstitial fluid, and maintaining the homeostasis of tissue fluid balance.

Kathleen Caron: A third really big one, which is sometimes underappreciated, is that lymphatics are the key vessels within the intestine that absorb lipid, and so all of our dietary lipids are absorbed through lymphatic vessels as opposed to the blood vasculature. Those three hallmark functions of lymphatics are the cornerstone of what they do throughout our body. But when you start to look into different organs and recognizing the different extrinsic and intrinsic forces that govern the function of these endothelial cells and different organs, you start to realize that they're even more complex, and that brings us to the heart.

Kathleen Caron: The heart just has this beautiful network of lymphatic vessels that begin in the subendocardial space, and then project out and cover the subepicardial surface of the heart. And because the heart is always pumping, and because lymphatic vessels don't have an intrinsic mechanism for the flow of fluid through them, they rely on the movement of the tissue that they're in to help propel the fluid. So, this really raises the question of how are lymphatics functioning physically within a myocardium that is pumping with a very strong extrinsic force, and what is the function of those vessels if the heart is a very dense, thick organ that is not necessarily prone to edema necessarily as maybe our peripheral tissue and our skin is?

Kathleen Caron: We've been studying this for many years now, and we've had several studies exploring genetic factors that are important for the growth and development of cardiac lymphatics. That's the focus of this paper today. They're quite unique and very different vessels.

Cindy St. Hilaire: Reading your paper, I definitely learned a lot about lymphatics in general. One of the things I was thinking about, obviously, you're looking at VE-Cadherin, which is an endothelial cell marker. When I think of VE-Cadherin, and when I think of endothelial cells, my mind goes primarily to those that are in arteries and veins. In those conduits, their role is to really keep a tight seal to keep things out. But in the lymphatic system, it's very different, so how exactly different are the endothelial cells in the lymphatic tissue, and are they different, say, in the cardiac lymphatics versus, like you said, the mesenteric lymphatic?

Kathleen Caron: Lymphatics are very different than the blood vasculature. First of all, the lymphatic vasculature has key differences in terms of its architecture and structure. The lymphatic endothelial cells themselves, as they exist in vessels, don't put down a basement membrane, and in general, the dermal capillaries or the initial collector lymphatics that are the ones that are taking in fluid also don't have smooth muscle cells surrounding them like our typical vasculature does. All of this is guided and precedented by the differences in gene expression patterns of these very specialized endothelial cells.

Kathleen Caron: They also have very different cell-cell junctions. So when we think of a blood endothelial cell, we typically think of these tight junctions that bring them together, but the lymphatic endothelial cells have oak leaf shaped overlapping junctions. They're really beautiful to see on an EM, and they're very different than the blood vasculature, because, Cindy, as you mentioned, the function is very different. You're supposed to let things leak out, and big things too, right, like immune cells and large proteins.

Cindy St. Hilaire: One of the neat things that really made your study possible is this really nice PROX1 inducible CRE that you crossed with the flox-cadherin5 gene. I was wondering a little bit about that protein. Is that one of these, I guess, markers that allows lymphatic EC to be a lymphatic EC, and how specific is that protein for those specific ECs?

Natalie Harris: The PROX1 CRE that we use is based off of the PROX1 transcription factor, which we consider to be one of the master transcription factors of lymphatics. In fact, that was one of the very first lymphatic specific transcription factors that help maintain the lymphatic identity. So in this case, PROX1 turns on from blood endothelial cells, because many lymphatics are of venous origin, so actually, PROX1 turning on is a hallmark of them becoming a lymphatic endothelial cell.

Natalie Harris: Those are really great CRE specifically to look at lymphatics in this case, and it actually is a perfect model system because VE-Cadherin itself is only expressed in lymphatics and blood vessels, and then we have PROX1 as our free driver. Therefore, it will only be lymphatic, so it's a very specific lymphatic knockout of VE-Cadherin.

Cindy St. Hilaire: That's so wonderful when we discover things that are so specific like that. So using this really nice model that's also Tamoxifen inducible, you then have control to look at things temporally. One of the neat things that you did was you looked at this in terms of an embryonic level knockout, but then another one postnatally, and then another one, it was an adult mouse, which not a lot of people do that intricate, temporal spacing of things. So I was wondering if you could just share with us what you were thinking behind doing that, and then really importantly, what those different models actually taught you about the cardiac lymphatics?

Kathleen Caron: That's a great question, Cindy. It would take me 20 minutes to answer. It really represents work by all of the co-authors. Really, it's the first effort to look at the different stages. That's because the growth and development of lymphatics, particularly within the myocardium, differs a lot during embryogenesis, and then the vessels themselves are quiescent in an adult animal. Then of course, we were interested in seeing what might happen in an injured myocardium, and that was also part of the study.

Kathleen Caron: We felt that it was important to address the changing and dynamic role of this protein in a developing lymphatic, because it's growing and forming these nascent vessels, and then as it's starting to remodel an early life, and then in adulthood when it's in a quiescence state. That was the rationale for looking at this. It was also... Sometimes, science just takes you where it takes you, and it was a co-author of ours, and collaborator of ours, who had noted a phenotype in the hearts of these animals that he generated and suggested that maybe it would be a good idea to look early in development. Then as one thing leads to another, you start looking later in development and so on and so forth, so the science just kind of…

Cindy St. Hilaire: Sometimes tells you where to go on its own.

Kathleen Caron: Exactly. It was a long project.

Natalie Harris: Part of the reason too is that the cardiac lymphatics have been shown to have a little bit of a different development and maintenance and pruning cycle than some of the other lymphatics. Some other lymphatics are totally fully formed in embryonic development, but the cardiac lymphatics have been shown to develop through birth and a little bit postnatally as well. That makes them a little bit unique in the sense that their maturation is very prolonged, so that's part of the reason as well we wanted to look both in embryonic development as well as that postnatal period.

Cindy St. Hilaire: That's so interesting. There are a lot of little nuggets that my antennas would perk up as I read your paper, really neat observations. One of them was that I think it was the postnatal and the adults. There was lymphatic endothelial cells in the cardiac tissue were disrupted. They were discontinuous and fragmented, yet there was no cardiac edema. I thought that was interesting because normally, you'd think about any of these mice with lymphatic issues. You think of edema. You think of swelling, and yet it wasn't happening in the heart. What do you think that means either about the lymphatic system in the heart or in lymphatics as a whole?

Kathleen Caron: That's a really great question, and one that we think about all the time. I think it goes back to the first question or the first comment about the really remarkable differences in the functions of lymphatics and different tissues, right? And within the myocardium, because it is continuously moving and pumping with great force, the extrinsic forces within that tissue will help to mitigate the formation of edema. This is not to say that you can't get myocardial edema, and we've actually developed surgical models in our lab to form myocardial edema in mice.

Kathleen Caron: It is a very common clinical condition in humans as well, but the lymphatics themselves being fully invested within this myocardium probably are being regulated differently in their function in draining fluid than, for example, the lymphatics that you might have in the skin or in your thigh or in other organs in your body. The fact that there wasn't edema, even though you had leaky vessels, didn't alarm us too much because we knew and sensed that with this constant pressure and pumping of the myocardium, that in itself helps to keep the tissue fluid balanced.

Natalie Harris: That might be another reason why we're not seeing such extremes in edema, and then going back to what Kathrine said, again, because lymphatics have multiple functions, perhaps it's more in the immune cell realm or even other functions we haven't uncovered yet.

Cindy St. Hilaire: One of the other neat observations you had was that you were doing a myocardial infarction model on the adult animals, and you noticed that the infarct size and the fibrosis was indeed larger in the knockouts, but the cardiac function wasn't exactly affected. What does this mean, and were you surprised by this?

Kathleen Caron: Yeah, we were surprised. We absolutely were surprised, and we think that's actually one of the key big reveals for the field. To balance this, to counterbalance the absence of a phenotype, that was really remarkable to us, and I hope to many others as well, is that other studies including work from our lab and Paul Riley's lab and Eva Brackinham's lab have very convincingly shown in multiple different ways that if you stimulate lymphangiogenesis after injury, if you have a model, either genetic or induced, where there are more lymphatics for whatever reason, that's a beneficial thing. That's a great thing, and having more lymphatics is positive and beneficial to improving heart repair, and mitigating heart injury, and helping in the context of myocardial infarction.

Kathleen Caron: Of course, it was really surprising that now we have a mouse model where we essentially have little to no lymphatics with very little to no function, and yet the ejection fractional shorting of the heart was doing just fine. I think that was a big moment and a big discovery for us, but very convincing. Then I think it leads us to really asking while more might be better, what really could be the critical function of the lymphatics in an injured myocardium? As Natalie just mentioned previously, it might be related to immune cell trafficking. Paul Riley's group has made some really seminal discoveries in that regard.

Natalie Harris: It's just very interesting, because it's really against everything that you would expect from, again, all the previous studies. It just goes to show again that the lymphatics are so heterogeneous in their organ level function that that's really worth exploring more, because maybe if you can figure out strategies to selectively target certain beds, you can really do a treat on the disease by disease, organ by organ basis. That makes the lymphatics just really cool in my opinion, because they are so different, but it's all the same system, so it's just a very interesting organ, in my opinion.

Kathleen Caron: I should also say serendipitously or right about a few months ago... Shout out to Mark Kahn's lab at University of Pennsylvania. They had a recent paper, I believe, in JCI that had a similar finding to ours. It's always gratifying when another lab says, "Oh, wow, really?" Their study was very different than ours and on a different series of signaling molecules, but similarly, they ablated or reduced cardiac lymphatics through different mechanisms, and then had an injury model. Also, were rather surprised to see that it didn't have this negative effect.

Cindy St. Hilaire: It's so neat. The whole observations that you saw with these knockouts was a paper in itself, but the next half of the paper, you dig into the mechanism, which is also interesting. Can you share a little bit about the links that you found between VE-Cadherin and the VEGF receptor signaling, and is your mechanism you think specific to all lymphatic ECs or even all ECs, or is it specific just to the cardiac lymphatic ECs?

Kathleen Caron: Yes, the mechanism, I find one of the funnest parts of this paper, because I think it really synergizes a lot of the key signaling molecules within our field. Also, I think it bridges together a G-protein-coupled-receptor signaling pathway that my lab has been interested in for decades now, and that is a pathway with the VEGFR3 signaling pathway. I think that's been a big open question in the field. How do these two critical requisite signaling paradigms for lymphatics converge together to maintain lymphatic function development?

Kathleen Caron: I think we've really made some really great inroads in the study, and VE-Cadherin is central to that because it forms a structural scaffold to keep a GPCR signaling pathway in register with the receptor tyrosine kinase signaling pathway, and basically allow for the transactivation of these two really powerful pathways. The mechanism really is gratifying to be able to finally pull how these molecules all interface together and regulate one another.

Natalie Harris: It's very interesting in the fact that VE-Cadherin, it's not necessarily like a lymphatic-specific molecule, but a lot of work in terms of VE-Cad has been more in studying mechanosensing and mechanotransductions. That's where a lot of little nuggets about maybe our mechanism has occurred that we know from really just on protein level studies that VE-Cadherin does interact with VEGFR2 and VEGFR3 by the transmembrane domain interaction. That was clue number one, and then clue number two is that we know that a lot of different mechanical signals that might affect VEGFR3 happened in the presence of VE-Cad.

Natalie Harris: So in a sense, this particular paper is just piecing together a lot of these nuggets of information, and it all makes sense. One thing that you were saying in terms of maybe specific to the heart, going back to some of the earlier studies on these papers on these mice, that we found very vessel-bed-specific effects. One of the vessel beds that is really impacted is the lacteals and the mesentery, so the gut lymphatics. We do know that these lymphatic beds are very sensitive to VEGFC. In fact, they require constant VEGFC signaling. So if you're not having VEGFR3 stable at the membrane to receive these signals, it makes sense if you would have really extreme effects. That might be, again, some of the case in the heart as well. We do know after a cardiac injury, we do see an increase in things like adrenomedullin, and an increase in VEGFC has been shown to increase lymphangiogenesis, so perhaps also the heart, the gut lymphatics also has a special requirement for VEFGR3 signaling.

Cindy St. Hilaire: So in terms of, I guess, the future of this line of research and maybe thinking about translation, what do you see as maybe a role for this in terms of developing therapeutic strategies or even preventative measures, I guess, specifically in the cardiac lymphatic area?

Natalie Harris: Like we mentioned earlier, there's been a lot of studies in mice that have looked at increasing lymphangiogenesis post-injury, so it would be interesting to see more when those hit the clinical end, and if you're seeing similar effects. Then the other thing that's interesting about lymphatics, you can think of them as both a target and also as a drug delivery route. There's a huge, huge field totally dedicated to using the lymphatics to deliver drugs like nanoparticles. That's very big in the cancer realm, and pretty much for any kind of drug delivery, if you can imagine using that as a super highway to deliver drugs as well.

Natalie Harris: That could be a potential avenue in terms of the heart as well, getting a more specific administration of cardiovascular drugs to the heart. So whether or not we're thinking of them as being modulated by disease, we can also use them to modulate the disease itself by delivering drugs as well, so it's interesting. You can think of the lymphatics as a therapeutic target and as a therapeutic administrator. That's going to be really interesting to see where the field goes.

Cindy St. Hilaire: I like that, a new super highway to deliver drugs. Thank you so much, soon to be Dr Harris and Dr Caron from UNC Chapel Hill. This was a wonderful conversation and a beautiful paper. Congratulations on all the hard work.

Kathleen Caron: Well, thanks so much, Cindy, and to the whole Circ Research team. We really appreciate your advocacy for our work and giving us this wonderful opportunity.

Natalie Harris: Thank you so much.

Cindy St. Hilaire: That's it for the highlights from our January issues of Circulation Research. Thank you for listening. Please check out the CircRes Facebook page, and follow us on Twitter and Instagram with the handle @CircRes and #DiscoverCircRes. Thank you to our guests, Natalie Harris and Dr Kathleen Caron. This podcast was produced by Ashara Ratnayaka, edited by Melissa Stoner, and supported by the editorial team of Circulation Research. Some of the copy text for highlighted articles was provided by Ruth Williams. I'm your host, Dr Cindy St. Hilaire, and this is Discover CircRes, your on-the-go source for the most up-to-date and exciting discoveries in basic cardiovascular research.

Cindy St. Hilaire: This program is copyright of the American Heart Association, 2022. The opinions expressed by speakers on this podcast are their own and not necessarily those of the editors or of the American Heart Association. For more information, visit ahajournals.org.

This month on Episode 31 of Discover CircRes, host Cynthia St. Hilaire highlights two original research articles featured in the December 3 issue of Circulation Research. This episode also features a conversation with Drs Xavier Revelo, and Jop van Berlo from the University of Minnesota about their study, Cardiac Resident Macrophages Prevent Fibrosis and Stimulate Angiogenesis.

Article highlights:

Tong, et al. Alternative Mitophagy Protects Obesity Hearts

Soetkamp, et al. Myofilament Phosphorylation in CDC Treated HFpEF

Cindy St. Hilaire: Hi, and welcome to Discover CircRes, the podcast of the American Heart Association's journal, Circulation Research. I'm your host, Dr Cindy St. Hillaire from the Vascular Medicine Institute at the University of Pittsburgh. And today I'll be highlighting two articles presented in our December 3rd issue of CircRes, and I'll also speak with doctors, Xavier Revelo, and Jop van Berlo from University of Minnesota about their study, Cardiac Resident Macrophages Prevent Fibrosis and Stimulate Angiogenesis.

Cindy St. Hilaire: The first article I want to share is titled, Alternative Mitophagy Protects the Heart Against Obesity-Associated Cardiomyopathy. The first doctor is Ming Ming Tong, and the corresponding author is Jun Sadoshima from Rutgers University. People with obesity or diabetes have an increased risk of developing cardiomyopathy, a condition which can eventually lead to heart failure. One of the major pathological features of obesity-related cardiomyopathy at the cellular level is a decrease in mitochondrial function. This decrease in mitochondrial function is likely due to a decrease in the canonical mitophagy pathway, which is a process by which dysfunctional mitochondria are degraded. However, a new process termed alternative mitophagy was recently discovered. When mice were fed a high fat diet for 24 weeks after only eight weeks, canonical mitophagy ceased. However alternative mitophagy steadily increased over the 24 weeks. Alternative mitophagy is regulated via the protein ULK1 and Rab9. The team went on to show that suppressing alternative mitophagy by knocking out ULK1, or expressing a loss of function, Rab9 mutant exacerbated the high fat diet induced cardiac dysfunction. Over expression of Rab9 in mouse hearts increased the alternative mitophagy pathway and protected the animals from cardiac dysfunction. These results suggest that pharmacological boosting of this ULK1 Rab9 mediated alternative mitophagy pathway might be a treatment strategy for preventing obesity related cardiomyopathy.

Cindy St. Hilaire: The second article I want to share is titled Myofilament Phosphorylation in Stem Cell Treated Diastolic Heart Failure. The first doctor is Daniel Soetkamp and the corresponding author is Jenny Van Eyk from Cedar Sinai Medical Center. Weakness, fatigue and troubled breathing are among the symptoms experienced by someone suffering from heart failure with preserved ejection fraction, which is frequently called HFpEF. The pathology of the condition includes hypertrophy, fibrosis and stiffening of the heart and hyperphosphorylation of the cell sarcomere proteins. Because this hyperphosphorylation is a key contributor to HFpEF pathology, and because cardio sphere derived stem cells or CDCs have shown promise as a potential HFpEF treatment, this group investigated whether CDC treatment reduces phosphorylation levels of the sarcomere proteins in the heart. They found that administering CDCs to rats with HFpEF decreased the associated protein hyperphosphorylation, compared with that seen in untreated animals. Bioinformatic analysis revealed that protein kinase C or PKC is a prime suspect behind the phosphorylation. The authors suggest that CDCs alleviate HFpEF in part by reversing PKC-induced phosphorylation, and that PKC inhibition may be a desirable alternative treatment strategy, especially as it avoids regulatory issues associated with cell-based therapies.

Cindy St. Hilaire: Today, I have Dr Xavier Revelo and Dr Jop van Berlo from the University of Minnesota and they're with me to discuss their study, Cardiac Resident Macrophages Prevent Fibrosis and Stimulates Angiogenesis. And this article is in our December 3rd issue of Circ Res. So, thank you both so much for joining me today.

Jop van Berlo: Thanks for having us.

Cindy St. Hilaire: Absolutely. So, your study is investigating the contribution of resident and monocyte drug of macrophages in cardiac remodeling, specifically in hypertrophy remodeling. So can you just introduce the topic of cardiac hypertrophy in humans, why that's not great to have, and then maybe tell us a little bit about what is known or what was known about the role of inflammatory cells in that hypertrophic remodeling.

Jop van Berlo: Yeah, so absolutely. Cardiac hypertrophy is not a disease in and of itself in humans, but it is often a consequence of pathologies that can happen in patients, such as high blood pressure, hypertension or aortic valve stenosis, or if you've had a myocardial infarction the remaining myocardial may also become hypertrophic. We know that cardiac hypertrophy has downsides to it. People can develop sudden cardiac death when they have hypertrophic heart disease. We notice from population studies like the Framingham Heart Study and other studies, but it also increases the chance of developing heart failure later on. So even though cardiac hypertrophy by itself is not a disease, it is contributing to the cardiac pathology that can develop in patients and that can contribute to the development of heart failure.

Cindy St. Hilaire: Great. So, what's the base level of knowledge of what is known regarding inflammatory cells in cardiac hypertrophy or cardiac hypertrophic remodeling?

Xavier Revelo: So previous forecast focused on the role of infiltrating cells, specifically monocyte-derived macrophages, and generally these cells are pro-inflammatory and they aggravate the progression of heart failure. With Jop, we focus on, and what we think is exciting is the role of cardiac resident macrophages. And so, in our experiments, we decided to look at what's the role of these cardiac meta macrophages during pressure overload.

Cindy St. Hilaire: That's a perfect segue to my next question, which is you obviously modeled this in mice, you used mice as your model and the method that you used to induce this hypertrophy is a technique called Transverse Aortic Constriction. So how does that actually work in a mouse and are there certain pros and cons to using that as a model for cardiac hypertrophy and, does it really recapitulate well, what happens in humans?

Jop van Berlo: So, you're absolutely right that we use model systems to mimic what happens in humans and every model system has pros and cons to it. What we're trying to do here is to induce essentially acute cardiac pressure overload in a mouse model by inducing a constriction of the transverse aorta, right between the anomia and the left carotid artery. And we do this by ligating a needle on top of the transverse aorta that is of a specific size. And then we pull the needle out of the ligation and that immediately induces constriction. This is known to induce cardiac hypertrophy, and there are thousands of papers about this model as a model to induce cardiac hypertrophy. I think Howard Rockman was the first to publish this as a model of cardiac hypertrophy. Over the past decades, most of the research has focused on how cardiomyocytes within the heart respond to their stress and how they become hypertrophic. And I think what is new about our study is that instead of really focusing on the cardiomyocyte, we are focusing more on the non-cardiomyocyte compartments early after this stress is induced on the heart.

Cindy St. Hilaire: That was one of the things I liked about this paper. We read about TAC a lot, the transaortic constriction model, but a lot of it is looking at either just the fibrotic cells or the scarring or the cardiomyocytes. So, this was, I thought a really nice unique take. So, one of the things I'm wondering is what are the functional differences between the systemic macrophages and these resident macrophages? I guess, resident to the cardiac tissue. And how does one tell the difference between these cells in the mice, but also in the humans? What is the human equivalent of those cells?

Xavier Revelo: So, these cells, they rest in the macrophages in the cardiac tissue. One of the key differences from circulatory cells is the origin of the cells. In the heart, these cells self-renew, and they are from embryonic origins, as opposed to circulatory immune cells that come from the bone marrow. In terms of similarities between mice and humans, there are some markers that we can use to specifically study the cardiac resident macrophages. And these markers fortunately seem to be consistent between people and mice, which is advantages.

Cindy St. Hilaire: That is good. That's always nice when it works out that way. So, you, you actually answered my next question, which was, are these residents macrophages a) able to self-replicate or are they from their own source? And so, regarding that developmental origin, how far apart are these lineages of the circulating monocytes versus the resident or the cardiac resident? How similar and how different, and how far back on the tree do they diverge? If we know it?

Xavier Revelo: It's a complicated question.

Jop van Berlo: And it's an active area of study right now, not just by us, but also by many other groups.

Xavier Revelo: So, what we know is that regardless of origin, the cells are myeloid cells. So, they're the same lineage within the big family of immune cells. Having said that, the function of the cells is dictated by the origin, as well as the issue of residency. I forgot a second part of your question.

Cindy St. Hilaire: I'm wondering how much they diverge functionally from the circulating monocytes?

Xavier Revelo: They do. It seems like the tissue factors and the residency dictates the function of the cells in general. This is a general comment. Resident cells seem to have a protective role. Sometimes they help with the repair and healing as opposed to infiltrating cells that come into the tissue and they cause inflammation, generally they aggravate disease progression.

Jop van Berlo: But what I also find fascinating about these resident macrophages is they are not only found in the heart, but they're also found in all organs, and they all come from developmental origins. But if you compare the macrophages between these different organs, they resemble the organ itself more than macrophages between organs and that's based on recent work where people have compared resident macrophages from different organs. And I think that's just fascinating how this develops in the heart, but also in other organs as a way to protect specific organs from potentially dangerous signals.

Cindy St. Hilaire: Yeah, that's so interesting. So, it's almost like their niche, their new residential home, is really informing their function. So, there's some kind of back and forth between that environment and the cell itself.

Jop van Berlo: That's what we presume, but I don't think we truly understand how the niche is important in dictating the function of these resident macrophages. And I think we need to do a lot more research into how the niche of tissue resident macrophages has formed and how that then dictates the differentiation of these resident macrophages to give rise to certain functionalities.

Cindy St. Hilaire: Maybe you can summarize in a couple short sentences or so what, what your key findings were.

Jop van Berlo: The main findings of our study is that very early after the induction of acute cardiac pressure overload, there is a high level of inflammation happening in the heart. And this allows the replication of resident macrophages and our study showed that these resident macrophages are really important for a protective mechanism within the heart to allow the heart to deal with this increased pressure in a heart. And what they do is they stimulate the formation of new block vessels, also known as angiogenesis and furthermore, they inhibit the formation of scar tissue or fibrosis, and we used different ways to substantiate these conclusions.

Xavier Revelo: We studied cardiac-resident macrophages as one population. But one thing we learned in this study is that these macrophages are highly diverse. And so, using our techniques, we discover that within cardiac macrophages, we have 11 different subsets. And so, our future studies will be aiming at understanding the precise role of these different subsets that we think have different roles in pressure overload.

Cindy St. Hilaire: One of the things I was thinking about is these 11 subsets, do they represent kind of end stage fully differentiated resident macrophages, meaning 11 different types, or are they kind of representing maybe the different stages that get to the one end type? Do we have a sense of what's going on?

Xavier Revelo: I don't think it's completely understood my take on that is that these different subsets they can represent different activation states or functional subsets that we don't really understand why is that we have this diversity?

Jop van Berlo: I think one of the aspects that we as a field need to work on is to better understand that complexity of immune cells that reside within an organ and associate that complexity to specific functionalities. And right now, the field is mostly lacking in technologies that allow us to do this. For example, we cannot culture these resident macrophages right now. We don't know the proper culturing conditions that allow us to test functional differences between subsets of macrophages. We don't have very good genetic tools to dissect these specific subsets of macrophages. And I think those are important areas that the field and us of course need to work on in the coming years.

Cindy St. Hilaire: Every layer of discovery, just brings like 10 more layers complexity, or 11 more co-layers of complexity in this case.

Jop van Berlo: Which is why we all love science!

Cindy St. Hilaire: Exactly, exactly. It's a drug that, that keeps on giving. So, one of your experiments, I forget which number, I think figure five or six or something like that, but in wild type mice, you went on to use an anti CD115 antibody. And because that treatment others, as well as yourselves has shown depletes the resident macrophages. And, and one thing I thought was really interesting. I just want to hear how you unpack it. And that is in the wild type mice that were treated with the anti CD115 antibody. You found that the depletion of the resident macrophages exacerbated the adverse remodeling and it increased fibrosis, it decreased angiogenesis, but when you did the same thing in a CCR2 knockout mouse in that mouse, they don't have the circulating macrophages, but they also don't have the resident macrophages. They were protected from the increased fibrosis, but there was no change in the angiogenesis. And I was just wondering if you could unpack these results for me and kind of talk about the competing roles of the resident and the non-resident macrophage in this pathogenesis.

Jop van Berlo So I think you highlight a really important experiment that we performed that try to dissect the protective versus damaging effects of different subsets of macrophages within the heart. We know that if you delete the receptor CCR2, that circulating monocytes cannot extravasate and enter the tissue in response to the cytokine CCL2 that is produced by the myocardium. So, using the CCR2 knockout, we essentially blocked the invasion of circulating monocytes into the myocardium to become monocyte-derived macrophages. And we knew from the literature that, especially the monocyte-derived macrophages, were pro fibrotic. So, we wanted to discern the effects on fibrosis between resident macrophages and monocyte derived macrophages. So, we were happy to indeed see that when we blocked extravasation of circulating monocytes and blocked them to become macrophages, that we indeed reduce the amount of fibrosis that we observed within the heart.

I think the difficulty here that we observed that we don't have a very good explanation for right now are the effects on angiogenesis. And I think what this highlights is that there are many, many more complexities than just the resident and recruited macrophages on the development of angiogenesis because when we block tissue resident macrophages, are we actually depleting tissue resident macrophages? We didn't completely block the development of angiogenesis. We merely inhibited this by a little bit. And so, I think there are many more actions happening within the heart in response to stress than just the immune cells. And I think it highlights how complex a living organ really is. And we always try to do reductionist experiments to try to understand the functioning of specific aspects of that organ, but it's much more complex than just one cell type doing one thing and another cell type doing another thing.

Xavier Revelo: One potential explanation to this complexity is the fact that when we deplete resident macrophages, the monocyte-derived macrophages, the infiltrating macrophages, they can replenish those resident macrophages. And so, whether there's a difference between the original resident macrophages compared to the replacing macrophages is unknown. And so, all these complexities can perhaps explain that different phenotypes that we observed in terms of angiogenesis.

Cindy St. Hilaire: What do your findings suggest about potential therapies or you even potential therapeutic targets? Is it possible in a human to be able to target one or the other macrophage population? I know a lot of your experiments, because it's an experiment, you're targeting the depletion of macrophages before to see the effects, but are we able to possibly activate or stimulate their production, post MI for example?

Xavier Revelo: Yeah, absolutely. So, thinking about cardiomyocyte independent interventions that can enhance the preparation process of any stressed heart, we could see potential in manipulating resident macrophages, specifically enhancing the functions of these resident macrophages that will help us heal and prevent fibrosis and enhance angiogenesis. So, we think that future studies need to look at what factors can be manipulated to enhance the function and survival of these resident macrophages.

Jop van Berlo: One important aspect of our study that we don't highlight is that after this large increase in tissue resident macrophages, that we observed within the first week after cardiac pressure overload, these cells actually disappear. And right now, we don't really know the signals that are important for mediating that disappearing of cells. And we don't know this whether maintenance of these signals could improve longer beneficial effects of tissue resident macrophages.

Cindy St. Hilaire: Interesting. I guess we know some questions you're going to start to ask in the future.

Jop van Berlo: Absolutely. There's always more questions to answer in science.

Cindy St. Hilaire: Well, great. Well, Dr Revelo, Dr van Berlo. Thank you so much for joining me today. Congrats on a wonderful paper and we look forward to these future studies.

Jop van Berlo: Thank you.

Xavier Revelo: Thank you.

Cindy St. Hilaire: That's it for the highlights from the December 3rd issue of Circulation Research. Thank you for listening. Please check out the CircRes Facebook page and follow us on Twitter and Instagram with the handle at @CircRes and #DiscovererCircRes. Thank you to our guests, Dr Xavier Revelo and Jop van Berlo. This podcast is produced by Ishara Ratnayaka, edited by Melissa Stoner and supported by the editorial team of Circulation Research. Some of the copy text for highlighted articles is provided by Ruth Williams. I'm your host, Dr Cindy St. Hillaire. And this is Discover CircRes, your-on-the-go source for the most exciting discoveries in basic cardiovascular research. This program is copyright of the American Heart Association, 2021. The opinions expressed by speakers in this podcast are their own and not necessarily those of the editors or the American Heart Association for more information visit aha journals.org.

This month on Episode 30 of Discover CircRes, host Cynthia St. Hilaire highlights four original research articles featured in the October 29 and November 12 issues of Circulation Research. This episode also features a conversation with Dr Elisa Klein from the University of Maryland about her study, Laminar Flow on Endothelial Cells Suppresses eNOS O-GlcNAcylation to Promote eNOS Activity.

Article highlights:

Subramani, et al. CMA of eNOS in Ischemia-Reperfusion

Liu, et al. Macrophage MST1 Regulates Cardiac Repair

Van Beusecum, et al. GAS6/Axl Signaling in Hypertension

Pati, et al. Exosomes Promote Efferocytosis and Cardiac Repair

Cindy St. Hilaire: Hi and welcome to Discover CircRes, the podcast of the American Heart Association's Journal Circulation Research. I'm your host, Dr Cindy St. Hilaire from the Vascular Medicine Institute at the University of Pittsburgh and today I'll be highlighting articles presented in our October 29th and November 12th issues of Circulation Research. I also will speak with Dr Elisa Klein from the University of Maryland about her study, Laminar Flow on Endothelial Cells Suppresses eNOS O-GlcNAcylation to Promote eNOS Activity.

Cindy St. Hilaire: The first article I want to share is titled, Chaperone-Mediated Autophagy of eNOS in Myocardial Ischemia Reperfusion Injury. The first author is Jaganathan Subramani and the corresponding author is Kumuda Das from Texas Tech University Health Sciences Center. Reestablishing blood flow to ischemic heart muscle after myocardial infarction is critical for restoring muscle function but the return of flow itself can cause damage, a so-called reperfusion injury. The generation of reactive oxygen species or ROS and loss of nitric oxide or NO both contribute to reperfusion injury.

Reperfusion injury is exacerbated when the NO producing enzyme, endothelial nitric oxide synthase or eNOS, produces damaging super oxide anions instead of NO. This switch in eNOS function is caused by glutathionylation of the enzyme, termed SG-eNOS. But how long this modification lasts and how it is fixed is unclear. This group used an in vitro model of ischemia reperfusion where human endothelial cells are exposed to several hours of hypoxia followed by reoxygenation. In this model, they found the level of SG-eNOS steadily increases for 16 hours and then sharply decreases. By blocking several different cellular degradation pathways, they discovered that this decrease in S-G eNOS was due to chaperone mediated autophagy with the chaperone protein, HSC70, being responsible for SG-eNOS destruction. Importantly, this team went on to show that pharmacological D-glutathionylation of eNOS in mice promoted NO production and reduced reperfusion injury, suggesting this approach may be of clinical benefit after myocardial infarction.

Cindy St. Hilaire: The second article I want to share is titled Macrophage MST1/2 Disruption Impairs Post-Infarction Cardiac Repair via LTB4. The first author is Mingming Liu and the corresponding author is Ding Ai and they're from Tianjin Medical University. Myocardial infarction injures the heart muscle. These cells are unable to regenerate and instead a non-contractile scar forms and that fibrotic scar can lead to heart failure.

Cardiomyocytes specific inhibition of the kinase MST1 can prevent infarction induced death of the cells and preserve the heart function, suggesting that it may have clinical utility. However, MST1 also has anti-inflammatory properties in macrophages. So inhibition of MST1 in macrophages may delay inflammation resolution after MI and impair proper healing. Thus, targeting this enzyme for therapy is not a straightforward process. This study examined mice lacking MST1 in macrophages and found that after myocardial infarction, the inflammatory mediator leukotriene B4 was upregulated in macrophages and the animal's heart function was reduced compared to that of wild type controls. Blocking the action of leukotriene B4 in mice reduced infarction injuries in the hearts of MST1-lacking animals and enhanced repair in the injured hearts of wild type animals given an MST1 inhibitor. The results suggest that if MST1 inhibition is used as a future post infarction regenerative therapy, then leukotriene B4 blockade may prevent its inflammatory side effects.

Cindy St. Hilaire: The next article I want to share is titled Growth Arrest Specific-6 and Axl Coordinate Inflammation and Hypertension. The first author is Justin Beusecum and the corresponding author is David Harrison and they're from Vanderbilt University. Inflammation contributes to hypertension pathology but the links of this relationship are unclear. It's thought one trigger of inflammation may be the hypertension-induced mechanical stretch of vascular endothelial cells. Mechanical stretch causes endothelial cells to release factors that convert circulating monocytes into inflammatory cells. And one such factor is the recently identified Axl and Siglec-6 positive dendritic cells, also called AS DCs.

AS DCs produce a large amount of inflammatory cytokines but little is known about the role of AS DCs or their cytokines in hypertension. This group found elevated levels of AS DCs in hypertensive people compared to normal tensive individuals. Mechanical stretch of human endothelial cells promoted the release of GAS6, which is an activator of the AS DC cell surface kinase, Axl. This stretch induced GAS6 release also promoted conversion of co-cultured monocytes to AS DCs. Inhibition of GAS6 or Axl in the co-cultured system prevented conversion of monocytes to AS DCs. This team went on to show that hypertensive humans and mice have elevated levels of plasma GAS6 and that blocking Axl activity in mice attenuated experimentally induced hypertension and the associated inflammation. This work highlights a new signaling pathway, driving hypertension associated inflammation and identifies possible targets to treat it.

Cindy St. Hilaire: The last article I want to share is titled Novel Mechanisms of Exosome-

Mediated Phagocytosis of Dead Cells in Injured Heart. The first author is Mallikarjun Patil and Sherin Saheera and the corresponding author is Prasanna Krishnamurthy from the University of Alabama, Birmingham. After myocardial infarction inflammation must quickly be attenuated to avoid excessive scarring and loss of muscle function. Macrophage mediated efferocytosis of dead cells is a critical part of this so-called inflammation resolution process. And resolution depends in part on the protein. MFGE8. MFGE8 helps macrophages engage with eat me signals on the dead cells and loss of macrophage MFGE8 delays inflammation resolution in mice. Because stem cell-derived exosomes promote cardiac repair after infarction and are anti-inflammatory and express MFGE8, this group hypothesized that perhaps part of a stem-cell derived exosomes proresolven activity may be due to boosting macrophage efferocytosis.

They showed that stem cell derived exosomes did indeed boost efferocytosis of apoptotic cardiomyocytes in vitro and in vivo. An in vitro experiments showed that if exosomes lacked MFGE8 then efferocytosis by macrophages was reduced. Furthermore, after myocardial infarction in mice, treatment with MFGE8 deficient exosomes did not reduce infarct size and did not improve heart function compared to control exosomes. These results suggest MFGE8 is important for the cardioprotective effects of stem cell-derived exosomes. And that this protein may be of interest for boosting efferocytosis after myocardial infarction and in other pathologies where inflammation is not readily resolved.

Cindy St. Hilaire So today, Dr Elisa Klein from the Department of Biomedical Engineering at the University of Maryland is with me to discuss her study Laminar Flow on Endothelial Cells Suppresses eNOS O-GlcNAcylation to Promote eNOS Activity and this article is in our November 12th issue of Circulation Research. So Dr Klein, thank you so much for joining me today.

Elisa Klein: Thank you for having me.

Cindy St. Hilaire: Yeah. So broadly your study is investigating how blood flow patterns specifically, kind of, laminar and oscillatory flow, how those blood flow patterns impact protein modifications and activity. So before we, kind of, get to the details of the paper, I was wondering if you could just introduce for us the concept of blood flow patterns, how they change in the body naturally but then how they might influence or contribute to disease pathogenesis in the vessels?

Elisa Klein: Sure. So obviously we have blood flow through all of our vessels and since we are complex human beings, we have complex vascular beds that turn and that split or bifurcate. And so every place we get one of these bifurcations or a turn in a vessel, the blood flow can't quite make that turn or split perfectly. So you get a little area where the flow is a oscillatory or what we call disturbed. There's lots of different kinds of disturbed flow. And the reason why that's important is because you tend to develop atherosclerotic plaques at locations where the blood flow is disturbed. So in my lab, we look a lot at what it is about that disturbed flow that makes the endothelial cells there dysfunctional and that leads to the atherosclerotic plaque development.

Cindy St. Hilaire: That is so interesting. So I can picture how this is happening in a mouse at the bifurcation of different arteries but how are you able to model this in vitro? Can you describe the setup and then also how that setup can mirror the physiological parameters?

Elisa Klein: Sure. So we have a couple of different systems we can use to model this and they all have their advantages and disadvantages, right? So a few years ago we made a system that's a parallel plate flow chamber. So you basically have your cells that you see that on a microscope slide and you use a gasket that's a given shape and that either drives the flow… Usually it drives the flow straight across the cells. So that's a nice laminar steady flow. And we see that the cells align and they produce nitric oxide in that type of flow which are measures that they are responding to the flow in vitro. So, a few years ago we made a device that actually makes the flow zigzag as it goes across the endothelial cells. And that creates these little pockets of disturbed flow and we did that in our parallel plate flow chamber.

And that parallel plate flow chamber is really good for visualizing the cells. So you can stick it on a microscope. You can see what's happening, we can label for specific markers but it's not good for doing the things that we did in this Circ Research paper, where we want it to measure metabolism, because you need a lot more cells to measure metabolism and we needed a better media to cell ratio, so less media and more cells. So for this one, we designed and built a cone-and-plate device. So what it is, it's a cone and you spin that cone on top of a dish of endothelial cells and that cone produces flow. So it's going around in a circle. And if we just make it go around in a circle, it'll produce a steady laminar flow but if we oscillated it, so basically we kind of turn it back and forth, it'll make this oscillating disturbed flow. And then we have our dish of cells.

We do this in a 60-millimeter dish and then we have a small amount of media in there and a lot of cells. And we can culture the cells in there for a while.

Cindy St. Hilaire: That is so neat. And so I'm assuming that then your cone system is very tuneable. You could either speed it up, slow it down or change that oscillatory rate with different, I guess, shifts of it?

Elisa Klein: Yeah, that's exactly right. So we can do all those things. It's programmable with a motor and so we can run whatever type of flow we want.

Cindy St. Hilaire: That's great. So before your study, what was known regarding this link between hemodynamics and endothelial cell dysfunction and also endothelial cell metabolism? Because I feel like that's a really interesting space that a lot of people look at, kind of, metabolism and EC dysfunction or they just look at shear stress and EC dysfunction and you're, kind of, combining the three. So what was kind of the knowledge gap that you were hoping to investigate?

Elisa Klein: Yeah, so we're really interested in macrovascular endothelial cell dysfunction. So this pro atherosclerotic phenotype that you can get in endothelial cells. And most of the work on endothelial cell metabolism had actually been done in the context of angiogenesis. So how much energy and how do cells get their energy to make new blood vessels? And that's more of a microvascular thing. So there was a study that came out before ours, actually, before we started this study, that was looking at how steady laminar flow could decrease endothelial cell glycolysis. And so that was after 72 hours of flow and they showed some gene expression changes at that time. Our study is shorter than that and we were still able to see a decrease in glycolysis in our cells in laminar flow. Before we started this study, no one had really looked at disturbed flow. So in the meantime, there are a few other papers that came out showing that the cells don't decrease glycolysis when they're in disturbed flow but not so much connecting them back to this function of making nitric oxide.

Cindy St. Hilaire: So we were kind of dancing to the topic of O linked N acetylglucosamine or how do you say it?

Elisa Klein: GlcNAC.

Cindy St. Hilaire: GlcNAC? O- GlcNAC. So, O- GlcNAC is a sugar drive modification and I think it's added to Syrian and three Indian residues and proteins.

Elisa Klein: Yup, that's right.

Cindy St. Hilaire: Okay, good. And that modification, it does help dictate a protein's function. And you were investigating the role of this moiety on endothelial nitric oxide synthase or eNOS and so what exactly does this GlcNAC do for eNOS' function and under what conditions or disease states is this modification operative?

Elisa Klein: Yeah. So there's some really important studies from a little bit ago that showed that eNOS gets GlcNAcylated in animals with diabetes, right? So if you have constantly high sugar levels, you get this modification of eNOS. The thought was that eNOS gets GlcNAcylated at the same site where it gets phosphorylated. But a more recent study came out and said, well, maybe that's not the case but it definitely gets GlcNAcylated somewhere where it affects this phosphorylation site. So it may be near it and prevent the folding or prevent the phosphorylation site availability. So if the eNOS gets GlcNAcylated, the thought is that it can't get phosphorylated and therefore it can't make nitric oxide.

Cindy St. Hilaire: And so an interesting thing about this GlcNAcylation, which is probably the hardest thing I've ever said on this podcast, is that it's integrated with lots of different things. Obviously you need glycolysis and the substrates from the breakdown of sugars to make that substrate but also the enzymes that make that substrate are required. And so what's known about that balance in endothelial cells? Is there much known regarding the metabolic rate of the cells and this N-Glcynation?

Elisa Klein: Yeah. So endothelial cells are thought to be highly glycolytic in terms of how they use glucose but they definitely take up glutamine to fuel the tricarboxylic acid or TCA cycle. And another paper came out a few years ago showing that quiescent and endothelial cells metabolize a lot of fatty acids. So they're fueling their energy needs that way. So there wasn't a lot known about GlcNAcylation in endothelial cells.

A lot of this work has been done in cancer cells, which are also highly glycolytic but their metabolism actually seems like it's maybe more diverse than people have thought for a long time. So the weird thing about GlcNAcylation, which if you're used to working with phosphorylation there's a thousand different enzymes that can phosphorolate right. But with GlcNAcylation there's one enzyme that's known to put the GlcNAC on and one enzyme that's known to take it off. And so they're global, right? So in our studies, if we say, okay, we're going to knock down that enzyme, you're effecting every single protein in the cell that's GlcNAcylated. And obviously ourselves in particular, we're not a big fan of that. Especially once you put them in flow, they were, like, nope, we're not going to make it.

Cindy St. Hilaire: Well, and that's a perfect segue to my next question because your results show that this flow really did not alter the expression of these enzymes that either add or subtract to the moiety. And rather it was the Hexosamine Biosynthetic Pathway that was decreased itself. So can you maybe give us a quick primer on what that is exactly and how that pathway feeds into the glycosylation... I think you wrote in the paper of over 4,000 proteins? So how would that fit in and why eNOS then?

Elisa Klein: Yeah, so the Hexosamine Biosynthetic Pathway is one of these branch pathways that comes off glycolysis and there are these numbers sometimes there are these pathways out there and people say for the HBP in particular, 2% to 5% of the glucose that's going down through glycolysis gets shunted off into the HBP. We've done a lot of looking to try and figure out exactly where that 2% to 5%-

Cindy St. Hilaire: Yeah, what exact percentage?

Elisa Klein: Yeah, but some percentage of it comes down and we really thought there were going to be changes in these enzymes that do the GlcNacylation, we thought there might be changes in the localization of the proteins and it's possible that those things do occur. We just couldn't detect them in our cells. And in the end, what we showed was the main thing was that when you have cells and steady laminar flow, you just decreased glycolysis. And therefore, that 2% to 5% goes down. So you seem to make less of this UDP- GlcNAC, which is the substrate that gets put on to eNOS in this case. The really strange thing that we could not explain despite a lot of work and obviously we don't get to put all of our experiments that didn't work in the paper-

Cindy St. Hilaire: The blood, sweat and tears gets left out. So-

Elisa Klein: Exactly. So we tried really hard to figure out why it was eNOS specifically, right? Because in steady laminar flow, you see a lot of these like GlcNAcylated proteins and a lot of them didn't change but eNOS changed hugely, essentially this GlcNAcylation just went away for the cells and steady laminar flow. So we couldn't quite answer that. We're still working on that part of the question and looking at some of the other proteins that maybe get GlcNAcylated more in this case and trying to figure out what they are.

Cindy St. Hilaire: I thought one of the cool results in your paper was one of the last ones. It was the one in healthy mice. In that you looked at healthy mice, just normal C57 black 6 mice that were 10 weeks old. So they just, kind of, reached maturity but you looked at their kind of these bifurcations and you looked at the inner aortic arch where there is more disturbed flow and you saw, similar to your in vitro studies, that there was this higher level of O-GlcNAcylation compared to the outer arch in the descending order. So my question is, these are healthy mice that are relatively young, they're not even full adults yet. That takes a couple more months. And so what are your thoughts about the role of this O-GlcNAcylation specifically on eNOS in driving atherogenesis. Where do you think this is happening in the disease process? It appears if it's in these wild type mice, it's already happening early. So where do you think this is most operative in the disease pathogenesis?

Elisa Klein: I mean, I think it's very early, the effects of disturbed flow on endothelial cells. I can't imagine that there's a time when it's not having an effect on the cells. So I teach college students and I tell them all the time you think you're invincible now but these choices you're making today are going to affect your cardiovascular future in 50 years, which is very hard to accept. So I think it's very early in the process and I think it's only made worse by the things that we eat, in particular, that changed our blood sugar and our blood fatty acids and things like that. And our lab is looking into this more to try and see how when you change your blood metabolites then how does that then also affect this GlcNAcylation and the endothelial cell metabolism and then how does that affect endothelial cell function?

Cindy St. Hilaire: Yeah. And it's funny, it's really making me think of those, kinds of, extreme diets like keto diets and things like that where you're just like depleting sugar. And obviously there's lots of controversy in that field, but if you just think about the sugar aspect what is that doing to those EC cells? Why do you think endothelial cells have this response? Meaning why do you think it is that they've adapted to induce a metabolic shift in response to disturbed flow? Because, obviously it's not going to be perfect laminar flow everywhere. So what do you think it is that provides some sort of advantage in the shift?

Elisa Klein: That's a really good question. I haven't thought about the advantage that it might provide. There are a lot of things that are going on in this area of disturbed flow. So there is the shear stress, the differential shear stress that the cells are experiencing. There's also transport issues, right? So if you have this area of disturbed flow, you have blood and the contents of the blood, including the white blood cells and the red blood cells, everything else that's, kind of, sitting around in that area and not getting washed downstream as quickly. So it is possible that maintaining glycolysis provides energy for repair or for protecting the endothelial cell from some sort of inflammatory insult or something like that, that's happening in the area of disturbed flow. And I feel like I just read something recently, it was in a different genre but... if they stopped the increased glycolysis or stop the metabolic shifts, it actually was worse.

Right? So I also believe that we treat humans for a single metabolic change, right? So if you have diabetes, I'm going to give you this drug and if you have high triglycerides, I'm going to give you this drug. But it's possible that if you have this metabolic abnormality, your body shifts the rest of your metabolism to protect the cells because of that metabolic abnormality. And so part of what we do as engineers is try and build computational models or we can take into account some of this complexity. So that's a really interesting question and my guess is that there are some protective aspects of this maintenance of high glycolysis and disturbed flow.

Cindy St. Hilaire: Yeah, maybe it would be perfectly fine until we get athero and then it all goes awry. So in terms of... obviously it's early days and I know you're a bioengineer but in terms of translational potential, what do you think your findings suggest about future potential therapies or future targets for which we can use to develop therapies? Is modulating this O-GlcNAcylation itself, a viable option?

Elisa Klein: I don't think that modulating it is a super viable option, right? Because as I said, when we tried to change those enzymes ourselves did not enjoy going through flow or anything else. So it's very hard to change it overall. What I think is these things that are coming out about how metabolism may shift for endothelial cells when they're activated versus when they're quiescent, right? So when laminar flow or cells are quiescent, they decrease glycolysis, they increase fatty acid oxidation. Those things are important to take into consideration when you are treating a person who has a metabolic disorder. So that's the biggest translational piece that I think is, how do we give therapies that modify the metabolism of a cell holistically instead of trying to hit one pathway in particular.

We have done some studies where we tried to give endothelial cells something to inhibit a specific metabolic pathway and you see the cell shifts its entire metabolism to account for that. So we're starting to look at some of these other drugs like statins or metformin that do change endothelial cell metabolism, possibly even the SGLT2 inhibitors and trying to see not just how they change glycolysis but how they change metabolism as a whole and how that then affects endothelial cell function.

Cindy St. Hilaire: So what are you going to do next on this project?

Elisa Klein: So on this project, so we have some stuff in the works like I said on statins and how statins work together. And one of our big goals is to sort of build a comprehensive metabolic model of the endothelial cell. So this study really focused on glucose but there are other things that endothelial cells metabolize, glutamine, and fatty acids, and trying to look at some of those and then seeing how changes in the glycolytic pathway may affect some of those other pathways. We also have some really nice mass spec data part of which is in this paper but part of which is going to go into our next work, which is looking at how laminar flow impacts some of the other side branch pathways that are in metabolism and coming off of glycolysis as well as the TCA cycle, right? So we don't think of endothelial cells as being big mitochondrial energy producers but they do use their mitochondria. And so we think it's really interesting and part of our goal of building an endothelial cell model and then hopefully a model of the complexity of the whole vascular wall.

Cindy St. Hilaire: Wow. That would be amazing. Well, Dr Elisa Klein from the University of Maryland, thank you so much for joining me today. This is an amazing study and I'm looking forward to seeing hopefully more of your future work.

Elisa Klein: Thank you so much. It was a pleasure.

Cindy St. Hilaire: That's it for the highlights the from October 29th and November 12th issues of Circulation Research. Thank you for listening. Please check out the CircRes Facebook page and follow us on Twitter and Instagram with the handle @CircRes or #DiscoverCircRes. Thank you to our guest, Dr Elisa Klein. This podcast is produced by Asahara Ratnayaka, edited by Melissa Stoner and supported by the editorial team of Circulation Research. Some of the copy texts for highlighted articles is provided by Ruth Williams. I'm your host, Dr Cindy St. Hilaire, and this is Discover CircRes, your on-the-go source for the most exciting discoveries and basic cardiovascular research. This program is copyright of the American Heart Association, 2021. The opinions expressed by speakers on this podcast are their own and not necessarily those of the editors or of the American Heart Association. For more information, visit AHAjournals.org.

This month on Episode 29 of Discover CircRes, host Cynthia St. Hilaire highlights four original research articles featured in the September 17th and October 1st issues of Circulation Research. This episode also features conversations with BCVS Outstanding Early Career Investigator Award finalists, Dr Jiangbin Wu from the University of Rochester, Dr Chen Gao from UCLA, and Dr Chris Toepfer from Oxford University.

Article highlights:

Raftrey, et al. Dach1 Extends Arteries and Is Cardioprotective

Zhang, et al. Blood Inflammatory Exosomes and Stroke Outcome

Joyce, et al. Cardiovascular Health and Epigenetic Age

Liu, et al. Wls Suppresses Fibrosis in Heart Regeneration

Cindy St. Hilaire: Hi, and welcome to Discover CircRes, the podcast of the American Heart Association's journal, Circulation Research. I'm your host, Dr Cindy St. Hilaire from the Vascular Medicine Institute at the University of Pittsburgh. And today, I'll be highlighting articles presented in our September 17th and October 1st issues of Circulation Research. I also am going to speak with the BCVS Outstanding Early Career Investigator Award finalists, Dr Jiangbin Wu from the University of Rochester, Dr Chen Gao from UCLA, and Dr Chris Toepfer from Oxford University.

Cindy St. Hilaire: The first article I want to share is titled, Dach1 Extends Artery Networks and Protects Against Cardiac Injury. The first author is Brian Raftrey, and the corresponding author is Kristy Red-Horse from Stanford University. Coronary artery disease occurs when blood vessels supplying the heart develop atherosclerotic plaques that limit blood flow, which prevents oxygen and nutrients from reaching the cardiac tissue and often leads to a heart attack or cardiac arrest. The suggested strategy for treating coronary artery disease is to promote the growth of new blood vessels to compensate for the dysfunctional ones. Several factors are known to control coronary blood vessel development, including the transcription factor, DACH1. In mice lacking DACH1, embryonic coronary artery development is stunted. But whether increasing DACH1 protein levels boosts heart vessel development, and whether this would work in mirroring coronary arteries, were unanswered questions.

Cindy St. Hilaire: This group engineered inducible gain-of-function DACH1 mice and found that DACH1 over expression in the embryo boosted coronary artery development. The team then used the same model to induce DACH1 in adult mice for six weeks. While there was no apparent differences in the artery growth between the animals and the controls under normal conditions, after myocardial infarction, the mice over expressing DACH1 had better recovery and survival with increased artery growth and heart function. The results paved the way for studying the mechanisms of DACH1-mediated protection, and how they might be leveraged as potential coronary artery disease treatments.

Cindy St. Hilaire: The second article I want to share is titled Circulating Pro-Inflammatory Exosomes Worsen Stroke Outcomes in Aging. The first author is Hongxia Zhang, and the corresponding author is Kunlin Jin from University of North Texas Health Science Center. Aging is associated with declining tissue function and an assortment of health issues. But in rodents at least, certain factors, including the plasma of youthful animals and the exosomes of stem cells, can have rejuvenating effects on old animals. Exosomes are small membrane-bound particles containing cellular contents that circulate in the blood after they're released from cells. This group has shown that as rats age, the animals' serum exosomes accumulate pro-inflammatory mediators, such as C3a and C3b.

Cindy St. Hilaire: When these aged rats were subjected to stroke, and then injected with serum exosomes isolated from either old or young rats, those receiving youthful exosomes fared much better in terms of infarct size and sensory motor deficits, while those receiving aged exosomes fared worse. The team went on to show that injected exosomes accumulate at the site of stroke injury, but those from old donors caused more neuronal damage, as seen by reduced synaptic function. Preventing C3a activity on microglia reversed the effects of the old exosomes and improved stroke outcome, suggesting that such modulation of inflammatory molecules might be a treatment strategy for stroke.

Cindy St. Hilaire: The next article I want to share is titled Epigenetic Age Acceleration Reflects Long-Term Cardiovascular Health. The first author is Brian Joyce, and the corresponding author is Donald Lloyd-Jones. And they're from Northwestern University. DNA methylation is an epigenetic modification that regulates gene transcription. Studies of young and old individuals have shown that at certain locations in the genome, methylation status is highly correlated with age. These methylation patterns are also linked to measures of cardiovascular health, including blood pressure, cholesterol level and body mass index. This suggests that if a person has particularly good or particularly poor cardiovascular health, their DNA may appear younger or older than the individual's actual age.

Cindy St. Hilaire: This group tested the hypothesis that people with poor cardiovascular health exhibit methylation changes more commonly found in elderly individuals than those with good cardiovascular health. And if so, DNA methylation patterns might be useful for predicting future cardiovascular risk.

Cindy St. Hilaire: The team examined DNA methylation of over a thousand individuals enrolled in a prospective heart health cohort, testing them around age 40 and then again at around age 45. Changes in methylation status were then compared to individuals' cardiovascular health scores over a longer period. Sure enough, faster epigenetic changes did correlate with poor cardiovascular health later in life. Data from the second cohort of individuals supported the initial findings. This study indicates that DNA methylation status may be an early biomarker that signals cardiovascular issues, and may therefore allow for prompt implementation of treatment and prevention strategies.

Cindy St. Hilaire: The last article I want to share is titled, Yap Promotes Noncanonical Wnt Signaling from Cardiomyocytes for Heart Regeneration. The first author is Shijie Liu, and the corresponding author is James Martin. And they're from Baylor College of Medicine. After a heart attack, cardiomyocytes are destroyed and replaced with a fibrotic scar that interferes with the contractile function of the heart. While adult mouse and human hearts are similar in this regard, the hearts of newborn mice possess greater regenerative capacity, and this regeneration capacity persists for approximately one week. The transcription factor YAP is known to regulate regenerative processes in neonatal hearts of mice. And its deletion eliminates regeneration, and its over-activation in adult cardiomyocytes reduces fibrosis.

Cindy St. Hilaire: These experiments suggest cardiomyocytes transmit signals to cardiac fibroblasts. Wntless protein regulates the release of Wnt signaling molecules and also is a target of YAP. Mice that lack Wntless in their cardiomyocytes appear to have normal heart development and function. However, their neonatal regenerative capacity was impaired. In the weeks after heart injury, the mice that lack Wntless had reduced heart function, increased scar size and increased numbers of activated cardiac fibroblasts compared with that seen in controls. The study indicates that Wntless is critical to the regeneration of cardiac tissue, and may perhaps be leveraged to minimize scarring after heart attacks.

Cindy St. Hilaire: I'm really excited to have with me today the three finalists of the BCVS Outstanding Early Career Investigator Award. The first person I'm going to be speaking with is Jiangbin Wu, who is a research assistant professor at the Aab Cardiovascular Research Institute at the University of Rochester. Thank you so much for joining me today.

Jiangbin Wu: Thank you.

Cindy St. Hilaire: And congratulations, actually. I know this is a highly competitive award that gets a lot of applications, so congrats on becoming a finalist. Before we get to your abstract, which is related to mitochondria and calcium influx in cardiomyocytes, I was wondering if you could share a bit about yourself. Maybe what your research path was, and what brought you to study cardiomyocytes and the mitochondria that are within them?

Jiangbin Wu: Yeah. Right now, I'm an assitant professor at Cardiovascular Research Institute of University of Rochester. Previous, I was actually studying in the cancer field and also some kind of mitochondria work in some cancer cells. Although when I came to the University of Rochester and I switched to cardiovascular and then we are working on a kind of microRNA[at the initial. The way we screen for these is just by doing the RNA-Seq is target the microRNA. and then we start to study the function of these genes, and found that it's a mitochondria calcium channel regulator.

Cindy St. Hilaire: The title of your abstract is FAM210A Maintains Cardiac Mitochondrial Homeostasis Through Regulating LETM1-Dependent Calcium Efflux. So before we unpack what all those words in the abstract title mean, could you tell me how you ended up focusing on FAM210A? What does this protein do, and why'd you focus on it?

Jiangbin Wu: Yeah. As I mentioned that we just gathered this protein actually is by some kind of chance as a microRNA target. And this protein full name is family with similarity 210 A, actually is a family of proteins. This is just one of them. And the way discover is localized in mitochondria in the membrane. And also, there is some other people's report is in mitochondria. And we want to sort out its function inside the mitochondria and in the cardiac background. So we do some kind of omics or mass spec to get its interlocking interacting proteins. And then we found LETM1. It's a calcium channel inside the mitochondria in the membrane. So we figured out is, this FAM210 protein regulate LETM1 function in calcium, pump calcium is part of the mitochondria matrix. And I think this is a very important, because calcium overload is always happening in the very heart of the cardiomyocytes.

Cindy St. Hilaire: That's a perfect segue, because my next question was really what is the gap in knowledge that your study was trying to address? Were you really focused on just the function of this one protein, or what was the greater goal of this study?

Jiangbin Wu: Actually, the function this protein is the initial step. Our final aim is to use this protein, to over expression this protein in the heart failure patient or in some kind of heart failure models to do the, sort of do the work in some heart failure patients.

Cindy St. Hilaire: Maybe a gene therapy approach, or if there's a pharmacological way to up regulate this protein?

Jiangbin Wu: Yeah, because we've proposed that the self expression of this proteins will reduce the calcium overloading cardiomyocytes, which is a major cause for the cardiomyocytes death in heart failure process. So over expression will reduce this kind of process. And then it will make the cardiomyocytes survival in the failure heart.

Cindy St. Hilaire: That is interesting. I mean, obviously you were using a mouse knockout model, so you know what's driving the expression down in that case. But in humans, what do we know about the regulation of this protein? Is anything known, or any known causes that cause its reduction in expression?

Jiangbin Wu: Actually, we do. Its expression in heart failure is slightly increased in heart failure. So we feel it's a kind of some kind of compensating effect to try to save the heart from failing.

Cindy St. Hilaire: Interesting. It's just not turned on early enough, in that case then.

Jiangbin Wu: Yeah. And for the regulating protein for this one, I think we find microRNA can suppress its expression, but not too many other influences on these regulator proteins.

Cindy St. Hilaire: That is so interesting. So what's next? What are you going to do next on this project?

Jiangbin Wu: Yeah. I think currently, we are just at the start to do some kind of therapeutic effect that use to these proteins. I think we will do more deep in the therapeutic effects for over expression of these genes in... Currently, we are working on mouse models. Maybe in different heart failure models to prove that it's very benefiting to the heart failure patients.

Cindy St. Hilaire: Wonderful. Well, congratulations on an excellent study. Really looking forward to your presentation, which is coming up shortly, and really looking forward to your future research in this field.

Jiangbin Wu: Okay, thank you.

Cindy St. Hilaire: So I also have with me, Dr Chris Toepfer, who's another finalist for the BCVBS outstanding early career investigator award. He's a principal investigator from the University of Oxford, and his abstract is titled, Defining Diverse Disease Pathway Mechanisms Across Thick And Thin Filament, Hypertrophic Cardiomyopathy Variance. So congratulations, Chris, and thank you for joining me today.

Chris Toepfer: Thank you very much. It's great to be here.

Cindy St. Hilaire: Before we start to discuss your abstract, I was wondering if you could just share a little bit about yourself. Maybe your career path, and how you came to study hypertrophic cardiomyopathy?

Chris Toepfer: Yeah, sure. I guess this story gets longer and longer every time somebody asks it,right, in your career?

Cindy St. Hilaire: That's a good thing.

Chris Toepfer: Yeah. I started out as an undergraduate in London, and actually during the second year of my undergraduate degree, I fell into a lab kind of out of interest. It was starting to study cardiac muscle mechanics. And that was the lab of Professor Michael Ferenczy. And ended up, after I finished my undergraduate degree, I joined him for a PhD. I had a PhD program that also took me overseas to the NIH to work with Dr James Sellers, who was a muscle motor protein biochemist. And we really, I sort of really fell in love, with the idea of studying disease of multiple levels, and understanding how the heart would function from the basic molecule up to the entire organ and looking at different systems in between.

Chris Toepfer: And that's what led me to then, so my postdoctoral position to seek out a completely different direction in some ways, but something that could also extend how we could look at the heart. And that's where I moved to Boston to work with Christine and Jonathan Seidman. I'm looking at more of the genetic basis then of hypertrophic cardiomyopathy rather than just, sort of more diffusely the mechanisms underlying cardiac muscle contraction. And then two years ago, I moved back to the UK to Oxford to sets up my own group, which has been fun during the pandemic as you can imagine.

Cindy St. Hilaire: It's hard enough starting up a lab under normal times. I can't imagine doing it during a pandemic.

Chris Toepfer: And we are now completely focused on stem cell models and CRISPR CAS engineering, and trying to understand hypertrophic cardiomyopathy in a dish.

Cindy St. Hilaire: That's wonderful. And actually I looked at your CV. We actually overlapped a little bit. I was doing my postdoc at NIH in the NHLBI while you were there for your graduate school. So I too fell in love with kind of the starting with the human as the model path of research. So maybe you can kind of fill in all the listeners in who aren't cardiomyopathy experts. So what is, I guess, in a nutshell, hypertrophic cardiomyopathy, and what gap in knowledge was your study specifically addressing?

Chris Toepfer: So in general, about one in 500 people have hypertrophic cardiomyopathy. And for those that are genetically linked, a lot of them are in the key contractile proteins of the heart, the drive muscle contraction. And what you often see in those people is they have thickened hearts. And what happens is actually the heart begins to be too hard, and it actually relaxes very poorly in between beats.

Chris Toepfer: So what we are really trying to understand in this disease and with this abstract was how are different forms of hypertrophic cardiomyopathy created? Because it can be a couple of different forms. There are different proteins involved that have very vastly different functional mechanisms within the cell. So would this, we went away, we generated some stem cell models where we could then differentiate into cardiomyocytes. Model the disease in a dish. And we made kind of a group of good methods to go and look at what was happening inside the cells. And then we could screen drugs against what's happening inside those cells, so that was kind of the idea of what we were looking at, at the time. And what's fallen out of all of that is a drug now called Melacamptin that's starting to get to the clinic, which addresses some of these underlying mechanisms we were beginning to study. So that's what I'll talk about a bit later on in our session today.

Cindy St. Hilaire: It's great. One of the things you focused on in the abstract is comparing these thick and thin filament variants. What are the implications of those, I guess, in the human disease state, but also in how you could design or use your stem cells as a model, and were any of the results that you found surprising?

Chris Toepfer: So I think what was the really key finding that we saw was that the thick filament variants seemed to be switching myosin, which is a molecular motor that drives cardiac muscle contraction very much to arm"ON". And my sort of analogy to that is they're all very sort of bodybuilder like. Myosin switched on, ready to go to work causing way too much contraction. And the compound that we were using at the time Myocamptin, we could turn those off and resolve the disease. Whereas with the thin filament variants, they were operating through a completely different mechanism. And when we tried to treat them with the same compound, they wouldn't always salvage disease. So though the face of it, they look the same in the dish, in that they contracted too much, relaxed very poorly. You're clearly doing it via complete different mechanism. And that's what we're starting to dig into now. And that's what we'll be talking about.

Cindy St. Hilaire: Yeah. And that's actually kind of the question I was going to finish up with you. What are the, I guess translational implications? No, yes. You're using this drug. Is that only good for thick filament-like variants? And are you going to be able to screen patients to tell which variant they have, and therefore if this or that drug might be useful?

Chris Toepfer: So we're in a real golden age now for genomics where I guess patients can come into the clinic and they can be sequenced and you could maybe tell them now what might be the underlying cause of their disease. I am not a clinician, but what we, as a basic scientist can say is, well, we can go away and try and understand whether this variant you may have in your genome is causative of disease. And if it is what mechanism that may fall under, what may be causing them to have this phenotype?

Chris Toepfer: And I think what we can do is we can try and then bin the subpopulations of variants, and try and find novel drugs or novel pathways that we could try and find drugs for to treat the disease, and to differentiate them from each other. So I think it's too early to say whether Mylocamptin will be able to sort this for everybody, I guess we will find out in the next years. But I think already we can start thinking about, well, what would be the next step after this? We can bring precision medicine even further. And that's, I think the goal where we're heading towards.

Cindy St. Hilaire: Well, that's wonderful, and this is a wonderful abstract. I'm really looking forward to seeing the full study and your presentation later on. And thank you so much for joining.

Chris Toepfer: No. Yeah. Thank you for having me. I'm really looking forward to it later on.

Cindy St. Hilaire: Great. Dr Chen Gaol is the third finalist for the BCBS Outstanding Early Career Investigator Award. She's an assistant researcher at UCLA, and her abstract is titled, Functional Impact of RBFox1C in Cardiac, Pathological Remodeling through Targeted MRNA Stability Regulation. So congratulations, and thank you so much for joining me today.

Chen Gal: Absolutely, thank you for having me.

Cindy St. Hilaire: Before we jump into your abstract, could you share with us a little bit about your career path, and how you came to study the role of RNA binding proteins, I guess specifically in pathological cardiac remodeling?

Chen Gal: Yes, I think my research over the years has been into the very basic questions, which is I'm interested in looking at how the RNA is being regulated. For example, how the RNA is being spliced, is being ideated, and how the RNA is being degraded if it's ever been translated into protein. And the second half of my research is of course, physiological driven, because I'm interested in different type of cardiac disease, starting from the traditional heart attack to the now more emerging medical need, which is the cardiometabolic disease. So I was trained as a molecular biologist. I started in molecular biology Institute at UCLA. My PhD supervisor is Dr Yibin Wang, who first introduced me to understand there is actually a whole new world of R regulation at a post-transcription level.

Chen Gal: So at that time we basically utilized the R sequencing. Just look for the easiest to heart, and try to understand how these RNA are differentially spliced in the heart. And I was so interested in understanding more about a cardiology. So I decided, even if I move out to my postdoc research I still want to continue working in the heart, although at a totally different angle. And that is when I started to really try to understand different aspects of RNA regulation. So now I am starting to be a junior faculty, establishing my own lab. And I really wanted to understand more how different steps of our metabolism is regulated.

Cindy St. Hilaire: Really timely research. And I really like how you are doing a great job combining extremely basic biochemical processes with advanced disease states. An extra, that's why this abstract made it as a finalist. So congrats on that. So your study was focused on the RNA binding protein, RB Fox one, which has several isoforms. And so can you tell us which isoform you were looking at, and why you were interested in that particular isoform?

Chen Gal: Yes, actually I've studied about ISO form of RPFox1. It itself, is actually subject to alternative splicing, while generating one nuclear, and another simosolic isoform. Where I was a PhD student, I was very simple minded, just trying to screen for the R binding protein that actually is expressed in the diseased heart. So RBFox1 is at least at a transcriptional level, the only one that we identify to be to decreased in the fatal heart. The nuclear function, the nucelo ISO form of RPFox1 is mainly regulating alternative splicing. But it is when I was studying this nuclear function of the RBFox1, I identified there is actually another isoform where she is in the set ourselves based on the different of c terminal domains of the RFox1. So I was just wondering, apparently you shouldn't be regulating and splicing anymore. I just move on to another layer of RA regulation. And then what I found most interesting is these RBFox1 is regulating the R stability, which is something that we'll talking about later today.

Cindy St. Hilaire: That's great. So to do this study, you actually created a new knockout mouse model where you specifically deleted this one C isoform. What was kind of the baseline and maybe the disease state phenotypes that you saw in that mouse?

Chen Gal: The result and phenotype so far is very striking. We utilize the CAS nine CRISPR technology simply because for, we were lucky the settle the Fox warehouse, one extra axon. So that does allow us to coach the lox P side, just blanking in that particular AXA. And in theory we could across it with different CRE, and to generate either cardiac or different tissue, specifically knock out. Even at a baseline we see a decreased cardiac function when we inactivate this isoform in the adult heart. And when we look at the gene expression profile is, I call mind-blowing type of experience, because turns out this gene not only is regulating some of the inflammatory genes, but also is helping involve protein translation and delivery metabolism, which I hope in the future will set us on the path to really understand the role of this RP Fox1. Not only into HFpEF, but also in the cardiometabolic disorder.

Cindy St. Hilaire: Yeah, that's great. It's so rewarding when you do this one really big kind of risky experiment, and it turns into not just one interesting path to study, but multiple. One of the things that you mentioned in the abstract is clip seek. I was wondering if you could tell us a little bit about this technology, and how you used it in your study?

Chen Gal: Yeah. I think one of the rewarding parts for me focusing on the R metabolism is really driving different accounting and sequencing tools, and utilize that in the heart. So cardiomyocyte has been traditionally viewed now to be very easy to work with type of model comparing helo cells, right? And I think in the field, we are still so short of knowledge, what type of the cutting-edge tools that we can use in the heart. My research involved clip seek, which is to use UV crosslinking the RNA with the R binding protein. So that will allow us to understand which are the RNA targets that are directly interacting with the RNA binding protein. I'm also using great seek, which is to find dynamically label the recency size to RNA. And that will allow us to look forward to RA degradation profile at a global level in the baseline or under disease. So I thought those are really cool technologies, and that's something that makes me excited about my work on a daily basis.

Cindy St. Hilaire: Yeah, that's wonderful. So what's next? What are you going to do after this initial study? What's the next question you're going to go after?

Chen Gal: Yeah, like I mentioned, I'm interested in, honestly, different type of heart disease, not just the stress induced heart failure, but also the recent years, I started to branch out a little bit to understand more of the biology of HFpEF. For example, how the R binding protein that we are studying right now is playing a role in the development of HFpEF. Or we actually understand very little about them, the micromechanism for HFpEF development, right. What are the RNA splicing profile in the cardio metabolic disorder on account? We also find differential regulation of R stability in the HfPEF compared to the HFpEF compared to the HFrEF. So I thought those are really interesting questions that I would like to pursue in the future.

Cindy St. Hilaire: That's great and best of luck in those future studies.

Chen Gal: Thank you.

Cindy St. Hilaire: Before we leave, I was wondering if you could share with us any advice that you would give to a trainee, maybe something that you wish you knew ahead of time in this kind of early career stage.

Chen Gal: I consider myself a really, really lucky person. And if I have one word to give to the younger people, younger than me, is to find great mentors for your career. And luckily our field has a lot of good mentors who are ready to help us every single step of our career. For example, my PhD supervisor, Dr Wang. And I have met a lot of good mentors inside and outside of UCLA. I'm pretty sure this is the same thing for Chris, who is trained by Dr Seidman, and everybody know how great a mentor she is. So I think having a great mentor will help you every step of your career development to making sure you're always on the right track. And that, that is also something that you will do when we have our own lab, because we want to be great mentors for our trainees as well.

Cindy St. Hilaire: I know. That's something I strive for too, is to emulate my amazing mentors that I've had. What do you think is a good quality for a good mentor? Like what's one of the, I guess key features that you look for in someone that you would like to be your mentor?

Chen Gal: For me, I think my mentors are all cheerleaders. They never try to push me to move out one career path versus the other. They are good listeners, and they are also my role models.

Cindy St. Hilaire: That's wonderful. Chris, what's a piece of advice that you would like to share with trainees that your former self wish you knew of?

Chris Toepfer: I think it's very important to echo the message of a good mentorship, and a good lab environment that allows you to flourish and really helps you to grow yourself to the future. And also helps you understand the bits of you that you could actually grow as well, a little bit better. So you become a more rounded scientist. I think something that's really important or something that I've always found very infectious is to find mentorship and mentors that are also incredibly enthusiastic about you as an individual, as well as the science. I think that that can really drive you. And I think that's also an important thing to have in yourself, to have, to find that question for yourself that really drives you and you can be really enthusiastic about.

Cindy St. Hilaire: I totally agree. Well, thank you again for joining me today. Congratulations on being a finalist, and I wish everyone the best of luck in their presentations later on at BCBS.

Chen Gal: Thank you so much.

Jiangbin Wu: Thank you.

Chris Toepfer: Thank you very much.

Cindy St. Hilaire: That's it for the highlights from the September 17th and October 1st issues of Circulation Research. Thank you for listening. Please check out the CircRes Facebook page, and follow us on Twitter and Instagram with the handle @CircRes and #Discover CircRes. Thank you to our guests, BCBS Outstanding Early Career Investigator Award Finalists, Dr Jaobing Wu, Dr Chen Gal, and Dr Chris Toepfer. And a special congratulations to Dr Toepfer who won this year's competition. This podcast is produced by Asahara Ratnayaka, edited by Melissa Stoner, and supported by the editorial team of circulation research. Some of the copy texts for highlighted articles is provided by Ruth Williams. I'm your host, Dr Cindy St. Hilaire. And this is Discover CircRes, you're on the go source for the most exciting discoveries in basic cardiovascular research. This program is copyright of the American heart association, 2021. The opinions expressed by speakers in this podcast are their own and not necessarily those of the editors or of the American heart association. For more information, please visit AHAjournals.org