Discover CircRes

This month on Episode 18 of the Discover CircRes podcast, host Cindy St. Hilaire highlights four featured articles from the October 23 and November 6 issues of Circulation Research. This episode features an in-depth conversation with Drs Eric Boilard from the Université Laval in Quebec, Canada, and Fadila Guessous from Mohammed VI University of Health Sciences in Casablanca, Morocco regarding their study titled Platelets Can Associate with SARS-Cov-2 RNA and Are Hyperactivated in COVID-19.

Article highlights:

Feng , et al. No Contribution of EMPs to Endothelium

Lin, et al. Step Count and Predicted CVD Risk

González-Hernández, et al; Sox17 in Developmental Coronary Arteriogenesis

Khawaja, et al; HIV Antiretrovirals Alter Endothelial Activation

Dr Cindy St. Hilaire: Hi, 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 four articles selected from the late October and early November issues of Circ Res. Dr Milka Koupenova, who is a platelet expert at the University of Massachusetts Medical School, will join me to interview Drs. Eric Boilard from the Université Laval in Quebec, Canada, and Fadila Guessous from Mohammed VI University of Health Sciences in Casablanca, Morocco. They're here to discuss their study, Platelets Can Associate with SARS-CoV-2 RNA and are Hyperactivated in COVID-19.

Dr Cindy St. Hilaire: The first article I want to share is titled No Evidence for Erythro-Myeloid Progenitor-Derived Vascular Endothelial Cells in Multiple Organs. The first authors are Teng Feng and Zibei Gao, and the corresponding author is Hui Zhang from ShanghaiTech University in Shanghai, China.

Dr Cindy St. Hilaire: In the early stages of mammalian embryogenesis, a population of cells called mesoderm-derived angioblasts, gave rise to self-renewing intraembryonic endothelial cells, that go on to populate the entire vasculature of the growing fetus. Recent studies have suggested that there is an additional embryonic source of vascular endothelial cells that is derived from erythro-myeloid progenitor cells, also called EMPs, that are found in organs such as the liver, the lung, the heart, and the hindbrain.

Dr Cindy St. Hilaire: Evidence of an EMP cell source for vascular endothelial cells stemmed from the use of mirroring cell lineage tracking models. However, the authors of this study argue that these former conclusions were based on flawed genetic tools. To mark erythro-myeloid progenitor cells, the initial study had used a set of supposedly EMP-specific transgenes. However, transgenes are notoriously leaky, meaning, that cells other than EMPs may have been marked. To more accurately mark EMP derived cells, this study used a reporter that they integrated into the endogenous loci of a gene, CSFR1, which is specifically expressed in EMPs. Using this model, they failed to find any evidence of EMP derived vascular endothelial cells in any organ, thus they consider it unlikely that EMPs give rise to vascular endothelial cells. Knowing the true origin of vascular endothelial cells is essential, as it informs the design, development of treatments, and regenerative medicine approaches for vascular diseases.

Dr Cindy St. Hilaire: The second article I want to highlight is titled Association of Habitual Physical Activity with Cardiovascular Disease Risk. First author is Mayank Sardana and the corresponding author is Honghuang Lin, and they're at Boston University School of Medicine. Everybody knows that exercise is very good for overall cardiovascular health and current government guidelines suggest a minimum of 150 minutes of moderate to vigorous exercise per week is necessary to keep hearts healthy. There's an ever-growing interest in the use of smart watches and fitness trackers to assess an individual's activity, and many people believe that 10,000 steps a day is a good health goal. However, it's unknown whether the data obtained from smartwatches actually aligns with cardiovascular health prediction.

Dr Cindy St. Hilaire: To address this gap, this study recruited participants from the Framingham Heart Study to wear Apple smart watches for a month. The participants had also undergone a 10 year atherosclerotic cardiovascular disease risk assessment based on their age, sex, cholesterol levels, blood pressure, diabetes diagnosis, and smoking history. The step count and activity data from 903 participants found that for every 1,000 average daily step increases, there was an associated 0.18% reduction in predicted cardiovascular disease risk. This association was weaker but still significant after adjusting for body mass indices. The authors conclude that like other measures of activity, those recorded by smart watches are correlated with better cardiovascular health.

Dr Cindy St. Hilaire: The third article I want to share is titled, SOX17 Controls Emergence and Remodeling of Nestin-Expressing Coronary Vessels. The first author is Sara González-Hernández and the corresponding author is Joan Isern, and they're from Centro Nacional de Investigaciones Cardiovasculares in Madrid, Spain. The exact signals and mechanisms that regulate coronary vascular development are not fully characterized, yet defining these pathways could provide valuable insights into both life threatening congenital coronary abnormalities as well as the neovascularization process that occurs after myocardial injury. To examine coronary vasculature development in more detail, this team created a transgenic reporter system, which used an enhancer region within the Nestin gene to specifically label mouse coronary artery endothelial cells.

Dr Cindy St. Hilaire: Cells were isolated from developing embryos at stages of vessel sprouting and vessel remodeling, embryonic days 13.5 and 17.5 respectively. And transcriptional differences were assessed between coronary and endocardial endothelial cells. They found that the transcription factor SOX17 was more highly expressed in these coronary endothelial cells compared with the endocardial endothelial cells, and that expression of SOX17 increased between these two time points that hearts were collected. Coronary specific deletion of SOX17 in genetically engineered mice lead to severe defects in arterial remodeling, confirming the transcription factor's role in coronary vessel development. The team suggested that perhaps SOX17 could be a future therapeutic target for conditions, where promoting coronary artery regeneration or remodeling might be of clinical benefit.

Dr Cindy St. Hilaire: The last article I want to share with you before we switch to our interview is titled, HIV Antivirals Affect Endothelial Activation and Endothelial-Platelet Crosstalk. The first author is Akif Khawaja, and the corresponding author is Michael Emerson from the Imperial College, London. Infection with HIV is not the death sentence that it once was. With a regime of continuous anti-retroviral medications, the disease can be managed for the long term. Now that people with HIV are living longer, it is clear that they are at a greater risk of developing cardiovascular disease, possibly due to the off-target effects of these drugs. One HIV treatment, Abacavir Sulfate or ABC, was found to cause a 90% increase in the relative risk of myocardial infarction. ABC has been proposed to cause endothelial dysfunction, however a mechanism by which this may occur has not been established. To see if anti-retrovirals effect endothelial cell function, this group tested three drugs; ABC, as well as tenofovir alafenamide or tenofovir disoproxil fumarate, TAF and TDF respectively, on human endothelial cells in culture.

Dr Cindy St. Hilaire: They found that treatment with ABC, but not TDF or TAF, caused endothelial cells to produce more cell adhesion protein, ICAM-1, and also tissue factor, both of which promote blood clotting. Endothelial-derived microparticles, or EMPs, are a biomarker of vascular dysfunction, and these were produced in the ABC treated cells, not in the TDF or TAF treated cells. These EMPs also promoted the increase of ICAM-1 and tissue factor expression, as well as the activation of platelets which can induce blood clotting. These results suggest that altering HIV treatments to avoid or minimize endothelial damage could help to reduce the cardiovascular disease risk in HIV patients.

Dr Cindy St. Hilaire: Today, I'm excited to have with me Drs. Eric Boilard from Université Laval in Quebec, Canada, Fadila Guessous from Mohammed VI University of Health Sciences in Casablanca, Morocco, and they are here to discuss their study, Platelets Can Associate with SARS-Cov-2 RNA and Are Hyperactivated in COVID-19. Also, with me today is Dr Milka Koupenova from the University of Massachusetts Medical Center in Worcester Mass, and she's an expert on platelet virus interactions, and she also wrote the editorial that's accompanying this article in our November 6th issue. So thank you, everyone, for joining me today.

Dr Eric Boilard: Thank you for inviting us. I look forward to reading the editorial.

Dr Milka Koupenova: I hope you like it Eric.

Dr Cindy St. Hilaire: Yeah, we're spanning a lot of time zones and we're actually having a little bit of technical difficulties so hopefully Fadila is going to be able to pop in. We can certainly see her on our Zoom call right now. But with that, we're many months into this pandemic now and it is blatantly clear that COVID does not just present with pulmonary symptoms. Many organ systems can be affected, such as the circulatory and neurological systems, but one thing that connects all the parts of the body is the blood system, and before we get into the details of the study, I would love to hear how this collaboration between Quebec and Morocco happened. And then, could you summarize the major findings?

Dr Eric Boilard: We both, in Morocco and in Quebec, we had pretty much the same working hypothesis that platelets may be contributing to the overwhelming inflammation in COVID-19 and why. As you know, in Morocco the pandemic hit before us in Canada, and they had a committee that studied patient samples and studies were going on. Whereas us in Quebec, in fact to be frank, in Quebec City, we didn't really have the first wave even. So we were very excited to evaluate our hypothesis but we had no clues other than the actual virus. We actually were working with the virus and human platelets from healthy individuals, but no samples from patients. Clinicians in Morocco were very willing to contribute and to perform working on patients so that's how Younes Zaid, the first author of the manuscript, and Fadila then contacted us given our past work on platelets and viruses and the collaboration was initiated.

Dr Cindy St. Hilaire: That's wonderful. I love hearing about these multi-institute collaborations. Team science is really the future. I think it really elevates everything.

Dr Eric Boilard: Despite the fact that the distance and I mean, it was through Zoom, but we've been doing weekly meetings with the two labs since March.

Dr Cindy St. Hilaire: So, can you maybe give us a quick summary of the major findings of your study? Then we'll dig into the details of it.

Dr Eric Boilard: Sure. When we actually started the work, what we knew about COVID-19 was that the severity was likely due to the overwhelming inflammation due maybe to a cytokine storm, so we knew that. It was reported that patients with COVID-19 had a lower platelet count and although thrombocytopenia in the patients was very mild or modest, and did not require transfusion, at that time, our hypothesis was that platelets could contribute to inflammation and therefore could release cytokines and other molecules from their granules in COVID-19. So it's only a couple of weeks after that became more of use that thrombosis and cardiovascular manifestations could also contribute to morbidity and mortality.

Dr Eric Boilard: Our design was to look at platelet activation in vitro. So there were studies where we performed aggregation assays with platelets from COVID-19 patients, some were severe and non-severe. There were assays where we actually evaluated a number of molecules and plasma from the patients. We looked at granular components, like PF4 and serotonin, that were greatly increased in COVID-19, both in non-severe and severe patients. We looked at vesicles, or microparticles, released from platelets that were also increased, and cytokines. We look at up to 42 cytokines, if I remember well, in the plasma of the patients and we also look at these cytokines in the platelets from the patients and we found less cytokines in the platelets when there was inflammations, which may suggest that platelets have released their cytokine cargo. I mentioned that platelets were hyperactivated so they bound better on collagen and they aggregated faster with suboptimal concentration of thrombin.

Dr Milka Koupenova: I find this dysregulation of cytokines in the platelets specifically very interesting and the fact perhaps that you see that certain cytokines are decreased in platelets and increased in plasma, basically can suggest that maybe platelets are the ones that are contributing to the overall cytokine storm, as you said. My question for you is, what do you think is the contribution of platelets to the dysregulated immune cell response in immune cell activation during this particular infection?

Dr Eric Boilard: That's a good question. There are studies that show that platelets could interact with leukocytes. The leukocytes migrate to organs through lungs in this case, so one possibility is that molecules derive from platelets and cytokines but also their interaction with leukocytes could further promote their migration to lungs or adhesion to vasculature. There are studies that suggest that NETs form in COVID-19 and could contribute thrombosis and NETs can implicate interaction between platelets and neutrophils. Or molecules derived from platelets that are neutrophils so these might be some of their contributions.

Dr Milka Koupenova: You are reporting changes of IL-7 in platelets which is particularly interesting because they're responsible for T and B cell development and activation. I was wondering if you could comment on that particular fact and how that may be practically responsible for these responses in these patients that come and present with symptoms.

Dr Eric Boilard: Yeah, it's a good question. If we look at the different cytokines that were in platelets, we found that many were reduced. That pointed to their release in COVID-19 and some we had expected, such as some broad factors and there was TGF, CD40 ligands inside the cytokine but we expect that its stored in platelets that was released. That made sense to us. IL7, like you said, it was not really reported in platelets and when we look at the 40... broad area of cytokines using the multiplex, that increased in platelets during COVID-19. So this one was increased. Does it mean that platelets translated but some of it is released but some remain in platelets? We have no explanation for this. Does it impact lymphocytes or B cells?

Dr Fadila Guessous: If we go further and in terms of seeing the front they know what was downstream, IL7 production in general does not look like for psychometry, what happens to B cells and T cells in our complex. So we just look at the panel of the 48 cytokines, but we didn't go really further.

Dr Cindy St. Hilaire: I kind of have a little bit of a more basic question regarding cytokines and where they're coming from. I'm going to guess it's definitely not known for COVID but in maybe any viral infection that can lead to a cytokine storm, is it known what the relative contributions of cytokines are from the platelets, from the leukocytes, and from inflammatory cells? Is there any evidence of which is the bigger player? I mean I know all the different camps have ideas, but has anyone looked at that?

Dr Fadila Guessous: We were talking about this actually with Eric. If I had to rephrase your question, like what's the starting point, right in terms of cytokine storms?

Dr Fadila Guessous: We don't know. We don't know because when it started in China, everyone was talking about the big cytokine storm. Everyone was talking about monocytes, about neutrophils, so on and so forth but the platelets we were just saying there was thromboembolism in the lungs, right? But no one was talking about cytokine production by the platelets. I think we were the first ones, Eric please correct me if I'm mistaken, we were the first ones to show, when we started, to show that there was the cytokine release by platelets. How big of the extent of the contribution of platelets to the cytokine storm, we don't know, but we have to figure out first what's the key player? The first one to respond, right?

Dr Cindy St. Hilaire: Lots of projects.

Dr Fadila Guessous: Well the big question, actually we have this big contribution in terms of the cytokine release and activation of platelets, but is it the starting point? We don't know.

Dr Cindy St. Hilaire: I guess related to that, you noted that you saw very little ACE2 expression in the platelets which most people appreciate is the entry mechanism of the virus into the platelets. Can you discuss that a little bit more, maybe explain it a little bit more and what that might mean? Your findings versus what other people have seen versus potential other means of entry?

Dr Eric Boilard: Yeah sure. As you mentioned, ACE2 is the putative receptor for the virus so that made sense to us to examine whether the receptor may be expressed by platelets. I don't think we mentioned it but we found that there were some... at least for some patients there were SARS-CoV-2 RNA associated with platelets and that's why in part we look at ACE2 RNA expression by platelets. It seems simple but it wasn't that simple. We were actually using through PCR and different approaches and primers we got different answers using intron-spanning and non-intron-spanning primers from that, the receptor was presented so that in fact we ended up concluding that there was little or no ACE2 RNA in platelets of COVID-19 individuals that we examined but that there was some DNA that could be contaminating, potentially due to extracellular DNA maybe provided by extracellular vesicles[inaudible 00:19:30] or something but either way we're speculating.

Dr Eric Boilard: One intriguing aspect of the study is that the SARS-CoV-2 RNA was not found on platelets from every patient, it was only in 20% of them and we unfortunately were not able to look at the RNA of these positive patients to determine whether these patients were positive for ACE2 RNA and potentially in some donor cells in some patients, there might be some levels of expression and we are not excluding this.

Dr Eric Boilard: So yeah, you ask whether it was consistent with other studies? There was the study by Manny that was published, in Blood where they also could not find the receptor but our studies contrast with the one that was more recently reported by Zhang in Journal Of Hematology & Oncology. In this study they found in fact that a robust expression of ACE2, both protein and RNA, and it was both in humans and mouse so clearly there's still work to do to determine whether ACE2 is present or not, but there could be other means of interaction between the SARS-CoV-2 and platelets if there is indeed an interaction. We speculated that maybe in some patients there could be antibodies against other coronaviruses, you know the cold, the viruses that give the minor cold symptoms and that in some of the patients these cross-reacting antibodies could promote the interaction with platelets through their receptor for IgG , that could be a means.

Dr Eric Boilard: There are other receptors that are expressed by platelets, there's probably several. I'm sure we have a list, but one could be the CD147. It's a receptor that is known to be expressed by platelets and that was suggested to interact with SARS-CoV-2, at least it does interact with SARS-CoV, the first one, CoV-1. So these are potential candidates. So there's work to do.

Dr Fadila Guessous: What we found was that in Eric's lab, that you have this 20% of aged people that have this SARS-CoV RNA in place, in 20% of aged people. So should we look only at aged people for this ACE2? If they have the receptor for ACE2, maybe? It's like 20%, only aged. And this 20% of people we investigated, they were only aged. Not other people. So do we... probably, we have to go further and investigate more of this age group for ACE2.

.

Dr Milka Koupenova: So if I can make a comment, despite the fact if ACE2 was expressed or not, right? Could it explain the profound thrombotic response that you see? What's your take on it?

Dr Eric Boilard: Personally, I doubt it. I think the disease starts in the lungs and this is where the damages are made, and that it may expand to blood vessels and then multiple players can then from a place of activation damages themself, the lack of O2 in some patients that are in ICU. And then of course the activation of the other cells, the cytokines themselves. So there are numerous ways I will say that can be to play for activation. In fact, the study by us, the one in Blood, they use plasma from the COVID-19 patient and I would assume there was no actual virus particle in this plasma. And they incubated it with platelets, and platelets were then activated. So there are molecules in COVID-19 that circulate in blood that can activate platelets, and that adds to the vasculature damage trauma. I would think that that could come from principle factors.

Dr Milka Koupenova: Which would explain why is it so dysregulated, which brings it to the next question now with a favorite question in the literature. Should we be using antiplatelet drugs? Your opinion, Eric and Fadila?

Dr Fadila Guessous: Well with my collaborator, Younes Zaid you know, and first author on this paper, actually they are going for that. They are having small trial giving antiplatelets treatment to a few patients in our lab. You will hear the story. I don't know if Eric will allow me to disclose this? I don't know because Younes is not here, but they are using heparin for now and for the treatment, but now they are trying antiplatelet treatment as a small trial.

Dr Milka Koupenova: So when do they give antiplatelet drugs? At what stage of the disease?

Dr Cindy St. Hilaire: I think maybe the way to ask the question is: If you were going to use antiplatelet therapy for changing the course of the disease, where do you think is the best time point within the disease, from the day you get infected to symptom presentation and well after that, what time point do you think it's most critical, the platelet function?

Dr Fadila Guessous: It's a good question. It's a good question because the trial is done in in our lab and I didn't have insight. What time is the time point. Honestly, I don't have an answer for that in time point. Because I have a... they started the trial-

Dr Cindy St. Hilaire: No, but I guess, based on what we know and what you've found in your study, where would you think it's more critical, if we even can speculate on that?

Dr Fadila Guessous: Yeah, the symptoms. I would give them from the beginning of the symptoms for me. Because otherwise, when you are in the cytokine storm and you have all this [thromboembolism in the lungs and everything, it's too late.

Dr Eric Boilard: And if I can add something. When we think of immune cells or B cells, D cells, we don't say anti-lymphocytes or anti-neutrophil therapy. But when we come to platelets, we think that the molecules that are known to impact some of the platelet activities, we think of aspirin, clopidogrel, coagulation, and we call it anti-platelets but. But platelets, I mean, they can do a lot independently of from vaccine, independently of EDP, and sometimes we forget that they are more than just these poor thrombotic cells and that there are different studies that has been published by different groups and us that found that you can use, for example, COX-1-deficient platelets. So they won't make thromboxane yet, they can make lot of IL1 and be pro inflammatory, although they will not make thromboxane. So we actually use mice where were we use a variety of anti-platelet therapies but yet their platelets were able to come out inflammation so there's no one drug that can get all the platelet functions at once, so it's important to remember that.

Dr Cindy St. Hilaire: It sounds like maybe some of them could possibly make things like a cytokine storm almost worse, if we're targeting the wrong anti-activity?

Dr Eric Boilard: All depends on how platelets are activated. Whether we are activating a GP pathway, or a nighttime pathway or both at the same time, and the environment where platelets are localized and activated so that they all back the response to this.

Dr Cindy St. Hilaire: So you're saying it's complicated.

Dr Eric Boilard: Yeah, I think it's not simple.

Dr Milka Koupenova: Anything complicated, it's platelets. So in a way I think that perhaps what we need to acknowledge is targeting platelets for whatever response. I completely agree that platelets should be targeted at one point but what drugs should be used, is the question? What specific receptor or what specific response? And if we manage to figure some form of controlling the inflammatory response, that specific, what you see, that huge amount of cytokine dysregulation that will be great. But if you're targeting platelet-specific function when it comes to thrombosis, I'm a bit concerned that there has to be an exact time in which that's important. And the reason why is because if you have this damage in the endothelium, as you see reproducing the virus in the endothelial cells, and if platelets are not doing their own function then perhaps a lot more virus is leaking into the circulation. And that could be why you see some patients that have it, some patients that don't have it, which is a question that I want to ask.

Dr Milka Koupenova: Why do you think some patients have it and some patients don't have it? And do you think that perhaps as the virus gets processed into a platelet and digested, we are missing a time point? Because we're not taking platelets from patients at the same time after infection. We have no way of doing that. So what is your take on the fact that you have virus in some platelets? And from your study, perhaps, if we rely on the numbers, the people who have more severe symptoms seem to have a little bit more. Correct me if I'm wrong.

Dr Eric Boilard: Actually, the older people have more chance of having it but it was not in the more severe people. In fact, even among the non-severe patients, there was 20% of them that had platelets with positive RNA. So that didn't correlate with severity or any outcomes. The only correlation we could identify was age.

Dr Milka Koupenova: So do you think that perhaps in older individuals there are problems would be degradation of the virus, and hence they are not initiating the proper response?

Dr Eric Boilard: Yeah, potentially. Maybe there's somehow an indication of the virus from the organs and it's a destination or it's not captured fast enough on the first varrier of the immune cells in the lungs. Right now we don't have the evidence that it's actually the virus that is in platelets, right? The RNA and there's still work to determine if the virus is there.

Dr Milka Koupenova: So practically, in summary, we don't know what it is. Although, I mean, with a bias, me probably like you, and you're very nice, you're cautious, but I also believe that the virus... and it's a belief. That the virus gets inside of platelets.

Dr Fadila Guessous: How, Milka? How?

Dr Milka Koupenova: There could be some issue that we don't see, and as Eric says, it's some form of an age factor. There could be other receptors. Platelets are tricky, they don't act like every regular cell so they may have evolved to have other receptors that maybe they are not as functional in the ither cells but they're platelet specific. There is the process that from all these cytokines perhaps, platelets are just sucking in things by micropinocytosis, which has happened. The thing that's important to think and mention is that if a virus is not going into a platelet by receptor-specific mechanism, perhaps it's locating to the wrong compartment, and then not introducing the proper immune response. Perhaps that's the reason of why you're catching it only in 20%. This is all very, very speculative.

Dr Eric Boilard: Yeah. And are you putting in a contribution of megakaryocytes] in the lungs where they will burst …

Dr Milka Koupenova: Yes. That it. Absolutely, it's quite possible. That's a collaboration then you should perhaps consider with Dr Craig Morrell. Which is very interesting, right? We don't know. We were never able to figure, even with our full studies, we also saw, what is it, four out of 18? So it's like 25% of the platelets had RNA for a flu but when you look what the antibody of the virus you find that a lot more platelets have it. So is it that we're missing some form of a process that the virus quickly gets digested and it initiates a response? I don't know until we get the right tools. But currently we are struggling with tools, practically, to assess that question.

Dr Eric Boilard: Yet it's impressive what's been done since March with the Covid.

Dr Milka Koupenova: Oh, some of them is good. Some of them is really important science and some of them is questionable that raises more questions.

Dr Eric Boilard: Absolutely, yeah.

Dr Milka Koupenova I personally think it's very important what your study is showing because it gives a base of truly thinking about how platelets maybe truly acting as an immune cell at the beginning. I know that Eric and I might be a little bit biased when it comes to that, but when does the thrombotic response stop? And when does the immune response stop of a platelet? And is it necessary to stop and to be all together? How do we push one and doesn't stop the other? These are the questions that we in the field truly need to assess and that's what I really like about your study, is that it raises these important questions.

Dr Fadila Guessous: Before the call, I was looking at the paper from University of Verona. I mean, I was hoping they will find more answers to the same question that is a favorite from University of Verona, and actually they are also more speculative. They didn't answer the question, the same questions we are asking.

Dr Milka Koupenova: Well, until we have the virus on hand, the actual virus, it's a little hard for us. And then the other point is even if you have the virus on hands, you're doing all of these in vitro studies, how do you assess the entire immune response? Can we think of platelets just as platelets without being in cells, right? What is your take on that, Eric or Fadila? Can you think of platelets just as platelets when it comes to mediating a response to a viral infection? Because we think okay we do this particular thing with platelets and then they mediate this and they mediate that, but perhaps the interaction between platelets and all the immune cells at the beginning of the response is super important. And I do struggle sometimes with the assessment of what amount is just platelets and what is the communication between each immune cell and platelet. It's very hypothetical.

Dr Eric Boilard: I agree that I see platelets as communicators to other cells. I mean, they're so numerous and there are bags with cytokines and other molecules and RNA and micro RNAs and it can impact for different organs, tissues, cells. The fact that they were activated, even in the non-severe patients, all the non-severe patients we analyzed, platelets were there, they were activated. So there was something going on, although thrombosis was not of use in these patients, so they made plates front row and the front part of the disease and the pathogen as a cell.

Dr Milka Koupenova: So is there a way of somehow, as you perceive in your future studies, to get platelets from people who get the virus but don't have any symptoms, and compare their cytokine profile of platelets to what you see? Because we focus so much on the people who get infected, logically, right? But truly to assess what is the difference, if we can figure why those patients are not having this profound response, right?

Dr Eric Boilard: Something that we dream of. The fact that collecting platelet from someone who has no symptoms is…Because we, if they have no symptoms, then they don't come to us. They don't stop-

Dr Fadila Guessous: They don't show up at the hospital.

Dr Milka Koupenova: And so you have to recruit them by tests, right? Because we screen, for instance, our institution screens. But it's hard, right? No one wants to take blood from a…

Dr Fadila Guessous: You get this kind of calls, people are asymptomatic, they don't have any symptom, they won't show up at the hospital. We used whatever we had in our recruitment from the hospital.

Dr Eric Boilard: In fact, you can maybe go through the transfusion work, the studies on transfusion where they actually collect blood from people and people sign that they have no symptoms when they do give blood. And there are studies from China and more recent ones in France where they then went back to this bags of... these were plasma, but maybe one could do it with platelet concentrates. And then go back to these bags where people had signed they had no symptoms when they gave blood, and screened platelets for RNA and then... but you have dozens of dozens of samples to test, but they are available. And maybe someone could find that many were positive but they were asymptomatic, using transfusion studies, maybe. But recruiting people from outside, asking we want people with no symptoms for…

Dr Cindy St. Hilaire: I know we're doing sentinal testing of our students, so. Somehow the group doing that needs to couple with platelets studies.

But that was wonderful. Thank you Dr Boilard, Geussous, and Koupenova for joining me today. This has been an amazing interview. It's a wonderful study. Good luck on the future research and thank you for moving the field forward on this.

Dr Eric Boilard: Thank you very much for inviting us. It was great and we are pleased to interact with you. Thank you.

Dr Milka Koupenova: Very nice to meet you Fadila, Eric.

Dr Fadila Guessous: Thank you so much for having us. It's really it's a pleasure to have this interview with you and of course I have to say that really it has been a wonderful collaboration with Eric and Younes in our lab. I am from Casablanca so we had really many people involved in this project and everyone, we worked really, really hard to get this very... I mean, you can see the time, it's from March to now, to get this paper out it was really a big effort. A big effort from the Moroccan side and from Eric's team in Quebec City and so we are really very pleased to have this wonderful collaboration. Thank you so much for having us.

Dr Cindy St. Hilaire: That's it for the highlights from the late October and early November 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, Drs. Eric Boilard and Fadila Guessous, as well as Dr Milka Koupenova for co-hosting the interview with me.

Dr Cindy St. Hilaire: This podcast is produced by Rebecca McTavish and Ishara Ratnayaka, edited by Melissa Stoner, and supported by the editorial team of Circulation Research. Some of the copy text for the 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 month on Episode 17 of the Discover CircRes podcast, host Cindy St. Hilaire highlights four featured articles from the September 25 and October 9 issues of Circulation Research. This episode features an in-depth conversation with Drs David Dichek, Sina Gharib and Tomáš Vaisar regarding their study titled Parallel Murine and Human Plaque Proteomics Reveals Pathways of Plaque Rupture.

Article highlights: Cai, et al. Single Cell RNA-Seq in Arteriosclerosis Schuhmann, et al. CD84 in Ischemic Stroke VanOudenhove, et al. Gene Regulatory Dynamics of Developing Human Heart Nie, et al. Periostin is a Target to Treat PH

Dr Cindy St. Hilaire: Hi, 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 four articles selected from the late September and early October issues of CircRes. I'll also be interviewing Drs David Dichek, Sina Gharib, and Tomáš Vaisar regarding their study titled Parallel Murine and Human Plaque Proteomics Reveals Pathways of Plaque Rupture. The first article I want to share is titled Single Cell RNA Sequencing of Allograft Cells in Transplant Arteriosclerosis. The first author is Jingjing Cai and the corresponding author is Qingbo Xu from Zhejiang university in Hangzhou, China. Arteriosclerosis is a major contributor to organ transplant failure. The thickening and stiffening of arteries within the grafts results in diminished blood flow supplies and diminished organ function. While it is well understood that atherosclerosis is an inflammatory disease, the details of the cellular and molecular players on this transplant-specific pathology are lacking. Now, Cai and colleagues used single cell RNA sequencing to identify a consensus of cells and cytokines in sclerotic transplanted aortas in mice. Two weeks after transplant, the grafted vessels exhibited signs of arteriosclerosis and by four weeks, this remodeling had worsened significantly. Analyzing the RNA transcripts of over 12,000 individual cells isolated at both two and four weeks, the team discovered that the number of T-cells was greatly increased throughout the process. An early abundance of macrophages gave way to a later wave of B cells and there was also evidence of the development of tertiary lymphoid tissue. They further found that chemokine CCL121 was up regulated after transplant, both the mRNA in the tissues, as well as the protein levels in the animal's blood. The authors then went on to show that blocking CCL121 or its attracting partner, CXC3, significantly delayed arteriosclerosis in the grafted vessel. Hence, this work not only defines the cellular and molecular drivers of arteriosclerosis in grafted vessels, but highlights potential molecular targets for future therapeutic interventions. The second article I want to share is titled CD84 Links T-cell and Platelet Activity in Cerebral Thrombo-inflammation in Acute Stroke. The first author is Michael Schuhmann and the corresponding author is David Stegner, and the work was completed at University of Würzburg in Germany. Ischemic stroke is caused by the occlusion of cerebral blood vessels and it is a leading cause of death and disability worldwide. Despite treatments to degrade or remove clots such as mechanical thrombectomy, infarct size itself can continue to grow even when blood perfusion is re-established. Thrombo-inflammatory processes are thought to mediate this worsening injury, with both T-cells and activated platelets playing a role. Because both T-cells and activated platelets express CD84, which is a self-binding adhesion molecule involved in lymphocyte activation, this team tested the hypothesis that CD84 might mediate stroke inflammatory processes. They went on to show that mice lacking CD84 have smaller infarct sizes with reduced T-cell inflammation after stroke than wild-type animals. Furthermore, mice that specifically lack CD84 in either T-cells or platelets also experienced smaller infarcts. The team went on to show that CD84 promotes T cell migration in vitro. And then in patients with stroke, high expression of CD84 in platelets was associated with poor outcomes. Together, these results suggest that activated CD84-secreting platelets encourage inflammatory T cell migration to the infarct site. And that blocking CD84 activity could be a novel therapeutic strategy for minimizing inflammatory injury after stroke. The third article I want to share is titled Epigenomic and Transcriptomic Dynamics During Human Heart Organogenesis. The first author is Jennifer VanOudenhove and the corresponding author is Justin Cotney. And they're from the University of Connecticut. Congenital heart defects, or CHDs, are common birth abnormalities and while some genes have been linked to congenital heart defects, the majority, close to 60%, have unknown etiologies. It's thought that multiple genetic and environmental factors contribute to congenital heart defects. One of which could be variations in both cis and trans regulatory regions of the genome. To find such heart specific regulatory regions, this team examined heart tissue from human embryos obtained four to eight weeks after conception. They performed chromatin immunoprecipitation experiments to scour the heart genomes for histone modifications associated with increased or decreased gene transcription. They also performed transcriptome analysis to see whether the genomic regions identified by chip corresponded with the activity status of nearby genes. In total, the team found more than 12,000 previously unknown enhancers that were enriched for binding sites for heart specific transcription factors, some of which included GATA, MEF2 and Nkx. These binding sites tended to be close to genes activated in the heart. Many of the regions also contain sequence variations that have been associated with atrial fibrillation. These newly identified sites are potential congenital heart defect candidate loci and the authors have now made their data readily available so that other investigators may study it. The last article I want to share with you before we switch to our interview is titled Periostin: a Potential Therapeutic Target for Pulmonary Hypertension? The first author is Xiaowei Nie from the Shenzhen Third People's Hospital and the corresponding authors are Jingyu Chen and Jin-Song Bian from the Wuxi People's Hospital and the National University of Singapore, respectively. Pulmonary hypertension, or PH for short, is a life-threatening disease where an excess in the proliferation of vascular smooth muscle cells and the deposition of extracellular matrix thickens the walls of the lung vasculature, which leads to an increase in pulmonary blood pressure and ultimately contributes to right heart failure. Vasodilatory medications can be used to treat the symptoms of the disease. However, these medications do not prevent or reverse the underlying pathogenic remodeling. This study now suggests that drugs targeting the secreted extracellular matrix protein, periostin, might be a potential therapeutic strategy for the treatment of pulmonary hypertension. Periostin is an abundant protein in the lung arteries of pulmonary hypertension patients. And it is thought to be involved in cell adhesion and wound healing mechanisms, such as the proliferation and the migration of smooth muscle cells. The team confirmed increased production of periostin in patient lungs, and also found the same to be true for mice with an induced model of pulmonary hypertension. They also showed that genetic deletion of periostin attenuated pulmonary hypertension in mice, while suppression of periostin via RNA inhibition could even reverse pathological vessel thickening and the subsequent right ventricle hypertrophy. The team went on to identify factors HIF-1a and TrkB as factors that mediate periostin's effects in cultured arterial cells. And they suggest that blocking either of these factors or by blocking periostin itself could be a novel strategy for the treatment of pulmonary hypertension patients. Drs Tomáš Vaisar, Sina Gharib, and David Dichek from the University of Washington in Seattle, Washington are here with me today and we're going to discuss their recent study titled Parallel Murine and Human Plaque Proteomics Reveals Pathways of Plaque Rupture. Thank you all so much for joining me today and congratulations on this beautiful and interesting study. So this is an atherosclerotic study, but unlike many in the field, it's really looking at the end stage event called plaque rupture. So for those listeners who are unfamiliar with the term, plaque rupture is when an atherosclerotic plaque degrades and its contents are exposed to the circulation, which can then induce a clotting event and lead to all sorts of adverse pathologies, myocardial infarction, transient ischemic events, stroke. So I'm wondering if we if maybe you can give us a little bit of background about what's known and what really is unknown at least before your study regarding plaque rupture.

Dr David Dichek: So the pathogenesis of acute myocardial infarction and stroke was really unknown for many years. And the idea that it was due to acute thrombosis was really confirmed by a study probably 30 years ago, that did angioscopy in the coronary arteries, proximal to a myocardial infarction, and visualized actual clot so that the clot was confirmed to be associated with the acute event. At that point, the question became why would a coronary artery form a clot? And that led to identification of, or histologic studies that identified ruptured caps of atherosclerotic plaques, exposure of the blood to the thrombogenic contents of the lesion, and a thrombus. However, it was not known what the initiating event was in rupture of the plaque cap. And there were a lot of hypotheses and a lot of nice work, but it does still remain an unknown. A significant amount of focus has been devoted to the possibility that proteolysis is the initiating event. And that was sort of the takeoff point from our study because we had developed a mouse model where proteolysis clearly was associated with rupture of plaque caps. And we decided we wanted to get more into the biochemistry of what was going on and go beyond the histology. So that was really what led up to our study.

Dr Cindy St. Hilaire: It's really interesting. So, mice are really good and obviously really useful, very well-known model systems to study atherosclerosis and particularly the initial drivers and maybe the mechanisms of the disease pathogenesis, but like many models systems, they're not perfect. So I'm wondering if you could discuss the limitations of murine model systems and specifically for this study, how you were able to overcome some of those limitations.

Dr David Dichek: So the limitations of mouse models of plaque rupture are that essentially none of them duplicate the histology of human plaque rupture, particularly the thrombus that occurs on top of the plaque rupture. So there are various mouse models where caps are disrupted, but there's not acute thrombosis. It has been argued in the vascular community as to whether these models are authentic models of plaque rupture, because they don't have the superimposed thrombosis. And the counter argument is well, mice aren't people, they have different hemostatic and coagulation factors that may be differentially regulated. The hemodynamics of small mouse arteries is different from human mouse arteries. And the fact that you don't get a thrombus doesn't necessarily mean that you're not modeling the process that would cause it. So we really accepted that argument as being valid and felt that the occurrence of frank plaque rupture, and that was in our Circulation paper in 2010, in these lesions in the mice, really validated it as an authentic model of cap disruption. And so I agree it's arguable, whether this is an authentic model. But we actually took that issue head on by saying, well, is the biochemistry of the ruptured plaque similar to the biochemistry of a ruptured human plaque? And that if there were similarities that we would gain more confidence in our model being an authentic model of plaque rupture and that it matched not only the histology, but also the biochemistry.

Dr Cindy St. Hilaire: One of the main tools you used, you used shotgun proteomics, which I think is just a great name for it. And also a algorithmic learning tool or analysis tool called proteomaps. I was wondering if you could give us a little bit of background about the proteomic aanalysis involved, what does that entail? Especially, when you're comparing a teeny tiny mouse plaque to a larger human plaque and then how that analysis was done?

Dr Tomáš Vaisar: So the shotgun proteomics term was coined by John Yates, but way back in the early 1990s. And it's essentially the way how you enumerate the proteins and in more recent forms, you even quantify the abundance of the proteins in a very complex mixture. So shotgun proteomics essentially takes a protein sample. And in this case it was an extract of the tissue and uses protease, trypsin typically, to cut the proteins down to peptides, which are relatively small and relatively well behaved compared to intact proteins. And then using tandem mass spectrometry combined with liquid chromatography separation, basically aims to sequence every single of those peptides or majority of the peptides. And then based from the identification of the sequence of individual peptides piece back together like a jigsaw puzzle, what was the protein present in the original mixture?

Dr Cindy St. Hilaire: That's so interesting. I know it's been around for a while. I'm always impressed by it.

Dr Tomáš Vaisar: And then the other approach we use it's called Proteomap developed by Ben Cravatt, collaborator on this paper at La Jolla. And that approach uses basically the shotgun approach but as a first step uses gel electrophoresis, SDS PAGE electrophoresis to fractionate the very complex mixture to size segments.

Dr Tomáš Vaisar: So you run your complex mixture on a gel, you slice it by size and then run shotgun proteomics experiment on each of those slices after gel digestion. And then Ben developed a set of tools where you identify the proteins and their abundance in each of the bands based on the size and the way it's applied to formation or mapping of proteolytic events is based on the idea of that intact protein will show up at the molecular weight of the intact protein but if it was cleaved by a protease, it will also show up at the molecular weight, which is smaller corresponding to the fragments formed by proteolysis. Then you use set of bioinformatics tools to piece this all together and generate the Proteomaps.

Dr Cindy St. Hilaire: You pick it apart, throw it in the machine and then put it back together. That's so cool. It's so amazing. So what were the main findings that you were able to pull out of your comparisons? And I think you had three main groups, if I understood it right? There's the transgenic mouse that has the plaques that don't rupture, and then there's the atherosclerotic mouse that had the transgenic bone marrow, and then you had the human. Can you tell us a little bit about the different groups you compared and then what ultimately you found?

Dr David Dichek: Sure. Yes. You are absolutely right. We had what we refer to as the straight transgenic mice that are either transgenic for macrophage overexpressed uPA or not. We also had older mice who had advanced atherosclerosis and receive bone marrow transplants from mice that either had uPA overexpression in bone marrow or not and then we had the human plaque. So those were the three groups. So what we found was that looking at the proteome of those three groups, we were able to find some common biological processes, and this was really Sina's work, but taking the proteomics data and analyzing it with sophisticated bioinformatics tools. We looked not only at the overlap in specific proteins among the models, but the overlap in biological processes, because it may be in different species that there are different proteins, different actors carrying out the same roles. And that's been described in other systems as well. So we were able to identify not only common biological processes, but surprisingly, we were able to identify decreases in specific category of proteins, basement membrane proteins that were common to two of the models, the straight transgenic and the human model and loss of these proteins certainly has a plausible role in precipitating plaque rupture. So I think one aspect of the analysis that's worthy of note is that we initially thought we would observe more profound changes in the bone marrow transplant mice because they had more advanced atherosclerosis. And in fact, we found fewer changes than in the straight transgenic mice, but thinking about it after letting the data talk to us, rather than trying to impose our own on the data-

Dr Cindy St. Hilaire: Always a good idea.

Dr David Dichek: ... was that the straight transgenic mice were telling us we've been overexpressing urokinase for 20 weeks since we were conceived, and the bone marrow transplant mice had it for only eight weeks. And indeed they had far less loss of basement membrane proteins and far fewer changes in their plaques than the mice that had expressed it for a longer time. And so when one placed the three groups in a chronology of exposure to protease activity with the bone marrow transplant mice, being the shortest exposure than the straight transgenic mice, and then the humans who've had decades of exposure, it really tells you a nice chronological story about the biological processes leading to plaque rupture. And I think that's a generally applicable lesson and can be applied to other problems in cardiovascular biology. And that is when you have a biological process for which you can't get human tissue until after it's occurred because you can't go in and biopsy.

Dr Cindy St. Hilaire: I have that problem with valve calcifications that you can't take your valve out early.

Dr David Dichek: If you can get a mouse model that duplicates the pathology, then you have access to the steps leading up to the event. And that's what we tried to construct in this study. And really it was really Sina's analyses that allowed us to make those connections.

Dr Sina Gharib: Yeah. Of course, David was kind of the mastermind behind the design of the experiments on the developing of genetic models and Tomáš is a renowned expert in proteomics analysis. And I kind of joined more on the bioinformatics component of this study, tried to put some of the large data that was being generated together. And as David and Tomáš mentioned, of course atherosclerosis is a very complex disease with many, many components. And then of course the mouse model doesn't quite capture all the different pathophysiological events that happen. So one of the aims of this study was to try to integrate and merge the findings from these model without coming a priori with a bias or a pathway or a candidate gene, we decided to do a relatively unbiased shotgun proteomics approach, we actually do for everything. So the challenge then was how to put it all together. And as Tomáš mentioned, there are statistical tools to try to identify a relative abundance of proteins. But, a few things that pure biologists often don't have to encounter is, you're not one or two different proteins, you're looking at thousands of proteins. So there are issues, statistical issues, such as multiple comparisons. If you looked for changes, you're going to find changes just by random chance. So a lot of statistical adjustments had to be made to ensure that those were adjusted for. This are also pathways and processes that were coming out of these results. There's many different pathways that were interrogated. And again, statistically, you want to adjust for the fact that many of those could have been there by random chance. So there's a fair amount of statistical methods that need to be applied for this data. We also did somewhat more sophisticated pathway analysis where we develop networks based on the differential expressed proteins between the ruptured and unruptured plaques to identify connection among these proteins and identify hubs which are highly connected nodes that could potentially drive the biology of a network. So other types of kind of deeper statistical analysis was done, which are maybe more hypothesis-generating because we actually did not follow up on some of these candidates, but I think they really do provide a map or framework to then pursue more mechanistic experiments to see what happens if we knocked out this highly connected node at the plaque rupture site to see if we can either stabilize or manipulate the biology as plaque rupture.

Dr Cindy St. Hilaire: Yeah. I mean, that's really the strength of these unbiased approaches is you can come up with so many more novel targets and pathways that might be contributing. So they're just really great. So one thing I found really interesting, you mentioned that you saw a clear distinction in the proteome and I think it was specifically talking about the human samples because they were large enough to see ruptured area versus non ruptured area, but you really saw a distinct difference in the proteomes of the ruptured area of the plaque versus the non ruptured area of the plaque. And obviously the models you were using are overexpressing a protease. So of course there's a role for proteolysis in this process, which you've now firmly established, but I'm wondering if there are other processes that might also erode the basement membrane. And did you pull up anything that might suggest of other things that are happening or even are there other hypothesis out there that we could test with an approach like yours?

Dr David Dichek: I think the pathways that came up have probably all been implicated previously. We have processes like inflammation and complement activation, immune response, thrombosis. That's a post-hoc event. I think what was most unexpected was the decrease in the abundance to basement membrane proteins rather than collagens. So collagen has become the sort of signature protein of stable and unstable plaques and used as a surrogate, people do Picrosirius red staining. It's easy to detect with a histochemical stain, you don't even need an antibody. And surprisingly, we found very few differences in collagens and actually no differences in the type 1 or type 3 collagen, which are thought to be the primary stabilizers of the plaque cap. They weren't significantly different in between ruptured and stable areas of the same plaque. So that was certainly a big surprise.

Dr Cindy St. Hilaire: Yeah. Because that would indicate that it's not necessary... We always say thinning of the cap, which obviously we know that there's remodeling, it can get thinner. But you kind of found that the contents were the same, but it's the basement really that was eroded.

Dr David Dichek: Yeah. The basement membrane proteins were lost. It used to be said in physiology that if you discovered something new, you should just go to the German literature and go back 30 years and it had already been described. And so, looking back in the literature, there are actually the work of Jean-Baptiste Michelle in France and a scientist in Finland, Petri Kovanen, have actually focused on the potential role of basement membrane in unstable atherosclerosis many years ago, but it was kind of buried in the collagen hypothesis. And I think it needs resurrection.

Dr Cindy St. Hilaire: Well, I think this paper has done that so well done there. That's great.

Dr Tomáš Vaisar: Would be worthwhile to know that of course, the way you prepare the samples may affect what exactly you're seeing. But we've done very careful characterization of the sample preparation of the extraction procedure to focus, to enrich the exosomal matrix proteins because of this collagen hypothesis. And even with that, we basically saw no difference.

Dr David Dichek: Yeah. I think that's an excellent point. If we hadn't found collagen in our extracts, we would not be able to conclude a lot about it. And how you do the extraction, how you process the samples here can really influence what you find. We call it unbiased, but there are technical biases that enter, especially in sample preparation, but our extraction process really was able to extract collagens as well as elastin, which is really infamous for being a-

Dr Cindy St. Hilaire: Difficult.

Dr David Dichek: ... I really think we were getting a good sampling of the matrix here.

Dr Cindy St. Hilaire: I don't know iif there is an answer for this question, but it's something I'm always thinking about. We always talk about athero being so prevalent because there's no kind of evolutionarily the way to tamp it down, it happens later in life. But can you think of any advantage that the vasculature would have in eroding the basement membrane or altering proteases in a response? I was just trying to think, is this just harnessing a wound healing process that's gone awry or could this ever be protective at all in any way?

Dr David Dichek: Well, I think you hit the nail on the head, at least according to my bias. It's a healing response gone awry and that you can really draw out the pathways, basement membrane digestion release of chemotactic peptides as part of the inflammatory response, attraction of more inflammatory cells and then a potential healing response that unfortunately results in digestion of the matrix, which has a morbid or fatal consequence rather than physiologic remodeling. And you're right, that's not selected against. It's selected for, in settings in earlier life infections, for example, perhaps neoplasia, but it's not selected against in late life because people are post reproductive.

Dr Cindy St. Hilaire: So what's next for these studies? What questions are you going to attack next with either these models or with some of your proteomic findings?

Dr David Dichek: Well, we were just talking about that recently, Tomáš and I, and I think we'd like to look at... For one study, we're interested in doing, plaques that are high risk based on MRI imaging, which is really very well developed here at the University of Washington. And many of those patients have endarterectomies and they don't have ruptured plaques. So they are in a high risk group. So they undergo a endarterectomy for that. Not because they've had a plaque rupture and those plaques might be particularly instructive because they're pre event and won't have the healing response to thrombotic response. And it would be really interesting to see if our studies were confirmed. So that's one direction we're going in.

Dr Cindy St. Hilaire: That would be amazing. Luckily, you have access to a whole bunch of human tissue for those kinds of really high impact studies.

Dr Sina Gharib: I just wanted to point out that one of the advantages of doing proteomics and being part of the scientific community is that we made all this data available in the manuscript for other researchers to access and confirm. So, really probably the best way to procced with this is to have other investigators replicate our findings and expand on it. So I just want to bring that up because all of that data that was generated has been included within the supplements of this manuscripts and it's accessible to any scientist who wants to pursue further.

Dr David Dichek: Yeah, I would add one other direction we'd like to go is we still like to know what the substrates are. We think their disappearance based on their abundance is due to proteolysis. But boy, would it be exciting if we could detect fragments. We were unable to do that in the study, probably because they were lost either in vivo or in processing. Technical advancements in that area, and maybe Tomáš can speak to that, might enable us to actually find more direct evidence of proteolysis.

Dr Tomáš Vaisar: Yeah, I mean, to start with, it's really hard to determine physiological substrates of proteases. There's a huge amount of literature identifying proteolytic substrates in vitro, but the physiological substrates are really extremely hard to determine, and especially physiologic in vivo confirming that because in vitro, in a tube, you can mix whatever you want and you modify the ratio of proteins to protease substrate, and you can cleave almost everything with anything. It's a little exaggeration, but it's close. While the physiology substrates in the really complex milia of tissues is extremely hard. And so there has been several approaches developed and one of them is the Proteomaps. The other one is an approach called TAILS developed by Chris Overall at UBC that uses the idea of formation of the neo termini and then tagging the neo termini. So that in the actual sample, you can specifically detect these neo termini formed. But even with that approach, it's really hard to determine what are actual physiological substrates. And on top of that, what are the cleavage sites of the proteases?

Dr Cindy St. Hilaire: And I guess the third being, if those substrates are cleaved, are they circulating and can we detect them in a blood sample? That would be, I guess, the gold standard. Well, thank you all so much for joining me today. Congratulations on this really very cool study. Being into human and translational work, I always love mouse studies that bring in lots of human samples. So congratulations on that. And I look forward to your future publications on this.

Dr Tomáš Vaisar: Thanks a lot.

Dr David Dichek: Thanks.

Dr Cindy St. Hilaire: That's it for the highlights from the late September and early October 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 #DiscoverCircRes. Thank you to our guests, Drs David Dichek, Sina Gharib, and Tomáš Vaisar. This podcast is produced by Rebecca McTavish and Ishara Ratnayake, edited by Melissa Stoner and supported by the editorial team of Circulation Research. Some of the copy texts 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 up-to-date and exciting discoveries in basic cardiovascular research.

This month on Episode 16 of the Discover CircRes podcast, host Cindy St. Hilaire highlights four featured articles from the August 28 and September 11 issues of Circulation Research. This episode features an in-depth conversation with Drs Andrew Murphy and Michelle Flynn from The Baker Heart and Diabetes Institute at Monash University in Melbourne, Australia regarding their study Transient Intermittent Hyperglycemia Accelerates Atherosclerosis By Promoting Myelopoiesis.

Article highlights:

Fish, et al. KRAS Mutations Cause Arteriovenous Malformations

Ehling, et al. B55a in Vascular Biology

Barrett, et al. Platelet Activity and Vascular Health in COVID-19

Furmanik, et al. Nox5 in VSMC Phenotype and Calcification

Cindy St. Hilaire: Hi. Welcome to Discover CircRes, the podcast to 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 share with you four articles selected from our late August and early September issues of Circulation Research. I'm also going to speak with Drs Andrew Murphy and Michelle Flynn from The Baker Heart and Diabetes Institute at Monash University in Melbourne, Australia regarding their study Transient Intermittent Hyperglycemia Accelerates Atherosclerosis By Promoting Myelopoiesis. So first, the highlights.

The first article I'm sharing with you is titled Somatic Gain of KRAS Function in the Endothelium is Sufficient to Cause Vascular Malformations that Require MEK but not PI 3-Kinase Signaling. First authors are Jason Fish, Carlos Perfecto Flores-Suarez, and Emily Boudreau. And the corresponding authors are Jason Fish and Joshua Wythe, and they're from University of Toronto and Baylor College of Medicine.

Arterial venous malformations, or AVMs, are tangles of blood vessels in which the arteries are directly connected to the veins without going through the capillary bed. These are thought to be present from birth and when they occur in the brain, they can cause an array of symptoms such as headaches or seizures, but they are also the leading cause of hemorrhagic stroke in children and young adults. This is because the venous system is not muscularized to respond to the pressure forces that are exerted on arteries.

These pressure forces cause distension and eventual leakage at the site of AVMs. Vessel tissue recovered from patients undergoing AVM repair has been shown to contain sematic gain of function mutations in the protein RAS GTPase, which is encoded by the gene, KRAS. However, whether these gain of function mutations directly cause AVMs has not been established. This study now shows that endothelial cells with constitutive expression of gain of function KRAS mutants in mice and zebra fish causes vascular malformations and cranial hemorrhages. Inhibiting a MEK kinase, which is a downstream mediator of RAS signaling, prevented hemorrhages in the mutant KRAS carrying fish. In vitro studies also showed that overactive RAS GTPase protein caused excessive angiogenic behavior of endothelial cells. Together, this work confirms the link between gain of function KRAS mutations and brain AVMs, and suggests that MEK inhibition could be a potential strategy for nonsurgical treatment.

The second article I want to share with you is titled B55a/PP2A Limits Endothelial Cell Apoptosis During Vascular Remodeling: A Complimentary Approach To Kill Pathological Vessels. The first author is Manuel Ehling and the corresponding author is Massimiliano Mazzone. And the work was completed at Leuven Center for Cancer Biology in Belgium. Building a mammalian vascular system is a dynamic process that is dependent on both growth of new vessels, as well as the pruning of unwanted ones. But while much is known about molecular mechanisms underlying angiogenesis, comparatively little is understood about the mechanisms regulating vascular pruning. This study discovered that suppression of the protein phosphatase 2 subunit, B55A, is a key protein regulating the pruning process. They found that in mouse vascular development, B55a is widely expressed. However, in adult mice expression is restricted only to sites of active angiogenesis.

Deletion of B55a in mice caused death in mid to late stages of embryogenesis as a result of vascular problems that appeared to be due to excessive vessel pruning. Switching off B55a in adult mice when the vascular development is for the most part complete did not cause any apparent problems. They did find though, that inhibition of B55a significantly delayed growth of tumors that form from the injection of cancerous cells. Inhibition of B55a produced tumors with less dense vasculature and reduced metastatic potential. Thus, the author suggests that ramping up blood vessel pruning, be it inhibition of B55a, could be a novel strategy for limiting tumor growth.

The next article I want to share is titled Platelet and Vascular Biomarkers Associated With Thrombosis and Death in COVID-19. The first author is Tessa Barrett and the corresponding author is Jeffrey Berger, and they're from New York University. Our knowledge of the complications of COVID-19 is evolving every day. Laboratory testing done to date suggests that approximately 30% of hospitalized COVID-19 patients go on to develop thrombotic events. Platelets are central characters in both arterial and venous thrombosis, and it is known that virus platelet interactions can stimulate a pro-coagulant and inflammatory state during a viral infection. Further, recent studies have reported COVID-19 patients have hyperactive platelets and autopsies of COVID-19 patients exhibit micro and macro thrombi across vascular beds, even in patients without clinical thrombosis.

This group then hypothesized that biomarkers of platelet activation are associated with incident thrombosis or death in COVID-19 patients. To test this, they randomly selected 100 COVID-19 positive patients and analyzed banked samples collected on the day of the COVID-19 diagnosis to investigate in vivo platelet activity, as well as vascular health biomarkers. They show for the first time that biomarkers of platelet activation at the time of diagnosis are associated with thrombosis or death in patients hospitalized with COVID-19. Their findings suggest platelet activation mechanisms may contribute to adverse events and highlight the potential role of antiplatelet therapy in this disease.

The last article I want to share with you before we switch to our interview is titled Reactive Oxygen-Forming Nox5 Links Vascular Smooth Muscle Cell Phenotypic Switching and Extracellular Vesicle-Mediated Vascular Calcification. The first authors are Malgorzata Furmanik and Martijn Chatrou. And the corresponding author is Leon Schurgers from Maastricht University in The Netherlands. Vascular calcification is an active process regulated by several mechanisms, including vascular smooth muscle cell apoptosis, osteochondral genic transdifferentiation, extracellular vesicle release, and cellular senescence. In healthy adult arteries, smooth muscle cells maintain a contractile phenotype. However, various insults such as oxidative or mechanical stress, can induce smooth muscle cells to lose their contractility and this process of de-differentiation is termed phenotypic switching. And phenotypic switching is thought to precede the development of vascular disease. Patients with conditions such as chronic kidney disease have mineral imbalances in their circulation and also exhibit higher levels of vascular calcification.

However, the mechanisms behind these observations are not well defined. This group found that extracellular calcium can enter the smooth muscle cells via extracellular vesicles and this increased cytosolic calcium concentration. Increased calcium induces expression and activity of Nox5 in NADPH oxidase. Activation of Nox5 increased production of reactive oxygen species, which in turn decreased contractile marker expression, and also promoted calcification in vitro. Intracellular calcium signaling also further enhanced extracellular vesicle secretion, and decreased extracellular vesicle uptake. This then promoted the accumulation of extracellular vesicles in the extracellular matrix, which is a mechanism that promotes calcification. Together, these data suggest that mineral imbalances, such as those seen in chronic kidney disease patients, contribute to loss of smooth muscle cell contractility, which promotes osteochondral genic transdifferentiation.

For the interview portion today, I have with me Drs Andrew Murphy and Michelle Flynn from the Baker Heart and Diabetes Institute and Monash University in Melbourne Australia. And we're going to be discussing their manuscript titled Transient Intermittent Hyperglycemia Accelerates Atherosclerosis by Promoting Myelopoiesis. But really I like the running title, which is Hyperglycemic Spikes Accelerate Atherosclerosis. So thank you both very much for joining me today.

Michelle Flynn: Thanks for having us.

Cindy St. Hilaire: So before we start to 'stalk a bit about what the details of this study is, could you maybe give us a little primer on what you've done that led up to this study?

Andrew Murphy: Yeah, so this really was a continuation of a study that began actually when I was in my postdoc in Allan Tall lab and working with Ira Goldberg's lab with the postdoc Prabhakara R Nagareddy there. We've shown along with Ed Fisher's group at NYU, that mice that had established atherosclerotic lesions that were then made diabetic, failed to have lesion regression compared to those that were non-diabetic with normalized plasma cholesterol levels. We showed that if we gave an SGLT-2 inhibitor to normalize glucose that regression then started to occur. And then we found that this was primarily driven by myelopoiesis, suddenly increased production of monocytes, which through that entered the plaque. And so from that, that was in the hyperglycemic model which is sort of a very rare patient group these days, because most people are on well-controlled glucosteroid drugs. And really the SGLT-2 inhibitors have been a game changer in that scenario. And what we were trying to do with this study was bring it into a more clinically relevant setting that might show the potential importance of glucose on a much larger population.

Cindy St. Hilaire: Excellent. Maybe you could give us an introduction to the link between what's known about diabetes and cardiovascular disease and the interplay?

Michelle Flynn: So diabetic and pre-diabetic patients actually account for 65% of all cardiovascular deaths, which really indicates that diabetes itself plays a major factor alongside other things like obesity and hypercholesterolemia. And so we've previously shown that hyperglycemia was actually driving atherosclerosis in a chronic hyperglycemic setting. So given that kind of vascular disease actually affects both diabetic and pre-diabetic patients, we suspected that it may not just be chronic hyperglycemia or really intense hyperglycemia that could be driving this issue. And so what we were actually looking at in this paper was how more transient levels of hyperglycemia, which actually occur quite often in both diabetic patients and pre-diabetic patients, how much this can contribute to cardiovascular disease.

Andrew Murphy: I guess this link between poor glucose control and cardiovascular disease is obviously very well established. The interesting part is that HbA1c only predicts part of the risk. If you look at fasting blood glucose, again, that's only partially responsible, but if you look at postprandial or two hour glucose loads, you'll see that that is more predictive of cardiovascular events than the other two measures. And it seems to be a continuum. So even if you are a healthy or non-diabetic individual, you obviously still have those postprandial events and depending how high that goes up is thought to be a predictive of future cardiovascular outcomes. And so obviously that's worse than people with pre-diabetes and then again worse with people that have actual, full blown diabetes.

Cindy St. Hilaire: And what is a transient hyperglycemic event? What would do that in maybe you and me who don't have diabetes versus someone who has diabetes or is pre-diabetic?

Michelle Flynn: So essentially what we're modeling with this transient hyperglycemia is that postprandial increase in glucose after you have a meal, which in people who have impaired glucose tolerance is going to be more pronounced than in someone who has a normal glucose tolerance.

Cindy St. Hilaire: Got it. And so how did you test this in the mice?

Michelle Flynn: We did this by developing a novel model of transient hyperglycemia. So we used ordinary wild type mice that weren't diabetic, and we injected them with glucose intraperitoneally, which then increased blood glucose levels in the plasma after about 15 minutes up to about 15 to 20 millimolar. And then after about two hours, this decreased back down to baseline levels. So this was very similar to what you actually see in a postprandial event. And by doing this four times throughout the day, we were able to mimic what you might see in a patient who has had several meals across the day who has impaired glucose tolerance.

Andrew Murphy: One other advantage with the model that we used was that we were trying to really isolate the effects of glucose. And so by injecting glucose intraperitoneally in otherwise healthy mice, it bypasses the incretin response, which we know loses efficacy, I guess, in people that are diabetic. And so we were just really mimicking acute glucose rises that would occur after a meal. And then obviously in this wild type mouse the insulin response would then kick in to clear the glucose so it really tests that glucose hypothesis.

Cindy St. Hilaire: So it's really digging in deeply on the actual sugar component, not just eating in general or other aspects. So in some of your experiments, or I guess in actually most of them, you show that the injection of glucose, it increased the plaque size in these mice, but it didn't alter the cholesterol levels. So can you explain a bit what's going on there? A little bit about the mechanism you discovered and kind of specifically introducing RAGE and the S100A8 and A9 axis?

Michelle Flynn: Yeah, so what we showed was that regardless of cholesterol levels, we were seeing an increase in clot plaque size, and this was actually driven by the monocytes and neutrophils which were increased in the circulation of these mice. And then these are able to infiltrate into the plaque where they promote plaque progression. And what we found was that the increase in monocytes and neutrophils was due to an increase in their production within the bone marrow.

And this was in turn due to the signaling by a protein heterodimer of S100A8 and A9, which signals via the receptor RAGE in the bone marrow on the progenitors of these cells, which induces their proliferation and differentiation. And then that produces an increase in the production of those immune cells, which promote plaque progression.

Cindy St. Hilaire: Interesting. So it's really independent of kind of the basic thing that everyone thinks about, or I guess as non-scientists think about, is cholesterol. The public really focus on cholesterol, but what your study's showing is there's this whole other glucose mediated immune arm to it. What else does this S100A8-A9 regulate?

Andrew Murphy: So S100A8 and A9 has some intracellular roles, which may direct the development of the model itself, but really a lot of its extracellular roles and so on is promoting sterile inflammation, chemotaxis, so activation of local immune cells. And in the context of diabetes and obesity, many of other diseases, it can signal via RAGE, as Michelle said, but it can also signal by TLR4. And so it seems as though in those diseases driven mainly by glucose, such as the modeling of postprandial hyperglycemia or all kinase in general, it will signal via RAGE, but we've also shown in the setting of obesity that it will signal via TLR4 to stimulate things like interleukin 1 beta. We've also had a paper just recently in Circulation with Prabhakara Nagareddy's group where we've shown post myocardial infarction that prime neutrophils in the heart to eventually release IL1-beta and cause myelopoiesis in that way.

Cindy St. Hilaire: Wow, so this is really kind of an early activator of a much bigger immune response, whether it's in atherosclerosis or MI or probably, I don't know, a handful other things, I guess, right?

Andrew Murphy: It seems to be really important when neutrophils are involved. So in a setting of an MI, we know that they come into the heart very early and become activated and it really makes them about 40% of the cytosol proteins of the cells. So when it degranulates or lyses, they are kind of neutral, at least in the predominant protein.

Cindy St. Hilaire: Okay. So this is released in NETs in NETosis then?

Andrew Murphy: That's what we're sort of discovering so far. So I guess all I can say is, stay tuned, this is a story for another day.

Cindy St. Hilaire: Okay. That's really interesting though.

Andrew Murphy: We haven't looked in gglucose driven events yet.

Cindy St. Hilaire: Yeah and actually one of the interesting things I've learned from your study, I had known about GLUT1 and that GLUT1 was I guess the constituently active of the glucose transporters, but I didn't realize it was so high on neutrophils and that neutrophils were so dependent metabolically on glucose. Can you maybe tell a little bit more about that story?

Michelle Flynn: Yes. So the neutrophil itself is actually very highly dependent on glycolysis because it doesn't actually have many mitochondria. So compared to most cells, they have very few mitochondria so they can't really rely upon the oxidative phosphorylation for their general metabolism. And so they predominantly rely on glucose coming into the cell and then being shuttled through glycolysis to generate their energy. And yeah this does seem to be predominantly due to uptake of glucose through GLUT1.

Cindy St. Hilaire: And then that excess glucose, the byproduct, is reactive oxygen species and upregulation and this cascade of-

Michelle Flynn: Yes, yes that's correct.

Cindy St. Hilaire: Okay, great. So currently we use HbA1c as a biomarker for overall kind of glucose regulation in diabetic patients. And based on your studies and perhaps the studies of others, would neutrophil numbers or even S100A8 or A9 be a better metric to figure out where a pre-diabetic or even a healthy patient is in terms of their glucose tolerability?

Michelle Flynn: Yeah. That could actually be an interesting marker to look at. Given that neutrophils and S100 are also associated with obesity and diabetes in general, and as well as the risk for cardiovascular disease. So with the progression of diabetes, you could expect that the levels of these would increase as well.

Andrew Murphy: We've shown previously when we first discovered that the S100 was important in diabetes, that in the Pittsburgh study with Trevor Orchard's group, he had followed people with type one diabetes for 20 years, that those that did develop a cardiovascular event had a higher S100A8 and A9 levels and that correlated with neutrophils. And so it certainly seems to be a marker of predictive outcomes. And so those that do have poorer glycemic control will have higher neutrophils. That's well known. And so perhaps you're right that probably in combination with HbA1c or things like two hour post glucose challenges, S100A8 and A9 and perhaps neutrophil counts would also be a nice predictive measure of potential cardiovascular outcomes of that person.

Cindy St. Hilaire: Wow. That'd be really great because you could then maybe kind of more fine tune and predict which patients might be more or less susceptible to cardiovascular events.

Andrew Murphy: That's right. Yeah. I think one other important aspect would be if HbA1c is deemed to be relatively well under control, yet you still have a high level of S100A8 and A9, that perhaps those transient spikes are contributing. You're not picking that up in the HbA1c, which looks like the average over approximately a month. And so that could be a nice way to add value onto that score.

Cindy St. Hilaire: Interesting. I didn't realize it was that stable about over a month. All right. So I'm relatively healthy. I'm not pre-diabetic, but if I eat a whole bunch of cake or a whole bunch of ice cream or drink a lot of beer, does that create un me a transient hyperglycemic event that is of the same range we're talking about and what do your findings suggest for people who are relatively healthy and things we should be aware about regarding eating habits and things like that?

Andrew Murphy: Yeah. I think it's a really good question. And it's sort of hard to give you an exact answer to that right now. We need to look at that in people, model these sort of same spikes in people, but what we I guess don't know yet, even in the preclinical models is how high and how long does that glucose have to be? And I think that's one of the most important questions first. So is there a danger zone where these neutrophils start be the innate senses of hyperglycemia that start to then release S100A8 and A9 to cause these downstream events? But what our data does show is that if you're doing this, having a binge night or a binge day once a week for your life, then that's probably not going to be a great thing.

Cindy St. Hilaire: Yeah. All right. So you need to figure out is one scoop of ice cream okay, but two not so great.

Andrew Murphy: Maybe if it's two different flavors it'll be okay.

Cindy St. Hilaire: Maybe, right? That's great. So, I mean, is there a way we could potentially therapeutically target this signaling axis or is it too ubiquitous in terms of what it regulates? Is there a way to harness what you've found potentially in the clinic?

Michelle Flynn: Yeah, so there's an inhibitor of S100A8 and A9 that prevents its binding to RAGE. It's currently approved as an Orphan Drug for systemic sclerosis in both the US and the UK. And that drug itself, we tested in our preclinical mouse model, and we found that it was in fact able to prevent their production of these immune cells, as well as prevent the accelerated atherosclerosis in response to these transient hypoglycemic spikes.

Andrew Murphy: So another sort of line of thinking that we're exploring is that we could actually target neutrophil metabolism itself. And so now we're sort of understanding, are there certain proteins that are more abundantly expressed in neutrophils and not other cells in the body that would regulate glycolysis? I know that might sound a bit of a pie in the sky sort of idea, because glycolytic pathway's quite regulated, but there we have found some proteins that are rich in neutrophils and not other cells that may be responsible for the early steps of glycolysis. And so whether that can be harnessed or not, we'll have to see in the future, but it might be a way of more directly targeting neutrophils rather than approaching that's important in sterile inflammation.

Cindy St. Hilaire: That makes sense. That is such a cool idea and this is really such a beautiful story. It's one of those papers that you just read it and it's just such a logical progression, but it's also really interesting and I really appreciated all those bone marrow transplants. I did those in grad school, so well done. It's a beautiful story. And then I'm just really happy that you published it with us. So thank you so much for joining me today.

Andrew Murphy: Yeah thanks for having us.

Michelle Flynn: Thank you.

Cindy St. Hilaire: That's it for highlights from the late August and early September issues of Circulation Research. Thank you so much for listening. Please check out the Circulation Research Facebook page and follow us on Twitter and Instagram with the handle @CircRes and #DiscoverCircRes. Thank you to our guests, Drs Andrew Murphy and Michelle Flynn. This podcast is produced by Rebecca McTavish and 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, your on-the-go source for the most exciting discoveries in basic cardiovascular research.

This month on Episode 15 of the Discover CircRes podcast, host Cindy St. Hilaire highlights three featured articles from the July 31 and August 14 issues of Circulation Research. This episode features an in-depth conversation with Drs Venu Venna and Juneyoung Lee from the Department of Neurology at the McGovern Medical School at the University of Texas Health Science Center at Houston regarding their study Gut Microbiota-Derived Short-Chain Fatty Acids Promote Post-Stroke Recovery in Aged Mice. This episode also includes a brief discussion with BCVS Outstanding Early Career Investigator Award competition finalists, Drs Shyam Bansal from Ohio State University, Emmanouil Tampakakis from Johns Hopkins University, and Yang Zhou from the University of Alabama, Birmingham.

Article highlights:

Veys, et al. GLUT1 in Angiogenesis and BBB Integrity

Zhang, et al. Self-Renewal of Local Macrophages Attenuates DiCM

Lerchenmüller, et al. CITED4 in Cardiac Remodeling

Dr Cindy St. Hilaire: Hi. 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 share with you three articles selected from the late July and early August issues of Circulation Research. I'm also excited to share with you my discussions with Drs Venugopal Venna and Juneyoung Lee, who are from the group of Louise McCullough at the University of Texas Health Science Center, regarding their study Gut Microbiota-Derived Short-Chain Fatty Acids Promote Post-Stroke Recovery in Aged Mice.

I also speak with the finalists of the BCVS Outstanding Early Career Investigator Award, Shyam Bansal from Ohio State University, Emmanouil Tampakakis from Johns Hopkins University, and Yang Zhou from the University of Alabama, Birmingham.

So first the highlights. The first article I'm sharing with you is titled Role of the GLUT1 Glucose Transporter in Postnatal CNS Angiogenesis and Blood-Brain Barrier Integrity. The first author is Koen Veys and the corresponding author is Katrin De Bock from ETH Zurich.

The primary energy source for the brain is glucose and the blood vessel endothelial cells which from the blood-brain barrier supplied glucose to the brain via the glucose transporter protein GLUT1. Patients with genetic mutations in GLUT1 have neurological problems, including seizures, movement disorders, and delayed neurological development. Low GLUT1 levels in the blood-brain barrier have also been linked to Alzheimer's disease in humans and have been known to exacerbate the disease in a mouse model.

In this study, the group examined the role of GLUT1 in blood-brain barrier endothelial cells in more detail. They found that while structural integrity of the blood-brain barrier remained intact, inhibiting the activity of GLUT1 in newborn mice impaired aspects of normal blood vessel growth in the brain, and inhibiting GLUT1 in adult mice led to progressive neuron loss, behavioral abnormalities, reduced movement, seizures, and signs of inflammation.

The results highlight GLUT1's importance in the brain endothelial cells, and the role of GLUT1 in glucose utilization in overall brain function.

The second article I want to share with you is titled Self-Maintenance of Cardiac Resident Reparative Macrophages Attenuates Doxorubicin-induced Cardiomyopathy Through the SR-A1-c-Myc Axis. The first authors are Hanwen Zhang, Andi Xu, Xuan Sun, and the corresponding author is Qi Chen and the work was completed at Nanjing Medical University in China.

Doxorubicin and it's analogues are commonly used chemotherapeutic agents. However, the use of these drugs is limited by dose-dependent cardiotoxicity. Doxorubicin-induced cardiomyopathy presents with dilated and poorly functioning left ventricle in the absence of abnormal loading conditions. This may induce cardiac systolic dysfunction.

Accumulating clinical evidence suggests that inflammation contributes to doxorubicin-induced cardiomyopathy pathogenesis. Several studies suggest that the inhibition of cardiac inflammation can improve cardiac function; however, the underlying mechanisms remain unclear.

This group wanted to explore the role of cardiac resident macrophages during doxorubicin-induced cardiomyopathy progression. They found that cardiac resident macrophages were vulnerable to doxorubicin insult but that monocyte-derived macrophages survived. Further, these surviving monocyte-derived macrophages exhibited a proinflammatory phenotype which contributed to impaired cardiac function.

Scavenger receptors are expressed on macrophages and help to modulate their inflammatory response. Global, or myeloid-specific deletion of class A1 scavenger receptor, also called SR-A1, inhibited proliferation of resident reparative macrophages and this inhibition exacerbated cardiomyopathy.

At the mechanistic level, this group identified that the transcription factor c-Myc mediated the effect of SR-A1 in reparative macrophage proliferation in doxorubicin-induced cardiomyopathy.

The last article I want to share with you before we switch to our interviews is titled CITED4 Protects Against Adverse Remodeling in Response to Physiological and Pathological Stress. The first author is Carolin Lerchenmüller and the corresponding author is Anthony Rosenzweig, and they're from Massachusetts General Hospital.

Exercise is good for the heart. It increases cardiac mass which is called physiological hypertrophy, which appears to induce cardiac benefits. However, pathological stimuli, such as hypertension and aortic stenosis, can lead to pathological hypertrophy which is associated with adverse outcomes and can lead to heart failure. Cardiac CITED4 is a protein that is induced by exercise and is sufficient to cause physiological hypertrophy and mitigate adverse ventricular remodeling after ischemic injury. However, the role of endogenous CITED4 in response to physiological or pathological stress is unknown. To understand the role of endogenous cardiomyocyte CITED4, this groups generated cardiomyocyte specific knockouts of CITED4.

These mice were analyzed at baseline. They were subjected to a swimming protocol which provided physiological stimuli or they underwent transverse aortic constriction, also called TAC, which causes pressure overload and served as the pathological stimulus for heart remodeling.

CITED4 knockout mice developed modest cardiac dysfunction and dilation in response to exercise. After TAC, these knockouts developed severe heart failure with left ventricular dilation and impaired cardiomyocyte growth.

The study goes on to show that CITED4 protects against pathological cardiac remodeling by regulating mTOR activity and also a network of microRNAs which control cardiomyocyte to fibroblast crosstalk.

So for our interview of this episode, I have with me Drs Venugopal Venna and Juneyoung Lee from the Department of Neurology at the McGovern Medical School at the University of Texas Health Science Center at Houston.

Today we're going to be discussing their manuscript titled Gut Microbiota-Derived Short-Chain Fatty Acids Promote Post-Stroke Recovery in Aged Mice. Thank you both very much for joining me today.

Dr Venu Venna: Thank you Cindy for having us. It's a pleasure.

Dr Juneyoung Lee: Yeah, thank you for the opportunity.

Dr Cindy St. Hilaire: It's a wonderful paper. Actually I really enjoyed the nice graphical abstract, that really made a good visual of what this papers about, so I encourage everyone to go take a peek at that. Could you introduce yourselves and tell us a little bit about your lab group?

Dr Venu Venna: Yeah, sure, I'm Venu Venna, it's my third year at McGovern Medical School UT Health, and we are a part of a large research group in the Department of Neurology here. This is headed by Dr Louise McCullough. She's also a co-corresponding author on this paper.

Unfortunately she's not here today, but it's basically her idea and her initiative that led us to drive this huge project. We are very excited to share with you more details today.

Dr Juneyoung Lee: Hi, my name is Juneyoung Lee. I'm postdoctoral fellow here and I'm working with Dr McCullough and Dr Venna.

Dr Cindy St. Hilaire: So this manuscript is testing the general hypothesis that the gut microbiome can influence stroke recovery but before we dig into the details of your study, can you give us a little bit of background about what the microbiota gut brain axis is?

Dr Venu Venna: That's a great question. Thank you for asking that. So recent advances in 16S sequencing, metagenomics, and metabolic analysis lead us to specifically identify the role of gut microbiota. We have... Everybody consists of large number of microbiota in the gut, so particularly the microbiota's role is largely remains unknown, as of now.

The recent advances helped us to understand whether it's for communication of the microbiome, how it actually influences our health, and how the metabolites that are released by the microbiota can actually influence the brain-gut.

So this is where the concept of microbiota gut-brain axis continues to evolve and we rely on 16S metagenomics, as well as metabolomics to understand if the microbiome itself has a specific role in the stroke recovery and stroke in this paper.

Dr Cindy St. Hilaire: Great, so I know that previous research by you specifically, and also you mentioned your fellow corresponding author, Dr Louise McCullough, your prior work has shown that stroke can cause aberrant changes in the gut regarding things like motility, permeability, activation of gut-immune cells. So this to me suggested that aberrant signaling can come from the brain and affect the gut, but your study is kind of now flipping that.

You want to ask the question is changing the gut microbiome after the stroke also beneficial? So there's kind of a chicken and egg type conundrum going on. Is there a preceding event, is it the stroke that alters the gut microbiome primarily, or is the gut microbiome maybe deficient in different people and therefore their stroke outcomes are different?

Dr Venu Venna: Yeah, I mean this is a very new emerging field and what's very interesting about this is the brain gut microbiota axis, it's a bi-directional axis. In this case, what we think is if we have a stroke, it may actually directly influence the gut.

There is a brain-gut axis. At the same time, the changes in the microbiome can actually trigger an inflammatory state where it can actually contribute to the worst stroke outcomes. It's a chicken and egg relationship as you rightly mentioned, but at the same time what is not known is whether if we can simply manipulate the microbiota, can you actually improve the stroke outcomes or can you improve the age associated outcomes?

Because what we found in previous studies is age itself causes changes in the microbiome.

Dr Cindy St. Hilaire: Interesting, so just being young or old, if you were to compare those microbiomes of old individuals and young individuals, you see differences that are I guess negatively impactful on things like stroke and disease?

Dr Venu Venna: That's exactly right, so the more imaging data coming out from the literature, not just our group, but all other groups, on humans and animal studies, do suggest that age itself is associated with changes in the gut microbiome.

Dr Cindy St. Hilaire: So the overall goal of this specific study was to determine if replacing the gut microbiota of an older mouse with the microbiota from a younger mouse would help in the recovery after an ischemic stroke. Can you talk about the design of that study and the different aspects that you had to consider when designing these experiments?

Dr Venu Venna: Yeah, absolutely. This was a great question. The initial experiments, like what we were trying to do before, was whether we can actually even manipulate the microbiome in an aged animal. In our previous study, what we did is we took a young animal and we transplanted the biome from an aged animal. We used a combination of antibiotics to actually deplete the existing biome and that's what gave us susceptibility to transplant.

Once you transplant the biome into a donor from a host, so the biome can actually sustain for quite a bit of time. This gave us an opportunity to study the direct role of microbiome. Later, what we did was we subjected these animals to the stroke and then what we found is when we induced this stroke in an animal that received aged biome, despite being young, the animal that received aged biome, can itself contribute to the worst stroke outcomes and increased mortality.

In this follow-up study, what we decided to do was can we even manipulate the microbiome after stroke? So this is particularly important because most of the clinical patients don't come into medical attention until after stroke. Transplanting the microbiome or even manipulating the microbiome after stroke can have a broader clinical relevance.

In this particular study we decided to see if we can actually manipulate the microbiome after several days or several hours after the stroke happens. We decided to test if we can wait for three days. This is a particular time where we can actually see the infarcts get mature and all the injury in all groups of animals are same, and then we transplanted the aged animals with the young microbiome.

So this gives us an opportunity to actually study the role of microbiome, independent of infarct. Meaning, all animals have a similar degree of injury, so now whatever the beneficial affects you are seeing because of the microbiome transplant, are potentially due to, not because of the size of the injury, because they have a smaller injury they have better recovery, but it's basically because their infarcts are the same and whatever you're seeing is because of the manipulation of the microbiome.

Dr Cindy St. Hilaire: Interesting. Juneyoung, would you like to tell me a little bit about what you found then? Using these interesting fecal transplant models, what are the key results that you found in this study?

Dr Juneyoung Lee: Great question. As Dr Venna explained, we treated young biome to aged stroke mice, after stroke. We found that young biome contributes to better behavior outcomes and they regulate the immune system in the brain and the gut and increase the short term brain-gut axis in the aged stroke recipient mice.

One interesting finding is that we found dominant T-cells, which are very small number of T-cells in the host, but they secrete proinflammatory cytokines which is IL-17. Cytokines exacerbate new inflammation in the brain so if we treat the young biome, we found that the level of proinflammatory cytokine IL-17 decrease cytokines compared to aged biome.

Dr Cindy St. Hilaire: You also focused on short-chain fatty acids, SCFA producing bacteria. What is it about these short-chain fatty acids that are beneficial and what are the signaling pathways that you found to be activated or things that were present that helped to promote better stroke recovery?

Dr Juneyoung Lee: Short-chain fatty acids are key metabolites produced by bacterial fermentation of dietary fiber in the gut. These are suspected to play an important role in microbiota gut-brain crosstalk. Also, in our previous study we found that young fecal biome has higher levels of short-chain fatty acid compared to aged biome so we think that the short-chain fatty acid has a beneficial role in our mild level stroke.

Dr Cindy St. Hilaire: So are you focusing more on identifying the metabolites or trying to move into humans? What do you think the next step of this vein of research is?

Dr Venu Venna: So what we think is right now, this is a very interesting and fascinating finding, even for us. We're trying to understanding what other metabolites could be involved and what other ways as you previously asked, what other pathways these bacteria itself are triggering or contributing to actually enhance this recovery, that's what we are seeing from the young microbiome.

As a future direction, we are also seeing if this transplant of biome can have a broader therapeutic relevance, meaning is it only specific to the stroke related outcomes or can it be beneficial in large settings of other age relate diseases like what we are seeing, again as I mentioned before like age related diseases such as... Many age related diseases like cognitive dementia, or Parkinson's disease, any neurodegenerative disease.

Dr Cindy St. Hilaire: Well thank you, Drs Venu Venna and Juneyoung Lee for joining me today. I really appreciate it and congratulations again on this wonderful story.

Dr Venu Venna: Thank you very much for having us, Cindy, and for this work I would like to acknowledge the funding agency. This work is funded by NIH and also the American Heart Association, both for my Scientist Development Grant and also as well as for Juneyoung Lee's postdoctoral fellowship. This funding helped us to perform these highly innovative studies in gut microbiome axis.

Dr Cindy St. Hilaire: Wonderful. Yes, well, we love seeing AHA funded research published in AHA journals, so thank you.

Right so now we're going to have our interview with the BCVS Outstanding Early Career Investigator Award competition finalists. I have with me today, Shyam Bansal from Ohio State University, Emmanouil Tampakakis from Johns Hopkins University, and Yang Zhou from the University of Alabama, Birmingham.

So congratulations to all of you for being recognized for your outstanding science. These topics are great. The timing of T-cells activity in chronic heart failure, sympathetic neuron signaling, circadian genes and cardiomyocyte proliferation, and the identification of a transcription factor that helps promote maturation of reprogrammed cardiomyocytes.

So, Dr Bansal, your abstract that's recognized, is titled Novel Inhibitors For Temporal Modulation Of T-lymphocytes During Chronic Heart Failure. Where was this study conducted and where are you now?

Dr Shyam Bansal: Right now I'm at Ohio State University. I joined here in July 2019 and I've been setting up my lab. While doing that, we conducted all this work. This work, most of it is done here, and we have been looking to identify certain inhibitors that can be used for T-cell modulation.

Dr Cindy St. Hilaire: Excellent so why should I care about T-cells in the heart? And what did you all find in this paper?

Dr Shyam Bansal: The right question is why shouldn't you? T-cells are coming out to be involved in almost every chronic disease. We have heard about CAR T-cell therapy. Recently it revolutionized the whole cancer research field. The heart failure and cardiovascular diseases has also been realizing the importance of T-cells. They're important in a way because they are kind of a two-edged blade. They are protective because we need them to initiate those wound healing cascades so the tissue can regain its original function. But then, too much activation of T-cells can be injurious and lead to autoimmune reactions.

In 2017 I published a paper during my postdoc with Dr Sumanth Prabhu at UAB where we showed that these T-cells get activated during chronic heart failure.

It's a double-sided activation to get activated immediately after injury but then they go down and they come back again, during chronic heart failure. That's where the two-edged blade comes into picture. If you alter these T-cells during this acute phase, during the cardiac infarction, the animals always do worse, right? They are protective because they are needed for wound healing pathways.

Dr Cindy St. Hilaire: We can't just stop them at the start, we need to fine tune.

Dr Shyam Bansal: Yes.

Dr Cindy St. Hilaire: So what's this temporal aspect you looked at?

Dr Shyam Bansal: So that's what we found in my postdoc in 2017 paper. If you inhibit these during the chronic phase, in mouse, in rodents, it was whole weeks after infarction, then you can actually stop maladaptive remodeling. You can complete shut it down, it doesn't get better but you at least shut it down completely.

We did those studies by using some antibodies, again CD4+ T-cells, and using genetic mouse models.

Dr Cindy St. Hilaire: So do you think anything that you found can quickly or soon translate to humans?

Dr Shyam Bansal: That's exactly what we did after we came here, right? So we compared what happens during this chronic heart failure, what happens to these T-cells. We identified one molecular pathway that's associated with receptor signaling, being activated in these T-cells. The interesting thing is, these T-cells came from male mice, not from females. Still, they had strong activation of the surge in receptor signaling.

We found a drug molecule that can activate another pathway that inhibits this pathway, so indirectly we're able to inhibit this pathway. We did those studies and found that we can actually stop T-cells from getting activated during chronic heart failure and when we do that, this drug can actually, again, inhibit left ventricular remodeling significantly.

Dr Cindy St. Hilaire: Wow.

Dr Shyam Bansal: And if you give this drug early in myocardial infarction, again, animals died.

Dr Cindy St. Hilaire: It's going to be very important to fine tune when that drug could potentially be administered to humans.

Dr Shyam Bansal: Yes, and that's the first drug in our knowledge that can actually target specific antigen activated T-cells.

Dr Cindy St. Hilaire: Super exciting, well congratulations again. Well done and well earned. Dr Tampakakis, your study is titled Sympathetic Innervation Negatively Regulates Postnatal Cardiomyocyte Proliferation Through Circadian Genes. So where was this conducted and what position are you in now?

Dr Emmanouil Tampakakis: This research was conducted at Johns Hopkins University and I'm currently part of the... I'm Assistant Professor within the Division of Cardiology in the School of Medicine. Pretty much for my curiosity and the fact that we know a lot about the role of neurons for adult heart disease but we really don't know what much about neurons are doing at the neonatal stages in heart development.

We know at least in preterm babies where the innervation is really affected, some of them do develop changes in their heart geometry, and there might be a role there, plus there is some data to suggest that the autonomic nervous system does manipulate or does affect the neonatal heart regeneration.

The role of neurons to me was really intriguing.

Dr Cindy St. Hilaire: So this is linking together sympathetic nervous signaling, circadian genes, and postnatal cardiomyocyte proliferation. Why do I care about all these things fitting together?

Dr Emmanouil Tampakakis: Yes, so apart from the fact that it's fascinating knowing that each individual organ has in their body its own circadian genes that regulate actually, several functions. Without being affected by the central nervous system and what's happening in the hypothalamus, which to me is really fascinating, is we really don't know much about what actually regulates and synchronizes the circadian cycle of the heart.

We, in this study, showing that actually the innervation that happens in neonatal stages, already aaffects how certain genes are circulating within the heart. That appears to be through one of the adrenergic pseudoephedrine way that the sympathetic nerves are secreted.

Again, by affecting this, we are showing that there is more proliferation of neonatal cardiomyocytes which can be important for disease at later time points, and we're also showing that if you mess up two specific circadian genes, Period 1 and Period 2, that are transcription regulators, and are some of the masterminds of this phenomenon, you can actually still affect neonatal cardiomyocyte proliferation which can be important for diseases like heart degeneration and whether we're thinking about manipulating other pathways to induce more regeneration and induce healing in the heart.

The novelty of our work is we see that there's a link between that cell cycle and the circadian genes, at least at neonatal time points when myocytes proliferate a little more.

Dr Cindy St. Hilaire: So neat. Congratulations again, it's a wonderful story and I'm really happy it's being recognized. And Dr Zhou, you're being recognized for your work that's titled TBX20 Activates Cardiac Maturation Gene Programs Promoting Direct Human Cardiac Reprogramming. So where was this study started and where are you now?

Dr Yang Zhou: So I started as an Assistant Professor of Biomedical Engineering at UAB in January 2019. Before I moved to Birmingham, I did my postdoc training at the University of North Carolina at Chapel Hill in Dr Li Qian's lab. I basically studied direct cardiac programming which directly convert non-myocyte cell type to the functional cardiomyocytes.

I did a lot of work and found the epigenetic barriers and I find that the features of these direct programming cells and almost in the mouse cells, but we know that we have to move that to the human cells, so then when I moved to Birmingham and then I studied the cardio programming from the cells.

Dr Cindy St. Hilaire: Excellent, so your study is looking for ways to really kind of push the direct conversion of cardiomyocytes into a more fully differentiated state. Why is that an important question and what did you find in this study?

Dr Yang Zhou: Yeah, it is still challenging to gather a functional beating cardiomyocytes from human fibroblast by the direct reprogramming method. We want to get the functional cardiomyocytes to do the cell therapy. Also this method has promise to do the in situ heart regeneration because we use the transcription factors we can inject these factors to the injured heart then directly convert those cardiac fibroblasts into the cardiomyocytes.

So we have to study, we have to know how to get the functional work that cardiomyocytes.

Dr Cindy St. Hilaire: That is so neat. So really you're hoping to harness those fibroblasts in the heart that everyone kind of ignores because they're not contractile and you're hoping to really take them and transition them to these fully functioning beating cardiomyocytes.

Dr Yang Zhou: Right, so we know that the stuff we are coding are very important for the contractility, the myocyte contractility, so we find that a lot of missing protein expression in the current direct programming cells, so my hypothesis is they might be missing key regulators and can promote expression of those coding in the genes.

My computational analysis of the transcription data, I find that this T-box, transcription factor Tbx20, that can highly promote those unexpressed genes in the reprogrammed human cardiomyocyte.

Dr Cindy St. Hilaire: That's wonderful. Well congratulations again on some excellent work. So I want to ask you all, early career question, you're all within I think the first couple years of starting up your lab, and we're in the midst of a pandemic which means none of us are in the labs. Maybe staff is at reduced numbers, but first, how's it going?

And second is, you're kind of still fresh in terms of transitioning. So I'm wondering if there's any one piece of advice that you'd like to share with maybe someone who's in the middle of transitioning or just about to.

Or if there's something you wish you knew ahead of time that you'd love to tell your pre-faculty self?

Dr Emmanouil Tampakakis: Yeah, I don't know if I can give advice, already I think I'm too junior to do this. I would say that-

Dr Cindy St. Hilaire: What, too traumatized?

Dr Emmanouil Tampakakis: Maybe. Probably, or will be traumatized, but I would say for me at least, the things that kept me sane during this is my son, who's three and a half years old and lives in a complete different world, so that helps me balance what's happening out there.

Dr Cindy St. Hilaire: That's so important.

Dr Emmanouil Tampakakis: Probably some good alcohol at the end of the day but both those two things combined actually helped me maintain my sanity. In terms of advice, I would say to try to enjoy science. Try to stay focused and productive and at the end of the day, enjoy what you do. I think that if you are creative and if you like what you do, find the right people to collaborate and work with and you can hope that you will do well.

Dr Cindy St. Hilaire: I agree. Excellent advice.

Dr Shyam Bansal: I also have two kids, three years and eight years old, so we were at home for two and a half months or so. I think those kids were really helpful in keeping my sanity because the weather was getting better so we had to put in a swing set for them, get some play items and stuff, so they kept me busy. That was good that way.

The advice that I will have for junior investigators is be collaborative. Try to see how you can help others because when you help others, others are ready to help you as well. Remember science is a collaborative field, the more you collaborate with people, the more you get to know many more stuff.

Dr Cindy St. Hilaire: I think that's so important.

Dr Shyam Bansal: I was able to get done a lot of whatever work we presented. I was able to set up my lab, get some work done, and be at a position that I was able to summit my first abstract to BCVS for my independent own work, just because I had good collaborations here. I had good people who helped me stand on my feet and obviously I was working and helping them also.

Dr Cindy St. Hilaire: Yeah, and it also sounds like you have good colleagues so that's another key to it.

Dr Shyam Bansal: Yeah, I'm really lucky that way. Our whole department is really great. We have several senior faculty who are always ready to help us out in whatever issues we have, personal, professional, scientific, they're always here for us.

Dr Cindy St. Hilaire: That's great. Yang, how about you?

Dr Yang Zhou: I think the pandemic is very challenging for our junior faculty and for research and career developments but we have to balance between the work and the family, like kids. You might have an issue because we have two PIs in our lab.

Dr Cindy St. Hilaire: Oh gosh.

Dr Yang Zhou: Yeah, but I learned a lot these two years before this position. I think the most important thing I feel like is you have to talk to people. You always can find people that can help you to find answers. You need a mentor because they are more senior, they have more experience, even in this pandemic, if you find someone to share even just your feelings, that's very helpful.

Dr Cindy St. Hilaire: We're almost lucky that this happened now where we can have platforms like Zoom, and Adobe Connect, where we can have these virtual conferences because at least on all the different committees I'm on, ATVB and BCVS, we can have these discussions and break-out sessions, so I think it's really... We're lucky it's happening now and not 1997 when there's no video.

Dr Yang Zhou: Concerns... We all feel that the University and the Department, they all responded very quickly and they have much more support here than before.

Dr Emmanouil Tampakakis: As a more senior, what advice can you give us as a more senior person?

Dr Cindy St. Hilaire: Oh gosh, more senior? Well thank you. My advice? I definitely agree on the collaboration, I think that's key. Finding sponsors is equally important, someone who's going to go to bat for you. Finding a safety net where you can send someone a half-baked game page and have them tell you just how bad it is and be honest and be willing to give you that kind of critical feedback is really important.

Building your network is key, and getting involved in societies and getting people to know you independently from your former mentor, I think is really critical. Yes, you want to collaborate but you also got to make sure that you have your own path in the sand, to make sure you can move forward independently, and have fun while you're doing it, like you said.

Great, well I wish you all the best of luck. Congratulations again on being recognized and I'll see you on the BCVS webinar.

That's it for our highlights from the late July and early August issues from Circulation Research. Thank you for listening. Please check out the Circulation Research Facebook page and follow us on Twitter and on Instagram with the handle @CircRes and #discoverCircRes.

Thank you to our guests, Drs Venu Venna and Juneyoung Lee, and to the BCVS Outstanding Early Career Investigator Finalists, Shyam Bansal, Emmanouil Tampakakis, and Yang Zhou. This podcast is produced by Rebecca McTavish and Ishara Ratnayake, edited by Melissa Stoner, and supported by the editorial team at 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 month on Episode 14 of the Discover CircRes podcast, host Cindy St. Hilaire highlights four featured articles from the July 3 and July 17 issues of Circulation Research. This episode also features an in-depth conversation with Dr. Brenda Ogle and Drs. Molly Kupfer and Wei-Han Lin regarding their study, In Situ Expansion, Differentiation and Electromechanical Coupling of Human Cardiac Muscle in a 3D Bioprinted, Chambered Organoid.

Article highlights:

Wei, et al. Palmitoylation Cycling and Endothelial Maturity

van Ouwerkerk, et al. Functional Variant Elements in Atrial Fibrillation Models

Ibarrola, et al. Aldosterone in MVP

Sharma, et al. Atherosclerosis Regression Requires Regulatory T Cells

Cynthia St. Hilaire: Hi, 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 share with you four articles selected from our July issues of Circulation Research, as well as have a discussion with Dr. Brenda Ogle and the first authors, Molly Kupfer and Wei-Han Lin, regarding their study, In Situ Expansion, Differentiation and Electromechanical Coupling of Human Cardiac Muscle in a 3D Bioprinted, Chambered Organoid. So first, the highlights.

The first article I want to share with you is titled, "Endothelial Palmitoylation Cycling Coordinates Vessel Remodeling in Peripheral Artery Disease." The first author is Xiaochao Wei, and the corresponding author is Clay Semenkovich from Washington University, St. Louis. Peripheral artery disease, or PAD for short, is a vascular occlusive disease of the lower extremities. It affects more than 2 million individuals globally, and its prevalence is ever increasing as our population ages. While statin therapy can be useful for combating coronary artery disease in peripheral artery disease patients, it does not prevent or reduce PAD patients' rates of lower extremity amputation.

So looking to gain insights into the mechanisms underlying PAD, this team focused on the findings that circulating fibronectin and the dietary saturated fatty acid, palmitate, are associated with peripheral artery disease. They found this interesting as lipid modification proteins has been implicated in infections, premature aging, cancer and diabetes. One such protein modification is palmitoylation, which is the formation of a thioester bond between palmitate sand cysteine. Acyl-protein thioesterase 1, or APT1, is a depalmitoylase enzyme, which removes the fatty acid palmitate from protein.

Using mouse models with inactivated endothelial APT1, as well as cell systems in arterial samples from humans with end stage peripheral artery disease, they tested whether deficiencies in palmitoylation cycling promotes endothelial instability, which is a hallmark of chronic arterial occlusive diseases. They discovered that as many as 10% of all proteins are palmitoylated. They found deficiency of APT1 in endothelial cells disrupts vascular homeostasis, in part by altering the intracellular trafficking of the small GTPase R-Ras. Impaired R-Ras membrane trafficking was rescued by modifying the palmitoylated R-Ras molecule to promote dissociation from membranes. These observations identify palmitoylation cycling as a potential therapeutic target in the treatment of peripheral vascular disease.

The second article I want to highlight is titled, "Identification of Functional Variant Enhancers Associated with Atrial Fibrillation." The first author is Antoinette van Ouwerkerk, and the corresponding authors are Antoine de Vries and Vincent Christoffels, And they're from UMC Amsterdam. As we heard in our podcast last month with our interview with Dr. David McManus, atrial fibrillation, or AFib, is the most common form of arrhythmia, and is a major risk for heart failure, dementia, and stroke, and sudden death. Genome-wide association studies have revealed more than a hundred genetic loci linked to this condition, and many of these loci are found in non-coding regions, which are enriched for transcription factor binding sites and epigenetic modification sites, suggesting that these loci could potentially have gene regulatory roles.

To test this idea, they use the method called self-transcribing active regulatory region sequencing, or STARR-seq, which is a method used to identify the sequences that act as transcriptional enhancers in a direct quantitative and genome-wide manner. They use STARR-seq to screen 12 of the strongest AFib linked regions of the genome, which contain more than 1600 individual aphid linked genetic variance, and they did this in cultured rat atrial monocytes. From this screen, they found approximately 400 regulatory elements, of which 24 exhibited variant-specific differences in regulatory activity. For one of these elements, upstream of the gene HCN4, deletion of the orthologous element in mice caused diminished transcriptional activity of the gene. Moreover, these variant-containing mice had brachycardia and sinus node dysfunction, both components of arrhythmia. This proof of principle study confirms that such a regulatory element screen could provide insight into the consequences of variants associated with AFib, or for that matter, many other diseases.

The next article I want to share with you is titled, "A New Role for the Aldosterone/Mineralocorticoid Receptor Pathway in the Development of Mitral Valve Prolapse." The first author is Jaime Ibarrola, and the corresponding author is Natalia López-Andrés, and their work was completed at Sanitaria de Navarra in Pamplona, Spain. Mitral valve prolapse is a condition where blood leaks back into the left atrium of the heart, and it is the most common form of heart valve defects. The underlying pathology includes an overabundance of cells in the valve leaflet, so-called valve interstitial cells, or VICs. These activated VICs overproduce extracellular matrix protein, and the combination of increased numbers of VICs and increased amounts of extracellular matrix proteins contributes to the impairment of the structural integrity of the valve leaflet. The increase in VICs is due to excess proliferation, but also transformation of valve endothelial cells, so the cells that line the leaflet, valve endothelial cells, into mesenchymal like VICs.

As a driver of endothelial to mesenchymal transition, aldosterone was suspected to play a role. Aldoesterone increased expression of VIC activation markers in cultured valve endothelial cells and increased production of certain extracellular matrix protein components. Spironolactone, an aldosterone inhibitor, prevented these effects, and importantly, prevented valve remodeling in a mouse model of mitral valve prolapse. The team showed that valve tissue from mitral valve prolapse patients taking aldosterone receptor inhibitors displayed less evidence of VIC activation and lower production of disease-regulated extracellular matrix components, than those not taking the drugs. These exciting results suggest aldosterone antagonists, already used for certain patients with heart failure or high blood pressure, may also benefit those with mitral valve prolapse.

The last article I want to share before we switch to our interview, is titled, " Regulatory T Cells License Macrophage Pro-Resolving Functions During Atherosclerosis Regression." The first author is Monika Sharma, and the corresponding author is Kathryn Moore, and they're from New York University. Atherosclerosis is a chronic inflammatory condition characterized by the buildup of fatty deposits in the artery walls, and monocytes and macrophages can infiltrate into these fatty deposits and contribute to the formation of plaque. Cholesterol-lowering drugs, like statins, promote the reduction of low-density lipoproteins in the blood, which can help to slow plaque growth, but they do not reverse disease progression.

One possibility for changing the course of the disease is to develop therapies that can reduce plaque inflammation, and therefore, progression. With that goal in mind, this team investigated how the immunosuppressive activity of regulatory T cells, or Tregs, may influence the functions of plaque monocytes and macrophages. Using mouse models in which the disease can be reversed through aggressive lipid lowering, they found that depletion of the Treg population caused an increase in the numbers of monocytes and macrophages in the plaques, and resulted in poorer plaque regression. Indeed, these monocytes and macrophages proliferated more, remained in the plaques longer, and were less likely to adopt an anti-inflammatory pro-plaque resolving M2-like phenotype than plaque macrophages in mice with normal Treg numbers. Together, these results highlight the importance of Tregs for promoting plaque regression, and suggest future therapies aimed at boosting these cells, or indeed, M2 macrophages may enable atherosclerosis remission.

Okay, so now we're going to switch over to the interview portion of our podcast. I have with me Dr. Brenda Ogle, who is a professor of biomedical engineering, and first authors Molly Kupfer and Wei-Han Lin, and they're from the University of Minnesota. And today we're going to be discussing their manuscript titled, "In Situ Expansion, Differentiation and Electromechanical Coupling of Human Cardiac Muscle in a 3D Bioprinted, Chambered Organoid." So thank you all for joining me today.

Brenda Ogle: Thank you.

Molly Kupfer: Thanks for having us.

Wei-Han Lin: Thank you.

Cynthia St. Hilaire: Great. I'm glad we can all do this remotely and nice and safe for COVID.

So Dr. Ogle, you're the PI of the group, but Molly and Wei-Han, what stages of career are you at?

Molly Kupfer: I just recently completed my PhD, so this work is sort of the culmination of that.

Cynthia St. Hilaire: Oh, congratulations!

Molly Kupfer: Yeah. Thank you.

Cynthia St. Hilaire: Well done. Circ Research is a great thesis publication. Congratulations.

Molly Kupfer: Thank you.

Cynthia St. Hilaire: Wei-Han, how about you?

Wei-Han Lin: So I'm a BME PhD student at the University of Minnesota. And I got my master degree in chemical engineering, but in Taiwan, and now I'm working with professor Brenda Ogle on cardiac tissue engineering stuff.

Cynthia St. Hilaire: Excellent. So this is a beautiful paper. It's stunning. It has all sorts of wonderful parts, biological, biomechanical, great imaging, and essentially you created a 3D bio-ink that can be used to print and make a living pump, kind of a heart in a dish. And it's something that you're calling this human chambered muscle pump, or ChaMP, which I think is a great name. Can you please describe exactly what that is and why did you want to go about trying to make it?

Molly Kupfer: Yeah, it might help if I give a little bit of context to this. So since the beginning, one of the central questions that the lab has been exploring is how do the cells of the heart interact with their environment, or the extracellular matrix, as we call it? We know that these interactions that occur at the cellular level are absolutely critical for cardiac function, both at the tissue and the organ level. And based on years of research studying how the extracellular environment modulates cellular function, we have now sought to apply what we've learned in order to engineer functional human cardiac tissues by recapitulating those very critical interactions in vitro.

And actually, back in 2017, we published another study in Circulation Research, where we generated these contractile patches of cardiac tissue using a form of light-based 3D printing that allowed us to fabricate scaffolds with really high resolution micron-level features that were distributed in a way that mimics the native extracellular environment. And what we found is that by organizing the extracellular matrix in that way, we enabled the cells to organize themselves in the scaffold and form connections with each other and with the scaffold itself. And this was critical to achieving synchronous electromechanical function of the tissue as a whole. But these were very small millimeter scale tissues, and so for this new study, we sought to create something on a larger scale where you could incorporate some new geometric features such as chambers and the capacity for perfusion.

And as you mentioned, using our knowledge of the interactions between cells and the extracellular matrix, we developed this unique bio-ink that could be used as a vehicle to 3D print these centimeter scale chambered tissue structures that are based on the geometry of the human heart. And so the tissues that resulted from this, the human chambered muscle pumps, or hChaMPs, exhibit thick, contiguous muscularization. They demonstrate electrical connectivity and pump function. And notably, this is the first time that this level of function and muscularization has been achieved in an engineered cardiac tissue of this level of geometric complexity.

Cynthia St. Hilaire: So can you maybe talk a little bit about what do you mean by an ink, exactly? Is it actually printed? Is this like a printer that I could buy on Amazon? Obviously there's a huge biological component, but what are the actual technical things that you had to develop to make this chamber happen?

Molly Kupfer: Yes. So we did use an extrusion-based 3D printing, which is similar to probably what people normally think about with 3D printing. Traditionally, it's been with plastics. In this case, we're printing with a bio-ink, which is essentially a formulation of proteins and other materials that we encapsulate the cells in, and then after that, we extrude it from a nozzle in a specific formulation or shape in order to create the structure.

Cynthia St. Hilaire: So that's interesting. So in this mix, the cells are already in there as opposed to, I guess, some other things that people tend to call scaffolds where you kind of print that and then seed it?

Molly Kupfer: Mm-hmm (affirmative). And in the example of the paper I discussed from 2017, that was an example where we printed a scaffold and put the cells in. But in this case, for such a large and complex structure, we actually mix the cells in prior to printing, and then we create the structure.

Cynthia St. Hilaire: Wow. What's the timeframe of that? Like the cells, you got to digest them and mix things up and then print it. The cells, are they happy?

Molly Kupfer: Yeah, that's a good question. So the actual printing process is quite fast, maybe a couple of minutes for this particular scale. We have to prepare, culture, the cells in advance and we're working with human-induced, pluripotent stem cells, so it takes time to grow them up, and then yes, we do detach them and singularize them, and we then mix them with the components. But overall, the actual printing process is relatively quick. Then it's a matter of maintaining the structure and culturing it and doing the differentiation as we did. And that takes weeks to do over time. But the actual process of making it, initially, is quite quick.

Brenda Ogle: Challenging thing about this project was the fact that mature cardiac muscle does not transfer well. Meaning when you move it from a dish to an ink and then print it and ask it to start beating again, it doesn't typically happen. And that is because cardiomyocytes don't proliferate well, or make more of each other, and they also don't move well, or migrate. And so the premise on which most of this paper relies is on printing the stem cells first, letting them expand, sort of like they do with development, and then encouraging them to specify into cardiac cell types.

Cynthia St. Hilaire: What's the bigger good that can come out of this? Why do we want to be able to do this in vitro, or even ex vivo heart in a dish?

Brenda Ogle: The value is pretty tremendous because, suddenly we have a human model system in which we can perfuse volume, so volume can go in and come out, in which the cells experience those volume metric and fluid-induced forces that we haven't been able to study human cells in this way ever before. In the context of human disease, this is the first time we'll be able to look at onset of a particular disease, what was happening with onset, and then progression. And I think that is what is going to transform this field.

Cynthia St. Hilaire: So what was the first one like? I'm thinking back to my graduate school and also my postdoc where I was involved in some disease discovery and I have a very vivid memory of the Western blot that proved the mutation that we found. And I literally ran down the hall holding the film. I'm imagining, maybe I'm projecting too much, but what was seeing that first one beat like?

Molly Kupfer: You're not projecting. I feel like that well describes my experience. We had some early experiences where we would start to see beating areas under the microscope, but I think the moment, for me, was, I think there was one night I was working in the lab and I had some plates out, I was looking at stuff under the microscope going through just the mundane lab tasks, and I think I sort of saw it at the corner of my eye in the dish, something was moving. And that was the first time. Like I had watched parts of these things beat under a microscope all the time. I spent years looking at cardiomyocytes under a microscope, but that was the first time, for these hChaMPs, where I could actually see it moving just by my eye.

Cynthia St. Hilaire: Wow.

Molly Kupfer: And that was a really cool moment.

Wei-Han Lin: Yeah. I was mostly working on the printing side, so the first time I realized the heart started beating, it's more like a shock to me, because I'm always printing the models or just the mold. But then really seeing those cells, or the whole structure, start to beat, was quite amazing.

Cynthia St. Hilaire: Could you please tell me a bit about the 3D printing aspect of it? Is it like a shell like the outside of a balloon, or does it have an interior structure that helps dictates where the cell go? Can you explain what the printing is?

Wei-Han Lin: So the structure we are printing is derived from MRI image stacks on a real human heart. And the image stack was segmented and reduce the size by 10 times, and then we convert the stack into the STL file, which is the standard operating format. And then we modify the model a little bit to make it into two chambers and with two vessels, and two connected chambers with two openings. And this is the heart we are using for the study.

Cynthia St. Hilaire: Got it. So it's got kind of the big picture items of the heart. It's got two tubes going in and it's got two chambers and the fluid can flow between all of those aspects in a specific flow pattern.

Wei-Han Lin: Exactly.

Cynthia St. Hilaire: You said you have to differentiate them in a dish and you're adding different factors to do that. Do the cells like being in that scaffold, or do they want to seep out of that structure or is there something about the bio-ink that they're happy there?

Molly Kupfer: You know, I think this bio-ink was, to a certain extent, optimized or designed such that the cells would be able to continue to attach and grow and remodel. So basically, for the most part, these components are biological materials. Some of them are just proteins. Some of them are proteins that have been modified with photo cross-linkable elements, but they still have these moieties that the cells can attach to. And over time we do see some remodeling and some extracellular matrix gets degraded and some gets deposited.

Cynthia St. Hilaire: So have you gone to the next steps of something like single cell seq and trying to see what kind of cells you're getting in this? Or even maybe inputting different, the scaffold is getting one differentiation protocol, but are you possibly able to prime IPS cells such that they're maybe halfway to a vascular cell, or halfway to a cardiomyocyte cell, and then put them in the bio-ink?

Brenda Ogle: That's a really interesting idea. I'm going to take that one.

Cynthia St. Hilaire: Give me an acknowledgment.

Brenda Ogle: So we've been thinking about that, the context of if expansion of IPS cells is the best way, for many cell types, how do we get multiple cell types and organize them? And you can imagine even just printing in specific areas, different cell types.

Cynthia St. Hilaire: Oh, sure.

Brenda Ogle: But the other thing we've thought about is delivering differentiation factor spatially.

So almost printing a cell, but then printing that. depot of a factor, in the area that we wanted or in an arrangement that we want, and then releasing it when we want. And it's challenging for stem cell differentiation, because you really need no release, and then basically zero order release for two or three days, and then no release again.

Cynthia St. Hilaire: Right.

Brenda Ogle: So it's a challenging drug delivery problem, but we've been thinking a lot about it. Now priming the cells beforehand is another interesting approach.

Cynthia St. Hilaire: Well, that's wonderful. I just want to congratulate you all again.

Brenda Ogle: Thank you so much for having us.

Cynthia St. Hilaire: Yeah, thank you so much.

Wei-Han Lin: Thank you so much.

Cynthia St. Hilaire: That's it for our highlights from the July issues of Circulation Research. Thank you so much for listening. Please check out the Circulation Research Facebook page and follow us on Twitter and on Instagram with the handle @CircRes and #discovercircres. Thank you to our guests, Dr. Brenda Ogle, Dr. Molly Kupfer and Wei-Han Lin. This podcast is produced by Rebecca McTavish and Ishara Ratnayake, edited by Melissa Stoner and supported by the editorial team of Circulation Research. Some of the copy texts 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 month on Episode 13 of the Discover CircRes podcast, host Cindy St. Hilaire highlights three featured articles from the June 5 issue of Circulation Research and gives listeners an inside scoop on the cutting-edge ideas in the June 19th Compendium on Atrial Fibrillation. This episode also features an in-depth conversation with Dr David McManus on emerging technologies for identifying AFib.

Article highlights:

Zhang, et al. ACEI/ARB on COVID-19 in patients with hypertension

Sakamoto, et al. ERR Signaling and Cardiac Maturation

Xie, et al. CIRP Governs the Heart Rate Response to Stress

Cindy St. Hilaire: Hello and welcome to Discover CircRes, the podcast for 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 share with you three articles selected from the June 5th issue of Circulation Research as well as give you an overview of the Compendium on Atrial Fibrillation also coming out in June. We'll follow that by having a discussion with Dr David McManus regarding his review on the emerging technologies for identifying AFib in the general population. So first, the highlights.

The first article I'm sharing with you is titled Association of Inpatient Use of Angiotensin-Converting Enzyme Inhibitors and Angiotensin II Receptor Blockers with Mortality Among Patients with Hypertension Hospitalized with COVID-19. The first author is Peng Zhang and the corresponding author is Hongliang Li and they're from Wuhan University in Wuhan, China.

Patients with hypertension have increased risk of death from COVID-19. While the high blood pressure itself is likely to contribute to this, concerns have been raised that medications used to treat hypertension, specifically ACE inhibitors and Angiotensin II receptor blockers may worsen coronavirus infection. Research in animals showed that these drugs increased expression of ACE-2, the protein on lung epithelial cells that is used by the virus to gain entry into the host cell where it can then replicate.

However, other conflicting evidence has shown that these drugs might reduce lung injury in pneumonia patients, which is also a complication of COVID. To weigh up the benefits and risks of ACE inhibitors and Angiotensin II receptor blockers, Zhang and colleagues performed a retrospective analysis of 1,128 patients with COVID-19 and hypertension who are treated at nine hospitals in Hubei Province, China. Of the patients, 188 took the hypertensive medicine during their hospital stay and 940 did not. The ages, sexes and comorbidities of the two groups were very similar.

After 28 days of follow up, 99 of the patients had died, seven from the group taking the hypertensive medications, equivalent to 3.7% and 92 from the group that did not or 9.8%. The team concludes that treatment of hypertension patients with hypertension medications does not increase risk of COVID-19 mortality and may even reduce the threat. However, a much larger sample size would be necessary to fully confirm.

The second article I want to highlight is titled A Critical Role For Estrogen Related Receptor Signaling and Cardiac Maturation. The first authors are Tomoya Sakamoto and Timothy Matsuura and the corresponding author is Daniel Kelly from the University of Pennsylvania.

From fetal to postnatal development, the human heart goes through significant changes, including the expansion of mitochondrial numbers, a change in fuel utilization within the mitochondria and replacement of fetal contractile proteins for the adult ones. Further, there is increases in ion uptake and release. Transcription factor estrogen-related receptor was known to drive postnatal mitochondrial biogenesis and now this group has shown that it also drives these developmental changes.

They developed a genetic model to knock down expression of estrogen-related receptor in early postnatal mice. When the animals were five weeks old, they performed transcriptomic analysis. In mice lacking estrogen-related receptor, there was a reduction in expression of genes involved in ion channeling in handling, fatty acid oxidation, which is the major metabolic process in the adult heart, as well as adult versions of the contractile proteins. By contrast, expression of genes and coding field contractile proteins and factors, specifically those involved in glycolysis, was upregulated. In heart failure, cardiomyocytes can revert to fetal-like cells. The authors, therefore, suggest that boosting estrogen-related receptor might be a way to counteract such pathology as well as a way to induce and study cardiomyocyte maturation and cultured progenitor cells.

The next article I want to share with you is titled Cold-Inducible RNA-Binding Protein Prevents the Excessive Heart Rate Response to Stress by Targeting Phosphodiesterase. The first authors are Duanyang Xie and Li Geng and the corresponding author is Yi-Han Chen and they're from the China Ministry of Education.

During a fight-or-flight situation, also known as the acute stress response, the heart rate increases rapidly due to the effects of adrenergic signaling on the cells in the sinoatrial node, which is the heart's pacemaker. Within sinoatrial node cells, levels of the signaling factor, cyclic AMP, ramp up and this, in turn, increases the cell's calcium handling and contraction rate, but excessive or prolonged racing heartbeat can be damaging and it is unclear what keeps the system in check. This group has now shown that cold-induced RNA-binding protein or CIRP puts the brakes on the heart by regulating cyclic AMP levels.

The team showed that while baseline rates between wild-type and CIRP-deficient rats were the same, triggering the adrenergic signaling via treatment with isoproterenol caused CRP-deficient rat hearts to beat faster for longer than in the wild-type counterparts. Cardiac tissue from the CIRP-lacking rats showed higher than usual levels of cyclic AMP after isoproterenol treatment. This was due to lower than usual levels of phosphodiesterase, the enzyme that normally degrades cyclical AMP. The team went on to show that CIRP normally binds and stabilizes phosphodiesterase's messenger RNA and sharing a ready supply of the enzyme to restrain cyclic AMP signaling. As well as revealing this crucial control mechanism, the work highlights CIRP as a potential new target for future heart rate lowering medications.

The last thing I want to share with you before we switch to our interview is our Atrial Fibrillation Compendium. Atrial fibrillation, or AFib, is a major cause of morbidity and mortality globally. There have been significant advances in the detection, management and treatment of AFib over the past two decades. However, the burden of the disease continues to increase. This Compendium on AFib features articles on epigenetics and transcriptional networks underlying atrial fibrillation, inflammasomes and proteostasis, novel molecular mechanisms associated with atrial fibrillation, emerging technologies with the identification of atrial fibrillation, epidemiology of atrial fibrillation in the 21st century, how will genetics inform the critical care of AFib, how will machine learning inform the clinical care of AFib, population-based screening for AFib, the molecular basis of AFib pathophysiology and therapy, the genetics of AFib in 2020, GWAS genome sequencing, polygenetic risk and beyond, is there hope for animal models of AFib and ablating AFib in 30 minutes, new technologies for safer and more efficient pulmonary vein isolation.

Okay. So we're now going to switch over to the interview portion of the podcast. I have with me, Dr David McManus, who is a professor of medicine in the Division of Cardiology and the Department of Medicine at UMass Medical Center in Worcester, Mass and he's also a cardiac electrophysiologist. And today, we're going to be discussing his recent Review on the emerging technologies for identifying atrial fibrillation, also known AFib, So thank you so much for taking the time to speak with me today.

David McManus: My pleasure. Thanks for inviting me.

Cindy St. Hilaire: Yeah, so before we dig into the review and about the emerging technologies for AFib, can you maybe give me a brief explanation of what is AFib, who gets it and what's the spectrum of disease severity in the patients that do get AFib?

David McManus: Sure, so atrial fibrillation is the world's most common sustained heart rhythm problem. It is associated with a number of different health conditions in terms of risk factors. The biggest risk factor for getting this rhythm problem is age, so it's most common in people over the age of 40 and it increases ... in fact, it doubles in terms of the incidents with each decade of life. So as you get into your 80s and 90, it's really quite common.

The additional risk factors are kind of common things that you might imagine and a few things you might not, things like diabetes and high blood pressure that are also risk factors forgetting plaque in your heart arteries are also risk factors for AFib, but some other risk factors that are a little more controversial with respect to heart artery disease, things like alcohol consumption, even if it's red wine, which otherwise might seem to have some benefit, is actually a risk factor too for AFib. In fact, in the old days, some doctors used to refer to AFib as holiday heart because of its association with acute alcohol intake around the holidays.

So, the fact is that AFib is related to some health behaviors like drinking. It's also related to a condition called sleep apnea or sleep-disordered breathing. Weight is associated with getting AFib. So all these things combined with your genetics, your family history and your age to contribute to getting this disease, so those are the most common risk factors.

The question about why it is important to diagnose? It was a disease that people sort of treated like gray hair for a long time. Something that you might not want to have, but is not particularly impactful, but some really important studies, especially in the 70s and 80s, started to establish a link between atrial fibrillation and clot-based strokes and so that is a very strong relationship that exists between people who get AFib and a much higher risk of having a clot form in the heart and traveling to the brain.

Cindy St. Hilaire: Which is obviously very dangerous. So how often does AFib go undetected? Because I think that's kind of at the core of using this new technology. Once you get AFib, do you know you have it right away?

David McManus: So, that's exactly right. The fact is that we don't truly know, right? Because by definition it's undiagnosed. We don't really know how long your average person goes before they're diagnosed and I think it varies a lot, but it's important to know a couple of things about AFib to sort of talk around the perimeter of this answer and try to kind of hone in a bit.

So first off, some people who develop AFib don't feel it or they have very minimal symptoms or they have symptoms that come and go so quickly. By the time they get in for an evaluation, the arrhythmia is gone so it can be what's called paroxysmal in its early stages, which means it can come and go. The duration of that AFib can be minutes or hours or even in some cases, seconds, and therefore elude a diagnosis.

The other thing is symptoms from AFib, when they do exist, are not always the sensation of palpitation, a sensation of an irregularity. Some people just feel short of breath when they go up a flight of stairs and-

Cindy St. Hilaire: Which you can associate with age.

David McManus: ... they may attribute that symptom to being older. Yeah. Right. They may just think "I'm getting tired because I'm older," or "I'm out of shape." And so the simple answer is, I guess, I'd start with this statistic. A significant minority of patients with atrial fibrillation present with a stroke as their first clear manifestations, so they come in with stroke. The estimates on that vary considerably, but at least one in five patients who present with a stroke have a first diagnosis of AFib at the time of that stroke and about 5% of patients with AFib overall present with stroke as their first manifestation.

Those are two different statistics to kind of come at it different ways and that's what you're trying to prevent. You want to make the diagnosis of AFib before the stroke because we have a lot of really good treatments that can prevent stroke if you know you have it.

Cindy St. Hilaire: Right and so I think that gets to this idea of maybe screening patients of a certain age. I don't know what that age cutoff would be. But when you look at the guidelines right now, there is no clear guideline. The US Preventative Task Force says there's no good data to screen patients beforehand. I think the AHA and the ACC just don't have any guidelines regarding screening. But yet in Europe and Australia, they do perform opportunistic screening for AFib patients and this is in the clinic. This is now with ECGs. They screen for patients over 65. So based on this statistic you said that 20% of stroke patients had undetected AFib, why is there not a specific guideline? Where does that come from?

David McManus: I think you're spot on that there's a lot of controversy about this right now and that's good because the reason I think it's controversial is there's some emerging technologies and opportunities that didn't exist before. Because pretty clearly, before doing a conventional 12-lead EKG in the office, did not offer sufficient benefit over and above usual care to demonstrate to groups like the US Preventative Service Task Force, which issues the guidance around things like breast cancer screening and colon cancer screening, so they have to weigh costs, financial costs, harms from false-positive tests. And so, the reason there's controversy is because what we had previously was a pretty suboptimal situation for screening. We had intermittent tests, which were of significant costs and they were spaced so far apart and required coming in for clinical care that there was really no benefit to doing them over and above taking someone's pulse in the office.

But there's no question that there are groups. There's a group called the AF-SCREEN Group, for example, that is really challenging the research community and clinicians to revisit some of these assumptions about screening, given new technologies and how we might thoughtfully use them in a pharmacy, for example, or in a clinic or at home with commercial technologies to study that. Because, really, to make a recommendation that screening is clearly beneficial, you have to do some kind of a study or studies that show that not only can you diagnose more of the arrhythmia, but that by diagnosing it, you can do something about it and that that action, in this case, anticoagulation, leads to a reduction in stroke and without a significant increase in harm from that treatment and that's somewhat controversial because this is a disease…the technologies that we have now are creating new diseases, right?

So in a sense, we've created the new disease, undiagnosed AFib. And so, okay, we found it. Now, is it kind of like cancer where finding it earlier maybe has a different prognosis than finding it later in terms of risk? Some people think so. And in that case, you really have to prove that finding it early and treating that early form with the treatment that you have for the late form works as well and doesn't cause harm. So that's kind of where we are right now is there a number of really big studies going on that are hoping to help inform this more, which is pretty cool.

Cindy St. Hilaire: Yeah. And so on that note, my parents got new supplemental insurance. They're both retired and this new insurance came with a nurse that dropped by your house and just kind of did a wellness check. And that nurse came and checked my mom's pulse, asked her how she was feeling and checked it again and said, "You're going to your doctor. You're in AFib," and she had no symptoms of that. She's 69, she's very healthy, she's active. And essentially in the course of a couple of months, she went from what she thought was normal to not being able to take one step up one stair because her AFib got so bad and she obviously saw a cardiologist and they got her on blood thinners. And eventually, after two cardioversions, things stuck and it's now in control, but one of the things that we did was we immediately bought her an at-home KardiaMobile heart monitor. That is essentially what you're reviewing now and I'm wondering if you could maybe give us a little bit of information about this.

So, there's multiple devices out there. There's the KardiaMobile. There's also the Apple Watch and the Fitbit that are starting to get these kinds of technologies. And so for us, it was at least reassuring to see her heart rate was something and now it's getting more normal with the medicine. And now even after the cardioversion, it's been a couple of months, she checks it once a day to make sure it's still functioning as it should. So can you tell us a little bit about these and about what this might mean for the future of AFib and clinical management?

David McManus: Sure. So you are using an FDA-cleared technology. You've referenced the AliveCor KardiaMobile. It's in a recent survey that the Heart Rhythm Society did. One of the most common ones that's purchased or advised to patients to buy by their cardiologists and healthcare providers and I think it was cleared about 2015. And what it is is it's a credit card-sized device that gets you a 1-lead EKG and it records about 30 seconds. And then that data, it can be transmitted directly to your cardiologist.

Cindy St. Hilaire: She actually would bring her phone in and show them, "Look at my last week."

David McManus: Yeah, you can do it that way or, like many of my patients are, just constantly emailing them or putting them in their electronic health record, which speaks to a whole other ball of wax. But that is a very valuable tool for people who are at risk for AFib or know they have it and want to monitor themselves for things like recurrences, to check their rhythm, check their rate, so that's an FDA-cleared device. And it also provides an automated read so that she is able to see at the top of the EKG what the computer thinks her heart rhythm is and that is a really nice technology that's been fairly well studied in a variety of different settings, including people like your mom who have had a prior cardioversion, to look for recurrences. So that's one approach that exists, which is for intermittent monitoring. And that's also in essence, the same approach that one of the two tools that an Apple Watch has embedded in it.

So the Apple Watch 4 and 5 are also FDA-cleared for similar analysis. So the Apple Watch has a EKG on the bevel of the watch. The thing you turn to change the time, not really in an Apple Watch, but in an old-fashioned watch. And so you can kind of put your finger on it and again, create that same circuit in your body to record a single-lead EKG and that similarly can present a 30-second EKG strip, so it's very similar to what AliveCor's KardioMobile does. Just in the watch. But again, that's an EKG-based approach and those approaches are not the only technologies that are out there.

There are a number of other devices that have CE marking designation in Europe. Other devices that are starting to become available in the US and we go through some of the performance of those devices in our review. And generally speaking, the ECG approach, the advantage is it's one of the more accurate approaches to AFib detection, but it does require, at least right now, that you intermittently check yourself.

In contrast, there's a movement afoot, and Apple has a separate FDA clearance, to use the lens and video camera on the back of the watch, that was previously used to measure your heart rate, to analyze the skin color changes that happen when your blood is flowing in and out of your wrist, to your hand. It looks at that skin color change, records it and analyzes it for irregularity. So it's an essence, like someone checking your pulse and it's called pulse plethysmography and that recording is similar to what other groups are analyzing. For example, there's something called FibriCheck, which is an app that is FDA cleared for AFib detection. But again, you put your finger on the camera of your iPhone or Android and it analyzes the pulse.

Cindy St. Hilaire: So a patient has to actively say, "I'm going to check this right now," as opposed to a background assessment?

David McManus: Yeah, so just to be clear. The background assessment is the automated sort of pulse check that the Apple Watch is doing and then it can prompt you to perform your own EKG, so that's the difference between kind of an intermittent-check approach versus a more near-continuous ... it's not truly continuous, but it's a near-continuous approach

Cindy St. Hilaire: So are there any drawbacks to people more regularly performing these in their kitchen? As a clinician, what are the drawbacks?

David McManus: It really is exciting and I think there are a lot of good reasons to pursue this. As you know, I'm sort of an early adopter of this idea and so I definitely would side with you that I think there were a lot of good reasons to be using these technologies. I just want to highlight though that there are a couple of issues.

So when commercial technology companies build medical-grade tech intended to diagnose or detect rhythm problems, those rhythm problems come to the clinics in a very different way and from a different type of patient, so they're not necessarily contextualized the same way a workup would happen if you came into the doctor, they prescribed a medical-grade patch monitor or a traditional monitor or did another type of medical test on you. You're kind of on your own. And so when you get the diagnosis or you get the possible diagnosis, you have very little information, and it can be anxiety-provoking. In many cases, especially in younger people ... in fact, the majority of people who have Apple Watches are at really low risk from having a complication from AFib. So now, you're making an upstream diagnosis and you may not do anything about it other than introduce a word onto their chart. And so, yeah, I do think there's some things that warrant further study and evaluation about some of the unintended downstream consequences of making diagnoses earlier and worrying people about a condition.

David McManus: Now, some have argued that an early diagnosis, even if you wouldn't put someone on a blood thinner, as you call it or an anticoagulant ... Like for example, maybe you change your health behaviors, maybe you lose some weight or you get more active or you stopped drinking so much. So I happen to think that early diagnosis is a good thing, but I do think that we don't really have robust care-management systems across the country that can support people who are at home. It's really hard right now for your doctor to, on top of seeing 30 patients in the office, to find the time to respond to your new test that he didn't order or she didn't order.

Cindy St. Hilaire: I got this blip on my strips.

David McManus: Yeah, what do I do about this? Well, okay, now I got to see you, what tests are ordered, what's the process and I think there's a lot of opportunity for us to, especially in the COVID era, redesign how we're delivering heart care and integrating these technologies become a facilitator as opposed to a burden, so I think there's a lot of interest in incorporating them.

But right now, at least, they're kind of separate from your chart, in the electronic chart and your doctor has to sort of find a way of reviewing on your smartphone, in the office, finding extra time to do that. They're not really paid to do that, how do you protect the safety of that information and et cetera, et cetera. So there are all these sort of little, but they seem little, but they're actually kind of important downstream implications. So we talk a little bit about, and this is kind of a unique part of this Review, the clinical actionability of device-detected AFib. There's no debating the fact that AFib is bad, but the real impact of device-detected AFib remained something that we really need to define and so there's a lot of interesting work going on in this area.

Cindy St. Hilaire: And so, because we're still all at home because of the COVID epidemic, there's been some things in the news regarding some of these wearable technologies being able to detect or track trends in swaths of patient health. Where do you see this going in terms of either things like COVID and epidemics or even things regarding AFib and we always see those maps from the AHA with hotspots of diabetes, things like that, so how do you think that this kind of technology can help transition the future of medical care, specifically in the US?

David McManus: I think it's really exciting because everybody has a smartphone and that crosses age, sex, race, occupation, religion and I think people are increasingly understanding the connections between health behaviors and their heart health. And I think, for example, just using your heat map example, that there's a stroke belt and a diabetes belt and different areas in the United States, also tremendous rural health disparities, that mobile devices have a really remarkable opportunity to help us understand what is going on, what is driving these sort of risks? Is it stress, is it alcohol, food consumption, nutrition, activity, sleep, all the things we talked about?

And whether it be AFib or other cardiovascular conditions, these wearable devices and mobile devices and digital technologies allow for quantifying different health behaviors and mood and opinion and activity in ways that our regular in-person exams that we do, when we see you for an hour or two every 365 days, we just really don't quantify. And I look forward to a time in the near future where your vital signs that are presented for your visit with your doctor over the internet is your activity, your blood pressure from your watch or heart rate from your watch or EKG, your oxygen levels at any of a myriad of other things that these devices can impact or will in the near future.

Cindy St. Hilaire: Yeah, I agree. I think it's great. And I think also it helps to empower the patient.

David McManus: Oh, for sure. I mean, if done well, it really connects you as a patient to your health more and it also, if done well, could connect you better to your healthcare team. I mean, a lot of people are afraid in the healthcare community of this technology replacing them, but that only will happen if we don't incorporate it as a tool into our relationships with our patients. I think if it's done in that way, it's a facilitator. It actually makes your mom maybe feel more connected to her cardiologist to be able to kind of run that list.

Cindy St. Hilaire: Yeah and she understands what she's looking at more, so it's been wonderful.

Well, thank you so much. This was an excellent Review; it was really timely and thank you again for the contribution and for taking the time to speak with me today.

David McManus: My pleasure and thanks for the invitation and I hope people will read the Review.

Cindy St. Hilaire: Great. Wonderful. Well, thank you so much, David.

David McManus: Good luck to you and your mom.

Cindy St. Hilaire: So that's it for the highlights from our June issues of Circulation Research and our compendium on atrial fibrillation. Thank you for listening.

Please check out the Circulation Research Facebook page and follow us on Twitter and Instagram with the handle @CircRes and #DiscoverCircRes. Thank you to our guests, Dr David McManus.

This podcast is produced by Rebecca McTavish and 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. 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 month on Episode 12 of the Discover CircRes podcast, host Cindy St. Hilaire highlights three featured articles from the May 8 issue of Circulation Research and gives listeners an inside scoop of the cutting edge ideas in the May 22nd Compendium on Obesity. This episode also features an in-depth conversation with Dr Eduardo Marbán concerning COVID-19 and its effects on the heart.

Article highlights:

Roberts et al. LYN Regulates Monocyte Heterogeneity and Lifespan

Lu, et al. Acute Hyperglycemia Activates CaMKII-ROS Pathway

Yan, et al. Epicardium and Atrial Cardiomyopathy

Transcript Dr Cindy St. Hilaire: Hi. 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 share with you articles selected from the May 8th issue of Circulation Research as well as give you a hint at the cutting-edge ideas in the May 22nd Compendium on Obesity. We'll also have discussion with Dr Eduardo Marbán from the Smidt Heart Institute at Cedar Sinai Medical Center about his Review on COVID-19 and its effects on heart. So, first the highlights.

The first article I'm sharing with you is titled Deep Phenotyping by Mass Cytometry and Single Cell RNA Sequencing reveals LYN Regulated Signaling Profiles Underlying Monocyte Subset Heterogeneity and Lifespan. The first authors are Morgan Roberts and Maunish Barvalia and the corresponding author is Kenneth Harder and they're from the University of British Columbia. Monocytes can be separated into two main groups, conventional monocytes which enter tissues from the bloodstream and differentiate into macrophages, and patrolling monocytes, which developed from conventional monocytes but tend to remain in the blood vessel walls where they can scavenge cells and tissue debris.

It's thought that patrolling monocytes help to prevent a range of diseases like atherosclerosis by helping to clean up the vessel walls. Studies in mice harboring genetic mutations in a gene called Nr4a1 cause mice to have less than normal numbers of patrolling monocytes. In these mice, the development of atherosclerosis is exacerbated.

In addition to Nr4a1, this group has now identified another factor regulating the survival of patrolling monocytes, the tyrosine kinase LYN, L-Y-N. Genetic deficiency of LYN caused the upregulation of Nr4a1 and other genes involved in patrolling monocytes development and survival. This in turn led to the accumulation of patrolling monocytes in the blood, also in the bone marrow, spleen, and the aorta. Loss of LYN was also protective against atherosclerosis in mouse models of this disease. These results not only provide novel insights into patrolling monocyte biology, but also suggest that targeting LYN could offer novel treatments for diseases like atherosclerosis, where boosting the patrolling monocyte numbers could be beneficial.

The second article I want to highlight is titled Hyperglycemia Acutely Increases Cytosolic Reactive Oxygen Species via O-linked GlcNAcylation and CaMKII Activation in Mouse Ventricular Myocytes. The first author is Shan Lu and the corresponding author is Don Bers, and they're from the University of California, Davis. Diabetes affects more than 400 million people worldwide and puts these individuals at a higher risk for developing heart failure. When heart failure does occur, the outcomes for these patients with diabetes are likely to be far worse than for individuals without the diabetic condition. Both heart failure and diabetes have been associated with excessive production of reactive oxygen species and also with increased activation of a protein kinase in the cells of the heart called CaMKII.

Both ROS and CaMKII are induced by hypoglycemia, where there is an increased amount of extracellular glucose levels in the blood. This study shows that reactive oxygen species in CaMKII are causally linked. When CaMKII was inhibited or genetically deleted in mouse cardiomyocytes, high extracellular glucose levels were unable to induce reactive oxygen species production, which is what would normally occur. The team also discovered that O-GlcNAcylation post-translational modification of CaMKII is induced by the extracellular glucose and this modification is necessary for the enzyme's reactive oxygen species- boosting effects.

Lastly, they found that the enzyme NADPH oxidase 2 or NOX2 was the source of this CaMKII induced reactive oxygen species. This work uncovers the molecular pathway linking hyperglycemia, cardiomyocyte-damaging reactive oxygen species production, and it helps explain why heart failure pathology is exacerbated in diabetic patients.

The next article I want to share with you is Reactivation of the Epicardium at the Origin of Myocardial Fibro-Fatty Infiltration During the Atrial Cardiomyopathy. The first author is Nadine Suffee and the corresponding author is Stéphane Hatem and they're from Inserm in Montpellier, France. Fatty tissue surrounding the heart is linked to an increased risk for atrial fibrillation, which is the most common form of arrhythmia. It seems that a combination of fat cells, which are called adipocytes and the fibroblast localized within the heart's epicardium, builds up and expand into the subepicardial layers, and this is a feature that is called fibro-fatty infiltration.

These fibro-fatty infiltrations cause disturbances to the electrical rhythms that regulate the heart beating. Although generally quiescent in the adult heart, epicardial cells possess the ability to proliferate and have been shown that they harbor the ability to differentiate into adipocytes and fibroblast. This team hypothesized that the epicardial cells were the source of the damaging fibro-fatty infiltrations. Sure enough, when they looked at human heart sections, they found that within the epicardial layer, there were cells that were expressing fibroblast and adipocyte progenitor cell markers.

In culture, these epicardial cells with fibroblast progenitor markers could be differentiated into fibroblasts by treatment with angiotensin II and cells with the adipocyte progenitor markers could be differentiated into adipocytes by treatment with atrial natriuretic peptide. The team also showed that these epicardial fibro-fatty infiltrations occurred in a mouse model of atrial cardiomyopathy. Together this work highlights the pathogenesis of epicardial fibro-fatty infiltrations and suggest a novel model in which to study its progression to AFib.

The last thing I want to share with you before we switch to our interview with Dr Marbán is that the May 22nd issue of Circulation Research is our Obesity Compendium. Obesity is a major threat to cardiovascular health worldwide. While early studies focused on body mass index as a generalized measure of obesity and focused on the BMIs relation to cardiovascular disease, studies within the last decade have now tried to more fully understand adipose tissue physiology and the overall impact of obesity on cardiovascular disease.

The articles in this compendium are obesity phenotypes, diabetes and cardiovascular diseases, basic mechanisms of diabetic heart disease, leukocyte heterogeneity and adipose tissue including obesity, an eclectic cast of cellular actors orchestrates innate immune responses and the mechanisms driving obesity and the metabolic perturbation, metabolic inflammation and insulin resistance in obesity, genetic insights into the relationship between Type 2 diabetes and coronary heart disease, metabolomics and proteomics in Type 2 diabetes, metabolic and molecular imaging in diabetic cardiomyopathy and treatment of obesity and mitigating metabolic risk.

This compendium reflects the collective work of leading investigators in the space of diabetes, cardiometabolic disease, and cardiovascular disease with the ultimate goal of providing a summary of selected aspects of obesity and metabolic physiology central to cardiovascular disease development.

So, I have with me here today, Dr Eduardo Marbán, the founder of the Smidt Heart Institute at Cedars-Sinai Medical Center in Los Angeles, California. He's a leading physician scientist in the fields of electrophysiology, cardiac progenitor cells, and next generation cell-free therapeutics. Dr Marbán, thank you very much for taking the time out of your busy schedule to speak with us today about your article COVID-19 and the Heart, which is now freely available on the Circulation Research webpage.

Dr Eduardo Marbán: It's my pleasure to talk to you Cynthia.

Dr Cindy St. Hilaire: First off, how are you and how are things at your hospital center in LA?

Dr Eduardo Marbán: We seem to have dodged the bullet here in the sense that we were pretty progressive in terms of quarantine and stay at home orders. Given that, we seem to have peaked at a level that is very manageable in terms of our surge capacity. So, we feel for those who are worse off, but at least knock on wood here, we seem to be surviving so far.

Dr Cindy St. Hilaire: Yeah, that's similar to how we are in Pittsburgh. We shut down about the same time that Philadelphia, who was already surging was shutting down. So, we are feeling safe but still prepared. So, I was extremely excited to read this article because as we know, cardiac injury is happening in between 20% to 30% of the COVID-19 patients and cardiac injury is also the cause of about 40% of the COVID-19 related deaths. So, my first question is, what are the types of cardiac injuries or events that you're seeing in these COVID-19 patients and are there any particular characteristics that the subpopulation of patients shares that's different from non-cardiac injury COVID patients?

Dr Eduardo Marbán: What seems to be extremely common in COVID-19 patients is elevations of circulating biomarkers, things like troponin I, troponin T, BNP as an indicator of heart failure, but what's much less certain is whether these biomarker elevations have any clinical significance. At the level of isolated case reports, there's fulminant myocarditis, ventricular tachycardia, arrhythmias, occasional acute coronary syndromes, but there seems to be a disconnect between the almost ubiquitous nature of the circulating biomarker elevations and the relative rarity of clinical events.

Dr Cindy St. Hilaire: So, do these patients, do a majority of them have a history of cardiovascular disease or is this all new developments? Do we know?

Dr Eduardo Marbán: Underlying cardiovascular disease, diabetes, hypertension, and recently obesity and, of course age, have all been implicated as general risk factors for being critically ill with COVID, but there's no specific indication epidemiologically yet that those with underlying cardiovascular disease have a particular predilection to manifesting worse heart symptoms or signs during COVID-19. It makes sense that that would be the case, but so far, the epidemiology is somewhat more general.

Dr Cindy St. Hilaire: When you were first writing this article, I'm sure between then and now we even have more epidemiological data points that are constantly changing.

Dr Eduardo Marbán: Since the article was published online on April 7th, I've given four updated versions of the webinar to various audiences. Every time we do so, the slides need to change subtly. It's a very rapidly evolving field.

Dr Cindy St. Hilaire: Yeah, that's amazing. In the first SARS outbreak, which was in 2002-2003, scientists discovered that this type of Coronavirus enters the cell by binding to angiotensin converting enzyme II as a receptor. So, ACE2 as it's called. It's not a receptor in the canonical sense of the word, but it's a cell surface enzyme and it's involved in the renin angiotensin aldosterone system, which regulates a handful of cardiovascular homeostatic processes and is quite frankly, rather complicated. So, I don't want to talk specifically about that, but I'm wondering if you could tell us a little bit about what ACE2 is, what cells it's found on, and what that might mean for the implications of this virus and its effects on the cardiovascular system?

Dr Eduardo Marbán: Well as you correctly stated, ACE2 is central to cardiac physiology in the sense that it creates the bioactive form of angiotensin. In so doing, its regulation is central to that of blood pressure, human dynamics. What is less appreciated and to me was a bit of a revelation is the fact that it's expressed fairly richly on the surface of epithelial cells of the lung and the SARS-CoV virus family seems to have co-opted the presence of that in order to create a handy sort of hook to get into the cells in the first place. Whether there are broader ranging implications of ACE2 other than the particular mode of entry into the cell for a viral infection is a topic of great speculation at this point.

Dr Cindy St. Hilaire: Yeah. In some of my preparation for this and also just my curiosity regarding this virus and the vascular system, when you look at things like the human protein atlas, you can see that ACE2 is highly expressed, not only on the lung epithelial like you say, but they're also expressed on cardiovascular cells in nearly all of the tissue. I'm thinking of cells like the smooth muscle cell and the endothelial cell. Is the virus binding to ACE2 positive cells part of the reason for the cardiac events or these cardiac events secondary to systemic toxicity? So, I guess the real question is, do we know anything about the direct versus the indirect effects of the virus on the heart?

Dr Eduardo Marbán: No question in vitro that SARS-CoV can infect cardiac myocytes and most surely almost any other cell that expresses these two on its surface. In vivo, how frequently that happens as opposed to triggering secondary cardiac damage due to the systemic inflammation is uncertain, but I can tell you from the various case reports that have actually analyzed human tissue either at autopsy or an endomyocardial biopsy in cases of fulminant myocarditis, the frequency of direct viral infection seen either by culturing viral particles or more frequently by electron microscopy and visualization of inclusion bodies within cells points to perhaps a third of the cases being due to direct infection and two thirds of the cases likely being due to some bystander effect of systemic inflammation.

Dr Cindy St. Hilaire: Interesting. So, are the phenotypes different between those patients where it seems to be direct versus indirect? Does the myocarditis appear similar or the cytokine profiles, anything like that?

Dr Eduardo Marbán: There are too few patients to make really good conclusions about whether or not the phenotypes differ greatly when there's direct versus indirect cardiac involvement, but certainly from the literature as it exists now, there's no reason to believe that we could outsmart the clinical picture. They all look pretty much the same from the bedside.

Dr Cindy St. Hilaire: So from the first SARS outbreak, do we know anything about the long-term effects of this type of viral infection on the cardiovascular system or on the heart specifically?

Dr Eduardo Marbán: Yeah. COVID-19 of course the follow-up is limited to a few months since the first cases probably didn't emerge until late October early November and weren't really recognized as such until late '19 early 2020, but for SARS from the 2002-2003 epidemic, some of the long lasting sequelae are unanticipated and include hypertension, hyperlipidemia, pulmonary fibrosis, avascular necrosis. So, it seems that even when a patient is out of the woods, perhaps they're not really out of the woods in terms of long-term sequelae. We need to be watchful for long-term sequelae in COVID-19 survivors. They're going to be many more of them than there were from the SARS epidemic.

Dr Cindy St. Hilaire: So, one of the things that's come out recently, which I've been really mulling about because my background is vascular biology and specifically smooth muscle cells and endothelial cells, but one of the findings is about the later stage or more sick patients. These are patients who are going on ventilators and about 50% of them going on the ventilators are dying and/or just not responding to ventilator therapy as doctors expect. So, just to give a little background about ventilators, they're normally used when a patient's blood oxygen level drops too low.

So, normal levels are between 95% and 100%. However, patients with pneumonia or acute respiratory symptoms are put on ventilators sometimes when their oxygen drops below 90%, but some of the COVID-19 patients are exhibiting blood oxygen levels at 70% or sometimes even lower, but they don't have outward signs of distress and they can still hold conversations. So, I'm wondering if you can give me any insight into possibly what's going on there with the lens of vascular remodeling, what might be happening to the vasculature in the lung that is unique to this ventilator response and COVID response?

Dr Eduardo Marbán: The observation you described is common that sometimes a patient will be profoundly hypoxemic but chatting away or surfing the internet as if nothing were happening. We're not used to seeing this in other cases of ARDS or viral sepsis where the patient usually is in extremis by the time the blood oxygen levels get that low. It begs the question as to whether perhaps there's something about the cerebral circulation, and this is complete and rampant speculation. Whether there's something about the cerebral circulation that makes it somewhat resistant to the effects of systemic hypoxia, perhaps there's a compensatory vasodilation that occurs that compensates for the otherwise deadly systemic hypoxemia. It would be quite interesting to monitor oxygen tensions within the cerebral parenchyma to test that, but all I can say with any certainty right now is that the clinical observation is robust. We see this not infrequently in patients who in the sort of clinical jargon have no right to look that good.

Dr Cindy St. Hilaire: Yeah. Yeah. It's like your numbers, you really have those numbers? Yeah. There's just so many questions. It's really unprecedented. So, I guess we've been talking a lot about the disease itself and the symptoms and the pathogenesis, but I want to switch to ask about potential therapies. There's been several therapies that have been suggested by a variety of people and there's, I don't even know how many clinical trials. I looked a week ago and there's really a great response of pharmaceutical companies and university hospital systems trying what they can with the tools they have. So, things like antivirals, HIV protease inhibitors, inhibitory antibodies, and even antimalarial drugs have been suggested that they could possibly work. So, I'm wondering if you could give us some insight from a cardiovascular standpoint, what are the potential implications or potential adverse side effects of using these different therapies off label and what might that mean for the heart in addition to treating the viral infection?

Dr Eduardo Marbán: You're correct in the explosion of clinical trials in this area or at least, clinical interventions. At our IRB, as of today, there are 56 active COVID protocols. Imagine nobody even cared about COVID until mid-February, right?

Dr Cindy St. Hilaire: That's just at Cedars-Sinai.

Dr Eduardo Marbán: Yeah. Now, we have 56 active protocols. So, not all of those are interventional. Some of them are epidemiological or biomarker studies, but still there's an incredible plethora. You're right, the approaches of targeted anything from the viral infection to the viremia to the downstream consequences of viral infection including the hyper inflammation and cytokine storm. The rationale for anti-malarials is actually fairly thin and resides on in vitro observations that actually were just from February that SARS-CoV-2 infection in vitro is somewhat retarded by exposure to hydroxychloroquine. This didn't come out of the blue. There had been an extensive literature and quite controversial literature, I should say, that anti-malarials might be useful in influenza and other infections.

In a very general sense, there was a lot of hype created by early in vitro studies, which turned out to be neutral or in some cases even harmful clinically, but this has led to an almost universal adoption of hydroxychloroquine in patients with COVID-19 coupled sometimes with the antibacterial agent azithromycin for which the rationale is even thinner. There's no reason to believe that an antibacterial per se would help in a viral infection, but azithromycin is said to have antioxidant properties, which may or may not potentiate the effects of hydroxychloroquine, but for sure what they do together is prolong repolarization of the heart and lead to a clinical syndrome known as prolonged QT, which is a known substrate for toxic arrhythmias like polymorphic ventricular tachycardia. So, in prescribing some of these agents, one needs to weigh the uncertain benefits against the very certain risk that they entail.

Dr Cindy St. Hilaire: Yeah. I think that's a really important point. I think one of the scary things that has the potential of happening during this crisis is too quick of a jump to conclusions. While there is a need for as rapid a response as possible, we still need to make sure that we're taking in all the scientific information we have and that that science is good and strong. I think one of the things that you mentioned in the Review is the lack of power in some of those initial anti-malarial studies. I think it's really important thing I want to emphasize that it's an emergency, but we still need to make proper good scientific decisions.

Dr Eduardo Marbán: Well, one of the problems is that hydroxychloroquine and other agents in some cases, remdesivir and you know, you choose, have gotten so popular and hyped that there's almost no possibility of being an ethical clinical trial because the patients want to be on them. So, it may be easier in some settings than in others, but it's certainly not going to be a trivial thing to sort out the true risk benefit ratio of these drugs in this illness.

Dr Cindy St. Hilaire: So right now, doctors and scientists, we're all in crisis mode, but once things settled down, we could really start to sit down and think about more mechanistic questions that might be able to be tested that will really help us flush out our understanding of COVID-19 disease pathogenesis and its effects on the cardiovascular system. So, what do you see after this initial crisis is under control, what do you see as the immediate next questions that basic scientists and translational scientists need to address that can help the next time that this comes again?

Dr Eduardo Marbán: First of all, it's quite clear that we've all become consumed by COVID-19 and SARS-CoV-2. We can't think of anything else often. It's really hard to even focus on work from the laboratory that doesn't have to do with SARS-CoV-2 and COVID. It's so ubiquitous in public perception and the way we're living our lives that it just makes it incredibly difficult to think about anything else. I think there's going to be a correction in which we're going to get frankly tired of SARS-CoV-2 and COVID and want to think about other things, but among the lasting questions and the ones that will have greater biological merit above and beyond how to deal with this particular virus and this particular pandemic are the following. What is the role of ACE2 in human biology? Clearly here, there's an experiment of nature in which this surface enzyme has been co-opted for viral entry and a tremendous amount of speculation surrounds the question of whether high ACE2 values are protective and detrimental and ACE inhibitors and angiotensin receptor blockers might be detrimental or beneficial.

All of these fundamental mechanisms need to be sorted out and now there's motivation to do so because of the epidemic. Some of this work is easier than others and those institutions that happen to have a BSL-3 level facility for being able to directly study the effects of the virus on various tissues should do so with alacrity because it's a limited resource right now where the number of questions really far exceed the ability to answer them just physically. Another question which I think is going to be motivated by our experience with COVID-19 is that of the mechanisms of cytokine storm and hyper thrombotic states. These are things that characterize the critically ill patient with COVID-19.

Dr Cindy St. Hilaire: Can you just explain what is a cytokine storm? What does that exactly mean?

Dr Eduardo Marbán: So, patients who are critically ill with COVID-19 manifest a late stage of the illness, which is often fatal, in which circulating levels of various inflammatory biomarkers, interleukin 6, C-reactive protein, ferritin being among them, but basically anything that goes up in an inflammatory state. And some of these appeared not to just be markers of inflammation. Something like C-reactive protein is probably just a biomarker of inflammation, but interleukin 6 for example, is a highly bioactive cytokine that itself probably causes tremendous tissue injury and there's some enthusiasm for the use of anti IL6 antibodies and anti IL6 receptor antibodies to treat the critically ill with some anecdotal dramatic success I should say. So perhaps the cytokine storm isn't just a marker of those who are critically ill, perhaps it's causative.

If that presumption is real, then it makes good sense to target the cytokine storm, but from a scientific point of view, what causes it in the first place? How does a viral infection lead to massive production of cytokines and inflammatory biomarkers and how can that be mitigated? One of the ways of dealing with that is by understanding precisely how it happens in the first place and there's not that much literature on it. There's a recent study which I found quite provocative that glucose metabolism and the whole process known as O-GlcNAcylation might actually be a trigger in the production of cytokines during viral infections like COVID-19, but I think understanding how it happens will lead to much more targeted therapeutics and perhaps enable us to eventually divorce the infection from the overreaction. Really what's happening is friendly fire. The body's immune system is turning against itself in a sort of vain effort to control the virus. Sometimes the viremia is actually almost gone by the time that these inflammatory biomarkers increase, and the cytokine storm surges.

Dr Cindy St. Hilaire: So, it's almost like the inflammatory response reaches some point beyond which it doesn't need virus anymore. It is just full force feeding forward and causing more damage by itself.

Dr Eduardo Marbán: Yeah, exactly. It's almost as if there's an eroding cliff and even though the river may be back down to normal levels, the cliff is still unstable and the whole hillside could come crashing down.

Dr Cindy St. Hilaire: Are there long terms effects of that? I wonder how long that would last after the infection or is it only during a viral titer in the system?

Dr Eduardo Marbán: Well, you raised yet another interesting question to the extent that patients who have survived SARS- CoV-2 infection develop long-term sequelae, what's the mechanism of those long-term sequelae? Why should patients who are previously well develop hyperlipidemia and hypertension after the infection, if in fact they do, so are any of these related to micro thrombotic events? It's quite conceivable.

Dr Cindy St. Hilaire: Great. Well, thank you so very much for taking the time to speak with me today. I don't think I found a ton of answers. I found a lot more questions, but hopefully as this develops and we get it under control, maybe we can talk again and talk about some of those new mechanistic findings and potential therapies.

Dr Eduardo Marbán: Absolutely. You're welcome, and I hope you and all the listeners stay safe during this pandemic.

Dr Cindy St. Hilaire: You too, and your clinical team.

That's it for highlights from the May 10th and May 22nd Obesity Compendium issues of Circulation Research. Thank you so much for listening. Please check out the Circulation Research Facebook page and follow us on Twitter and Instagram with the handle @CircRes and #DscoverCircRes. Thank you to our guest, Dr Eduardo Marbán. This podcast is produced by Rebecca McTavish, 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 Discover CircRes, your on-the-go source for the most up-to-date and exciting discoveries in basic cardiovascular research.

This month on Episode 111 of the Discover CircRes podcast, host Cindy St. Hilaire highlights three featured articles from the March 27 issue of Circulation Research and talks with Dr. Matthias Nahrendorf and Dr. Maximilian Schloss about their article Modifiable Cardiovascular Risk, Hematopoiesis and Innate Immunity.

Article highlights:

Liu et al. Genetics of Transposition of the Great Arteries

Park et al. Mild Lipid Abnormalities and ASCVD in the Young

Yan, et al. Gut Flora Adjusts Blood Pressure By Corticosterone

Transcript

Cindy St. Hilaire: Hello 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 share with you articles selected from the March 27th issue of Circulation Research, as well as give you a hint of the cutting-edge ideas in the Compendium on atherosclerosis. We'll also have a discussion with Dr Maximilian Schloss and Matthias Nahrendorf about their article On Modifiable Cardiovascular Risk, Hematopoiesis And Innate Immunity. So, first the highlights.

The first article I'm sharing with you is titled Exome-Based Case Control Analysis Highlights the Pathogenic Role of Ciliary genes and Transposition of the Great Arteries Exome-Based Case-Control Analysis Highlights the Pathogenic Role of Ciliary Genes in Transposition of the Great Arteries. The first authors are Xuanyu Liu and Wen Chen and the corresponding author is Zhou Zhou from Peking Union Medical College in Beijing, China. In normal healthy hearts, the aorta develops from the left ventricle and the pulmonary arteries from the right ventricle, but in the common congenital heart malformation called transposition of the great arteries or TGA, the plumbing of these two major vessels is switched. Thus, the pulmonary arteries arise from the left ventricle and the aorta from the right.

This is a life-threatening condition, requires surgery in the earliest days of life and currently, the genetic etiology of this congenital disease is largely unknown. To identify the genetic drivers of transposition of the great arteries, the authors of this study performed whole exome sequencing of 249 TGA patients and, in 66 cases, they were actually able to do exome sequencing on their parents as well. The analysis identified 82 candidate genes in which the allele variant or mutation that was found in TGA patients was predicted to alter protein function.

Interestingly, a quarter of these mutations or variants were found to be in genes that are involved in cilia function. So, the cilium is an organelle that's found on all eukaryotic cells and is in the shape of a slender protuberance that projects from the much larger cell body. Recently, cilia have been identified as playing a central role in the pathogenesis of congenital heart diseases, and it has been suggested that congenital heart disease may be a new class of ciliopathy. Transposition of the great arteries has been hypothesized to arise from disturbances in the left right patterning during embryo development, and cilia are required cellular organelles and they are essential for left-right axis determination in early development. These findings add to the growing body of evidence that has identified a role of cilia genes and congenital heart disease and may lead to future prenatal diagnostic screenings.

The next article I want to highlight is titled Mildly Abnormal Lipid Levels, but Not High Lipid Variability, Are Associated with Increased Risk of Myocardial Infarction and Stroke in 'Statin-Naive' Young Population: A Nationwide Cohort Study. The first author is Jun-Bean Park and the corresponding author is Hyung-Kwan Kim from Seoul National University Hospital in Seoul in the Republic of Korea. High levels of lipids in the blood increase a person's risk of cardiovascular disease, and evidence suggests that this risk builds over lifetime. However, in young adults, and in this case, young adult means any individual between 20 and 39 years of age. In young adults, relatively little evidence is available that identifies individuals at high risk for atherosclerotic cardiovascular diseases, except for very high LDLC levels.

Variability in lipid levels has recently emerged as a predictor of adverse clinical outcomes and lipid level variability may be causally linked with the atherosclerotic cardiovascular disease risk. This is because theoretically, high lipid levels can induce fluctuations in the atherosclerotic plaque composition. These fluctuations result in plaque instability and rupture and ultimately, plaque related clinical events, such as myocardial infarction. However, high lipid level variability may merely reflect other risk factors or confounders for atherosclerotic cardiovascular diseases, including unhealthy lifestyle and unrecognized comorbidities. This study therefore examined health data of close to two million Korean individuals aged 20 to 39. None of them had ever been treated for high cholesterol with statins nor had any of them suffered any myocardial infarctions or stroke.

Over a four-year period, the subjects had undergone at least three lipid measurements as part of their general health assessments and then they were followed for a further four years or until death. The data showed that high baseline lipid levels was linked with an increased risk of adverse cardiovascular events, and in particular, myocardial infarctions. They also found that individuals who exhibited high lipid variability, so sometimes getting high readings, sometimes getting low readings, these individuals who exhibited high variability and lipid level measurements were not at any greater risk of such cardiovascular events. While such up and downs have previously been linked to cardiovascular disease, this study argues that perhaps statin use in other cohorts may have contributed to the variability and thus confounded research interpretation, an issue that was specifically avoided in this study. Together the results indicate that lipid in young adults can indeed indicate future cardiovascular risk and therefore suggest lipid-lowering strategies could be beneficial for this age group.

The next article I want to share with you is titled Intestinal Flora Modulates Blood Pressure by Regulating the Synthesis of Intestinal-Derived Corticosterone in High Salt-Induced Hypertension. The first author is Xuefang Yan and the corresponding authors are Zhe Wang and Qunye Zhang from Shandong University in China. Hypertension is highly prevalent in the adult population all over the world and it is a major risk factor for heart disease and stroke. A high salt diet can help to drive hypertension pathogenesis, but complete details about the mechanisms by which high salt intake shapes vascular pathology are lacking.

Recent studies show that fecal transfer from salt hypertensive to salt normotensive animals can lead to hypertension in the recipients, and this suggests that perhaps gut flora may play a role in hypertension. In the article by Yan and colleagues, they show that rats on a high salt diet have altered gut flora profiles and in particular that levels of the bacterium, Bacteroides fragilis, was reduced. Analysis of intestinal metabolites and substrates in high salt diet fed rats also showed that levels of arachidonic acid, which is produced by this bacterium, were low and levels of the stress hormone, corticosterone, which regulates blood pressure, were elevated.

The team went on to show that supernatants from this bacterial culture could prevent corticosterone production in the intestinal tissue of high salt fed mice as could direct treatment with arachidonic acid. Moreover, both B. fragilis and arachidonic acid were found to be lower in the feces of humans with hypertension compared to that of healthy controls. The results suggest B. fragilis and arachidonic acid normally curb corticosterone production and could therefore be novel targets for hypertension treatment strategies.

The last thing I want to mention before we switch to our interview is the Circulation Research Compendium on Atherosclerosis. The last compendium on this topic was back in 2016 and this new compendium provides the most cutting-edge ideas in the field. The topics highlighted in this compendium are polygenic scores to assess atherosclerotic risk, clinical perspectives, and basic implications, epigenetic reader proteins and cardiovascular transcriptional programs, sex as a biological variable in atherosclerosis, neutrophil extracellular traps in cardiovascular diseases, CD31 as a therapeutic target in athero, interleukin-1 and the inflammasome as therapeutic targets in cardiovascular disease, non-coding RNAs in vascular diseases, intracellular aspects of macrophage immunometabolism in atherosclerosis, single cell RNA sequencing in atherosclerosis, vaccination strategies and immune modulation in atherosclerosis and we have an update from the group leading the One Brave Idea. Please check out this compendium.

All right. So, now we're going to switch over to our interview portion of the podcast. I have with me today Dr. Matthias Nahrendorf, who is a professor at the Center of Systems Biology at Massachusetts General Hospital Research Institute and Harvard Medical School and his research fellow, Dr. Maximilian Schloss. Today, we're going to be discussing the article Modifiable Cardiovascular Risk, Hematopoiesis, Innate Immunity, which is part of our Compendium on Atherosclerosis. Circulation Research puts together two to three compendiums annually and this current one is the Compendium on Atherosclerosis. We will have two additional compendiums planned for 2020. One on Obesity, Metabolic Syndrome and Cardiovascular Disease and that should come out in late May and another on Atrial Fibrillation scheduled for June. So stay tuned.

So, thank you very much for being with me here today, Matthias and Maximilian.

Matthias Nahrendorf: Thanks for having us.

Maximilian Schloss: Thanks for having us.

Cindy St. Hilaire: So, I really enjoyed this review article. I actually learned a lot. I also really liked your cartoons at the end, so maybe we can talk about those a little bit later, but what it's on is essentially the role of inflammation and cardiovascular disease and years of study, which have recently culminated in the completion of the CANTOS trial, have showed us that reducing inflammation can help reduce cardiovascular events. When we look at the factors that we know drive cardiovascular disease, it's things like bad diet choices, lack of exercise, stress, and inadequate or disrupted sleep and in this article you make the more nuanced argument that these modifiable factors are in fact influenced by the innate immunity. So, before we dig too deep into what you are really discussing in this article, could you maybe give us a brief introduction to the role of innate immunity and cardiovascular disease initiation and progression?

Matthias Nahrendorf: Sure. Yeah. So, I think one very instructive experiment that had been done by more than one lab actually almost two decades ago, is stopping innate immune cells from migrating to atherosclerotic plaque by deleting the chemokine MCP-1 or the chemokine receptor CCR2 in mice that have a propensity to develop atherosclerosis. What became apparent is that these mice, despite having very high blood cholesterol levels, they don't really develop atherosclerosis. This really led the whole field now almost 20 years ago, to the insight that it's not only the hypercholesterolemia, it's also the immune system that contributes to the disease. So, innate immune cells, most numerous neutrophils and monocytes then in tissue also macrophages and they're meant to defend us against infections and they support healing. In this particular setting, they are probably doing more harm than good because they promote inflammation in areas where inflammation shouldn't be i.e., in the vessel wall.

Maximilian Schloss: Yeah, I would add that what Matthias said is that basically it's all about a balance between necessary inflammation and too much inflammation. If we take, for instance, myocardial infarction as an example, we need a certain amount of inflammation, local inflammation. We need a recruitment of innate immune cells like neutrophils and monocytes and eventually macrophages, to do their job. For instance, phagocytizing a dying cardiomyocytes or inducing fibrosis. So in this example, we need inflammation, but what we see in different models where we can manipulate inflammation being at reducing or increasing inflammation, we can see that if we do either/or then wound healing and scar formation is impaired. I think that's all we are interested in studying not only the mechanisms, how inflammation can be increased or decreased, but also what is actually the perfect balance in view also of finding ways of improving outcomes in cardiovascular patients.

Cindy St. Hilaire: One of the things in my research, so I focus on cardiovascular calcification, which is very hard to do in a mouse. They don't like to calcify similarly like they don't like to make plaque without a proper genetic background. Are there aspects of the mouse versus the human innate immune system that are very different? I mean I know specific receptors are slightly different, but in general, are they matched up pretty well or is there things that are quite different about them?

Matthias Nahrendorf: I think the answer is both and there are very important parallels and then there are very important differences. So, one important difference is just if you look at sheer numbers and the contribution of immune cells in the blood and, possibly also in the plaque, can be quite different. So, recent studies that use unbiased profiling in human plaques, for instance, say that there's quite a lot of lymphocytes and we still have to understand whether this is due to the retrieval or if it says species difference or the situation, but I think there are important differences. On the other hand, I think that it really make sense to study mice because a lot of the important discoveries about the immune system in the setting have translated to humans.

Cindy St. Hilaire: Like the IL-1 beta story.

Matthias Nahrendorf: That's right. Yeah.

Cindy St. Hilaire: So, actually one of the topics that you started out with in your article is on the role of hematopoiesis in cardiovascular disease. You had a beautiful paper years ago actually with my colleague at University of Pittsburgh, Partha Dutta, who's right down the hall from me, where you guys showed that myocardial infarction itself further exacerbates atherosclerotic plaques mid part through recruiting monocytes from the spleen and mobilizing the immune system. So, I'm wondering, what are the role of the cells when they get mobilized? You talk about these modifiable risk factors of stress and sleep interruption, unhealthy diet. So, how can these risk factors help or promote this mobilization of hematopoietic cells?

Matthias Nahrendorf: Yeah. So, I think that early on when we thought about going down this road and studying these risk factors, even before going there, you realize that the cells that we're interested in, innate immune cells are very short lived. So they live on the order of hours or days. So, they're really produced just in time. That's different to lymphocytes and resident macrophages, which have much longer lifespans. So, this really triggered the insight that we should look at production and release because it's a just in time supply situation. So, what we were wondering is whether in the setting of cardiovascular disease, whether production rates are increased and we now know and a number of labs have studied hematopoiesis in this setting including Fil Swirski, Alan Tall, and some others.

We now know that this is really the case, so hematopoiesis increases in chronic atherosclerosis. It increases in acute myocardial infarction and increases in heart failure. What we don't know is what mechanisms actually ramp up blood cell production and we're beginning to understand that the sympathetic nervous system is involved. But I think we only see the tip of the iceberg here. That's why we wanted to study modifiable risk factors, because if you look at others such as high cholesterol, once the insight was gained that lowering cholesterol is helpful, we had the statins which make a huge change. So, we hope to repeat that.

Cindy St. Hilaire: Maximilian, one of the things that you brought up is this balance. The inflammation's a little bit good and then it's a little bit bad or a lot bad. So, where is that good and bad spectrum in terms of mobilizing hematopoiesis or hematopoietic cells?

Maximilian Schloss: Yeah. I think that depends a bit on the disease type or we're talking about a chronic disease or an acute disease? For instance, to stay at the example of myocardial infarction, once cardiomyocytes become ischemic, they will release certain chemokines and cytokines into the blood, which then circulate to the bone marrow and tell the cells that leukocytes need to leave the bone marrow to enter the blood circulation system and then go to the heart to fulfill their very important functions there. Once the cells leave the bone marrow, the bone marrow need to reproduce themselves, then this process starts of hematopoiesis and there we can go back again to the concept of a balance. Of course, there is a certain beneficial physiological need of cell production, but one sees mechanisms so to say maybe go out of control and too many leukocytes are produced and released to the blood.

Then that again impairs patient outcome. There are very many papers, clinical papers, who have shown that leukocyte counts after myocardial infarction have a certain U shape relationship with the outcome. That I think is best described that if leukocyte counts are very high, that they actually negatively correlate with the outcome of MI patients. If you look at the bone marrow specifically, there are certain mechanisms, which we know, and what we are more closely looking at now, what are actually the modifier of this process, what are the signals which tell these cells to secrete more hematopoietic factors or quiescence factors? I think that's what also the Review is a little bit about.

Cindy St. Hilaire: Yeah, it's great. So, you were speaking about that kind of U-shaped curve in the release of these cells. Do we know based on some of the other things you spoke about, I guess I'm thinking about like diet or exercise or sleep in contributing to that release after an event like myocardial infarction. Is that known yet or has anyone looked into that?

Matthias Nahrendorf: Yeah. So, I think we're in the very beginning of understanding what's happening acutely. There's more knowledge on the chronic side and this is what we've been working on. Often the things that influenced the chronic situation can be quite different from what happens acutely. So I think in general, we're just beginning to understand what happens in acute myocardial infarction. Well, we know for instance is that exercise doesn't compromise the release and supply of leukocytes that's necessary in acute infection or acute myocardial infarction. So, if the mouse or the individual was exercising before the event, that may reduce overall leukocyte levels, but not to a degree that it's harmful.

Cindy St. Hilaire: Yeah. You can't exercise your way beyond a certain point.

Matthias Nahrendorf: Maybe that's also possible. If you run more than one marathon a day, I'm sure that's…

Cindy St. Hilaire: That will do something else.

Matthias Nahrendorf: Yeah.

Cindy St. Hilaire: Actually, so one of the interesting things that I saw in the article was when you were talking about diet and the role of diet in innate immunity, which is something I really never thought about, and you did bring up things like intermittent fasting. Can you discuss what's known at least scientifically about how that kind of diet timing can impact the immune system and therefore maybe cardiovascular disease?

Matthias Nahrendorf: So, that's a very emerging field. There's very little known about this. I think it's very interesting because very relevant and a lot of people are excited about it, but it's basically, from what I know, it's mostly two papers that were published, I think both in Cell, and they say that intermittent fasting leads to a decline of cells that are in circulation. So, that's a very exciting observation. I think it's similar insight as to discovering that immune cell levels circulate the circadian rhythms, which had been discovered a while ago. So, I think there's definitely an impact and we're just beginning to understand why this is and what regulates it.

Cindy St. Hilaire: Yeah, that segues nicely into the next thing I was wondering about and that is we all know not enough sleep, you get tired, your brain's not focused and stuff like that, but it really does impact the inflammatory system and also cardiovascular disease. So how is sleep involved in this innate immunity cardiovascular disease progression?

Matthias Nahrendorf: The way we approached this was actually thinking about lifestyle factors and their impact on cardiovascular disease. Maybe a decade ago, Fil, who's our middle author on this Review, and I started thinking about lifestyle factors and what struck us is that the association of some of these risk cardiovascular events is really high. So, if you look at sleep or if you look at psychosocial stress, psychosocial stress has an odds ratio of 2.4 for premature myocardial infarction. That is right on scale with all these powerful risk factors that everybody knows about like hypertension, but then what isn't really clear or maybe not entirely, is whether or not these risk factors also act via the innate immune system and that's where we were coming from.

I think at this point it's pretty clear that they do have an influence via the immune system. What I think what we've done is we uncovered a couple mechanisms that lead to the activation or dampening of inflammation depending on what you look at, but we don't really understand the broader network. I think there's a lot of work to be done looking into these pathways, which is exciting because I think that we can learn from nature what's dangerous and what's helpful. That this is how humans learn to fly. So, I think that observing what leads to cardiovascular disease, which behaviors are really harmful, will maybe lead us to new ways of mitigating it.

Cindy St. Hilaire: Yeah. Also, I think all of this, it's interesting. We all went after smoking for decades, stop smoking, reduce cardiovascular risk and maybe it's stress and sleep is the next smoking.

Matthias Nahrendorf: Smoking was so successful, right? I mean if you look 50 years back, it was promoted as this healthy thing that you should do. Then people really started to learn how bad it is and now we're at a time where smoking is declining and has declined and we see the results. Lung cancer is really on the decline. So, I think that's a good example how understanding health effects of behavior can be really helpful.

Maximilian Schloss: I think one thing I would like to add is when you ask more general question about innate immunity and when we talk about sleep and sleeping habits, I think what's generally quite interesting to know is that the immune system or these leukocyte numbers in circulation, they oscillate quite dramatically over the course of a day in a healthy human being and also in mouse models. I think one aspect also among others to consider is when we have unhealthy steeping habits, like for example, going to bed late or being a shift worker, drinking for example before going to bed. Then this will also confuse a system on the circadian entrainment, which then subsequently will lead to other problems.

I also think another thing is that what you were mentioning with the fasting is what we learned from this similar to these extreme circadian patterns seen when we fast or when a mouse is fasting, then monocyte levels drop into extreme low levels and these monocytes hone back into the bone marrow. I think this is interesting because it shows how dynamic actually a system like innate immune cells actually is. So, it's a very delicate system which responds to sleep disruption, exercise, diets in a very dramatic way.

Cindy St. Hilaire: All right, I'm going to bed early tonight and eating a good dinner. Well, this was a wonderful Review. I really enjoyed reading it. I really do think it's introducing the next targets that we have to go after in modifying cardiovascular disease. Thank you so much for taking the time to speak with me today.

Matthias Nahrendorf: Thank you.

Maximilian Schloss: Thank you so much.

Cindy St. Hilaire: That's it for our highlights from the March 27th and Compendium issue of Circulation Research. Thank you so much for listening. This podcast is produced by Rebecca McTavish, 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. Thank you to our guests, Max Schloss and Matthias Nahrendorf. 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.

This month on Episode 10 of the Discover CircRes podcast, host Cindy St. Hilaire highlights four featured articles from the February 28 and March 13, 2020 issues of Circulation Research and talks with Dr Mary McDermott about her article Cocoa to Improve Walking Performance in Older People With Peripheral Artery Disease: The Cocoa-Pad Pilot Randomized Clinical Trial.

Article highlights:

Rykaczewska, et al. PCSK6 Is a Key Protease in Vascular Injury

Lebek, et al. SDB Induces Arrhythmias via CaMKII and Late Ina

Mueller, et al. Brain Damage With Heart Failure

Napierski, et al. Cut and Paste of cMyBP-C Domains In Situ

Transcript

Cindy St. Hilaire: Hi. Welcome to Discover CircRes, the podcast of the American Heart Association's journal Circulation Research. I'm your host, Dr Cindy St. Hilaire, and I'm from the Vascular Medicine Institute at the University of Pittsburgh.

Today I'm going to share with you four articles selected from the February 28th and the March 13th issues of Circulation Research as well as have an in-depth discussion with Dr Mary McDermott, who is the corresponding author of the study COCOA-PAD Pilot Randomized Clinical Trial. So first, the highlights.

The first article I'm sharing with you is titled PCSK6 Is a Key Protease in the Control of Smooth Muscle Cell Function in Vascular Remodeling. The first authors are Urszula Rykaczewska, Bianca Suur, Samuel Röhl, and the corresponding author is Ljubica Matic from the Karolinska Institute in Stockholm, Sweden.

The family of proprotein convertase subtilisins/kexins, or PCSKs case for short, are a group of proteases whose role in vascular disease was only recently recognized. Humans with gain- and loss-of-function mutations in PCSK9 exhibit very high or very low levels of cholesterol, respectively, and this information was leveraged for the development of novel, albeit extremely expensive, drugs for regulating cholesterol. However, the role of other members of the PCSK family in cardiovascular disease is not known.

This group previously found that PCSK6 was one of the most enriched molecules in human carotid artery plaques as compared to normal arteries, while other PCSK family members did not show the same trend. This prompted the group to further explore the role of PCSK6 in vascular disease. They used a very integrative approach drawing from several independent human biobanks for genetic information, conducting in situ functional investigations using human tissue, also conducting in vivo animal models of vascular injury, including using the PCSK6 knockout mice, as well as ex vivo and in vitro mechanistic studies.

And they found that PCSK6 was a key modulator of smooth muscle cell function in vascular remodeling and atherosclerosis through a very novel mechanism implicating MMP14 and MMP2 activation upon cytokine stimulation. Future studies will investigate the role of PCSK6 on atherosclerotic plaque remodeling and instability because, as we know, plaque rupture can have devastating consequences.

The second article I will highlight is titled Enhanced CaMKII-Dependent Late I Na Induces Atrial Pro-Arrhythmic Activity in Patients with Sleep-Disordered Breathing. The first author is Simon Lebek, and the corresponding author is Stefan Wagner, from the University Hospital Regensburg in Regensburg, Germany.

Sleep-disordered breathing is an umbrella term for any chronic condition involving the complete or partial interruption of breathing during sleep, and this is commonly called sleep apnea. Aside from daytime sleepiness, people with sleep-disordered breathing run the risk of developing arrhythmia, such as atrial fibrillation.

Arrhythmias are an electrical problem as opposed to a mechanical one. And at the cellular level, arrhythmias are associated with increased activity of the enzyme calcium/calmodulin-dependent protein kinase 2, or CaMKII, and this protein regulates cellular electrophysiology. Despite the role of CaMKII in propagating electrical signals in the heart, its activity has not been investigated in sleep-disordered breathing patients.

This group now shows in a study that used 113 patients undergoing heart surgery that those with sleep-disordered breathing have higher CaMKII in biopsied myocardium than those without the condition. Furthermore, this CaMKII increase was associated with other pro-arrhythmic alterations to the tissue, including increased reactive oxygen species production, enhanced phosphorylation of a major sodium channel, and consequent late firing of sodium currents.

Importantly, these alterations could be prevented by pharmacological inhibition of CaMKII, suggesting that such an inhibitor could be a novel treatment strategy for patients with sleep-disordered breathing to reduce their arrhythmia risk.

The next article I want to share with you is titled Brain Damage with Heart Failure: Cardiac Biomarker Alterations and Gray Matter Decline. The first authors are Karsten Mueller and Friederike Thiel, and the corresponding author is Matthias Schroeter, and the work was completed at the Max Planck Institute for Human Cognitive and Brain Sciences in Leipzig, Germany.

Heart failure leads to decreased blood flow due to a reduced pumping efficiency of the heart, and as a consequence, this can cause insufficient oxygen supply to the tissues, including the brain. Cardiovascular insults, including heart failure, increase the risk for the development of neurological diseases later in life, such as vascular dementia and Alzheimer's disease. Patients with heart failure can show neurological symptoms such as fatigue, nausea, and dizziness. However, the long-term consequences of the effects of heart failure on brain integrity are not well understood.

However, several studies suggest that structural changes in the gray matter can occur. This study sought to identify correlations between cardiovascular biomarkers and structural gray matter changes in the brain. They found that patients who suffered from heart failure undergo detrimental brain structural changes. Reduced gray matter density in several regions of the brain correlated with decreased ejection fraction at baseline and increased NT-proBNP, which is a heart failure biomarker. While these observations might reflect structural brain damage in areas that are related to cognition, whether these structural changes facilitate the development of cognitive alterations will need to be proven in future longitudinal studies.

The last article I want to share with you before we switch to our interview is titled A Novel "Cut And Paste" Method for In Situ Replacement of Cardiac Myosin Binding Protein C Reveals a New Role for This Protein in the Regulation of Contractile Oscillations. The first author is Nathaniel Napierski, and the corresponding author is Samantha Harris, and they're from the University of Arizona.

Actin and myosin are the respective thin and thick filament proteins that allow for muscle contraction, including in the cardiomyocytes, the muscle cells of the heart. Cardiac myosin binding protein C is a critical protein that regulates heart contraction, but the mechanisms by which this protein affects actin and myosin are only partially understood. One reason for this is that cardiac myosin binding protein C localization on the thick filaments may be a key component of contraction, but most in vitro studies cannot spatially replicate arrangements of cardiac myosin binding protein C within the sarcomere.

To address this technical gap, this group created a novel hybrid genetic/protein engineering approach that allows for rapid manipulation of cardiac myosin binding protein C in sarcomeres of permeabilized myocytes isolated from genetically engineered Spy-C mice in situ. So essentially, they can do some gene editing in tissue in situ. Using this approach, they were able to rapidly remove and replace cardiac myosin binding protein C. Deletion of this protein fully recapitulates effects obtained using traditional knockout and transgenic mouse models of cardiac myosin binding protein C.

However, the ability to rapidly remove and replace this protein identified a new regulatory role for cardiac myosin binding protein C where it functions to dampen contractile oscillations. The novel cut and paste approach should be very useful in testing these new hypotheses of the role of cardiac myosin binding protein C function as well as in defining the role of how spontaneous contractile oscillations affect cardiac contractility during both health and disease.

Okay. Now we're going to switch over to the interview portion of the podcast. I have with me Dr Mary McDermott from the Departments of Medicine and Preventative Medicine at Northwestern University's Feinberg School of Medicine. And today we're going to be discussing her manuscript titled Cocoa to Prevent Walking Performance in Older People With Peripheral Artery Disease: The COCOA-PAD Pilot Randomized Clinical Trial. Thank you for joining me.

Mary McDermott: Oh, it's a pleasure to be here.

Cindy St. Hilaire: Before we dig into the details and the nitty-gritty of the study, could you maybe first explain to us what peripheral artery disease is and perhaps maybe why it's so pernicious?

Mary McDermott: Sure. So peripheral artery disease is atherosclerosis of the arteries that supply the legs, and it is a problem because it causes great difficulty with walking. People with peripheral artery disease, or PAD, can typically walk only at one or two blocks before they have to stop because of symptoms or weakness or pain or tightness in their lower extremities, in their legs. And it's also difficult to treat because we have very few medical therapies available that are effective.

Cindy St. Hilaire: So this is really something that you only know it's there until you're feeling the adverse symptoms.

Mary McDermott: That's correct.

Cindy St. Hilaire: That sounds very difficult to treat clinically. This study was called the COCOA-PAD study, and it was a double-blind pilot, randomized clinical trial, and it was designed to test the hypothesis that daily cocoa consumption for about six months improves or prevents the decline in something called the six-minute walk distance test. So my first burning question is, is it okay to eat a lot of chocolate every day? And then my second question is, what is the significance of the six-minute walk test? And maybe you could tell us a little bit about this trial's design.

Mary McDermott: Sure. Maybe I'll go in reverse order for those questions.

Cindy St. Hilaire: Sure.

Mary McDermott: So the trial design, it was a randomized clinical trial. 44 participants with peripheral artery disease were randomized to receive either the cocoa beverages, we asked them to take three a day, or a placebo control, which was very much like the intervention except that the placebo did not have cocoa or cocoa flavanols. And participants were followed for six months, and at baseline and six-month follow-up, we measured the six-minute walk test, and we also did muscle biopsy on those who consented to that, and we also measured lower extremity perfusion with MRI.

Now, the six-minute walk test is a test that's very well-validated in patients with peripheral artery disease, and it's really a measure of walking endurance. The way you conduct it is you need a hundred-foot hallway. We use standardized instructions. We actually use a script where the research assistant reads the script with the instructions, and the goal of the test is for the participant to walk as many lengths as they can in the six minutes. And often what you see in people with peripheral artery disease is they start out fine, but after maybe a few hundred-foot lengths, they start to slow down or they start to limp, and many of them cannot finish the six-minute walk without having to stop and rest. If they need to stop, then they can start walking again.

Cindy St. Hilaire: That's interesting. Is it a fatigue or is it a pain, or what is prompting them to stop?

Mary McDermott: It's symptoms in the legs or the hips classically, and it can be either of those symptoms that you mentioned. It may be a fatigue or a weakness. Some people will say, "I don't have pain. My legs just get weak, and I can't keep walking." Others will have pain. Many will have tightness or burning. And it's interesting, some people will get symptoms mainly in their feet or ankles, others will get it classically in the calves, but many will get it in the hips. And the location depends in part on the location of the atherosclerosis and where they're experiencing the ischemia.

Cindy St. Hilaire: Interesting. And so ultimately this is due to the atherosclerotic plaque blocking blood flow?

Mary McDermott: Exactly. Right. So when they go to walk, their muscles are not getting an adequate oxygen supply, and that causes these symptoms or weakness in the legs.

Cindy St. Hilaire: Interesting. What's so special about cocoa, and what are these flavonoids that you mentioned?

Mary McDermott: Sure. Cocoa actually comes from the cacao plant, and in that plant there's cocoa, but also something called cocoa flavonoids, and this is a nutritional substance. And there's a variety, but in cocoa, epicatechin is the most prevalent flavonoid.

And flavonoids have health benefits that include improving blood flow by causing dilation of vessels, but also, they've been shown to have favorable effects on muscles, skeletal muscle. And so this is particularly potentially helpful in peripheral artery disease because, obviously, patients with PAD have difficulty with blood flow because of those atherosclerotic blockages, but also they've been shown to have skeletal muscle abnormalities, probably because their leg muscles aren't getting enough oxygen. So they develop loss of muscle mass, they develop mitochondrial dysfunction and other abnormalities in their muscle that also make it hard for them to walk. So cocoa and cocoa flavonoids are an attractive therapy in PAD because they both can improve blood flow and improve the health of the skeletal muscle in the legs.

Cindy St. Hilaire: Interesting. So it's kind of a two-pronged approach to possibly helping these patients. What was the scientific evidence out there that the flavonoids or maybe even just dark chocolate may be beneficial, and how was your study different from other studies?

Mary McDermott: Some of the evidence comes from animal studies where it's clearly been shown to improve skeletal muscle mitochondrial activity and muscle growth and also blood flow. But there were also some preliminary studies in humans, a couple of them really small sample sizes of patients with heart failure, showing improvements in skeletal muscle health. But there was one trial published about five years ago in patients with PAD where the PAD patients were given one dose of dark chocolate or one dose of milk chocolate, and that one dose helped them achieve increased walking distance on a treadmill about two to three hours later. But to our knowledge, no prior studies had tested whether a daily dose of cocoa could improve six-minute walk or improve skeletal muscle or blood flow.

Cindy St. Hilaire: So should I eat chocolate every day?

Mary McDermott: Well, there's a couple of important things about chocolate. First of all, most of the chocolate that you can buy at the store is not the type we used in our study. Oftentimes chocolate is alkalized, and what that does is it makes it taste better, but it also removes some of those cocoa flavonoids that are thought to be responsible for the health benefits.

The cocoa that we studied was rich in the cocoa flavanols. It had not been alkalized, and it was more of the dark chocolate. So if you want to eat it for health benefits, you need to read the label, and it should tell you whether the chocolate has been alkalized.

The other thing to take note of is, of course, many forms of chocolate come with a lot of calories or sugars, so that can be problematic if it leads to weight gain. The chocolate that we used in our study and the placebo added about 180 calories per day to the diet, and prior to starting the study we did a little bit of diet counseling with all the participants, and we helped them identify drinks or foods they were eating that maybe could be removed so that they could take the 180 calories without gaining weight. And we did not find weight gain in either group in this study.

Cindy St. Hilaire: That's good. That's good. One of your results I found interesting was that it showed that this daily supplementation of cocoa in the diet improved the six-minute walk test at a timeframe that was shortly after the chocolate dosing, but not 24 hours after. Can you maybe talk about that result and what the implications for that mean?

Mary McDermott: Sure. Because of that prior trial that I mentioned, which indicated that cocoa had an acute effect, we were interested in separating out the acute and the chronic effects. So we did two six-minute walk tests at six-month follow-up. The first was performed two and a half hours after the final cocoa dose, and the second was performed 24 hours later.

And we saw the biggest benefit at the time point that was two and a half hours after the final cocoa dose. The benefit was about 42 meters favoring the cocoa intervention. When participants came back 24 hours later, the difference between the intervention and the placebo was only 18 meters, and that didn't quite achieve statistical significance in our primary analyses.

Now, we were a little surprised by the difference in those findings. One possible explanation is that cocoa has both the chronic and acute benefit and that first measurement reflected both the acute and the chronic benefits. So that's one possible explanation.

Another is, interestingly, we found that the placebo group had a bit of a learning effect between the two-and-a-half-hour time point and the 24-hour time point, and it's possible that that explained the diminishment in the difference of the 24-hour time point. But we didn't see that learning effect in the cocoa group, so that didn't quite make sense.

In my mind, the best explanation is there may be both an acute and a chronic effect, and we saw the benefit of both of those at that first time point.

Cindy St. Hilaire: Interesting. And a learning effect, by that you just mean the patients just learned to do the test better?

Mary McDermott: Yeah. So they got more comfortable with it between the first and the second measurement, which were just 24 hours apart. Prior study in peripheral artery disease patients has not shown a learning effect. But in the prior study, the six-minute walk tests were performed one or two weeks apart. And to my knowledge, no one's ever tested it just 24 hours apart.

Cindy St. Hilaire: Interesting. Very interesting. What was really the most challenging aspect of this study? Can you talk about some of the limitations also?

Mary McDermott: Sure. I'd say the biggest limitation was the sample size. This was a pilot study. It's not a definitive result. There were 44 people, so that is certainly a limitation. And perhaps related to that, we did see some imbalances at baseline between the two groups in terms of BMI and prevalence of African-Americans between the two groups. Our analyses do adjust for those differences to try to overcome that potential difference.

With regard to challenges, well, recruiting for studies of peripheral artery disease is always a challenge because the patients are limited in their own mobility, and it can be hard for them to come in for the study visits. A study like this requires multiple visits at baseline and follow-up.

Another potential challenge is that the adherence rate was about 64% in the intervention group versus closer to 80% in the placebo group. We don't know-

Cindy St. Hilaire: Oh, interesting. What do you think that is?

Mary McDermott: We don't know exactly why. It's possible that the cocoa intervention had a different taste and maybe was not as palatable, but since participants only had their own drink, we didn't ask them to compare, and we can't say that for sure. That could've been just due to chance.

Cindy St. Hilaire: Sure. Wow. Well, hopefully, a future study can help figure that out. Speaking of that, what would be next really in terms of kind of translating this study into either a bigger study or really translating it to the clinic? What do you see for this moving forward?

Mary McDermott: Well, couple things. I think most immediately, because there are so few therapies for peripheral artery disease and because cocoa has essentially no side effects with the caveat being the potential for weight gain, that it would be reasonable to recommend it to patients who are really symptomatic and can't seem to get better with standard options such as exercise or maybe in addition to exercise.

But I do think before we can reach a definitive conclusion, a definitive trial is needed. We have applied, we have submitted a grant application to obtain funding to do a larger study, but we'll need to wait and see how that goes.

Cindy St. Hilaire: Well, hopefully, that gets funded because I would love any excuse to eat a little more chocolate, even if it's non-alkalized. Well, great. Well, thank you so much for joining me today, Dr McDermott. This is a wonderful study, and I wish you the best of luck on that next funding to do a larger study.

Mary McDermott: Well, thank you so much. I really appreciate your interest in this work. Thank you.

Cindy St. Hilaire: That's it for highlights from the February 28th and March 13th issues of Circulation Research. Thank you so much for listening.

This podcast is produced by Rebecca McTavish, 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. Thank you to our guest, Dr Mary McDermott. I'm your host, Dr Cindy St Hilaire, and this is Discover CircRes, your source for the most up-to-date and exciting discoveries in basic cardiovascular research.

This month on Episode 9 of the Discover CircRes podcast, host Cindy St. Hilaire highlights four featured articles from the January 31 and February 14, 2020 issues of Circulation Research and talks with Dr Joe Miano and DrThomas Quertermous about their article Coronary Disease-Associated Gene TCF21 Inhibits Smooth Muscle Cell Differentiation by Blocking the Myocardin-Serum Response Factor Pathway.

Article highlights:

Wang, et al. Multi-Omics Integration Study of AF

Heianza, et al. Antibiotics and Risk of Mortality

Dikalova, et al. Sirt3 Reduces Hypertension and Vascular Dysfunction

Hu, et al. Lipid Overload Acetylates Drp1 in the Heart

Transcript Dr St. Hilaire: Hi, welcome to Discover CircRes, the podcast of the American Heart Association's journal, Circulation Research. I'm your host, Dr Cindy St. Hilaire, and I'm from the Vascular Medicine Institute at the University of Pittsburgh.

Today I'm going to share with you four articles that we selected from the January 31st and February 14th issues of Circulation Research. I'm also going to have a discussion with corresponding authors, Drs. Joe Miano and Thomas Quertermous about their study on the role of TCF21 and smooth muscle cell lineage specificity in coronary artery disease. So first, the highlights.

The first article I'm sharing with you is titled, Integrative Omics Approach to Identifying Genes Associated with Atrial Fibrillation. First author is Biqi Wang, and the corresponding author is Honghuang Lin from Boston University School of Medicine in Boston, Massachusetts. Atrial fibrillation, or Afib, is the most common form of heart arrhythmia and in the US alone there's somewhere between three and six million individuals with this condition. AFib can be either idiopathic or inherited, and genome-wide association studies, or GWAS studies, have identified hundreds of genetic loci that are linked to AFib. However, these loci explained only a small percentage of inherited cases. This suggests that there are many more AF related genes yet to be discovered.

To try and identify these elusive a-fibrillated loci, this study integrated data from previously performed transcriptome, epigenome and GWAS studies. The TWAS and EWAS, as the transcriptome and epigenome-wide studies are short-handedly called, was collected from more than 150 Afib patients, and over 2,000 control individuals. While existing GWAS data, that's genomic data, was collected from tens of thousands of AFib and control participants. By combining and analyzing the data from these TWAS, EWAS, AND GWAS studies, the team was able to identify an additional 1700 genes that were associated with AFib. Now this is compared to the original 206 loci that were identified by the GWAS studies alone.

Many of these new genes are involved in cardiac development as well as the regulation of the heart and the muscle cells. The additional gene hunting power afforded by co-analyzing multiple Omics data is not only helpful for approaching AFib but is really setting a platform upon which future studies might be done to provide novel insights for numerous other diseases of complex ideology.

The second article I will highlight is titled, Duration and Life-Stage of Antibiotic Use and Risks of All-Cause and Cause-Specific Mortality, a Prospective Cohort Study. The first author is Yoriko Heianza, and the corresponding author is Lu Qi from Tulane University in New Orleans, Louisiana.

So microbiome is a word that is used to describe all of the microbes; the bacteria, the fungi, the protozoa, and the viruses that live on and also live inside the human body. And how our microbiome influences human health as well as disease state is a new and hot research topic.

Alterations to the gut microbiome have been suggested to influence the risk of developing certain chronic diseases, including cancer and cardiovascular disease. There are many factors that influence the constituents of the gut microbiome; things such as your diet, your environment, your stress level, but another factor that can significantly alter the gut microbiome is the use of antibiotics. So there's preliminary evidence that suggests long-term antibiotic use may be linked to increased mortality in adult women, and now this study defined that link in more detail.

The authors performed a large-scale population study of antibiotic use in middle aged and older women with a follow up period of 10 years. Over 37,000 women who were in middle age or in late age at the start of the study show that long durations of antibiotic use, which was defined as using antibiotics over two or more months, was associated with increased risk of all-cause mortality and of cardiovascular disease-related mortality in late adulthood, even after adjusting for risk factors such as age, lifestyle, diet and obesity. While no such association was apparent in middle-aged women, the risk for older women was more pronounced if they had also used antibiotics during middle life. And middle life is defined as between the age of 40 and 59 years of age.

This suggests that risk of mortality due to antibiotic use may be cumulative. While antibiotics are unquestionably beneficial for saving lives, the link is not necessary causative, and the results indicate a potential risk may exist that could be factored into prescription decisions. Obviously, there's much more details that need to be worked out, but this is quite a provocative study. While antibiotics unquestionably saved lives and the link is not necessarily causative, the results indicate a potential risk may exist that could be factored into prescription decisions.

Moving to a metabolism theme, the next article I want to share with you is titled, Mitochondrial Deacetylase Sirt3 Reduces Vascular Dysfunction and Hypertension While Sirt3 Depletion in Essential Hypertension Is Linked to Vascular Inflammation and Oxidative Stress. The first author is Anna Dikalova and the corresponding author is Sergey Dikalov, and the work was completed at Vanderbilt University.

Hypertension affects about a third of the global adult population. That's a huge number of individuals. It's a risk factor for stroke, myocardial infarction and heart failure. Although blood pressure lowering treatments are widely available, hypertension remains uncontrolled in about 30% of patients who are on those treatments. A thorough understanding of the complex pathophysiology of the condition would facilitate the search for much needed alternate treatments for this third of patients with hypertension.

To that end, these investigators studied the role of Sirt3, which is an enzyme that tends to be at the lower than usual levels in blood vessels of patients with hypertension. Sirt3 regulates metabolic and antioxidant functions, and alterations in either of these functions can contribute to cardiovascular disease and vascular dysfunction. The team showed that mice genetically engineered to over express Sirt3 had healthier blood vessels and lower blood pressure than control animals who were subjected to experimentally induced hypertension. By contrast, Sirt3 depletion was shown to cause vascular inflammation and increased signs of vascular aging in mice. The team also confirmed that humans with hypertension exhibit low levels of Sirt3; however, the mechanism causing Sirt3 to be low in certain people is not clear. These data suggest that boosting Sirt3 may be potential therapy for hypertension; however, of course, more studies must be conducted to thoroughly investigate this.

The last article I want to share with you before we switch to our interview is titled, Increased Drp1 Acetylation by Lipid Overload Induces Cardiomyocyte Death and Heart Dysfunction. The first author is Qingxun Hu and the corresponding author is Wang from the University of Washington School of Medicine in Seattle, Washington.

In the heart, fat molecules are the main energy source. However, excessive lipids caused from diet induced dyslipidemia, AKA eating too much fat, can lead to cardiomyocyte dysfunction. It's known that lipid overload in the heart can cause increased activity of dynamin-related protein one, or Drp1. Drp1 is an enzyme that regulates mitochondrial fission, but exactly how Drp1 becomes activated due to lipid overload is entirely unclear. The authors of this paper confirmed that Drp1 activity and mitochondrial fission are abnormally increased in the hearts of mice fed a high-fat diet, and these mice also exhibit signs of heart dysfunction. They show similar effects in monkeys who were fed a high-fat diet. Interestingly, Drp1 mRNA was not altered in the hearts of mice. However, Drp1 protein acetylation was increased. So this suggests post-translational modifications are regulating its activity in dyslipidemia.

The team went on to perform experiments on cultured rat cardiomyocytes, and they found that incubation with saturated fatty acid palmitate led to the acetylation of Drp1, and thus its activation. And this activation resulted in an excess of mitochondrial fission, which reduced cell viability. By contrast, mutation of Drp1 to prevent its acetylation protected the cells. Together, the results reveal the mechanism of how dyslipidemia can contribute to heart cell dysfunction. Further, this data suggests that Drp1 activity or acetylation state could be novel targets for treating obesity-related heart disease.

Okay, we're now going to switch over to the interview portion of the podcast. I have with me Dr Thomas Quertermous, the William G Erwin Professor of Medicine and the Director of Research in the division of cardiovascular medicine at Stanford University. And Dr Joe Miano, Professor and Jay Harold Harrison Distinguished University Chair in vascular biology at the Medical College of Georgia at Augusta University. And today we're going to be discussing their manuscript titled Coronary Disease Associated Gene TCF21 Inhibits Smooth Muscle Cell Differentiation by Blocking the Myocardin-Serum Response Factor Pathway. So welcome to both of you. Thank you for joining me.

Dr Miano: Thank you.

Dr Quertermous: Thank you.

Dr St. Hilaire: So I'm going to start with you, Dr Quertermous. You've been taking a genomics approach to identify factors that contribute to coronary artery disease. And I'm wondering if you could just give us a brief summary of your work thus far and how it brought you to this current study?

Dr Quertermous: Well, as you know, the classical risk factors for coronary artery disease and vascular disease in general really only contribute about 30% of the total risk and the remainder has not been studied, and not been investigated, and can't currently be targeted by therapeutics. So the goal is to try and better understand what are the molecular mechanisms in the blood vessel wall that must contribute the remainder of the risk. And so with the advent of genome-wide association studies and the identification of genes and loci, we've been able to begin to uncover the signaling pathways and mechanisms of disease risk.

Dr St. Hilaire: And so the one we're most interested in today, this TCF21, you pulled that up out of one of your GWA studies, or how did we get to this?

Dr Quertermous: Well, it's an interesting story. I first cloned that gene about 15 years ago when I was trying to understand vascular development, and it's a basic helix loop helix factor, and I was, well, a number of labs at that point in time were cloning this class of transcription factor to try and better understand developmental processes, and so it was one of a number of bHLH proteins that we cloned at the time. I did some work on it and then named it Pod-1 at that point in time, and then lost interest, and went away from it.

And then I was involved in the cardiogram genome-wide association study for coronary artery disease, and I was sitting at my desk one night, and I was watching the hits coming in, you know, as we were doing the association, as we were doing the analysis, and I saw this gene, TCF21, and I thought, "Well, I don't really know what the heck that gene is." And so I was going back and forth between our data and a spreadsheet on the web, and I saw that I had published on this gene, and I was like, "Wow, I didn't even know that I had written a paper about this gene." And then it became clear that it was the bHLH factor I'd cloned some time ago. And then knowing what I knew about the development, that this gene is involved in early processes that lead to the formation of the coronary artery, and in particular the development of smooth muscle cells, then I became super interested, and I said, "Okay, my gene, I'm coming back to you. You and me are going to have a great future together." And that was really how I got started.

Dr St. Hilaire: It re-found you.

Dr Quertermous: It found me, I guess in this case, yes, and so I began then to work very seriously, because it's hard to try and understand mechanisms. And so we had a good starting point. We had a transcription factor so I could quickly identify the targets downstream of that, and I can link it into some cell biology that I already had some insights into.

Dr St. Hilaire: That's a really neat story. I like that. It's kind of penicillin-esque.

Dr Quertermous: Thank you.

Dr St. Hilaire: Dr Miano, those of us familiar with smooth muscle cells appreciate that they are plastic, that they have this ability to kind of switch their phenotypes per se, and those of us familiar with that also then know about the myocardin and serum response factor pathway. But for our listeners who are less familiar with that, could you maybe give a brief background about what myocardin SRF pathway is and what smooth muscle cell phenotype modulation is?

Dr Miano: Sure. I wish I could say, as my colleague said, that I cloned one of those factors, but I didn't. I've been interested in SRF since I was a graduate student actually. Actually went to Eric Olson's lab to look for what we affectionately called back then SmyoD, which stands for smooth muscle myo D. So at that time, we didn't understand what the factors were, even the signaling, that directed cells to become differentiated smooth muscle cells. So I went to Eric's lab looking for SmyoD. Of course I didn't find it. I found some other things. Worked a little bit on SRF, but it was actually Daiju Wong in 2000 or 2001 who in a Cell paper described an elegant a way of finding myocardin, what he called myocardin. So SRF myocardin is a transcriptional switch that is necessary and sufficient to make just about any cell a smooth muscle cell.

So when myocardin is not present, then smooth muscle cells lose their differentiated state and they become another cell type, depending on who SRF talks to. And so how does a factor that binds a very discrete element like the CArG box, how does it confer cell identity or specific cell states? And it does so through its interaction with these cofactors, one of which is myocardin. And as this paper describes so elegantly, what Tom did in his lab, is that this TCF1 transcription factor, which is DNA binding, unlike myocardin, it does a similar thing in that it competes for SRF binding with myocardin, so it binds myocardin, prevents myocardin's ability to bind SRF, and thereby directs a new program of gene expression.

Dr St. Hilaire: Interesting. So it's kind of helping to fine tune that transcriptional regulation. So I always think of smooth muscle cells, they're kind of always in a contractile state when they're healthy, and it's when they're in either unhealthy, or diseased, or a stress state that they're in that more proliferative-like state. And Dr Quertermous, your previous studies have shown that TCF21 is required for the De-differentiation, and proliferation, and migration of smooth muscle cells. However, there was one sentence in the paper that I was slightly confused on and I'm hoping that you can expand about the bigger role of TCF21. And what it said was that TCF21 expression is protective towards human coronary artery disease. And so the data in the paper show that TCF21 inhibits smooth muscle cell contractility. So can you maybe reconcile the bigger mutations or things you identified in the GWAS with the functional activities you're seeing that you presented in the paper, and maybe talk about the timeline in the continuum of atherosclerosis where TCF is maybe good or maybe bad?

Dr Quertermous: So this paper is one of a duo of papers, honestly, that the other paper being published in Nature Medicine almost exactly the same time, and so that paper sort of described some of the aspects of TCF21 at a population level and shows that if you look at all of the single base pairs in the genome that regulate disease risk at 6q23.2 and also regulate expression, you can gain an idea of what's the directionality of the expression of TCF21. And those data suggests that the more TCF21 you have, the less your risk of developing coronary artery disease. And Joe and other scientists have worked for a long, long time to characterize this process and characterize the plasticity of this cell type. And note that one can switch the cell back and forth between being a contractile0differentiated cell to a de-differentiated cell, and elegant work by Gary Owens and a number of investigators have profiled the phenotype of the cells that the smooth muscle cell can become if it undergoes this differentiation process.

It's not been able to know though up until this point in time whether that's a good process or a bad process. I mean, 15, 10 years ago we thought smooth muscle cells are proliferating, they're creating a space-occupying lesion, they're decreasing the lumen of the blood vessel, and that's got to be a bad thing. And in honesty, I think over the past three or four years, it's been increasingly clear that perhaps the smooth muscle cells are actually doing a good thing. They are stabilizing the lesion, they're creating the fibrous cap, and there's been some nice work correlating the number of smooth muscle cells in the plaque to the risk that that plaque is going to rupture.

Dr St. Hilaire: Yeah, that was kind of my next question. Do you think there's more nuance to it's not just contractile, and synthetic? There's much more broader scope and it's not so much a good or a bad smooth muscle cell.

Dr Quertermous: I think it depends on the circumstances I guess, but it's important that the smooth muscle cell be able to migrate into the plaque, and begin to produce matrix components which stabilize the plaque, and to form the fibrous cap, and I think if the smooth muscle cell remains in the media as a contractile cell, it's really not able to do those things, right? And so the human genetics data, looking at the directionality, the expression, the different alleles and their expression patterns, and what is the risk allele at? In this region of the genome, it's pretty clear that more TCF21 is good, and what TCF21 does is to promote this phenotypic modulation.

And so that suggests that the process as a whole is good. Not just the gene, but what it does. It's really not possible that TCF21 does anything else in the blood vessel wall. It's primarily restricted to the smooth muscle cell. It's not expressed in macrophages, or endothelial cells, or the other cell types that we think are important in the pathophysiology of the disease process. So putting everything together, it looks for the most part, like this is a positive force in the blood vessel wall, this gene and this process.

Dr St. Hilaire: Interesting. And speaking to the vessel wall, I thought one of the very cool and really key experiments in the paper was taking your mechanistic in vitro studies into the mouse, and Dr Miano, maybe you could tell us a little bit about how you were able to do that and mutate these smooth muscle specific CArG boxes in a mouse model.

Dr Miano: Well, that's a really good question. Again, it's a history. We've wanted to edit CArG boxes, well, mutate back then, for a long, long time, but it wasn't until the CRISPR craze took a foothold that we really recognize now the power of harnessing that and doing the experiments we wanted to do for so long. And so we've previously published on a CArG box in the first intron of the calponin locus, and found that, to our surprise, that a subtle mutation in that element completely abolished expression of calponin into the smooth muscle. So Tom and I were working in parallel and unbeknownst to me, Tom was working on this SRF enhancer in the second intron, and we've known for quite a while that SRF is auto regulated by itself, and there's CArG boxes in it 5-prime promoter, and there's CArG boxes in the interior of the locus as well, including the one in the second intron that Tom describes in the paper here. And so what we've been doing is using CRISPR in the mouse to make these subtle edits in these CArG boxes around the SRF locus. And unlike the affirmation calponin model I just described, if we mutate the two proximal CArG boxes of SRF, we don't see a lot of change in SRF expression. That was really surprising to us, because studies from Bob Schwartz' lab in Houston two decades ago showed those were important, at least in an artificial reporter assay for the autoregulatory loop that he first described.

So we moved interior to this CArG box that's really the focus of this paper, highly conserved, much more so than other CArG boxes, and we first deleted the region, which we often do with CRISPR, and found there was a decrease. But we'd like to do more subtle things with CRISPR, which is really the power of this new editing technology. And so we went in and made just, I think it was like four or five base substitutions to create a novel restriction cipher ease in genotyping. And we reported in the paper, you can see that, to compliment Tom's group's data, that in vivo, indeed, that CArG box by itself, nothing else altered within the locus, did cause a, I would call, a substantial decrease in expression of this important regulator. And so that was really our main contribution to this paper.

Dr St. Hilaire: Yeah. I know the opportunities are endless, but also complicated and expensive, and I thought this was a beautiful addition to really confirming those mechanistic studies. So I think my next big question is, if TCF21 is, so important and protective, and perhaps it's upregulation is beneficial, what is regulating it, and do we know how we can potentially modulate this?

Dr Quertermous: That's a great question. That's a great question. And so we know a few things; we certainly know that platelet-derived growth factor stimulation of smooth muscle cells will upregulate TCF21, which is sort of surprising. I mean, it's not so surprising, I guess. So we've spent a little time working on that, and there's a micro RNA which regulates the expression level of TCF21, but we haven't spent a lot more time than that, honestly. We spent a lot more time downstream trying to figure out what's the mechanism by which TCF21 works to suppress the smooth muscle contractile phenotype and activate this more de-differentiated migratory phenotype that the smooth muscle cell adopts.

So we've not gone upstream, but your question's a really, really good one. We certainly mapped where TCF21 binds across the genome and we've mapped the variation that regulates its expression, and so we've made progress in that direction, and as I said, identified things which are downstream. But we definitely need to spend more time upstream, and I think that's the area of this intersection of molecular science and genomic science, that there are not many groups that really spend much time up above the gene trying to understand. And so we've not spent enough time doing that, and I think that as a community we've not spent enough time doing that, because I think that's where the big payoff can come in terms of therapeutics.

Dr St. Hilaire: To that end, I think I'll end with that question. What do you think is the best way that we could leverage your findings in the clinic? Would it be to focus more on the downstream or to try to identify these more upstream factors in TCF21?

Dr Quertermous: Well, I think both open up opportunities, right? If we can understand how TCF21 works and what's downstream, and we can activate those processes and activities, then that's good. If we can figure out what's above TCF21, that would be good as well. The danger there is that TCF21 does a lot of things in a lot of different cells in the body.

Dr St. Hilaire: So it'd be a little bit harder to focus onto a smooth muscle cell in a plaque than perhaps some of the downstream effects of TCF21?

Dr Quertermous: Correct. Right. That's my worry. It's sort of like thinking about TGF beta and you wouldn't really want to try and manipulate TGF beta.

Dr St. Hilaire: That's a whole another can of worms.

Dr Quertermous: Yeah, it gets you into a lot of difficulties, I think. So we're really pretty focused downstream now and thinking that we can find specific opportunities there that are resident in that smooth muscle cell in the blood vessel that may not be active in other cell types. So that's really our thinking and that's the way we're going.

Dr St. Hilaire: Wonderful. Well, thank you so much to both of you for joining me today. I learned a lot and I really thought this was a beautiful, complex, but well-done study, so thank you very much.

Dr Miano: Thank you, Cindy.

Dr Quertermous: Thank you so much for calming us down, I guess.

Dr St. Hilaire: Well, that's it for our highlights from the January 31st and February 14th issues of Circulation Research. Thank you so much for listening. This podcast is produced by Rebecca McTavish, edited by Melissa Stoner, and supported by the Editorial team of Circulation Research. Some of the copy texts for the highlighted articles was provided by Ruth Williams. Thank you to our guests, Drs Thomas Quertermous and Joseph Miano. I'm your host, Dr Cindy St. Hilaire, and this is Discover CircRes, your source for the most up-to-date and exciting discoveries in basic cardiovascular research.