August 2022 Discover CircRes

This month on Episode 39 of Discover CircRes, host Cynthia St. Hilaire highlights four original research articles featured in the August 5th and 19th issues of the journal. This episode also features an interview with Dr Annet Kirabo and Dr Ashley Pitzer from Vanderbilt University on their article, Dendritic Cell ENaC-Dependent Inflammasome Activation Contributes to Salt-Sensitive Hypertension.   Article highlights:   Jain, et al. Role of UPR in Platelets   Orlich et al: SRF Function in Mural Cells of the CNS   Xue et al: Gut Microbial IPA Inhibits Atherosclerosis   Wang et al: Endothelial ETS1 on Heart Development   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'm going to be highlighting articles from our August 5th and August 19th issues of Circulation Research. I'm also going to have a chat with Dr Annet Kirabo and Dr Ashley Pitzer from Vanderbilt University about their study, Dendritic Cell ENaC-Dependent Inflammasome Activation Contributes to Salt-Sensitive Hypertension.   But before I get to the interview, I first want to share an article from our August 5th issue, and that article is titled, Unfolded Protein Response Differentially Modulates the Platelet Phenotype. The first author of this study is Kanika Jain and the corresponding author is John Hwa from Yale University. Self-stress can lead to protein misfolding, and the accumulation of misfolded proteins can lead to a reduction in protein translation and may alter gene transcription, a process collectively known as the unfolded protein response, or UPR. UPR is well documented in nucleated cells; however, it has not been studied in platelets, which are anuclear, but do have a rapid response to cellular stress. In this study, they investigated the UPR in anucleate platelets and explore its role, if any, in platelet physiology and function.   They found that treating human and mouse platelets with various stressors caused aggregations of misfolded proteins and induction of UPR-specific factors. Oxidative stress, for example, induced the UPR kinase PERK, while an endoplasmic reticulum stressor induced the transcription of the UPR factor XBP1. The team went on to study the UPR in platelets from people with type II diabetes, which is a population in which platelet mediated thrombosis is a major complication. They showed that protein aggregation and upregulation of the XBP1 pathway in diabetic patient platelets correlated with disease severity. Furthermore, treating the diabetic patient platelets with a chemical chaperone that helps to correct protein misfolding reduced protein aggregations and prevented the cells prothrombotic activation. This work confirms that even without transcription, platelets display stress-induced UPR, and that targeting this response may be a way to reduce thrombotic risk in diabetic patients.   Cindy St. Hilaire:        The second article I want to share with you is from our August 5th issue and is titled, Mural Cell SRF Controls Pericyte Migration, Vessel Patterning and Blood Flow, and it was led by Michael Orlich from Uppsala University in Sweden. Blood vessels are lined with endothelial cells and surrounded by mural cells. Vascular smooth muscle cells are the mural cells in the case of veins and arteries, and pericytes are the mural cells in the case of capillaries. In the capillaries, pericytes maintain blood-brain and blood-retina barrier function and can mediate vascular tone, similar to smooth muscle cells. While these pericytes and smooth muscle cells are related, they have distinct roles and characteristics.   To learn more about the similarities and the differences between pericytes and smooth muscle cells, this group examined how each would be affected by the absence of SRF in the other. SRF is a transcription factor, essential for nonvascular or visceral smooth muscle cell function. In visceral smooth muscle cells, SRF drives expression of smooth muscle actin and other smooth muscle genes. Using mice engineered to lack SRF in mural cells, they show that SRF drives smooth muscle gene expression in these pericytes and smooth muscle cells, and its loss from smooth muscle cells causes atrial venous malformations and diminishes vascular tone. In pericytes, loss of SRF impaired cell migration in angiogenic sprouting. In a mouse model of retinopathy, activation of SRF drove pathological growth of pericytes. This work not only highlights the various functions of SRF in mural cell biology, but it also suggests that it has a role in pathological capillary patterning.   Cindy St. Hilaire:        The third article I want to share is from our August 19th issue of Circulation Research and is titled, Gut Microbially Produced Indole-3-Propionic Acid Inhibits Atherosclerosis by Promoting Reverse Cholesterol Transport and its Deficiency Is Causally Related to Atherosclerotic Cardiovascular Disease. The first authors are Hongliang Xue and Xu Chen, and the corresponding author is Wenhua Ling from Sun Yat-Sen University in Guangzhou, China. Recent studies provide evidence that disorders in the gut microbiota and gut microbiome derived metabolites affect the development of atherosclerosis. However, which and how specific gut microbial metabolites contribute to the progression of atherosclerosis and the clinical relevance of these alterations remain unclear. Gut microbiome derived metabolites, such as short-chain fatty acids and trimethylamine N-oxide, or TMAO, have been found to correlate with atherosclerotic disease severity.   This study has now found that serum levels of indole-3-propionic acid, or IPA, are lower in atherosclerosis patients than controls. The team performed unbiased metagenomic and metabolomic analyses on fecal and serum samples from 30 coronary artery disease patients and found that, compared with controls, patients with atherosclerosis had lower gut bacterial diversity, depletion of species that commonly produce IPA and lower levels of IPA in their blood. Examination of a second larger cohort of atherosclerosis patients confirmed this IPA disease correlation. The team also showed serum IPA was reduced in a mouse model of atherosclerosis, and that supplementing such mice with dietary IPA could slow disease progression. Analysis of the macrophages from these mice showed that IPA increased cholesterol efflux, and the team went on to elucidate the molecular steps involved. The results of this study not only unraveled the details of IPA's influence on atherosclerosis, but suggest boosting levels of this metabolite could slow atherosclerotic disease progression.   Cindy St. Hilaire:        The last article I want to share is also from our August 19th issue, and it's titled, Endothelial Loss of ETS1 Impairs Coronary Vascular Development and Leads to Ventricular Non-Compaction. The first author is Lu Wang and the corresponding author is Paul Grossfeld, and they are at UCSD. Congenital heart defects, or CHDs, are present in nearly 1% of the human population. In some cases, the heart defects result from a genetic error, which can give researchers clues to its etiology. Jacobson syndrome is a complex condition caused by deletions from one end of chromosome 11, and the occurrence of a congenital heart defect in this syndrome has been associated with the loss of the gene ETS1. ETS1 is an angiogenesis promoting transcription factor, but how ETS1 functions in heart development was not known.   Wang and colleagues now show that both global or endothelial-specific loss of ETS1 in mice caused differences in embryonic heart development that ultimately led to a muscular wall defect known as ventricular non-compaction. The mice also had defective coronary vasculogenesis associated with decreased abundance of endothelial cells in the ventricular myocardium. RNA sequencing of ventricular tissue revealed that, compared with controls, mice lacking ETS1 had reduced expression of several important angiogenesis genes and upregulation of extracellular matrix factors, which together contributed to the muscular and vascular defects.   Cindy St. Hilaire:        Today I have with me, Dr Annet Kirabo and Dr Ashley Pitzer, both from Vanderbilt University, and we're going to talk about their paper, Dendritic Cell ENaC-Dependent Inflammasome Activation Contributes to Salt-Sensitive Hypertension. This article is in our August 5th issue of Circulation Research. Thank you both so much for joining me today.   Annet Kirabo:             Yeah, thank you so much for having us.   Ashley Pitzer:              Yeah, thank you for having us.   Cindy St. Hilaire:        Yeah, it's a great paper. I think we're all familiar with hypertension and this idea that too much salt is bad for our cardiovascular system. When I was a kid, my grandparents had those salt replacements on their kitchen table, Mrs. Dash and whatever. But, like you said in the start of your paper, the exact mechanism by which salt intake increases blood pressure and also increases cardiovascular risk, it's not really well understood, and you guys are focusing on the contribution of immune responses in this process or in this pathogenesis. Before we dig into the details of your paper, I was wondering if you could give us a little bit of background about what's known regarding the role of inflammation in this salt-sensitive hypertension pathogenesis.   Annet Kirabo:             Yeah. It's difficult to know where begin to from, but the role of inflammation in cardiovascular disease have been known for many, many decades. Right now, Dr David Harrison showed more than 10 years ago that T cells contribute to hypertension, but the mechanisms were not known. Back when I was a post doc in David Harrison's lab, we discovered a new mechanism, how immune cells are activated in inflammation and hypertension, whereby we found that there is increased oxidative stress in antigen-presenting cells. This leads to formation of oxidative products known as arachidonic acid or lipid products known as isolevuglandin, or IsoLGs. These IsoLGs are highly, highly reactive and they adapt to lysines on proteins. This is a covalent binding, which leads to permanent alteration of proteins, and so these proteins act as neoantigens that are presented as self-antigens to T cells, leading to an autoimmune-like state in hypertension.   Annet Kirabo:             We found that these antigen-presenting cells are activated and they start producing a lot of cytokines that paralyze T cells to IL-17 producing T cells that contribute to hypertension. And so, when I started my lab back in 2016, we discovered that excess dietary salt profoundly activates this pathway, and we found for the first time that these antigen-presenting cells, they express ENaC, the epithelial sodium channel, and sodium goes into these antigen-presenting cells and activates the NADPH oxidase, which is an enzyme which produces this reactive oxygen species, leading to this IsoLG formation, which I've talked about, and leading to inflammation.   So, three years ago when Ashley joined my lab, she had extensively studied the inflammasome in her PhD program, and she suggested why don't we look at the role of the inflammasome in this pathway and how IsoLG may contribute to this. In her paper that we are discussing right now, she found that in a dependent manner, sodium enters the cell and activates this pathway, and the NLRP3 inflammasome is involved in this process.   Cindy St. Hilaire:        That's such a wonderful story that fits together so many pieces. One of the things you talk about, which I guess I didn't even appreciate myself is, there are certain individuals out there who are more salt-sensitive than others.   Annet Kirabo:             Yeah.   Cindy St. Hilaire:        What is that difference? Do we know the root cause of that? And then also, how many individuals are we talking about are salt-sensitive?   Annet Kirabo:             Salt-sensitive blood pressure, it is a variable trait and it's normally distributed in the population, but it happens more in some individuals than others. It happens even in 25% of people without any hypertension. These people go to that doctor, that doctor thinks they're normal, they don't have any hypertension, but these people can be at a risk of sudden heart attack or cardiovascular risk or even a stroke, simply because when they eat a salty meal, their blood pressure will go up.   Cindy St. Hilaire:        Yeah, that's one of my questions. How much salt are we talking about here? And not only how much in a meal, but a sustained amount? How bad is a miso soup a day?   Annet Kirabo:             Yes. The American Heart Association and the World Health Organization have recommendations. American Heart Association recommends one spoon per day. We have refused to adapt to this recommendation, but that is the recommendation that they have recommended per day to eat. But this is difficult because most of the salt, as you know, is already in our food through processing in our processed foods and we don't have any control over how much salt we have, and there's also a lot of adding of salt at a table.   Cindy St. Hilaire:        Ashley, your background was more the inflammasome. What were your thoughts entering into this project? Did you have much of a hypertension background?   Ashley Pitzer:              No. My graduate thesis focused mainly on endothelial dysfunction and cardiovascular disease, and so it was a pretty easy segue. But it was just with Annet, so excited about the project and showing me all the data and this robust IL-1 beta production that she was seeing after these immune cells being exposed to high salt, I, with my inflammasome background, was immediately like, this could be playing a role. And so it was, like I said, a pretty easy transition and, as is in the paper, we're doing human studies. All of my research back in grad school was very basic research, so it was very exciting to see how our research was being translated with people having this condition and potentially finding mechanisms where we can target this to help actual people.   Cindy St. Hilaire:        I think a lot of us who are not in the hypertension field, and maybe this was you before you joined Annet's lab, we really only kind of think of the kidneys and the blood vessels when we think about hypertension, but studies like this are changing that. And I think a lot of Annet's earlier work, as well as the work of others, have shown a role for this epithelial sodium channel as an important player in this salt-induced hypertension. New to me, it's not just found in the kidney, which I totally did not appreciate that. And it's this channel sensing the salt that can trigger this IL-1 beta production that does a whole bunch of other things.   Cindy St. Hilaire:        What are those other things? What are those cells that are affected and where is this happening? Obviously it's not just kidney cells, but is it only in the kidney or are these systemic cells? What do we think is happening?   Ashley Pitzer:              That's the question, is, where is this happening? There's been studies at Vanderbilt by Jens Titze and his lab showing, where are these immune cells sensing the salt? And so they've shown that sodium accumulates in the skin, a huge argument is for they're sensing the sodium in the kidney because that's where a lot of it is being processed. But these immune cells travel through the whole body, so they're seeing it where there are the highest amounts of sodium concentration, and so I would argue it's in the kidney.   Annet Kirabo:             Indeed, because we're now collaborating with Tina Kon, and we have recently published with her a paper in the International Journal of Science, where we have done sodium MRI and we find this accumulation of sodium in the kidney even much more than in the skin. And we know that the kidney is where sodium is highly concentrated. So the working hypothesis in the lab is that these immune cells can be activated wherever they are, in the lymph nodes or not, in other tissues, but they can travel to the kidney.   We find that in high salt, if you feed high salt to the mouse, the endothelium in the kidney becomes dysfunctional and it expresses molecules, chemoattractants, that attract these immune cells in the kidney. We think that the high salt accumulation in the kidney can activate these, and then these immune cells are activated and they produce cytokines. Dr Steve Crowley showed that they can produce IL-1 beta, which induces activation of sodium channels that can be induced. We have also actually found that even IL-17 can be produced by these immune cells in the kidney and they can activate sodium channels in the kidney, leading retention of sodium and water and hypertension.   Cindy St. Hilaire:        Very cool. You used a lot of mice in this paper. Can you tell us, I just want to know a little bit about the models you chose to use, but also how similar is hypertension in mouse and humans? Obviously for atherosclerosis, we have to do lots of things to get them to form a plaque. Is hypertension similar in a mouse and do mice also show this salt-sensitive phenotype?   Annet Kirabo:             That is an extremely important point. If you read our paper, we use a slightly different approach. Most people do benchside to bed approach. We did the opposite. We did a bed to benchside approach.   Cindy St. Hilaire:        Always smart.   Annet Kirabo:             Yeah. We first started humans, and then with some references, we went to the mice, because I think when it comes to salt-sensitive blood pressure, mice are different from humans. In fact, if we look in the lab, we find that female mice are protected from salt-sensitive blood pressure, but we find that in the humans, it's the opposite. Females are more prone to salt-sensitive hypertension. Those are studies that we are doing right now. We haven't published. But we know that it can be different.   The model we use most of the time in the lab, the C57 mice, are resistant to salt-sensitive hypertension. These C57 mice would rather die before they raise their blood pressure in response to salt. We can induce salt-sensitivity in these mice like in the paper that we are discussing. When we induce the endothelial dysfunction using L-NAME and we wash it out, then these mice, when you give them, subsequently, salt, suggests that they become salt-sensitive. But we also have a salt-sensitive mouse model that we use, the 129/SV mouse. So we use several models to kind of prove the same thing over and over again with the findings that we found in humans.   Cindy St. Hilaire:        And you used a technique, which I'm a little bit familiar with, but I'd like to hear, A, about it from you, but also your experience in using it, and that is CITE-seq. So, how does that work?   Ashley Pitzer:              That was with our human study where we actually had patients come in, who were hypertensive, took them off medication for 2 weeks. They come in, we get baseline samples, we give them a salt load on one day, and then the next day we completely salt deplete them.   Cindy St. Hilaire:        How much is a salt load? Like a Big Mac? What's a salt load?   Ashley Pitzer:              Yeah, it's pretty much just like eating Lays chips all day. It's a lot of salt. It's a very salty meal.   Annet Kirabo:             And then in addition, we also infuse saline too.   Cindy St. Hilaire:        Oh, wow.   Annet Kirabo:             Because these people, when they come into the hospital, some them have already eating high salt. This approach is to just maximize the whole system so that then when we sort deplete everybody, it's at the same level and it's just to unify the whole process. But sorry, Ashley, you go ahead.   Ashley Pitzer:              With the CITE-seq, we're able to take different patients on different days. So we take samples each day, and we can give each sample a barcode, basically. Give them a barcode, we can pool them all together, process them, and we can sequence their RNA, we can probe for a certain amount of protein expression as well. So then when we analyze, we can look at protein expression, so you get the translation and the transcription for each person on each day, and then you're able to compare. And so you get this huge picture and it's a lot of data.   Cindy St. Hilaire:        How long did it take you to sort through?   Ashley Pitzer:              Well, we have a statistician who does all of that, because my wheelhouse is here and it is on a different planet. So we have somebody who helps us with that who does an unbiased approach. And then once he does an analysis, gives us back what are the things that are changing the most, and one of those was IL-1 beta.   Annet Kirabo:             As you can see, our list is huge, this is a massive input of so many collaborators. We have computational people on there that help us with this. I can't even begin to learn these techniques, but with all this collaboration and the resources at Vanderbilt, these things are possible. And so, this is a really powerful approach where you can combine protein expression and you get the specific cells that express the genes and you couple the channel type to the gene expression.   Annet Kirabo:             We actually found that not all monocytes are the same. There's a specific class that of monocytes, A small class of monocytes that is so angry, and the inflammasome is activated and producing this IL-1 beta, and that is enough to contribute to this phenotype of salt-sensitive hypertension, which dynamically changed according to blood pressure, suggesting that this is a targetable salt-sensitive blood pressure, even in normotensive people, is a targetable trait. And because these monocytes are in blood, can we get a blood sample and routinely diagnose salt-sensitive blood pressure so that doctors are aware and they can appropriately advise patients.   Cindy St. Hilaire:        This was samples obviously taken from a blood draw, right? So they're circulating.   Annet Kirabo:             It was a blood draw, yes.   Cindy St. Hilaire:        What do you think about these immune cells, perhaps, native in the kidney? Do you think the small population of angry cells, like you said, is escaping from the kidney environment? What do you think?   Annet Kirabo:             When I was a post-doc in David Harrison's lab, we found that the most angry dendritic cells that contribute to this inflammation and hypertension are monocyte-derived. So that's why in the human study we focused on monocytes, because there are so many subtypes of dendritic cells, plasmacytoid dendritic, classical dendritic cells. We have studied all of these subtypes, and we have focused on monocyte-derived dendritic cells because they're the ones that seem to be contributing to this phenotype the most.                                     Cindy St. Hilaire:        You guys focused in on the NLRP3 inflammasome, which, obviously it's a really critical component broadly for the innate immune system. Do you think that this is going to be a targetable approach that can be leveraged for hypertension? Or do you think it's too broad? What do you think about that as a therapeutic potential?   Ashley Pitzer:              Even when you look in our paper, and we use a knockout model, where we use a completely global knockout model, put them on high salt, and we give them back only dendritic cells that are from wild-type mice, so they have that NLRP3, that have been exposed to high salt. We were able to increase blood pressure, but I also did, in mice, where I gave them an IL-1 beta neutralizing antibody, similar to canakinumab, which is the CANTOS trial, and there's not much of a difference. There is, but it's minor. It's very minor.   Ashley Pitzer:              So, to be able to target in specific cell types in humans one thing, it's very difficult, and maybe one day we can get there. But I think it at least gives us a better idea of what is the full picture, what's the big mechanism going on with immune cells? In part of our human study, we are looking at something to try and be able to identify who is salt sensitive. So if anything, we're able to sit here and potentially have a way of identifying salt-sensitive patients, where, right now, all we can do is have them come in like we do and do a 3-day study, and not everybody can do that.   Annet Kirabo:             To add onto that, perhaps you know, we are talking about precision medicine. This is an era of precision medicine where you need to really tailor treatments if we can get there, and I think this is one way. CANTOS trial. They had no way of knowing who is salt-sensitive and who is not, it was a global approach, and the lack of differences in blood pressure might be explained that this IL-1 beta pathway is targetable in a specific population whose blood pressure is probably driven by inflammation. There are so many, many mechanisms that drive hypertension, and so perhaps we need to focus this on salt-sensitive people, and maybe we can really use this approach to target. Plus, this is ENaC-dependent.   As you know, amiloride has lost favor in the clinic as a treatment of hypertension, because in the majority, it's not effective. But studies have shown that in Black men, for example, who had been categorized salt-resistant, when they give them amiloride, their blood pressure went down, and yet it's not effective in the majority of the people.   So, can we bring back, can we take another look at amiloride. As our studies indicate that blockade of ENaC is anti-inflammatory and it's also antioxidant agent, can we at least bring back amiloride and look at it again and we focus it for specific populations of people that may be more prone to salt-sensitive hypertension?   Here we have so many targets for potential precision treatment of salt-sensitive potential in this paper. You can target SGK1, which we know is possible, we listed a number of clinical trials that they have used NLRP3 inflammasome inhibitors, you can use amiloride for these people, and you can also potentially scavenge IsoLGs.     Cindy St. Hilaire:        What was the most challenging aspect of this study? There's a lot of moving parts, so what was the biggest challenge? And then, also, what was the most surprising part or the most pleasantly surprising part?   Ashley Pitzer:              You have to think, most of this was going on right when the pandemic hit. And right before that, we had started our human recruitment for the human study. And so that put a little bit of a time damper on it.   Ashley Pitzer:              Other than that, it was just, we were finding one thing, developing a new experiment, doing it again, doing it again. And honestly, what was the most surprising and rewarding was just seeing the same thing in, because we took just PBMCs from normotensive patients, treated them with high salt, and saw the changes that we did with the inflammasome. And to see that exactly again in an in vivo model of giving patients high salt and seeing the same thing, it was very rewarding and confirmed that, okay, we're on the right path. Seeing the same thing over and over and over again, it kind of reaffirms that you had a good idea.   Annet Kirabo:             I might add, one of the most challenging was, initially, the computational. Oh, part of the pandemic I was, the pandemic hit, I had a baby during the pandemic, and it was my time to leave my home, and then all these things were going on. We had a clinical trial where patients had to come in. Vanderbilt was so super supportive ,even checking for COVID-19. Our patients could not have COVID-19. We needed to check them.   Cindy St. Hilaire:        Yeah.   Annet Kirabo:             They also had to check for COVID-19. And so during that time, I realized, wait, I need learn computation analysis. I realized I cannot learn, and then reached out to collaborators that helped. That was extremely challenging. And then the other challenging thing that we faced later during the pandemic is vaccinations. In our criteria, these people cannot be vaccinated for reasons. We've studied inflammation, hypertension, and so vaccination was confounding. And even COVID-19 is even more for confounding. So we had this exclusion criteria where we could not recruit anyone.   Annet Kirabo:             Everybody was having COVID, everybody was being vaccinated, and everybody was in that exclusion criteria, so it was difficult to get people. We have had some slow down, but right now it's beginning to build up.   Cindy St. Hilaire:        So, what's next? What's the next question?   Annet Kirabo:             We have so many.   Cindy St. Hilaire:        That means it was a great study. If you have more, that means it was a great study.   Annet Kirabo:             Yeah. This study and us, it kind of warms. The inside seat just opened up, we have primary data in the genetic regulation of ENaC, we have primary data where we found. We are trying to figure out the specific ENaC channel in these antigen-presenting cells. We don't know. We found that ENaC delta, for example, it's not found in a kidney or you talked about a kidney contribution versus immune cells. ENaC delta is not found in the kidney, but we have primary data that show that ENaC delta is the most correlated with cardiovascular risk, is the most correlated with kidney disease and all forms of hypertension. So now we're like, ENaC delta expressed in the immune cells, not in the kidney, it is the one that is most involved in cardiovascular disease, so how are we going to tell the world that.   Cindy St. Hilaire:        Yeah, very cool.   Annet Kirabo:             Those cells, not necessarily the kidney. The kidney plays a part because the cells are going there, but it's very, very exciting. Plus a number of other lines that we are investigating.   Cindy St. Hilaire:        It's great. Well, congratulations, again, on this publication, on just getting all this done with what sounds like extremely difficult patient recruitment. So, Dr Kirabo and Dr Pitzer, thank you so much for joining me today and I'm looking forward to these next studies on maybe ENaC delta.   Annet Kirabo:             Thank you. Thank you so much.   Ashley Pitzer:              Thank you for having us.   Cindy St. Hilaire:        That's it for the highlights from the August 5th and August 19th 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 hashtag Discover CircRes. Thank you to our guests, Dr Annet Kirabo and Dr Ashley Pitzer.   This podcast is produced by Ashara Ratnayaka, edited by Melissa Stoner, and supported by the editorial team of Circulation Research. Some of the copy text for the highlighted articles is provided by Ruth Williams. I'm your host, Dr Cindy St. Hilaire, and this is Discover CircRes, your on the go source for the most exciting discoveries in basic cardiovascular research. This program is copyright of the American Heart Association 2022. Opinions expressed by speakers in this podcast are their own, and not necessarily those of the editors or of the American Heart Association. For more information, visit ahajournals.org.