Dec 17, 2020
This month on Episode 19 of the Discover CircRes podcast, host Cindy St. Hilaire highlights three featured articles from the December 4 issue of Circulation Research. This episode features an in-depth conversation with Drs Mete Civelek and Redouane Aherrahrou, from the University of Virginia regarding their study titled Genetic Regulation of Atherosclerosis-Relevant Phenotypes in Human Vascular Smooth Muscle Cells.
Zahreddine, et al. Tamoxifen and E2 Effects On Reendothelialization
Zheng, et al. Arterial Stiffness Preceding Diabetes
Galang, et al. ATAC-seq Identifies Novel Isl1 SAN Enhancer
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 will be highlighting three articles selected from the December 4’th issue of Circ Res. Drs Mete Civelek and Redouane Aherrahrou, from the University of Virginia, are here to discuss their study, Genetic Regulation of Atherosclerosis-Relevant Phenotypes in Human Vascular Smooth Muscle Cells.
Cindy St. Hilaire: The first article I want to share is titled, Tamoxifen Accelerates Endothelial Healing by Targeting Estrogen Receptor-alpha in Smooth Muscle Cells. The first author is Rana Zahreddine, and the corresponding author is John Francois Arnal and they're from INSERM and the University of Toulouse, France. For breast cancers that contain high levels of estrogen receptor, a standard treatment is to give drugs that block either estrogen production or the receptor itself, such as tamoxifen. However, estrogen can elicit beneficial vascular protective effects, so treatment with tamoxifen might increase the risk of cardiovascular disease. Depending on the tissue, tamoxifen can both antagonize or activate estrogen receptor, so it's role in cardiovascular disease is unclear.
Cindy St. Hilaire: Some evidence even suggest tamoxifen might have protective effects, such as promoting vascular endothelial healing. Zahreddine and colleagues now show that while might suffering damage to the endothelial lining of a blood vessel have improved healing when treated with tamoxifen, or with estrogen, those suffering perivascular injury, that is to say, the injury that affects both the endothelial layer as well as the surrounding smooth muscle cell layer, heal only in response to estrogen. This suggests tamoxifen's healing effects might require smooth muscle cells. In mice, lacking the estrogen receptor and smooth muscle cells, they found estrogen, but not tamoxifen, healed endovascular injuries. While in mice lacking estrogen receptor and endothelial cells alone, they found the opposite. This work reveals nuances in the molecular actions of tamoxifen that should inform further assessment of its risk and benefits for use in patients.
Cindy St. Hilaire: The second article I want to share is titled, Arterial Stiffness Proceeding Diabetes, A Longitudinal Study. The first authors are Mengyi Zheng and Xinyuan Zhang and the corresponding authors are Xiang Gao and Shouling Wu, from Pennsylvania State University and North China University of Science and Technology. As a person ages, their risk of developing diabetes and cardiovascular disease increases. Aging is also linked to increase in arterial stiffness and high blood pressure, but how all these individual conditions affect and influence each other is not entirely clear. For example, while arterial stiffness and diabetes tend to correlate, whether one increases the risk, or the other, or the risk relationship or whether the risk relationship is bi-directional, is unknown.
Cindy St. Hilaire: To assess the interplay between these disease states, Zheng and colleagues studied diabetes and arterial stiffness in a cohort of 8,956 Chinese people between 2010 and 2015, none of whom had had diabetes or cardiovascular disease at the outset of the study. With repeated measures of fasting glucose levels, which is an indicator of diabetes, and pulse wave velocity, which is a measure of arterial stiffness, the team found that participants with a higher baseline arterial stiffness were more likely to develop diabetes during the five-year period than those with lower stiffness levels. Out of the original cohort of just over 8,900 individuals, a total of 979 individuals developed diabetes during the study. Higher baseline glucose levels did not predict future arterial stiffness; this suggests a risk relationship that is a one-way street. While the results require confirmation in additional cohorts, this finding is the first to identify the pathological mechanisms linking arterial stiffness to diabetes.
Cindy St. Hilaire: The third article I want to share is titled, ATAC-Seq Reveals an ISL1 Enhancer That Regulates Sinoatrial Node Development and Function. The first author is Giselle Galang, Ravi Mandla, and Hongmei Ruan. And the corresponding author is Vasanth Vedantham, from the University of California, San Francisco. Pacemaker cells, of the sinoatrial node, establish and control the rhythmic contractions of the heart. These cells differ from regular cardiomyocytes in their transcription profiles, but how this transcriptional profile is established and maintained is not fully understood. To investigate the epigenetic landscape defining pacemaker cell fate, Galang and colleagues have employed a technique called ATAC-Seq, which identifies areas of the genome with accessible open chromatin structures, which is an indication of transcriptional activity. The team compared the genomes of pacemaker cells with atrial cardiomyocytes, and found a number of pacemaker cell-specific accessible loci that had both large numbers of transcription factor binding sequences and enhancer activity, when assayed in mice.
Cindy St. Hilaire: The team went on to specifically characterize one novel enhancer upstream of the gene, encoding ISL1, which is a key transcription factor for pacemaker cell identity. They showed that deleting the enhancer caused under development of the Sinoatrial node and arrhythmias in mice. They also noted that single cell nucleotide polymorphisms at the equivalent loci in humans, have been linked to variations in resting heart rate. The report verifies ATAC-Seq as an effective tool for identifying pacemaker enhancers and will launch future studies into how such enhancers function in heart development and disease.
Cindy St. Hilaire: Okay, so today with me is Dr Mete Civelek and Dr Redouane Aherrahrou, from the University of Virginia, and they're here to discuss their paper titled, Genetic Regulation of Atherosclerosis-Relevant Phenotypes in Human Vascular Smooth Muscle Cells. And this article is featured in our December 4th issue. So thank you both so much for being here with me today.
Mete Civelek: Great to talk to you Cindy. Thank you for choosing our paper.
Cindy St. Hilaire: Yeah, and seeing you over Zoom, I wish these were in person, but...
Redouane Aherrahrou: Thank you for having us.
Cindy St. Hilaire: Yeah. Great. So, before we dig into the details of this paper, which I think is a really nice paper, one of the things I like about it is that it couples GWAS with some functional things, which is obviously super important for figuring out what is important in that GWAS data. So, before we dig into the nitty-gritty of the paper, could you maybe explain what a GWAS study is, and what the strengths and weaknesses are, in terms of using that as an approach to figure out disease related pathophysiology?
Mete Civelek: So, we know that coronary artery disease, or these cardio-metabolic diseases, have a genetic component, and in the past we used to do linkage studies, studying families, but in the last 15 years or so, because of the developments in technology, we can do these genome-wide association studies. And essentially what they do is they look at a population and some of the people in the population will have coronary artery disease and some people will not have coronary artery disease, will be otherwise healthy. And then you study the genetic variance across the entire genome and look for frequency differences in the people with the healthy phenotype and people who have the disease. And of course you do some statistical tests to find if this frequency differences is indeed statistic, the different between these two groups and you identify essentially loci that are associated with the disease.
Mete Civelek: But I see GWAS as almost a detective work, you say something like, okay, let's say there was a murder in the United States, and then now you do GWAS of course to find the murder, right? But what that tells you is, okay, the murder occurred in, let's say Pittsburgh or Charlottesville or Washington DC, sure, it narrows down the scope of where you're going to look at, but it doesn't tell you exactly what happened and where it happened and things like that. And so after GWAS there many more questions to answer looking at the molecular mechanism of the locus, the tissue or cell type of action, the gene, which is being affected by the locus to affect the phenotype. So, it's very good at narrowing down possibilities and coming up with hypotheses, but then the real work begins.
Cindy St. Hilaire: I was wondering actually, as you were saying that, have there ever been... I guess like false discoveries, where people have really focused in, on a loci, because it came up maybe in one or multiple studies, but then maybe it didn't prove to be causative or they still can't figure it out. Are there examples of that?
Mete Civelek: The most obvious example is actually the 9p21 locus.
Cindy St. Hilaire: Interesting. That's the one I was thinking of actually.
Mete Civelek: Which has been associated with coronary artery disease susceptibility in all kinds of studies and in kinds of populations, this signal itself is real, what it's doing is been a lot of debate. Some people think that it's affecting the CDKN2A and 2B genes nearby.
Cindy St. Hilaire: Is that p21 or p16?
Mete Civelek: One is p21 and one is p16, but I can't remember which one.
Cindy St. Hilaire: Yeah, I can't either.
Mete Civelek: Right. And then there's a non-coding RNA in that region called lnRNA. Some people think it's affecting and lncRNA expression. Some people think it's affecting isoform abundance, so that's just probably the most famous locus in our field, in terms of figuring out what it's doing. Yeah.
Cindy St. Hilaire: Well, at least it's probably causing a lot of people to think of a lot of good questions to ask, so that's exciting. In your study, you state that you want to focus on the impact of coronary artery disease associated variants in atherosclerosis-relevant smooth muscle cell phenotypes, and the phenotypes you wanted to focus on were calcification, which is my personal favorite. So calcification, proliferation, and migration. So I was wondering why you wanted to focus on these phenotypes and then what kind of functional assays did you do?
Redouane Aherrahrou: So, the reason we choose those phenotypes because they are playing important role in the disease. So, for example, during the advanced stage of the disease, smooth muscles cells, they proliferate and migrate to make the fibrous cap. So the fibrous cap is actually stabilize the plaque against the rupture, and also during the advanced stage of the disease, the calcification also happening, a lot of people believe that the calcification also contribute to instability of the fibrous cap. So that's why we focus on those three phenotype, migration, proliferation, and calcification.
Cindy St. Hilaire: Interesting, and so I think you'd said you had about 150 patients in your study. Does that mean you did these functional assays in 150 different cell lines? Or how did you do that?
Redouane Aherrahrou: That's a good question. So we conducted actually, our assays from 150 healthy and multi-ethnic donors, so those people actually did die from motorcycle and car accidents, and the doctors actually use the chunk of the aorta where we'll actually isolate these cells from, and then they are actually healthy enough to use for the heart transplantation.
Cindy St. Hilaire: Wow. And so were you introducing known SNPs or SNPs that are pulled out of GWAS into the cells, or did the cells already have the SNPs available? How was the correlation done between functionality and SNPs?
Redouane Aherrahrou: That's a great question. So we actually use the natural SNPs that already exist in those donors. And we ask the question how the genetic variants of those donor affects migration, proliferation, and calcification phenotypes.
Mete Civelek: So we essentially perform a GWAS in a dish-
Cindy St. Hilaire: Yeah, that's kind of what I was thinking-
Mete Civelek: That's the bottom line, you just culture these cells and do these phenotypic characterizations, which you cannot do in healthy living human beings of course, and then just the naturally occurring genetic variation in these individuals, in these donors, to essentially calculate the association between the genetic variants and then these phenotypes.
Cindy St. Hilaire: And you were using aortic smooth muscle cells, right?
Mete Civelek: Yes.
Cindy St. Hilaire: Do you think... this is one thing I always think about, especially because kind of harping back to Mark Majesky's early work with the chick embryo and developmental origins. Do you think if you had coronary arteries from the same individuals that the smooth muscle cells would respond similarly?
Mete Civelek: This is a really good question, partially, yes and partially, no. I'll give you one specific example, for example, one of the loci that is associated with coronary artery disease is over a transcription factor called TCF21. And TCF21 is actually playing an important role in smooth muscle cell phenotypes, and that transcription factor is expressed only in coronary artery smooth muscle cells, but not in aortic smooth muscle cells.
Cindy St. Hilaire: Interesting.
Mete Civelek: This was something that Dr Tom Quertermous from Stanford showed. So presumably we are capturing some of the genetic variation that's important in coronary artery disease as some of it was probably missing because we're using aortic smooth muscle cells.
Cindy St. Hilaire: Yeah. That is so neat. I really like that heat map you had, I think it was figure 4 because you really lined up along the SNPs that were identified in these patients you looked at the effect of that SNPs on a specific function test, you did, and you did, was it 11 functional tests?
Mete Civelek: 12 different functional tests.
Cindy St. Hilaire: 12?
Mete Civelek: Yes.
Cindy St. Hilaire: It's an amazing amount of work really. Well, how long have you been working on this project?
Mete Civelek: Redouane, why don't you answer this question?
Cindy St. Hilaire: Or do you not want to talk about that?
Redouane Aherrahrou: Of course, it's not easy actually to culture and characterize 151 smooth muscle because you expect sometimes, you capture them, some of them, they will not grow, some of them they get contaminated, and you have to perform it again. And also, you cannot do the same experiment for all of them at the same time. So what we did actually, before we started the experiments, we decided to take a smooth muscle cell from one donor, and expanded many times and then we use the same donor to run each time for all the experiments, just to count for the batch and environment effects.
Cindy St. Hilaire: Yep.
Redouane Aherrahrou: So it took me actually almost one and a half year, to finish the characterization for all 151 smooth muscle cell. At that time I was also using also two incubators and then you can imagine, when you put the incubator-
Cindy St. Hilaire: It's full.
Redouane Aherrahrou: ...and I try to finish that, and then I again, start the experiment again to finish the other batch.
Cindy St. Hilaire: Oh God. Yeah, my lab also... we work only in primary human tissues from vessels, but also from valves. So my staff will certainly appreciate all your efforts for this paper.
Mete Civelek: And you can also imagine there was this group of undergraduates, trailing-
Cindy St. Hilaire: An army yeah.
Mete Civelek: Redouane wherever he goes….they were helping him out with many aspects.
Cindy St. Hilaire: Oh, sure that's amazing-
Mete Civelek: He mentored, I think maybe five, six different undergraduate students throughout this project and they're all part of the paper. Yeah.
Cindy St. Hilaire: That's excellent. So towards the end of the study, you guys really focused on a gene, MIA3. So can we talk a little bit about that? What is this gene? What its normal function? Is it known? And then what did you find out in relation to smooth muscle cells?
Mete Civelek: Well, I'll start and Redouane you can continue. So let me just walk back just a little bit to tell you how we kind of decided to focus on that. So we identified the 79 loci that are associated with smooth muscle cell phenotypes and coronary artery disease. So we wanted to show at least some kind of a validation and so we looked for loci that are not associated with lipids because we thought they will maybe not be important in smooth muscle cells. And then we then looked for loci out of those, who affect a nearby gene expression in aorta, in smooth muscle cells, but not in endothelial cells and in monocyte so we thought that will give us confidence-
Cindy St. Hilaire: So kind of enriching for this smooth muscle cell?
Mete Civelek: And this MIA3 popped up and there was only one study actually that showed that, in codes for protein that localizes to the ER exit site and affects these COPII carriers, which secrete collagen into extracellular matrix. Well, collagen as you know, is important in cell stability and what smooth cell muscle cells produced. So, that's how we decided to focus on that gene. And I will let Redouane describe I guess, what we did with that gene.
Redouane Aherrahrou: Yeah. So after the function and mutation, we come up with this gene. So the first thing we did, because we found that the genetic variety in this locus affects actually proliferation in smooth muscle cell. So to what it did at least, this is actually SNP that's affecting the proliferation of smooth muscle cell by this gene, we down-regulate this gene actually in smooth muscle cell using two different shRNA, and we found actually the downregulation of this gene lead to affect the proliferation in sense that dominant regulation of this gene will affect the proliferation.
Redouane Aherrahrou: And also we found that the same genetic variance also in this gene lead to lower periphery, migration, sorry, the expression of this gene in small as I said, and then also in the aortic tissue. And interestingly, we did not see in the monocytes macrophages or other cell type, actually in the aortic tissue suggesting that this genetic variance affecting the coronary artery disease via smooth muscle cell.
Mete Civelek: And we also collaborated with Renu Virmani's group at CVPath Institute, stained lesions, coronary artery lesions, which have these thick caps and thin caps as you very well know that is really relevant to plaque stability, and show that the thin caps have fewer smooth muscle cells which were positive for this protein MIA3, which was all in line with our genetic findings, because our genetic findings basically show that lower expression of this gene was associated with increased susceptibility to coronary artery disease.
Cindy St. Hilaire: Interesting. I wonder if it could be correlated with plaque rupture, right. If there's less smooth muscle cell, obviously, then a thin fibrous cap is nothing that anybody wants. So is the proliferation of the smooth muscle cell almost protective in a sense, that's one of the things people are starting to think about in calcification, [Alina has these amazing imaging studies, where she looks at matrix vesical accumulation and calcification mitosis. And really the field has noticed, with work by Linda Demer also, the field has noticed that a large, huge chunk of calcification seems to be much less risky, I guess, compared to the microcalcifications, and I wonder if MIA3 might be like that too, if you can have just enough smooth muscle cell proliferation to kind of keep that cap thicker, is that more protective?
Mete Civelek: I think that's a really good point that you are raising, because some of the answer is, in that figure 4 in the paper that you mentioned. What we find is that these loci, when people have the risk allele of these loci, so obviously people are at higher risk for coronary artery disease, some of them are associated with higher proliferation, but some of them associated with lower proliferation, same with calcification, same with migration. So it's really difficult to say, at least just looking at the genetic loci, yes, higher proliferation is always better.
Cindy St. Hilaire: Yeah.
Mete Civelek: There's probably this really delicate balance that allows for plaque stability.
Cindy St. Hilaire: Yeah, it's reminisent, I guess, of the IO1-beta story, right?
Mete Civelek: Exactly.
Cindy St. Hilaire: Gary Owens and colleagues have really shown that well. The role of it in early versus late plaque is different and it's complicated. That's what we learned.
Mete Civelek: I agree. And it's specific to MIA3, that locus is also associated with myocardial infarction.
Cindy St. Hilaire: Oh, interesting.
Mete Civelek: So indeed there is a possibility that really affects plaque stability.
Cindy St. Hilaire: Yeah. So Mete, say you and I are in the same faculty class in that we both started-
Mete Civelek: Yes we are. We are classmates.
Cindy St. Hilaire: 2015 we started our labs, and this is obviously a huge undertaking and really starting a project like this... when you're new, it's really a risk. You're proposing to collect 150 smooth muscle cell lines and characterize them all functionally and it turned out amazingly, but can you maybe talk about the early days in this project's development, and was there ever a moment where you're like, "What the heck am I doing? Is this going to work or..." Just kind of maybe talk to us a bit about that.
Mete Civelek: That's a really excellent question. To be totally honest with you, I was really lucky to have recruited Redouane to the lab, but he and I worked together when I was a postdoc and when he was a PhD student, as part of this little consortium. So I knew he was going to work hard on his part and he was very driven-
Cindy St. Hilaire: He had magic hands.
Mete Civelek: And he really want to do this project. So, I knew that it was going to work, but of course he and I both had these moments of, "Are we sure of what we're doing, and why are we doing this? Are we going to get something out of it?" And it was both of us kind of pumping each other, if you will say like, "Yep, this is going to work, we know it's going to work, we have faith in this," but I should also say that my lab also works on adipose tissue biology, and I already had another kind of a safe project going on in that realm.
Cindy St. Hilaire: So, that's funny, I started my lab that way too. I kind of had the project that was the direct continuation of my K grant, and then this kind of high risk, high reward project on valves and you know what, I think that's something that’s smart, it's kind of you have two tracks of research and hopefully one works and then the other one will work. And if they don't work at the same time, hopefully the other one can fill in the gaps, so...
Mete Civelek: I totally agree with you, but truly, Redouane made a big difference in this project, imagine a postdoc, who's doing nothing but cell culture for two years, hoping that something is going to come out, that's a big risk for him too, it certainly paid off and it's paying off because he has two other papers in the pipeline from this project.
Cindy St. Hilaire: Wonderful. That's excellent. So what's the future for this project? What's kind of the next question you're going to ask if you don't mind sharing.
Mete Civelek: Oh no, not at all. The most obvious one is looking at gene expression. So we have cultured these cells under two distinct conditions, one is the more contractile phenotype, one is the more productive phenotype. And we did RNA-Seq from, again 150 of these individuals in both conditions and we did what is known as eQTL mapping, so looking at the effect of the genetic loci on gene expression. In a separate project, we also actually collected media from these cells and looked at secreted proteins in the media and we're also finding the genetic loci that are affecting secreted protein, because as you very well know, smooth muscle cells secrete proteins to stabilize plaque stability. So those two papers are Redouane's next projects. And he's almost finished with one and has finished the analysis of the other ones, so hopefully-
Cindy St. Hilaire: That's exciting.
Mete Civelek: ...more papers coming out in the next six months or so. Oh, I should have said the paper was chosen for the Genomic and Precision Medicine Counci; Young Investigator Award, so Redouane is competing-
Cindy St. Hilaire: Wonderful, also you are in that, excellent. Thank you both so much for joining me today. This was a lovely paper, it was actually inspiring. It made me think about some way to think about my calcification studies.
Mete Civelek: Thank you so much, Cindy. This was really wonderful.
Cindy St. Hilaire: Absolutely. Thank you.
That's it for the highlights from the December 4th issue of Circulation Research. Thank you very 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 Mete Civelek and Redouane Aherrahrou. 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 texts for highlighted articles is provided by Ruth Williams. I'm your host, Dr Cindy St. Hilaire. And this Discover CircRes, your on the go source, for the most up-to-date and exciting discoveries in basic cardiovascular research.