February 2022 Discover Circ Res

This month on Episode 33 of Discover CircRes, host Cynthia St. Hilaire highlights two original research articles featured in the February 4 issue of Circulation Research. In addition, she previews Circulation Research’s Compendium on Women and Cardiovascular Health, featured in the February 18th issue. This episode also features a conversation with Dr Alastair Poole and Dr Laura Corbin from the University of Bristol and Dr Stephen White from the Manchester Metropolitan University about their study, Epigenetic Regulation of F2RL3 Associates with Myocardial Infarction and Platelet Function.   Article highlights:   Samargandy, et al. Blood Pressure Trajectories and Menopause   Gilchrist, et al. Research Goes Red Registry   Cindy St. Hilaire:        Hi, and welcome to Discover CircRes, the podcast of the American Heart Association's Journal, Circulation Research. I'm your host, Dr Cindy St. Hilaire from the Vascular Medicine Institute at the University of Pittsburgh and today I'm going to be highlighting articles from our February issues of Circulation Research. I'm also going to speak with Dr Alastair Poole and Dr Laura Corbin from the University of Bristol and Dr Stephen White from the Manchester Metropolitan University about their study, Epigenetic Regulation of F2RL3 Associates with Myocardial Infarction and Platelet Function.   Cindy St. Hilaire:        The first article I want to share is titled Trajectories of Blood Pressure in Midlife Women: Does Menopause Matter? The first author is Saad Samargandy, and the corresponding author is Samar El Khoudary from the University of Pittsburgh. Blood pressure increases with age, but after midlife, the rate of increase for women generally exceeds that for men. This observation has led to debate over whether menopause might influence the blood pressure trajectory.   Cindy St. Hilaire:        To find out, this group examined data on over 3300 women of diverse ethnicity enrolled in the Study of Women's Health Across the Nation, or SWAN study. The women began the study between 42 and 52 years old, and they had 17 follow-up visits at roughly one-year intervals. At these visits, blood pressure, hormone levels, weight and other health parameters were measured.   Cindy St. Hilaire:        Analysis of the data revealed women fell largely into three blood pressure trajectory groups. Those with low blood pressure before menopause and accelerated blood pressure after menopause, those with a linear increase linked to age, and those with high blood pressure before and a slower ascent afterwards. White, Chinese and Japanese women were more likely to be in the low to accelerated group, as were those with early menopause, while Latino and Black women were more likely to have high blood pressure in general. Together, the results indicate that for many women, menopause itself does not accelerate age-related blood pressure increase, and that women of menopausal age should be advised of this risk and have their blood pressure monitored regularly.   Cindy St. Hilaire:        The second article I want to highlight is titled Research Goes Red: Early Experience With a Participant-Centric Registry. The first author is Susan Gilchrist and the corresponding author is Jennifer Hall from the American Heart Association. Cardiovascular disease is a leading cause of death for men and women alike, but there are particular factors such as pregnancy and menopause that may specifically influence the genesis, presentation and management of the condition in women.   Cindy St. Hilaire:        With that in mind, for the past two decades, the AHA's Go Red for Women campaign has been raising awareness of and driving research into women's cardiovascular health issues. The latest Go Red initiative, an online platform called Research Goes Red, was launched in 2019 with the aim of empowering women to contribute to health research by, among other things, taking part in health surveys. In the last two years, the platform has garnered 15,000 registered individuals between the ages of 30 and 60. It has deployed six targeted health surveys and prompted two AHA-funded research studies based on participant responses: one on perimenopausal weight gain, and one on the use of social media to engage young women in cardiovascular disease awareness. While Research Goes Red has successfully amassed middle aged participants, the authors say that future goals should include increasing the number and the diversity of the registrants and encouraging researchers to use the registry not just for data, but for identifying potential trial participants.   Cindy St. Hilaire:        I want to now mention the 15 articles that are featured in our Compendium on Women and Cardiovascular Disease that is featured in our February 18th issue of Circulation Research. And this also happens to correspond with February being the American Heart Month. So Susan Cheng and colleagues present A Scientific Imperative As Seen Through a Sharpened Lens: Sex, Gender and the Cardiovascular Condition. Genetic, molecular and cellular determinants of sex-specific cardiovascular traits is discussed by Teemu Niiranen and colleagues. Bonnie Ky et al. describe sex-specific cardiovascular risks of cancer and its therapies. Sex differences and similarities in valvular heart disease is presented by Francis Delling and colleagues. Cecile Lahiri and colleagues wrote about the cardiovascular implications of immune disorders in women. Joshua Smith and colleagues discuss sex differences in cardiac rehabilitation outcomes.   Cindy St. Hilaire:        Pregnancy and reproductive risk factors of cardiovascular disease in women is reviewed by Michael Honigberg and colleagues. The impact of sex and gender on stroke is presented by Kathryn Rexrode and colleagues. Ersilia DeFilippis and colleagues cover heart failure subtypes and cardiomyopathies in women. Demilade Adedinsewo and colleagues wrote about cardiovascular disease screening in women, leveraging artificial intelligence, and digital tools. Sexual dimorphism in cardiovascular biomarkers, clinical research implications, is discussed by Jennifer Ho and colleagues. Connie Hess et al. review sex differences in peripheral artery disease. Janet Wei and colleagues provide an update on coronary arterial function and disease in women with non-obstructive coronary arteries. Sex differences in myocardial and vascular aging is presented by Hongwei Ji and colleagues. And lastly, arrhythmias in female patients, incidence, presentation and management, is reviewed by Andrea Russo and colleagues.   Cindy St. Hilaire:        Today I have with me Drs  Alastair Poole and Laura Corbin from the University of Bristol and Dr Stephen White from the Manchester Metropolitan University. And they're here with me to discuss their study, Epigenetic Regulation of F2RL3 Associates with Myocardial Infarction and Platelet Function. And this article is in our February 4th issue of Circ Res. Well, Drs Corbin, Poole and White, thank you so much for joining me today.   Laura Corbin:             Thank you very much.   Stephen White:            Thank you.   Alastair Poole:            Thanks.   Cindy St. Hilaire:        So this is a really neat study. It's bringing in a couple different fields. It's investigating what I'm calling a Venn diagram of these intersecting topics all related to cardiovascular disease: cigarette smoking, epigenetic modification and platelet activation. So can you maybe give us a little bit of background on the status of the field and how these three topics intersected at the start of your study?   Laura Corbin:             So yeah, our working hypothesis was based on existing literature and it was really to look at whether smoking-induced epigenetic DNA hypermethylation of F2RL3 could increase risk of myocardial infarction and whether the route to that could be through platelet function. So there's quite a lot of literature going back probably to around about 2015 that shown that there are changes to the methylome in response to smoking. And DNA methylation is a way of cells controlling gene expression, but without having to actually make changes in the DNA sequence itself. So this could be a really important way that we know that smoking increases the risk of a number of cardiovascular diseases, but we don't really know how that happens. And one way that that could happen is through changes to methylation.   Cindy St. Hilaire:        What is known about how cigarette smoke impacts the status of DNA methylation? How has that switched or changed? Maybe when someone is actively smoking, when someone quits, is it dynamic? What is known about that relationship?   Laura Corbin:            Okay. So yeah, going back to about 2015, there was a number of studies that looked at methylation across the whole genome. So in a hypothesis-free untargeted manner, developments and technology meant that we could look at many, many sites across the genome at the same time. And so studies were done to look at changes that were associated with smoking. And what was found was those changes, actually quite a lot of changes across the genome in a number of different genes, but not really anything much beyond that. So F2RL3 was one of the first sites to be identified as being associated, methylation at that site associated with smoking. And it was replicated in several studies.   Laura Corbin:            And it was also showing that there was a dose-response relationship. So the more a person smoked, the less methylated that site appeared to be. And then there's been some work done already, but we also did it in our paper to show that those methylation marks actually hang around for quite a long time once somebody quits smoking. But also that there's a lot of variation within an individual, so even if you smoke, it doesn't necessarily mean that you'll definitely have low methylation, there's still variation. So there's other factors that are involved in that.   Cindy St. Hilaire:        So you were looking at a specific population of patients, can you tell us a little bit about that group of patients you were looking at? And you mentioned the variability in the amount of smoke they were exposed to, do you know that information? And I guess one of the base questions I had is I'm in Pittsburgh, which back in the '80s and earlier was a steel mill town that had a lot of pollution. And so I'm wondering if you're able to clearly separate out a cigarette smoker from maybe someone who is a light cigarette smoker, but lives in a more polluted area?   Laura Corbin:             Okay. So there's two parts of the study that were looking at this in a human context, so in a whole person context. One of those was using data from the Copenhagen City Heart Study, and that's the one where we looked at the relationship between smoking and methylation and then between methylation and myocardial infarction. So that study is great because it's been tracking people over time and so we're able to use the samples that were collected before they had their cardiovascular event and look at methylation at that point. So we know that the event occurs after that point, which is important. And so we were able to verify in that population that we did see an association between smoking and methylation. We were able to show that it was a dose-dependent relationship. So if we look at something like in that dataset, we had things like the intensity of which people smoke, so pack years is one of the things that we looked at. And it did appear to correspond in an approximately linear fashion.   Laura Corbin:             So we don't really know, I don't think, at this point, what impact other environmental exposures would have on the methylome and how that would interact with the cigarette smoking. That's actually a really interesting point that we'll probably come onto later about whilst we were looking here at the smoking effect on this methylation site, in the second part of the work, we were able to show that even in non-smokers, there's variability in methylation at this site, and that can still have impacts on the biology downstream. So yeah, it's an interesting point.   Stephen White:            Just to maybe just jump in, there's very good amounts of literature now that show quite a good correlation between changes in air quality and cardiovascular events. So smogs, wildfires and so on, clearly correlate with an increase in cardiovascular events. But actually the opposite's also been observed in the more recent COVID lockdowns, where reduction in air pollution also mirrored a reduction in the number of cardiovascular events. So I think you raise a really interesting point about is it cigarette smoke alone or does air quality in general play an effect? And clearly it does play an effect, although we didn't correlate that within this current dataset.   Cindy St. Hilaire:        Your study looked at DNA methylation patterns at cytosine, phosphate, guanidine or CPG sites in the genome. Can you tell us a little bit more about what these islands are and how they change throughout maybe different cells in the body, but also maybe in the same cell, but throughout the course of life or the course of, in this case, cigarette exposure?   Stephen White:            So if we just want to focus in on our study, what we showed was that exposure to cigarette smoke changes endothelial cell methylation. It also changes megakaryocyte methylation patterns in the same way. And I think one of the surprising things was that only 48 hours of exposure to cigarette smoke significantly changes the methylation pattern of the F2RL3 locus. So it's quite a dynamic event, but it does show that these can be quite rapidly regulated. And Laura's really nice work shows that the methylation on cessation of smoking, that pattern does actually go down, but it's a 20-year process. So it looks like it can be rapidly induced, but may actually remain as a methylation mark for a considerable length of time.   Stephen White:            And I think one of the things we did in our study was actually to triangulate not only the observational data and the association data in patients, but actually start to look at a mechanism of how that might actually relate to changes in gene expression. So we showed that this particular CPG site is right next to a binding site for a transcription factor, and transcription factors are the cell’s way of regulating how much of a particular gene is expressed. And we show that changes in methylation changed the binding of this transcription factor and therefore change the amount of this particular gene that was made.   Cindy St. Hilaire:        Yeah. Actually, I want to start to talk about that locus you were interested in. So what was known about the F3RL2 locus? How big is it, but also what genes are there and what did you start to investigate with your in vitro modeling?   Stephen White:            So I think when we started, we had the observation that a change in methylation at the F2RL3 locus was associated with the risk of cardiovascular events. And then it was a detective expedition into the gene using various in silico analyses that identified the methylation site that we are interested in, or most interested in, is right next to a transcription factor binding site.   Stephen White:            So we then went on to show that binding of that transcription factor is sensitive to methylation, that if we would just excise that piece of DNA, we can show that that has the ability to regulate F2RL3 expression or the expression of a reporter gene. And then if you knocked out the transcription factor binding site, you lose that regulation. So it was a series of detective work and experimental steps that allowed us to put a mechanism behind the observation that changes in methylation might truly affect the level of gene expression of the F2RL3, otherwise known as PAR4 to platelet biologists. So get that in there.   Alastair Poole:            I first came across it when another member of our team actually mentioned it to me over a casual conversation actually a few years ago that F2RL3 gene was regulated in this way. To me as a platelet biologist, F2RL3 didn't mean a lot, but when I was told then it was the gene that encodes PAR4, it meant everything. And so platelet biologists, we talk about PAR4, which is of course the protein product of the F2RL3 gene. And PAR4 is one of several really key receptors on a platelet surface that responds to, in this case, to changes in thrombin generation, thrombin activity, which is of course the major effectively end product of the coagulation cascade.   Alastair Poole:            So it's what couples coagulation and platelet biology together, thrombin. And there are two major receptors on platelets that operate in response to changes in thrombin and that's PAR1 and PAR4. And they're both very important genes, but yeah, really interestingly, you have this rather selective effect on PAR4 and the paper actually shows it is indeed a selective effect on PAR4 as opposed to PAR1 in terms of epigenetic regulation of its responsiveness to PAR4 activation.   Cindy St. Hilaire:        So I want to tap back onto something that Laura had mentioned briefly, and that is talking about your platelet assays where you isolated platelets from a specific subset of the patients. And I believe it was figure three, and you looked at patients who in adolescence had exhibited differences in the methylation pattern at the site in the F3RL2 locus. What do we know about that innate or early-age change? And then I would love to hear more about this actual experiment, how you looked at the patients earlier versus current and what the thinking was behind that.   Laura Corbin:            So yeah, this part of the work was done in a birth cohort study called the Avon Longitudinal Study of Parents and Children, which is based at the University of Bristol. And this is a really great study, a great resource that we have, and in fact, it's open to all researchers so anyone could use it, where mothers were recruited during pregnancy, which was in around 1991 to 1992. And then those children that were born from those pregnancies have then been followed up ever since.   Laura Corbin:             So that was the data that we were able to use for this part of the study. And what we wanted to do was to look at how this could work functionally, so look at the platelet function, but we really wanted at this point to step away from the smoking. Because obviously if you're going to look at platelet function in smokers versus non-smokers, it's incredibly difficult then to say that that's coming through a specific pathway, because we know that smoking induces lots and lots of changes in methylation, in proteins, all sorts of things going on. So we couldn't see a way of doing that part of the experiment with a comparison of smokers versus non-smokers. But what we know is that there's natural variation in methylation across all sites, including F2RL3.   Laura Corbin:             So we had historic data from earlier time points, so two earlier time points from when the children were under 20. And we looked at those measures for F2RL3 and then just simply ranked people according to whether they had high or low methylation, and then used those two ranks together to then work out who had a consistently high versus consistently low level. And then we invited participants back into the clinic to have samples taken from those up and lower ends of the distribution. At that point, we were just really hoping that that methylation pattern would continue because this was then, I think they aged about 24 by the time we did this work, so it was some time after. And we restricted our selection just to people who were non-smokers, so never smokers based on the information they provided, but also asking them when they came in for that clinic just to verify that they were non-smokers.   Laura Corbin:             And then we had a look at the methylation again. This time we looked across four sites in the region, which are the sites presented in the paper. And luckily for us, there was still that mean difference between the high group and the low group. But what we were able to then do is to compare people with high and low methylation, but without all of the trouble of isolating that pathway in amongst all the other smoking effects. And also not just the smoking effects, but the other confounding factors that come with smoking. So we know that smoking is correlated with a lot of other lifestyle factors. So if you ever do a smoking versus non-smoking comparison, it's really hard to work out exactly which bits are coming from smoking and which pathways it might be going down. So this was the idea behind this part of the study was to just really zoom in on F2RL3 methylation in the absence of all of the other noise in the other experimental designs.   Laura Corbin:             So yeah, the natural variation we see in the non-smoking healthy participants in this part of the study is actually quite a lot less than we see when we look at smokers compared to non-smokers, but it was still enough to then go on and look at the platelet function. And then the differences we saw in the platelet’s responses, there is nothing pathological there. It was just very subtle changes in the response when stimulated in the lab.   Alastair Poole:            The only other thing I could add would be that platelets are very complicated cells. Every cell of the body is very complicated. Platelets are certainly very complicated. PAR4, F2RL3, is just one of very many components of the platelet that modulate its activity. So platelets are controlled by multiple forces sort of thing, at which F2RL3 and PAR4 is just one of them. So biology is very good at compensating for one level going up in one part of a pathway and going down compensatory wise in another part of a pathway. There isn't necessarily a direct relationship between one pathway enhancement and an overall effect because of the compensation.   Cindy St. Hilaire:        Why would it be easy?   Alastair Poole:            Yeah. Yeah. It's just very complex, the biology. So yeah, I completely get what you're saying, Laura, that we obviously don't want to frighten people that maybe they've got a propensity to enhance thrombosis based upon a single gene methylation difference because it will be much more complex than that.   Cindy St. Hilaire:        Yeah. I think that's one of the beautiful things about your study is with the luck of having this sample population, you were able to ask these really precise questions that... You can't just start a study now and ask that sort of question. So it was really elegant in that sense.   Cindy St. Hilaire:        Do we know the mechanism of how cigarette smoke induces these methylation changes, or maybe even the specific components of the smoke? And I guess I'm thinking that in terms of vaping that's becoming more and more popular, obviously the company selling those products want to advertise them as safer, but it comes down to is it all of the mixture of the cigarette smoke or is it one component that we know impacts the methyltransferases and demethyltransferases in this process?   Alastair Poole:            Those are two follow-on routes of our study that I have to say that we discussed previously amongst ourselves and identified those as definitely very important follow-up areas. So do e-cigs have similar effects and that's a study that definitely needs to be done. We have done a little bit of work to try to investigate that initially, but I think that's a very important follow-on study. But yeah, you're also right that one of the key things that we want to understand and is, the missing piece in a way, is how is methylation at a molecular mechanistic level altered by smoking? Steve, I don't know whether you have any further details to add to that.   Stephen White:            I think one of the key molecular pathways seems to be the antioxidant response. And so that's largely controlled by another transcription factor called NRF2. And so if you think about smoking or poor air quality, all of those things do combine through this particular pathway that senses free radical damage, free radical stress. So as Alastair said, it's an area we are going to carry on to look at and it's a big area of my own lab's investigations. But oxygen stress is probably the mediating factor, but the actual nuts and bolts about how the demethylase is targeted to this particular locus is still an area of active investigation.   Cindy St. Hilaire:        All right, well, I will be on the lookout for those future studies because it's a really interesting topic, just the whole interplay of all of this. Are there any translational implications for these findings? Do you think potentially we could screen patients, say, to see their methylation status? I don't know if megakaryocytes are easy to isolate, but is it in a circulating cell, would this possibly be able to be turned into a screening tool or a diagnostic tool to predict thrombic events in patients?   Alastair Poole:            It is possible. I think it would not be possible to isolate megakaryocytes very easily. There are a small number in the peripheral circulation, but the majority are not in the peripheral circulation. But we and others have used other blood cells as proxy measures. So actually, the gene methylation changes that we identified here come from other leukocytes, white blood cells, and those effectively are a cell that are exposed to smoke in the same way, or the smoke products in the same way. So we'd use a proxy cell for that.   Alastair Poole:            Yes, I suppose it is possible. As you say, there's a natural variation in methylation status of that gene and there's, layered on top of that, a smoking induced. And I suppose that it would be an interesting further investigation to understand whether, effectively, your natural methylation status of that gene happened to give you an enhanced risk of a cardiovascular event. The work we've done seems to suggest that that may well be the case and therefore you could imagine possibly a personalized medicine approach that might include understanding the methylation status of F2RL3 as part of that.   Cindy St. Hilaire:        Well, it was a beautiful study. I love these studies that bring in lots of different fields or specialties to ask interesting questions. So Dr Corbin and Dr Poole from the University of Bristol and Dr White from Manchester Metropolitan University, thank you so much joining me today.   Stephen White:            Thank you. Our pleasure.   Alastair Poole:            Thank you.   Laura Corbin:             I'd also just like to acknowledge all of our co-authors as it really was a big team effort, especially the guys who are not represented on the call today, which is the folk from the Copenhagen City Heart Study, and also to all of the participants of that study and the Children of the '90s Study that contributed to the work. Thanks very much.   Cindy St. Hilaire:        That's it for the highlights from our February issues of Circulation Research. Thank you so much for listening. Please check out the CircRes Facebook page and follow us on Twitter and Instagram with the handle @CircRes and the hashtag #DiscoverCircRes. Thank you to our guests, Drs  Alastair Poole, Laura Corbin and Stephen White.   Cindy St. Hilaire:        This podcast is produced by Ashara Ratnayaka, edited by Melissa Stoner, and supported by the editorial team of Circulation Research. Some of the copy text for highlighted articles is provided by Ruth Williams. I'm your host, Dr Cindy St. Hilaire, and this is Discoverer CircRes, your on-the-go source for the most exciting discoveries and basic cardiovascular research.   Cindy St. Hilaire:        This program is copyright of the American Heart Association 2022. The opinions expressed by speakers of this podcast are their own and not necessarily those of the editors or of the American Heart Association. For more information, please visit ahajournals.org.