Coronary Microvascular Disease: the Next Frontier in Ischemic Heart Disease
Coronary Microvascular Disease: the Next Frontier in Ischemic Heart Disease
Marie Guerraty, MD, PhD, is currently an instructor in cardiovascular medicine @pennmedicine.
Objectives:
1. Understand why Coronary Microvascular Disease (CMVD) is an important and prevalent clinical entity.
2. Draw from existing genomic and developmental research to gain insight into CMVD.
3. Understand how transcriptional coregulator Friend of GATA 2 (FOG2) may play a role in CMVD.
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0:00 Introduction
2:24 Coronary Arterial Circulation
7:23 CAD GWAS = Ischemic Heart Disease GWAS
12:32 Phenome-Wide Association Study of non
15:26 Discrete left heart catheterization
16:44 Matched cohorts for deeper phenotypic
24:34 Using CRISPR/Cas9 genome
27:08 Angiogenic effects of
33:37 TWIST1 in Smooth Muscle Cells
38:00 TWIST1 Summary
46:40 Acknowledgements
Content
0.05 -> So when we get started.
4.55 -> How do we get started so it's my pleasure to introduce Marie Garrity who's visiting us from U Penn?
12.49 -> Marie did her undergraduate at Duke and majored in mathematics and Minored in biology and then I went to Upenn, where she got her MD and PhD.
24.87 -> And then she stayed on at U Penn for residency and fellowship and now as an instructor and has been focused in on studying microvascular disease at multiple levels, so studying at a very fundamental level and then also studying it in translation. TLE small animal models and actually even in patients using dynamic pad image so join me in welcoming.
60.99 -> Good morning can you hear me well?
63.23 -> Thanks so much for the invitation to be here, so that I can share some of this work with you. I'm going to be telling you a little bit about coronary microvascular disease and how I think it's next frontier in Ischemic Heart do.
74.73 -> So after 40 years of progress in decreasing cardiovascular mortality, these efforts in the progress has stalled recently.
83.85 -> Can see the curve? Which was coming down nicely hasn't actually just stalled it's rebounded up?
89.31 -> And there are many reasons why this has happened one is health care disparities were not able to get the right treatments to the right patients at the right time.
96.95 -> Also rising rates of obesity and diabetes that contribute to this.
101.1 -> But there's also a contribution from coronary microvascular disease and the fact that we've been under appreciating a significant portion of the coronary microvasculature.
110.26 -> Because of these curves. It's pretty clear that we need new out of the box ways of approaching ischemic heart disease new therapies new imaging modalities new diagnostic motel.
120.69 -> So the court hearing microvasculature is structurally and functionally pretty complex.
126.75 -> It's composed of arteries and veins aliphatic's, but mostly arteries of different sizes that have pretty specialized functions.
136.97 -> The larger conduit vest.
139.2 -> That are greater than 500 microns in diameter are the named coronary arteries. The RCA the left Cirque that we imaged with coronary angiograms. These vessels develop coronary after sclerosis, but functionally they don't do all that much.
153.88 -> The micro vessels on the other hand, is where all the Physiology happens. This is where all the action is happening, so these are the vessels that are a little bit smaller, so 200. One hundred microns and actually all the way down to the capillary level.
165.81 -> These are the blood vessels that regulate blood flow to the cardio myocyte.
170.36 -> These are the blood vessels that phase of the flow and these are the blood vessels that are sensing the local metabolic environment and regulating blood flow to meet that need.
181.44 -> So for 4050 years we've been studying coronary artery disease, which effects these larger conduit vessels and we've been studying coronary after sclerosis. The cell types involved in coronary Arthur Sclerosis and treating coronary Atheros Grossis.
195.24 -> And I think the next phase of treating ischemic heart disease is going to be focused on the coronary microvasculature these smaller vessels.
203.9 -> We know a little bit about the pathophysiology of coronary microvascular.
208.66 -> We know that there are some structural changes. There is lumen, obstruction or vascular wall thick name and get immune cell infiltration under vessels. We know that you can get dysfunction of the vessels and endothelial dysfunction.
223.85 -> And we've all seen what this can look like clinically so patients present with a mizore with Angela. They have some sort of objective evidence of this kimia either on stress test or they have big proponents.
238.69 -> They get a coronary angiogram, which shows that there's actually no obstructive coronary artery disease and then clinicians put the pieces together and say that this must be microvascular disease.
249.66 -> So one way of thinking about corner Microvax for diseases. A ski Mia in the absence of obstructive coronary artery disease.
257.66 -> There's a lot of political evidence that this is a pretty important disease process.
263.45 -> So there's a recent growing interest from the women's a ski Mia syndrome evaluation study that was summarized in the 2017 White paper.
271.69 -> And this talks about a ski Mia with no obstructive coronary disease as I know have.
277.69 -> This paper that came out of Europe looked at 12,000 patients that had convincing exertional angina symptoms. They were convincing enough for cardiologist to feel that these patients needed a cat.
290 -> And when they got their cap 60% of women and 30% of men actually had no obstructive coronary disease.
296.49 -> Yet these patients had worsened outcomes compared to control patients, so they don't have obstructive CD that they have something that increases their cardiovascular risk.
307.71 -> And then more recently this is the verdict trial that came out in 2018. The entire purpose of this, randomized, controlled clinical trial was to enroll patients that were having an N Stemi and randomized them to captain revascularization at 24 or 848 hours. There is no difference in the timing, but what I found really interesting is that one third of these patients actually didn't have any obstructive coronary artery disease. So we're seeing these patients pop up in a lot of these big trials that we've been doing.
337.07 -> And I think CMVD is an important clinical entity to address now for the following reason.
342.43 -> So it directs contributes directly to cardiac morbidity and mortality patients have symptoms. From this they present to the emergency room with this and they can die from this.
353.05 -> CMPD also worsens outcomes for patients that have coronary artery disease.
357.67 -> Or patients that have an MI.
360.17 -> The biology of coronary microvascular disease is very closely related to collateral formation, which we know to be productive in ischemic heart disease.
368.63 -> There's also recent growing evidence that coronary microvascular disease and pathology may be part of the pathogenesis for heart failure with preserved ejection fraction and for help. At this point we?
379.64 -> Are still just trying to understand how it's developing to try to come up with targeted therapies that might perhaps be upstream of the clinical syndrome that were seen.
388.77 -> And we also know that microvascular function plays a really important role in various cardiomyopathies like Hypertrophic Cardiomyopathy.
397.13 -> Technological advances in cardiac imaging are now making this field, iaccessible to clinicians were finally able to measure surrogate markers of the coronary microvascular with cardiac pet and MRI and some echo modalities.
410.91 -> But yet we still have limited evidence based treatments and actually know targeted therapies.
418.46 -> So, in a perfect world, the next step would be to do an unbiased assessment of coronary microvascular disease and come up with some targets so we could think about doing a genome wide Association study for CMVD to enroll patients that have CMVD and patients that don't have CMVD genotyped them or sequence. Their genomes and look to see if we can find some jeans that might be related to CMVD.
443.3 -> But we don't live in a perfect world. We don't have any coronary microvascular disease. Jiwa studies and it's actually pretty hard to design. These studies because it's hard to phenotype these patients. The phenotype is a lot of different data that is brought together by the clinician.
458.96 -> But we do have our thousands and thousands and thousands and hundreds of thousands of patients that have been enrolled in coronary artery disease, Jiwa stuff.
467.57 -> These are actually really ischemic heart disease study.
470.83 -> For example, if you look at the pro parties enrollment criteria patients got enrolled in this coronary artery disease trial. If they had one of two events that could have an MI or symptomatic acute coronary syndrome, so to have an MI.
485.87 -> I put in bold criteria that you could meet if you only had microvascular disease. You had to come in with typical ischemic chest, pain and leak some proponent.
495.69 -> You didn't necessarily have to have coronary artery disease.
499.2 -> Similarly to be classified as having symptomatic acute coronary syndrome. You could present with an unstable anjanette type picture and have chest pain at rest with dynamic easy G changes and that would get you.
511.36 -> So there are clearly some coronary microvascular disease patients that are clumped into these ischemic heart disease Jiwa study.
521.37 -> Additionally, I've been really interested in the fact that developmental programs are often re capitulated in disease States and we think about this. A lot in heart failure when fetal programs are turned on and there's a switch in metabolism back to glucose so we think about this a little bit in Cardiology, but it's always fascinated me that there is a lot of cardiac development that we are not drawing from to get clues about what's going on in disease states.
546.38 -> So my approach to see MBD is based on those 2 things one that coronary artery disease, Jiwa slow sigh.
553.65 -> Are actually ischemic heart disease low cyan contained in those 200 and some jeans that we have right now work 200 and some low side. We actually have a lot of information about coronary microvascular disease.
565.17 -> And their jeans that are involved in coronary development that we're learning about that might be really relevant in coronary microvascular did.
573.41 -> So based on this framework, I focused on 2 molecules that I'll tell you a little bit about one is fog 2, which is a cardio myocyte transcriptional Co regulator.
584.52 -> And the other one twist. One is a transcription factor that is active in under feeling cells, but also in smooth muscle cells?
593.17 -> So far up to her friend of gratitude in a really, really neat molecule. If you knock it out in mouse embryos. They don't develop coronary microvascular so it's crucial to develop coronary.
605.08 -> But if you look at the role of fog, too, and adults.
608.35 -> If you conditionally delete so if you turn off log 2 in an adult mouse within weeks. The microvasculature of that mass received so you get capillary rarefaction and the whole vascular tree instead of being nice and plump and covering the whole heart like you see up here.
625.75 -> Becomes very, very thin.
629.4 -> And, of course, this results in heart failure in the mouse.
632.55 -> The mechanism of this is that fog 2 is regulating an angiogenic program.
637.44 -> So far, 2 is increasing expression of pro angiogenic factors like veg F&FG F2 and 9.
644.21 -> The Cloud II is also decreasing Anja static fact times like the temps and different collagen factors. So Phog 2 in mouse development and fog too. In adult melt coronary microvascular homeostasis plays a really important role in regulating these programs.
663.84 -> So this, let us do the hypothesis that fog, too might play a role in adult human coronary microvascular.
671.52 -> So, in order to set out to see if this is true. We started doing some Feebas analysis. So you are all familiar with Geoss analysis, where we take a single phenotype like coronary artery disease or serum LDL levels.
685.73 -> And we checked the entire genome color coded by chromosome to see if there's an Association between any locus and the phenotype of interest.
695.36 -> He was is it converse of that it's a phenome wide Association study so we have a genetic variant or a group of genetic variance and we look at all ICD 9 and 10 diagnosis. Codes color coded by organ system to see if that variant might be associated with some sort of phenotype by ICD 9 diagnosis.
716.57 -> And in order to do this we used the Penn Medison Bio Bank, which is a collection of patients that have been enrolled at Penn for this particular study. We had about 12,000 patients and these are patients that have interacted with the health care system and consented to be enrolled so they're not clinical trial patients they're not particularly well. Phenotyped we may or may not have cardiac testing on them. They may or may not be seen once in the emergency room or maybe they've been followed by cardiologist every six months for years, so we kind of get what we get with those 12,000.
746.71 -> But we do have whole exome sequencing information on those draft applications.
752.87 -> So it asked the question talk to my irrelevant to adult human coronary microvascular disease. We looked at 34 non synonymous variance in the fog to Gene and the coding region. In fact to Jinan. The 3 prime UTR and of those, 34 variants. We actually found one that in FiOS analysis was associated with human phenotype and that phenotype was an ICD 9 diagnosis of Afro Sclerosis, which is a great diagnosis because it's not really something you give someone when they have chest pain.
783.54 -> When they're presenting to an emergency room kind of an image and finding.
788.26 -> So this variant, which is Rs 28374544 is actually an ETA G substitution or mutation or variation and this results in an amino acid switch from this area into a glycine.
801.19 -> Picture of protein it's actually pretty big and this is where that variant manifests and what's interesting is that fog too has been studied in the context of mouse development and also congenital heart disease. There are different fog to mutations that have been associated with complex congenital heart disease and this is one of them, actually so when we stumbled upon this finding we went back and looked and said. Hey people have been talking about this mutation because they think it's associated with congenital heart disease specifically TGA.
834 -> Into Trala G.
837.43 -> But what was really interesting is what this variant looked like in our population.
841.96 -> The first of all it was really well represented. We had 44 patients that had that were homozygous for the minor allele that had a Gigi Gina type, we had 550 hundreds I guess patients.
853.87 -> And we had almost 11,000 EA or controller, wildtype patients.
859.74 -> This represented a minor allele frequency of .03, which is actually in line with the minor little frequency of this variant, not not just in the Penn Medison Bio Bank. But in some of these other databases like the exact database.
874.07 -> It's really neat is if you look at more homogeneous populations like populations of African patients. You see that this variant is much more highly represented and if you look at populations that are Caucasian or Asian this variant is not really present at all, so this variant is actually pretty African American specific in our population.
898.52 -> The other thing we found those pretty interesting is that in our adult population. This variant was indeed associative complex congenital heart disease so of our 44 homozygous patients. We had two complex congenital heart disease patients. One tetralogy in one TGA that had been repaired when they were when they were born.
917.66 -> We also saw one in our headers average population. But what's really interesting is 42 of our 44 patients did not have congenital heart disease?
926.88 -> So what did those patients look like so when we looked at discrete left heart catheterization himos for these patients. We saw no difference. No difference in systolic aortic blood pressure during invasive left heart Cath. We saw no difference in LVEP or wedge pressures orare pressures. We saw no difference in heart rate for these patients when we looked at echo parameters for those patients on whom we had echoes. We saw that the EF was the same in all these patients stroke volume left neutral.
957.23 -> Volume and diameters and Wall thickness is where all the same, so we weren't seeing a lot of differences to explain why diagnosis. Atheros grosses might have gone up. But we also were not focusing on vascular phenotypes for these patients.
971.94 -> When we looked at what these patients had they were actually presenting to the emergency room with chest pain, so patients with the GGG neotype actually presented much more frequently than the other. Gina types, and if you look at the survival curve. These patients actually had different survivals. So we didn't see any difference in actual mi's we didn't see any difference in outpatient engine of presentations or heart failure. So we did see a difference in patients coming in to the emergency room with chest pain.
1004.39 -> The problem with that set of data is that it's not matched so the 44 GG patients are actually mostly African-American, there 60% female and may vary in a risk profile in the risk factor profile. So to do deeper phenotyping. We have to generate some matched cohorts. So we took our 44. GG participants and then we found 88 heterozygous and 88. A participants that were matched based on age, gender and race and as you can see, there 93% African American.
1037.55 -> The cardiovascular risk factor profile for these groups is actually pretty similar.
1042.44 -> We did see a difference in the history of the diagnosis of Athero Sclerosis. Here, which is reassuring because that was our initial fee lost signal to begin with.
1055.03 -> So we looked at coronary artery disease in these different cohorts. We manually abstracted coronary angiograms by blinded reviewer.
1063.32 -> And just counted the number of obstructed vessels that they had, and what we saw was that patients with the GG genotype had fewer expected vessels. So they were less likely to have 3 vessel disease more likely to have no vessel disease.
1078.25 -> We then looked at whether these patients were meeting clinical criteria for CMD D so a little bit like that slide. I showed you earlier patients had to have symptoms of angina or MI. Zor exertional dyspnea. They had to have symptoms that were not due to another cost of a patient had a restrictive. Cardiomyopathy didn't really matter that they were desnick it was not attributed to CMPD.
1101.09 -> We considered whether or not they had a positive stress test, but this was not a requirement to count them as having CMVD but they had to have a cat and that Cath had to have relatively clean coronary arteries and we use these pretty strict criteria for participants again for many of them. We didn't have enough data to include them in this count. But what we found is 3 of our 44 participants that were Gigi actually met those criteria whereas only two of the header is aigis and none of the EA participants met those criteria.
1133.73 -> So to get yet another measure of microvascular floor function, we turned to their coronary angiogram.
1141.45 -> Used to play.
1150.78 -> So when you inject contrast dye into the coronaries it can flow quickly or it can flow slowly and if there is not an obstruction. Proximately, the coronary microvasculature and the resistance vessels are determining the rate of which that contrast dye flows.
1165.42 -> And in some patients.
1168.25 -> The phase much, much slower than another patience. Here's a pretty normal. One and in this one. There's a little bit of low flow. If this is really extreme.
1177.62 -> Then we call it a low flow phenomenon, and there have been cohorts published looking at patients whose flow on current ram is really low. But there's actually a pretty dynamic. There's a gradient so some patients are totally normal. Some have low flow where you can look at them and you see that the die is barely into its way along and this one. It's noticeable but not terribly severe.
1199.38 -> So the Timmy frame count is a way to quantify how quickly the die is moving down the vessel.
1205.73 -> And what we found is when we counted the number of frames. It takes a die to move from a proximal landmark to an established distal landmark as a semi quantitative measure of microvascular flow.
1217.33 -> The GG participants had altered my cardio blood flow by this criteria.
1222.58 -> So.
1224.47 -> Reassured that there might be something going on with these patients that fob 2 May, actually be affecting the coronary microvasculature, we dug a little bit deeper.
1233.49 -> So this ada gene mutation at the 1968 base position results in a searing to glycine switch. So when we did some peptide modeling of this small region, which again is right here.
1244.97 -> We found that if you have a polar Serien at that residue then you maintain this loop structure.
1252.27 -> But if you switch that polar Siri into a glycine, which is the smallest amino acid and it can introduce a lot of mobility into a peptide.
1259.33 -> Suddenly, you lose that secondary structure.
1262.65 -> So we then decided to look at fog 2 wild type and the fog to variant or mutant to see what it might be doing in cells, so for a lot of these experiments. We use the cell line AC16 party. Maya sites actually human cell line, which is great because it has human genome in there. It also is a cell line that grows really nicely like HeLa cells. It is pre contractile. So if you're studying circa Merrick Biology. It's not very useful. But if you're studying transcriptional programs in cardiomyocytes actually green sauce.
1293.72 -> And we put into these cells.
1296.18 -> Bob 2 or mutant fog to to see what fob 2 might be doing.
1301.58 -> And it actually wasn't doing too much so fob to as a transcriptional Co regulator so even if you increase the amount of fog 2 or mutant Bob too. In a cell 1000 fold, actually doesn't do too much. We didn't see a significant difference in most of the known targets of fog too.
1320.12 -> But since Part 2 as a transcriptional Co regulator it needs a partner needs a binding partner to actually have transcriptional activity.
1328.86 -> So this made us think that we should find a good binding partner for 5. Two Gata 4 is known to be a binding partner for fog too. But we hypothesis hypothesize that flopped, too, may also interact with him.
1341.62 -> So far, 2 is expressed at the same time frame and development as half and they actually share a really important target in veg F.
1351.3 -> So I'm sure most of you have heard about his by now. the Nobel Prize was just awarded to Francis Crick. Peter Radcliffe and Bob Kaylin for their discoveries in half and it's because he is such an amazing cellular pathway, so in the presence of hypoxemia.
1366.51 -> The hip protein is stabilized and you get a buildup of the Hipc protein. The hip protein can then go to the nucleus and it leads to transcription of a bunch of jeans. You can classify those jeans two ways. Angiogenesis and mitochondrial function jeans and this actually makes great sense of yourself your cell and you're not getting enough oxygen. You want to do these 2 things you want to increase angiogenesis get more blood vessels to bring you more oxygen, but you also want to decrease your reliance on oxidative phosphorylation and decrease your utilization of oxygen.
1397.46 -> You want to switch away from oxidative phosphorylation, so hip regulates both of these that promotes angiogenesis, but it also dampens down mitochondrial function.
1406.79 -> And if you're a cardiologist or you have a cardiac centric view of the world. One of these is really good.
1412.56 -> And one of these is really bad.
1414.39 -> And when they manipulated hip and mice actually the result is that bad things happen. So if you overexpress hip sure you got more Energe Genesis, but you end up getting heart failure because you dampen down, mitochondrial function. Similarly, if you don't have enough if you don't get appropriate angiogenesis, so manipulating if at that level is not a viable option for the heart.
1440.27 -> So what half dozen these AC16 cells is the same thing that it doesn't pretty much every self so if you.
1447.89 -> Treat the cells with hypoxia over a matter of hours, the hip protein builds up. It goes to the nucleus and you get.
1455.33 -> Transcription of these target.
1457.59 -> The Jeffay is an androgenic target and PDK won an being up 3 or metabolic targets so the transcription factor just goes and does its thing.
1468.32 -> We wanted to ask the question does fog to affect how hip does this job in Korea Maya sites.
1474.57 -> So to do that, we use, CRISPR CAS, 9 genome editing to make cells deficient in fog too.
1480.15 -> So AC 16, Curry Maya sites like all Cardi mind sites are diploid or more. So it's actually pretty hard to knock down all the alleles but we were able to knock down 1, two or 3 of Belial's so these are knocked down not knockout cell lines and you can see from here is an example of a control. There's some fog to expression and in a lot of these knockdown cell lines. The fog to RNA expression is decrease. Similarly, fog to protein is expressed at low Basel levels in the control.
1510.18 -> And in the knockdown cell lines fog 2 is decreased. This doesn't really affect if the cell has.
1518.16 -> And we saw something really interesting.
1520.56 -> So when we took these knockdown cell lines and we treated them with hypoxemia. We saw no difference in how the cells responded to these metabolic jeans, so expression of PDK one went up over hours being up. It went up over hours and Cox for one, which is another metabolic factor went up stayed the same over several hours.
1543.05 -> Angiogenic factors on the other hand, it's something pretty neat, so veg. F usually increases in response to increased hypoxia and half. But in the fog to knock down cell lines. We saw that this response was pretty blunted. Similarly, we looked at adrenaline. Edulan and saw the same response and we also looked at some of the Ange PT one and 2 jeans and saw something similar.
1567.75 -> So, In other words, it looks like modulating fog 2 actually affected how hip induced its angiogenic in metabolic target jeans. So instead of just having this model where hypoxia leads to increased hip and we get both of these arms. It seems that fog, too, may help if target, some of its target jeans and not all.
1590.85 -> So there are clearly a lot of really interesting therapeutic potentials that may come from this.
1596.15 -> But if we go back now to our fog 2 and our father to mute and say OK well. When we overexpress 52. Wild type in mutant. We didn't see too much. But what if we do that in the context of hypoxia.
1608.32 -> And then we started seeing some interesting differences, so.
1611.56 -> If you
1612.97 -> Transfect the cells with an empty vector versus the fog 2 versus the fog to mute and you start to see that there's a pretty big difference and how these cells upregulate angiogenic jeans in response to hypoxia.
1628.52 -> To further characterize this we didn't just look at the gene expression in the cells. We took conditioned media from the cells that had been treated so the cells are making more of a Jeff and Angie PT one, but what are they sick reading into the media? What signals might be getting out and we found a difference in some of the HP teas and some other inflammatory markers, so we can take that soup. That's different between fog 2 and fog to mutant and put it on endothelial cells to see all right if his cardio myocyte is expressing fog 2.
1660.07 -> Or if it's expressing the mutant form of log 2, does that affect how it signals. The neighboring vascular cells to make her support vessels and Innertube formation angiogenesis acid. We saw that there actually this is what they look like. But we saw that the fog 2 and the 5 two mutant behave differently to cells that had been treated with fog 2 or the fog to mutant formed different amount of branches of segments the length of the branches were different.
1687.43 -> And so we started seeing some subtle angiogenic difference between these.
1693.52 -> So to summarize this part we saw that in humans. This fog 2 variant results in a CMVD like phenotype.
1701.26 -> Fog 2 May serve as a cofactor for hip and modulate how hip induces its angiogenic targets.
1708.18 -> And how it may do that differently from its metabolic targets.
1712.09 -> He saw that the mutant enters a hip induced angiogenic program in vitro.
1718.35 -> But we haven't yet determined what the effect of this mutation might be in terms of coronary microvascular Physiology, which is what we need to do to tie it back to the patients, so moving forward. We have to take the wild type and the mutant and start to evaluate them in the context of a model system so we can actually see what these different angiogenic factors mean for the vasculature.
1742.72 -> In order to evaluate that model system. We have to have a way to quantitatively assess the coronary microvasculature in Mysore your model system of choice, so the direct goal of this is to find a non invasive method to quantify microvascular function and then use this to phenotype mice that have that a 1968 Gene Mutation.
1763.21 -> There are lots of cardiac imaging options for Mikes, including MRI pet an echo we have used coronary Doppler of the mouse on left coronary to get coronary flow reserve, it's really technically challenging.
1776.43 -> And we're also working to develop mouse Specht.
1780.32 -> So this is some Mail spect imaging that we've done using a pimple collimator and this is very similar to what we do clinically with cardiac pet So what you can do is take pretty short frame so these are 10 second frames.
1794.08 -> An image the mouse heart as technetium 99 M sestamibi is injected Venus Lee.
1800.7 -> So you can see what the curves do here. The The Blue curve is the blood pool, which is this little red box in there, so initially there's no activity.
1810.45 -> And then as the treats are reaches the.
1813.91 -> Left ventricle cavity, you see a huge burst in activity and then overtime as a tracer gets taken up by the myocardium and other tissues. The activity in the blood pool goes down.
1824.47 -> And similarly in the myocardium, which is this blue region of interests. You can see that there's no update and then as you get the bolus of the tracer.
1833.57 -> You get steady uptake into the myocardium you can model this to actually get a K1 value and try to link that back to myocardial blood form. This is what we do with our patients in cardiac pet.
1844.73 -> But it's really tricky in mice, so some people, including groups here have described using Inter myocardial blood volume as an alternate measure of the coronary microvascular and this is based on the idea that there is a relationship between flow and volume actually and a lot of the volume into my party with blood volume is actually contained in the coronary micro circulation.
1869.75 -> So to leverage this in mice what we did is we labeled red blood cells with technetium 99 pyrophosphate and we can image the same regions of interest, the blood pool and the LV.
1884.85 -> And traffic to be overtime.
1887.08 -> And we can give the mouse a hyperemic insult whether it's more exploring to vasodilator diaper to Malta Visa, dialing what you can see is that the blood pool have constant the amount of uptake in the left ventricle goes up.
1900.51 -> And this means that the mice are able to increase under hyperemic conditions there. Microvascular blood volume about 22% which is similar to values that have been found in dogs.
1914.35 -> So initially the goal of some of the image. Ng work is to have a quantitative noninvasive readout to assess the mutant mouse that I'm going to study next, but this imaging modality actually has thousands of different applications. It would be incredibly useful to study potential therapies for coronary microvascular disease and to also evaluate other molecular gene targets that might be involved or might play a role in the pathogenesis of microvascular disease.
1943.2 -> And now I'll get to the twist, one story, which is an example of this so the genome wide Association studies that have been performed for coronary artery disease have given us 200 and some loci that are associated coronary disease. Lots of groups have spent a lot of effort to do functional. Genomic studies and figure out how this variant identified by G Wason people actually effects different cell types to cause coronary artery disease.
1970.8 -> This is one locus that has been described 210-7595 and then this is its position on the genome so it's located here between each deck 9 and twist one and it's actually this variant has been linked to each deck 9 and there has been a lot of research in the role of each deck 9 in coronary disease and some other phenotypes.
1992.79 -> Because it's geographically closer.
1996 -> But we showed in our studies that this locus is actually regulating twist 1 gene expression as well as H Dec 9.
2005.32 -> So we found that participants that had the minor allele for this invariant actually had more twist. One transcripts than the GG Gina type.
2018.58 -> So we looked at whether twist one, maybe playing a role in endothelial smooth muscle cells, and given that the expression of twist. One is much greater and smooth muscle cells, and a lot of the regulatory regions are actually enhancers that are active in smooth muscle cells?
2033.31 -> We examined the role of twist, one in smooth muscle cells, and what we found in a proliferation assay is that if you overexpress twist, one which is this pink line number of cells goes up so twist, one promotes smooth muscle cells pull iteration.
2050.86 -> Conversely, if you use siRNA to decrease the endogenous amounts of twist one that sell really don't grow?
2058.58 -> So twist, one is really important for smooth muscle cells to grow and Pull Ephorate.
2064.54 -> A different smooth muscle cell phenotype is calcification, so smooth muscle cells have the ability to become osteoblast like inform calcifications and one way to test that is, you can take smooth muscle cells in culture and give them a phosphate. Rich media and they will develop these little black classifications that are stained by owls are in red.
2083.66 -> So when we did that we found that if we overexpress twist, one we decrease the number of calcifications.
2091.48 -> Conversely, if we decrease the amount of twist, one by using an siRNA against which one we increase the number of calcifications.
2100.57 -> So this was a pretty interesting result that twist one may play a pretty significant role in modulating smooth muscle cell phenotypes.
2109.95 -> We next reached out to a mouse model to see if we could understand what this might mean in Vivo. So we took myosin heavy chain 11 cream ice so these are mites that are have flocks twist. One allele and when you combine the Korean the flocks. You remove the twist 1 gene only from mature smooth muscle cells.
2131.13 -> And then we take these cells. These mice that have twist one deleted from their smooth muscle cells, and we give them an atherogenic stimulus.
2140.83 -> And the first thing we found is that there is actually no statistically significant difference in the amount of Athero Sclerosis. So the twist. One knockout mice only had 2 hits. One deleted from a tiny subset of their smooth muscle cells, and then this did not affect how much after sclerosis developed over a few months.
2159.51 -> But when we looked a little bit closer. We saw something really interesting so these are immuno fluorescent sections of the ascending aorta and these are from a wild type mouse where you have the shoulder region of an atheroma that has developed in the artery and then you have the middle of the plaque here.
2176.3 -> And what you see is that there are a lot of smooth muscle cells in the vessel wall when we staying with SM22A.
2182.33 -> But there are also some smooth muscle cells, overlying this after all.
2187.05 -> In the middle of the League. Legion you have a lot of SM22A positive smooth muscle cells, overlying the after.
2194.55 -> In the knockout mouse, we didn't see that so if you look at the shoulder region.
2199.29 -> You don't see a lot of smooth muscle cells there. There's still a lot of smooth muscle cells around the vessel ball, but not as many overlying the atheroma and shoulder region.
2208.36 -> And when we looked mid lesion.
2210.62 -> We also didn't see the same number of smooth muscle cells.
2215.88 -> Then when we looked at calcific calcification just like we had done in.
2221.4 -> So this is a cross section at the base of the aortic root so you have a little bit of the aortic valve. Leaflet Here and then this is the root of the big hunk of atheroma and if you look at the sub endocardial calcifications. We don't really see any here, but you do see a lot in the knockout mouse, so the knockout mouse is forming subendocardial calcifications, whereas the wild. Type is not you also see some difference differences here invalid classification, where they're both forming calcifications.
2252.95 -> At the knockout mouse is clearly forming more aortic valve calcifications in the wild type notes.
2261.59 -> When we quantify these we saw that there was a difference. So it's not just these pictures, but when you quantify over a large cohort. We see that so it looks like twist. One may play an important role in smooth muscle cells in vivo and that role maybe to affect plaque morphology.
2282.16 -> So we found that this Locus Rs 210-7595 is associated with increased twist 1 levels.
2291.05 -> And then we evaluated what twist one might be doing in smooth muscle cells in our in vitro studies. We showed that twist one might increase proliferation and decrease calcification and similarly in our in vivo model. We saw that the twist. One knockout had fewer smooth muscle cells, overlying lesions and had more calcification. So the way we put this together is that twist one may actually modulate the developmental stage are the phenotype of smooth muscle cells.
2322.13 -> And smooth muscle cells are known to be really important in.
2325.28 -> Coronary disease and they're able to modulate their phenotype and promote or protect against after sclerosis based on.
2333.2 -> So if you have a smooth muscle cell that is driven to be more proliferate if it will grow but it will not have the potential reform calcifications.
2342.6 -> If you don't have twist one you push the cells towards a more developed more and more specialized function. They become more contractile and you can even push them to be osteoblast like inform calcifications. But these cells don't really proliferate well so twist. One is playing a role. We think in coronary artery disease by modulating how the smooth muscle cells behaved and again a lot of in vivo stuff. I showed you is based on deleting twist, one from a tiny subset of smooth muscle cells.
2373.33 -> But we haven't deleted twist, one from larger populations and then may be able to see a more dramatic Phoenix.
2380.12 -> So if we want to connect this finding back to human disease increase twist. One has actually been associated with stable coronary artery disease, so of the 200 low side that we have found for CD people have been doing sub phenotyping of those, saying that coronary artery disease that includes MI and stable angina is actually pretty heterogeneous group, so if you look at a locus you can tell whether or not, that Locus is associated with a more stable CD phenotype. Warren MI phenotype and this is one paper that did that, so they found for example, that the Aveo Locus is associated with.
2414.35 -> Am I over CD the twist one or H Dec 9 Locust on the other hand, is actually associated with stable symptoms.
2422.26 -> So it's possible that twist one may contribute to plaque stability and lack patience form larger plaques that therefore don't rupture and they get a stable CD type simple.
2433.63 -> Alternately and this is where I think there's a lot of interesting potential twist. One effects, smooth muscle cell phenotype and the smooth muscles are smooth muscle cells are really important in regulating coronary microvascular tone. So it's possible that if we disrupt where on this spectrum. The smooth muscle cells in the coronary microvasculature sit. We may lose some microvascular regulation and then end up with dysfunctional microvasculature, so another aim of developing the mouse imaging is to be able to evaluate the role.
2464.62 -> Of these coronary artery disease, G walks loci jeans in the context of microvascular disease starting with twist one.
2473.98 -> So, In summary coronary microvascular disease contributes to the burden of ischemic heart disease. It's an understudied area of coronary vascular biology and it has pretty direct and definitely widespread clinical relevance.
2488.46 -> So my approach to coronary microvascular disease has been to use known coronary artery disease. Giwa's loci and jeans and also jeans that are known to play a role in coronary development.
2500.11 -> We found that a variant of Father 2 is associated with human CMVD and may affect how hip regulates an angiogenic program.
2509.44 -> And we also showed that fog, too, maybe acting as a cofactor for him.
2514.62 -> Bob tude because of that, maybe a really interesting therapeutic target since it allows us to manipulate part of the Hipc Pathway without actually directing a transcription factor.
2526.55 -> And twist wine we show is a smooth muscle cell phenotype regulator and this approach in general methodology could be applied to lots of jeans and different targets to gain novel insight into coronary microvascular disease and potentially find new therapies.
2540.48 -> So a lot of people have helped with this project and I'm happy to answer any question.
2557.15 -> Study.
2564.66 -> What was did you find other other potential targets besides when you must have do you have any give us any insight into some of those other targets well. One of the targets that meets both of those criterias enosse and I know there's been a lot of interest in that so that's a really good example of a coronary artery disease. G wash gene and Locus. That, clearly, plays an important role in the micro better and I haven't pursued that or studied that but I think that's another pretty low hanging fruit for that specifically a lot of the coronary artery disease, Jiwa slow sigh.
2596.67 -> Also affect lipid levels and LDL and we know that those in some not necessarily direct way affect the microvasculature so I think there's a lot of.
2605.55 -> Of interesting biology that we can link that either known or unknown. A lot of the G last targets actually aren't even known their jeans that we don't even really understand their function so whether or not they may play a role in microvascular disease is wide open.
2620.11 -> I have 2 questions one is that about your genetics and that is the fuck to that. Usually the common variant in African American you compare it with the actual general population of control. I think black and white together, yes so.
2641.26 -> I think it would be more validated if you would have actually compared to the black controls than not at all, and then if you have done for only correction or not I don't know what would be happy value, but another concept is that a link to the microvascular disease and you I could testing 2. Neural Christine's. At least 1. In fact, basically expressed in a new request an actually involve epicardial vessels specifically in your not calling.
2671.37 -> Talk to I don't know what's hypothenar called you had on there was a targeted. I don't know we need more teachers. That was so the fog to conditional knockout that I showed was built fuse work and they conditionally deleted fog to actually from cardio my sites and Ophelia cells and smooth muscle cells, and they found that it's cardio myocyte fog too. That is important for this phenotype. It actually doesn't do much in a new feeling smooth muscle cells, so that we're not talking also intended Thilo muscle cells. So is it seems to be more League.
2701.57 -> 2 actually macrovascular because they lot of your features are macro. Vascular also the 3rd question are hobbies aren't asking too many question is that the common barrier in the second gene that was in the functional variant. I mean, meaning that a lot of areas, but they may not be exactly functional. They may be associated with another variant tags actually functional. You guys were come to find what is the function we did some fine mapping to determine if this was indeed the lead step?
2732.18 -> I don't know with 100% certainty that it's lead Snip and it's hard to differentiate whether that's lead snip or whether there are 4 lead snips in that appetite block that are really important. We did do some studies where we did. Lucifer, a study so we mutated that single residue that single on base pair and actually saw that it affected how that region worked as an enhancer so you're right. I think we don't have definitive proof that it's the lead. Snip buy it based on insilico modeling of what it would do to that enhancer region. I think we're.
2764.71 -> Confident that it's probably very important.
2769.73 -> A wonderful work back to the 5 two story so some of the effects of protein target protein expression or target. Gene encoding protein expression was pretty dramatic and maybe even more dramatic than effects on RNA level. So I was wondering whether there was any again. The problem with glasses looking at low sign coding regions, etc. Whether any non coding. Indirect effects of fact 2. Not so much as a transcriptional correct ya later if.
2804.74 -> But affecting non coding. Rnas my current is and that could have a sort of a multiplicity effect on numerous jeans. I think that's a really good point because there is a very long anti sense, Tran script that gets made for that distal part where a lot of these variants would also hit the role of that anti sense transcript is not really known. So it could absolutely have a role in that and then the other interesting thing to your point.
2834.23 -> The Locus in the fog to gene that's been associated in coronary disease, Jiwa Studies is actually intronic and far from this so I looked at coding variance as low hanging fruit for the fee was but figuring out the genetics of why this intronic variantes, but this isn't the one that's been linked with coronary artery disease it. Some intronic variance so figuring out what they're doing, and whether they're acting through 5, two or weather like you're saying they're regulating link RNA that then goes on and effects a different gene and I think that's why the field of functional genomics is so.
2866.9 -> Challenging and frustrating.
2870.48 -> First of all welcome and free talk to work either kind of far field. These questions, so one is that there is the 1st question related to back that there had been associations. Clinically, maybe with regards to differences by sex as well as in patients who might have stress response differences as well, and query much faster and how would you explore that in these models and the 2nd question really relates to?
2901.65 -> Next steps and you know, obviously there's been a lot of interest in medallion randomization kind of projects UK biobank as an example do you see this moving in that direction and what are your next steps personally so I think to address the sex issue? I think there's a really interesting interplay between gender and microvascular disease because traditionally historically cardiac syndrome. X was a woman's disease. And now in some in some clinical studies. We've actually found that for example, views cardiac.
2932.39 -> At the rate of microvascular dysfunction by cardiac pet is the same in both genders. But I think might be going on is that men tend to form more visible focal coronary artery disease, so for that huge segment of the clinical population that has both microvascular disease ansidei. The men are getting treated because they have something that just looks a little more prominent so they get binned is having coronary artery disease. They could put an aspirin a statin. Maybe beta blocker and they're treated more aggressively and that's why we see differences in outcomes.
2963.4 -> I don't think there is necessarily a huge difference in whether men or women are having coronary my professor dysfunction. There are some physiologic differences, so myocarde resting myocardial blood flow. For example, is higher in women and MVFR tends to be higher in African American so there are some gender and racial differences to the corner capacitor and whether that is from something like a fog to variant that is more expressed in African Americans that remains to be seen, but I think it's a really interesting area to investigate to see if maybe some of the disparities actually have a?
2995.07 -> Biological source and then I think moving forward, the framework of looking at coronary artery disease. Jiwa studies and getting a subset of those that are involved in coronary microvascular disease uses things like the UK by?
3008.38 -> Have.
3009.54 -> Data that's been collected and resources that exist and trying to find additional targets so that is one area that I would definitely love to pursue an then the fog, too, and twist stories specifically figuring out how they affect microvascular function in a mouse model and what that translates back to humans to mean I think is another direct area that I'd love to work.
3032.04 -> Muscle cell Phone.
3038.37 -> And its potential in the myogenic response as it relates to the microcirculation and there is some data that integrins are involved in my agenda. Christmas have you looked at changes? No. I think that the very easy. Next step for this is to just look at the mice that have these differences and do broader profiling to see are the hearts of these mice expressing different smooth muscle cell factors like integrins are they expressing different PDG apps and things like that.
3068.63 -> Very nice.
3079.23 -> Smooth muscle problem or inflammatory problem or what information I think it's a car problem, so a lot of the fog to work has been very cardio myocyte centric and I think we know that hypertension diabetes. Poor periodontal held all of these things hypercholesterolemia are vascular toxic so they affected with human cells. They infect macrophages. They affect inflammatory cells effects with muscle cells, but what I'm interested is in understanding how the cardio myocyte.
3109.24 -> Hopes to regenerate against it because I think it's very important to treat hypertension. It's important to treat diabetes, but
3116.15 -> One step further is you already have the vascular insults. What can we learn about the cardio myocyte that promotes collateral ization that promotes healthy compens atory response to those vascular.
3133.07 -> Full follow Marians passing and then based on the clinical autopsy study.
3148.69 -> 2nd.
3183.76 -> We haven't looked specifically in those models to answer that question. But I think your question raises really interesting things about what do we know about the pathophysiology of CMVD and some of these are from human autopsy samples and some are from mouse models like a hypertensive diabetic rap for example.
3206.84 -> And there's clearly evidence of smooth muscle cell proliferation and that's why you get some of the Lumina obstruction and some of that orchila cities that form but I think we don't know. And when you mentioned endothelial dysfunction on the autopsy samples that's hard to assess and less you're doing you know stick chemistry and finding markers. It suggests that, but one of the things that field is missing is the link between these structural abnormalities and the functional problems that we see and we measured. We evaluate in patients that we think are giving them the syndrome so I think there's still a pretty big disconnect between those.
3238.25 -> And that disconnect also exists in coronary artery disease.
3241.49 -> I think we've been able to bridge a little bit better.
3245.47 -> 2nd question sorry 2nd question in regard to the upstream pluck the town's German corner artery was collected this based on the clinical result there's no correlation.
3259.42 -> Can you comment on that?
3267.5 -> Large corner artery plug and the town's tomb. Microvessel did disease. They can coexist an overlap, but they don't necessarily have to when I think about it a little bit like aortic stenosis and aortic sclerosis. So these are endothelial cells that line, some sorts with muscle cell or valve interstitial cell group of cells and they come from different embryological origins. There are exposed to different mechanical forces and that's why we end up seeing pretty different.
3299.59 -> Manifestations of
3301.47 -> The person's underlying hypertension diabetes hypercholesterolemia, so some people will develop really bad aortic stenosis and really bad coronary disease. Some will have one or the other same thing with CMVD. Some some people will show up with only CMVD and the rest of their coronary tree looks pristine their valves are completely pristine, but I think they're just 3 different disease processes that affect 3 different regions because there are different developmental origins and they have different forces that affect them, so certainly they can coexist.
3331.49 -> But we see that they can also exist in isolation in patients.
3337.36 -> Thanks, I was a great talk, um, I was interested to understand recognizing that microvascular disease is sorely underdiagnosed and it's been estimated that about 30% of women with ischemic syndromes.
3349.74 -> Go undiagnosed in may have microvascular disease. I mean, there's this search to identify women that we should be doing more advanced testing in and who may have a microvascular disease.
3363.69 -> In your few S work are you seeing.
3368.44 -> Clinical the clustering of clinical factors like obesity hypertension in that group with the Mutation is that specific to African American women or should we be kind of thinking more broadly, I think.
3386.77 -> The fee was studies don't specifically address that I think it's a really interesting intersection of how risk factors are affecting the different genders and certainly as you bring another risk factors like obesity how that plays out, but I do think that as we start to do more routine clinical imaging, where we assess the microvasculature like cardiac pet or as we do more.
3407.64 -> Timmy frame counts for clinical cats, which nobody does, but in theory, we could I think will start to understand more about what you're saying there's been some studies that have looked specifically at your question and I think the results differ so in some studies. There isn't a huge difference between men and women and other studies have actually found that there's an increased prevalence of risk factors in women and these women are coming in and having decreased on VFR by cardiac pet.
3434.12 -> I wonder if you can help us with.
3445.61 -> If you
3447.24 -> Normal stuff.
3454.19 -> Group.
3456.89 -> Not for sure that makes perfect sense now based on the full correlation between microvascular disease.
3464.15 -> Is there a test order and said this is a favor?
3478.1 -> I I think I think cardiac pets are really good option for that, so the sensitivity and specificity for evaluating those perfusion defects is much better than specs. So you're not likely to save for a woman. Oh, this is just breast artifacts in or the quality of the perfusion imaging is just better, but Additionally you get microvascular data. You get myocardial blood flow and blood flow reserve and I think that information as we collect more and more of it will be able to answer your question much better.
3506.78 -> And that is that you can actually look to see is very number one number.
3539.13 -> Yeah, yeah, yeah, the question.
3547.69 -> Email.
3551.07 -> Email happy to talk about for for I think the match culverts were about 60% female and that's interesting in and of itself because the population. About 5050 in the medicine bio thing. So why is 60% female interesting question?
3568.05 -> B.
3569.42 -> The fog 2 studies were not done in female mice. So it's a great question of what is the role of fog too, and maintaining the corner market passenger specifically in mice and we know now that more and more journals are requiring us to look at these phenotypes in both genders that there's huge variability in the manifestation of these transgenic phenotypes and the two genders.
3591.42 -> Alright, thank you.
Source: https://www.youtube.com/watch?v=tmHO1FSkHck