Hal Dietz, M.D., presents at the Johns Hopkins POTS Grand Rounds on November 11, 2021.
so a few quick disclosures but the only one really of relevance is um the scientific advisory board for I two bioscience. Um they did um license some of our intellectual property and are planning a clinical trial for vascular Ehlers Danlos syndrome that I'll describe briefly. So my lab has focused on aortic aneurysm for the last 30 years. Um as you know, aortic aneurysm is defined as a zone of enlargement of the Aorta in genetic presentations of aortic aneurysm. Um these lesions are generally in the thorax and the majority of them um specifically involved the base of the aorta at the sinuses about salva with very rapid taper to a normal dimension in the more distal a sending a word up now, common presentations of aortic aneurysm have been very difficult to model and study because they integrate many unknown genetic contributions but also a complex environmental factors. So we made the very deliberate decision to initially focus on rare Mendel ian presentations of aortic aneurysm and then to use the foothold that we can establish in disease pathogenesis to try to reach out to more common but complex presentations of disease. Another advantage of focusing on mandalorian presentations is that it allows us to create mouse models of aortic aneurysm. Um this is one of many examples that I'll discuss but you can see that um it nicely recapitulates the very dramatic enlargement at the sinuses of val val salva, I'm compared to a wild type littermates. So um there are many. Now Mendel Ian presentations of aortic aneurysm that have been defined um in broad terms they can be split into three different groups. Um Those conditions that relate to alterations in the transforming growth factor beta signaling pathway. Um A number of conditions that specifically relate to alterations in structural extra cellular matrix proteins. And finally uh a growing group of conditions that relate to alterations in the vascular smooth muscle cell contract. I'll apparatus if you just consider these very broad groupings. Um you can make some predictions about phenotype. For example, in the T G. F beta related conditions, they tend to be syndrome IQ with involvement of many body systems and the aneurysms tend to focus specifically at the aortic root. Um With matrix related conditions. Again, you tend to see syndrome IQ features, but the aneurysms more broadly involve the arterial system. Um And then in non syndrome IQ presentations um uh they are more typically related to the smooth muscle contract, I'll apparatus. Um and you can see tears of blood vessels or dissections without prior debilitation. So we've focused on Marfan syndrome. Um for many years, as you know, Marfan syndrome is a systemic disorder of connective tissue with dominant inheritance and a prevalence of about one in 5000. The syndrome is highly variable, with cardinal features in the ocular skeletal and cardiovascular systems, including dislocation of the ocular lens um and overgrowth of the long bones but most importantly, progressive dilatation of the root of the aorta that essentially will lead to aortic tear and early death if left untreated In 1991, we were able to show that mutations causing Marfan syndrome occur in the gene encoding the connective tissue protein fiber all in one. So what do we know about fiber line one? We knew that the protein aggregates to form complex extra cellular structures called microfiber roads and that microfiber holds cluster around the maturing ends of elastic fibers during embryonic growth. So the simple spatial and temporal relationship led to the firm belief that you need a lattice of microfiber holds to make an elastic fiber. And if you think about it that really voted poorly for the development of treatments, it suggested that Children with Marfan syndrome are born with inadequate elastic fibers and that they therefore have an obligate structural predisposition for their tissues to fail later in life. So this week tissues model did a reasonable job of explaining islands, dislocation, perhaps explaining aortic root enlargement. Um and pulmonary emphysema, which can also be seen in Marfan syndrome. But we also realized that it didn't do a very good job at explaining things like bone overgrowth or the cranial facial features of Marfan syndrome or the low muscle mass and low fat stores that are so characteristic of the condition. Each of these findings was more suggestive of altered cellular performance. Now to summarize about five years of work from many groups in a single slide, it later became known that microfiber holds composed of fibra win one serve a second important regulatory function in that they bind the large latent complex of the cytokine. T G F beta and suppress T G F beta release or activation in the presence of inadequate microfiber holds. As seen in Marfan syndrome. We reason that there would be inadequate matrix sequestration of latent T G F beta and on that basis promiscuous activation of the cytokine. This would lead to increased engagement of the cell surface receptor for T G F beta and an intracellular signaling cascade that includes phosphor relation of smash bad two, or smash three. That then partner with mad for trans locate to the nucleus and mediate T G F beta transcription responses. Most importantly, we showed that the high T G F beta signaling in mouse models of Marfan syndrome had direct fanatic pick consequences such as emphysema, aortic aneurysm, thickening and dysfunction of the mitral valve and skeletal muscle myopathy. Because each of these findings could be greatly attenuated or even prevented by treating fibra and one deficient Marfan mice with a pan specific T. G F beta blocking antibody, they still had inadequate microfiber roads, still had abnormal elastic fibers. But these manifestations were greatly improved simply by blocking T G F beta. Now around this time we asked, is there a drug and even better an FDA approved drug that might mimic some of these protective effects. Our attention turned to the angiotensin two type one receptor blocker class of medications with one example being the drug laws Artan because so called A RBS had been previously shown to attenuate excessive T G F beta signaling in rodent models of chronic kidney disease. So how does Lasarte network angiotensin two, signaling through its type one receptor Can increase production of TGF beta Liggins, the receptor subunits for TGF beta and also important activators of TGF beta like thrombosis respondent one. In contrast signaling of angiotensin two through its type two receptor is thought to antagonize many of these 81 mediated events. So we reason that selective blockade of the 81 pathway would attenuate T G F beta signaling in a direct manner And might also be beneficial by shunting signaling through the protective 82 pathway. We went on to test this theory in mouse models of Marfan syndrome. Um Here I show you the aortic root growth rate of abnormal mice, so called wild type mice. In Marfan mice that received a placebo. You see a very rapid rate of aortic root growth if you gave the beta adrianne ergic receptor blocker propranolol that lowers blood pressure but does not address T. G. F. Data. We see some improvement in aortic growth rate but no improvement in aortic wall architecture. It's still very thick, there's still fragmentation of elastic fibers and there's still excessive T G F beta signaling as shown by nuclear accumulation of phosphor elated smell bad too. But no observer. Um could distinguish Lazard untreated Marfan mice from their normal counterparts by any functional or histological parameter, including complete normalization of aortic growth rate and preservation of aortic wall architecture with direct evidence for blunting of T. G. F. Beta activity. So we went on to try this strategy in Children with the most severe presentation of Marfan syndrome. They show unrelenting growth of the aortic root despite maximal treatment with beta blockers or ace inhibitors reaching surgical endpoints or death in early childhood. Um These are the aortic growth curves for the first two such Children that we treated with lows Artan showing a dramatic and sustained plateau in aortic size. Once this medication was started in total, we treated about 19 such Children with A. R. Bs and saw a dramatic 90% reduction um in aortic group growth rate. After starting laws Artan, there have been many trials of A. R. Bs um in Marfan syndrome since that time. Um All of them have either shown uh that they are protected that Lasarte in is protective um or that it is as good as other antihypertensive therapies. Um but recently a meta analysis of seven trials with over 1500 patients really nicely demonstrated the potential of this therapeutic strategy with a dramatic decrease in aortic root and ascending aortic growth when compared to placebo and also a dramatic improvement in growth rate when added on top of beta blocker therapy. Um A dutch group recently reported an eight year follow up of their trial patients. And not only do you see uh decline in aortic root growth rate but also reduced rate of important out clinical outcomes including operation aortic tear or all cause mortality. So back around 2003 or so, um my good friend and colleague Bart Lois and I recognized and described a new aortic aneurysm syndrome that had some features in common with Marfan syndrome, like curvature of the spine, practice deformity, long fingers and aortic root aneurysm but also a number of unique features such as hyper Tillery is um a cleft palate or a bifurcated uvula, cranial sin, osteoporosis, club foot deformity and select forms of congenital heart disease. Most importantly, these individuals show a diffuse arterial, opened the with widespread arterial tortuous city and aneurysms not only at the root, but all throughout the arterial tree. These aneurysms tend to rupture at small dimensions and young ages when compared to Marfan syndrome, We've had the chance to care for over 500 families with this condition that's now referred to as Louise Dietz syndrome. So based upon what we had learned about Marfan syndrome, the reason that this condition would also relate to altered T. G. F beta signaling and indeed we and others found that all presentations of Lois Dietz syndrome relate to mutations in genes that encode direct defectors of T G F beta signaling such as the T G F beta Liggins, T G F beta two or beta three. Either subunit of the T G F beta receptor or the intracellular signaling intermediates Mad two or smash and three. We also had high incentive to understand a rare condition called sprints in Goldberg syndrome, that shows virtually all of the craniofacial skeletal cutaneous and cardiovascular manifestations of Marfan syndrome and Louise did syndrome with the added feature of highly penetrated developmental disability. And we were able to show that that's caused by mutations in the gene encoding ski on the Sloan Kettering institute proto oncogene that functions to directly suppress the T. G. F beta transcription or response. So um Elena, gallo and uh and uh Dave lock in the lab went on to make and characterized mouse models of Louise Dietz syndrome by uh using um site directed neurogenesis um and introducing mutations that had been observed in people with this condition. As we had seen in Marfan syndrome, there's a clear signature for excessive T. G. F beta activity in the aortic root of Lois Dietz syndrome mice. Um Elena was able to show us a very dramatic response to a R. Bs such as laws are written with complete normalization of aortic root growth rate and preservation of aortic wall architecture. And in this context this protection was not seen by treatment with beta blockers that simply lowered blood pressure. Um Ben Kang in the lab went on to create mouse models of sprints and Goldberg syndrome. Um As we expected, these animals showed greatly accelerated aortic root growth in combination with skeletal and craniofacial manifestations of Princeton Goldberg syndrome. Um And again uh as expected we saw high expression of all T. G. F. Beta target genes um that we assessed in the aortic root of sprints and Goldberg syndrome mice. Ben was able to show an equally dramatic response to Lasarte hidden um in this condition again both influencing aortic growth rate um and aortic wall architecture. So the take home um is that these data suggest a broadly relevant mechanism for syndrome IQ presentations of aneurysm. That is T. G. F. Beta um and treatment strategy um that is A R. Bs um in these conditions. And you know, as we have now been able to parse out different ideologies of sprints and Goldberg syndrome and uh Lois deeds and Marfan syndrome. Um It has allowed us to begin to make rules regarding patient management. For example, in Princeton Goldberg syndrome we see low incidence and severity of vascular manifestations um if present vascular issues, focus at the aortic root. So we image very intermittently and reserve medical management and less specifically indicated In Marfan Syndrome. We see highly penetrate route aneurysms but they tend to follow the rules with the risk of tear only becoming significant once the aortic root dimension reaches about 5cm. So we image by echocardiography on a yearly basis. We initiate medical therapy early in life, anticipating later problems. Um And then we begin to image the entire aorta only in adulthood or after route surgery. In Louie's Dietz syndrome. We know it's a completely different beast. Very, I'm sorry, very aggressive vascular disease throughout the entire arterial tree even at young ages. Um We know that the disease associated with mutations in the genes encoding the T G F beta receptor is more severe for example, than the disease caused by mutations in the genes encoding T. G F beta Liggins. Um But we think in all of the presentations of Lois Dietz syndrome, surgery should occur at dimensions less than 4.5 centimeters uh and less than four centimeters in types one or two the receptor associated conditions. We image right from the get go using M. R. A. Or C. T. A. Because of where the aneurysms can be found. So we thought we were making good progress but we realized that we had only focused up to this point on one aspect of T. G. F beta signaling. So called canonical signaling or smack dependent signaling. But it's also known that ligand activated T. G F beta receptors can stimulate so called non canonical cascades most prominently including the mid engine activated protein, kindnesses, junk P 38 and er Kirk we also knew that signaling through the angiotensin two receptor can directly stimulate ERK activation. So we in hope of finding the achilles heel of these diseases. We decided to look for evidence of excessive stimulation of non canonical signaling cascades and really were shocked to see the extent of excessive activation of the ERK pathway in Marfan mice compared to wild type littermates. At this point we didn't know if ERK activation was simply a marker of disease or an actual driver of disease. To assess this, we gained access to an orally bio available potent and selective ERK inhibitor called R. D. E. A 1 19 and saw that this was every bit as effective as laws are written in suppressing abnormal aortic growth and indeed had the expected effect on suppressing ERK activation without any suggestion of uh an effect on any of the other signaling cascades under consideration. So we had a new landmark that irk is bad. Um It we were able to show that ERK activation in Marfan syndrome is both T G F beta and 81 dependent. And we wondered if we could use this new information to address particularly vulnerable or underserved patient populations, populations among such people are women with Marfan syndrome who choose to become pregnant. What you observe is an extreme risk of aortic dissection that has historically been attributed to the high he mode I nam IQ stress seen in pregnancy but it seemed odd that the vast majority of the sections were occurring within the weeks to months after delivery and that the risk did not seem to be altered by cesarean section or antihypertensive agents. So jen hibachi in the lab asked you know what initiates toward the end of pregnancy is maintained after delivery and might synergize with some of these biochemical events that we had associated with aneurysm and dissection. The hypothesis that came to mind was the hormone oxytocin, which is needed to initiate uterine contraction and milk letdown. It's released, peaks at the end of pregnancy and is sustained during breastfeeding and its receptor is up regulated in the aorta. In response to pregnancy. We became particularly excited when we learned that the mechanism that by which oxytocin oxytocin stimulates peripheral tissues is through ERK activation. So we needed a model to test the relevance of oxytocin um to pregnancy. Associated vascular disease. And fortunately our colleague Francesco Ramirez had created a particularly severe mouse model of Marfan syndrome In our hands. With this mouse model. Um we saw that in the week, in the weeks after pregnancy. In the 1st 30 postpartum days, You essentially saw 90% death due to aortic tear and rupture. Um there was no death leading up to pregnancy, I'm sorry, leading up to delivery but all in the postpartum period. This is very different from male Marfan mice or female Marfan mice that had never been pregnant. So we asked a very simple question, what would happen if you allowed these mice to become pregnant, allowed them to deliver but simply took the pups away right after birth to prevent lactation induced oxytocin release. And the answer was that that simple modification transition survival from about 10% all the way up to about 80%. We then asked well what would happen even better if we treated these mice with a very selective oxytocin receptor antagonist quite remarkably. That did not fully block the ability to lactate. In fact these mice on oxytocin receptor antagonists were able to support their pups through lactation and yet they never died. Um We saw essentially 100% survival um in mice receiving an oxytocin receptor antagonist outcome correlated perfectly with the level of ERK activation in the aortic wall which was high in Marfan syndrome, very high in Marfan mice that were lactating and brought back down to baseline by removing the pups or by treating with the oxytocin receptor blocker. So on this basis we tried an FDA approved ERK inhibitor called traumatized NIB and saw that this also achieved very remarkable protection in our mouse model. So through a variety of experiments some of which I don't have time to show today. Um We've had the ability to define an access for vascular disease in Marfan syndrome. Um That includes angiotensin signaling T. G. F beta signaling and oxytocin signaling. This ultimately collapses on a cascade that involves the P. L. C. Or phosphor like a C. I. P. Three protein kinase C. And ultimately irked cascade. We've poked and prodded this pathway every way we could imagine with three manipulations that we predicted would worsen aneurysm and that indeed turned out to be the case and with six different provocations that we predicted would be protective. Um And that all of them also proved to have their expected effect, including things like a P. K. C inhibitor. Um as I mentioned, anarchy inhibitor and this also suggested that the antihypertensive agent hydrolyzed scene would be protective because it works at least in part by blocking I. P three mediated P. K. C activation. This was potentially important because unlike many of these drugs that I've talked about, hydra lysine is known to be safe to use during pregnancy. We went back to our animal model of pregnancy associated aortic dissection and indeed were able to show that hydra lysine is protective. So this is something that could potentially transition to the care of women with Marfan syndrome in the near future. So what I've talked about is um a somewhat hypothesis constrained approach to these diseases, you know, making an observation, making the next derivative prediction and then testing it. But we wondered if there was a way a discovery based method that might take us to someplace unanticipated. Um and the possibility presented itself in the form of a number of exceptional families that showed discrete intra familial clinical variability despite having a defined fibra win one mutation that was present in uh you know that we could define to be present in all of the shaded family members. So in these pedigrees um everyone shaded in black or yellow has the same underlying Marfan mutation. The people shaded in black have classic severe Marfan syndrome with aortic surgery or tear or early death. The people shaded in yellow have no cardiovascular manifestation of Marfan syndrome, even in old age, even into their fifties, sixties and seventies. So we decided to ask, how does nature protect some but not all people with a Marfan mutation and are strong hypothesis was that there was a genetic modifier at work. Um So we performed a standard genetic um technique linkage analysis and asked not if we could map the gene for Marfan syndrome. We knew that already, but we asked if we could map the gene Um that is capable of protecting someone from the cardiovascular consequences of Marfan Syndrome. And we saw this very significant um signal on human chromosome six um far away from the Marfan gene on chromosome 15 that routinely segregated with protection in these families. Um there are 32 genes in this interval, but one kind of jumped out at us as a strong candidate. First of all, it's expressed in the Aorta that was important. Second of all, um we could find that there was a difference in the level of expression of the gene and protein um in people in these families who had mild disease compared to those who had severe disease. The people with more severe disease had higher levels of expression of this gene called map three K four. You know, a third of all, It encodes a mid engine activated protein kinase keen something that activates molecules like irk junction P 38. So we didn't have the opportunity to find more of these exceptional families on a quick basis. So we returned to our mouse model. We found that if we disrupt just one copy of the Map three K four gene, it was sufficient to fully normalize aortic root growth rate in these mice in association with the normalization of the level of P. K. C. And ERK activation in the aortic wall. Around the same time, Jeff Doyle in the lab had a really startling observation. He found that if he put our Marfan mutation In one strain of mouse that's called the Black six strain, it's just the inbred experimental strain that's commonly used in biomedical research. He found that the aneurysms as you breaded into this pure Black Six background, the aneurysms disappeared, they still had the Marfan mutation, but there was no longer a cardiovascular consequence. In contrast, if you put the same Marfan mutation On an ultra pure 129 background and you know, just another mouse background that that's you commonly used in research, then you see these giant aortic root aneurysms and also early death due to tear of aneurysms. There's no difference in blood pressure, heart rate or other parameters between the black six and 1 29 strains. So it's not a there's no trivial explanation about one back, why one background is so protective and the other one is associated with such vulnerability. If you look at the signaling consequences in the predisposed vulnerable 1 29 background, every signaling cascade that we've talked about is dramatically accentuated, including Smadar activity, Earth P. K. C. If you look at the protected Black Six background, you can see all of these signaling abnormalities are either greatly attenuated or fully normalized. Um So it really uh suggested that there was a strong relevance of this signaling cascades to this mechanism of modification. So um we also found that even in the extremely predisposed 129 background there was full responsiveness to Lasarte in or two ERK inhibitors. So again suggesting that we had bumped into something um that is modifying known events, predictable biochemical events. So we had had high incentive to learn how nature was doing this um with the hope that if we knew her strategy it could be mimicked using medications. So what Jeff did is he bred together the two strains, he made mixed mice that were part black six and part 1 29. And then he introduced a Marfan mutation into those mixed mice and stratified them. He said okay, these mixed mice are behaving just like the protected black six parental strain and these other mixed mice are behaving just like the vulnerable 1 29 parental strain. And he asked, well what genes at what chromosomal locations are are consistently segregating from the predisposed 1 29 background um to the offspring. The mixed offspring that show big aortas. And in that way he showed that these modifier genes can be localized to only two positions in the mouse genome. There's one on chromosome five And another on mouse chromosome 11. So when we looked at these and went through our filtering exercise, what's expressed in the aorta, what shows sequence differences between these strains? It really quickly narrowed down to two strong candidate genes on mouse chromosome five. There's a gene called mm P 17 that's known to regulate ERK activation. It's highly expressed in the aorta. And there is a very dramatic sequence change between the two strains with the stop code on in one strain being changed to the code unfortunate ripped often in the other, causing a a c terminal extension of the protein on mouse chromosome 11. Map two K six jumped out with similar reasoning, oh it does activate miraj inactivated protein kindnesses. And really interestingly this map two K six protein is activated by map three K four. The human modifier that we had just identified. So by considering two different species, it appeared that we had stumbled onto a pathway of modification and again there's a very dramatic amino acid substitution that differentiates the two mouse strains. So what Jeff did is he took these mice um He bred them to mice that had knockout alleles of mm P. 17 and map two K. Six. And in that way he could sequentially remove one and then both copies of both of these genes. And that was the only thing that he was selecting for in these mixed mice. He realized that if he knocked out both copies of both genes and only these two genes that was sufficient to make the mice behave just like the fully protected pure Black six strain. If he's selected for intact copies of these two genes and intact 1 29 copies of just these two genes. These mice these mixed mice behaved identically to the severe vulnerable pure 129 parental strain. So this really proved that these are the two modifier genes. Um in uh you know in the in this background difference. Um if you knock out just those two genes um you can see that you fully normalize P ERK activation. P 38 activation. All the mitigation activated protein kinase signaling abnormalities. Also fully normalized Smadar activation. So what about MPP 17. That was a new player. Um and in reading through the literature we learned that mm P 17 is a matrix is a cell membrane anchored protea ace. Um It does have pros geologic activity but independent of that activity, it also has the ability to activate the epidermal growth factor receptor which goes on to activate wrasse raf mech and ultimately our old friend Earth. And in fact in the predisposed 1 29 mice we could show directly that there was excessive activation of the E. G. F. Receptor, excessive phosphor relation compared to the protected Black Six strain here. If you knocked out only mm P. 17 you could fully eliminate that excessive activation of the E. G. F. Receptor as you'd predict from this model. So we were able to gain access to an FDA approved E. G. F. Receptor antagonist called er latino nib and could show that that was dramatically protective against abnormal aortic growth. Um in the predisposed 1 29 mouse background. And indeed treatment with her lot Nib was able to normalize the abnormal biochemical Activity in the Aortic Wall of the Predisposed 1 29 Mice. So we really um I think had made a good progress in defining the biochemical events that culminate. Um In aortic aneurysm and aortic tear. We had a number of therapeutic strategies that were available. Some immediately available because they involved FDA approved drugs um and others that were really appealing targets for clinical development. And I'm gonna end by talking about my least favorite connective tissue disorder called vascular Ehlers Danlos syndrome. It's caused by a deficiency of type three collagen due to dominant mutations in the col three a one gene. Now these folks have aneurysms um and ruptures of any muscular artery notably the aortic root is typically spared in vascular E. D. S. In fact if you're seeing highly penetrate aortic root aneurysms you need to be thinking about other diagnoses these aneurysms or I'm sorry these arteries often tear without any prior enlargement which is particularly frustrating. So you can see a patient do an M. R. A. Or C. T. A. Uh document that all the blood vessels looked fine. And unfortunately you should not be surprised if you learn that they had a fatal vascular rupture the next day or the next week. There's also a high risk of rupture of the intestines and pregnant uterus. Um There's a high risk of operative complications due to tissue fragility. Um So really uh a condition where we're struggling to be able to try to make a difference. So um Caitlyn Bowen and juan Calderone um in the lab went on to make knock in mouse models of vascular Ehlers Danlos syndrome. We introduced the most common type of mutation that is a glass icing substitution in the collaborationist domains Of Type three Collagen. Um we intentionally made mutations of different severity. So one that we predicted would be mild and indeed shows a median survival of 400 days. Um and one that we predicted would be severe with a median survival of only 40 days. All these premature deaths we can document or caused by arterial and more most commonly aortic rupture. So when we looked at the vascular wall in the these individuals we really didn't see all that much. You know there were occasional breaks in elastic fibers but nothing like we see in Marfan syndrome or Lois Dietz syndrome. The aortic wall is a little bit thin. There's a slight reduction in the amount of collagen shown as the blue stain amusing try chrome or shown as this fluorescent stain using pick or a serious red. Um You know, similar to what's seen in the histology of patients with vascular E. Ds. Perhaps uh a modest reduction in the amount of collagen. So Our initial instinct was first trial is art and it's worked every time before and um these are survival curves. Um what I'm showing you here is the performance of our severe mouse model with about half the mice being dead within the 1st 40 days of life shown in orange. We found that if we treated those mice with lazard and it didn't make a bit of difference, they were dying at the exact same pace. We tried beta adrianne ergic receptor blockers like propranolol or tunnel all. They made no difference. We tried calcium channel blockers and as we had seen in Marfan syndrome, they actually accelerate the rate of aortic tear and rupture. Um Despite the fact that they have the, you know, predictable effect of lowering blood pressure, we decided to test an atypical beta blocker called celebra lol because some small studies in France had suggested that that that specific agent might have particular benefit for people with vascular EDS. But the results were unequivocal in both are severe and are mild model of vascular E. Ds. The use of cell liberal all greatly accelerated death due to aortic rupture. In fact in our mild model we would typically not see any death in the in the in the in during this interval. Um But in the mice treated with cell liberal all about half mice had died um within that 40 day period. So the clear conclusion was that we needed new ideas. So what Caitlin did is she took a vulnerable segment of the aorta was not enlarged, it was not torn, but we knew it was the most likely place for an event to happen. And she sequenced virtually all of the RNA molecules within the aortic wall to monitor gene expression. And she was able to come up with this profile of jeans and and RNA is that showed altered expression in vascular Ehlers Danlos syndrome. Compared to normal mice. Um The yellow signal means that there's more expression in the vascular E. Ds mice. The red signal means that there's less expression, but this is a very reproducible pattern. And we asked a computer tell us what molecules are most likely to cause this altered gene expression signature signature. And the clear answer in fact the only answer was that it likely involved a G protein coupled receptor, specifically one that used the G alpha sub Q sub unit allowing it to signal through the P. L. C. P. K. C. ERK pathway, you know. So we had stumbled back onto the same pathway, albeit likely activated through a different mechanism. So we did our usual tricks. We asked well if this is true, if this is really important then if we block P. K. C. With molecules like roe bucks a store nor Enza Storin, it should be protective. If this is really important. Then if we block ERK activation with drugs like Kobe metin IB that should also be protective and that indeed very dramatically was the case. In fact this was the first evidence that we had that there is anything capable of dramatically altering the natural history of vascular E. D. S. You can see that with the P. K. C. Inhibitor, Roebuck's historian survival jumps back up to almost complete um That the same thing is true scene with ERK inhibition. We could document the relevant biochemical events in the aortic wall. Um So um you know the first inkling of a treatment strategy um we wanted to try to understand what was the upstream signaling event. Um We knew it wasn't the angiotensin two type one receptor which is the right type of G protein coupled receptor. But we had already tried Lasarte in and we knew that didn't work. Um We ultimately also um looked at oxytocin signaling because the oxytocin receptor is also the right kind of of G protein coupled receptor. We see the same high risk of pregnancy associated vascular death in vascular E. Ds as we have seen in Marfan syndrome here. If you simply take the pups away, you achieve 100% survival through the vulnerable period and beyond. And you could also mimic that with an oxytocin receptor blocker. So we know that at least one GPC are um that is relevant to this disease is the oxytocin receptor but we also have direct evidence of relevance of other G protein coupled receptors like the enzo. I'm sorry, the endothelial receptor. So we decided to try hydrolyzed scene because of the same reasoning that we had used in Marfan syndrome. It would block P. K. C activation. We started the trial and everything looked great, 100% survival out to day 40 and untreated mice, about 40% had already died. And then the bottom fell out. You know, suddenly we saw this burst of death despite the use of hydra lazin causing these mice to almost catch up to the untreated mice. When we stratified this by sex, we saw that some of it was going on in female vascular E. DS Mice, but it was greatly exaggerated in male mice and specifically starting at around day 40-45 of life. If you ask what's going on at that time. The answer is puberty. Um So this led us to the hypothesis that there might be a role for male sex hormones. This was very important because in patients with vascular E. D. S. Um adolescence is a point of great vulnerability for um young men. Um So this is what really rang true with an unmet clinical need. So what we did is we treated the mice with hydra listen to get them to puberty. And then added a potent androgen receptor antagonist called by colluding meid and found that this was able to allow these mice to get through this vulnerable period. Um We also realized that parents of adolescent boys with vascular ups, we're not going to be very eager to start a highly potent androgen receptor blocker. We wondered if something more mild might work. And what came to mind is the diuretic spironolactone um which also has anti androgen activity. In fact it's used to treat a number of androgen related clinical states including acne and alopecia. We were thrilled to see that the combination of hydrolyzed scene plus spironolactone um could achieve complete protection through this the period of adolescence um and beyond. So we did our trick of breeding to the different mouse backgrounds. Remember in Marfan syndrome 1 29 mice were vulnerable and black six mice were protected Here. It's exactly the opposite. Black six mice are highly vulnerable. That's the data that I've shown you up to now. But if we put a vascular E. DS mutation, the severe mutation on a 129 background. The mice lived through the period of vulnerability. They live to a year of age. They live to two years of age they get pregnant. They do all sorts of vulnerable you know of risky things. Um And yet they just survive and survive and survive. Um So nature has found a way of beating vascular E. D. S. In a very dramatic way curiously. It does not involve involve increasing the amount of collagen in the aortic wall and doesn't even involve increasing the structural integrity of the aortic wall. If we take aortic rings out from protected 1 29 mice they're just as biomechanically fragile As the vulnerable black six mice. So um Caitlyn did the same strategy that we asked. We described before. She defined what a vulnerable mouse looks like. Um She described what a protected mouse looked like. She then made mixed mice and stratified them As to whether they were behaving in a protected manner or 129 like or a vulnerable manner that is black six like. And then mapped the modifier by stratify ng these animals. And interestingly we bumped into one of the same chromosomes that we had found in Marfan syndrome. In fact the exact same map position that we had founded Marfan syndrome. In fact the exact same gene That we had found in Marfan Syndrome Map two K. 6. But now high activity is protective unlike in the Marfan experience where high activity made the aortic root vulnerable. We found that if you simply knock out the map two K. 16 in vascular E. D. S. Vulnerable 1 29 mice um that you then they then um I'm sorry in the protected 1 29 mice you then reintroduce vulnerability. So map two K. Six is doing something beneficial, something important and protective in vascular EDS. So we looked into what map two K. Six does and what it predominantly does is activate a protein called P. 38. What we know p 38 10 do is to activate proteins called phosphate ass's and specifically PP one and PP two A. That are known to deactivate P. K. C. And earth. So this would all make very good sense if hye map two K six activity in the descending a ward of the vulnerable segment was associated with high p. activity that would be supportive. And that's indeed what we saw in the 1 29 protected background. There's high levels of activation of P 38 compared to the vulnerable Black Six background. So we could satisfy that prediction. Next thing we'd predict is high activity of these phosphate ass's. And that's exactly what Caitlin found. Um when she took protein extracts and monitored phosphate based activity. It's greatly increased in the protected 129 background. So we can verify that the final prediction is that that high phosphate latest activity should be keeping P. K. C. And ERK activation low at basal levels. So in the vulnerable Black six background we see high P. K. C. And ERK activation in vascular E. Ds mice. Um But on the protected background with the high phosphate taste activity. There's absolutely none of that. In fact the level of activity is slightly lower than in wild type or normal animals. So really now we've defined a full Cascade of protection. Um and p 38 activators or agonists and phosphates. Activators or agonists have now emerged as new treatment strategies. What about people while we're just starting to look at tissue from patients with vascular E. Ds. But we certainly see the high levels of P. K. C. And ERK activation in the aorta or in the iliac artery compared to healthy blood vessels. Um And that's where this company i to BioPharma comes in. They now plan to launch a large multinational Phase three clinical trial of the P. K. C. Inhibitor ends a store in in vascular E. Ds patients. So last slide a confluence of clinical observation hypothesis. Um discovery based methods um has led to a refined understanding of aortic wall homeostasis. Um and aortic wall vulnerability. We have many new promising therapeutic strategies including FDA approved drugs and drugs that are being explored for clinical development including P. K. C. Inhibitors. I'd like to end by acknowledging the really wonderful people. I have the privilege of working with every day. Those who made specific contributions to the story I told today are shown in Red would like to acknowledge my collaborators and other institutions and my funding sources and I thank you for your attention. I'd be happy to answer questions.
Facebook
Twitter
YouTube
LinkedIn
Instagram
E-News
BROADCASTMED