Dr. Jeffrey R. Fineman - Pulmonary Vascular Disease in Congenital Heart Disease: From Bench to Bedside

Good morning, everyone. Welcome to Combined Grand Rounds. And we are very pleased to welcome Dr. Jeff Feynman today. He urged me to give the short form of his CV. And indeed, I have to because the long form is too long. He is a professor of pediatric set, the University of California San Francisco, and Benning off Children's Hospital, where he's vice chair of pediatrics, his chief of critical care, he's the director of the Pulmonary Hypertension Service, and he's the director of the Translational Research Program. He has a lab that's been continuously funded by the NIH for the past 20 years, studying prenatal, transitional, and postnatal pulmonary blood flow and pulmonary vascular resistance. He's the associate editor of three journals. He's won a number of awards, and he is as modest and collegial as he has accomplished. Bob Schamberger encouraged us to the CDH Working Group to look around the country and examine other programs. And it's really Bob's credit that we got Jeff out here with some difficulty. And so Jeff, we look forward to hearing from you. And as you know, I ask you to describe both the work that you do as director of the Pulmonary Hypertension Service, in particular, as you care for patients with CDH at UCSF and the publications that you've done, but also to speak to us about your research into pulmonary hypertension and where you see the field today. So Jeff, welcome. Thank you very much, Dr. Burns. It's an absolute privilege to be here. And I think when we start talking about my work, you'll see that I think the landmark studies that really inspired me to go into the research field that I went into, actually all came from this institution. So it's a real honor to be here. Thanks. So as Jeff said, well, in terms of disclosures, the really relevant one is that the first part of this talk is one of those. This is what we do type of talks, which I really don't like to do. But Jeff had asked me to talk a little bit about how we built up our Pulmonary Hypertension Service and in particular, some of the approaches we have to the CDH patients. So I'll start with that and then I'd like to move into some of the work that we've done where we focus on Pulmonary Hypertension Associated with Congenital Heart Disease. I'll talk a little bit about the natural history and some of the work we've been doing with the underlying path of biology. So first in terms of the Pulmonary Hypertension Service, as many of you know in this audience, there's critical differences between adult and pediatric Pulmonary Hypertension with a significant difference being of the fact that there's really a lot of issues related to developmental biology and there's a lot of issues related to genetic predispositions and other genetic malformations, etc. And as this slide will show, there's significant differences in the epidemiology. This is data from the PPH network, the first 1,500 patients, which Dr. Mary Mullin, who's in the audience, really has done a tremendous job putting together. And Group 1 is Pulmonary Hypertension within that group of Congenital Heart Disease Group 2, as Pulmonary Hypertension associated with the left heart disease, Group 3 is associated with the lung disease and the adult epidemiology is very clear that Group 1 is clearly the most common followed by Group 2 and then Group 3. And as you can see by this data on the North American network that actually Group 3, that associated with hypoxia and lung disease, is the most common followed by Group 2. And if you look deeper into Group 3, obviously most of these patients are infants with either lung hypoplasia or chronic lung disease. So it's a very different type of disease in pediatric Pulmonary Vascular Disease and adults. So I think the adult model of a low in pulmonologist or cardiologist kind of taking care of these patients just is somewhat suboptimal in the pediatric world. And this is, I didn't have enough room to make any more circles, but obviously the pH service interacts with basically almost every service within a children's hospital. The multisupp specialties as you know, you can have rheumatologic-related disorders, GI-related disorders, hematologic-related disorders, pulmonary transplant, et cetera, that all end up with some form of clinically relevant pulmonary vascular disease. My biases in the purple, the things that are absolutely necessary to be part of a comprehensive pH service are in purple. Nursing is paramount, obviously, and having great nurse practitioners is really what makes our everyday life terrific and terrific for the patients. My biases that obviously this is a cardiovascular disease and cardiologists need to be paramount part of the service as well as pulmonologist, particularly given group three, etiology, and neonatology. And then I'd like to think when they get really sick that maybe the ICU helps a little bit considering them also an ICU doctor. And then obviously there's a lot of other folks, genetics is becoming more and more important, specialized pharmacy, et cetera, cardiac surgery, pediatric surgery, et cetera. So this is the kind of what we've done for whatever it's worth. Obviously, I think the point is that I feel that it has to be multidisciplinary and obviously collaborative. Consistent diagnostic and therapeutic approach, I think this is really what's helped our program the most. When I say this, I mean, for example, our cardiologist who's part of our service that does pH service comes to our weekly conferences, et cetera, Hythmnoe 2 is also an imager, so he actually sees and reads either officially or unofficially every single pH echo. So he knows the patients, he knows how they're doing clinically, he knows what their trajectory has been, and obviously over the years, reading 600 pH echoes a year, and then seeing their calf and seeing their clinical course is developed tremendous expertise in evaluating pH from an imaging standpoint. I think that's been extremely helpful. And on the cast's David title, as he's been retiring, he's really just focused on our pH diagnostic cast, and he too knows all the patients comes to our pH conference and is done all does all the pH casts. And having that kind of consistent input, I think, has been extremely helpful. Obviously we're a consult service, so we're not there all the time. We may have our opinions, but we have to respect all the people that are actually at the bedside all the time. And then I've mentioned this weekly clinical pH conference where all of the major players in the program come for what started out as two hours now, it's about three plus hours, and then our regional referral places will call into present patients or give us follow-up on patients. But we're all there, including obviously our nurse practitioners are extremely important, our pharmacist or nutritionist. So everyone knows about all the patients, and patients knows who's coming to clinic that week, knows who the casts are that week, and we have a nice respectful conversation. I think that's very helpful. I'd like to think that we're continuing to be dedicated to the academic and educational missions of the university, so we do have our rotations for residents and fellows. We dabble a little bit in some clinical related research, and we are also a big part of the weekly BPD working group. In terms of the multidisciplinary care of congeal diaphragmatic hernia, I'm a little uncomfortable because my approach is just to listen to Dr. Roberta Keller, who is a new natologist. She's been with the field treatment group from the very beginning. She did rotations when she was a medical student in the cardiac ICU, and she was actually our first rotator. And so she's very cardiovascular savvy, and she really is, it's been with the surgical group from the beginning, and she rounds on these patients every single day, even when she's not on pH service. And she, it's really her research. I'm lucky enough to have my name on many of her papers, but she's the driver of how we care for these CDH patients. So obviously it's a chronic multi-system disease, and therefore requires, I think, a multidisciplinary team approach. I think as you guys are all devoted to a very specific ventilation approach where to try to prevent acute and chronic ongoing lung injury, which is paramount to their outcome. I know there's a lot of focus on the vascular chair, but clearly preventing ongoing lung injury is paramount, and everyone's got their own approach to this. But as you guys have switched over to, we too take an approach of permissive oxygenation, permissive hypercatenia, and aggressive pulmonary toilet. Those are some of the settings that we use. I think one thing, one study that Roberter did that I think has been very helpful to us in terms of the daily care is to show that all of these patients with CDH obviously have elevated pulmonary vascular resistance, and it takes quite a long time for them to transition. So only about 50% of the patients, this is 140 patients that they echoed weekly, and only about 50% of the patients, their pediatric pressure is estimated to be less than two-thirds systemic levels by three weeks of age. So that has led us to kind of wait in terms of before we decided any kind of real chronic therapies. We tend to wait about six weeks before we, as you can see in the bottom there, assessment of long-term therapy. We usually wait around six weeks to assess them by a complete cardiocathodization before thinking about chronic therapies. Obviously, there's nothing novel here. We do like to maintain Dr. Patency to maintain cardiac output and protect the RV. The vasodilator therapy acutely, we use nitric oxide. We sometimes use isle-aprocyte. We have not had any issues with pulmonary hemorrhage today with that drug. I think the only thing that we may do a little bit differently from my discussions yesterday is we tend to avoid PD-5 and Hibir's sildenafil because of the significant GI side effects in one study by Dunbar's group in Colorado. There was about a 25% incidence of GI side effects with sildenafil, and that was all cumbers, and I'm sure that in this patient population, the incidence of reflux and other GI side effects is much higher. Because I have so much feeding problems and towering antrophides is so important to their recovery, we tend to avoid sildenafil. We use a lot of endocelan receptor antagonists, but sent in particular. We obviously follow their LFTs closely, but we have not had any significant problems with that. We do that for a couple of reasons. One is, as I said, we try to avoid this sildenafil. And two, we, as many of you know, sildenafil, and the thelan is up-regulated in CDH and Dr. Keller had a nice study showing that levels at a couple of weeks of age actually were predictive of outcomes. So we do think that there may be some indirect evidence that endothelan participates in the underlying pathobiology, so we like to target that therapy. We've only used remodule and chronically in 10 patients, 50% of survived and all but one are off remodule right now. Two of the non-survivors were kids that also had significant congenital heart disease, which, as you know, is a difficult population. So I'm going to stop there with the CDH if that's okay. I know we have a meeting this afternoon where we can talk about particulars and Roberta gave me a cheat sheet so I can tell you what she does. But I'd like to move on now to some of the work that I've been interested in, which is a pulmonary hypertension story of socioeconomic congenital heart disease. Just as a set up in a review and I know people in the audience know this a lot better than I do. Dr. Koolwick has written a tremendous amount about this, but one thing that's interesting about this form of pulmonary hypertension is that we actually understand the natural history of the disease without surgery, right? We're only a majority of idiopathic or primary pulmonary hypertension or toxin mediated, et cetera. They present when they're already in heart failure so we don't really see them with advanced disease. So this is kind of unique in the sense that we know what the underlying mechanisms are. And if you look at the natural history, I think there's lessons to be learned here. The pulmonary venous side of things is not well studied, not well characterized. Thus, everything seems to be variable. But the increased pulmonary blood flow, lesions are definitely better characterized. And what's striking, as you all know, is the lesions that give you increased flow and a direct pressure head like a trunkus arteriosus, if unrepaired, have 100% incidence of developing irreversible pulmonary vascular disease and it occurs very early in age. And then the AST on the other end of the spectrum just an increased flow without a direct pressure head to the pulmonary vasculature has about a 10 to 20% risk of developing irreversible pulmonary vascular disease. And if they do so, it's when they're much older. And I think that there's some interesting things that we can pull out of that. This is a cartoon that you know, the teagami after stealing it from Stacey Obyenis. And this just shows you the vascaromorophology. This would be representative ventricleaceptal defect. And the red there is the muscle layer. And I think the first thing that you will see is medial hypertrophy where that muscle layer gets thicker. And then you see abnormal extension of muscle down to the periphery. And then as things get more advanced, you actually see the loss of the small arteries are so called pruning. In a in a classic study that was performed here by Marlene Rubinowitz and the group here, they took on biopsies of patients undergoing corrective surgery for these type of lesions. And she showed that these grades of morphology was associated with perioperative reactivity. And there's a reversible form and then an irreversible form in a huge gray area. That's where all the discussions and fights and count conference are all about. And also as you know, if it goes on repaired, ultimately you end up getting either bi-directional or right to left shunting through the connection, which is the isomagocindrome. Paul would give a beautiful description of this in 1958. And it's really quite impressive. If you haven't read it, I put it on the Oprah's list of reading. It's really something. So just very briefly, I think this is a beautiful study by Menace. In their clinic, they looked at patients with congenital heart disease and pulmonary hypertension and compared them to their idiopaths over 13 years. And the point of this slide is that patients with pulmonary hypertension, associated with congenital heart disease generally do better than idiopathic pulmonary hypertension. So that's their survival curves. If you look at the bottom, their overall survival is 85% at 10 years and 77% at 20 years compared to the idiopaths in that group. And the numbers are large, 278. 46% at 10 years and 38% at 15 years. But if you break it down into subgroups of congenital heart disease, this is the more recent classification. You have the eyes and fingers, patients. You have left to right shunts with basically a pre-eyes and mingres, basically the same. Then you have a pulmonary hypertension with a congenital congenital heart disease. So a patient may be with a small atrial shunt that probably is not really the etiology of the pulmonary hypertension. These are probably idiopaths that happen to have a shunt. And then the fourth one is you thought that they had correctable reversible pulmonary vascular disease. The repair was fixed. The ICU did a tremendous job getting them through. But then they reemerged five or 10 years later with pulmonary vascular disease. And then if you look at the survival of those patients, 190 patients together, the eyes and mingres and the kind of pre-eyes and mingres are the two top curves. So they do the best by far. The coincidental defect doesn't do nearly as well. It's very similar to an idiopath. But if you look at that bottom curve there, that's the corrected one. So if you've been corrected and then you go on to have progressive disease, you do worse than any other group known. So that's why the fundamental question of who is reversible, who's not reversible. Can you make a reversible patient reversible? Is really crucial because you'd much rather leave them as an eyes and mingres. Go on to have eyes and mingres syndrome in terms of survival. Why eyes and mingres live longer is a whole other discussion. And Rebecca Camini has been studying that. We have a pop-off to prevent sudden deaths is certainly important. But we think the maintenance of a fetal RV phenotype is fundamental to that. That they never remodel because that RV has always been exposed to high pressure and high flow and that that is an adaptive actually RV and that they end up doing much better. So why study pulmonary hypertension associated with general heart disease? Well, as I was talking about, it's one of the few types of pulmonary hypertension where we actually understand the triggers of the disease. Clearly abnormal mechanical forces secondary to perturb flow and or pressure is paramount. There clearly could be genetic predispositions that we're just starting to understand more and more. And maybe unless well understood, the altered oxygen tension that these lesions present to the pulmonary vascular. There's clearly an ability to have a window into early disease which we don't often have in other types of pulmonary hypertension. It's really the only type of pulmonary hypertension that we can completely cure or the surgeons can completely cure. And there seems to be a window reversibility which is lost after a period of time and other ways that we can kind of either through biomarkers or just understand the fundamental path of biology. Can we get at what's reversible, what's irreversible and what can we make an irreversible and reversible and are there therapeutic targets there? So I was first got interested in this topic many years ago with two I think fundamental observations. One done here by Dr. Rabinoitz where those biopsies that she took in patients undergoing corrective repair, she did scanning EMs which you can see here. This is a, I don't know if I can use. Can you see that? Oh yeah. So here's a nice normal endothelial cell layer and this is a much perturbed endothelial cell layer. And then some of the other kids that had increased flow, they were young so they all had reversible disease but you can see early on at least anatomic aberrations of the endothelial cell layer. Then she went on to show that the well-brands factor in which was an indirect determinant of endothelial function was also perturbed in these patients. And then cellomire a few years later did I think a very simple but elegant study in the cath lab. He put lines in the pulmonary artery measured flow velocity so if there was an upward reflection now it would be indicative of vasodilation. And he had three groups. He had patients in these boxes here are just kids that were undergoing cardiac catheterization for I hope good reasons but they had a normal pulmonary vasculature. They had open boxes these children were young, they had increased pulmonary blood flow but normal pulmonary vasculature resistance, clearly reversible patients. And then these triangles these were older patients that had more severe disease and their calculated woods units was greater than eight. And then he the C1 is a control the C2 is a second baseline and then he gave increasing doses of astiocolein here one two and three and NP is nitropressi. So acetylcholine is what we call an endothelium dependent pulmonary vasodilator it requires the endothelial cells to make nitric oxide in order to dilate. And then sodium nitric press side is an endodona it actually donates nitric oxide so it does not require endothelial cell function. And so what they showed was if you had normal pulmonary vasculature you had nice those dependent relaxation test your colon and one dose of nitric press side you relaxed very nicely. The patients with advanced disease didn't relax to either astiocolein or nitropress side which makes sense they have a lot of muscle there that not going to be able to conclusively relax. But what was intriguing to me was this middle group that they were young they were infants they had normal resistance as I said. They dilate very well to nitropress side normally to nitropress side but it's significantly attenuated response to acetylcholine. That endothelial cell function even in these kids that are low risk seem to be perfectly fine have early endothelial dysfunction. And then an observation made again at this institution when Dr. Henley was here with Dr. Castaneda when they changed the approach of operating on trunkus arteriosus patients from waiting for them to get old and get sick and for their pulmonary vasculine resistance to drop to saying we can correct them when they're very young and what's let's try to do that. So when I first started many years ago these patients Paul Ebert was our surgeon you see us have so we got a lot of trunkus referrals and they had a tremendous amount of periopera pulmonary hypertension so the theory was let's wait for them to get symptomatic their pulmonary vasculine resistance is falling and therefore then take them to the operating room so they won't have as much pulmonary hypertension. And Dr. Henley Castaneda took the other approach saying well perhaps this waiting time and exposing the pulmonary vasculine to increase foe and pressure for four to six weeks is actually making things worse. So they operated on them sooner and if you look at groups A and C and compare those two which are the simple trunkus without associated abnormalities and operated with on within the first few months of life versus greater than three months of age you see the mortality is significantly greater if you operated on earlier and then more importantly if you look at before a month of age and after their pulmonary pressure even though it's coming going into the operating room is probably higher in the younger kids coming out of the operating room was actually lower than the older kids and the amount of pulmonary hypertensive crises was significantly lower in the younger kids. So this observation I thought was it was fascinating and it led us and others to kind of come up with this this theory or hypothesis that pressure and or flow result in an early endothelial injury and that results in a whole hodgepodge of things when you have that many arrows it's clear that you don't really know what's going on but there's aberrations and vasoactive factors etc etc all these things that end up with vasoconstriction and then either directly or indirectly vascular remodeling and obviously the focus on the endothelial cell has has been has barrel out of fruit because all of our all of our therapies currently are based on endothelial pathobiology and these are the three pathways that we now focus on blocking the endothelial pathway augmenting the nitric oxide pathway and augmenting the cross the cyclone pathway. So we were intrigued by these early these initial observations and we really wanted develop approach to see you know how early do you get endothelial aberrations and what potential mechanisms are what are the mechanisms of these and so we embarked on a journey about 20 years ago with the help of doctors handly and ready where we we created a congenital heart defect in the in the fetal lamb and the reason we felt strongly about having the communication in utero is not that we're changing fetal hemodynamics because obviously you're just stealing some flow from the doctors if we create we created an ear or a pulmonary window basically but there's dramatic changes that as you know that occurred birth and having the having this communication in place at the time of birth we thought was very very important because kids with large defects they transition differently similar to the CDH as a delayed transition so we thought it was very important to go through the process of actually creating the defect in utero and this is a diagram that Dr. Ready had made from our first publication and basically this is a cartoon of the left or economy see the pulmonary artery in the ascending aorta right next to it and you know the fetal the lamb at birth is about three kilos and we're putting in an 8 millimeter Gore-Tex graft with very little lines to it so it's side by side in asthmosis and we take the surgical clip off so you can imagine a three kilo baby with an 8 millimeter short shunches would have torrential pulmonary blood flow so you can't put this in postnatally they'll just if pulmonary vast resistance has already fallen they'll have torrential pulmonary blood flow you would never be able to recover them so it also allows us to put in a very very large communication and this is just what it looks like in real time and what we found is you know besides the tail and the fur they they look in behavior very much like like young kids with torrential pulmonary blood flow they fail to thrive sometimes they need the ng-feetings they this is an x-ray they have significant cardiomygally and CT findings have increased vascular markings the next two slides are about 15 years worth of work we focus on the endothione cascade as well as the nitric oxide cascade and what we what we showed at least in these lambs that there's a mark I'm sorry these coast didn't work out but there's marked elevation of endothione one and these these were in one week lambs so within the first five days of life actually the endothione cascade is markedly elevated the b receptor which is the endothelial receptor which is actually the good receptor it makes nitric oxide is markedly down regulated within the first week of life the bad receptor this with muscle cell receptor that's it causes intense phase of constriction is actually markedly up regulated by months four weeks of age and then we actually see a two two months of age the emergence of this b receptor that now shows up on this muscle cell and actually instead of mediating by is a dilation through nitric oxide it mediates the phase of constriction and this was subsequently shown in adults with CTF that they have b receptors now on this muscle cell so we've we've always felt that endothione was a major player in the pathobiology disease and that probably needs a little bit of our bias to use a lot more ERAs than than a lot of folks in terms of the nitric oxide cascade this is just a summary and basically these are some of the things that we think end up with decreased by available and we have uncoupling of the enzyme that make nitric oxide related to increased reactive oxygen species generation that's important mediated by endothione one and part mediated by mitochondrial dysfunction the reason I show you this is although there's tremendous amount of endothelial based targets now we do think that there's some areas in fact in animals p-paragonist or carnitine to normalize endothelial function the antioxidant similar to many other disease dates hasn't quite panned out but these and missing the animals have been helpful so I think there's still potential in the thelial based therapies so more recently we've been quite interested in kind of translating the natural history into pathobiology in other words why is it that an ASD with just flow and no pressure versus atroncus arteriosus have such a different natural history and can we try to sort out what's going on with different mechanical forces take a step back you know our whole approach to treating pulmonary hypertension is rather barbaric right I mean we've got a lot of new therapies we've improved outcomes we've made patients feel better but compared to other diseases basically what we say is if they've got mild disease we give them one drug if they've got moderate disease we give them two drugs and if they've got really bad disease we give them three drugs and that's basically our approach with no real focus on what's their underlying pathobiology can we target even our existing therapies to particular patients let alone come up with new patients so you know compare to like cancer it's it's barbaric it's like saying 40-year-old woman with breast cancer in a 75-year-old man with prostate cancer both have the same disease and we give them the same drugs that's basically what we're doing right now with pulmonary vascular disease so in a kind of our small way and focus on congenital heart disease we want to try to at least get to you are there different mechanical forces that preferentially regulate different genes and perhaps we can start to target our therapies within this subpopulation so so we created a second model where we just tied off the left pulmonary artery in utero and this is a the cartoon of that and so what we're trying to do more recently and this is pretty recent data none of this is published today but we're trying to compare fall alone which you may see in a single longer or other situations with with our kind of aorticulmonary window model which is direct pressure head plus increased pulmonary blood flow okay from this is just CT scans this is our shunt model this is what we'll call our LPA ligation model with no LPA and these are controls in terms of the hemodynamics I mean it's it's there's a little bit of an increased pressure PA pressure in the LPA model you see that I can so it's it's see it's a little bit higher than control 14 versus 19 but the shunt model has a much higher pressure and the flow is increased in both of them so it's not perfect but it's it's a it's a story so the first thing we did was we looked at pulmonary vascular activity as you know these patients are have increased pulmonary vascular reactivity to known stimuli such as hypoxia or agitation alpha ageneric agenergic receptor activation so we used classic hypoxia and U46619 as a a thrombodement remember it causes very potent for a visit constriction excuse me and I think what you can see as we previously shown in the shunt and model that they're hyperreactive to both hypoxia in this U46619 but the LPA ligation is somewhat of an intermediate phenotype when it comes to reactivity and then we just took isolated pulmonary arteries and exposed them to your epinephrine as another means increasing doses and here you can see that the out the increase full alone actually has normal reactivity to neuropanephrine control versus a shunt being exaggerated if you look at the morphology the muscle wall thickness is clearly greater in the shunt compared to LPA ligation or control then we we were actually able to culture these cells which is nice and they maintain their phenotype in terms of endosingling etc so this is some RNA sequencing data so the the light blue are shunt the red is LPA these are endothelial cells from these models and the dark blue is control what this shows you is that there's really nice clustering in terms of the genes that they're and they're very different and then this is the heat map showing shunt here LPA here control in the middle and you can see that there's very so that in this particular heat map red is up your up regulated genes and green is down regulated genes and you can see marked differences between the three three groups we have focused on angiogenesis and the thelial angiogenesis and apoptosis as two of our targets to start and these are just some venn diagrams so A and C so this is up regulated related to angiogenesis this is down regulated transcripts related to apoptosis and A and C are genes that are altered uniquely by pho and pressure in other words they're only up in the shunt and they're down in both the LPA model or the pho alone model and the controls and then B and D are altered both by pho alone and pho and pressure so shunt and LPA are similar but different than controls if that makes sense and then if you look at a marker of angiogenesis it's a made to gelinase a tube formation you can see that the endothelial cells are the shunts make make more have more angiogenesis with the LPA ligation kind of being an intermediate phenotype compared to control with their ability to proliferate the shunts are greater where in this case LPA ligation and control are the same and in terms of apoptosis the shunt and the thelial cells are apopt anti apoptotic with the pho alone being somewhat of an intermediate phenotype so we think that the shunts tend to be more angiogenic the endothelial cells they're they're more resistant to apoptosis they proliferate more and flow alone without the pressure had markedly changes these particular things we're just starting to look at some of the basic endothelial function things if you look at enos which is the prote is the enzyme that makes nitric oxide and is stimulated by pho not surprisingly both the shunted animals and the LPA animals have a marked abergulation of enos both message and protein but if you look at the enoma tabloids that are being made in the lung tissue as we've shown previously the shunted animals even though there's a lot of a lot of enzyme they're not making nitric oxide the enzyme is uncoupled in fact it's makes super oxide instead but here flow alone not only is there a lot an upregulation of nitric oxide but in fact they do make of nitric oxide synthase but in fact they're making nitric oxide so they don't seem to have the uncoupled we've also focused on endothelian and here you can see that in the shunted animal these are just the levels in the lung that endothelian levels are upregulated and there's a little bit of an intermediate in the flow alone this is the gene prepro ET1 that makes endothelian before it has to be cleaved a couple of times and you can see a marked upregulation in the shunt and not in the LPA so where I'm trying to get here is it seems like the pressure head seems to be stimulating endothelian one so perhaps in a situation where you don't have a pressure head but just flow wouldn't endothelian receptor antagonists really be useful you know that that's kind of the focus of where we're trying to go if you take these endothelial cells and shear them in fact so this is static you shear them actually with physiologic shear and the thelian levels actually go down but if you stretch them which would be the mechanical force associated with pressure in fact endothelian levels go way up so we've done some more RNA-seq analysis and we actually think that endothelian drives many of the anti-apoptotic and the angiogenic processes so these are just genes associated with endothelian one that are also associated with anti-apoptotic processes and in the dark voice I hope you can appreciate that many of these genes are actually upregulated in the shunt but either not upregulated at all in the flow alone or kind of intermediate compared to controls and then similarly so that's anti-apoptosis and this is angiogenesis similarly these are again their genes associated with ET1 that appear to be upregulated in the shunt preferentially and then these last couple of slides are just data that we've just gotten so I don't know how to really make pretty slides so they're not that good looking but we did a major gel acid looking at a tube formation within without endothelian and without endothelian receptor antagonist so this is the normal cells, H-match normal cells these are from the flow alone cells and these are the shunted cells and you can see as I showed you earlier that shunted animals the endothelial cells have more endogenesis if you add endothelian nothing really happens to the normal cells but the flow alone cells actually jump right off and they are equal to shunt's ability to and cause endogenesis adding endothelian extra endothelian endothelian one to shunted animals actually doesn't change anything so it's almost as if it's a primed phenotype this flow and then conversely if you give BQ-788 which is an endothelian B receptor antagonist you can drop down the endogenesis to control levels suggesting that it's an ETB receptor mediated so a lot of the mechanical forces in vitro data is fundamentally flawed by the fact that you can't really separate pressure and flow you can shear but they're closed systems and then the more you shear you definitely develop pressure within the chamber so Juan Wacharis who's an abroient engineer at UCSD was kind enough to and this is the picture of the first device he made but it looks much nicer now but he's made a microfluidic monosel culture chamber for us that actually can isolate and change shear and pressure independently and so we just started working with this these data actually these last two slides just came that were sent to me last night so one thing is so we're only able to do a QPCR on it so we can only do RNA analysis at this point but the blue here is endothelial cells that we've been cultured from the proximal pulmonary artery and all the data that I've shown you the cell culture data to date is from the proximal pulmonary artery but as many of you know that there may be differences in in the in locations so we've also now successfully cultured micro vascular endothelial cells which are in red and so the first thing statically we've shown that there are there are differences this is pre pro ET1 and this is RNA for in the vce1 which is a major converting enzyme that makes converts it into its active form so there seems to be some location differences and again this is all very preliminary but then what we've done is we've looked at changing pressure so we have physiologic shear you know under all these conditions and then we gave you know normal pressure pressure head of 20 and then we increased it to 50 and I think what you can appreciate the blue here is endothelialin 1 that in the this is the proximal cells as an up regulation of ET1 with increase with increasing pressure and EC1 is pretty much flat and then in the micro vascular cells there is an up regulation of endothelialin 1 with going from 20 but no further up regulation with higher pressure but EC1 the converting enzyme goes up rather dramatically suggesting that you know that the pressure head does seem to stimulate some of the endothelialin production and it may stimulate it differently in different areas of the along with the with the precursor being more stimulated proximally and the converting enzyme being stimulated more distally again very very plumbinary so kind of summarize feel that combined mechanical forces of pressure and flow result in much more significant vascular modeling vascular reactivity and the therial proliferation angiogenesis anti-apoptosis and marked endothelial dysfunction where flow alone is somewhat of an intermediate phenotype and the future of all this we hope and there's a lot of data to go through here particularly with the RNA-seq data is that we want to get to therapy that being informed by the physiologic ideology of the pulmonary vascular disease as opposed to the disease severity and then in general I think the therapeutic goals I think we can optimize our endothelial based therapies although that's come a long long line the last decade there's very very little on smooth muscle cell based therapies currently there's a couple of ongoing trials but there's a lot of work to be done there to improve outcome there needs to be a lot more work focused on the RV because as you know you die not because your pulmonary vascular resistance hits 25 or 30 you die because your RV fails and there needs to be a lot more focus on our potential RV based therapies and then there's a lot coming coming out now in genetic predispositions and as we figure out what that particular gene signals we can potentially get at potential therapeutic targets more when we're going to withstand for us doing a tremendous amount of work focus on BMPR2 related to this and then again future directions that we must be informed by underlying path of biology as opposed to disease severity this is a picture of the pH group which really makes it fun to come to work every day and I mentioned Roberta this is hythomacoreologist David Elizabeth Colgoyser and Claire Parker are the heart and soul of our program there are nurse practitioners and you know what we do compared to what they do is ridiculous they really are great but a social worker nutritionist a specialized pharmacist who really focuses on on the drugs it's all a great team then in terms of further thank you's Ena de Tia has was here taught me everything I know about clinical pH and really got me started got me out of the lab and seeing patients that weren't just in the ICU and I hold a great you know gratitude to Ian he's a constant continued constant mentor I'm in the lab Dr. Ready got me started Sanji Dittars done intraments amount of the RNA-seq work Rebecca Camille works as a lot of the RV work and Andrew Reddington helps us a lot with that and then a special shout out to Abrudoff who's continues to be a mentor to many of us in the field of pediatric cardiology and I'll stop there thanks very much so your work into after listening to you present your work over the last 20 years I sat here and I thought you know there's a lot of variability as to when people choose to can relate CDH patients but after listening to your work in my head I'm thinking I think I want to can relate especially the highest risk category on VA act most sooner to bypass the pulmonary and let them sit there for a couple of weeks now this is highly speculative but what do you say to that I say it's highly speculative it's interesting I find it interesting that you know and I'm not you know I am not intimately involved in ECMO in ECMO decisions related to CDH let me just say that I find it interesting that the overwhelming majority of the time we we can relate VV as opposed to VA and I would agree I think it's intriguing your your your thought I there are many reasons that I think resting resting the the heart and lung with a VA cannulation would be hopeful second question do you guys mostly cannulae VV or VA? VA but the question is are we as aggressive as cannulating soon we're kind of looking at our own practice right now as you'll hear the satan in as to how long we let the baby go spontaneously versus converting to VA the second question is this precision medicine lots of hope and lots of hype if you look forward five ten years in the treatment of pH where are we? I think I think that I don't have the slide with me but I think you know the last decade has been tremendous in terms of the number of therapies that we now have and if you look at registry survival data it's dramatically improved having said that it's still the three different cascades and the different drugs are really just different formulations and but it is it's changed our world being able to give things subcutaneously inhaled etc once a day versus three times a day suspensions versus pills that's helped but we really have so much we can go so much further than that I think that the genetic world is really opening up with pomeric hypertension I really think that that's that's going to that's going to open up a lot of new targets for us I think we've talked about this if we can I think almost every kid warrants genetic testing and there are new genes coming out all the time and I would argue that all that a majority of the ASTs in fact may be idiopaths and happen to have an AST and they if you if the adults did more genetic testing they'd see that they probably that they may have genetic predisposition but I think the more we learn about the genetics of the disease and then we function we focus on what what's singling abnormalities these genes connect to that that will give us new targets to go after so I see this I see in the next decade things continuing to prove dramatically as I said in the last slide I think we're going to get some smooth muscle cell based therapies some more RV based therapies again related to the the more that we learn about genetics and I think we'll you know we'll we'll start catching up a little bit to some of the other fields but we've got we've got to focus on underlying pathobiology at least at least even now when we're thinking about starting a drug if there's anything we know about the underlying pathobiology let that at least drive the decision because we're not going to be able to it's not like the adult world we're going to have data you know to drive every three therapeutic decisions so all we've got is is is what's known from the research right now not human-based research other questions for Dr. Feynman what role do you think there is for biopsy in these patients and do you think that we can do in vivo testing with biopsy samples to help with the precision medicine? yeah so the first part of the question is I think is an easy one the second one is I think from that's from the precision medicine standpoint I think it potentially could be very helpful right similar to what they're doing with cancers where they're doing genotyping of cancer we're not there obviously yet so until until we get there not sure which patient population you're talking about but I would say that short of the neonate that has severe disease of unclear etiology or the occasional patient where you think they may have pvod but the imaging isn't particularly clear and the the response of as a dilators is not particularly clear short of those situations I think along biopsy and the pathologist will tell you that along biopsy is not helpful it's the studies have shown that in terms of predicting reversibility for congel heart disease for example it is not predictive so in that setting I think it's it's definitely not helpful. Jeff I think it was an excellent talk thank you. Cool disclosure I worked with Jeff when I was a fellow you mentioned angiogenesis as part of the pathophysiology is the portrait of Dr. Folkman right now. Is anyone used angiogenesis block as it all is part of the therapy or the prevention of the progression of the disease? Not to my knowledge no I mean it's an interesting question because you kind of caught me because I'm not convinced that angiogenesis early on like this is is is necessarily maladaptive that in fact it could be an adaptive response to try to recruit and incorporate the flow and if you look at the vessel number of these so this is early disease right so if you look at the vessel number it's actually in number of arterios is actually increased in these shunted animals compared to their controls so it's not it's not advanced where you're actually using arterial number so I think the anti-apoptosis is part of the pathology but whether at this point angiogenesis is actually adaptive or maladaptive I think you can argue either one. Jeff thanks so much for a spectacular talk. You talked about the physiology associated with the left right shunt and particularly the pressure overload. One of the most difficult situations that we're faced with in CDH are patients with left to right shunt lesions or with congenital heart disease or even with a persistent pain ductus. Do you think that your results on the lay models should direct us to potentially different management of those patients? Well I'm hesitant to say anything that we do in the sheep is we should definitely translate to the to the to the patient right away. You know the as you know this the CDH with congenital heart disease is that that's a tough group. I think the lessons we learned were that group we I think we needed to be more aggressive earlier on with with very with aggressive pulmonary viso diabetotherapy so in this concept of waiting six weeks etc I think we got burnt with the first couple of those patients that we had and the last two that are surviving were that's the group that we tend to be very aggressive early on with other than that I don't know if I can make any any real correlations I think we do we do like to use maintained ductal pregnancy for for RV protection and cardiac output protection and occasionally we've had to go to the cast lab and enclosed the duct but in general it hasn't it hasn't really hurt us I think it's really hurting us and we're feeling good about the pulmonary viscages here so well Jeff thanks very much the fascinating talk thank you very much thank you