All right, everybody. We'll go ahead and get started. Thanks for being here for another medical grand rounds. It's a privilege to have Drs. Annex and Sharma from the Division of Cardiovascular Medicine to give us a case-based discussion on the emerging therapies in a patient with peripheral arterial disease. 

By way of brief introduction-- start with Dr. Annex. He's the Division Chief of Cardiovascular Medicine here at UVA, and also is the George A. Beller Medical Imaging Distinguished Professor in the Department of Cardiovascular Medicine. He's been at UVA since 2008, and he has a longstanding clinical and research interest in the field of peripheral arterial disease. He's performed and led numerous studies nationally on angiogenesis and gene therapy to treat PAD, and also served on numerous national programs related to the disease. He has more than 125 published papers related to the topic. 

And Dr. Sharma is an assistant professor in cardiovascular medicine. He's been at UVA since 2012. And since his arrival, he's directed the Vascular Medicine Clinic. His research emphasis is on the medical optimization of peripheral arterial disease. Drs. Annex and Sharma co-direct UVA's Vascular Medicine Fellowship Program. And please join me in welcoming Dr. Sharma first today. 

[APPLAUSE] 

We thank you. Can I use-- let's go ahead and-- this is working now? OK, this is working. Great. So thank you, everyone, for having us here today to talk about some of the emerging therapies in our approach to management of patients with peripheral arterial disease. 

So what I will be talking about primarily is more how things may be changing or are changing in antithrombotic approach, mainly to reduce cardiovascular event rates. I think it's maybe this one. Maybe to reduce cardiovasc-- so I'll be talking more about mainly how we can proceed reducing cardiovascular event rates in patients with peripheral arterial disease. And Dr. Annex will be talking more about what new is coming up in terms of symptom improvement in patients with PAD. 

So we'll start off with a scenario. I just want to you guys-- because this is a case-based scenario. Really, what would you do in these three patient examples that I've provided in front of you? 

So a 65-year-old with asymptomatic PAD has an ABI about 0.95. So how would you-- what kind of antiplatelet, anticoagulant-- what would you use in this scenario? Aspirin? 

Just one person, aspirin? Two people asp-- maybe a few people aspirin. Plavix? Ticagrelor? Something a little bit-- more other antiplatelet agents. 

How about dual antiplatelet therapy? Warfarin? Put them on a DOAC and aspirin plus morphine? No. OK. 

What about a symptomatic PAD? Same age, but now has known coronary disease, and has diabetes. Aspirin? Ticagrelor? Clopidogrel? 

What about dual antiplatelet therapy? OK. DOACs? DOAC plus aspirin? And warfarin? OK. 

65-year-old, PAD, has already had a fem-pop bypass and has diabetes. Aspirin? Ticagrelor? Clopidogrel? Aspirin plus clopidogrel or ticagrelor? OK. 

DOAC? DOAC plus aspirin? And warfarin? OK. So there's a variation-- a little bit of difference in even how all of you would actually treat these three examples. 

So PAD is not uncommon, by the way. This is fairly-- quite significantly under-diagnosed. About 200 million people worldwide have it. 

This is not the disease of the rich. Most-- in fact, 2/3 of PAD currently is actually in the low to middle income countries. And the prevalence is rising aggressively. And quite a few of these patients will require some kind of endovascular or surgical procedure in the long run, mainly for their symptoms. 

When you look at PAD outcomes, cardiovascular mortality or mortality in general, really, it's not the same based on what PAD you have. So just looking at examples. This is a meta-analysis that looked at ABIs and mortality. And as the ABI gets worse, the mortality actually does get worse. So cardiovascular event rates does go up in these patients. 

More recently-- this is the REACH Registry that looked at patients who had high risk events. And they had about more than 3,500 patients with PAD that they followed for a period of four years. And all of these patients were on statins. Majority were on aspirin. And even with that, they did have quite high rates of cardiovascular events, MI strokes, peripheral revascularization needs, and amputation rates. 

Another study that was done by the TIMI Group, really looking at antiplatelet agents, had actually reported outcomes. What they saw is, a large number of their patients, also who had PAD, who were already on statin therapy, who were on aspirin, quite a few were also on pyridines, actually still had high cardiovascular events rates, high rates of peripheral revascularization and amputations. 

And then when you move on to the really sick group of PAD patients, those are patients with CLI, which is Critical Limb Ischemia. These are patients who have really bad ABIs. Typically, they have rest pain or non-healing wounds. 

Wehn you look at their mortality. It's significantly high. They are one- or two-year mortalities. And looking at various different studies, ranges anywhere between 15% to 25% or so, as you can see in these two particular trials that primarily were enrolling patients with critical limb ischemia. 

What's also interesting is, PAD contributes to mortality really aggressively. We often take care of patients with coronary artery disease. Really, that's been the primary thing. Everybody understands these patients need to be on statins, aspirin-- so on. 

But what's interesting is, there are three different trials that I'm talking about here-- the PEGASUS, PRODIGY, and DAPT. They were primarily CAD trials, and they were looking at different antiplatelet agents. But what they looked at is, is when they looked at outcomes, MACE events-- cardiovascular death and mild stroke-- when CAD patients had a combination-- had PAD in addition to CAD, their cardiovascular event rates are significantly worse. 

So just as an example, PEGASUS here-- their MACE were about 8.4%. However, when CAD was combined with PAD, as in these patients who have CAD alone versus CAD with PAD, the cardiovascular event rates went up to 19%. This is consistently seen in all of these trials. 

More recently, the IMPROVE-IT trial that you guys must have looked at that was looking at use of ezetimibe for reduction in cardiovascular event rates. When you specifically look at just the event rates, what's interesting is that again, patients with CAD alone, versus when you combine CAD with PD-- so presence of polyvascular disease-- had much significant higher event rates. 

So here, they are looking at seven year out event rates in these patients-- about 29% event rate. When you have CAD alone, goes up to 40%. When you have CAD with diabetes or CAD with PAD, and a combination of polyvascular disease with diabetes, event rates are significantly higher compared to the other groups. So we have this habit of treating all of these groups the same way. Perhaps we need to-- can we do more for these other groups? 

And so when you look at the guidelines, which is last published in 2016, for PAD, specifically what they recommend is antiplatelet agents, especially aspirin. You could consider clopidogrel as their primary recommendation. In asymptomatic patients with PAD, they recommend maybe you can consider, as well. It's reasonable. What they suggest is dual antiplatelet therapy for reducing cardiovascular event rates, as of now. It's not well-established, but you could consider that in patients who have had lower extremity revascularization, primarily to reduce limb events. 

So really, a concept here. We just looked at all these different patient scenarios, combining CAD with PAD, looking at presence of diabetes. And the concept here-- what guidelines are recommending. And what we really do in clinical practice is just giving everybody the same thing. And really, it doesn't seem like when you have different event rates. Does one size truly fit all patients? 

And really, perhaps we are coming to an area where we really need to now risk stratify our individualized therapies in these patients. I'm not a hematologist, so I won't go through the entire coagulation cascade. But what we are talking about here is, what we concentrate when we look at PAD patients is looking at antiplatelet agents. 

But when you are looking at thrombosis in patients with PAD, there's really not just luminal stenosis that we are looking at, but we are looking at the presence of acute thrombosis, causing cardiovascular events. And this really involves not just the platelet aggregation aspect of it, but even the coagulation cascade beyond that. And so perhaps we need to consider anticoagulants in these group of patients that really didn't pan out much. 

That's because of WAVE Trial that was done quite a few years ago, where they looked at patients with symptomatic PAD which were randomized to aspirin alone versus warfarin and antiplatelet with warfarin versus antiplatelet alone. And really, we didn't see significant reduction in cardiovascular event rates when we looked at all PAD patients together. But there was significant increase in bleeding events. And this shut down any further anticoagulant research until recently. 

The reason why we have considered aspirin or antiplatelet agents in patients with PAD is it's initially driven by this meta analysis that was done quite awhile back by the ATC group. And what-- they had about 6,000 patients that did show that in PAD patients, use of antiplatelet agent-- majority of the time in this scenario, it was aspirin-- there was about a 23% reduction in cardiovascular event rates. And this has driven both guidelines for many years have suggested as using an aspirin. 

CAPRIE Trial was one of the initial studies done way back that looked at comparing different antiplatelet agents, and actually compared aspirin to clopidogrel in that scenario, and looked at atherosclerotic vascular disease. And interestingly, PAD was the only subgroup that actually showed about 8.7% relative risk reduction with the use of clopidogrel compared to aspirin. Again, a mild reduction-- 8.7% risk reduction is not a lot, but still something that was noted. And this is the reason why clopidogrel is given as an option in the ACC/AHA guidelines, in addition to aspirin. 

Now, given the fact that clopidogrel had really better outcomes at this trial, the EUCLID Trial, was performed in patients with PAD. They looked at just symptomatic PAD. About 30% of them did have concomitant CAD as well. And we actually participated in this trial. 

And this was very interesting when they looked at ticagrelor versus clopidogrel-- their event rates, in terms of PAD patients for MACE, was similar between both the groups. A little bit disappointing in the sense. We couldn't find at that moment any better antiplatelet agents to reduce the cardiovascular events rates in this group of patients. 

They did notice-- in their setup, they did some other secondary analysis. And what the EUCLID Trial did find out is when you start breaking down patients with PAD versus PAD plus CAD, or PAD with cerebral vascular disease, or the combination of the three, the MACE outcomes gets significantly worse as you look at polyvascular disease. And really, the concept of treating polyvascular disease separately, or differently than how you treat PAD, has risen from that. 

Another aspect to look at is, we've been talking about MACE event, but also looking at limb events. There are three different trials that have actually reported that patients with peripheral arterial disease, when you start looking at prior revascularization, patients who have received prior revascularization, stenting, bypass, and so on-- they seem to be the ones with the highest risk for limb events. All the three trials-- these trials have shown that. So-- 

And especially, it was more driven in the surgical bypass, but even endovascular, we see the same outcomes. And interestingly, use of additional antiplatelet therapies, so stronger antiplatelet therapies, as in the PEGASUS's use of ticagrelor or vorapaxar in the TRA2P-TIMI study, did show reduction in limb ischemia events. And what we're seeing, basically, is that-- from all these trials is evidence of prior peripheral revascularization increases the incidence of limb events by almost four times. 

And limb events are important. Because if you look at patients who had acute limb ischemia, look at their outcomes. They have significantly higher rate of cardiovascular events, about 12% death rate, about 24% recurrent acute limb ischemia, and high amputation rates, as well. 

So really, this brings up into the introduction into going back into use of DOACs now in patients with peripheral arterial disease. So the COMPASS Trial was a very interesting and a unique trial, because look at the doses that they used with rivaroxaban here. So they randomized their PAD patients to CAD these patients into three arms. 

One was aspirin alone, one was rivaroxaban, 5 milligrams twice a day, alone, and the third group was aspirin, 100 milligrams plus rivaroxaban, 2.5 milligrams twice a day-- looked at primary MACE outcomes, so that is cardiovascular outcomes. So cardiovascular death, stroke, and MI. And they also looked at MALE outcomes. That is major adverse limb events-- acute chronic limb ischemia, major amputations. 

And then when you specifically look at the PAD and the carotid subgroup, which at about 7,000 patients, they actually saw that there was a significant reduction in MACE when you compare aspirin alone versus aspirin plus rivaroxaban, 2.5 milligrams twice a day. There is a significant reduction, not only in the cardiovascular event rates, but reduction in amputation rates, need for vascular interventions, reduction in acute limb ischemia, need for vascular hospitalization. There was increased major bleeding that was reported. 

So when you looked at both the subgroups, really, that risk of bleeding with rivaroxaban use was about 3% versus 2% in the aspirin alone group. But when you look at the net clinical benefit that is combining MACE, MALE events, which are limb events and bleeding events together, you still again do notice that there was overall net clinical benefit for the use of this combination of aspirin plus rivaroxaban, 2.5 milligrams twice a day dosing. Again, in this group, if you see this particular chart, the patients who had undergone prior revascularization had the highest limb event rates. So again, this was a group that clearly showed to have much worse outcomes. 

Another trial that was done during this period of time is the PRODIGY Trial. This was, interestingly, a primarily a coronary disease trial. They were looking at coronary stenting and use of dual antiplatelet for a short duration of time, which is six months, versus prolonged period, which was about 24 months or more, and looking at MACE outcomes. 

And so what was interesting is overall, the trial, when they looked at CAD prospective, it was negative. But when they started breaking down patients into the PAD subgroup, so those who had coronary stenting and had pre-existing PAD, their MACE outcomes were significantly higher-- about 25%. If it was coronary disease stenting without PAD, it was significantly lower. 

And so prolonged therapy of dual antiplatelet therapy in these patients who had primary stenting with PAD, there was an absolute risk reduction of about 7% in them-- so a significant reduction in their overall MACE event rates. Now, in addition to MACE event rates, there were lower events of stent thrombosis. Interestingly, bleeding in this subgroup of patients was similar, whether they had a short course of dual antiplatelet therapy versus a prolonged course aspirin. And overall net clinical benefit was seen in this group of patients. 

And then lastly, asymptomatic PAD patients. We've often used aspirin. A lot of us did say that we would do that. This was an interesting study that was done as a community health registry. 

And so patients who had no known cardiovascular disease, save an ABI, they were asymptomatic. And then they were randomized to aspirin versus placebo. And they were followed for almost 10 years. And there was no difference in outcomes when you looked at patients who received placebo aspirin. 

Primarily because these low ABIs, asymptomatic PAD patients had very low event rates. In 10 years, they had less than 2% event rates, and so you really can't show much benefit if you don't have a lot of MIs-- of strokes, right? So that's one of the reasons. 

So what we are coming up to now is really looking at more of an individualized approach to PAD treatment. You have to look at patients. What increases their ischemic events, in addition to having PAD? As we have learned, the strongest ones are polyvascular disease and diabetes. 

What can increase their MALE event rates? The strongest factor is prior revascularization, especially if they have surgical revascularization. Endovascular as well, but particularly surgical revascularization. And then looking at what increases their risk of bleeding, and trying to risk stratify patients based on that. 

And when I see a patient with PAD, this is how I approach it now. If the patient's asymptomatic with mildly abnormal ABI, maybe aspirin, especially if they have coronary disease or I see athero in their cerebrovascular segments or so on. Otherwise, they may not even need a baby aspirin. 

If patients are symptomatic-- so then you're looking at bleeding risk firstly. If they have high bleeding risk, then usually antiplatelet therapy, mono antiplatelet therapy, aspirin, ticagrelor should be sufficient, or clopidogrel. Mostly, most of the time, if there's high bleeding risk, we'll just stick with the baby aspirin. 

If the bleeding risk is low, next thing is assess-- what's their MACE and MALE rate? So what's their cardiovascular risk? Presence of polyvascular disease, diabetes, primary revascularization to look for if they have had increases their limb event rates. If that's not the case, then again, monotherapy is probably sufficient. 

But if they do have high MACE or MALE event rates, then usually more intensive antithrombotic therapy would be beneficial in these patients, particularly a combination of aspirin and rivaroxaban. If these patients have had recent coronary or peripheral stenting, perhaps dual antiplatelet therapy for the initial period and then transition perhaps to [INAUDIBLE] rivaroxaban. 

In some scenarios, you may consider just a stronger mono antiplatelet therapy. But for the majority of the time, you would stick with the dual therapy approach. All right, so I think I'll stop here. 

All right. So as soon as my-- all right. So I'm going to take a very different approach here. And what I'm going to do is actually talk to you about how we develop drugs for PAD. We've heard a lot about antiplatelet agents. 

And by way of disclosure, though I have absolutely no intention of mentioning microRNA-93, in the event I do, I do have a very real conflict with that, as we have actually started a company and have entered a collaborative agreement with Daicchi. So if you hear me say microRNA-93 to anything, just don't believe a word I said. OK? 

And here's a slide I want to point out to complement what Aditya mentioned. The problem is, if you look at the distribution of symptoms in PAD, this is one of the major problems we're facing today. So if you look at patients that have a known low ankle brachial blood pressure index, more than half of them are actually asymptomatic-- completely asymptomatic, or have atypical symptoms. Only a third have typical claudication. 

And the group that Aditya mentioned about critical limb ischemia and the high event rate group, they represent less than 10% of our patients with peripheral arterial disease. This is obviously a real problem, because it's unlikely a patient's going to come in to you saying, boy, I heard about the TRA2P Trial, and I heard about this drug. 

Right? They're going to come into you reporting symptoms. And are they actually going to do that? And the problem is that they are likely not to. 

So what I-- why is that? And I think the problem is, the field has never really focused on the full problem in peripheral arterial disease. There's two problems in PAD. You can separate the two. And actually, on my very, very, very last data slide, I'll show you how we potentially are linking them today. 

So for example, one thing you could say, as a clinici-- if you're seeing patients, why should I be concerned about the PAD? Because patients will report symptoms, right? I've showed you that data. That's incorrect. 2/3 will have no symptoms. 

I only need to worry about patients with symptoms. I'm not going to show you the data. Outcomes, whether you have symptomatic PAD or asymptomatic PAD, are actually the same. 

Third is, the disease is stable. Again, work by Mary McDermott-- I'm not going to show you the data-- is showing that patients with PAD have an annual decline in their six-minute walk. If we follow a heart failure patient, and they have an annual decline in their six-minute walk, we're adding drugs. We're adding therapies. We're thinking about advanced approaches. 

But I think what may be the major one is the idea that treatments for PAD are easy. So who knows a treatment for PADs? Exercise. Very good, Mike. And it really works, especially if you use the stairs instead of the escalator here. 

The problem is that if you look at the benefit of exercise-- this is one of, I think, the most utilized slides, it gives the effects of benefits of supervised exercise-- a meta analysis of multiple trials. And the bottom line is that exercise training will increase walking by 180%. That sounds great, right? 

Except the average patient with claudication can walk one city block. So they can now go from one city block to two city blocks. And by the way, to get the benefits of exercise training, all you have to do is start a session of supervised exercise. 

Get on a treadmill. Walk till you'll have pain of 18 out of 20 on a Borg Scale. I didn't even know what a Borg Scale was. I thought it was something related to Star Trek. But there actually is a pain scale that you can use. 

You wait till they hit 18 out of 20. You stop them. They go on the side of the treadmill. They wait till the pain is bearable. They get back on it. 

They walk till that pain is 18 out of 20. They get off. They get back on. And all they have to do is do that for 45 minutes. 

And all they've got to do is do that three times a week for 12 weeks. And at the end of that, they will be able to go perhaps one extra city block. And it strikes me as an enormous problem-- is that at the end of the day, we don't have treatments for this disease. 

And as way of background, the last drug that was approved for the treatment of PAD was in 1999. The Boston Red Sox have won the World Series four times since the last drug was approved for peripheral arterial disease. As a New York Yankee fan, this is something I take very personal. OK? 

[LAUGHTER] 

So-- and I would actually pose to you that the fact that we don't have anything, and the fact that we don't have anything that improves blood flow to the leg, and we don't have anything that makes people any better, is one of the reasons that the disease is poorly recognized. And so our approach for years is to look-- this is a magnetic resonance angiogram. We don't do this anymore for a typical patient with peripheral arterial disease. 

And you will see that the blood flow that a leg should be down a single straight line. And there is an occlusion from the groin, with the main blood vessel recapitulating down below the leg. So the total occlusion in this patient is about this long, OK? Compare that to coronary artery disease, where we're dealing with blockages in the millimeter range. The burden of atherosclerosis is much higher. 

But really, about a decade and a half ago, we used this and looked at it and said, well, the problem is inadequate blood flow. Or inadequate perfusion is what we're saying today. And the greatest defect is at the most distal part of the limb. So cardiologists-- we tend to be very direct about things. If you're not getting enough blood, what do you do? Try to grow more blood vessels. 

So this was our approach for years. Angiogenesis is the growth and proliferation of new blood vessels from pre-existing vascular structures. But really, what we were trying to do in patients was therapeutic angiogenesis, which was the growth of new vessels for treatment of disorders of inadequate tissue perfusion-- a very different, actually, set of definitions. The top is what the laboratory is looking at. The bottom is what the patients actually care about. 

And perhaps the most extensively studied was vascular endothelial growth factor. We know about this a lot from people who we are giving a lot of these VEGF inhibitors-- VEGF blockers to cancer patients. It was originally identified from studies from Beth Israel and published in Science in 1983. And the receptor for this was cloned less than a decade later. Early human studies were done giving VEGF uncontrolled trials that showed enormous clinical benefit. 

But really, to date, there remains only one positive, large, randomized placebo-controlled trial, which we published now a long time ago in the Lancet, which is fiberglass growth factor. But the bottom line is, VEGF, vascular endothelial growth factor, as a therapeutic in humans does not work. I will, perhaps at the end, show you at least one new thinking as to why. 

In fact, most of the studies done-- this was one by Sanjay Rajagopalan-- was the first author. At the time we did this study, it was the largest gene therapy study for patients with peripheral arterial disease. Patients received intramuscular delivery of an adenovirus expressing VEGF 121. 

And the bottom line at the end of this 105-patient study-- if I looked at change in walking time in three groups, you could not make three groups look more identical if I gave you a piece of paper. And investigators are saying oh, no, no, no. This worked. We know this worked. 

Somehow, we're missing something, right? When a clinical trial doesn't work, what's the first thing you do? You blame the patient. Second thing you do is you blame something with the execution of the study. You never actually think that your drug didn't work. 

Well, here was the data. This is change in ankle brachial blood pressure index. 0.00 in the placebo group. 0.00 in the low dose group. 0.00 in the high dose group. I don't-- you don't need to be a biostatistician to know those three groups are absolutely no different from each other. 

So VEGF, despite some signal in the background, clearly did not work. Does anybody know what the original name of VEGF was? Matt's thinking. We didn't put that in the book chapter alone, now did we? OK. 

It was actually identified as vascular permeability factor, not vascular endothelial growth factor. And look what happened in this study. 10% of the patients in the low dose in the placebo group had clinically significant edema at 30 days, 20 in the high in the middle, and almost a third in the high dose group. 

Fortunately, because of our clinical design, this was not a safety issue. There were no adverse event rates, but that was in part because of the trial we designed. So what I would actually argue is we did indeed induce vascular permeability. We actually just simply did not induce therapeutic angiogenesis. 

Following that, there was a trial with a HIF-1-alpha transcription factor. And the bottom line to that trial-- almost identical. Absolutely no difference between placebo, low dose, and high dose groups. And I think largely, the field of gene therapy today looks like this, which is a traffic light in Rome. So you could try to cross, because there is some green. But I would do it with care. 

[LAUGHTER] 

So in fact, our approach with this has been-- we took a couple of different approaches. One is we could just close up shop, say, this is actually not going to work. But in fact, what our lab does-- and I will for the next 10 minutes or so take you through some laboratory data that we've generated, and take you through the approaches that we're using to do this. 

And really, we take three-prong approach. One is new ways to deliver agents-- really working with Chris Kramer over the past decade. New ways to assess agents in clinical trials. But the one that without a doubt is the most fun is to come up with new agents. So that's what we are largely looking at. 

And I'm going to now credit people that are doing the work. Some of them you remember. Ayotunde Dokun-- I don't know if he's announced yet, but he will be having a very exciting career change on July 1. Tao Wang's a former Fellow in the lab. Vijay Ganta's on our faculty now. And Surovi is at the Cleveland Clinic. All these are key people for the data I'm about to present to you. 

So we go at this by studying animal models. We actually study mouse models. And why do we do this? Because we can, in an animal, try the very best we can to recapitulate what we see in humans. 

And we try to look at angiogenesis, or the growth of blood vessels-- arteriogenesis, the growth of preformed arterioles-- as well as systemic and local responses. But almost every lab in the country can do this. But why-- what is it about our group? What have we been able to do? 

And our last four grants we've submitted to the NIH in four consecutive cycles have all been funded. And what I'd argue is, we do have a secret sauce. And that secret sauce is this-- that we've recognized from the very beginning that humans with PAD are actually quite heterogeneous. 

So if you want to fact check me, these are two patients who were seen back at Duke in 2004. This patient on the left was enrolled in our supervised exercise program. That's Jenny Robbins, one of the exercise technicians that at the Duke Center for Living. 

This patient on the right-- sorry for those eating. This is the remains of their foot. And these two patients were the same age, same gender, same risk factors, same ABI. And if I showed you their arteriogram, you would not be able to tell which patient actually stabilized with a below-the-knee amputation, versus which patient was actually quite stable for years and years. 

So what Ayotunde showed is that we can recapitulate the same thing in mice. There's a knee-bred mouse, C57BL/6 mice. They do fine following hind limb ischemia. BALB/c mice do terrible. We crossed these two strains-- generated an F1. 

The F1 response is the same as the wild type. And then we go ahead and look at the F1 crossing to BALB/c. We did 105 mice in five days with a very trained vascular surgeon, sent it for genome-wide linkage scan, which is probably pretty arcane right now, and this is what it looked like. 

We saw a single peak on the short arm of mouse chromosome 7, which had a LOD score of about 7, or 300 million to 1 that there was a gene or gene in this area that actually was involved in the clinical outcome. In fact, 50% of the variability in this complex, preclinical model was due to the genetic variation. By the way, my labs always tended to have cardiology Fellows, particularly interventional cardiology Fellows. And actually, for this type of surgery, our really good interventional cardiology Fellows sometimes couldn't even do this and execute it. 

So when we redrew the mouse map, it turns out a gene at the very peak of this is the interleukin-21 receptor. I immediately told my Fellow, I don't want to do anything with interleukins, because it involves inflammation and people are going to yell at me if I try to do that. Fortunately, he didn't listen to me, which is a common thing in my lab. Which is, they usually go on and prove I'm wrong. 

So what he did here is they looked at mice who responded well. They upregulated the interleukin-21 receptor. They showed that in the endothelial cell fraction, when they co-stain, the interleukin-21 receptor with endothelial cells, lo and behold, that's where the interleukin-21 receptor was located. 

We collaborated with Warren Leonard at the NHLBI, who sent us some wonderful reagents, including [INAUDIBLE] the interleukin-21 receptor. They had impaired perfusion recovery. He also provided us with a soluble interleukin-21 receptor that completely removed the ligand. They had reduced angiogenesis and perfusion recovery. 

We had one major problem, though, in the lab, which is there was only one published paper at the time we did these studies on interleukin-21 and angiogenesis. And it said the following-- "Interleukin-21 inhibitors angiogenic sprouting of endothelial cells." Right? 

So obviously, I've got a problem here. Because we're showing activation of this pathway is good for growing blood vessels. The only published paper tells us it's not. And what I'm going to do is spare you-- oops. I don't want that. 

I'll spare you all the experimental detail. It's in this paper. But the bottom line is, the same ligand and the same receptor have a completely different downstream signaling pathway, based on the conditions that you do the study. So always be careful when somebody tells you what an agent or a pathway does. Make sure you understand the context and the background of it. 

By the way, it's also nice to know that humans do the same thing. So we actually showed in human samples the interleukin-21 receptor also co-localized with endothelial cells. Doesn't necessarily prove anything, but if you're going to go test this, you might as well know that the receptor's there to be activated. 

What I'll do is quickly walk through. This is a new work and in a grant that's just been awarded. We're actually showing that interleukin-21 works in several animal models. Again, whether it's high-fat diet, or some mice that have impaired nitric oxide metabolism. 

This is even more strong data. We're actually looking to show that activation to this pathway works in models where VEGF has failed. In eNOS knock-out mice, VEGF has absolutely no effect in mice. We delivered the IL-21 plasmid. We reduced necrosis. This is what happens if we give the drug L-NAME, which blocks nitric oxide bioavailability. And interleukin-21 is able to reduce necrosis, even in the presence of nitric oxide inhibitors. 

And this data is going to show you that the pathway does not require the cononical Akt/eNOS/nitric oxide pathway. So clearly, we've developed a pathway entirely separate of VEGF-- entirely separate of a pathway that we've already shown doesn't work in people. It really does strike me as a better idea to try to take what we've learned from people, instead of trying to say, people did things wrong with the clinical trials. 

Is that actually good? I'll show you just a smidgen of laboratory data. Again, when you look at cell survival, VEGF's effects-- the amount above the green, our block with L-NAME, not IL-21. And if you go down here, when you get VEGF-mediated angiogenesis, you get a reduction in electrical resistance, or an increase in permeability. And I would like to argue that you'd rather form good, stable blood vessels that do not let things leak through them, like cancer blood vessels do when you're trying to treat peripheral arterial disease. 

I'll skip through that. Final thing in humans-- it turns out African-Americans with peripheral arterial disease have horrible PAD. And they also do not have this interleukin-21 receptor. 

Final last data slide-- I told you about VEGF. And in fact, recent work has shown the VEGF story may be much more complicated. There is a single exon switch in the eighth exon of VEGFA that turns VEGF from angiogenic to anti-angiogenic. 

This is a flow pattern of CD14 and CD16 on human monocytes. This is a very quick primer for what you need to know. If you have CD14, that's bad. If you have CD14, that's bad. CD16, that's good. 

Turns out, patients with PAD had this intermediate phenotype that has been reported by Peter Libby and others group to be unique to PAD. Would anybody like to guess where we find the VEGF165b isoform? We find it only in this subset of monocytes. And again, what I told you at the beginning-- we do not understand this link between peripheral arterial disease, the legs in life. And if we're going to make advances in this field, we have to begin to do that. 

So PAD driven by smoking and diabetes, a major health care problem that remains overlooked, can easily be missed. Your routine questions, unfortunately, don't work for screening. Attempts to promote therapeutic angiogenesis using classic approaches have failed. 

A lack of understanding, or the lack of the appreciation of human disease may account for that. And PAD remains poorly studied, and offers numerous research opportunities. So I will stop there. Thank you. 

[APPLAUSE]