 Good morning. Welcome to Grand Rounds. Everybody must still be at the Academy. It got done yesterday though, right? So, he's coming back this morning. Everybody's recovering from Academy, I guess. So, we got the last of our three visiting medical students today. We're gonna start with Romulo Albuquerque. He's been working with a doctor on body here. He's a medical student at the University of Kentucky and works with Dr. On Body's brother, Jay On Body. And his a lot of there doing work with, who also does work with VEGF. So, we'll start with him. After that will be David Gray, who's been working with Dr. Dries and Dr. Mamelis. Well, first, Romulo will be talking about a new member of the VEGF family and then David Gray, David Guy, rather, from Virginia Commonwealth, who's been working with Dr. Dries and Dr. Mamelis will present a patient with an anterior segment mass, a patient of Dr. Dries. And then lastly, we have Stuart Walther from Texas Tech. He's been working with Dr. Tasky and Dr. Dries and he'll round us out talking about CMV retinitis. Anyway, I'll turn the time over to Romulo. So, do I need to put the earpiece or will people hear me okay if I talk through this one? All right. Well, good morning. It's a pleasure to be here today and I'm very thankful for the opportunity to share with you some of my research efforts that I have in Kentucky. I'm... Although I come from the University of Kentucky, I'm not a native of Kentucky. I show on during the Feynman from New Mexico because of my last name I am not. I'm a native of Brazil and some of the Brazilians in the audience gonna pass the Albuquerque is a very common name in Brazil, very common last name. But I hope to excite you as I hope to get you as excited as I get about this new VEGF receptor that I have found. So the objectives of the talk today is to review briefly the history of angiogenesis of the field of angiogenesis. I also hope to define the two domains of angiogenesis and its molecular drivers. And I like to share with you how this discovery of this new receptor came about. And also maybe show you some data that convinces you of the biological role of this soluble VEGF receptor 2 and its clinical implications. So I, you know, as in describing the history of angiogenesis, I could not not talk about Dr. Fogman. So Dr. Fogman published a paper in 1971 where he proposed that if we understood how to modulate the growth of blood vessels, we could actually treat cancer and perhaps cure cancer. And his idea was published in this New England Journal of Medicine, paper in 71. And it's funny if you, if you PubMed angiogenesis before 71 or at 71, you get like three papers. And I did a PubMed search yesterday on angiogenesis, and there are nearly 50,000 papers published in angiogenesis. So his idea has really taken off and the 20 years following his, the launching of his idea, this field really thrived and in this very short amount of time we cloned VEGF. We made knockout mice that lacked VEGF and we really understood vascular biology and how vasculine does your growth factor plays a role in it. And I enjoy talking to ophthalmologists about angiogenesis because you guys are really in the forefront of this transformation. You're really practicing anti-vegeta therapy and seeing how many patients worldwide is actually benefiting from this idea of blocking blood vessel growth. So this is a table, a figure from the anchor trial showing that Lucentus has really changed the way that macular degeneration is treated. But ophthalmologists also have a tendency to really focus on one aspect of angiogenesis, which is hemangogenesis, which pertains to the growth of blood vessels and kind of neglect linked angiogenesis. And I'm really interested in this aspect of angiogenesis, and I'll tell you why. So more accurately what we know is that VGFA primarily drives hemangogenesis through activating VEGF receptor 1 and VEGF receptor 2. And it's actually VEGF-A is a target of Lucentus. On the other hand, we have a cousin molecule known as VEGF-C that drives lymph angiogenesis by primarily activating VEGF receptor 2 and VEGF receptor 3. And this field was kind of revolutionized with the discovery of soluble VEGF receptor 1 by Kando and Thomas in 1993. These guys show that this molecule is a splice variant of VEGF receptor 1. It's a very powerful inhibitor of VEGF-A, hence it's a very powerful inhibitor of hemangogenesis. Our laboratory in conjunction with Bala's laboratory actually showed that soluble VEGF receptor 1 is expressed in the cornea, and it's singularly essential to maintaining the cornea devoid of blood vessels. However, the concomitant absence of lymphatic vessels in the cornea is really not well understood. But while I was working in this paper that we published about soluble VEGF receptor 1, I actually made an observation that when looking for VEGF receptor 2 in the cornea, instead of finding the 230 kilodoutons band that corresponds to the full length VEGF receptor 2, what we found was a 75 kilodotons band in the cornea, which led us to ask the question, could this be soluble VEGF receptor 2, just like soluble VEGF receptor 1? And so we devised this hypothesis that there was a truncated form of VEGF receptor 2, and I've done some homework in gene analysis, and I actually showed that this truncated form was very likely to be a splice variant of VEGF receptor 2. The full length VEGF receptor 2. So I continued to do my homework and actually clone the mRNA that codes for soluble VEGF receptor 2, and if you translate the sequence, you actually get a protein that is composed of the six Ig-like domains that forms the actual cellular domain of VEGF receptor 2, and it also has a unique C-terminus, which we exploited to make an antibody that was capable of detecting soluble VEGF receptor 2, but not VEGF receptor, the full length VEGF receptor 2, which is shown here by this western blood, the 75 kilodotons band corresponding to soluble R2, but not the 230 kilodotons band corresponding to membrane bound VEGF receptor 2. So at this point, we were armed with tools to actually study the spatial distribution of soluble VEGF receptor 2, and primarily in the cornea, and inside the hybridization actually localized soluble VEGF receptor 2 to the cornea epithelium, and we actually devised antibodies, that antibody that I showed you, we actually studied the expression of soluble VEGF receptor 2 also spatially within the cornea, and you can see that it's very highly expressed at birth, so this is P0, and then it's expressed throughout the entire cornea thickness, but then its expression becomes confined to the cornea epithelium, and if you look at the conjunctiva corneal interface, there is a very clear demarcation of SVAGF receptor 2 here shown in green in the corneal epithelium, but not in the conjunctiva, and you can also see for this corneal flat mount that it's very highly expressed in the limbo area of the corneal, but not at the central corneal, not as much I should say. So at this point, we were convinced that we had established the existence of a new receptor, but we had no idea what it actually did. So I actually, in some preliminary studies, I showed that this receptor, soluble VEGF receptor 2, actually interacted with VEGF-C, physically interacted with VEGF-C, and then I also showed that in vitro assays that it blocked VEGF-C signaling, hence we hypothesized that this molecule would be anti-lynf angiogenic. Because it was so highly expressed at birth, then we were interested in understanding why it was its role in corneal development, and to do that, we created a mouse that was deficient in soluble VEGF receptor 2 in the cornea, and here's a demonstration that these knockout mice, they do not have soluble R2 in the cornea compared to the wild-type mice, which you can see the epithelium does express soluble R2, and these mice had a really an astonishing phenotype. These mice were born with their corneas spontaneously invaded by lymphatic vessels here, shown by this corneal flat mount, which was very, very astonishing phenotype, and it was very powerful because we were very excited about this because the blood vasculature was unchanged, and this was unprecedented, because the two vasculatures are usually very well coupled, so for the first time we were seeing uncoupling of these two vasculatures, which was unheard of, but why was I really excited? Well, so it's well known that regularization in the cornea is a very negative predictor of transplant survival, and the theory is that the lymphatic system actually here, shown as the afferent pathway, actually works to drain antigens and antigen presenting cells from the donor cornea that then migrate to the lymph node and activate the whole cascade of that ultimately leads to a transplant rejection. So maybe by modulating this afferent pathway, it could actually impact the survival of the corneal transplant, and this has never been really fully understood because the because these two vasculatures are coupled together, no one could ever really tell what was the particular role of lymphatic versus blood vessels in this context. So we, to really move this project forward, we looked at what was the role of SR2 in repartive corneal injury, in repartive lymph angiogenesis, and we actually showed that in using the suture induced corneal angiogenesis assay by placing two 11-oh sutures in the mouse cornea, and given time these blood vessels and lymphatic vessels would grow, so we actually showed that if I deleted soluble R2 from the cornea as I did in the developing mouse, you have a drastic increase in the levels on the density of lymphatic vessels in the cornea, but no change in blood vessels shown here in red, but you can see that there's a drastic increase in lymph angiogenesis, and then the opposite was also true. So if I over expressed soluble HG2 in the cornea, you saw a decrease in the levels of lymph angiogenesis, whereas the levels of blood vessel density was actually unchanged. So we're pretty convinced that we had now a tool to manipulate lymphatic vessels without really affecting blood vessels. And the ultimate proof would be to actually show that if I treated corneas with soluble VEGF receptor 2 and blocked lymphatic vessels from growing into the graft, that I would actually impact the survival of corneal allografts, and we use the mouse model of PKP, and you can see here this is a Kaplan-Meier curve showing, in the control group, you can see that at eight weeks post-transplant, that there is actually a 40% survival compared to a group in which we injected soluble VEGF receptor 2 immediately before transplantation, which doubled the survival rate. It was nearly all of the corneas survived. So and this was even the more astonishing because this survival was accomplished even in the presence of blood vessels crossing through the interface between the graft and the donor, really implicating that lymphatic vessels are the key negative predictor of transplant rejection. So I have really cherry-picked things from this paper that we published to be able to talk to you in the time that was allotted to me, but if you're interested, you can actually go ahead and read the full paper. It was published in Nature Medicine last last year, September, and we actually got the cover. We're pretty excited about that. So I will conclude and summarize by telling you that soluble VEGF-R2, I hope I showed you enough data to convince you that soluble VEGF-R2 is a splicing variant of VEGF-R2. That soluble VEGF-R2 is the first reported endogenous specific inhibitor of lymphatic vessel growth and also that soluble VEGF-R2 works to aid in the creation of an alien-thatic cornea and also that it inhibits reparative lymph intergenesis and it enhances the survival of cornea transplants. And this we've been moving this project forward and we're actually looking at fugue dystrophy. We I actually found out yesterday just got a paper accepted in which we're looking at cancer metastases and there's a lot of other models in which you're exploiting this molecule for other clinical applications. I would like to acknowledge all of my lab friends and our lab family, we call it. These guys have been critical in in helping me move this project forward. Good science is not done without good collaboration. So I'd like to acknowledge all of these collaborators around the world and the other thing that good science is not done without is money. So I need to acknowledge Fight for Sight, Research to Prevent Blindness, the MD-PAGD program at the Inverse of Kentucky and my biggest source of funding which was Hachia Krishnan-Bhati Balas Prada who really supported me financially and intellectually through this entire journey. With that, I will entertain any questions and you guys, if you like horses, you should come to Kentucky. Thank you. Sure. Actually, so the when I talked about maybe exploiting it in fuchs dystrophy one of the ideas is that I've been looking at, we have a library of corneas that I've been looking at and it appears that fuchs patients have higher expression of solivore to hence, lymphatic vessels cannot grow into the corneas, but if it could actually make lymphatic vessels grow into the corneas stroma even though there is endothelial dysfunction in edema, maybe we could prevent edema from forming with lymphatics coming to the corneas. So this is an idea we are actually exploring. Thank you very much.