 been a fabulous intern over at the VA. He's done an absolutely fantastic job. The interns, I don't think, have any idea what they're getting themselves into until they get over there. But he's done a fantastic job lining up our surgeries for us and making the transition to the Moran much easier. But he'll be talking about angiotensin-converting enzyme and corneal neovascularization. And I'll turn the time over to him. Thanks, Snow, for that kind introduction. I want to thank everybody for the opportunity to speak this morning. As an intern, I'm not drawing from a deep well of knowledge and experience, but I did want to take this opportunity to share with you some research that I had, the opportunity to be a part of as a third and fourth year medical student. And that was in this particular area looking at the potential connection between angiotensin converting enzyme and corneal neovascularization. So just by way of introduction, those of you with clinical experience know that ACE inhibitors, in alloprol being one, are among the most common anti-hypertensives prescribed for hypertension. And they have been since the 1960s when they were introduced. They act on the renin angiotensin system, which you can see outlined in this graphic over here. Specifically, they act at this step, which is the conversion of angiotensin-1 to angiotensin-2 by angiotensin-converting enzyme. And angiotensin-2 carries out most of the physiologic functions in this cascade and acts on two well-described receptors, the AT-1 receptor and the AT-2 receptor. Specifically, it has two broad areas of effects. One is by increasing aldosterone expression. And aldosterone works in the kidney to increase sodium absorption and thereby increasing fluid volume and blood pressure. It also has several aldosterone-independent effects, one being vasoconstriction, anti-diuretic hormone release, anti-diuretic hormone acts on the distal portions of the nephron to lead to free water resorption. And also down below, I put some more recently described actions of the downstream effects of angiotensin-2, which include cell proliferation, chemotaxis, and increased VEGF transcription, VEGF being vascular endothelial growth factor. So if you look at this group that I've outlined in yellow, it's quite interesting when you look at increased cell turnover, the migration of cells, and the elaboration of cytokines like VEGF, those are all key precursors to angiogenesis and neovascularization. So it's quite interesting to think that blockage of this cascade may affect those cellular activities as well. So just kind of a little background as to how these activities were described. So this is kind of an interesting case study as to how research works, which often starts with an intriguing observation, which leads to an idea, which leads to testing that idea first in a lab, in vitro, in petri dishes, then in animals, and then in humans. So it started about a little over 10 years ago in 1998 when a man named Lever and his colleagues made this interesting observation, just epidemiologically, that hypertensive patients undergoing long-term treatment with ACE inhibitors had a decreased incidence of breast and lung cancers. And here you can see that it's a little over half, 0.65 versus 1.1%. And this was very interesting. They didn't have a whole lot of background as to why this would be the case at the time, but it certainly tweaked a lot of ears and got a lot of people going in the lab as far as looking at why this would be the case. Fast forward about 10 years and we can find that individuals with certain ACE gene insertion deletion polymorphisms, which lead to increased ACE activity, have a 38 to 80% increased incidence of breast cancer. And this increased risk can be ameliorated or even reversed by ACE inhibitor therapy. So can you imagine going to your doctor and saying, I have a really strong history of breast cancer in my family, what can I do to decrease my risk? And most of us wouldn't think to give our patients an anti-hypertensive, but that's what this study appears to suggest may be a benefit in these certain individuals. Fast forwarding from the observation phase to the testing phase, multiple studies have demonstrated that ACE inhibitor therapy decreases in vitro, preneoplastic lesions, cellular proliferation, angiogenesis, and VEGF in human carcinomas in petri dishes in the lab. This is very interesting. This pretty recent research, 2007, 2008. So moving on from in vitro studies to models in animals, mouse models of lung, gastric, pancreatic carcinomas have demonstrated that the way that ACE inhibitors decrease angiogenesis in these models is by blocking transcription of VEGF. So the way that they did this is they looked at all these different models, and then they looked at circulating, circulating VEGF levels in these animals. And what they found is that in these animals, with these particular models of cancer, if they treated them with ACE inhibitors, that the VEGF levels in their sera was markedly reduced. So studies in both animal and human tumors have demonstrated a correlation between the extent of angiotensin receptor expression with VEGF expression in those tumors, tumor angiogenesis, and tumor invasiveness. So not only does it appear that this cascade that I showed on that second slide, the renin angiotensin system, affects the elaboration of VEGF, it also affects the clinical characteristics of the cancer, as in its ability to invade surrounding tissues, form new blood vessels, and elaborate these cytokines. So some of you may be asking, well, why is Daniel speaking so much about cancer when this test is supposed to be about blood vessel formation in the eye? So I did my training in Arkansas, so you have to forgive me, but the areas of research on cancers and neovascularization are really kissing cousins, and that's because these cancers, often when they have such high metabolic activity, they have such a demand for tissue, such a demand for blood, they have to recruit their own blood supply, that obviously is not a part of the normal anatomy, and one of the ways they do that is by elaborating these cytokines, VEGF being one, and it's a very potent stimulator of new blood vessel formation. This next bullet is a little bit closer to home for us, as eye care professionals, and that shows that a couple of recent clinical trials have shown up to a 65% reduction in the progression of diabetic retinopathy in patients who are in ACE inhibitors or ARB, and so this is from an article in the New England Journal of Medicine in 2009, and also the direct trial, which stands for the diabetic retinopathy Candisartan trial, and we've known for some time that in patients with diabetic retinopathy, particularly proliferative diabetic retinopathy, that if you look at VEGF levels within the vitreous, that they're quite elevated, so this does make some sense. Yes, they looked at both in these studies, they looked at progression of NPDDR and PDR, specifically they looked at progression of, well, obviously the earlier progression where they had more room to go forward was reduced more than the far advanced PDR patients, primarily yes, but even PDR was reduced in these studies. I'm not trying to suggest that it isn't, and that certainly is a possibility, and they addressed that in the study itself. So what can we take from all these lines of evidence? What's the take home point? I would suggest that the downstream cleavage products of the renin angiotensin systems via all these lines of evidence have been shown to promote angiogenesis in a wide variety of settings, from in vitro models to animal models, and thus inhibition of this cascade may represent a new therapy or direction for trying to block angiogenesis in vivo, and so that's what we decided to look at. So our question was this, will inhibition of ACE with enalapril block corneal angiogenesis in a rabbit model of VEGF induced corneal neovascularization? So again, we're kind of drawing from our animal companions on this planet to tell us a little bit about the human condition. So first, in order for this to be a feasible experiment, we have to show that there is a local renin angiotensin system in the cornea. If there is not, then we have no reason to believe that systemic treatment with enalapril would have any localized effect. And actually, local renin angiotensin systems have been well described elsewhere in the body, including cardiac lung, vascular, and particularly renal tissue. And even elsewhere in the eye, including the ciliary body, vitreous, coroid, and retina. But in perusing the literature before this study, it was really not all that well characterized in the cornea. So how did we decide to address that? Since we knew we were going to be using a rabbit animal model, we decided to look at rabbit corneal culture, specifically rabbit corneal fibroblast and rabbit corneal epithelial cells, which you can see over here. We extracted mRNA from these cells and then via standard microbiological techniques. We transformed that into DNA, amplified that DNA, and then ran specific primers to look for expression of specifically angiotensin converting enzyme, AT1 receptor and AT2 receptor expression. So what did we find? Up at the top, you can see, this is a standard gel of these PCR products. You can see a DNA ladder on the left. And what you can see is that this top one is the rabbit corneal fibroblast. We found ACE expression, AT1 receptor expression, and AT2 receptor expression. Of course, to prove that this is high quality gel, you have to run both positive and negative controls. So our positive control was beta actin, which is a standard housekeeping gene that's present in almost all cells. And then the negative control was the primers with just water. So you wouldn't expect any PCR products to be seen in that column, and that's exactly the case. So here in rabbit corneal fibroblast, we found all three elements of that initial cascade that we showed down here is the rabbit corneal epithelial cells. And interestingly, in the epithelial cells, we found AT1 receptor expression and AT2 receptor expression, but no angiotensin converting enzyme. And again, good control. So with this evidence in hand, we felt comfortable moving forward with the second phase of our experiment, including animals, being that systemic therapy in allopril may have a localized effect on the cornea, given the fact that all the players are present. So we decided to use New Zealand white rabbits, and we induced corneal neovascularization in these animals by implanting a veg eff pellet in their paracentral cornea. After that, these groups of rabbits were randomized into one of two conditions, the first of which received intramuscular water injections every day for 14 days, and the second of which received in allopril intramuscularly every day for 14 days. We used three milligrams per kilogram, which for those not familiar with rabbit dosaging, that's a relatively low dose, we got it from this paper in 1997. We had initially wanted to use a higher dose of the in allopril, but the IRB was a little bit concerned about inducing hypotension in the animals, which is legitimate, and so that's why we decided to go ahead and use that dose. We monitored the animals by a slit lamp microscopy at days four, nine, and 14 to monitor for the development of corneal neovascularization, and the way that we quantified that was by calculating the mean area of corneal neovascularization using image J software, which is provided by the NIH. So this is a little bit of insight into my life as a third and fourth year medical student in the lab. If today you think you're having trouble keeping your patient in the slit lamp, try a rabbit for a while and see how that goes. These are some representative images. You can see this VEGF pellet here with some new blood vessels coming in, and quite often what we see in corneal neovascularization is that these blood vessels arise from this plexus of vessels that runs around the limb towards that stimulator of the neovascularization in the first place. And then this other image is designed just to show that within the rabbit corneas, there was not a significant amount of edema or inflammation incited by this nearby VEGF pellet. So here we get to a little bit more of the meat of the study itself. So here we are at day four. On the left is the control treated animals. Remember that they received intramuscular water and then the inalipral treated animals on the right. So here you can see just this little fronds of neovascularization originating from the limbis and emanating towards that potent stimulator of neovascularization, that VEGF pellet. And even at day four, you can start to see that this amount of neovascularization is more in the control group relative to the treated group. Fast forwarding to day nine, you can see that these same vessels have progressed towards the VEGF pellet and in some cases are beginning to surround it and infiltrate it. You can see that this process is a little bit further along in the inalipral treated group. But if you look a little bit more closely, what you can see is that in the control group, there are increased number of blood vessels emanating from the limbis and also those vessels tend to branch more as they get closer to this VEGF pellet. So if you look at the inalipral treated group, you can see that there are fewer blood vessels originating from the limbis. And as these move towards the VEGF pellet, they demonstrate decreased branching. And obviously the more branching you have that would exponentially increase. So here we are at day 14, our last point. So what you can see is here in the control group, there's quite a lot of vessels that have encompassed that whole VEGF pellet. So relative to the inalipral treated group, you can see there's a whole lot more blood vessels emanating from the limbis and they demonstrate increased, again increased branching relative to the inalipral treated. So these are some immunohistochemical slides. We do not choose to quantify these results, but basically immunohistochemistry denotes taking an antigen antibody to a particular antigen on a cell type or a portion of a cell and then attaching to it a little fluorescent marker so that you can shine a light and see exactly what it has attached to. So in this particular immunohistochemistry slides, we've used DAPI to stain cell nuclei. And so those show up blue in these photos. And this is corneal stroma. So you can see a lot of cell nuclei. And we use lectin to stain vascular endothelium. So obviously it's very important that corneal stroma be clear and free of blood vessels to maintain a good vision. So any amount of neovascularization or red staining in these slides is abnormal. And so you can see that the VEGF pellet induced neovascularization of the corneal stroma in both treatment conditions. However, in the control group, you can see quite a bit more red staining diffusely throughout the corneal stroma relative to the enalaparal treated group. Those are corneal sections, which were stained with DAPI and lectin. I'm sorry, flat plates, flat mounts. No, we did not do that. We just took sections. So what are the results of this study? So we've already kind of gone over this first part, but a previously undescribed Green and Androgynous system exists within rabbit corneas. So specifically within rabbit corneal fibroblasts. That's where we saw all three of the players of this cascade. So angiotensin converting enzyme, AT1 receptors and AT2 receptors. Corneal epithelium again showed AT1 receptors and AT2 receptors, but no ACE. And then moving on to some of the quantification of the slides that we saw earlier of the images at the different time points. We saw that the VEGF pellet was quite good, surprising no one, at stimulating corneal neovascularization, yielding a mean area of corneal neovascularization in the control group of 1.8, 2.8, and 3.2 millimeters squared on the three tested time points, respectively. Enalaparal treatment, by contrast, decreased the amount of corneal neovascularization in these animals by 44% at four days, 28% at nine days, and 31% at 14 days. And you can see the actual hard numbers there. Exactly. Two of them were and one wasn't. We'll go over that in this slide here, which is a graphic representation of these same results. I know this is a little bit of a busy slide, but here on the y-axis you can see the blood vessel area a millimeter squared, x-axis is the time and days. The black bars represent the control animals, and the white bars represent the enalaparal treated animals. So there's a few things to notice about this. This size symbol here represents that at days nine and 14 there was statistically significantly more corneal neovascularization than was present on day four. So our VEGF pellet was doing a good job at inducing and continuing to induce corneal neovascularization as the experiment progressed. And you can see at all three time points as we saw in the photos and in the raw data that the enalaparal treated animals demonstrated less corneal neovascularization than their control counterparts. What this asterisk denotes is statistical significance. And so we can see that only at days four and 14 was that being statistically significant by two way and over testing. The Psi, I was saying that that represents that at days nine and 14 there is a statistically significantly more corneal neovascularization relative to our first tested time point. So what conclusions can we draw from this admittedly limited study? So we can say that there is the components of a previously undescribed Renan angiotensin system within rabbit corneas, specifically rabbit corneal fibroblasts. And it appears to play an important role in corneal angiogenesis. I say that because as we've seen treatment with enalaparal or blockade of this Renan angiotensin system statistically significantly reduced corneal neovascularization in a VEGF model in rabbits. So what future directions could we take? What could we look at from trying to build on this knowledge? So future studies are needed to explore the therapeutic potential of enalaparal in other animals and potentially in humans. It's important to note that if you look at the literature that corneal RAS expression is highly variable among different animals and even among mammals. If you look at rat and dog cornea where these studies have been done, they haven't found angiotensin one or ACE expression. And so in order for this to be applicable to humans, one would have to do the same experiment on human corneal tissue, which to my knowledge has not been done at this point in time. Obviously, I've already alluded to this, but we could use higher doses of enalaparal, which might induce a more robust response. And I elaborated our reasoning for using a relatively low dose before. Also, we used a very potent inducer of corneal neovascularization in VEGF. So one could use a number of different means to induce corneal neovascularization, including chemical, trauma, thermal, or a number of different inciting events. Those are all areas for future research. And lastly, I just wanna thank the staffs, the resident, everybody for making this a great form of rotation. I feel like I've learned a lot. I'm excited to be coming back. I'll be counting down the days until I get to come back. And in the meantime, I'll be counting down the days till I get to meet this little guy. My wife is 38 weeks pregnant with our first son, a boy. His name's gonna be Levi, so we'll be meeting him soon. I'll open it up to any questions. I have a, thank you. Yes, Dr. Olson. So, exactly. So, it's a very good point about looking at whether it would block formation of VEGF or whether it would block utility of already formed VEGF. And from looking at these studies of animal models of tumors, it's unclear whether the blockade occurs at the level of formation or utility, whether something about it blocked, but nevertheless, the VEGF were reduced. And that's a good point that it could well be decreased formation. And in fact, that might be the more likely response. But it wasn't clearly elaborated in these studies that I read up to this, this. So, so that's a good point. You know, it is. Right, so that would be, you know, statistically significant from our study is that at least in this model of VEGF, there's really not a whole, there are other ways that it could have blocked, but these appear that at least RAS blockade appears to disrupt the VEGF function within the eye and within other angiogenic models. Right, right, that's true, yeah, that's a good point. Right, no, that would be an excellent way to address both that question and the question Dr. Olsen posed, you know, to look to see the circulating levels of VEGF. And we did not specifically measure or quantify the circulating or the localized levels of VEGF either in the cornea or in the animal itself. That would be a very good point. Thank you very much.