 All right, I think we'll go ahead and get started. We have a good lineup today. A lot of our home and visiting medical students are presenting, so I'll introduce a couple of the presenters at a time, then we'll go from there. So our first presenter is gonna be Judd Kahoon, who's currently a fourth year medical student at the University of Utah. Judd and I go way back. We were students together as undergrads, as medical students, actually before that even, he taught my brother guitar. So that's another thing about Judd, if you don't know, he's a fantastic guitar player and teaches many prolific students. Did he get health back? Ha ha ha ha ha ha. Great question, and I'm glad you brought that up. Judd pursued a PhD in neuroscience, neuroscience, and he's now back finishing the MD portion of the MD-PhD. So we'll be happy to hear from him. Oh sorry, and then I'll also introduce Rebecca Genture, who is a fourth year student who is at Rutgers, Robert Wood Johnson Medical School in New Jersey, and she has also earned a PhD in biomedical engineering, and she'll be talking to us about trans-corneal electrical stimulation today. So we'll go from there. Thanks a lot. Thanks for inviting me to give this little talk. I gave a grand round a couple of years ago, and I guess I can kind of update kind of where the progress happened since then. I'll be talking about CompEng1, and a therapy that we're trying to use in diabetic retinopathy. So just kind of an overview of the talk. We'll discuss an intro to diabetic retinopathy, some of the methods we use to probe this, and then I'll focus just on a small segment of results regarding vascular inflammation. Basically, I just chose the results that I thought looked really cool and had kind of cool videos to see physiology in action so that we could enjoy that. Then we'll wrap up from there. Great, so diabetic retinopathy, my understanding of diabetic retinopathy is that, A, it's a big problem. It's the leading cause of blindness in the working age population. It's 93 million people worldwide that's a good chunk of folks. It costs a lot to treat, very expensive to treat, but there's also some indirect costs because this is happening to the working age population, so there's lost productivity, there's absenteeism with people having visual disturbances because of the diabetes. 35% of people, so over 80% of people with diabetes know the experience of diabetic retinopathy, and 80% of people with diabetic retinopathy don't even know they have it, as they can remain asymptomatic for a while despite the pathophysiology going on. Those numbers are expected to triple by 2030. So I try to conceptualize how this works and what's going on in relatively simple terms, and I just think of hyperglycemia inducing neurovascular damage. How is it inducing neurovascular damage just through some of the classical pathways of increased reactive oxygen species? Some cells are very sensitive to that. Advanced glycation end products, PTC pathway, and the polyalk pathway with sorbitol and increased osmotic. By neurovascular damage. I mean, vascular permeability in edema, the blood vessels become leaky. We'll take a look at some of that. There's capillary degeneration and non-perfusion where actual endothelial cells are gone, and the only thing left is an empty basement membrane through which no blood or oxygen or nutrients flow, and as you can imagine, that could lead to some neuroglial dysfunction and degeneration. So what's the problem then? The problem is that the current treatments are reactive. When I think about a retina that is undergoing vascular damage and vascular compromise, such as it happens in diabetic retinopathy, I picture the retina screaming that it needs to be fed, and what it screams is VEGF, and so some of our treatments for that are to just eliminate the screaming cells with PRP, and you can see we're just eliminating those cells that are telling us something bad is going on and ignoring the underlying problem. Secondarily, we could just eliminate the message that they're screaming, which happens to be VEGF, and ultimately we know that the best treatment for this would be to prevent the hyperglycemia causing that, and so we're, to further conceptualize what's happening to the endothelial cells, here's a capillary. We've got endothelial cells here in pink, pericytes, which are supporting cells for the endothelial cells, diagrammed in yellow, and the retina special because it's got this increased ratio, the highest ratio in the body of parasites to endothelial cells, a one-to-one ratio of some parts, and the pericytes are responsible for providing structural and trophic support, and one important signal that they send is angiopoietin one receptor. Type two is a tyrosine kinase receptor, which is kind of a secondary vascular maturation signal that comes after VEGF, where VEGF could induce neovascularization or angiogenesis, and ang1 through the type two receptor induces vascular maturation. It prevents the vessels from leaking and it helps them become more stable, and that's in part through this vascular endothelial calcium adhesion molecule, VECAD here, and this is about the only thing I want you to remember from the talk is angiopoietin one signaling through the type two receptor, the signaling molecules we can leave for another day. In a hyperglycemic state, you lose pericytes and their trophic signaling support. Angiopoietin one levels decrease, there's decreased signaling through the type two receptor, therefore those adherence molecules, VECAD herons, they become internalized and you have increased hyperpermeability. Additionally, diabetes leads to an inflammatory state in the retina where the VEGF levels and TNF alpha levels are increased, leading to adhesion of these, my representation of leukocytes sticking on to integrins and ICAM ones and eventually down the road, you get capillary degeneration itself with resulting poor perfusion and increased VEGF secretion. We hypothesized that if we had a model with persistent hyperglycemia and we restored signaling through the type two receptor, we could prevent the pathophysiologic sequelae of diabetic retinopathy. So, how did we study that? Briefly, I'll go over the methods here. We used a mirroring model of diabetic retinopathy, this is the akita mouse, which has a point mutation in the insulin gene, so it's a type one diabetic model, it doesn't secrete insulin, results in hyperglycemia. It mimics the early pathophysiologic presentation of diabetic retinopathy, including capillary loss, increased retinal hyperpermeability, and ganglion cell loss. Comp-ange one is a modified version of angiopoietin one, so the angiopoietin one that I described earlier, secreted by the parasites and acting on the type two receptor is a very long protein, and when it's been tried to use that specific protein in therapeutic applications before, this coil-coil domain results in hyper conglomeration, forming multimmers that fall out of solution and aren't therapeutically reliable. Some collaborators of ours developed this comp-ange one, which stands for cartilage oligometrics protein, basically replacing this portion of the protein which caused clumping and aggregation and falling out of solution with a portion that makes it much more soluble and actually much more potent while leaving the portion, the domain that interacts with the type two receptor. So this is the drug that we were using, and our delivery mechanism of this was to take the code for that protein and put it in a plasmid and use an adeno-associated virus, serotype two. The reason we decided to use this one is that we felt it was very translationally relevant as multiple clinical trials have used AAV2 for retinal purposes with an intravitrile or sub-retinal injection. So the setup of the experiments went something like this. We took mice and we had four groups of mice. We had a non-diabetic mouse and then two controlled diabetic mice treated with either a sham injection or a sham virus that expressed GFP so we could light up the retina and see where it was being expressed and then finally our control group. So these are the four different kind of representations I'll show you here. And the important thing to remember is with these mice we treated them with a single intravitrile injection, not repeat injections, not a systemic injection, but one intravitrile injection of this AAV2 construct and then monitored them over the course of four months. The end of our experiments took place when the mice were six months old and today I'll focus on the inflammatory cycle of leukocyte adhesion and some extravagant gravitation. All right, so in terms of results from this, what we were looking for, I'll focus in on the vascular results and specifically we'll talk about the vascular function in terms of inflammation. So what we're looking at here is an in vitro treatment where we have an endothelial monolayer where we tried to make a blood vessel on a dish basically. And here we've got a bird's eye view of some endothelial cells back down here and then these little white dots represent leukocytes. Roll the leukocytes under constant pressure and a regular density from the top to the bottom. And we'll take a look at that one more time. And these leukocytes are rolling, they're flowing from the top to the bottom and not much is happening, this is a control situation. When we add one inflammatory side of the endothelial cells, you can see these leukocytes grab a hold, stick and roll and there's much more adhesion going on in this inflammatory state. When we add Compang1 plus that inflammatory cytokine, the adhesion and the rolling is greatly reduced. That's all well and good for our blood vessels that we mock created in a dish. How does this work in vivo? So we took, oh we quantified that and found it significant. So we quantified that with acridine orange leukocyte fluoride. And what we do here is we label leukocytes with acridine orange. Just give us the best view possible. Using a Heidelberg Spectralis we anesthetize the mice and plop them up there and we can watch the leukocytes as they travel up the artery through the capillaries and then back down the vein. So we can track these and measure them and look at the flow time, the response time and how well they're flowing. So here's our control animal and you'll see a wash of white blood cells come up this arterial, cross through this capillary here. We can go back and take a look one more time. And here is an inflammatory response. This is a diabetic mouse. Here's two veins coming back in to the central portion here. And I'll play this video one more time so that we can see leukocytes rolling as they come through here, adhering, much slower transit times. You can see them plugged up at these bifurcations pretty well. Here's our other control mouse. This is a GFP mouse. You can see the GFP staining of the renal ganglion cells and their axons and they had increased rolling as well. And finally, with our Compang1 treated mice, the leukocytes were flowing much more cleanly through the arterials, the capillaries and then back down through the veins. We were able to quantify this showing that Compang1 normalized the velocity as well as decreased the rolling in our diabetic mice. So that's just a snippet. I think my time is up and the take home point would be that Compang1 prevented leukocytes adhesion and an inflammatory response as well as extravasation despite persistent leukemia. Again, lots of people helped out with this project and this is just a small slice of it. I'd like to acknowledge them. And I'll thank you and I'll take any questions at this point. Yeah, Dr. Olson. Fascinating work. And to make it even more germane, sadly with the incidence of diabetic complications and diabetes going like this, and with macular degeneration improvement in the visual quality due to our VEGF inhibitors, there are many people who think if not, everybody agrees with them one or two years of working. Yeah, yeah, that's still an interesting. So all of the big breakthrough on that is critical. I'll just point out that I know that the Hagen group have been looking at their human eyes with diabetes with absolutely normal histories, absolutely normal, relatively normal. There's already profound changes in up-and-down regulation of vascular homeostatic mechanisms that are way out of whack. So this is something that happens early on. Eventually, I think we'll come up with these that are saying, oh, this person is, we don't change this, they are gonna get it. There's definitely a lot of silent things that are happening. We'll start treating it and doing it. It's gonna be very, very easy. Definitely. Thank you. The light's on real fast before we go to our next speaker. And we should have done this a while ago. But, and then it's our, Zach, we're mostly Zach, partly because of the introduction that's been around with us. It's there, it's there. Hey, welcome.