Hello, everyone. On behalf of American Diabetes Association, Eye Health Interest Group leadership team is my great honor to introduce our today's speaker, Dr. Maria Grant. She's Ivor and Alston Callaham and our chair in the Department of Ophthalmology and Visual Sciences at the University of Alabama at Birmingham. Dr. Grant is a leading clinician scientist in the field of diabetic leptopathy and has made several seminal contributions. Honestly, the list of them is so large, if I start talking about everything, we'll have no time for her to give her presentation. So I will try to be very brief and mention just a few of her achievements. Her work has established IGF-1 as an angiogenic retinal factor. Maria has shown homing of hemipatic stem-presenter cells to the newly formed retinal vasculature and has identified molecular defects in diabetic cells resulting in aberrant homing and lack of repair. She has also shown bone marrow neuropathy as a mechanism of stem cell dysfunction in diabetic retinopathy. And now her work has documented normalization of these stem cells using gene therapy. Again, I don't want to forget that Maria is also the first one to show circadian clogged gene dysfunction in diabetic retinopathy. And now she's working on the heart area of role of gut retinal excess in diabetic retinopathy. So the list will go on and on and on, but I'll have to stop it here. And I'll give Dr. Grant an opportunity to talk to us about molecular mechanism of diabetic retinopathy. Before she starts, I would just want to add that please put your questions in the chat box and Dr. Grant will address them after she's done presenting her talk. Thank you, Maria. It's all yours. Okay. Thank you. Thank you, Renu, for the kind introduction. And I'd like to thank the ADA for their inviting me to present today. on the mechanisms of diabetic retinopathy. So I'd like to just give you an overview. I'm going to spend a few minutes talking about clinical and histological features of diabetic retinopathy and then a general overview of some key pathogenic mechanisms that have historically been at the centerpiece of understanding the pathogenesis of this disease. And then in the last half of the presentation, I'm really going to talk about some new mechanisms with regard to the role of cholesterol crystals in diabetic retinopathy, a new look at systemic inflammation, and then a topic that, as Renu mentioned, that the lab has been focusing on recently is the gut retinal axis and how it impacts diabetic retinopathy. So all of you are very familiar with the concept that diabetic retinopathy is a vascular disease. And everyone is aware of how the endothelium has high junctions and that the parasites serve to protect the endothelium and regulate blood flow. As the pathogenesis of the disease continues, though, there is endothelial loss, parasite loss, and basement membrane thickening, and ultimately leading to vasodegeneration. And as you can look in the endoscopic image here on the top, this is very classic that everyone sort of thinks of retinopathy as a vascular disease because the image is so striking, the vascular changes, as well as the concept that there's lipid exudate leaving these vessels. And as you can see, this circinate pattern surrounding the macula right here. And then on the trypsin digest of a human retina, these are both from patients with nonproliferative diabetic retinopathy. You can see the arrows pointing areas of vasodegeneration. It's also striking to see the microaneurysms, or little outpouchings where parasites and there's disruption of the natural configuration of the vessels. And this is from a review by Alan Stitt's group. And as you can see on the top panel on the left, there's electron microscopy showing a nice healthy vessel, the endothelium shown as E, and parasites, P. And there's a nice – whoops, sorry, my mouse is a little bit trigger-happy here. And if you look down in panel C, this is also a trypsin digest, or a flat mount of the retina. And you can see that collagen IV labels the extracellular matrix. You look down at E, there's evidence here of GFAP, which is a marker of the inflammation or not, of the astroglia, of the astrocytes. And typically, this is what is seen in a healthy retina. In contrast, if you look over to B, D, and F, you will see that the basal membrane is thick, and that on isolectin B, which is in panel D, that same area, which you can see very clearly with the collagen staining, is lost, meaning that the endothelial cells from that structure have disappeared. And in panel F, you can see the much more pronounced GFAP staining. This now involves not just the astrocytes on the surface of the retina, but also the mulliglia that go through the entire retina. On the right-hand panels, you can see on the top the microglia. And they have a beautiful ramified morphology. But if you look in the diabetic retina, the morphology is changed. The nucleus is much bigger, and the processes have become blunted. And as you can see in B2, there's evidence of much more of the microglia going throughout the retina, not just in the first or anterior part. Now, work from Dr. Antonetti and Gardner at Michigan, they really began to put an emphasis on the notion that it's not just a microvascular disease, but that the neurons are playing a critical role. And in this beautiful paper that they published in the New England Journal of Medicine in 2012, they really emphasized the idea that there is loss of this communication between the neurons and the vasculature. And this is leading to the pathology, and it actually can precede the typical microvascular pathology that we see on fundoscopic exam, and which has been the focus of much of our understanding of diabetic retinopathy. So if you look in panel B, what can be seen is that the parasites become apoptotic, the endothelial cells become disrupted, there's leakage of blood. And because the parasites lose their healthy nature, there's less PDF, or platelet-derived growth factor, to maintain the vascular structure. And in turn, there's activation of microglia, as we saw on the previous slide, and also that there's the beginning notion of white blood cells actually adhering to the endothelium, and this can cause vasoobstruction. So the loss of PDF can cause breakdown of the barrier. Ultimately, there's also vasodegeneration that can lead to ischemia, that leads to neovascularization. And the group, Tom Gardner's group, really emphasized the idea that there's insulin signaling within the retina, and this loss of insulin receptor signaling can be the result of basically the metabolic disturbances, as well as from damage from the fact that the lack of insulin results in loss of a very important neurotrophic factor, because insulin is much like IGF-1, in that it helps sustain key features of the neurons. So you can lose the neurons, that this loss of synaptic connections between the neural cells and also the muller glia can lead to additional pathology. So just to quickly look at a few of the historically relevant pathways, this is glucose-mediated process where glucose via aldose reductase generates sorbitol, sorbitol via sorbitol dehydrogenase forms fructose, and fructose then can lead to the formation of advanced glycation end products. All of this does indeed lead to increased oxidative stress, and there's been considerable evidence over the years that while aldose reductase inhibitors can prevent neuropathy, they did not prevent retinopathy in dog models of diabetic retinopathy, and that most of the clinical trials that used aldose reductase inhibitors actually produced negative results. So how about advanced glycation end products? Here we can see that endothelium is a victim of much of the pathology. There are receptors for these advanced glycation end products on the surface of endothelium, and when these, what are called RAGE, or receptor for AGEs, are activated, they get internalized, and they can actually result in the transcription, increased production of key transcription factors that can turn, such as NF-kappa B, can activate the production of key immune regulators, such as ICAM, ECAM, E-selectin, BCAM, and BGF, as well as inflammatory cytokines. In addition, RAGE is known, or activation of RAGE is known to reduce endothelial nitric oxide activity and result in less nitric oxide, and therefore, less vascular perfusion. So AGEs, while they're found in increased amounts, there's been different types of inhibitors that have been successful in partially preventing diabetic retinopathy. However, when there was a large, randomized, double-blinded, placebo-controlled, multi-center trial in type 1 patients with nephropathy, the AGE inhibitor slowed down the progression of both nephropathy and retinopathy, suggesting that this is still a very viable mechanism for improving this disease. Now, hyperglycemia can induce oxidative stress. That's a mechanism that is mediated in part by the AGEs that I just talked about. But also, NADPH oxidase, or what we call NOX, NOX 1, 4, and 5, are present on endothelium. They produce more reactive oxygen species that can contribute to tight junction pathology. It can result in retinal vasculopathy, increased VEGF, and blood retinal barrier disruption, and vascular leakage, ultimately leading to neovascularization. So in this kind of busy slide, I just want to recap a few of the things that I talked about. In the top part of this, you can see, I'm not sure how well my pointer is showing, but all the classic biochemical mechanisms that are glucose-mediated, such as the polyol pathway, hexosamine pathway, PQC, or protein kinase C activation, and the AGEs that I mentioned, all lead to oxidative stress. Oxidative stress can have, in turn, a wide spectrum of pathology. If you look at the section here on the left, the emphasis on neuroglial pathology is that, once again, introducing this idea that even before microvascular pathology occurs, the neurons in the retina, the ganglion cells, the bipolar cells, the photoreceptor cells, are experiencing pathology. And ultimately, there's loss of their function. And these cells have a very important role in neurovascular coupling. So that pathology is ongoing. There's also, central to what I'm going to be talking about, is the role of inflammation. And even though I mentioned how the AGEs can activate certain inflammatory cytokine production through NF-kappa B, there's multiple mechanisms. The microglial cells themselves can cause dramatic release of inflammatory cytokines. And then, as we mentioned earlier, leukostasis has an important role in preventing perfusion. Now, all of this, lack of perfusion, ultimately leading to increased VEGF, or vascular endothelial growth factor, which has been at the centerstone of treatments now, because we've been using different strategies to reduce VEGF, using either small molecules or antibodies that can reduce its expression, given intravitrially. And it has a very profound beneficial effect on the treatment of diabetic retinopathy, as well as diabetic macular edema. So, how can we look at retinal damage? Let's take a step back clinically. And there's good evidence that hyperglycemia, through the DCCT and EDIC studies, show that this is really important in the pathogenesis. And all the mechanisms that I mentioned earlier are driven by high glucose. However, dyslipidemia also has had a central role. And the DCCT and the ACCORD study have placed an emphasis on this. So, let's talk about dyslipidemia for a minute. Lipids represent a large percentage of the weight of the mammalian retina. And they're very unique, and they play a critical role in the function. But when we're looking at these big clinical trials, what have we learned from them? Well, for one thing, the severity of retinopathy in both the DCCT and EDIC cohort was positively associated with triglyceride levels and negatively associated with HDL, suggested that perhaps serum markers such as the lipids could be diagnostic. The ACCORD study demonstrated that if you paraffin fibrate with simvastatin for intensive dyslipidemia therapy, there's a reduction in progression of DR over the four years of the study. Now, in turn, there's other studies such as the FIELD study and the ACCORD-I study where the beneficial effects of fin fibrate were independent of changes in serum lipid levels. And also, the WEST-DAR study showed no association between cholesterol, HDL, and DR. So, when we're looking at these systemic lipids, are we really looking at the right lipids or are we looking at the wrong ones? And so, I'd like to bring a little bit of attention to the work that we're doing in collaboration with Irina Piklyova and Julia Busek's lab where we're looking at retinal cholesterol. And in retinal pathology, as you can imagine, because there's high serum cholesterol, there's an influx of cholesterol into the retina. And this can lead to pathology. In turn, because of this influx, there's disruption of the reverse cholesterol transporters such as ABCA1 and ABCG1. And thus, there's less cholesterol getting out of the retina. And as you can see here in the panels on the right, that if you look at the amount of lipid in the diabetic versus non-diabetic control, there's a dramatic increase in the amount of cholesterol. So, what happens here? When we try to use therapies to restore homeostasis by either activating LXR or using agents such as alpha cyclodextrin, we can remove cholesterol, pathological cholesterol. Now, in the retina, there's also changes where if they're not removed, there can be an increase of cholesterol. And this accumulation of cholesterol can lead to crystal formation. So, let's go back for one step. Where is the cholesterol coming from? It can be coming through the coracoplasm. It's delivered to retinal cells like the photoreceptors for their use. So, this is part of normal homeostasis. And that there's an important role in the, as I mentioned earlier, in LXR or the varicose receptor, mediating the reverse cholesterol transport of this cholesterol from the retina when it gets to be too high out into the back into the circulation. So, in normal mice, there's more biosynthesis of cholesterol than there is uptake from the serum. Now, in contrast, in the diabetic retina, things change. There's a disruption of the blood-retinal barrier, both at the leakage in the anterior retinal circulation as well as in the RP, and there's more cholesterol coming in. There's the amount of cholesterol that's coming in actually inhibits or blocks the natural generation of these reverse cholesterol transports. And these cholesterol crystals can form, and they can promote inflammation. And so, if you look at the six-month retina here at the bottom, that you can see that in the diabetic animal, and we suspect that this is the case in the human, that there's an increase in serum cholesterol uptake and reduced biosynthesis. And this is also shown here on the other panels. So, is there some evidence that there can be abnormal cholesterol crystal formation in the retina? Well, there's beautiful work from Christine Curcio's group and others showing that in AMD, you get this typical onion sign, and you can see it in panel H, where these cholesterol crystals can actually, you can kind of sort of imagine seeing these long-shaped crystals here in the bottom. And this is seen in other diseases, and this onion sign represents really sub-RP, or retinal pigment epithelial cholesterol crystal precipitation, and intra-retinal, these hyper-reflective foci that are attributed to the retinal pigment epithelial cells actually being filled with lipid, as well as lipid-filled monocytes forming these onion sign. So, the onion sign is also seen in aspects of the inner retina of the diabetic, not to the same degree, but there's been evidence of this sort of hyper-reflective areas, and they also reflect changes that are compatible in patients that have diabetic macular edema. So, if you look at cholesterol crystals, we actually did this, and Julia Busek has actually spearheaded all of this work, you can look at these crystals, and actually, you can see them, they're artificially colorized in this panel, but you can see they're sharp, they're also some blunt end, but they're clearly present in the retina. And if you look at this panel, this is beautiful work that's done by her graduate student, Tim O'Doringer, who basically was able to show in the retina that these crystals are present, and you can see here in red that there's a microglia, that's shown here, this is IBA positive cell, that is actually wrapped around this cholesterol crystal, trying to dissolve it, to destroy it, to remove it as part of its function. But these crystals can remain, and they do cause activation of complement, and activation of complement in turn leads to the inflammasome being activated, resulting in caspase-1 formation and cell death. And we feel strongly, and this is work that Sarah, I'm sorry, Sandra Hammer and Tim Doruler in Julia's lab did, and this is under, it's actually in press in Diabetologia, showing this important role of complement activation and inflammasome activation in response to cholesterol crystals, promoting development of DR or diabetic retinopathy. So let's move on to inflammation. Inflammation has a very important role in retinal pathology, and there's activation, as we've seen, of resident microglia. There's myeloid monocytic cells that come in from the bone marrow that contribute to the pathology. They then can cause activation of endothelial cells as well as activation of the microglia, and this whole series of pro-inflammatory cytokines that are generated, and also adhesion molecule expression, which in turn sort of has a feed forward where more endothelial cells express these selectins, VCAM, and there's higher attachment of leukocytes that are circulating. So why is this important? Well, and how does this all occur? Well, I'm going to spend the last part of this presentation talking to you about the importance of the bone marrow and how the bone marrow and the gut communicate and how the bone marrow gets its signals from the gut. And why is this? Well, the bone marrow cells, these inflammatory cells, have what's called TLRs on their surface. They're toll-like receptors, and they are actually responsive to systemic levels of microbial products. And when there is systemic administration of a bacterial product, such as LPS, it can actually activate the bone marrow, causing self-renewal of the stem cells, their mobilization, their proliferation. And this supports the idea that these products leave the gut and get into the bone marrow. So they leave the gut, get into the systemic circulation, and reach the bone marrow. Additional evidence exists that when antibiotics are used in mice or in humans, it actually has a dramatic effect on myelopoiesis. And if you have germ-free mice, these are mice that have absolutely no bacteria, they have defective myelopoiesis. So let me show you a little bit of the data. This is not work done in my lab, but I think it really emphasizes the importance of how the gut communicates with the bone marrow. And here in the panels on the left, you can see there's germ-free mice, mice that receive three species of bacteria, mice that receive 20 to 100 species, those are the LCMs, and then our typical SPF mice that have over 100 species of bacteria. And what you can see along the y-axis is that as the gut gets more complex, that is, as there's more species of bacteria, there's actually more myeloid cells in the circulation. This is also shown in beautiful work from Joshi et al, showing that they did a very similar experiment. They gave either PBS or LPS, either at 2X or 4X, and in animals that had their cells labeled with BRDU. So BRDU basically is diluted out as stem cells proliferate more. So more proliferation means lower BRDU. And this suggests that in all the stem cell populations studied here, the HSCs, the myeloid progenitor cells, and the LSKs and the LKs, these are lineage-negative positive cells, all these different population of stem cells, the ones that, the animals that receive the 4X LPS had much less BRDU, which means much more proliferation, showing that these LPS or these gut microbial products have a dramatic effect on the bone marrow. So we did a study, and this is work done by my graduate student, Jason Floyd, where he took a large cohort of type 2 diabetics and compared them to age-matched controls. And you can see here that the neutrophil content is much higher in a number of neutrophils in the type 2s. And if you break this down into the different cohorts with regard to diabetic retinopathy, the patients that have non-proliferative diabetic retinopathy and diabetic macular edema together have the highest levels of neutrophils as well as the highest neutrophil-to-lymphocyte ratio. And these are suggestive of strong meta-inflammation in this cohort of diabetic patients. Now, if you look at a plasma marker, this is peptidoglycan, which is a gut microbial antigen that is generated by the bacteria. You can use it as a biomarker of gut permeability. And you can see here in this same cohort of patients that peptidoglycan is highest in the diabetic patients that have PBR. Now, this suggests that these patients that have pathology also have gut leakage. There was a nice review paper that came out last year by Thakur et al. And they put together this whole notion of how does gut dysbiosis impact the eye? And in the last part of this talk, I'm just going to emphasize a few of the sections related to this. We talked about LPS, endotoxemia in the leaky gut causing activation of TLRs in the white blood cells. I'm going to show you some data where we have activation in endothelial cells. Also, the gut is a source of short-chain fatty acids that can control inflammation and also impact insulin sensitivity. So the bacteria themselves, if we lose butyrate-producing bacteria, we lose the ability of these bacteria to cause or improve insulin sensitivity. So there's reduced insulin sensitivity. I'm going to show you data about neuroprotective secondary bile acids and their role in the gut as well as the role of ACE2 deficiency in the pathogenesis of diabetic retinopathy. So there's, at many different levels, the gut can regulate what's going on in the retina. The microbes themselves can activate T cells. They can cause changes in the Th17 cells. These cells are typically inside the gut, but they can be activated to mobilize into the circulation. And data that we presented previously at Arvo were able to show that these cells can go to the retina, where Th17 cells can actually orchestrate the behavior of the innate and adaptive immune cells within the retina. So let's go back a little bit to, in a little more depth, understanding what is going on. So there are certain bacteria that are known to be downregulated and these are considered important in that these bacteria tend to reduce pro-inflammatory cytokines. And these are Lactobacillus, Bacteroidetes, Rosaviria, and Fecali bacterium. There are also other bacteria that are good bacteria that when they are upregulated, such as Acromantia and Bacteroidetes, can actually increase tight junction expression in the intestinal epithelium, making the barrier more robust. There's considerable studies throughout the world showing that if you do fecal transplantation in a type two patient, you improve insulin resistance. In addition, there's also work by Doss et al that showed that there are a direct link in that there are fewer anti-inflammatory bacteria and there are increase, well, one particular pro-inflammatory bacteria, Shigella, in diabetic patients with retinopathy. This study was done in South India, so there may be issues with regard to certain types of diet and maybe this isn't necessarily applicable throughout the world. And certainly there's a need for more studies such as what was done there in a different cohort of individuals. Now, there's also evidence in the United States that the ratio of Bacteroidetes to Firmicutes can predict the development of vision-threatening diabetic retinopathy. And work I'm gonna tell you about in a minute, but I wanted to bring out this point that this bile acid that is generated in the gut by certain bacterias can actually help promote repair of the vasculature by affecting the bone marrow to produce more of these vascular reparative cells. So TUDCA is very, very important. And also, I think to keep in mind for everyone in this interest group, to keep in mind that there are many factors that influence the microbiome and the gut. The antibiotic usage in patients, certain anti-diabetic drugs, the classic is metformin. Metformin has dramatic effects on the microbiome in a very, very favorable way by increasing the bacteria that increase your chain fatty acids. Of course, your diet is important. Your microbiome changes depending on the season. There's local geographic issues, ethnicity, and age of individuals. So it's a very, very difficult to necessarily make widespread generalizations about which bacteria are beneficial or deleterious. And so I'd just like you to keep that in mind. Okay, so we did a study back several years ago where we did what was called intermittent fasting, and you're all familiar with it. And this was in a model of type 2 diabetes where the animals were actually restricted. So one day they were able to eat their typical diet. The next day they were fasted. And in this cohort, I'd like to show you that when we looked at their retinas, now the ad-lib cohort were those animals that had free access to food throughout the time. And you can see that here in the blue bar, there's an increase in the amount of A-cellular capillaries. This is a hallmark of diabetic retinopathy. It's a hallmark showing vasodegeneration. And yet the two cohorts, this is the cohort on the intermittent fasting, had a reduced level of A-cellular capillaries when you compared it to the diabetic animal on the ad-lib. And there was no real difference in the control cohort, and we wouldn't expect that. What was interesting too is that in the panel here, F, these IBA cells, that is, these are actually within the retina. We looked at the amount of inflammation. These are the activated microglia that I showed you in the beginning of this presentation. There are higher numbers in the diabetic animals that receive the ad-lib regimen. Oops, I'm sorry. Whenever I use my mouse, it kind of goes, okay. And you can see in purple that the animals, these are the animals on the intermittent fasting. So these were sacrificed either in the feeding phase or the fasting phase, but they were having this intermittent fasting for seven months. So it was a prolonged protocol. They had reduced levels of IBA within their retinas. And CD45 is another, it's kind of a very nonspecific, but it does indicate any hematopoietic cell. And you can see here in the blue that this is the ad-lib cohort, had more pro-inflammatory, or at least CD45 cells coming into the retina. And those on intermittent fasting had reduced levels. So we then looked, knowing that fasting could have a dramatic effect on the microbiome, we looked at the microbiome in each of these cohorts. And you can see it's really clear here in the lower part. I just like you to focus on the bottom three pie graphs. And let me not jump ahead here. Okay. If you look at there, you can see that in green, this is firmicutes. And in the diabetic on the ad-lib has a much lower or smaller level of firmicutes and a much higher amount of bacteroidetes shown in red. And then as you go through and you look at the intermittent fasting cohort, the amount of green to red ratio changes dramatically. So this ratio goes, the F to B ratio goes from 0.25 on the bottom here to 1.72 or 2.33, suggesting that indeed we can dramatically impact the gut microbiome in this cohort of animals by this protocol, by this fasting protocol. And what we found, we did a lot of work, but we did, as you can imagine, we studied the different bacteria. And when we looked at what bacteria changed, what was really interesting was that the bacteria that actually modified the secondary bile acids were increased with the intermittent fasting protocol. And one particular bile acid, TUDCA or tyrosodioxycholate, went from being very low in the ad-lib group here in blue, to much higher in the group that were on the intermittent fasting. Now, why is this important? Well, TUDCA has been known for over 4,000 years to be neuroprotective. It was used for what was considered to treat an Alzheimer type condition in many decades before. And it's a very, very high in the bile acids of black bear. So it has been a medicinal for many, many years. So TUDCA, and many people much more recently have looked at the beneficial effects of TUDCA in the retina. And as I mentioned earlier, TUDCA has a very positive effect on the bone marrow by increasing the number of vascular reparative cells. So TUDCA increased with IF. And so we went on to study what is the mechanism? Well, this is a little cartoon in the interest of time to tell you what happened. TUDCA increases, TUDCA goes into the systemic circulation and it can activate receptors. These are TGR5 on the retina, on the ganglion cells. And when TUDCA activates these receptors, it actually decreases inflammation, specifically pro-inflammatory cytokines such as TNF. So as you know, in diabetes, TNF is increased as are other cytokines. And then by blocking their production with TUDCA, we can reduce inflammation. Thus, largely by restructuring the microbiome in the intermittent fasting cohort, we generated these secondary bile acids such as TUDCA that were protective to the retina. So then the very last part of this presentation is on the renin-angiotensin system. And this is an area that my lab has been very interested in for quite a long time. And at the center of all this is ACE2, or angiotensin converting enzyme two. And as you all know, angiotensin in health, which is a circulating protein, is degraded by ACE2 to form this peptide called ANG1-7. And it activates the mass receptor to have a very beneficial effects, protective effects. It's anti-fibrotic, it's vasodilatory, it's also an antioxidant. In diabetes, ACE2 levels are low. And what happens is there's too much activation of AT1 or the angiotensin receptor one. And so there's a shift or an imbalance in this axis towards the vasodilatorious axis where there's vasoconstriction, cell proliferation, hypertrophy, and fibrosis. And what you may not realize, and I think this is in the context of COVID and everything that we experienced as a global experience, it's quite, makes a lot of sense why the virus had so many GI effects because ACE2 is expressed almost 200-fold higher in the small intestine than anywhere else in the body. And so we spent a lot of time studying ACE2 in the small intestine to understand what indeed it did. Well, before we go on today, I just want to tell a little bit of data with regard to ACE2 and the RAS axis. If you look at, this is work that's now published in Cirque Research. If you look at a cohort of patients that have, are controls, diabetics without retinopathy, diabetics with NPR, NPDR, and PDR, angiotensin II, which is the precursor for the beneficial ANG1-7, but in and of itself, as you all know, ANG2 is vasoconstrictive, causes problem through the AT1 receptor, and causes many blood pressure, and fibrosis, and vasoconstriction, et cetera. It's higher in PDR patients. And when we measure in the same identical patients, we measure the gut markers, peptidoglycan, it's higher. Whoops, it's higher. PDR is higher peptidoglycan. Fatty acid binding protein is another biomarker of what's going on in the gut. It's higher in these patients with PDR and higher in the non-PDR as well compared to those with no retinopathy. And then LPB is similar to LPS. It's just the binding protein of LPS. It's more stable in the plasma. And you can see that the patients that have retinopathy have higher levels of LPB. And those that have PDR have the highest compared to the other cohorts. So we wanted to try to understand, well, what is this peptidoglycan doing? And how is it having an effect? And what we were able to show is that this peptidoglycan actually activates a non-canonical TLR or toll-like receptor 2 on endothelial cells. And because I apologize, my mouse is not working. I'm gonna try to use my finger here. But basically when we take these cells and we culture them and we look at them, whoops, under, I'm gonna not use it. I'm gonna just focus on the bottom here, sorry. When we take them in culture and we expose them, these are human retinal endothelial cells to peptidoglycan 100 nanograms per mil. You can see that there's disruption of the tight junctions and there's actually this loss of the nice beautiful green staining, which is P1-20-catenin and blue is the nuclei of these cells. So what happens is, and if you just look at the little cartoon at the bottom, it sort of says everything. What happens is TLR2, when it's activated by these gut peptides, such as peptidoglycan, activates a non-canonical pathway in myoD88-Arno and this GTPA is called ARF6. And it results in the internalization of E-cadherin. It's a process of endocytosis. So that what happens with endocytosis of V-cadherin, you actually get degradation of P1-20 or this beautiful or very, very important surface protein that helps maintain tight junctions. So there's a direct effect of these gut microbial peptides through this TLR2 pathway to cause vascular leakage. This is what we saw in vitro. And when we did additional studies, now this was in type one animals. We were able to show that what happened was, in addition to having abnormalities with regard to the immune system as far as different phenotypes, the myeloid angiogenic cells or these very vasoreparative cells were markedly reduced in diabetes. And this is something we've known for many, many years. But what we didn't know is the impact of these cells on the gut. And so in these animals, what we did, we took the diabetic animals and we actually looked at levels of ACE2 and the animals that were diabetic had the lowest levels of ACE2. And we then were able to restore the barrier in the gut by giving back these myeloid angiogenic cells. And while what we did is by giving back these myeloid angiogenic cells, we were able to eliminate the leakage from the gut. And there was less of these gut microbial antigens getting into the circulation to activate these TLRs on the surface of the endothelium. So there was a direct connection in between the diabetic animals basically using the cell therapy to replace the levels of these cells in the circulation, and these cells are vascular reparative, but instead of repairing perhaps the retina directly, we were focusing on repairing the gut, and we were able to show that the gut barrier with impact was restored and there was less gut microbial antigens in the circulation and less activation and less permeability in the endothelium of the retina. So, in the last, very last slide, I want to talk about ACE2 going back to it. The gut is a source of angiotensin 2, just like the circulation, and interestingly, bacteria can convert, generate angiotensin 2, and thus if there is ACE2 around and soluble ACE2, which as I mentioned before, the gut is the highest source of ACE2 throughout the body, and so here, let me just point here on the little cartoon in the interest of time, you look here, this ACE2 is actually on the surface of the epithelium. ACE2 can be cleaved. It can convert angiotensin 2 to angio1,7. Angio1,7 then can act on the mass receptor, and what we were able to show is when you activate this mass receptor, you actually reduce the expression of SGLT1 and GLUT2. These are both the critical transporters of glucose from the gut to the circulation, and these are regulated by a member of the angiotensin system, and this is published in the paper that just came out in CERC research, and importantly, it's something you may not be aware of, but I know everyone here is very interested in the role of incretins. Well, incretins have their systemic effect to reduce hyperglycemia, but what you may not appreciate is the fact that ACE2 dimerizes with a tryptophan transporter, and these actually regulate the expression of GLP1 and GIP, and what happens in diabetes is that you get disruption of this barrier. You lose the barrier, and you get leakage of these into the systemic circulation, as I've talked about, and that what you can do, and this is something that we have done before, is that you can actually give a probiotic, and this is work that we did with Kyu-Hang Lee from University of Florida. You can make a probiotic that produces either ANG1-7 or soluble ACE2, and you can increase the amount of ACE2 being produced, and it can, of course, have a good effect on the gut for absorption, but it also has a beneficial effect, and in the paper, the CERC research paper, we're able to show that GLP1 and GIP, not only are they incretins that have effects on the levels of glucose by modulating the beta cells, but they have direct effects on blocking inflammation within the gut, and so by using probiotics such as the one that we have, the ACE2-producing probiotic, we can promote a healthier gut, reducing inflammation. We can preserve the natural function of ACE2. We can also activate the mass receptor to reduce the amount of hyperglycemia, and so just to wrap things up here, I would like to just give you an overview. I think we talked about the fact that the prevalence of diabetic retinopathy and DR-related blindness is increasing, while other types of blindness are actually decreasing, and there are multiple mechanisms that are responsible for the pathogenesis of DR. We talked about hyperglycemia leading to a reactive oxygen species, inflammation, leukostasis, parasite loss, hyperperfusion, and ischemia. We also talked about neurogliopathology, loss of neurons, and loss of neurovascular coupling. We also learned the idea that this neurogliopathology is in part responsible for VEGF production and hyperpermeability, and this is the mainstay of management now with targeting VEGF, and that there's also loss of vasoreparative mechanisms, leading to vasodegeneration that contributes to ischemia. Those are multiple well-established mechanisms. I'd like to just emphasize once again that cholesterol crystals are new now, even though this idea has been around in the field of atherosclerosis, it's new to the retina and how we observe complement activation and flammasome activation. I'd like you to consider how important the gut is in not only managing patients' general health and metainflammation, but that it can have an important role in diabetic complications, such as diabetic retinopathy, and that the future actually holds the possibility of using probiotics, not just to replace bacteria, but to have the bacteria produce important enzymes that are needed for physiologic function that are lost in diabetes, such as ACE2. I'd like to just thank my lab. The work that I presented was work that Ram Prasad did in the lab, as well as two of the graduate students that have recently left and graduated, Jason Floyd and Bright, Bede Dinko. I'd like to thank my collaborators, Julia Busek and Irina Piklyova, the work that I presented on the cholesterol crystals, and Kyu Hong Lee, who has been a great collaborator for many years in generating the probiotics that we studied. Thank you for your attention. Thank you very much, Julia. Maria, that was a great presentation. You touched on so many different aspects, and it's really very, very interesting. You talked about standard mechanisms, and then went to gut-retina axis, stem cells, intermittent fasting, cholesterol crystal, and everything. You tell them together in the end, which is so beautiful. It's really very, very interesting. If you have any questions, please put in the chat box, and I will ask Dr. Maria. I will start with a very simple, maybe a naive question. You said by intermittent fasting, you can increase Tadka. For people like us who like to eat, can't do intermittent fasting, how can you increase Tadka in those kind of patients? That's a great question. Women only need to fast 14 hours a day, whereas men, 16 hours. The intermittent fasting, which I'm sure many people are familiar with, the protocol is such that you don't have to do what we did in the mice, that we get the same sort of benefits by just restricting during each day our feeding time. I think it's not that unreasonable to eat from eight to six. Basically, that's a pretty reasonable time to be able to eat, so not eating too late. Just for women, at least 14 hours of fasting gives you the same beneficial metabolic benefits that is much more rigorous in animals. Also, unfortunately, men have 16 hours, but if you fast for 16 hours and then the remaining time, you're allowed to eat. You pretty much can eat whatever you want. Those studies have shown benefit with regard to changes in the microbiome in humans as well. It's just a little clearer in animals. We have a couple of questions. We have a question from Susanna Park. How long was the intermittent fasting in mice before you noted changes in gut bacteria? Yeah, it's a good question. The study was a prevention study, so we did it seven months. However, we have done time-restricted feeding in animals for shorter periods of time and been able to reduce inflammation. So, we sacrificed the animals at seven months because, classically, in the DBDB, you have to have at least six months to have the endpoint of acetyl capillaries. That was a hard endpoint that we used, so we want to make sure that the animals that were diabetic had changes in the retina and that with the intermittent fasting protocol, we could prevent them. That's why we had that particular time point. We have a question from Mike Dennis. I was surprised to see that the increase in TATCA and farm bacteria ratio in DBD mice within intermittent fasting was absent in TBM control. Does that suggest the benefits are not driven by intermittent fasting, per se, but rather preventing hyperphagia? Yeah, I think that's a very good question. We did not look at that, per se. We didn't really look at the total food intake after. I can't really answer that. In so many things that we've done with these animals, it seems like in the controls, if everything is under homeostasis, then you don't need an intervention. We have seen that the probiotics even in the cohort and controls aren't necessarily beneficial. They are beneficial in the diabetic because they replace something that is missing, but in the normal healthy animal, there's good levels of ACE2. I think it applies to that same thing, that intermittent fasting didn't necessarily have a benefit in the controls, but it did in the experimental cohort. I think the hyperphagia is certainly a reasonable thing to consider, but we can't confirm that. It's a great question, but I don't have an answer. Okay, then there's a question from Siddharth Suneel Kumar. Are there specific TLRs on myeloid cells that increase in your diabetic patient samples? I'm sorry, can you say that again? Specific TLRs on myeloid cells that increase in the diabetic patient samples. Okay. In the myeloid cells, we have looked at other things in the TLRs. We do look at TLR2, TLR4. We don't see necessarily a change in the expression, but we don't look at the activation because we do this by flow. I think it's a great question. We've looked at CD38, which is also important for regulating so many things, and that is definitely changes. If we can inhibit CD38, we have a different profile in the human samples, but the TLRs are mostly the activation state rather than surface expression. However, in endothelial cells, I didn't have a chance to present it, but if you take and expose endothelial cells to higher levels of peptidoglycan, they actually increase their expression of TLRs. In the endothelium, it definitely responds by increasing the TLRs. In the white blood cells, in the humans, the question is an excellent question, but I can't answer because we sample a patient at one time point typically, and so we don't do repeated measures on that same patient, so I can't answer it, but it's a great question. This question, again, is from Lee. Is retina's endothelial cell more susceptible to oxidative stress than other endothelial cells? If it is, why? Oh, you know, it's a great question. I can't answer it. I don't know that the retina, I would say the retina is not any more susceptible. I think it's, I think we study the retina. I think it's, it's probably, it, I don't know how the retinal endothelial activation state of the retinal endothelial cells compared to the heart microvasculature or to the endothelium of the vasoderm. I think because we have a tight, you know, that or the brain, but because of the presence of the barrier, the importance of the blood-retinal barrier and the blood-brain barrier, they're very distinct, and if anything, I would say they're less sensitive. They're more resilient because they need to be. They have to be to protect the retina and to protect the brain. So, you know, I do think they're special. I think they're protected, not so much more sensitive. I mean, I don't know the answer, but that's how I would interpret it. Okay. Another question. Can you elaborate on the mechanism of activation of the NLRP3 inflammasomes? Yes. Well, can I elaborate? I can elaborate that in the context of the cholesterol crystals, we think it's going through complement and that there's the activation, that the complement is sort of like the initiator of that inflammasome activation. Typically, in our experience, and this might go back to the previous question, that if you look at inflammasome activation in white blood cells, which can very much be susceptible to cholesterol crystals, they actually may be more susceptible than the endothelium. The endothelium may be more protected, and that is just some work that we published about a couple of years ago, and it goes back to the fact that, you know, the same triggers can have distinct effects. The white blood cells respond much more aggressively compared to the endothelial cells. The question, is hyperglycemia change in gut microbiota composition is potentially harmful due to the change in gut ecosystem? I'm sorry, the eco, can you repeat that again? This change, hyperglycemia may induce change in gut microbiota. Is it harmful due to change in the gut ecosystem? So, relating gut microbiota with gut ecosystem. Oh, to the gut ecosystem. Yes, I mean, I think I didn't have a lot of time to explain that all, but the gut ecosystem is much more than just hyperglycemia, and, you know, there are, the whole, the concept of dysbiosis is much more than just high glucose, even though the high glucose is one of the major mediators. It's, you know, the bacteria, it's just so complicated. I, you know, I don't think I can do justice to answering that question, but it's just, you know, incredibly complicated. And yes, the amount of glucose you absorb affects, obviously, what goes into the systemic circulation. I can say one thing that I think is interesting is that the amount of variation in the glucose. So, like the glycemic excursions, the high glycemic excursions tend to have more damage to the gut epithelium, epithelium, and endothelium than maintaining just hyperglycemia. So, you know, I think that's where we know that these glycemic excursions are worse for patients. We know that the glycemic excursions have an impact on a ROS production, have an impact on VEGF production. That's been done by very many different investigators, but the concept of excursions being more, so the glycemic excursions and what you eat to cause that, you know, high glycemic glucose. So, like if you eat a diet that is more, minimizes the glycemic excursions, so a diet that tends to be more, you know, with brown rice, whole wheat bread, et cetera, versus the white bread diet, you're probably having a more beneficial effect on your epithelium because you're not having such high changes in glucose, which definitely have direct effects on epithelium and endothelium. I don't know if I answered that. Sure. Two more questions I'm going to have it here. Are the probiotic ACE2 bacteria effect on the retina mediated by gut permeability changes, bone marrow changes, or direct effect in the retina? This is from Julia Busey. Yeah, no, that's a great question. My feeling based on our data on the mass receptor being activated directly in the gut, I think if you directly target the gut and reduce gut permeability, then you're going to have less of these gut microbial antigens getting down into the bone marrow to cause this myelopoiesis, this excess of white blood cell production. And I think you'll have less of the, when you have less of the gut microbial antigens and you're going to have less activation of TLR2 in the retina. So for sure, by targeting the gut, which we know it does have a definitely protective by restoring permeability, basically barrier characteristics, both at the epithelium and endothelium level, and by improving the incretin production, all those things that it does, we're having direct gut effects. That being said, the bacteria do indeed produce enough of the ACE2, the soluble ACE2, and the particular soluble ACE2 that we used also gets into the systemic circulation. And I didn't have time to talk about that, but the levels are increased. And so yes, indeed, you could have more, you could have direct beneficial effects from the soluble ACE2 that gets into the systemic circulation. And that could get across the blood retinal barrier because of the defects and improve intrinsically ACE2 levels in the retina, which we've shown and others can be down in diabetic retinopathy and you can restore them. Okay. The last question, that's from Jenna Stanley, for therapeutic development, is there a specific probiotic panel that should be employed? Oh, I think it's a great question. And I think, yes, there are, if you, there's work done at University of South Florida in Tampa, where they're personalizing your gut microbiome. And so it's really personalizing, they're basically studying your microbiome and determining what are the types of foods that you should eat. And it's getting to the point that, yes, they can identify deficiencies that perhaps you need more acromancia, perhaps you need more of the rosaberry. There are a whole series of bacteria that are beneficial that are deficient, but they may not be deficient every diabetic. So I think this may be a case where personalized medicine has a very new and important role. And the point of the work that's done at University of South Florida is really to try to optimize diet so that people lose weight or eating the best diet. But it's also providing us really important information about the differences in everyone. There's so many unique differences that you can't really make a generalization. Like I don't think we can go to Whole Foods or to the pharmacy and get a probiotic, you know, like one probiotic solution will treat all of us in a beneficial way. So I think that, yeah, I think it's a great question. I think the future is going to hold a lot of that's a place where personalized medicine, I think it's going to really blossom. Thank you, Maria, for such an excellent presentation. And thank you, everyone, for joining. And everybody said excellent talk, great talk, which I didn't repeat. So it was really a wonderful talk and learned a lot from your talk. Thank you so much. And thanks, everyone. Thank you. Thank you, everyone. Bye.

diabetic retinopathy

glucose-mediated processes

oxidative stress

inflammation

neurogliopathy

cholesterol crystals

complement activation

gut microbiome

probiotics

personalized medicine