During this live webinar, Dr. Kazlauskas discusses how anti-VEGF agents provide benefit by temporarily reducing the level of VEGF and overcoming vascular permeability as well as the variable patient response to VEGF.

Lecturer: Dr. Andrius Kazlauskas, University of Illinois Eye & Ear Infirmary, Chicago, USA


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DR KAZLAUSKAS: So this lecture is going to be about VEGF and anti-VEGF and eye diseases. I’m a professor at the University of Illinois, in Chicago, in the department of ophthalmology and physical sciences. I have no financial disclosures to report, and the summary of this presentation is on the following slide. Here are the points: That anti-VEGFs, which I will be calling aVEGFs, provide benefit by temporarily reducing VEGF. Patients’ response to VEGF is variable. aVEGF’s therapeutic benefit is due to overcoming leakage, especially in diseases such as diabetic macular edema and proliferative diabetic retinopathy, and in the wet or age-related form of macular degeneration, or wet AMD. And we’ll talk about why anti-VEGF is effective, and why it is… The mechanism behind or underlying its effectiveness. We’ll also talk about new ways to overcome leakage in pathological blood vessels, the results of some phase two clinical trials, indicating that activating type II is a way to potentiate the effects of anti-VEGFs. So that’s an overview of the presentation, and now we’ll go through it. So here’s an overview and concepts in angiogenesis. Vascular endothelial cell growth factor, or VEGF, was originally identified as a factor that tumor cells secreted, and caused blood vessels to leak. So it was originally called vascular permeability factor. And a number of years later, a different group identified a factor from tumor cells, based on the factor’s ability to make endothelial cells proliferate. And they not only identified the factor. They also cloned the factor, meaning that they determined the cDNA for this factor, and they called it — because they had purified it based on its ability to make cells proliferate, endothelial cells proliferate — they called it vascular endothelial growth factor, and that’s the name that stuck with this molecule. The picture on the left here indicates that it has a number of jobs in the eye. It helps the retinal vasculature develop, and it also drives pathogenesis of a number of diseases that we’ll be talking about. So what VEGF can do is two different but overlapping things. One of the things that it can do is it can promote the growth of new blood vessels. So this was originally identified in the context of tumor genesis. Tumors secrete many factors, one of which is VEGF. And what VEGF does is it instructs the physiological blood vessels of the host in which the tumor is developing to form new blood vessels, and the tumor needs those new blood vessels for its metabolic demands. Both the uptake of nutrients and the disposition or getting rid of some waste products as well. The other thing it can do, besides making new blood vessels, it can cause blood vessels to leak. As you can see in this diagram, this is an endothelial cell. This is the endothelial cell vessel. This is the junction between two abutting cells. The endothelial cell sits on a basement membrane, which it shares with pericytes, which are mesenchymal cells that line the blood vessels, and then this is the inside of the vessel, the lumen, where the blood is flowing, and what VEGF does is it instructs the endothelial barrier to relax, such that fluid in the vessels can leak into the tissue. And so there are some definitions that are important, that we’ll go over. So I’m gonna talk about permeability and leakage, and what I mean by that is the following: So leakage is different from hemorrhage, which results when the vessels rupture, and that allows the tissue to be exposed to the entire contents of the vasculature. So the blood, the proteins, the cells. Everything that’s in the circulation can spill into the tissue, when it’s a hemorrhage. Leakage is also different from extravasation, which allows immune cells to exit from the circulation. So inflammation is an example of both leakage and extravasation. And so leakage results in accumulation of fluid and proteins from the circulation into the tissue. Okay. I’m no longer able to advance my slides. Let me see. Yes, that works great. I just want to get rid of this other little… Okay, perfect. So we’re gonna talk a lot about the ways that excessive VEGF is treated in eye diseases, and there are four approaches to do that in the clinic that are commonly used. They involve the names indicated on top, and what these agents are is they’re all directed against VEGF. Not VEGF receptors, not signaling events downstream of VEGF receptors, but at the VEGF itself. And they attack the VEGF in different ways. This one was developed first. It’s an aptamer, a synthetic oligonucleotide, which was selected for its ability to recognize one of the forms of VEGF-A. Bevacizumab is a monoclonal antibody, which recognizes all of the forms of VEGF-A. Ranibizumab is a segment of the bevacizumab, and what it does is recognizes the same group of the VEGF family. All of the VEGF-A isoforms. Whereas aflibercept is a trap. It’s the extracellular domain of VEGF receptors. Both VEGF receptor one and two have been fused to make a high affinity trap, which combine all forms of VEGF-A, and also two other VEGF family members. So the two points here are that there are a number of ways to neutralize VEGF ligand that are used clinically. The efficacy of these ligands, when you compare the efficacy of these ligands, although they’re not identical, they have profound similarities between especially these three, and when you ask what is the common target of all three of the effective agents — and the answer is VEGF-A. So the conclusion one can draw, that neutralization of VEGF-A alone is sufficient to the therapeutic benefit of the most effective anti-VEGFs that are used in the clinic today. Okay. And so the way these drugs are administered are intravitreally, meaning that the drug is injected with a needle into the vitreous of the eye. In the case of retinal diseases, where the retinal vasculature is leaking or proliferating, the access of the drug to the target tissue is not very complicated, but in other diseases, such as wet age-related macular degeneration, where the pathological blood vessels are behind the retina, underneath the RPE layer, as you can see from this diagram — so this is a diagram of an eye, obviously. This is the retina in a patient that’s developing wet AMD. Here is the neural retina. These cells are the RPE cells. This is the Bruch’s membrane. It’s a basement membrane upon which the RPE cells sit, and provide the outer retinal barrier to the retina. And you can see that… And then this vasculature beneath the retina is the choroidal vasculature, and you can see the pathological blood vessel is moving from the choroid into the retina, where it will form a vascular bed, a pathological vascular bed, underneath the RPE cells, and above the Bruch’s membrane. So when the drug is injected into the vitreous, as you can see here, it has to get through the retina, through the RPE, in order to access these pathological blood vessels. And when the drug was being developed, it wasn’t obvious that an antibody, which is a large molecule, would be able to penetrate the neural retina. And so experiments to test that are shown in the bottom. Here we see pictures of a rabbit retina that was injected with an antibody. This is the vitreal side of the retina. And after one month, the retina… The animal was sacrificed. The retina was sectioned. Here a section was stained with a reagent that recognizes the injected antibody. And you can see that the signal, which is this white substance here, this signal for the antibody indicates that the antibody was able to move into the vitreous. This is an uninjected eye, serving as a negative control, which makes it obvious where the signal or the antibody that was injected is. So antibody that’s injected into the vitreous is able to get through the retina and access the pathological blood vessels, which are even behind the retina, as is the case in AMD. So this is the way the drugs that are usually used are administered. There are two prominent, dominant features of anti-VEGF. One of them is that it works. And so here is a piece of data from these studies, these anchor studies, that were done in a way that patients were treated every month with either the standard of care at that time, which was photodynamic therapy, and you can see that patients with wet AMD, their vision declined over this time course, when they were treated with the best possible drug at the time. And anti-VEGF was an experimental therapy. And you can see that it not only prevented the decline of vision. It also improved patients’ vision. This outcome that’s being measured is the BCVA, best corrected vision, so it’s how well patients can read an eye chart. And you can see that once they’ve been treated with the drug, their ability to read that eye chart improves, as opposed to declining, which was the standard of care, which was the best achievable outcome with the standard of care at the time. So this demonstrates convincingly that it works well when patients are treated on a monthly treatment protocol. On the right, you can see the results of a different study, in which the patients were treated in a different way. After three monthly injections — so they were injected three times, once per month, and then after that three-month loading period, the patients were… The treatment response was — the vision of the patients was monitored. And you can see that some of the patients, who received no more injections, their vision improved and continued to stay that way, whereas other patients, after a period of good vision, their vision started to decline. Whereas other patients without any further treatment… Their vision started to decline much more rapidly. So the outcome of this study indicated that if patients are treated aggressively, then some of them will have a declining vision. So some patients need monthly treatment, in order to maintain good visual acuity. Other patients don’t need any additional therapy, and you can see that after those three injections, their vision stayed good for a whole year. So in some cases, the anti-VEGF, the three injections of anti-VEGF, results in long-term benefit. And then this patient group shows something in between. So the patient response is variable, and one of the challenges in anti-VEGF therapy these days is to know how your patient is going to respond, and it is not clear at this point what to look for, besides their vision. There’s no way to predict the response of a patient, except for monitoring their vision and knowing when to readminister the drug. Okay, so how does anti-VEGF work? As expected, it should be working by reducing the level of VEGF, because it’s directed against VEGF. When antibodies bind their target, they typically neutralize them, they prevent them from doing their job, which we’ll see in the next couple of slides is binding and activating the VEGF receptor on endothelial cells. So here’s a study that was done that measured the concentration of VEGF in patients with neovascular AMD. It measured the level of VEGF in the aqueous humor, instead of the vitreous, because aqueous humor sampling is less invasive than vitreal sampling. And you can see that, of the patients, the mean level of VEGF was approximately seventy picograms. They were injected with anti-VEGF, ranibizumab in this case, and their level of VEGF declined to undetectable levels, as expected. Now, the half-life of ranibizumab is two and a half days in the vitreous, and when you inject this amount of the drug, then you expect it to last for approximately thirty days. So the blue arrow indicates the theoretical half-life of the drug, and how long the patient’s VEGF level should be suppressed. And you can see that in fact it was suppressed for this amount of time. But the suppression extended for a bit longer, and it took about 50 days until the level of VEGF rebounded to the pretherapy levels. So two points here. The anti-VEGF, it reduces the level of VEGF in the vitreous, or at least in the aqueous humor. We expect that to reflect the vitreal levels. And the duration of action, or the duration of efficacy, is a little bit longer than the half-life of the drug in the vitreous. And that could be because the drug then soaks into the retina, and it takes a little bit longer for the drug to move through the retina and leave the eye, at which point it is not expected to be effective anymore. Now, so this is what the drug does to the level of VEGF. Let’s take a look at what happens to the symptoms, the patient’s symptoms. So this dark black line, on this graph, indicates the percentage of patients who have VEGF suppressed. So after they were treated, all of the patients had VEGF suppressed in their eye, and then over time, what happened is: All of the patients returned back to the pretherapy levels of VEGF. And you can see… So these are the visual parameters of the patient. One is the thickness, one is the visual acuity, and those outcomes decline once the VEGF level has returned back to the pretherapy level. Both the morphological and the functional outcomes were good when VEGF was low, and deteriorated once the level of VEGF rebounded. So as expected, neutralizing VEGF is the reason why… As the VEGF, anti-VEGF loses its efficacy, the VEGF comes back, then what happens is the symptoms also come back. Another point that can be made from this slide is that VEGF is an effective biomarker, biomarker efficacy of the drug. Okay. So because current approaches only transiently reduce the level of VEGF, second generation approaches seek to enduringly suppress VEGF. One would expect from the slides that we’ve looked at so far that if you could keep the VEGF levels down longer, you should also maintain better vision for the patient longer as well. So long-term suppression of VEGF is expected to result in a long-term clinical benefit. Now, as we consider the strategy of making a more durable VEGF, is there potentially a downside of accomplishing that? Preclinical studies demonstrate that VEGF is neuroprotective, and indicate that keeping VEGF suppressed for a long time would have potential negative side effects. Here’s a piece of data from preclinical data, in which preclinical experiments, demonstrating what else VEGF might be doing in the eye, besides causing pathology… So these are sections from mice that were genetically modified so that LACZ, which is a reporter enzyme that turns tissue blue was driven by the VEGF encoder. So in these mice, whenever VEGF is expressed, you can stain the tissue blue. And what this shows is that layers of the retina in which VEGF are being produced include the ganglion cell layer and the internuclear layer. So the point is that, in a healthy eye, in a healthy mouse eye, there are a number of cell types within the retina that produce VEGF. So the bottom section here is another reporter mouse, where LacZ was driven by the VEGF receptor, instead of the VEGF promoter. And this mouse tells us which cell types are expressing VEGF receptor, and potentially responding to the VEGF that is being made by these cell types. And as you can see, there are multiple cell types within the retina. Not only the endothelial cells. There are multiple cell types within the retina that express VEGF receptors. And this is a healthy animal, so if you were to suppress VEGF production or neutral use VEGF with antibodies for a long time in mice, you would expect to cause deleterious issues, and in fact, that’s what happened. This research group reported that endogenous VEGF is required for visual function. There’s evidence for survival. VEGF has a survival role are on Muller cells and photoreceptors. And how about in humans? In humans, this is a picture from this paper, in which they concluded that VEGF inhibits complement proteins in the eye and in the kidneys. Complement is part of the innate immune system. And its job is to kill cells that have been infected, or in some other ways not good for the viability of the organism. And the way that the immune system — complement, which is part of the innate immune system — is held in check is by a number of complement inhibitors. So the pathway is always on, and complement inhibitors are what suppress the activity of that complement system. And what drives expression of those complement inhibitors is VEGF. Or one of the things that drives expression of those complement inhibitors is VEGF. So in the normal scenario, VEGF is activating its receptor, and that’s instructing the cells to make complement inhibitors, and that keeps the level of complement activity at the appropriate level in things like endothelial cells. If one were to constitutively or at least long-term reduce the level of VEGF, that would be expected to cause less production of complement inhibitors, therefore more active complement, and that could lead to the demise of cells by the innate immune system. So one expected or potential undesirable consequence of long-term suppression of VEGF would be vulnerability to complement activation. And in fact, there are clinical studies that report that geographic atrophy progresses in patients treated with anti-VEGF, although it’s difficult to know from these studies whether that’s the natural history of the disease, versus a side effect of anti-VEGF, since a control is not possible in clinical studies of this design. So I’d like to point out that, whereas there are definite caveats of long-term treatment, long-term treatment with anti-VEGF, long-term suppression of VEGF, the current clinical benefit, improvement of vision, for instance, far outweighs any negative side effects that have been thus far detected by the use of anti-VEGF for ocular diseases in the clinic. But if we take a step back from everything that we’ve been talking about, one question that is obvious is: Why does the eye produce VEGF under adverse conditions? And if we’re going to suppress that VEGF in those adverse conditions, what are we going to counter, that the eye is trying to accomplish? This is an important and as yet unanswered question that needs to be considered, as we design strategies to manipulate the levels of VEGF and the responsiveness in the eye. Okay. So let’s ask questions. The question of variability. So what are the possible reasons why response to anti-VEGF is variable? It’s a clinically relevant question. And this is especially true for diseases such as diabetic macular edema. Here we have a picture of an edematous DME eye. You can see these are the drusens, in AMD, they’re called drusens, although these sort of precipitates also accumulate in patients with diabetic retinopathy. You can see the hemorrhages that are accumulating here, the microaneurysms, and here is an OCT picture of an eye, of a person treated — not treated with anti-VEGF. This is baseline. And this is the region of the macula, and you can see that this big bump here is a problem, and that’s highly associated, closely associated with why the patient has a poor BCVA, or best corrected visual acuity of 20/10, which means they don’t see very well. When you give them a dose of anti-VEGF, you see the swelling has gone down and the vision has improved. This moves further in a good direction, and eventually the patient can get a non-swollen retina and a good visual acuity of 20/30. So what can be done so that all patients can respond this way is what drives us to understand why there is variability. So here, before we go on and ask this other question, let’s have this polling question. The question is: Anti-VEGF is effective in the eye because it, A, causes regression, B, reduces retinal edema associated with leaking blood vessels, C, reduces fibrosis, which compromises vision and can cause retinal detachment, or because it reduces intraocular pressure. So please choose the best answer. Okay, so I see your answers. And it’s a split. Not really a split. 63% of you answered that it reduces retinal edema associated with leakage. And yes, that is the correct answer in the eye. In diseases like diabetic retinopathy. And in wet AMD, there is evidence that anti-VEGF does not cause regression of vessels. Instead, unlike its action in cancer, the beneficial action in the eye seems to be associated much more tightly with reducing leakage, as opposed to causing pathological blood vessels to regress. Okay. So now back to this question of why does anti-VEGF not work in everybody. So a plausible reason for the variability is because VEGF is not the only agent elevated in vitreous of patients with leaking blood vessels. So there’s more than one thing that can make blood vessels leak. Some of these things include cytokines and members of the bradykinin pathway, some of which are elevated in patients with eye diseases. For instance, here we have a study, a table from a study in which patients with diabetic macular edema were treated with either triamcinolone, which is a steroid, and reduces the level of cytokines, or bevacizumab, which is the full length anti-VEGF antibody. And you can see here that if we look first in the anti-VEGF-treated group at the preinjection and the postinjection level of cytokines and VEGF, before we look at the data, note that there are cytokines, in addition to VEGF, in patients with DME, and if you treat them with bevacizumab, an anti-VEGF, the level of cytokines don’t change, because that’s not what anti-VEGF recognizes. Instead, the level of VEGF crashes, and that’s exactly what it’s supposed to do. In contrast, if you take a look at what happens to the level of cytokines versus VEGF in eyes treated with the steroid triamcinolone, the answer is: The level of the cytokines goes down, and this one especially, whereas the level of anti-VEGF does not — sorry, the level of VEGF does not go down. So triamcinolone is effective in DME, but it doesn’t change the level of VEGF. Rather, it changes the level of cytokines. Which leads us to suspect that cytokines promote the permeability of retinal vessels. Similarly, a group has published a proteomics paper, where they compared proteins that are present in the vitreous of patients with different types of diabetic retinopathy, and what they learned is that carbonic anhydrase is higher in patients with DME than in other types of diabetic retinopathy. So carbonic anhydrase is an enzyme which changes the pH of the vitreous slightly. And at that lower pH, the conversion of prekallikrein to kallikrein goes better. And what kallikrein does is it generates bradykinin, and bradykinin is capable of causing leakage of blood vessels. So here is another agent which is present in eyes of patients with DME, and could be contributing to the leakage and the inability of anti-VEGF to stop the leakage. So, again, the main point here is that vitreous contains multiple agents that induce permeability. Not only VEGF. And that’s a potential reason why not all patients respond the same way to anti-VEGF. Yes, so here we’re gonna push this point a little bit further. Is anti-VEGF’s therapeutic benefit all about permeability? In diseases like DME and non-proliferative diabetic retinopathy, that seems to be clearly the case. Because in these diseases, there are no pathological blood vessels for anti-VEGF to regress. Instead, there are just leaking blood vessels. Dysfunctional blood vessels. But you don’t want to regress those blood vessels. Those are the retinal vessels that the retina depends on. So here the therapeutic benefit of anti-VEGF is not about regression. It is about calming or overcoming the permeability. And even in patients with the proliferative form of diabetic retinopathy, which there are new pathological blood vessels that have entered the vitreous at the interface with the retina, when studies have been done, asking: Does anti-VEGF cause these pathological blood vessels to regress, the answer is no. It does not cause them to regress. And the idea that anti-VEGF is therapeutic because it suppresses permeability is further reinforced for this particular disease. Now, so let’s stop here for a minute, and consider another eye disease, for which anti-VEGF is being tested, but has not yet been approved, because the data are not yet… Those studies are not complete, so those decisions cannot yet be made. But unlike diabetic retinopathy and wet AMD, retinopathy of prematurity involves the regression of blood vessels. So in this disease, patients are born preterm, and need to be exposed to high oxygen, because their lungs aren’t fully developed, and this causes overgrowth of the retinal vasculature, which again isn’t developed fully, because the baby hasn’t come to term. And in those cases, anti-VEGF is used to suppress this angiogenesis, of which there is too much. So anti-VEGF here is being used to regress blood vessels, unlike DME, in which there is no regression of blood vessels. Rather, it’s just the overcoming the leakage of those blood vessels, which is anti-VEGF’s function. And before we go further, just as a side note, I would like to say that VEGF therapy is being used in certain diseases, which are ischemic. For example, in ischemic heart disease, the problem is that there aren’t enough blood vessels. So VEGF can be used to promote the formation of vessels. So in the eye, anti-VEGF is good to stop leakage, but anti-VEGF in the eye with different diseases even can be used to induce regression of vessels, and still other pathological scenarios VEGF — not anti-VEGF — VEGF can be used to promote the growth of blood vessels. So the anti-VEGF, even therapeutic VEGF field is broader and different in different organs of the body. Okay, so we talked about whether it’s a good idea to improve anti-VEGF, and definitely there are initiatives to get that done. One of the ways to do that is to increase the durability of the drug by making a smaller molecular weight compound. For instance, single chain antibodies, and modifying the anti-VEGF so that it would stay in the vitreous longer, to increase its half-life. There’s also combo therapy going on, and we’re gonna talk about that combo therapy in a minute. I’m just looking at the time. We’re gonna move along here. So attempts to improve anti-VEGF therapy include anti-VEGF and anti-PDGF or anti-PDGFR. So the strategy is to prevent the maturation of blood vessels. It will neutralize PDGF, which recruits pericytes and allows them to mature. So combo therapy is better because it will reduce the dependence on anti-VEGF. A couple of drug companies tried this, and found that combo therapy was not better than monotherapy, and this particular approach to combo therapy has been abandoned by those companies. So another initiative that seems to be going better is combo therapy to improve anti-VEGF by using anti-VEGF and enforcement of the barrier function of the endothelium. And in order to talk about that, we’re gonna have to get into the details of the mechanisms of how VEGF works. But before we do that, here’s another polling question. Please answer the best answer. Anti-VEGF-based therapy is effective for most patients afflicted with: Glaucoma, dry AMD, dry eye, or wet AMD. Okay, you guys are right on the ball. One hundred percent response said wet AMD, which is the correct answer. So let’s move forward with the VEGF basics. So far, we’ve been talking about VEGF-A, and that’s because it’s a therapeutically relevant member. And again, we came to that conclusion by comparing the four anti-VEGFs and the common denominator between them is VEGF-A. So that’s the therapeutically relevant member. But in fact, there are seven members all together. And each of them binds to a receptor. There’s a lot of overlap between the receptors. But that’s the relationship that the ligands have with their receptors. And VEGF-A is actually… There’s eight isoforms of VEGF. Those are due to RNA splicing, is where that variability comes from. All eight isoforms are similar, in that they have the part of VEGF that binds to the receptors. The variability lies within exons 6, 7, and 8, which alter the ability to bind to either heparin or neuropilin. There’s also the 165 variant. Obviously there’s a different amino acid sequence there, but no one has been able to demonstrate how that difference in the C terminus alters the biochemical functions of VEGF, its ability to activate its receptor, and how this molecule, this isoform of VEGF, is antiangiogenic remains a mystery. So the VEGF-C and D family members promote lymph angiogenesis instead of blood angiogenesis. So lymphedema, an example of which is shown here, is caused by lymphatic dysfunction. So unlike anti-VEGF therapies in the eye, therapies for lymphedema which are being considered would be to use VEGF-C therapeutically, to add VEGF-C to these patients, for instance, in a gene therapy scenario, which would promote the formation of lymphatics, and that’s considered — is currently being considered as a way to cure patients of lymphedema. VEGF-B promotes fatty acid uptake in the endothelium. VEGF-B also promotes nerve regeneration in the cornea. So you can see that the other VEGF family members are doing very different things than VEGF-A, which is controlling the blood vessels. Both their formation and their permeability. And VEGF-A is being considered as an approach to overcome ischemia in heart disease. When there’s ischemia, it’s often caused because the blood vessels are blocked and the blood vessels downstream of that blockage die, and then the tissue surrounding those blood vessels, which were dependent on those blood vessels for nutrient exchange, become ischemic. So therapies that increase the level of VEGF, that would cause more of these blood vessels to form, are being considered. Here’s a great review article concerning VEGF-A as a therapeutic agent. Not anti-VEGF, but VEGF-A as a therapeutic agent. So how does VEGF work? How does it talk to cells? As we saw earlier, VEGF will bind to a receptor. That receptor has an extracellular domain that’s specific for one or more of the VEGF family members. The receptor has a transmembrane domain that anchors it to the membrane of the cell. Here we’re gonna talk about endothelial cells. And there’s an intracellular portion that encodes a tyrosine kinase that is activated upon ligand bonding. What I mean by that is that the kinase activity of the enzyme increases. It’s a kinase, so it phosphorylates proteins. That changes their conformation, which then is instructive for engaging signal transduction pathways. So again, dimerization, phosphorylation changes the conformation of an enzyme from low to high activity. In the case of receptor with tyrosine kinase, it’s low to high. That’s not always the case for how phosphorylation changes the enzymatic activity. You can see the kinase here in this diagram, which is relevant to VEGF receptors, in its inactive state is monomeric. The ligand is a dimer and causes the dimerization of the receptor. This juxtaposition of two receptors in an appropriate way allows the kinase domain to assume an active conformation, whereupon it will autophosphorylate and further lock the enzyme in an active conformation, whereupon the enzyme will phosphorylate substrates. One of the substrates is the enzyme itself, and that creates docking sites to recruit signaling molecules. It’ll also phosphorylate other proteins, which are its substrates, as well. So the activated receptor triggers signaling events, and changes in gene expression, which direct cellular responses. Okay, so here are illustrations of these two points. The first point is that the receptor, once it binds ligand, it gets activated, it phosphorylates itself and other molecules, it engages signaling pathways such as these listed here, these traverse the cytoplasm and get to the nucleus, where it changes gene expression, and those gene expression changes then lead to the production of new proteins which instruct the cells, permit the cells, to do different things. For instance, in the case of angiogenesis, one of the new proteins that can be made are proteases, which will then cause remodeling of the matrix that the endothelial cell sits on, and the changes in that matrix then permit the endothelial cells to move from a quiescent to an angiogenic state. And as we’ll see later, changes in that extracellular matrix can change, whether the endothelial cells can maintain the barrier function. So these are the cellular processes intrinsic to angiogenesis. They involve the exposure of the vasculature to angiogenic factors. The basement membrane is degraded by matrix metalloproteinases and other types of proteases, that allows the endothelial cells to proliferate and migrate. Tubes will form. You can see here new tubes are forming. And then they will stabilize by the recruitment of other cell types, such as pericytes. Okay, so I believe that this is our last polling question. Please choose the best answer. VEGF is a growth factor that acts via G protein-coupled receptors to promote cell survival, reduces fluctuations in blood glucose, promotes permeability of blood vessels, is a major driver of pathology with no known role in physiology. So the correct answer, which wasn’t unanimous, is that it promotes the permeability of blood vessels. Obviously VEGF does other things as well, but of the group of four that we could choose, that was the best answer. It doesn’t reduce fluctuations of blood glucose. That’s not the system that it regulates. It acts via tyrosine kinase, not a G protein-coupled receptor. And it has a major role in physiology. Just FYI, if you make a mouse that is missing even one allele of VEGF, the mouse cannot make it through development. Typically genes, when they’re deleted in mice, you can delete one allele, but then if it’s going to be a problem, it’s when both alleles are eliminated. Here, for VEGF, even one allele deletion is incompatible with embryogenesis, meaning that even a 50% reduction during embryogenesis causes the animal to die. So it is important for physiology. Okay, so… There are two — so how is it — so what the rest of this talk is gonna be about, and we’re almost done, is about permeability. Because that’s the big problem in the eye — at least with the diseases that we’re focusing on, what VEGF drives. So how do blood vessels within the eye, how does the retina within the eye, preserve the blood-retina barrier? And there are two barriers within the eye. One is at the level of the vessels. You can see that the endothelial cells form the barrier that keeps the substance of the circulation within the vessel and away from the tissue, and then there’s another barrier in the eye, and that’s the outer blood-retinal barrier. That’s where the RPE cells juxtapose each other, and that also forms a barrier within the eye. And so there’s several ways to get across the inner blood-retinal barrier. The most obvious one is to get between the cells. But they can get inside cells in various ways. We’ll be focusing on how VEGF permits the barrier between cells to relax. So one of the cell types that helps vessels mature are pericytes, and one of the things that pericytes do is that they prevent transcellular transport of molecules. You can see that here are pictures. These are electron micrographs of a mouse that’s a normal mouse, and you can see this is the pericyte. This is the endothelial cell. And the arrows point to three kinds of vessels or vesicles. One is at the vascular lumen. So this is the blood. And this is at the vascular lumen. There are these sorts of vessels. Then within the cells there are vessels, and at the abluminal side, once you get to the pericyte… I’m sorry. This is the endothelial cell. This is the pericyte. I had that backwards. And once you get through the endothelial cell, then the pericyte, the things that are being transported from the blood through the endothelial cell, meets the basement membrane and the pericyte here. And so this experiment was done with a normal mouse, and a mouse that’s been modified so that it doesn’t have pericytes. What happens is there are a lot more of these vesicles on the inside of cells. A lot more vesicles on the abluminal surface, indicating that pericytes need to be there, to prevent the transcellular transport of vesicles from the blood into the tissue. So that’s how pericytes are influencing the permeability. They’re regulating the flow of molecules through the endothelial cells. What VEGF is doing is it’s governing this cell-cell junction between the pericytes, and there are two types of junctions that can be affected. The tight junctions and the adherens junctions. And VEGF acts mostly on the adherens junctions. The adherens junctions are a complex of proteins that reach out between cells to touch each other, and they also are connected to proteins on the inside, and most importantly, those connections, those accessory proteins on the inside of the cell, connected to the cytoskeleton, which is what anchors the cell-cell junction proteins. So that’s the scaffold, which is important for permeability. So VEGF relaxes the inner blood-retinal barrier by causing phosphorylation of components of the adherens junctions. You can see it’s activated by its ligand, turns on signaling events, which leads to phosphorylation within the adherens junctions, and that compromises their ability to set up the barrier. As mentioned earlier, the components of the endothelial cell junction include parts of the junctions that were being phosphorylated. We looked at that a few minutes ago, and also the scaffold, the cytoskeleton, on which the junction stands. And there’s one molecule here in this junction, this adherens junction. It’s very important. That molecule is a phosphatase. It dephosphorylates and reverses that functional modification, and it’s important for these molecules to be able to make these cytoskeletal scaffolds, which are essential for permeability. So not only is this phosphatase important, but there’s also another kinase that’s present in adherens junctions. That kinase is Tie2, also an RTK, receptor tyrosine kinase. It’s got ligands that activate it, a tyrosine kinase domain that gets turned on, which means the kinase activity is increased, and it phosphorylates a number of proteins, including itself. And Tie2 not only has ligands that activate it, but it has ligands that suppress its activity. Inhibiting a phosphatase is one of the ways to activate Tie2. So the receptor tyrosine kinases, such as VEGF, that we’ve discussed so far, are ones which were thought to have very low basal activity. Meaning when there’s no ligand, the kinase activity is off. And only when the ligand, VEGF, is present does the kinase activity go up. Tie2 may not fit into that simple story. Tie2 instead may be constitutively on, and VE-PTP, this phosphatase, associates with it, and dephosphorylates, and that’s the way that the kinase is kept off, by the presence of VE-PTP. So when its ligand, Ang2, binds to Tie2, it pushes the phosphatase away, and that’s what lets the kinase come on. Ang2 is a negative regulator of Tie2, because when it binds it’s unable to push this away, so it can keep dephosphorylating and keep it off, and it can prevent Ang1 binding because they want to bind in the same place. So these basic studies have recently been translated to the clinic in a couple of exciting phase 2 clinical trials, which both indicate that activating type II appears to potentiate the therapeutic benefit of anti-VEGF. For instance, one study, done by Roche, a phase 2 study called Boulevard, they combined anti-VEGF with anti-Ang2, so that’s a way of activating Tie2. This approach resulted in a greater improvement in BCVA, retinal thickness, and ETDRS score, as compared with aVEGF monotherapy. And there was a phase 2 study where they combined anti-VEGF with an inhibitor called this, and that was more effective than anti-VEGF alone. So the attempts to improve anti-VEGF — we talked about increased durability, and more recently, with a combination of closing the barrier, or preventing the barrier from opening, by neutralizing VEGF, and then this activation of Tie2 may be functioning by closing the barrier. So those two approaches. Now, I would like to point out that this work with Tie2, phase two studies, are very exciting. They complement one another. But the phase three trials are ongoing, and their results are not yet known. So whether these things will become therapies or not remains to be seen. Okay, so the mechanisms to enforce the inner barrier are to prevent opening of the barrier and approaches to do that are to antagonize VEGF, because what VEGF is doing is it’s turning on signaling pathways that mediate phosphorylation. And the other way to enforce the barrier is to close the barrier, which seems to be done by activating Tie2, and that activates the scaffold on which the junctional proteins can stand and push those endothelial cells up against one another, so that the barrier would be enforced. Okay, so here’s the summary of the talk. We started with that. And we’ve gone through it, and hopefully these are self-evident at this point, that anti-VEGF provides benefit by temporarily reducing the level of VEGF. Patients’ response to VEGF is variable. That’s a clinically relevant and actively pursued question. Anti-VEGF’s therapeutic benefit is due to overcoming leakage in diseases like DR and wet AMD, and new approaches are being pursued to overcome leakage, one of which seems promising, and that’s to activate Tie2. So that’s the end of the talk.


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July 2, 2018

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