 Good morning everyone. Welcome to Grand Rounds today. If you don't mind going ahead and having a seat. We'll go ahead and get started. I'm very pleased to introduce one of our retina surgeons. With us today we'll be speaking Dr. Hartnett and this should be a very interesting topic for us all. I think it would be a great review for the residents as well. Should be talking about retinopathy of prematurity from both the clinical and laboratory aspect. Thank you, Kenden. All right. So I have no relevant financial disclosures and I may be discussing about a baston as it relates to a new clinical trial. So in this talk I'd like to first give a definition on how we classify and manage retinopathy of prematurity and then discuss a little bit about how we've come to recognize that ROP is really a complex disease having environmental genetic and perhaps epigenetic associations. And then I'm going to discuss a little bit about research findings in our laboratory and end with a new clinical trial that's combining pediatric ophthalmology and pediatric retina in a multi-center fashion. So retinopathy of prematurity is one of the leading causes of childhood blindness worldwide. It only occurs in preterm infants. Full term infants do not develop ROP although there are conditions that look like ROP in full term infants like familial exudative vitriol retinopathy. It's thought that in the United States about 16,000 preterm infants develop ROP yearly and even with our management strategies the numbers are still similar that approximately 550 still become blind. Our goal is to prevent stage 5 ROP or total retinal detachment and this is a picture of an eye with stage 5 ROP so here's the iris and the posterior synechia and although it looks like there's a cataract the lens is clear it's that there's a retrolental fibrous membrane behind which is a total retinal detachment. So first classification how do we classify ROP? So based on a number of studies mainly the international classification of retinopathy of prematurity we use several parameters the zone, the stage, the presence of plus disease and in the past the extent of stage although that's become less important with time. So the zone it gives an idea of I think of it as how mature the retinal vascular development has become. Vessels start to pour out or precursors develop into vessels from the optic nerve at about 16 weeks gestation and then move out toward the auricirata. So zone 1 is considered a circle centered around on the optic nerve and the radius is twice the distance between the optic nerve and the macula. Zone 2 is also centered on the optic nerve a circle the radius of which is the distance from the optic nerve to the nasal auricirata and zone 3 is the temporal crescent. The stages of ROP are how severe it is and it's really usually we think of it as the appearance between the vascular and avascular retina. So these are retcam images of human infants these are all different infant eyes and showing the optic nerve here this is a left eye the macula immature vessels growing out and then we see just a faint line here and avascular retina that's stage 1. Stage 2 we see some volume developing along that line avascular retina peripherally. Stage 3 we have this blood vessel growth into the vitreous into vitreous neovascularization and stage 4 can be partial retinal detachment either involving the fovea in stage 4b or missing the fovea in stage 4a. And as I mentioned stage 5 was total retinal detachment. Now I wanted to put this slide up to discuss the differences we often hear about animal models of retinopathy prematurity and then human retinopathy prematurity the animal models are all limited. First of all they're not in preterm infants are preterm animals they're in full term animals and none of the animal models develop what we see in stage 4 stage 5 retinopathy prematurity. So in the human we really think of two phases we have this vascular phase where we have severe retinopathy prematurity we have blood vessel growth into the vitreous we have something called plus disease which is a dilation and tortuosity of the vessels currently that's a qualitative assessment based on our memory of an image from multicenter clinical trials as to whether or not the severity of dilation and tortuosity makes true plus disease. So this is what most of the animal models mimic and we rarely get into the fibrovascular form of ROP where the blood vessels start to play less of a role and we start to see vitreous membranes and pre-retinal membranes that cause traction on the retina and pull the retinal off into retinal detachment and that's shown here in stage 4 ROP. So the fibrovascular phase we really don't have any way of modeling that in animals and so most of what we know is based on our experience in human infants. So we have the stages and I think back on the first slide where I was showing the stages I presented the four stages. Stage 4 ROP is when the fibrovascular phase starts where retinal detachment occurs and this just gives an idea of what actually occurs because we often look at images like this. This is a red cam image of an infant eye showing some dilation and tortuosity of the veins. The veins being pulled to this elevated area. This is actually retina that's pulled up into a fold and this white area is fibrous tissue. So a cross section might look like this. So we see how the fibrous membranes pull the retina off and how we know when we're looking at a flat two dimensional image is that the vessels around the optic nerve appear out of focus when we're focused on the fibrous membrane here. And then stage 5 ROP as I mentioned which is total retinal detachment. So here is an example this is the example I showed in the first slide the white membrane behind which is a total retinal detachment. So this would be if we were to take a ultrasound through the eye or could look through the eye with X-ray vision this is kind of what we would see. So a total retinal detachment and even with the ability to reattach the retina surgically the visual acuity in these infants is often very poor. You know less than 2200 less than legal blindness and so we do everything possible to try to prevent this. So what is our current management of retinopathy of prematurity? So retinopathy of prematurity as I said develops only in preterm infants it develops after birth. And there are certain characteristics that put a preterm infant at greater risk of developing severe ROP. In the United States based on a number of studies we found that infants born less than or equal to 1500 grams or less than or equal 30 weeks gestation are the ones at high risk for developing some ROP. Also any infants that fall outside these parameters but have an unstable course. So basically anyone that the neonatologist feel are at risk either high oxygen, not feeding well, lots of infections. Any time that there's an infant that there's some concern about poor postnatal waking we will take a look and screen these infants. ROP develops after birth and we don't have any symptoms from the preterm infant to clue us in to look at their eyes. So we automatically do examinations at between four to six weeks chronologic age or 31 weeks post menstrual age and the post menstrual age is the sum of the chronologic and gestational age in weeks. So for example if you had a 24 week gestational age baby who is about four months old or 16 weeks gestation that baby would be 40 weeks post menstrual age. So when you say the first exam is four to six weeks chronologic or 31 weeks well which is it? It's the older of the two. So if you had a 23 week gestational age baby who is six weeks old so therefore 29 weeks post menstrual age you would wait in additional two weeks to examine that baby and all this is based on studies and information that we've gotten from multi center trials. Now these parameters differ in other areas of the world where ROP occurs in infants born at larger birth weights or at older gestational ages. So screening occurs they do screening at earlier time points. Then based on the examination we do follow up examinations and these can be every one to two weeks and we do this until retinal development is complete. So beyond zone three to the aura serata or until severe retinopathy or prematurity occurs. And severe retinopathy or prematurity is stage three ROP with plus disease or stage two and I'll show you some of the early or some of the parameters that we use for that now. If severe ROP occurs laser treatment is performed and the patients are followed very carefully for regression of the neovascular or the vascular components of ROP. If the vascular components persist we do more laser. If fibro vascular components start to develop that's when we consider surgery for progressive stage for ROP. And in all these patients when we get the retina attached the most important thing is visual rehabilitation. To make sure that the infant can see and see as a child and infants with ROP are more likely to be myopic and also to have stroke business. So it's important to work very closely with pediatric ophthalmology. So what about this I said about severe ROP. So what is severe ROP and how did we come up with those terms. So in the multi-center cryotherapy trial that was to determine whether cryotherapy to the peripheral avascular retina would reduce the risk of a bad outcome in infants with severe ROP. And so they chose a threshold of severity where there was a 50% risk of a bad outcome in that infant if nothing was done. And so that threshold ROP was basically any infant in zone one or two with stage three that's the intra vitriol neovascularization having five contiguous or a total clock hours. So that's where the extended stage comes in. And then plus disease that dilation and tortuosity in four out of four quadrants. And it was found in that study that cryotherapy significantly reduced the risk of retinal detachment or poor outcome in infants with threshold ROP. But there were still a lot of infants that went on to develop stage five ROP or did not have good vision. And so the question was well maybe we should go earlier and then also around that time we had developed the technology to deliver laser with an indirect delivery system. So in the cryotherapy study we didn't have that ability and that's why it wasn't probably recommended or tried in that study. So based on the early treatment retinopthera prematurity study they developed or they divided pre-threshold ROP into two types type one or two and found that type one where there's a 15% risk of a bad outcome in natural history that those infants did better if they had laser treatment to the avascular retina. So this is when we now consider laser in most cases so any infant was zone one and stage three without plus disease and then the other two criteria have plus disease. Zone one any ROP or zone two stage two or three with plus disease. And here the definition of plus disease also changed. Plus disease was defined as two quadrants have dilated into tortuous vessels and the severity of the plus disease is not quite as severe as the picture that we know from the cryo ROP study. We prefer laser to cryotherapy. In some cases we can't get laser in because there's blood or the visualization to the retina is not good. We have better tools now. Cryotherapy tends to cause more inflammation to the eye and can also be associated with plaques in the macula post-operatively and these can interfere with visual development. I want to mention another form of severe ROP that we're seeing more of and that's aggressive posterior ROP. So the typical zone two stage three severe ROP tends to occur around 37 weeks post-menstrual age regardless of birth weight or gestational age of the infant. Aggressive posterior ROP however develop we often see that on the first or second exam. So this could be 33 to 35 weeks post-menstrual age. It's generally in zone one or posterior zone two and there are four quadrants of plus disease. So this does not tend to progress through the standard stage one, stage two, stage three but rather we look in and we see dilated tortuous vessels and this flat neovascularization which can be difficult to detect. So flat neovascularization is right here actually and it just looks like a brush fuzzy border. So how do we know that that's neovascularization? Well I guess we don't really accept that when we treat these infants with laser. So say for example we were treating with laser up to this point. What happens is there's regression and these blood vessels tend to regress and about 37 weeks post-menstrual age so the time when a typical post or typical stage two zone two, stage three ROP would develop neovascularization. We see new formation of intra vitreous neovascularization into the area where the new avascular zone developed. So here just shows this is actually after laser treatment. So this patient had been treated with laser here. There was an avascular area of flat neovascularization here and neovascularization then developed and new laser was placed in. So I often tell parents who have children or infants with aggressive posterior ROP that their infant will likely need more than one laser treatment. So this is neovascular. And that's definitely vascular activity and the best way to treat that is with laser. But then as we start to develop this progressive stage four ROP with fibrous changes, so what are the features that make us think about surgery for progressive stage four ROP? There can be ridge thickening at the junction of vascular and avascular retina and I reviewed a number of infants who this was years ago before we became a little more aggressive about doing lens sparing protractomies for these infants. And the features that I found predicted progression of stage four ROP were six clock hours of ridge thickening at the junction of vascular and avascular retina. Any type of fibro vascular proliferation or vitreous condensation and two of four quadrants of plus disease. And sometimes it appears that the plus disease comes back, so it regresses after laser and then reforms. And then others have found that any neovascularization, even in the treated areas of the avascular zone, that's another thing that should be watched and may indicate the need for surgery. So what surgery do we like to do in these infants? The lens is important in visual development. If we remove the lens in an infant, they can have a terrible aphake at Gambliopia. So we always try to save the lens and the type of surgery we do is a two port lens sparing protractomy when at all possible. And we try to remove the vectors of vitreous that are pulling the retina off between the ridge and the anterior part of the eye, the circumferential vector here, and also the ridge to the lens. And it's important to remember that in the preterm or in any infant, the pars plana, pars placata region is not mature. So whereas in an adult human, we might have four millimeters of a safe zone to enter into the vitreous cavity without tearing the retina. In a newborn full term infant, we have .87 millimeters, and in the preterm infants, it's even smaller. So we're going about a half a millimeter posterior to the limbus trying to avoid the lens and the retina. So we don't take this casually. This is not an easy decision to make to operate on these infant eyes. And yet if we wait, then this area of retina gets pulled up to the lens and it becomes virtually impossible to safely repair the retina without removing the lens. And remember also, if we get a hole in the retina in an infant eye, unlike in the adult, it's an inoperable retinal detachment often because the vitreous is not able to be mechanically removed from the retina at this point. So we take these decisions very seriously and I usually ask for multiple opinions to make sure that I'm keeping myself, you know, that I'm doing, that we're doing the best thing for the infant. And then as I mentioned, stage five, prevent it with careful screening, repeat examinations, and prompt treatment. I often, though, do offer surgery to reattach the retina. And my reasoning is that, you know, in research, we're doing so much to restore vision with computer chips, with various ways. We're learning more about ways that we may be able to provide hope for this infant 20 years from now with vision. And this is especially when it's bilateral, which it often is. So I will offer various ways to anatomically reattach the retina, either with a pars placata of the trectomy, often removing the lens or an open sky of the trectomy. And with an open sky of the trectomy, what we do is we remove the cornea and place it into, like, tissue culture, menia. And then we get this, and then we take the lens out with the cryoprobe and we see this pre-retinal membrane, and underneath it is the retina. So you have to do sharp dissection, like this, to remove the membrane from the retina so that you're left with the retina like this. And then we can use viscoelastic to help push back the retina. Sometimes we drain some retinal fluid and then we replace the same cornea. So that's one of the ways that we have to treat stage five ROP, but we're always trying to prevent it because that's when our outcomes are best. So now I'm going to just start to switch to what we know about ROP and then talk a little bit about what's going on in the lab. We've come to recognize that ROP is a complex disease, and what that means is that it has a number of factors that go into its pathogenesis. There are environmental associations, there are genetic associations, and we're getting more evidence that there may be epigenetic associations as well. There's variability and risk, so ROP can manifest differently throughout the world. In India, for example, where there are not the resources to implement oxygen monitoring and regulation, they often have the type of ROP that we saw in the 1940s with very high oxygen-induced retinopathy and ROP. So it's almost a different type of condition than what we have here. And they also see ROP in bigger babies that are born at an older gestational age and probably for that reason because oxygen plays a strong role. Frequency is different worldwide in Latin America and South America. ROP is one of the leading causes of visual impairment in infants, whereas in the U.S., it's not. And that, again, is because the countries are able to save the preterm infants and they may have variability in regulating and monitoring oxygen. There may be other reasons as well. Prenatal care, nutrition. Environmental associations that we know are affecting, they can affect ROP oxygen. We've seen that. I just mentioned how it differs throughout the world. Oxidative stress, maybe. I'll go through that briefly. And then nutrition is being recognized as an important factor in the risk of ROP. And then there are genetic associations. At least one paper suggested that 70 percent of the variants was due to genetics, but we don't have any strong candidate genes yet. And we want to be able to study genetics for that reason. So what about the role of oxygen? So in the United States, there was the initial ROP epidemic of the 1940s that was really associated with unregulated oxygen at birth. And when it became recognized through really animal models that oxygen was a culprit, oxygen was restricted, which reduced ROP, but then there was a higher incidence of infant mortality and cerebral palsy. And then over the decades, there was the technology for oxygen monitoring and regulation. And so with that also came greater survival of low birth weight infants. ROP virtually disappeared for a while, and then it came back to the second epidemic when we had lower and lower birth weight infants and younger gestational age infants in the 90s. And it became recognized that other oxygen stresses were important in ROP besides hyperoxyiberth. For example, fluctuations in oxygen, that was a high risk for ROP, and perhaps supplemental oxygen during the course of the infant's time in the NICU. What about oxidative stress? Well, oxidative stress has long been linked to ROP based on oxygen levels. So high oxygen can increase reactive oxygen species by donating electrons to oxygen and causing superoxide radical. But it's also been argued that low oxygen levels can increase reactive oxygen species generation because it tends to slow down the electron transport chain and you end up with more electron donors. So hypoxia may also increase ROS. And then the fluctuations with ischemia reperfusion may also increase ROS. And in the preterm infant, the infant is in utero. Oxygen is about 30 to 40 millimeters mercury. And so when the infant is born and placed into sometimes they were put into 100% oxygen at the time of birth, that was definitely hypoxia. And so it's not clear if that plays a role, but it may. The infants have been measured for oxidative compounds and they're found to have higher oxidative compounds when they're preterm than full term infants. There's also reduction in antioxidative enzymes. They don't have the reserve to be able to handle oxidative stress. And in a meta analysis from a number of studies done decades ago on trying vitamin E to reduce the risk of ROP, they found when they put all the numbers together, they found that there was reduced stage three in those infants with vitamin E. But I think vitamin E has risks and that's why it's not currently being used. I think those trials were stopped. The role of nutrition, there have been a number of studies done in the last decade that have found that low serum insulin-like growth factor is associated with poor postnatal weight gain in preterm infants and also with larger avascular areas of retina and severe ROP. And so in Sweden they developed a reference model based on weekly IGF-1 levels in the serum and safe postnatal weight gain for infants without severe ROP. And then they used this and whenever the infant's parameters fell beyond what their numbers were each week, an alarm went off. And so they then correlated the number of times of the alarms with those infants that went on to develop severe ROP. And they found 100% sensitivity in Sweden, but it's not been generally found to be as valuable in other areas of the world but still valuable. 90% in Brazil. And there have been several studies in the United States but they've been retrospective. And the goal is to do a prospective study to test this. So why do this? Because what's happening, especially in these countries that have so much ROP without the oxygen regulation and monitoring, is that they don't have enough people to do the examinations. And a lot of infants that don't develop severe ROP need examination. So how do they best use the resources? And this is one model that's being tested. As I mentioned in genetics, there's a retrospective study of monozygotic and dizygotic twins that's 70% of the variance in susceptibility to ROP is due to genetic factors alone. But no one gene strongly is associated with increased risk. And I'm working with Magdi Angelis here and with Kanyost and with the neonatology and pediatric ophthalmology and to be able to determine genetics in ROP. We've looked at studies done so far. There have been a number of candidate genes but there really has not been one candidate gene that's been found to be like a complement factor H in AMD. So nothing that really stands out as being helpful yet. And then I'll just briefly mention about epigenetic factors. So epigenetics can be things like if the DNA, if the proteins around the DNA, the histones interfere with the ability of having the gene transcribed, then that gene will not be able to be translated into protein. And what's been found is that intrauterine growth restriction can lead to dysregulation of angiogenic factors based on differences in being male or female. Rob Lane is doing a lot of this work in multiple organs and we're collaborating with him to look in the retina as well. And human's IUGR can increase the risk of diabetes later and obesity and risk can seem to be passed on to later generations. We've found in the retina that IUGR had a variability in the regulation of retinal IGF1 receptor and VEGF receptor one based on sex. And we're doing more studies with this right now. I don't have enough to be able to even conclude and tell you what it means. Okay, so I'm going to switch now and talk a little bit about what's going on in our laboratory. Some of this I've presented briefly before and some of it is new. So so the way that we think about R.O.P. is based on the avascular retina because that's a risk for severe R.O.P. It's a risk for intravitrile neovascularization. And it's long been thought to be the source of angiogenic factors. So based on what we see in nature, we've developed a mental model of what goes on in R.O.P. And that when a baby is born preterm, the peripheral retina is a vascular. Now 90% of these will vascularize their avascular retina and be fine and never develop R.O.P. But 10% will develop a severity of R.O.P. where there's intravitrious neovascularization. Even if we look at this group and look at the natural history, 50% will have regression of neovascularization with ongoing vascularization of a vascular retina. So we know that that's possible. And that's been our goal from the start is that we're not interested in just inhibiting neovascularization, but we want to redirect the blood vessel growth appropriately. And as I mentioned, R.O.P. is complex, there are genetic and environmental factors involved. So we have different models based on what the question is that we're trying to answer. So the for genetics, the hyperoxy induced vaso obliteration model that was developed by Lois Smith and Pat DeMory is probably the most well known. And in this mice are brought at postnatal day seven into high constant oxygen for five days and then placed into room air following that. And although these oxygen levels create arterial oxygen levels that are not consistent with what preterm infants develop, I mean, it's probably like over 300 millimeters mercury. But it does develop. It's it is still a very useful model for studying genetic genetic mechanisms because you can use use transgenic animals. The retinal flat mound here shown lectin stained. When we flatten the retina, it comes out looking like a clover leaf because the retinas round. We see central obliterated retina, and then these endothelial buds in the vasculature. So it doesn't really look like R.O.P. either but it's a very useful model. The model the 50 10 fluctuating model was developed by John Penn and the supplemental oxygen part of it was added by Bruce Berkowitz. And in this rat pups are put laced into 50% oxygen for 24 hours and then cycle down to 10% oxygen for 24 hours. This cycling is continued to day 14. When they're brought up from 10% into 21% or 28% oxygen. So these oxygen extremes create arterial oxygen levels in the rat that are similar to transcutaneous oxygen levels in a human infant that develops severe R.O.P. It also has fluctuation in oxygen, which is more similar to what the preterm infant experiences. And the retinal flat mound has the appearance more similar to severe R.O.P. with the peripheral avascular retina and this introvitory is the vascularization at the junction. So we mentioned about angiogenic factors being important in introvitory's new vascularization and certainly one of the ones that has become recognized as very important as vascular endothelial growth factor. It's associated with severity of a number of adult and infant diseases. It's regulated by oxygen and that's relevant in the preterm infant. And if we inhibit the bioactivity of VEGF we can reduce the severity of disease and this includes R.O.P. But VEGF is essential to retinal vascular development and therefore we have to remember that inhibiting it might have an adverse effect on the developing vasculature because it is an angiogenic inhibitor. So in the first part I'm going to show you some of the work we did to determine the effective relevant oxygen stresses on expression of VEGF to gain insight into the development of avascular retina and subsequently introvitory is neovascularization. So we used a number of outcomes. We can take whole retinas out and we can measure protein by Western blot and we can measure the mRNA by using real-time PCR. But we can also measure several parameters in the retinas themselves. So this is from a postnatal 14 room air race rat pup. This is a retina flattened sting with lectin showing that the retina is fully vascularized in the interplexus. In the 5010 OIR model at postnatal day 14 there would be about 33% avascular retina in the flat mouth. At postnatal day 18 in the 5010 OIR model we see that there's characteristic avascular retina about 25% and we see that almost if we were to measure by clock hours there are almost 12 clock hours of introvitory on the avascularization. And I'll also mention that in this model there's always regression of disease. So it gives us the opportunity to look at what things might be causing regression which is what we would like. So we first measured the VEGF mRNAs and protein in room air in 5010 OIR model at different postnatal day ages to find out the association of the VEGF with a vascular retina and introvitory on the avascularization. Now I'll mention that in the mouse model when the animals are in high oxygen and they have vaso obliteration in the central retina VEGF is very low in the retina. And then when they have endothelial budding into the vitreous VEGF is very high. So we thought well maybe since we are in the rat model and we're going from low oxygen at 10% to high oxygen at 21% maybe we would see a similar pattern. But we didn't find that. So here is VEGF 164 which is the most prevalent mRNA and we found this is full change in expression relative to beta actin. And using an ANOVA that accounts for multiple comparisons we found that both older developmental age as well as the model compared to room air were significantly associated with increased VEGF 164. And then when we looked at the protein we had a similar relationship and there were also several significant days based on post hoc protected T test evaluation including post natal day 14 when a vascular retina persisted in the model but room air retinas were fully vascularized. And at post natal day 18 when the vascularization was present in the model. So in the summary VEGF was increased at post natal day 14 in the model when a vascular retina existed compared to room air. So this is a little different than what we saw in the mouse model. It was also increased at P18 when intravitrile neovascularization was present. So then we asked the question whether if we inhibited the bioactivity of VEGF would we reduce intravitrile neovascularization but would it also cause persistent avascular retina or maybe even more avascular retina since VEGF is important in retinal development. And we found that neutralizing VEGF reduced clock hours of intravitrile neovascularization. So this is with a neutralizing antibody to VEGF. So similar to Avastin but not Avastin and an IgG control. And we found that it did not interfere with a vascular retina. So more avascular retina is high on this slide. And then when we used a receptor tyrosine kinase inhibitor for VEGF receptor 2 which is the angiogenic receptor we found a similar pattern that there was a significant reduction this time area of intravitrile neovascularization but it did not interfere with ongoing retinal vascular development. So that was a little bit unexpected and you know based on what we were getting I was we wondered G could VEGF VEGF receptor 2 signaling actually contribute to a vascular retina. So it's too much VEGF causing a vascular retina. And so how could that be? So we went back to what occurs in vascular development and when endothelial cells divide they undergo mitosis they set up a cleavage plane and the angle between the cleavage plane and the long axis of the vessel predicts where the daughter endothelial cells will migrate. Such that a 90 degree angle predicts vessel elongation, a zero degree angle predicts vessel widening and perhaps we thought that if you had a lot of irregular angles that maybe it would cause disoriented mitosis and sort of a pattern like what we see in intravitrile neovascularization. So studying VEGF is challenging because a single allele or either receptor knockout is lethal. So we went we collaborated with Vicky Bouch who has an embryonic stem cell model at the University of North Carolina and she has a VEGF receptor 1 knockout. So what that does VEGF receptor 1 combined VEGF but it doesn't have a very strong receptor tyrosine kinase to signal cell events and it binds the VEGF keeping it from VEGF receptor 2. So when we knock it out all the VEGF goes into VEGF receptor 2 and we get increased VEGF receptor 2 signaling. And indeed we found that this caused disoriented mitosis in the embryonic stem cell model and it was similar to the appearance of what we see with intravitrile neovascularization and further it could be rescued with a soluble flit one that's VEGF receptor 1 soluble form transgene linked to a promoter that was specific for endothelial cells. So we took it back to the model and we asked the question whether or not VEGF if we neutralized the bioactivity if we could reduce some of the tortuosity or disorientation of the dividing endothelial cells. And so this we used a neutralizing end of the body to VEGF and we took lectin state flat mounts. Here is the cleavage plane measured cleavage angles and then on this graph this is a number of mitosis and these are the angles the cleavage angles grouped by 10 degrees. And just to give a sort of a summary that the elongation would be toward 90 degrees dilation would be toward zero degrees. And we found that in veins that the neutralizing antibody tended to cause the veins to be less dilated than the IgG control. And when we looked at arterial tortuosity using the sort of a pre version of ROP tool we found that the neutralized nanobody significantly reduced arterial tortuosity compared to control or to the non-injected. So we've so far we've found that increased VEGF receptor 2 signaling disorders dividing vascular cells and contributes to dilation and tortuosity and may have an effect in plus disease. And now we're studying in the lab whether excess VEGF would disorder normal developmental angiogenesis and contribute to a vascular retina by permitting endothelial cells to divide outside the plane of the retina and proliferate in a pattern that looks like intravitrial nevascularization. And the clinical studies have provided support for the hypothesis and that anti-VEGF does reduce intravitrial nevascularization and permits ordered angiogenesis. But long term and safety results are lacking and more studies are warranted. So, you know, I don't know. Okay, I think, are we okay? We can go for 10 minutes. Okay. So, so I've just, we have, this is our current working hypothesis for the lab and we just showed that VEGF receptor 2 signaling leads to some, or we're studying disordered angiogenesis which increases a vascular retina. Some of this I've presented before but now I want to go through another mechanism because it's, a vascular retina is going to have multiple mechanisms. There's not going to be one bullet that cures it. And so I'm going to take you through this pathway where we find that VEGF receptor signaling actually activates the jack stack pathway and contributes to a vascular retina. This is the work of Haibou Wang who's a research associate in our lab and also a number of people in my lab. So the signal transducers and activators are transcription, stat and cytokine activation. These are, this is an important pathway whereby you have a cytokine receptor with two of these jacks. A cytokine can activate and then cause stats to form a dimer and they can translocate into the nucleus and then either with stimulators or repressors affect transcription of genes. And the cytokine growth factor can be erythropoietin, interferon, interleukins, possibly others growth factors like VEGF and it can also be activated by hypoxia, reactive oxygen species or supplemental oxygen. So it seemed like a good pathway to study. And our hypothesis was that jack stat signaling would contribute to a vascular retina through either apoptosis or death of endothelial cells or by affecting growth factor expressions such as VEGF. So we first looked at the, so when stat three is activated becomes phosphorylated. So jack stack goes through stat three and can be phosphorylated. So we first looked at the amount of phosphorylated stat three in retinas in room air and in the model at postnatal day 14 when a vascular retina was present. And basically we found that the model had increased phosphorylated stat three compared to room air. And this is just showing the western blots. Furthermore, we could use a chemical jack two, jack three inhibitor and we found that it significantly reduced phosphorylated stat three when we gave it as an intraperitoneal injection into these animals. We also found that when we use this inhibitor and inhibited stat three, it reduced the area of a vascular retina in the ROP model. So stat three activation or phosphorylation contributed to a vascular retina. And so then we wanted to set out to understand the mechanisms. Could it be affecting downstream VEGF expression? So if you, that would then lead to a vascular retina either by inhibiting VEGF or could it be causing apoptosis? So in this case we use the model at postnatal day 14 and we use AG490 which inhibits stat three versus PBS control but we found that there was no effect on either VEGF or on cleat caspase three which is downstream of two of the apoptotic pathways. So it didn't seem to be caused by either the mechanisms that we thought. So then we started to think about erythropoietin because it's been, it's shown to be neuroprotective, it can be thrombogenic, it can protect against hyperoxy-induced avascular retina in the mouse model of hyperoxy-induced vasodiliteration. But there have been studies where use of erythropoietin has been associated with severe ROP in preterm infants. So there's controversy as to its role in ROP. So we first measured erythropoietin in animals in the 5010 OIR model treated with either AG490 or PBS and we found that when we inhibited stat three that we had increased erythropoietin. And here I'm just kind of showing you what the signaling pathway might be. So jack stat seems to lead to decrease erythropoietin. And so then we asked, well, is VEGF involved because we know that VEGF is important. So we looked at animals that were treated with a neutralizing antibody to VEGF at a dose that we knew decreased intravitrile neovascularization or control and we measured phosphorylated stat three. And what we found is that in the neutralizing antibody to VEGF there was a significant reduction in phosphorylated stat three compared to the IGG injection. And these are intravitrile injections and the way we do these experiments we don't inject the fellow eye to look for crossover effect. So this suggested that VEGF was upstream of jack stat in this model. And we took these same eyes and probe for erythropoietin and we found that the neutralizing antibody increased VEGF. So this looked like VEGF was activating jack stat to cause decreased erythropoietin. And then finally we injected erythropoietin into the 5010 OIR model compared to control and we found that it reduced a vascular retina. So that's where we are right now and we're studying in vitro we're using in models of endothelial cells and Mueller cells to get at mechanisms. But stat three does seem to be activated after fluctuating oxygen in the rat model at postnatal day 14 when a vascular retina persists. It contributes to a vascular retina and VEGF appears to be upstream of stat three. And then stat three signaling seems to reduce erythropoietin and this is associated with a vascular retina. So our current working hypothesis is that VEGF fluctuations in oxygen may activate jack street to decrease erythropoietin in the retina and lead to a vascular retina. So our ongoing studies, anti-VEGF is not the only answer. Additional clinical studies are warranted. We had a grand rounds here talking about a clinical trial using a Vastin in infants with severe ROP compared to laser. So we need more clinical studies done. Erythropoietin may increase intravitrious neovascularization in models but it depends on the timing, apparently that it's given. And when we, stat three activation in our model reduced erythropoietin, we think that perhaps the neuroprotective effect of erythropoietin may be beneficial though in ROP by promoting vascular retina if it's given earlier. So all these things we are studying and it may mean that ROP is something that will require multiple drugs to treat it. And the update on the clinical trial is probably the most exciting thing is that pediatric ophthalmology and pediatric retina are coming together to form a multicenter clinical trial to study a Vastin versus laser in severe ROP and we'll be using photographs too to be able to characterize the severity of ROP rather than just mental images of what we see when we examine the infants. And then I wanted to acknowledge our funding sources and also the people in my laboratory who have done all this work. This is Yanchao Zhang, Shamie Kanakar, Manabu Makwaski, Haibo Wang, Tyler Smith, and Aichi Nishimura. Thank you. And any questions? Yes. Well, I think that could be the case. I mean, we're still, you know, it's evolving. ROP infants are younger and smaller and then new medications are being used for other complications that they have, so it is kind of an evolving field. And I think what you're seeing is very accurate and perhaps what we need is to have multiple tools kind of to be able to target this and figure out what's safe and what's not safe. Yes. No, no, that, in fact, those studies we gave intraperitoneal to the POPs. And usually, I don't know, is it given, how do you give it in infants? Is it sub-Q? So it's not, but it probably, you know, the studies that showed severity were retrospective studies. And I always question that because it's when do you take the infant? If the infant, you know, maybe those infants had they had EPO would have lived. You know, it might be those questions so when you say, well, we're gonna look at all infants that have survived to the time when they have ROP examinations, you're taking out a number of infants who may have died because they didn't have EPO. I mean, so I think that it depends on how the trial. Yeah, yes, anemia prematurity. And I don't know about any, are there other reasons that, we have our neonatal group here that came too as well. Thank you. Besides anemia prematurity, do you give it for brain? Right, I know of one study, I think it may have been retrospective just by looking in the literature that EPO was associated when infants had EPO, they had better cognitive scores or better mental development scores later on when they were children. And I think that in Europe, are they starting a study for EPO on preterm infants? I thought I heard that, but I couldn't find it on the PubMed. But I think it would be a good, I mean, I think what would be interesting is to do EPO and then if they get severe ROP, anti-vegeta, because maybe the EPO would be neuroprotective. We should try that in our model. And may not cause some of the problems that we're concerned about with a vastin into the vitreous, which then gets systemic and may have a negative effects on other organs. Oh yeah, yeah. So I don't know, maybe I shouldn't have you.