Bax expression impacts postnatal retinal vascular development and hyperoxia sensitivity.

Apoptosis plays prominent roles during organ development, maturation and homeostasis. In the retina, Bcl-2 family members function through the intrinsic cell death pathway with vital roles during vascular development and hyperoxia-mediated vessel obliteration during oxygen induced ischemic retinopathy (OIR). Bim, a BH3 only protein Bcl-2 family member, binds and activates Bax and/or Bak to facilitate apoptosis. In some systems deletion of both Bax and Bak are required to prevent cell loss, such as regression of ocular hyaloid vasculature. We previously showed Bim expression significantly impacts normal retinal vascular development and sensitivity to hyperoxia. Mice deficient in Bim (Bim-/-) show increased retinal vascular density and are protected from hyperoxia mediated vessel obliteration. Since Bim activates Bax, here we determined the impact lack of Bax expression has on these processes. Compared to Bax+/+ mice, retinas from Bax-/- mice had significantly increased numbers of retinal endothelial cells and pericytes. We also demonstrated that hyperoxia-mediated vessel obliteration during OIR was significantly decreased in the absence of Bax. Although the increased endothelial cell numbers were comparable to that of Bim-/- mice, the increased numbers of pericytes were not to the extent noted in Bim-/- mice. These changes were supported by partial protection of retinal vessels from hyperoxia in Bax-/- mice compared to that noted in Bim-/- mice. Thus, Bim-Bax driven pathway is sufficient to remove excess endothelial cells but not pericytes during postnatal retinal vascularization and hyperoxia-mediated vessel obliteration. Thus, additional Bim-mediated pathway(s) are required for removal of pericytes and hyperoxia-mediated vessel obliteration.

Bim Expression Influences Choroidal Endothelial Cell Characteristics and Their Response to Therapeutic Intervention.

In the aging population, choroidal vessels grow through the Bruch's membrane, resulting in a loss of central vision due to choroidal neovascularization (CNV). During active neovascularization, CNV is associated with inappropriate levels of apoptosis in multiple cell types, including choroidal endothelial cells (ChECs). Bim is a pro-apoptotic member of the Bcl-2 family. It is essential for cell apoptosis due to exposure to drugs such as dexamethasone or decreased pro-survival factors, including vascular endothelial growth factor (VEGF). To better elucidate the cell autonomous contribution of Bim expression in the integrity and neovascularization of the choroidal vasculature, we isolated ChECs from wild-type and Bim-deficient (Bim-/-) mice. ChECs lacking Bim expression demonstrated increased expression of VEGF, osteopontin, and the inflammatory cytokines Rantes/Ccl5 and IL6. Bim-/- ChECs were more proliferative and demonstrated an increased capacity to undergo capillary morphogenesis. Anti-VEGF had a diminished capacity to disrupt capillary morphogenesis in Bim-/- ChECs. In vivo, utilizing the mouse laser photocoagulation model, anti-VEGF treatment mitigated CNV in wild-type but not Bim-/- mice. We also tested other modalities that are thought to not require the intrinsic death pathway for their function and showed that propranolol, anti-CTGF, and the TSP1-mimetic peptide ABT898 mitigated CNV in mice lacking Bim expression to varying degrees. Thus, in ChECs, Bim expression could impact the effectiveness of treatment modalities that require the intrinsic death pathway to mitigate CNV.

Artesunate mitigates choroidal neovascularization and scar formation.

Angiogenesis, although required during eye development, has a causative effect in many ocular diseases. Aberrant neovascularization contributes to the progression of neovascular age-related macular degeneration (nAMD), a vision-threaten disease in aging Americans. Since increased amounts of vascular endothelial growth factor (VEGF) drives neovascularization during the pathogenesis of nAMD the standard of care are anti-VEGF therapies attempt to disrupt this vicious cycle. These current anti-VEGF therapies try to maintain vascular homeostasis while abating aberrant neovascularization but regrettably don't prevent fibrosis or scar formation. In addition, some patients demonstrate an incomplete response to anti-VEGF therapy as demonstrated by progressive vision loss. Here, we show choroidal endothelial cells (ChEC) incubated with artesunate demonstrated decreased migration and inflammatory and fibrotic factor expression, which corresponded with decreased sprouting in a choroid/retinal pigment epithelium (RPE) explant sprouting angiogenesis assay. To assess the efficacy of artesunate to curtail neovascularization in vivo, we utilized laser photocoagulation-induced rupture of the Bruch's membrane to induce choroidal neovascularization (CNV). Artesunate significantly inhibited CNV and the accompanying fibrotic scar, perhaps due in part to its ability to inhibit mononuclear phagocyte (MP) recruitment. Thus, artesunate shows promise in inhibiting both CNV and fibrosis.

Cytochrome P450 1B1 Expression Regulates Intracellular Iron Levels and Oxidative Stress in the Retinal Endothelium.

Cytochrome P450 (CYP) 1B1 is a heme-containing monooxygenase found mainly in extrahepatic tissues, including the retina. CYP1B1 substrates include exogenous aromatic hydrocarbons, such as dioxins, and endogenous bioactive compounds, including 17ß-estradiol (E2) and arachidonic acid. The endogenous compounds and their metabolites are mediators of various cellular and physiological processes, suggesting that CYP1B1 activity is likely important in maintaining proper cellular and tissue functions. We previously demonstrated that lack of CYP1B1 expression and activity are associated with increased levels of reactive oxygen species and oxidative stress in the retinal vasculature and vascular cells, including retinal endothelial cells (ECs). However, the detailed mechanism(s) of how CYP1B1 activity modulates redox homeostasis remained unknown. We hypothesized that CYP1B1 metabolism of E2 affects bone morphogenic protein 6 (BMP6)-hepcidin-mediated iron homeostasis and lipid peroxidation impacting cellular redox state. Here, we demonstrate retinal EC prepared from Cyp1b1-deficient (Cyp1b1-/-) mice exhibits increased estrogen receptor-α (ERα) activity and expresses higher levels of BMP6. BMP6 is an inducer of the iron-regulatory hormone hepcidin in the endothelium. Increased hepcidin expression in Cyp1b1-/- retinal EC resulted in decreased levels of the iron exporter protein ferroportin and, as a result, increased intracellular iron accumulation. Removal of excess iron or antagonism of ERα in Cyp1b1-/- retinal EC was sufficient to mitigate increased lipid peroxidation and reduce oxidative stress. Suppression of lipid peroxidation and antagonism of ERα also restored ischemia-mediated retinal neovascularization in Cyp1b1-/- mice. Thus, CYP1B1 expression in retinal EC is important in the regulation of intracellular iron levels, with a significant impact on ocular redox homeostasis and oxidative stress through modulation of the ERα/BMP6/hepcidin axis.

Inhibition of retinal neovascularization by a PEDF-derived nonapeptide in newborn mice subjected to oxygen-induced ischemic retinopathy.

Retinopathy of prematurity (ROP) is a growing cause of lifelong blindness and visual defects as improved neonatal care worldwide increases survival in very-low-birthweight preterm newborns. Advancing ROP is managed by laser surgery or a single intravitreal injection of anti-VEGF, typically at 33-36 weeks gestational age. While newer methods of scanning and telemedicine improve monitoring ROP, the above interventions are more difficult to deliver in developing countries. There is also concern as to laser-induced detachment and adverse developmental effects in newborns of anti-VEGF treatment, spurring a search for alternative means of mitigating ROP. Pigment epithelium-derived factor (PEDF), a potent angiogenesis inhibitor appears late in gestation, is undetected in 25-28 week vitreous, but present at full term. Its absence may contribute to ROP upon transition from high-to-ambient oxygen environment or with intermittent hypoxia. We recently described antiangiogenic PEDF-derived small peptides which inhibit choroidal neovascularization, and suggested that their target may be laminin receptor, 67LR. The latter has been implicated in oxygen-induced ischemic retinopathy (OIR). Here we examined the effect of a nonapeptide, PEDF 336, in a newborn mouse OIR model. Neovascularization was significantly decreased in a dose-responsive manner by single intravitreal (IVT) injections of 1.25-7.5 µg/eye (1.0-6.0 nmol/eye). By contrast, anti-mouse VEGFA164 was only effective at 25 ng/eye, with limited dose-response. Combination of anti-VEGFA164 with PEDF 336 gave only the poorer anti-VEGF response while abrogating the robust inhibition seen with peptide-alone, suggesting a need for VEGF in sensitizing the endothelium to the peptide. VEGF stimulated 67LR presentation on endothelial cells, which was decreased in the presence of PEDF 336. Mouse and rabbit eyes showed no histopathology or inflammation after IVT peptide injection. Thus, PEDF 336 is a potential ROP therapeutic, but is not expected to be beneficial in combination with anti-VEGF.

Tunicamycin-induced photoreceptor atrophy precedes degeneration of retinal capillaries with minimal effects on retinal ganglion and pigment epithelium cells.

Endoplasmic reticulum (ER) stress is recognized as a contributing factor to various ocular neurovascular pathologies including retinitis pigmentosa, glaucoma, and diabetic retinopathy (DR). ER stress in particular is implicated in the development of DR, which is significantly influenced by inflammation driven retinal vascular degeneration and dysfunction. Ultimately, loss of vision occurs if left untreated. However, the identity of the target cells and their temporal involvement in diabetes-mediated dysfunction need further investigation. Early diabetes-induced stress in photoreceptor cells is proposed as the driver of inflammatory mediated neurovascular changes during diabetes. Although tunicamycin induced ER stress results in photoreceptor loss, its consequences for retinal vascular degeneration and retinal ganglion (RGC) and pigment epithelium (RPE) cell loss remains unclear. Here we show intravitreal delivery of tunicamycin primarily induced ER stress in photoreceptor cells resulting in their loss by apoptosis. This was concomitant with induced expression of the unfolded protein response marker CHOP in these cells. We also demonstrated significant degeneration of retinal capillaries following the loss of photoreceptor cells with minimal impact on loss of RGC and RPE cells. However, activation of retinal microglial and Muller cells were noticeable. Thus, our data support the notion that ER stress mediated dysfunction and/or loss of photoreceptor cells in response to inflammation and oxidative stress could precede retinal vascular and neuronal dysfunction and degeneration.

Novel anti-angiogenic PEDF-derived small peptides mitigate choroidal neovascularization.

Abnormal migration and proliferation of endothelial cells (EC) drive neovascular retinopathies. While anti-VEGF treatment slows progression, pathology is often supported by decrease in intraocular pigment epithelium-derived factor (PEDF), an endogenous inhibitor of angiogenesis. A surface helical 34-mer peptide of PEDF, comprising this activity, is efficacious in animal models of neovascular retina disease but remains impractically large for therapeutic use. We sought smaller fragments within this sequence that mitigate choroidal neovascularization (CNV). Expecting rapid intravitreal (IVT) clearance, we also developed a method to reversibly attach peptides to nano-carriers for extended delivery. Synthetic fragments of 34-mer yielded smaller anti-angiogenic peptides, and N-terminal capping with dicarboxylic acids did not diminish activity. Charge restoration via substitution of an internal aspartate by asparagine improved potency, achieving low nM apoptotic response in VEGF-activated EC. Two optimized peptides (PEDF 335, 8-mer and PEDF 336, 9-mer) were tested in a mouse model of laser-induced CNV. IVT injection of either peptide, 2-5 days before laser treatment, gave significant CNV decrease at day +14 post laser treatment. The 8-mer also decreased CNV, when administered as eye drops. Also examined was a nanoparticle-conjugate (NPC) prodrug of the 9-mer, having positive zeta potential, expected to display longer intraocular residence. This NPC showed extended efficacy, even when injected 14 days before laser treatment. Neither inflammatory cells nor other histopathologic abnormalities were seen in rabbit eyes harvested 14 days following IVT injection of PEDF 336 (>200 µg). No rabbit or mouse eye irritation was observed over 12-17 days of PEDF 335 eye drops (10 mM). Viability was unaffected in 3 retinal and 2 choroidal cell types by PEDF 335 up to 100 µM, PEDF 336 (100 µM) gave slight growth inhibition only in choroidal EC. A small anti-angiogenic PEDF epitope (G-Y-D-L-Y-R-V) was identified, variants (adipic-Sar-Y-N-L-Y-R-V) mitigate CNV, with clinical potential in treating neovascular retinopathy. Their shared active motif, Y - - - R, is found in laminin (Ln) peptide YIGSR, which binds Ln receptor 67LR, a known high-affinity ligand of PEDF 34-mer.

Negative regulators of angiogenesis: important targets for treatment of exudative AMD.

Angiogenesis contributes to the pathogenesis of many diseases including exudative age-related macular degeneration (AMD). It is normally kept in check by a tightly balanced production of pro- and anti-angiogenic factors. The up-regulation of the pro-angiogenic factor, vascular endothelial growth factor (VEGF), is intimately linked to the pathogenesis of exudative AMD, and its antagonism has been effectively targeted for treatment. However, very little is known about potential changes in expression of anti-angiogenic factors and the role they play in choroidal vascular homeostasis and neovascularization associated with AMD. Here, we will discuss the important role of thrombospondins and pigment epithelium-derived factor, two major endogenous inhibitors of angiogenesis, in retinal and choroidal vascular homeostasis and their potential alterations during AMD and choroidal neovascularization (CNV). We will review the cell autonomous function of these proteins in retinal and choroidal vascular cells. We will also discuss the potential targeting of these molecules and use of their mimetic peptides for therapeutic development for exudative AMD.

Endoglin regulates the activation and quiescence of endothelium by participating in canonical and non-canonical TGF-ß signaling pathways.

Endoglin (Eng) is an auxiliary receptor for transforming growth factor-ß (TGFß), with important roles in vascular function. TGFß regulates angiogenesis through balancing the pro-proliferative and pro-differentiation signaling pathways of endothelial cells (EC). However, the contribution of endoglin to these TGFß activities, and more specifically modulation of EC phenotype, remains elusive. Mutations in endoglin cause hereditary hemorrhagic telangiectasia-1 in humans. The Eng+/- mice are viable and exhibit some of the vascular defects seen in humans with endoglin haploinsufficiency. In the present study we show that haploinsufficiency of endoglin results in attenuation of retinal neovascularization during oxygen-induced ischemic retinopathy. Although the importance of endoglin expression in angiogenesis and vascular development has been demonstrated, the underlying mechanisms remain obscure. To gain detailed insight into the cell autonomous regulatory mechanisms that affect angiogenic properties of EC, we prepared retinal EC from Eng+/+ and Eng+/- Immorto mice. The Eng+/- EC were more adherent, less migratory, and failed to undergo capillary morphogenesis. Aortic sprouting angiogenesis was similarly attenuated in aortas from Eng+/- mice. In addition, Eng+/- EC expressed increased levels of VEGF but reduced expression of endothelial NO synthase and NO production. Mechanistically, these changes were consistent with sustained activation of mitogen-activated protein kinase (MAPK) pathways, and aberrant Smad-dependent signaling pathways in Eng+/- EC. Taken together, our results underscore the importance of endoglin in both canonical and non-canonical TGFß signaling pathways modulating both the activation and quiescence of the endothelium during angiogenesis.

Bone morphogenetic protein 7 regulates reactive gliosis in retinal astrocytes and Müller glia.

PURPOSE: The focus of this study was to determine whether bone morphogenetic proteins (BMPs) trigger reactive gliosis in retinal astrocytes and/or Müller glial cells. METHODS: Retinal astrocytes and the Müller glial cell line MIO-M1 were treated with vehicle, BMP7, or BMP4. Samples from the treated cells were analyzed for changes in gliosis markers using reverse transcriptase - quantitative PCR (RT-qPCR) and western blotting. To determine potential similarities and differences in gliosis states, control and BMP-treated cells were compared to cells treated with sodium peroxynitrite (a strong oxidizing agent that will bring about some aspects of gliosis). Last, mature mice were microinjected intravitreally with BMP7 and analyzed for changes in gliosis markers using RT-qPCR, western blotting, and immunohistochemistry. RESULTS: Treatment of retinal astrocyte cells and Müller glial cells with BMP7 regulated various reactive gliosis markers. When compared to the response of cells treated with sodium peroxynitrite, the profiles of gliosis markers regulated due to exposure to BMP7 were similar. However, as expected, the profiles including the oxidative agent and growth factor were not identical. Treatment of cells with BMP4, however, showed an attenuated response in comparison to peroxynitrite and BMP7 treatment. Injection of BMP7 into the mouse retina also triggered a reactive gliosis response 7 days after injection. CONCLUSIONS: BMP7 induced changes in levels of mRNA and protein markers typically associated with reactive gliosis in retinal astrocytes and Müller glial cells, including glial fibrillary acidic protein (GFAP), glutamine synthetase (GS), a subset of chondroitin sulfate proteoglycans (CSPGs), matrix metalloproteinases (MMPs), and other molecules.

The sustained delivery of resveratrol or a defined grape powder inhibits new blood vessel formation in a mouse model of choroidal neovascularization.

The objective of this study was to determine whether resveratrol or a defined, reconstituted grape powder can attenuate the formation of new blood vessels in a mouse model of choroidal neovascularization (CNV). To accomplish this objective, C57BL/6J mice were randomized into control or treatment groups which received either resveratrol or grape powder by daily oral gavage, resveratrol or grape powder delivered ad libitum through the drinking water, or resveratrol by slow release via implanted osmotic pumps. A laser was used to rupture Bruch's membrane to induce CNV which was then detected in sclerochoroidal eyecups stained with antibodies against intercellular adhesion molecule-2. CNV area was measured using fluorescence microscopy and Image J software. Ad libitum delivery of both resveratrol and grape powder was shown to significantly reduce the extent of CNV by 68% and 57%, respectively. Parallel experiments conducted in vitro demonstrated that resveratrol activates p53 and inactivates Akt/protein kinase B in choroidal endothelial cells, contributing to its anti-proliferative and anti-migratory properties. In addition resveratrol was shown to inhibit the formation of endothelial cell networks, augmenting its overall anti-angiogenic effects. The non-toxic nature of resveratrol makes it an especially attractive candidate for the prevention and/or treatment of CNV.

Adenosine Receptors Expression in Human Retina and Choroid with Age-related Macular Degeneration.

Purpose: Adenosine signaling modulates ocular inflammatory processes, and its antagonism mitigates neovascularization in both newborns and preclinical models of ocular neovascularization including age-related macular degeneration (AMD). The adenosine receptor expression patterns have not been well characterized in the human retina and choroid. Methods: Here we examined the expression of adenosine receptor subtypes within the retina and choroid of human donor eyes with and without AMD. Antibodies specifically targeting adenosine receptor subtypes A1, A2A, A2B, and A3 were used to assess their expression patterns. Quantitative real-time PCR analysis was used to confirm gene expression of these receptors within the normal human retina and choroid. Results: We found that all four receptor subtypes were expressed in several layers of the retina, and within the retinal pigment epithelium and choroid. The expression of A1 receptors was more prominent in the inner and outer plexiform layers, where microglia normally reside, and supported by RNA expression in the retina. A2A and A2B showed similar expression patterns with prominent expression in the vasculature and retinal pigment epithelium. No dramatic differences in expression of these receptors were observed in eyes from patients with dry or wet AMD compared to control, with the exception A3 receptors. Eyes with dry AMD lost expression of A3 in the photoreceptor outer segments compared with eyes from control or wet AMD. Conclusion: The ocular presence of adenosine receptors is consistent with their proposed role in modulation of inflammation in both the retina and choroid, and their potential targeting for AMD treatment.

Bim is responsible for the inherent sensitivity of the developing retinal vasculature to hyperoxia.

Apoptosis plays an important role in development and remodeling of vasculature during organogenesis. Coordinated branching and remodeling of the retinal vascular tree is essential for normal retinal function. Bcl-2 family members, such as bim not only influence apoptosis, but also cell adhesive and migratory properties essential during vascular development. Here we examined the impact of bim deficiency on postnatal retinal vascularization, as well as retinal neovascularization during oxygen-induced ischemic retinopathy (OIR) and laser-induced choroidal neovascularization. Loss of bim expression was associated with increased retinal vascular density in mature animals. This was mainly attributed to increased numbers of pericytes and endothelial cells. However, the initial spread of the superficial layer of retinal vasculature and, the appearance and density of the tip cells were similar in bim+/+ and bim-/- mice. In addition, hyaloid vessel regression was attenuated in the absence of bim. Furthermore, in the absence of bim retinal vessel obliteration and neovascularization did not occur during OIR. Instead, normal inner retinal vascularization proceeded independent of changes in oxygen levels. In contrast, choroidal neovascularization occurred equally well in bim+/+ and bim-/- mice. Together our data suggest bim expression may be responsible for the inherent sensitivity of the developing retinal vasculature to changes in oxygen levels, and promotes vessel obliteration in response to hyperoxia.

Bim Expression Promotes the Clearance of Mononuclear Phagocytes during Choroidal Neovascularization, Mitigating Scar Formation in Mice.

Inflammation is increasingly recognized as an important modulator in the pathogenesis of neovascular age-related macular degeneration (nAMD). Although significant progress has been made in delineating the pathways that contribute to the recruitment of inflammatory cells and their contribution to nAMD, we know little about what drives the resolution of these inflammatory responses. Gaining a better understanding of how immune cells are cleared in the choroid will give a novel insight into how sustained inflammation could influence the pathogenesis of nAMD. The pro-apoptotic Bcl-2 family member Bim is a master regulator of immune cell homeostasis. In its absence, immune cell lifespan and numbers increase. Most therapeutic regimes that squelch inflammation do so by enhancing immune cell apoptosis through enhanced Bim expression and activity. To test the hypothesis that Bim expression tempers inflammation during the pathogenesis of nAMD, we used the mouse laser-induced choroidal neovascularization (CNV) model in which inflammation acts as a facilitator of CNV. Here, we showed minimal to no change in the recruitment of F4/80-, CD80-, CD11b-, and Iba1-positive myeloid-derived mononuclear phagocytes to the site of laser photocoagulation in the absence of Bim expression. However, the resolution of these cells from the choroid of Bim-deficient (Bim -/-) mice was significantly diminished following laser photocoagulation. With time, we noted increased scar formation, demonstrated by collagen I staining, in Bim -/- mice with no change in the resolution of neovascularization compared to wild-type littermates. We also noted that mice lacking Bim expression in mononuclear phagocytes (BimFlox/Flox; Lyz2-Cre (BimMP) mice) had delayed resolution of F4/80-, CD80-, CD11b-, and Iba1-positive cells, while those lacking Bim expression in endothelial cells (BimFlox/Flox; Cad5-Cre (BimEC) mice) had delayed resolution of only CD11b- and Iba1-positive cells. Both BimMP and BimEC mice demonstrated increased scar formation, albeit to differing degrees. Thus, our studies show that resolving inflammation plays an important role in moderating scar formation in nAMD, and it is impacted by Bim expression in both the endothelium and mononuclear phagocyte lineages.

Bim Expression Modulates Branching Morphogenesis of the Epithelium and Endothelium.

Branching morphogenesis is a key developmental process during organogenesis, such that its disruption frequently leads to long-term consequences. The kidney and eye share many etiologies, perhaps, due to similar use of developmental branching morphogenesis and signaling pathways including cell death. Tipping the apoptotic balance towards apoptosis imparts a ureteric bud and retinal vascular branching phenotype similar to one that occurs in papillorenal syndrome. Here, to compare ureteric bud and retinal vascular branching in the context of decreased apoptosis, we investigated the impact of Bim, Bcl-2's rival force. In the metanephros, lack of Bim expression enhanced ureteric bud branching with increases in ureteric bud length, branch points, and branch end points. Unfortunately, enhanced ureteric bud branching also came with increased branching defects and other undesirable consequences. Although we did see increased nephron number and renal mass, we observed glomeruli collapse. Retinal vascular branching in the absence of Bim expression had similarities with the ureteric bud including increased vascular length, branching length, segment length, and branching interval. Thus, our studies emphasize the impact appropriate Bim expression has on the overall length and branching in both the ureteric bud and retinal vasculature.

Fingolimod (FTY720), a Sphinogosine-1-Phosphate Receptor Agonist, Mitigates Choroidal Endothelial Proangiogenic Properties and Choroidal Neovascularization.

Neovascular or wet age-related macular degeneration (nAMD) causes vision loss due to inflammatory and vascular endothelial growth factor (VEGF)-driven neovascularization processes in the choroid. Due to the excess in VEGF levels associated with nAMD, anti-VEGF therapies are utilized for treatment. Unfortunately, not all patients have a sufficient response to such therapies, leaving few if any other treatment options for these patients. Sphingosine-1-phosphate (S1P) is a bioactive lipid mediator found in endothelial cells that participates in modulating barrier function, angiogenesis, and inflammation. S1P, through its receptor (S1PR1) in endothelial cells, prevents illegitimate sprouting angiogenesis during vascular development. In the present paper, we show that, in choroidal endothelial cells, S1PR1 is the most abundantly expressed S1P receptor and agonism of S1PR1-prevented choroidal endothelial cell capillary morphogenesis in culture. Given that nAMD pathogenesis draws from enhanced inflammation and angiogenesis as well as a loss of barrier function, we assessed the impact of S1PR agonism on choroidal neovascularization in vivo. Using laser photocoagulation rupture of Bruch's membrane to induce choroidal neovascularization, we show that S1PR non-selective (FTY720) and S1PR1 selective (CYM5442) agonists significantly inhibit choroidal neovascularization in this model. Thus, utilizing S1PR agonists to temper choroidal neovascularization presents an additional novel use for these agonists presently in clinical use for multiple sclerosis as well as other inflammatory diseases.

Regulation of angiogenesis by histone chaperone HIRA-mediated incorporation of lysine 56-acetylated histone H3.3 at chromatin domains of endothelial genes.

Angiogenesis is critically dependent on endothelial cell-specific transcriptional mechanisms. However, the molecular processes that regulate chromatin domains and thereby dictate transcription of key endothelial genes are poorly understood. Here, we report that, in endothelial cells, angiogenic signal-mediated transcriptional induction of Vegfr1 (vascular endothelial growth factor receptor 1) is dependent on the histone chaperone, HIRA (histone cell cycle regulation-defective homolog A). Our molecular analyses revealed that, in response to angiogenic signals, HIRA is induced in endothelial cells and mediates incorporation of lysine 56 acetylated histone H3.3 (H3acK56) at the chromatin domain of Vegfr1. HIRA-mediated incorporation of H3acK56 is a general mechanism associated with transcriptional induction of several angiogenic genes in endothelial cells. Depletion of HIRA inhibits H3acK56 incorporation and transcriptional induction of Vegfr1 and other angiogenic genes. Our functional analyses revealed that depletion of HIRA abrogates endothelial network formation on Matrigel and inhibits angiogenesis in an in vivo Matrigel plug assay. Furthermore, analysis in a laser-induced choroidal neovascularization model showed that depletion of HIRA significantly inhibits neovascularization. Our results for the first time decipher a histone chaperone (HIRA)-dependent molecular mechanism in endothelial gene regulation and indicate that histone chaperones could be new targets for angiogenesis therapy.

CYP1B1 expression promotes the proangiogenic phenotype of endothelium through decreased intracellular oxidative stress and thrombospondin-2 expression.

Reactive species derived from cell oxygenation processes play an important role in vascular homeostasis and the pathogenesis of many diseases including retinopathy of prematurity. We show that CYP1B1-deficient (CYP1B1(-/-)) mice fail to elicit a neovascular response during oxygen-induced ischemic retinopathy. In addition, the retinal endothelial cells (ECs) prepared from CYP1B1(-/-) mice are less adherent, less migratory, and fail to undergo capillary morphogenesis. These aberrant cellular responses were completely reversed when oxygen levels were lowered or an antioxidant added. CYP1B1(-/-) ECs exhibited increased oxidative stress and expressed increased amounts of the antiangiogenic factor thrombospondin-2 (TSP2). Increased lipid peroxidation and TSP2 were both observed in retinas from CYP1B1(-/-) mice and were reversed by administration of an antioxidant. Reexpression of CYP1B1 in CYP1B1(-/-) ECs resulted in down-regulation of TSP2 expression and restoration of capillary morphogenesis. A TSP2 knockdown in CYP1B1(-/-) ECs also restored capillary morphogenesis. Thus, CYP1B1 metabolizes cell products that modulate intracellular oxidative stress, which enhances production of TSP2, an inhibitor of EC migration and capillary morphogenesis. Evidence is presented that similar changes occur in retinal endothelium in vivo to limit neovascularization.

Targeted Thrombospondin-1 Expression in Ocular Vascular Development and Neovascularization.

Tight regulation of positive and negative regulators of angiogenesis is essential, particularly in the eye where their dysregulation can lead to vision loss. Thrombospondin-1 (TSP1) is a matricellular protein that negatively regulates angiogenesis and inflammation in the eye. It aids ocular vascular homeostasis such that its loss contributes to increased retinal vascular density and pathologic ocular neovascularization. Our previous studies demonstrated that mice globally lacking TSP1 expression had increased retinal vascular density, decreased hyperoxia-induced retinal vessel loss, and increased choroidal neovascularization. Here we determined the impact to the ocular vasculature of endothelial cell, pericyte, or astrocyte loss of TSP1 expression. Only lack of TSP1 expression in endothelial cells was sufficient to increase choroidal neovascularization with mice lacking expression in pericytes or astrocytes not demonstrating a significant impact. Although the global TSP1 knockout mice demonstrated increased retinal vascular density, individual cell type loss of TSP1 resulted in decreased retinal endothelial cell numbers before and/or after vascular maturation in a cell type specific fashion. Retinas from mice lacking TSP1 expression in endothelial cells, pericytes or astrocytes were not protected from retinal vessel regression in response to hyperoxia as we previously observed in the global knockout. Thus, modulation of TSP1 expression in individual cell types demonstrates a response that is unique to the role TSP1 plays in that cell type of interest, and their coordinated activity is critical for vision.

Caffeine Inhibits Choroidal Neovascularization Through Mitigation of Inflammatory and Angiogenesis Activities.

Adenosine receptors (AR) are widely expressed in a variety of tissues including the retina and brain. They are involved in adenosine-mediated immune responses underlying the onset and progression of neurodegenerative diseases. The expression of AR has been previously demonstrated in some retinal cells including endothelial cells and retinal pigment epithelial cells, but their expression in the choroid and choroidal cells remains unknown. Caffeine is a widely consumed AR antagonist that can influence inflammation and vascular cell function. It has established roles in the treatment of neonatal sleep apnea, acute migraine, and post lumbar puncture headache as well as the neurodegenerative diseases such as Parkinson and Alzheimer. More recently, AR antagonism with caffeine has been shown to protect preterm infants from ischemic retinopathy and retinal neovascularization. However, whether caffeine impacts the development and progression of ocular age-related diseases including neovascular age-related macular degermation remains unknown. Here, we examined the expression of AR in retinal and choroidal tissues and cells. We showed that antagonism of AR with caffeine or istradefylline decreased sprouting of thoracic aorta and choroid/retinal pigment epithelium explants in ex vivo cultures, consistent with caffeine's ability to inhibit endothelial cell migration in culture. In vivo studies also demonstrated the efficacy of caffeine in inhibition of choroidal neovascularization and mononuclear phagocyte recruitment to the laser lesion sites. Istradefylline, a specific AR 2A antagonist, also decreased choroidal neovascularization. Collectively, our studies demonstrate an important role for expression of AR in the choroid whose antagonism mitigate choroidal inflammatory and angiogenesis activities.