Pathological Retinal Neovascularization Research Articles

In Vitro Effects of Bevacizumab Treatment on Newborn Rat Retinal Cell Proliferation, Death, and Differentiation

Vascular endothelial growth factor (VEGF) is an important signal protein in vertebrate nervous development, promoting neurogenesis, neuronal patterning, and glial cell growth. Bevacizumab, an anti-VEGF agent, has been extensively used for controlling pathological retinal neovascularization in adult and newborn patients, although its effect on the developing retina remains largely unknown. The purpose of this study was to investigate the effect of bevacizumab on cell death, proliferation, and differentiation in newborn rat retina. Retinal explants of sixty 2-day-old Lister hooded rats were obtained after eye enucleation and maintained in culture media with or without bevacizumab for 2 days. Immunohistochemical staining was assessed against proliferating cell nuclear antigen (PCNA, to detect cell proliferation); caspase-3 and beclin-1 (to investigate cell death); and vimentin and glial fibrillary acidic protein (GFAP, markers of glial cells). Gene expressions were quantified by real-time reverse-transcription polymerase chain reaction. Results from treatment and control groups were compared. No significant difference in the staining intensity (on immunohistochemistry) of PCNA, caspase-3, beclin-1, and GFAP, or in the levels of PCNA, caspase-3, beclin-1, and vimentin mRNA was observed between the groups. However, a significant increase in vimentin levels and a significant decrease in GFAP mRNA expression were observed in bevacizumab-treated retinal explants compared with controls. Bevacizumab did not affect cell death or proliferation in early developing rat retina but appeared to interfere with glial cell maturation by increasing vimentin levels and downregulating GFAP gene expression. Thus, we suggest anti-VEGF agents be used with caution in developing retinal tissue.

Abstract 19123: Brag2, An Arf6 Gef, Regulates Integrin-dependent Adhesion in Endothelial Cells and is Involved in Angiogenesis

β1-integrins are essential for angiogenesis. However, the mechanisms regulating integrin function in endothelial cells (EC) and their contribution to angiogenesis are unclear. Brag2 is a guanine nucleotide exchange factor for the small GTPase ARF6, which is involved in the traffic of receptors and in the regulation of actin cytoskeleton. Here, we demonstrate that siRNA-mediated silencing of Brag2 reduced basal and VEGF-induced activation of Arf6 and significantly blocked basal and VEGF-induced angiogenic sprouting (by 40 ± 7 %), tube formation and 3-dimensional migration of EC. Transfection with Brag2 siRNA increased focal adhesions and enhanced adhesion of EC on β1-integrin ligands, such as fibronectin and collagen. In line with these results, silencing of Brag2 significantly increased the surface expression of the α5β1-integrin. Moreover, silencing of Brag2 significantly reduced the endocytosis of β1-integrins and specifically of the active (matrix bound) α5β1-integrin present in focal adhesions, suggesting that Brag2 may contribute to the disassembly of focal adhesions during migration via β1-integrin-endocytosis. Strikingly, in vivo silencing of the Brag2 orthologues in zebrafish embryos with morpholinos perturbed vascular development by leading to defects of the parachordal vessels, the dorsal longitudinal anastomotic vessel and the intersomitic vessels. Beyond developmental angiogenesis, in vivo silencing of Brag2 with intravitreal injection of shRNA significantly reduced pathological retinal neovascularization in the model of retinopathy of prematurity (32 % inhibition) and pathological choroidal neovascularization in a model of laser-induced choroidal neovascularization. These data reveal that the Arf6 GEF, Brag2 is essential for developmental and pathological angiogenesis probably by mediating EC migration and angiogenic sprouting through regulation of adhesion by promoting integrin internalization.

Effects of bevacizumab on newborn rat retinal proteoglycans gene expression and on cell proliferation, death and differentiation, in vitro

AbstractPurpose VEGF is an important in nervous development, promoting neurogenesis and glial cell growth. Bevacizumab (BVZ), an anti‐VEGF agent, has been extensively used for controlling pathological retinal neovascularization in adult and newborn patients, although its effect on the developing retina remains largely unknown. In this study we investigate BVZ effect on proteoglycans and on cell death, proliferation and differentiation in newborn rat retina.Methods Retinal explants of sixty 2‐day‐old Lister hooded rats were maintained in culture media with or without BVZ for 2 days. Immunohistochemical staining was assessed against proliferating cell nuclear antigen (PCNA), caspase‐3 and beclin‐1 and vimentin and glial fibrillary acidic protein (GFAP). Gene expressions for PCNA, caspase‐3, beclin‐1, vimentin, GFAP and for proteoglycans were quantified by real‐time reverse‐transcription polymerase chain reaction. Results from treatment and control groups were comparedResults No significant difference in staining intensity of PCNA, caspase‐3, beclin‐1 and GFAP or in the mRNA levels of PCNA, caspase‐3, beclin‐1, vimentin, neurocan and phosphacan was observed. However, a significant increase in vimentin levels, decrease in GFAP and in syndecan‐3 mRNA expression were observed in BVZ‐treated retinas compared to controls.Conclusion Bevacizumab did not affect cell death or proliferation in early developing rat retina but appeared to interfere with glial cell maturation (by interfering with vimentin and GFAP). It can also alter syndecan‐3 gene expression. Thus, we suggest anti‐VEGF agents be used with caution in developing retinal tissue.

Propranolol Inhibition of β-Adrenergic Receptor Does Not Suppress Pathologic Neovascularization in Oxygen-Induced Retinopathy

Retinopathy of prematurity (ROP) is a leading cause of blindness in children and is, in its most severe form, characterized by uncontrolled growth of vision-threatening pathologic vessels. Propranolol, a nonselective β-adrenergic receptor blocker, was reported to protect against pathologic retinal neovascularization in a mouse model of oxygen-induced retinopathy (OIR). Based on this single animal study using nonstandard evaluation of retinopathy, clinical trials are currently ongoing to evaluate propranolol treatment in stage 2 ROP patients who tend to experience spontaneous disease regression and are at low risk of blindness. Because these ROP patients are vulnerable premature infants who are still in a fragile state of incomplete development, the efficacy of propranolol treatment in retinopathy needs to be evaluated thoroughly in preclinical animal models of retinopathy and potential benefits weighed against potential adverse effects. Retinopathy was induced by exposing neonatal mice to 75% oxygen from postnatal day (P) 7 to P12. Three routes of propranolol treatment were assessed from P12 to P16: oral gavage, intraperitoneal injection, or subcutaneous injection, with doses varying between 2 and 60 mg/kg/day. At P17, retinal flatmounts were stained with isolectin and quantified with a standard protocol to measure vasoobliteration and pathologic neovascularization. Retinal gene expression was analyzed with qRT-PCR using RNA isolated from retinas of control and propranolol-treated pups. None of the treatment approaches at any dose of propranolol (up to 60 mg/kg/day) were effective in preventing the development of retinopathy in a mouse model of OIR, evaluated using standard techniques. Propranolol treatment also did not change retinal expression of angiogenic factors including vascular endothelial growth factor. Propranolol treatment via three routes and up to 30 times the standard human dose failed to suppress retinopathy development in mice. These data bring into question whether propranolol through inhibition of β-adrenergic receptors is an appropriate therapeutic approach for treating ROP.

Candesartan, an angiotensin II type 1 receptor antagonist, inhibits pathological retinal neovascularization by downregulating VEGF receptor-2 expression

Several studies have examined the anti-angiogenic effects of angiotensin II type 1 (AT1) receptor antagonists; however, the mechanisms underlying these effects are currently unclear. In the present study, we examined the efficacy and the mechanism of candesartan, an AT1 receptor antagonist, in suppressing pathological retinal neovascularization. We used an in vivo murine oxygen-induced retinopathy (OIR) model and also studied the in vitro proliferation and migration of human retinal microvascular endothelial cells (HRMECs) induced by vascular endothelial growth factor (VEGF)-A. The regulation of angiogenesis-associated genes such as hypoxia-inducible factor (HIF-1α), VEGF-A, VEGF receptor-1, and VEGF receptor-2 was evaluated with real-time RT-PCR in the OIR model. In the OIR model, candesartan suppressed the pathological neovascularization in a dose-dependent manner, but did not prevent the physiological angiogenesis. However, candesartan did not inhibit VEGF-A-induced proliferation or migration in HRMECs in the in vitro study. When administered interperitoneally in the OIR model, candesartan reduced the upregulation of VEGF receptor-2 in the retina, but had no effects in the other angiogenesis-related genes, such as HIF-1α, VEGF-A, and VEGF receptor-1. These findings indicate that candesartan inhibited the retinal pathological neovascularization, at least in part, by suppressing the expression of VEGF receptor-2, independent of VEGF signaling cascade. Therefore, candesartan may be a useful therapeutic target for the inhibition of retinal neovascularization that has a low risk of serious side effects.

MEF2C Ablation in Endothelial Cells Reduces Retinal Vessel Loss and Suppresses Pathologic Retinal Neovascularization in Oxygen-Induced Retinopathy

Ischemic retinopathies, including retinopathy of prematurity and diabetic retinopathy, are major causes of blindness. Both have two phases, vessel loss and consequent hypoxia-driven pathologic retinal neovascularization, yet relatively little is known about the transcription factors regulating these processes. Myocyte enhancer factor 2 (MEF2) C, a member of the MEF2 family of transcription factors that plays an important role in multiple developmental programs, including the cardiovascular system, seems to have a significant functional role in the vasculature. We, therefore, generated endothelial cell (EC)-specific MEF2C-deficient mice and explored the role of MEF2C in retinal vascularization during normal development and in a mouse model of oxygen-induced retinopathy. Ablation of MEF2C did not cause appreciable defects in normal retinal vascular development. However, MEF2C ablation in ECs suppressed vessel loss in oxygen-induced retinopathy and strongly promoted vascular regrowth, consequently reducing retinal avascularity. This finding was associated with suppression of pathologic retinal angiogenesis and blood-retinal barrier dysfunction. MEF2C knockdown in cultured retinal ECs using small-interfering RNAs rescued ECs from death and stimulated tube formation under stress conditions, confirming the endothelial-autonomous and antiangiogenic roles of MEF2C. HO-1 was induced by MEF2C knockdown in vitro and may play a role in the proangiogenic effect of MEF2C knockdown on retinal EC tube formation. Thus, MEF2C may play an antiangiogenic role in retinal ECs under stress conditions, and modulation of MEF2C may prevent pathologic retinal neovascularization.

Polyethylene Glycol-Modified Pigment Epithelial-Derived Factor: New Prospects for Treatment of Retinal Neovascularization

Pathological retinal neovascularization and choroidal neovascularization are major causes of vision loss in a variety of clinical conditions, such as retinopathy of prematurity, age-related macular degeneration, and diabetic retinopathy. Pigment epithelial-derived factor (PEDF) has been found to be the most potent natural, endogenous inhibitor of neovascularization, but its application is restricted because of its instability and short half-life. Polyethylene glycol (PEG) has been used as a drug carrier to slow clearance rate for decades. The present study investigated PEGylated-PEDF for the first time and evaluated its long-term effects on preventing angiogenesis in vitro and in vivo. PEG showed lower cytotoxicity to human umbilical vein endothelial cells (HUVECs). In vitro, PEGylated-PEDF inhibited HUVEC proliferation, migration, tube formation, and vascular endothelium growth factor secretion and induced HUVEC apoptosis in a dose-dependent manner, and it showed a statistically significant difference compared with the PEDF treatment group. In vivo, PEGylated-PEDF had a long-lasting effect in both plasma and retinal concentrations. In an oxygen-induced retinopathy model, one intravitreous injection of PEGylated-PEDF after mouse pups were moved into room air resulted in a significant difference in the inhibition of retinal neovascularization, which decreased the nonperfusion area, compared with the PEDF-treated group. Our present study demonstrated for the first time the long-term inhibitory effects of PEGylated-PEDF on the prevention of neovascularization in vitro and in vivo. These data suggest that PEGylated-PEDF could offer an innovative therapeutic strategy for preventing retinal neovascularization.

Wnt Signaling Mediates Pathological Vascular Growth in Proliferative Retinopathy

Ischemic proliferative retinopathy, characterized by pathological retinal neovascularization, is a major cause of blindness in working-age adults and children. Defining the molecular pathways distinguishing pathological neovascularization from normal vessels is critical to controlling these blinding diseases with targeted therapy. Because mutations in Wnt signaling cause defective retinal vasculature in humans with some characteristics of the pathological vessels in retinopathy, we investigated the potential role of Wnt signaling in pathological retinal vascular growth in proliferative retinopathy. In this study, we show that Wnt receptors (Frizzled4 and low-density lipoprotein receptor-related protein5 [Lrp5]) and activity are significantly increased in pathological neovascularization in a mouse model of oxygen-induced proliferative retinopathy. Loss of Wnt coreceptor Lrp5 and downstream signaling molecule dishevelled2 significantly decreases the formation of pathological retinal neovascularization in retinopathy. Loss of Lrp5 also affects retinal angiogenesis during development and formation of the blood-retinal barrier, which is linked to significant downregulation of tight junction protein claudin5 in Lrp5(-/-) vessels. Blocking claudin5 significantly suppresses Wnt pathway-driven endothelial cell sprouting in vitro and developmental and pathological vascular growth in retinopathy in vivo. These results demonstrate an important role of Wnt signaling in pathological vascular development in retinopathy and show a novel function of Cln5 in promoting angiogenesis.

A PEDF-Derived Peptide Inhibits Retinal Neovascularization and Blocks Mobilization of Bone Marrow-Derived Endothelial Progenitor Cells

Proliferative diabetic retinopathy is characterized by pathological retinal neovascularization, mediated by both angiogenesis (involving mature endothelial cells) and vasculogenesis (involving bone marrow-derived circulating endothelial progenitor cells (EPCs)). Pigment epithelium-derived factor (PEDF) contains an N-terminal 34-amino acid peptide (PEDF-34) that has antiangiogenic properties. Herein, we present a novel finding that PEDF-34 also possesses antivasculogenic activity. In the oxygen-induced retinopathy (OIR) model using transgenic mice that have Tie2 promoter-driven GFP expression, we quantified Tie2GFP+ cells in bone marrow and peripheral blood by fluorescence-activated cell sorting (FACS). OIR significantly increased the number of circulating Tie2-GFP+ at P16, correlating with the peak progression of neovascularization. Daily intraperitoneal injections of PEDF-34 into OIR mice decreased the number of Tie2-GFP+ cells in the circulation at P16 by 65% but did not affect the number of Tie2-GFP+ cells in the bone marrow. These studies suggest that PEDF-34 attenuates EPC mobilization from the bone marrow into the blood circulation during retinal neovascularization.

Nanoceria Inhibit the Development and Promote the Regression of Pathologic Retinal Neovascularization in the Vldlr Knockout Mouse

Many neurodegenerative diseases are known to occur and progress because of oxidative stress, the presence of reactive oxygen species (ROS) in excess of the cellular defensive capabilities. Age related macular degeneration (AMD), diabetic retinopathy (DR) and inherited retinal degeneration share oxidative stress as a common node upstream of the blinding effects of these diseases. Knockout of the Vldlr gene results in a mouse that develops intraretinal and subretinal neovascular lesions within the first month of age and is an excellent model for a form of AMD called retinal angiomatous proliferation (RAP). Cerium oxide nanoparticles (nanoceria) catalytically scavenge ROS by mimicking the activities of superoxide dismutase and catalase. A single intravitreal injection of nanoceria into the Vldlr-/- eye was shown to inhibit: the rise in ROS in the Vldlr-/- retina, increases in vascular endothelial growth factor (VEGF) in the photoreceptor layer, and the formation of intraretinal and subretinal neovascular lesions. Of more therapeutic interest, injection of nanoceria into older mice (postnatal day 28) resulted in the regression of existing vascular lesions indicating that the pathologic neovessels require the continual production of excessive ROS. Our data demonstrate the unique ability of nanoceria to prevent downstream effects of oxidative stress in vivo and support their therapeutic potential for treatment of neurodegenerative diseases such as AMD and DR.

PLD1-dependent PKCγ activation downstream to Src is essential for the development of pathologic retinal neovascularization

AbstractVascular endothelial growth factor (VEGF) appears to be an important mediator of pathologic retinal angiogenesis. In understanding the mechanisms of pathologic retinal neovascularization, we found that VEGF activates PLD1 in human retinal microvascular endothelial cells, and this event is dependent on Src. In addition, VEGF activates protein kinase C-γ (PKCγ) via Src-dependent PLD1 stimulation. Inhibition of Src, PLD1, or PKCγ via pharmacologic, dominant negative mutant, or siRNA approaches significantly attenuated VEGF-induced human retinal microvascular endothelial cell migration, proliferation, and tube formation. Hypoxia also induced Src-PLD1-PKCγ signaling in retina, leading to retinal neovascularization. Furthermore, siRNA-mediated down-regulation of VEGF inhibited hypoxia-induced Src-PLD1-PKCγ activation and neovascularization. Blockade of Src-PLD1-PKCγ signaling via the siRNA approach also suppressed hypoxia-induced retinal neovascularization. Thus, these observations demonstrate, for the first time, that Src-dependent PLD1-PKCγ activation plays an important role in pathologic retinal angiogenesis.

Docosahexaenoic Acid Improves the Nitroso-Redox Balance and Reduces VEGF-Mediated Angiogenic Signaling in Microvascular Endothelial Cells

Disturbances to the cellular production of nitric oxide (NO) and superoxide (O(2)(-)) can have deleterious effects on retinal vascular integrity and angiogenic signaling. Dietary agents that could modulate the production of these signaling molecules from their likely enzymatic sources, endothelial nitric oxide synthase (eNOS) and NADPH oxidase, would therefore have a major beneficial effect on retinal vascular disease. The effect of ω-3 polyunsaturated fatty acids (PUFAs) on angiogenic signaling and NO/superoxide production in retinal microvascular endothelial cells (RMECs) was investigated. Primary RMECs were treated with docosahexaenoic acid (DHA) or eicosapentaenoic acid (EPA) for 48 hours. RMEC migration was determined by scratch-wound assay, proliferation by the incorporation of BrdU, and angiogenic sprouting using a three-dimensional model of in vitro angiogenesis. NO production was quantified by Griess assay, and phospho-eNOS accumulation and superoxide were measured using the fluorescent probe dihydroethidine. eNOS localization to caveolin-rich microdomains was determined by Western blot analysis after subfractionation on a linear sucrose gradient. DHA treatment increased nitrite and decreased superoxide production, which correlated with the displacement of eNOS from caveolar subdomains and colocalization with the negative regulator caveolin-1. In addition, both ω-3 PUFAs demonstrated reduced responsiveness to VEGF-stimulated superoxide and nitrite release and significantly impaired endothelial wound healing, proliferation, and angiogenic sprout formation. DHA improves NO bioavailability, decreases O(2)(-) production, and blunts VEGF-mediated angiogenic signaling. These findings suggest a role for ω-3 PUFAs, particularly DHA, in maintaining vascular integrity while reducing pathologic retinal neovascularization.

Pathological Neovascularization Is Reduced by Inactivation of ADAM17 in Endothelial Cells but Not in Pericytes

Pathological neovascularization is a critical component of diseases such as proliferative retinopathies, cancer and rheumatoid arthritis, yet much remains to be learned about the underlying causes. Previous studies showed that vascular endothelial growth factor (VEGF)-A activates the membrane-anchored metalloproteinase ADAM17 (a disintegrin and metalloproteinase 17) in endothelial cells, thereby stimulating crosstalk between VEGF receptor 2 and extracellular signal-regulated kinase. These findings raised interesting questions about the role of ADAM17 in angiogenesis and neovascularization in vivo. The objective of this study was to inactivate ADAM17 in endothelial cells or in pericytes to determine how this affects developmental angiogenesis, pathological retinal neovascularization and heterotopic tumor growth. We generated animals in which floxed ADAM17 was removed by Tie2-Cre in endothelial cells, or by smooth muscle (sm) Cre in smooth muscle cells and pericytes. There were no evident developmental defects in either conditional knockout strain, but pathological retinal neovascularization and growth of heterotopically injected tumor cells was reduced in Adam17flox/flox/Tie2-Cre mice, although not in Adam17flox/flox/sm-Cre mice. Moreover, lack of ADAM17 in endothelial cells decreased ex vivo chord formation, and this could be largely restored by addition of the ADAM17 substrate HB-EGF (heparin-binding epidermal growth factor-like growth factor). Finally we found that ADAM17 is important for the VEGF receptor 2 stimulated processing of several receptors with known functions in endothelial cell biology. These results provide the first evidence for a role for ADAM17 in pathological neovascularization in vivo. Because ADAM17 does not appear to be required for normal developmental angiogenesis or vascular homeostasis, it could emerge as a good target for treatment of pathological neovascularization.

Endothelial Progenitor Cells (EPCs) Mobilized and Activated by Neurotrophic Factors May Contribute to Pathologic Neovascularization in Diabetic Retinopathy

Diabetic retinopathy is characterized by pathological retinal neovascularization. Accumulating evidence has indicated that high levels of circulating endothelial progenitor cells (EPCs) are an important risk factor for neovascularization. Paradoxically, the reduction and dysfunction of circulating EPCs has been extensively reported in diabetic patients. We hypothesized that EPCs are differentially altered in the various vasculopathic complications of diabetes mellitus, exhibiting distinct behaviors in terms of angiogenic response to ischemia and growth factors and potentially playing a potent role in motivating vascular precursors to induce pathological neovascularization. Circulating levels of EPCs from diabetic retinopathy patients were analyzed by flow cytometry and by counting EPC colony-forming units, and serum levels of neurotrophic factors were measured by enzyme-linked immunosorbent assay. We found increased levels of nerve growth factor and brain-derived neurotrophic factor in the blood of diabetic retinopathy patients; this increase was correlated with the levels of circulating EPCs. In addition, we demonstrated that retinal cells released neurotrophic factors under hypoxic conditions to enhance EPC activity in vitro and to increase angiogenesis in a mouse ischemic hindlimb model. These results suggest that neurotrophic factors may induce neoangiogenesis through EPC activation, leading to the pathological retinal neovascularization. Thus, we propose that neovascularization in the ischemic retina might be regulated by overexpression of neurotrophic factors.

Pleiotropic Effects of YC-1 Selectively Inhibit Pathological Retinal Neovascularization and Promote Physiological Revascularization in a Mouse Model of Oxygen-Induced Retinopathy

Vascular endothelial growth factor (VEGF) and inducible nitric-oxide synthase (iNOS) have been implicated in ischemia-induced retinal neovascularization. Retinal ischemia has been shown to induce VEGF and iNOS expression. It has been postulated that one of the crucial consequences of iNOS expression in the ischemic retina is the inhibition of angiogenesis. Furthermore, iNOS was shown to be overexpressed in Müller cells from patients with diabetic retinopathy. YC-1, a small molecule inhibitor of hypoxia-inducible factor (HIF)-1 alpha, has been shown to inhibit iNOS expression in various tissue models. Our aim was to assess the pleiotropic effects of YC-1 in an oxygen-induced retinopathy (OIR) mouse model and evaluate its therapeutic potential in HIF-1- and iNOS-mediated retinal pathologies. Dual-injections of YC-1 into the neovascular retinas decreased the total retinopathy score, inhibited vaso-obliteration and pathologic tuft formation, and concomitantly promoted physiological retinal revascularization, compared with dimethyl sulfoxide (DMSO)-treated group. Furthermore, YC-1-treated retinas exhibited a marked increase in immunoreactivities for CD31 and von Willebrand factor and displayed significant inhibition in HIF-1alpha protein expression. Furthermore, YC-1 down-regulated VEGF, erythropoietin, endothelin-1, matrix metalloproteinase-9, and iNOS message and protein levels. When hypoxic Müller and neuoroglial cells were treated with YC-1, iNOS mRNA and protein levels were reduced in a dose-dependent fashion. We demonstrate that YC-1 inhibits pathological retinal neovascularization by exhibiting antineovascular activities, which impaired ischemia-induced expression of HIF-1 and its downstream angiogenic molecules. Furthermore, YC-1 enhanced physiological revascularization of the retinal vascular plexuses via the inhibition of iNOS mRNA and protein expressions. The pleiotropic effects of YC-1 allude to its possible use as a promising therapeutic iNOS inhibitor candidate for the treatment of retinal neovascularization.

TERT-siRNA inhibits oxygen-induced retinal neovascularization in mice

To investigate the inhibitory effect of small interfering RNA (siRNA) targeting TERT on murine retinal neovascularization and explore the feasibility of potential therapeutic approach in retinal vascular disease. Two recombinant plasmids TERT siRNA (pSIREN-mTERT-1) and negative plasmid (pSIREN-mTERT-N) were constructed and 80 seven-day-old C57BL/6J mice were divided randomly into therapeutic group (A), negative plasmid group (B), oxygen-induced retinopathy group (C) and normal control group (D), 20 mice in each group. Group A, B and C were exposed to 75% +/- 2% oxygen for 5 days and then to room air, which induced mice retinal neovascularization. Groups A and B were injected two kinds of the above recombinant plasmid into the murine vitreous on the 12th day. The mice of group D were raised in normal oxygen circumstance. On the 19th day, 2% Evens blue angiography was used to observe the pattern of the retinal vascular. Expression of TERT mRNA were confirmed by reverse-transcription polymerase chain reaction (RT-PCR) and Real-time PCR. Histological observation and vascular endothelial cells counting were used to examine the effects of siRNA on the retinal neovascularization. Retinal flat after Evans blue angiography indicated that the vessels of group A formed a fine radial branching pattern, which was similar to normal mice. In group A, the retinal neovascularization reduced and the structure of retina were more regular than group B and C. At the same time the large vessels were distorted, neovascular clusters proliferated and fluorescence leaked in the middle and periphery area in group B and C. RT-PCR and Real-time PCR showed the expression of TERT mRNA was downregulated in group A compared with groups B and C (P < 0.05). Paraffin tissue slice with hematoxylin-eosin staining showed that the average counts of vascular endothelial cells which break through the inner limiting membrane in group A were less than groups B and C, the differences were significant (P < 0.05). Pathologic retinal neovascularization can be inhibited by specific TERT siRNA in vivo, which may be a novel efficient strategy against proliferative vasculopathies.

Hypoxia-Induced Annexin A2 Expression Regulates Oxygen-Induced Retinopathy.

Abstract 471Annexin A2 (A2), a Ca2+-dependent membrane binding protein that usually complexes with a S100 protein, p11, regulates fibrinolysis by serving as an endothelial cell co-receptor for plasminogen and tissue plasminogen activator to promote plasmin generation. Compared to A2+/+ mice, A2-/- mice have reduced post-hyperoxic retinal neovascularization in the model of oxygen-induced retinopathy (OIR) that mimics human retinopathy of prematurity. Here, we sought to determine the mechanism by which A2 contributes to the pathogenesis of OIR. A2+/+ mouse pups (P7) were placed with their dams in a high O2 (75%) chamber for 5 consecutive days (until P12), and then rapidly shifted to room air (21% O2) for several more days. Mouse retinas were isolated over a range of time points for analysis of neovascularization, A2 protein and mRNA expression, fibrin deposition, cell proliferation and apoptosis assays. In post-hyperoxic A2+/+ mice, we noted extensive retinal neovascularization along with a more than 10-fold increase of VEGFA compared to retinas of mice maintained in room air. Both A2 protein and mRNA levels increased 2-4-fold in mouse retinas with OIR in comparison with expression of either CD31 or VE-cadherin 5. p11 and hypoxia-inducible factor-1α (HIF-1α) protein, but not mRNA levels, also increased 2-4-fold during OIR. Because increased A2 expression was predominantly associated with retinal neovessels, we examined the response of cultured endothelial cells to hypoxia, and observed a 2-4-fold increase in A2 protein and mRNA. Although HIF-1α stabilization was not affected by A2 deficiency, upregulation of A2 by hypoxia was HIF-1α dependent. Expression of A6, on the other hand, was not upregulated by hypoxia. Neither erythropoietin (EPO) nor VEGFA signaling changed A2 expression. In addition, hypoxic endothelial cells translocated 50% more A2 protein to the cell surface, generated 30% more plasmin, and migrated twice as efficiently. While stability of A2 mRNA was not changed by hypoxia-related stimuli, we observed direct binding between HIF-1α and the A2 promoter by electrophoretic mobility shift assay, and a 3-4-fold increased A2 promoter activity in the presence of HIF-1α and HIF-1β by luciferase reporter assay. Promoter activation was abrogated when the HIF-1α binding site within the A2 promoter was mutated. By immunofluorescent staining of post-hyperoxic P17 retinal sections, we found that infiltration of endothelial cells and pericytes was 2-fold greater in A2+/+ mice as compared to A2-/- mice. In addition, there was significantly less fibrin deposition, more macrophage recruitment, less apoptosis and greater cell proliferation in P17 post-hyperoxic retinas of A2+/+ mice as compared to A2-/- mice. In conclusion, annexin A2 is upregulated by hypoxia through direct HIF-1α-mediated transcriptional regulation. Upregulated A2 appears to enhance plasmin generation, facilitate directed migration of vascular endothelial cells, and enable the recruitment of supporting cells (e.g. pericytes) and inflammatory cells (e.g. macrophages). It also reduces fibrin deposition, inhibits apoptosis, and promotes cell proliferation, thereby promoting pathologic oxygen-induced retinal neovascularization. Disclosures:No relevant conflicts of interest to declare.

Thiazolidinediones Reduce Pathological Neovascularization in Ischemic Retina Via an Adiponectin-Dependent Mechanism

Background- The insulin-sensitizing agents referred to as thiazolidinediones (TZDs) possess antiatherogenic and anti-inflammatory actions that contribute to protection against diabetic macrovascular complications. However, little is known about the effects of TZDs on retinal microvessel disorders. To investigate whether TZDs modulate retinal vessel formation in a mouse model of oxygen-induced retinopathy. Neonatal mice were subjected to ischemia-induced retinopathy to produce pathological neovascular tuft formation. Pioglitazone, 10 mg/kg per day, rosiglitazone, 10 mg/kg per day, or vehicle was given by gavage once a day from postnatal day 7 to postnatal day 17. Systemic treatment of wild-type (WT) mice with TZDs led to a significant decrease in pathological retinal neovascularization during ischemia compared with vehicle treatment, which was accompanied by increased plasma levels of the fat-derived hormone adiponectin (APN). In contrast to WT mice, TZDs had no effects on ischemia-induced pathological retinal vessel formation in APN-knockout (KO) mice. Pioglitazone reduced tumor necrosis factor (TNF) alpha expression in ischemic retina in WT mice but not in APN-KO mice. Furthermore, pioglitazone increased plasma APN levels in TNF-alpha-KO mice but did not affect ischemia-induced pathological retinal neovascularization in this strain. These data show that TZDs attenuate pathological retinal microvessel formation through APN-mediated modulation of TNF-alpha production.

The development of the rat model of retinopathy of prematurity

Retinopathy of prematurity (ROP) is a potentially blinding disease affecting premature infants. ROP is characterized by pathological ocular angiogenesis or retinal neovascularization (NV). Models of ROP have yielded much of what is currently known about physiological and pathological blood vessel growth in the retina. The rat provides a particularly attractive and cost effective model of ROP. The rat model of ROP consistently produces a robust pattern of NV, similar to that seen in humans. This model has been used to study gross aspects of angiogenesis. More recently, it has been used to identify and therapeutically target specific genes and molecular mechanisms involved in the angiogenic cascade. As angiogenesis occurs as a complication of many diseases, knowledge gained from these studies has the potential to impact nonocular angiogenic conditions. This article provides historical perspective on the development and use of the rat model of ROP. Key findings generated through the use of this model are also summarized.

Maintaining retinal astrocytes normalizes revascularization and prevents vascular pathology associated with oxygen‐induced retinopathy

Astrocytes are well known modulators of normal developmental retinal vascularization. However, relatively little is known about the role of glial cells during pathological retinal neovascularization (NV), a leading contributor to vision loss in industrialized nations. We demonstrate that the loss of astrocytes and microglia directly correlates with the development of pathological NV in a mouse model of oxygen-induced retinopathy (OIR). These two distinct glial cell populations were found to have cooperative survival effects in vitro and in vivo. The intravitreal injection of myeloid progenitor cells, astrocytes, or astrocyte-conditioned media rescued endogenous astrocytes from degeneration that normally occurs within the hypoxic, vaso-obliterated retina following return to normoxia. Protection of the retinal astrocytes and microglia was directly correlated with accelerated revascularization of the normal retinal plexuses and reduction of pathological intravitreal NV normally associated with OIR. Using astrocyte-conditioned media, several factors were identified that may contribute to the observed astrocytic protection and subsequent normalization of the retinal vasculature, including vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF). Injection of VEGF or bFGF at specific doses rescued the retinas from developing OIR-associated pathology, an effect that was also preceded by protection of endogenous glia from hypoxia-induced degeneration. Together, these data suggest that vascular-associated glia are also required for normalized revascularization of the hypoxic retina. Methods developed to target and protect glial cells may provide a novel strategy by which normalized revascularization can be promoted and the consequences of abnormal NV in retinal vascular diseases can be prevented.