Laser-induced Choroidal Neovascularization Model Research Articles

Efficient Delivery of siRNA by Atelocollagen in a Murine Laser-Induced Choroidal Neovascularization Model

Purpose: Previous studies have shown that small interfering RNAs (siRNAs) could suppress angiogenesis via stimulation of toll-like receptor-3 (TLR3). The purpose of this study was to determine the efficacy of atelocollagen to deliver siRNA without TLR3 stimulation in the laser-induced choroidal neovascularization (CNV) model. Methods: CNV was induced by laser injury in C57BL/6J mice and volumes were measured 7 days later. Nontargeted siRNA, 21-nucleotide (nt) siRNA-Luc (Luciferase) and 21-nt siRNA-Vegfa were injected into the vitreous following injury. Atelocollagen was incubated with naked 21-nt siRNAs before injection. To block TLR3 endosomal activity, chloroquine was injected intravitreously after laser injury. Results: The mean CNV volumes were significantly smaller in the naked siRNA-Luc, naked siRNA-Vegfa, or siRNA-Vegfa/atelocollagen complex compared with PBS, atelocollagen or siRNA-Luc/atelocollagen complex-injected mice (p < 0.05). Conclusion: These findings demonstrate that atelocollagen may deliver siRNA without nonspecific TLR3 stimulation in the murine laser-CNV model.

Activation of Rap1 inhibits NADPH oxidase‐dependent ROS generation in retinal pigment epithelium and reduces choroidal neovascularization

Activation of Rap1 GTPase can improve the integrity of the barrier of the retina pigment epithelium (RPE) and reduce choroidal neovascularization (CNV). Inhibition of NADPH oxidase activation also reduces CNV. We hypothesize that Rap1 inhibits NADPH oxidase-generated ROS and thereby reduces CNV formation. Using a murine model of laser-induced CNV, we determined that reduced Rap1 activity in RPE/choroid occurred with CNV formation and that activation of Rap1 by 2'-O-Me-cAMP (8CPT)-reduced laser-induced CNV via inhibiting NADPH oxidase-generated ROS. In RPE, inhibition of Rap1 by Rap1 GTPase-activating protein (Rap1GAP) increased ROS generation, whereas activation of Rap1 by 8CPT reduced ROS by interfering with the assembly of NADPH oxidase membrane subunit p22phox with NOX4 or cytoplasmic subunit p47phox. Activation of NADPH oxidase with Rap1GAP reduced RPE barrier integrity via cadherin phosphorylation and facilitated choroidal EC migration across the RPE monolayer. Rap1GAP-induced ROS generation was inhibited by active Rap1a, but not Rap1b, and activation of Rap1a by 8CPT in Rap1b(-/-) mice reduced laser-induced CNV, in correlation with decreased ROS generation in RPE/choroid. These findings provide evidence that active Rap1 reduces CNV by interfering with the assembly of NADPH oxidase subunits and increasing the integrity of the RPE barrier.

Rap1 GTPase Activation and Barrier Enhancement in RPE Inhibits Choroidal Neovascularization In Vivo

Loss of barrier integrity precedes the development of pathologies such as metastasis, inflammatory disorders, and blood-retinal barrier breakdown present in neovascular age-related macular degeneration. Rap1 GTPase is involved in regulating both endothelial and epithelial cell junctions; the specific role of Rap1A vs. Rap1B isoforms is less clear. Compromise of retinal pigment epithelium barrier function is a contributing factor to the development of AMD. We utilized shRNA of Rap1 isoforms in cultured human retinal pigment epithelial cells, along with knockout mouse models to test the role of Rap1 on promoting RPE barrier properties, with emphasis on the dynamic junctional regulation that is triggered when the adhesion between cells is challenged. In vitro, Rap1A shRNA reduced steady-state barrier integrity, whereas Rap1B shRNA affected dynamic junctional responses. In a laser-induced choroidal neovascularization (CNV) model of macular degeneration, Rap1b−/− mice exhibited larger CNV volumes compared to wild-type or Rap1a−/−. In vivo, intravitreal injection of a cAMP analog (8CPT-2′-O-Me-cAMP) that is a known Rap1 activator significantly reduced laser-induced CNV volume, which correlated with the inhibition of CEC transmigration across 8CPT-2′O-Me-cAMP-treated RPE monolayers in vitro. Rap1 activation by 8CPT-2′-O-Me-cAMP treatment increased recruitment of junctional proteins and F-actin to cell-cell contacts, increasing both the linearity of junctions in vitro and in cells surrounding laser-induced lesions in vivo. We conclude that in vitro, Rap1A may be important for steady state barrier integrity, while Rap1B is involved more in dynamic junctional responses such as resistance to junctional disassembly induced by EGTA and reassembly of cell junctions following disruption. Furthermore, activation of Rap1 in vivo inhibited development of choroidal neovascular lesions in a laser-injury model. Our data suggest that targeting Rap1 isoforms in vivo with 8CPT-2′-O-Me-cAMP may be a viable pharmacological means to strengthen the RPE barrier against the pathological choroidal endothelial cell invasion that occurs in macular degeneration.

The Role of Macrophage Class A Scavenger Receptors in a Laser-Induced Murine Choroidal Neovascularization Model

Laser-induced choroidal neovascularization (CNV) is a widely used model to mimic many features of CNV resulting from wet AMD. Macrophages have been implicated in the pathogenesis of AMD. Class A scavenger receptors, scavenger receptor-A (SR-A) and macrophage receptor with collagenous domain (MARCO), are expressed on macrophages and are associated with macrophage function. The goal of this study is to examine the role of macrophage scavenger receptors in immune cell recruitment and the formation of CNV. Laser photocoagulation was performed in wild-type and knockout mice with deletion of SR-A (SR-A(-/-)), MARCO (MARCO(-/-)), or both SR-A and MARCO double knockout (DKO). Immune cell recruitment at different time points and CNV lesions at 14 days after laser treatment were evaluated through immunostaining and confocal microscopy. Microarray analysis was performed in eyes 1 day after laser injury. Wild-type eyes showed higher chemokine/receptor expression compared with knockout eyes after laser injury. Scavenger receptor deficiency markedly impaired the recruitment of neutrophils and macrophages to CNV lesions at 1- and 3-days post laser injury, respectively. Significantly reduced CNV volumes were found in the eyes from scavenger receptor knockout mice compared with wild-type mice. The deficiency of scavenger receptors impairs the formation of CNV and immune cell recruitment. Our findings suggest a potential role for scavenger receptors in contributing to CNV formation and inflammation in AMD.

Exosomes from Retinal Astrocytes Contain Antiangiogenic Components That Inhibit Laser-induced Choroidal Neovascularization

Exosomes released from different types of host cells have different biological effects. We report that exosomes released from retinal astroglial cells (RACs) suppress retinal vessel leakage and inhibit choroidal neovascularization (CNV) in a laser-induced CNV model, whereas exosomes released from retinal pigmental epithelium do not. RAC exosomes inhibit the migration of macrophages and the tubule forming of mouse retinal microvascular endothelial cells. Further, we analyzed antiangiogenic components in RAC exosomes using an angiogenesis array kit and detected several endogenous inhibitors of angiogenesis exclusively present in RAC exosomes, such as endostatin. Moreover, blockade of matrix metalloproteinases in the cleavage of collagen XVIII to form endostatin using FN-439 reverses RAC exosome-mediated retinal vessel leakage. This study demonstrates that exosomes released from retinal tissue cells have different angiogenic effects, with exosomes from RACs containing antiangiogenic components that might protect the eye from angiogenesis and maintain its functional integrity. In addition, by identifying additional components and their functions of RAC exosomes, we might improve the antiangiogenic therapy for CNV in age-related macular degeneration and diabetic retinopathy.

Long-term efficacy of ciliary muscle gene transfer of three sFlt-1 variants in a rat model of laser-induced choroidal neovascularization

Inhibition of vascular endothelial growth factor (VEGF) has become the standard of care for patients presenting with wet age-related macular degeneration. However, monthly intravitreal injections are required for optimal efficacy. We have previously shown that electroporation enabled ciliary muscle gene transfer results in sustained protein secretion into the vitreous for up to 9 months. Here, we evaluated the long-term efficacy of ciliary muscle gene transfer of three soluble VEGF receptor-1 (sFlt-1) variants in a rat model of laser-induced choroidal neovascularization (CNV). All three sFlt-1 variants significantly diminished vascular leakage and neovascularization as measured by fluorescein angiography (FA) and flatmount choroid at 3 weeks. FA and infracyanine angiography demonstrated that inhibition of CNV was maintained for up to 6 months after gene transfer of the two shortest sFlt-1 variants. Throughout, clinical efficacy was correlated with sustained VEGF neutralization in the ocular media. Interestingly, treatment with sFlt-1 induced a 50% downregulation of VEGF messenger RNA levels in the retinal pigment epithelium and the choroid. We demonstrate for the first time that non-viral gene transfer can achieve a long-term reduction of VEGF levels and efficacy in the treatment of CNV.

A Novel Platelet-Activating Factor Receptor Antagonist Inhibits Choroidal Neovascularization and Subretinal Fibrosis

Choroidal neovascularization (CNV) is a critical pathogenesis in age-related macular degeneration (AMD), the most common cause of blindness in developed countries. To date, the precise molecular and cellular mechanisms underlying CNV have not been elucidated. Platelet-activating factor (PAF) has been previously implicated in angiogenesis; however, the roles of PAF and its receptor (PAF-R) in CNV have not been addressed. The present study reveals several important findings concerning the relationship of the PAF-R signaling with CNV. PAF-R was detected in a mouse model of laser-induced CNV and was upregulated during CNV development. Experimental CNV was suppressed by administering WEB2086, a novel PAF-R antagonist. WEB2086-dependent suppression of CNV occurred via the inhibition of macrophage infiltration and the expression of proangiogenic (vascular endothelial growth factor) and proinflammatory molecules (monocyte chemotactic protein-1 and IL-6) in the retinal pigment epithelium–choroid complex. Additionally, WEB2086-induced PAF-R blockage suppresses experimentally induced subretinal fibrosis, which resembles the fibrotic subretinal scarring observed in neovascular AMD. As optimal treatment modalities for neovascular AMD would target the multiple mechanisms of AMD-associated vision loss, including neovascularization, inflammation and fibrosis, our results suggest PAF-R as an attractive molecular target in the treatment of AMD.

Cathepsin proteases promote angiogenic sprouting and laser-induced choroidal neovascularisation in mice

Cysteine cathepsins are a family of proteases involved in intracellular protein turnover and extracellular matrix degradation. Cathepsin B (Ctsb) and cathepsin Z (Ctsz) promote tumorigenesis and Ctsb is a known modulator of tumor angiogenesis. We therefore investigated the angiomodulatory function of these cathepsins in vitro as well as in a mouse model of laser-induced choroidal neovascularization (laser-CNV). Ctsb−/−, Ctsz−/−, Ctsb/Ctsz double-knockout (Ctsb/z DKO), and wild type (WT) mice underwent argon laser treatment to induce choroidal neovascularization (CNV). The neovascularized area was quantified individually for each lesion at 14 days after laser coagulation. In vitro the effects of cathepsin inhibitors on angiogenesis were analysed by endothelial cell (EC) spheroid sprouting and EC invadosome assays. Retinas from cathepsin KO mice did not show gross morphological abnormalities. In the laser CNV model, however, Ctsb/z DKO mice displayed a significantly reduced neovascularized area compared to WT (0.027 mm2 vs. 0.052 mm2; p = 0.012), while single knockouts did not differ significantly from WT. In line, VEGF-induced EC spheroid sprouting and invadosome formation were not significantly altered by a specific cathepsin B inhibitor alone, but significantly suppressed when more than one cathepsin was inhibited. Our results demonstrate that laser-CNV formation is significantly reduced in Ctsb/z DKO mice. In line, EC sprouting and invadosome formation are blunted when more than one cathepsin is inhibited in vitro. These results reveal an angiomodulatory potential of cathepsins with partial functional redundancies between different cathepsin family members.

Expression of insulin-like growth factor 1 receptor in rat retina following optic nerve injury

To investigate the apoptosis in retinal ganglion cells (RGCs) and insulin-like growth factor 1 receptor (IGF-1R) in the retina following optic nerve crush. Healthy Wistar rats (N = 70) were divided into two groups: a normal control group and an optic nerve injury group. Immunohistochemistry and flow cytometry were performed to detect the expression of IGF-1R and to measure the apoptosis of RGCs, respectively. Immunohistochemistry revealed that at 1 hr after optic nerve injury, IGF-1R immunoreactivity began to increase and reached a maximal level at 24 hr (p < 0.05), where it remained elevated up to 14 days after injury. RGC apoptosis in the normal control group was 0.53%, while the apoptosis rate in the optic nerve injury group increased over time. The apoptosis rate in the optic nerve injury group was 1.4% at 1 hr, 4.4% at 6 hr, 5.2% at 12 hr and reached a maximal level (8.5%) at 24 hr. Subsequently, the rate declined to 1.9% 7 days after injury and 0.9% 2 weeks after injury. The IGF-1R immunereactivity in the retina increased after optic nerve injury. IGF-1R may regulate the apoptosis and regeneration of RGCs at different stages after optic nerve injury.

Transscleral Sustained Vasohibin-1 Delivery by a Novel Device Suppressed Experimentally-Induced Choroidal Neovascularization

We established a sustained vasohibin-1 (a 42-kDa protein), delivery device by a novel method using photopolymerization of a mixture of polyethylene glycol dimethacrylate, triethylene glycol dimethacrylate, and collagen microparticles. We evaluated its effects in a model of rat laser-induced choroidal neovascularization (CNV) using a transscleral approach. We used variable concentrations of vasohibin-1 in the devices, and used an enzyme-linked immunosorbent assay and Western blotting to measure the released vasohibin-1 (0.31 nM/day when using the 10 μM vasohibin-1 delivery device [10VDD]). The released vasohibin-1 showed suppression activity comparable to native effects when evaluated using endothelial tube formation. We also used pelletized vasohibin-1 and fluorescein isothiocyanate-labeled 40 kDa dextran as controls. Strong fluorescein staining was observed on the sclera when the device was used for drug delivery, whereas pellet use produced strong staining in the conjunctiva and surrounding tissue, but not on the sclera. Vasohibin-1 was found in the sclera, choroid, retinal pigment epithelium (RPE), and neural retina after device implantation. Stronger immunoreactivity at the RPE and ganglion cell layers was observed than in other retinal regions. Significantly lower fluorescein angiography (FA) scores and smaller CNV areas in the flat mounts of RPE-choroid-sclera were observed for the 10VDD, VDD (1 μM vasohibin-1 delivery device), and vasohibin-1 intravitreal direct injection (0.24 μM) groups when compared to the pellet, non-vasohibin-1 delivery device, and intravitreal vehicle injection groups. Choroidal neovascularization can be treated with transscleral sustained protein delivery using our novel device. We offer a safer sustained protein release for treatment of retinal disease using the transscleral approach.

Attenuation of Choroidal Neovascularization by β2-Adrenoreceptor Antagonism

To determine whether β-adrenergic blockade inhibits choroidal neovascularization (CNV) in a mouse model of laser-induced CNV and to investigate the mechanism by which β-adrenoreceptor antagonism blunts CNV. Mice were subjected to laser burns, inducing CNV, and were treated with daily intraperitoneal injections of propranolol hydrochloride. Neovascularization was measured on choroidal-scleral flat mounts using intercellular adhesion molecule 2 immunofluorescence staining. The effect of β-adrenoreceptor signaling on expression of vascular endothelial growth factor (VEGF) was investigated using primary mouse choroidal endothelial cells (ChECs) and retinal pigment epithelial (RPE) cells. These cells were incubated with β-adrenoreceptor agonists and/or antagonists and assayed for Vegf messenger RNA and protein levels. University of Wisconsin School of Medicine and Public Health. Wild-type 6-week-old female C57BL/6j mice. Inhibition of CNV after propranolol treatment and Vegf messenger RNA and protein expression after treatment with β-adrenoreceptor agonists and antagonists. Propranolol-treated mice demonstrated a 50% reduction in laser-induced CNV. Treatment with norepinephrine bitartrate stimulated Vegf messenger RNA expression and protein secretion in ChECs and RPE cells. This effect was blocked by β2-adrenoreceptor antagonism and mimicked by β2-adrenoreceptor agonists. Attenuation of CNV is achieved by β-adrenergic blockade. The β2-adrenoreceptors regulate VEGF expression in ChECs and RPE cells. Antagonists of β-adrenoreceptors are safe and well tolerated in patients with glaucoma and cardiovascular disease. Thus, blockade of β-adrenoreceptors may provide a new avenue to inhibit VEGF expression in CNV.

JNK inhibition reduces apoptosis and neovascularization in a murine model of age-related macular degeneration

Age-related macular degeneration (AMD) is the leading cause of registered blindness among the elderly and affects over 30 million people worldwide. It is well established that oxidative stress, inflammation, and apoptosis play critical roles in pathogenesis of AMD. In advanced wet AMD, although, most of the severe vision loss is due to bleeding and exudation of choroidal neovascularization (CNV), and it is well known that vascular endothelial growth factor (VEGF) plays a pivotal role in the growth of the abnormal blood vessels. VEGF suppression therapy improves visual acuity in AMD patients. However, there are unresolved issues, including safety and cost. Here we show that mice lacking c-Jun N-terminal kinase 1 (JNK1) exhibit decreased inflammation, reduced CNV, lower levels of choroidal VEGF, and impaired choroidal macrophage recruitment in a murine model of wet AMD (laser-induced CNV). Interestingly, we also detected a substantial reduction in choroidal apoptosis of JNK1-deficient mice. Intravitreal injection of a pan-caspase inhibitor reduced neovascularization in the laser-induced CNV model, suggesting that apoptosis plays a role in laser-induced pathological angiogenesis. Intravitreal injection of a specific JNK inhibitor decreased choroidal VEGF expression and reduced pathological CNV. These results suggest that JNK1 plays a key role in linking oxidative stress, inflammation, macrophage recruitment apoptosis, and VEGF production in wet AMD and pharmacological JNK inhibition offers a unique and alternative avenue for prevention and treatment of AMD.

Tissue Kallikrein Attenuates Choroidal Neovascularization via Cleavage of Vascular Endothelial Growth Factor

To investigate the antiangiogenic properties of tissue kallikrein in a murine model of laser-induced choroidal neovascularization (CNV). CNV was induced in male C57BL/6J mice by laser photocoagulation. The animals received daily subcutaneous injections of tissue kallikrein (50 μg/kg) or vehicle control for 2 days before the laser photocoagulation, and this treatment continued until sample collection. Seven days after laser injury, the CNV size was quantified. The levels of monocyte chemoattractant protein (MCP)-1, intercellular adhesion molecule (ICAM)-1, and interleukin (IL)-6 were assessed by enzyme-linked immunosorbent assay 3 days after laser injury. Cleavage of mouse VEGF with tissue kallikrein was assessed in vivo and in vitro. The protein levels of bradykinin were assessed in the RPE-choroid complexes and hearts. A significant decrease in CNV size was observed in animals treated with tissue kallikrein (27,168.3 ± 2432.2 μm(2)) compared with vehicle-treated controls (36,374.6 ± 3204.1 μm(2), P < 0.05). Tissue kallikrein treatment significantly reduced MCP-1, ICAM-1, and IL-6 levels in RPE-choroid complexes. Furthermore, immunoblotting showed the bands, presumably corresponding to the fragmented VEGF(164) protein, in the samples of both mouse VEGF preincubated with tissue kallikrein and RPE-choroid complexes obtained from animals treated with tissue kallikrein. In addition, bradykinin was unchanged in the RPE-choroid complexes of animals treated with tissue kallikrein, whereas the level of bradykinin was increased in the heart obtained from these experimental animals. The current data indicate that kallikrein exhibits antiangiogenic properties by cleaving VEGF(164) in a laser-induced CNV model.

Pathogenic Role of the Wnt Signaling Pathway Activation In Laser-Induced Choroidal Neovascularization

Choroidal neovascularization (CNV) is a severe complication of AMD. The Wnt signaling pathway has been shown to mediate angiogenesis. The purpose of this study was to investigate the pathogenic role of the Wnt pathway in CNV and explore the therapeutic potential of a novel Wnt signaling inhibitor in CNV. Adult rats and mice were photocoagulated using diode laser to induce CNV. On the same day, the animals were intravitreally injected with a monoclonal antibody (Mab2F1) blocking LRP6 or nonspecific mouse IgG. The Wnt signaling activation and target gene expression in the eyecup were determined by Western blot analysis. Fundus angiography was used to examine leakage from the laser lesion. CNV areas were measured on choroidal flatmount using FITC-dextran. Levels of Wnt pathway components and Wnt target gene expression were elevated in both laser-induced CNV rat and mouse eyecups, suggesting activation of the Wnt pathway. Significant suppression of Wnt signaling was observed in the Mab2F1 treatment group. Mab2F1 decreased vascular leakage from CNV lesions and reduced the neovascular area in laser-induced CNV rats. Mab2F1 inhibited the hypoxia-induced activation of Wnt signaling in cultured RPE cells. Mab2F1 also ameliorated retinal inflammation and vascular leakage in the eyecups of very low-density lipoprotein receptor knockout mice, a model of subretinal neovascularization. The Wnt pathway is activated in the laser-induced CNV models and plays a pathogenic role in CNV. Blockade of Wnt signaling using an anti-LRP6 antibody has therapeutic potential in CNV.

Methallothionein-3 contributes to vascular endothelial growth factor induction in a mouse model of choroidal neovascularization

In the present study, we investigated possible roles of the zinc (Zn)-binding protein metallothionein-3 (MT3) and cellular Zn in a mouse model of laser-induced choroidal neovascularization (CNV) using wild-type (WT) and MT3-knockout (KO) mice. Quantitative RT-PCR was used for the detection of MT3 mRNA. CNV was induced in mice between 8 and 12 weeks of age by disrupting the Bruch's membrane using an argon laser. Fundus photography and fluorescein angiography (FA) were performed 2 weeks following laser photocoagulation. The possible connection between MT3 and vascular endothelial growth factor (VEGF) expression was explored by quantifying VEGF levels in WT and MT3-KO mouse retinas by enzyme-linked immunosorbent assay. The role of Zn in VEGF expression was tested in WT and MT3-KO cells treated with pyrithione, with or without additional Zn, using immunoblotting and fluorescence photomicrography. Following laser-treatment, MT3-KO mice exhibited substantially smaller areas of CNV compared to WT mice. In addition, retinal angiograms revealed less severe fluorescein leakage in MT3-KO mice than in WT mice. On day 14 following the induction of CNV, VEGF expression was markedly increased in WT mice, but remained unchanged in MT3-KO mice. Consistent with the possible involvement of Zn released from MT3, raising intracellular Zn levels increased VEGF levels and activated its receptor, Flk-1, in both WT and MT3-KO retinal cells. Present results demonstrated that neural retinal cells express high levels of MT3, which might play a role in the process of CNV development. Moreover, Zn released from MT3 may contribute to VEGF induction.

Inhibition of Choroidal and Corneal Pathologic Neovascularization byPlgf1-deGene Transfer

Ocular neovascularization (NV), the primary cause of blindness, typically is treated via inhibition of VEGF-A activity. However, besides VEGF-A, other proteins of the same family, including VEGF-B and placental growth factor (PlGF, all together VEGFs), have a crucial role in the angiogenesis process. PlGF and VEGF, which form heterodimers if co-expressed, both are required for pathologic angiogenesis. We generated a PlGF1 variant, named PlGF1-DE, which is unable to bind and activate VEGFR-1, but retains the ability to form heterodimer. PlGF1-DE acts as dominant negative of VEGF-A and PlGF1wt through heterodimerization mechanism. The purpose of our study was to explore the therapeutic potential of Plgf1-de gene in choroid and cornea NV context. In the model of laser-induced choroidal neovascularization (CNV), Plgf1-de gene, and as control Plgf1wt, LacZ, or gfp genes, were delivered using adeno-associated virus (AAV) vector by subretinal injection 14 days before the injury. After 7 days CNV volume was assessed. Corneal NV was induced by scrape or suture procedures. Expression vectors for PlGF1wt or PlGF1-DE, and as control the empty vector pCDNA3, were injected in the mouse cornea after the vascularization insults. NV was evaluated with CD31 and LYVE-1 immunostaining. The expression of Plgf1-de induced significant inhibition of choroidal and corneal NV by reducing VEGF-A homodimer production. Conversely, the delivery of Plgf1wt, despite induced similar reduction of VEGF-A production, did not affect NV. Plgf1-de gene is a new therapeutic tool for the inhibition of VEGFs driven ocular NV.

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.

Reduction of choroidal neovascularization in mice by adeno‐associated virus‐delivered anti‐vascular endothelial growth factor short hairpin RNA

Strategies leading to the long-term suppression of inappropriate ocular angiogenesis are required to avoid the need for repetitive monthly injections for treatment of diseases of the eye, such as age-related macular degeneration (AMD). The present study aimed to develop a strategy for the sustained repression of vascular endothelial growth factor (VEGF), which is identified as the key player in exudative AMD. We have employed short hairpin (sh)RNAs combined with adeno-associated virus (AAV) delivery to obtain the targeted expression of potent gene-regulatory molecules. Anti-VEGF shRNAs were analyzed in human retinal pigment epithelial (RPE) cells using Renilla luciferase screening. For in vivo delivery of the most potent shRNA, self-complementary AAV vectors were packaged in serotype 8 capsids (scAAV2/8-hU6-sh9). In vivo efficacy was evaluated either by injection of scAAV2/8-hU6-sh9 into murine hind limb muscles or in a laser-induced murine model of choroidal neovascularization (CNV) following scAAV2/8-hU6-sh9 subretinal delivery. Plasmids encoding anti-VEGF shRNAs showed efficient knockdown of human VEGF in RPEs. Intramuscular administration led to localized expression and 91% knockdown of endogenous murine (m)VEGF. Subsequently, the ability of AAV2/8-encoded shRNAs to impair vessel formation was evaluated in the murine model of CNV. In this model, the sizes of the CNV were significantly reduced (up to 48%) following scAAV2/8-hU6-sh9 subretinal delivery. Using anti-VEGF vectors, we have demonstrated efficient silencing of endogenous mVEGF and showed that subretinal administration of scAAV2/8-hU6-sh9 has the ability to impair vessel formation in an AMD animal model. Thus, AAV-encoded shRNA can be used for the inhibition of neovascularization, leading to the development of sustained anti-VEGF therapy.

Antiangiogenic Effects of Axitinib, an Inhibitor of Vascular Endothelial Growth Factor Receptor Tyrosine Kinase, on Laser-Induced Choroidal Neovascularization in Mice

Purpose: To investigate the effects of axitinib, an inhibitor of vascular endothelial growth factor receptors, on choroidal neovascularization (CNV) in an animal model of neovascular age-related macular degeneration (AMD).Methods: Experimental CNV lesions were induced in C57BL/6 mice by laser photocoagulation. Beginning 1 day after CNV induction, mice were treated with axitinib (5 mg/kg/day) or vehicle for 2 weeks. In other groups of mice, axitinib or vehicle treatment was started 7 days after the laser application to determine the effect of the drug on established CNV. Untreated mice were used as a baseline group. Two weeks after laser injury, the extent of CNV was assessed from choroidal flat mounts perfused with fluorescein-labeled dextran. Immunofluorescence staining with isolectin IB4 was also used to quantify the CNV lesions.Results: Orally administered axitinib inhibited CNV growth in the laser-induced CNV model. Axitinib caused a 70.1% inhibition of CNV lesions compared to vehicle-treatment (p < 0.001). Axitinib also caused a significant regression of established CNV, reducing the area by 71.1% compared to vehicle treatment (p < 0.001). Moreover, immunofluorescence staining showed that the area of isolectin IB4 labeled vessels was smaller in the axitinib-treated group compared to the vehicle-treated group (p < 0.001).Conclusions: Axitinib effectively inhibits the progression of CNV in an experimental animal model. These results suggest that axitinib could constitute a therapeutic alternative for the treatment of neovascular AMD.

Impacts of Hypoxia-Inducible Factor-1 Knockout in the Retinal Pigment Epithelium on Choroidal Neovascularization

Hypoxia-inducible factor (HIF)-1 is a key oxygen sensor and is believed to play an important role in neovascularization (NV). The purpose of this study is to determine the role of retinal pigment epithelium (RPE)-derived HIF-1α on ocular NV. Conditional HIF-1α knockout (KO) mice were generated by crossing transgenic mice expressing Cre in the RPE with HIF-1α floxed mice, confirmed by immunohistochemistry, Western blot analysis, and fundus fluorescein angiography. The mice were used for the oxygen-induced retinopathy (OIR) and laser-induced choroidal neovascularization (CNV) models. HIF-1α levels were significantly decreased in the RPE layer of ocular sections and in primary RPE cells from the HIF-1α KO mice. Under normal conditions, the HIF-1α KO mice exhibited no apparent abnormalities in retinal histology or visual function as shown by light microscopy and electroretinogram recording, respectively. The HIF-1α KO mice with OIR showed no significant difference from the wild-type (WT) mice in retinal levels of HIF-1α and VEGF as well as in the number of preretinal neovascular cells. In the laser-induced CNV model, however, the disruption of HIF-1α in the RPE attenuated the over expression of VEGF and the intercellular adhesion molecule 1 (ICAM-1), and reduced vascular leakage and CNV area. RPE-derived HIF-1α plays a key role in CNV, but not in ischemia-induced retinal NV.