Short hairpin RNA-mediated knockdown of VEGFA in Müller cells reduces intravitreal neovascularization in a rat model of retinopathy of prematurity.

Vascular endothelial growth factor (VEGF) A is implicated in aberrant angiogenesis and intravitreous neovascularization (IVNV) in retinopathy of prematurity (ROP). However, VEGFA also regulates retinal vascular development and functions as a retinal neural survival factor. By using a relevant ROP model, the 50/10 oxygen-induced retinopathy (OIR) model, we previously found that broad inhibition of VEGFA bioactivity using a neutralizing antibody to rat VEGF significantly reduced IVNV area compared with control IgG but also significantly reduced body weight gain in the pups, suggesting an adverse effect. Therefore, we propose that knockdown of up-regulated VEGFA in cells that overexpress it under pathological conditions would reduce IVNV without affecting physiological retinal vascular development or overall pup growth. Herein, we determined first that the VEGFA mRNA signal was located within the inner nuclear layer corresponding to CRALBP-labeled Müller cells of pups in the 50/10 OIR model. We then developed a lentiviral-delivered miR-30eembedded shRNA against VEGFA that targeted Müller cells. Reduction of VEGFA by lentivector VEGFA-shRNAetargeting Müller cells efficiently reduced 50/10 OIR up-regulated VEGFA and IVNV in the model, without adversely affecting physiological retinal vascular development or pup weight gain. Knockdown of VEGFA in rat Müller cells by lentivector VEGFA-shRNA significantly reduced VEGFR2 phosphorylation in retinal vascular endothelial cells. Our results suggest that targeted knockdown of overexpressed VEGFA in Müller cells safely reduces IVNV in a relevant ROP model.

Increased expression of multifunctional serine protease, HTRA1, in retinal pigment epithelium induces polypoidal choroidal vasculopathy in mice.

Age-related macular degeneration (AMD) is the leading cause of irreversible blindness in the elderly. Wet AMD includes typical choroidal neovascularization (CNV) and polypoidal choroidal vasculopathy (PCV). The etiology and pathogenesis of CNV and PCV are not well understood. Genome-wide association studies have linked a multifunctional serine protease, HTRA1, to AMD. However, the precise role of HTRA1 in AMD remains elusive. By transgenically expressing human HTRA1 in mouse retinal pigment epithelium, we showed that increased HTRA1 induced cardinal features of PCV, including branching networks of choroidal vessels, polypoidal lesions, severe degeneration of the elastic laminae, and tunica media of choroidal vessels. In addition, HTRA1 mice displayed retinal pigment epithelium atrophy and photoreceptor degeneration. Senescent HTRA1 mice developed occult CNV, which likely resulted from the degradation of the elastic lamina of Bruch's membrane and up-regulation of VEGF. Our results indicate that increased HTRA1 is sufficient to cause PCV and is a significant risk factor for CNV.

VEGF-mediated STAT3 activation inhibits retinal vascularization by down-regulating local erythropoietin expression.

Avascular, hypoxic retina has been postulated to be a source of angiogenic factors that cause aberrant angiogenesis and intravitreal neovascularization (IVNV) in retinopathy of prematurity. Vascular endothelial growth factor (VEGF) is an important factor involved. However, VEGF is also required for normal retinal vascular development, which raises concerns about inhibiting its activity to treat IVNV in retinopathy of prematurity. Therefore, understanding the effects that VEGF has on other factors in the development of avascular retina is important to prevent aberrant angiogenesis and IVNV. Here, we show that STAT3 was activated by increased retinal VEGF in the rat 50/10 oxygen-induced retinopathy model. Phospho-STAT3 colocalized with glutamine synthetase-labeled Müller cells. Inhibition of STAT3 reduced avascular retina and increased retinal erythropoietin (Epo) expression. Epo administered exogenously also reduced avascular retina in the model. In an in vitro study, hypoxia-induced VEGF inhibited Epo gene expression by STAT3 activation in rat Müller cells. The mechanism by which activated STAT3 regulated Epo was by inhibition of Epo promoter activity. Together, these findings show that increased retinal VEGF contributes to avascular retina by regulating retinal Epo expression through Janus kinase/STAT signaling. Our results suggest that rescuing Epo expression in the retina before the development of IVNV may promote normal developmental angiogenesis and, therefore, reduce the stimulus for later pathologic IVNV.

Anti-VEGF antibody leads to later atypical intravitreous neovascularization and activation of angiogenic pathways in a rat model of retinopathy of prematurity.

PURPOSE: Inhibiting VEGF improves adult retino/choroido-vascular diseases, but can lead to recurrent intravitreous neovascularization (IVNV), avascular retina (AVA), and retinal detachment in preterm infants with retinopathy of prematurity (ROP). We sought to understand causes of late-onset IVNV and AVA following anti-VEGF using an ROP model. METHODS: In the Penn model of ROP, postnatal day (p)12 pups received 1 µL intravitreal VEGFA164 antibody (anti-VEGF; 25-100 ng) or IgG control in each eye. Analyses included lectin-stained percent IVNV and AVA; VEGF protein, erythropoietin, phosphorylated extracellular signal-related kinases and signal transducer and activator of transcription-3 (p-STAT3); and immunohistochemistry of retinal sections for p-VEGFR2. Western blots of human retinal microvascular endothelial cells (hRMVECs) stimulated with VEGF or erythropoietin were analyzed for p-STAT3. Statistical analysis was performed with one-way ANOVA or two-tailed t-tests. RESULTS: At p18, 50 ng anti-VEGF reduced IVNV, and at p25, caused increased IVNV and AVA compared with controls. VEGF and p-VEGFR2 labeling increased following 100 ng anti-VEGF. Following 50 ng anti-VEGF, reduced p-STAT3 and increased erythropoietin occurred at p18. Erythropoietin or VEGF stimulated hRMVEC proliferation and STAT3 activation. In vivo, anti-VEGF reduced pup growth. CONCLUSIONS: Increases in erythropoietin and angiogenic signaling following anti-VEGF may account for recurrent IVNV. Anti-VEGF reduced pup growth. Research is needed regarding safety, dose, and type of antiangiogenic treatment for ROP.

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.