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

Rap1 GTPase Activation and Barrier Enhancement in RPE Inhibits Choroidal Neovascularization <i>In Vivo</i>

<div><p>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. <i>In vitro</i>, 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, <i>Rap1b^−/−</i> mice exhibited larger CNV volumes compared to wild-type or <i>Rap1a^−/−</i>. <i>In vivo</i>, 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 <i>in vitro</i>. 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 <i>in vitro</i> and in cells surrounding laser-induced lesions <i>in vivo</i>. We conclude that <i>in vitro</i>, 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 <i>in vivo</i> inhibited development of choroidal neovascular lesions in a laser-injury model. Our data suggest that targeting Rap1 isoforms <i>in vivo</i> 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.</p></div

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

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

Rap1B knockdown <i>in vitro</i> affects dynamic junctional responses.

<p>Monolayer re-formation and resistance to EGTA-induced junctional disassembly were assessed using the calcium switch assay. (A) Representative immunofluorescence localization of ZO-1 after 30 min EGTA to disrupt junctions (left column), followed by 1 or 3 hr of washout to synchronously initiate junctional re-assembly (middle and right columns). Note greater cell-free areas (gaps) and impaired recruitment of ZO-1 to cell-cell contacts in Rap1B shRNA RPE. Quantification of the monolayer gap area per image field is shown in the graph. Recovery of Rap1B shRNA monolayer integrity was delayed compared to Neg control and Rap1A shRNA. (* compared to Neg, ** compared to Neg and Rap1A shRNA). Pooled averages (multiple random fields) from n = 3 independent experiments ± SEM, normalized to gap area of Neg control monolayers after EGTA treatment. *, **p≤0.05 (B) Gap area during EGTA-induced junctional disassembly was quantified as in panel A. EGTA treatment caused larger gaps in Rap1B shRNA monolayers compared with control or Rap1A shRNA monolayers. Graph shows the mean of n = 4 fields ± SEM, representative experiment from 2 independent trials. *p≤0.05, 1B shRNA compared to both Neg and 1A shRNA. (C) Electrical impedance analysis of cell monolayer disruption induced by EGTA. Data points represent the average ± SD from quadruplicate wells for each condition. Representative trace of 3 independent experiments.</p

Schematic of cellular events in the laser CNV model.

<p>Laser treatment creates a breach in RPE and Bruch's membrane; RPE barrier integrity is compromised in cells adjacent to the lasered region. Inflammatory and wound healing events lead to activation of CECs from the choriocapillaris; CECs begin to migrate and transmigrate the lasered lesion as well as the adjacent RPE with compromised barrier integrity. In Type 2 CNV (shown), CECs proliferate and invade the subretinal space to form CNV. Type 1 (occult) CNV occurs when CECs remain sub-RPE (not shown in this model). 8CPT-cAMP injection post-laser inhibits CNV by promoting barrier integrity in the neighboring RPE, thereby reducing the lesion width through which CECs migrate. CEC junctional integrity may also be strengthened, contributing to the decreased CNV. Compared to WT, <i>Rap1b^−/−</i> RPE cell junctions are more easily disrupted, allowing greater CEC transmigration and increased CNV. 8CPT-cAMP treatment activates both Rap1 isoforms, which is associated with increased junctional resealing and limits RPE monolayer disruption through which CEC migration occurs.</p

Activation of total Rap1 enhances recruitment of junctional proteins and cortical F-actin both <i>in vitro</i> and <i>in vivo</i>.

<p>(A) 8CPT-cAMP treatment (250 µM, 1 hr) of cultured RPE monolayers decreases stress fibers and enhances cortical F-actin morphology. (B) Enhanced recruitment and linear junctional staining of ZO-1 with 8CPT-cAMP treatment compared to PBS control. Boxed areas are enlarged in the upper right inset to highlight differences in junctional staining pattern. Scale bar, 50 µm (C) Quantification of <i>in vitro</i> 8CPT-cAMP treatment. Percent of cells that show enhanced (linear) junctional localization of ZO-1 (average % of 10 fields/condition (>600 cells total counted). * p<0.01 (D) <i>In vivo</i>, eyes injected with 8CPT-cAMP also have enhanced linear junctional recruitment of proteins such as F-actin and β-catenin. (E) Quantification of junctional β-catenin in PBS vs. 8CPT-cAMP-injected eyes. Data plotted as average junctional pixel intensity from random cells per field, from n = 4 injected eyes (>100 cells). * p = 0.0324 compared to PBS-injected.</p

Rap1A knockdown <i>in vitro</i> impairs steady state monolayer integrity and decreases transepithelial electrical resistance.

<p>(A) RPE cells transduced with indicated shRNA constructs were grown on coverslips for 4–5 days to obtain steady state monolayers. Top row, representative immunofluorescence localization of the junctional marker ZO-1. Scale bar  = 50 µm. Boxed areas are enlarged to highlight intercellular gaps in Rap1A shRNA cell monolayers (bottom row). Scale bar  = 25 µm. Quantification of total monolayer gap area per image field (expressed as % of total field area) using image analysis software (ImageJ) was used as an indication of monolayer integrity. Graph shows the mean of n = 4 fields ± SEM, representative experiment from 2 independent trials. **p<0.01, 1A shRNA compared to both Neg and 1B shRNA. (B) TER was measured on cells prior to shRNA knockdown (day 0), and again after 5 days. Graph shows TER of negative control, Rap1A, or Rap1B shRNA treated cells, representing average TER normalized to control at t = day 0 from n = 4 independent experiments. Control shRNA and Rap1B shRNA cells increased TER after 5 days in culture, while Rap1A shRNA cells did not significantly increase TER from day 0 to day 5. * p≤0.01, NS =  not significant; **p≤0.01, TER of Rap1A shRNA monolayers significantly lower at steady state (day 5) compared to control and Rap1B shRNA.</p

Laser-induced CNV in WT vs. <i>Rap1a^−/−</i> and <i>Rap1b^−/−</i> mice.

<p>One week following laser, eyes were processed as RPE/choroid flat mounts and stained with AlexaFluor 568-labeled lectin to visualize the choroidal neovascular endothelial cells. (A) Representative confocal images from WT, <i>Rap1a^−/−</i>, and <i>Rap1b^−/−</i> (maximum projections). (B) Quantification shows that CNV volume is significantly greater in <i>Rap1b^−/−</i> compared to WT and <i>Rap1a^−/−</i> mice. Bars represent average lesion volume ± SEM from at least 6 individual mice per genotype (n = 17–26 lesions). * p<0.01, <i>Rap1b^−/−</i> compared to WT and <i>Rap1a^−/−</i>.</p