RESUMO
To determine the effect of a vascular endothelial growth factor receptor 2 tyrosine kinase (VEGFR2) inhibitor on intravitreous neovascularization (IVNV), endothelial tip cell filopodia, and intraretinal vascularization in a rat model of retinopathy of prematurity (ROP). Within 4h of birth, newborn Sprague-Dawley rat pups and their mothers were cycled between 50% and 10% oxygen daily until postnatal day (p)12. Pups were given intravitreous injections of VEGFR2 inhibitor, SU5416, or control (dimethyl sulfoxide, DMSO) and returned to oxygen cycling until p14, then placed into room air. Intravitreous neovascularization (IVNV), avascular/total retinal areas, and endothelial tip cell filopodial number and length were determined in lectin-labeled neurosensory retinal flat mounts. Cryosections or fresh tissue were analyzed for phospho-VEGFR1, phospho-VEGFR2, activated caspase-3, or phospho-beta3 integrin. Human umbilical venous (HUVECs) and human choroidal endothelial cells (ECs) were treated with VEGFR2 inhibitor to determine effect on VEGFR2 phosphorylation and on directed EC migration toward a VEGF gradient. Filopodial length and number of migrated ECs were also measured. Compared to control, the VEGFR2 inhibitor reduced VEGFR2 phosphorylation in HUVECs in vitro and clock hours and areas of IVNV but not percent avascular retina in vivo. Filopodial length and number of filopodia/EC tip cell were reduced in retinal flat mounts at doses that inhibited IVNV, whereas at lower doses, only a reduction in filopodial length/EC tip cell was found. There was no difference in phosphorylated beta3 integrin and cleaved caspase-3 labeling in VEGFR2 inhibitor-treated compared to control in vivo. Doses of the VEGFR2 inhibitor that reduced filopodial length and number of filopodia/migrating EC corresponded to reduced EC migration in in vitro models. VEGFR2 inhibitor reduced IVNV and filopodial number and length/EC tip cell without interfering with intraretinal vascularization. Reducing the number and length of filopodia/endothelial tip cell may reduce guidance cues for endothelial cells to migrate into the vitreous without interfering with migration into the retina toward a VEGF gradient.
Assuntos
Inibidores da Angiogênese/farmacologia , Células Endoteliais/efeitos dos fármacos , Indóis/farmacologia , Neovascularização Patológica/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Pseudópodes/efeitos dos fármacos , Pirróis/farmacologia , Vasos Retinianos/efeitos dos fármacos , Retinopatia da Prematuridade/tratamento farmacológico , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Corpo Vítreo/irrigação sanguínea , Animais , Animais Recém-Nascidos , Caspase 3/metabolismo , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Células Endoteliais/enzimologia , Humanos , Recém-Nascido , Integrina beta3/metabolismo , Neovascularização Patológica/enzimologia , Neovascularização Patológica/fisiopatologia , Fosforilação , Pseudópodes/enzimologia , Ratos , Ratos Sprague-Dawley , Vasos Retinianos/enzimologia , Vasos Retinianos/fisiopatologia , Retinopatia da Prematuridade/enzimologia , Retinopatia da Prematuridade/fisiopatologia , Fatores de Tempo , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
PURPOSE: To study the effects of vascular endothelial growth factor (VEGF) on endothelial nitric oxide synthetase (eNOS) and retinal vascular tortuosity and cleavage planes in a rat model of retinopathy of prematurity (ROP). METHODS: Within 4 hours of birth, pups and mothers were cycled between 50% and 10% oxygen daily. At postnatal day (p)12, pups received either intravitreous anti-rat neutralizing antibody to VEGF or control nonimmune rat IgG in one eye and returned to oxygen cycling until p14 when they were placed in room air (RA) for 4 days (50/10 oxygen-induced retinopathy [50/10 OIR]). Tortuosity indices and endothelial cleavage plane angles relative to the long axes of the major retinal vessels during anaphase were calculated from phosphohistone- and Alexa-isolectin-stained retinal flatmounts. Some retinas were processed for eNOS protein or phosphorylated/total eNOS. RESULTS: Retinas from 50/10 OIR had increased tortuosity over time with peaks at p12 and p14 (P < 0.001 vs. RA) before the development of intravitreous neovascularization, which peaked at p18. Compared with RA, eNOS/actin in 50/10 OIR retinas was increased at p12 (P = 0.0003) and p14 (P = 0.047). Inhibition of VEGF with a neutralizing antibody decreased tortuosity and caused endothelial mitosis cleavage planes to orient in favor of vessel elongation but did not affect eNOS protein or activation. CONCLUSIONS: In the 50/10 OIR model, a model with relevance to ROP, arteriolar tortuosity, and venous dilation are increased through VEGF, which influences the orientation of endothelial cell cleavage in major arterioles and veins, independent of eNOS.
Assuntos
Endotélio Vascular/patologia , Vasos Retinianos/patologia , Retinopatia da Prematuridade/metabolismo , Retinopatia da Prematuridade/patologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Animais Recém-Nascidos , Anticorpos/farmacologia , Arteríolas/patologia , Divisão Celular/efeitos dos fármacos , Modelos Animais de Doenças , Humanos , Recém-Nascido , Mitose/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Veia Retiniana/patologia , Vasos Retinianos/fisiopatologia , Retinopatia da Prematuridade/fisiopatologia , Fator A de Crescimento do Endotélio Vascular/imunologia , VasodilataçãoRESUMO
PURPOSE: To study the effects of intravitreous triamcinolone acetonide (TA) on neovascularization (NV), capillary density, and retinal endothelial cell (REC) viability in a model of oxygen-induced retinopathy (OIR). METHODS: Newborn rats exposed to OIR underwent intravitreous injections (right eye) at day 14 to achieve intravitreous concentrations of: dexamethasone (DEX) (0.3 mg/mL), triamcinolone (TA; 0.4-4 mg/mL), or PBS. Animals were removed to room air and at day 18, retinal flatmounts were assayed for clock hours of NV, percent peripheral avascular retina, capillary density, apoptosis, and VEGF protein. At day 15, retinas were assayed for insulin-like growth factor (IGF)-1 receptor phosphorylation (IGF-1Rphos). Human RECs exposed to TA were assayed for trypan blue exclusion or activated caspase-3. RESULTS: TA but not DEX or PBS reduced NV (ANOVA, P < 0.001), capillary density (ANOVA, P < 0.001), and systemic weight gain (ANOVA, P = 0.002). VEGF protein was not different between TA- and PBS-injected or noninjected groups. Apoptosis was not increased in vivo or in vitro between groups, but there was a dose-dependent toxic effect of TA on cultured RECs (P < 0.001). At day 15, retinas from the 4 mg/mL TA-injected OIR group had a trend toward reduced IGF-1Rphos compared with room air-raised PBS- or non-injected OIR groups. CONCLUSIONS: TA caused dose-dependent reductions in NV, retinal vascularization, and systemic weight gain associated with a reduction in IGF-1Rphos. Long-term studies are needed to assess TA toxicity in vivo. TA doses should be carefully considered before administering the drug in diseases with ongoing retinal vascular development, such as retinopathy of prematurity.