Intravitreal injection of resveratrol inhibits laser-induced murine choroidal neovascularization

Abstract

Aim: To determine the effects of intravitreal resveratrol (RSV) on murine laser-induced choroidal neovascularization (CNV).

Methods: The toxicity of RSV to choroidal endothelial cell (CEC) was measured using thiazolyl blue tetrazolium bromide (MTT) assay. Effects of RSV on choroidal endothelial cell (CEC) migration were evaluated with a modified Boyden chamber assay, while tube formation was evaluated in a 2-D gel assay. CNV was induced by laser photocoagulation in mice. The effects of intravitreal injection of RSV on CNV development were evaluated by fluorescein angiography (FA), confocal analysis of isolectin B4 labeled choroidal flat mounts, and histologic examination of CNV membranes. Immunostaining was used to analyze the expression and phosphorylation of vascular endothelial growth factor receptor 2 (VEGFR2).

Results: No significant cell toxicity was observed in CEC if the concentration of RSV was less than 200 µmol/L (P>0.05). RSV inhibited vascular endothelial growth factor (VEGF)-induced CEC migration (P<0.05) and tube formation (P<0.05) in vitro. Furthermore, intravitreal injection of RSV significantly inhibited laser induced CNV formation in mice. The FA leakage, CNV volume and CNV area analysis revealed that there were 41%, 45%, and 58% reduction in RSV-treated eyes (1.691±0.1032, 178 163±78 623 µm³ and 6508±619.0 µm², respectively) compared with those in control (2.724±0.08447, 379 676±98 382 µm³ and 16 576±2646 µm², respectively; P<0.05). Phospho-VEGFR2 expression was much weaker in the sections of CNV lesions in RSV injected mice compared with that in control (P<0.05).

Conclusion: Intravitreal injection of RSV exerts an inhibitory effect on CNV, which may through suppressing endothelial cell migration, tube formation and VEGFR2 phosphorylation.

Keywords: choroidal neovascularization; intravitreal injection; murine; resveratrol; tube formation; vascular endothelial growth factor receptor 2.

Figure 1. The effect of RSV on CEC cell survival

The CEC cells were treated with various concentrations of RSV (0, 10, 20, 50, 100, and 200 µmol/L) for 48h. MTT assay was performed to evaluate the cell survival. No significant differences in OD reading values were seen at the concentration of RSV from 10 to 100 µmol/L compared with control without RSV application (P>0.05). The cell survival was reduced up to 200 µmol/L of RSV addition (^aP<0.05). Values represent mean±SEM for 3 separate experiments (n=3).

Figure 2. RSV inhibits VEGF-induced CECs migration and tube formation

The bovine CECs were used for cell migration assay (A) and tube formation assay (C). RSV showed a significant inhibition on cell migration as shown by hematoxylin stained cells on the lower side of the Boyden chamber membrane (B, ^aP<0.05) and the extent of tube formation after staining with Calcein-AM (green; D, ^aP<0.05) induced by VEGF (50 ng/mL). In tube formation assay, the result was obtained from 3 gel wells per condition in each experiment.

Figure 3. Intravitreal injection of RSV inhibits laser-induced CNV in mice

Mice underwent retinal laser photo-coagulation in both eyes and intravitreal injection with RSV (2 µL, 100 µmol/L) or vehicle (0.1% DMSO) directly immediately after laser and on day 3. Fluorescein angiograms (FA) were performed at day 7 after laser surgery. On day 8, the eyes of euthanized mice were evaluated for histology and CNV volume analysis. RSV displayed an inhibitory effect on CNV formation, which was demonstrated by FA (A, B; ^aP<0.05); A, B: The results were from vehicle vs RSV; CNV volume analysis using isolectin B4-stained choroidal flat mounts (C, D; Scale bar, 100 µm; ^aP<0.05). C, D: The results were obtained from vehicle vs RSV: CNV lesion area on hematoxylin & eosin stained sections (E, F; Scale bar, 50 µm; ^aP<0.05), which include vehicle vs RSV.

Figure 4. RSV inhibits VEGFR2 phosphorylation in CNV lesions

Confocal immunofluorescent images of CNV lesion sections showed no significant difference in the total VEGFR2 (green) expression between RSV injection and control (A, C; P>0.05) but phospho-VEGFR2 (green) expression was much weaker in RSV injected mice compared with controls (B, D; ^aP<0.05). Nuclear counterstain (red) obtained using propidium iodide (PI). CNV lesion area in the sections is indicated by white rectangle. Scale bar: 100 µm.