Effects of bevacizumab on the neovascular membrane of proliferative diabetic retinopathy: reduction of endothelial cells and expressions of VEGF and HIF-1α

Xiao-Xia Han, Chang-Mei Guo, Yue Li, Yan-Nian Hui, Xiao-Xia Han, Chang-Mei Guo, Yue Li, Yan-Nian Hui

Abstract

Purpose: Anti-vascular endothelial growth factor (VEGF) agents have recently been used intravitreally during the perioperative period for proliferative diabetic retinopathy (PDR). However, the mechanism of theraputic effects of the agents remains unclear. This study aimed to investigate the effects of intravitreal bevacizumab (IVB) on retinal vascular endothelial cells and expressions of VEGF and hypoxia inducible factor-1α (HIF-1α) in PDR.

Methods: Twenty-four patients with PDR were enrolled and randomized to two groups. Twelve eyes of 12 patients of each group received either an intravitreal injection of 1.25 mg bevacizumab or a sham injection 6 days before vitrectomy. Neovascular membranes (NVMs) were collected during pars plana vitrectomy. The numbers of vascular endothelial cells in the NVMs were counted after staining with hematoxylin and eosin and von Willebrand. The expressions of VEGF and HIF-1α in the NVMs were detected through immunohistochemistry. Ten epiretinal membrane specimens from patients with proliferative vitreoretinopathy (PVR) without IVB treatment were set as an additional control.

Results: The number of vascular endothelial cells in NVMs of the IVB pretreated group was significantly lower than that of the sham group (21.5±3.94 versus 41.33±7.44, p=0.003). The IVB pretreated group also showed significantly lower levels of VEGF and HIF-1α in NVMs than those of the sham group (P(HIF-1α)=0.02, P(VEGF)<0.001). A stepwise regression analysis showed that IVB was a significant negative predictor for the numbers of vascular endothelial cells (β=-0.89, p<0.001) and the expressions of VEGF (β=-0.85, p<0.001) and HIF-1α (β=-0.64, p=0.001) in PDR patients. Epiretinal membranes of the PVR group showed negative staining of VEGF and HIF-1α.

Conclusions: Pretreatment with IVB in patients with PDR significantly decreased vascular endothelial cells and expressions of VEGF and HIF-1α, which further supports preoperative use of IVB in such patients.

Figures

Figure 1
Figure 1
Pretreatment with intravitreal bevacizumab (IVB) significantly reduced the numbers of vascular endothelial cells in neovascular membranes (NVMs) of the eyes with proliferative diabetic retinopathy (PDR). Hematoxylin and eosin stain (H&E; A, C, and E) and von Willebrand stain (B, D, and F) were applied to detect the vascular endothelial cells in the NVMs of PDR eyes (A-D) and epiretinal membranes of the eyes with proliferative vitreoretinopathy (PVR; E, F). The untreated group (A, B) shows a significantly more number of vascular endothelial cells when compared to the IVB pretreated group (C, D; p=0.003). The epiretinal membranes of PVR eyes were set as the control group. von Willebrand stain for vascular endothelial cells in the control group was negative (F). Figures were selected as representative data from three independent experiments. Scale bars: 200 μm.
Figure 2
Figure 2
Increased vascular endothelial cell count in eyes with proliferative diabetic retinopathy (PDR) was suppressed by pretreatment of intravitreal bevacizumab (IVB). The epiretinal membranes of proliferative vitreoretinopathy (PVR) eyes were set as the control group. Quantitative analysis for the vascular endothelial cell count in the neovascular membranes (NVMs) of PDR eyes and epiretinal membranes of PVR eyes was determined by counting positively stained cells in 10 representative fields. Each value represents means±SEM from three independent experiments (p=0.003).
Figure 3
Figure 3
Pretreatment of intravitreal bevacizumab (IVB) remarkably reduced the levels of hypoxia inducible factor-1 alpha (HIF-1α) and vascular endothelial growth factor (VEGF) in eyes with proliferative diabetic retinopathy (PDR). Immunohistochemistry for HIF-1α (A, C) and VEGF (B, D) was performed in the neovascular membranes (NVMs) of no IVB pretreated sham group (A, B) and IVB pretreated group (C, D). The stain of both HIF-1α and VEGF in the IVB pretreated group (C, D) was significantly lower than that of the no IVB pretreated sham group (A, B). Figures were selected as representative data from three independent experiments. Scale bars: 200 μm.
Figure 4
Figure 4
Pretreatment of intravitreal bevacizumab (IVB) remarkably reduced the expressions of hypoxia inducible factor-1 alpha (HIF-1α) and vascular endothelial growth factor (VEGF) in eyes with proliferative diabetic retinopathy (PDR). Quantitative analysis for the levels of HIF-1α and VEGF in the PDR eyes was measured by measuring the Average Optic Density values of each image using ImagePro-Plus (Media Cybernetics) software. The expression levels of both HIF-1α (p=0.02) and VEGF (p

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