Müller cell-derived VEGF is essential for diabetes-induced retinal inflammation and vascular leakage

Abstract

Objective: Vascular endothelial growth factor (VEGF-A or VEGF) is a major pathogenic factor and therapeutic target for diabetic retinopathy (DR). Since VEGF has been proposed as a survival factor for retinal neurons, defining the cellular origin of pathogenic VEGF is necessary for the effectiveness and safety of long-term anti-VEGF therapies for DR. To determine the significance of Müller cell-derived VEGF in DR, we disrupted VEGF in Müller cells with an inducible Cre/lox system and examined diabetes-induced retinal inflammation and vascular leakage in these conditional VEGF knockout (KO) mice.

Research design and methods: Leukostasis was determined by counting the number of fluorescently labeled leukocytes inside retinal vasculature. Expression of biomarkers for retinal inflammation was assessed by immunoblotting of TNF-alpha, ICAM-1, and NF-kappaB. Vascular leakage was measured by immunoblotting of retinal albumin and fluorescent microscopic analysis of extravascular albumin. Diabetes-induced vascular alterations were examined by immunoblotting and immunohistochemistry for tight junctions, and by trypsin digestion assays for acellular capillaries. Retinal integrity was analyzed with morphologic and morphometric analyses.

Results: Diabetic conditional VEGF KO mice exhibited significantly reduced leukostasis, expression of inflammatory biomarkers, depletion of tight junction proteins, numbers of acellular capillaries, and vascular leakage compared to diabetic control mice.

Conclusions: Müller cell-derived VEGF plays an essential and causative role in retinal inflammation, vascular lesions, and vascular leakage in DR. Therefore, Müller cells are a primary cellular target for proinflammatory signals that mediates retinal inflammation and vascular leakage in DR.

FIG. 1.

Analysis of VEGF and HIF-1α expression in normal and diabetic conditional VEGF KO mice. A and B: Immunoblotting analysis for VEGF (A) and HIF-1α (B) in retinas from conditional VEGF KO mice and WT controls 2 months after diabetes. C and D: Confocal microscopic analysis of immunostained retinas for VEGF expression (green) in conditional VEGF KO mice and WT controls subjected to a diabetic stress. Blue: nuclear staining (DAPI). Scale bar equals 40 μm. ONL, outer nuclear layer; INL, inner nuclear layer. Error bar: SEM. ***P < 0.001. ns, not significant. VEGF expression was significantly reduced in the retinas of conditional VEGF KO mice under normal or diabetic conditions. Although diabetes upregulated HIF-1α, no significant change in the levels of retinal HIF-1α was observed in diabetic conditional VEGF KO mice. (A high-quality digital representation of this figure is available in the online issue.)

FIG. 2.

Analysis of retinal inflammation in conditional VEGF KO mice 2 months after inducing diabetes. A and B: FITC-conjugated ConA staining for adherent leukocytes (arrows) in retinal microvasculatures of diabetic conditional VEGF KO mice and WT controls. Scale bar represents 100 μm. C: Quantification of adherent leukocytes in retinal vasculatures of diabetic conditional VEGF KO mice and WT controls. D and E: Immunoblotting analysis of ICAM-1 (D) and TNF-α (E) expression in conditional VEGF KO mice. F: Immunoblotting analysis of NF-κB p65 phosphorylation in diabetic retinas of conditional VEGF KO mice. Error bar: SEM. ***P < 0.001. Loss of Müller cell-derived VEGF caused a significant reduction in the number of adherent leukocytes, expression of ICAM-1 and TNF-α, and phosphorylated NF-κB p65 in diabetic retinas. ns, not significant.

FIG. 3.

Analysis of retinal vascular leakage in conditional VEGF KO mice 6 months after the onset of diabetes. A: Immunoblotting analysis of retinal extravascular albumin in conditional VEGF KO mice. B and E: Microscopic images of retinal flat-mounts (B and C) and enlarged areas (d and E) showing fluorescently labeled albumin (arrows) in diabetic retinas of WT and conditional VEGF KO mice after injection with FITC-labeled albumin. Scale bars represent 100 μm. F: Computer-assisted quantitative analysis of FITC-labeled albumin in diabetic retinas of conditional VEGF KO mice. Error bar: SEM. ***P < 0.001. Loss of Müller cell-derived VEGF caused a significant reduction in diabetes-induced retinal vascular leakage.

FIG. 4.

Analysis of tight junction protein expression in retinas of conditional VEGF KO mice 6 months after diabetes. A and B: Immunoblotting and quantification of tight junction protein occludin and ZO-1 in retinas of diabetic conditional VEGF KO mice. Error bar: SEM. ***P < 0.001. C–F: Confocal microscopic analysis of immunostained tight junction protein ZO-1 in retinal vasculature (C and D) from the diabetic retinas of conditional VEGF KO mice. Scale bars represent 50 μm. Loss of Müller cell-derived VEGF caused a significant attenuation of diabetes-induced depletion of tight junction proteins occludin and ZO-1. ns, not significant.

FIG. 5.

Retinal vascular histopathology in the conditional VEGF KO mice 6 months after inducing diabetes. A–C: Analysis of acellular capillaries (arrowheads) with trypsin digestion in diabetic retinas of the conditional VEGF KO mice and WT controls. Error bar: SEM. ***P < 0.001. Scale bar in B represents 50 μm. Scale bars in D–F represent 1 μm. D–F: Representative TEM images for basement membrane thickness in retinal vascular capillaries. Although the number of acellular capillaries was significantly decreased in diabetic conditional VEGF KO mice, no apparent change in the thickness (arrowheads) of basement membrane was observed among all mice examined.

FIG. 6.

Retinal morphology and morphometry in conditional VEGF KO mice 6 months after onset of diabetes. A and B: Morphology of H&E-stained retinal sections. C: Thickness of outer nuclear layer, inner nuclear layer, and total retina. D and E: Representative TEM images showing no detectable defects in diabetic retina of conditional VEGF KO mice. F: Average number of ganglion cells. Scale bar equals 50 μm in B and 1 μm in d and E. No detectable changes in retinal thickness, numbers of ganglion cells, and retinal ultrastructure were observed in diabetic conditional VEGF KO mice. Ch, choroid; CC, choriocapillaris; GC, ganglion cells; INL, inner nuclear layer; ns, not significant; ONL, outer nuclear layer; P, photoreceptors.