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

Wang J, Xu X, Elliott MH, Zhu M, Le YZ - Diabetes (2010)

Bottom Line: 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.Retinal integrity was analyzed with morphologic and morphometric analyses.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, Xiangya Hospital, Central South University, Changsha, China.

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.

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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.)
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Figure 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.)

Mentions: In an effort to generate human VMD2 promoter controlled tetracycline-inducible RPE-specific Cre mice (28), we identified a mouse line displaying Cre function predominantly localized to the retinal Müller cells (29). Further characterization demonstrated that this mouse line is feasible for conditional gene expression in Müller glia (19). The transgenic Cre mice (cre+) were mated with mice carrying a floxed VEGF (Vegfff) allele (18) to generate the conditional Müller glial VEGF KO mice. Using primary cultures derived from the conditional VEGF KO mice, we determined that there was a 66.5% reduction of VEGF expression in Müller cells (21). Immunoblotting assays demonstrated a significant decrease (43.2%) in VEGF expression in the retinas of conditional VEGF KO mice (cre+Vegfff) compared with WT (cre−Vegfff) controls (Fig. 1A), confirming that Müller cells are a major source of VEGF in the retina.


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

Wang J, Xu X, Elliott MH, Zhu M, Le YZ - Diabetes (2010)

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.)
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2927953&req=5

Figure 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.)
Mentions: In an effort to generate human VMD2 promoter controlled tetracycline-inducible RPE-specific Cre mice (28), we identified a mouse line displaying Cre function predominantly localized to the retinal Müller cells (29). Further characterization demonstrated that this mouse line is feasible for conditional gene expression in Müller glia (19). The transgenic Cre mice (cre+) were mated with mice carrying a floxed VEGF (Vegfff) allele (18) to generate the conditional Müller glial VEGF KO mice. Using primary cultures derived from the conditional VEGF KO mice, we determined that there was a 66.5% reduction of VEGF expression in Müller cells (21). Immunoblotting assays demonstrated a significant decrease (43.2%) in VEGF expression in the retinas of conditional VEGF KO mice (cre+Vegfff) compared with WT (cre−Vegfff) controls (Fig. 1A), confirming that Müller cells are a major source of VEGF in the retina.

Bottom Line: 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.Retinal integrity was analyzed with morphologic and morphometric analyses.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, Xiangya Hospital, Central South University, Changsha, China.

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.

Show MeSH
Related in: MedlinePlus