HMG-CoA reductase inhibitors (statin) prevents retinal neovascularization in a model of oxygen-induced retinopathy

Abstract

Purpose: Retinal neovascularization (RNV) is a primary cause of blindness and involves the dysfunction of retinal capillaries. Recent studies have emphasized the beneficial effects of inhibitors of HMG-CoA reductase (statins) in preventing vascular dysfunction. In the present study, the authors characterized the therapeutic effects of statins on RNV.

Methods: Statin treatment (10 mg/kg/d fluvastatin) was tested in a mouse model of oxygen-induced retinopathy. Morphometric analysis was conducted to determine the extent of capillary growth. Pimonidazole hydrochloride was used to assess retinal ischemia. Western blot and immunohistochemical analyses were used to assess protein expression levels and immunolocalization. Lipid peroxidation and superoxide radical formation were determined to assess oxidative changes.

Results: Fluvastatin treatment significantly reduced the area of the capillary-free zone (P < 0.01), decreased the formation of neovascular tufts (P < 0.01), and ameliorated retinal ischemia. These morphologic and functional changes were associated with statin effects in preventing the upregulation of VEGF, HIF-1 alpha, phosphorylated STAT3, and vascular expression of the inflammatory mediator ICAM-1 (P < 0.01). Superoxide production and lipid peroxidation in the ischemic retina were also reduced by statin treatment (P < 0.01).

Conclusions: These data suggest the beneficial effects of statin treatment in preventing retinal neovascularization. These beneficial effects appear to result from the anti-oxidant and anti-inflammatory properties of statins.

FIGURE 1

(A) Flatmounted mouse retinas stained with isolectin B4 to identify areas of neovascularization (arrows) and capillary-free zones (dotted white line). (B) Histograms representing the results of morphometric analysis of retinal flatmounts measuring the capillary-free areas (upper bars) and neovascular tufts (lower bars). OIR+PBS, sham ischemic mouse retina at postnatal day 17; OIR+F, ischemic mouse retina at postnatal day 17 injected with 10 mg/kg/d fluvastatin (P12–P17). *P < 0.01 vs. OIR+PBS; x = mean ± SE; n = 11. (C) Three-dimensional images (z-stacks, 20× magnification) of retinal flatmounts stained with isolectin B4 (Texas Red demonstrating retinal capillary morphology in the different treatment groups).

FIGURE 2

Assessment of retinal ischemia measured by identification of positive areas (FITC) for H-1 and counterstained with isolectin B4 (Texas Red) to identify (A) retinal midperiphery and (B) central retina. (C) Morphometric analysis measuring H-1–specific immunofluorescence in the whole retina. *P < 0.01 vs. P17; #P < 0.01 vs. OIR+PBS; x = mean ± SE; n = 8.

FIGURE 3

(A) Measurements of DCF-specific fluorescence to assess ROS production in murine retinas subjected to different treatments. DCF was incubated with retinal homogenates. To control the specificity of the reaction of 100 µL OIR+PBS, homogenates were also preincubated with 100 µL SOD (OIR+P+S) alone or with a mixture of 100 IU SOD and 1000 IU catalase (OIR+P+S+C). (B) Measurement of lipid peroxide levels to assess fluvastatin effects on oxidative stress in the ischemic retina. Lipid peroxides are measured as microgram MDA normalized by total protein. *P < 0.01 vs. P17; #P < 0.01 vs. OIR+PBS; x = mean ± SE; n = 8.

FIGURE 4

Measurement of VEGF and ICAM-1 expression by Western blot and immunohistochemical analyses. (A) Immunoblots showing VEGF-specific immunoreactivity and quantified as measurements of optical density (histogram). *P < 0.01 vs. P17; #P < 0.01 vs. OIR+PBS; x = mean ± SE; n = 5. (B) Immunoblot showing ICAM-1–specific immunoreactivity and measured as optical density units relative to actin. *P < 0.01 vs. P17; #P < 0.01 vs. OIR+PBS; x = mean ± SE; n = 5. (C) Immunohistochemical analysis demonstrating ICAM-1–specific immunofluorescence (FITC) and quantified by morphometric analysis (bar histograms). *P < 0.01 vs. P17; #P < 0.01 vs. OIR_PBS; x = mean ± SE; n = 3.

FIGURE 5

Fluvastatin effects on activation of STAT3 and HIF-1. (A) Western blot analysis showing immunoreactivity specific for the activated form of STAT3 phospho-Tyr705-STAT3 (PYSTAT3) and measured as optical density values normalized compared with total STAT3 protein (histogram). *P < 0.01 vs. P17; #P < 0.01 vs. OIR+PBS; x = mean ± SE; n = 5. (B) Western blot analysis showing immunoreactivity specific for HIF-1α, the inducible subunit of transcription factor HIF-1. Data are expressed as values of optical density normalized compared with actin (histogram). *P < 0.01 vs. P17; #P < 0.01 vs. OIR+PBS; x = mean ± SE; n = 5.