Statin Prevents VEGF and High Glucose–Dependent STAT3 Activation and STAT3–Dependent ICAM–1 Expression in Retinal Endothelial Cells and in Diabetic Rat Retinas

Abstract

Purpose: : Over expression of VEGF and the adhesion molecule ICAM–1 has been shown to cause retinal vascular inflammation and retinopathy. Preliminary studies indicated that statin treatment prevents the up–regulation of VEGF and ICAM–1 in the diabetic retina. We have previously shown that the transcription factor STAT3 is a key regulator of VEGF expression and activity suggesting its involvement in diabetic retinopathy. In this study we evaluated the role of STAT3 in VEGF– and/or high glucose–dependent expression of ICAM–1 in the retinal endothelium and tested the effects of statins in preventing these effects.

Methods: : Western blotting was used to measure ICAM–1 protein expression and phosphotyrosine–STAT3 (PYSTAT3). ICAM–1 and PYSTAT3 levels were assessed in streptozotocyn–induced diabetic rats (STZ–rat), STZ rats treated with simvastatin (5mg/Kg/day) and normoglycemic age–matched control rats. Retinal endothelial cells (REC) were cultured for 72 hours with 25mM glucose (HG) or for 24 hours with 20ng/ml VEGF165 in the presence or absence of 10µM simvastatin and compared to REC cultured in normal glucose medium (5mM glucose, NG). ICAM–1 production was also tested in REC infected with 30 m.o.i. of an adenovirus carrying a transcriptional inactive STAT3 mutant (AdSTAT3D) or the reporter gene green fluorescent protein (AdGFP).

Results: : In STZ–rats retinas, after 4 weeks of hyperglycemia, we found a significant elevation of PYSTAT3 and ICAM–1 expression, as compared to age–matched normoglycemic rats. Treatments of the STZ–rats with simvastatin prevented the increases in expression of ICAM–1 and PYSTAT3. In addition, treatment of REC with 10µM simvastatin also prevented HG– and VEGF–stimulated formation of PYSTAT3 and ICAM–1. Finally, in REC, HG– and VEGF–stimulated expression of ICAM–1 was blocked by infection of the cells with AdSTAT3D but not by infection with AdGFP.

Conclusions: : Our results indicate that increases in ICAM–1 in both the diabetic retina and in REC treated with HG or VEGF are associated with activation of STAT3. Moreover, STAT3 activity is required for VEGF or HG–dependent ICAM–1 expression in REC. In addition statin treatment prevents ICAM–1 expression and STAT3 activation in vivo and in vitro, suggesting that the statin action in down–regulating ICAM–1 expression is associated with its ability to inhibit HG– and VEGF–induced activation of STAT3.

Keywords: diabetic retinopathy • signal transduction: pharmacology/physiology • transcription factors