Role Of Protein Kinase C In Early Diabetic Retinopathy

Purpose To investigate (1) the role of PKC on the alteration of ET system, VEGF and BRB (2) the interventional effect of specific PKC inhibitor in early diabetic retinopathy.Methods The cultured HUVEC line and the SD rats model with STZ-induced diabetes were used. The measurement of PKC activities from membranous and cytosolic fractions, retinal and vitreous concentration of VEGF protein were conducted by ELISA. HUVEC line and the retinal tissues were analysed for the expression of ET-1, ET-3, ET-A, ET-B and VEGF mRNA by semi-quantitative RT-PCR. HUVEC line was immunostained for ET-1 and VEGF by immunocytochemistry. The retina and retinal vasculature isolated by trypsin digest technique was immunostained for ET-1 by immunohistochemistry. BRB breakdown was quantified using the Evans blue technique. The effect of a general PKC inhibitor, GF109203X, on the alteration of ET system, VEGF and BRB were observed in vivo and in vitro.Results (1) In the cultured HUVEC line, PKC specific activities from the membranous fraction were significantly increased by 45% in the media with high glucose (25 mM) compared with that in the media with low glucose (5.5 mM), and were reversed after exposure in the media with low glucose for an additional 48 h. Retinal PKC specific activities were significantly increased by 45% in the membranous fraction in 2-week diabetic rats compared with normal rats. The PKC specific activities from the cytosolic fraction were unchanged both in the cultured HUVEC line and in the retina of diabetic rats. (2) In the cultured HUVEC line, ET-1 mRNA and immunoreactivity were increased after exposure in the high glucose media, and were decreased after addition of GF109203X (final concentration 10^-5, 10^-6, 10^-7 M) in a dose-dependent manner. The retina from the diabetic rats showed increased ET-1 mRNA expression after 2 weeks, while no changes were found for ET-3, ET-A and ET-B. ET-1 immunoreactivity was also increased in the retinal vasculature of diabetic rats. Retinal ET-1 expression was decreased after intravitreal injection of GF109203X (10^-5, 10(-6), 10^-7 M) in a dose-dependent manner, whereas there were no...