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G.E. Lang, G.K. Lang, A. Baldysiak-Figiel; Effect of the PKC ß Inhibitor LY 379196 in Combination with Growth Factors on Proliferation of Bovine Retinal Vascular Endothelial Cells . Invest. Ophthalmol. Vis. Sci. 2003;44(13):3208.
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Purpose:Diabetes mellitus is associated with numerous long-term clinical complications such as retinopathy. It has been demonstrated that retinal vascular cells derived from diabetic animals with retinopathy have high expression levels and increased activation of the protein kinase Cß (PKCß) -dependent transduction pathway. The expression of VEGF, IGF-1 or bFGF is enhanced in diabetic retinopathy and the proliferative action of some of these growth factors has been attributed, at least in part, to the activation of PKCß. In this study, we investigated if LY 379196, a selective inhibitior of PKCß, is able to inhibit the VEGF, IGF-1 and bFGF-induced proliferation of bovine retinal microvascular endothelial cells (BREC). Methods:BREC were isolated using the Dynabeads-based method and cultured in ECGM-MV medium. Subsequently, BREC were detached with trypsin and seeded into 96-well-plates. Upon reaching confluency, medium was changed to serum free defined medium for 24h. The cells were stimulated for the next 48 h with VEGF (20 ng/ml), IGF-1 (20 ng/ml) and bFGF (10 ng/ml) alone or in combination with LY 379196 (1-100 nM). On the second day, the cells were pulsed with [3H]-thymidine for 24 h. [3H]-Thymidine incorporation was assessed by liquid scintillation counting as a direct measure of BREC proliferation. In all experiments, untreated serum free cultures were used as negative controls. Statistical analysis was performed using two-sample t-test. Results:VEGF, IGF-1 and bFGF significantly stimulated the proliferation of BREC (p<0.0001). PKCß inhibitor was able to inhibit the proliferation of BREC induced by VEGF (p<0.0001), IGF-1 (p=0.0001) and bFGF (p<0.0001) in vitro, as compared to the treatment with growth factor alone only at the concentration of 10 nM. Conclusions:Our data shows that the potent mitotic action of VEGF, IGF-1 and bFGF on BREC can be totally inhibited by LY 379196 and strongly argues for the importance of the PKCß-dependent pathway for induction of endothelial cell proliferation. Additionally, our findings point at LY 379196 as a putative potent antiproliferatory drug in treatment of proliferative retinopathy.
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