Effect of the PKC–ß inhibitor LY 379196 alone and in combination with growth factors on the proliferation of bovine choroidal vascular endothelial cells

Abstract

Abstract: : Purpose: Age–related macular degeneration (AMD) is associated with increased expression of the growth factors VEGF, IGF–1, and bFGF, which stimulate proliferation of choroidal endothelial cells. The proliferative action of these growth factors has been attributed, at least in part, to the activation of protein kinase C–ß (PKC–ß). In this study we investigated if LY 379196, a selective inhibitor of PKC–ß, is able to inhibit the VEGF, IGF–1, and bFGF–induced proliferation of bovine choroidal microvascular endothelial cells (BCEC). Methods: BCEC were isolated using the Dynabeads–based method and cultured in ECGM–MV medium. For proliferation assays, BCEC were seeded into 96–well–plates and grown to confluency. Subsequently, the cells were incubated in serum free defined medium for 24h and treated for another 48 h with LY 379196 (0.01 – 1000 nM) or with VEGF (20 ng/ml), IGF–1 (20 ng/ml) and bFGF (10 ng/ml) alone or in combination with LY 379196 (10 nM) in the presence of [3H]–thymidine for the last 24 h. [3H]–thymidine incorporation was assessed by liquid scintillation counting. In all experiments, untreated serum free cultures were used as negative controls. Statistical analysis was performed using two–sample t–test. Results: LY 379196 inhibited the proliferation of BCEC at the concentration of 10 – 1000 nM (p<0.01), while significantly increased cell proliferation was found in the presence of LY 379196 at the concentration of 0.01 – 1 nM (p<0.01). VEGF–, IGF–1–, and bFGF–induced BCEC proliferation (p<0.0001) was inhibited by LY 379196 at the concentration of 10 nM (p<0.0001). Conclusions:Our data show that LY 379196 has a concentration–dependent modulatory effect on BCEC proliferation and is able to abolish the potent mitotic action of VEGF, IGF–1, and bFGF, which are the key growth factors involved in the onset and progression of AMD. Our results strongly argue for the importance of the PKC–ß–dependent pathway in induction of endothelial cell proliferation. Additionally, our findings point at LY 379196 as a potent antiangiogenic drug candidate in treatment of neovascular AMD.

Keywords: age–related macular degeneration • choroid: neovascularization • proliferation