FAK and p38-MAP Kinase-Dependent Activation of Apoptosis and Caspase-3 in Retinal Endothelial Cells by α1(IV)NC1

PURPOSE. To determine the impact of the antiangiogenic factor alpha 1(IV)NC1 on vascular endothelial growth factor-mediated proangiogenic activity in mouse retinal endothelial cells (MRECs). METHODS. Primary culture of MRECs was established as previously described and was used to determine the effects of alpha 1(IV)NC1 on the proangiogenic activity of VEGF. Cell proliferation was evaluated using [H-3]-thymidine incorporation and 3,(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide colorimetric assays. Cell migration was determined using modified Boyden chamber and scratch wound assays and tube formation was assessed on basement membrane matrix (BMM). Intracellular signaling events Bcl-2/Bcl-x(L) and caspase-3/poly (ADP-ribose) polymerase (PARP) activities were evaluated in cells stimulated with VEGF and plated on type IV collagen-coated dishes. Apoptosis was assessed by measuring caspase activity and by performing quantitative fluorescence analysis using fluorescence-activated cell sorting assay. Subcutaneously injected VEGF induced in vivo neovascularization was studied with the BMM plug assay. RESULTS. VEGF-induced subconfluent MREC proliferation, migration, and tube formation were significantly inhibited by alpha 1(IV)NC1 at 1 mu M (P < 0.001). alpha 1(IV)NC1 induced MREC apoptosis is mediated by inhibition of Bcl-2 and Bcl-x(L) expression and activation of caspase-3/PARP through FAK/p38-MAPK signaling. In addition, alpha 1(IV)NC1 dose dependently inhibited VEGF-mediated neovascularization in vivo . CONCLUSIONS. alpha 1(IV)NC1 inhibited VEGF-mediated angiogenesis by promoting apoptosis and caspase-3/PARP activation and by negatively impacting FAK/p38-MAPK phosphorylation, Bcl-2, and Bcl-x(L) expression leading to MREC death. The endothelial-specific inhibitory actions of recombinant alpha 1(IV)NC1 may be of benefit in the treatment of a variety of eye diseases with a neovascular component. (Invest Ophthalmol Vis Sci. 2009; 50: 4567-4575) DOI:10.1167/iovs.09-3473