Extra Cellular Matrix Derived Metabolite Regulates Angiogenesis by FasL Mediated Apoptosis

Object: Antiangiogenic treatments are beginning to give promising outcomes in many vascular diseases including tumor angiogenesis. In this current study the antiangiogenic and pro-apoptotic actions of alpha 1(IV)NC1 and its N- and C-peptides alpha 1S1(IV)NC1, alpha 1S2(IV) NC1 were investigated in-vitro and in-vivo. Study Method: Endothelial cells (ECs) were treated with alpha 1(IV)NC1, alpha 1S1(IV)NC1, alpha 1S2(IV)NC1 and in-vitro proliferation, migration, tube formation and apoptotic assays were executed. FasL, Fas, Caspase-8, -3 and PARP activations were studied using immunoblotting analysis using specific antibodies. Also the in-vivo antiangiogenic and pro-apoptotic effects were tested using alpha 1(IV)NC1 in a mice model. Results: Like alpha 1(IV)NC1, its N- and C-terminal alpha 1S2(IV)NC1 and alpha 1S1(IV)NC1 domains posses anti-proliferative, proapoptotic activity and inhibit ECs migration and tube formation in-vitro. Both alpha 1S1(IV)NC1 and alpha 1S2(IV)NC1 domains promote apoptosis by activating FasL and down stream apoptotic events including activation of caspase-8, -3 and PARP cleavage in a dose dependent manner in-vitro in ECs. Tumors in mice showed apoptotic TUNEL positive microvasculature upon alpha 1(IV)NC1 treatment, indicating inhibition of tumor angiogenesis and tumor growth. Further, the antitumor activity of alpha 1(IV)NC1 was abrogated when caspase-3 inhibitor was used. These results conform additional properties of alpha 1(IV)NC1 as an endogenous angioinhibitor that induces apoptosis in-vitro and in-vivo by activating FasL mediated caspase-3. Significance: alpha 1(IV)NC1 and its N-and C-terminal alpha 1S1(IV)NC1 and alpha 1S2(IV)NC1 domains also posses pro-apoptotic and angioinhibitory activity in-vitro and in-vivo. alpha 1(IV)NC1 regulates tumor angiogenesis by activating FasL mediated apoptosis in-vitro and in-vivo. These results demonstrate that alpha 1(IV)NC1 and its peptides inhibit neo-vascular diseases.