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Raj K. Verma, Venugopal Gunda, Chandra S. Boosani, Sudhakar A. Yakkanti; Arresten Regulates Choroidal Angiogenesis By Fasl, Caspase-3 And Parp Mediated Apoptosis In-vitro And In-vivo. Invest. Ophthalmol. Vis. Sci. 2012;53(14):2949.
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Type IV collagen plays a crucial role in regulation of angiogenesis. Proteolytic degradation of type IV collagen in the vascular basement membrane (VBM) generates antiangiogenic molecules. We previously reported that the antiangiogenic non-collagenous (NC1) domain derived from α1 chain of type IV collagen, α1(IV)NC1 (arresten) induces apoptosis in endothelial cells. However, active site of arresten responsible for its antiangiogenic properties is not yet identified. In the present study, the N- and C-terminal regions of arresten were cloned as two individual subunits and studied their anti-angiogenic and pro-apoptotic properties.
Both the N- and C-terminal subunits of arresten were cloned in the pET22b (+) expression vector and expressed in Escherichia coli. Expressed subunits were purified using affinity and size exclusion chromatographies with simultaneous renaturation.
Our preliminary in-vitro results with mouse choroidal endothelial cells (MCECs) have shown inhibition of VEGF and bFGF induced endothelial cells proliferation, migration and tube formation by both the subunits. In addition, arresten promoted FasL mediated apoptosis through caspase-3 and PARP both in-vitro and in-vivo.
Both N- and C-terminal subunits of arresten exhibited antiangiogenic activity in MCECs by showing inhibitory effects on VEGF and bFGF induced proliferation, migration and tube formation. The FasL mediated apoptosis activation by arresten is also identified.
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