Proapoptotic and survival signaling pathways plays a role in thrombospondin type I repeat mediated apoptosis in human microvascular endothelial cells

Abstract

Abstract Introduction: 3 TSR/hTSP-1, a fragment of human thrombospondin is important in physiological and pathological angiogenesis. We investigated the molecular mechanisms for 3TSR/hTSP-1 mediated apoptosis in microvascular endothelial cells. Methods: Human dermal microvascular endothelial cells (HDMEC, passages 6/7) were treated with recombinant 3TSR/hTSP-1, and camptothecin for 24 hours.Ten thousand cells were assayed with FACS (Becton Dickinson, FACS Calibur) and Cell Quest software. Caspase-8, caspase-9, and caspase 3 activities in cell lysates were assayed (BD ApoAlert™ Caspase Fluorescent and Colorimetric Assay, BD Biosciences). Detection of cytochrome c release into the cytosolic cell fraction was performed using MBL cytochrome c kit. Activation of Akt was assayed by Western blot with phosphorylation specific Akt antibody (Cell Signaling Technolgy). Results: 3TSR/hTSP-1 induced up to 42% apoptosis at the concentration of 5 mM compared to 10% apoptosis seen in controls. 3TSR/hTSP-1 resulted in activation of caspase-8 and caspase-9 which triggered the caspase cascade, resulting in activation of apoptosis executioner caspase-3 and apoptosis. 3TSR/hTSP-1 (2 mM) still demonstrated a potent apoptosis-inducing effect even in the presence of high concentrations of VEGF (50 ng/ml). 3TSR/hTSP-1 reduced endogenous Akt activity significantly, and partially attenuated the phosphorylation of AKT473 and cFLIP(L) expression stimulated by VEGF, which suggests modulation of cell survival through modulation of PI3kinase/Akt signaling. Conclusions: Extrinsic and intrinsic signaling pathways play an independent role in 3TSR/hTSP-1-mediated apoptosis in endothelial cells. Akt signaling is also involved in the regulation of 3TSR/hTSP-1-mediated apoptosis in microvascular endothelial cells cultured in low serum or stimulated by VEGF.