Expression of Tissue Inhibitor of Metalloproteinase–3 Induces Endothelial Cell Apoptosis: A Caspase –Independent Mechanism

Abstract

Abstract: : Purpose: Tissue inhibitor of metalloproteinase–3 (TIMP3), one of the four known TIMPs, is an endogenous inhibitor of VEGF mediated angiogenesis by virtue of its ability to inhibit the binding of VEGF to its receptor VEGF receptor–2 (KDR). Since VEGF is a survival factor for endothelial cells we set out to examine if apoptosis played a role in the anti–angiogenic activity of TIMP–3. Methods: quantitated using standard assays. Results:TIMP–3 was expressed in monkey choroid endothelial cells, and porcine aortic endothelial cells using adenovirus vectors. Apoptosis was TIMP–3 can promote apoptosis in endothelial cells expressing KDR receptor (PAE/KDR) compared with cells lacking KDR but expressing PDGF(b) receptor (PAE/PDGFbR). TIMP–3 expression significantly increases the caspase activity in PAE/KDR cells compared to PAE/PDGFbR. However treatment with a pan–caspase inhibitor (Z–VAD–FMK) does not block apoptosis. Furthermore, expression of TIMP3 fails to lead to increased cleavage of focal adhesion kinase (FAK) in the cells undergoing apoptosis. These cells instead display an irregular FAK tyrosine phosphorylation and an incomplete formation of focal adhesion complexes. Conclusions: These data indicate a caspase–independent mechanism for TIMP3 mediated–apoptosis in endothelial cells, which may be a contributing factor for its anti–angiogenic activity.

Keywords: choroid: neovascularization • cell death/apoptosis • enzymes/enzyme inhibitors