Tumstatin Inhibits Retinal Endothelial Cell Invasion by Binding to the Collagen Binding Domain of Mmp2 and Inhibits Its Activation in-vitro and in-vivo

Abstract

Purpose: : The angioinhibitory activities of tumstatin are attributed in part due to its binding to specific integrins, independent of cellular invasion. Despite, its potent angioinhibitory activity, the molecular targets that regulate cellular invasion by tumstatin has not been investigated.

Methods: : Mouse retinal endothelial cells (MREC) were used to determine the effects of tumstatin on pro-angiogenic activity of bFGF. MREC proliferation was determined using [H³] thymidine incorporation and MTT assays. MREC migration was measured using Boyden chamber. MREC tube formation and invasion assays were assessed on Matrigel matrix. The intracellular signaling events related to FAK/Akt/mTOR/4E-BP1 phosphorylation was evaluated in MREC stimulated with bFGF and plated on fibronectincoated plate. Matrix metalloproteinase-2 (MMP-2) activation and MMP-2/tumstatin complex formation was studied using gelatin zymography, immunobloting and ISCO gradient analysis. Serum from alkali induced CNV (Choroidal neovascularization) mice, β3 and α3 integrin null endothelial cells secreting MMP-2 was analyzed using gelatin zymography.

Results: : Tumstatin inhibits MREC invasion and activation of MMP-2. Treatment with tumstatin also blocked MMP-2 activation mediated by both membrane-type-1 MMP (MT1-MMP) and 4-amino-phenyl mercuric acetate (APMA). ISCO gradient studies with recombinant MMP-2 confirmed that tumstatin inhibits proMMP-2 activation. Binding studies with collagen binding domain (CBD) of MMP-2 confirmed that tumstatin binds to CBD domain and results in inhibition of MMP-2 activation both in-vitro and in-vivo in CNV model.

Conclusions: : Angioinhibitory activities of tumstatin are mediated, in part, by inhibiting cellular invasion and MMP-2 activation, independent of integrins. Collectively our in-vitro and in-vivo data shows the first evidence demonstrating that tumstatin have dual functions in integrin dependent and independent suppression of CNV.

Keywords: extracellular matrix • signal transduction • choroid: neovascularization