Elastin-derived peptides enhance angiogenesis by promoting endothelial cell migration and tubulogenesis through upregulation of MT1-MMP

Abstract

Elastin-derived peptides display a wide range of biological activities in a number of normal and transformed cells but their involvement in angiogenesis has not been reported. In the present study, we show that κ-elastin and VGVAPG hexapeptide elastin motif accelerated angiogenesis in the chick chorio-allantoic membrane in an in vivo model. They also stimulated pseudotube formation from human vascular and microvascular endothelial cells in the matrigel and collagen models as well as cell migration in an in vitro wound healing assay. Confocal and scanning electron microscopy analyses revealed the main reorganization of actin filaments mediated by elastin-derived peptides and changes in cell shape that correlated with a decrease of the cell form factor determined by computerized image analysis. Such elastin-derived peptide effects were attributed to upregulation of proMT1-MMP and proMMP-2 expression and activation at both the mRNA and protein levels. Batimastat, an inhibitor of furin convertase and TIMP-2, but not TIMP-1, totally abolished the influence of elastin-derived peptides (EDPs) on cell migration and tubulogenesis, thus favoring the involvement of MT1-MMP in such processes. To assess its contribution to EDP-mediated angiogenesis further, we used a small interfering RNA (siRNA) approach for specifically silencing MT1-MMP in human microvascular endothelial cells. Four sets of 21 bp siRNA duplexes targeting MT1-MMP mRNA were synthesized by in vitro transcription. Two of them proved to inhibit MT1-MMP expression efficiently but did not affect MT2-, MT3- and MT5-MMP expression. Seventy-two hours after transfection with 25 nM siRNAs EDP-induced MT1-MMP expression at the mRNA and protein levels was decreased fourfold. In parallel, proMMP-2 activation was inhibited. A scrambled siRNA, used as a negative control, had no effect. Finally, the effect of elastin peptides on pseudotube formation in MT1-MMP-siRNA transfected cells was totally abolished. These data emphasise the crucial role of MT1-MMP in the elastin-induced angiogenic phenotype of endothelial cells.

Automated time-lapse microscopy and high-resolution tracking of cell migration

“…One of our main focuses was to refine the commonly used scratch or wound healing assay into a highly quantitative Super Scratch assay. The scratch assay before this work had not progressed beyond a single or few timepoint assay where measurements were made of distance between edges (Maschler et al 2005;Zhu et al 2004;Pratt et al 2005;Wadham et al 2003;Besson et al 2004;Cretu et al 2005;Hoang et al 2004;John et al 2004;Ray et al 2003;Lynch et al 2005;Yarrow et al 2004), number of cells entering a defined area (Petridis et al 2004;Nishio et al 2005;Zhu et al 2004;Piccolo et al 2002;Robinet et al 2005;Raftopoulou et al 2004;Zhu et al 2005), or qualitative assessment without quantitation (Gavert et al 2005;Yoshida et al 2004;Zhang et al 2005;Herren et al 2001;Lee et al 2004;Motegi et al 2003;Laurent-Matha et al 2005;Miao et al 2005). We have shown that such a method of analysis is not as accurate a representation of cell migration as tracking individual cells starting from the advancing front of the wound.…”

Mesenchymal Stem Cells Overexpressing CXCR4 Attenuate Remodeling of Postmyocardial Infarction by Releasing Matrix Metalloproteinase-9

“…Angiogenesis resulting from injury stimulus plays a key role in repairing the infarcted heart by expanding the existing vascular network. Previous study demonstrated that EGCG, a general inhibitor of MMPs, [24] inhibits angiogenesis [25]. However, the molecular mechanisms by which EGCG inhibits angiogenesis are still unclear; thus, MSC MP9 -129SvEv mice employed for this study further demonstrated the important role of MMP-9 in angiogenesis.…”

Pharmacomodulation of Broad Spectrum Matrix Metalloproteinase Inhibitors Towards Regulation of Gelatinases

“…generate elastin fragments i.e. elastokines with potent angiogenic activity (Robinet et al, 2005). At the opposite, proteolysis of plasminogen and  3 chain of collagen IV leads to the formation of angiostatin and tumstatin (Cornelius et al, 1998;Kessenbrock et al, 2010).…”