Cleaved high molecular weight kininogen inhibits tube formation of endothelial progenitor cells via suppression of matrix metalloproteinase 2

Abstract

Background and objective Endothelial progenitor cells (EPCs) contribute to postnatal neovascularization, thus promoting wide interest in their therapeutic potential in vascular injury and prevention of their dysfunction in cardiovascular diseases. Cleaved high molecular weight kininogen (HKa), an activation product of the plasma kallikrein-kinin system (KKS), inhibits the functions of differentiated endothelial cells including in vitro and in vivo angiogenesis. In this study, our results provided the first evidence that HKa is able to target EPCs and inhibits their tube forming capacity. Methods and results We determined the effect of HKa on EPCs using a three-dimensional vasculogenesis assay. Upon stimulation with vascular endothelial growth factor (VEGF) alone, EPCs formed vacuoles and tubes, and differentiated into capillary-like networks. As detected by gelatinolytic activity assay, VEGF stimulated secretion and activation of matrix metallopeptidase 2 (MMP-2), but not MMP-9, in the conditioned medium of 3D culture of EPCs. Specific inhibition or gene ablation of MMP-2, but not MMP-9, blocked the vacuole and tube formation by EPCs. Thus, MMP-2 is selectively required for EPC vasculogenesis. In a concentration-dependent manner, HKa significantly inhibited tube formation by EPCs and the conversion of pro-MMP-2 to MMP-2. Moreover, HKa completely blocked the association between pro-MMP- 2 and αvβ3 integrin, and its inhibition of MMP-2 activation was dependent on the presence of αvβ3 integrin. In a purified system, HKa did not directly inhibit MMP-2 activity. Conclusions HKa inhibits tube forming capacity of EPCs by suppression of MMP-2 activation, which may constitute a novel link between activation of the KKS and EPC dysfunction.

Anti-angiogenic and tumor-suppressive roles of candidate tumor-suppressor gene, Fibulin-2, in nasopharyngeal carcinoma

“…Downregulation of MMP2 was also observed in the transfectants by zymography and Q-PCR. The stimulation of MMP2 by VEGF has been reported in endothelial progenitor cells; knockdown of MMP2 by small interfering RNA and suppression of MMP2 by kininogen inhibits tube formation of endothelial progenitor cells in vitro (Wu et al, 2010). Downregulation of MMP2, which contributes to the angiogenesis inhibition of the FBLN2S stable transfectants, may be associated with the reduced expression of VEGF.…”

Regulation of Endothelial Progenitor Cell Function by Plasma Kallikrein-Kinin System

“…MMP-2 and MMP-9 participate in the mobilization of EPCs. Recently we have demonstrated that MMP-2 is selectively required for VEGF-stimulated vasculogenic differentiation of EPCs [11]. In the conditioned culture media of EPCs embedded in 3D collagen gel, VEGF selectively stimulated the secretion and activation of MMP-2, but not MMP-9, and VEGF-stimulated proMMP-2 expression and secretion are time-dependent, the secreted proMMP-2 was meanwhile converted into active form.…”

“…In the conditioned culture media of EPCs embedded in 3D collagen gel, VEGF selectively stimulated the secretion and activation of MMP-2, but not MMP-9, and VEGF-stimulated proMMP-2 expression and secretion are time-dependent, the secreted proMMP-2 was meanwhile converted into active form. As the specific inhibitor of MMP-2 (444244), but not MMP-9 (444278), concentration-dependently attenuated VEGFstimulated tube formation by EPCs, suggesting the role of MMP-2 activities in tube formation by EPCs [11]. The requirement of MMP-2 and MMP-9 expression for the tube formation by EPCs is examined by specific siRNA oligonucleotides.…”

“…The inhibitory effect of HKa may result from its inhibition of v 3 integrin function [7,9], and induction of apoptosis via its interaction with uPAR [10]. Because EPCs express high levels of uPAR, which is a HKa receptor, HKa may exerts inhibitory effect on EPCs [11]. In this chapter, we summarize the recent observations that the KKS regulates EPC function.…”

“…If EPCs are to be defined as true progenitor cells with postnatal vasculogenic potential, they should give rise to differentiated progeny with the capacity for vessel formation. In a 3D collage gel, EPCs, but not differentiated endothelial cells such as HAECs and HUVECs, exhibit strong capacity to form vacuoles and tubes in the presence of VEGF [11], suggesting that EPCs are unique in their stronger potential for tubular morphogenesis and are more sensitive to physiological growth factor stimulation.…”