Up-regulated Expression Of Matrix Metalloproteinases In Endothelial Cells Mediates Platelet Microvesicle-induced Angiogenesis

BackgroundNeovessel formation plays a great role throughout the life,which contributes to many physiological and pathophysiological processes,such as embryonic development,wound healing,atherosclerosis plaque destabilization,and tumour development.Neovessel formation,namely “vascularization”,includes the three modes: vasculogenesis,angiogenesis and arteriogenesis.Angiogenesis entails the sprouting of pre-existing vasculature to form new vessels,which requires many coordinated activities,such as endothelial cell(EC)proliferation,migration,and alignment to form vessel-like tube.Besides,ECs are thought to release a cocktail of angiogeneic factors,which account for their beneficial effects on the growth and restoration of tissues.It’s now clear that platelets paticipate in the vasculopathy.Platelet microvesicles(PMVs),also known as “platelet microparticles(PMPs)”,are shed from platelets upon activation and commonly defined as lipid membrane vesicles of 0.1-1 μm in size.PMVs,similar to platelets,possess important biological functions and are thus involved in various physiological and pathophysiological responses.In recent years,it has been acceptable that PMVs also play a great role in neovascularization by serving as a tool or mediator through which platelets exert their pro-angiogenic effects,as various molecules that have traditionally been considered soluble and are released by platelets,such as vascular endothelial growth factor(VEGF),platelet-derived growth factor(PDGF)and fibroblast growth factor(FGF),are in fact bound to PMVs.We previously demonstrated that SSL5-PMPs,staphylococcal superantigen-like protein 5(SSL5)-induced PMPs,provoke inflammatory mediator release in monocytes.Here we hypothesized that PMVs may promote angiogenesis by regulating EC secretion of pro-angiogenic factors,in addition to directly transporting angiogenic factors,which might be helpful to understand platelet more deeply and explore the suitable control measures for angiogenesis-associated disorders.Methods1.PMVs were prepared by stimulating platelets with thrombin receptor activator peptide-6(TRAP-6)in vitro.Flow cytometry was used to identify PMVs and BCA assay was exploited to quantify PMVs.Human umbilical vein endothelial cells(HUVECs)were cultured for experiments.Supernatants of centrifuged PMVs were set as vehicle control,while Tyrode’s buffer was used as blank control.The pro-angiogenic activity of PMVs in vitro was detected by Matrigel tube formation assay.CCK-8 assay was applied to evaluate the effect of PMVs on the cell proliferation.In addition,wound-healing assay and transwell chamber assay were used to assess the pro-migration effect of PMVs on HUVECs.2.HUVECs were cultured for experiments.Supernatants of centrifuged PMVs were set as vehicle control,while Tyrode’s buffer was used as blank control.The mRNA expression of pro-angiogenic factors secreted by HUVECs,such as hypoxia-induced factor 1α(HIF-1α),vascular endothelial growth factor-A(VEGF-A),fibroblast growth factor-2(FGF-2),interleukin-8(IL-8)and matrix metalloproteinases(MMPs),was examined by real-time quantitative PCR.The protein level of MMPs was assayed by western blot.The activity of MMP-2/9 in supernatant of culture medium was analysed by gelatin zymography.Futhermore,effects of MMP inhibitors on the PMV-enhanced tube formation and migration were also evaluated to identify the role of MMPs in the PMV-induced angiogenesis.To explore the potential molecular basis of PMVs and HUVECs interactions resulting in angiogenesis,neutralising antibodies were used to block the binding of CD40 L to CD40 or of P-selectin to PSGL-1.3.Matrigel plug assay was adopted to examine the effect of PMVs on angiogenesis in vivo.Results1.PMVs promote HUVEC tube formation in vitro.(1)PMVs enhanced HUVEC tube formation in a dose-dependent manner compared with the blank control and vehicle control.(2)Supernatants and PMVs promoted HUVEC proliferation and the enhancements of HUVEC proliferation were similar compared with the blank control.(3)PMVs led to dose-dependent promotion of HUVEC migration.2.The mechanism by which PMVs promote HUVEC tube formation in vitro.(1)HIF-1α,VEGF-A,PDGF m RNA levels remained unchanged,while IL-8 levels were significantly reduced in a dose-dependent manner.(2)PMVs dose-dependently stimulated MMP-2 and MMP-9 expression in HUVECs,while there was no change in MMP-7 and TIMP-1 production.Moreover,PMVs significantly stimulated synthesis and activation by HUVECs of MMP-2 and MMP-9 in a dose-dependent manner.(3)PMV-enhanced tube formation and migration were markedly inhibited by pan-MMP inhibitor GM6001.(4)MMP-2 and MMP-9 inhibitors both weakened the PMV-enhanced tube formation and migration,while the effecacy of MMP-2 inhibitor surpassed that of MMP-9 inhibitor.(5)CD40L-CD40 and P-selectin-PSGL-1 interactions were not involved in PMV-enhanced angiogenesis.3.The role and mechamism of PMVs in promoting angiogenesis in vivo.(1)PMVs promoted angiogenesis in vivo.(2)PMV-enhanced angiogenesis in vivo was also mediated by MMPs,while MMP-2 might play a more important role than MMP-9.ConclusionsIn the present study,we demonstrated that PMVs both significantly promote angiogenesis in vitro and in vivo.And the enhancement of PMVs on the cell migration surpasses supernatants,while the effects of PMVs and supernatants on EC proliferation are similar.The possible mechanism is that PMVs up-regulate the expression and gelatinolytic activity of MMP-2/9,which mediates the process of extracellular matrix(ECM)degradation and is in favor of the pro-angiogenic process in futher.Our findings reveal a novel mechanism by which PMVs promote angiogenesis,in addition to directly transporting angiogenic factors,which might be beneficial to understand platelet more deeply and explore the suitable control measures for angiogenesis-associated disorders.