Role Of SIRT1and MicroRNA-34a On Shear Stress Induced Endothelial Progenitor Cell Differentiation And Relative Mechanisms

Endothelial progenitor cells (EPCs) plays an important role in vascularrepair and maintenance of vascular homeostasis. Shear stress imposed byblood flow is one of the crucial modulators for EPC differentiation.However, the mechanobiological mechanism of EPC differentiationremains to be elucidated.Human umbilical cord blood-derived EPCs were exposed to laminarshear stress of15dyn/cm2by parallel plate chamber system. Shear stressenhanced EPC differentiation toward endothelial cells (ECs) whileinhibited to smooth muscle cells (SMCs). To determine the underlyingmechanisms of EPC differentiation induced by shear stress, the presentstudy is designed as the following two parts:In the first part, the roles of SIRT1in shear stress induced EPC differentiation were studied. Shear stress significantly activated Aktphosphorylation, augmented SIRT1expression and downregulatedhistone H3acetylation at lysine9(ac-H3K9), which reached the peak at6,12and24h, respectively. SIRT1siRNA in EPCs diminished theexpression of EC markers, KDR, VE-cadherin, vWF and CD31, butincreased the expression of SMC markers, α-SMA and sm22α, andresulted in upregulation of histone H3acetylation. However, resveratrol,an activator of SIRT1, had the opposite effects on both EPCdifferentiation and histone H3acetylation. Wortmannin, an inhibitor ofPI3K, suppressed the endothelial differentiation of EPCs, decreasedSIRT1expression and upregulated histone H3acetylation. In addition,SIRT1promoted in vitro tube formation of EPCs on collagen gels. Theseresults indicate that SIRT1plays a role in shear stress induced EPCdifferentiation into ECs and suggest that PI3K/Akt-SIRT1-ac-H3pathwaymay be involved in such a process.In the second part, the function of microRNA-34a (miR-34a) inshear stress induced EPC differentiation was investigated. Shear stressinduced miR-34a expression of EPCs in a time-dependent manner, with apeak at12h. The putative targets of miR-34a were predicted by threealgorithms, suggesting that FOXJ2might be one of the potential targetsof miR-34a involved in cell function regulation. Dual luciferase reporterassays showed that FOXJ2is the directly targeted by miR-34a. MiR-34a mimics and inhibitor treatments further confirmed the inverse correlationof miR-34a and FOXJ2expression. Also FOXJ2was suppressed by shearstress. Furthermore, miR-34a contributed to EPC differentiation byupregulating expressions of EC markers, KDR, VE-cadherin, vWF andCD31, and downregulating SMC markers α-SMA, sm22α, calponin andsmmhc. In addition, FOXJ2overexpression attenuated the endothelialdifferentiation of EPCs, while FOXJ2siRNA had the opposite effect. Inaddition, FOXJ2inhibited in vitro tube formation of EPCs in matrix gels.These data indicate that miR-34a participates in EPC differentiationinduced by shear stress, and miR-34a-FOXJ2may be involved in thus aprocess.In conclusion, the laminar shear stress within the physiological levelinduced EPC differentiation into EC lingeage whereas inhibited to SMCs,and PI3K/Akt-SIRT1-ac-H3K9, as well as miR-34a-FOXJ2pathway maybe crucial in this process. The data eluciated the molecular mechanism ofEPC response to flow stimuli, and provided a novel mechanobiologicalpotential orientation of vascular repair and relevant diseasesinvestigation.