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Flow shear stress plays important roles in modulating differentiation of endothelial progenitor cells (EPCs). MicroRNAs are crucial for diverse cellular processes, but the expressions and functions of microRNAs in EPCs responding to mechanical stimuli remain unclear. We sought to determine the effects of microRNA-34a (miR-34a) and a novel target Forkhead box j2 (Foxj2) on shear stress-induced EPC differentiation. Human umbilical cord blood-derived EPCs were exposed to laminar shear stress of 15 dyn/cm 2 with parallel plate flow chamber system. Real time RT-PCR showed that shear stress significantly increased miR-34a expression, which was accompanied by the endothelial differentiation of EPCs. Whereas Foxj2, a putative target of miR-34a predicted by multiple algorithms, was suppressed in this process. Dual luciferase reporter assays, as well as miR-34a mimics and inhibitor treatment were used to confirm the interplay between miR-34a and Foxj2. Our results revealed an inverse correlation of miR-34a and Foxj2 expressions implicated in the endothelial differentiation of EPCs. MiR-34a contributed to this process by up-regulating the expressions of endothelial cell markers, and down-regulating smooth muscular cell markers. In addition, Foxj2 overexpression attenuated endothelial differentiation of EPCs, while Foxj2 siRNA had the opposite effect. These data suggested a unique mechanism that shear stress induces the expression of miR-34a, which targets to Foxj2 and promotes endothelial differentiation of EPCs. The results provide new insights into miR-34a/Foxj2 on shear stress-induced EPC differentiation.

The arterial system is subjected to cyclic strain because of periodic alterations in blood pressure, but the effects of frequency of cyclic strain on arterial smooth muscle cells (SMCs) remain unclear. Here, we investigated the potential role of the cyclic strain frequency in regulating SMC alignment using an in vitro model. Aortic SMCs were subject to cyclic strain at one elongation but at various frequencies using a Flexercell Tension Plus system. It was found that the angle information entropy, the activation of integrin-beta1, p38 MAPK, and F/G actin ratio of filaments were all changed in a frequency-dependent manner, which was consistent with SMC alignment under cyclic strain with various frequencies. A treatment with anti-integrin-beta1 antibody, SB202190, or cytochalasin D inhibited the cyclic strain frequency-dependent SMC alignment. These observations suggested that the frequency of cyclic strain plays a role in regulating the alignment of vascular SMCs in an intact actin filament-dependent manner, and cyclic strain at 1.25 Hz was the most effective frequency influencing SMC alignment. Furthermore, integrin-beta1 and p38 MAPK possibly mediated cyclic strain frequency-dependent SMC alignment.

Aims Low shear stress (LSS) plays a significant role in vascular remodelling during atherogenesis, which involves migration, proliferation, and apoptosis of vascular smooth muscle cells (VSMCs). The aim of the present study is to elucidate the molecular mechanisms by which LSS induces vascular remodelling. Methods and results Using proteomic techniques, two-dimensional electrophoresis, and mass spectrometry, the protein profiles of Sprague–Dawley rat aorta cultured under two levels of shear stress, 5 and 15 dyn/cm2, were determined. The results showed a significantly lower expression of protein-Rho-GDP dissociation inhibitor alpha (Rho-GDIα) in the LSS vessels. Rho-GDIα signalling mechanisms and effects on VSMC migration and apoptosis were then studied to understand the role of Rho-GDIα in the LSS-induced vascular remodelling. A decrease in Rho-GDIα expression by using target small interfering RNA (siRNA) transfection caused increases in the phosphorylation of Rac1 and Akt and enhancements of VSMC migration and apoptosis. Treatment with the PI3K/Akt-specific inhibitor wortmannin significantly decreased Akt phosphorylation, but had no effect on Rho-GDIα expression and Rac1 phosphorylation. Wortmannin was able to reverse the Rho-GDIα siRNA-induced enhancement of VSMC migration, but not VSMC apoptosis. Conclusion The results indicate that the LSS-induced VSMC migration and apoptosis are mediated by a downregulation of Rho-GDIα. The effect of Rho-GDIα on VSMC migration is mediated by the PI3K/Akt pathway, but its effect on VSMC apoptosis is not.

Phenotype transformation of vascular smooth muscle cells (VSMCs) has been reported to be directly influenced by the frequency of mechanical strain. This study explored the effects of different frequencies of mechanical strain on expression of phenotype marker h1-calponin and the possible mechanism. VSMCs were subjected to cyclic strains of 10% elongation at 1 and 2 Hz for 24 h by using a Flexercell strain unit. The protein expression of h1-calponin was assessed by Western blotting and the possible protein kinases involved were evaluated by their specific inhibitor or targeted siRNA 'knock-down.' The results showed that cyclic strains modulated the expressions of h1-calponin, phospho-p38, Rac and Rho-guanine nucleotide dissociation inhibitor alpha (Rho-GDI alpha) in nonlinear frequency-dependent manners. This nonlinear frequency-dependent change of h1-calponin expression could be blocked by a specific p38 inhibitor, SB202190. The changed expression of phospho-p38 induced by the frequencies of cyclic strain was reversed by targeted siRNA 'knock-down' of Rac, while enhanced by targeted siRNA 'knock-down' of Rho-GDI alpha. These results suggest that the frequency-dependent expression of h1-calponin under cyclic strain is mediated at least partly by the regulation of Rac and Rho-GDI alpha expression on the activation of p38 pathway.

Phenotype transformation of vascular smooth muscle cells (VSMCs) is known to be modulated by mechanical strain. The present study was designed to investigate how different frequencies of mechanical strain affected VSMC phenotype. VSMCs were subjected to the strains of 10% elongation at 0, 0.5, 1 and 2 Hz for 24 h using a Flexercell strain unit. VSMC phenotype was assessed by cell morphology, measurement of two-dimensional cell area, Western blotting for protein and RT-PCR for mRNA expression of differentiation markers. Possible protein kinases involved were evaluated by Western blotting with their specific antibodies. The strains at certain frequencies could induce a contractile morphology in VSMC with almost perpendicular alignment to the strain direction. The strains also regulated protein and mRNA expression of several differentiation markers, as well as the activation of extracellular signal-regulated kinases (ERKs), p38 MAP kinase and protein kinase B (Akt) in a frequency-dependent manner. Furthermore, the inhibition of the p38 pathway could block the frequency-induced phenotype modulation of VSMCs, but not inhibition of ERK or Akt pathways. These results indicate that the frequency of cyclic strain can result in the differentiated phenotype of VSMCs, and it is mediated at least partly by the activation of the p38 pathway.

Vascular endothelial cells (ECs) are constantly exposed to blood flow-induced shear stress; these forces strongly influence the behaviors of neighboring vascular smooth muscle cells (VSMCs). VSMC migration is a key event in vascular wall remodeling. In this study, the authors assessed the difference between VSMC migration in VSMC/EC coculture under static and shear stress conditions. Utilizing a parallel-plate coculture flow chamber system and Transwell migration assays, they demonstrated that human ECs cocultured with VSMCs under static conditions induced VSMC migration, whereas laminar shear stress (1.5 Pa, 15 dynes/cm2) applied to the EC side for 12 h significantly inhibited this process. The changes in VSMC migration is mainly dependent on the close interactions between ECs and VSMCs. Western blotting showed that there was a consistent correlation between the level of Akt phosphorylation and the efficacy of shear stress-mediated EC regulation of VSMC migration. Wortmannin and Akti significantly inhibited the EC-induced effect on VSMC Akt phosphorylation and migration. These results indicate that shear stress protects against endothelial regulation of VSMC migration, which may be an atheroprotective function on the vessel wall.