Effect Of Mechanic Induced Vascular Endothelial Cells And Smooth Muscle Cells On Rolling Adhesion And Differentiation Of Endothelial Progenitor Cells

Endothelial progenitor cells (EPCs) is a population of pluripotent cells, which have a crucial role in maintaining the integrity of the endothelium. EPCs need multiple steps, mobilization, homing, capture, recruitment, rolling, adhesion, migration and differentiation, to repair the damage endometrial layer. ECs, VSMCs and hemodynamics play vital roles in modulating the adhesion, rolling and differentiation of EPCs, but the mechanisms have not been understood completely. Therefore, we should consider the influence of hemodynamics and adjacent ECs and VSMCs when studying adhesion and differentiation of EPCs.In the present study, human EPCs were isolated from cord blood and identified by double staining for the fluorescent labeled ac-LDL and UEA-lectin. Primary ECs and VSMCs were obtained from human umbilical veins by using trypsin-digest and tissue pieces-stick methods respectively.EPCs rolling observation system was set up with a parallel-plate glass flow chamber, where cultured ECs monolayer on the bottom. The rolling velocities of EPCs were recorded under different shear stress, 0.1 - 0.4 dyn/cm2, and without ECs, with ECs or with ECs treated with tumor necrosis factor arpha (TNFα), 10 ng/ml, for 6 hours, respetively. Moreover, EC adhesion molecule expressions, such as VCAM, ICAM, P-Selectin and E-Selectin, were detected by western blot.In the experiment of EPCs differentiation,EPCs were co-cultured indirectly with ECs or VSMCs, with the stretched ECs or VSMCs which were exposured to 5 % - 1.25 Hz-strain for 24 hours by an improved FX-4000T cell strain loading system. Then, Western blot and RT-PCR were used to examining the expressions of endothelial marker molecules, such as CD31, vascular endothelial growth factor receptor (KDR) and von Willebrand factor (vWF), and VSMCs marker molecules, Calponin,α-actin and SM22α, in EPCs respectively. Furthermore, EPC signal molecules, Akt, ERK and Sirt1, were also analyzed by Western blot.The results showed that shear stress pushed the captured EPC rolling on the ECs, and ECs reduced the rolling velocity of the captured EPCs. The expressions of VCAM, ICAM, P-Selectin and E-Selectin in EPCs were upregulated by TNFα, which resulted in a lower rolling velocity of EPCs. ECs promoted VCAM expression, but inhibited expressions ofα-actin and Calponin in EPCs. However, VSMCs decreased the mRNA levels of CD31, KDR and vWF, and increased Calponin and SM22αmRNA levels in EPCs. The stretched ECs reduced the mRNA level of CD31 and vWF in EPCs, whereas it rised SM22αand Calponin mRNA level. Furthermore, Akt,ERK and Sirt1 were activated in EPCs by ECs, and were suppressed by VSMCs and the stretched ECs or VSMCs.These results demonstrate that shear stress pushed the rolling of EPCs, and EPCs were captured by ECs associated with adhesion molecules, which reduced the rolling velocity of EPCs. Moreover, ECs promoted EPCs differentiation into ECs via activation of Akt, ERK and Sirt1. VSMCs and the stretched ECs or VSMCs promoted EPCs differentiation into VSMCs, and Akt and ERK might be involved in VSMCs mediated effects, but unrelated with Sirt1.