| Objective:To explore regulating effects of UrotensinⅡ(UII) on endothelial progenitor cells (EPCs) derived from rat bone marrow and its potential intracellular singal pathways.Method:Bone marrow from the femurs of the SD rats was removed and mononuclear cells were isolated by density gradient centrifugation. Cells were resuspended by EGM-MV BulletKit system and incubated at 37℃in 100% humidified air with 5% CO2. Morphology and growth characteristics of cells were observed by microscopy. Immunohistochemistry staining were performed to detect for the expressoion of von Willebrand factor (vWF) and CD133 and VEGFR-2. Experiment of uptaking DIL-ac-LDL and combining FITC-Lectin-UEA-1 was observed by laser sanning confocal microscope to identify endothelial cells characteristic. To better understand whether UII can regulate EPC function via a receptor-dependent pathway, RT-PCR and Western blot were performed to detect expression of the GPR14 in EPCs at mRNA and protein levels. Chemotaxis assays were performed using Transwell cell-culture chambers with UII (10-10-10-6 M). The activation of RhoA and the phosphorylation of myosin light chain (MLC) in EPCs were analyzed. The effets of Rho-kinase inhibitor Y-27632 and GPR14 receptor blocker Urantide to UII-induced migration of EPCs and the phosphorylation of MLC were observated. Proliferation assays were performed using WST-8 with UII (10-10-10-6M). The activation the phosphorylation of MAPK in EPCs were analyzed. The effets of MAPK inhibitor and GPR14 receptor blocker Urantide to UII-induced proliferation of EPCs and the phosphorylation of MAPK were observated.Result:Parts of cells start attaching after 48 hours. The morphology formed the shuttle, triangle, the spindle or irregular form. The attached cells were able to form cells "colonies" and line up in the core-like structure from 4 to 8 days, and get close to coalesce and exhibite the typical cobble stone morphology. The cells were positively stained for CD133, VEGFR-2 and vWF. Fluorescence microscopy showed that adherent cells took up DiI-acLDL and bound FITC-Lectin-UEA-1. EPCs express GPR14 at mRNA and protein levels. Fluorescence microscopy showed that cells were positively stained for GPR14. UII induces migration of EPCs in a concen- tration-dependent manner in a certain extent. The cell count assay revealed that 1.5-, 2.1-,2.9-,2.3-and 1.6-fold increase was detected in migration of EPCs induced by UII at the concentration of 10-10 M,10-9 M,10-8 M,10-7 M and 10-6 M, respectively (all p＜0.05 vs. control group). The Rho-kinase inhibitor, Y-27632 (10μM), abolished UII (10-8 M)-induced migration of EPCs (p＜0.05 vs. control group, p＜0.05 vs. UII group). UII (10-8 M) induced 3-fold increase in the amount of active GTP-bound RhoA in EPCs (p＜0.05, UII group vs. control group), and Urantide (10μM) abolished the effect of UII (p＜0.01 vs. UII group). UII induced 2.5-fold increase in MLC phosphorylation(p＜0.05, UII group vs. control group) which was blocked by Y-27632 (10μM) and Urantide(10μM) (p＜0.05, Y-27632 group vs. UII group). UII promotes proliferation of EPCs in a concentration-dependent manner in a certain range, and treatment with a GPR14 inhibitor or MAPK inhibitors suppressed UII-induced EPC proliferation. Results of the WST-8 assay showed 14.2%,48.8%,69.2%,46.3%, and 27.3% increases in proliferation in response to treatment with UII at concentrations of 10-10M,10-9M, 10-8M,10-7 M, and 10-6 M, respectively (all p＜0.05 vs. control group). The proliferative effect of UII on EPCs was inhibited by 62.4%,54.3%, and 49.2% in response to Urantide, SB203580, and PD98059, respectively. But the proliferative effect of UII on EPCs wasn't inhibited by SP600125(p＞0.05 vs. the 10-8 M UII group). Treatment with UII (10-8 M) increased the phosphorylation of p38MAPK (p＜0.05 vs. control group). This effect was inhibited by the inhibitors SB203580 (10μM) (p＜0.05 vs. UII group). Treatment with UII (10-8M) increased the phosphorylation of p44/42MAPK (p＜0.05 vs. control group). This effect was inhibited by the inhibitors PD98059 (10μM) (p＜0.05 vs. UII group).Conclusion:1.Cultured cells were considered EPCs which express specific receptor GPR14 of UII.2. UII induces migration of EPCs in a concentration-dependent manner. UII-induced migration is mediated by activation of the RhoA/Rho kinase pathway. 3.UII induces migration of EPCs in a concentration-dependent manner. UII-induced migration is mediated by activation of the RhoA/Rho kinase pathway. |
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