Figure 4.

Smooth muscle cell (SMC) induces vascular progenitor cells (VPCs) migration via activated GTPase Cdc42 and Rac1 and p38 signaling pathway. (A, B): Pull down assays were performed on VPCs treated with either CCL2 or CXCL1 for 5 minutes. The transwell assay was performed on VPCs that were pretreated with either vehicle, DMSO or ML141 (20 µM) (C), NSC23766 (50 µM) (D), SB203580 (10µM) (K) for 1 hour before migration toward either SMC conditioned medium or recombinant CCL2, CXCL1. Scale bars, 100µm. The quantification of transwell assay shows as the fold of changes compared with cell migrated toward each treatment pre‐treated with vehicle or DMSO. Western blotting was performed on SMC conditioned medium (E), CCL2 or CXCL1 (F), SMCs (transfected with CCL2 siRNA or CXCL1 siRNA) conditioned medium (G)‐treated VPCs for the detection of p‐p38 and t‐p38. (H): Cell lysates from CCR2 shRNA or CXCR2 shRNA transfected VPCs cultured in the SMC conditioned medium were harvested for the detection of the p‐p38 and t‐p38. Untreated cells served as the controls. (I, J): After pre‐treated with ML141, NSC23766 for 1 hour, VPC were stimulated by SMC conditioned medium or CCL2, CXCL1 before cell lysates were harvested for detection of the p‐p38 and t‐p38. All the blots shown are representative of three separate experiments. All graphs are shown as mean ± SEM. n = 3. *p < .05, **p < .01, ***p < .001. Abbreviations: CM, SMC conditioned medium; ctrl, control, serum free medium; DMSO, dimethyl sulfoxide; NCsh, noncoding shRNA; NCsi, noncoding siRNA; p‐p38, phosphorylated p38; shCCR2, CCR2 shRNA; shCXCR2, CXCR2 shRNA; siCCL2, CCL2 siRNA; siCXCL1, CXCL1 siRNA; t‐p38, total p38; vehicle, sterile distilled water.