Figure 3.

SMC conditioned medium induced VPC migration by up‐regulating expression of CCR2 and CXCR2. (A): Cluster grams show the expression of 84 chemokine receptors, chemokine and other chemotactic cytokines on the untreated smooth muscle cells, clones and Sca‐1⁺ VPCs individually. A row in the cluster represents a cell line, and relative level of expression for a gene is arranged in column. The magnitude of gene expression increases from green to red. The dendrogram of the different cell lines clustering is displayed aside and describes the degree of relatedness among different cell lines. Quantification was performed with three different batches of each cell line. The vascular progenitor cells were treated with or without SMC conditioned medium for 18 hours, untreated cells served as controls. n = 3. The mRNA (B, D) and cell‐surface protein (C, E) expression of CCR2 or CXCR2 was confirmed using real‐time quantitative PCR and flow cytometry. n = 3. (F): Vascular progenitor cells were infected with lentiviral short hairpin RNA (shRNA) (noncoding shRNA, CCR2 shRNA or CXCR2 shRNA) for ablation of CCR2 or CXCR2 before migration toward SMC conditioned medium. The noncoding shRNA served as the control. n = 3. Scale bars, 100 µm. All graphs are shown as mean ± SEM. *p < .05, **p < .01, ***p < .001. Abbreviations: CM, SMC conditioned medium; ctrl, control, serum free medium; NCsh, noncoding shRNA; shCCR2, CCR2 shRNA; shCXCR2, CXCR2 shRNA; SMC, smooth muscle cell; VPCs, vascular progenitor cells.