Fig. 3
β2AR gene transfer in EPCs upregulates cell proliferation, migration, and adhesion; NO production; and SDF-1/CXCR4 expression in vitro. a Proliferation was measured in cultured EPCs by using CCK8 assays after 24 h of stimulation with FENO (10−8 M). The rate of proliferation in the Ad5/β2AR EPC group was significantly higher than the rate in the non-Adv-EPC, non-Adv- EPC + FENO, or Ad5/EGFP-EPC groups (0.29 ± 0.05, 0.16 ± 0.00, 0.24 ± 0.03, and 0.15 ± 0.01, respectively; *P < 0.01 versus non-Adv EPCs or Ad5/EGFP-EPCs, # P < 0.01 versus non-Adv-EPCs, non-Adv-EPCs + FENO, or Ad5/EGFP-EPCs; n = 5 per group). b NO secretion was assayed in EPCs by using the nitrate reductase method after β2AR gene transfer. The production of NO was significantly higher in the Ad5/β2AR-EPCs than in the non-Adv-EPCs, non-AdvEPCs + FENO, or Ad5/EGFP-EPCs (74.42 ± 8.68, 24.01 ± 2.68, 46.77 ± 4.08, and 24.24 ± 3.71, respectively; *P < 0.01 versus non-Adv-EPCs or Ad5/EGFP-EPCs, and # P < 0.01 versus non-Adv-EPCs, non-Adv-EPCs + FENO, or Ad5/EGFP-EPCs; n = 5 per group). c and e Quantitative analyses (c) and representative photographs (e) showing the migratory activity observed in EPCs (*P < 0.01 versus non-Adv-EPCs + FENO or Ad5/EGFP EPCs + FENO; n = 5 per group). d and f Quantitative analyses (d) and representative photographs (f) of CM-DiI-labeled EPCs that adhered to human umbilical vein endothelial cells with or without stimulation with TNF-α (1 μg/ml; *P < 0.01 versus non-Adv-EPCs + TNF-α or Ad5/EGFP-EPCs + TNF-α; n = 5 per group). g Overexpression of β2AR increased the expression of SDF-1 and CXCR4 in EPCs. Representative photograph and quantification analysis for SDF-1 and CXCR4 expression in EPCs (*P < 0.01 versus non-Adv EPCs without FENO activation, # P < 0.01 versus non-Adv-EPCs with FENO activation, and NS versus non-Adv-EPCs without FENO activation, n = 5 per group). Ad5 adenovirus serotype 5, DiI-ac-LDL DiI-labeled acetylated low-density lipoprotein, EGFP enhanced green fluorescent protein, EPC endothelial progenitor cell, FENO fenoterol, NO nitric oxide, NS not significant, SDF-1a stromal cell-derived factor 1a, TNF-α tumor necrosis factor-α, β2AR β2 adrenergic receptor