Donor age affects confluent EPCs on phenotypic transition,proliferation and migration of smooth muscle cells

AIM: To explore the effects of confluent endothelial progenitor cells (EPCs) derived from young and aged rats on the phenotype conversion, proliferation and migration of vascular smooth muscle cells (SMCs). METHODS: Mononuclear cells were obtained from the bone marrow of young (1~2 month old) and aged (19 to 26 month old) Sprague-Dawley rats and cultured with medium DMEM/F12 (containing 15% fetal bovine serum, endothelial cell growth supplements (ECGs) 100 g/L, 1×10⁵ units/L of penicillin and streptomycin, respectively). EPCs were characterized as double positive for DiI-Ac-LDL uptake and lectin binding. Abdominal aorta was obtained from 1 to 2 month old Sprague-Dawley rats. Vascular SMCs were cultured by tissue explant method and identified by α-SM-actin immunofluorescence. In transwell co-culture system, the confluent EPCs located in the upper chamber and SMCs were seeded on the lower chamber. The experiments were divided into passage 3 SMCs group (P3), passage 4 SMCs group (P4), passage 4 SMCs co-culture with EPCs derived from young rats group (P4YE) and passage 4 SMCs co-culture with EPCs derived from aged rats group (P4AE). The protein expression of α-SM-actin and osteopontin was detected by Western blotting. [³H]-TdR incorporation assay was used to determine the proliferation. SMC migration was analyzed by scratch wound healing assay. RESULTS: Compared with P3 group, α-SM-actin expression in P4 group significantly decreased and osteopontin protein expression obviously increased, whereas no significant change was found in P4YE group. Compared with P4 group, confluent EPCs derived from young and aged rats both markedly increased α-SM-actin and decreased osteopontin expression in P4 SMCs. Compared with aged rat-derived EPCs, young rat-derived EPCs were more effectively to induce a delayed SMC phenotype transition (from contractile phenotype to a synthetic phenotype), and to inhibit SMC proliferation and migration. CONCLUSION: Co-culture of confluent EPC induces a delayed vascular SMC phenotype transition and inhibits SMCs proliferation and migration. Young rat derived EPCs are more effective to induce a delayed vascular SMC phenotype transition and has stronger inhibitory effects on SMCs proliferation and migration compared with that derived from aged rats.