ABSTRACT
Human adipose-derived stem cells (hADSCs) and human umbilical vein endothelial cells (HUVECs) co-cultured in vitro are widely used in adipose tissue engineering but exhibit various limitations. Chemokine (C-C motif) ligand 2 (CCL2) has been proved essential during adipogenesis and angiogenesis in vivo. We examined whether adipogenesis and angiogenesis could also be directly promoted by CCL2 in vitro. Cells were cultured with 0, 10, 50, and 100 ng/ml CCL2. The effects of CCL2 on adipogenesis of hADSCs, and lipid accumulation in the positive control group (hADSCs), blank control group (hADSCs + HUVECs), and experimental group (hADSCs + HUVECs + CCL2) in the hADSC and HUVEC direct co-culture system were evaluated by Oil Red O staining. Angiogenesis in the presence of CCL2 was evaluated by Matrigel tube formation assay. Angiogenic- and adipogenic-associated gene and protein expression in the co-culture system were measured by Quantitative Real-time Polymerase Chain Reaction and western blotting, respectively. All concentrations of CCL2 promoted hADSC adipogenic differentiation and HUVEC tube formation (P < 0.05). Following direct co-culture, the experimental group accumulated more lipid droplets than the positive control (P < 0.0001), whereas the latter showed better adipogenesis than the blank control group. 50 ng/ml CCL2 exhibited stronger adipogenic and angiogenic potential than other concentrations. After 72 h of direct co-culture, the mRNA expression of adipogenic differentiation (peroxisome proliferators-activated receptorsγ, CCAAT/enhancer binding protein-α, Leptin, and lipoprotein lipase) and angiogenic genes (vascular endothelial growth factor-A, vascular endothelial growth factor receptor 2, matrix metalloprotein (MMP) 9, and 14) in the experimental group was much higher than in the control (P < 0.05). The addition of 50 ng/ml CCL2 in the system resulted in elevated phosphorylated Protein kinase B/AKT expression. In summary, CCL2 directly promoted adipogenesis of hADSCs and angiogenesis of HUVECs under both mono-culture and co-culture condition in vitro possibly by enhancing AKT phosphorylation. An optimal concentration of 50 ng/ml CCL2 could improve the adipogenesis and angiogenesis of hADSC and HUVEC co-culture system.
