COX-2/PGE/VEGF signaling promotes ERK-mediated BMSCs osteogenic differentiation under hypoxia by the paracrine action of ECs.

Understanding the crosstalk between endothelial cells (ECs) and bone-marrow mesenchymal stem cells (BMSCs) in response to hypoxic environments and deciphering of the underlying mechanisms are of great relevance for better application of BMSCs in tissue engineering. Here, we demonstrated that hypoxia promoted BMSCs proliferation, colony formation, osteogenic markers expression, mineralization, and extracellular signal-regulated protein kinase (ERK) phosphorylation, and that PD98059 (ERK inhibitor) blocked hypoxia-induced osteogenic differentiation. Hypoxia enhanced ECs migration, cyclooxygenase 2 (COX-2) and integrin αβ expression, and prostaglandin E (PGE), vascular endothelial growth factor (VEGF) secretion. NS398 (selective COX-2 inhibitor) and LM609 (integrin αβ specific inhibitor) impaired the ECs response to hypoxia, and exogenous PGE partially reversed the effects of NS398. BMSCs: ECs co-culture under hypoxia upregulated BMSCs osteogenesis and ERK phosphorylation, as well as ECs migration, integrin αβ expression, and PGE and VEGF secretion. NS398 (pretreated ECs) lessened PGE, VEGF concentrations of the co-culture system. NS398-treated ECs and AH6809 (combined EP1/2 antagonist)/L-798106 (selective EP3 antagonist)/L-161982 (selective EP4 antagonist)/SU5416 [VEGF receptor (VEGFR) inhibitor]-treated BMSCs impaired the co-cultured ECs-induced enhancement of BMSCs osteogenic differentiation. In conclusion, hypoxia enhances BMSCs proliferation and ERK-mediated osteogenic differentiation, and augments the COX-2-dependent PGE and VEGF release, integrin αβ expression, and migration of ECs. COX-2/PGE/VEGF signaling is involved in intercellular BMSCs: ECs communication under hypoxia.