Nitric oxide mediates ultrasound-induced hypoxia-inducible factor-1α activation and vascular endothelial growth factor-A expression in human osteoblasts

Vascular endothelial growth factor (VEGF) is an important regulator for angiogenesis and endochondral bone formation. Although low-intensity pulsed ultrasound (US) has been recently used for accelerating fracture healing, the effect of US stimulation on angiogenic factor production by osteoblasts remains undetermined. Here, we found that US elevation of VEGF-A expression in human osteoblasts to be mediated by nitric oxide (NO) and hypoxia-inducible factor-1α (HIF-1α). Human osteoblasts were treated with or without US stimulation (200 μs pulse, 1 kHz at 30 mW/cm²) for 20 min. Cells were subjected to assessment of VEGF-A expression, NO production, nitric oxide synthase (NOS) catalytic activities, and HIF-1α transactivation. Results showed that US significantly increased VEGF-A mRNA and protein levels in 6 h. US augmentation of VEGF level was transcriptionally mediated. Early inhibition of NO production, but not calcium or prostaglandin E₂, significantly reduced US-enhanced VEGF-A levels. Osteoblasts responded to US treatment by increasing NO production, NOS catalytic activities, iNOS immunoexpression, nuclear HIF-1α activation, and binding to the VEGF-A promoter. Inhibition of NOS activity by N-nitro-L-arginine methyl ester (L-NAME) or blockade of guanylate cyclase activity by ODQ reduced US-augmented HIF-1α transactivation and VEGF-A levels. Conditioned medium harvested from US-treated osteoblasts promoted tube formation of human umbilical vein endothelial cells (HUVEC). Monoclonal VEGF-A antibody neutralization or L-NAME pretreatment reduced the promoting effect of conditioned medium on angiogenesis of HUVEC. Together, these findings show that NO plays an important role in mediating extracellular stimuli released by US and triggering intracellular response of osteoblasts to produce angiogenic factor after US treatment.