In vivo expression and regulation of genes associated with vascularization during early response of sutures to tensile force

Sutures are fibrous tissues that connect bones in craniofacial skeletal complexes. Cranio- and dentofacial skeletal deformities in infant and adolescent patients can be treated by applying tensile force to sutures to induce sutural bone formation. The early gene expression induced by mechanical stress is essential for bone formation in long bones; however, early gene expression during sutural bone formation induced by tensile force is poorly characterized. In vivo studies are essential to evaluate molecular responses to mechanical stresses in heterogeneous cell populations, such as sutures. In this paper we examined in vivo early gene expression and the underlying regulatory mechanism for this expression in tensile-force-applied cranial sutures, focusing on genes involved in vascularization. Tensile force upregulated expression of vascular factors, such as vascular endothelial growth factor (Vegf) and endothelial cell markers, in sutures within 3 h. The expression of connective tissue growth factor (Ctgf) and Rho-associated coiled-coil containing protein kinase 2 (Rock2) was also upregulated by tensile force. A CTGF-neutralizing antibody and the ROCK inhibitor, Y-27632, abolished tensile-force-induced Vegf expression. Moreover, tensile force activated extracellular signal-related kinase 1/2 (ERK1/2) signaling in sagittal sutures, and the ERK1/2 inhibitor, U0126, partially inhibited tensile-force-induced Ctgf expression. These results indicate that tensile force induces in vivo gene expression associated with vascularization early in tensile-force-induced sutural bone formation. Moreover, the early induction of Vegf gene expression is regulated by CTGF and ROCK2.