From skeleton to cytoskeleton: osteocalcin transforms vascular fibroblasts to myofibroblasts via angiotensin II and Toll-like receptor 4.

RATIONALE The expression of osteocalcin is augmented in human atherosclerotic lesions. How osteocalcin triggers vascular pathogenesis and remodeling is unclear. OBJECTIVE To investigate whether osteocalcin promotes transformation of adventitial fibroblast to myofibroblasts and the molecular mechanism involved. METHODS AND RESULTS Immunohistochemistry indicated that osteocalcin was expressed in the neointima of renal arteries from diabetic patients. Western blotting and wound-healing assay showed that osteocalcin induced fibroblast transformation and migration, which were attenuated by blockers of the renin-angiotensin system and protein kinase Cδ (PKCδ), toll-like receptor 4 (TLR4) neutralizing antibody, and antagonist and inhibitors of free radical production and cyclooxygenase-2. Small interfering RNA silencing of TLR4 and PKCδ abolished fibroblast transformation. Angiotensin II level in the conditioned medium from the osteocalcin-treated fibroblasts was found elevated using enzyme immunoassay. Culturing of fibroblasts in conditioned medium collected from differentiated osteoblasts promoted fibroblast transformation. The expression of fibronectin, TLR4, and cyclooxygenase-2 is augmented in human mesenteric arteries after 5-day in vitro exposure to osteocalcin. CONCLUSIONS Osteocalcin transforms adventitial fibroblasts to myofibroblasts through stimulating angiotensin II release and subsequent activation of PKCδ/TLR4/reactive oxygen species/cyclooxygenase-2 signaling cascade. This study reveals that the skeletal hormone osteocalcin cross-talks with vascular system and contributes to vascular remodeling.