From Skeleton to Cytoskeleton

Abstract

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 C␦ (PKC␦), 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 PKC␦ 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 IIrelease and subsequent activation of PKC␦/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. (Circ Res. 2012;111:e55-e66.) Key Words: fibroblast transformation Ⅲ osteocalcin Ⅲ angiotensin II Ⅲ protein kinase C Ⅲ toll-like receptor 4 V ascular dysfunction has traditionally been considered an "inside-out" process in blood vessels, 1 in which vascular inflammation is initiated at the intimal surface of the artery as a consequence of overproduction of inflammatory mediators such as reactive oxygen species (ROS) in the endothelium. Until recently, growing evidence has suggested an "outsidein" hypothesis, 1 proposing that inflammation may originate from the adventitial fibroblasts and then progress toward the intima, culminating in vascular wall thickening and dysfunction. Neointima formation is partially characterized by the acquisition of migratory and proliferative ability of fibroblasts after their transformation to myofibroblasts, signified by the induced expression of cytoskeletal proteins such as ␣-smooth muscle actin (␣-SMA) and the excessive synthesis and secretion of matrix components including fibronectin. 2 These phenotypic alterations allow adventitial fibroblasts to migrate toward the lumen, contributing to neointima formation and intimal thickening. 2,3 The subsequent narrowing of arterial lumen is one of the hallmark features of vascular remodeling. Adventitial fibroblasts can differentiate to myofibroblasts in response to vascular injury, as revealed by their localization in the neointima ...

Protein Kinase Cε-Calcineurin Cosignaling Downstream of Toll-Like Receptor 4 Downregulates Fibrosis and Induces Wound Healing Gene Expression in Cardiac Myofibroblasts

“…a proinflammatory, profibrotic agonist in myofibroblasts (62) on the one hand and also with the ability of AngII to mediate complex effects on COX-2 expression by other agonists via posttranslational increases in mRNA stability and decreased proteasomedependent degradation of COX-2 (63). Furthermore, AngII and TLR4 have been suggested to cooperate or be involved in a canonical pathway downstream of osteocalcin in the transformation of vascular myofibroblasts, which also involves COX-2 and PKC (64). Therefore, whereas AngII/AT1R-dependent signaling alone is profibrotic, TLR4/AT1R cross talk may modify the response to alter the fibrosis/wound healing balance.…”

Vein graft failure

“…[97] Other studies show that TLR-4 is integral to osteocalcin-induced myofibroblast transformation from adventitial fibroblast which involves the inflammatory mediators, protein kinase C-δ(PKC-δ) and cylco-oxygenase 2 (cox-2). [99] These studies highlight the link between innate immune activation and matrix turnover with respect to vascular remodeling.…”

Toll-Like Receptor 4 Upregulation by Angiotensin II Contributes to Hypertension and Vascular Dysfunction through Reactive Oxygen Species Production

“…Increased activation of the renin-angiotensin system (RAS) seems to be associated with the inflammatory state observed in hypertension, as well as with its associated vascular alterations [1], [5], [11], [12]. Angiotensin II (Ang II), the effector peptide of RAS, is able to induce Toll-like Receptor 4 (TLR4), and it seems that TLR4-dependent signaling pathway contributes to the proinflammatory effects of this humoral factor [13]–[19].…”