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.

Telmisartan inhibits vasoconstriction via PPARγ-dependent expression and activation of endothelial nitric oxide synthase.

AIMS: Telmisartan activates peroxisome proliferator-activated receptor-γ (PPARγ) in addition to serving as an angiotensin II type 1 receptor (AT(1)R) blocker. The PPARγ activity of telmisartan on resistance arteries has remained largely unknown. The present study investigated the hypothesis that telmisartan inhibited vascular tension in mouse mesenteric resistance arteries, which was attributed to an increased nitric oxide (NO) production through the PPARγ-dependent augmentation of expression and activity of endothelial nitric oxide synthase (eNOS). METHODS AND RESULTS: Second-order mesenteric arteries were isolated from male C57BL/6J, eNOS knockout and PPARγ knockout mice and changes in vascular tension were determined by isometric force measurement with a myograph. Expression and activation of relevant proteins were analysed by Western blotting. Real-time NO production was measured by confocal microscopy using the dye DAF. Telmisartan inhibited 9,11-dideoxy-11α,9α-epoxymethanoprostaglandin F(2α) (U46619)- or endothelin-1-induced contractions. An NOS inhibitor, N(G)-nitro-L-arginine methyl ester (l-NAME), or an inhibitor of soluble guanylate cyclase, 1H-[1,2,4]-oxadizolo[4,3-a]quinoxalin-1-one (ODQ), prevented telmisartan-induced inhibition of U46619 contractions. A PPARγ antagonist, GW9662, abolished telmisartan-induced inhibition. Likewise, the PPARγ antagonist rosiglitazone attenuated U46619-induced contractions. The effects of telmisartan and rosiglitazone were prevented by actinomycin-D, a transcription inhibitor. In contrast, losartan, olmesartan, and irbesartan did not inhibit contractions. The inhibition was absent in mesenteric arteries from eNOS knockout or PPARγ knockout mice. Telmisartan augmented eNOS expression, phosphorylation, and NO production, which were reversed by the co-treatment with GW9662. CONCLUSIONS: The present results suggest that telmisartan-induced inhibition of vasoconstriction in resistance arteries is mediated through a PPARγ-dependent increase in eNOS expression and activity that is unrelated to AT1R blockade.