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Objective: Increasing evidence suggests that osteocalcin (OC), one of the osteoblast-specific proteins, has been associated with atherosclerosis, but results are conflicting. The aim of this study was to elucidate the independent effect of uncarboxylated osteocalcin (ucOC), an active form of osteocalcin which has been suggested to have an insulin sensitizing effect, on vascular endothelial cells., Materials and Methods: We used human aortic endothelial cells and treated them with ucOC. Linoleic acid (LA) was used as a representative free fatty acid. Apoptosis was evaluated using various methods including a terminal deoxyribonucleotide transferase-mediated deoxyuridine triphosphate nick-end labeling analysis kit and Western blotting for cleaved caspase 3, cleaved poly (ADP-ribose) polymerase and Bcl-xL. The phosphorylations of Akt and endothelial nitric oxide synthase (eNOS) as well as the level of NO were measured to confirm the effect of ucOC on insulin signaling pathway., Results: Pretreatment of ucOC (30 ng/ml) prevented LA-induced apoptosis in insulin-stimulated endothelial cells; effects were abolished by pretreatment with the phosphatidylinositol 3-kinase (PI3-kinase) inhibitor, wortmannin. Treatment of ucOC (ranged from 0.3 to 30 ng/ml) significantly increased the phosphorylation of Akt and eNOS and nitric oxide secretion from endothelial cells in a PI3-kinase dependent manner., Conclusions: Our study is the first to demonstrate the independent effect of ucOC on vascular endothelial cells. Our results further suggest that ucOC could have beneficial effects on atherosclerosis., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Linoleic acid (LA), a dietary unsaturated fatty acid, has been known to increase the expression of adhesion molecules such as intercellular adhesion molecule-1 (ICAM-1) through the activation of nuclear factor-kappa B. Rho/Rho-kinase (ROCK) pathway mediates various cellular functions related to cardiovascular disease and affects the expression of ICAM-1. However, the exact mechanism underlying this action has not been fully elucidated. In this study, we aimed to find out the role of Rho/ROCK pathway in LA-induced ICAM-1 expression in human aortic endothelial cells (HAECs). We found that LA increased ICAM-1 expression and phosphorylation of ROCK and MYPT-1, a distal signal of ROCK. Y-27632, a ROCK inhibitor, suppressed ICAM-1 expression and phosphorylation of MYPT-1 induced by LA. The effect of LA on the increased phosphorylation of MYPT1 and expression of ICAM-1 was abolished by knocking down RhoA and ROCK2 protein level expression using small interfering RNA. LA increased NF-κB DNA-binding activity, which was inhibited with pretreatment with Y-27632. This study suggests that Rho/ROCK pathway plays a role in LA-induced ICAM-1 expression, which is possibly mediated by NF-κB in HAECs.

Vaspin is an adipocytokine recently identified in the visceral adipose tissue of diabetic rats and having anti-diabetic effects. We have recently shown that vaspin has anti-atherogenic effect through Akt-mediated inhibition of endothelial cell apoptosis. Decreased activity of endothelial nitric oxide synthase (eNOS) plays an important role in the pathogenesis of atherosclerosis. Asymmetric dimethylarginine (ADMA) is a well-known endogenous competitive inhibitor of eNOS and risk factor of cardiovascular diseases. The aim of this study was to examine whether vaspin might protect against atherosclerosis through its beneficial effects on the ADMA-eNOS system. Treatment of vaspin significantly increased NO secretion from endothelial cells and isolated aorta from Sprague-Dawley (SD) rats. Furthermore, treatment of vaspin prevented fatty acid-induced decrease in endothelium-dependent vasorelaxation in isolated aorta of SD rat. For the mechanism of vaspin-induced NO biosynthesis, vaspin activated the STAT3 signaling pathway and stimulated eNOS phosphorylation (Ser 1177), a marker of eNOS activation, through STAT3-dependent mechanism. Furthermore, vaspin treatment increased the expression of dimethylarginine dimethylaminohydrolase (DDAH) II, the responsible enzyme for the degradation of ADMA, leading to a reduction in ADMA levels. Vaspin-induced increase in DDAH II gene expression was through STAT3-mediated stimulation of DDAH II promoter activity. These results suggest that vaspin increases eNOS activity by reducing ADMA level through STAT3-mediated regulation of DDAH II expression. Our findings provide a novel molecular mechanism of antiatherogenic actions of vaspin.

Abstract: Vaspin, an adipocytokine recently identified in a rat model of type 2 diabetes, has been suggested to have an insulin-sensitizing effect. However, the exact mechanism underlying this action has not been fully elucidated. Furthermore, the specific function of vaspin is largely unknown, especially in vascular cells. We examined whether vaspin affects the insulin-signaling pathway in cultured endothelial cells and is capable of preventing free fatty acid (FFA)-induced apoptosis in endothelial cells through its insulin sensitizing effect, specifically, through its stimulatory effect on PI3-kinase/Akt signaling pathways. Vaspin significantly increased Akt phosphorylation and prevented the impairment of Akt phosphorylation by linoleic acid (LA) in insulin-stimulated endothelial cells, which effects were abolished by pretreatment with the PI3-kinase inhibitor, Wortmannin. Moreover, pretreatment with vaspin prevented LA-induced apoptosis in insulin-stimulated endothelial cells; this anti-apoptotic effect of vaspin was also eliminated by pretreatment with Wortmannin. The present study indicates that vaspin protects vascular endothelial cells from FFA-induced apoptosis through upregulation of the PI3-kinase/Akt signaling pathway. Our study is the first to demonstrate that vascular cells can be targets of vaspin. Our results further suggest that vaspin could have beneficial effects on the atherosclerosis. [Copyright &y& Elsevier]