Effect of SFRP5 (Secreted Frizzled-Related Protein 5) on the WNT5A (Wingless-Type Family Member 5A)-Induced Endothelial Dysfunction and Its Relevance With Arterial Stiffness in Human Subjects.

OBJECTIVE: SFRP5 (secreted frizzled-related protein 5) is an endogenous inhibitor of WNT5A (wingless-type family member 5a), which has been implicated in atherosclerosis. However, contradictory results have been reported about the role of SFRP5 in atherosclerosis. We aimed to investigate whether SFRP5 could restore WNT5A-induced endothelial dysfunction in vitro and ex vivo. In addition, we sought to determine whether the serum concentration of SFRP5 is associated with atherosclerosis in humans. APPROACH AND RESULTS: We measured endothelium-dependent vasorelaxation in the isolated thoracic aorta of Sprague-Dawley rats. In addition, we measured intracellular nitric oxide (NO) in human endothelial cells. The protein abundance of total and phosphorylated JNK (c-Jun N-terminal kinase), AKT (protein kinase B), and endothelial NO synthase was analyzed in human endothelial cells. Circulating SFRP5 and WNT5A levels and brachial-ankle pulse wave velocity were measured in 282 human subjects with type 2 diabetes mellitus. SFRP5 dose dependently restored Wnt5-induced impaired vasorelaxation in rat thoracic aorta by an endothelial NO synthase-dependent mechanism. SFRP5 treatment restored the WNT5A-induced reduction of NO production via endothelial NO synthase in human endothelial cells. WNT5A-induced changes in the phosphorylation of JNK, AKT, and endothelial NO synthase were ameliorated with SFRP5 administration. In humans with type 2 diabetes mellitus, the serum SFRP5 concentration positively correlated with brachial-ankle pulse wave velocity (r=0.146; P=0.024). Multivariate linear regression analysis demonstrated that the serum SFRP5 concentration was independently associated with brachial-ankle pulse wave velocity after adjustment for potential confounders [B (SE)=7.40 (3.35); P=0.028]. CONCLUSIONS: Our data suggest the possible compensatory action of SFRP5 against atherosclerosis under conditions of metabolic dysfunction.

Association of serum angiopoietin-like protein 2 with carotid intima-media thickness in subjects with type 2 diabetes.

BACKGROUND: Although recent animal studies have suggested that angiopoietin-like protein 2 (ANGPTL2), a novel inflammatory adipokine, is likely to be involved in the pathogenesis of atherosclerosis, in rodents, little is known regarding whether serum ANGPTL2 level is also associated with atherosclerosis in humans, especially in patients with type 2 diabetes. The aim of this study was to investigate whether serum ANGPTL2 concentration is associated with atherosclerosis by measuring carotid intima-media thickness (IMT) in subjects with type 2 diabetes without previous history of cardiovascular diseases. In addition, we examined the clinical and biochemical variables associated with serum ANGPLT2 concentration. METHODS: We measured the circulating ANGPTL2 level in 166 subjects (92 men and 74 women; mean age of 60.0 years) with type 2 diabetes. Measurements of carotid IMT were performed in all subjects. RESULTS: Serum ANGPTL2 concentration was positively correlated with carotid IMT (r = 0.220, p = 0.004). In multiple linear regression, serum ANGPTL2 concentration was independently associated with increased carotid IMT along with older age, male gender, and higher systolic blood pressure. Higher levels of hemoglobin A1c and high-sensitivity C-reactive protein were significantly associated with elevated serum ANGPTL2 concentration in subjects with type 2 diabetes. CONCLUSIONS: Serum ANGPTL2 concentration was significantly and positively associated with carotid atherosclerosis in patients with type 2 diabetes, suggesting that ANGPTL2 may be important in the atherosclerosis in humans.

Vaspin protects vascular endothelial cells against free fatty acid-induced apoptosis through a phosphatidylinositol 3-kinase/Akt pathway.

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.

RECK expression in osteosarcoma: correlation with matrix metalloproteinases activation and tumor invasiveness.

Osteosarcoma is a malignant tumor of bone characterized by its high metastatic potential. For the development of metastasis, activation of matrix metalloproteinases (MMPs) is required. A novel MMPs inhibitor, reversion inducing cysteine rich protein with Kazal motifs (RECK), is known to down-regulate MMPs and suppress the invasive and metastatic potential in many tumor-derived cell lines and some types of tumors. The expression of RECK and its role in tumor invasiveness have never been studied in osteosarcoma. We examined RECK mRNA expression and MMPs activation in osteosarcoma using quantitative real time PCR, gelatin zymography, invasion assay, and transfection experiments. RECK was expressed but down-regulated in osteosarcoma cells. Activation of pro-MMP-2 was observed in all samples, whereas activation of MMP-2 and pro-MMP-9 was detected in only 11% and 7% of the samples, respectively. MMP-9 was not activated in any of the samples. The level of RECK expression was inversely correlated with pro-MMP-2 activation, and overexpression of RECK by transfection resulted in decreased pro-MMP-2 activation and reduced tumor invasiveness. These findings suggest that RECK plays an important role in the invasiveness of osteosarcoma.

C1q/TNF-related protein-9 inhibits cytokine-induced vascular inflammation and leukocyte adhesiveness via AMP-activated protein kinase activation in endothelial cells.

Although recent studies have reported cardioprotective effects of C1q/TNF-related protein 9 (CTRP9), the closet adiponectin paralog, its role on cytokine-induced endothelial inflammation is unknown. We investigated whether CTRP9 prevented inflammatory cytokine-induced nuclear factor-kappa B (NF-κB) activation and inhibited the expression of adhesion molecules and a chemokine in the vascular endothelial cell. We used human aortic endothelial cells (HAECs) to examine the effects of CTRP9 on NF-κB activation and the expression of NF-κB-mediated genes, including intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and monocyte chemoattractant protein-1 (MCP-1). Tumor necrosis factor alpha (TNFα) was used as a representative proinflammatory cytokine. In an adhesion assay using THP-1 cells, CTRP9 reduced TNFα-induced adhesion of monocytes to HAECs. Treatment with CTRP9 significantly decreased TNFα-induced activation of NF-κB, as well as the expression of ICAM-1, VCAM-1, and MCP-1. In addition, treatment with CTRP9 significantly increased the phosphorylation of AMP-activated protein kinase (AMPK) and acetyl-CoA carboxylase (ACC), the downstream target of AMPK. The inhibitory effect of CTRP9 on the expression of ICAM-1, VCAM-1, and MCP-1 and monocyte adhesion to HAECs was abolished after transfection with an AMPKα1-specific siRNA. Our study is the first to demonstrate that CTRP9 attenuates cytokine-induced vascular inflammation in endothelial cells mediated by AMPK activation.

Dehydroepiandrosterone prevents linoleic acid-induced endothelial cell senescence by increasing autophagy.

BACKGROUND: Autophagy has emerged as a potentially important factor in the pathogenesis of atherosclerosis. Dehydroepiandrosterone (DHEA) is an adrenal steroid of great recent interest due to its anti-aging and anti-atherogenic effects; however, little is known about its role in autophagy and endothelial senescence. OBJECTIVE: The aim of this study was to investigate whether DHEA prevents linoleic acid (LA)-induced endothelial senescence by enhancing autophagy. MATERIALS/METHODS: After pre-treatement with or without DHEA prior to LA treatment in human aortic endothelial cells (HAECs), the level of senescence was compared by senescence-associated acidic ß-galactosidase (SA-ß-Gal) staining and hyperphosphorylated pRB (ppRB) protein level. Autophagy was detected by LC3 conversion and measuring the level of p62/SQSTM1 (sequestosome 1), a protein degraded by autophagy. The fusion of autophagosome and lysosome was confirmed by fluorescence microscopy. RESULTS: Pre-treatment with DHEA inhibited LA-induced endothelial senescence. DHEA increased the conversion of LC3-I to LC3-II and decreased the level of p62 in a time- and dose-dependent manner. Although both DHEA and LA treatment increased the conversion of LC3-I to LC3-II, treatment of LA increased p62 and decreased fusion of autophagosome and lysosome, which reflected decreased autophagic flux. However, pre-treatment with DHEA restored autophagic flux inhibited by LA. When we evaluated signaling pathways, we found that JNK activation involved in LC3 conversion induced by DHEA. CONCLUSION: DHEA prevents LA-induced endothelial senescence by restoring autophagy and autophagic flux through JNK activation.

Association of serum C1q/TNF-related protein-9 concentration with arterial stiffness in subjects with type 2 diabetes.

CONTEXT: Although recent animal studies have suggested that C1q/TNF-related protein-9 (CTRP9) is more likely to be involved in the regulation of vascular function, more specifically atherosclerosis, in rodents, little is known about whether serum CTRP9 level is associated with atherosclerosis in humans. OBJECTIVE: The aim of this study was to investigate whether serum CTRP9 concentration is associated with atherosclerosis by measuring brachial ankle pulse wave velocity (baPWV) in subjects with type 2 diabetes. In addition, we examined the clinical and biochemical variables associated with serum CTRP9 concentration. DESIGN AND METHODS: We measured circulating CTRP9 and total adiponectin levels in 278 subjects (169 men and 109 women; mean age of 58.3 years) with type 2 diabetes. Measurements of baPWV were performed in all subjects. RESULTS: Serum CTRP9 concentration was positively correlated with baPWV. This correlation was significant even after adjusting for total adiponectin levels. In multiple linear regression, serum CTRP9 concentration was independently associated with increased baPWV. Female gender, higher body mass index, and homeostatic model assessment of insulin resistance were significantly associated with elevated serum CTRP9 concentration in subjects with type 2 diabetes. CONCLUSIONS: Serum CTRP9 concentration was significantly and positively associated with arterial stiffness in patients with type 2 diabetes, suggesting that CTRP9 might be important in the regulation of arterial stiffness in humans.

The preventive effect of uncarboxylated osteocalcin against free fatty acid-induced endothelial apoptosis through the activation of phosphatidylinositol 3-kinase/Akt signaling pathway.

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.

The role of Rho/Rho-kinase pathway in the expression of ICAM-1 by linoleic acid in human aortic endothelial cells.

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 increases nitric oxide bioavailability through the reduction of asymmetric dimethylarginine in vascular endothelial cells.

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.