Muneyoshi Okada

Omentin, a novel adipokine, induces vasodilation in rat isolated blood vessels.

Omentin is a recently identified adipose tissue-derived cytokine and is implicated in obesity-related cardiovascular disorders. In the present study, we tested the hypothesis that omentin could directly affect vascular reactivity of isolated blood vessels. In endothelium-intact rat isolated aorta, pretreatment with omentin (300 ng/ml, 30 min) inhibited noradrenaline (NA; 1 nM-1 microM)-induced concentration-dependent contraction. In NA (100 nM)-pre-contracted aorta, omentin (1-300 ng/ml) directly induced an endothelium-dependent relaxation. While a nitric oxide (NO) synthase (NOS) inhibitor, N(G)-nitro-l-arginine methyl ester (100 microM, 30 min) inhibited the relaxation, a PI3K/Akt inhibitor, LY294002 (10 microM, 30 min) or a tyrosine kinase inhibitor, genistein (30 microM, 30 min) was ineffective. Omentin (300 ng/ml, 5 min) induced a phosphorylation of endothelial NOS at serine 1177 but not a phosphorylation of Akt at serine 473. Omentin (1-300 ng/ml) also relaxed NA pre-contracted mesenteric artery. Present study for the first time demonstrated that omentin has a vasodilating effect on isolated blood vessels, which is mediated through endothelium-derived NO.

Omentin plays an anti-inflammatory role through inhibition of TNF-α-induced superoxide production in vascular smooth muscle cells.

Omentin is a recently identified adipocytokine and its effect in vasculature is largely unknown. Here we examined the effects of omentin on smooth muscle cells (SMCs) inflammatory states. Western blotting was performed to analyze inflammatory signal transduction in cultured SMCs. Phosphorylation of nuclear factor-κB (NF-κB), p38 and JNK, and expression of vascular cell adhesion molecule (VCAM)-1 and cyclooxygenase-2 were not induced by omentin (50-300ng/ml, 20min or 24h). On the other hand, tumor necrosis factor-α (TNF-α; 10ng/ml, 20min)-induced phosphorylation of p38 and JNK was significantly inhibited by omentin pretreatment in a concentration-dependent manner (50-300ng/ml, 30min). TNF-α (24h)-induced expression of VCAM-1 was also significantly inhibited by omentin pretreatment in a concentration-dependent manner. Both inhibitor of p38 (SB203580) and JNK (SP600125) significantly inhibited TNF-α-induced VCAM-1 expression. Omentin (300ng/ml, 30min) inhibited TNF-α (1h)-induced nicotinamide adenine dinucleotide phosphate oxidase activity as determined by lucigenin assay. An antioxidant drug, N-acetyl-l-cysteine significantly inhibited TNF-α-induced phosphorylation of p38 and JNK. Furthermore, omentin (300ng/ml, 30min) significantly inhibited TNF-α (24h)-induced monocytic cells adhesion to SMCs. In rat isolated thoracic aorta, omentin (300ng/ml, 30min) inhibited TNF-α (24h)-induced VCAM-1 expression. The present results demonstrate for the first time that omentin plays an anti-inflammatory role by preventing the TNF-α-induced VCAM-1 expression in SMCs. It is suggested that omentin inhibits TNF-α-induced VCAM-1 expression via preventing the activation of p38 and JNK at least in part through inhibition of superoxide production.

Methylglyoxal mediates vascular inflammation via JNK and p38 in human endothelial cells

Methylglyoxal (MGO) is a reactive metabolite of glucose. Since the plasma concentration of MGO is increased in diabetic patients, MGO is implicated in diabetes-associated vascular endothelial cells (ECs) injury, which might be responsible for atherosclerosis. In the present study, we examined effects of treatment of human umbilical vein ECs with MGO on EC morphology and inflammatory responses. MGO (24 h) induced cytotoxic morphological changes in a concentration-dependent manner (0-420 microM). MGO induced mRNA and protein expression of cyclooxygenase (COX)-2 in a concentration (0-420 microM)- and time (6-24 h)-dependent manner. COX-2 induction was associated with increased PGE(2) release. Acute treatment with MGO (20 min) induced concentration-dependent (0-420 microM) activation of JNK and p38 MAP kinase but not ERK or NF-kappaB. Both the JNK inhibitor SP600125 and the p38 inhibitor SB203580 prevented the MGO induction of COX-2. However, inhibiting JNK and p38 or COX-2 was ineffective to the morphological damage by MGO (420 microM, 24 h). EUK134, a synthetic combined superoxide dismutase/catalase mimetic, had no effect on MGO-induced COX-2. Present results indicated that MGO mediates JNK- and p38-dependent EC inflammatory responses, which might be independent of oxidative stress. On the other hand, MGO-induced morphological cell damage seems unlikely to be associated with COX-2-PGE(2).

HDAC4 mediates development of hypertension via vascular inflammation in spontaneous hypertensive rats

Histone deacetylases (HDACs) are transcriptional corepressors. Our recent study demonstrated that HDAC4 protein specifically increases in mesenteric artery from spontaneous hypertensive rats (SHR) compared with Wistar Kyoto rats (WKY). Vascular inflammation is important for pathogenesis of hypertension. We examined whether HDAC4 affects vascular inflammatory responses and promotes hypertension. In vivo, blood pressure, reactive oxygen species (ROS) production, and VCAM-1 expression in isolated mesenteric artery were elevated in young SHR (7 wk old) compared with age-matched WKY, which were prevented by long-term treatment of SHR with an HDACs inhibitor, trichostatin A (TSA; 500 μg·kg(-1)·day(-1) for 3 wk). In isolated mesenteric artery, the increased angiotensin II-induced contraction in SHR was reversed by TSA. The endothelium-dependent relaxation induced by ACh in SHR was augmented by TSA. In cultured rat mesenteric arterial smooth muscle cells (SMCs), expression of HDAC4 mRNA and protein was increased by TNF-α (10 ng/ml). TSA (10 μM, pretreatment for 30 min) inhibited VCAM-1 expression and NF-κB phosphorylation induced by TNF (10 ng/ml, 24 h or 20 min) in SMCs. HDAC4 small interfering RNA inhibited TNF-induced monocyte adhesion, VCAM-1 expression, transcriptional activity of NF-κB, and ROS production in SMCs. The present results demonstrated that proinflammatory effects of HDACs may mediate the further development of hypertension in SHR. It is also suggested in cultured vascular SMCs that TNF-induced HDAC4 mediates vascular inflammation likely via VCAM-1 induction through ROS-dependent NF-κB activation.

Vaspin prevents methylglyoxal-induced apoptosis in human vascular endothelial cells by inhibiting reactive oxygen species generation

Vaspin (visceral adipose tissue‐derived serine protease inhibitor) is a novel adipocytokine found in visceral white adipose tissues of obese type 2 diabetic rats. We have previously shown that vaspin has anti‐inflammatory and antimigratory effects in vascular smooth muscle cells. Methylglyoxal (MGO) is an active metabolite of glucose and mediates diabetic vascular complications including endothelial cell (EC) apoptosis. Nonetheless, effects of vaspin on MGO‐induced apoptosis of vascular EC remain to be determined. We investigated the effects of vaspin on MGO‐induced apoptosis of human umbilical vein ECs (HUVECs).

Histone Deacetylase 4 Controls Neointimal Hyperplasia via Stimulating Proliferation and Migration of Vascular Smooth Muscle Cells

Histone deacetylases (HDACs) are transcriptional coregulators. Recently, we demonstrated that HDAC4, one of class IIa family members, promotes reactive oxygen species–dependent vascular smooth muscle inflammation and mediates development of hypertension in spontaneously hypertensive rats. Pathogenesis of hypertension is, in part, modulated by vascular structural remodeling via proliferation and migration of vascular smooth muscle cells (SMCs). Thus, we examined whether HDAC4 controls SMC proliferation and migration. In rat mesenteric arterial SMCs, small interfering RNA against HDAC4 inhibited platelet-derived growth factor (PDGF)-BB–induced SMC proliferation as determined by a cell counting and bromodeoxyuridine incorporation assay as well as migration as determined by Boyden chamber assay. Expression and activity of HDAC4 were increased by PDGF-BB. HDAC4 small interfering RNA inhibited phosphorylation of p38 mitogen–activated protein kinase and heat shock protein 27 and expression of cyclin D1 as measured by Western blotting. HDAC4 small interfering RNA also inhibited PDGF-BB–induced reactive oxygen species production as measured fluorometrically using 2′, 7′-dichlorofluorescein diacetate and nicotinamide adenine dinucleotide phosphate oxidase activity as measured by lucigenin assay. A Ca2+/calmodulin-dependent protein kinase II inhibitor, KN93, inhibited PDGF-BB–induced SMC proliferation and migration as well as phosphorylation of HDAC4. In vivo, a class IIa HDACs inhibitor, MC1568 prevented neointimal hyperplasia in mice carotid ligation model. MC1568 also prevented increased activation of HDAC4 in the neointimal lesions. The present results for the first time demonstrate that HDAC4 controls PDGF-BB–induced SMC proliferation and migration through activation of p38 mitogen–activated protein kinase/heat shock protein 27 signals via reactive oxygen species generation in a Ca2+/calmodulin-dependent protein kinase-dependent manner, which may lead to the neointimal hyperplasia in vivo.

A novel adipocytokine, nesfatin-1 modulates peripheral arterial contractility and blood pressure in rats

Nesfatin-1 is a novel adipocytokine which exerts not only anorexigenic but also hypertensive roles through acting on hypothalamus melanocortin-3/4 receptors. Although it is logical to hypothesize that nesfatin-1 could also affect the contractile reactivity of peripheral blood vessels, it still remains to be examined. The present study was performed to test the hypothesis. In both endothelium-intact and -denuded mesenteric artery of rats, acute treatment with nesfatin-1 (10nM, 30min pretreatment) had no influence on the noradrenaline- and 5-hydroxytryptamine-induced concentration-dependent contractions. Chronic treatment of mesenteric artery with nesfatin-1 (10nM, 3days) using organ-culture method had also no influence on the agonists-induced contractions. In contrast, nesfatin-1 (10nM, 30min) significantly inhibited the sodium nitroprusside (SNP)-induced relaxations of smooth muscle in mesenteric artery. A membrane permeable cyclic GMP (cGMP) analog, 8-bromo-cGMP-induced relaxations were not affected by nesfatin-1. Consistently, the SNP-induced cGMP production in smooth muscle was impaired by nesfatin-1. Intravenous application of nesfatin-1 to rats not only increased blood pressure but also impaired the SNP-induced decreases in blood pressure. The present study for the first time reveals that nesfatin-1 affects peripheral arterial blood vessel and inhibits the nitric oxide donor-induced smooth muscle relaxations via impairing the cGMP production. The results are the first to demonstrate that nesfatin-1 modulates blood pressure through directly acting on peripheral arterial resistance.

A novel adipocytokine, chemerin exerts anti-inflammatory roles in human vascular endothelial cells

Chemerin is a recently identified adipocytokine which plays a role on inflammation and adipocytes metabolism. However, its function in vasculature is largely unknown. We examined the effects of chemerin on vascular endothelial inflammatory states. Treatment of human umbilical vein endothelial cells with chemerin (300 ng/ml, 20 min) induced phosphorylation of Akt (Ser473) and endothelial nitric oxide (NO) synthase (eNOS) (Ser1177). Consistently, chemerin increased intracellular cyclic GMP content. Pretreatment with chemerin (1-300 ng/ml, 24 h) significantly inhibited phosphorylation of nuclear factor (NF)-κB p65 (Ser536) and p38 as well as vascular cell adhesion molecule (VCAM)-1 expression induced by tumor necrosis factor (TNF)-α (5 ng/ml, 20 min-6 h). Inhibitor of NF-κB or p38 significantly inhibited the TNF-α-induced VCAM-1 expression. Chemerin also inhibited TNF-α-induced VCAM-1 expression in rat isolated aorta. Moreover, chemerin significantly inhibited monocytes adhesion to TNF-α-stimulated endothelial cells. The inhibitory effect of chemerin on TNF-α-induced VCAM-1 was reversed by a NOS inhibitor. Conversely, an NO donor, sodium nitroprusside significantly inhibited TNF-α-induced VCAM-1. The present results for the first time demonstrate that chemerin plays anti-inflammatory roles by preventing TNF-α-induced VCAM-1 expression and monocytes adhesion in vascular endothelial cells. The effect is mediated via inhibiting activation of NF-κB and p38 through stimulation of Akt/eNOS signaling and NO production.

Death-Associated Protein Kinase 3 Mediates Vascular Inflammation and Development of Hypertension in Spontaneously Hypertensive Rats

Death-associated protein kinase (DAPK) is a Ca2+/calmodulin-regulated serine/threonine kinase that mediates cell death. Our recent study demonstrated that DAPK3 protein increases in the mesenteric artery from spontaneously hypertensive rats compared with Wistar Kyoto rats. Pathogenesis of hypertension is modulated at least in part by vascular inflammation. We examined whether DAPK3 mediates vascular inflammatory responses and development of hypertension. In rat mesenteric arterial smooth muscle cells, small interfering RNA against DAPK3 inhibited vascular cell adhesion molecule 1 expression and monocyte adhesion induced by tumor necrosis factor-&agr;. DAPK3 small interfering RNA inhibited phosphorylation of c-Jun amino-terminal kinase, p38, and Akt, as well as reactive oxygen species (ROS) production induced by tumor necrosis factor-&agr;. In human umbilical vein endothelial cells, expressions of vascular cell adhesion molecule 1, endothelial selectin, and cyclooxygenase 2, as well as ROS production induced by tumor necrosis factor-&agr;, were inhibited by DAPK inhibitor. In vivo, blood pressure, ROS production, inflammatory molecule expression (vascular cell adhesion molecule 1 and endothelial selectin), and hypertrophy in isolated mesenteric artery were elevated in spontaneously hypertensive rats (10 weeks old), which were prevented by long-term treatment with a DAPK inhibitor (500 µg/kg per day for 6 weeks). In isolated mesenteric artery, the increased angiotensin II–induced contraction and the impaired acetylcholine-induced endothelium-dependent relaxation in spontaneously hypertensive rats were reversed by a DAPK inhibitor. The present results for the first time demonstrated in cultured smooth muscle cells and endothelial cells that DAPK3 mediates tumor necrosis factor–induced inflammatory responses via ROS-dependent mechanisms. It is also suggested that DAPK3 mediates the development of hypertension in spontaneously hypertensive rats likely via ROS-dependent inflammation, hypertrophy, and hypercontractility.

Prostaglandin E2 Promotes Wound-Induced Migration of Intestinal Subepithelial Myofibroblasts via EP2, EP3, and EP4 Prostanoid Receptor Activation

Intestinal subepithelial myofibroblasts (ISMFs) are mesenchymal cells that reside in the subepithelial region throughout the intestine. When the intestine is damaged, the migratory and mitotic responses of ISMFs are crucial for wound closure. However, their mechanism of action remains unknown. We have investigated the role of cyclooxygenase (COX) and its metabolite prostaglandin E2 (PGE2) in the wound repair process of bovine ISMFs. The action of a mechanical scratch in a layer of ISMFs in cell culture elevated the levels of both COX-2 mRNA expression and PGE2 secretion 1 and 6 h after the event. After 24 h ISMFs had migrated to and reduced the wounded area around the site of the scratch. Treatment with the COX-1/2 inhibitor indomethacin, the COX-2 inhibitor 3-(4-methylsulphonylphenyl)-4-phenyl-5-trifluoromethylisoxazole (CAY10404), or E prostanoid receptor 2 to 4 (EP2–EP4) antagonists significantly inhibited wound repair. Conversely, inhibition of wound closure by indomethicin was reversed by treatment with PGE2 or agonists of the receptors EP2, EP3, or EP4 but not of EP1. Although EP2 to EP4 stimulation did not influence ISMF proliferation, it did stimulate ISMF migration in the transwell cell migration assay. It is noteworthy that cell migration stimulated by EP2 and EP4 was inhibited by the tyrosine kinase receptor inhibitor genistein and also by (Z)-3-[2,4-dimethyl-5-(2-oxo-1,2-dihydro-indol-3-ylidenemethyl)-1H-pyrrol-3-yl]-propionic acid (SU6668). However, cell migration stimulated by EP3 was unaffected. Reverse transcription-polymerase chain reaction showed EP2 or EP4 stimulation elevated the level of mRNA expression for fibroblast growth factor-2, which stimulates ISMF migration. Collectively, COX-2-dependent PGE2 secretion promotes wound healing by ISMFs. PGE2-EP3 signaling may directly stimulate ISMF migration. PGE2-EP2/4 signaling indirectly stimulates ISMF migration by elevating the level of growth factor secretion.