Paraoxonase-1 suppresses experimental colitis via the inhibition of IFN-γ production from CD4 T cells.

Crohn's disease (CD) is a chronic inflammatory disorder of the gastrointestinal tract, where excessive Th1 cell responses are observed. We performed experiments to identify immunologically bioactive proteins in human plasma and found that paraoxonase (PON)-1, which has esterase activity and is associated with high-density lipoproteins, inhibited the IFN-γ production by both murine and human differentiating Th1 cells. Trinitrobenzene sulfonic acid-induced colitis was attenuated by the administration of PON-1. The beneficial effects of PON-1 were associated with a reduced ratio of IFN-γ-producing CD4 T cells in the mesenteric lymph nodes and decreased production of T cell-related cytokines in the colon. PON-1 inhibited the TCR-induced activation of ERK-MAPK signaling and the nuclear translocation of NF-κB in CD4 T cells. Interestingly, an excessive CD4 T cell response was observed in PON-1-deficient mice under physiological and pathological conditions. Additionally, the efficacy of PON-1 or G3C9-C284A (G3C9), which shows a higher esterase activity than PON-1, on colitis was similar to that of an anti-TNF-α mAb, which is a clinically used CD treatment. Moreover, G3C9 more effectively suppressed CD4(+)CD45RB(high) cell transfer-induced chronic colitis in mice than did PON-1, and the efficacy of G3C9 against the colitis was similar to that of the anti-TNF-α mAb. Therefore, PON-1 (or G3C9) administration may be clinically beneficial for CD patients.

Eukaryotic elongation factor 2 kinase regulates the development of hypertension through oxidative stress-dependent vascular inflammation.

Eukaryotic elongation factor 2 kinase (eEF2K) is a Ca2+/calmodulin-dependent protein kinase. We recently demonstrated that eEF2K protein increases in mesenteric artery from spontaneously hypertensive rats (SHR). Pathogenesis of hypertension is regulated in part by vascular inflammation. We tested the hypothesis whether eEF2K mediates vascular inflammatory responses and development of hypertension. In vascular endothelial cells, small interfering RNA (siRNA) against eEF2K inhibited induction of VCAM-1 and endothelial-selectin as well as monocyte adhesion by TNF-α (10 ng/ml). eEF2K siRNA inhibited phosphorylation of JNK and NF-κB p65 as well as reactive oxygen species (ROS) production by TNF-α. In vascular smooth muscle cells, eEF2K siRNA also inhibited VCAM-1 induction and phosphorylation of JNK and NF-κB by TNF-α. In vivo, increased blood pressure in SHR and ROS production, induction of inflammatory molecules, and hypertrophy in SHR superior mesenteric artery were reduced by an eEF2K inhibitor NH125 (500 µg·kg(-1)·day(-1)). In SHR superior mesenteric artery, impairment of ACh-induced relaxation was normalized by NH125. The present results for the first time demonstrate that eEF2K mediates TNF-α-induced vascular inflammation via ROS-dependent mechanism, which is at least partly responsible for the development of hypertension in SHR.

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.

A novel adipocytokine, vaspin inhibits platelet-derived growth factor-BB-induced migration of vascular smooth muscle cells.

Vaspin is a novel adipocytokine originally identified in visceral white adipose tissues of Otsuka Long-Evans Tokushima fatty rats, an animal model of type 2 diabetes. We have previously shown that vaspin has anti-inflammatory effects in vascular smooth muscle cells (SMCs). SMCs migration is an important process for development atherosclerosis. However, effects of vaspin on SMCs migration remain to be clarified. Rat mesenteric arterial SMCs were treated with platelet-derived growth factor (PDGF)-BB (10 ng/ml, 90 min) in the absence or presence of vaspin (0.01-10 ng/ml, pretreatment for 2h). SMCs migration was evaluated by a Boyden chamber assay. Western blotting was performed to analyze cellular signals. Reactive oxygen species (ROS) generation was fluorometrically measured using 2',7'-dichlorofluorescein diacetate. Vaspin significantly inhibited PDGF-BB-induced SMCs migration. Vaspin significantly inhibited PDGF-BB-induced phosphorylation of p38 and heat shock protein (HSP) 27 as well as ROS generation. SMCs migration was blocked by an inhibitor of p38 or an anti-oxidant drug, N-acetyl-l-cysteine (NAC). NAC significantly inhibited the PDGF-BB-induced phosphorylation of p38 and HSP27. In addition, vaspin inhibited PDGF-BB-induced actin cytoskeletal reorganization (lamellipodia formation) as revealed by a rhodamine phalloidin staining. The present study for the first time revealed that vaspin inhibits PDGF-BB-induced SMCs migration through inhibiting p38/HSP27 signals via preventing the ROS generation.

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.

Long-term methylglyoxal treatment impairs smooth muscle contractility in organ-cultured rat mesenteric artery.

Methylglyoxal (MGO), a metabolite of glucose accumulates in vascular tissues of hypertensive rats. We recently showed that short-term (30min) treatment with MGO inhibits noradrenaline (NA)-induced smooth muscle contraction in rat aorta and mesenteric artery. In the present study, long-term effect of MGO was examined using organ culture method. The contractility, morphology, and protein expression of rat mesenteric artery after organ culture with MGO for 3 days were examined. MGO (4 and 42µM) inhibited NA (0.1nM to 3µM) or KCl (72.7mM)-induced contraction. The inhibitory effect was higher in endothelium-denuded than endothelium-intact artery. An anti-oxidant drug, N-acetyl-l-cysteine (NAC; 1mM) or an inhibitor of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX), gp91ds-tat (0.1µM) prevented the inhibitory effect of MGO. MGO increased superoxide production as detected by lucigenin assay. In the medial layer of the arteries cultured with MGO, apoptotic morphological change was observed, and NAC or gp91ds-tat prevented it. MGO significantly increased expression of a homolog of gp91(phox), NOX1 but not gp91(phox) as determined by Western blotting. An NF-κB inhibitor, pyrrolidine dithiocarbamate prevented the MGO-induced NOX1 expression. MGO had no effect on protein expression of p22(phox), p67(phox), p47(phox), as well as superoxide dismutase (SOD)-1, SOD-2 and SOD-3. Present results indicate that long-term MGO treatment has an inhibitory effect on contractility of isolated blood vessel, which is likely mediated via increased NOX1-derived superoxide production and subsequent apoptosis.

Exploring mechanisms of diabetes-related macrovascular complications: role of methylglyoxal, a metabolite of glucose on regulation of vascular contractility.

Methylglyoxal (MGO) is a metabolite of glucose. MGO binds to and modifies arginine, lysine, and cysteine residues in proteins, which leads to formation of a variety of advanced glycation end-products (AGEs) such as argpyrimidine and N(ε)-(carboxyethyl)lysine. The concentration of MGO significantly increases in plasma from diabetic patients. Increased plasma MGO level seems to be associated with diabetic microvascular complications. In addition, MGO accumulates in large vascular tissues from spontaneous hypertensive rats, which is associated with increased blood pressure. Although it is logical to hypothesize that MGO could directly affect vascular reactivity, available reports are very limited. Our group has examined effects of MGO on vascular reactivity (contraction and relaxation) and explored underlying mechanisms. In this review article, we summarized our recent findings on 1) short-term effects of MGO, 2) long-term effects of MGO, and 3) effects of MGO accumulation in arterial walls on vascular reactivity. These findings may provide further mechanistic insights into the pathogenesis of diabetes-related macrovascular complications including hypertension.

Methylglyoxal accumulation in arterial walls causes vascular contractile dysfunction in spontaneously hypertensive rats.

Methylglyoxal (MGO) is a metabolite of glucose and perhaps mediates diabetes-related macrovascular complications including hypertension. In the present study, we examined if MGO accumulation affects vascular reactivity of isolated mesenteric artery from spontaneously hypertensive rats (SHR). Five-week-old SHR were treated with an MGO scavenger, aminoguanidine (AG), for 5 weeks. AG partially normalized increased blood pressure in SHR. In mesenteric artery from SHR treated with AG, increased accumulation of MGO-derived advanced glycation end-products was reversed. In mesenteric artery from SHR, AG normalized impaired acetylcholine (ACh)-induced relaxation and increased angiotensin (Ang) II-induced contraction. Reactive oxygen species (ROS) production increased in SHR mesenteric artery, and acute treatment with a nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) inhibitor augmented ACh-induced relaxation. Protein expression of NOX1 and Ang II type 2 receptor (AT2R) increased in SHR mesenteric artery, which was normalized by AG. Acute treatment with an AT2R blocker but not a NOX inhibitor normalized the increased Ang II-induced contraction in SHR mesenteric artery. The present results demonstrate that MGO accumulation in mesenteric artery may mediate development of hypertension in SHR at least in part via increased ROS-mediated impairment of endothelium-dependent relaxation and AT2R-mediated increased Ang II contraction.

Treatment with IL-27 attenuates experimental colitis through the suppression of the development of IL-17-producing T helper cells.

Inflammatory bowel disease (IBD) represents a group of chronic inflammatory diseases characterized by inflammation and relapsing gastrointestinal disorders. Recent studies have shown that Th17 cells, which are well known as key mediators of chronic inflammation, have a pivotal role in onset and development of IBD in humans and mice, alike. In recent years, it has been reported that IL-27, which is an IL-12-related heterodimeric cytokine consisting of EBI3 and p28 subunits, act directly on naive T cells to suppress the differentiation of Th17 cells. However, effects of exogenous IL-27 on the IBD are not well elucidated. To clarify the suppressive effect of IL-27 treatment on IBD, we applied the flexible linking method to EBI3 and p28 subunits and generated a single-chain human IL-27 (scIL-27). scIL-27 inhibited xenogenic mouse Th17 cell differentiation in vitro, indicating that scIL-27 also acts in mouse immune systems. In a 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced mouse acute colitis model, subcutaneous scIL-27 treatment significantly improved the colon length, extent of necrosis, and ulceration and thickened epithelium and several pathological scores in a dose-dependent manner. scIL-27 clearly suppressed several inflammatory cytokines, including IL-17, in inflamed colon, except for anti-inflammatory cytokine IL-10. The mesenteric lymph node cells from scIL-27-treated mice also exhibited a reduced inflammatory response and, furthermore, a lower population of Th17 cells than those of PBS-treated mice. Finally, we showed the therapeutic efficacy of scIL-27 on TNBS-induced colitis even after active colitis was established. These results suggest new possible therapeutic approaches for IBD, including disorders such as Crohn's disease and ulcerative colitis.

Exploring calmodulin-related proteins, which mediate development of hypertension, in vascular tissues of spontaneous hypertensive rats.

Calmodulin (CaM) is associated with a variety of cell functions including inflammation, apoptosis, and muscular contraction. It is recently clarified that some CaM-related proteins are responsible for cardiovascular diseases. We therefore explored CaM-related proteins that mediate hypertensive vascular diseases. Expression levels of six CaM-related proteins with almost unknown function in blood vessels were examined in aorta and mesenteric artery from spontaneously hypertensive rats (SHR) and Wistar Kyoto rats (WKY) by Western blotting. In aorta from SHR, eukaryotic elongation factor (eEF)2 kinase (eEF2K) and death-associated protein kinase (DAPK)3 protein increased compared with WKY, while Ca(2+)/CaM-dependent protein kinase IIδ, histone deacetylases (HDAC)4 and HDAC5 protein decreased. In mesenteric artery from SHR, eEF2K, HDAC4 and DAPK3 protein increased compared with WKY, while HDAC5 decreased. Our findings demonstrate that expression levels of several CaM-related proteins are changed in vascular tissues of SHR and suggest that CaM-related proteins might be at least in part related to the pathogenesis of hypertensive vascular diseases.

Omentin, a novel adipocytokine inhibits TNF-induced vascular inflammation in human endothelial cells.

Omentin is a recently identified adipocytokine with insulin-sensitizing effect. While lack of omentin may be related to the pathogenesis of obesity-related cardiovascular diseases, its effect in vasculature is largely unknown. We examined effects of omentin on vascular endothelial inflammatory states. Western blotting was performed to analyze inflammatory signal transduction in cultured vascular endothelia cells. The cyclic guanosine monophosphate (cGMP) content was measured using enzyme immunoassay. Treatment of human umbilical vein endothelial cells with omentin (300 ng/ml, 20 min) induced phosphorylation of 5'-AMP-activated protein kinase (AMPK) (Thr 172) and endothelial nitric oxide (NO) synthase (eNOS) (Ser 1177). Consistently, omentin increased the cGMP level. Pretreatment with omentin (300 ng/ml, 30 min) significantly inhibited the phosphorylation of JNK as well as expression of cyclooxygenase (COX)-2 by TNF-α (5 ng/ml, 20 min-24h). An inhibitor of JNK significantly inhibited the TNF-α-induced COX-2 expression. Inhibitory effect of omentin on TNF-α-induced COX-2 was reversed by a NOS inhibitor. The present results demonstrate for the first time that omentin plays an anti-inflammatory role by preventing the TNF-α-induced COX-2 expression in vascular endothelial cells. Omentin inhibits COX-2 induction via preventing the JNK activation presumably through activation of AMPK/eNOS/NO pathways.

Vaspin prevents TNF-α-induced intracellular adhesion molecule-1 via inhibiting reactive oxygen species-dependent NF-κB and PKCθ activation in cultured rat vascular smooth muscle cells.

Vaspin (visceral adipose tissue-derived serine protease inhibitor) was recently identified as a novel adipocytokine with insulin-sensitizing effects. We hypothesized that vaspin could play a role in vascular inflammation. To test the hypothesis, we investigated the effects of vaspin on TNF-α-stimulated vascular smooth muscle cells (SMCs) focusing on inflammatory signal transduction. Vascular SMCs from mesenteric artery of male Wistar rats were treated with TNF-α (5-10 ng/ml, 20 min-6 h) in the absence or presence of vaspin (1-300 ng/ml, pretreatment for 24 h). Western blotting was performed to analyze the cellular signal. Reactive oxygen species (ROS) generation was fluorometrically measured using 2',7'-diclorofluorescein diacetate. Vaspin alone treatment had no effect on vascular SMCs morphology and cellular signal. Vaspin significantly decreased the TNF-α-induced monocyte adhesion to SMCs. Vaspin significantly inhibited the protein expression of intracellular adhesion molecule (ICAM)-1 and the phosphorylation of NF-κB and protein kinase C (PKC)θ induced by TNF-α. Both of NF-κB and novel PKC inhibitors significantly attenuated the TNF-α-induced ICAM-1 expression. Moreover, vaspin inhibited TNF-α-induced ROS generation. An anti-oxidant, N-acetyl-L-cysteine blocked the TNF-α-induced activation of NF-κB, PKCθ and expression of ICAM-1. The present results demonstrated for the first time that vaspin inhibits TNF-α-induced expression of ICAM-1 via preventing the ROS generation and subsequent activation of NF-κB and PKCθ. Consequently, vaspin could play inhibitory roles on inflammatory states of vascular SMCs.

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.

Methylglyoxal augments angiotensin II-induced contraction in rat isolated carotid artery.

Methylglyoxal (MGO), a metabolite of glucose, accumulates in vascular tissues of a hypertensive animal. In the present study, we examined the effect of MGO on angiotensin (Ang) II-induced contraction of rat carotid artery. Treatment of carotid artery with MGO (420 µM, 30 min) significantly augmented Ang II (0.1 to 30 nM)-induced concentration-dependent contraction. The effect was abolished by the removal of endothelium. BQ-123 (1, 5 µM), an endothelin A-receptor blocker, had no effect on the MGO-induced enhancement of Ang II-induced contraction. AL8810 (1 µM), a prostaglandin F(2α)-receptor blocker, or SQ29548 (1 µM), a thromboxane A(2)-receptor blocker, was also ineffective. However, tempol (10 µM), a superoxide scavenger, and catalase (5000 U/mL), which metabolizes hydrogen peroxide to water, significantly prevented the effect of MGO. Combined MGO and Ang II treatment increased reactive oxygen species (ROS) production. Apocynin (10 µM) or gp91ds-tat (3 µM), an inhibitor of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, significantly prevented the effect of MGO. Gp91ds-tat or an Ang II type 1-receptor (AT1R) blocker, losartan (10 µM), prevented the MGO-mediated increased ROS production. The present study revealed that MGO augments Ang II-induced contraction by increasing AT1R-mediated NADPH oxidase-derived superoxide and hydrogen peroxide production in endothelium of rat carotid artery.

Methylglyoxal enhances sodium nitroprusside-induced relaxation in rat aorta.

The concentration of methylglyoxal (MGO), a metabolite of glucose, increases in plasma of type II diabetic patients as well as in tissues of hypertensive rats. We have previously shown that MGO inhibited noradrenaline (NA)-induced smooth muscle contraction in rat aorta. However, the effect of MGO on relaxing responses in isolated blood vessel remains to be clarified. Thus, we examined if MGO affects acetylcholine (ACh)- or sodium nitroprusside (SNP)-induced vasodilation on NA (100 nM)-induced pre-contraction in rat thoracic aorta. Treatment of endothelium-intact aorta with MGO (420 microM, 30 min) did not change ACh (1 nM - 3 microM)-induced endothelium-dependent relaxation. In contrast, treatment of endothelium-denuded aorta with MGO shifted the concentration-response curve for SNP (0.1 - 300 nM) to the left. MGO increased reactive oxygen species (ROS) production in smooth muscle on analysis of protein carbonylation. Anti-oxidant agents such as tempol (10 microM), catalase (5000 U/mL), and nitric oxide synthase inhibitor, N(G)-nitro-L-arginine methylester (100 microM) had no effect on MGO-induced enhancement of SNP-induced relaxation. However, iberiotoxin (100 nM), a large-conductance Ca(2+)-activated K(+) (BK(Ca))-channel inhibitor, significantly prevented the effect. The present study revealed that MGO enhanced SNP-induced relaxation in a ROS-independent manner via in part opening smooth muscle BK(Ca) channels.

CV-159, a unique dihydropyridine derivative, prevents TNF-induced inflammatory responses in human umbilical vein endothelial cells.

CV-159, a 1,4-dihydropyridine derivative, has Ca(2+) antagonistic and anti-calmodulin actions. An early feature of atherosclerosis is vascular endothelial inflammatory change. We examined whether CV-159 has protective effects against endothelial inflammatory responses. After pretreatment of human umbilical vein endothelial cells (ECs) with CV-159 (10 microM, 30 min), TNF-alpha (10 ng/ml) was applied for 20 min or 24 h. Expressions of inflammatory markers and activation of inflammatory signal molecules were examined by Western blotting. Reactive oxygen species (ROS) generation was examined by using 2',7'-dichlorodihydrofluorescein diacetate. CV-159 inhibited TNF (24 h)-induced expression of e-selectin but not vascular cell adhesion molecule-1 and intercellular adhesion molecule-1. CV-159 inhibited TNF (20 min)-induced phosphorylation of JNK, p38, and NF-kappaB p65 (Ser536). A JNK inhibitor, SP600125, and a p38 inhibitor, SB203580, inhibited TNF-induced e-selectin expression. An antioxidant drug, N-acetyl-L-cysteine (NAC), inhibited TNF-induced e-selectin expression. NAC inhibited TNF-induced phosphorylation of JNK and p38 but not NF-kappaB. CV-159 inhibited TNF-induced ROS generation. Our results indicate that in ECs CV-159 specifically inhibits TNF-induced e-selectin expression through inhibition of JNK, p38, and NF-kappaB phosphorylation. It is suggested that CV-159 prevents activation of JNK and p38 through inhibition of ROS, while it prevents activation of NF-kappaB via a ROS-independent manner.

Mechanisms underlying the anti-inflammatory effects of the Ca2+/calmodulin antagonist CV-159 in cultured vascular smooth muscle cells.

CV-159 is a unique dihydropyridine Ca(2+) antagonist with an anti-calmodulin (CaM) action. A pathogenic feature of atherosclerosis is vascular inflammatory change. In the present study, we examined whether CV-159 exerts protective effects on smooth muscle inflammatory responses. After pretreatment of rat mesenteric arterial smooth muscle cells (SMCs) with CV-159 (0.1 - 10 microM, 30 min), TNF-alpha (10 ng/ml) was applied for 20 min or 24 h. CV-159 inhibited TNF (24 h)-induced vascular cell adhesion molecule (VCAM)-1 as determined by Western blotting. CV-159 inhibited TNF (20 min)-induced phosphorylation of Akt (Ser473) and NF-kappaB p65 (Ser536). An Akt inhibitor, LY294002, and an NF-kappaB inhibitor, pyrrolidine dithiocarbamate, inhibited TNF-induced VCAM-1. An antioxidant drug, N-acetyl-L-cysteine (NAC) inhibited TNF-induced VCAM-1. NAC also inhibited TNF-induced phosphorylation of Akt and NF-kappaB. Furthermore, CV-159 inhibited TNF-induced reactive oxygen species (ROS) production as determined fluorometrically using dichlorodihydrofluorescein diacetate. A CaM inhibitor, W-7, and a calcium/CaM-dependent protein kinase type II inhibitor, KN93, inhibited TNF-induced VCAM-1. W-7 and KN93 inhibited TNF-induced phosphorylation of Akt but not NF-kappaB. The present results indicate that in vascular SMCs, CV-159 inhibits TNF-induced VCAM-1 through inhibition of NF-kappaB and Akt phosphorylation. CV-159 prevents NF-kappaB phosphorylation by inhibiting ROS, while it prevents Akt phosphorylation by inhibiting both ROS and CaM.

Effects of captopril and telmisartan on matrix metalloproteinase-2 and -9 expressions and development of left ventricular fibrosis induced by isoprenaline in rats.

The aim of this study was to clarify the effects of renin-angiotensin system (RAS) blockade by captopril, an angiotensin converting enzyme inhibitor, and telmisartan, an angiotensin II type 1 receptor antagonist, on matrix metalloproteinase (MMP)-2 and MMP-9 expressions and development of left ventricular (LV) fibrosis induced by isoprenaline in rats. Rats were treated with subcutaneous injection of isoprenaline (5 mg/kg/d) and with oral administration of captopril (30 mg/kg/d) or telmisartan (3 mg/kg/d) for 1 or 7 d. Hearts were excised at the day 2 and day 8. Degree of fibrosis was evaluated by Azan staining. MMP-2 and MMP-9 expressions were analyzed by Western blotting. Localization of MMP-9 expression in LV section was detected by immunohistochemical staining. At the day 8, myocardial fibrosis was observed in LV section from isoprenaline-treated rats. Captopril but not telmisartan significantly enhanced the isoprenaline-induced myocardial fibrosis. MMP-9 expression at the day 2 and MMP-2 expression at the day 8 increased significantly in LV from isoprenaline-treated rats. Captopril had no influence on the MMP-2 expression, but significantly augmented the isoprenaline-induced MMP-9 expression. Telmisartan had no effect on the isoprenaline-induced MMP-2 and MMP-9 expressions. In immunohistochemical staining, MMP-9 positive-interstitial cells were extensively observed in LV sections from isoprenaline + captopril-treated rats at the day 2. The present study reveals that RAS blockade by captopril and telmisartan does not have suppressive effects on isoprenaline-induced MMP-2 and MMP-9 expressions as well as LV fibrosis. Furthermore, captopril may enhance LV fibrosis through promoting isoprenaline-induced MMP-9 expression in cardiac interstitial cells.

Antimuscarinic action of doxorubicin does not involve free-radical formation in isolated guinea pig hearts.

It has been proposed that the cardiotoxicity of anthracycline anticancer drugs involves free-radical formation. One early manifestation of toxicity appears to be caused by the antimuscarinic actions of these drugs. Accordingly, we examined whether the antimuscarinic action of one of these drugs, doxorubicin, is altered by antioxidants. In electrically stimulated left atrial muscle preparations obtained from guinea pig hearts, doxorubicin significantly increased the tissue concentration of thiobarbituric acid-reactive substance indicating increased lipid peroxidation. This effect of doxorubicin was significantly suppressed by the antioxidants alpha-tocopherol, dexrazoxane, and epigallocatechin gallate. Carbachol produced a concentration-dependent negative inotropic effect in our atrial preparations. Doxorubicin caused a seemingly parallel rightward shift of the concentration-response curve for carbachol. Neither alpha-tocopherol, dexrazoxane, nor epigallocatechin gallate reversed this effect of doxorubicin. The results indicate that in extirpated heart tissue, doxorubicin causes lipid peroxidation through the formation of free radicals. However, this effect of doxorubicin is unrelated to its antimuscarinic action.