Angiotensin II-induced vascular endothelial dysfunction through RhoA/Rho kinase/p38 mitogen-activated protein kinase/arginase pathway

Enhanced vascular arginase activity impairs endothelium-dependent vasorelaxation by decreasing l-arginine availability to endothelial nitric oxide (NO) synthase, thereby reducing NO production. Elevated angiotensin II (ANG II) is a key component of endothelial dysfunction in many cardiovascular diseases and has been linked to elevated arginase activity. We determined signaling mechanisms by which ANG II increases endothelial arginase function. Results show that ANG II (0.1 μM, 24 h) elevates arginase activity and arginase I expression in bovine aortic endothelial cells (BAECs) and decreases NO production. These effects are prevented by the arginase inhibitor BEC (100 μM). Blockade of ANG II AT(1) receptors or transfection with small interfering RNA (siRNA) for Gα12 and Gα13 also prevents ANG II-induced elevation of arginase activity, but siRNA for Gαq does not. ANG II also elevates active RhoA levels and induces phosphorylation of p38 MAPK. Inhibitors of RhoA activation (simvastatin, 0.1 μM) or Rho kinase (ROCK) (Y-27632, 10 μM; H1152, 0.5 μM) block both ANG II-induced elevation of arginase activity and phosphorylation of p38 MAPK. Furthermore, pretreatment of BAECs with p38 inhibitor SB-202190 (2 μM) or transfection with p38 MAPK siRNA prevents ANG II-induced increased arginase activity/expression and maintains NO production. Additionally, inhibitors of p38 MAPK (SB-203580, 5 μg·kg(-1)·day(-1)) or arginase (ABH, 8 mg·kg(-1)·day(-1)) or arginase gene knockout in mice prevents ANG II-induced vascular endothelial dysfunction and associated enhancement of arginase. These results indicate that ANG II increases endothelial arginase activity/expression through Gα12/13 G proteins coupled to AT(1) receptors and subsequent activation of RhoA/ROCK/p38 MAPK pathways leading to endothelial dysfunction.     

Mechanism of endoplasmic reticulum stress-induced vascular endothelial dysfunction

Background

We recently reported that ER stress plays a key role in vascular endothelial dysfunction during hypertension. In this study we aimed to elucidate the mechanisms by which ER stress induction and oxidative stress impair vascular endothelial function.

Methodology/principal findings

We conducted in vitro studies with primary endothelial cells from coronary arteries stimulated with tunicamycin, 1 μg/mL, in the presence or absence of two ER stress inhibitors: tauroursodeoxycholic acid (Tudca), 500 μg/mL, and 4-phenylbutyric acid (PBA), 5 mM. ER stress induction was assessed by enhanced phosphorylation of PERK and eIF2α, and increased expression of CHOP, ATF6 and Grp78/Bip. The ER stress induction increased p38 MAPK phosphorylation, Nox2/4 mRNA levels and NADPH oxidase activity, and decreased eNOS promoter activity, eNOS expression and phosphorylation, and nitrite levels. Interestingly, the inhibition of p38 MAPK pathway reduced CHOP and Bip expressions enhanced by tunicamycin and restored eNOS promoter activation as well as phosphorylation. To study the effects of ER stress induction in vivo, we used C57BL/6J mice and p47phox^−/− mice injected with tunicamycin or saline. The ER stress induction in mice significantly impaired vascular endothelium-dependent and independent relaxation in C57BL/6J mice compared with p47phox^−/− mice indicating NADPH oxidase activity as an intermediate for ER stress in vascular endothelial dysfunction.

Conclusion/significance

We conclude that chemically induced ER stress leads to a downstream enhancement of p38 MAPK and oxidative stress causing vascular endothelial dysfunction. Our results indicate that inhibition of ER stress could be a novel therapeutic strategy to attenuate vascular dysfunction during cardiovascular diseases.     

Hyperglycemia reduces nitric oxide synthase and glycogen synthase activity in endothelial cells.

Hyperglycemia is considered a primary cause of diabetic vascular complications. A hallmark of vascular disease is endothelial cell dysfunction characterized by diminished nitric-oxide (NO)-dependent phenomena such as vasodilation, angiogenesis, and vascular maintenance. This study was designed to investigate the effects of a high level of D-glucose on endothelial NO response, oxidative stress, and glucose metabolism. Bovine aortic endothelial cells (BAECs) were pretreated with a high concentration of glucose (HG) (22 mmol/L) for at least 2 weeks and compared with control cells exposed to 5 mmol/L glucose (NG). The effect of chronic hyperglycemia on endothelial NO-synthase (eNOS) activity and expression, glycogen synthase (GS) activity, extracellular-signal-regulated kinase (ERK 1,2), p38, Akt expression, and Cu/Zn superoxide-dismutse (SOD-1) activity and expression were determined. Western blot analysis showed that eNOS protein expression decreased in HG cells and was accompanied by diminished eNOS activity. The activity of GS was also significantly lower in the HG cells than in NG cells, 25.0+/-17.4 and 89+/-22.5 nmol UDP-glucose.mg protein(-1)x min(-1), respectively. Western blot analysis revealed a 40-60% decrease in ERK 1,2 and p38 protein levels, small modification of phosphorylated Akt expression, and a 30% increase in SOD-1 protein expression in HG cells. Although SOD expression was increased, no change was observed in SOD activity. These results support the findings that vascular dysfunction due to exposure to pathologically high D-glucose concentrations may be caused by impairment of the NO pathway and increased oxidative stress accompanied by altered glucose metabolism.     

High glucose accelerates MCP-1 production via p38 MAPK in vascular endothelial cells

In diabetes mellitus (DM), hyperglycemia causes cardiovascular lesions through endothelial dysfunction. Monocyte chemoattractant protein-1 (MCP-1) is implicated in the pathogenesis of cardiovascular lesions. By using human umbilical vein endothelial cells, we investigated the effect of hyperglycemia on MCP-1 production and its signaling pathways. Chronic incubation with high glucose increased mRNA expression and production rate of MCP-1 in a time (1-7 days)- and concentration (10-35 mM)-dependent manner. Chronic exposure to high glucose resulted in enhancement of generation of reactive oxygen species (ROS), as determined by increasing level of 2,7-dichlorofluorescein (DCF), and subsequent activation of p38 mitogen-activated protein kinase (MAPK). Neither c-Jun NH(2)-terminal kinase nor extracellular signal-regulated kinase1/2 was affected. SB203580 or FR167653, p38 MAPK specific inhibitors, completely suppressed MCP-1 expression. Catalase suppressed p38 MAPK phosphorylation and MCP-1 expression. These results indicate that hyperglycemia can accelerate MCP-1 production through the mechanism involving p38 MAPK, ROS-sensitive signaling pathway, in vascular endothelial cells.     

Activation of p38 has opposing effects on the proliferation and migration of endothelial cells

Pathological conditions such as hypertension and hyperglycemia as well as abrasions following balloon angioplasty all lead to endothelial dysfunction that impacts disease morbidity. These conditions are associated with the elaboration of a variety of cytokines and increases in p38 activity in endothelial cells. However, the relationship between enhanced p38 activity and endothelial cell function remains poorly understood. To investigate the effect of enhanced p38 MAPK activity on endothelial cell function, we expressed an activated mutant of MEK6 (MEK6E), an upstream regulator of p38. Expression of MEK6E activated p38 and resulted in phosphorylation of its downstream substrate, heat shock protein 27 (Hsp27). Activation of p38 was not sufficient to induce apoptosis; however, it did induce p38-dependent cell cycle arrest. MEK6E expression was sufficient to inhibit ERK phosphorylation triggered by growth factors and integrin engagement. MAPK phosphatase-1 (MKP-1) expression was increased upon p38 activation, and expression of a "substrate-trapping" MKP-1 was sufficient to restore ERK activity. Activation of p38 was sufficient to induce cell migration, which was accompanied by alterations in actin architecture characterized by enhanced lamellipodia. Co-expression of a mutant form of Hsp27, lacking all three phosphorylation sites, reversed MEK6E-induced cell migration and altered the cytoskeletal changes induced by p38 activation. Collectively, these results suggest that cellular decisions regarding migration and proliferation are influenced by p38 activity and that prolonged activation of p38 may result in an anti-angiogenic phenotype that contributes to endothelial dysfunction.     

p38 MAPK-dependent eNOS upregulation is critical for 17beta-estradiol-mediated cardioprotection following trauma-hemorrhage

Studies have shown that p38 MAPK and nitric oxide (NO), generated by endothelial NO synthase (eNOS), play key roles under physiological and pathophysiological conditions. Although administration of 17beta-estradiol (E2) protects cardiovascular injury from trauma-hemorrhage, the mechanism by which E2 produces those effects remains unknown. Our objective was to determine whether the E2-mediated activation of myocardial p38 MAPK and subsequent eNOS expression/phosphorylation would protect the heart following trauma-hemorrhage. To study this, male Sprague-Dawley rats underwent soft-tissue trauma (midline laparatomy) and hemorrhagic shock (mean blood pressure 35-40 mmHg for 90 min), followed by fluid resuscitation. Animals were pretreated with specific p38 MAPK inhibitor SB-203580 (SB; 2 mg/kg), and nonselective NO synthase inhibitor NG-nitro-l-arginine methyl ester (l-NAME; 30 mg/kg) 30 min before vehicle (cyclodextrin) or E2 (100 microg/kg) treatment, followed by resuscitation, and were killed 2 h thereafter. Cardiovascular performance and other parameters were measured. E2 administration following trauma-hemorrhage increased cardiac p38 MAPK activity, eNOS expression and phosphorylation at Ser(1177), and nitrate/nitrite levels in plasma and heart tissues; these were associated with normalized cardiac performance, which was reversed by SB administration. In addition, E2 also prevented trauma-hemorrhage-induced increase in cytokines (IL-6 and TNF-alpha), chemokines (macrophage inflammatory protein-2 and cytokine-induced neutrophil chemoattractant-1), and ICAM-1, which was reversed by l-NAME administration. Administration of E2 following trauma-hemorrhage attenuated cardiac tissue injury markers, myeloperoxidase activity, and nitrotyrosine level, which were reversed by treatment with SB and l-NAME. The salutary effects of E2 on cardiac functions and tissue protection following trauma-hemorrhage are mediated, in part, through activation of p38 MAPK and subsequent eNOS expression and phosphorylation.     

Negative modulation of eNOS by laminin involving post‐translational phosphorylation

Nitric oxide (NO) regulates the vascular tone, and influences survival and apoptosis of endothelial cells (ECs). NO is produced by nitric oxide synthase (NOS) and eNOS is the constitutive enzyme in the endothelium. Though the extracellular matrix (ECM) has been reported to regulate various EC functions, the role of ECM in the regulation of eNOS is not clear. The present study was designed to analyze if laminin‐1 (Ln‐1), the major glycoprotein of the basement membrane, can regulate eNOS. The activity of eNOS was significantly low in ECs maintained on Ln‐1 as compared to those on Col I and polylysine. Reversal of the effect of Ln‐1 on treatment with inhibitor of p38 MAPK and changes in Thr and Ser phosphorylation in purified eNOS suggested that eNOS activity in cells maintained on Ln‐1 is negatively regulated by post‐translational phosphorylation at Ser and Thr residues by recruiting p38 MAPK pathway. Increase in eNOS activity and induction of apoptosis upon inhibition of p38 MAPK and reversal of this on inhibition of NOS by L ‐NAME suggested that increased NO induced apoptosis in ECs maintained on Ln‐1 when p38 MAPK was inhibited. These results suggest that Ln contributes to survival of ECs by negatively modulating eNOS in a p38 MAPK dependent pathway. J. Cell. Physiol. 219: 123–131, 2009. © 2008 Wiley‐Liss, Inc.     

Critical role of hydrogen peroxide signaling in the sequential activation of p38 MAPK and eNOS in laminar shear stress

Laminar shear stress (LSS) is a protective hemodynamic regulator of endothelial function and limits the development of atherosclerosis and other vascular wall diseases related to pathophysiological generation of reactive oxygen species. LSS activates several endothelial signaling responses, including the activation of MAPKs and eNOS. Here, we explored the mechanisms of activation of these key endothelial signaling pathways. Using the cone/plate model we found that LSS (12 dyn/cm²) rapidly promotes endothelial intracellular generation of superoxide and hydrogen peroxide (H₂O₂). Physiological concentrations of H₂O₂ (flux of 0.1 nM/min and 15 μM added extracellularly) significantly activated both eNOS and p38 MAPK. Pharmacological inhibition of NADPH oxidases (NOXs) and specific knockdown of NOX4 decreased LSS-induced p38 MAPK activation. Whereas the absence of eNOS did not alter LSS-induced p38 MAPK activation, pharmacological inhibition and knockdown of p38α MAPK blocked H₂O₂- and LSS-induced eNOS phosphorylation and reduced ^?NO levels. We propose a model in which LSS promotes the formation of signaling levels of H₂O₂, which in turn activate p38α MAPK and then stimulate eNOS, leading to increased ^?NO generation and protection of endothelial function.     

Telmisartan Activates Endothelial Nitric Oxide Synthase via Ser1177 Phosphorylation in Vascular Endothelial Cells

Because endothelial nitric oxide synthase (eNOS) has anti-inflammatory and anti-arteriosclerotic functions, it has been recognized as one of the key molecules essential for the homeostatic control of blood vessels other than relaxation of vascular tone. Here, we examined whether telmisartan modulates eNOS function through its pleiotropic effect. Administration of telmisartan to mice significantly increased the phosphorylation level of eNOS (Ser1177) in the aortic endothelium, but administration of valsartan had no effect. Similarly, telmisartan treatment of human umbilical vein endothelial cells significantly increased the phosphorylation levels of AMP-activated protein kinase (Thr172) and eNOS and the concentration of intracellular guanosine 3′,5′-cyclic monophosphate (cGMP). Furthermore, pretreatment with a p38 mitogen-activated protein kinase (p38 MAPK) inhibitor suppressed the increased phosphorylation level of eNOS and intracellular cGMP concentration. These data show that telmisartan increases eNOS activity through Ser1177 phosphorylation in vascular endothelial cells mainly via p38 MAPK signaling.     

Endothelial nitric-oxide synthase enhances lipopolysaccharide-stimulated tumor necrosis factor-alpha expression via cAMP-mediated p38 MAPK pathway in cardiomyocytes

The purpose of this study was to investigate the role of endothelial nitric-oxide synthase (eNOS), cAMP, and p38 MAPK in tumor necrosis factor-alpha (TNF-alpha) expression induced by lipopolysaccharide (LPS). LPS dose- and time-dependently induced phosphorylation of p38 MAPK and TNF-alpha expression in neonatal mouse cardiomyocytes. TNF-alpha expression was preceded by p38 MAPK phosphorylation, and selective inhibition of p38 MAPK abrogated LPS-induced TNF-alpha expression. Deficiency in eNOS decreased basal and LPS-stimulated TNF-alpha expression in cardiomyocytes. NOS inhibitor l-NAME attenuated LPS-induced p38 MAPK phosphorylation and TNF-alpha production in wild-type cardiomyocytes, whereas NO donor 2,2'-(hydroxynitrosohydrazono)bis-ethanamine (DETA-NO) (2 microm) or overexpression of eNOS by adenoviral gene transfer restored the response of eNOS(-/-) cardiomyocytes to LPS. These effects of NO were mediated through cAMP-dependent pathway based on the following facts. First, deficiency in eNOS decreased basal levels of intracellular cAMP, and DETA-NO elevated intracellular cAMP levels in eNOS(-/-) cardiomyocytes. Second, a cAMP analogue 8-Br-cAMP mimicked the effect of NO in eNOS(-/-) cardiomyocytes. Third, either inhibition of cAMP or cAMP-dependent protein kinase attenuated LPS-stimulated p38 MAPK phosphorylation and TNF-alpha production in wild-type cardiomyocytes. In conclusion, eNOS enhances LPS-stimulated TNF-alpha expression in cardiomyocytes. Activation of p38 MAPK is essential in LPS-stimulated TNF-alpha expression. Moreover, the effects of NO on LPS-stimulated TNF-alpha expression are mediated through cAMP/cAMP-dependent protein kinase-dependent p38 MAPK pathway in neonatal cardiomyocytes.     

Extracellular Signal-regulated Kinase Mediates Expression of Arginase II but Not Inducible Nitric-oxide Synthase in Lipopolysaccharide-stimulated Macrophages

The mitogen-activated protein kinases (MAPK) have been shown to participate in iNOS induction following lipopolysaccharide (LPS) stimulation, while the role of MAPKs in the regulation of arginase remains unclear. We hypothesized that different MAPK family members are involved in iNOS and arginase expression following LPS stimulation. LPS-stimulated RAW 264.7 cells exhibited increased protein and mRNA levels for iNOS, arginase I, and arginase II; although the induction of arginase II was more robust than that for arginase I. A p38 inhibitor completely prevented iNOS expression while it only attenuated arginase II induction. In contrast, a MEK1/2 inhibitor (ERK pathway) completely abolished arginase II expression while actually enhancing iNOS induction in LPS-stimulated cells. Arginase II promoter activity was increased by ∼4-fold following LPS-stimulation, which was prevented by the ERK pathway inhibitor. Arginase II promoter activity was unaffected by a p38 inhibitor or JNK pathway interference. Transfection with a construct expressing a constitutively active RAS mutant increased LPS-induced arginase II promoter activity, while transfection with a vector expressing a dominant negative ERK2 mutant or a vector expressing MKP-3 inhibited the arginase II promoter activity. LPS-stimulated nitric oxide (NO) production was increased following siRNA-mediated knockdown of arginase II and decreased when arginase II was overexpressed. Our results demonstrate that while both the ERK and p38 pathways regulate arginase II induction in LPS-stimulated macrophages, iNOS induction by LPS is dependent on p38 activation. These results suggest that differential inhibition of the MAPK pathway may be a potential therapeutic strategy to regulate macrophage phenotype.     

Anti-oxidant and anti-inflammatory mechanisms of amlodipine action to improve endothelial cell dysfunction induced by irreversibly glycated LDL

Amlodipine, alone or in combination with other drugs, was successfully used to treat hypertension. Our aim was to evaluate the potential of amlodipine (Am) to restore endothelial dysfunction induced by irreversibly glycated low density lipoproteins (AGE-LDL), an in vitro model mimicking the diabetic condition. Human endothelial cells (HEC) from EA.hy926 line were incubated with AGE-LDL in the presence/absence of Am and the oxidative and inflammatory status of the cells was evaluated along with the p38 MAPK and NF-κB signalling pathways. The cellular NADPH activity, 4-hydroxynonenal (4-HNE) and 3-nitrotyrosine levels in the culture medium and the adhesion of human monocytes to HEC were measured by chemiluminescence, UHPLC, Western Blot and spectrofluorimetric techniques. The gene expression of NADPH subunits (p22^phox, NOX4), eNOS and inflammatory molecules (MCP-1, VCAM-1) were determined by Real Time PCR, while the protein expression of p22^phox, MCP-1, iNOS, phospho-p38 MAPK and phospho-p65 NF-κB subunit were measured by Western Blot. Results showed that in HEC incubated with AGE-LDL, Am led to: (i) decrease of the oxidative stress: by reducing p22^phox, NOX4, iNOS expression, NADPH oxidase activity, 4-HNE and 3-nitrotyrosine levels; (ii) decrease of the inflammatory stress: by the reduction of MCP-1 and VCAM-1 expression, as well as of the number of monocytes adhered to HEC; (iii) inhibition of ROS-sensitive signalling pathways: by decreasing phosphorylation of p38 MAPK and p65 NF-κB subunits. In conclusion, the reported data demonstrate that amlodipine may improve endothelial dysfunction in diabetes through anti-oxidant and anti-inflammatory mechanisms.     

Mechanisms of shear stress-induced endothelial nitric-oxide synthase phosphorylation and expression in ovine fetoplacental artery endothelial cells

Placental blood flow, nitric-oxide (NO) levels, and endothelial NO synthase (eNOS) expression increase during human and ovine pregnancy. Shear stress stimulates NO production and eNOS expression in ovine fetoplacental artery endothelial (OFPAE) cells. Because eNOS is the rate-limiting enzyme essential for NO synthesis, its activity and expression are both closely regulated. We investigated signaling mechanisms underlying pulsatile shear stress-induced increases in eNOS phosphorylation and protein expression by OFPAE cells. The OFPAE cells were cultured at 3 dynes/cm2 shear stress, then exposed to 15 dynes/cm2 shear stress. Western blot analysis for phosphorylated ERK1/2, Akt, p38 mitogen activated protein kinase (MAPK), and eNOS showed that shear stress rapidly increased phosphorylation of ERK1/2 and Akt but not of p38 MAPK. Phosphorylation of eNOS Ser1177 under shear stress was elevated by 20 min, a response that was blocked by the phosphatidyl inositol-3-kinase (PI-3K)-inhibitors wortmannin and LY294002 but not by the mitogen activated protein kinase kinase (MEK)-inhibitor UO126. Basic fibroblast growth factor (bFGF) enhanced eNOS protein levels in static culture via a MEK-mediated mechanism, but it could not further augment the elevated eNOS protein levels otherwise induced by the 15 dynes/cm2 shear stress. Blockade of either signaling pathway changed the shear stress-induced increase in eNOS protein levels. In conclusion, shear stress induced rapid eNOS phosphorylation on Ser1177 in OFPAE cells through a PI-3K-dependent pathway. The bFGF-induced rise in eNOS protein levels in static culture was much less than those observed under flow and was blocked by inhibition of MEK. Prolonged shear stress-stimulated increases in eNOS protein were not affected by inhibition of MEK- or PI-3K-mediated pathways.     

Angiogenic activity of sesamin through the activation of multiple signal pathways

The natural product sesamin has been known to act as a potent antioxidant and prevent endothelial dysfunction. We here found that sesamin increased in vitro angiogenic processes, such as endothelial cell proliferation, migration, and tube formation, as well as neovascularization in an animal model. This compound elicited the activation of multiple angiogenic signal modulators, such as ERK, Akt, endothelial nitric oxide synthase (eNOS), NO production, FAK, and p38 MAPK, but not Src. The MEK inhibitor PD98059 and the PI3K inhibitor Wortmannin specifically inhibited sesamin-induced activation of the ERK and Akt/eNOS pathways. These inhibitors reduced angiogenic events, with high specificity for MEK/ERK-dependent cell proliferation and migration and PI3K/Akt-mediated tube formation. Moreover, inhibition of p38 MAPK effectively inhibited sesamin-induced cell migration. The angiogenic activity of sesamin was not associated with VEGF expression. Furthermore, this compound did not induce vascular permeability and upregulated ICAM-1 and VCAM-1 expression, which are hallmarks of vascular inflammation. These results suggest that sesamin stimulates angiogenesis in vitro and in vivo through the activation of MEK/ERK-, PI3K/Akt/eNOS-, p125^FAK-, and p38 MAPK-dependent pathways, without increasing vascular inflammation, and may be used for treating ischemic diseases and tissue regeneration.     

Downregulation of human endothelial nitric oxide synthase promoter activity by p38 mitogen-activated protein kinase activation.

Human endothelial nitric oxide synthase (eNOS) plays a crucial role in maintaining blood pressure homeostasis and vascular integrity. eNOS gene expression may be upregulated by a signaling pathway, including PI-3Kgamma--> Jak2--> MEK1 --> ERK1/2--> PP2A. It remains unclear whether other mitogen-activated protein kinase (MAPK) family members, such as JNK, p38 kinase, and ERK5/BMK1, also modulate eNOS gene expression. Our purpose, therefore, is to shed light on the effect of the p38 MAPK signaling pathway on the regulation of eNOS promoter activity. The results showed that a red fluorescent protein reporter gene vector containing the full length of the human eNOS promoter was first successfully constructed, expressing efficiently in ECV304 cells with the characteristics of real time observation. The wild-types of p38alpha, p38beta, p38gamma, and p38delta signal molecules all markedly downregulated promoter activity, which could be reversed by their negative mutants, including p38alpha (AF), p38beta (AF), p38gamma (AF), and p38delta (AF). Promoter activity was also significantly downregulated by MKK6b (E), an active mutant of an upstream kinase of p38 MAPK. The reduction in promoter activity by p38 MAPK could be blocked by treatment with a p38 MAPK specific inhibitor, SB203580. Moreover, the activation of endogenous p38 MAPK induced by lipopolysaccharide resulted in a prominent reduction in promoter activity. These findings strongly suggest that the activation of the p38 MAPK signaling pathway may be implicated in the downregulation of human eNOS promoter activity.     

The involvement of tyrosine kinases, cyclic AMP/protein kinase A, and p38 mitogen-activated protein kinase in IL-13-mediated arginase I induction in macrophages: its implications in IL-13-inhibited nitric oxide production

In macrophages, L-arginine can be used by NO synthase and arginase to form NO and urea, respectively. Therefore, activation of arginase may be an effective mechanism for regulating NO production in macrophages through substrate competition. Here, we examined whether IL-13 up-regulates arginase and thus reduces NO production from LPS-activated macrophages. The signaling molecules involved in IL-13-induced arginase activation were also determined. Results showed that IL-13 increased arginase activity through de novo synthesis of the arginase I mRNA and protein. The activation of arginase was preceded by a transient increase in intracellular cAMP, tyrosine kinase phosphorylation, and p38 mitogen-activated protein kinase (MAPK) activation. Exogenous cAMP also increased arginase activity and enhanced the effect of IL-13 on arginase induction. The induction of arginase was abolished by a protein kinase A (PKA) inhibitor, KT5720, and was down-regulated by tyrosine kinase inhibitors and a p38 MAPK inhibitor, SB203580. However, inhibition of p38 MAPK had no effect on either the IL-13-increased intracellular cAMP or the exogenous cAMP-induced arginase activation, suggesting that p38 MAPK signaling is parallel to the cAMP/PKA pathway. Furthermore, the induction of arginase was insensitive to the protein kinase C and p44/p42 MAPK kinase inhibitors. Finally, IL-13 significantly inhibited NO production from LPS-activated macrophages, and this effect was reversed by an arginase inhibitor, L-norvaline. Together, these data demonstrate for the first time that IL-13 down-regulates NO production through arginase induction via cAMP/PKA, tyrosine kinase, and p38 MAPK signalings and underline the importance of arginase in the immunosuppressive activity of IL-13 in activated macrophages.     

Activation of endothelial nitric-oxide synthase by the p38 MAPK in response to black tea polyphenols

Black tea improves endothelial function in patients with coronary artery disease. We sought to determine the responsible components of black tea and elucidate the underlying cell signaling mechanisms. We exposed porcine aortic endothelial cells to components of black tea and found that the polyphenol fraction acutely enhanced nitric oxide bioactivity. This effect involved endothelial nitric-oxide synthase (eNOS) phosphorylation at Ser-1177 and dephosphorylation at Thr-495, consistent with increased eNOS activity. These effects were calcium-dependent, as removal of extracellular calcium prevented eNOS phosphorylation at Ser-1177, whereas inhibition of intracellular calcium mobilization with TMB-8 blunted Thr-495 dephosphorylation. Black tea polyphenol-induced eNOS activation appeared dependent upon the phosphatidylinositol 3-kinase-Akt pathway, as it was significantly inhibited by LY294002 and a dominant negative Akt, respectively. Pharmacological inhibition of p38 mitogen-activated protein kinase (p38 MAPK) with either SB202190 or SB203580 as well as overexpression of a dominant negative p38 MAPKalpha attenuated both eNOS activation and phosphorylation changes in response to black tea polyphenols. Inhibition of p38 MAPKalpha also blunted Akt activation in response to black tea polyphenols, suggesting that p38alpha MAPK is upstream of Akt in this pathway. Finally, a constitutively active mutant of MKK6bE, an upstream kinase for p38 MAPK, enhanced both the basal and stimulated activity of Akt, leading to increased eNOS activity. Taken together, these data identify the p38 MAPK as an upstream component of Akt-mediated eNOS activation.     

Flavonoids inhibit high glucose-induced up-regulation of ICAM-1 via the p38 MAPK pathway in human vein endothelial cells

Recently, several flavonoids have been shown to have cardioprotective, cancer preventive, or anti-inflammatory properties. However, the specific mechanisms underlying their protective effects remain unclear. We aimed to investigate the different effects of three representative flavonoids—hesperidin, naringin, and resveratrol—on intracellular adhesion molecule-1 (ICAM-1) induction in human umbilical vein endothelial cells (HUVECs) by using high-glucose (HG) concentrations and the possible underlying molecular mechanisms. In HG-induced HUVEC cultures, the effects of three different flavonoids on ICAM-1 production and p38 phosphorylation were examined in the presence or absence of inhibitors targeting the mitogen-activated protein kinase (MAPK) signal transduction pathway. HG stimulation of HUVECs increased the levels of the adhesion molecules ICAM-1 and endothelial selectin (E-selectin). Pretreatment with all the three flavonoids drastically inhibited ICAM-1 expression in a time-dependent manner, but did not alter VCAM-1 and E-selectin expressions. Moreover, we investigated the effects of flavonoids on the MAPK signal transduction pathway, because MAPK families are associated with vascular inflammation under stress. These flavonoids did not block HG-induced phosphorylation of extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK), but completely inhibited the HG-induced phosphorylation of p38 MAPK. SB202190, an inhibitor of p38 MAPK, also inhibited the HG-induced enrichment of ICAM-1. This study demonstrated that hesperidin, naringin, and resveratrol reduced the HG-induced ICAM-1 expression via the p38 MAPK signaling pathway, contributing to the inhibition of monocyte adhesion to endothelial cells.