Rubbing salt into wounded endothelium: sodium potentiates proatherogenic effects of TNF-α under non-uniform shear stress.

Increased consumption of sodium is a risk factor for hypertension and cardiovascular diseases. In vivo studies indicated that high dietary sodium may have a direct negative influence on endothelium. We investigated the effects of high sodium on the endothelial activation during early steps of atherogenesis. Endothelial cells (HUVECs) grown in a model of arterial bifurcations were exposed to shear stress in the presence of normal or high (+ 30 mmol/l) sodium. Adherent THP-1 cells, and the adhesion molecule expression were quantified. Sodium channel blockers, pathways' inhibitors, and siRNA against tonicity-responsive enhancer binding protein (TonEBP) were used to identify the mechanisms of sodium effects on endothelium. ApoE-deficient mice on low-fat diet received water containing normal or high salt (8% w/v) for four weeks, and the influence of dietary salt on inflammatory cell adhesion in the common carotid artery and carotid bifurcation was measured by intravital microscopy. In vitro, high sodium dramatically increased the endothelial responsiveness to tumour necrosis factor-α under non-uniform shear stress. Sodium-induced increase in monocytic cell adhesion was mediated by reactive oxygen species and the endothelial nitric oxygen synthase, and was sensitive to the knockdown of TonEBP. The results were subsequently confirmed in the ApoE-deficient mice. As compared with normal-salt group, high-salt intake significantly enhanced the adhesion of circulating CD11b+ cells to carotid bifurcations, but not to the straight segment of common carotid artery. In conclusion, elevated sodium has a direct effect on endothelial activation under atherogenic shear stress in vitro and in vivo, and promotes the endothelial-leukocyte interactions even in the absence of increased lipid concentrations.

CD40/CD154 system and pro-inflammatory cytokines in young healthy male smokers without additional risk factors for atherosclerosis.

OBJECTIVE AND DESIGN: Atherosclerosis, as an inflammatory disease, is characterized by pathologically altered levels of cytokines. We investigated whether smoking affects the CD40/CD154 system and pro-inflammatory cytokines in young males without other risk factors for atherosclerosis. SUBJECTS: Young male smokers (n=13) and 14 non-smoking controls were investigated. METHODS: The differences in CD40/CD154 system and serum cytokines between the groups were measured using flow cytometry and ELISA. RESULTS: In smokers, there was a strong trend (P<0.06) for increased CD40 expression on platelets as compared with non-smokers. However, there were no significant differences in CD40 expression on monocytes or in CD154 expression on platelets and T-cells between smokers and non-smokers. There was a strong trend for increased platelet-monocyte aggregates in smokers (P<0.06). Also, smokers had slightly but not significantly elevated hsCRP and IL-6 levels, and slightly decreased TNF-alpha and MCP-1. Interestingly, IL-18, a cytokine which has the ability to promote both Th1 and Th2 responses, was significantly decreased in smokers group (P=0.03 vs controls). CONCLUSIONS: In young healthy males, smoking is not associated with dramatic changes in CD40/CD154 system. However, cigarette smoke alters the secreted cytokine profile, leading to significant decrease in systemic IL-18 levels.

Elevated VEGF-plasma levels in young patients with mild essential hypertension.

BACKGROUND: Growing evidence shows that inflammation plays a pivotal role in the pathophysiology of essential hypertension (EH). Vascular endothelial cell growth factor (VEGF) is currently discussed as a possible mediator of inflammation. To investigate the hypothesis that VEGF plays a role as an inflammatory mediator in EH we performed the present pilot study of young patients in a very early stage of EH. MATERIALS AND METHODS: 15 young patients with mild EH [33.8 +/- 7.3 years, systolic blood pressure (SBP): 143.8 +/- 10.5 mmHg, diastolic blood pressure (DBP): 88.2 +/- 11.1 mmHg, mean arterial pressure (MAP) 106.6 +/- 10.4 mmHg] and 15 healthy controls (31.7 +/- 10.6 years) were examined. Blood was drawn from a peripheral vein and serum levels of VEGF, monocyte-chemoattractant-protein (MCP)-1, high-sensitivity C-reactive protein (hsCRP), interleukin (IL)-6, and tumour-necrosis-factor (TNF)-alpha were measured via commercially available enzyme-linked immunoassays. RESULTS: Hypertensives showed increased plasma levels of VEGF (P < 0.05) and MCP-1 (P < 0.05). VEGF positively correlated with MAP (r = 0.46, P < 0.05) and MCP-1 (r = 0.63, P < 0.01). Multivariate analysis demonstrated VEGF to be an independent predictor of MCP-1 levels. Furthermore, hypertensives had higher levels of hsCRP (P < 0.01), IL-6 (P < 0.001) and TNF-alpha (P < 0.05). IL-6 levels correlated with SBP (r = 0.59, P < 0.001), DBP (r = 0.67, P < 0.001) and MAP (r = 0.46, P < 0.001). A significant positive correlation was also found between hsCRP levels and SBP (r = 0.39, P < 0.05). CONCLUSIONS: This pilot study demonstrates that in an early state of EH, inflammatory pathways have already been activated. Besides classical pro-inflammatory cytokines, VEGF serum levels are significantly elevated. The positive correlation of VEGF with MCP-1 is suggestive for the already described induction of MCP-1 via VEGF.

Platelets contribute to enhanced MCP-1 levels in patients with chronic heart failure.

BACKGROUND: Increasing scientific data suggest a role for inflammation in chronic heart failure (CHF), but up to now the exact mechanisms are still not clear. Recently, platelets were identified as inducing inflammation partly by releasing cytokines. This new aspect necessitates further studies about the contribution of platelets for the inflammatory setting of CHF. METHODS: 50 CHF patients (mean 66.9 (SD 12.6) years, mean EF 22.1% (SD 9.1)) and 25 healthy controls (mean 63.6 (SD 10.2) years) were examined. MCP-1 serum levels were measured via EIA, expression of platelet CD154 by flow cytometry. In in-vitro experiments activated platelets were cocultured with human umbilical vein endothelial cells (HUVEC) in the presence and absence of anti-CD154 antibodies. MCP-1 in the supernatants was measured by EIA. RESULTS: CHF patients showed significantly enhanced MCP-1 levels (median: 191.8; 25th centile: 153.7; 75th centile: 227.1 pg/ml vs median: 101.0; 25th centile: 86.7; 75th centile: 117.5 pg/ml, p<0.001). MCP-1 levels positively correlated with severity of CHF. In the cell coculture model activated platelets were able to significantly induce MCP-1 release from HUVEC in a CD154-dependent manner. Furthermore, CHF patients showed enhanced platelet CD154 expression with a positive correlation with MCP-1 levels. Aspirin therapy had no influence on either CD154 expression or MCP-1 levels. CONCLUSIONS: Platelets can contribute to enhanced MCP-1 levels in CHF. MCP-1 is markedly elevated in serum of CHF patients showing a direct correlation with the severity of symptoms and the degree of left ventricular dysfunction. Further studies are required to test whether MCP-1 blocking or sophisticated anti-platelet strategies may represent new therapeutic options in CHF.

Increased prolactin in acute coronary syndromes as putative Co-activator of ADP-stimulated P-selectin expression.

Prolactin and leptin are newly recognized platelet co-stimulators due to enhancement of ADP-induced platelet aggregation. The aim of our study was to assess whether both hormones prolactin and leptin play a role as co-activators of platelet activation in patients with acute coronary syndromes. Twenty-one patients with acute coronary syndromes, 10 with stable angina pectoris and 10 controls were studied. Patients with acute coronary syndromes showed significantly higher prolactin and leptin values and a significant increased P-selectin expression on platelets compared to patients with stable angina pectoris or controls. However, patients with acute myocardial infarction as a subgroup of acute coronary syndromes showed the highest prolactin levels as well as ADP stimulated P-selectin expression. In the myocardial infarction subgroup prolactin values showed a significant correlation to ADP stimulated P-selectin expression on platelets (r (2)=0.41; p=0.025), whereas leptin was not correlated. Our data indicate an association between increased prolactin values and enhanced P-selectin expression on platelets in patients with acute coronary syndromes. Therefore, the stress hormone prolactin could be a co-stimulator of platelet activation in these patients. In contrast, the putative platelet activator leptin does not seem to play a major role in acute coronary syndromes.

Dose-dependent disintegration of human endothelial monolayers by contrast echocardiography.

Biological effects on endothelium induced by contrast ultrasound (US) may be relevant for transferring drugs into the tissue. An in vitro tissue-mimicking phantom was developed to simulate clinical precordial echocardiography of three modalities (two-dimensional (2DE), pulsed wave (PW), and Power Doppler echocardiography) with gradual increases of acoustic output (mechanical index (MI) 0.0-1.6 and thermal index soft tissue (TIS) 0.0-1.3, respectively; transmit-frequency 1.8 MHz in second harmonic mode (SHI) by 2DE, 1.8 MHz for PW-Doppler, and 3.2 MHz for Power Doppler) as well as contrast agent (CA) concentrations (0.002-4 mg/mL Levovist). Disintegration of the endothelial monolayer was quantitatively analyzed by counting intercellular gaps in light microscopy. No gaps were observed in CA application without sonication. Only few gaps appeared at sonication without CA application in 2DE at MI=1.6 and in PW- and Power Doppler at TIS > or =0.4 and MI > or =0.4. The number of gaps increased significantly with the gradual increase of US output and to a comparably lesser but also significant extent with CA concentrations. Diagnostic contrast echocardiography may induce endothelial disintegrations dependent on US output as well as on CA concentrations. This aspect might be helpful in further in vivo series on local drug delivery.

Delay of neutrophil apoptosis in acute coronary syndromes.

Apoptosis of polymorphonuclear neutrophils (PMN) is currently discussed as a key event in the control of inflammation. This study determined PMN apoptosis and its underlying mechanisms in controls (C), patients with stable (SAP) or unstable angina (UAP), and with acute myocardial infarction (AMI). Blood was drawn from 15 subjects of each C, SAP, UAP, and AMI. Apoptosis was measured by flow cytometry in isolated PMN (propidium iodide staining) and PMN from whole blood (CD16, FcgammaRIII). Serum cytokines were determined by enzyme-linked immunosorbent assay. Apoptosis of isolated PMN was delayed significantly in acute coronary syndromes (ACS) as compared with SAP or C (C, 51.2+/-12.6%; SAP, 44.9+/-13.6%; UAP, 28.4+/-10.1%; AMI, 20.3+/-8.5%; AMI or UAP vs. SAP or C, P<0.001). These results were confirmed by measurement of PMN apoptosis in cultured whole blood from patients and controls. Moreover, serum of patients with ACS markedly reduced apoptosis of PMN from healthy donors. Analysis of patients' sera revealed significantly elevated concentrations of tumor necrosis factor alpha, interferon-gamma (IFN-gamma), granulocyte macrophage-colony stimulating factor (GM-CSF), and interleukin (IL)-1beta in ACS (vs. C and SAP). IFN-gamma, GM-CSF, and IL-1beta significantly delayed PMN apoptosis in vitro. Furthermore, coincubation of PMN with adenosine 5'-diphosphate-activated platelets significantly inhibited PMN apoptosis as compared with coculture with unstimulated platelets. This study demonstrates a pronounced delay of PMN apoptosis in UAP and AMI, which may result from increased serum levels of IFN-gamma, GM-CSF, and IL-1beta and from enhanced platelet activation. Therapeutical modulation of these determinants of PMN lifespan may provide a new concept for the control of inflammation in ACS.

Upregulation of CD40-CD40 ligand (CD154) in patients with acute cerebral ischemia.

BACKGROUND AND PURPOSE: Inflammation and hypercoagulability contribute to the development of acute cerebral ischemia. Both can be mediated by the CD40 system. This study investigated whether the CD40 system and related mediators are upregulated in patients with transient ischemic attack (TIA) or stroke. METHODS: Seventeen patients with TIA, 60 patients with complete stroke, and 15 control subjects were investigated. CD154 and P-selectin were analyzed on platelets and CD40 on monocytes during and 3 months after acute cerebral ischemia by double-label flow cytometry. Blood concentrations of soluble CD154 and monocyte chemoattractant protein-1 (MCP-1) were evaluated. RESULTS: Our main findings are as follows: (1) patients with acute cerebral ischemia showed a significant increase of CD154 on platelets and CD40 on monocytes compared with controls; (2) plasma levels of soluble CD154 were significantly higher in these patients; (3) these patients had significantly higher numbers of prothrombotic platelet-monocyte aggregates; (4) the chemoattractant MCP-1 was significantly elevated in cerebral ischemia; and (5) at 3 months' follow-up, upregulation of CD154 still persisted in patients with previous acute cerebral ischemia. CONCLUSIONS: Patients with acute cerebral ischemia show upregulation of the CD40 system, which might contribute to the known proinflammatory, proatherogenic, and prothrombotic milieu found in these patients.

Upregulation of CD40 and CD40 ligand (CD154) in patients with moderate hypercholesterolemia.

BACKGROUND: Hypercholesterolemia, a risk factor for cardiovascular disease, is associated with inflammation and hypercoagulability. Both can be mediated by the CD40 system. This study investigated whether the CD40 system is upregulated in patients with moderate hypercholesterolemia and whether it is influenced by therapy with a hydroxymethylglutaryl coenzyme A (HMG-CoA) reductase inhibitor. METHODS AND RESULTS: Fifteen patients with moderate hypercholesterolemia and 15 healthy control subjects were investigated. CD154 and P-selectin were analyzed on platelets and CD40 was analyzed on monocytes before and under therapy with the statin cerivastatin by double-label flow cytometry. Blood concentrations of soluble CD154 and monocyte chemoattractant protein-1 (MCP-1) were evaluated. Our main findings were as follows. Patients with moderate hypercholesterolemia showed a significant increase of CD154 and P-selectin on platelets and CD40 on monocytes compared with healthy subjects. Soluble CD154 showed a nonsignificant trend for higher plasma levels in patients. A positive correlation was found for total or LDL cholesterol and CD154, but not for CD40 on monocytes. The latter was upregulated in vitro by C-reactive protein, which was found to be significantly elevated in patients with moderate hypercholesterolemia. CD154 on platelets proved to be biologically active because it enhanced the release of MCP-1, which was markedly elevated in an in vitro platelet-endothelial cell coculture model and in the serum of patients. Short-term therapy with a HMG-CoA reductase inhibitor significantly downregulated CD40 on monocytes and serum levels of MCP-1. CONCLUSION: Patients with moderate hypercholesterolemia show upregulation of the CD40 system, which may contribute to the known proinflammatory, proatherogenic, and prothrombotic milieu found in these patients.

Patients with acute coronary syndromes express enhanced CD40 ligand/CD154 on platelets.

OBJECTIVE: To investigate whether CD40L/CD154 on platelets and soluble CD40L/CD154 may play a role in the inflammatory process of acute coronary syndromes. DESIGN AND SETTING: Observational study in a university hospital. PATIENTS: 15 patients with acute myocardial infarction, 25 patients with unstable angina, 15 patients with stable angina, and 12 controls. MAIN OUTCOME MEASURES: CD40L/CD154 on platelets, P-selectin/CD62P on platelets, soluble CD40L/CD154 serum concentrations. RESULTS: Mean (SD) CD40L/CD154 expression on platelets was 6.2 (2.8) MFI (mean fluorescence intensity) in the infarct group, 11 (3.3) MFI in the unstable angina group (p < 0.001 v infarction), 3.6 (0.9) MFI in the stable angina group (p < 0.01 v infarction; p < 0.001 v unstable angina), and 3.2 (1.0) MFI in the controls (p < 0.01 v infarction; p < 0.001 v unstable angina; NS v stable angina). Soluble CD40L/CD154 concentration was 5.2 (1.1) ng/ml in the infarct group, 4.2 (0.7) ng/ml in the unstable angina group (p < 0.001 v infarction), 2.9 (1.0) ng/ml in stable angina group (p < 0.001 v infarction and unstable angina), and 3.0 (0.5) ng/ml in the controls (p < 0.001 v infarction and unstable angina; NS v stable angina). At a six months follow up, there was lower expression of CD40L/CD154 on platelets in patients with unstable angina (12.3 (3.6) v 3.8 (1.2) MFI, p < 0.0001) and acute myocardial infarction (6.2 (2.8) v 3.5 (0.8) MFI, p < 0.01) compared with their admission values six months earlier. Patients with unstable angina who needed redo coronary angioplasty (PTCA) or who had recurrence of angina were characterised by increased CD40L/CD154 expression on platelets compared with the remainder of the study group (recurrence of angina: 12.7 (3.2) v 9.7 (1.6) MFI, p < 0.05; re-do PTCA: 14.3 (4.2) v 10.3 (2.1) MFI, p < 0.05). CONCLUSIONS: Both CD40L/CD154 on platelets and soluble CD40L/CD154 are raised in patients with unstable angina and myocardial infarction. These findings suggest that CD40-CD40L/CD154 interactions may play a pathogenic role in triggering and propagation of acute coronary syndromes.

Monocytes coexpress endothelial and macrophagocytic lineage markers and form cord-like structures in Matrigel under angiogenic conditions.

OBJECTIVES: It has been shown that circulating human non-adherent CD34+ cells coexpressing vascular endothelial growth factor (VEGF)-R2 and AC133 have the capacity to differentiate into adherent mature endothelial cells. However, prior studies have demonstrated that a much bigger subset of primary adherent mononuclear cells can also form endothelial progenitor cells (EPC). To determine the origin of the latter cell population we tested the hypothesis: do monocytes as a firmly adherent and plastic cell type have the potential to differentiate into an endothelial phenotype. METHODS: CD34-/CD14+ monocytes were isolated from human peripheral blood by adherence separation and magnetic bead selection (purity >90%) and cultured on fibronectin-coated plastic dishes (medium containing VEGF 10 ng/ml, basic fibroblast growth factor (bFGF) 2 ng/ml, insulin like growth factor (IGF-1) 1 ng/ml, 20% fetal calf serum). RESULTS: After 2 weeks of culture, using fluorescence activated cell analysis we observed a new expression of the endothelial markers von Willebrand factor (vWf), VE-cadherin (VE) and ec-NOS in 45.2, 12.4 and 9.8% of the cells, respectively. The proportion of cells expressing these markers further increased after 4 weeks (94.2, 89.7 and 58.8% of these cells, respectively). The proportion of CD45 expressing cells remained unchanged during this period. However, after 14 days the specific macrophage antigen CD68 was newly expressed in 62% of the analysed cells with a further increase to 90% after 28 days of culture. In three-dimensional gel (Matrigel the formation of cord- and tubular-like structures was observed. CONCLUSION: The present data indicate that under angiogenic stimulation macrophages develop an endothelial phenotype with expression of specific surface markers and even form cord- and tubular-like structures in vitro suggesting that this cell population may be recruited for vasculogenesis.

Decreased plasma concentrations of L-hydroxy-arginine as a marker of reduced NO formation in patients with combined cardiovascular risk factors.

Patients with metabolic syndrome represent a group with extensive cardiovascular risk factors for the development of atherosclerosis, which may be preceded by an impairment of endothelial function. Endothelial dysfunction is characterized by a reduced availability of bioactive nitric oxide, the principal mediator of endothelium-dependent vasodilation. In the present study we assessed NO synthesis in vivo by measuring the NO-related amino acids L-arginine and L-citrulline and in particular the stable intermediate compound N(omega)-hydroxy-L-arginine (L-NHA) in patients with metabolic syndrome by using high-performance liquid chromatography (HPLC) analysis. As a prerequisite to our study, we measured the amino acid concentrations in 31 healthy volunteers to investigate gender and age differences. To prove whether blood drawn from peripheral veins reflects plasma concentrations of the whole vessel system, several blood samples from different regions were obtained from patients undergoing elective left and right heart catheterization. In the latter group, no significant differences were noted among the plasma concentrations between the different sample sites. In healthy volunteers, there were no significant differences in plasma concentrations of any one specific amino acid between males and females or age groups. The main finding of the study is that the intermediate product of NO synthesis, L-NHA, is significantly reduced in the plasma samples of patients with a metabolic syndrome as compared with samples from healthy control subjects. The plasma concentrations of the NO precursor L-arginine and the end product of NO synthesis, L-citrulline, were unchanged. In conclusion, our results suggest that plasma levels of L-NHA are independent of age and gender and are not different at various locations within the vascular system. In a group of patients at high risk for the development of atherosclerosis, we found reduced plasma concentrations of L-NHA, either caused by a decreased endothelial NO synthase activity or caused by an increased breakdown of L-NHA by pathways independent of NO synthase, resulting in a reduced availability of L-NHA for NO synthesis.

Priming of superoxide anion in polymorphonuclear neutrophils by brain natriuretic peptide.

In acute coronary syndromes such as unstable angina and myocardial infarction, serum concentration of brain natriuretic peptide, a cardiac hormone with potent vasodilatatory, natriuretic and diuretic activities, is elevated. Little is known about the effect of elevated BNP plasma concentration on free radical-mediated tissue damage in these states. We investigated the influence of human BNP 32 and its fragment BNP 7-32 on the production of superoxide anion by PMN, a major cause for myocardial damage. Although BNP showed itself no stimulatory potential on superoxide anion release in PMN, it enhanced significantly the stimulatory potential of cell stimuli such as fMLP or phorbol 12-myristate 13-acetate (PMA) in PMN. Thus our data show that the cardiac-derived hormone BNP influences an important function of PMN. This 'priming' effect of BNP on PMN may contribute to the tissue damage occuring during acute coronary syndromes.

Beta-blockers reduce the release and synthesis of endothelin-1 in human endothelial cells.

BACKGROUND: Endothelins play an important role in cardiovascular diseases, and clinical trials have shown a reduction in endothelin levels after long-term treatment of chronic heart failure with beta-adrenergic antagonists. It is not known, however, whether this effect is caused by haemodynamic changes associated with the use of beta-adrenergic antagonists or by direct interaction of beta-blockers with human endothelial cells. The aim of this study was to determine whether beta-adrenergic antagonists have an influence on endothelin-1 (ET-1) synthesis and release in human endothelial cells. METHODS: Pretreatment of cultured endothelial cells from human umbilical veins (HUVECs) with different concentrations of the non-selective beta-blocker propranolol, the beta 1-blocker metoprolol and the beta 1-blocker and beta 2-agonist celiprolol (all 10(-7)-10(-4) mol L-1) was found to reduce ET-1 production. This ET-1-reducing effect was even more pronounced in thrombin-stimulated cells (10(-5) mol L-1 of propranolol, metoprolol and celiprolol: 19% +/- 5.8%, 25% +/- 4% and 37% +/- 5.2% respectively). RESULTS: Quantitative reverse transcriptase polymerase chain reaction and Northern blotting confirmed an inhibitory effect of the beta-blocker on biosynthesis. Furthermore, the ET-1-reducing effect of propranolol, metoprolol and celiprolol was not due to a compensatory increase in prostacyclin and was not reversible by N-nitro-L-arginine. CONCLUSION: The effect of beta-adrenergic antagonists on ET-1 production of the endothelium may at least partially explain the efficacy of beta-blockers in the treatment of diseases such as advanced heart failure, essential hypertension as well as acute coronary syndromes.

Role of Ca2+-ATPase inhibitors in activation of cytosolic phospholipase A2 in human polymorphonuclear neutrophils.

In the present study, we investigated the involvement of Ca2+-signaling and protein kinases in the effect of Ca2+-ATPase inhibitors on the activation of cytosolic phospholipase A2 (cPLA2) in human polymorphonuclear neutrophils. We found that activity and mobility on electrophoresis gels of the cPLA2 protein were significantly increased by f-Met-Leu-Phe (fMLP), 12-myristate 13-acetate (PMA) and the Ca2+-ATPase inhibitors, thapsigargin and cyclopiazonic acid. This effect was completely suppressed by staurosporine. Calphostin C partially inhibited the fMLP- and PMA-induced cPLA 2 activation, but had no influence on thapsigargin- and cyclopiazonic acid-treated cells. Thapsigargin and cyclopiazonic acid also showed no effect on protein kinase C activity. However, the thapsigargin- and cyclopiazonic acid-induced cPLA2 activation was completely inhibited by the tyrosine kinase inhibitor, erbstatin, and Ca2+ chelator, EGTA. In addition, the cPLA2 activity was reduced after pretreatment with the mitogen-activated protein kinase kinase inhibitor PD98059. The arachidonic acid release was significantly reduced in cells pretreated with the cPLA2 inhibitor, AACOCF3. Furthermore, we found that the human neutrophil cPLA2 cDNA contain a Ca2+-dependent-lipid binding domain which shares homology to several other enzymes such as protein kinase C and phospholipase C. Our results suggest that tyrosine kinases and the MAP kinase cascade are involved in Ca2+-ATPase inhibitor-induced activation and phosphorylation of cPLA2. Protein kinase C is not required in this event.

Angiotensin II-induced superoxide anion generation in human vascular endothelial cells: role of membrane-bound NADH-/NADPH-oxidases.

BACKGROUND: Angiotensin II (ANG II) mediated hypertension accelerates atherosclerosis (AS) and thereby increases the incidence of myocardial infarction (MI). On the other hand, superoxide anion (O2-) is involved in the modification of low density lipoproteins, inhibition of prostacyclin (PGI2) formation and breakdown of nitric oxide. These events finally lead to rapid progression of AS and MI. In the present study, we investigate whether ANG II can induce O2- release from human vascular endothelial cells (HVECs) and the possible mechanisms involved. METHODS AND RESULTS: The expression of ANG receptors subtype-1 (AT-1) and subtype-2 (AT-2) were identified by using reverse transcription polymerase chain reaction and sequence analysis. The O2- production was dose-dependently increased in HVECs treated with ANG II (10(-7)-10(-9) M) and with a maximum rate after 1 h of incubation. This event was significantly inhibited by pretreatment of cells with the specific AT-1 blocker losartan (10(-7) M) and to a lesser extent by the specific AT-2 receptor blocker PD123319 (10(-7) M). The combined incubation of both receptor blockers was even more effective. In addition, our lucigenin-enhanced chemiluminescence assay showed that the activity of plasma membrane-bound NADH-/NADPH-oxidases derived from ANG II-treated cells was also significantly increased, this effect was reduced in cells pretreated with losartan or to lesser extent by PD123319. However, the activity of xanthine oxidase remained unchanged in response to ANG II. Furthermore, the basal O2- release from HVECs was inhibited in cells treated with angiotensin-converting enzyme (ACE) inhibitor, Lisinopril (10(-6) M), and this event could be reversed by ANG II. CONCLUSION: ANG II induces O2- release in HVECs via activation of membrane-bound NADH-/NADPH-oxidase, an effect, that is mediated by both AT-1 and AT-2 receptors. This suggests that acceleration of AS and MI in ANG II-mediated hypertension may at least be due to ANG II-induced O2- generation from vascular endothelial cells. In this case, the ACE inhibitors and the ANG receptor antagonists may act as causative "antioxidants".

Involvement of tyrosine kinases, Ca2+ and PKC in activation of mitogen-activated protein (MAP) kinase in human polymorphonuclear neutrophils.

1. Activation of mitogen-activated protein (MAP) kinase is an early response to a wide variety of stimuli and plays an important role in the regulation of cellular functions. In the present study we investigated the activation of MAP kinase in human polymorphonuclear neutrophils (PMNs). 2. Activity of MAP kinase and protein kinase C (PKC) was measured radiometrically from the rate of phosphorylation of specific peptide substrates. Protein phosphorylation was measured by immunoprecipitation and Western blot analysis. 3. N-Formyl-Met-Leu-Phe (fMLP), phorbol 12-myristate, 13-acetate (PMA) and the Ca2+-ATPase inhibitors thapsigargin (Tg) and cyclopiazonic acid (CPA) increased MAP kinase activity significantly. The tyrosine kinase inhibitors erbstatin and herbimycin A partially inhibited the effects of fMLP and PMA, and completely abolished the effects of both Tg and CPA. The specific PKC inhibitor calphostin C suppressed activation of MAP kinase produced by fMLP and PMA, but had no effect on that produced by Tg and CPA. Tg and CPA were without effect on PKC activity. 4. Immunoprecipitation and Western blot analysis indicated that the 42 and 44 kDa tyrosine-phosphorylated proteins found after stimulation of PMNs were both members of the MAP kinase family. Pretreatment of PMNs with staurosporine, EGTA or erbstatin significantly reduced the tyrosine phosphorylation of MAP kinase(s). 5. These results suggest that in human PMNs, MAP kinase can be stimulated in both a PKC-dependent and a PKC-independent manner. The Ca2+ signal leads to activation of tyrosine kinases, which contribute to the activation of MAP kinase. However, a PMA-sensitive Ca2+-independent pathway also exists. Mobilization of Ca2+ and activation of PKC synergistically induce maximal MAP kinase activation and tyrosine phosphorylation.