Rapid reactive oxygen species production by mitochondria in endothelial cells exposed to tumor necrosis factor-alpha is mediated by ceramide.

Tumor necrosis factor (TNF)-alpha increases mitochondrial reactive oxygen species (ROS) production in tumor cells and hepatocytes. However, whether TNF-alpha stimulates mitochondrial ROS production in endothelial cells (EC) has not yet been reported. We studied the effect of TNF-alpha on mitochondrial ROS generation in EC and the signaling pathways involved. Cultured human umbilical vein EC (HUVEC) were studied by fluorescence microscopy, using dichlorodihydrofluorescein diacetate (DCFH-DA) as a marker of ROS production and propidium iodide uptake for cell viability. TNF-alpha increased DCFH oxidation in HUVEC dose-dependently. To determine the source of ROS, the mitochondrial respiratory chain inhibitors rotenone + thenoyltrifluoroacetone (TTFA), which inhibit electron entry to ubiquinone, and antimycin A (AA), a blocker of ubisemiquinone, were used. Rotenone and TTFA inhibited (n = 7, P < 0.05), whereas AA increased (118% in 3 min; n = 4, P < 0.01) ROS generation in HUVEC. In contrast, ROS production was not abolished by the nicotinamide adenine dinucleotide phosphate-dependent oxidase inhibitor diphenylene iodonium, by the xanthine oxidase inhibitor allopurinol, nor by the nitric oxide and cyclooxygenase pathway inhibitors N(omega)-nitro-L-arginine and mefenamic acid. In addition, TNF-alpha-induced ROS production was inhibited by the acidic sphingomyelinase inhibitor desipramine (5 microM; -80%, n = 4, P < 0.01) and totally blocked by the ceramide-activated protein kinase (CAPK) inhibitor dimethylaminopurine (1 mM; n = 6, P < 0.05). Thus, TNF-alpha induces mitochondrial ROS production in HUVEC that primarily occurs at the ubisemiquinone site and is mediated by ceramide-dependent signaling pathways involving CAPK.

Activation of NADPH oxidase by AGE links oxidant stress to altered gene expression via RAGE.

Engagement of the receptor for advanced glycation end products (RAGE) by products of nonenzymatic glycation/oxidation triggers the generation of reactive oxygen species (ROS), thereby altering gene expression. Because dissection of the precise events by which ROS are generated via RAGE is relevant to the pathogenesis of complications in AGE-related disorders, such as diabetes and renal failure, we tested the hypothesis that activation of NADPH oxidase contributed, at least in part, to enhancing oxidant stress via RAGE. Here we show that incubation of human endothelial cells with AGEs on the surface of diabetic red blood cells, or specific AGEs, (carboxymethyl)lysine (CML)-modified adducts, prompted intracellular generation of hydrogen peroxide, cell surface expression of vascular cell adhesion molecule-1, and generation of tissue factor in a manner suppressed by treatment with diphenyliodonium, but not by inhibitors of nitric oxide. Consistent with an important role for NADPH oxidase, although macrophages derived from wild-type mice expressed enhanced levels of tissue factor upon stimulation with AGE, macrophages derived from mice deficient in a central subunit of NADPH oxidase, gp91phox, failed to display enhanced tissue factor in the presence of AGE. These findings underscore a central role of NADPH oxidase in AGE-RAGE-mediated generation of ROS and provide a mechanism for altered gene expression in AGE-related disorders.

Extracellular matrix and growth factors during heart growth.

The effects of growth factors on tissue remodeling and cell differentiation depend on the nature of the extracellular matrix, the type and organization of integrins, the activation of metalloproteinases and the presence of secreted proteins associated to the matrix. These interactions are actually poorly known in the cardiovascular system. We describe here: 1) the main components of extracellular matrix within the cardiovascular system; 2) the role of integrins in the transmission of growth signals; 3) the shift in the expression of the components of the extracellular matrix (fibronectin and collagens) and the stimulation of the synthesis of metalloproteinases during normal and hypertrophic growth of the myocardium; 4) the effects of growth factors, such as Angiotensin II, Fibroblast Growth Factors (FGF), Transforming Growth Factor-beta (TGF-beta), on the synthesis of proteins of the extracellular matrix in the heart.

The extracellular matrix and the cytoskeleton in heart hypertrophy and failure.

Cell characteristics and phenotype depend on the nature of the extracellular matrix, the type and organization of integrins and cytoskeleton. The interactions between these components are poorly known at the myocyte level and during cardiac remodeling associated with cardiac hypertrophy and heart failure. We analyze here the nature and organization of extracellular matrix (ECM) proteins, cytoskeleton and integrins and their regulation by growth factors, such as angiotensin II, in normal myocyte growth and in pathological growth (hypertrophy) of the myocardium and heart failure.

Hydrogen peroxide decreases pHi in human aortic endothelial cells by inhibiting Na+/H+ exchange.

Postischemic endothelial dysfunction may occur as a result of the effects of endogenous oxidants like hydrogen peroxide. Since endothelium-dependent vasodilator function may be affected by pHi, the effect of hydrogen peroxide on endothelial pHi was examined. Hydrogen peroxide (100 micromol/L for 10 minutes) decreased pHi from 7.24+/-0.01 to 7.02+/-0.02 and inhibited recovery from an ammonium chloride-induced intracellular acid load in carboxy SNARF 1 (c-SNARF 1)-loaded human aortic endothelial cells in bicarbonate-free solution. Prior inhibition of Na+/H+ exchange with 5-(N-ethyl-N-isopropyl)amiloride (10 micromol/L), by removal of extracellular Na+, or by glycolytic inhibition with iodoacetic acid blocked the subsequent effect of hydrogen peroxide on pHi. A 2-minute exposure to 100 micromol/L H2O2 decreased intracellular ATP levels by approximately 40%; this was prevented by 3-aminobenzamide and nicotinamide (1 mmol/L each), inhibitors of the DNA repair enzyme poly(ADP-ribose) polymerase. Both 3-aminobenzamide and nicotinamide significantly inhibited the hydrogen peroxide-induced intracellular acidification and the effect of hydrogen peroxide on recovery from an intracellular acid load. Hydrogen peroxide decreases pHi in human endothelial cells by inhibiting Na+/H+ exchange. This appears to be mediated by activation of the DNA repair enzyme poly(ADP-ribose) polymerase and subsequent depletion of intracellular ATP. Since a decrease in pHi in this range may alter the activity of NO synthase or affect the synthesis of vasodilator prostaglandins, the effect of hydrogen peroxide on the endothelial Na+/H+ exchanger may be important in the pathogenesis of postischemic endothelial dysfunction.

Paracrine regulation of cardiac myocytes in normal and septic heart.

A paracrine pathway for the regulation of cardiac contractile function by nonmuscle cells is documented in the heart. Coronary and endocardial endothelium release several diffusible agents, such as prostaglandins, endothelin-1, and nitric oxide, with an action on cardiac myocyte function. Cardiac diseases involving an immune or inflammatory mechanism, such as endotoxic shock, are now seen as conditions in which cross-talk between different cell types in the heart is clearly implicated. The potential biological relevance of inducible nitric oxide synthase in the myocardium, and the subsequent production of nitric oxide has been proposed as a mechanism of the cardiac depression observed in septic shock. In addition to cardiac myocytes, activated microvascular endothelial cells and cardiac endothelial cells may contribute to nitric oxide generation and, ultimately, to the depression of myocardial contractile activity during sepsis. This article reviews the local intercellular communication between cardiac myocytes and endothelial cells in the normal heart and discusses some of the mechanisms potentially claimed to depress heart function in sepsis.

Hydrogen peroxide induces intracellular calcium oscillations in human aortic endothelial cells.

BACKGROUND: Because the vascular endothelium is exposed to oxidant stress resulting from ischemia/reperfusion and from the products of polymorphonuclear leukocytes or monocytes, studies were performed to examine the effect of hydrogen peroxide (1 micromol/L to 10 mmol/L) on endothelial Ca2+ signaling. METHODS AND RESULTS: At low concentrations (1 to 10 micromol/L), hydrogen peroxide did not affect intracellular Ca2+ concentration in subconfluent, indo 1-loaded human aortic endothelial monolayers. At a concentration of 100 micromol/L hydrogen peroxide, intracellular free Ca2+ gradually increased from 125.3+/-6.8 to 286.3+/-19.9 nmol/L over 4.2+/-0.9 minutes before repetitive Ca2+ oscillations were observed, consisting of an initial large, transient spike of approximately 1 micromol/L followed by several spikes of decreasing amplitudes at a frequency of 0.7+/-0.1 min-1 over 12.0+/-1.1 minutes. After these oscillations, intracellular Ca2+ reached a plateau of 543.4+/-64.0 nmol/L, which was maintained above baseline levels for >5 minutes and then partially reversible on washout of hydrogen peroxide in most monolayers. Intracellular Ca2+ oscillations were typically observed when monolayers were exposed to 100 to 500 micromol/L hydrogen peroxide. Higher concentrations of hydrogen peroxide (1 and 10 mmol/L) increased intracellular Ca2+ but only rarely (2 of 6 monolayers at 1 mmol/L) or never (at 10 mmol/L) stimulated intracellular Ca2+ oscillations. Removal of Ca2+ from the buffer either before hydrogen peroxide stimulation or during an established response did not block intracellular Ca2+ oscillations in response to 100 micromol/L hydrogen peroxide, but prior depletion of an intracellular Ca2+ store with either caffeine, histamine, or thapsigargin abolished Ca2+ oscillations. CONCLUSIONS: Hydrogen peroxide induces concentration-dependent intracellular Ca2+ oscillations in human endothelial cells, which results from release of an endoplasmic reticulum Ca2+ store. Because oxidant production appears to occur in the micromolar range in the postischemic/anoxic endothelium and is associated with impaired endothelium-dependent relaxation, the effects of micromolar concentrations of hydrogen peroxide on endothelial Ca2+ signaling described in the present study may be important in the pathogenesis of postischemic endothelial dysfunction.

Trophic effect of human pericardial fluid on adult cardiac myocytes. Differential role of fibroblast growth factor-2 and factors related to ventricular hypertrophy.

Pericardial fluid (PF) may contain myocardial growth factors that exert paracrine actions on cardiac myocytes. The aims of this study were (1) to investigate the effects of human PF and serum, collected from patients undergoing cardiac surgery, on the growth of cultured adult rat cardiac myocytes and (2) to relate the growth activity of both fluids to the adaptive changes in overloaded human hearts. Both PF and serum increased the rate of protein synthesis, measured by [14C]phenylalanine incorporation in adult rat cardiomyocytes (PF, +71.9 +/- 8.2% [n = 17]; serum, +14.9 +/- 6.5% [n = 13]; both P < .01 versus control medium). The effects of both PF and serum on cardiomyocyte growth correlated positively with the respective left ventricular (LV) mass. However, the magnitude of change with PF was 3-fold greater than with serum (P < .01). These trophic effects of PF were mimicked by exogenous basic fibroblast growth factor (FGF2) and inhibited by anti-FGF2 antibodies and transforming growth factor-beta (TGF-beta), suggesting a relationship to FGF2. In addition, FGF2 concentration in PF was 20 times greater than in serum. On the other hand, the LV mass-dependent trophic effect, present in both fluids, was independent of FGF2 concentration or other factors, such as angiotensin II, atrial natriuretic factor, and TGF-beta. These data suggest that FGF2 in human PF is a major determining factor in normal myocyte growth, whereas unidentified LV mass-dependent factor(s), present in both PF and serum, participates in the development of ventricular hypertrophy.

pH dependence of neutrophil-endothelial cell adhesion and adhesion molecule expression.

Neutrophil adhesion to the vascular endothelium is enhanced during tissue ischemia and/or inflammation, conditions that are associated with tissue acidosis. This study examined the effects of hypercarbic acidosis (10 or 20% CO2) and of hypocarbic alkalosis (0% CO2) on human neutrophil CD18 and human aortic endothelial cell intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and E-selectin expression quantified by flow cytometry. Acidosis with 20% CO2 for 4 h decreased ICAM-1 to 60.6 +/- 9.7% of control. In contrast, alkalosis with 0% CO2 for 4 h enhanced ICAM-1 expression to 143.8 +/- 10.1% of control. There was no pH dependence of VCAM-1 or E-selectin expression. Tumor necrosis factor-alpha (TNF-alpha; 10 ng/ml) increased endothelial ICAM-1, E-selectin, and VCAM-1; under these conditions, acidosis with 20% CO2 blunted both ICAM-1 and E-selectin surface expression compared with 5% CO2-, TNF-alpha-treated cells. Hypercarbic acidosis with 20% CO2 increased neutrophil CD18 expression and enhanced neutrophil adhesion. This latter effect was inhibited by neutrophil pretreatment with an anti-CD18 monoclonal antibody. In contrast, when only endothelial cells were preincubated with the hypercarbic buffer, neutrophil adhesion diminished to 55.6 +/- 7.8% of control. The results suggest that acidosis generated during tissue ischemia/inflammation may induce CD18-mediated neutrophil adhesion despite a decrease in ICAM-1 expression.

Endoplasmic reticulum Ca2+ depletion unmasks a caffeine-induced Ca2+ influx in human aortic endothelial cells.

Intracellular Ca2+ pools contribute to changes in cytosolic [Ca2+] ([Ca2+]i), which play an important role in endothelial cell signaling. Recently, endothelial ryanodine-sensitive Ca2+ stores were shown to regulate agonist-sensitive intracellular Ca2+ pools. Since caffeine binds the ryanodine Ca2+ release channel on the endoplasmic reticulum in a variety of cell types, we examined the effect of caffeine on [Ca2+]i in human aortic endothelial cell monolayers loaded with the fluorescent probe indo 1. Under baseline conditions, 10 mmol/L caffeine induced a small increase in [Ca2+]i from 86 +/- 10 to 115 +/- 17 nmol/L (mean +/- SEM); this effect was similar to that of 5 mumol/L ryanodine and was unaffected by buffer Ca2+ removal. After depletion of an intracellular Ca2+ store by the irreversible endoplasmic reticulum Ca(2+)-ATPase inhibitor thapsigargin (1 mumol/L), ryanodine did not affect [Ca2+]i. In contrast, caffeine induced a large rapid increase in [Ca2+]i (176 +/- 19 to 338 +/- 35 nmol/L, P < .001) after thapsigargin exposure; this effect of caffeine was only observed when extracellular Ca2+ was present. A similar increase in [Ca2+]i was induced by caffeine after depletion of ryanodine- and histamine-sensitive Ca2+ stores or after pretreatment with the endoplasmic reticulum Ca(2+)-ATPase inhibitor cyclopiazonic acid (10 mumol/L). Thus, under baseline conditions the effect of caffeine on [Ca2+]i is similar to that of ryanodine and appears to be due to the release of an intracellular store. However, after depletion of an endoplasmic reticulum Ca2+ store, caffeine, but not ryanodine, stimulates Ca2+ influx, resulting in a large increase in [Ca2+]i.(ABSTRACT TRUNCATED AT 250 WORDS)

Initial contact and subsequent adhesion of human neutrophils or monocytes to human aortic endothelial cells releases an endothelial intracellular calcium store.

BACKGROUND: Increases in both leukocyte and endothelial cytosolic free [Ca2+] may be involved in intercellular adhesion by regulating the affinity of surface adhesion molecules or by facilitating transendothelial leukocyte migration. The purpose of this study was to examine the effect of initial contact and subsequent adhesion of human neutrophils or monocytes on human aortic endothelial [Ca2+]. METHODS AND RESULTS: Endothelial monolayers were loaded with the fluorescent Ca2+ indicator indo 1 and exposed to isolated human peripheral blood neutrophils or to a cultured human monocyte cell line. A rapid, fourfold to fivefold increase in endothelial cytosolic [Ca2+] occurred within seconds of leukocyte contact. No increase in endothelial [Ca2+] occurred on contact of 18.25-microns inert microspheres, isolated red blood cells, or suspensions of cultured human aortic endothelial cells. In experiments performed on monolayers grown in 1-mm2 capillary flow tubes, the increase in endothelial cytosolic [Ca2+] on initial leukocyte contact was found to be related to the subsequent resistance to leukocyte detachment during exposure to arterial levels of shear stress (13.4 dyne.cm-2). The increase in endothelial cytosolic [Ca2+] during leukocyte contact was not inhibited in Ca(2+)-free buffer but was abolished by prior depletion of an endoplasmic reticulum Ca2+ store by thapsigargin. Pretreatment of neutrophils with R15.7, a specific monoclonal antibody to the adhesion protein CD-18, inhibited the increase in endothelial cytosolic [Ca2+] on neutrophil contact. CONCLUSIONS: Initial contact leading to subsequent adhesion of human leukocytes to human aortic endothelial cells releases an endothelial intracellular Ca2+ store. This may, in part, be mediated by specific adhesion proteins and may in turn regulate the affinity of surface adhesion molecules or facilitate transendothelial migration of leukocytes.

A functional ryanodine-sensitive intracellular Ca2+ store is present in vascular endothelial cells.

The presence of the ryanodine receptor was recently demonstrated in vascular and endocardial endothelium, but its function has not been established. We investigated whether functional ryanodine-sensitive Ca2+ stores are present in cultured endothelial cells from rat aorta (RAECs), human aorta (HAECs), human umbilical vein (HUVECs), and bovine pulmonary artery (BPAECs) and what role these may play in intracellular Ca2+ regulation. Under resting conditions, HAECs, BPAECs, and HUVECs demonstrated a slow increase in intracellular Ca2+ (indexed by indo 1 fluorescence) on exposure to 5 mumol/L ryanodine, whereas RAECs did not. However, after an initial bradykinin exposure in RAECs, ryanodine markedly blunted the rapid increase in Ca2+ on a second exposure to bradykinin. In HUVECs, ryanodine in buffer with 1.5 mmol/L Ca2+ did not inhibit the agonist-sensitive Ca2+ increase, whereas it blunted the rapid increase in Ca2+ on histamine exposure in buffer with 5 mmol/L Ca2+, suggesting that increasing [Ca2+] enhances the binding of ryanodine to its receptor. Thus, functional ryanodine-sensitive Ca2+ stores are present in vascular endothelial cells. These appear to be involved in regulation of Ca2+ storage and release from agonist-sensitive intracellular compartments.

Endothelial cell Ca2+ increases upon tumor cell contact and modulates cell-cell adhesion.

The signal transduction mechanisms involved in tumor cell adhesion to endothelial cells are still largely undefined. The effect of metastatic murine melanoma cell and human prostate carcinoma cell contact on cytosolic [Ca2+] of bovine artery endothelial cells was examined in indo-1-loaded endothelial cell monolayers. A rapid increase in endothelial cell [Ca2+] occurred on contact with tumor cells, but not on contact with 8-microns inert beads. A similar increase in endothelial cell [Ca2+] was observed with human neutrophils or monocyte-like lymphoma cells, but not with endothelial cells, red blood cells, and melanoma cell-conditioned medium. The increase in endothelial cell [Ca2+] was not inhibited by extracellular Ca2+ removal. In contrast, endothelial cell pretreatment with thapsigargin, which releases endoplasmic reticulum Ca2+ into the cytosol and depletes this Ca2+ store site, abolished the cytosolic [Ca2+] rise upon melanoma cell contact. Endothelial cell pretreatment with the membrane-permeant form of the Ca2+ chelator bis-(O-aminophenoxyl)ethane-N,N,N',N'-tetraacetic acid blocked the increase in cytosolic [Ca2+]. Under static and dynamic flow conditions (0.46 dyn/cm2) bis-(O-aminophenoxyl)ethane-N,N,N',N'-tetraacetic acid pretreatment of bovine pulmonary artery endothelial cell monolayers inhibited melanoma cell adhesion to the endothelial cells. Thus, tumor cell contact with endothelial cells induces a rapid Ca2+ release from endothelial intracellular stores, which has a functional role in enhancing cell-cell adhesion.