Treatment Of Bovine Aortic Endothelial Cells Research Articles

In vitro treatment of lipopolysaccharide increases invasion of Pasteurella multocida serotype B:2 into bovine aortic endothelial cells.

Pasteurella multocida serotype B:2 causes hemorrhagic septicemia in cattle and buffalo. The invasion mechanism of the bacterium when invading the bloodstream is unclear. This study aimed to characterize the effects of immunomodulatory molecules, namely dexamethasone and lipopolysaccharide, on the invasion efficiency of P. multocida serotype B:2 toward bovine aortic endothelial cells (BAECs) and the involvement of actin microfilaments in the invasion mechanism. The results imply that treatment of BAECs with lipopolysaccharide at 100 ng/mL for 24 h significantly increases the intracellular bacteria number per cell (p < 0.01) compared with those in untreated and dexamethasone-treated cells. The lipopolysaccharide-treated cells showed a significant decrease in F-actin expression and an increase in G-actin expression (p < 0.001), indicating actin depolymerization of BAECs. However, no significant differences were detected in the invasion efficiency and actin filament reorganization between the dexamethasone-treated and untreated cells. Transmission electron microscopy showed that P. multocida B:2 resided in a vacuolar compartment of dexamethasone-treated and untreated cells, whereas the bacteria resided in cellular membrane of lipopolysaccharide-treated cells. The results suggest that lipopolysaccharide destabilizes the actin filaments of BAECs, which could facilitate the invasion of P. multocida B:2 into BAECs.

Abstract 16962: Angiotensin II Limits NO Production by Upregulating Arginase through a Mitogen Activated Protein Kinase-Activating Transcription Factor-2 pathway

Nitric oxide (NO) produced by endothelial nitric oxide synthase (NOS) is needed for normal vascular function. During hypertension, diabetes or aging, elevated levels of arginase (ARG) can compete with NOS for available L-arginine thus reducing vascular NO production. Elevated angiotensin II (Ang II) is a key participant of endothelial dysfunction in many cardiovascular diseases and has been linked to elevated ARG activity. In this study we explored the signaling pathway leading to increased ARG expression/activity in responses to Ang II in bovine aortic endothelial cells (BAEC). Treatment of BAEC with Ang II (10 -7 M, 24 hrs) caused a 40±6% increase (p&lt;0.05) in ARG activity. This was accompanied by 30±8% decrease (p&lt;0.05) in NO production. Our studies indicate involvement of p38 mitogen activated protein kinase (MAPK) in Ang II-induced ARG upregulation and reduced NO production as p38 MAPK inhibitor SB202190 (2 μM) prevented the Ang II-induced increase in ARG activity. Our mouse studies also show involvement of p38 MAPK in Ang II-induced vascular dysfunction associated with elevated ARG activity and ARG expression. Ang II (42 µg/kg/h, sc, 2 wks) caused impaired EC-dependent vasorelaxation in mouse aorta (55±7% vs. 75±8% for control). This impairment was prevented by treatment with p38 inhibitor SB203580 (5µg/kg/day). Ang II also caused a 6.2 fold increase in vascular ARG activity/expression that was completely prevented by p38 MAPK inhibition. Additionally, treatment of BAEC with Ang II causes phosphorylation of activating transcription factor-2 (ATF-2) by 10 minutes with a peak at 30 minutes (3.9 fold). Transfection of BAEC with ATF-2 siRNA prevents Ang II-induced increases in ARG activity/expression and maintains NO production. These results indicate that ATF-2 is necessary for enhanced expression of ARG by Ang II. Collectively our results indicate that Ang II increases endothelial ARG activity/expression through a p38 MAPK-ATF-2 pathway leading to reduced NO production and endothelial dysfunction. Targeting these signaling steps might be therapeutic points for preventing vascular endothelial dysfunction associated with elevated ARG activity/expression.

2-Deoxy-D-glucose treatment of endothelial cells induces autophagy by reactive oxygen species-mediated activation of the AMP-activated protein kinase.

Autophagy is a cellular self-digestion process activated in response to stresses such as energy deprivation and oxidative stress. However, the mechanisms by which energy deprivation and oxidative stress trigger autophagy remain undefined. Here, we report that activation of AMP-activated protein kinase (AMPK) by mitochondria-derived reactive oxygen species (ROS) is required for autophagy in cultured endothelial cells. AMPK activity, ROS levels, and the markers of autophagy were monitored in confluent bovine aortic endothelial cells (BAEC) treated with the glycolysis blocker 2-deoxy-D-glucose (2-DG). Treatment of BAEC with 2-DG (5 mM) for 24 hours or with low concentrations of H2O2 (100 µM) induced autophagy, including increased conversion of microtubule-associated protein light chain 3 (LC3)-I to LC3-II, accumulation of GFP-tagged LC3 positive intracellular vacuoles, and increased fusion of autophagosomes with lysosomes. 2-DG-treatment also induced AMPK phosphorylation, which was blocked by either co-administration of two potent anti-oxidants (Tempol and N-Acetyl-L-cysteine) or overexpression of superoxide dismutase 1 or catalase in BAEC. Further, 2-DG-induced autophagy in BAEC was blocked by overexpressing catalase or siRNA-mediated knockdown of AMPK. Finally, pretreatment of BAEC with 2-DG increased endothelial cell viability after exposure to hypoxic stress. Thus, AMPK is required for ROS-triggered autophagy in endothelial cells, which increases endothelial cell survival in response to cell stress.

Citrus Polyphenol Hesperidin Stimulates Production of Nitric Oxide in Endothelial Cells while Improving Endothelial Function and Reducing Inflammatory Markers in Patients with Metabolic Syndrome

Hesperidin, a citrus flavonoid, and its metabolite hesperetin may have vascular actions relevant to their health benefits. Molecular and physiological mechanisms of hesperetin actions are unknown. We tested whether hesperetin stimulates production of nitric oxide (NO) from vascular endothelium and evaluated endothelial function in subjects with metabolic syndrome on oral hesperidin therapy. DESIGN, SETTING, AND INTERVENTIONS: Cellular mechanisms of action of hesperetin were evaluated in bovine aortic endothelial cells (BAEC) in primary culture. A randomized, placebo-controlled, double-blind, crossover trial examined whether oral hesperidin administration (500 mg once daily for 3 wk) improves endothelial function in individuals with metabolic syndrome (n = 24). We measured the difference in brachial artery flow-mediated dilation between placebo and hesperidin treatment periods. Treatment of BAEC with hesperetin acutely stimulated phosphorylation of Src, Akt, AMP kinase, and endothelial NO synthase to produce NO; this required generation of H(2)O(2). Increased adhesion of monocytes to BAEC and expression of vascular cell adhesion molecule-1 in response to TNF-α treatment was reduced by pretreatment with hesperetin. In the clinical study, when compared with placebo, hesperidin treatment increased flow-mediated dilation (10.26 ± 1.19 vs. 7.78 ± 0.76%; P = 0.02) and reduced concentrations of circulating inflammatory biomarkers (high-sensitivity C-reactive protein, serum amyloid A protein, soluble E-selectin). Novel mechanisms for hesperetin action in endothelial cells inform effects of oral hesperidin treatment to improve endothelial dysfunction and reduce circulating markers of inflammation in our exploratory clinical trial. Hesperetin has vasculoprotective actions that may explain beneficial cardiovascular effects of citrus consumption.

Resveratrol recruits rat muscle microvasculature via a nitric oxide-dependent mechanism that is blocked by TNFα

Resveratrol, a polyphenol found in many plants, has antioxidant and anti-inflammatory actions. It also improves endothelial function and may be cardioprotective. Tumor necrosis factor-α (TNFα) causes oxidative stress and microvascular endothelial dysfunction. Whether resveratrol affects microvascular function in vivo and, if so, whether inflammatory cytokines antagonize its microvascular action are not clear. In cultured bovine aortic endothelial cells (BAECs), resveratrol (100 nM) increased the phosphorylation of protein kinase B (Akt), endothelial nitric oxide (NO) synthase (eNOS), and ERK1/2 within 15 min by more than twofold, and this effect lasted for at least 2 h. Treatment of BAECs with TNFα (10 ng/ml) significantly increased the NADPH oxidase activity and the production of hydrogen peroxide and superoxide. Pretreatment of cells with resveratrol (100 nM) prevented each of these. Injection (ip) of resveratrol in rats potently increased muscle microvascular blood volume (MBV; P = 0.007) and flow (MBF; P < 0.02) within 30 min, and this was sustained for at least 2 h. The phosphorylation of Akt in liver or muscle was unchanged. Superimposed systemic infusion of L-NAME (NOS inhibitor) completely abolished resveratrol-induced increases in MBV and MBF. Similarly, systemic infusion of TNFα prevented resveratrol-induced muscle microvascular recruitment. In conclusion, resveratrol activates eNOS and increases muscle microvascular recruitment via an NO-dependent mechanism. Despite the potent antioxidant effect of resveratrol, TNFα at concentrations that block insulin-mediated muscle microvascular recruitment completely neutralized resveratrol's microvascular action. Thus, chronic inflammation, as seen in type 2 diabetes, may limit resveratrol's vasodilatory actions on muscle microvasculature.

Down-regulation of neprilysin (EC3.4.24.11) expression in vascular endothelial cells by laminar shear stress involves NADPH oxidase-dependent ROS production

Neprilysin (NEP, neutral endopeptidase, EC3.4.24.11), a zinc metallopeptidase expressed on the surface of endothelial cells, influences vascular homeostasis primarily through regulated inactivation of natriuretic peptides and bradykinin. Earlier in vivo studies reporting on the anti-atherosclerotic effects of NEP inhibition and on the atheroprotective effects of flow-associated laminar shear stress (LSS) have lead us to hypothesize that the latter hemodynamic stimulus may serve to down-regulate NEP levels within the vascular endothelium. To address this hypothesis, we have undertaken an investigation of the effects of LSS on NEP expression in vitro in bovine aortic endothelial cells (BAECs), coupled with an examination of the signalling mechanism putatively mediating these effects. BAECs were exposed to physiological levels of LSS (10dynes/cm2, 24h) and harvested for analysis of NEP expression using real-time PCR, Western blotting, and immunocytochemistry. Relative to unsheared controls, NEP mRNA and protein were substantially down-regulated by LSS (≥50%), events which could be prevented by treatment of BAECs with either N-acetylcysteine, superoxide dismutase, or catalase, implicating reactive oxygen species (ROS) involvement. Employing pharmacological and molecular inhibition strategies, the signal transduction pathway mediating shear-dependent NEP suppression was also examined, and roles implicated for Gβγ, Rac1, and NADPH oxidase activation in these events. Treatment of static BAECs with angiotensin-II, a potent stimulus for NADPH oxidase activation, mimicked the suppressive effects of shear on NEP expression, further supporting a role for NADPH oxidase-dependent ROS production. Interestingly, inhibition of receptor tyrosine kinase signalling had no effect. In conclusion, we confirm for the first time that NEP expression is down-regulated in vascular endothelial cells by physiological laminar shear, possibly via a mechanotransduction mechanism involving NADPH oxidase-induced ROS production.

Protein interactions with cdc37 in agonist stimulated bovine aortic endothelial cells

Previously we have demonstrated that the cell division cycle 37 homolog, cdc37, interacts directly with the endothelial nitric oxide synthase (eNOS) and inhibits eNOS activity in vitro. In addition, cdc37 regulates interactions between other key regulators of eNOS activity including Hsp90 and Akt. Therefore, we investigated changes cdc37 interactions with eNOS, Hsp90, and Akt in bovine aortic endothelial cells (BAECs) stimulated with either vascular endothelial growth factor (VEGF) or bradykinin (BK). Treatment of BAECs with VEGF or BK for 0, 1, 5, 15, 30, or 60 min resulted in a biphasic change in cdc37/eNOS interaction. Cdc37 interaction with eNOS decreased upon stimulation but returned to basal levels by 15 min and then decreased by 30 and 60 min. Agonist stimulation also resulted in a transient increase in Hsp90 association with cdc37, however, no significant changes in cdc37 and Akt interaction were observed. These data coupled with our previous findings suggest that cdc37 initially inhibits basal eNOS activity through direct interaction and may play a role in agonist stimulated eNOS activity through its interaction with Hsp90. Supported by NIH R15 HL082649 (MBH) and an American Physiological Society Summer Undergraduate Research Fellowship (KEH).

Green tea polyphenols down-regulate caveolin-1 expression via ERK1/2 and p38MAPK in endothelial cells

Caveolin-1 (Cav-1), a negative regulator of endothelial nitric oxide synthase (eNOS), influences various aspects of the cardiovascular functions. We had reported that a high-fat diet up-regulated aortic Cav-1 expressions in rats. In this study, we investigated the effects of green tea polyphenols (GTPs) on endothelial Cav-1 expression and phosphorylation in vitro. Bovine aortic endothelial cells (BAECs) were treated with 4 μg/ml GTPs for 0, 4, 8, 12, 16 and 24 h, and with 0, 0.04, 0.4, 4 and 40 μg/ml GTPs for 16 h, respectively. Cav-1 protein and mRNA were detected using Western blot and reverse transcriptase polymerase chain reaction. Cav-1 protein expression was down-regulated after treatment of BAECs with 4 μg/ml GTPs for 12, 16 and 24 h. And decrease in the level of Cav-1 mRNA was observed after GTP treatment for 4 and 8 h. GTPs (0.04–4 μg/ml) down-regulate Cav-1 protein expressions and mRNA levels dose dependently. PD98059, an inhibitor of extracellular signal-regulated kinase 1/2 (ERK1/2), up-regulated Cav-1 expression in BAECs alone and abolished the down-regulation effects of GTPs in BAECs while pretreatment with it. Inhibition of p38 mitogen-activated protein kinase (p38MAPK) with SB203580, which down-regulates Cav-1 expression in BAECs alone, deteriorated the Cav-1 down-regulating effects by GTPs. In addition to the effects on expression of Cav-1, GTP treatment inhibited phosphorylation of Cav-1 [tyrosine 14 (Tyr14)]. These data indicate that GTPs down-regulate gene expression of Cav-1 time- and dose- dependently via activating ERK1/2 and inhibiting p38MAPK signaling.

Abstract 1915: Simvastatin Regulates Insulin Signaling in Endothelial Cells via Activation of AMP-Activated Protein Kinase

Statins exert pleiotropic effects upon the cardiovascular system, but the role of statins in modulation of insulin signaling remains incompletely defined. In these studies, we explore the effects of simvastatin on insulin pathways in bovine aortic endothelial cells (BAEC), and propose a novel mechanism linking these pathways that involves AMP-activated protein kinase (AMPK). Treatment of BAEC with 100 nM insulin promotes the rapid phosphorylation of Akt (3.8±0.2-fold increase; n=4; p&lt;0.01), glycogen synthase kinase-3β (GSK3β, 2.7±0.5-fold increase; n=4; p&lt;0.01), as well as that of critical substrates in the mammalian target of rapamycin (mTOR) axis including mTOR (2.8±0.2-fold increase; n=4; p&lt;0.02) and p70S6K (3.2±0.5-fold increase; n=4; p&lt;0.02). However, in cells pretreated with simvastatin (10 μM for 24 hr), these insulin-dependent phosphorylations are significantly attenuated (62±10%, 48±12%, 53±9%, and 58±13% reduction in the phosphorylation of Akt, GSK3β, mTOR, and p70S6K, respectively; p&lt;0.02; n=4). Given our findings on the modulation of these key metabolic enzymes by simvastatin, we explored the effects of insulin and simvastatin on AMPK. We found that insulin promotes a rapid (within minutes) and robust increase in phosphorylation of both AMPK and its substrate acetyl CoA carboxylase (2.5±0.4-fold increase; n=4; p&lt;0.01). Importantly, transfection of BAEC with siRNA targeting AMPK results in enhanced insulin-dependent phosphorylation of Akt (57±9% enhancement compared to cells transfected with control siRNA; n=4; p&lt;0.015), as well as significant increases in phosphorylation of GSK3β, mTOR, and p70S6K (55±12% enhancement; n=4; p&lt;0.02), indicating a key regulatory role for AMPK in insulin-dependent metabolic signaling. Importantly, in cells transfected with AMPK siRNA, simvastatin treatment no longer attenuates insulin signaling to these substrates, suggesting that AMPK is required for the regulation of insulin pathways by statins. Taken together, these results establish that simvastatin modulates critical insulin responses in endothelial cells, and provide new insights into the roles of AMPK in linking insulin- and statin-regulated metabolic signaling pathways in the vascular wall.

Myoferlin Regulates Vascular Endothelial Growth Factor Receptor-2 Stability and Function

Myoferlin and dysferlin are members of the ferlin family of membrane proteins. Recent studies have shown that mutation or genetic disruption of myoferlin or dysferlin promotes muscular dystrophy-related phenotypes in mice, which are the result of impaired plasma membrane integrity. However, no biological functions have been ascribed to myoferlin in non-muscle tissues. Herein, using a proteomic analysis of endothelial cell (EC) caveolae/lipid raft microdomains we identified myoferlin in these domains and show that myoferlin is highly expressed in ECs and vascular tissues. The loss of myoferlin results in lack of proliferation, migration, and nitric oxide (NO) release in response to vascular endothelial growth factor (VEGF). Western blotting and surface biotinylation experiments show that loss of myoferlin reduces the expression level and autophosphorylation of VEGF receptor-2 (VEGFR-2) in native ECs. In a reconstituted cell system, transfection of myoferlin increases VEGFR-2 membrane expression and autophosphorylation in response to VEGF. In vivo, VEGFR-2 levels and VEGF-induced permeability are impaired in myoferlin-deficient mice. Mechanistically, myoferlin forms a complex with dynamin-2 and VEGFR-2, which prevents CBL-dependent VEGFR-2 polyubiquitination and proteasomal degradation. These data are the first to report novel biological activities for myoferlin and reveal the role of membrane integrity to VEGF signaling.

Epigallocatechin Gallate, a Green Tea Polyphenol, Mediates NO-dependent Vasodilation Using Signaling Pathways in Vascular Endothelium Requiring Reactive Oxygen Species and Fyn

Green tea consumption is associated with reduced cardiovascular mortality in some epidemiological studies. Epigallocatechin gallate (EGCG), a bioactive polyphenol in green tea, mimics metabolic actions of insulin to inhibit gluconeogenesis in hepatocytes. Because signaling pathways regulating metabolic and vasodilator actions of insulin are shared in common, we hypothesized that EGCG may also have vasodilator actions to stimulate production of nitric oxide (NO) from endothelial cells. Acute intra-arterial administration of EGCG to mesenteric vascular beds isolated ex vivo from WKY rats caused dose-dependent vasorelaxation. This was inhibitable by L-NAME (NO synthase inhibitor), wortmannin (phosphatidylinositol 3-kinase inhibitor), or PP2 (Src family kinase inhibitor). Treatment of bovine aortic endothelial cells (BAEC) with EGCG (50 microm) acutely stimulated production of NO (assessed with NO-specific fluorescent dye DAF-2) that was inhibitable by l-NAME, wortmannin, or PP2. Stimulation of BAEC with EGCG also resulted in dose- and time-dependent phosphorylation of eNOS that was inhibitable by wortmannin or PP2 (but not by MEK inhibitor PD98059). Specific knockdown of Fyn (but not Src) with small interfering RNA inhibited both EGCG-stimulated phosphorylation of Akt and eNOS as well as production of NO in BAEC. Treatment of BAEC with EGCG generated intracellular H(2)O(2) (assessed with H(2)O(2)-specific fluorescent dye CM-H(2)DCF-DA), whereas treatment with N-acetylcysteine inhibited EGCG-stimulated phosphorylation of Fyn, Akt, and eNOS. We conclude that EGCG has endothelial-dependent vasodilator actions mediated by intracellular signaling pathways requiring reactive oxygen species and Fyn that lead to activation of phosphatidylinositol 3-kinase, Akt, and eNOS. This mechanism may explain, in part, beneficial vascular and metabolic health effects of green tea consumption.

Requirement of Transmembrane Transport for S-Nitrosocysteine-dependent Modification of Intracellular Thiols

S-nitrosothiols have been implicated as intermediary transducers of nitric oxide bioactivity; however, the mechanisms by which these compounds affect cellular functions have not been fully established. In this study, we have examined the effect of S-nitrosothiol transport on intracellular thiol status and upon the activity of a target protein (caspase-3), in bovine aortic endothelial cells. We have previously demonstrated that the specific transport of amino acid-based S-nitrosothiols (S-nitroso-L-cysteine and S-nitrosohomocysteine) occurs via amino acid transport system L to generate high levels of intracellular protein S-nitrosothiols (Zhang, Y., and Hogg, N. (2004) Proc. Natl. Acad. Sci. U. S. A. 101, 7891-7896). In this study, we demonstrate that the transport of S-nitrosothiols is essential for these compounds to affect intracellular thiol levels and to modify intracellular protein activity. Importantly, the ability of these compounds to affect intracellular processes occurs independently of nitric oxide formation. These findings suggest that the major action of these compounds is not to liberate nitric oxide in the extracellular space but to be specifically transported into cells where they are able to modify cellular functions through nitric oxide-independent mechanisms.

Interactions of peroxynitrite with uric acid in the presence of ascorbate and thiols: Implications for uncoupling endothelial nitric oxide synthase

It has been suggested that uric acid acts as a peroxynitrite scavenger although it may also stimulate lipid peroxidation. To gain insight into how uric acid may act as an antioxidant, we used electron spin resonance to study the reaction of uric acid and plasma antioxidants with ONOO −. Peroxynitrite reacted with typical plasma concentrations of urate 16-fold faster than with ascorbate and 3-fold faster than cysteine. Xanthine but not other purine-analogs also reacted with peroxynitrite. The reaction between ONOO − and urate produced a carbon-centered free radical, which was inhibited by either ascorbate or cysteine. Moreover, scavenging of ONOO − by urate was significantly increased in the presence of ascorbate and cysteine. An important effect of ONOO − is oxidation of tetrahydrobiopterin, leading to uncoupling of nitric oxide synthase. The protection of eNOS function by urate, ascorbate and thiols in ONOO −-treated bovine aortic endothelial cells (BAECs) was, therefore, investigated by measuring superoxide and NO using the spin probe 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethyl-pyrrolidine (CMH) and the NO -spin trap Fe[DETC] 2. Peroxynitrite increased superoxide and decreased NO production by eNOS indicating eNOS uncoupling. Urate partially prevented this effect of ONOO − while treatment of BAECs with the combination of either urate with ascorbate or urate with cysteine completely prevented eNOS uncoupling caused by ONOO −. We conclude that the reducing and acidic properties of urate are important in effective scavenging of peroxynitrite and that cysteine and ascorbate markedly augment urate's antioxidant effect by reducing urate-derived radicals.

Vascular Endothelial Growth Factor (VEGF)-A165-induced Prostacyclin Synthesis Requires the Activation of VEGF Receptor-1 and -2 Heterodimer

We previously reported that vascular endothelial growth factor (VEGF)-A(165) inflammatory effect is mediated by acute platelet-activating factor synthesis from endothelial cells upon the activation of VEGF receptor-2 (VEGFR-2) and its coreceptor, neuropilin-1 (NRP-1). In addition, VEGF-A(165) promotes the release of other endothelial mediators including nitric oxide and prostacyclin (PGI(2)). However, it is unknown whether VEGF-A(165) is mediating PGI(2) synthesis through VEGF receptor-1 (VEGFR-1) and/or VEGF receptor-2 (VEGFR-2) activation and whether the coreceptor NRP-1 potentiates VEGF-A(165) activity. In this study, PGI(2) synthesis in bovine aortic endothelial cells (BAEC) was assessed by quantifying its stable metabolite (6-keto prostaglandin F(1alpha), 6-keto PGF(1alpha)) by enzyme-linked immunosorbent assay. Treatment of BAEC with VEGF analogs, VEGF-A(165) (VEGFR-1, VEGFR-2 and NRP-1 agonist) and VEGF-A(121) (VEGFR-1 and VEGFR-2 agonist) (up to 10(-9) m), increased PGI(2) synthesis by 70- and 40-fold within 15 min. Treatment with VEGFR-1 (placental growth factor and VEGF-B) or VEGFR-2 (VEGF-C) agonist did not increase PGI(2) synthesis. The combination of VEGFR-1 and VEGFR-2 agonists did not increase PGI(2) release. Pretreatment with a VEGFR-2 inhibitor abrogated PGI(2) release mediated by VEGF-A(165) and VEGF-A(121), and pretreatment of BAEC with antisense oligomers targeting VEGFR-1 or VEGFR-2 mRNA reduced PGI(2) synthesis mediated by VEGF-A(165) and VEGF-A(121) up to 79%. In summary, our data demonstrate that the activation of VEGFR-1 and VEGFR-2 heterodimer (VEGFR-1/R-2) is essential for PGI(2) synthesis mediated by VEGF-A(165) and VEGF-A(121), which cannot be reproduced by the parallel activation of VEGFR-1 and VEGFR-2 homodimers with corresponding agonists. In addition, the binding of VEGF-A(165) to NRP-1 potentiates its capacity to promote PGI(2) synthesis.

Detection of intracellular superoxide formation in endothelial cells and intact tissues using dihydroethidium and an HPLC-based assay.

Recently, it was demonstrated that superoxide oxidizes dihydroethidium to a specific fluorescent product (oxyethidium) that differs from ethidium by the presence of an additional oxygen atom in its molecular structure. We have adapted this new HPLC-based assay to quantify this product as a tool to estimate intracellular superoxide in intact tissues. Ethidium and oxyethidium were separated using a C-18 column and quantified using fluorescence detection. Initial cell-free experiments with potassium superoxide and xanthine oxidase confirmed the formation of oxyethidium from dihydroethidium. The formation of oxyethidium was inhibited by superoxide dismutase but not catalase and did not occur upon the addition of H(2)O(2), peroxynitrite, or hypochlorous acid. In bovine aortic endothelial cells (BAEC) and murine aortas, the redox cycling drug menadione increased the formation of oxyethidium from dihydroethidium ninefold (0.4 nmol/mg in control vs. 3.6 nmol/mg with 20 microM menadione), and polyethylene glycol-conjugated superoxide dismutase (PEG-SOD) significantly inhibited this effect. Treatment of BAEC with angiotensin II caused a twofold increase in oxyethidium formation, and this effect also was reduced by PEG-SOD (0.5 nmol/mg). In addition, in the aortas of mice with angiotensin II-induced hypertension and DOCA-salt hypertension, the formation of oxyethidium was increased in a manner corresponding to superoxide production estimated on the basis of cytochrome c reduction. Detection of oxyethidium using HPLC represents a new, convenient, quantitative method for the detection of superoxide in intact cells and tissues.

Sphingosine 1-phosphate effect on endothelial cell PAF synthesis: role in cellular migration.

Sphingosine 1-phosphate (S1P) and vascular endothelial growth factor (VEGF) are two inflammatory mediators capable of promoting endothelial cell (EC) migration and angiogenesis. As VEGF inflammatory effect is mediated by the synthesis of endothelial platelet-activating factor (PAF) which is also contributing to VEGF chemotactic activity, we wanted to assess if S1P can trigger PAF synthesis in EC and if S1P-induced migration is PAF-dependent. Treatment of bovine aortic EC (BAEC) with S1P (10(-10)-10(-6) M) increased dose- and time-dependently the synthesis of PAF by up to 3.3-fold above the basal level, with a maximal amount of PAF detected at 20 min post-stimulation. This biological response was attenuated by inhibiting p38 mitogen-activated protein kinase (MAPK), cytosolic or secreted phospholipase A(2) (cPLA(2), sPLA(2)) activity, suggesting that p38 MAPK activation by S1P promotes the conversion of membrane phospholipids into PAF through the combined activation of cPLA(2) and sPLA(2). Interestingly, pretreatment of BAEC with extracellular PAF receptor antagonists (BN52021, 10(-5) M and CV3988, 10(-6) M) reduced by up to 42% the cellular migration induced by S1P (10(-6) M). These data demonstrate the capacity of S1P to induce PAF synthesis, which contributes in part to S1P chemotactic activity.

Interactions of Peroxynitrite, Tetrahydrobiopterin, Ascorbic Acid, and Thiols: IMPLICATIONS FOR UNCOUPLING ENDOTHELIAL NITRIC-OXIDE SYNTHASE

Tetrahydrobiopterin (BH4) serves as a critical co-factor for the endothelial nitric-oxide synthase (eNOS). A deficiency of BH4 results in eNOS uncoupling, which is associated with increased superoxide and decreased NO* production. BH4 has been suggested to be a target for oxidation by peroxynitrite (ONOO-), and ascorbate has been shown to preserve BH4 levels and enhance endothelial NO* production; however, the mechanisms underlying these processes remain poorly defined. To gain further insight into these interactions, the reaction of ONOO- with BH4 was studied using electron spin resonance and the spin probe 1-hydroxy-3-carboxy-2,2,5-tetramethyl-pyrrolidine. ONOO- reacted with BH4 6-10 times faster than with ascorbate or thiols. The immediate product of the reaction between ONOO- and BH4 was the trihydrobiopterin radical (BH3.), which was reduced back to BH4 by ascorbate, whereas thiols were not efficient in recycling of BH4. Uncoupling of eNOS caused by peroxynitrite was investigated in cultured bovine aortic endothelial cells (BAECs) by measuring superoxide and NO* using spin probe 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethyl-pyrrolidine and the NO*-spin trap iron-diethyldithiocarbamate. Bolus ONOO-, the ONOO- donor 3-morpholinosydnonimine, and an inhibitor of BH4 synthesis (2,4-diamino-6-hydroxypyrimidine) uncoupled eNOS, increasing superoxide and decreasing NO* production. Exogenous BH4 supplementation restored endothelial NO* production. Treatment of BAECs with both BH4 and ascorbate prior to ONOO- prevented uncoupling of eNOS by ONOO-. This study demonstrates that endothelial BH4 is a crucial target for oxidation by ONOO- and that the BH4 reaction rate constant exceeds those of thiols or ascorbate. We confirmed that ONOO- uncouples eNOS by oxidation of tetrahydrobiopterin and that ascorbate does not fully protect BH4 from oxidation but recycles BH3. radical back to BH4.

Inhibitory effect of adrenomedullin on basal and tumour necrosis factor alpha-stimulated endothelin-1 synthesis in bovine aortic endothelial cells is independent of cyclic AMP

Adrenomedullin (ADM) is a potent vasodilator and reverses the vasoconstrictor action of endothelin-1 (ET-1). These studies aimed to determine the effect of ADM on ET-1 synthesis in bovine aortic endothelial cells (BAEC) and to identify the possible mechanisms involved. In this cell model, ADM increased cyclic AMP production by BAEC with threshold concentrations of 100 pM and an ec 50 of 1 nM. This effect was not blocked by co-treatment with the CGRP type 1 receptor antagonist CGRP 8–37. ADM caused a potent concentration-dependent inhibition of ET-1 release that was correlated with reduced preproET-1 mRNA levels. This reached a maximal reduction of 70% compared to basal levels after 2 and 6 hr exposure of BAEC to 1 nM ADM, with significant decreases at concentrations as low as 10 pM. However, a 100-fold discrepancy between the threshold ADM concentration for cyclic AMP production and inhibition of ET-1 release was observed. Treatment of BAEC with tumour necrosis factor alpha (TNFα; 10 ng/mL) caused a 2-fold increase over basal ET-1 release. ADM caused a more marked reduction in stimulated ET-1 synthesis with a threshold of 1 pM, and suppression of ET-1 release to basal levels at 100 nM. 8-Bromo cyclic AMP, showed no concentration-dependent inhibition of ET-1 release, yet caused a 50% reduction in TNFα-stimulated intercellular adhesion molecule-1 (ICAM-1) mRNA levels. Thus, physiological ADM concentrations inhibit ET-1 synthesis independently of cyclic AMP in BAEC at the level of preproET-1 mRNA expression. The high sensitivity of this inhibition implicates ADM as an important physiological regulator of endothelial ET-1 production.

NC1 Domain of Human Type VIII Collagen (α 1) Inhibits Bovine Aortic Endothelial Cell Proliferation and Causes Cell Apoptosis

Endostatin, a natural angiogenesis inhibitor, had been identified for years. It opened a new approach for cancer therapy. Sequence analysis reveled that endostatin is the NC1 domain (non-triple-helical domain) of collagen XVIII. In this report, the cDNA of NC1 domain of type VIII collagen (α 1) was cloned and expressed as soluble form in Escherichia coli. The recombinant protein was purified with Ni-NTA agarose column and named as vastatin. It inhibited the proliferation of bovine aortic endothelial (BAE) cell stimulated by basic fibroblast growth factor (bFGF) in a dose-dependent manner. The ED50 of vastatin was 0.6 μg/ml, while the ED50 of endostatin was 0.5 μg/ml. Treatment of BAE cell with vastatin caused G0–G1 arrest and cell apoptosis. It is interesting that sequence analysis showed that there was only about 12% amino acid sequence homology between vastatin and endostatin. The structure–function relationship of these angiogenesis molecules remains to be elucidated.

Overexpression of Cytochrome P450 CYP2J2 Protects against Hypoxia-Reoxygenation Injury in Cultured Bovine Aortic Endothelial Cells

CYP2J2 is abundant in human heart and its arachidonic acid metabolites, the epoxyeicosatrienoic acids (EETs), have potent vasodilatory, antiinflammatory and cardioprotective properties. This study was designed to examine the role of CYP2J2 in hypoxia-reoxygenation-induced injury in cultured bovine aortic endothelial cells (BAECs). Early passage BAECs were exposed to 24-h hypoxia followed by 4-h reoxygenation (HR). HR resulted in cell injury, as indicated by significant increases in lactate dehydrogenase (LDH) release and trypan blue stained cells (p < 0.01) and was associated with a decrease in CYP2J2 protein expression. Transfection of BAECs with the CYP2J2 cDNA resulted in increased CYP2J2 expression and arachidonic acid epoxygenase activity, compared with cells transfected with an irrelevant green fluorescent protein (GFP) cDNA. HR induced significant injury in GFP-transfected BAECs, as indicated by increases in LDH release and trypan blue-stained cells (p < 0.01); however, the HR-induced injury was markedly attenuated in CYP2J2-transfected cells (p < 0.01). HR increased cellular 8-iso-prostaglandin F(2alpha) (p < 0.05), and decreased eNOS expression, L-arginine uptake and conversion, and nitrite production (p < 0.01) in GFP-transfected BAECs. CYP2J2 transfection attenuated the HR-induced increase in 8-iso-prostaglandin F(2alpha) (p < 0.05) and decreased the amount of extracellular superoxide detected by cytochrome c reduction under normoxic conditions (p < 0.05) but did not significantly affect HR-induced decreases in eNOS expression, L-arginine uptake and conversion, and nitrite production. Treatment of BAECs with synthetic EETs and/or epoxide hydrolase inhibitors also showed protective effects against HR injury (p < 0.05). These observations suggest: (1) HR results in endothelial injury and decreased CYP2J2 expression; (2) transfection with the CYP2J2 cDNA protects against HR injury; and (3) the cytoprotective effects of CYP2J2 may be mediated, at least in part, by antioxidant effects.