Cultured Porcine Aortic Endothelial Cells Research Articles

High‐ but not low‐dose folic acid improves endothelial function in coronary artery disease

While folic acid (FA) reduces plasma homocysteine (Hcy), whether the simultaneous improvement in endothelial function is dependent on Hcy lowering per se is questionable. In the present study the relationship between FA dose, Hcy lowering and endothelial function in patients with coronary artery disease (CAD) was investigated. Eighty-four patients with CAD received either 400 microg FA or 5 mg placebo daily for a 6-week treatment period. A further 44 patients with CAD received either 100 mg kg(-1) day(-1) of betaine or placebo for a 6-week treatment period. Flow-mediated dilatation (FMD), a measure of endothelial function, was assessed before and after the 6-week periods. Isometric tension and Western blotting were used to investigate the effect of FA on endothelial function and endothelial nitric oxide synthase (eNOS) dimerization in isolated rabbit aortic rings and cultured porcine aortic endothelial cells (PAEC), respectively. Both 400 micro g day(-1) and 5 mg day(-1) FA significantly increased plasma folate and decreased plasma Hcy. The FMD improved significantly after 6 weeks' treatment of 5 mg day(-1) FA but did not correlate with the reduction in Hcy. There was no change in FMD in either the 400 micro g FA or placebo group. In a subgroup analysis of 11 patients in the betaine group, despite a reduced Hcy, a significant impairment in FMD was observed. In the in vitro studies FA, but not betaine, reversed methionine-induced endothelial dysfunction. Moreover, the FA promoted eNOS dimerization in cultured PAEC. These data suggest that FA dose-dependently improves endothelial function in CAD via a mechanism independently of Hcy lowering. It may involve promotion of eNOS dimerization.

The Endothelium-Derived Hyperpolarizing Factor, H2O2, Promotes Metal-Ion Efflux in Aortic Endothelial Cells: Elemental Mapping by a Hard X-ray Microprobe

Hydrogen peroxide (H(2)O(2)) is a physiologic oxidant implicated in vascular cell signaling, although little is known about the biochemical consequences of its reaction with endothelial cells. Submicrometer-resolution hard X-ray elemental mapping of cultured porcine aortic endothelial cells (PAEC) has provided data on the global changes for intracellular elemental density within PAEC and indicates an efflux of metal ions and phosphorus from the cytoplasm after H(2)O(2) treatment. The synchrotron-radiation-induced X-ray emission experiments (SRIXE) show that H(2)O(2)-treated cells are irregularly shaped and exhibit blebbing indicative of increased permeability due to the damaged membrane. The SRIXE results suggest that H(2)O(2)-induced damage is largely restricted to the cell membrane as judged by the changes to membrane and cytoplasmic components rather than the cell nucleus. The SRIXE data also provide a mechanism for cell detoxification as the metal-ion efflux resulting from the initial H(2)O(2)-mediated changes to cell membrane potentially limits intracellular metal-mediated redox processes through Fenton-like chemistry. They may also explain the increased levels of these ions in atherosclerotic plaques, regardless of whether they are involved in plaque formation. Finally, the SRIXE data support the notion that cultured endothelial cells exposed to H(2)O(2) respond with enhanced cellular metal-ion efflux into the extracellular space.

Hydrogen peroxide restrains endothelium-derived nitric oxide bioactivity—Role for iron-dependent oxidative stress

Vascular diseases are characterized by impairment of endothelial-derived nitric oxide (NO) bioactivity and increased vascular levels of hydrogen peroxide (H 2O 2). Here we examined the implications of H 2O 2 for agonist-stimulated endothelial NO bioactivity in rabbit aortic rings and cultured porcine aortic endothelial cells (PAEC). Vessels pre-treated with H 2O 2 exhibited impaired endothelial-dependent relaxation induced by acetylcholine or calcium ionophore. In contrast, H 2O 2 had no effect on endothelium-independent relaxation induced by a NO donor, indicating a defect in endothelium-derived NO. This defect was not related to eNOS catalytic activity; treatment of PAEC with H 2O 2 enhanced agonist-stimulated eNOS activity indicated by increased eNOS phosphorylation at Ser-1177 and de-phosphorylation at Thr-495 and enhanced conversion of [ 3H]-L-arginine to [ 3H]-L-citrulline that was prevented by inhibitors of Src and phosphatidylinositol-3 kinases. Despite activating eNOS, H 2O 2 impaired endothelial NO bioactivity indicated by attenuation of the increase in intracellular cGMP in PAEC stimulated with calcium ionophore or NO. The decrease in cGMP was not due to impaired guanylyl cyclase as H 2O 2 treatment increased cGMP accumulation in response to BAY 41-2272, a NO-independent activator of soluble guanylyl cyclase. At concentrations that impaired endothelial NO bioactivity H 2O 2 increased intracellular oxidative stress and size of the labile iron pool in PAEC. The increase in oxidative stress was prevented by the free radical scavenger's tempol or tiron and the iron chelator desferrioxamine and these antioxidants reversed the H 2O 2-induced impairment of NO bioactivity in PAEC. This study shows that despite promoting eNOS activity, H 2O 2 impairs endothelial NO bioactivity by promoting oxidative inactivation of synthesized NO. The study highlights another way in which oxidative stress may impair NO bioactivity during vascular disease.

Mechanical and Chemical Regulation of Endothelial Cell Polarity

See related article, pages 939–946 McCue et al1 showed by a combination of in vitro and in vivo studies that shear stress regulates planar cell polarity (PCP) of endothelial cells (ECs) and that glycogen synthase kinase 3β (GSK3β) plays a significant role in this process. The application of shear stress at 15 dyn/cm2 to cultured porcine aortic ECs caused tubulin acetylation and stabilization of microtubules (MTs), downstream polarization of microtubule-organizing centers (MTOCs), as well as MT orientation and EC elongation in the shear direction. The importance of tubulin dynamics in these shear-induced changes was demonstrated by their elimination by the MT-disrupting agent nocodazole and also the MT-stabilizing agent taxol. A novel finding is the role of GSK-3β in shear-induced MT orientation. GSK-3β is a regulatory serine/threonine kinase2 that has high activity under basal state, and its activity is inhibited after serine-9 phosphorylation (p-GSK-3β); this would lead to downstream signaling3 and MT stabilization,4 eg, through the binding of adenomatous polyposis cell protein (APC).4,5 GSK-3β can be inhibited by lithium and SB415286, which interfere with the binding of ATP to GSK-36 rather than acting directly on GSK-3 phosphorylation. The evidence that GSK-3β plays a role in mediating the shear-induced MT orientation and cell shape change is as follows. First, shear stress caused an increase in p-GSK-3β (at 1 and 4 hours, though waned at 24 hours). Second, inhibition of GSK-3β by LiCl and SB415286 blocked the shear-induced EC elongation. SB415286 was also shown to reverse the shear-induced downstream localization of MTOCs to become upstream. Third, overexpression of a constitutively active GSK-3β (GSKS9A), which cannot be phosphorylated, attenuated tubulin acetylation, blocked cell elongation, and caused MTOC distribution to be more random. Although these results provide evidence that the shear-induced PCP and cell elongation are …

Folic acid reverses endothelial dysfunction induced by inhibition of tetrahydrobiopterin biosynthesis

While folic acid has been shown to reverse endothelial dysfunction, the exact underlying mechanism remains elusive. Here, folic acid reversed both the endothelial dysfunction and increased production of superoxide following depletion of rabbit aortic ring tetrahydrobiopterin (BH 4) levels with 2,4-diamino-6-hydroxy-pyrimidine (DAHP) and N-acetyl-5-hydroxy-tryptamine (NAS). Incubation with l-nitroarginine methyl ester also attenuated the production of superoxide. DAHP and NAS reduced BH 4 concentrations in both aorta and cultured porcine aortic endothelial cells. Folic acid had no effect on BH 4 concentrations in either preparation. The superoxide anion scavenger Tiron but not folic acid reversed the endothelial dysfunction produced in aortic rings by inhibition of copper–zinc superoxide dismutase with diethyldithiocarbamic acid. Neither folic acid nor its metabolite 5-methyltetrahydrofolate prevented the in vitro oxidation of BH 4. This study demonstrates that folic acid reverses the endothelial dysfunction induced by BH 4 depletion independently of either the regeneration or stabilization of BH 4 or an antioxidant effect.

Carbon Monoxide From Heme Oxygenase-2 Is a Tonic Regulator Against NO-Dependent Vasodilatation in the Adult Rat Cerebral Microcirculation

Although the brain generates NO and carbon monoxide (CO), it is unknown how these gases and their enzyme systems interact with each other to regulate cerebrovascular function. We examined whether CO produced by heme oxygenase (HO) modulates generation and action of constitutive NO in the rat pial microcirculation. Immunohistochemical analyses indicated that HO-2 occurred in neurons and arachnoid trabecular cells, where NO synthase 1 (NOS1) was detectable, and also in vascular endothelium-expressing NOS3, suggesting colocalization of CO- and NO-generating sites. Intravital microscopy using a closed cranial window preparation revealed that blockade of the HO activity by zinc protoporphyrin IX significantly dilates arterioles. This vasodilatation depended on local NOS activities and was abolished by CO supplementation, suggesting that the gas derived from HO-2 tonically regulates NO-mediated vasodilatory response. Bioimaging of NO by laser-confocal microfluorography of diaminofluorescein indicated detectable amounts of NO at the microvascular wall, the subdural mesothelial cells, and arachnoid trabecular cells, which express NOS in and around the pial microvasculature. On CO inhibition by the HO inhibitor, regional NO formation was augmented in these cells. Such a pattern of accelerated NO formation depended on NOS activities and was again attenuated by the local CO supplementation. Studies using cultured porcine aortic endothelial cells suggested that the inhibitory action of CO on NOS could result from the photo-reversible gas binding to the prosthetic heme. Collectively, CO derived from HO-2 appears to serve as a tonic vasoregulator antagonizing NO-mediated vasodilatation in the rat cerebral microcirculation.

Xanthoangelol D Isolated from the Roots of Angelica keiskei Inhibits Endothelin-1 Production through the Suppression of Nuclear Factor-.KAPPA.B

Transcription factor nuclear factor-kappa B (NF-kappaB) has been demonstrated to be important in regulating various gene expressions such as cytokines, adhesion molecules, and endothelin-1 (ET-1) in vascular endothelial cells. In the present study, we show the effects of xanthoangelol, xanthoangelol D, E, and F, which isolated from the root of Angelica keiskei KOIDZUMI (Umbelliferae), on NF-kappaB activation and ET-1 gene expression in cultured porcine aortic endothelial cells (PAECs). Treatments of xanthoangelol D but not xanthoangelol, xanthoangelol E and F markedly suppressed both of basal and tumor necrosis factor-alpha (TNF-alpha)-induced NF-kappaB activation in PAECs. To clarify the mechanism of xanthoangelol D-induced suppression on NF-kappaB activation, we evaluated the effects of xanthoangelol D on phosphorylation and degradation of IkappaBalpha, an inhibitory protein bound to NF-kappaB, and obtained evidence that xanthoangelol D selectively suppresses the phosphorylation of IkappaBalpha rather than the degradation of phosphorylated IkappaBalpha. In addition, xanthoangelol D significantly attenuated basal and TNF-alpha-induced prepro ET-1 mRNA expression in PAECs. These results suggest that xanthoangelol D may be useful for the treatment of various vascular diseases involved NF-kappaB activation.

A study of HLA-G1 protection of porcine endothelial cells against human NK cell cytotoxicity

Human natural killer (NK) cells, which can directly lyse porcine endothelial cells, play an important role in xenotransplantation. HLA-G is a nonclassical major histocompatibility complex (MHC) class I molecules that has been implicated in protecting susceptible target cells from lysis by NK cells. The objective was to study the effect of protecting porcine endothelial cells transfected with HLA-G1 from human NK cell lysis. Methods The recombinant expression vector pcDNA3-HLA-G1 was transfected into primary cultured porcine aortic endothelial cells (PAECs) by lipofection. Surface expression of HLA-G1 in transected PAECs was confirmed by an immunofluoresence technique. Peripheral blood mononuclear cells (PBMC) and NK cell line (NK92) were used as NK effects cells with pcDNA3-HLA-G1-transfected PAECs as targets in a MTT method using pcDNA3 transfection as a negative control. Results Expression of HLA-G1 on PAECs conferred significant protection against NK-mediated lysis. The rate of NK92 cytotoxicity was reduced to 41.5% ± 14.0% from 75.3% ± 10.5% in the control group ( P < .01). Similarly the rate of the PBMC cytotoxicity among different donors ( n = 7) was reduced to 45.4% ± 12.1% in contrast to 74.6% ± 11.2% in the control group ( P < .05). Conclusions HLA-G1 molecules can directly protect xenogeneic PAECs against attack by human NK cells. These results indicate that the expression of HLA-G1 on the porcine cell surface may provide a new approach to overcome NK-mediated immunity to xenografts.

Interference of the polyphenol epicatechin with the biological chemistry of nitric oxide- and peroxynitrite-mediated reactions

The formation of reactive nitrogen species in mammalians has both beneficial and undesirable effects. Nitric oxide (NO) production in endothelial cells leads to vascular smooth muscle relaxation, but if reactive nitrogen species are generated in high amounts by cells under inflammatory conditions they are toxic. Flavonoids like (−)-epicatechin show an inverse association of their intake with diseases thought to be associated with overproduction of reactive nitrogen species. We found that the formation of cyclic GMP in cultured porcine aortic endothelial cells was not affected by up to 1 mM (−)-epicatechin. Half maximal inhibition of interferon-γ/lipopolysaccharide induced nitrite accumulation in murine macrophages required about 0.5 mM of the flavonoid. In contrast, nitration of free tyrosine triggered by 0.1 and 1 mM authentic peroxynitrite was inhibited by (−)-epicatechin with ic 50 values of 6.6 and 28.0 μM, respectively. The presence of 15 mM sodium bicarbonate had no significant effect. Nitration of protein-bound tyrosine in phorbol 12-myristate 13-acetate treated HL-60 cells in the presence of nitrite was inhibited by (−)-epicatechin at a similar concentration range ( ic 50=10 –100 μM). Myeloperoxidase activity of phorbol 12-myristate 13-acetate stimulated HL-60 cells was inhibited by (−)-epicatechin with an ic 50 value of 77.4 μM. Epicatechin inhibited dihydrorhodamine oxidation by 50 μM authentic peroxynitrite and 1 mM 3-morpholino-sydnonimine with ic 50 values of 11.8 and 0.63 μM, respectively. Our data suggest that at up to 0.1 mM (−)-epicatechin preferentially inhibits NO-related nitration and oxidation reactions without affecting NO synthesis and cyclic GMP signaling.

Involvement of Ca2+/calmodulin-dependent protein kinase II in endothelial NO production and endothelium-dependent relaxation.

Nitric oxide (NO) is synthesized from l-arginine by the Ca(2+)/calmodulin-sensitive endothelial NO synthase (NOS) isoform (eNOS). The present study assesses the role of Ca(2+)/calmodulin-dependent protein kinase II (CaMK II) in endothelium-dependent relaxation and NO synthesis. The effects of three CaMK II inhibitors were investigated in endothelium-intact aortic rings of normotensive rats. NO synthesis was assessed by a NO sensor and chemiluminescence in culture medium of cultured porcine aortic endothelial cells stimulated with the Ca(2+) ionophore A23187 and thapsigargin. Rat aortic endothelial NOS activity was measured by the conversion of l-[(3)H]arginine to l-[(3)H]citrulline. Three CaMK II inhibitors, polypeptide 281-302, KN-93, and lavendustin C, attenuated the endothelium-dependent relaxation of endothelium-intact rat aortic rings in response to acetylcholine, A23187, and thapsigargin. None of the CaMK II inhibitors affected the relaxation induced by NO donors. In a porcine aortic endothelial cell line, KN-93 decreased NO synthesis and caused a rightward shift of the concentration-response curves to A23187 and thapsigargin. In rat aortic endothelial cells, KN-93 significantly decreased bradykinin-induced eNOS activity. These results suggest that CaMK II was involved in NO synthesis as a result of Ca(2+)-dependent activation of eNOS.

Effects of cytochrome P450 inhibitors on agonist-induced Ca2+ responses and production of NO and PGI2 in vascular endothelial cells.

Production of endothelium-dependent vascular relaxing factors, such as nitric oxide (NO) and prostaglandin I2 (PGI2), and endothelium-derived hyperpolarizing factor (EDHF), is regulated in part by changes in intracellular Ca2+ concentration ([Ca2+]i) in vascular endothelial cells (ECs). Cytochrome P450 (CYP), shown to mediate endothelium-dependent hyperpolarization via epoxyeicosatrienoic acids, is one of the candidates for EDHF. In this study we tested the hypotheses that CYP might be involved in EC Ca2+ signaling and that CYP activity might be linked with production of vasodilating factors other than EDHF. To this end, structurally different CYP inhibitors including SKF 525A, econazole and miconazole were tested on primary cultured porcine aortic endothelial cells. Intracellular Ca2+ concentration was measured using the fluorescent Ca2+ indicator fura-2/AM. Bradykinin (BK, 10 nM) and thapsigargin (TG 1 microM) provoked large biphasic increases in [Ca2+], which consist of Ca2+ release from intracellular stores and transplasmalemmal Ca2+ entry. SKF 525A dose-dependently (30-100 microM) inhibited BK- and TG-stimulated Ca2+ entry, but not intracellular Ca2+ store release. Econazole (10 microM) and miconazole (10 microM) had the same effect as SKF 525A on the Ca2+ entry. SKF 525A also dose-dependently inhibited BK-stimulated production of NO and PGI2, assessed by measuring cGMP and 6-keto-PGF(1alpha) concentration. These data suggest that, in addition to its regulation of EDHF production, CYP also contributes to the regulation of other endothelium-dependent vasorelaxing factors by modifying EC Ca2+ signaling.

Involvement of Na(+)/Ca(2+) exchanger in endothelial NO production and endothelium-dependent relaxation.

Endothelial nitric oxide (NO) synthase (eNOS) is controlled by Ca(2+)/calmodulin and caveolin-1 in caveolae. It has been recently suggested that Na(+)/Ca(2+) exchanger (NCX), also expressed in endothelial caveolae, is involved in eNOS activation. To investigate the role played by NCX in NO synthesis, we assessed the effects of Na(+) loading (induced by monensin) on rat aortic rings and cultured porcine aortic endothelial cells. Effect of monensin was evaluated by endothelium-dependent relaxation of rat aortic rings in response to acetylcholine and by real-time measurement of NO release from cultured endothelial cells stimulated by A-23187 and bradykinin. Na(+) loading shifted the acetylcholine concentration-response curve to the left. These effects were prevented by pretreatment with the NCX inhibitors benzamil and KB-R7943. Monensin potentiated Ca(2+)-dependent NO release in cultured cells, whereas benzamil and KB-R7943 totally blocked Na(+) loading-induced NO release. These findings confirm the key role of NCX in reverse mode on Ca(2+)-dependent NO production and endothelium-dependent relaxation.

Intracellular Ca2+ homeostatic regulation and 4-hydroxynonenal-induced aortic endothelial dysfunction.

The aldehydic lipid peroxidation product 4-hydroxynonenal (HNE) is known to compromise erythrocyte passive Ca2+ permeability and to irreversibly inhibit the plasma membrane (Ca2+ + Mg2+)-ATPase and Ca2+-transport. To measure the effects of HNE on passive and active Ca2+ transport in endothelial cells, we first characterized 45Ca2+ uptake and efflux in cultured porcine aortic endothelial cells (PAEC). PAEC exchanged 45Ca2+ to a cumulative near-isotopic equilibrium of about 4.5 pmole 45Ca2+/10(6) cells in 120 min at 37 degrees C. This Ca2+ pool was diminished by thapsigargin, cyclopiazonic acid, oligomycin B, and sodium azide. In contrast, ouabain enhanced Ca2+ uptake capacity from 5.17 to 5.77 pmole/10(6) cells. Accumulated 45Ca2+ was extruded at rate of 8.7 fmole 45Ca2+/10(6) cells/min or shunted rapidly by the ionophore A23187. HNE increased total 45Ca2+ accumulation in a time- and concentration-dependent manner by as much as 562% with an EC50 of 64.0 wM. Concomitant morphological analysis of PAEC revealed vacuolization, nuclear swelling, cell shrinking, and cell detachment. Initial structural changes, such as vacuolization, began well before any changes in Ca2+ accumulation were observed. These functional and morphological changes indicate that HNE significantly increases intracellular Ca2+ accumulation in vascular endothelium, which may explain the cytotoxic effects associated with HNE exposure and provide further evidence that atherogenic effects of HNE may, in part, be caused by disturbances in Ca2+ homeostasis.

Evaluation of endothelin-converting enzyme inhibitors using cultured cells.

AbstractThe concept that endothelin (ET-1) biosynthesis is dependent on specific hydrolysis of the intermediate big endothelin-1 (big ET-1) by an endopeptidase referred to as endothelin-converting enzyme (ECE) originates from the initial description of this peptide by Yanagisawa and colleagues (1). The search for the putative endothelin-converting enzyme soon became the focus of intense research (2). The underlying goal was the drive to identify the target for development of specific ECE inhibitors that could be used therapeutically for treating ET-1 dependent pathologies. These investigations were conducted both on the endogenous production of ET-1 by endothelial cells, and by using cell or tissue homogenates with exogenous big ET-1 as the substrate (2,3). The first clear evidence of a specific physiologically relevant ECE came from studies of the effect of phosphoramidon (N-α-L-rhamnopyranosyloxyhydroxyphosphinyl- L-Leu-L-Trp) on cultured porcine aortic endothelial cells (4). This was an important breakthrough as it showed that high concentrations of phosphoramidon suppressed ET-1 levels and resulted in increased levels of big ET-1 (4). This inverse relationship between ET-1 and big ET-1 production is an important characteristic of biosynthetic processes as the accumulation of the enzyme’s substrate demonstrates the specific inhibition of a key enzymatic step, in this case the physiological ECE. Subsequently other studies have confirmed the effect of phosphoramidon on ET-1 synthesis in a variety of human cell types by using high performance liquid chromatography (HPLC) (5) or “double recognition site” sandwich immunoassays (6).KeywordsHigh Performance Liquid ChromatographyAffinity PurificationHigh Performance Liquid Chromatography SystemCoating BufferSandwich ImmunoassayThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Effects of SK&F 96365 and mefenamic acid on Ca2+ influx in stimulated endothelial cells and on endothelium-derived hyperpolarizing factor-mediated arterial hyperpolarization and relaxation.

This study was undertaken to assess how Ca2+ influx into endothelial cells via Ca2+-permeable nonselective cation channels (NSCCs) is important in vascular responses mediated by endothelium-derived hyperpolarizing factor (EDHF). In cultured porcine aortic endothelial cells, the sustained increases in the intracellular Ca2+ concentration ([Ca2+]i) elicited by bradykinin and cyclopiazonic acid, which were strongly dependent on the presence of extracellular Ca2+, were suppressed by the NSCC blockers, SK&F 96365 and mefenamic acid. In porcine coronary artery with intact endothelium, bradykinin elicited a rapid fall in the membrane potential, followed by sustained hyperpolarization with a slow decay. In the presence of SK&F 96365 or mefenamic acid, the peak amplitude was severely reduced and the decay phase of hyperpolarization to bradykinin was greatly accelerated, which was apparently similar to the response obtained in Ca2+-free medium. Cyclopiazonic acid caused sustained hyperpolarization in an extracellular Ca2+-dependent manner, an effect which was markedly diminished by SK&F 96365 and mefenamic acid. In rings of coronary artery precontracted with U46619, bradykinin and cyclopiazonic acid produced endothelium-dependent relaxations even in the presence of N(G)-nitro-L-arginine and indomethacin. SK&F 96365 and mefenamic acid significantly attenuated the relaxant responses. These results indicate that the increase in [Ca2+]i of endothelial cells due to Ca2+ entry via NSCCs plays a crucial role in the maintenance of the EDHF-mediated vascular responses.

Hypoxia-induced upregulation of eNOS gene expression is redox-sensitive: a comparison between hypoxia and inhibitors of cell metabolism.

Several papers report a hypoxia-induced upregulation of the endothelial nitric oxide synthase (eNOS) mRNA expression. Since there is no known hypoxia-sensitive element binding site in the eNOS promoter, we reasoned that the effect of hypoxia could be simulated by a metabolically elicited alteration of the redox state. Therefore, cultured porcine aortic endothelial cells (PAEC) were exposed to hypoxia (1-10% O(2)) or inhibitors of cellular energy metabolism including rotenone, 2, 4 dinitrophenol (DNP) and 2-deoxyglucose for 6 to 24 h. Additionally, cells were treated with lactate and nicotinic acid to alter the cellular NAD(P)H/NAD(P) ratio without changes of energy supply. The cellular NAD(P)H/NAD(P) ratio was used as an index of the cellular redox state and determined using the MTT-assay. Hypoxia increased eNOS mRNA transcription and MTT-reduction in a manner inversely proportional to pO(2). Exposure to rotenone, DNP, and lactate increased the NAD(P)H/NAD(P) ratio, MTT-reduction, and eNOS mRNA also in parallel. In contrast, 2-deoxyglucose and nicotinic acid attenuated both MTT-reduction and eNOS mRNA expression. In order to study a potential role of the redox regulated transcription factor complex AP-1 in hypoxia-induced eNOS mRNA transcription, c-jun expression was determined and decoy experiments were performed. c-jun expression paralleled changes of eNOS mRNA expression and MTT-reduction. Furthermore, in the presence of oligodeoxynucleotides corresponding to the AP-1 binding sites of the eNOS promoter, the hypoxia and chemically induced eNOS mRNA expression was completely abolished. We propose that hypoxia, by altering cellular metabolism, leads to an increase in the cellular NAD(P)H/NAD(P) ratio which favors enhanced eNOS expression by redox-sensitive AP-1 mediated transcriptional control.

Adherence of human monocytes and NK cells to human TNF-alpha-stimulated porcine endothelial cells.

Discordant xenograft models undergoing delayed rejection response are characterized by xenograft infiltration with host monocytes and NK cells, associated with the release of large quantities of pro-inflammatory cytokines, such as TNF-alpha. In the present study, human monocytes (PBMo)/NK cells (PBNK) isolated from peripheral blood and cultured porcine aortic endothelial cells (PAEC) treated with recombinant human TNF-alpha (rhTNF-alpha) were used to investigate their adhesive interactions and mAbs against porcine E-selectin, human CD11a and CD49d were used to test their relative contributions to such intercellular adhesions. The PBMo exhibited significantly greater adherence to resting (unstimulated) PAEC than PBNK. The rhTNF-alpha upregulated E-selectin and vascular cell adhesion molecule-1 (VCAM-1) expression on PAEC and augmented the adhesiveness of PAEC for PBMo and PBNK in a time- and dose-dependent manner. In mAb blocking assays, anti-E-selectin, anti-CD11a and anti-CD49d mAbs did not inhibit PBMo adherence to rhTNF-alpha-stimulated PAEC when used singly, but resulted in a maximal inhibitory effect when used in combination. Regarding PBNK, anti-E-selectin mAb had no marked influence on PBNK adherence. The combined use of anti-CD11a and anti-CD49d mAbs produced additive reduction in the PBNK binding to rhTNF-alpha-stimulated PAEC, even to far below baseline (unstimulated) levels. Therefore, it is concluded that human TNF-alpha promotes the adhesiveness of PAEC for human monocytes and NK cells and that the mechanism underlying the increased adherence differs for PBMo and PBNK.

Inhibitory effect of insulin on bradykinin-induced venodilation.

Excessive insulin is one of the risk factors of hypertension and arteriosclerosis despite its vasodilative properties shown in recent studies. Although many vasoactive substances contribute and interact with each other in the development of hypertension, the interactions between insulin and other vasoactive substances have yet to be elucidated. To assess the effect of insulin on the action of bradykinin. The vasodilating effect of bradykinin was evaluated, with or without coadministration of insulin, in human dorsal hand veins of healthy volunteers. In cultured porcine aortic endothelial cells, the bradykinin-induced increase of intracellular calcium was also investigated before and after insulin administration. Insulin significantly attenuated bradykinin-induced increase in intracellular calcium and venodilation in cultured endothelial cells and human dorsal hand veins, respectively. These findings suggest that insulin attenuates the bradykinin-induced calcium elevation in endothelial cells and may decrease the production of vasodilative substances from endothelial cells, resulting in the reduction of vasodilation. This effect may contribute to the development of hypertension in patients with hyperinsulinaemia.

Pharmacological and immunohistochemical characterization of calmodulin-stimulated (Ca(2+)+Mg(2+))-ATPase in cultured porcine aortic endothelial cells.

Plasma membrane (Ca(2+)+Mg(2+))-ATPase and Ca(2+) transport activities, best characterized in human erythrocytes, are stimulated by calmodulin and thought to play a crucial role in the termination of cellular Ca(2+) signaling in all cells. In plasma membranes isolated from cultured porcine aortic endothelial cells, the (Ca(2+)+Mg(2+))-ATPase was not readily measured. This is in part because of an overabundance of nonspecific Ca(2+)- and/or Mg(2+)-activated ecto-5'-nucleotide phosphohydrolases. Moreover, addition of exogenous calmodulin (10(-9) to 10(-6) mol/L) produced no measurable stimulation of ATPase activities, suggesting a permanently activated state or, alternatively, a complete lack thereof. To establish and verify the presence of a calmodulin-regulated (Ca(2+)+Mg(2+))-ATPase activity in these endothelial cells, immunohistochemical localization using a monoclonal mouse anti-(Ca(2+)+Mg(2+))-ATPase antibody (clone 5F10) was applied to intact pig aorta endothelium, cultured endothelial monolayers, and isolated endothelial plasma membrane fractions. This approach clearly demonstrated Ca(2+) pump immunoreactivity in each of these preparations. To confirm functional calmodulin stimulation of the (Ca(2+)+Mg(2+))-ATPase, 10(-5) mol/L calmidazolium (R24571) was added to the isolated plasma membrane preparation, which lowered the (Ca(2+)+Mg(2+))-ATPase activity from 143.0 to 78.15 nmol P(i)/mg protein x min(-1). This calmidazolium-reduced activity could then be stimulated 113.1+/-0.8% in a concentration-dependent manner by the addition of exogenous calmodulin (10(-7) to 2 x 10(-6) mol/L) with an EC(50) of 3.45+/-0.04 x 10(-7) mol/L (n=4). This represents a competitive lowering of the apparent calmodulin affinity by approximately 100 compared with other unopposed calmodulin-stimulated processes. Together, these findings support evidence for the presence of a calmodulin-stimulated plasma membrane (Ca(2+)+Mg(2+))-ATPase activity in cultured porcine aortic endothelial cells.

Homocysteine-induced inhibition of endothelium-dependent relaxation in rabbit aorta: role for superoxide anions.

Hyperhomocysteinemia is associated with endothelial dysfunction, although its mechanism is unknown. Isometric tension recordings and lucigenin chemiluminescence were used to assess the effects of homocysteine exposure on endothelium-dependent and -independent relaxation in isolated rabbit aortic rings and superoxide anion (O(2)(-)) production by cultured porcine aortic endothelial cells, respectively. Homocysteine (0.1 to 10 mmol/L) produced a significant (P<0.001) concentration- and time-dependent inhibition of endothelium-dependent relaxation in response to both acetylcholine and the calcium ionophore A23187. Only the intracellular O(2)(-) scavenger 4,5-dihydroxy-1,3-benzene disulfonic acid (Tiron, 10 mmol/L) significantly (P<0.001) inhibited the effect of homocysteine on acetylcholine- and A23187-induced relaxation. Incubation of porcine aortic endothelial cells with homocysteine (0.03 to 1 mmol/L for up to 72 hours) caused a significant (P<0.001) time-dependent increase in the O(2)(-) released by these cells on the addition of Triton X-100 (1% [vol/vol]), with levels returning to values comparable to those of control cells at the 72-hour time point. These changes in O(2)(-) levels were associated with a time-dependent increase in endothelial cell superoxide dismutase activity, becoming significant (P<0.001) after 72 hours. Furthermore, the homocysteine-induced increase in endothelial cell O(2)(-) levels was completely inhibited (P<0.001) by the concomitant incubation with either Tiron (10 mmol/L), vitamin C (10 micromol/L), or vitamin E (10 micromol/L). These data suggest that the inhibitory effect of homocysteine on endothelium-dependent relaxation is due to an increase in the endothelial cell intracellular levels of O(2)(-) and provide a possible mechanism for the endothelial dysfunction associated with hyperhomocysteinemia.