Homocysteine and atherothrombosis.
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Amino Acid Metabolism, Inborn Errors↗
Biomedical subjects
G N Welch
Publications and source records attributed to G N Welch.
Stimulation of endothelial nitric oxide production by homocyst(e)ine.
Hyperhomocyst(e)inemia, characterized by accelerated atherosclerosis, is believed to induce endothelial cell injury and promote atherothrombosis by supporting the generation of hydrogen peroxide. Earlier observations in our laboratory demonstrated that in vitro nitrosation of homocyst(e)ine (HCY) prevents the generation of hydrogen peroxide. We, therefore, hypothesized that stimulating the production of nitric oxide (NO) by endothelial cells would detoxify HCY by forming the corresponding S-nitrosothiol, S-nitroso-homocysteine. In an attempt to prove this hypothesis, media containing 1 mM L-arginine, 1 microM bradykinin, a known NO agonist, and one of the biologically relevant thiols (HCY, cysteine, or glutathione) at concentrations of 0, 0.05, 0.5 and 5.0 mM were incubated with bovine aortic endothelial cells (BAEC) for 0.5, 1 and 4 h. S-nitrosothiol (RSNO) concentrations were measured by photolysis-chemiluminescence. Nitric oxide synthase (eNOS or isoform 3) activity and Nos 3 steady-state mRNA levels were determined by the conversion of [3H]L-arginine to [3H]L-citrulline and Northern analysis, respectively. Results demonstrate that increasing concentrations of HCY, and not cysteine or glutathione, in the presence of bradykinin at 0.5, 1, and 4 h led to significant (P < 0.05 by ANOVA) time- and dose-dependent increases in RSNO produced by BAEC. Cells exposed to 1 microM calcium ionophore A23187 in the presence of 5.0 mM HCY also produced a time-dependent increase in RSNO compared to control (P < 0.05 by ANOVA). In an attempt to determine if de novo synthesis was occurring, BAEC were treated with bradykinin following a 4 h pretreatment with HCY. Pretreatment with HCY followed by stimulation also led to a time- and dose-dependent increase in RSNO production (P < 0.05 by ANOVA). Using high performance liquid chromatography with electrochemical detection, S-nitroso-homocysteine was identified following treatment of BAEC with HCY and bradykinin. The increase in RSNO production in the presence of bradykinin and HCY at 4 h occurred concomitantly with a 78% increase in eNOS activity and a 58% increase in steady-state Nos 3 mRNA, with no change in Nos 3 mRNA half-life, compared to control. A partial explanation for HCY's unique ability to support an increase in NO production was demonstrated by showing that the t1/2 of HCY in media was greater than that of cysteine or glutathione. These data show that, in the presence of an NO agonist, HCY increases RSNO production in a time- and dose-dependent fashion that is reflected by an increase in eNOS activity and Nos 3 transcription. These results suggest that stimulation of endogenous NO, or provision of an exogenous NO donor, may ameliorate endothelial cell injury and thereby decrease the atherothrombotic risk of hyperhomocyst(e)inemic states.
Animals↗
Homocyst(e)ine decreases bioavailable nitric oxide by a mechanism involving glutathione peroxidase.
Hyperhomocyst(e)inemia is believed to injure endothelial cells in vivo through a number of mechanisms, including the generation of hydrogen peroxide (H2O2). Earlier in vitro studies demonstrated that homocyst(e)ine (Hcy) decreases the biological activity of endothelium-derived relaxing factor and that this decrease can be reversed by preventing the generation of hydrogen peroxide. Here we show that Hcy treatment of bovine aortic endothelial cells leads to a dose-dependent decrease in NOx (p = 0.001 by one-way analysis of variance) independent of endothelial nitric-oxide synthase activity or protein levels and nos3 transcription, suggesting that Hcy affects the bioavailability of NO, not its production. We hypothesized that, in addition to increasing the generation of H2O2, Hcy decreases the cell's ability to detoxify H2O2 by impairing intracellular antioxidant enzymes, specifically the intracellular isoform of glutathione peroxidase (GPx). To test this hypothesis, confluent bovine aortic endothelial cells were treated with a range of concentrations of Hcy, and intracellular GPx activity was determined. Compared with control cells, cells treated with Hcy showed a significant reduction in GPx activity (up to 81% at 250 microM Hcy). In parallel with the decrease in GPx activity, steady-state GPx mRNA levels were also significantly decreased compared with control levels after exposure to Hcy, which appeared not to be a consequence of message destabilization. These data suggest a novel mechanism by which Hcy, in addition to increasing the generation of hydrogen peroxide, may selectively impair the endothelial cell's ability to detoxify H2O2, thus rendering NO more susceptible to oxidative inactivation.
Animals↗
Homocysteine, oxidative stress, and vascular disease.
First recognized in patients with rare inborn errors of metabolism, the association of elevated plasma homocysteine concentrations with atherosclerosis and thrombosis now seems relevant to the general population as well. The mechanism of injury appears to involve oxidative damage to endothelial cells. Vitamin supplementation can normalize homocysteine levels and may lower the incidence of atherothrombotic vascular disease.
Age Factors↗
Hyperhomocyst(e)inemia and atherothrombosis.
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Animals↗
High-dose heparin decreases nitric oxide production by cultured bovine endothelial cells.
BACKGROUND: Abrupt cessation of heparin therapy can lead to a recrudescence of thrombosis and acute ischemia. Endothelial NO is an important endogenous inhibitor of platelet-mediated thrombosis, yet biochemical studies examining the effect of heparin on NO production by the endothelium have heretofore been lacking. METHODS AND RESULTS: In an attempt to address the effect of heparin on endothelial cell production of NO, confluent bovine aortic endothelial cells (BAECs) on microcarrier beads were incubated in the presence or absence of heparin. Results indicate that BAECs incubated with heparin were less able to inhibit platelet aggregation than control cells (P<.005 by ANOVA) and that this effect correlated with a decrease in NO production (36% decrease for heparin compared with control, P<.05). Dextran sulfate evoked the same response (67% decrease, P<.0001 compared with control), suggesting that the decrease in NO after heparin treatment is secondary to its negative charge rather than to a specific polysaccharide sequence. The decrease in NO production by heparin was accompanied by a 72% decrease in steady-state Nos 3 mRNA as well as a 49% decrease in immunodetectable endothelial NO synthase (eNOS) protein. CONCLUSIONS: These data show that high-dose heparin at concentrations achieved in some acute cardiovascular settings increases in vitro platelet aggregation in media conditioned by endothelial cells by decreasing endothelial NO production through a mechanism that involves a decrease in steady-state Nos 3 mRNA and eNOS protein. These observations suggest a possible mechanism by which to explain in part the prothrombotic effects of heparin.
Animals↗
S-nitrosothiol detection.
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Capillary Action↗
Homocysteine, EDRF, and endothelial function.
Nitric oxide, a heterodiatomic molecule, serves as an endothelium-dependent vasodilator and an antithrombotic agent in the vasculature. Because of its reactivity under physiological conditions, accumulating data suggest that nitric oxide forms adducts with several classes of biologic compounds, one of which is the sulfhydryl functionality, to form thionitrites or S-nitrosothiols. In this overview, we will examine the effects of S-nitrosothiols in the vascular system as modulators of tone and inhibitors of platelet activation to understand better what role these compounds may play in physiological and pathological states. With this background, we will review the role nitric oxide (EDRF) plays in helping to ameliorate vascular injury by homocysteine.
Animals↗
Effect of heparin on fibrinolytic activity and platelet function in vivo.
Heparin, a polyanionic glycosaminoglycan, is used routinely before the induction of cardiopulmonary bypass. Earlier observations in our laboratory suggested that the postoperative bleeding that occurs, despite neutralization of heparin with protamine, is secondary to hypothermia and dilutional anemia during bypass. An additional, potential mechanism for excessive bleeding following cardiopulmonary bypass is that heparin activates the fibrinolytic system, which may, in turn, adversely affect hemostasis. To understand better the effects of heparin administration on the fibrinolytic system in vivo, we simulated the anticoagulant regimen of cardiopulmonary bypass by administering increasing doses of intravenous heparin to five adult baboons over 60 min. We measured fibrinolytic parameters serially following heparinization and demonstrated that heparin induces activation of the fibrinolytic system. We showed that the fibrinolytic system was activated in vivo as evidenced by an increase in plasmin activity and immunoreactive plasmin light chain, as well as an increase in immunoreactive fibrinogen fragment E in vitro. These results demonstrate that the fibrinolytic system is activated in vivo by the administration of heparin during cardiopulmonary bypass. These data suggest that, despite administration of a neutralizing agent such as protamine, heparin may contribute to postoperative bleeding complications following cardiopulmonary bypass surgery owing principally to its longer lived effects on the fibrinolytic system.
Animals↗
The vascular biology of S-nitrosothiols, nitrosated derivatives of thiols.
Nitric oxide (NO) is a simple heterodiatomic molecule with a broad range of biologic actions. In the cardiovascular system NO serves as an endothelium-dependent vasodilator, an antithrombotic agent, an antiproliferative molecule, and a regulator of cardiac contractility. Owing to its reactivity under physiologic conditions, accumulating data suggest that NO forms derivatives with several classes of biologic compounds. One group of biochemical functionalities that serves this role is that of thiols, which can form thionitrites or S-nitrosothiols with NO. In this paper we will examine the effects of the biologically known and the chemically produced S-nitrosothiols on the cardiovascular system in order to understand better what role these compounds may play in physiologic and pathologic states.
Animals↗
S-nitrosothiols: chemistry, biochemistry, and biological actions.
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Animals↗
Glutathione peroxidase potentiates the inhibition of platelet function by S-nitrosothiols.
GSH peroxidase (Px) catalyzes the reduction of lipid hydroperoxides (LOOH), known metabolic products of platelets and vascular cells. Because interactions between these cells are modulated by nitric oxide (NO) and LOOH inactivate NO, we investigated the effect of GSH-Px on the inhibition of platelet function by the naturally occurring S-nitrosothiol, S-nitroso-glutathione (SNO-Glu). Concentrations of SNO-Glu that alone did not inhibit platelet function (subthreshold inhibitory concentrations) were added to platelet-rich plasma together with GSH-Px (0.2-20 U/ml); this led to a dose-dependent inhibition of platelet aggregation with an IC50 of 0.6 U/ml GSH-Px. In the presence of subthreshold inhibitory concentrations of SNO-Glu, the LOOH, 5-hydroperoxy-6,8,11,14-eicosatetraenoic acid, increased platelet aggregation, an effect reversed by GSH-Px. Glutathione and SNO-Glu were equally effective as cosubstrates for GSH-Px. Incubation of SNO-Glu with GSH-Px for 1 min led to a 48.5% decrease in the concentration of SNO-Glu. Incubation of SNO-Glu with serum albumin led to the formation of S-nitroso-albumin, an effect enhanced by GSH-Px. These observations suggest that GSH-Px has two functions: reduction of LOOH, thereby preventing inactivation of NO, and metabolism of SNO-Glu, thereby liberating NO and/or supporting further transnitrosation reactions.
Blood Platelets↗
Inhibition of neointimal proliferation in rabbits after vascular injury by a single treatment with a protein adduct of nitric oxide.
Endothelium-derived relaxing factor is important for vascular homeostasis and possesses qualities that may modulate vascular injury, including vasodilation, platelet inhibition, and inhibition of smooth muscle proliferation. S-nitrososerum albumin is a naturally occurring adduct of nitric oxide (NO) with a prolonged biologic half-life and is a potent vasodilator and platelet inhibitor. Given the avidity of serum albumin for subendothelial matrix and the antiproliferative effects of NO, we investigated the effects of locally delivered S-nitroso-bovine serum albumin (S-NO-BSA) and a polythiolated form of bovine serum albumin (pS-BSA) modified to carry several S-nitrosothiol groups (pS-NO-BSA) on neointimal responses in an animal model of vascular injury. Locally delivered S-NO-BSA bound preferentially to denuded rabbit femoral vessels producing a 26-fold increase in local concentration compared with uninjured vessels (P = 0.029). pS-NO-BSA significantly reduced the intimal/medial ratio (P = 0.038) and did so in conjunction with elevations in platelet (P < 0.001) and vascular cGMP content (P < or = 0.001). pS-NO-BSA treatment also inhibited platelet deposition (P = 0.031) after denuding injury. Comparison of BSA, S-NO-BSA, pS-NO-BSA, and control revealed a dose-response relationship between the amount of displaceable NO delivered and the extent of inhibition of neointimal proliferation at 2 wk (P < or = 0.001). Local administration of a stable protein S-nitrosothiol inhibits intimal proliferation and platelet deposition after vascular arterial balloon injury. This strategy for the local delivery of a long-lived NO adduct has potential for preventing restenosis after angioplasty.
1-Methyl-3-isobutylxanthine↗
In vivo transfer of nitric oxide between a plasma protein-bound reservoir and low molecular weight thiols.
Plasma albumin reacts with nitric oxide (NO) to form the bioactive adduct, S-nitroso-albumin (S-NO-albumin). The limited intracellular access of S-NO-albumin suggests the need for a vascular transfer mechanism of NO from a large plasma S-NO-albumin pool to effect biologic function. To study the role of low molecular weight (LMW) thiols in NO transfer in vivo, we administered intravenous S-NO-albumin (1-300 nmol/kg) to rabbits before and after an intravenous infusion of L-cysteine or N-acetyl-L-cysteine. S-NO-albumin produced dose-dependent hypotension that was significantly augmented by prior infusion of either LMW thiol. LMW thiol infusion significantly accelerated the rate of onset and reduced the duration of action of the hypotension induced by S-NO-albumin. The hemodynamic effects of S-NO-albumin after pretreatment with LMW thiols were mimicked by administration of the corresponding LMW S-nitrosothiol. The transfer of NO from albumin to L-cysteine was directly measured in rabbit plasma using a novel technique that couples high performance liquid chromatography to electrochemical detection. These data demonstrate that NO exchange between plasma protein thiol-bound NO and available LMW thiol pools (transnitrosation) occurs in vivo.
Acetylcysteine↗
Homocysteine-induced nitric oxide production in vascular smooth-muscle cells by NF-kappa B-dependent transcriptional activation of Nos2.
Increased plasma levels of homocysteine are an independent risk factor for atherothrombosis. While the endothelial cytotoxicity of homocysteine has been attributed to oxidative stress associated with the reactivity of the thiol group, the oxidative effect of homocysteine on vascular smooth-muscle cells has not been investigated. Recent evidence suggests that expression of inducible nitric oxide synthase (iNOS), or Nos2 gene product, in vascular smooth-muscle cells may, in part, promote atherosclerosis by increasing local oxidative stress. We therefore hypothesized that homocysteine contributes to atherosclerosis by affecting cytokine-induced production of nitric oxide (NO) by vascular smooth-muscle cells. Confluent rat aortic smooth-muscle cells were exposed to a range of concentrations of homocysteine for 4 hr, then were treated with interferon-gamma, interleukin-1 beta, and lipopolysaccharide to induce iNOS. Media NOx content (nitrite plus S-nitrosothiol) was measured over 24 hr using the Saville reaction. As compared to controls, 5, 50, and 500 microM homocysteine produced a dose-dependent increase in media NOx content, an effect that was primarily a consequence of increased S-nitrosothiol production. iNOS enzyme activity and iNOS protein levels were increased significantly in the homocysteine-treated cells as compared with controls. Northern analysis showed that homocysteine treatment increased steady-state Nos2 mRNA levels by 61% at 6 hr as compared with controls, an effect that was not caused by changes in message stability. By electrophoretic mobility shift assay, homocysteine activated NF-kappa B and also potentiated cytokine activation of NF-kappa B. These data demonstrate that exposure of vascular smooth-muscle cells to pathophysiologically relevant concentrations of homocysteine prior to cytokine stimulation leads both to an increase in NO production and to an NF-kappa B-mediated increase in Nos2 transcription. Upregulation of Nos2 may contribute to the inflammatory response that characterizes early atherogenesis and may, in part, account for the adverse vascular effects of hyperhomocysteinemia.
Animals↗