European Heart Journal (2000) 21, 967974 doi:10.1053/euhj.1999.1914, available online at http://www.idealibrary.

com on

Review Article Homocysteine and atherothrombosis mechanisms for injury

J. Thambyrajah and J. N. Townend
Division of Medical Sciences (Cardiology), University of Birmingham, U.K.

Introduction
Homocysteine, a sulphur-containing amino acid, is an intermediate formed during the catabolism of the essential dietary amino acid methionine. Approximately 80% of plasma homocysteine is protein bound. Only a small amount exists as free reduced homocysteine, as the majority of the unbound portion is oxidized to form diamers (homocystine) or combined with cysteine to form mixed disulphides[1,2]. The homocysteine theory of atherosclerosis arose from the observation that diseases such as homozygous homocystinuria, which are characterized by severe hyperhomocysteinaemia (>01 mmol . l 1), are associated with premature vascular disease[3,4]. Mildly elevated levels (0015 003 mmol . l 1) have been found in up to 7% of the population[5,6]. Epidemiological evidence suggests that even such slight elevation of plasma homocysteine is a risk factor and possibly a causative agent for atherosclerotic disease. Evidence for this includes: 1. Cross-sectional studies showing an association between plasma homocysteine and atherosclerotic disease[710]. 2. Prospective nested case-control data showing that elevated levels of plasma homocysteine precede the development of atherosclerotic disease[11,12]. 3. The strong predictive value of plasma homocysteine concentration as an indicator of future mortality in patients with established coronary artery disease[13]. While it should be acknowledged that not all epidemiological studies have found homocysteine to be an independent risk factor for arterial disease[14,15] further support for the homocysteine theory of atherosclerosis
Key Words: Homocysteine, endothelial function and atherosclerosis.
Revision submitted 9 August 1999, and accepted 18 August 1999. Correspondence: Dr J. Thambyrajah, Department of Cardiovascular Medicine, Division of Medical Sciences, University of Birmingham, B15 2TH, U.K. 0195-668X/00/120967+08 $35.00/0

comes from experiments investigating possible mechanisms of homocysteine-induced atherogenesis. This article seeks to review the evidence for these mechanisms, which are summarized in Fig. 1, and include: v endothelial injury v platelet activation v smooth muscle proliferation v oxidative modication of low-density lipoproteins v endothelialleukocyte interactions

Endothelial injury
The endothelium possesses many properties that facilitate vascular homeostasis and maintain tissue perfusion. The response to injury hypothesis proposes that the primary event in atherogenesis is endothelial damage and dysfunction which disrupts these properties resulting in platelet and leukocyte adhesion, thrombosis, smooth muscle proliferation, vasospasm, lipid accumulation and ultimately atheroma[1618].

Endothelial damage
Using the release of radio-labelled Chromium 51 and the detachment of endothelial cells from conuent monolayers as measures of in vitro endothelial damage, homocysteine concentrations between 110 mmol . l 1 were found to result in dose-dependent damage of human umbilical vein endothelial cells[19]. Increased permeability to albumin of bovine aortic endothelial cell monolayers exposed to 05 mmol . l 1 homocysteine in the presence of copper provided further evidence of endothelial damage[20]. This appears to be mediated by oxidative stress. Hydrogen peroxide generated by the copper catalysed auto-oxidation of homocysteine[21] was implicated in the mechanism of toxicity by the demonstration of a reduction in endothelial damage with the addition of catalase[19,20]. In addition, endothelial cells incubated with homocysteine were subject to increased DNA strand scission and depleted cellular NAD+ levels[22], an
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Homocysteine

Oxidant stress by: 1. Generation of free radicals 2. Inhibition of glutathione peroxidase

Endothelial injury

Nitric oxide degradation

Smooth muscle proliferation NITRIC OXIDE BIOAVAILABILITY Vasoconstriction Leukocyte and platelet adhesion and activation

Endothelial defences

Nitric oxide synthesis

Damaged /dysfunctional endothelium

TF GAG Protein C/TM tPa

Thrombogenesis Haemostatic mechanisms Factor V Factor Va

Figure 1 Potential adverse eects of homocysteine. GAG, Glycosaminoglycan-Antithrombin III anticoagulant pathway; Protein C/TM, Protein C-Thrombomodulin anticoagulant pathway; TF, Tissue factor; tPa, tissue plasminogen activator. eect similar to that seen following incubation with hydrogen peroxide[23]. As well as the induction of DNA degradation, homocysteine also inhibits DNA synthesis in human umbilical vein endothelial cells in a concentration-dependent manner[22,24]. These eects on DNA may not only reduce the ability of endothelial cells to regenerate following damage but may also reduce the synthesis of the enzymes participating in the remethylation and transsulphuration pathways of homocysteine metabolism. Thus, the targeting of DNA represents a mechanism for the amplication of homocysteinemediated endothelial damage. The homocysteine concentrations used by Wall et al.[19] and by Berman et al.[20] were between three and six hundred times greater than pathophysiological concentrations in vivo (0015003 mmol . l 1). However, in the presence of copper at levels normally found in human serum, the concentration of homocysteine required to induce the same degree of human and bovine endothelial cell lysis was lower at 01 mmol . l 1[25]. Although this concentration is close to that found in patients with homocysteinuria, it is still up to six times greater than the levels on which the epidemiological evidence for the homocysteine theory of atherosclerosis is based. Homocysteine-induced endothelial damage has also been demonstrated in animal studies. In rats fed on a
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methionine-rich diet resulting in elevated homocysteine concentrations, the aortic intima showed degeneration and desquamation of endothelial cells [26]. In baboons, chronic intravenous infusion of homocysteine caused vascular endothelial damage and intimal lesions which were said to be similar to atherosclerotic lesions in man[27].

Endothelial dysfunction
Nitric oxide Endothelial-derived nitric oxide regulates vessel tone, inhibits platelet activation, adhesion and aggregation[28,29], limits smooth muscle proliferation and modulates endothelialleukocyte interactions[30]. The initial response of bovine aortic endothelial cells exposed to homocysteine, after the induction of nitric oxide synthesis with bradykinin or a calcium ionophore, is to increase nitric oxide synthesis in a dose-dependent manner[31]. Homocysteine reacts with nitric oxide to form S-nitroso-homocysteine[32], which has some of the properties of nitric oxide. It markedly inhibits platelet aggregation, is a potent vasodilator and does not support hydrogen peroxide generation[33]. This represents a protective mechanism against the adverse eects of homocysteine. Whilst the exposure of bovine aortic

Review endothelial cells to homocysteine for 15 min stimulated the release of nitric oxide and subsequent formation of S-nitroso-homocysteine, both prolonged exposure]33] and high homocysteine concentrations[29] resulted in impaired nitric oxide production. Thus, chronically elevated homocysteine levels appear to result in a selfperpetuating cycle that progressively overwhelms the capacity of endothelial cells to reduce the toxicity of homocysteine. Homocysteine also reduces nitric oxide activity by oxidative degradation[25,34] and by inhibiting glutathione peroxidase[21,35] (see section entitled Glutathione peroxidase). In-vivo studies have also demonstrated impairment of vascular nitric oxide activity by homocysteine, probably as a result of oxidant stress. L-arginine, the natural precursor of nitric oxide, reduces mean blood pressure and lowers blood viscosity when infused in healthy subjects[36]. These responses were impaired in healthy volunteers by acute hyperhomocysteinaemia (003 mmol . l 1) induced by a methionine load, an eect prevented by the co-administration of antioxidant vitamins[37]. In healthy arteries, reactive hyperaemia following transient occlusion results in nitric oxide dependent vasodilatation which can be measured with high-resolution ultrasound[38,39]. Patients with moderate hyperhomocysteinaemia (002004 mmol . l 1) as well as homozygous homocysteinuria had signicantly impaired endotheliumdependent ow-mediated vasodilatation compared to controls[4042]. An oral methionine load producing an acute increase in plasma homocysteine concentration to 003 mmol . l 1 also caused transiently impaired owmediated vasodilatation in healthy volunteers[43,44]. This decreased ow-mediated vasodilatation, but not the acute elevation in plasma homocysteine, was prevented by both pre-treatment with vitamin C[45] and folic acid[46]. Thus antioxidant vitamins and folic acid can prevent homocysteine-induced endothelial dysfunction probably by modulating cellular oxidative metabolism. Folic acid may have an additional eect by stimulating nitric oxide production[47,48]. This in vitro and in vivo evidence suggests that homocysteine decreases nitric oxide bioavailability by promoting oxidative degradation and inhibiting endothelial synthesis. This represents a potent mechanism by which homocysteine could contribute to the pathogenesis of atherogenesis and thrombosis. Glutathione peroxidase Endothelial glutathione peroxidase catalyses the reduction of both hydrogen and lipid peroxides to their corresponding alcohol[21,49] and represents a mechanism for cellular defence against oxidant stress. It also prevents the oxidative inactivation of nitric oxide[35]. The activity of glutathione peroxidase in bovine aortic endothelial cells was reduced by up to 81% by 025 mmol . l 1 homocysteine. There was a parallel reduction in glutathione peroxidase mRNA levels, suggesting that the mechanism of action involves the suppression of cellular expression of glutathione peroxi-

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dase[35]. Signicantly, homocysteine is alone amongst thiols in its ability to inhibit glutathione peroxidase activity in vitro. This unique property of homocysteine may serve to explain its greater toxicity compared to cysteine, a thiol-containing amino acid, which is also capable of generating free radicals and is present in serum at concentrations four times higher than homocysteine[35]. Haemostasis The vascular endothelium maintains a nonthrombogenic surface by the glycosaminoglycanantithrombin III and thrombomodulin-protein C anticoagulant pathways[50,51], the synthesis of plasminogen activators[5254] and the inhibition of platelet activation by ecto-ADPase, prostacyclin and nitric oxide[52]. The endothelium also synthesises clotting factors and the balance between procoagulant and anticoagulant mechanisms is vital to the maintenance of vascular haemostasis. There is evidence that homocysteine may upset this balance and predispose to thrombogenesis via a number of mechanisms. (a) Stimulation of procoagulant factors After treatment with homocysteine, at a minimum concentration of 05 mmol . l 1, bovine aortic endothelial cells and human umbilical vein endothelial cells exhibited enhanced Factor V activity. The mechanism of action involves the induction of a protease endothelial cell activator of Factor V and oers a mechanism for the promotion of coagulation by homocysteine in the absence of thrombin[55]. Homocysteine at a concentration of 0306 mmol . l 1 also increased the transcription and activity of tissue factor[56]. (b) Inhibition of anticoagulant mechanisms Porcine aortic endothelial cell expression of heparin-like glycosaminoglycan was reduced in a dose and time dependent manner by incubation with homocysteine concentrations of 011 mmol . l 1[57]. Reduced functional antithrombin III has also been demonstrated in a small number of patients with homocysteinuria[58,59]. The exposure of human and bovine endothelial cells to homocysteine concentrations of 06125 mmol . l 1 resulted in a reduction in activated protein C, which was explained by the competitive inhibition by homocysteine of thrombin to thrombomodulin (a component of the protein C pathway) binding[60]. Others have suggested that homocysteine directly and irreversibly inactivates both thrombomodulin and protein C[61,62]. Homocysteine may also act indirectly to decrease cell surface expression of thrombomodulin by impairing its transit along the secretory pathway[61,62]. Thrombomodulindependent protein C activation was 35% lower in the ex vivo thoracic aorta of monkeys with diet induced hyperhomocysteinaemia compared to controls[63]. (c) Impairment of brinolysis Endothelial cells incubated with homocysteine concentrations of 175 mmol . l 1 showed a dose-related
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J. Thambyrajah and J. N. Townend scribed. Homocysteine concentrations of 1 mmol . l 1 resulted in increased production of the vasoconstrictor and thrombogenic agent thromboxane, from platelet rich plasma, an eect not seen with other thiols[68]. An increased urinary excretion of thromboxane metabolites was found in 11 homocysteinuric patients when compared to healthy controls[73]. In baboons with hyperhomocysteinaemia, intimal lesion formation but not endothelial damage was reduced by antiplatelet therapy[27] suggesting that homocysteine-induced endothelial damage stimulates the release of platelet derived mitogenic factors which results in smooth muscle proliferation.

decrease in both tissue-plasminogen activator activity and membrane binding sites. Other thiol-containing substances did not signicantly reduce the binding of tissue-plasminogen activator suggesting that this eect was specic to homocysteine[54]. Lipoprotein(a) is an atherogenic lipoprotein which reduces brinolysis by competitively inhibiting the binding of plasminogen with brin. Homocysteine at a concentration of 0008 mmol . l 1 promoted lipoprotein(a) binding to brin, reducing the activation of plasminogen[64]. (d) Modulation of plateletendothelial interactions Platelet activation results in thrombosis and smooth muscle proliferation and is therefore important in the pathogenesis of atherothrombosis[16,17]. Ecto-ADPase catalyses the breakdown of ADP, the mediator responsible for the nal common pathway of platelet activation. High (supra-pathophysiological) homocysteine concentrations (10 mmol . l 1) have been shown to inhibit the ecto-ADPase activity of human umbilical vein endothelial cells[65]. Prostacyclin production by ex-vivo rat thoracic aorta was inhibited by homocysteine concentrations of 110 mmol . l 1[66]. The physiological relevance of these ndings is dubious as they are based on the eect, on animal tissue, of extremely high homocysteine concentrations. Others, using human tissue, have failed to detect any in vitro eect of homocysteine or serum from homocysteinuric patients on prostacyclin production at lower homocysteine levels (02 1 mmol . l 1)[67,68]. While the eects of homocysteine on endothelial ADPase and prostacyclin production appear unlikely to be of pathophysiological signicance, homocysteine may activate platelets by reducing endothelial nitric oxide bioavailability. The prolonged exposure (>4 h) of endothelial cells to homocysteine resulted in the progressive loss of nitric oxide mediated inhibition of platelet aggregation[33]. In addition, an acute increase in homocysteine in healthy subjects promoted platelet aggregation that was reduced by the co-administration of the antioxidant vitamins C and E, suggesting that altered platelet function may be secondary to oxidative stress[37].

Smooth muscle proliferation

Vascular intimal smooth muscle proliferation with subsequent formation of extracellular matrix collagen is an integral component of atherosclerosis[17]. This process may be directly stimulated by homocysteine or may be secondary to the mitogenic eect of endothelial and/or platelet-derived growth factors released by homocysteine-induced endothelial cell damage. Homocysteine at a concentration of 001 mmol . l 1 induced a dose and time dependent increase in collagen expression by human vascular smooth muscle cells[74]. Tsai et al. demonstrated that homocysteine concentrations of 01 mmol . l 1 increased DNA synthesis in both rat and human aortic smooth muscle cells and this was subsequently followed by proliferation[24,75]. This same concentration of homocysteine also resulted in a 25% reduction in DNA synthesis in human umbilical vein endothelial cells[24]. Smooth muscle cells are less susceptible to homocysteine-induced damage compared to endothelial cells[19,25] and these contrasting eects on the endothelial and smooth muscle cells may be an important mechanism in the development of atheroma. The signal pathway for the stimulation of DNA synthesis in smooth muscle cells by homocysteine involves cyclins and cyclin-dependent kinases. Cyclins trigger the re-entry of quiescent cells into the cell cycle[76,77] and their association with cyclin-dependent kinases is required for DNA replication[78,79]. Homocysteine concentrations of 01 mmol . l 1 resulted in an increase in mRNA of cyclin D1 and A in rat aortic smooth muscle cells[24,75]. The eect on cyclin A mRNA was mediated at transcriptional level and accompanied by increased cyclin A protein[75]. Rats fed with varying doses of homocysteine showed a dose-dependent increase in aortic cyclin-dependent kinase mRNA and protein, an eect not seen with cysteine. This was associated with an increase in smooth muscle proliferation in the aortic wall[80].

Direct effects on platelets

The eects of homocysteine on platelet kinetics and function are contentious. Early studies on platelet kinetics in four homocysteinuric patients and in a baboon model suggested an increased turnover of platelets[27,69] but were not conrmed by subsequent studies[70,71]. Although McDonald demonstrated increased platelet adhesion in homocysteinuric patients[72], there is no evidence of a direct in vitro eect of homocysteine on platelet adhesion[70,71] or aggregation[33]. Furthermore, no signicant dierences in tests of in vitro platelet function were detected between homocysteinuric patients and healthy controls[73]. In vitro abnormalities in platelet arachidonic acid metabolism that favour thrombogenesis have been deEur Heart J, Vol. 21, issue 12, June 2000

Oxidative modication of low-density lipoproteins

Oxidatively modied low-density lipoproteins are believed to be an important mediator of cholesterol-

Review induced atherosclerosis. Although homocysteine has been suggested to cause lipid peroxidation[81,82], there is little convincing support from a review of the available evidence. Thiobarbituric acid, a measure of the products of lipid peroxidation, was increased when human umbilical vein endothelial cells were incubated with 5 mmol . l 1 homocysteine and copper. Whilst the lipophilic antioxidant DPPD prevented the accumulation of thiobarbituric acid, it did not alter the degree of cellular lysis implying that lipid peroxidation is not a mechanism of homocysteine-induced cellular toxicity[83]. There was no signicant dierence in parameters of in vitro copper-catalysed oxidization of low-density lipoproteins between patients with homocysteinuria and matched controls[84,85]. In vivo studies have shown no signicant dierence in the products of lipid peroxidation in the serum[86] and the plasma[84] of patients with homocysteinuria compared to normal controls. Indeed, Dudman et al. found that the concentration of lipid peroxidation products was decreased in patients with homocysteinuria[87]. Although the precise interaction between homocysteine and lipoproteins remains to be claried, there is little available evidence to suggest that at pathophysiological concentrations homocysteine promotes oxidative modication of low-density lipoproteins.

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Endothelialleukocyte interactions
Leukocyte adhesion to endothelium is an important part of the inammatory process of atherogenesis[16]. Increased adhesion and trans-endothelial migration of neutrophils was demonstrated in-vitro in human umbilical vein endothelial cells monolayers pre-treated with homocysteine concentrations of 005 mmol . l 1 and in-vivo in rats following homocysteine infusion[88]. This was accompanied by in-vitro evidence of endothelial damage reected by the release of radiolabelled chromium 51 and monolayer detachment, an eect mediated by hydrogen peroxide[88]. The mechanism for homocysteine induced endothelialleukocyte interactions may involve the neutrophil docking protein complex CD11b/CD18[88] and endothelial expression of monocyte chemoattractant protein-1[89]. Although, elevated plasma levels of circulating endothelial adhesion molecules ICAM-1 and VCAM-1 have been demonstrated in healthy volunteers following an oral methionine load[37], in-vitro endothelial cell expression of P-selectin[88], E-selectin, ICAM-1 or VCAM-1[90] were not increased by homocysteine.

04 mmol . l 1[4]. However, many of the in vitro studies described above demonstrated adverse eects of homocysteine at concentrations of 110 mmol . l 1, making the pathophysiological signicance of these experiments dicult to interpret. There are two possibilities to explain the relative lack of evidence of adverse eects at lower concentrations of homocysteine. Firstly, the methods used may not be sensitive enough to identify abnormalities at lower homocysteine concentrations. Secondly, patients have a lifetime exposure to homocysteine as opposed to the limited exposure period of the in vitro experiments and consequently this may restrict the amount of damage generated by homocysteine. Most studies have been based on free reduced homocysteine which is capable of generating free radicals. However, this form comprises only 14% of total plasma homocysteine and similar eects have rarely been found with the more abundant oxidized homocystine. A concentration of 10 mmol . l 1 of reduced homocysteine in vitro is actually 50 000 times higher than that found in patients with premature vascular disease[91]. Some of the adverse eects of homocysteine, such as its ability to generate reactive oxygen species, have also been observed with other thiol-containing amino acids like cysteine[92]which have not been associated with vascular disease. Proposed mechanisms that are based on general actions of thiol groups are not suciently specic to explain the possible increased vascular risk attributed to homocysteine. Despite these criticisms, there are several plausible mechanisms for homocysteine induced atherogenesis and thrombosis: v Endothelial cellular and DNA damage v Reduced activity of glutathione peroxidase v Stimulation of procoagulant and impairment of anticoagulant and brinolytic pathways v Mitogenic eect on smooth muscle proliferation v Promotion of endothelialleukocyte interactions Many of these eects can be explained by a reduction in nitric oxide bioavailability and it is possible that this is the nal common pathway of homocysteine induced vascular toxicity. The relevance of much of the experimental data to patients with hyperhomocysteinaemia remains unclear and further work must be performed at lower concentrations of homocysteine to examine the specic mechanisms of atherogenesis and thrombosis. An understanding of these mechanisms will aid the development of new strategies for possible prevention and treatment of atheroma by lowering plasma homocysteine.
J. Thambyrajah and J. N. Townend are supported by the British Heart Foundation.

Conclusion
The epidemiological evidence for the homocysteine theory of atherosclerosis is based largely upon pathophysiological homocysteine concentrations of 001 003 mmol . l 1[8,11,12]. Patients with homozygous homocysteinuria have plasma concentrations up to

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