Low-Dose Homocystine Enhances Proliferation and Migration of Bv2 Microglia Cells.

Homocysteine (Hcy) is a non-essential amino acid that is derived from the breakdown of dietary methionine. Hyperhomocysteinemia (HHcy) is an independent risk factor for a variety of chronic diseases, especially neurodegenerative conditions. To better understand the role of HHcy in the pathogenesis of neurodegenerative disorders, we investigated the effect of Hcy on the proliferation and activation of microglia Bv2 cells. Cells were treated with six different Hcy concentrations: 0, 50, 100, 300, 500, and 1000 µM for different time periods (8, 12, 16, 24, and 48 h). The morphology of Bv2 cells was observed, and cell activity and proliferation were detected. Cell migration and secretion of pro-inflammatory cytokines were detected by the scratch wound assay, the transwell assay, and ELISA, respectively. The effect of Hcy on Bv2 proliferation occurred earlier (<24 h, especially 16 h) after treatment with concentrations between 100 and 300 µM, and there was no cytotoxicity to Bv2 cells. Meanwhile, functional assays suggested that Hcy not only promoted Bv2 secretion of the pro-inflammatory cytokines IL-1ß, TNF-α, and IL-6, but also enhanced Bv2 migration and invasion, with 100 µM being the most effective concentration. In summary, Bv2 proliferation and activation can be promoted by short-term treatment with low-dose Hcy.

Decreased fibronectin production significantly contributes to dysregulated repair of asthmatic epithelium.

RATIONALE: Damage to airway epithelium is followed by deposition of extracellular matrix (ECM) and migration of adjacent epithelial cells. We have shown that epithelial cells from children with asthma fail to heal a wound in vitro. OBJECTIVES: To determine whether dysregulated ECM production by the epithelium plays a role in aberrant repair in asthma. METHODS: Airway epithelial cells (AEC) from children with asthma (n = 36), healthy atopic control subjects (n = 23), and healthy nonatopic control subjects (n = 53) were investigated by microarray, gene expression and silencing, transcript regulation analysis, and ability to close mechanical wounds. MEASUREMENTS AND MAIN RESULTS: Time to repair a mechanical wound in vitro by AEC from healthy and atopic children was not significantly different and both were faster than AEC from children with asthma. Microarray analysis revealed differential expression of multiple gene sets associated with repair and remodeling in asthmatic AEC. Fibronectin (FN) was the only ECM component whose expression was significantly lower in asthmatic AEC. Expression differences were verified by quantitative polymerase chain reaction and ELISA, and reduced FN expression persisted in asthmatic cells over passage. Silencing of FN expression in nonasthmatic AEC inhibited wound repair, whereas addition of FN to asthmatic AEC restored reparative capacity. Asthmatic AEC failed to synthesize FN in response to wounding or cytokine/growth factor stimulation. Exposure to 5', 2'deoxyazacytidine had no effect on FN expression and subsequent analysis of the FN promoter did not show evidence of DNA methylation. CONCLUSIONS: These data show that the reduced capacity of asthmatic epithelial cells to secrete FN is an important contributor to the dysregulated AEC repair observed in these cells.

Homocysteine inhibits arterial endothelial cell growth through transcriptional downregulation of fibroblast growth factor-2 involving G protein and DNA methylation.

Homocysteine (Hcy) contributes to atherogenesis and angiostasis by altering the phenotype of arterial endothelial cells (ECs). The present study was aimed at elucidating potential mechanisms by which Hcy can slow EC proliferation and induce EC apoptosis, thereby disrupting endothelial integrity. Given the strong mitogenic and antiapoptotic properties of fibroblast growth factor (FGF)2, we examined whether Hcy can modulate its expression. In cultured human coronary and bovine aortic ECs, Hcy exerted time- and concentration-dependent (0 to 500 micromol/L) reduction of the mRNA and protein levels of FGF2, whereas vascular endothelial growth factor expression was not affected until Hcy reached a proapoptotic 500 micromol/L. By testing a panel of signal transduction inhibitors, we found that the Hcy-induced downregulation of FGF2 was specifically attenuated by pertussis toxin, an inhibitor of Gi protein signaling. Hcy induced cell cycle arrest at the G(1)/S transition and increased TUNEL-positive apoptotic cells in a graded manner. These effects were effectively counteracted by exogenous FGF2. Reporter gene assays showed that Hcy downregulated FGF2 by transcriptional repression of the gene promoter encompassed in a CpG dinucleotide-rich island. This region was heavily methylated at the cytosine residues by Hcy despite decreased methylation potential (S-adenosylmethionine to S-adenosylhomocysteine ratio). Normal levels of FGF2 transcription were restored to ECs simultaneously exposed to Hcy and 5-aza-deoxycytidine. We conclude that homocysteine disrupts the growth and survival of ECs through a G protein-mediated pathway associated with altered promoter DNA methylation and the transcriptional repression of FGF2.

Growth promotion by homocysteic acid.

Homocysteic acid, H03SCH2CH2CHNH2CO2H, promotes growth of hypophysectomized rats, assayed by observation of increased thickness of epiphyseal cartilage of the tibia and by observation of tail growth. Doses of homocysteic acid as low as 1 microgram per day for 4 days in the tibia assay and 2.5 milligrams per kilogram per day for 5 weeks in the tail assay were effective in promoting growth. Serum somatomedin activity, determined by the porcine cartilage disk assay, was also increased by homocysteic acid. These findings relate an area of sulfur amino acid metabolism to the physiological action of growth hormone, accelerated growth in homocystinuria, initiation of arteriosclerosis, and control of cellular growth.

Comparative studies on homocysteine and its metabolite-homocysteine thiolactone action in blood platelets in vitro.

Homocysteine (Hcy), an intermediate formed during the catabolism of the essential dietary amino acid methionine, and its cyclic thioester, homocysteine thiolactone (TL) formed from Hcy in plasma, may be implicated in pathological haemostasis and atherosclerosis. The mechanism by which TL exerts the prothrombotic effect and influences blood platelets remains unclear. Activation of blood platelets plays an important role in prothrombotic events. The aim of our study was to establish and compare the influence of a reduced form of homocysteine (at final doses of 10-100 microM) and its cyclic thioester, homocysteine thiolactone (0.1-1 microM), on platelet activation induced by thrombin (platelet aggregation), on platelet protein modifications (determined by parameters such as level of protein carbonyl groups, 3-nitrotyrosine residues in proteins) and on superoxide anion radicals ( O2-*) generation using the model system in vitro. We have observed that TL, like its precursor, Hcy, stimulates the generation of O2* in platelets and causes an augmentation of platelet aggregation induced by thrombin. Our present results in vitro also demonstrate that Hcy (10-100 microM) and TL at lower doses than Hcy (0.1-1 microM) cause modification of platelet proteins: diminished formation of carbonyl groups and distinctly decreased tyrosine nitration in platelet proteins after thrombin stimulation, but increased platelet aggregation induced by thrombin. TL like Hcy (at concentrations corresponding to concentrations in blood during hyperhomocysteinemia) modifies platelet responses to an important physiological agonist--thrombin.

Antioxidant effects of methionine, alpha-lipoic acid, N-acetylcysteine and homocysteine on lead-induced oxidative stress to erythrocytes in rats.

Lead, widely used in industry, is a great environmental health problem. Many studies have examined its effects on the health of both humans and animals. Experimental studies have shown that sulphur-containing antioxidants have beneficial effects against the detrimental properties of lead. The present study was designed to investigate markers of oxidative stress (hemoglobin (Hb) in whole blood, malondialdehyde (MDA) in sera; superoxidase dismutase (SOD) and glutathione peroxidise (GSH-Px) in erythrocyte hemolysate and vitamins A and E in plasma) in rats given lead (2000ppm) with or without sulphur-containing antioxidants (l-methionine (Met) (100mg/kg/day), N-acetylcysteine (NAC) (800mg/kg/day), l-homocysteine (Hcy) (25mg/kg/day), lipoic acid (LA) (50mg/kg/day)) in their water for 5 weeks. In the lead group, Hb and plasma vitamin E levels were significantly lower whereas MDA levels were significantly higher compared to controls (p<0.05). Hb levels in lead-methionine and lead-LA groups were significantly higher than the lead group (p<0.01). MDA levels were reduced in all groups compared to the lead group (p<0.01). There was a decrease below control values in erythrocyte SOD (p<0.01) and GSH-Px (p<0.05) levels in the lead-LA group. Plasma vitamin A levels were significantly high in lead-methionine group compared to lead group (p<0.01). In conclusion, the data suggests that oxidative stress induced by lead is reduced by sulphur-containing compounds.

A collagen defect in homocystinuria.

The biochemical mechanism accounting for the connective tissue abnormalities in homocystinuria was explored by examining the effects of various amino acids known to accumulate in the plasma of patients with this disease on cross-link formation in collagen. Neutral salt solutions of purified, rat skin collagen, rich in cross-link precursor aldehydes, were polymerized to native type fibrils by incubating at 37 degrees C in the presence of homocysteine, homocystine, or methionine. After the polymerization was completed, each sample was examined for the formation of covalent intermolecular cross-links, assessed indirectly by solubility tests and directly by measuring the cross-link compounds after reduction with tritiated sodium borohydride and hydrolysis. Collagen solutions containing homocysteine (0.01 M-0.1 M) failed to form insoluble fibrils. Furthermore, much less of the reducible cross-links, Delta(6,7) dehydrohydroxylysinonorleucine, Delta(6,7) dehydrohydroxylysinohydroxynorleucine, and histidino-dehydrohydroxymerodesmosine were formed in the preparations containing homocysteine as compared with the control and the samples containing methionine or homocystine. The content of the precursor aldehydes, alpha-aminoadipic-delta-semialdehyde (allysine) and the aldol condensation product, was also markedly diminished in tropocollagen incubated with homocysteine. It is concluded that homocysteine interferes with the formation of intermolecular cross-links that help stabilize the collagen macromolecular network via its reversible binding to the aldehydic functional groups. Analysis of the collagen cross-links in skin biopsy samples obtained from three patients with documented homocystinuria showed that the cross-links were significantly decreased as compared with the age-matched controls, supporting the tentative conclusions reached from the in vitro model studies. In addition, the solubility of dermal collagen in non-denaturing solvents was significantly increased in the two patients examined, reflecting a functional defect in collagen cross-linking. Although the concentration of homocysteine used in this study to demonstrate these effects in vitro is clearly higher than that which is observed in homocystinuric's plasma, the data do suggest a possible pathogenetic mechanism of connective tissue defect in homocystinuria.

Homocystine-induced arteriosclerosis. The role of endothelial cell injury and platelet response in its genesis.

The atherogenic mechanism of homocystinemia has been defined by measuring endothelial cell loss and regeneration, platelet consumption, and intimal lesion formation in a primate model. Three groups of baboons were studied: (a) 8 control animals; (b) 15 animals after 3 mo of continuous homocystinemia; and (c) 11 animals after 3 mo of combined homocystinemia and oral treatment with dipyridamole. Experimental homocystinemia caused patchy endothelial desquamation comprising about 10% of the aortic surface despite a 25-fold increase in endothelial cell regeneration. Neither endothelial cell loss nor regeneration was changed significantly by dipyridamole. Homocystine-induced vascular deendothelialization produced a threefold increase in platelet consumption that was interrupted by dipyridamole inhibition of platelet function. All homocystinemic animals developed typical arteriosclerotic or preatherosclerotic intimal lesions composed of proliferating smooth muscle cells averaging 10-15 cell layers surrounded by large amounts of collagen, elastic fibers, glycosaminoglycans, and sometimes lipid. Intimal lesion formation was prevented by dipyridamole therapy. We conclude that homocystine-induced endothelial cell injury resulted in arteriosclerosis through platelet-mediated intimal proliferation of smooth muscle cells that can be prevented by drug-induced platelet dysfunction.

Adverse vascular effects of homocysteine are modulated by endothelium-derived relaxing factor and related oxides of nitrogen.

Elevated levels of homocysteine are associated with an increased risk of atherosclerosis and thrombosis. The reactivity of the sulfhydryl group of homocysteine has been implicated in molecular mechanisms underlying this increased risk. There is also increasingly compelling evidence that thiols react in the presence of nitric oxide (NO) and endothelium-derived relaxing factor (EDRF) to form S-nitrosothiols, compounds with potent vasodilatory and antiplatelet effects. We, therefore, hypothesized that S-nitrosation of homocysteine would confer these beneficial bioactivities to the thiol, and at the same time attenuate its pathogenicity. We found that prolonged (> 3 h) exposure of endothelial cells to homocysteine results in impaired EDRF responses. By contrast, brief (15 min) exposure of endothelial cells, stimulated to secrete EDRF, to homocysteine results in the formation of S-NO-homocysteine, a potent antiplatelet agent and vasodilator. In contrast to homocysteine, S-NO-homocysteine does not support H2O2 generation and does not undergo conversion to homocysteine thiolactone, reaction products believed to contribute to endothelial toxicity. These results suggest that the normal endothelium modulates the potential, adverse effects of homocysteine by releasing EDRF and forming the adduct S-NO-homocysteine. The adverse vascular properties of homocysteine may result from an inability to sustain S-NO formation owing to a progressive imbalance between the production of NO by progressively dysfunctional endothelial cells and the levels of homocysteine.

The role of hydrogen sulfide in homocysteine-induced cardiodynamic effects and oxidative stress markers in the isolated rat heart.

This study aimed to assess the role of H2S in homocysteine-induced cardiodynamic effects in the isolated rat heart. The hearts were retrogradely perfused according to the Langendorff technique. The maximum and minimum rates of pressure in the left ventricle (dp/dt max, dp/dt min), systolic and diastolic left ventricular pressures (SLVP, DLVP), heart rate (HR), and coronary flow (CF) were measured. A spectrophotometrical method was used to measure the following oxidative stress markers: index of lipid peroxidation (thiobarbituric acid reactive substances, TBARS), nitrite level (NO2-), superoxide anion radicals (O2•-), and hydrogen peroxide (H2O2) concentrations. The administration of 10 µmol/l DL-homocysteine (DL-Hcy) alone decreased dp/dt max, SLVP, and CF but did not change any oxidative stress parameters. The administration of 10 µmol/l DL-propargylglycine (DL-PAG) decreased all cardiodynamic parameters and increased the concentration of O2•-. The co-administration of DL-Hcy and DL-PAG induced a significant decrease in all estimated cardiodynamic parameters and decreased the concentration of NO2- and O2•- but increased the levels of TBARS and H2O2. Homocysteine shows a lower pro-oxidative effect in the presence of hydrogen sulfide (H2S), which indicates a potential anti-oxidative capacity of H2S.

Disruption of thymidylate synthesis and glycine-serine interconversion by L-methionine and L-homocystine in Raji cells.

Excessive concentrations of L-methionine inhibited the folate-dependent de novo synthesis of thymidylic acid (TMP) in Raji cells, demonstrating the usefulness of this cell line for the study of methionine-folate antagonism. The effect was also produced by L-homocystine but not by other amino acids including D-methionine and L-ethionine, suggesting that this effect is exerted by a common intermediate of methionine and homocystine metabolism. L-Methionine, L-homocysteine, S-adenosylmethionine (SAM), and S-adenosylhomocysteine (SAH) are not inhibitors of thymidylate synthase activity. On the other hand the capacity of the cells to incorporate serine 3-carbon and glycine 2-carbon into DNA is impaired by the presence of L-methionine or L-homocystine. Studies with cell-free extracts demonstrated that the glycine cleavage enzyme is inhibited by 45% by L-methionine, L-homocysteine, SAM or SAH. Serine hydroxymethylase on the other hand was slightly stimulated by these sulfur-containing compounds and this stimulation was shown to occur in the intact cell as well. These findings suggest that when levels of L-methionine metabolites are elevated, there is an increase in the use of glycine to maintain the intracellular concentration of serine, which is required for homocysteine detoxification by conversion to cystathionine. The reduction in TMP synthesis caused by excess L-methionine or L-homocystine may result from increased utilization of one-carbon units for serine synthesis.

Copper-mediated LDL oxidation by homocysteine and related compounds depends largely on copper ligation.

Oxidation of low-density lipoprotein (LDL) is thought to be a major factor in the pathophysiology of atherosclerosis. Elevated plasma homocysteine is an accepted risk factor for atherosclerosis, and may act through LDL oxidation, although this is controversial. In this study, homocysteine at physiological concentrations is shown to act as a pro-oxidant for three stages of copper-mediated LDL oxidation (initiation, conjugated diene formation and aldehyde formation), whereas at high concentration, it acts as an antioxidant. The affinity for copper of homocysteine and related copper ligands homocysteine, cystathionine and djenkolate was measured, showing that at high concentrations (100 microM) under our assay conditions, they bind essentially all of the copper present. This is used to rationalise the behaviour of these ligands, which stimulate LDL oxidation at low concentration but generally inhibit it at high concentration. Albumin strongly reduced the effect of homocystine on lag time for LDL oxidation, suggesting that the effects of homocystine are due to copper binding. In contrast, copper binding does not fully explain the pro-oxidant behaviour of low concentrations of homocysteine towards LDL, which appears in part at least to be due to stimulation of free radical production. The likely role of homocysteine in LDL oxidation in vivo is discussed in the light of these results.

Effect of caspase 9 related signaling molecules on the apoptosis of human vascular endothelial cell induced by homocysteine.

OBJECTIVE: To understand the role of mitochondria associated signaling pathway in the apoptosis of human vascular endothelial cell induced by homocysteine (Hcy). METHODS: The mRNA and protein expression levels of the up-stream signaling molecules of caspase 3, Bcl 2, caspase 9, and cytosolic cytochrome-c, were investigated. The in vitro cultured human umbilical vein endothelial cells with homocysteine at different concentrations were incubated for 24 h. The expressions of Bcl 2 and caspase 9 at mRNA and protein levels were analyzed by reverse transcription-polymerase chain reaction(RT-PCR) and Western blot. Cytochrome-c in cytoplasm was also detected by Western blot. RESULTS: The expression levels of three signaling molecules were all down-regulated by homocysteine at both mRNA and protein levels in a dose-dependent manner. CONCLUSION: Homocysteine could affect the formation of apoptosome through repressing the expression of Bcl 2 gene and release of cytochrome-c from mitochondria. Decreasing of apoptosome could disturb the activation of caspase 9. The results also indicate that the mitochondria pathway is not the major signaling pathway involved in Hcy-induced apoptosis.

Hyperhomocyst(e)inemia and endothelial dysfunction in IDDM.

OBJECTIVE: While elevated blood levels of homocyst(e)ine represent an independent risk factor for macrovascular disease, we assessed the link between hyperhomocyst(e)inemia and diabetic microvascular diseases. RESEARCH DESIGN AND METHODS: Plasma levels of homocyst(e)ine and thrombomodulin (TM), markers of endothelial cell damage, were measured before and 3 h after oral methionine loading in 75 patients with IDDM and 40 healthy control subjects matched for sex and age. Exclusion criteria were hyperlipidemia, hypertension, smoking, or positive family history for cardiovascular disease. RESULTS: IDDM patients had higher pre- and postload plasma levels of homocyst(e)ine than did healthy control subjects (12.0 vs. 7.7 mumol/l and 27.6 vs. 16.0 mumol/l; P < 0.001). Of 75 IDDM patients, 26 had plasma homocyst(e)ine levels above the normal range (means +/- 2 SD of values obtained in the control group). These IDDM patients with hyperhomocyst(e)inemia had higher plasma TM levels (62.2 vs. 38.2 ng/ml, P < 0.001), higher albumin excretion rates (485 vs. 115 mg/l, P < 0.005), and a higher prevalence of late diabetic complications (nephropathy, 76 vs. 33%; retinopathy, 69 vs. 51%; neuropathy, 57 vs. 41%; and macroangiopathy, 57 vs. 33%) compared with IDDM patients with normal plasma homocyst(e)ine. In vitro experiments with human umbilical vein cells showed an increased release of TM into the culture supernatant only when endothelial cells were pretreated with advanced glycation end product (AGE)-albumin before L-homocystine was added. A synergistic action of homocyst(e)ine and AGEs might contribute to vascular complications in patients with diabetes. CONCLUSIONS: Hyperhomocyst(e)inemia is common in nephropathic diabetic patients and may contribute to the enhanced morbidity and mortality from cardiovascular diseases characteristically observed in IDDM patients with diabetic nephropathy.

L-Homocysteine and L-homocystine stereospecifically induce endothelial nitric oxide synthase-dependent lipid peroxidation in endothelial cells.

Atherothrombotic cardiovascular disease associated with hyperhomocysteinemia has been proposed to result, at least in part, from increased vascular oxidative stress. Here we characterize one mechanism by which homocyteine may induce a vascular cell type-specific oxidative stress. Our results show that L-homocysteine at micromolar levels stereospecifically increases lipid peroxidation in cultured endothelial cells, but not in vascular smooth muscle cells or when medium is incubated in the absence of cells. Consistent with these observations, homocysteine also increases the formation of intracellular reactive oxygen species. The pro-oxidant effect of homocysteine can be fully replicated by an equivalent concentration of homocystine (i.e., an oxidized form of homocysteine), but not with cysteine or glutathione. Homocyst(e)ine-dependent lipid peroxidation is independent of H(2)O(2) and alterations in glutathione peroxidase activity, but dependent on superoxide. Mechanistically, the pro-oxidant effect of homocysteine appears to involve endothelial nitric oxide synthase (eNOS), as it is blocked by the eNOS inhibitor L-N(G)-nitroarginine methyl ester. Thus, homocyst(e)ine actively promotes oxidative stress in endothelial cells via an eNOS-dependent mechanism.

Effects of dietary folic acid supplementation on cerebrovascular endothelial dysfunction in rats with induced hyperhomocysteinemia.

This study shows, for the first time, that hyperhomocysteinemia induces endothelial dysfunction in a rat brain, and that this can be alleviated by dietary folic acid supplementation. Our experiments examined the effects of folic acid supplementation on the endothelial nitric oxide synthase (eNOS) expression in the hyperhomocysteinemic rat brain, and related the observed changes in eNOS expression to the expression of the cell adhesion molecule and the glucose transporter protein. The animals were raised on an experimental diet containing 0.3% homocystine for 2 weeks and then they were placed either on a 0.3% homocystine, 0.3% homocystine with 8 mg/kg folic acid, or folic acid (8 mg/kg) diet for 2 weeks. The cerebrovascular eNOS activity was examined immunohistochemically. Cerebral levels of eNOS, glucose transporter-1 (GLUT-1), and the vascular cell adhesion molecule-1 (VCAM-1) proteins were evaluated by Western blot analysis. At 4 weeks, the homocystine diet induced a fourfold increase in plasma homocysteine (control: 6.5+/-0.4 micromol/l, homocystine: 26.2+/-2.5 micromol/l), and a reduction in the cerebral eNOS and GLUT-1 expression levels with a concomitant increase in the level of VCAM-1 expression. Dietary folic acid supplementation caused a significant decrease in the plasma homocysteine levels, a concomitant increase in the hyperhomocysteinemia-induced reduction in the cerebral eNOS and GLUT-1 expression levels, and a decrease in the hyperhomocysteinemia-induced VCAM-1 expression levels.

Elevated levels of homocysteine increase IL-6 production in monocytic Mono Mac 6 cells.

Hyperhomocysteinemia is a risk factor for atherosclerosis and thrombosis. The aim of this study was to analyze if exposure of monocytic cells to increased levels of homocysteine (HCY) induces the accumulation of inflammatory mediators. Interleukin (IL)-6 production by monocytic cell line Mono Mac 6 (MM6) was 1.7-fold increased in the presence of 50 micromol/l HCY and 3.5-fold with 200 micromol/l HCY. Incubation with homocystine resulted in a comparable dose-dependent increase, but neither cysteine nor methionine stimulated IL-6 accumulation. Elevated homocysteine concentrations did not affect the production of IL-8 and monocytic chemotactic protein-1 (MCP-1) in MM6. Furthermore, lipopolysaccharide (LPS) stimulation of MM6, cultured with elevated HCY (200 micromol/l) levels, resulted in a 3.5-fold increased response after 18 h, whereas no effect on LPS-induced IL-8 and MCP-1 response was observed. In conclusion, increased concentrations of homocysteine induce IL-6 accumulation in monocytic cells. After treatment with homocysteine, monocytic cells become more susceptible to endotoxin. This study is in favor of an association between homocysteine and monocytic IL-6 production.

Effect of homocysteine on cytokine production by human endothelial cells and monocytes.

BACKGROUND: Hyperhomocysteinaemia is an independent risk factor in the development of cardiovascular disease. Although homocysteine has been shown to affect endothelial cell function, the mechanisms by which it induces disease states are still poorly understood. Here, we report the ability of homocysteine to influence inflammatory cytokine/chemokine production by human saphenous vein endothelial cells, peripheral blood monocytes and monocyte-derived macrophages. METHODS: Human saphenous vein endothelial cells, peripheral blood monocytes and monocyte-derived macrophages were treated with homocysteine (0.1-5 mmol/L) for 4 and/or 24h. Tumour necrosis factor (TNF)-alpha, interleukin (IL)-1beta, IL-6 and IL-8 production was measured in the cell culture media using commercially available enzyme-linked immunosorbent assays. RESULTS: Interleukin-6 production by human saphenous vein endothelial cells was significantly stimulated following a 24-h treatment with homocysteine, whilst IL-8 concentrations were inhibited after both 4- and 24-h treatments. Homocysteine was also found to stimulate IL-1beta production by human peripheral blood monocytes and TNF-alpha production by monocyte-derived macrophages. CONCLUSIONS: Overall, results from this study suggest that homocysteine alters the profile of cytokine/chemokine production by endothelial cells and macrophages. This altered profile may be important in the inflammatory events that initiate or enhance the development of atherosclerotic lesions.