Vein graft disease in a knockout mouse model of hyperhomocysteinaemia

A major reason for vein graft failure after coronary artery bypass grafting is neointimal hyperplasia and thrombosis. Elevated serum levels of homocysteine (Hcy) are associated with higher incidence of cardiovascular disease, but homocysteine levels also tend to increase during the first weeks or months after cardiac surgery. To investigate this further, C57BL/6J mice (WT) and cystathionine‐beta‐synthase heterozygous knockout mice (CBS+/?), a mouse model for hyperhomocysteinaemia, underwent interposition of the vena cava of donor mice into the carotid artery of recipient mice. Two experimental groups were examined: 20 mice of each group underwent bypass surgery (group 1: WT donor and WT recipient; group 2: CBS+/? donor and CBS+/? recipient). After 4 weeks, the veins were harvested, dehydrated, paraffin‐embedded, stained and analysed by histomorphology and immunohistochemistry. Additionally, serum Hcy levels in CBS knockout animals and in WT animals before and after bypass surgery were measured. At 4 weeks postoperatively, group 2 mice showed a higher percentage of thrombosis compared to controls, a threefold increase in neointima formation, higher general vascularization, a lower percentage of elastic fibres with shortage and fragmentation in the neointima, a lower percentage of acid mucopolysaccharides in the neointima and a more intense fibrosis in the neointima and media. In conclusion, hyperhomocysteinaemic cystathionine‐beta‐synthase knockout mice can play an important role in the study of mechanisms of vein graft failure. But further in vitro and in vivo studies are necessary to answer the question whether or not homocysteine itself or a related metabolic factor is the key aetiologic agent for accelerated vein graft disease.     

Plasma homocysteine is related to albumin excretion rate in patients with diabetes mellitus: a new link between diabetic nephropathy and cardiovascular disease?

Summary The high risk of cardiovascular disease in patients with diabetes mellitus, particularly in those with nephropathy, is not completely explained by classical risk factors. A high plasma homocysteine concentration is an independent risk factor for cardiovascular disease but information on its association with diabetes is limited. Fasting homocysteine concentrations were measured in the plasma of 165 diabetic patients (75 with insulin-dependent [IDDM]; 90 with non-insulin-dependent diabetes [NIDDM]) and 56 non-diabetic control subjects. Other measurements included the prevalence of diabetic complications, glycaemic control, lipid and lipoprotein levels, vitamin status and renal function tests. Patients with NIDDM had higher homocysteine levels than control subjects, whereas IDDM patients did not (9.2 ± 4.5 vs 7.7 ± 2 μmol/l, p < 0.01; and 7.0 ± 3 vs 7.4 ± 2 μmol/l, NS). Univariate correlations and multiple regression analysis showed albumin excretion rate to be the parameter with the strongest independent association with homocysteine. Patients with both types of diabetes and nephropathy had higher plasma homocysteine concentrations than those without nephropathy. Increases of homocysteine in plasma were related to increases in the severity of the nephropathy. Fasting hyperhomocysteinaemia was considered as the mean of the plasma homocysteine for all control subjects (7.5 ± 2.1 μmol/l) + 2 SD (cut-off =11.7 μmol/l). Nephropathy was present in 80 % of diabetic patients with fasting hyperhomocysteinaemia. In conclusion, increases in fasting homocysteine in diabetic patients are associated with increased albumin excretion rate, especially in those with NIDDM, thus providing a potential new link between microalbuminuria, diabetic nephropathy and cardiovascular disease. [Diabetologia (1998) 41: 684–693] Received: 4 August 1997 and in final revised form: 4 February 1998     

Hyperhomocysteinaemia-induced atrial remodelling in hypertensive rats

Studies from our laboratory and others indicate that, in addition to its purported atherothrombotic effects, hyperhomocysteinaemia (Hhe) is a powerful stimulus for ventricular remodelling, dysfunction and clinical heart failure. Because changes in atrial structure and function can impact on cardiac function in progressive ventricular remodelling and dysfunction, we conducted experiments to examine structural and functional remodelling of the atria in the hyperhomocysteinaemic hypertensive rat, a previously described model of ventricular diastolic dysfunction. Atrial muscle preparations were isolated from hearts of spontaneously hypertensive rats that were fed control, intermediate Hhe-inducing or severe Hhe-inducing diet for 10 weeks. Atrial developed tension, +dT/dt(max) and -dT/dt(max) were found to increase in parallel with levels of Hhe and ventricular diastolic dysfunction. Post-rest developed tension and the maximum developed tension observed in the presence of isoproterenol were also increased significantly in both Hhe groups compared with control. These results indicate that Hhe increases both basal and maximal contractile function in atrial muscle. Atrial structural remodelling was characterized by increased interstitial fibrosis in both Hhe groups. These data suggest that Hhe-associated changes in atrial structure and function may act to maintain ventricular filling in Hhe-induced diastolic dysfunction.     

Severe methylenetetrahydrofolate reductase deficiency revealed by a pulmonary embolism in a young adult

Deficiency in methylenetetrahydrofolate reductase (MTHFR), the enzyme involved in the remethylation of homocysteine to methionine using methyltetrahydrofolate as cofactor, induces hyperhomocysteinaemia, homocysteinuria, hypomethioninaemia and low methylfolate levels. Diagnosis usually occurs during infancy because of various neurological abnormalities. We report MTHFR deficiency diagnosed in an adult woman after a pulmonary embolism. Her adult sister, intellectually retarded, suffered from the same disease. Molecular analysis of the MTHFR gene exhibited four different mutations (two missense mutations, one exon skipping and C677T). The impact of these mutations was analysed through the biological abnormalities in the parents and children.     

The use of an in vivo model to study the effects of hyperhomocysteinaemia on vascular function

BACKGROUND/AIMS: To use an in vivo rat model of hyperhomocysteinaemia (HHCy) to study its impact on vascular function. METHODS: Twenty rats were fed either a control or HHCy-inducing diet for 10 wk. The response of aortic rings to contraction with phenylephrine, and relaxation to acetylcholine (endothelium-dependant relaxation) or sodium nitroprusside (endothelium-independent relaxation) was analyzed. The results were compared using an analysis of variance (ANOVA). RESULTS: There was a significant elevation of HCy in the treated group (20.5 versus 1.6 micromol/L, P = 0.004). There was no significant difference between the two groups in blood pressure measurements (ANOVA, P = 0.152). In a dose-dependant manner, phenylephrine elicited significantly greater contraction in aorta taken from HHCy rats than that taken from controls (ANOVA, P < 0.001), acetylcholine elicited significantly less percentage relaxation in aorta taken from HHCy rats than from controls (ANOVA, P = 0.003) and though sodium nitroprusside stimulated less percentage relaxation in aorta taken from HHCy rats than controls, this did not reach significance (ANOVA, P = 0.051). CONCLUSIONS: In diet induced hyperhomocysteinaemic rats, there is enhanced vascular contraction in response to phenylephrine and impaired endothelium-dependant relaxation in response to acetylcholine.     

Mecobalamin

Background: Mecobalamin, one of the coenzyme forms of vitamin B₁₂, acts as an important cofactor in the activities of B₁₂-dependent methyltransferases. Since the discovery of mecobalamin, it has been applied mainly in the treatment of hyperhomocysteinaemia and peripheral neuropathy. However, there is still lack of a systemic review on the clinical administration of mecobalamin and its potential mechanism. Objective: To review the mechanism, clinical efficacy and safety of mecobalamin in the treatment of hyperhomocysteinaemia and peripheral neuropathy. Methods: First, the potential mechanism, pharmacokinetics and metabolism of mecobalamin were clarified. In addition, the clinical administration including efficacy, safety and tolerability of mecobalamin as monotherapy or combined therapy in the treatment of hyperhomocysteinaemia and peripheral neuropathy were also detailed. Results/conclusions: Although both monotherapy and combined therapy can lower plasma/serum homocysteine levels and improve the neuropathic symptoms, combined therapy with other B vitamins seems to be more effective. However, more precise, double-blind and randomised control studies are necessary to confirm the efficacy of mecobalamin on hyperhomocysteinaemia, peripheral neuropathy interaction, and cardiovascular, neurological and osteoporotic mortality or morbidity.     

The VITA Project: C677T mutation in the methylene-tetrahydrofolate reductase gene and risk of venous thromboembolism

We evaluated the hypothesis that a common polymorphism of the methylenetetrahydrofolate reductase gene (C677T), which results in increased levels of plasma homocysteine, may be a putative risk factor for venous thromboembolism (VT). Sixty-five cases of VT and 130 controls, both identified within the framework of an epidemiologic survey on thrombophilia, the Vicenza Thrombophilia and Arteriosclerosis (VITA) Project, were genotyped for the mutation. No increased risk of VT was found in carriers of the mutation. We conclude that screening for the C677T mutation of the methylenetetrahydrofolate reductase gene should not be recommended in unselected patients with VT.     

The methylenetetrahydrofolate reductase gene C677T polymorphism in patients with homozygous sickle cell disease and stroke

Homozygosity for the methylenetetrahydrofolate reductase (MTHFR) gene C677T polymorphism may cause hyperhomocysteinaemia, a recognized risk factor for stroke, in individuals with folate deficiency. Homozygous sickle cell (SS) disease is associated both with increased demands for folic acid and a tendency to develop stroke. We therefore investigated a possible role of the MTHFR C677T polymorphism in SS disease patients with stroke. Investigation of the frequency of the polymorphism in 48 patients with stroke and in 48 age-, sex- and racially-matched SS controls without stroke failed to reveal a difference between the groups (Fisher exact test, P = 0.99). Homozygosity for the MTHFR C677T polymorphism is unlikely to be a risk factor for stroke in this population with SS disease.     

Folic acid 5 or 15 mg/d similarly reduces plasma homocysteine in patients with moderate-advanced chronic renal failure

BACKGROUND: Hyperhomocysteinaemia is an independent risk factor for cardiovascular disease with a remarkable prevalence in patients with chronic renal failure (CRF). Low doses of folic acid (FA) with or without vitamin B6 and B12 has been shown to effectively reduce plasma homocysteine (Hcy). The aim of this study was to compare the short-term effects of two different oral doses of FA (5 vs 15 mg/d) on plasma Hcy levels in subjects suffering from moderate-severe CRF. METHODS: A double-blind, double-dummy, comparative, two-stage randomised study was performed. Seventeen patients aged 45-71 years, with glomerular filtration rates between 15.4-50 mL/min 1.73/m2 were randomly assigned to receive FA 5 mg/d (FA-5, n: 8) or FA 15 mg/d (FA-15, n: 9) for 30 days. At the end of this 30-day double-blind period, all the participants were placed on FA 5 mg/d (open period), for 5 additional months. Both groups were also supplemented with vitamins B1, B6 and B12 throughout the trial. Blood samples were drawn at 0, 15, 30, 90 and 180 days to assess Hcy, complete blood count (CBC) and sequential multichannel analysis (SMA). Chest X-ray and a 12-lead electrocardiogram (ECG) were also performed. RESULTS: Plasma Hcy (mean +/- SEM) decreased from 27.9 +/- 1.4 (baseline) to 15.1 +/- 0.6, 13.3 +/- 0.9, 14.1 +/- 0.5 and 13.8 +/- 0.5 micromol/L (FA-5) and from 28.8 +/- 2.7 to 15.6 +/- 1.2, 14.4 +/- 1.3, 13.0 +/- 0.7 and 13.1 +/- 0.6 micromol/L (FA-15) at days 15, 30, 90 and 180, respectively. (P < 0.01 from day 15 to 180 vs baseline for both groups with no differences between them). Renal function remained stable throughout the entire period of the study in all but one patient in whom it deteriorated to pre-end stage disease. No adverse cardiovascular events developed during the trial. CONCLUSION: Both folic acid doses induced a significant and similar decrease in plasma Hcy in subjects with moderate-severe chronic renal failure. The possible dose-related effect of this approach in reducing the risk of accelerated sclerotic vascular disease and cardiovascular events in this especially vulnerable population should be a matter of further investigation.     

Lipid peroxidation and susceptibility of low-density lipoprotein to in vitro oxidation in hyperhomocysteinaemia

Abstract. The pathobiochemical mechanism of arteriosclerosis in hyperhomocysteinaemia has not yet been elucidated. In vitro studies have shown that the cytotoxic properties of homocysteine can be ascribed to its generation of reactive oxygen species. We studied lipid peroxidation, both in vivo and in vitro , in 10 homozygous cystathionine synthase-deficient (CSD) patients and in a control group of 10 healthy subjects of comparable age and sex. The susceptibility of low-density lipoprotein (LDL) from hyperhomo-cysteinaemic patients to oxidation was determined in vitro by continuously measuring the conjugated diene production induced by incubation with copper ions. Oxidation resistance (expressed as lag time), maximal oxidation rate, and extent of oxidation (expressed as total diene production) of LDL from CSD patients were not significantly different from those of LDL from controls. Furthermore, the time needed to reach maximal diene production, i.e. t(max), was similar for LDL from patients and controls. In addition, the vitamin E concentrations in LDL of CSD patients and controls were similar. The mean concentration (± SD) of plasma thiobarbituric acid reactive substances (TBARS), an indicator of in vivo lipid peroxidation, was 2.2 ± 0.7 μmol L^-1 in CSD patients, a lower value than that measured in the matched controls (50± 2.0 μmol L^-1). Investigation of in vivo and in vitro parameters of lipid peroxidation shows that the increased risk of arteriosclerosis in hyperhomocysteinaemia is unlikely to be due to increased lipid peroxidation.