Brain Advance Access originally published online on November 7, 2003
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Brain, Vol. 127, No. 1, 212-219, 2004
© 2004 Guarantors of Brain
doi: 10.1093/brain/awh023
Homocysteine is a risk factor for cerebral small vessel disease, acting via endothelial dysfunction
1 Department of Clinical Neurosciences, St George’s Hospital Medical School, 2 Department of Haematology, Guy’s and St Thomas’s Trust, St Thomas’s Hospital, 3 Department of Medical Genetics and 4 Department of Clinical Biochemistry, St George’s Hospital, London and 5 Department of Neurology, St James’s University Hospital, Leeds
Correspondence to: Professor Hugh Markus, Department of Clinical Neurosciences, St George’s Hospital Medical School, London SW17 ORE, UK E-mail: h.markus{at}sghms.ac.uk
 |
Summary |
|---|
 Top Summary IntroductionMethodsResultsDiscussionReferences |
|---|
Cerebral small vessel disease (SVD) causes focal lacunar infarction and more diffuse ischaemia, referred to as leukoaraiosis. Endothelial dysfunction has been proposed as a causal mechanism in the disease. Homocysteine is toxic to endothelium. We determined whether elevated homocysteine levels and the methylene tetrahydrofolate reductase (MTHFR) C677T polymorphism are risk factors for SVD as a whole, and for two different SVD subtypes: isolated lacunar infarction and ischaemic leukoaraiosis. We also determined whether any association was mediated by endothelial dysfunction, as assessed by circulating endothelial markers. One hundred and seventy-two Caucasian patients with SVD and 172 community controls of similar age and sex were studied. Serum homocysteine measurement and MTHFR genotyping was performed. Levels of intercellular adhesion molecule 1 (ICAM1) and thrombomodulin were measured in a subgroup. Mean homocysteine levels were higher in SVD than controls [14.55 µmol/l [95% confidence interval (CI) 13.78–15.35] versus 12.01 µmol/l (95% CI 11.42–12.64), P < 0.0005]. Homocysteine was a stronger risk factor in those with ischaemic leukoaraiosis [12.92 (95% CI 4.40–37.98), P < 0.0005) per µmol increase in log homocysteine concentration (P < 0.0005)] in comparison with isolated lacunar infarction [4.22 (95% CI 1.29–13.73), P = 0.02] after controlling for both conventional risk factors and age. The MTHFR 677T allele was a risk factor only in the ischaemic leukoaraiosis group [odds ratio (OR) 2.02 (95% CI 1.31–3.1), P = 0.001]. Inclusion of the endothelial markers ICAM1 and thrombomodulin in a logistic regression model resulted in the association between homocysteine and SVD no longer being significant. In conclusion, hyperhomocysteinaemia is an independent risk factor for SVD, particularly ischaemic leukoaraiosis, and this effect may be mediated via endothelial dysfunction. Homocysteine-lowering therapy may be particularly effective in this subgroup.
Key Words: cerebral small vessel disease; homocysteine; MTHFR; endothelial dysfunction; cerebrovascular disease
Abbreviations: CI = confidence interval; ICAM1 = intercellular adhesion molecule 1; MTHFR = methylene tetrahydrofolate reductase; OR = odds ratio; SVD = small vessel disease; TM = thrombomodulin
Received April 9, 2003. Revised July 25, 2003. Accepted August 25, 2003.
|
Introduction |
|---|
|
TopSummaryIntroductionMethodsResultsDiscussionReferences |
|---|
Lacunar infarction, resulting from disease of the cerebral small vessels, accounts for a quarter of ischaemic strokes. Infarcts can be isolated or accompanied by diffuse changes, referred to as leukoaraiosis, in the periventricular white matter seen on CT or MRI. Both lacunar infarction and leukoaraiosis are thought to be caused by cerebral small vessel disease (SVD). At autopsy, thickening and hyaline deposition of the small perforating end-arterioles supplying the white matter can be seen, and in some cases of larger symptomatic lacunar infarction localized microatheroma was found at the origin of the deep perforating arterioles (Fisher, 1968, 1979). The neuropathological appearance corresponding to leukoaraiosis is neuronal loss, ischaemic demyelination and gliosis (Pantoni and Garcia, 1997
). Both clinical and pathological studies support the hypothesis that lacunar infarction and ischaemic leukoaraiosis represent different forms of small vessel disease. It is hypothesized that if ischaemia is acute this causes small focal regions of parenchymal damage in perforating arteriole territories (lacunar infarction), while if it is more chronic it results in diffuse ischaemic injury (leukoaraiosis). Leukoaraiosis itself is a radiological definition and can also be caused by non-ischaemic pathologies, although ischaemia is believed to account for the vast majority of cases. The term ‘ischaemic leukoaraiosis’, defined as radiological leukoaraiosis with a clinical lacunar syndrome, has been introduced to identify a group of patients in whom leukoaraiosis is likely to have an ischaemic basis (Jones et al., 1999).
The pathogenesis of cerebral SVD is not fully understood. Hypertension is the major risk factor but fails to account for much of the risk (Boiten and Lodder, 1995). It has been proposed that newer risk factors, including genetic predisposition, are important (Carmelli et al., 1998). Several lines of evidence have suggested that chronic endothelial dysfunction plays a pivotal role. This may be responsible for breakdown of the blood–brain barrier (Tomimoto et al., 1996; Lin et al., 2000) and impaired cerebral autoregulation (Bakker et al., 1999; Terborg et al., 2000), which have been observed in SVD. Endothelial dysfunction can be assessed in vivo by measuring soluble plasma markers (Cines et al., 1998). These are released into the circulation in response to endothelial activation by a number of different proatherogenic stimuli. Surface expression of intercellular adhesion molecule 1 (ICAM1) is a precondition for the adhesion and transendothelial migration of lymphocytes (Bevilacqua et al., 1989) and blood levels reflect an endothelial inflammatory response. Thrombomodulin (TM) is normally expressed on the endothelial cell surface, where, with thrombin, it regulates the activity of protein C. Increased plasma levels are thought to reflect endothelial damage (Ishii et al., 1991). Levels of both ICAM1 and TM are elevated in patients with SVD (Kario et al., 1996; Fassbender et al., 1999a; Hassan et al., 2003). Homocysteine could also be an important risk factor for SVD. This hypothesis is biologically plausible because homocysteine has been shown to have important effects on endothelial function in vitro (Wall et al., 1980; Stamler et al., 1993) and in vivo (Tawakol et al., 1997; Woo et al., 1997). Moderate hyperhomocysteinaemia can be caused by genetic or environmental factors or a combination of both. The most frequent genetic defect involves the enzyme methylene tetrahydrofolate reductase (MTHFR). A common polymorphism (C677T) is associated with hyperhomocysteinaemia, the highest levels of homocysteine being found in those with the TT genotype (Frosst et al., 1995).
Whilst many studies have described the relationship between homocysteine and stroke in general (Hankey and Eikelboom, 2001), and intra- or extracranial large vessel disease (Yoo et al., 1998; McQuillan et al., 1999; Spence et al., 1999), there have been fewer studies which have specifically examined the role of homocysteine in SVD or its different subtypes. Some studies have suggested that homocysteine is a more potent risk factor for SVD in comparison with other stroke subtypes (Evers et al., 1997; Fassbender et al., 1999b), whilst others have not been able to confirm this (Lindgren et al., 1995; Eikelboom et al., 2000). Potential explanations for these disparate findings include small sample sizes and differences in study design, particularly the selection of controls. Heterogeneity within SVD could also be an issue, particularly if homocysteine was more closely related to the presence of diffuse leukoaraiosis or focal lacunar infarction. Therefore in this study we determined whether homocysteine (and the MTHFR C677T polymorphism) is a risk factor for SVD as a whole, or for different SVD subtypes. We also examined if the effects of homocysteine were mediated by endothelial dysfunction, determined using the circulating markers of endothelial function ICAM1 and TM.
|
Methods |
|---|
|
TopSummaryIntroductionMethodsResultsDiscussionReferences |
|---|
Study population
One hundred and seventy-two consecutive Caucasian patients with cerebral SVD attending outpatient stroke clinics were enrolled. Cerebral SVD was defined as a presentation with a clinical lacunar syndrome (Bamford et al., 1987
) with a compatible lesion on neuroimaging. In the cerebral SVD group many patients (particularly those with leukoariosis) had other features associated with SVD, including cognitive impairment (8%), gait disorder excluding hemiplegia (16%), and a past or current history of mood disturbance (22%). All patients had standard stroke investigation, including brain imaging, and carotid artery imaging with duplex or magnetic resonance angiography. Exclusion criteria included subcortical infarction 
1.5 cm diameter, cortical infarction of any size, a potential source of cardiac source of embolism (Adams et al., 1993), and large vessel cerebrovascular disease, defined as carotid or vertebral artery stenosis >50%. One hundred and seventy-two Caucasian community controls without a history or signs of cerebrovascular disease were also recruited by sampling of family doctor lists from the same geographical regions as the patients. Sampling was stratified to provide a distribution of age and sex similar to that in the patient group. Brain imaging was not performed in controls.
All patients and controls were reviewed by one physician and underwent a standardized clinical assessment. Hypertension was defined as a systolic blood pressure >160 mm Hg or diastolic pressure >95 mm Hg (World Health Organization–International Society of Hypertension, 1993), or current treatment with antihypertensive drugs. A positive smoking history was recorded for those who had smoked at any time in their lives. Diabetes mellitus was defined as a previous diagnosis of insulin or non-insulin-dependent diabetes. The study protocol was approved by local research ethics committees and informed consent was obtained from all participants.
Blood sampling and laboratory methods
In all patients, non-fasting blood was taken at least 2 months after the last clinical ischaemic event because both depression of homocysteine levels (Stein and McBride, 1998) and transient elevation of some endothelial markers have been reported in the first few weeks after acute stroke (Fassbender et al., 1995; Lindsberg et al., 1996). Twenty millilitres of blood was obtained and divided into polypropylene tubes for serum and siliconized glass tubes containing 0.105 M sodium citrate for plasma collection. Specimens were centrifuged at 3000 g for 10 min and the isolated serum and plasma was stored at –80°C. Total serum homocysteine concentration was measured using reversed-phase high-performance liquid chromatography with fluorescent detection after manual preparation of derivatives. The intra-assay (inter-assay) coefficients of variation were 3% (5%). DNA was extracted from leucocytes and MTHFR genotyping was performed as described previously (Frosst et al., 1995). Circulating markers of endothelial function were measured using enzyme-linked immunosorbent assay-based commercially available kits in the first consecutive 110 cases and 50 controls. The methods and results of these assays have been reported in detail in a related study concerning the role of endothelial dysfunction in cerebral SVD (Hassan et al., 2003).
Assessment of scans
MRI scans were performed in 142 patients (82.5%). In all of the cases with MRI, axial T2-weighted images were evaluated blind to clinical and laboratory data by a single observer using a semiquantitative rating scale. The Fazekas scale was used to score leukoaraiosis as this scale been shown to reflect pathological severity of cerebral SVD in a post-mortem validation study (Fazekas et al., 1993). An additional category was included to allow differentiation of more severe cases of leukoaraiosis. Leukoaraiosis was rated as: 1 = absent or mild (equivalent to Fazekas periventricular score 
2); 2 = moderate (Fazekas scale 3); 3 = severe (more than half of the hemispheric white matter involved). In addition we scored the number of lacunar infarcts: 1 = 2 lesions; 2 = 3–5 lesions; 3 = >5 lesions. Separate scores were generated for small (5 mm maximal diameter) and large (6–14 mm maximal diameter) focal lesions.
Subtyping of SVD
To explore possible pathogenic differences between patients with isolated lacunar infarcts and those with ischaemic leukoaraiosis, cerebral SVD patients with MRI were subtyped according to their scan appearances. Isolated lacunar infarction was defined as at least one focal lesion and a leukoaraiosis score of 1 (absent or mild). Ischaemic leukoaraiosis was defined as at least one focal lesion and a leukoaraiosis score of 2–3. Twenty MRI scans were also randomly selected for re-evaluation by the same observer. There was perfect repeatability (
= 1.0) for allocation of subtype. For the MRI disease scores, the repeatability (weighted kappa) was good for the leukoaraiosis category ( = 0.85), and moderate for both the large ( = 0.58) and small focal lesion categories ( = 0.43).
Statistical analysis
Natural logarithmic transformation was used to normalize distributions prior to analysis by parametric tests. Otherwise non-parametric tests were used. Homocysteine levels are expressed as geometric means with the 95% confidence interval (CI) for the mean. Separate analyses were performed in those with MRI to determine differences between SVD subtypes and association with extent of disease. The 
2 test was used to compare proportions and the unpaired t test or analysis of variance to compare normally distributed data between two or more groups. Post hoc tests were performed using Bonferroni’s method. Logistic regression analysis was performed to determine the relationship between homocysteine and SVD presence. Linear regression was used to determine the association between homocysteine and SVD grade. Association between MTHFR genotype and presence of SVD was tested according to additive (TT versus CT versus CC), dominant (TT + CT versus CC) and recessive (TT versus CC + CT) models in univariate and multivariate analyses. The –2 log likelihood ratio was used to assess logistic regression models where SVD was the dependent variable and homocysteine a covariate, before and after inclusion of endothelial markers. Repeatability measures for phenotype assignment and MRI grading scales was based on scores (Altman, 1991)
|
Results |
|---|
|
TopSummaryIntroductionMethodsResultsDiscussionReferences |
|---|
Characteristics of cases and controls
SVD patients and controls were well matched for age and gender, but hypertension, smoking and diabetes were more common in the SVD group (Table 1). Of the 142 cases with MRI, 52 were subtyped as isolated lacunar infarction (36.6%) and 90 as ischaemic leukoaraiosis (63.4%). In these 142, the severity of leukoaraiosis was scored as grade 1 in 52 patients (36.6%), grade 2 in 68 (47.9%) and grade 3 in 22 (15.5%). For large focal lesions (6–14 mm) the scores were as follows: grade 1, 109 cases (76.8%); grade 2, 25 cases (17.6%); grade 3, eight cases (5.6%). For small focal lesions (
5 mm) the distribution was: grade 1, 17 (12.0%); grade 2, 11 (7.7%); grade 3, 114 (80.3%).
|
Relationships between homocysteine and conventional risk factors
In the whole study population, there were significant associations between homocysteine levels and age (r = 0.20, P < 0.0005) and serum creatinine (r = 0.35, P < 0.0005). Elevated homocysteine levels were associated with male gender (13.83 µmol/l, 95% CI 13.14–14.57 versus 12.40 µmol/l, 95% CI 11.76–13.08 in females; P = 0.005), hypertension (13.90 µmol/l, 95% CI 13.28–14.60 versus 12.32 µmol/l, 95% CI 11.63–13.05; P = 0.002), and smoking (ever smoked 13.64 µmol/l, 95% CI 13.01–14.3 versus 12.40 µmol/l, 95% CI 11.67–13.19 in never smokers; P = 0.02). Vitamin status was available in a subgroup of cases; there were significant negative correlations between homocysteine and both serum folate (n = 45,
= –0.34, P = 0.024) and serum B12 (n = 35, = –0.38, P = 0.025).
Homocysteine levels in cases and controls
Mean homocysteine levels were higher in patients with SVD than in controls (14.55 µmol/l, 95% CI 13.78–15.35, versus 12.01 µmol, 95% CI 11.42–12.64; P < 0.0005). The difference remained significant after controlling for age, sex, hypertension, diabetes mellitus, smoking history, history of myocardial infarction, serum cholesterol, creatinine concentration and MTHFR genotype (P < 0.0005). The odds ratio (OR) for SVD increased with increasing quartile of homocysteine with the lowest quartile as reference (P < 0.0005 for trend) (Table 2). After adjustment for risk factors and creatinine, the OR associated with homocysteine was 8.34 (95% CI 3.63–19.14) per 1 µmol increase in log concentration (P < 0.0005). This was only slightly reduced (to 7.91, 95% CI 3.93–18.44; P < 0.0005) after additionally controlling for MTHFR genotype.
|
Homocysteine and SVD subtype
There were significant differences in homocysteine levels among the subtypes of SVD and controls (analysis of variance, P < 0.0005). Homocysteine levels were higher amongst patients with ischaemic leukoaraiosis (15.15 µmol /l, 95% CI 14.13–16.24) compared with both those with isolated lacunar infarction (13.14 µmol/l, 95% CI 11.99–14.39; P = 0.04) and controls (12.01 µmol/l, 95% CI 11.42–12.64; P < 0.0005). After adjustment for conventional risk factors and creatinine, and both before and after additional adjustment for MTHFR genotype, homocysteine levels remained significantly higher amongst ischaemic leukoaraiosis and isolated lacunar infarction in comparison with controls (Fig. 1). For isolated lacunar infarction, homocysteine was associated with an OR of 4.22 (95% CI 1.29–13.73, P = 0.02) per 1 µmol increase in log homocysteine concentration after adjustment for risk factors and creatinine, and 4.00 (95% CI 1.21–13.19, P = 0.02) after additional adjustment for MTHFR genotype. For ischaemic leukoaraiosis the adjusted OR was 12.92 (95% CI 4.40–37.98, P < 0.0005), and 11.05 (95% CI 3.66–33.38, P < 0.0005) after additional adjustment for MTHFR genotype.
|
Homocysteine and extent of SVD
Homocysteine levels correlated with the grade of leukoaraiosis (P = 0.02) and large focal lesions (P = 0.04), but not with the grade of small focal lesions (P = 0.80). After adjustment for risk factors and creatinine, both leukoaraiosis and large focal lesions remained associated with homocysteine levels (P = 0.02 and P = 0.006, respectively), and (P = 0.048 and P = 0.008 respectively) after additional adjustment for MTHFR genotype.
MTHFR genotype and homocysteine concentration
The MTHFR genotype was determined in 170 cases and 170 controls. In two cases and two controls DNA could not be amplified. MTHFR genotype was in Hardy–Weinberg equilibrium amongst the entire study population (2 = 1.00, P = 0.61), cases (2 = 1.48, P = 0.48) and controls (2 = 0.00, P = 1.00). In the whole study group, there was a graded increase in homocysteine concentration across the MTHFR genotypes (P = 0.005 for trend) with homocysteine concentrations highest in those with the TT genotype (Fig. 2). Following adjustment for vascular risk factors and creatinine the trend remained significant (P = 0.02).
|
MTHFR genotype and SVD
There was a significant difference in MTHFR genotype distribution between cases and controls [TT 33 (19.4%), CT 71 (41.8%), CC 66 (38.8%) in cases versus TT 16 (9.41%), CT 73 (42.94%) and CC 81 (47.6%) in controls;
2 = 7.46, P = 0.02]. According to an additive model, the OR for SVD associated with the 677T allele was 1.47 (95% CI 1.08–2.00, P = 0.01). Using a recessive model the OR was 2.32 (95% CI 1.22–4.40, P = 0.01). There was no association with the MTHFR 677T allele according to a dominant model (OR 1.19, 95% CI 0.95–1.59, P = 0.14). The MTHFR 677T allele remained associated with the presence of disease following adjustment for vascular risk factors and serum creatinine (additive model, OR 1.54, 95% CI 1.09–2.18, P = 0.01; recessive model, OR 2.72, 95% CI 1.32–5.60, P = 0.007].
MTHFR genotype and SVD subtype
The MTHFR polymorphism was associated with the ischaemic leukoaraiosis subtype, but there was no significant association with isolated lacunar infarction (Table 3). Consistent with this, the MTHFR genotype was over-represented in patients with moderate or severe white matter changes (grade 2 or above) on MRI (2 = 11.37, P = 0.02), whilst there was no difference in genotype distribution across the different grades of focal lesions scored (large lesions, 2 = 3.30, P = 0.51; small lesions, 2 = 2.83, P = 0.59).
|
Homocysteine and endothelial markers
Homocysteine levels correlated with TM (r = 0.37, P < 0.0005) and ICAM1 (r = 0.18, P = 0.02). In a logistic regression model, inclusion of endothelial markers improved the predictive model for the presence of SVD (decrease in –2 log likelihood), but homocysteine was no longer independently associated with SVD (Table 4).
|
|
Discussion |
|---|
|
TopSummaryIntroductionMethodsResultsDiscussionReferences |
|---|
This study confirms that homocysteine is an independent risk factor for SVD as a whole, but extends previous findings to suggest that it is a much stronger risk factor for the ischaemic leukoaraiosis subtype. Furthermore, we found evidence of a dose–response relationship, with increasing homocysteine concentrations associated with greater risk of developing SVD. We also found that homocysteine levels were associated with the markers of endothelial dysfunction ICAM1 and TM. Inclusion of these markers as covariates reduced the association with homocysteine but improved the overall logistic regression model for prediction of SVD. These findings are consistent with the hypothesis that endothelial dysfunction is an important mechanism through which homocysteine mediates its effects in SVD. The effects of homocysteine could include direct endothelial damage or stimulation of an endothelial inflammatory response, which have been noted previously in SVD (Kario et al., 1996
; Fassbender et al., 1999a).
Our findings are compatible with the known effects of homocysteine. In a number of in vitro and animal experiments, homocysteine has been shown to lead to endothelial cell injury and functional abnormalities in the release of endothelial nitric oxide (Wall et al., 1980; Stamler et al., 1993; Lentz et al., 1996). In human subjects, high levels of homocysteine impair nitric oxide-mediated blood flow responses (Tawakol et al., 1997; Woo et al., 1997) and previous studies have confirmed the association between homocysteine and circulating markers of endothelial dysfunction (Rohde et al., 1999).
In comparison with large vessel atherosclerosis (Yoo et al., 1998; McQuillan et al., 1999; Spence et al., 1999), the role of homocysteine in SVD is less well established, but the results of our study are consistent with earlier reports (Fassbender et al., 1999b; Matsui et al., 2001) that homocysteine is an important risk factor for SVD. In addition, we found homocysteine to be associated with the extent of disease, correlating with grade of both large focal lesions and leukoaraiosis. There was no association with the grade of small focal lesions 5 mm. This may reflect inconsistency in rating small T2 hyperintensities or heterogeneous aetiology, since very small lesions can equally represent perivascular spaces, gliosis and demyelination as well as ischaemic lacunar infarcts (Awad et al., 1986). However, focal hyperintensities >5 mm are likely to represent true lacunar infarcts associated with cerebral SVD (Braffman et al., 1988).
Cerebral SVD may represent a clinical and pathological spectrum, with different mechanisms underlying isolated lacunar infarcts and diffuse leukoaraiosis. It has been hypothesized that localized microatheroma at the origin of perforating arterioles may in part be responsible for some cases of isolated lacunar infarction, whilst diffuse arteriosclerosis is the predominant pathology in the leukoaraiosis group (de Jong et al., 2002). Consistent with this heterogeneity, we found differing associations between both homocysteine and the MTHFR genotype and between the two subtypes of SVD. Homocysteine levels were highest among ischaemic leukoaraiosis patients, whilst a more modest increase was seen in those with isolated lacunar infarcts. Therefore homocysteine could be more important in the development of small vessel arteriosclerosis than microatheroma. The differences in pathogenesis may explain the controversy concerning the relative importance of homocysteine in large and small vessel diseases. Some reports have concluded that homocysteine is particularly damaging to small penetrating arteries (Evers et al., 1997; Fassbender et al., 1999b), whilst other studies have found lower levels of homocysteine in SVD compared with other stroke subtypes (Lindgren et al., 1995; Eikelboom et al., 2000). The inconsistencies could reflect different SVD case mixes and the methods used to subtype patients. Interestingly, our ischaemic leukoaraiosis group were similar to the group studied by Fassbender and colleagues (Fassbender et al., 1999b). They similarly found very high levels of homocysteine among patients with subcortical arteriosclerotic encephalopathy, characterized by diffuse ischaemic white matter changes, cognitive impairment and lacunar stroke. In this group of patients, hyperhomocysteinaemia could predispose to cognitive decline and gait disturbance through direct excitotoxic and pro-apoptotic effects on white matter tracts as well as predisposing to small vessel ischaemic demyelination (Lipton et al., 1997; Kruman et al., 2000; Seshadri et al., 2002; Vermeer et al., 2002).
There is strong evidence from twin (Carmelli et al., 1998) and family history (Polychronopoulos et al., 2002) studies to suggest that genetic influences are important in the pathogenesis of SVD. Given the importance of homocysteine in SVD, we tested a commonly occurring polymorphism in the MTHFR gene (C677T). As previously demonstrated (Frosst et al., 1995), we found that the 677T allele influenced homocysteine levels, but it was also significantly over-represented in the ischaemic leukoaraiosis group but not in the isolated lacunar infarction group. This is consistent with our findings of a greater elevation of serum homocysteine levels in this group. The role of the MTHFR gene in stroke and vascular diseases in general remains controversial, but our study supports the notion that separation of phenotypes according to pathogenic basis is important in establishing underlying molecular genetic risk factors.
We did not specifically exclude the presence of white matter hyperintensities in controls by brain imaging but used a representative community control population. The requirement for brain imaging markedly reduces the recruitment rate and can introduce bias in control recruitment. Even if our controls did have asymptomatic cerebral SVD, this would not alter our conclusions; in contrast, their inclusion would be expected to reduce the magnitude of association reported with homocysteine and MTHFR genotype.
Random homocysteine was measured, as it was not practical to obtain fasting samples on patients who were seen in an outpatient setting. However, we were still able to replicate previously reported associations between homocysteine and a number of lifestyle and cardiovascular risk factors including blood pressure, male sex, smoking history and serum creatinine concentration (Eikelboom et al., 2000; Matsui et al., 2001).
Our results may have important clinical implications. Current treatment of patients with cerebral SVD is limited. Homocysteine levels can be modified using folate or B12 (Homocysteine Lowering Trialists’ Collaboration, 1998), which could be particularly effective in patients with SVD for a number of reasons. First, homocysteine levels in SVD correlated with low B12 and folate, consistent with earlier findings (Fassbender et al., 1999b). Secondly, the MTHFR 677TT genotype was over-represented in SVD, and it has been found that folate status particularly influences homocysteine levels in individuals with this genotype (Jacques et al., 1996; Malinow et al., 1997). Promisingly, lowering of homocysteine with folic acid has been shown to have beneficial effects on endothelial dysfunction, including improvement in TM levels (Constans et al., 1999) and endothelium-dependent blood flow responses (Chambers et al., 2000). Given our findings, if vitamin supplementation is effective it may well offer varying therapeutic benefit in different stroke subtypes and could be particularly beneficial in patients with leukoaraiosis.
|
Acknowledgements |
|---|
We wish to thank Professor Martin Brown, Dr Daffyd Thomas and Dr Dennis Briley for referring patients into the study, Kiran Parmar for her assistance with the endothelial marker assays and Linda Porteous for help with data collection. This work was funded by a Stroke Association (UK) Project Grant. A.H. also received support from St James’s Trust for Nervous System Diseases. Linda Porteous is supported by NHS research and development funding from St George’s Hospital.
|
References |
|---|
|
TopSummaryIntroductionMethodsResultsDiscussionReferences |
|---|
Adams HP Jr, Bendixen BH, Kappelle LJ, Biller J, Love BB, Gordon DL, et al. Classification of subtype of acute ischemic stroke. Definitions for use in a multicenter clinical trial. TOAST. Trial of Org 10172 in Acute Stroke Treatment. Stroke 1993; 24: 35–41.
Altman DG. Practical statistics for medical research. London: Chapman and Hall; 1991. p. 404–8.
Awad IA, Johnson PC, Spetzler RF, Hodak JA. Incidental subcortical lesions identified on magnetic resonance imaging in the elderly. II. Postmortem pathological correlations. Stroke 1986; 17: 1090–7.
Bakker SL, de Leeuw FE, de Groot JC, Hofman A, Koudstaal PJ, Breteler MM. Cerebral vasomotor reactivity and cerebral white matter lesions in the elderly. Neurology 1999; 52: 578–83.
Bamford J, Sandercock P, Jones L, Warlow C. The natural history of lacunar infarction: the Oxfordshire Community Stroke Project. Stroke 1987; 18: 545–51.
Bevilacqua MP, Stengelin S, Gimbrone MA Jr, Seed B. Endothelial leukocyte adhesion molecule 1: an inducible receptor for neutrophils related to complement regulatory proteins and lectins. Science 1989; 243: 1160–5.
Boiten J, Lodder J. Risk factors for lacunar infarction. In: Donnan G, Norrving B, Bamford J, Bogousslavsky J, editors. Lacunar and other subcortical infarctions. Oxford: Oxford University Press; 1995. p. 56–69.
Braffman BH, Zimmerman RA, Trojanowski JQ, Gonatas NK, Hickey WF, Schlaepfer WW. Brain MR: pathologic correlation with gross and histopathology. 1. Lacunar infarction and Virchow–Robin spaces. AJR Am J Roentgenol 1988; 151: 551–8.
Carmelli D, DeCarli C, Swan GE, Jack LM, Reed T, Wolf PA, et al. Evidence for genetic variance in white matter hyperintensity volume in normal elderly male twins. Stroke 1998; 29: 1177–81.
Chambers JC, Ueland PM, Obeid OA, Wrigley J, Refsum H, Kooner JS. Improved vascular endothelial function after oral B vitamins: an effect mediated through reduced concentrations of free plasma homocysteine. Circulation 2000; 102: 2479–83.
Cines DB, Pollak ES, Buck CA, Loscalzo J, Zimmerman GA, McEver RP, et al. Endothelial cells in physiology and in the pathophysiology of vascular disorders. Blood 1998; 91: 3527–61.
Constans J, Blann AD, Resplandy F, Parrot F, Renard M, Seigneur M, et al. Three months supplementation of hyperhomocysteinaemic patients with folic acid and vitamin B6 improves biological markers of endothelial dysfunction. Br J Haematol 1999; 107: 776–8.[CrossRef][Web of Science][Medline]
de Jong G, Kessels F, Lodder J. Two types of lacunar infarcts: further arguments from a study on prognosis. Stroke 2002; 33: 2072–6.
Eikelboom JW, Hankey GJ, Anand SS, Lofthouse E, Staples N, Baker RI. Association between high homocyst(e)ine and ischemic stroke due to large- and small-artery disease but not other etiologic subtypes of ischemic stroke. Stroke 2000; 31: 1069–75.
Evers S, Koch HG, Grotemeyer KH, Lange B, Deufel T, Ringelstein EB. Features, symptoms, and neurophysiological findings in stroke associated with hyperhomocysteinemia. Arch Neurol 1997; 54: 1276–82.
Fassbender K, Mossner R, Motsch L, Kischka U, Grau A, Hennerici M. Circulating selectin- and immunoglobulin-type adhesion molecules in acute ischemic stroke. Stroke 1995; 26: 1361–4.
Fassbender K, Bertsch T, Mielke O, Muhlhauser F, Hennerici M. Adhesion molecules in cerebrovascular disease. Evidence for an inflammatory endothelial activation in cerebral large- and small-vessel disease. Stroke 1999a; 30: 1647–50.
Fassbender K, Mielke O, Bertsch T, Nafe B, Froschen S, Hennerici M. Homocysteine in cerebral macroangiography and microangiopathy. Lancet 1999b; 353: 1586–7.[CrossRef][Web of Science][Medline]
Fazekas F, Kleinert R, Offenbacher H, Schmidt R, Kleinert G, Payer F, et al. Pathologic correlates of incidental MRI white matter signal hyperintensities. Neurology 1993; 43: 1683–9.
Fisher CM. The arterial lesions underlying lacunes. Acta Neuropathol (Berl) 1968; 12: 1–15.[Medline]
Fisher CM. Capsular infarcts: the underlying vascular lesions. Arch Neurol 1979; 36: 65–73.
Frosst P, Blom HJ, Milos R, Goyette P, Sheppard CA, Matthews RG, et al. A candidate genetic risk factor for vascular disease: a common mutation in methylenetetrahydrofolate reductase. Nature Genet 1995; 10: 111–3.[CrossRef][Web of Science][Medline]
Hankey GJ, Eikelboom JW. Homocysteine and stroke. Curr Opin Neurol 2001; 14: 95–102.[CrossRef][Web of Science][Medline]
Hassan A, Hunt B, O’Sullivan M, Parmar K, Bamford J, Briley D, et al. Markers of endothelial dysfunction in lacunar infarction and ischaemic leukoaraiosis. Brain 2003; 126: 424–32.
Homocysteine Lowering Trialists’ Collaboration. Lowering blood homocysteine with folic acid based supplements: meta-analysis of randomised trials. BMJ 1998; 316: 894–8.
Ishii H, Uchiyama H, Kazama M. Soluble thrombomodulin antigen in conditioned medium is increased by damage of endothelial cells. Thromb Haemost 1991; 65: 618–23.[Web of Science][Medline]
Jacques PF, Bostom AG, Williams RR, Ellison RC, Eckfeldt JH, Rosenberg IH, et al. Relation between folate status, a common mutation in methylenetetrahydrofolate reductase, and plasma homocysteine concentrations. Circulation 1996; 93: 7–9.
Jones DK, Lythgoe D, Horsfield MA, Simmons A, Williams SC, Markus HS. Characterization of white matter damage in ischemic leukoaraiosis with diffusion tensor MRI. Stroke 1999; 30: 393–7.
Kario K, Matsuo T, Kobayashi H, Asada R, Matsuo M. ‘Silent’ cerebral infarction is associated with hypercoagulability, endothelial cell damage, and high Lp(a) levels in elderly Japanese. Arterioscler Thromb Vasc Biol 1996; 16: 734–41.
Kruman II, Culmsee C, Chan SL, Kruman Y, Guo Z, Penix L, et al. Homocysteine elicits a DNA damage response in neurons that promotes apoptosis and hypersensitivity to excitotoxicity. J Neurosci 2000; 20: 6920–6.
Lentz SR, Sobey CG, Piegors DJ, Bhopatkar MY, Faraci FM, Malinow MR, et al. Vascular dysfunction in monkeys with diet-induced hyperhomocyst(e)inemia. J Clin Invest 1996; 98: 24–9.[Web of Science][Medline]
Lin JX, Tomimoto H, Akiguchi I, Matsuo A, Wakita H, Shibasaki H, et al. Vascular cell components of the medullary arteries in Binswanger’s disease brains: a morphometric and immunoelectron microscopic study. Stroke 2000; 31: 1838–42.
Lindgren A, Brattstrom L, Norrving B, Hultberg B, Andersson A, Johansson BB. Plasma homocysteine in the acute and convalescent phases after stroke. Stroke 1995; 26: 795–800.
Lindsberg PJ, Carpen O, Paetau A, Karjalainen-Lindsberg ML, Kaste M. Endothelial ICAM-1 expression associated with inflammatory cell response in human ischemic stroke. Circulation 1996; 94: 939–45.
Lipton SA, Kim WK, Choi YB, Kumar S, D’Emilia DM, Rayudu PV, et al. Neurotoxicity associated with dual actions of homocysteine at the N-methyl-D-aspartate receptor. Proc Natl Acad Sci USA 1997; 94: 5923–8.
Malinow MR, Nieto FJ, Kruger WD, Duell PB, Hess DL, Gluckman RA, et al. The effects of folic acid supplementation on plasma total homocysteine are modulated by multivitamin use and methylene tetrahydrofolate reductase genotypes. Arterioscler Thromb Vasc Biol 1997; 17: 1157–62.
Matsui T, Arai H, Yuzuriha T, Yao H, Miura M, Hashimoto S, et al. Elevated plasma homocysteine levels and risk of silent brain infarction in elderly people. Stroke 2001; 32: 1116–9.
McQuillan BM, Beilby JP, Nidorf M, Thompson PL, Hung J. Hyperhomocysteinemia but not the C677T mutation of methylenetetrahydrofolate reductase is an independent risk determinant of carotid wall thickening. The Perth Carotid Ultrasound Disease Assessment Study (CUDAS). Circulation 1999; 99: 2383–8.
Pantoni L, Garcia JH. Pathogenesis of leukoaraiosis: a review. Stroke 1997; 28: 652–9.
Polychronopoulos P, Gioldasis G, Ellul J, Metallinos IC, Lekka NP, Paschalis C, et al. Family history of stroke in stroke types and subtypes. J Neurol Sci 2002; 195: 117–22.[CrossRef][Web of Science][Medline]
Rohde LE, Hennekens CH, Ridker PM. Cross-sectional study of soluble intercellular adhesion molecule-1 and cardiovascular risk factors in apparently healthy men. Arterioscler Thromb Vasc Biol 1999; 19: 1595–9.
Seshadri S, Beiser A, Selhub J, Jacques PF, Rosenberg IH, D’Agostino RB, et al. Plasma homocysteine as a risk factor for dementia and Alzheimer's disease. New Engl J Med 2002; 346: 476–83.
Spence JD, Malinow MR, Barnett PA, Marian AJ, Freeman D, Hegele RA. Plasma homocyst(e)ine concentration, but not MTHFR genotype, is associated with variation in carotid plaque area. Stroke 1999; 30: 969–73.
Stamler JS, Osborne JA, Jaraki O, Rabbani LE, Mullins M, Singel D, et al. Adverse vascular effects of homocysteine are modulated by endothelium-derived relaxing factor and related oxides of nitrogen. J Clin Invest 1993; 91: 308–18.[Web of Science][Medline]
Stein JH, McBride PE. Hyperhomocysteinemia and atherosclerotic vascular disease: pathophysiology, screening, and treatment. off. Arch Intern Med 1998; 158: 1301–6.
Tawakol A, Omland T, Gerhard M, Wu JT, Creager MA. Hyperhomocyst(e)inemia is associated with impaired endothelium-dependent vasodilation in humans. Circulation 1997; 95: 1119–21.
Terborg C, Gora F, Weiller C, Rother J. Reduced vasomotor reactivity in cerebral microangiopathy: a study with near-infrared spectroscopy and transcranial Doppler sonography. Stroke 2000; 31: 924–9.
Tomimoto H, Akiguchi I, Suenaga T, Nishimura M, Wakita H, Nakamura S, et al. Alterations of the blood–brain barrier and glial cells in white-matter lesions in cerebrovascular and Alzheimer’s disease patients. Stroke 1996; 27: 2069–74.
Vermeer SE, van Dijk EJ, Koudstaal PJ, Oudkerk M, Hofman A, Clarke R, et al. Homocysteine, silent brain infarcts, and white matter lesions: The Rotterdam Scan Study. Ann Neurol 2002; 51: 285–9.[CrossRef][Web of Science][Medline]
Wall RT, Harlan JM, Harker LA, Striker GE. Homocysteine-induced endothelial cell injury in vitro: a model for the study of vascular injury. Thromb Res 1980; 18: 113–21.[CrossRef][Web of Science][Medline]
Woo KS, Chook P, Lolin YI, Cheung AS, Chan LT, Sun YY, et al. Hyperhomocyst(e)inemia is a risk factor for arterial endothelial dysfunction in humans. Circulation 1997; 96: 2542–4.
World Health Organization–International Society of Hypertension. Summary of 1993 World Health Organization–International Society of Hypertension guidelines for the management of mild hypertension. Subcommittee of WHO/ISH Mild Hypertension Liaison committee. BMJ 1993; 307: 1541–6.
Yoo JH, Chung CS, Kang SS. Relation of plasma homocyst(e)ine to cerebral infarction and cerebral atherosclerosis. Stroke 1998; 29: 2478–83.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  L. Paternoster, W. Chen, and C. L.M. Sudlow Genetic Determinants of White Matter Hyperintensities on Brain Scans: A Systematic Assessment of 19 Candidate Gene Polymorphisms in 46 Studies in 19 000 Subjects * Supplemental References Stroke, June 1, 2009; 40(6): 2020 - 2026. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. S. Thompson and A. M. Hakim Living Beyond Our Physiological Means: Small Vessel Disease of the Brain Is an Expression of a Systemic Failure in Arteriolar Function: A Unifying Hypothesis Stroke, May 1, 2009; 40(5): e322 - e330. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. Pieters, J. Staals, I. Knottnerus, R. Rouhl, P. Menheere, A. Kessels, and J. Lodder Periventricular White Matter Lucencies Relate to Low Vitamin B12 Levels in Patients With Small Vessel Stroke Stroke, May 1, 2009; 40(5): 1623 - 1626. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W. G. Christen, R. J. Glynn, E. Y. Chew, C. M. Albert, and J. E. Manson Folic Acid, Pyridoxine, and Cyanocobalamin Combination Treatment and Age-Related Macular Degeneration in Women: The Women's Antioxidant and Folic Acid Cardiovascular Study Arch Intern Med, February 23, 2009; 169(4): 335 - 341. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
U. Khan, C. Crossley, L. Kalra, A. Rudd, C. D.A. Wolfe, P. Collinson, and H. S. Markus Homocysteine and Its Relationship to Stroke Subtypes in a UK Black Population: The South London Ethnicity and Stroke Study Stroke, November 1, 2008; 39(11): 2943 - 2949. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L Kalra, E Iveson, C Rambaran, R Sherwood, P Chowienczyk, J Ritter, A Shah, and T Forrester Homocysteine, migration and early vascular impairment in people of African descent Heart, September 1, 2008; 94(9): 1171 - 1174. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. O'Sullivan Leukoaraiosis Practical Neurology, January 1, 2008; 8(1): 26 - 38. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Francis, S. Raghunathan, and P. Khanna The role of genetics in stroke Postgrad. Med. J., September 1, 2007; 83(983): 590 - 595. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 U. Khan, L. Porteous, A. Hassan, and H. S Markus Risk factor profile of cerebral small vessel disease and its subtypes J. Neurol. Neurosurg. Psychiatry, July 1, 2007; 78(7): 702 - 706. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L.-K. Yang, K.-C. Wong, M.-Y. Wu, S.-L. Liao, C.-S. Kuo, and R.-F. S. Huang Correlations Between Folate, B12, Homocysteine Levels, and Radiological Markers of Neuropathology in Elderly Post-Stroke Patients J. Am. Coll. Nutr., June 1, 2007; 26(3): 272 - 278. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. L. Schwartz, K. R. Bailey, T. Mosley, D. S. Knopman, C. R. Jack Jr, V. J. Canzanello, and S. T. Turner Association of Ambulatory Blood Pressure With Ischemic Brain Injury Hypertension, June 1, 2007; 49(6): 1228 - 1234. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. N. Rowan, H. O. Dickinson, S. Stephens, C. Ballard, R. Kalaria, and R. Anne Kenny Homocysteine and post-stroke cognitive decline Age Ageing, May 1, 2007; 36(3): 339 - 343. [Full Text] [PDF]
|
|
|
|
 |
|
 G. A. Prathapasinghe, Y. L. Siow, and K. O Detrimental role of homocysteine in renal ischemia-reperfusion injury Am J Physiol Renal Physiol, May 1, 2007; 292(5): F1354 - F1363. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
U. Khan, A. Hassan, P. Vallance, and H. S. Markus Asymmetric Dimethylarginine in Cerebral Small Vessel Disease Stroke, February 1, 2007; 38(2): 411 - 413. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Opherk, N. Peters, M. Holtmannspotter, A. Gschwendtner, B. Muller-Myhsok, and M. Dichgans Heritability of MRI Lesion Volume in CADASIL: Evidence for Genetic Modifiers Stroke, November 1, 2006; 37(11): 2684 - 2689. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. B. Goldstein, R. Adams, M. J. Alberts, L. J. Appel, L. M. Brass, C. D. Bushnell, A. Culebras, T. J. DeGraba, P. B. Gorelick, J. R. Guyton, et al. Primary Prevention of Ischemic Stroke: A Guideline From the American Heart Association/American Stroke Association Stroke Council: Cosponsored by the Atherosclerotic Peripheral Vascular Disease Interdisciplinary Working Group; Cardiovascular Nursing Council; Clinical Cardiology Council; Nutrition, Physical Activity, and Metabolism Council; and the Quality of Care and Outcomes Research Interdisciplinary Working Group: The American Academy of Neurology affirms the value of this guideline. Circulation, June 20, 2006; 113(24): e873 - e923. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. B. Goldstein, R. Adams, M. J. Alberts, L. J. Appel, L. M. Brass, C. D. Bushnell, A. Culebras, T. J. DeGraba, P. B. Gorelick, J. R. Guyton, et al. Primary Prevention of Ischemic Stroke: A Guideline From the American Heart Association/American Stroke Association Stroke Council: Cosponsored by the Atherosclerotic Peripheral Vascular Disease Interdisciplinary Working Group; Cardiovascular Nursing Council; Clinical Cardiology Council; Nutrition, Physical Activity, and Metabolism Council; and the Quality of Care and Outcomes Research Interdisciplinary Working Group: The American Academy of Neurology affirms the value of this guideline. Stroke, June 1, 2006; 37(6): 1583 - 1633. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. A. Armstrong, S. N. Bevan, K. T. Gormley, H. S. Markus, S. A. Koblar, J. Jannes, A. M. Hamilton-Bruce, and S. A. Koblar Tissue Plasminogen Activator -7351C/T Polymorphism and Lacunar Stroke * Response: Stroke, February 1, 2006; 37(2): 329 - 330. [Full Text] [PDF]
|
|
|
|
 |
|
 I. C. Duncan and J. M. Terblanche Spontaneous Isolated Posterior Communicating Artery Dissection in a Young Adult with Hyperhomocysteinemia AJNR Am. J. Neuroradiol., September 1, 2005; 26(8): 2030 - 2032. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H.-K. Kuo, F. A. Sorond, J.-H. Chen, A. Hashmi, W. P. Milberg, and L. A. Lipsitz The Role of Homocysteine in Multisystem Age-Related Problems: A Systematic Review J. Gerontol. A Biol. Sci. Med. Sci., September 1, 2005; 60(9): 1190 - 1201. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. D. Napoli and F. Papa C-Reactive Protein and Cerebral Small-Vessel Disease: An Opportunity to Reassess Small-Vessel Disease Physiopathology? Circulation, August 9, 2005; 112(6): 781 - 785. [Full Text] [PDF]
|
|
|
|
|
|
S. Cronin, K. L. Furie, and P. J. Kelly Dose-Related Association of MTHFR 677T Allele With Risk of Ischemic Stroke: Evidence From a Cumulative Meta-Analysis Stroke, July 1, 2005; 36(7): 1581 - 1587. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. B. Wright, M. C. Paik, T. R. Brown, S. P. Stabler, R. H. Allen, R. L. Sacco, and C. DeCarli Total Homocysteine Is Associated With White Matter Hyperintensity Volume: The Northern Manhattan Study Stroke, June 1, 2005; 36(6): 1207 - 1211. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Hoshi, K. Kitagawa, H. Yamagami, S. Furukado, H. Hougaku, and M. Hori Relations of Serum High-Sensitivity C-Reactive Protein and Interleukin-6 Levels With Silent Brain Infarction Stroke, April 1, 2005; 36(4): 768 - 772. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Singhal, S. Bevan, T. Barrick, P. Rich, and H. S. Markus The influence of genetic and cardiovascular risk factors on the CADASIL phenotype Brain, September 1, 2004; 127(9): 2031 - 2038. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Homocysteine May Be Associated with Small-Vessel Disease Journal Watch Neurology, March 12, 2004; 2004(312): 4 - 4. [Full Text]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||