Targeted delivery of rosuvastatin enhances treatment of hyperhomocysteinemia-induced atherosclerosis using macrophage membrane-coated nanoparticles

Rosuvastatin (RVS) is an excellent drug with anti-inflammatory and lipid-lowering properties in the academic and medical fields. However, this drug faces a series of challenges when used to treat atherosclerosis caused by hyperhomocysteinemia (HHcy), including high oral dosage, poor targeting, and long-term toxic side effects. In this study, we applied nanotechnology to construct a biomimetic nano-delivery system, macrophage membrane (Møm)-coated RVS-loaded Prussian blue (PB) nanoparticles (MPR NPs), for improving the bioavailability and targeting capacity of RVS, specifically to the plaque lesions associated with HHcy-induced atherosclerosis. In vitro assays demonstrated that MPR NPs effectively inhibited the Toll-like receptor 4 (TLR4)/hypoxia-inducible factor-1α (HIF-1α)/nucleotide-binding and oligomerization domain (NOD)-like receptor thermal protein domain associated protein 3 (NLRP3) signaling pathways, reducing pyroptosis and inflammatory response in macrophages. Additionally, MPR NPs reversed the abnormal distribution of adenosine triphosphate (ATP)-binding cassette transporter A1 (ABCA1)/ATP binding cassette transporter G1 (ABCA1)/ATP binding cassette transporter G1 (ABCG1) caused by HIF-1α, promoting cholesterol efflux and reducing lipid deposition. In vivo studies using apolipoprotein E knockout (ApoE^(−/−)) mice confirmed the strong efficacy of MPR NPs in treating atherosclerosis with favorable biosecurity, and the mechanism behind this efficacy is believed to involve the regulation of serum metabolism and the remodeling of gut microbes. These findings suggest that the synthesis of MPR NPs provides a promising nanosystem for the targeted therapy of HHcy-induced atherosclerosis.

Levels of serum homocysteine in depressive patients Self-correlation factor analysis and comparison with healthy subjects

BACKGROUND： Data indicate that the levels of serum homocysteine in depressive patients are higher than those in normal subjects. OBJECTIVE： To investigate the levels of serum homocysteine in patients with major depressive disorder, to determine whether serum homocysteine levels differ with sex, family history, or drug treatment, and to compare depressive patients with normal subjects. DESIGN： Non-randomized concurrent control trial. SETTING： Mental Heath Center of Shandong Province. PARTICIPANTS： Forty in-patients （23 males and 17 females, 18-63 years old） with major depressive disorder were selected from the Mental Health Center of Shandong Province from January to October 2006. All selected patients met the depressive diagnostic standard of Chinese Classification of Mental Disorder （3^rd Edition, CCMD-3）, and total scores evaluated by the 17-item Hamilton Rating Scale for Depression （HRSD） were ≥ 20. Meanwhile, 36 healthy subjects （20 males and 16 females, 18-60 years old） were enrolled as controls; their total 17-item HRSD scores were ≤ 7. All selected subjects provided consent, and the study was approved by the local ethics committee. METHODS： Fasting venous blood （3 mL） was drawn in both groups at 8：00 in the morning. The levels of serum homocysteine were determined by a fluorescence polarization immunoassay （FPIA）. The 17-item HRSD was also compiled from the patients when entering groups. The higher the scores were, the more severe the depression was. Enumeration data for both groups were compared by Chi-square test, measurement data were compared by t-test, and correlations were detected using Pearson and Spearman correlation analysis. MAIN OUTCOME MEASURES： ① Levels of serum homocysteine; ② incidence of hyperhomocysteinemia （HHcy）; ③ correlation between HRSD17 scores and levels of serum homocysteine in depressive patients. RESULTS： Forty depressive patients and 36 control subjects were included in the final analysis without any loss of participants. ① Levels of serum homocysteine and HHcy detection rate： the levels of serum homocysteine in the depressive patients were significantly higher than those in the control group （t = 4.377, P=0.000）. Hhcy detection rates were 42% （17/40） and 10% （4/36） in depressive group and control group, respectively. There was a significant difference between two groups （x^2 = 10.912, P = 0.001）. In the depressive group, there were no differences in serum homocysteine levels between males and females, before and after treatment, or between patients with positive or negative family histories of depression （t = 0.217-0.520, P 〉 0.05）. ② Correlation analysis： the HRSD17 scores in the depressive group were positively correlated with levels of serum homocysteine （r = 0.724, P = 0.000）. CONCLUSION： ① The increase in serum homocysteine levels may play an important role in the pathogenic mechanism of depressive disorder. ② The higher the levels of serum homocysteine are, the more severe the depressive disorder is. ③ There are no significant differences in serum homocysteine levels between patients of different sex or family history, or before and after drug treatment, among depressive patients.

Elevated Homocysteine and C-reactive Protein Levels Independently Predict Worsening Prognosis after Stroke in Chinese Patients

Increased plasma total homocysteine (tHcy) and high sensitivity C-reactive protein (hsCRP) levels are independent risk factors for cardiovascular disease.However, the predictive value of tHcy in combination with hsCRP in patients with stroke is not known.To determine the relationship between tHcy and hsCRP, we enrolled 291 patients with first-onset stroke (196 ischemic and 95 hemorrhagic).Plasma tHcy and hsCRP levels were measured and subsequent vascular events and deaths were determined over a 5-year period.Using the arbitrary cutoff for tHcy (【18 μmol/L and ≥18 μmol/L) and hsCRP (【1 mg/L, 1-3 mg/L and 】3 mg/L), the patients were divided into 6 groups.Survival analysis showed that the probability of death or new vascular events during a 5-year follow-up increased according to tHcy and hsCRP levels (P【0.01).The relative risk (RR) of death or new vascular events was 4.67 (95% CI, 1.96 to 11.14, P=0.001) in patients with high tHcy (≥18 μmol/L) and hsCRP (】3 mg/L) compared with those with low tHcy (【18 μmol/L) and hsCRP (【1 mg/L).The increased tHcy level (≥18 μmol/L) combined with increased hsCRP level (】3 mg/L) was still significantly associated with the risk of death or new vascular events (RR, 4.10, 95% CI, 1.61 to 10.45, P=0.003) even when adjusted for other risk factors at inclusion.The combination of increased tHcy and hsCRP levels had a stronger predictive value than increased hsCRP alone or increased tHcy level alone.Further studies are required to evaluate the potential decrease in risks associated with lowering both Hcy and hsCRP levels in patients that present with both increased tHcy and hsCRP.

Increased levels of homocysteine in patients with ulcerative colitis

AIM: To investigate serum levels of homocysteine (Hcys) and the risk that altered levels carry for thrombosis development in ulcerative colitis (UC) patients. METHODS: 55 UC patients and 45 healthy adults were included. Hcys, vitamin B12 and folic acid levels were measured in both groups. Clinical history and thrombo- embolic events were investigated. RESULTS: The average Hcys level in the UC patients was 13.3 ± 1.93 μmmol/L (range 4.60-87) and was higher than the average Hcys level of the control group which was 11.2 ± 3.58 μmmol/L (range 4.00-20.8) (P < 0.001). Vitamin B12 and folic acid average values were also lower in the UC group (P < 0.001). Whenmultivariate regression analysis was performed, it was seen that folic acid deficiency was the only risk factor for hyperhomocysteinemia. Frequencies of thromboembolic complications were not statistically significantly different in UC and control groups. When those with and without a thrombosis history in the UC group were compared according to Hcys levels, it was seen that there were no statistically significant differences. A negative linear relationship was found between folic acid levels and Hcys. CONCLUSION: We could not find any correlations between Hcys levels and history of prior thromboembolic events.

Plasma Homocysteine and Gene Polymorphisms Associated with the Risk of Hyperlipidemia in Northern Chinese Subjects

Objective To examine the relationship between occurrence of hyperlipidemia, plasma homocysteine and polymorphisms of methylenetetra hydrofolate reductase （MTHFR） gene and methionine synthase （MS） gene. Methods A total of 192 hyperlipidemia patients were selected and divided into hypercholesterolemia group, hypertriglyceridemia group, and combined hyperlipidemia group. Another 208 normal individuals were selected as control. Total plasma homocysteine （tHcy） concentration was measured by high-performance liquid chromatography （HPLC）. Lipid profiles were measured for all subjects The polymorphisms of MTHFR gene C677T and MS gene A2756G were analyzed by PCR-RFLP. Results The tHcy concentration in the combined hyperlipidemia patients was significantly higher than that in the control （15.95μmol/L vs 13.43 μmol/L, P〈0.05）. The prevalence of hyperhomocysteinemia （HHcy） in the combined hyperlipidemia group was significantly higher than that in the control （42.2% vs 23.0%, P=0.015）, with the odds ratio （OR） of 3.339 （95%CI： 1.260-8.849）. The hyperlipidemia patients with HHcy had a higher concentration of total cholesterol （TC） than that in the normal tHcy patients （5.67±0.95 mmol/L vs 5.47±0.92 retool/L, P=0.034）. There was no significant difference in genotype or allele frequencies of MTHFR C677T between the hyperlipidemic and control groups. The hyperlipidemia patients with MTHFR CT/TT genotype had a higher concentration of triglyceride （TG） than those with CC genotype （2.24±1.75 mmol/L vs 1.87±0.95 mmol/L, P〈0.05）. Individuals with CT/TT genotype had a higher concentration of tHcy than those with 677CC genotype both in the hyperlipidemia group （12.61±1.24μmol/L vs 11.20±1.37 μmol/L, P〈0.05） and in the control group （14.04±1.48 μmol/L vs 12.61±1.24 μmol/L, P〈0.05）. The percentage of MS 2756 GG/AG genotype in the combined hyperlipidemia group was significantly higher than that in the control （26.7% vs 13.0%, P=0.012）, with the OR of 3.121 （95%C1： 1.288-7.65/）. The hyperlipidemia patients with MS 2756AG/GG genotype had a higher concentration of TC （5.87±0.89 mmol/L vs 5.46±0.93 retool/L, P〈0.05） and LDL-C （3.29±0.81 mmol/L vs 2.94±0.85 retool/L, P〈0.05） than those with AA genotype. However, individuals with 2756AG/GG genotype showed no significant difference in tHcy among those with AA genotype. Conclusion HHcy and MS A2756G mutation may be the risk factors for combined hyperlipidemia. Further study is needed to confirm the role of HHcy and MS A2756G mutation in the development of hyperlipidemia.

Association between Serum Homocysteine and Oxidative Stress in Elderly Patients with Obstructive Sleep Apnea/hypopnea Syndrome

Objective Elderly patients with obstructive sleep apnea/hypopnea syndrome （OSAHS） has a higher risk of cardiovascular and cerebrovascular disease. However, changes of homocysteine （Hey） as markers of cardiovascular and eerebrovascular disease associated with OSAHS and their mechanism have not been elucidated so far. This study aims to investigate the changes of both serum Hcy and oxidative stress and their possible links with OSAHS in elderly patients. Methods Based on polysomnogram （PSG） and age, 83 patients with OSAHS were recruited and divided into elderly-OSAHS （n=32） and non-elderly OSAHS groups （n=51）. Fifty two subjects without OSAHS were divided into elderly control （n=29） and non-elderly control groups （n-23）. A total of 135 subjects were included in the present study. All subjects were recorded for PSG variables and the contents of homocysteine （Hcy）, malonaldehyde （MDA）, and glutathione （GSH） which were detected after sleep. Serum homocysteine was measured by cyclophorase. MDA and GSH were measured by speetrophotometer. Results （1） The serum levels of Hcy showed significant difference among the four groups （P〈0.05）. The concentrations of Hcy in elderly OSAHS patients were higher than in other groups, while those in the elderly control group were higher than in the non-elderly control; the concentrations in the non-elderly OSAHS group were higher than in the non-elderly control. （2） The concentrations of MDA and GSH changed at an equal pace with Hcy in the four groups. （3） Multielement linearity regression analysis indicated a statistically significant relationship between Hcy concentration and age, MDA, GSH, and apnea hypopnea index （AHI）. Conclusions （1） The concentrations of Hcy and oxidative stress have increased with advancing age. （2） The concentrations of Hcy and oxidative stress have further increased in the elderly patients with OSAHS. （3） Oxidative stress might cause high-level serum Hcy in the elderly patients with OSAHS.

Study on the Relationship between Plasma Homocysteine and Acute Cerebral Vascular Disease

The levels of plasma homocysteine were determined by using high-performance liquid chromatographic method. It was found that plasma homocysteine levels were significantly higher in the patients with stroke than that in the controls. There was no correlation between plasma homocys- teine levels and hypertension, smoking, concentrations of blood glucose or hypertriglyceridesemia. It was suggested that hyperhomocysteinemia may be an independent risk factor for acute cerebral vascu- lar disease.

Hyperhomocysteinemia independently causes and promotes atherosclerosis in LDL receptor-deficient mice

Background Hyperhomocysteine is an independent risk factor of coronary heart disease （CHD）. However, whether hyperhomocys teine affects the progression of atherosclerosis is unclear. In the present study, we examined the effect of hyperhomocysteine on the forma tion of atherosclerosis in low-density lipoprotein receptor-deficient （LDLr ） mice. Methods Forty-eight 7-week-old LDLr/ mice were assigned to the following groups： mice fed a standard rodent diet （control group）, mice fed a high-methionine diet （high-methionine group）, mice fed a high-fat diet （high-fat group）, and mice fed a diet high in both methionine and fat （high-methionine and high-fat group）. At the age of 19, 23, and 27 weeks, four mice at each interval in every group were sacrificed. Results At the end of the study, mice did not show atherosclerotic lesions in the aortic sinus and aortic surface until 27 weeks old in the control group. However, atherosclerotic lesions developed in the other three groups at 19 weeks. The amount of atherosclerotic lesions on the aortic surface was lower in the high-methionine group than in the high-fat group （P 〈 0.001）. Atherosclerotic lesions on the aortic surface in the high-methionine and high-fat group were the most severe. The mean area of atherosclerotic lesions in the aortic sinus compared with atherosclerotic lesions on the aortic surface was lower in the high-methionine group than in the high-fat group （P 〈 0.001）. Atherosclerotic lesions in the aortic sinus in the high-methionine and high-fat group were the most severe. Conclusions Homocysteinemia accelerates atherosclerotic lesions and induces early atherosclerosis independently in LDLrmice. Reducing the level of homocysteinemia may be beneficial for prevention and treatment of CHD.

Genome-wide DNA hypermethylation and homocysteine increase a risk for myopia

AIM: To test for the association between genome-wide methylation and myopia in human and mice. METHODS: Long interspersed nucleotide element 1(LINE-1) methylation levels were used to surrogate genome-wide methylation level. We first tested for the association between high myopia(<-6 D) and LINE-1 methylation in leukocytes in 220 cases and 220 control subjects. Secondly, we validated the results of LINE-1 methylation in eyes from the form deprivation myopia(FDM) mice. Furthermore,we calculated the correlation of LINE-1 methylation levels between leukocyte DNA and ocular DNA in the mice. We also tested whether dopamine can alter LINE-1 methylation levels. RESULTS: The LINE-1 methylation level was significantly higher in the myopic human subjects than controls. The upper and middle tertiles of the methylation levels increased an approximately 2-fold(P≤0.002) risk for myopia than the lower tertile. Similarly, FDM mice had high LINE-1 methylation levels in the leukocyte, retina and sclera, and furthermore the methylation levels detected from these three tissues were significantly correlated. Immunohistochemical staining revealed higher levels of homocysteine and methionine in the rodent myopic eyes than normal eyes. Dopamine treatment to the cells reduced both LINE-1 methylation and DNA methyltransferase levels. CONCLUSION: LINE-1 hypermethylation may be associated with high myopia in human and mice. Homocysteine and methionine are accumulated in myopic eyes, which may provide excess methyl group for genome-wide methylation.

Endogenous hydrogen sulfide and ERK1/2-STAT3 signaling pathway may participate in the association between homocysteine and hypertension

Background Homocysteine(Hcy)is a risk factor for hypertension,although the mechanisms are poorly understood.Methods We first explored the relationship between Hcy levels and blood pressure(BP)by analyzing the clinical data of primary hypertensive patients admitted to our hospital.Secondly,we explored a rat model to study the effect of Hcy on blood pressure and the role of H2S.An hyperhomocysteinemia(HHcy)rat model was induced to explore the effect of Hcy on blood pressure and the possible mechanism.We carried out tissue histology,extraction and examination of RNA and protein.Finally,we conducted cell experiments to determine a likely mechanism through renin-angiotensin-aldosterone system(RAAS)and extracellular signal-regulated kinase 1/2(ERK1/2)signaling pathway.Results In primary hypertensive inpatients with HHcy,blood pressure was significantly higher as compared with inpatient counterparts lacking HHcy.In the rat model,blood pressure of the Wistar rats was significantly increased with increases in serum Hcy levels and decreased after folate treatment.Angiotensin converting enzyme 1(ACE1)expression in the Wistar Hcy group was enhanced comparing to controls,but was decreased in the Wistar folate group.Angiotensin II receptor type 1(AGTR1)levels in the kidney tissue increased in the Wistar folate group.Both serum H2S and kidney cystathionineγ-lyase decreased with elevated levels of serum Hcy.In vitro,increased concentrations and treatment times for Hcy were associated with increased expression of collagen type 1 and AGTR1.This dose and time dependent response was also observed for p-STAT3 and p-ERK1/2 expression.Conclusion Endogenous H2S might mediate the process of altered blood pressure in response to changes in serum Hcy levels,in a process that is partly dependent on activated RAAS and ERK1/2-STAT3 signaling pathway.

Association between serum homocysteine and arterial stiffness in elderly： a community-based study

Background Arterial stiffness and homocysteine are both powerful predictors of cardiovascular disease, especially in older popula tions. Previous studies have investigated the association of homocysteine with arterial stiffness in human subjects, while the relationship between homocysteine and arterial stiffness in the elderly is still indefinite. The current study examined the association of homocysteine with arterial stiffness in Chinese community-based elderly persons. Methods We related serum levels of homocysteine to two measures of arte- rial stiffness （carotid-femoral pulse wave velocity （PWV） and carotid-radial PWV） in 780 participants （46.3% men, mean age 71.9 years （ranging 65-96 years old）） from two communities of Beijing, China. Arterial stiffness were measured within two days of the time of bio- marker measurement. Results In multiple-adjusted models, homocysteine levels was strongly associated with the carotid-femoral PWV （standardized 13 = 0.13, P 〈 0.001）, even after adjustment for classical risk factors of cardiovascular disease. The association is also stronger when the carotid-femoral PWV is elevated above normal, whereas no significant association with homocysteine was observed for ca-rotid-radial PWV. Conclusions In Chinese elderly persons, serum homocysteine levels are associated with alterations of aortic stiffness.

Factors influencing ischemic cerebrovascular disease complicated by hyperhomocysteinemia

BACKGROUND： Hyperhomocysteinemia, as an important risk factor for ischemic cerebrovascular disease is receiving increasing attention. OBJECTIVE： To analyze whether differences of gender, age, cerebrovascular disease typing, and disease conditions exist when ischemic cerebrovascular disease occurs together with hyperhomocysteinemia. DESIGN： A controlled observation. SETTING： Department of Neurology, Tianjin Huanhu Hospital. PARTICIPANTS： A total of 601 acute ischemic cerebrovascular disease inpatients, comprising 386 males and 215 females, aged 33-90 years old, were admitted to the Department of Stroke, Tianjin Huanhu Hospital between August 2005 and April 2007, and were recruited for this study. All included patients consisted of 342 aged patients （≥ 60 years old） and 92 middle-aged and young patients （〈 60 years old）. Among these patients, 48 suffered from transient cerebral ischemic attack, 138 from lacunar cerebral infarction, 273 from atherosclerotic stroke, 38 from cardiogenic cerebral infarction, 44 from agnogenic ischemic stroke, and 6 from other factor-induced ischemic strokes. All included inpatients corresponded to the diagnosis criteria of acute ischemic cerebrovascular disease, formulated in the 4^th National Working Conference of Cerebrovascular Disease, and were confirmed as acute ischemic cerebral infarction by CT and/or MRI examinations. Informed consents of laboratory measurements were obtained from all subjects, and this study was approved by the Hospital＇s Ethics Committee. METHODS： Following admission, 2 mL venous blood was collected from each fasting patient on the third morning. Plasma homocysteine level was measured by an enzymatic cycling assay with a CX5 reader （Beckman, USA）. Plasma homocysteine levels ≥ 16μ mol/L were defined as hyperhomocysteinemia. Clinical neurological function deficit scoring was also performed for each ischemic stroke patient using Chinese stroke scales. Scores ranged from 0 45 （0-15： mild neurological function deficits, 16-30： moderate neurological deficits, and 31-45： severe neurological deficits）. The scores positively correlated with severity of stroke. MAIN OUTCOME MEASURES： Incidence of ischemic cerebrovascular disease patients complicated by hyperhomocysteinemia and the effects of patient age and gender; plasma homocysteine levels of each type of ischemic cerebrovascular disease; and effects of ischemic cerebrovascular disease conditions on plasma homocysteine levels. RESULTS： All 601 inpatients with acute ischemic cerebrovascular disease were included in the final analysis. The detection rate of homocysteine was significantly higher in aged patients than in middle-aged and young patients （ x^2 = 5.353 0, P 〈 0.05）. The incidence of hyperhomocysteinemia was significantly higher in male patients than in female patients （ x^2 = 9.484 4, P 〈 0.05）. There was no significant difference in the incidence of hyperhomocysteinemia among various types of ischemic cerebrovascular diseases （P 〉 0.05）. No significant difference in incidence of hyperhomocysteinemia existed between mild, moderate, and severe cerebrovascular disease patients （P 〉 0.05）. CONCLUSION： There is a greater chance of ischemic cerebrovascular disease complicated by hyperhomocysteinemia in older, male patients.

Homocysteine aggravates cerebral hemorrhage in rats: Correlation with oxygen-free radical production and cell apoptosis in tissue surrounding hematoma

BACKGROUND： Previous studies have demonstrated that homocysteine is an independent risk factor for ischemic stroke, as determined by detection of apoptosis and oxygen-free radical scavengers following cerebral ischemia. However, the mechanisms of homocysteine remain unclear Several reports have addressed the effects of homocysteine on ischemic stroke. OBJECTIVE： To analyze the effects of homocysteine on apoptosis, intracellular superoxide dismutase （SOD） activity, and malondialdehyde content in tissue surrounding hematoma in rats with cerebral hemorrhage, and to determine the action pathway of malondialdehyde following cerebral hemorrhage. DESIGN, TIME AND SETTING： The randomized, controlled, animal experiment was performed at the Laboratory of Molecular Biology, Hospital Affiliated to Luzhou Medical College, China from April 2007 to April 2008. MATERIALS： In situ apoptosis detection kit （Roche, Mannheim, Germany）, SOD detection kit and malondialdehyde detection kit （Nanjing Jiancheng Bioengineering Institute, China）, and homocysteine （Sigma, St Louis, MO, USA） were used in the present study. METHODS： A total of 75 Sprague Dawley rats were equally and randomly assigned to sham surgery model, and homocysteine groups. Autologous blood was infused into the caudate putamen of rats to establish models of cerebral hemorrhage in model and homocysteine groups. Homocysteine was injected directly into the brain through the skull at the hematoma hemisphere at 30 minutes after model induction in the homocysteine group. MAIN OUTCOME MEASURES： At 6, 12, 24, and 72 hours, as well as 1 week, post-surgery, neurological deficits were observed in each group. Brain water content was measured using the dry-wet weight method. Cell apoptosis in tissue surrounding the hematoma was detected utilizing terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling （TUNEL）. SOD activity and malondialdehyde content in tissue surrounding the hematoma were respectively measured using the xanthine oxidase and thiobarbituric acid methods. RESULTS： Neurological function was similar between model and homocysteine groups following cerebral hemorrhage （P 〉 0.05）. Brain water content was increased at 12 hours post-surgery, peaked at 3 days, and remained unchanged at 7 days in the model group. Brain edema was not significantly aggravated following homocysteine intervention （P 〉 0.05）, but SOD activity significantly decreased and malondialdehyde content significantly increased （P 〈 0.05）. The number of apoptotic cells increased in rats with cerebral hemorrhage at 12 hours （P 〈 0.05）, and numbers peaked at 72 hours following model establishment （P〈 0.05）. The time of peak value was identical between model and homocysteine groups. Brain water content was negatively associated with SOD activity （rmodel group =-0.448, P 〈 0.05; rhomocysteine group =-0.612, P 〈 0.05）, but was positively associated with malondialdehyde content （rmodel group = 0.542, P 〈 0.05; rhomocysteine group = 0.684, P 〈 0.05） in brain tissues surrounding the hematoma following surgery in model and homocysteine groups. CONCLUSION： Homocysteine aggravates neurological dysfunction and brain edema in rats with cerebral hemorrhage. The mechanisms of action are likely associated with production of oxygen-free radical and cellular apoptosis following cerebral hemorrhage.

Effect of advanced age on plasma homocysteine levels and its association with ischemic stroke in non-valvular atrial fibrillation

Background Elevated homocysteine （Hcy） has been reported to be associated with cardiovascular events in atrial fibrillation （AF） pa- tients, while the age-related expression pattern of plasma Hcy in AF remains unknown. The study was aimed to investigate the effect of ad- vanced age on plasma Hcy levels and its association with ischemic stroke in non-valvular AF patients. Methods A total of 2562 consecu- tive patients with non-valvular AF and 535 controls were enrolled and divided into six age groups. Plasma Hcy levels were analyzed among different age groups, and the effect of advanced age on Hcy was investigated. Results Plasma Hcy levels did not show any difference among groups aged below 65 years, while it increased sharply in patients aged 65-74 years and aged over 75 years （15.7 ±4.6 μmol/L, 17.1 ±4.9 μmol/L, both P 〈 0.01 compared with the first four age groups）. Hcy was much higher in AF patients than in controls at the same age group （all P 〈 0.05）. The proportion of patients with hyperhomocysteinemia increased gradually with age from 32.3%, 29.2%, 31.2%, 32.4%, 45.9%, to 51.4% in six age groups. The concentration of Hcy in AF patients with ischemic stroke increased progressively with age, and was higher than those without stroke at the same age. Logistic regression analysis demonstrated that age 65-74 years [odds ratios （OR）： 1.742, 95% confidence interval （CI）： 1.223-2.482, P = 0.002] and age ≥ 75 years （OR： 2.637, 95% CI： 1.605-4.335, P 〈 0.001） were significantly independent predictors of elevated plasma Hcy levels. Conclusions Advanced age was significantly associated with elevated Hcy levels, which may provide a possible explanation for the progressive increase in ischemic stroke especially in elderly AF patients.

Association Between Homocysteine Level and Methylenetetrahydrofolate Reductase Gene Polymorphisms in Type 2 Diabetes Accompanied by Dyslipidemia

Objective To investigate the association between total homocysteine(tHcy)level in plasma and methylenetetrahydrofblate reductase(MTHFR)C677T and A1298C genetic polymorphisms in a Chinese Han nationality population with type 2 diabetes mellitus(T2DM)accompanied by dyslipidemia.Methods This case-control study enrolled T2DM patients with dyslipidemia and without dyslipidemia respectively.Sanger dideoxy-mediated chain-termination method was used to detect the gene polymorphisms of MTHFR C677T and A1298C.Plasma tHcy and lipid levels were measured as well.The genotype frequency and allele frequency between the dyslipidemia and non-dyslipidemia groups were compared by using Chi-square test.Plasma tHcy level ofT2DM patients who carried the different genotypes was compared by Student's t test.Results Finally,82 T2DM patients with dyslipidemia and 94 ones without dyslipidemia were included in this study.There was a significant correlation between tHcy level and MTHFR C677T gene polymorphism inT2DM patients(t=2.27,P=0.02).Moreover,the plasma tHcy level in the dyslipidemia patients who carried MTHFR 677TT genotype was significantly higher than that in those with CT+CC genotype(13.62+6.97 vs.10.95+3.62pmol/L,t=2.2O,P=0.03);while for patients without dyslipidemia,comparison of the tHcy level between those who carried the above two alleles showed no significantly difference(13.34±6.03 vs.12.04±5.09μmol/L,t=1.08,P=0.29).Conclusion MTHFR 677TT genotype might associate with higher tHcy level in T2DM patients with dyslipidemia.

Homocysteine is associated with the progression of non-culprit coronary lesions in elderly acute coronary syndrome patients after percutaneous coronary intervention

Background The influence of homocysteine （Hcy） on the migration and proliferation of vascular smooth muscle cells has been well established. However, the impact of Hcy levels on the progression of non-culprit coronary lesions （NCCLs） is controversial. This study aims to evaluate whether the plasma level of Hcy is related to the progression of NCCLs after percutaneous coronary stent implantation in elderly patients with acute coronary syndrome （ACS）. Methods A total of 223 elderly patients （〉 65 years old） with ACS undergoing stent im- plantation and follow-up coronary angiography were enrolled. Laboratory determination comprised of blood sample evaluation for Hcy was carried out before baseline coronary intervention. The patients were classified into two groups according to the blood Hcy tertiles （〉 15 mmol/L or 〈 15 mmol/L）. Patients were followed up for 12.2 months. NCCL progression was assessed by three-dimensional quantitative coronary angiography. Results A significantly higher ratio of NCCL progression was observed in the group with baseline Hcy concentrations above 15 mmol/L compared to the group with concentrations below 15 mmol/L （41/127, 32.3% vs. 14/96, 14.6%, P = 0.002）. Multivariate Cox regression analysis showed that Hcy and diabetes mellitus were independent risk factors for NCCL progression. The crude haz- ard ratio （HR） of NCCL progression for Hcy level was 1.056 （95% CI： 1.01-1.104, P = 0.015）. The adjusted HR of NCCL progression for Hcy level was 1.024 （95% CI： 1.007-1.042, P = 0.007）. The adjusted HR of NCCL progression for diabetes mellitus was 1.992 （95% CI： 1.15-3.44, P = 0.013）. Conclusions Hcy is an independent risk factor for NCCL progression after 12 months of follow-up in elderly patients with ACS who has undergone percutaneous coronary stenting.

High levels of homocysteine downregulate apolipoprotein E expression via nuclear factor kappa B

AIM: To investigate the effect of high homocysteine(Hcy) levels on apolipoprotein E(apoE) expression and the signaling pathways involved in this gene regulation.METHODS: Reverse transcriptase polymerase chain reaction(RT-PCR) and Western blot were used to assess apo E expression in cells treated with various concentrations(50-500 μmol/L) of Hcy. Calcium phosphatetransient transfections were performed in HEK-293 and RAW 264.7 cells to evaluate the effect of Hcy on apoE regulatory elements [promoter and distal multienhancer 2(ME2)]. To this aim, plasmids containing the proximal apoE promoter [(-500/+73)apoE construct] alone or in the presence of ME2 [ME2/(-500/+73)apoE construct] to drive the expression of the reporter luciferase gene were used. Co-transfection experiments were carried out to investigate the downstream effectors of Hcymediated regulation of apoE promoter by using specific inhibitors or a dominant negative form of IKβ. In other co-transfections, the luciferase reporter was under the control of synthetic promoters containing multiple specific binding sites for nuclear factor kappa B(NF-κB), activator protein-1(AP-1) or nuclear factor of activated T cells(NFAT). Chromatin immunoprecipitation(ChI P)assay was accomplished to detect the binding of NF-κB p65 subunit to the apoE promoter in HEK-293 treated with 500 μmol/L Hcy. As control, cells were incubated with similar concentration of cysteine. NF-κB p65 proteins bound to DNA were immunoprecipitated with anti-p65 antibodies and DNA was identified by PCR using primers amplifying the region-100/+4 of the apoE gene. RESULTS: RT-PCR revealed that high levels of Hcy(250-750 μmol/L) induced a 2-3 fold decrease in apoE m RNA levels in HEK-293 cells, while apo E gene expression was not significantly affected by treatment with lower concentrations of Hcy(100 μmol/L). Immunoblotting data provided additional evidence for the negative role of Hcy in apoE expression. Hcy decreased apoE promoter activity, in the presence or absence of ME2, in a dose dependent manner, in both RAW 264.7 and HEK-293 cells, as revealed by transient transfection experiments. The downstream effectors of the signaling pathways of Hcy were also investigated. The inhibitory effect of Hcy on the apo E promoter activity was counteracted by MAPK/ERK kinase 1/2(MEK1/2) inhibitor U0126, suggesting that MEK1/2 is involved in the downregulation of apoE promoter activity by Hcy. Our data demonstrated that Hcy-induced inhibition of apoE took place through activation of NF-κB. Moreover, we demonstrated that Hcy activated a synthetic promoter containing three NF-κB binding sites, but did not affect promoters containing AP-1 or NFAT binding sites. ChI P experiments revealed that NF-κB p65 subunit is recruited to the apoE promoter following Hcy treatment of cells.CONCLUSION: Hcy-induced stress negatively modulates apoE expression via MEK1/2 and NF-κB activation. The decreased apo E expression in peripheral tissues may aggravate atherosclerosis, neurodegenerative diseases and renal dysfunctions.

Association between serum homocysteine and arterial stiffness：role of anti-hypertensive drugs

To the Editor I read the article of Zhang, et al. with great interest. They investigated the association of homocysteine with arterial stiffness in Chinese community-based elderly persons. The carotid-femoral pulse wave velocity （PWV） was significantly higher in the high homocyteine group than in the normal one, however, there was no differences in carotid-radial PWV between the high homocyteine group and the normal one. Homocysteine levels were strongly associated with the carotidfemoral PWV even after adjustment for classical risk factors of cardiovascular disease. I congratulate the authors for this important study. However, I want to make minor criticism for this study from the methodological aspect.

Homocysteine-induced Enhanced Expression of Tissue Factor in Human Vascular Smooth Muscle Cells

The homocysteine （Hcy）-induced tissue factor （TF） expression in human vascular smooth muscle cells （VSMCs） and the effect of Hcy on the activity of nuclear factor-kappaB （NF-кB） and the expression of inducible nitric oxide synthase （iNOS） were investigated. Human umbilical artery VSMCs were cultured by tissue explanting method, identified by α-actin immunohistochemistry, and incubated with different concentrations of Hcy/PTDC （NF-кB inhibitor）. Semi-quantitative RT-PCR was performed to detect the expression of TF mRNA in VSMCs. Flow cytometry was used to assay the expression of TF protein on the surface of VSMCs and the expression of iNOS in VSMCs. Western blot was carried out to detect the expression of NF-кB protein in nuclei. The results showed that Hcy could induce VSMCs expressing TF mRNA significantly after the VSMCs were incubated with Hcy at concentrations of 10, 100, 500 μmol/L respectively. There was low expression level of TF protein on the surface of the resting VSMCs and Hcy could also induce VSMCs expressing TF pro- tein on the cell surface in different concentrations. Additionally, Hcy could rapidly induce the activation of NF-кB and this effect could be significantly inhibited by PDTC. Hcy alone could not induce the expression of iNOS in VSMCs. It was concluded that Hcy could significantly induce the expression of TF in VSMCs and enhance the activation of NF-ΚB, subsequently mediate TF gene expression and protein synthesis. NF-кB-mediated expression of TF in VSMCs might be the important mechanism of atherosclerosis and thrombosis induced by Hcy.

Hyperhomocysteinemia induces injury in olfactory bulb neurons by downregulating Hes1 and Hes5 expression

Hyperhomocysteinemia has been shown to be associated with neurodegenerative diseases; however, lesions or histological changes and mechanisms underlying homocysteine-induced injury in olfactory bulb neurons remain unclear. In this study, hyperhomocysteinemia was induced in apolipoprotein E-deficient mice with 1.7% methionine. Pathological changes in the olfactory bulb were observed through hematoxylin-eosin and Pischingert staining. Cell apoptosis in the olfactory bulb was determined through terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling （TUNEL） staining. Transmission electron microscopy revealed an abnormal ultrastructure of neurons. Furthermore, immunoreactivity and expression of the hairy enhancer of the split 1 （Hesl） and Hess were measured using immunohistochemistry, immunofluorescence, and western blot assay. Our results revealed no significant structural abnormality in the ol- factory bulb of hyperhomocysteinemic mice. However, the number of TUNEL-positive cells increased in the olfactory bulb, lipofuscin and vacuolization were visible in mitochondria, and the expression of Hes1 and Hes5 decreased. These findings confirm that hyperhomocyste- inemia induces injury in olfactory bulb neurons by downregulating Hes1 and Hes5 expression.