LCZ696 Possesses a Protective Effect Against Homocysteine (Hcy)-Induced Impairment of Blood-Brain Barrier (BBB) Integrity by Increasing Occludin, Mediated by the Inhibition of Egr-1.

Homocysteine (Hcy) is a non-essential amino acid produced from methionine. It has been reported that high concentrations of Hcy are related to the pathogenesis of neurodegenerative diseases and induce the disruption of the blood-brain barrier (BBB) by triggering oxidative stress and inflammation. LCZ696 is a novel antihypertensive agent that has been recently reported to possess promising anti-inflammatory properties. However, whether it has a protective effect on the BBB disruption is still unknown. For the first time, in this study, we aim to investigate whether LCZ696 exerts anti-inflammatory effects on Hcy-induced injury in brain endothelial cells and explore its neuroprotective properties. In in vivo experiments, we found that treatment with LCZ696 ameliorated oxidative stress by reducing malondialdehyde (MDA) and increasing glutathione (GSH). Furthermore, LCZ696 downregulated the excessive release of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) at mRNA and protein levels. Importantly, it reversed the disruption of the BBB induced by Hcy stimulation. In the in vitro human brain microvascular endothelial cell (HBMVEC) experiments, compared to the control, the permeability of the endothelial monolayer was significantly enlarged, the expression level of occludin declined, and Egr-1 upregulated by the introduction of Hcy, and these were all reversed by the treatment with LCZ696. Lastly, we found that the protective effects of LCZ696 against Hcy-induced reduction of occludin and hyper-permeability of the endothelial monolayer were greatly abolished by the overexpression of Egr-1. Taken together, we found that LCZ696 protected against Hcy-induced impairment of BBB integrity by increasing the expression of occludin, all mediated by the inhibition of Egr-1.

Utilization of B12 for the treatment of chronic migraine.

Chronic migraine is a particular classification of a headache that is typically unilateral and pulsatile and lasts for at least 3 months. Owing to its high prevalence and detrimental impact on personal, social, and economic aspects of patient lives, much desire has gone into fully understanding the pathogenesis of migraine, and to search for therapeutic agents. In addition to current therapeutics such as triptans, ergotamine, and monoclonal antibodies targeting calcitonin gene-related peptide receptors, vitamin B12 has been investigated for its possible use as a prophylactic agent for migraines. Specifically, the observed effects of vitamin B12 on nitric oxide and homocysteine prompt further investigation of its underlying mechanisms in migraine pathophysiology. In this comprehensive review, we provide a brief overview of migraines and current therapies while focusing on the promising role of vitamin B12 as a possible treatment option for chronic migraine management.

A novel nutritional supplement to reduce plasma homocysteine in nonpregnant women: A randomised controlled trial in The Gambia.

BACKGROUND: Infant DNA methylation profiles are associated with their mother's periconceptional nutritional status. DNA methylation relies on nutritional inputs for one-carbon metabolic pathways, including the efficient recycling of homocysteine. This randomised controlled trial in nonpregnant women in rural Gambia tests the efficacy of a novel nutritional supplement designed to improve one-carbon-related nutrient status by reducing plasma homocysteine, and assesses its potential future use in preconception trials. METHODS AND FINDINGS: We designed a novel drink powder based on determinants of plasma homocysteine in the target population and tested it in a three-arm, randomised, controlled trial. Nonpregnant women aged between 18 and 45 from the West Kiang region of The Gambia were randomised in a 1:1:1 allocation to 12 weeks daily supplementation of either (a) a novel drink powder (4 g betaine, 800 µg folic acid, 5.2 µg vitamin B12, and 2.8 mg vitamin B2), (b) a widely used multiple micronutrient tablet (United Nations Multiple Micronutrient Preparation [UNIMMAP]) containing 15 micronutrients, or (c) no intervention. The trial was conducted between March and July 2018. Supplementation was observed daily. Fasted venepuncture samples were collected at baseline, midline (week 5), and endline (week 12) to measure plasma homocysteine. We used linear regression models to determine the difference in homocysteine between pairs of trial arms at midline and endline, adjusted for baseline homocysteine, age, and body mass index (BMI). Blood pressure and pulse were measured as secondary outcomes. Two hundred and ninety-eight eligible women were enrolled and randomised. Compliance was >97.8% for both interventions. At endline (our primary endpoint), the drink powder and UNIMMAP reduced mean plasma homocysteine by 23.6% (-29.5 to -17.1) and 15.5% (-21.2 to -9.4), respectively (both p < 0.001), compared with the controls. Compared with UNIMMAP, the drink powder reduced mean homocysteine by 8.8% (-15.8 to -1.2; p = 0.025). The effects were stronger at midline. There was no effect of either intervention on blood pressure or pulse compared with the control at endline. Self-reported adverse events (AEs) were similar in both intervention arms. There were two serious AEs reported over the trial duration, both in the drink powder arm, but judged to be unrelated to the intervention. Limitations of the study include the use of a single targeted metabolic outcome, homocysteine. CONCLUSIONS: The trial confirms that dietary supplements can influence metabolic pathways that we have shown in previous studies to predict offspring DNA methylation. Both supplements reduced homocysteine effectively and remain potential candidates for future epigenetic trials in pregnancy in rural Gambia. TRIAL REGISTRATION: Clinicaltrials.gov Reference NCT03431597.

Hydrogen sulfide suppresses homocysteine-induced glial activation and inflammatory response.

Neuro-inflammation plays a critical role in hyperhomocysteinemia (HHcy)-associated neurodegenerative disorders. Hydrogen sulfide (H2S) has been suggested as an endogenous neuromodulator and potent anti-inflammatory molecule. In present study, we have investigated the effect of NaHS supplementation (a H2S source) on inflammatory response in animals subjected to HHcy. NaHS adminstration restored the decreased levels of H2S and polysulfides with a concomitant increase in the activity of cystathionase (CSE) and cystathionine ß-synthase (CBS) in the brain regions of HHcy animals. NaHS supplementation reduced the expression of glial fibrillary acidic protein (GFAP) and ionized calcium binding adaptor molecule 1 (Iba1) suggesting attenuation of astrocyte and microglia activation in HHcy animals. Tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6) and monocyte chemoattractant protein-1 (MCP-1) were decreased in the cortex and hippocampus of HHcy animals following NaHS supplementation. Moreover, NaHS supplementation also decreased the TNF-α, IL-6 and MCP-1 in the serum of HHcy animals. NaHS supplementation reduced nitrite levels, 3-nitrotyrosine (3-NT) modified proteins and inducible nitric oxide synthase (iNOS) in the cortex and hippocampus of HHcy animals. However, NaHS administration increased endothelial nitric oxide synthase (eNOS) expression in brain regions of Hcy treated animals. Expression of platelet endothelial cell adhesion molecule (PECAM) was decreased in the microvessels from HHcy animals supplemented with NaHS. Furthermore, HHcy-induced memory deficits assessed by Morris water maze and novel object recognition test were reversed by NaHS administration. Taken together, the findings suggest that NaHS supplementation ameliorates Hcy-induced glia mediated inflammatory response and cognitive deficits. Therefore, H2S may be a novel therapeutic molecule to treat HHcy associated neurological disorders and neuro-inflammatory conditions.

Homocysteine and small vessel stroke: A mendelian randomization analysis.

OBJECTIVE: Trials of B vitamin therapy to lower blood total homocysteine (tHcy) levels for prevention of stroke are inconclusive. Secondary analyses of trial data and epidemiological studies suggest that tHcy levels may be particularly associated with small vessel stroke (SVS). We assessed whether circulating tHcy and B vitamin levels are selectively associated with SVS, but not other stroke subtypes, using Mendelian randomization. METHODS: We used summary statistics data for single-nucleotide polymorphisms (SNPs) associated with tHcy (n = 18), folate (n = 3), vitamin B6 (n = 1), and vitamin B12 (n = 14) levels, and the corresponding data for stroke from the MEGASTROKE consortium (n = 16,952 subtyped ischemic stroke cases and 404,630 noncases). RESULTS: Genetically predicted tHcy was associated with SVS, with an odds ratio of 1.34 (95% confidence interval [CI], 1.13-1.58; p = 6.7 × 10-4 ) per 1 standard deviation (SD) increase in genetically predicted tHcy levels, but was not associated with large artery or cardioembolic stroke. The association was mainly driven by SNPs at or near the MTHFR and MUT genes. The odds ratios of SVS per 1 SD increase in genetically predicted folate and vitamin B6 levels were 0.49 (95% CI, 0.34-0.71; p = 1.3 × 10-4 ) and 0.70 (95% CI, 0.52-0.94; p = 0.02), respectively. Genetically higher vitamin B12 levels were not associated with any stroke subtype. INTERPRETATION: These findings suggest that any effect of homocysteine-lowering treatment in preventing stroke will be confined to the SVS subtype. Whether genetic variants at or near the MTHFR and MUT genes influence SVS risk through pathways other than homocysteine levels and downstream effects require further investigation. Ann Neurol 2019;85:495-501.

Pioglitazone restores the homocysteine­impaired function of endothelial progenitor cells via the inhibition of the protein kinase C/NADPH oxidase pathway.

Homocysteine (Hcy) has been shown to impair the migratory and adhesive activity of endothelial progenitor cells (EPCs). As a peroxisome proliferator­activated receptor γ agonist, pioglitazone (PIO) has been predicted to regulate angiogenesis, and cell adhesion, migration and survival. The aim of the present study was to determine whether PIO could inhibit Hcy­induced EPC dysfunctions such as impairments of cell migration and adhesion. EPC migration and adhesion were assayed using 8.0­µm pore size Transwell membranes and fibronectin­coated culture dishes, respectively. Hcy at a concentration of 200 µM was observed to markedly impair cell migration and adhesiveness, and PIO at a concentration of 10 µM attenuated the Hcy­mediated inhibition of EPC migration and adhesion. The mechanism of these effects may be through the inhibition of protein kinase C (PKC) and reactive oxygen species production. The expression levels of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase subunits, NADPH oxidase 2 (Nox2) and p67phox, were upregulated by Hcy, with a peak in levels following treatment with a concentration of 200 µM. PIO downregulated the expression levels of Nox2 and p67phox via the PKC signaling pathway. Furthermore, the mechanism of PIO associated with downregulating the p67phox and Nox2 subunits of NADPH oxidase was verified. Thus, PKC and NADPH oxidase may serve a major role in the protective effects of PIO in EPCs under conditions of high Hcy concentrations.

The intervention of enalapril maleate and folic acid tablet on the expressions of the GRP78 and CHOP and vascular remodeling in the vascular smooth muscle cells of H-hypertensive rats with homocysteine.

OBJECTIVE: GRP78 and CHOP play essential roles in endoplasmic reticulum stress (ERS) of the vascular smooth muscle cells. We aim to investigate the effect of enalapril maleate and folic acid tablet on the expressions of GRP78 and CHOP and vascular remodeling in a homocysteine (HCY)-treated hypertensive rat model. MATERIALS AND METHODS: The hypertensive rat model was established with the technique of coarctation in the abdominal aorta, and the blood pressure of the rat was measured with the non-destructive tail-cuff method two weeks after operation. Thirty-six rats with hypertension were randomly divided into 3 groups (n=12 in each group). The control group received common diet and double distilled water, methionine group received 30 g/L methionine diet and double distilled water, while enalapril maleate and folic acid tablet group received 30 g/L methionine diet and 0.2 mg.kg-1.d-1 solution of enalapril maleate and folic acid tablet. Samples were collected at week 4 and week 8 for analysis. The plasma homocysteine was measured by homocysteine detector; MAP was detected through carotid artery incubation and aortic media thickness was determined by image analyses software. The expression of GRP78 and CHOP in the vascular smooth muscle cells were identified by immunohistochemistry and Western blot. RESULTS: Compared with the control group, the concentration of HCY in the serum of rats in methionine group was increased significantly after 4 weeks (p < 0.01), and even more significant after 8 weeks (p < 0.01). Compared with that of methionine group, the level of HCY in enalapril maleate and folic acid tablet group rats was significantly decreased (p < 0.01). The level of MAP in methionine group was increased significantly after 8 weeks compared with that of control group (p < 0.05). However, the MAP in enalapril maleate and folic acid tablet group was decreased significantly compared with that of methionine group. Compared with control group, the media thickness of vascular smooth muscle of rats in the methionine group was increased significantly (p < 0.05) while was statistically reduced in the enalapril maleate and folic acid tablet group (p < 0.05). The expressions of GRP78 and CHOP in methionine group were significantly elevated compared to that of control in a time dependent manner (p < 0.05), which were remarkably down regulated in enalapril maleate and folic acid tablet group compared with that in methionine group. CONCLUSIONS: The administration of enalapril maleate and folic acid tablet can maintain the normal state of cells via the alleviation of ERS and vascular damages, reduction of HCY and the thickness of arterial media as well as the improvement of vascular remodeling.

Probiotics Affect One-Carbon Metabolites and Catecholamines in a Genetic Rat Model of Depression.

SCOPE: Probiotics may influence one-carbon (C1) metabolism, neurotransmitters, liver function markers, or behavior. METHODS AND RESULTS: Male adult Flinders Sensitive Line rats (model of depression, FSL; n = 22) received Lactobacillus helveticus R0052 and Bifidobacterium longum R0175 (109 or 1010 colony-forming units per day) or vehicle for 10 weeks. The controls, Flinders Resistant Line rats (FRL, n = 8), only received vehicle. C1-related metabolites were measured in plasma, urine, and different tissues. Monoamine concentrations were measured in plasma, hippocampus, and prefrontal cortex. Vehicle-treated FSL rats had higher plasma concentrations of betaine, choline, and dimethylglycine, but lower plasma homocysteine and liver S-adenosylmethionine (SAM) than FRLs. FSL rats receiving high-dose probiotics had lower plasma betaine and higher liver SAM compared to vehicle-treated FSL rats. FSLs had higher concentrations of norepinephrine, dopamine, and serotonin than FRLs across various brain regions. Probiotics decreased plasma dopamine in FSLs in a dose-dependent manner. There were no detectable changes in liver function markers or behavior. CONCLUSIONS: Probiotics reduced the flow of methyl groups via betaine, increased liver SAM, and decreased plasma dopamine and norepinephrine. Since these changes in methylation and catecholamine pathways are known to be involved in several diseases, future investigation of the effect of probiotics is warranted.

Probucol reverses homocysteine induced inflammatory monocytes differentiation and oxidative stress.

Reactive oxygen species have been demonstrated to involve in homocysteine-induced Ly-6Chi monocytes differentiation. Probucol is an anti-oxidant agent that has been used to treat atherosclerosis. We sought to evaluate the effect and potential mechanism of probucol on homocysteine-induced inflammatory monocytes differentiation. The primary mouse splenocytes suspensions were initiated by recombinant interferon-γ and cultured with L-homocysteine in the presence or absence of probucol. The cells were co-incubated with monoclonal antibodies to CD11b-PE and Ly-6C FITC. Flow cytometry analysis was performed on BD FACS caliber. Data were analyzed using the FlowJo software. Mononuclear cells were gated according to the lower granular and larger size, distinguished with granulocytes and lymphocytes. Monocytes were defined as CD11b+ mononuclear cells and further divided into three groups based on their Ly-6C expressions, Ly-6Chi, Ly-6Cmid and Ly-6Clow subsets. The productions of reactive oxygen species in monocytes subsets were detected by 2',7'-dichlorofluorescein-diacetate (DCFH-DA) containing monocytes were marked as DCFH-DA+ cells in both Ly-6C+ and Ly-6C- subsets. The activity of nicotinamide adenine dinucleotide phosphate oxidase in THP-1 cells was measured by assay kit on enzyme-labelling instrument. L-homocysteine promoted inflammatory monocytes differentiation and its reactive oxygen species productions in dose-dependent manner. Probucol dose-dependently suppressed the differentiation and reactive oxygen species productions of inflammatory monocytes induced by L-homocysteine. Furthermore, the increased NADPH oxidase activity induced by L-homocysteine was significantly reversed by probucol in THP-1 cells. Probucol prevented L-homocysteine-induced inflammatory monocytes differentiation and its reactive oxygen species generation probably through inhibiting NADPH oxidase activity.

Role of homocysteine and folic acid on the altered calcium homeostasis of platelets from rats with biliary cirrhosis.

Previously, we have found that intracellular calcium homeostasis is altered in platelets from an experimental model of liver cirrhosis, the bile-duct ligated (BDL) rat; these alterations are compatible with the existence of a hypercoagulable state. Different studies indicate that cholestatic diseases are associated with hyperhomocysteinemia; thus, we hypothetized that it could contribute to those platelet alterations. In the present study, we have investigated the role of homocysteine (HCY) in platelet aggregation and calcium signaling in the BDL model. The effect of chronic folic acid treatment was also analyzed. Acute treatment with HCY increased the aggregation response to ADP and calcium responses to thrombin in platelets of control and BDL rats. Capacitative calcium entry was not altered by HCY. Chronic treatment with folic acid decreased platelet aggregation in control and BDL rats, but this decrease was greater in BDL rats. In folic acid-treated rats, thrombin-induced calcium entry and release were decreased in platelet of control rats but unaltered in BDL rats; however, capacitative calcium entry was decreased in platelets of control and BDL rats treated with folic acid. Reactive oxygen species were produced at higher levels by BDL platelets after stimulation with HCY or thrombin and folic acid normalized these responses. HCY plays a role in the enhanced platelet aggregation response of BDL rats, probably through an enhanced formation of ROS. Folic acid pretreatment normalizes many of the platelet alterations shown by BDL rats.

Endoplasmic Stress Inhibitors for Homocysteine Induced Cardiovascular Disease.

Cardiovascular disease (CVD) remains a major cause of death and disability worldwide, thus preventing and inhibiting CVD remains a health priority. Several lines of pharmacological interventions have not met with great success, thus inhibition of novel cellular stress pathways could be a novel therapeutic avenue to treat CVD. This review will focus on homocysteine and endoplasmic reticulum stress linked to mitochondria function, and possible therapeutic avenues for treatment.

Polyphenols and Polypeptides in Chinese Rice Wine Inhibit Homocysteine-induced Proliferation and Migration of Vascular Smooth Muscle Cells.

The beneficial effect of Chinese rice wine on atherosclerosis has been proved, but the exact components that have the cardiovascular protective effect are still unknown. This study aimed to explore the exact ingredients in Chinese rice wine that could inhibit homocysteine (Hcy)-induced vascular smooth muscle cell (VSMC) proliferation and migration. VSMCs were divided into 7 groups: control, Hcy (1 mmol/L), Hcy + oligosaccharide, Hcy + polypeptides, Hcy + polyphenols, Hcy + alcohol, and Hcy + Chinese rice wine. methyl thiazolyl tetrazolium (MTT) assay, Transwell chambers, and wound-healing assay were used to test the proliferation and migratory ability of the VSMCs. Western blot and gelatin zymography were used to investigate the expressions and activities of metal matrix proteinase 2/9 (MMP-2/9) and tissue inhibitor of metalloproteinase 2 (TIMP-2) in VSMCs. Polypeptides and polyphenols in the Chinese rice wine reduced the proliferation and migration ability of the VSMCs. Furthermore, they also decreased the expression and activity of MMP-2/9 but had no obvious impact on the expression of TIMP-2 in each group. This study further confirms that polypeptides and polyphenols in the Chinese rice wine could inhibit Hcy-induced proliferation and migration of VSMCs and maintain the balance between matrix metalloproteinases (MMPs) and TIMPs.

Oral N-acetylcysteine reduces plasma homocysteine concentrations regardless of lipid or smoking status.

BACKGROUND: Elevated total plasma homocysteine (tHcy) is considered to be an independent cardiovascular disease risk factor, although tHcy lowering by B-vitamins improves only certain clinical endpoints. N-acetylcysteine (NAC), a thiol-containing antioxidant, acutely lowers tHcy and possibly also blood pressure. However, to our knowledge, at present no conclusive long-term evaluation exists that controls for factors such as hyperlipidemia, smoking, medication, and disease stage, all of which affect the thiol redox state, including tHcy. OBJECTIVE: We reanalyzed 2 double-blind, placebo-controlled trials in unmedicated middle-aged men, one in a hyperlipidemic group (HYL group; n = 40) and one in a normolipidemic group (NOL group; n = 42), each stratified for smokers and nonsmokers. DESIGN: We evaluated the effect of 4 wk of oral NAC (1.8 g/d) on tHcy (primary endpoint), plasma thiol (cysteine), and intracellular glutathione concentrations as well as on blood pressure. The HYL group had total cholesterol >220 mg/dL or triglycerides >150 mg/dL. RESULTS: NAC treatment significantly (P = 0.001, multivariate analysis of variance for repeated measures) lowered postabsorptive plasma concentrations of tHcy by -11.7% ± 3.0% (placebo: 4.1% ± 3.6%) while increasing those of cysteine by 28.1% ± 5.7% (placebo: 4.0% ± 3.4%) with no significant impact of hyperlipidemia or smoking. Moreover, NAC significantly decreased systolic (P = 0.003) and diastolic (P = 0.017) blood pressure within all subjects with a significant reduction in diastolic pressure in the HYL group (P = 0.008) but not in the NOL group. An explorative stepwise multiple regression analysis identified 1) post-treatment cysteine as well as 2) pretreatment tHcy and 3) albumin plasma concentrations as being significant contributors to tHcy reduction. CONCLUSIONS: Four weeks of oral NAC treatment significantly decreased plasma tHcy concentrations, irrespective of lipid or smoking status, and lowered systolic blood pressure in both normolipidemic and hyperlipidemic men, with significant diastolic blood pressure reductions in the HYL group only. Increased oral intake of cysteine may therefore be considered for primary or secondary prevention of vascular events with regard to the 2 independent risk factors of hyperhomocysteinemia and arterial hypertension.

Emodin Inhibits Homocysteine-Induced C-Reactive Protein Generation in Vascular Smooth Muscle Cells by Regulating PPARγ Expression and ROS-ERK1/2/p38 Signal Pathway.

Atherosclerosis is an inflammatory disease. As an inflammatory molecule, C-reactive protein (CRP) plays a direct role in atherogenesis. It is known that the elevated plasma homocysteine (Hcy) level is an independent risk factor for atherosclerosis. We previously reported that Hcy produces a pro-inflammatory effect by inducing CRP expression in vascular smooth muscle cells (VSMCs). In the present study, we observed effect of emodin on Hcy-induced CRP expression in rat VSMCs and molecular mechanisms. The in vitro results showed that pretreatment of VSMCs with emodin inhibited Hcy-induced mRNA and protein expression of CRP in a concentration-dependent manner. The in vivo experiments displayed that emodin not only inhibited CRP expression in the vessel walls in mRNA and protein levels, but also reduced the circulating CRP level in hyperhomocysteinemic rats. Further study revealed that emodin diminished Hcy-stimulated generation of reactive oxygen species (ROS), attenuated Hcy-activated phosphorylation of ERK1/2 and p38, and upregulated Hcy-inhibited expression of peroxisome proliferator-activated receptor gamma (PPARγ) in VSMCs. These demonstrate that emodin is able to inhibit Hcy-induced CRP generation in VSMCs, which is related to interfering with ROS-ERK1/2/p38 signal pathway and upregulating PPARγ expression. The present study provides new evidence for the anti-inflammatory and anti-atherosclerotic effects of emodin.

Betaine prevents homocysteine-induced memory impairment via matrix metalloproteinase-9 in the frontal cortex.

Betaine plays important roles that include acting as a methyl donor and converting homocysteine (Hcy) to methionine. Elevated plasma Hcy levels are known as hyperhomocysteinemia (HHcy) and contribute to impairments of learning and memory. Although it is commonly known that betaine plays an important role in Hcy metabolism, the effects of betaine on Hcy-induced memory impairment have not been investigated. Previously, we demonstrated the beneficial effects of betaine on acute stress and lipopolysaccharide-induced memory impairment. In the present study, we investigated whether betaine ameliorates Hcy-induced memory impairment and the underlying mechanisms of this putative effect. Mice were treated with Hcy (0.162mg/kg, s.c.) twice a day for nine days, and betaine (25mg/kg, s.c.) was administered 30min before the Hcy injections. The memory functions were evaluated using a spontaneous alternation performance test (Y-maze) at seven days and a step-down type passive avoidance test (SD) at nine and ten days after Hcy injection. We found that betaine suppressed the memory impairment induced by repeated Hcy injections. However, the blood concentrations of Hcy were significantly increased in the Hcy-treated mice immediately after the passive avoidance test, and betaine did not prevent this increase. Furthermore, Hcy induces redox stress in part by activating matrix metalloproteinase-9 (MMP-9), which leads to BBB dysfunction. Therefore, we tested whether betaine affected MMP-9 activity. Interestingly, treatment with betaine significantly inhibited Hcy-induced MMP-9 activity in the frontal cortex but not in the hippocampus after acute Hcy injection. These results suggest that the changes in MMP-9 activity after betaine treatment might have been partially responsible for the amelioration of the memory deficits and that MMP-9 might be a candidate therapeutic target for HHcy.

Acetyl-l-carnitine prevents homocysteine-induced suppression of Nrf2/Keap1 mediated antioxidation in human lens epithelial cells.

Previous studies have revealed that high levels of serum homocysteine (Hcy) are closely associated with the development of juvenile and age-related cataracts. An increased concentration of Hcy is likely to induce gene specific demethylation in DNA promoter regions. The aim of the present study was to prevent this demethylation by administering acetyl-l-carnitine (ALCAR) to human lens epithelial cells (HLECs). Different concentrations of Hcy were used to treat HLECs for 3, 6, 12 and 24 h and the findings were used to determine the optimum dose to induce endoplasmic reticulum (ER) stress. Similarly, the concentration of ALCAR was standardized. The production of reactive oxygen species (ROS) and the percentage of cells undergoing cell death were measured. The levels of antioxidants, ER stress-associated proteins, mRNA levels of nuclear factor erythroid-2-related factor 2 (Nrf2), Kelch-like ECH-associated protein 1 (Keap1) and promoter DNA methylation of the Keap1 gene were also assessed. Hcy was observed to induce ER stress, produce ROS and lead to cell death. However, administration of ALCAR prevented these effects to a significant degree. Additionally, western blot analysis revealed that ALCAR increased the levels of antioxidant proteins, including catalase, superoxide dismutase, glutathione peroxidase, Nrf2, Keap1 and glutathione. Similarly, the reverse transcription-quantitative polymerase chain reaction experiments on Nrf2 and Keap1, as well as the bisulfite genomic DNA sequencing analysis revealed a preventive effect of ALCAR against Hcy-induced ER stress. The ER stress-induced activation of the unfolded protein response is responsible for demethylation of Keap1 promoter DNA to activate the expression of the Keap1 protein, which then increases the targeting of Nrf2 for proteosomal degradation. This decrease in Nrf2 activity represses the transcription of numerous antioxidant enzyme genes and alters the redox-balance towards lens oxidation. However, treatment with ALCAR led to significant protection from these effects. The present results suggested that ALCAR either prevents or ameliorates the actions of the antioxidant system in HLECs at the level of the protein and the gene. Further advanced studies are required for the development of ALCAR as an anti-cataract agent.

Effects of homocysteine lowering with B vitamins on cognitive aging: meta-analysis of 11 trials with cognitive data on 22,000 individuals.

BACKGROUND: Elevated plasma homocysteine is a risk factor for Alzheimer disease, but the relevance of homocysteine lowering to slow the rate of cognitive aging is uncertain. OBJECTIVE: The aim was to assess the effects of treatment with B vitamins compared with placebo, when administered for several years, on composite domains of cognitive function, global cognitive function, and cognitive aging. DESIGN: A meta-analysis was conducted by using data combined from 11 large trials in 22,000 participants. Domain-based z scores (for memory, speed, and executive function and a domain-composite score for global cognitive function) were available before and after treatment (mean duration: 2.3 y) in the 4 cognitive-domain trials (1340 individuals); Mini-Mental State Examination (MMSE)-type tests were available at the end of treatment (mean duration: 5 y) in the 7 global cognition trials (20,431 individuals). RESULTS: The domain-composite and MMSE-type global cognitive function z scores both decreased with age (mean ± SE: -0.054 ± 0.004 and -0.036 ± 0.001/y, respectively). Allocation to B vitamins lowered homocysteine concentrations by 28% in the cognitive-domain trials but had no significant effects on the z score differences from baseline for individual domains or for global cognitive function (z score difference: 0.00; 95% CI: -0.05, 0.06). Likewise, allocation to B vitamins lowered homocysteine by 26% in the global cognition trials but also had no significant effect on end-treatment MMSE-type global cognitive function (z score difference: -0.01; 95% CI: -0.03, 0.02). Overall, the effect of a 25% reduction in homocysteine equated to 0.02 y (95% CI: -0.10, 0.13 y) of cognitive aging per year and excluded reductions of >1 mo per year of treatment. CONCLUSION: Homocysteine lowering by using B vitamins had no significant effect on individual cognitive domains or global cognitive function or on cognitive aging.