Identification of Novel Antimicrobial Compounds Targeting Mycobacterium tuberculosis S-Adenosyl-L-Homocysteine Hydrolase Using Dual Hierarchical In Silico Structure-Based Drug Screening.

The emergence of multidrug-resistant and extensively drug-resistant Mycobacterium tuberculosis (M. tuberculosis) has become a major medical problem. S-adenosyl-L-homocysteine hydrolase (MtSAHH) was selected as the target protein for the identification of novel anti-TB drugs. Dual hierarchical in silico Structure-Based Drug Screening was performed using a 3D compound structure library (with over 150 thousand synthetic chemicals) to identify compounds that bind to MtSAHH's active site. In vitro experiments were conducted to verify whether the nine compounds selected as new drug candidates exhibited growth-inhibitory effects against mycobacteria. Eight of the nine compounds that were predicted by dual hierarchical screening showed growth-inhibitory effects against Mycobacterium smegmatis (M. smegmatis), a model organism for M. tuberculosis. Compound 7 showed the strongest antibacterial activity, with an IC50 value of 30.2 µM. Compound 7 did not inhibit the growth of Gram-negative bacteria or exert toxic effects on human cells. Molecular dynamics simulations of 40 ns using the MtSAHH-Compound 7 complex structure suggested that Compound 7 interacts stably with the MtSAHH active site. These in silico and in vitro results suggested that Compound 7 is a promising lead compound for the development of new anti-TB drugs.

Pirfenidone alleviates vascular intima injury caused by hyperhomocysteinemia.

OBJECTIVES: Hyperhomocysteinemia (HHcy) can induce vascular inflammatory and oxidative damage and accelerate intimal hyperplasia. This study investigated the protective effect of pirfenidone (PFD) on the recovery process of injured endothelial arteries during HHcy. MATERIALS AND METHODS: Thirty rabbits were randomly separated into three groups: A control group (n=10, standard rabbit chow), a model group (n=10, control diet plus 30 g methionine/kg food), and a PFD group (n=10, model diet plus oral administration of 90 mg/day of PFD). After 14 weeks of arterial injury, histopathological changes were determined. Plasma homocysteine (Hcy) concentrations, lipid profiles and oxidant and antioxidant status were evaluated. Macrophage infiltration was assessed using immunohistochemical staining. RESULTS: PFD supplementation decreased macrophage infiltration of iliac artery significantly without changes in blood lipids and Hcy concentrations. Compared with the model group, PFD restored superoxide dismutase and glutathione peroxidase activities and reduced malondialdehyde and reactive oxygen species levels. A high-methionine diet significantly increased neointimal area and the ratio between neointimal and media area. Systemic administration of PFD inhibited neointimal formation. CONCLUSIONS: PFD can partly alleviate intimal hyperplasia by inhibiting inflammatory and oxidative stress response induced by HHcy during endothelial injury. It may be a potential therapeutic agent for the prevention and treatment of endothelial injury-associated diseases such as atherosclerosis.

Tetrahydroxystilbene glucoside attenuated homocysteine-upregulated endothelin receptors in vascular smooth muscle cells via the ERK1 /2 /NF-κB signaling pathway.

2,3,5,4'-Tetrahydrostilbene-2-o-ß-d-glucoside (TSG) is the main active component of Polygonum multiflorum Thunb. It has effects on hypertension. However, the mechanism is unclear. Current research is devoted to exploring the mechanism of TSG improving HHcy-induced hypertension. The mice received a subcutaneous injection of Hcy in the presence or absence of TSG for 4 weeks. Blood pressure (BP) was measured using a noninvasive tail-cuff plethysmography method. Levels of plasma Hcy and endothelin-1 were measured using ELISA. Rat SMA without endothelium was cultured in a serum-free medium in the presence or absence of TSG with or without Hcy. The contractile response to sarafotoxin 6c or endothein-1 was studied using a sensitive myography. The levels of protein were detected using Western blotting. The results showed that TSG lowered HHcy-elevated BP and decreased levels of plasma Hcy and endothelin-1 in mice. Furthermore, the results showed that TSG inhibited Hcy-upregulated ET receptor expression and ET receptor-mediated contractile responses as well as the levels of p-ERK1/2 and p-p65 in SMA. In vivo results further validate the in vitro results. In conclusion, TSG can decrease the levels of plasma Hcy and ET-1 and downregulate Hcy-upregulated ET receptors in VSMCs by inhibiting the ERK1/2 /NF-κB/ETB2 pathway to lower the BP.

Omega 3 fatty acids - Potential modulators for oxidative stress and inflammation in the management of sickle cell disease.

OBJECTIVE: Sickle cell disease is characterized by clinical complications resulting in vaso-occlusive crisis with prominent attributes of oxidative stress, inflammation, and pain. Inflammation is an integral part of this disease which further exacerbates the pain during a crisis. Omega-3 fatty acids are known to possess anti-inflammatory and anti-aggregatory properties and assist in diminishing the slow physiological inactivation. METHODS: A pilot nutritional interventional study was conducted wherein forty-three children with sickle cell disease aged 5-16 years were supplemented with omega-3 fatty acids for a period of six months. Analysis of oxidative stress, as well as inflammatory parameters, was done pre and post-supplementation. RESULTS: Increased free oxygen radical transference values depicting free radical generation is enhanced in these patients along with a reduced antioxidant defense, as seen by decreased free oxygen radical defense values. Supplementation with omega-3 fatty acids for a period of six months significantly reduced the inflammatory marker homocysteine in all patients, whereas high sensitive C reactive protein was significantly reduced only in females of the age group 11-16years. Simultaneously a significant reduction in oxidative stress parameters with a concomitant increase of antioxidant defense was observed in all patients. CONCLUSION: The authors' findings suggest the regulatory effects of omega-3 fatty acids as cellular activators in alleviating the complications due to sickle cell disease. Omega-3 fatty acids hold promise as future therapeutic candidates in patients with sickle cell disease.

Adequate Intake and Supplementation of B Vitamins, in Particular Folic Acid, can Play a Protective Role in Bone Health.

In vitro and animal model studies have shown that vitamin B (VB) deficiency has negative consequences on bone as a result of direct or mediated activity of hyperhomocysteinemia. However, there are still no precise indications regarding a possible VB role in order to maintain bone health. So, the aim of this narrative review was to consider state of the art correlation between VB dietary intake, blood levels and supplementation and bone health (bone mineral density (BMD), bone turnover markers and fractures risk) in humans. This review includes 29 eligible studies. Considering VB blood levels, the 14 studies considered have shown that low serum folate can be a risk factor for reduced BMD and fractures in the elderly, particularly women; no independent association was found for other VB. Studies that evaluate the relationship between VB dietary intake and BMD are only 2; one, conducted on 1869 women, demonstrated a positive effect of folate intake on BMD. Another demonstrated a dose-dependent inverse relationship between vitamin B6 dietary intake and risk of hip fracture, but only for 35298 female participants. Regarding the relationship between BV supplementation and bone health (9 studies with only VB and 4 with other nutrients), all studies that considered patients with hyperhomocysteinemia or with low folate blood levels, are in agreement in demonstrating that folate supplementation (500mcg- 5mg) is useful in improving BMD. In conclusion, a request for folate and homocysteine blood levels in elderly patients with osteopenia/osteoporosis is mandatory. For patients with hyperhomocysteinemia or with low folate blood levels, folate supplementation (500mcg-5mg) is crucial.

Protective role of selenium on MPP+ and homocysteine-induced TRPM2 channel activation in SH-SY5Y cells.

Homocysteine is an intermediate product of biochemical reactions occurring in living organisms. It is known that drugs that increase dopamine synthesis used in Parkinson's disease (PD) cause an increase in the plasma homocysteine level. As the plasma homocysteine level increases, the amount of intracellular free calcium ion ([Ca2+]i) and oxidative stress increase. As a result, it contributes to the excitotoxic effect by causing neurodegeneration. TRPM2 cation channel is activated by high [Ca2+]i and oxidative stress. The role of TRPM2 in the development of neuronal damage due to the increase in homocysteine in PD has not yet been elucidated. In current study, we aimed to investigate the role of the TRPM2 and selenium (Se) in SH-SY5Y neuronal cells treated with homocysteine (HCT) and MPP . SH-SY5Y cells were divided into four groups: control, MPP, MPP + HCT, and MPP + HCT + Se. The results of plate reader assay, confocal microscope imaging, and western blot analyses indicated upregulation of apoptosis, [Ca2+]i, mitochondrial membrane depolarization, caspase activation, and intracellular ROS values in the cells. The MPP + HCT group had considerably higher values than the other groups. The MPP + HCT + Se group had significantly lower values than all the other groups except the control group. In addition, incubation of MPP + HCT and MPP + HCT + Se groups with TRPM2 antagonist 2-APB increased cell viability and reduced intracellular calcium influx and apoptosis levels. It is concluded that the activation of TRPM2 was propagated in HCT and MPP-induced SH-SY5Y cells by the increase of oxidative stress. The antioxidant property of Se regulated the TRPM2 channel activation and neurodegeneration by providing intracellular oxidant/antioxidant balance.

Lycium barbarum polysaccharide protects against Homocysteine-induced Vascular smooth muscle cell proliferation and phenotypic transformation via PI3K/Akt pathway.

Lycium barbarum polysaccharide (LBP) is an alkaloid extracted from lycium barbarum. LBP is the active component of lycium barbarum used to treat hypertension, atherosclerosis and other cardiovascular diseases in Chinese traditional medicine. However, the underlying cellular and molecular mechanisms of LBP- mediated activity in vascular disease remain poorly understood. In the present study, we showed the protective effect of LBP in vascular smooth muscle cells. Our results indicate that LBP significantly reduces the proliferation of VSMCs caused by Homocysteine (Hcy) and inhibits the phenotypic transformation of VSMCs caused by Hcy, from contractile to synthetic. LBP inhibited the protein expression of PI3K and Akt caused by Hcy, and increased the expression of miR-145. The results indicate that LBP exhibits substantial therapeutic potential for the treatment of Hcy-induced VSMCs proliferation and phenotypic transformation through inhibition of PI3K/Akt signaling pathway.

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.

Folic acid ameliorates homocysteine-induced angiogenesis and portosystemic collaterals in cirrhotic rats.

INTRODUCTION AND OBJECTIVES: Liver cirrhosis is characterized by increased intrahepatic resistance, splanchnic vasodilation/angiogenesis, and formation of portosystemic collateral vessels. Collaterals can cause lethal complications such as gastroesophageal variceal hemorrhage. Homocysteine is linked to vascular dysfunction and angiogenesis and higher levels have been reported in cirrhotic patients. It is also known that folic acid supplementation reverses the effects of homocysteine. However, the treatment effect in cirrhosis has yet to be investigated. MATERIAL AND METHODS: Liver cirrhosis was induced in Sprague-Dawley rats with common bile duct ligation (CBDL). The CBDL rats randomly received (1) vehicle; (2) dl-homocysteine thiolactone (1g/kg/day); (3) dl-homocysteine thiolactone plus folic acid (100mg/kg/day); or (4) folic acid. On the 29th day, hemodynamic parameters, liver and renal biochemistry, protein expressions of proangiogenic factors, mesenteric vascular density and portosystemic shunting were evaluated. RESULTS: In the cirrhotic rats, homocysteine increased mesenteric vascular density and the severity of shunting. It also up-regulated the protein expressions of mesenteric vascular endothelial growth factor (VEGF) and phosphorylated-endothelial nitric oxide synthase (p-eNOS). These effects were reversed by folic acid treatment (P<0.05). CONCLUSION: Folic acid ameliorated the adverse effects of homocysteine in the cirrhotic rats, which may be related to down-regulation of the VEGF-NO signaling pathway.

Homocysteine, Thioretinaco Ozonide, and Oxidative Phosphorylation in Cancer and Aging: A Proposed Clinical Trial Protocol.

The objective of the proposed clinical interventional trial is to demonstrate the efficacy of a novel therapeutic strategy in subjects with cancer and hyperhomocysteinemia. Following discovery of abnormal homocysteine thiolactone metabolism in cultured malignant cells, thioretinamide, the amide synthesized from retinoic acid and homocysteine thiolactone, and thioretinaco, the complex formed from cobalamin and thioretinamide, were demonstrated to have antineoplastic, anticarcinogenic, and anti-atherogenic properties in animal models. Retinol, ascorbate, and homocysteine thiolactone are necessary for biosynthesis of thioretinamide and thioretinaco by cystathionine synthase and for formation of thioretinaco ozonide from thioretinamide, cobalamin, and ozone. Thioretinaco ozonide is required for prevention of abnormal oxidative metabolism, aerobic glycolysis, suppressed immunity, and hyperhomocysteinemia in cancer.The pancreatic enzyme therapy of cancer promotes catabolism of proteins, nucleic acids, and glycosaminoglycans with excess homocysteinylated amino groups resulting from abnormal accumulation of homocysteine thiolactone in malignant cells. Dietary deficiencies of pyridoxal, folate, cobalamin, and nitriloside contribute to hyperhomocysteinemia in cancer, and in protein energy malnutrition. A deficiency of dietary sulfur amino acids downregulates cystathionine synthase, causing hyperhomocysteinemia.The organic sulfur compound diallyl trisulfide increases hydrogen sulfide production from homocysteine in animal models, inhibits Stat3 signaling in cancer stem cells, and produces apoptosis of malignant cells. The furanonaphthoquinone compound napabucasin inhibits Stat3 signaling and causes mitochondrial dysfunction, decreased oxidative phosphorylation, and apoptosis of malignant cells. The protocol of the proposed clinical trial in subjects with myelodysplasia consists of thioretinamide and cobalamin as precursors of thioretinaco ozonide, combined with pancreatic enzyme extracts, diallyl trisulfide, napabucasin, nutritional modification to minimize processed foods, vitamin supplements, essential amino acids, and beneficial dietary fats and proteins.

In vitro evaluation of dietary compounds to reduce mercury bioavailability.

Mercury in foods, in inorganic form [Hg(II)] or as methylmercury (CH3Hg), can have adverse effects. Its elimination from foods is not technologically viable. To reduce human exposure, possible alternatives might be based on reducing its intestinal absorption. This study evaluates the ability of 23 dietary components to reduce the amount of mercury that is absorbed and reaches the bloodstream (bioavailability). We determined their effect on uptake of mercury in Caco-2 cells, a model of intestinal epithelium, exposed to Hg(II) and CH3Hg standards and to swordfish bioaccessible fractions. Cysteine, homocysteine, glutathione, quercetin, albumin and tannic reduce bioavailability of both mercury species. Fe(II), lipoic acid, pectin, epigallocatechin and thiamine are also effective for Hg(II). Some of these strategies also reduce Hg bioavailability in swordfish (glutathione, cysteine, homocysteine). Moreover, extracts and supplements rich in these compounds are also effective. This knowledge may help to define dietary strategies to reduce in vivo mercury bioavailability.

Negative modulation of the GABAA ρ1 receptor function by l-cysteine.

l-Cysteine is an endogenous sulfur-containing amino acid with multiple and varied roles in the central nervous system, including neuroprotection and the maintenance of the redox balance. However, it was also suggested as an excitotoxic agent implicated in the pathogenesis of neurological disorders such as Parkinson's and Alzheimer's disease. l-Cysteine can modulate the activity of ionic channels, including voltage-gated calcium channels and glutamatergic NMDA receptors, whereas its effects on GABAergic neurotransmission had not been studied before. In the present work, we analyzed the effects of l-cysteine on responses mediated by homomeric GABAA ρ1 receptors, which are known for mediating tonic γ-aminobutyric acid (GABA) responses in retinal neurons. GABAA ρ1 receptors were expressed in Xenopus laevis oocytes and GABA-evoked chloride currents recorded by two-electrode voltage-clamp in the presence or absence of l-cysteine. l-Cysteine antagonized GABAA ρ1 receptor-mediated responses; inhibition was dose-dependent, reversible, voltage independent, and susceptible to GABA concentration. Concentration-response curves for GABA were shifted to the right in the presence of l-cysteine without a substantial change in the maximal response. l-Cysteine inhibition was insensitive to chemical protection of the sulfhydryl groups of the ρ1 subunits by the irreversible alkylating agent N-ethyl maleimide. Our results suggest that redox modulation is not involved during l-cysteine actions and that l-cysteine might be acting as a competitive antagonist of the GABAA ρ1 receptors.

Synergistic Toxicity of the Neurometabolites Quinolinic Acid and Homocysteine in Cortical Neurons and Astrocytes: Implications in Alzheimer's Disease.

The brain of patients affected by Alzheimer's disease (AD) develops progressive neurodegeneration linked to the formation of proteins aggregates. However, their single actions cannot explain the extent of brain damage observed in this disorder, and the characterization of co-adjuvant involved in the early toxic processes evoked in AD is essential. In this line, quinolinic acid (QUIN) and homocysteine (Hcy) appear to be involved in the AD neuropathogenesis. Herein, we investigate the effects of QUIN and Hcy on early toxic events in cortical neurons and astrocytes. Exposure of primary cortical cultures to these neurometabolites for 24 h induced concentration-dependent neurotoxicity. In addition, QUIN (25 µM) and Hcy (30 µM) triggered ROS production, lipid peroxidation, diminished of Na+,K+-ATPase activity, and morphologic alterations, culminating in reduced neuronal viability by necrotic cell death. In astrocytes, QUIN (100 µM) and Hcy (30 µM) induced caspase-3-dependent apoptosis and morphologic alterations through oxidative status imbalance. To establish specific mechanisms, we preincubated cell cultures with different protective agents. The combined toxicity of QUIN and Hcy was attenuated by melatonin and Trolox in neurons and by NMDA antagonists and glutathione in astrocytes. Cellular death and morphologic alterations were prevented when co-culture was treated with metabolites, suggesting the activation of protector mechanisms dependent on soluble factors and astrocyte and neuron communication through gap junctions. These findings suggest that early damaging events involved in AD can be magnified by synergistic toxicity of the QUIN and Hcy. Therefore, this study opens new possibilities to elucidate the molecular mechanisms of neuron-astrocyte interactions and their role in neuroprotection against QUIN and Hcy.

Endothelial Cells Inhibit the Angiotensin II Induced Phenotypic Modulation of Rat Vascular Adventitial Fibroblasts.

The phenotypic modulation of vascular adventitial fibroblasts plays an important role in vascular remodeling. Evidence have shown that endothelial cells and adventitial fibroblasts interact under certain conditions. In this study, we investigated the influence of endothelial cells on the phenotypic modulation of adventitial fibroblasts. Endothelial cells and adventitial fibroblasts from rat thoracic aorta were cultivated in a co-culture system and adventitial fibroblasts were induced with angiotensin II (Ang II). Collagen I and alpha smooth muscle actin (α-SMA) expression and migration of adventitial fibroblasts were analyzed. Ang II upregulated the expression of collagen I and α-SMA and the migration of adventitial fibroblasts. Adventitial fibroblasts-endothelial cells co-culturing attenuated the effects of Ang II. Homocysteine-treated endothelial cells, which are functionally impaired, were less inhibitory of the phenotypic modulation of adventitial fibroblasts. Supplementation of endothelial cells with L-arginine (L-Arg) or 8-bromoguanosine 3',5'-cyclic monophosphate (8-BrcGMP) enhanced the trends, while with L-NG-nitroarginine methyl ester (L-NAME) or 1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) the opposite effect was observed. Under the influence of Ang II, adventitial fibroblasts were prone to undergo phenotypic modulation, which was closely related to vascular remodeling. Our study showed that endothelial cells influenced fibroblast phenotypic transformation and such effect would be mediated through the nitric oxide (NO)/cGMP signaling pathway. J. Cell. Biochem. 118: 1921-1927, 2017. © 2017 Wiley Periodicals, Inc.

The effect of N-acetylcysteine supplementation on serum homocysteine levels and hepatic and renal oxidative stress in homocysteine thiolactone-treated rats.

The effect of N-acetylcysteine (NAC) (1 g/kg body weight/day) on serum homocysteine (Hcy) levels, insulin resistance (IR), and hepatic and renal prooxidant-antioxidant balance was evaluated in rats treated with homocysteine thiolactone (HcyT) (500 mg/kg body weight/day for 6 weeks). Reactive oxygen species (ROS), malondialdehyde (MDA), glutathione, ferric reducing antioxidant power, and superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities were determined in the liver and kidney. HcyT elevated serum Hcy levels and caused IR, but liver and kidney function tests remained unchanged. HcyT increased ROS and MDA without any change in hepatic antioxidants, but it elevated renal SOD and GSH-Px activities. NAC decreased serum Hcy, hepatic and renal ROS and MDA levels, and renal SOD and GSH-Px activities in rats with high Hcy levels. However, it did not ameliorate IR. Our results indicate that NAC supplementation may be effective in decreasing Hcy levels and Hcy-induced hepatic and renal oxidative stress.

Black tea protects against hypertension-associated endothelial dysfunction through alleviation of endoplasmic reticulum stress.

Hypertensive patients have been found to be associated with elevated levels of homocysteine, known as hyperhomocysteinemia. Homocysteine (Hcy) can induce endoplasmic reticulum (ER) stress in endothelial cells. This study aims to investigate whether black tea (BT) protects against hypertension-associated endothelial dysfunction through alleviation of ER stress. Rat aortae and cultured rat aortic endothelial cells were treated with Hcy, BT extract, and theaflavin-3,3'-digallate (TF3). Male Sprague Dawley rats were infused with angiotensin II (Ang II) to induce hypertension and orally administrated with BT extract at 15 mg/kg/day for 2 weeks. Hcy impaired endothelium-dependent relaxations of rat aortae and led to ER stress in endothelial cells, which were ameliorated by co-incubation of BT extract and TF3. The blood pressure of Ang II-infused rats and plasma Hcy level were normalized by BT consumption. Impaired endothelium-dependent relaxations in renal arteries, carotid arteries and aortae, and flow-mediated dilatations in third-order mesenteric resistance arteries were improved. Elevations of ER stress markers and ROS level, plus down-regulation of Hcy metabolic enzymes in aortae from Ang II-infused rats were prevented by BT treatment. Our data reveal the novel cardiovascular benefits of BT in ameliorating vascular dysfunctions, providing insight into developing BT into beneficial dietary supplements in hypertensive patients.

Homocysteine accelerates senescence of endothelial cells via DNA hypomethylation of human telomerase reverse transcriptase.

OBJECTIVE: Homocysteine can accelerate the senescence of endothelial progenitor cells or endothelial cells (ECs) via telomerase inactivation and length shortening. However, the underlying mechanism is unclear. Here, we investigated whether homocysteine promotes endothelial senescence by reducing the expression and activity of human telomerase reverse transcriptase (hTERT) by DNA methylation to reduce ECs telomerase activity. APPROACH AND RESULTS: When compared with primary human umbilical vein endothelial cells grown under standard conditions, ECs with chronic homocysteine treatment showed accelerated upregulation of p16, p21, and p53, markers of cellular senescence, during 6 to 10 passages. Interestingly, homocysteine-stimulated but not angiotensin II-stimulated ECs senescence could be reversed by hypermethylation induced by folic acid or s-adenosylmethionine supplementation. Meanwhile, homocysteine promoted the shortening of telomere length specifically related to restoration of hTERT transcriptional expression and CCCTC-binding factor binding sites with hTERT promoter hypomethylation, as detected by quantitative real-time polymerase chain reaction, Western blot, methylation-specific polymerase chain reaction, and bisulfite sequencing assay. Electrophoretic mobility shift assay and chromatin immunoprecipitation results showed that homocysteine-reduced telomere activity and homocysteine-induced EC senescence might contribute to hTERT promoter demethylation by increasing CCCTC-binding factor repression and interfering in the SP1 binding to the demethylated hTERT promoter, which might relate with reduced of DNA methyltransferase 1. Furthermore, the CCCTC-binding factor-dependent mechanism of homocysteine-reduced hTERT expression via DNA demethylation was confirmed in aortic endothelia of mice with hyperhomocysteine levels. CONCLUSIONS: CCCTC-binding factor and SP1 cross talk may contribute to homocysteine-reduced hTERT DNA methylation and expression in endothelial senescence.

Epigallocatechin-3-gallate inhibits homocysteine-induced apoptosis of endothelial cells by demethylation of the DDAH2 gene.

Our previous study showed that hypermethylation of dimethylarginine dimethylaminohydrolase 2 contributes to homocysteine-induced apoptosis of human umbilical vein endothelial cells. Epigallocatechin-3-gallate is a green tea-derived phenol which has been proved beneficial on atherosclerosis. It was demonstrated that epigallocatechin-3-gallate inhibits DNA methyltransferase activity and reactivates methylation-silenced genes in cancer cells. The aim of this study was to address whether epigallocatechin-3-gallate could induce DNA demethylation of the dimethylarginine dimethylaminohydrolase 2 gene, contributing to prevent endothelial cells from apoptosis induced by homocysteine. Human umbilical vein endothelial cells (ATCC, CRL-2480) were treated with homocysteine (1 mM) for 48 hours with or without epigallocatechin-3-gallate (20 µM) or 5-Aza (DNA methyltransferase inhibitor, 5 µM). Apoptosis rate of human umbilical vein endothelial cells was assayed by flow cytometry with an annexin V-FITC apoptosis detection kit. The mRNA and protein expression level of dimethylarginine dimethylaminohydrolase 2 and DNA methyltransferase 1 were detected by real-time PCR and Western blot, respectively. DNA methylation level of dimethylarginine dimethylaminohydrolase 2 was assayed by methylation specific PCR. The binding level of DNA methyltransferase 1 in the promoter of dimethylarginine dimethylaminohydrolase 2 was determined by chromatin immunoprecipitation-quantitative real-time PCR. It was shown that the apoptosis rate was decreased significantly in human umbilical vein endothelial cells treated with homocysteine compared with the control. Furthermore, the mRNA and protein level of dimethylarginine dimethylaminohydrolase 2 were downregulated, the dimethylarginine dimethylaminohydrolase 2 gene promoter was hypermethylated, and the DNA methyltransferase 1 mRNA and protein level were increased in human umbilical vein endothelial cells treated with homocysteine. Chromatin immunoprecipitation-quantitative real-time PCR revealed that homocysteine-induced binding of DNA methyltransferase 1 to the dimethylarginine dimethylaminohydrolase 2 promoter was increased. Pretreatment with epigallocatechin-3-gallate or 5-Aza inhibited such effects of homocysteine. In conclusion, epigallocatechin-3-gallate exerted protective effects on homocysteine-induced apoptosis in human umbilical vein endothelial cells by inhibiting promoter hypermethylation of the dimethylarginine dimethylaminohydrolase 2 gene and inducing dimethylarginine dimethylaminohydrolase 2 expression. These effects may be due to the decreased DNA methyltransferase 1 expression and binding of DNA methyltransferase 1 to the dimethylarginine dimethylaminohydrolase 2 promoter induced by epigallocatechin-3-gallate. This research suggests that modulating the epigenetic processes might be a novel plausible way for treatment of atherosclerosis.

Methoxistasis: integrating the roles of homocysteine and folic acid in cardiovascular pathobiology.

Over the last four decades, abnormalities in the methionine-homocysteine cycle and associated folate metabolism have garnered great interest due to the reported link between hyperhomocysteinemia and human pathology, especially atherothrombotic cardiovascular disease. However, clinical trials of B-vitamin supplementation including high doses of folic acid have not demonstrated any benefit in preventing or treating cardiovascular disease. In addition to the fact that these clinical trials may have been shorter in duration than appropriate for modulating chronic disease states, it is likely that reduction of the blood homocysteine level may be an oversimplified approach to a complex biologic perturbation. The methionine-homocysteine cycle and folate metabolism regulate redox and methylation reactions and are, in turn, regulated by redox and methylation status. Under normal conditions, a normal redox-methylation balance, or "methoxistasis", exists, coordinated by the methionine-homocysteine cycle. An abnormal homocysteine level seen in pathologic states may reflect a disturbance of methoxistasis. We propose that future research should be targeted at estimating the deviation from methoxistasis and how best to restore it. This approach could lead to significant advances in preventing and treating cardiovascular diseases, including heart failure.

Homocysteine alters the osteoprotegerin/RANKL system in the osteoblast to promote bone loss: pivotal role of the redox regulator forkhead O1.

In this study we determined the molecular mechanisms of how homocysteine differentially affects receptor activator of nuclear factor-κB ligand (RANKL) and osteoprotegerin (OPG) synthesis in the bone. The results showed that oxidative stress induced by homocysteine deranges insulin-sensitive FOXO1 and MAP kinase signaling cascades to decrease OPG and increase RANKL synthesis in osteoblast cultures. We observed that downregulation of insulin/FOXO1 and p38 MAP kinase signaling mechanisms due to phosphorylation of protein phosphatase 2A (PP2A) was the key event that inhibited OPG synthesis in homocysteine-treated osteoblast cultures. siRNA knockdown experiments confirmed that FOXO1 is integral to OPG and p38 synthesis. Conversely homocysteine increased RANKL synthesis in osteoblasts through c-Jun/JNK MAP kinase signaling mechanisms independent of FOXO1. In the rat bone milieu, high-methionine diet-induced hyperhomocysteinemia lowered FOXO1 and OPG expression and increased synthesis of proresorptive and inflammatory cytokines such as RANKL, M-CSF, IL-1α, IL-1ß, G-CSF, GM-CSF, MIP-1α, IFN-γ, IL-17, and TNF-α. Such pathophysiological conditions were exacerbated by ovariectomy. Lowering the serum homocysteine level by a simultaneous supplementation with N-acetylcysteine improved OPG and FOXO1 expression and partially antagonized RANKL and proresorptive cytokine synthesis in the bone milieu. These results emphasize that hyperhomocysteinemia alters the redox regulatory mechanism in the osteoblast by activating PP2A and deranging FOXO1 and MAPK signaling cascades, eventually shifting the OPG:RANKL ratio toward increased osteoclast activity and decreased bone quality.