Different Concentrations Of Hcy Research Articles

Naringenin induces neuroprotection against homocysteine-induced PC12 cells via the upregulation of superoxide dismutase 1 expression by decreasing miR-224-3p expression.

Naringenin is a flavonoid compound with antioxidant effects. It is used to treat oxidative stress-related diseases, but its mechanism is unclear. In this experiment, we explored whether naringenin can increase the expression of superoxide dismutase 1(SOD1), reduce the oxidative stress of PC12 cells induced by homocysteine (Hcy), and decrease the apoptosis of PC12 cells induced by Hcy by inhibiting the expression of mir-224-3p. Different concentrations of Hcy (1, 3, 5, 8, and 10 mmol/L) was used to analyze effect of homocysteine on PC12 cells. A total of 5 mmol/L Hcy was used to induce the excitatory and neurotoxicity model of PC12 cells in vitro. The cells were divided into normal control, Hcy induction, Hcy + Naringenin (25 μM), Hcy + Naringenin (50 μM), Hcy + Naringenin (75 μM), Hcy + Naringenin (100 μM), and Hcy + Naringenin (150 μM) groups. The relative survival rate and activities of the PC12 cells were determined by the MTT method, and the apoptosis rate of the PC12 cells was determined by using flow cytometry. The Western blot method was used to determine the expressions of SOD1, Bax, Caspase-3, Caspase-8, and Bcl-2 in the PC12 cells induced by Hcy. The expressions of SOD1 mRNA and miR-224-3p in the Hcy-induced PC12 cells were determined by RT-PCR. Results found that Hcy increased the expression of miR-224-3p in a dose-dependent manner but decreased that of SOD1 mRNA and protein. Hcy also increased oxidative stress in the PC12 cells and the proapoptotic proteins Bax, Caspase-3, and Caspase-9. Furthermore, it decreased the expression of anti-apoptotic protein Bcl-2 and the activity and survival rate of the HT22 cells, but it increased the apoptosis of the PC12 cells. The treatment of Hcy-induced PC12 cells with different concentrations of naringenin for 24 h decreased the expression of miR-224-3p in a dose-dependent manner and increased the expressions of SOD1 mRNA and protein. The treatment also decreased the oxidative stress in the PC12 cells and the expressions of pro-apoptotic proteins Bax, Caspase-3, and Caspase-9; increased the expression of anti-apoptotic protein Bcl- 2; decreased the apoptosis of the PC12 cells; and increased the PC12 cells.The results suggest that Naringenin can decrease the apoptosis and oxidative stress of PC12 cells induced by Hcy and increase the activities and survival rates of PC12 cells. The mechanism may be related to naringenin decreasing the expression of miR-224-3p in PC12 cells induced by Hcy and increasing the expressions of SOD1 mRNA and protein.

Impact of Herpud1 in the homocysteine-induced phenotypic switching of vascular smooth muscle cells

Objective: To investigate the impact of homocysteine inducible endoplasmic reticulum(ER) protein with ubiquitin like domain 1 protein (Herpud1) in the homocysteine (Hcy) -induced phenotypic switching of vascular smooth muscle cells (VSMCs). Methods: VSMCs were derived from thoracic aortic artery of male Sprague Dawley rats and cultured VSMCs (4-7 passage) were treated with various concentrations of Hcy (0, 100, 500 and 1 000 μmol/L) and applied to immunofluorescence to observe the morphological changes of VSMCs via SM-actin staining. Western blot was used to detect the expression of VSMCs phenotypic markers, including Osteopontin, Calponin and smooth muscle myosin heavy chain (SM-MHC) and the expression of endoplasmic reticulum stress (ERS) related proteins, including C/EBP-homologous protein (CHOP), inositol-requiring kinase 1 (IRE-1) and glucose regulating protein 78 (GRP78) in the absence and presence of non-selective inhibitor of ERS, 4-phenylbutyric acid (4-PBA, 2 mg/ml). The Herpud1 mRNA and protein levels were determined in Hcy-stimulated VSMCs treated with 4-PBA or transfected with specific siRNA targeting Herpud1. Results: Compared with the control group, SM-actin staining results showed that the shape of VSMCs treated with different concentrations of Hcy for 24 hours changed from long fusiform into round form, arrangement of myofilament became irregular and the most significant alteration was found in the 500 μmol/L Hcy group. After intervention of 24 hours, various concentration of Hcy increased protein expression of Osteopontin, and reduced Calponin and SM-MHC protein expressions in VSMCs (all P<0.05). In addition, the results showed that Hcy increased the expression of CHOP, IRE-1 and GRP78 in a dose-dependent manner, which could be reversed by 4-PBA treatment (all P<0.05). However, 4-PBA inhibited Hcy induced upregulation of Osteopontin and downregulation of Calponin and SM-MHC, suggesting that ERS was involved in Hcy-induced phenotypic switching of VSMCs. Herpud1 protein was mostly expressed in the cytoplasm and was also expressed in the nucli, both in the control, Hcy and Hcy+4-PBA groups. Moreover, Hcy increased mRNA and protein levels of Herpud1 (P<0.05), whereas treatment with 4-PBA could significantly reduce Hcy-induced upregulation of Herpud1 (P<0.05). Furthermore, knockdown of Herpud1 abrogated the effects of Hcy on VSMCs phenotype markers. Conclusion: Herpud1 plays an important role in Hcy-induced phenotypic switching of VSMCs.

Homocysteine induces human mesangial cell apoptosis via the involvement of autophagy and endoplasmic reticulum stress.

Homocysteine (Hcy) level characterizes a progressive increase in chronic kidney disease (CKD). In fact, Hcy accumulation is considered to be a crucial biochemical culprit in CKD progression, but the mechanism underlying this remains poorly understood. This study investigated the role of Hcy in glomerular mesangial cell (MC) apoptosis and the potential involvement of autophagy and endoplasmic reticulum (ER) stress in this process, shedding light on Hcy toxicity in kidney disease. Human mesangial cells (HMCs) were incubated with different concentrations of Hcy for different times. Flow cytometry was used to determine the proportion of apoptotic cells and western blotting was used to analyze protein levels after the administration of Hcy, endoplasmic reticulum inhibitor 4-phenylbutyric acid (4-PBA), and Atg5 siRNA. The results demonstrated that the cell viability gradually decreased and the proportion of HMCs undergoing apoptosis increased with increasing Hcy concentration and prolonged incubation time. Meanwhile, levels of the apoptosis-related proteins Bax and cleaved caspase-3 were significantly increased, while ER stress-related proteins such as ATF4, CHOP, GRP78, and phospho-eIF2α significantly increased. Levels of cleaved LC3, and beclin1 and Atg5 proteins also increased, accompanied by p62 degradation, indicating autophagy activation. 4-PBA effectively inhibited ER stress and reversed Hcy-induced apoptosis and autophagy. Moreover, Atg5 siRNA alleviated Hcy-induced apoptosis. Taken together, these results suggest that Hcy induces HMC apoptosis in a dose- and time-dependent manner via the activation of Atg5-dependent autophagy triggered by ER stress. This study suggests a novel strategy against Hcy toxicity in kidney injury and should help in clarifying the pathogenesis of CKD.

Hyperhomocysteinemia Causes Chorioretinal Angiogenesis with Placental Growth Factor Upregulation

Hyperhomocysteinemia is a risk factor for atherosclerosis, which may also be associated with retinal vascular disease, diabetic retinopathy, retinal vein occlusion, and glaucoma. For this study, we established a hyperhomocysteinemia animal model to explore homocysteine (hcy)-related choroidal angiogenesis and possible related factors. We injected Sprague Dawley (SD) rats with different concentrations of hcy and performed color fundus imaging, fluorescein angiography, image-guided optical coherence tomography, and retinal histology to observe the retinal and choroidal changes. Subsequently, we observed prominent choroidal vasculature with congested and tortuous retinal and choroidal vessels in fundus angiographies of the hyperhomocysteinemia animal model. In the histological study, the choroidal capillaries proliferated in the hcy-treated eyes, mimicking choroidal neovascularization. Disrupted retinal pigment epithelium (RPE), abnormal branching vascular network (BVN), and polyp-like structures were also observed in the hcy-treated eyes. Furthermore, we found that placental growth factor (PlGF), but not vascular epithelial growth factor (VEGF), was the key mediating factor of this phenomenon. Our findings suggest that hyperhomocysteinemia might cause choroidal angiogenesis.

Homocysteine induces oxidative stress to damage trabecular meshwork cells.

The aim of the present study was to investigate the effect of homocysteine (Hcy) in on human trabecular meshwork cells (HTMCs). A total of 41 patients with primary open-angle glaucoma (POAG) and 53 patients with senile cataracts (control group) were recruited. Plasma and aqueous humor samples were collected and the Hcy concentrations were determined using enzymatic cycling assays. In cell experiments, normal HTMCs were passaged and randomly divided into a blank control group, a normal HTMC group and experimental groups, which were treated with different concentrations of Hcy. The HTMC activities were detected using the Cell Counting Kit-8 method and the HTMC mitochondrial membrane potential (MMP) was detected using JC-1 staining. Reactive oxygen species (ROS) released by trabecular meshwork cells was detected using flow cytometry and superoxide dismjutase-1 (SOD1) expression was detected using immunoblotting. The results revealed that the concentration of Hcy in the plasma and aqueous humor of the POAG group (14.44±0.86 and 1.60±0.27 µmol/l, respectively) was significantly higher compared with the control group (10.82±0.29 and 0.69±0.39 µmol/l). All tested concentrations (30, 100, 300 and 1,000 µmol/l) of Hcy reduced the MMP in HTMCs and inhibited HTMC proliferation in a dose-dependent manner. ROS production by HTMCs significantly increased with increased concentrations of Hcy, whereas SOD1 expression significantly decreased in a dose-dependent manner. In summary, patients with POAG were demonstrated to have increased concentrations of Hcy in the plasma and aqueous humor. High concentrations of Hcy in HTMCs induced an oxidative stress state, thereby further inhibiting HTMC proliferation. The results of the present study demonstrate that Hcy may be a potential treatment target in patients with POAG.

Homocysteine‑induced oxidative stress through TLR4/NF‑κB/DNMT1‑mediated LOX‑1 DNA methylation in endothelial cells.

Atherosclerosis (AS) is a progressive disease of multifactorial origin, which occurs in response to endothelial injury. Increased homocysteine (Hcy) is considered a major cause of endothelial dysfunction, oxidative stress and DNA methylation; however, the mechanisms remain to be fully elucidated. The aim of the present study was to investigate whether Hcy causes injury to endothelial cells (ECs) by the effect of lectin‑like oxidized‑low density lipoprotein receptor‑1 (LOX‑1) DNA methylation through toll‑like receptor 4(TLR4)/nuclear factor (NF)‑κB/DNA methyltransferase (DNMT)1. The ECs were treated with different concentrations of Hcy, and it was found that Hcy promoted the expression of TLR4, leading to EC injury. The effect of oxidative stress was analyzed by measuring superoxide dismutase, malondialdehyde and hydrogen peroxide in the ECs. In addition, the association between NF‑κB and DNMT1 was examined by treatment of the ECs with pyrrolidine dithiocarbamate (PDTC). The results suggested that Hcy induced LOX‑1 DNA hypomethyaltion to promote the expression levels of LOX‑1. Taken together, Hcy injured the ECs through the effect of methylation and trans‑sulfuration metabolism of LOX‑1 through TLR4/NF‑κB/DNMT1. Following injury to the ECs, lipids, particularly ox‑LDL, accumulated in the sub‑endothelial layer to promote the formation of AS.

Hyperhomocysteinemia Alters Retinal Endothelial Cells Barrier Function and Angiogenic Potential via Activation of Oxidative Stress

Hyperhomocysteinemia (HHcy) is associated with several human visual disorders, such as diabetic retinopathy (DR) and age-related macular degeneration (AMD). Breakdown of the blood-retinal barrier (BRB) is linked to vision loss in DR and AMD. Our previous work revealed that HHcy altered BRB in retinal endothelial cells in vivo. Here we hypothesize that homocysteine (Hcy) alters retinal endothelial cell barrier function and angiogenic potential via activation of oxidative stress. Human retinal endothelial cells (HRECs) treated with and without different concentrations of Hcy showed a reduction of tight junction protein expression, increased FITC dextran leakage, decreased transcellular electrical resistance and increased angiogenic potential. In addition, HRECs treated with Hcy showed increased production of reactive oxygen species (ROS). The anti-oxidant N-acetyl-cysteine (NAC) reduced ROS formation and decreased FITC-dextran leakage in Hcy treated HRECs. A mouse model of HHcy, in which cystathionine-β-synthase is deficient (cbs−/−), was evaluated for oxidative stress by dichlolorofluorescein (DCF), dihydroethidium (DHE) staining. There was a marked increase in ROS production and augmented GSH reductase and antioxidant regulator NRF2 activity, but decreased antioxidant gene expression in retinas of hyperhomocysteinemic mice. Our results suggest activation of oxidative stress as a possible mechanism of HHcy induced retinal endothelial cell dysfunction.

Homocysteine mediates transcriptional changes of the inflammatory pathway signature genes in human retinal pigment epithelial cells.

To test whether homocysteine (Hcy) can influence the transcriptional profile, we hypothesized that Hcy can lead to the induction of proinflammatory molecules in the retinal cells of aging people. An unbiased in vitro inflammatory pathway focused study was designed employing retinal pigment epithelial (RPE) cell line, ARPE-19. Cells were cultured in the presence or absence of Hcy to capture target genes' expression profile. Three different concentrations of Hcy were added in the culture medium of confluent monolayers. cRNAs were made from the isolated total RNAs and the labeled cRNA probes were hybridized to microarrays specific for human disease pathway inflammatory cytokines, chemokines and their receptor gene micro-array panels as per manufacture's recommendations. Two Hcy up-regulated molecules: IL6 and CEBPB were further validated via Western blot analysis. Hcy's effect on ARPE-19 cellular morphology and genomic DNA integrity were also evaluated. Gene microarray analyses of RPE cells in response to Hcy treatment revealed alterations in the expressions of several inflammatory gene transcripts such as CCL5, CEBPB, IL13RA2, IL15RA, IL6, IL8 and CXCL3 that were up-regulated. The transcripts for C3, CCL2, IL11RA and IL18 genes exhibited down-regulation. The IL6 and CEBPB expressions were subsequently validated at the protein levels. Treatment of the retinal cells with increasing Hcy concentration influenced their density in culture however their morphology and DNA integrity remained unaffected. These findings suggest that Hcy can potentially mediate the expression of chemokines, cytokines and interleukins receptors in the retinal cells without having any debilitating effects on their morphology and the genomic DNA integrity.

Homocysteine Induces Glial Reactivity in Adult Rat Astrocyte Cultures.

Astrocytes are dynamic glial cells associated to neurotransmitter systems, metabolic functions, antioxidant defense, and inflammatory response, maintaining the brain homeostasis. Elevated concentrations of homocysteine (Hcy) are involved in the pathogenesis of age-related neurodegenerative disorders, such as Parkinson and Alzheimer diseases. In line with this, our hypothesis was that Hcy could promote glial reactivity in a model of cortical primary astrocyte cultures from adult Wistar rats. Thus, cortical astrocytes were incubated with different concentrations of Hcy (10, 30, and 100μM) during 24h. After the treatment, we analyzed cell viability, morphological parameters, antioxidant defenses, and inflammatory response. Hcy did not induce any alteration in cell viability; however, it was able to induce cytoskeleton rearrangement. The treatment with Hcy also promoted a significant decrease in the activities of Na+, K+ ATPase, superoxide dismutase (SOD), and glutathione peroxidase (GPx), as well as in the glutathione (GSH) content. Additionally, Hcy induced an increase in the pro-inflammatory cytokine release. In an attempt to elucidate the putative mechanisms involved in the Hcy-induced glial reactivity, we measured the nuclear factor kappa B (NFκB) transcriptional activity and heme oxygenase 1 (HO-1) expression, which were activated and inhibited by Hcy, respectively. In summary, our findings provide important evidences that Hcy modulates critical astrocyte parameters from adult rats, which might be associated to the aging process.

Influence of hydrogen sulfide on zymogen activation of homocysteine-induced matrix metalloproteinase-2 in H9C2 cardiocytes.

To observe the influence of different concentrations of homocysteine (Hcy) and hydrogen sulfide (H2S) on the secretion and activation of matrix metalloproteinase-2 (MMP-2) in cardiocytes so as to search for new ways to fight against myocardial tissue fibrosis. Cardiocytes H9C2 was cultured invitro and different concentrations of Hcy and H2S were added for 6-h and 24-h cultivation. MTT cell proliferation assay was applied to test the activation change of cardiocytes H9C2 after affecting by different concentrations of Hcy and H2S. ELISA and MTT were employed to detect the expression and enzymatic activity of MMP-2. The H9C2 cell inhibition of activity was more significant with 1000μmol/L of Hcy as compared with other concentrations (P<0.001). With 2.5-100.0μmol/L Hcy and 0.1, 1.0 and 10.0mmol/L H2S, the activity of H9C2 did not change significantly (P>0.05). Hcy with concentrations of 10, 50 and 100μmol/L could increase the quantity of MMP-2 secreted by cardiocytes H9C2, and the interaction strength was concentration-dependent (P<0.05). After interacting with 100μmol/L of Hcy for 6h, the zymogen activation effect of MMP-2 was stronger than that of the 2.5-25μmol/L group (P<0.05). After interacting with Hcy and H2S (1.0mmol/L) for 6h and 24h, the activation effect of MMP-2 was stronger than those interacted with 10, 25, 50 and 100μmol/L of Hcy (P<0.05). Hcy can increase the production of MMP-2 secreted by H9C2 cell and improve its zymogen activation. Besides, the interaction strength is concentration-dependent; while H2S can up-regulate the activation of MMP-2 and co-promote the activation of MMP-2 with Hcy as well.

Endoplasmic reticulum oxidoreductin 1α mediates hepatic endoplasmic reticulum stress in homocysteine-induced atherosclerosis.

Endoplasmic reticulum (ER) stress is emerging as an important modulator of different pathological process and as a mechanism contributing to homocysteine (Hcy)-induced hepar injury. However, the molecular event that Hcy-induced ER stress in the hepar under the atherosclerosis background is currently unknown. Endoplasmic reticulum oxidoreductin 1α (ERO1α) plays a crucial role in maintaining ER stress function. In this study, we determined the expression of ERO1α in the hepar in hyperhomocysteinemia and the effect of ERO1α in hepacytes ER stress in the presence of Hcy. HHcy model was established by feeding the methionine diet in apolipoprotein-E-deficient (ApoE-/-) mice, and the hepatocytes were incubated with folate and different concentrations of Hcy. Our results showed that Hcy triggered ER stress characterized by an increased contents of glucose-regulated protein 78 (GRP78), protein kinase RNA-like ER kinase (PERK), activating transcription factor (ATF) 6 and X-box binding protein-1 (XBP-1). The ERO1α expressions in HHcy mice and Hcy-treated hepatocytes were decreased compared with those in ApoE-/- group and control hepacytes (P < 0.05), respectively. Knocking-down the expression of ERO1α with small-interfering RNA significantly augmented Hcy-induced ER stress. Meanwhile, the expressions of ER stress-related factor including GRP78, PERK, ATF6 and XBP-1, were significantly decreased when the ERO1α gene was over-expressed in hepacytes. Our results suggested that ERO1α may be involved in Hcy-induced hepar ER stress, and the inhibition of ERO1α expression can accelerate this process.

The synergistic effect of homocysteine and lipopolysaccharide on the differentiation and conversion of raw264.7 macrophages.

BackgroundMacrophages play pivotal roles in the progression of atherosclerosis (AS) and their heterogeneous differentiation patterns have been studied extensively. The classical subtype of activated macrophage, M1, promotes the progression of AS. Conversely, the alternative subtype of activated macrophage, M2, is regarded as a repressor of AS. Homocysteine (Hcy) may influence macrophage subtype polarization both in vivo and in vitro. Homocysteinemia (HHcy) is an independent risk factor in coronary heart disease and the effect of Hcy on macrophage differentiation has not been studied until now.MethodsDifferent concentrations of Hcy in combination with a fixed concentration of lipopolysaccharide (LPS, 200 ng/mL) were used to treat RAW264.7 macrophages. Real-time PCR was used to detect and quantify RNA transcripts indicative of M1 and M2 differentiation. The efficacy and specificity for each chemical stimulant in inducing macrophage differentiation were also investigated. The M2 macrophages (anti-inflammatory subtype) induced using classical methods (IL-4, 10 ng/mL) were also treated with different concentrations of Hcy complemented with LPS. The synergistic effect of Hcy and LPS in the converting the M2 subtype to M1 was also studied.ResultsMacrophages can be induced to differentiate towards M1 by a combination of Hcy with LPS, with the strongest effect observed at an Hcy concentration of 50 μmol/L. After inducing macrophages to the M2 subtype using IL-4, treatment with both Hcy and LPS could elicit conversion from the M2 to M1 subtype.ConclusionCombined treatment with Hcy and LPS can induce the polarization of cultured RAW264.7 macrophages into the pro-inflammatory subtype, as well as promote subtype conversion from anti-inflammatory to pro-inflammatory.

Homocysteine-mediated cholesterol efflux via &amp;lt;italic&amp;gt;ABCA1&amp;lt;/italic&amp;gt; and &amp;lt;italic&amp;gt;ACAT1&amp;lt;/italic&amp;gt; DNA methylation in THP-1 monocyte-derived foam cells

Homocysteine (Hcy) has been recognized as a prevalent risk factor for cardiovascular events. Cholesterol-loaded foam cells are a central component of atherosclerotic lesions. ATP-binding cassette transporter A1 (ABCA1), which mediates the efflux of cellular cholesterol and phospholipids, is the rate-limiting step in lipid metabolism. Acyl-coenzyme A:cholesterol acyltransferase-1 (ACAT1) promotes accumulation of cholesterol ester in macrophages, thereby resulting in the foam cell formation, a hallmark of early stage in atherosclerosis. In this study, cultured monocyte-derived foam cells were incubated with clinical relevant concentrations of Hcy for 24 h. Both increased number of foam cells and accumulation of cholesterol were found, and the mRNA and protein expression levels of ABCA1 were decreased, while ACAT1 expression was increased in the presence of Hcy. Furthermore, the DNA methylation level of ABCA1 gene was increased whereas ACAT1 DNA methylation was decreased by using different concentrations of Hcy. Moreover, our results showed that DNA methyltransferase (DNMT) activity and DNA methyltransferase 1 (DNMT1) mRNA expression were increased by Hcy. It is indicated that DNA methylation has the function to regulate the expression of ABCA1 and ACAT1 via DNMT. In conclusion, these results suggest that ABCA1 and ACAT1 DNA methylation induced by Hcy may play a potential role in ABCA1 and ACAT1 expression and the accumulation of cholesterol in monocyte-derived foam cells.

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.

E0176 The effects of rosuvastatin on the expression of homocysteine-induced expression of matrix metalloproteinase-2 (MMP-2) and cell migration in rat vascular smooth muscle cells

ObjectiveThe aim of this study was to investigate the effects of rosuvastatin on the expression of homocysteine-induced expression of matrix metalloproteinase-2 (MMP-2) and cell migration in rat vascular smooth muscle...

Homocysteine harasses the imprinting expression of &amp;lt;italic&amp;gt;IGF2&amp;lt;/italic&amp;gt; and &amp;lt;italic&amp;gt;H19&amp;lt;/italic&amp;gt; by demethylation of differentially methylated region between &amp;lt;italic&amp;gt;IGF2&amp;lt;/italic&amp;gt;/&amp;lt;italic&amp;gt;H19&amp;lt;/italic&amp;gt; genes

Homocysteine (Hcy) can induce proliferation of vascular smooth muscle cells (VSMCs), which is a key event in the genesis of the lesions of atherosclerosis. Insulinlike growth factor 2 (IGF2) and H19 are two important regulating molecules of cell proliferation. The role of Hcy in the proliferation of smooth muscle cell by regulating IGF2 and H19 has not been shown or analyzed so far. This study aims to investigate the potential impact of Hcy on gene imprinting of IGF2 and H19. Cultured human umbilical VSMCs were treated with different concentrations of Hcy. The DNA methylation status of VSMCs was assayed by nested methylationspecific polymerase chain reaction (PCR). The mRNA levels of H19, IGF2, and CCCTC-binding factor (CTCF) were detected by reverse transcription PCR, and the protein expression of IGF2 by Western blotting. The results showed that the Hcy treatment resulted in hypomethylation of the sixth CTCF-binding site upstream of H19 of VSMCs. The expression of H19 was increased, whereas the IGF2 mRNA and protein were decreased, the CTCF expression increased with the increase in Hcy concentration. These data indicated that Hcy could induce hypomethylation of the sixth CTCF-binding sites upstream of H19, which is an important regulating area for the imprinting expression of IGF2 and H19. The increased CTCF expression may be a potential mechanism for the demethylation modification of DNA, which resulted from the Hcy treatment.

Growth hormone–releasing peptide ghrelin inhibits homocysteine-induced endothelial dysfunction in porcine coronary arteries and human endothelial cells

Ghrelin, a novel growth hormone-releasing peptide, is implicated to play a protective role in cardiovascular tissues. However, it is not clear whether ghrelin protects vascular tissues from injury secondary to risk factors such as homocysteine (Hcy). This study investigated the effect and potential mechanisms of ghrelin on Hcy-induced endothelial dysfunction. Porcine coronary artery rings were incubated for 24 hours with ghrelin (100 ng/mL), Hcy (50 microM), or ghrelin plus Hcy. Endothelial vasomotor function was evaluated using the myograph tension model. The response to the thromboxane A(2)analog U46619, bradykinin, and sodium nitroprusside was analyzed. Endothelial nitric oxide synthase (eNOS) expression was determined using real-time polymerase chain reaction and immunohistochemistry staining, and superoxide anion production was documented lucigenin-enhanced chemiluminescence analysis. Human coronary artery endothelial cells (HCAECs) were treated with different concentrations of Hcy, ghrelin, or antighrelin receptor antibody for 24 hours, and eNOS protein levels were determined by Western blot analysis. Maximal contraction with U46619 and endothelium-independent vasorelaxation with sodium nitroprusside were not different among the four groups. However, endothelium-dependent vasorelaxation with bradykinin (10(-6) M) was significantly reduced by 34% with Hcy compared with controls (P < .05). The addition of ghrelin to Hcy had a protective effect, with 61.6% relaxation, which was similar to controls (64.7%). Homocysteine significantly reduced eNOS expression, whereas ghrelin cotreatment effectively restored eNOS expression to the control levels. Superoxide anion levels, which were increased by 100% with Hcy, returned to control levels with ghrelin cotreatment. Ghrelin also effectively blocked the Hcy-induced decrease of eNOS protein levels in HCAECs in a concentration-dependent manner. Antighrelin receptor antibody effectively inhibited the effect of ghrelin. Ghrelin has a protective effect in the porcine coronary artery by blocking Hcy-induced endothelial dysfunction, improving eNOS expression, and reducing oxidative stress. Ghrelin also shows a protective effect on HCACEs from the Hcy-induced decrease in eNOS protein levels. The effect of ghrelin is receptor-dependent. Thus, ghrelin administration may have beneficial effects in the treatment of vascular disease in patients with hyperhomocysteinemia.

Ligands of Peroxisome Proliferator-activated Receptor Inhibit Homocysteine induced DNA Methylation of Inducible Nitric Oxide Synthase Gene

Homocysteine (Hcy) is a risk factor for atherosclerosis. It is generally accepted that inducible nitric oxide synthase (iNOS) is a key enzyme in the regulation of vascular disease. The aim of the present study is to investigate the effects of peroxisome proliferator-activated receptor ligands on iNOS in the presence of Hcy in human monocytes. Foam cells, induced by oxidize low density lipoprotein (ox-LDL) and phorbol myristate acetate (PMA) in the presence of different concentrations of Hcy, clofibrate and pioglitazone in human monocytes for 4 d, were examined by oil red O staining. The activity of iNOS was detected by real-time quantitative reverse transcription-polymerase chain reaction and Western blot analysis. The capability of DNA methylation was measured by assaying endogenous C5 DNA methyltransferase (C5MTase) activity, and the iNOS promoter methylation level was determined by quantitative MethyLight assays. The results indicated that Hcy increased the activity of C5MTase and the level of iNOS gene DNA methylation, resulting in a decrease of iNOS expression. Clofibrate and pioglitazone could antagonize the hcy effect on iNOS expression through DNA methylation, resulting in attenuation of iNOS transcription. These findings suggested that Hcy decreased the expression of iNOS by elevating iNOS DNA methylation levels, which can repress the transcription of some genes. Peroxisome proliferator-activated receptor alpha/gamma ligands can down-regulate iNOS DNA methylation, and could be useful for preventing Hcy-induced atherosclerosis by repressing iNOS expression.