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

  • Authors:
    • Sheng‑Chao Ma
    • Yin‑Ju Hao
    • Yun Jiao
    • Yan‑Hua Wang
    • Ling‑Bo Xu
    • Cai‑Yan Mao
    • Xiao‑Ling Yang
    • An‑Ning Yang
    • Jue Tian
    • Ming‑Hao Zhang
    • Shao‑Ju Jin
    • Hua Xu
    • Yi‑Deng Jiang
    • Hui‑Ping Zhang
  • View Affiliations

  • Published online on: October 10, 2017     https://doi.org/10.3892/mmr.2017.7753
  • Pages: 9181-9188
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Abstract

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.

Introduction

Atherosclerosis (AS), the underlying cause of cardiovascular disease, is characterized by multiple key events, including endothelial cell (EC) injury, conversion of lesion-resident macrophages into foam cells, and smooth muscle cell proliferation (1). EC injury is the key initiating step in the formation of AS (2), which is induced by multiple mechanisms, including oxidative stress, endoplasmic reticulum stress, and insulin resistance. Oxidative stress has increasingly been investigated to explain EC injury (3). Homocysteine (Hcy) is an independent risk factor for AS and is involved as an early atherosclerotic promoter, which enhances EC injury (4). However, the potential causative role of Hcy in EC injury by oxidative stress remains to be fully elucidated.

EC injury can be triggered by oxidized low-density lipoprotein (ox-LDL) in AS, and associated investigations have indicated that ox-LDL and its receptor, lectin-like ox-LDL receptor-1 (LOX-1), are important in the development of EC injury (5). LOX-1 is a scavenger receptor, which allows the uptake of ox-LDL into ECs, and the expression of this receptor is involved in the formation of atherosclerotic vascular lesions; LOX-1 involved at various steps in the pathogenesis of AS and expressed at high levels in atherosclerotic lesions (6,7). Simultaneously, it has been suggested that anti-LOX-1 antibody significantly suppresses EC injury in the absence of hypertension (8). Based on genetic and functional investigations, LOX-1 may be a novel biomarker and target in EC injury of AS. One of main mechanisms involved in AS induced by Hcy involves DNA methylation (9), and our previous study suggested that Hcy induced the hypomethylation of genes associated with AS, including platelet-derived growth factor (10). Therefore, it was hypothesized that Hcy injures ECs through mediating LOX-1 DNA methylation, however, the mechanism remains to be elucidated.

DNA methylation is an epigenetic change, which arises from the addition of a methyl group at the carbon-5 position of cytosine residues, this process is mediated by DNA methyltransferases (DNMTs), a family of enzymes, including DNMT1, DNMT3a and DNMT3b (11). DNMT1, the principal DNMT in mammalian cells, is a large dynamic enzyme with multiple regulatory features, which can control DNA methylation in cells (12). A number of studies have reported that DNMT1 is an alternative mechanism of DNA methylation, and nuclear factor-κB (NF-κB) is one of the transcriptional factors regulating the transcription of several genes involved in numerous critical pathways (13,14), this transcriptional factor is potentially targeted at various levels. An associated study has suggested that bortezomib results in the downregulation of DNMT1 via the specificity protein-1 (SP1)/NF-κB pathway and induces genomic DNA hypomethylation in leukemia cells (15). NF-κB is a key transcription factor pathway in several key biological processes, including inflammation, apoptosis and immune responses, which is a key in the toll-like receptor 4 (TLR4) signaling pathway (16). TLR4 is a pattern-recognizing receptor, forming the first line of defense. A previous study suggested that the lipopolysaccharide (LPS) -induced inflammatory response may be mediated through the reduced expression of TLR4 to suppress the activation of NF-κB (17,18). Simultaneously, TLR4/NF-κB is important in monocyte-endothelium adhesion, at least in part.

The aim of the present study was to investigate whether elevated Hcy levels are associated with EC injury and to examine whether Hcy-induced oxidative stress occurs through TLR4/NF-κB/DNMT1-mediated LOX-1 DNA methylation in ECs.

Materials and methods

EC culture

The CRL-1730 EC line was purchased from American type culture collection (Manassas, VA, USA). The cells were treated with different concentrations (0, 50, 100, 200 and 500 µmol/l) of Hcy (Sigma; Merck Millipore, Darmstadt, Germany) or 100 µmol/l Hcy with 30 µmol/l vitamin B12 and 30 µmol/l folic acid at 37°C in an incubator with 5% CO2 for 72 h. The ECs were divided into a further three groups: untreated cells, endothelial cells treated with Hcy (100 µmol/l), endothelial cells treated with Hcy (100 µmol/l) and 10 µmol/l pyrrolidine dithiocarbamate (PDTC; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany).

Cell viability assessment

Methylthiazoletetrazolium (MTT) (Sigma-Aldrich; Merck KGaA) was used for the evaluation of cell viability. The cells were grown in 96-well microtiter plates at a density of 1×104 cells in 200 µl per well. When cells grew to 80% confluence, 20 µl MTT (5 mg/ml) was added to each well and incubated at 37°C for 4 h. The supernatant was discarded and 150 µl dimethylsulfoxide was added to each well. Following incubation for 10 min, the plates were read on a microplate reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA) at 490 nm.

Measurement of NF-κB, DNMT1 and ox-LDL concentrations using ELISA

The cells were collected and samples were determined using the following ELISA kits, NF-κB (cat. no. DY1795) and ox-LDL (cat. no. DYC4299) obtained from R&D Systems, Inc. (Minneapolis, MN, USA), and the DNMT1 ELISA kit (cat. no. ab113469; Abcam, Cambridge, UK) according to the manufacturers' protocols.

Measurement of superoxide dismutase (SOD), malondialdehyde (MDA) and hydrogen peroxide (H2O2) concentrations via colorimetry

The levels of SOD (Nanjing Jiancheng Bioengineering Institute, Nanjing, China), MDA (Nanjing Jiancheng Bioengineering Institute) and H2O2 (Nanjing Jiancheng Bioengineering Institute) were determined using colorimetry according to the manufacturer's protocol. The absorbance was read on a microplate reader at 550 nm.

Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis

Total RNA was isolated from the cultured cells using a Tri-Reagent kit (Invitrogen; Thermo Fisher Scientific, Inc.). The SYBR Green kit (Fermentas; Thermo Fisher Scientific, Inc.) was then used for RT-qPCR analysis. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was applied as an internal control for TLR-4, LOX-1 and DNMT1 in the ECs. The primers are listed in Table I and the reaction system was: 2X SYBR mixture 25 µl, Forward primer 1 µl, Reverse primer 1 µl, cDNA 2 µl and Rnase-free water up to 50 µl. The thermal cycler (Funglyn Biotech, Inc., Toronto, ON, Canada) conditions comprised an initial activation step at 95°C for 5 min, followed by a 2-step PCR program of 95°C for 15 sec, annealing temperatures for 15 sec and at 72°C for 30 sec for 30 cycles. Subsequently, the relative changes in the mRNA expression levels of TLR-4, LOX-1 and DNMT1 were determined by fold-change analysis, in which the degree of change was calculated as 2−ΔΔCq, where Cq = (Cqgene - CqGAPDH) treatment - (Cqgene - CqGAPDH) control (19).

Table I.

Primer sequences of TLR-4, LOX-1, DNMT1 and GAPDH for reverse transcription-quantitative polymerase chain reaction analysis.

Table I.

Primer sequences of TLR-4, LOX-1, DNMT1 and GAPDH for reverse transcription-quantitative polymerase chain reaction analysis.

GenePrimerSequence (5′-3′)Temperature (°C)Length (bp)
TLR4Forward ATAAGTGTCGAACTCCCTC51138
Reverse GCTCATTCCTTACCCAGT
LOX-1Forward AATGATAGAAACCCTTGC46132
Reverse TTCCCAGTTAAATGAGCC
DNMT1Forward GGAGCCCAGCAAGAGTA51141
Reverse GGGAGACACCAGCCAAAT
GAPDHForward AGAAGGCTGGGGCTCATTT51146
Reverse AGGGGCCACAGTCTTCG

[i] TLR4, toll-like receptor 4; LOX-1, lectin-like oxidized-low density lipoprotein receptor-1; DNMT1, DNA methyltransferase 1; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Western blot analysis

Total proteins were isolated from the cells using cell lysis buffer (Keygen Biotech Co., Ltd., Nanjing, China). Equal amounts of protein (~80 µg) and known molecular weight marker were loaded onto 12% sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE) and were transferred to PVDF membrane by electrophoresis at 300 mA for 50 min at 4°C, the membrane was then blocked in 10 ml 5% skimmed milk for 2 h at room temperature with gentle agitation on a platform shaker. The LOX-1 and TLR-4 proteins were detected using LOX-1 (cat. no. sc-66155) and TLR-4 (cat. no. sc-13593) antibodies were obtained from Santa Cruz Biotechnology, Inc., (Dallas, TX, US) diluted 1:500, and β-actin protein was detected using a rabbit anti-human β-actin antibody (cat. no. sc-70319, Santa Cruz Biotechnology, Inc.) diluted 1:2,000; all primary antibodies were incubated at 4°C. The secondary antibody (goat anti-mouse IgG-HRP, cat. no. sc-2031, 1:2,000; Santa Cruz Biotechnology, Inc.) was added for 2 h at room temperature. The protein bands were visualized and analyzed by the Gel Documentation and Analysis System ChemiDoc XRS system with Image Lab software, version 4.1 (Bio-Rad Laboratories, Inc.) and calculated by the gray value of the bands.

Nested touchdown methylation-specific PCR (ntMSP) analysis

Total genomic DNA was extracted from the cultured cells using a DNA isolation kit (Sigma-Aldrich; Merck Millipore) according to the manufacturer's protocol, and genomic DNA (1 µg) was bisulfite modified (Sigma-Aldrich; Merck Millipore). The detection of methylation levels was conducted as previously described (20). The reaction conditions of PCR were as follows: 94°C for 45 sec, 68.3°C for 45 sec, and 72°C for 45 sec for 20 cycles, followed by a 0.5°C decrease of 53.3°C every cycle for 20 cycles. The second step of PCR was performed with conventional PCR primers under the following reaction conditions: 94°C for 45 sec, 67°C for 45 sec, and 72°C for 45 sec for 20 cycles, followed by a 0.5°C decrease of 52°C every cycle for 20 cycles, and ending with extension at 72°C for 5 min. Primer sequences are listed in Table II.

Table II.

Primer sequences of lectin-like oxidized-low density lipoprotein receptor-1 for nested touchdown methylation-specific polymerase chain reaction analysis.

Table II.

Primer sequences of lectin-like oxidized-low density lipoprotein receptor-1 for nested touchdown methylation-specific polymerase chain reaction analysis.

PrimerSequence (5′-3′)Length (bp)
Left outerTTAGTATTGTGGG251
AGGTTGAGGTAG
Right outerTAAAATTTCACCC
TTATTACCCAAA
Left MTTGAAAATATAAA137
ATAATTAGTCGG
Right MTAAATTACAATAA
CATAATCTCG
Left UTTGAAAATATAAA139
ATAATTAGTTGG
Right UAATAAATTACAATA
ACATAATCTCAAC

[i] M, methylated; U, unmethylated.

Statistical analysis

Prism 5.0 (GraphPad Software, Inc., La Jolla, CA, USA) was used for data processing. Experiments were performed at least in triplicate. Results were expressed as mean ± standard error of the mean ( ±S). Two-way analysis of variance was used for comparisons between groups and additional analysis was performed using Student-Newman-Keuls test for multiple comparisons within treatment groups. P<0.05 was considered to indicate a statistically significant difference.

Results

Hcy impairs EC viability

Following incubation of the ECs with Hcy for 72 h, the viability of ECs was determined using an MTT assay. As shown in Fig. 1, Hcy affected EC viability in a dose-dependent manner, and found that cell activities were decreased by 21, 28, and 56% in the 100, 200, and 500 µmol/l Hcy group, respectively (P<0.05 or P<0.01). When treated with folic acid and vitamin B12, the viability of the ECs was increased by 1.15-fold, compared with that in the 100 µmol/l Hcy group. Taken together, these results suggested that Hcy induced EC injury.

Hcy decreases the activity of SOD and increases levels of MDA, H2O2 and ox-LDL in ECs

Hcy is an independent risk factor for several major pathologies in cardiovascular disease, and elevated Hcy is important in various pathologies by increasing the production of H2O2, which affects the antioxidant defense systems (21). In the present study, H2O2 concentrations were increased in the 100, 200 and 500 µmol/l Hcy groups (P<0.05 and P<0.01). The results also demonstrated that the activity of the antioxidant enzyme SOD was markedly decreased in parallel with the levels of MDA in the Hcy-treated cells. Taken together, these results suggested that Hcy injures ECs by oxidative stress. Ox-LDL is considered to be a biomarker of in vivo oxidative stress in AS, and elevated oxidative stress and superoxide anion formation in vascular cells can promote the conversion of LDL to ox-LDL, contributing to endothelial dysfunction and AS. In the present study, the change in the levels of ox-LDL was consistent with the trend observed for the oxidative stress indicators, increasing by 1.44-, 1.54- and 2.1-fold in the 100, 200 and 500 µmol/l Hcy groups, respectively (P<0.01; Table III). The levels decreased by 20.2% in the folate and vitamin B12-treated cells.

Table III.

Levels of SOD, MDA, H2O2 and ox-LDL in endothelial cells.

Table III.

Levels of SOD, MDA, H2O2 and ox-LDL in endothelial cells.

GroupSOD (U/mg prot)MDA (nmol/mg prot) H2O2 (mmol/l)ox-LDL (mg/l)
0 µmol/l 156.28±10.35 24.52±2.29 11.63±1.67 0.72±0.06
50 µmol/l 148.23±7.40 27.90±2.00 16.49±1.58 0.87±0.09
100 µmol/l 135.96±6.25a 30.71±1.58a 21.44±2.61b 1.04±0.12b
200 µmol/l 111.89±6.18a 37.49±5.09a 36.81±3.42a 1.11±0.18a
500 µmol/l 103.40±4.89a 39.23±3.81a 46.03±4.15a 1.50±0.20a
100 µmol/l+V+F 146.06±3.60 28.66±2.62 15.01±2.23 0.83±0.06

{ label (or @symbol) needed for fn[@id='tfn3-mmr-16-06-9181'] } 100 µmol/l+V+F group, co-incubation with 100 µmol/l Hcy, 30 µmol/l folate and 30 µmol/l vitamin B12 for 72 h. Data expressed as the mean ± standard deviation.

a P<0.01

b P<0.05, compared with the 0 µmol/l Hcy group. Hcy, homocysteine; SOD, superoxide dismutase; MDA, malondialdehyde; H2O2, hydrogen peroxide; ox-LDL, oxidized low density lipoprotein.

Hcy promotes expression levels of TLR4 and LOX-1 in ECs

TLR4 is a central mediator of the innate immune response, which is important in the defense mechanism against microorganisms (22). In the present study, the mRNA levels if TLR4 were upregulated by 16.6-, 18.7- and 30.3-fold in the 100, 200 and 500 µmol/l Hcy groups, compared with that in the untreated group, respectively. In addition, the protein expression of TLR4 was increased by 1.8-, 1.9- and 2.0-fold in the 100 and 200 and 500 µmol/l Hcy groups, respectively (P<0.01; Fig. 2A and B). The expression of TLR4 was decreased when the cells were treated with folic acid and vitamin B12. As the level of TLR4 increased with the increase in Hcy, TLR4 may be important in EC injury induced by Hcy.

LOX-1 is a type II membrane glycoprotein in ECs, which has been implicated in atherosclerotic plaque formation, progression and destabilization (23). When the cells were treated with different concentrations of Hcy for 72 h, the mRNA expression of LOX-1 was promoted in the Hcy groups (P<0.05 and P<0.01; Fig. 2C), and decreased when the cells were treated with folic acid and vitamin B12. The protein levels of LOX-1 were increased by 1.7-fold in the 100 µmol/l Hcy group (Fig. 2A and C) and when the cells were treated with folic acid and vitamin B12, the expression levels of LOX-1 decreased by 26%. These findings suggested that Hcy promoted the expression of LOX-1, leading to EC injury.

Hcy induces LOX-1 DNA hypomethylation in ECs. In the present study, Hcy suppressed the levels of LOX-1 DNA methylation by in the 100, 200 and 500 µmol/l Hcy groups (Fig. 3A and B; P<0.01) while, when treated with folate and vitamin B12, LOX-1 DNA methylation levels were increased, compared with 100 µmol/l Hcy treated cells. These data also suggested that the mRNA levels of DNMT1 were decreased in the Hcy-treated cells (Fig. 3C), and the protein expression of DNMT1 was suppressed in cells treated with increasing concentrations of Hcy (Fig. 3D). Taken together, Hcy appeared to decrease the levels of LOX-1 DNA methylation by reducing the levels of DNMT1. Combined with the results obtained on the expression levels of LOX-1, these results suggested that Hcy may induce LOX-1 DNA hypomethylation to promote the expression levels of LOX-1.

NF-κB decreases the levels of DNMT1 in ECs

Hcy can promote the secretion of NF-κB, and the present study found that Hcy induced the levels of NF-κB in ECs by 1.6-, 1.9- and 2.3-fold in the 100, 200 and 500 µmol/l Hcy groups, respectively (Fig. 4A; P<0.05 and P<0.01), whereas the level was decreased by 28.8% following treatment of the cells with folate and vitamin B12 (P<0.05). Previous studies have suggested that the NF-κB signaling pathway is potentially targeted at various levels, including nuclear translocation, DNA binding and via methyl transferases. The present study hypothesized that NF-κB may be associated with DNMT1. Therefore, the cells were treated with pyrrolidine dithiocarbamate (PDTC), which suppresses the activity of NF-κB, to confirm the association between NF-κB and DNMT1. The results suggested that, the PDTC-induced suppression of the activity of NF-κB led to increased levels of DNMT1 (Fig. 4B and C). Taken together, these findings suggested that Hcy may result in DNA hypomethylation by inducing the activation of NF-κB and further decreasing the expression of DNMT1.

Discussion

Studies have confirmed that elevated levels of Hcy are a cause of EC injury and can promote the formation of AS (24,25). In the present study, it was demonstrated that Hcy induced EC injury, which was the main mechanism in the development of AS induced by Hcy.

Oxidative stress is a condition in which the balance between the production of reactive oxygen species (ROS) and level of antioxidants is significantly disturbed and results in damage to cells by excessive ROS production. O2•-, H2O2 and OH•, as the ROS produced, are sensitively controlled by several antioxidant enzymes, including SOD and MDA (26). Accumulating evidence suggests that elevated plasma Hcy affects the oxidant-antioxidant balance in the body following endothelial injury (27). In the present study, H2O2 concentrations were higher in the Hcy groups and the activity of antioxidant enzyme SOD was decreased, whereas the levels of MDA were increased in the cells under hyperhomocysteinemia. Taken together, these results suggested that oxidative stress induced the imbalance of redox reactions and that Hcy may injure ECs, which produce high levels of ROS through oxidative stress imbalance. This evidence provides scope for subsequent investigations.

LOX-1 was originally identified as the major receptor for ox-LDL in ECs. A feedback exists involving ox-LDL and LOX-1, in which ox-LDL can induce the secretion of LOX-1, then more ox-LDL can be uptaken by LOX-1 in early phase of AS (28). The present study suggested that Hcy promoted ox-LDL and LOX-1 expressions in ECs. Hcy is a sulfur amino acid that can induce oxidative stress by trans-sulfuration and increasing evidence suggests that oxidative stress occurs in response to EC injury, which can oxidize LDL to produce ox-LDL. LOX-1 is one of the scavenger receptors and Hcy has been shown to upregulate scavenger receptors, as reported for the expression of CD36 at atherogenic lesion sites in apoE−/− female mice (29). However, the regulation of gene expression is affected by various factors under elevated Hcy, including DNA methylation, which is a focus with AS.

DNA methylation serves as an important mechanism which controls gene expression in AS (30). As shown in the present study, the levels of LOX-1 DNA methylation were significantly decreased in the Hcy groups. Hcy is involved in a one-carbon transfer reaction, which is also important for DNA methylation. In Hcy metabolism, S-adenosylmethionine (SAM) is a metabolic intermediate, which is synthesized from methionine catalyzed by methionine adenosyltransferase, providing methyl group moieties in several transmethylation reactions (31). SAM is converted into S-adenosylhomocysteine (SAH), which is the sole metabolic precursor of Hcy in a reversible reaction catalyzed by SAH hydrolase and can inhibit DNMT1. It has been confirmed that DNA methylation patterns depend on DNMT1, and the results of the present study found that Hcy decreased the levels of DNMT1.

NF-κB is a key transcription factor which is responsible for several biological processes, and it has been identified as a member of the structurally-related eukaryotic transcription factor family and regulates inducible gene expression (32). In the present study, data showed that Hcy increased the levels of NF-κB, whereas DNMT1 was suppressed in the Hcy groups. In order to further examine the association between NF-κB and DNMT1, PDTC, an antioxidant suppressing the activity of NF-κB, was used, and it was found that NF-κB downregulated DNMT1. A possible mechanism for this involves the translocation of NF-κB to the nucleus, binding to specific DNA sequences and promoting the transcription of target genes. DNMT1 has been shown to interact with the transcription factors Sp1, Sp3 and signal transducer and activator of transcription (STAT) 3, with an STAT3-DNMT1-HDAC1 complex binding to the promoter of phosphatase-1 (33), and Sp1/NF-κB DNA-binding decreasing the expression of DNMT1. Investigations of DNA hypomethylation have suggested that Hcy induces oxidative stress to activate NF-κB, possibly by generating ROS (34). Taken together, NF-κB/DNMT1 may be key in Hcy-induced DNA methylation. NF-κB is capable of migrating into the nucleus and activating the transcription of target genes, contemporaneously. It is also involved in the pro-inflammatory response, a first line of defense against infectious diseases, whereas TLR4 is involved in the induction of innate immune and inflammatory responses. A previous study has demonstrated that TLR4-mediated signaling pathways mainly stimulate the activation of NF-κB (35) and the present study showed that Hcy promoted the mRNA and protein expression of TLR4. According to previous studies and the present study, TLR4/NF-κB/DNMT1 may be involved in Hcy-induced LOX-1 DNA hypomethylation.

In our previous study, it was demonstrated that folic acid and vitamin B12 are important in regulating the metabolic process of Hcy (20), and Ma et al (36) showed that supplementation of folic acid and vitamin B12 in patients with hyperhomocysteinemia (HHcy) reduced the levels of Hcy, suggesting folic acid supplementation may be useful in reducing Hcy levels in high risk patients with HHcy, but may also significantly improve endothelial dysfunction in patients with coronary artery disease. In the present study, it was found that, following supplementation with folate and vitamin B12, the damaging effect of Hcy on the ECs was inhibited, and this may be an important method for the remittance of AS caused by Hcy.

In conclusion, the accumulated evidence suggests that Hcy injures ECs via oxidative stress and that TLR4/NF-κB/DNMT1 may be involved in Hcy-induced EC injury by mediating LOX-1 DNA hypomethylation. These findings may be significant in the treatment of EC injury associated with gene expression due to the hypomethylation of gene regulatory regions, with TLR4/NF-κB/DNMT1 identified as a novel component in the mechanism. Taken together, the findings reveal a novel role of Hcy in the pathogenesis of AS.

Acknowledgements

This study was supported by grants from the National Natural Science Foundation of China (grant nos. 81570452, 81460121, 81560120, 81560084, 81560086, 81660047, 81660088 and 81760139).

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December-2017
Volume 16 Issue 6

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Online ISSN:1791-3004

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Copy and paste a formatted citation
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Spandidos Publications style
Ma SC, Hao YJ, Jiao Y, Wang YH, Xu LB, Mao CY, Yang XL, Yang AN, Tian J, Zhang MH, Zhang MH, et al: Homocysteine‑induced oxidative stress through TLR4/NF‑κB/DNMT1‑mediated LOX‑1 DNA methylation in endothelial cells. Mol Med Rep 16: 9181-9188, 2017
APA
Ma, S., Hao, Y., Jiao, Y., Wang, Y., Xu, L., Mao, C. ... Zhang, H. (2017). Homocysteine‑induced oxidative stress through TLR4/NF‑κB/DNMT1‑mediated LOX‑1 DNA methylation in endothelial cells. Molecular Medicine Reports, 16, 9181-9188. https://doi.org/10.3892/mmr.2017.7753
MLA
Ma, S., Hao, Y., Jiao, Y., Wang, Y., Xu, L., Mao, C., Yang, X., Yang, A., Tian, J., Zhang, M., Jin, S., Xu, H., Jiang, Y., Zhang, H."Homocysteine‑induced oxidative stress through TLR4/NF‑κB/DNMT1‑mediated LOX‑1 DNA methylation in endothelial cells". Molecular Medicine Reports 16.6 (2017): 9181-9188.
Chicago
Ma, S., Hao, Y., Jiao, Y., Wang, Y., Xu, L., Mao, C., Yang, X., Yang, A., Tian, J., Zhang, M., Jin, S., Xu, H., Jiang, Y., Zhang, H."Homocysteine‑induced oxidative stress through TLR4/NF‑κB/DNMT1‑mediated LOX‑1 DNA methylation in endothelial cells". Molecular Medicine Reports 16, no. 6 (2017): 9181-9188. https://doi.org/10.3892/mmr.2017.7753