WITHDRAWN: Mechanism of extracellular­superoxide dismutase DNA methylation in a hyperhomocysteinemia­induced atherosclerosis in a mouse model.

Ahead of Print article withdrawn by publisher.

Homocysteine-induced proliferation of vascular smooth muscle cells occurs via PTEN hypermethylation and is mitigated by Resveratrol.

Vascular smooth muscle cell (VSMC) proliferation is a primary pathological event in the development of atherosclerosis (AS), and the presence of homocysteine (Hcy) acts as an independent risk factor for AS. However, the underlying mechanisms remain to be elucidated. Phosphatase and tensin homologue on chromosome 10 (PTEN), is endogenously expressed in VSMCs and induces multiple signaling networks involved in cell proliferation, survival and inflammation, however, the specific role of PTEN is still unknown. The present study detected the proliferation ratio of VSMCs following treatment with Hcy and Resveratrol (RSV). In the 100 µM Hcy group, the proliferation ratio increased, and treatment with RSV decreased the proliferation ratio induced by Hcy. Reverse transcription­quantitative polymerase chain reaction and western blotting were used to analyze PTEN expression, RSV treatment was associated with decreased PTEN expression levels in VSMCs. PTEN levels were decreased in Hcy treated cells, and the proliferation ratio of VSMCs were increased following treated with Hcy. To study the mechanism of regulation of PTEN by Hcy, the present study detected PTEN methylation levels in VSMCs, and PTEN DNA methylation levels were demonstrated to be increased in the 100 µM Hcy group, whereas treatment with RSV decreased the methylation status. DNA methyltransferase 1 is important role in the regulation of PTEN methylation. Overall, Hcy impacts the methylation status of PTEN, which is involved in cell proliferation, and induces the proliferation of VSMCs. This effect is alleviated by treatment with RSV, which exhibits an antagonistic mechanism against Hcy.

Homocysteine inhibits endothelial progenitor cells proliferation via DNMT1-mediated hypomethylation of Cyclin A.

Endothelial progenitor cells (EPCs) contribute to neovasculogenesis and reendothelialization of damaged blood vessels to maintain the endothelium. Dysfunction of EPCs is implicated in the pathogenesis of vascular injury induced by homocysteine (Hcy). We aimed to investigate the role of Cyclin A in Hcy-induced EPCs dysfunction and explore its molecular mechanism. In this study, by treatment of EPCs with Hcy, we found that the expression of Cyclin A mRNA and protein were significantly downregulated in a dose-dependent manner. Knockdown of Cyclin A prominently reduced proliferation of EPCs, while over-expression of Cyclin A significantly promoted the cell proliferation, suggesting that Hcy inhibits EPCs proliferation through downregulation of Cyclin A expression. In addition, epigenetic study also demonstrated that Hcy induces DNA hypomethylation of the Cyclin A promoter in EPCs through downregulated expression of DNMT1. Moreover, we found that Hcy treatment of EPCs leads to increased SAM, SAH and MeCP2, while the ratio of SAM/SAH and MBD expression decrease. In summary, our results indicate that Hcy inhibits Cyclin A expression through hypomethylation of Cyclin A and thereby suppress EPCs proliferation. These findings demonstrate a novel mechanism of DNA methylation mediated by DNMT1 in prevention of Hcy associated cardiovascular disease.

Homocysteine­induced oxidative stress through TLR4/NF­κB/DNMT1­mediated LOX­1 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 lectin­like oxidized­low density lipoprotein receptor­1 (LOX­1) DNA methylation through toll­like 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 NF­κB and DNMT1 was examined by treatment of the ECs with pyrrolidine dithiocarbamate (PDTC). The results suggested that Hcy induced LOX­1 DNA hypomethyaltion to promote the expression levels of LOX­1. Taken together, Hcy injured the ECs through the effect of methylation and trans­sulfuration metabolism of LOX­1 through TLR4/NF­κB/DNMT1. Following injury to the ECs, lipids, particularly ox­LDL, accumulated in the sub­endothelial layer to promote the formation of AS.

Aberrant promoter methylation of multiple genes in VSMC proliferation induced by Hcy.

Vascular smooth muscle cell (VSMC) proliferation is a primary pathological event in atherosclerosis (AS), and homocysteine (Hcy) is an independent risk factor for AS. However, the underlying mechanisms are still lagging. Studies have used the combination of methylation of promoters of multiple genes to diagnose tumors, thus the aim of the current study was to investigate the role of methylation status of several genes in VSMCs treated with Hcy. CpG islands were identified in the promoters of platelet­derived growth factor (PDGF), p53, phosphatase and tensin homologue on chromosome 10 (PTEN) and mitofusin 2 (MFN2). Hypomethylation was observed to occur in the promoter region of PDGF, hypermethylation in p53, PTEN and MFN2, and hypomethylation in two global methylation indicators, aluminium (Alu) and long interspersed nucleotide element­1 (Line­1). This was accompanied by an increase in the expression of PDGF, and reductions of p53, PTEN and MFN2, both in mRNA and protein levels. An elevation of S­adenosylmethionine (SAM) and a reduction of S­adenosylhomocysteine (SAH) and the SAM/SAH ratio were also identified. In conclusion, Hcy impacted methylation the of AS­associated genes and global methylation status that mediate the cell proliferation, which may be a character of VSMCs treated with Hcy. The data provided evidence for mechanisms of VSMCs proliferation in AS induced by Hcy and may provide a new perspective for AS induced by Hcy.

[Corrigendum] Integration of gene expression and DNA methylation profiles provides a molecular subtype for risk assessment in atherosclerosis.

After the publication of the article, the authors noted that they had made an error regarding the order of one of their names. For Professor Zheng Yu, the given name is Yu and the family name is Zheng, therefore he should be listed as Professor Yu Zheng in the author list and as the corresponding author. [the original article was published in the Molecular Medicine Reports 13: 4791-4799, 2016; DOI: 10.3892/mmr.2016.5120].

Integration of gene expression and DNA methylation profiles provides a molecular subtype for risk assessment in atherosclerosis.

The aim of the present study was to identify an effective method for detecting early­phase atherosclerosis (AS), as well as to provide useful DNA methylation profiles to serve as biomarkers for the detection of AS. A total of 300 individuals (150 AS patients and 150 healthy subjects) were recruited for peripheral blood DNA methylation analyses at 12 gene promoter loci using nested methylation­specific polymerase chain reaction in a test set. Based on the test set, the promoter methylation of TIMP metallopeptidase inhibitor 1 (TIMP1), ATP binding cassette subfamily A member 1 (ABCA1), and acetyl-CoA acetyltransferase 1 (ACAT1) were determined to be candidate biomarkers; demonstrating the highest sensitivity (88%) and specificity (90%). The biomarkers that were candidates for early AS detection were validated in an independent validation set (n=100). In the validation set, the combination of TIMP1, ABCA1 and ACAT1 methylation achieved sensitivity, specificity and coincidence rate values of 88, 70 and 79%, respectively. In the current pilot study, the patterns of DNA methylation of AS­associated genes were observed to be significantly altered in the peripheral blood of AS patients. Thus, the AS-specific methylation of the three­gene panel (TIMP1, ABCA1, and ACAT1) may serve as a valuable biomarker for the early detection of AS.

A regulatory circuit involving miR-143 and DNMT3a mediates vascular smooth muscle cell proliferation induced by homocysteine.

Accumulating evidence has suggested that homocysteine (Hcy) is an independent risk factor for atherosclerosis (AS). Hcy can promote vascular smooth muscle cell (VSMC) proliferation, which is pivotal in the pathogenesis and progression of AS. The aim of the present study was to investigate the epigenetic regulatory mechanism of microRNA (miR)­143­mediated VSMCs proliferation induced by Hcy. The results of a 3­(4,5­dimethylthiazol­2­yl)­2,5­diphe­nyltetrazolium bromide assay revealed that VSMC proliferation was increased by 1.39­fold following treatment with 100 mM Hcy, compared with the control group. The levels of miR­143 were markedly downregulated in the Hcy group, compared with the control group, as determined using reverse transcription­quantitative polymerase chain reaction analysis. In addition, the level of miR­143 methylation was increased markedly in the VSMCs treated with Hcy, compared with the control, and was reduced following transfection with DNA methyltransferase (DNMT)3a small interfering RNA, determined using methylation­specific­PCR. The activities of DNMT3a luciferase were also altered accordingly in VSMCs transfected with pre­miR­143 and miR­143 inhibitor, respectively. In addition, the expression of miR­143 was observed to be inversely correlated with the mRNA and protein expression of DNMT3 in the VSMCs. Taken together, these findings suggest that DNMT3a is a direct target of miR­143, and that the upregulation of DNMT3 is responsible for the hypermethylation of miR­143 in Hcy-induced VSMC proliferation.

High-methionine diets accelerate atherosclerosis by HHcy-mediated FABP4 gene demethylation pathway via DNMT1 in ApoE(-/-) mice.

Homocysteine (Hcy) is an independent risk factor for atherosclerosis, but the underlying molecular mechanisms are not known. We investigated the effects of Hcy on fatty acid-binding protein 4 (FABP4), and tested our hypothesis that Hcy-induced atherosclerosis is mediated by increased FABP4 expression and decreased methylation. The FABP4 expression and DNA methylation was assessed in the aorta of ApoE(-/-) mice fed high-methionine diet for 20weeks. Over-expression of FABP4 enhanced accumulation of total cholesterol and cholesterol ester in foam cells. The up-regulation of DNA methyltransferase 1 (DNMT1) promoted the methylation process and decreased FABP4 expression. These data suggest that FABP4 plays a key role in Hcy-mediated disturbance of lipid metabolism and that DNMT1 may be a novel therapeutic target in Hcy-related atherosclerosis.

[The effect of miR-124 on homocysteine-induced atherosclerosis via promoter region DNA methylation in ApoE(-/-) mice].

The aim of the present study is to explore the role of miR-124 and its promoter region DNA methylation in homocysteine (Hcy)-induced atherosclerosis. ApoE(-/-) mice were fed with hypermethionine diet for 16 weeks to duplicate hyperhomocysteinemia model. Meanwhile, a normal control group (C57BL/6J mice fed with normal diet, N-control) and a model control group (ApoE(-/-) mice fed with normal diet, A-control) were set. The degree of atherosclerosis was observed by HE and oil red O staining. Automatic biochemical analyzer was used to detect the serum levels of Hcy. Foam cell model was duplicated and oil red O staining was used to confirm whether the model was successfully established. And foam cells were stimulated with 0, 50, 100, 200, 500 µmol/L Hcy and 50 µmol/L Hcy + 10 µmol/L AZC respectively. Real-time quantitative PCR (RT-qPCR) was used to detect the expressions of miR-124 in mice aorta and foam cells; Nested landing methylation specific PCR (nMS-PCR) was used to detect the levels of miR-124 promoter DNA methylation in mice aorta and foam cells. Meanwhile, the effects of DNA methylation inhibitor AZC on miR-124 expression were observed at the cellular level. The effect of miR-124 promoter DNA methylation status on lipid accumulation in foam cells was observed by oil red O staining. The results showed that compared with model control group, the serum levels of Hcy in high methionine group were significantly increased (P < 0.01) and developed aortic atherosclerotic plaque, the expression of miR-124 was markedly decreased (P < 0.01), while the levels of miR-124 promoter DNA methylation were significantly increased (P < 0.01). Given different levels of Hcy, the expression of miR-124 in foam cells was decreased, while the levels of miR-124 promoter DNA methylation were increased in a dose-dependent manner (P < 0.05, P < 0.01). AZC reversed the results of mentioned indices as above markedly (P < 0.05). Downregulation of miR-124 may play a role in Hcy-induced atherosclerosis and its promoter DNA methylation status may be an important mechanism in this process.

Aberrant DNA methylation of the PDGF gene in homocysteine­mediated VSMC proliferation and its underlying mechanism.

It is well established that homocysteine (Hcy) is an independent risk factor for atherosclerosis (AS), which is characterized by vascular smooth muscle cell (VSMC) proliferation. However, the molecular mechanism underlying AS in VSMCs is yet to be elucidated. The aim of this study was to investigate the potential involvement of aberrant DNA methylation of the platelet­derived growth factor (PDGF) gene in Hcy­mediated VSMC proliferation and its underlying mechanism. Cultured human VSMCs were treated with varying concentrations of Hcy. VSMC proliferation, PDGF mRNA and protein expression and PDGF promoter demethylation showed a dose­dependent increase with Hcy concentration, suggesting an association among them. Cell cycle analysis revealed a decreased proportion of VSMCs in G0/G1 and an increased proportion in S phase, indicating that VSMC proliferation was increased under Hcy treatment. Furthermore, S­adenosylhomocysteine (SAH) levels were observed to increase and those of S­adenosylmethionine (SAM) were observed to decrease. The consequent decrease in the ratio of SAM/SAH may partially explain the hypomethylation of PDGF with Hcy treatment. Folate treatment exhibited an antagonistic effect against Hcy­induced VSMC proliferation, aberrant PDGF methylation and PDGF expression. These data suggest that Hcy may stimulate VSMC proliferation through the PDGF signaling pathway by affecting the epigenetic regulation of PDGF through the demethylation of its promoter region. These findings may provide novel insight into the molecular association between aberrant PDGF gene demethylation and the proliferation of VSMCs in Hcy­associated AS.

Knockdown of PIK3R1 by shRNA inhibits the activity of the splenic macrophages associated with hypersplenism due to portal hypertension.

Phosphatidylinositol 3-kinase (PI3K) plays a central role in the metabolic actions of insulin. One 85 kDa regulatory subunit of PIK3 is encoded by phosphoinositide-3-kinase, the regulatory subunit 1 (PIK3R1). Our previous study has demonstrated that PIK3R1 was up-regulated significantly in the splenic macrophage (MΦ) of portal hypertensive spleen. In the present study, RNA interference specific to PIK3R1 was employed to investigate its inhibitive effects on the activity of MΦ associated with hypersplenism due to portal hypertension (HS-PHT). The expression of PIK3R1 in the spleen was detected by immunohistochemical staining. Plasmid vector pGenesil-1 expressing specific small hairpin RNA (shRNA) against PIK3R1 and the scrambled shRNA control was constructed. MΦ were isolated and purified by anchored cultivation from patients with HS-PHT (HS-PHT-MΦ) and traumatic rupture of the spleen (Con-MΦ). After transfection into MΦ, PIK3R1 expression at both the mRNA and the protein level was examined by real-time polymerase chain reaction and Western blot. The activities of MΦ were determined, and the expression and activity of NF-κB were also detected. Immunohistochemistry revealed expression and cellular distribution of PIK3R1 in the spleen. The PIK3R1-shRNA was successfully synthesized and cloned into the plasmid vector pGenesil-1, and specifically suppressed PIK3R1 expression at both the mRNA and the protein level. After transfection into HS-PHT-MΦ and Con-MΦ, PIK3R1 knockdown inhibited the viability of MΦ, reduced the phagocytic rate, the rate of antigen-presenting positive cells, the metabolic rate, and the secretion of IL-1ß and TNF-α (all p<0.05), and decreased the expression and activity of NF-κB. Our data showed that the knocking down of PIK3R1 with shRNA produced by pGenesil-1 led to inhibition of viability and to decreased activity of MΦ associated with HS-PHT in vitro. Therefore, it is tempting to speculate that PIK3R1 might play a considerable role in the pathogenesis of HS-PHT, and inhibition of PIK3R1 expression might be a novel therapeutic strategy for HS-PHT.

[Experimental study on the in vitro resistance to Toxoplasma gondii by murine lymphocyte].

AIM: To study the activities of in vitro resistance to the tachyzoite of Toxoplasma gondii by murine lymphocytes. METHODS: The rat's splenocyte culture method was used to observe the effects of lymphocytes themselves and lymphocytes together with Mandelic( MA) on the invasion and proliferation of T. gondii in lymphocytes. At the same time acetylspiramycin was used as positive control. RESULTS: As compared with other somatic cells, the lymphocytes invaded by T. gondii still inhibited and killed the toxoplasma organisms in the presence of immunity, the effect safety dose of MA on inhibition of the invasion of T. gondii was not notable while the inhibition of the proliferation of T. gondii in lymphocytes was remarkable. CONCLUSION: Cell-mediated immunity(CMI) was an important factor that host resists the T. gondii infection. So, we should pay attention to improving the organism's CMI and take proper medicine so as to enhance the effect of lymphocyte's resistance to T. gondii.