ABSTRACT
Ahead of Print article withdrawn by publisher.
ABSTRACT
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.
Subject(s)
DNA Methylation/drug effects , Epigenesis, Genetic , Homocysteine/pharmacology , Myocytes, Smooth Muscle/drug effects , Promoter Regions, Genetic , Alu Elements , Atherosclerosis/genetics , Atherosclerosis/metabolism , Atherosclerosis/pathology , Cell Proliferation/drug effects , CpG Islands , Female , GTP Phosphohydrolases/genetics , GTP Phosphohydrolases/metabolism , Humans , Long Interspersed Nucleotide Elements , Mitochondrial Proteins/genetics , Mitochondrial Proteins/metabolism , Models, Biological , Muscle, Smooth, Vascular/cytology , Muscle, Smooth, Vascular/drug effects , Muscle, Smooth, Vascular/metabolism , Myocytes, Smooth Muscle/cytology , Myocytes, Smooth Muscle/metabolism , PTEN Phosphohydrolase/genetics , PTEN Phosphohydrolase/metabolism , Platelet-Derived Growth Factor/genetics , Platelet-Derived Growth Factor/metabolism , Primary Cell Culture , S-Adenosylhomocysteine/metabolism , S-Adenosylmethionine/metabolism , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolism , Umbilical Veins/cytology , Umbilical Veins/drug effects , Umbilical Veins/metabolismABSTRACT
Hyperhomocysteinemia (HHcy) is an independent risk factor for the development of atherosclerosis (AS), according to overwhelming number of clinical and epidemiological studies. However, the underlying pathogenic molecular mechanisms by which HHcy promotes AS remain to be fully elucidated. Fatty acid binding protein 4 (FABP4) has been shown to be important in macrophage cholesterol trafficking. The objective of the present study was to determine whether homocysteine (Hcy) accelerates AS through regulating FABP4, and then mediates cholesterol accumulation in macrophages. Hcy concentrations of 0, 50, 100, 200 and 500 µM, and 100 µM Hcy+30 µM vitamin B12 (VB12)+30 µM folic acid (FA) were respectively added to cultured THP1 monocytederived macrophages for 24 h. The levels of FABP4, which acts as a key factor connecting cellular lipid accumulation to inflammation, were determined using reverse transcriptionquantitative polymerase chain reaction (RTqPCR) and western blot analyses in the macrophages. The present study used a nested touchdown methylationspecific PCR assay to detect the DNA methylation status of the FABP4 promoter region. In addition, the FABP4 gene fragment was inserted into the cloning vector, pcDNA3.1EGFP, to construct the recombinant plasmid, pcDNA3.1EGFP/FABP4, which was identified using restriction endonuclease digestion analysis and DNA sequencing. The pcDNA3.1EGFP/FABP4 expression plasmid was transfected into THP1 monocytederived macrophages, mediated by liposome reagent, following which the expression levels of FABP4 were detected using RTqPCR and western blot analyses. The present study also determined the intracellular accumulation of total cholesterol in the macrophages. The results indicated that Hcy decreased the levels of FABP4 promoter methylation, but increased the mRNA and protein expression levels of FABP4 in the macrophages, compared with the control group (0 µM Hcy). However, no dosedependent changes were observed with increasing concentrations of Hcy. The recombinant fluorescent eukaryotic expression vector, pcDNA3.1EGFP/FABP4, was successfully constructed and effectively expressed in the THP1 macrophages. The results also showed that FABP4 accelerated the accumulation of cholesterol in the macrophages. Taken together, the results of the present study suggested that FABP4 DNA hypomethylation induced by Hcy may be involved in the overexpression of FABP4, thereby inducing cholesterol accumulation in macrophages.
