title: FABP4-mediated homocysteine-induced cholesterol accumulation in THP-1 monocyte-derived macrophages and the potential epigenetic mechanism.
abstract: 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 THP‑1 monocyte‑derived macrophages for 24 h. The levels of FABP4, which acts as a key factor connecting cellular lipid accumulation to inflammation, were determined using reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR) and western blot analyses in the macrophages. The present study used a nested touchdown methylation‑specific 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, pcDNA3.1‑EGFP, to construct the recombinant plasmid, pcDNA3.1‑EGFP/FABP4, which was identified using restriction endonuclease digestion analysis and DNA sequencing. The pcDNA3.1‑EGFP/FABP4 expression plasmid was transfected into THP‑1 monocyte‑derived macrophages, mediated by liposome reagent, following which the expression levels of FABP4 were detected using RT‑qPCR 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 dose‑dependent changes were observed with increasing concentrations of Hcy. The recombinant fluorescent eukaryotic expression vector, pcDNA3.1‑EGFP/FABP4, was successfully constructed and effectively expressed in the THP‑1 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.
authors.affiliation: Department of Cardiothoracic Surgery, West China Hospital of Sichuan University, Chengdu, Sichuan 610000, P.R. China.
authors.forename: Yideng
authors.initials: Y
authors.lastname: Jiang
authors.affiliation: School of Basic Medical Sciences, Ningxia Medical University, Yinchuan, Ningxia 750004, P.R. China.
authors.forename: Shengchao
authors.initials: S
authors.lastname: Ma
authors.affiliation: Medical Experimental Center, General Hospital of Ningxia Medical University, Yinchuan, Ningxia 750004, P.R. China.
authors.forename: Huiping
authors.initials: H
authors.lastname: Zhang
authors.affiliation: School of Basic Medical Sciences, Ningxia Medical University, Yinchuan, Ningxia 750004, P.R. China.
authors.forename: Xiaoling
authors.initials: X
authors.lastname: Yang
authors.affiliation: Medical Experimental Center, General Hospital of Ningxia Medical University, Yinchuan, Ningxia 750004, P.R. China.
authors.forename: Guan Jun
authors.initials: GJ
authors.lastname: Lu
authors.affiliation: School of Basic Medical Sciences, Ningxia Medical University, Yinchuan, Ningxia 750004, P.R. China.
authors.forename: Hui
authors.initials: H
authors.lastname: Zhang
authors.affiliation: School of Basic Medical Sciences, Ningxia Medical University, Yinchuan, Ningxia 750004, P.R. China.
authors.forename: Yangyang
authors.initials: Y
authors.lastname: He
authors.affiliation: School of Basic Medical Sciences, Ningxia Medical University, Yinchuan, Ningxia 750004, P.R. China.
authors.forename: Fanqi
authors.initials: F
authors.lastname: Kong
authors.affiliation: School of Basic Medical Sciences, Ningxia Medical University, Yinchuan, Ningxia 750004, P.R. China.
authors.forename: Anning
authors.initials: A
authors.lastname: Yang
authors.affiliation: School of Basic Medical Sciences, Ningxia Medical University, Yinchuan, Ningxia 750004, P.R. China.
authors.forename: Hua
authors.initials: H
authors.lastname: Xu
authors.affiliation: School of Basic Medical Sciences, Ningxia Medical University, Yinchuan, Ningxia 750004, P.R. China.
authors.forename: Minghao
authors.initials: M
authors.lastname: Zhang
authors.affiliation: Medical Experimental Center, General Hospital of Ningxia Medical University, Yinchuan, Ningxia 750004, P.R. China.
authors.forename: Yun
authors.initials: Y
authors.lastname: Jiao
authors.affiliation: School of Basic Medical Sciences, Ningxia Medical University, Yinchuan, Ningxia 750004, P.R. China.
authors.forename: Guizhong
authors.initials: G
authors.lastname: Li
authors.affiliation: School of Basic Medical Sciences, Ningxia Medical University, Yinchuan, Ningxia 750004, P.R. China.
authors.forename: Jun
authors.initials: J
authors.lastname: Cao
authors.affiliation: Department of Cardiothoracic Surgery, West China Hospital of Sichuan University, Chengdu, Sichuan 610000, P.R. China.
authors.forename: Yuexia
authors.initials: Y
authors.lastname: Jia
authors.affiliation: Department of Cardiothoracic Surgery, West China Hospital of Sichuan University, Chengdu, Sichuan 610000, P.R. China.
authors.forename: Shaoju
authors.initials: S
authors.lastname: Jin
authors.affiliation: Medical Experimental Center, General Hospital of Ningxia Medical University, Yinchuan, Ningxia 750004, P.R. China.
authors.forename: Jun
authors.initials: J
authors.lastname: Wei
authors.affiliation: Department of Cardiothoracic Surgery, West China Hospital of Sichuan University, Chengdu, Sichuan 610000, P.R. China.
authors.forename: Yingkang
authors.initials: Y
authors.lastname: Shi
mesh_terms: D002460:Cell Line; D002784:Cholesterol / Q000378:metabolism*; D019175:DNA Methylation; D044127:Epigenesis, Genetic; D050556:Fatty Acid-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects; D006710:Homocysteine / Q000378:metabolism* / Q000494:pharmacology; D006801:Humans; D008264:Macrophages / Q000187:drug effects / Q000378:metabolism*; D009000:Monocytes / Q000378:metabolism; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism
chemical_list: C493410:FABP4 protein, human; D050556:Fatty Acid-Binding Proteins; D012333:RNA, Messenger; D006710:Homocysteine; D002784:Cholesterol