The comprehensive effects of hyperlipidemia and hyperhomocysteinemia on pathogenesis of atherosclerosis and DNA hypomethylation in ApoE<sup>−/−</sup> mice

Abstract

Atherosclerosis (AS) is a disease induced by multiple factors, including genetic and environmental elements. The aim of the present study is to investigate the comprehensive effects of high cholesterol, high methionine diet, and apolipoprotein E deficiency (ApoE(-/-)) on the pathogenesis of AS. ApoE(-/-) mice were fed with high cholesterol and methionine diet for 15 weeks to induce hyperlipidemia and hyperhomocysteinemia. The methylation levels of genomic DNA (gDNA) and B1 repetitive elements in aortic tissues were measured by both methylation-dependent restriction analysis and nested methylation-specific polymerase chain reaction (PCR). Methylation sequence-bias pattern was assayed by DNA methyl-accepting capacity with restriction endonuclease digestion. The mRNA expression of DNA methyltransferase-1, 3 (DNMT1, 3) was detected by real-time PCR. The concentrations of S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH) were determined by high-performance liquid chromatography. The results showed hypomethylation of gDNA and B1 repetitive elements. The mRNA expression of DNMT1 was reduced. The levels of SAM, SAH, and SAM/SAH ratio were increased. The atherosclerotic lesion areas strongly correlated with the risk factors. The distribution of DNA demethylation was preferred to non-CpG islands, which may suggest the major impact of hypomethylation on DNA integrity and genomic instability. Overall, our data unequivocally showed that the comprehensive role of high cholesterol, high methionine diet, and ApoE(-/-) is not uniformly consistent with the role of a single risk factor. The DNA methylation pattern in AS is quite complex and depends on genetic background and many involved risk factors.

Ratio of S-adenosylmethionine to S-adenosylhomocysteine as a sensitive indicator of atherosclerosis

“…In the present study, it was found that the methylation levels of pivotal AS-associated genes were altered. B1 repetitive elements are the sequences of short interspersed nucleotide elements in mice, corresponding to the Alu sequence in humans (19). Increasing evidence suggests that B1 repetitive element hypomethylation is associated with gene transcriptional activity in the pathogenesis of cardiovascular diseases, particularly AS (43).…”

“…However, the area of the atherosclerotic lesions was decreased by 39.4% in the Meth-F group, compared with the Meth group, (P<0.05). The atherosclerotic lesions in the Meth group were the most severe and widespread, which suggested HHcy may have had an effect in accelerating the development of AS, whereas folate and vitamin B 12 modulated the effect of HHcy on the formation of atherosclerotic plaques (19,20).…”

“…Liu et al (18) found that MCP-1 is a potent chemokine and important in AS, in addition to reporting that the hypomethylation of CpG sites in the MCP-1 promoter region may be important in the vascular complications of type 2 diabetes. In our previous studies, it was demonstrated that B1 repetitive element hypomethylation is involved in the pathogenesis of AS (19), in which EC-SOD, FABP4, MCP-1 and B1 repetitive elements were analyzed to determine their DNA methylation levels to confirm the association between the methylation status of these genes and the development of AS.…”

“…To quantify plaques by stereological methods, ~10 evenly spaced sections of each aortic segment were sampled. The blocks were exhaustively sectioned at 10 µM and stained with hematoxylin and eosin (H&E; Boster Biological Technology, Ltd., Wuhan, China) (19,20) as follows: The aortas were separated carefully and then put into 4% (w/v) formalin for 24 h. The samples subsequently underwent washing with water, dehydration with a Citadel tissue processor (Thermo Fisher Scientific, Inc.), clearing of surplus liquid from tissues, embedding using a Histostar tissue embed-ding system (Thermo Fisher Scientific, Inc.), coating with paraffin wax, slicing (thickness, 4 µM) on a HM340E microtome (Thermo Fisher Scientific, Inc.) and H&E staining. The sampled sections of the aorta were projected onto a table top at 40x magnification using a BX50F4 microscope (Olympus Corporation, Tokyo, Japan).…”

FABP4-mediated homocysteine-induced cholesterol accumulation in THP-1 monocyte-derived macrophages and the potential epigenetic mechanism

“…The RT-qPCR reaction was performed using an FTC-3000 real-time PCR detection system (Funglyn, Toronto, Canada), according to the manufacturer's protocol, as follows: 94˚C for 10 min, 50 cycles at 94˚C for 15 sec, at the annealing temperature of 53˚C for 30 sec and 72˚C for 30 sec. The RNA level of the gene was acquired from the value of the quantification cycle (Cq) of the RT-qPCR associated with that of GAPDH using the following formula: ΔCq, ΔCq = Cq GAPDH -Cq gene (15). The final results were expressed as N-fold differences in the target gene expression, relative to the calibrator, termed 'N target ', which was determined as follows: N target = 2 ΔCqsample -ΔCqcalibrator , where the ΔCq values of the calibrator and sample were determined by subtracting the Cq value of the target gene from the Cq value of GAPDH, as previously described (11).…”