Inhibiting DNA Methylation by 5-Aza-2′-deoxycytidine Ameliorates Atherosclerosis Through Suppressing Macrophage Inflammation

Abstract

Inflammation marks all stages of atherogenesis. DNA hypermethylation in the whole genome or specific genes is associated with inflammation and cardiovascular diseases. Therefore, we aimed to study whether inhibiting DNA methylation by DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine (5-aza-dC) ameliorates atherosclerosis in low-density lipoprotein receptor knockout (Ldlr(-/-)) mice. Ldlr(-/-) mice were fed an atherogenic diet and adminisered saline or 5-aza-dC (0.25 mg/kg) for up to 30 weeks. 5-aza-dC treatment markedly decreased atherosclerosis development in Ldlr(-/-) mice without changes in body weight, plasma lipid profile, macrophage cholesterol levels and plaque lipid content. Instead, this effect was associated with decreased macrophage inflammation. Macrophages with 5-aza-dC treatment had downregulated expression of genes involved in inflammation (TNF-α, IL-6, IL-1β, and inducible nitric oxidase) and chemotaxis (CD62/L-selectin, chemokine [C-C motif] ligand 2/MCP-1 [CCL2/MCP-1], CCL5, CCL9, and CCL2 receptor CCR2). This resulted in attenuated macrophage migration and adhesion to endothelial cells and reduced macrophage infiltration into atherosclerotic plaques. 5-aza-dC also suppressed macrophage endoplasmic reticulum stress, a key upstream signal that activates macrophage inflammation and apoptotic pathways. Finally, 5-aza-dC demethylated liver X receptor α (LXRα) and peroxisome proliferator-activated receptor γ1 (PPARγ1) promoters, which are both enriched with CpG sites. This led to overexpression of LXRα and PPARγ, which may be responsible for 5-aza-dC's anti-inflammatory and atheroprotective effect. Our findings provide strong evidence that DNA methylation may play a significant role in cardiovascular diseases and serve as a therapeutic target for prevention and treatment of atherosclerosis.

The Histone Demethylase UTX Promotes Brown Adipocyte Thermogenic Program Via Coordinated Regulation of H3K27 Demethylation and Acetylation

“…The fluorescence intensity was detected using a Perkin Elmer VICTOR 3 plate reader (Perkin Elmer, Waltham, MA) with excitation 545 nm and emission 575 nm. For TG content, lipid extraction from BAT1 brown adipocytes was conducted as we previously described (33). The lipid extraction was dissolved in 2% Triton X-100 and used for triglyceride quantification using a TG kit/L-Type TG M (Wako Chemicals, Wako, Richmond, VA).…”

A therapeutic T cell receptor mimic antibody targets tumor-associated PRAME peptide/HLA-I antigens

“…PRAME protein expression alone is not sufficient for Pr20 binding, but IFN-γ can enhance Pr20 binding in PRAME + solid tumors and enhance ADCC. Interestingly, neither PRAME mRNA levels nor PRAME protein levels correlate with Pr20 cell-surface 51 Cr-labeled target leukemia or lymphoma cells were incubated with healthy donor PBMCs at an effector/target ratio of 50:1. Pr20M or an afucosylated isotype control Ab was added at the indicated concentration.…”

“…Data represent 3 independent experiments. (B) Melanomas were pretreated with 10 ng/ml IFN-γ for 72 hours or left untreated before 51 Cr-ADCC assay was used to determine specific lysis by Pr20M. Samples were assayed in 3 technical replicates, and data are representative of 3 experiments per cell line.…”

“…It would be important to understand whether these agents could enhance antigen expression and synergize with TCRm therapy. However, these epigenetic drugs can also have context-dependent effects on immune cell function and therefore must be evaluated carefully to ensure they do not also inhibit the effector cells required for TCRm-mediated cytotoxicity (49,51).…”

“…Cancer cell lines used as ADCC target cells were incubated with 50 μCi of 51 Cr for 1 hour at 37°C and washed 3 times to remove free 51 Cr. Indicated concentrations of Pr20M or matched isotype control hIgG1 (afucosylated Eureka Therapeutics ET901) were incubated with target cells and fresh PBMCs at effector/target ratios of 50:1 for 6 hours at 37°C.…”