RNA N6-methyladenosine modulates endothelial atherogenic responses to disturbed flow in mice

Atherosclerosis preferentially occurs in atheroprone vasculature where human umbilical vein endothelial cells (HUVECs) are exposed to disturbed flow. Disturbed flow is associated with vascular inflammation and focal distribution. Recent studies have revealed the involvement of epigenetic regulation in atherosclerosis progression. N6-methyladenosine (m6A) is the most prevalent internal modification of eukaryotic mRNA, but its function in endothelial atherogenic progression remains unclear. Here, we show that m6A mediates the EGFR signaling pathway during EC activation to regulate the atherosclerotic process. Oscillatory stress (OS) reduced the expression of METTL3, the primary m6A methyltransferase. Through m6A sequencing and functional studies, we determined that m6A mediates the mRNA decay of the vascular pathophysiology gene EGFR which leads to EC dysfunction. m6A modification of the EGFR 3'UTR accelerated its mRNA degradation. Double mutation of the EGFR 3'UTR abolished METTL3-induced luciferase activity. Adenovirus-mediated METTL3 overexpression significantly reduced EGFR activation and endothelial dysfunction in the presence of OS. Furthermore, TSP-1, an EGFR ligand, was specifically expressed in atheroprone regions without being affected by METTL3. Inhibition of the TSP-1/EGFR axis by using shRNA and AG1478 significantly ameliorated atherogenesis. Overall, our study revealed that METTL3 alleviates endothelial atherogenic progression through m6A-dependent stabilization of EGFR mRNA, highlighting the important role of RNA transcriptomics in atherosclerosis regulation.

Phage Display Preparation of Specific Polypeptides in Atherosclerotic Foam Cells

Atherosclerosis and related complications are the most common causes of death in modern societies. Macrophage-derived foam cells play critical roles in the initiation and progression of atherosclerosis. Effective, rapid, and instrument-independent detection in the early stage of chronic atherosclerosis progression could provide an opportunity for early intervention and treatment. Therefore, as a starting point, in this study, we aimed to isolate and prepare foam cell-specific polypeptides using a phage display platform. The six target polypeptides, which were acquired in this study, were evaluated by ELISA and showed strong specificity with foam cells. Streptavidin coupled quantum dots (QDs) were used as fluorescence developing agents, and images of biotin-modified polypeptides specifically binding with foam cells were clearly observed. The polypeptides obtained in this study could lay the foundation for developing a rapid detection kit for early atherosclerosis lesions and could provide new materials for research on the mechanisms of foam cell formation and the development of blocking drugs.

The Nutritional Supplement L-Alpha Glycerylphosphorylcholine Promotes Atherosclerosis

L-alpha glycerylphosphorylcholine (GPC), a nutritional supplement, has been demonstrated to improve neurological function. However, a new study suggests that GPC supplementation increases incident stroke risk thus its potential adverse effects warrant further investigation. Here we show that GPC promotes atherosclerosis in hyperlipidemic Apoe−/− mice. GPC can be metabolized to trimethylamine N-oxide, a pro-atherogenic agent, suggesting a potential molecular mechanism underlying the observed atherosclerosis progression. GPC supplementation shifted the gut microbial community structure, characterized by increased abundance of Parabacteroides, Ruminococcus, and Bacteroides and decreased abundance of Akkermansia, Lactobacillus, and Roseburia, as determined by 16S rRNA gene sequencing. These data are consistent with a reduction in fecal and cecal short chain fatty acids in GPC-fed mice. Additionally, we found that GPC supplementation led to an increased relative abundance of choline trimethylamine lyase (cutC)-encoding bacteria via qPCR. Interrogation of host inflammatory signaling showed that GPC supplementation increased expression of the proinflammatory effectors CXCL13 and TIMP-1 and activated NF-κB and MAPK signaling pathways in human coronary artery endothelial cells. Finally, targeted and untargeted metabolomic analysis of murine plasma revealed additional metabolites associated with GPC supplementation and atherosclerosis. In summary, our results show GPC promotes atherosclerosis through multiple mechanisms and that caution should be applied when using GPC as a nutritional supplement.

If Mediterranean Diet Could Slow Atherosclerosis Progression, Could Specific Herbal Supplement Achieve the Same?

Dietary Control has been emphasized as an important means to prevent the development of atherosclerosis. It is a form belief without scientific evidence until the early report of the Study on Coronary Diet Intervention with olive oil and Cardiovascular Prevention (CODIOPREV Study on Mediterranean Diet). The current study is using Surrogate market IMT as the objective indicator, to compare the CORDIOPREV volunteers with known cardiovascular disease, which have demonstrated objective reduction in IMT thickness after 5 to 7 years of Mediterranean diet consumption. A study done in Hong Kong since 2003 using a simple twin herb formula for the prevention of progress of atherosclerosis in 4 different groups of patients with cardiovascular diseases, and using the same surrogate marker IMT as the objective indicator, showed comparable results within a much shorter period of 6-12 months. Extensive laboratory tests have also been completed to verify the bioactivities of the twin herb formula on anti-inflammation, anti-oxidation, endothelial protection and regenerative capacity related to atherosclerosis. It is suggested that before more potent therapeutic measures become available to slow atherosclerosis progression are available. The purpose of the paper is to compare the Mediterranean Diet for long term consumption and selective herbal preparations for shorter term or intermittent scheduled intake, and recommend to high-risk individuals.

The Differential Effects of HDL Subpopulations on Lipoprotein Lipase (LPL)-Mediated VLDL Catabolism

High-density lipoprotein (HDL) subpopulations functional assessment is more relevant for HDL anti-atherogenic activity than cholesterol level. The aim of the study was to assess the impact of HDL-2 and HDL-3 on lipoprotein lipase (LPL)-mediated very-low-density lipoprotein (VLDL) catabolism related to hypertriglyceridemia development. VLDL and HDLs were isolated from serum by ultracentrifugation. VLDL was incubated with LPL in the absence and presence of total HDL or HDL subpopulations. Next, VLDL remnants were separated, and their composition and electrophoretic mobility was assessed. Both HDL subpopulations increased the efficiency of triglyceride lipolysis and apolipoprotein CII and CIII removal from VLDL up to ~90%. HDL-3 exerted significantly greater impact than HDL-2 on apolipoprotein E (43% vs. 18%, p < 0.001), free cholesterol (26% vs. 18%, p < 0.05) and phospholipids (53% vs. 43%, p < 0.05) removal from VLDL and VLDL remnant electrophoretic mobility (0.18 vs. 0.20, p < 0.01). A greater release of these components was also observed in the presence of total HDL with a low HDL-2/HDL-3 cholesterol ratio. Both HDL subpopulations affect VLDL composition during lipolysis, but HDL-3 exhibited a greater effect on this process. Altered composition of HDL related to significant changes in the distribution between HDL-2 and HDL-3 can influence the VLDL remnant features, affecting atherosclerosis progression.

PET-Based Imaging with 18F-FDG and 18F-NaF to Assess Inflammation and Microcalcification in Atherosclerosis and Other Vascular and Thrombotic Disorders

Positron emission tomography (PET) imaging with 18F-fluorodeoxyglucose (FDG) represents a method of detecting and characterizing arterial wall inflammation, with potential applications in the early assessment of vascular disorders such as atherosclerosis. By portraying early-stage molecular changes, FDG-PET findings have previously been shown to correlate with atherosclerosis progression. In addition, recent studies have suggested that microcalcification revealed by 18F-sodium fluoride (NaF) may be more sensitive at detecting atherogenic changes compared to FDG-PET. In this review, we summarize the roles of FDG and NaF in the assessment of atherosclerosis and discuss the role of global assessment in quantification of the vascular disease burden. Furthermore, we will review the emerging applications of FDG-PET in various vascular disorders, including pulmonary embolism, as well as inflammatory and infectious vascular diseases.

Stimulation of the PD-1 Pathway Decreases Atherosclerotic Lesion Development in Ldlr Deficient Mice

Aim: Signaling through the coinhibitory programmed death (PD)-1/PD-L1 pathway regulates T cell responses and can inhibit ongoing immune responses. Inflammation is a key process in the development of atherosclerosis, the underlying cause for the majority of cardiovascular diseases. Dampening the excessive immune response that occurs during atherosclerosis progression by promoting PD-1/PD-L1 signaling may have a high therapeutic potential to limit disease burden. In this study we therefore aimed to assess whether an agonistic PD-1 antibody can diminish atherosclerosis development.Methods and Results: Ldlr−/− mice were fed a western-type diet (WTD) while receiving 100 μg of an agonistic PD-1 antibody or control vehicle twice a week. Stimulation of the PD-1 pathway delayed the WTD-induced monocyte increase in the circulation up to 3 weeks and reduced T cell activation and proliferation. CD4+ T cell numbers in the atherosclerotic plaque were reduced upon PD-1 treatment. More specifically, we observed a 23% decrease in atherogenic IFNγ-producing splenic CD4+ T cells and a 20% decrease in cytotoxic CD8+ T cells, whereas atheroprotective IL-10 producing CD4+ T cells were increased with 47%. Furthermore, we found an increase in regulatory B cells, B1 cells and associated atheroprotective circulating oxLDL-specific IgM levels in agonistic PD-1-treated mice. This dampened immune activation following agonistic PD-1 treatment resulted in reduced atherosclerosis development (p &lt; 0.05).Conclusions: Our data show that stimulation of the coinhibitory PD-1 pathway inhibits atherosclerosis development by modulation of T- and B cell responses. These data support stimulation of coinhibitory pathways as a potential therapeutic strategy to combat atherosclerosis.