TRAK2, a novel regulator of ABCA1 expression, cholesterol efflux and HDL biogenesis.

Aims: The recent failures of HDL-raising therapies have underscored our incomplete understanding of HDL biology. Therefore there is an urgent need to comprehensively investigate HDL metabolism to enable the development of effective HDL-centric therapies. To identify novel regulators of HDL metabolism, we performed a joint analysis of human genetic, transcriptomic, and plasma HDL-cholesterol (HDL-C) concentration data and identified a novel association between trafficking protein, kinesin binding 2 (TRAK2) and HDL-C concentration. Here we characterize the molecular basis of the novel association between TRAK2 and HDL-cholesterol concentration. Methods and results: Analysis of lymphocyte transcriptomic data together with plasma HDL from the San Antonio Family Heart Study (n = 1240) revealed a significant negative correlation between TRAK2 mRNA levels and HDL-C concentration, HDL particle diameter and HDL subspecies heterogeneity. TRAK2 siRNA-mediated knockdown significantly increased cholesterol efflux to apolipoprotein A-I and isolated HDL from human macrophage (THP-1) and liver (HepG2) cells by increasing the mRNA and protein expression of the cholesterol transporter ATP-binding cassette, sub-family A member 1 (ABCA1). The effect of TRAK2 knockdown on cholesterol efflux was abolished in the absence of ABCA1, indicating that TRAK2 functions in an ABCA1-dependent efflux pathway. TRAK2 knockdown significantly increased liver X receptor (LXR) binding at the ABCA1 promoter, establishing TRAK2 as a regulator of LXR-mediated transcription of ABCA1. Conclusion: We show, for the first time, that TRAK2 is a novel regulator of LXR-mediated ABCA1 expression, cholesterol efflux, and HDL biogenesis. TRAK2 may therefore be an important target in the development of anti-atherosclerotic therapies.

Methylomic and transcriptomic characterization of postoperative systemic inflammatory dysregulation.

In this study, we define and validate a state of postoperative systemic inflammatory dysregulation (PSID) based on postoperative phenotypic extremes of plasma C-reactive protein concentration following major abdominal surgery. PSID manifested clinically with significantly higher rates of sepsis, complications, longer hospital stays and poorer short, and long-term outcomes. We hypothesized that PSID will be associated with, and potentially predicted by, altered patterns of genome-wide peripheral blood mononuclear cell differential DNA methylation and gene expression. We identified altered DNA methylation and differential gene expression in specific immune and metabolic pathways during PSID. Our findings suggest that dysregulation results in, or from, dramatic changes in differential DNA methylation and highlights potential targets for early detection and treatment. The combination of altered DNA methylation and gene expression suggests that dysregulation is mediated at multiple levels within specific gene sets and hence, nonspecific anti-inflammatory treatments such as corticosteroids alone are unlikely to represent an effective therapeutic strategy.

Vitamin D3 replacement enhances antigen-specific immunity in older adults.

Introduction: Ageing is associated with increased number of infections, decreased vaccine efficacy and increased systemic inflammation termed inflammageing. These changes are reflected by reduced recall responses to varicella zoster virus (VZV) challenge in the skin of older adults. Vitamin D deficiency is more common in the old and has been associated with frailty and increased inflammation. In addition, vitamin D increases immunoregulatory mechanisms and therefore has the potential to inhibit inflammageing. Objectives: We investigated the use of vitamin D3 replacement to enhance cutaneous antigen-specific immunity in older adults (≥65 years). Methods: Vitamin D insufficient older adults (n = 18) were administered 6400IU of vitamin D3/day orally for 14 weeks. Antigen-specific immunity to VZV was assessed by clinical score assessment of the injection site and transcriptional analysis of skin biopsies collected from challenged injection sites pre- and post-vitamin D3 replacement. Results: We showed that older adults had reduced VZV-specific cutaneous immune response and increased non-specific inflammation as compared to young. Increased non-specific inflammation observed in the skin of older adults negatively correlated with vitamin D sufficiency. We showed that vitamin D3 supplementation significantly increased the response to cutaneous VZV antigen challenge in older adults. This enhancement was associated with a reduction in inflammatory monocyte infiltration with a concomitant enhancement of T cell recruitment to the site of antigen challenge in the skin. Conclusion: Vitamin D3 replacement can boost antigen-specific immunity in older adults with sub-optimal vitamin D status.

Recruitment of inflammatory monocytes by senescent fibroblasts inhibits antigen-specific tissue immunity during human aging.

We have previously shown that healthy older adults exhibit reduced cutaneous immune responses during a varicella zoster virus (VZV) antigen challenge that correlated with a nonspecific inflammatory response to the injection itself. Here we found that needle damage during intradermal injections in older adults led to an increase in the number of cutaneous senescent fibroblasts expressing CCL2, resulting in the local recruitment of inflammatory monocytes. These infiltrating monocytes secreted prostaglandin E2, which inhibited resident memory T cell activation and proliferation. Pretreatment of older participants with a p38 mitogen-activated protein kinase inhibitor in vivo decreased CCL2 expression and inhibited monocyte recruitment and secretion of prostaglandin E2. This coincided with an increased response to VZV antigen challenge in the skin. Our results point to a series of molecular and cellular mechanisms that link cellular senescence, tissue damage, excessive inflammation and reduced immune responsiveness in human skin and demonstrate that tissue-specific immunity can be restored in older adults by short-term inhibition of inflammatory responses.

DNA methylation patterns from peripheral blood separate coronary artery disease patients with and without heart failure.

AIMS: Natriuretic peptides are useful for diagnosis and prognostication of heart failure of any cause. Now, research aims to discover novel biomarkers that will more specifically define the heart failure phenotype. DNA methylation plays a critical role in the development of cardiovascular disease with the potential to predict fundamental pathogenic processes. There is a lack of data relating DNA methylation in heart failure that specifically focuses on patients with severe multi-vessel coronary artery disease. To begin to address this, we conducted a pilot study uniquely exploring the utility of powerful whole-genome methyl-binding domain-capture sequencing in a cohort of cardiac surgery patients, matched for the severity of their coronary artery disease, aiming to identify candidate peripheral blood DNA methylation markers of ischaemic cardiomyopathy and heart failure. METHODS AND RESULTS: We recruited a cohort of 20 male patients presenting for coronary artery bypass graft surgery with phenotypic extremes of heart failure but who otherwise share a similar coronary ischaemic burden, age, sex, and ethnicity. Methylation profiling in patient blood samples was performed using methyl-binding domain-capture sequencing. Differentially methylated regions were validated using targeted bisulfite sequencing. Gene set enrichment analysis was performed to identify differences in methylation at or near gene promoters in certain known Reactome pathways. We detected 567 188 methylation peaks of which our general linear model identified 68 significantly differentially methylated regions in heart failure with a false discovery rate <0.05. Of these regions, 48 occurred within gene bodies and 25 were located near enhancer elements, some within coding genes and some in non-coding genes. Gene set enrichment analyses identified 103 significantly enriched gene sets (false discovery rate <0.05) in heart failure. Validation analysis of regions with the strongest differential methylation data was performed for two genes: HDAC9 and the uncharacterized miRNA gene MIR3675. Genes of particular interest as novel candidate markers of the heart failure phenotype with reduced methylation were HDAC9, JARID2, and GREM1 and with increased methylation PDSS2. CONCLUSIONS: We demonstrate the utility of methyl-binding domain-capture sequencing to evaluate peripheral blood DNA methylation markers in a cohort of cardiac surgical patients with severe multi-vessel coronary artery disease and phenotypic extremes of heart failure. The differential methylation status of specific coding genes identified are candidates for larger longitudinal studies. We have further demonstrated the value and feasibility of examining DNA methylation during the perioperative period to highlight biological pathways and processes contributing to complex phenotypes.