Local M-CSF (Macrophage Colony-Stimulating Factor) Expression Regulates Macrophage Proliferation and Apoptosis in Atherosclerosis.

OBJECTIVE: Previous studies have shown that deficiency of M-CSF (macrophage colony-stimulating factor; or CSF1 [colony stimulating factor 1]) dramatically reduces atherosclerosis in hyperlipidemic mice. We characterize the underlying mechanism and investigate the relevant sources of CSF1 in lesions. Approach and Results: We quantitatively assessed the effects of CSF1 deficiency on macrophage proliferation and apoptosis in atherosclerotic lesions. Staining of aortic lesions with markers of proliferation, Ki-67 and bromodeoxyuridine, revealed around 40% reduction in CSF1 heterozygous (Csf1+/-) as compared with WT (wild type; Csf1+/+) mice. Similarly, staining with a marker of apoptosis, activated caspase-3, revealed a 3-fold increase in apoptotic cells in Csf1+/- mice. Next, we determined the cellular sources of CSF1 contributing to lesion development. Cell-specific deletions of Csf1 in smooth muscle cells using SM22α-Cre (smooth muscle protein 22-alpha-Cre) reduced lesions by about 40%, and in endothelial cells, deletions with Cdh5-Cre (VE-cadherin-Cre) reduced lesions by about 30%. Macrophage-specific deletion with LysM-Cre (lysozyme M-Cre), on the other hand, did not significantly reduce lesions size. Transplantation of Csf1 null (Csf1-/-) mice bone marrow into Csf1+/+ mice reduced lesions by about 35%, suggesting that CSF1 from hematopoietic cells other than macrophages contributes to atherosclerosis. None of the cell-specific knockouts affected circulating CSF1 levels, and only the smooth muscle cell deletions had any effect on the percentage monocytes in the circulation. Also, Csf1+/- mice did not exhibit significant differences in Ly6Chigh/Ly6Clow monocytes as compared with Csf1+/+. CONCLUSIONS: CSF1 contributes to both macrophage proliferation and survival in lesions. Local CSF1 production by smooth muscle cell and endothelial cell rather than circulating CSF1 is the primary driver of macrophage expansion in atherosclerosis.

Cell-specific discrimination of desmosterol and desmosterol mimetics confers selective regulation of LXR and SREBP in macrophages.

Activation of liver X receptors (LXRs) with synthetic agonists promotes reverse cholesterol transport and protects against atherosclerosis in mouse models. Most synthetic LXR agonists also cause marked hypertriglyceridemia by inducing the expression of sterol regulatory element-binding protein (SREBP)1c and downstream genes that drive fatty acid biosynthesis. Recent studies demonstrated that desmosterol, an intermediate in the cholesterol biosynthetic pathway that suppresses SREBP processing by binding to SCAP, also binds and activates LXRs and is the most abundant LXR ligand in macrophage foam cells. Here we explore the potential of increasing endogenous desmosterol production or mimicking its activity as a means of inducing LXR activity while simultaneously suppressing SREBP1c-induced hypertriglyceridemia. Unexpectedly, while desmosterol strongly activated LXR target genes and suppressed SREBP pathways in mouse and human macrophages, it had almost no activity in mouse or human hepatocytes in vitro. We further demonstrate that sterol-based selective modulators of LXRs have biochemical and transcriptional properties predicted of desmosterol mimetics and selectively regulate LXR function in macrophages in vitro and in vivo. These studies thereby reveal cell-specific discrimination of endogenous and synthetic regulators of LXRs and SREBPs, providing a molecular basis for dissociation of LXR functions in macrophages from those in the liver that lead to hypertriglyceridemia.

Epigenome-wide association in adipose tissue from the METSIM cohort.

Most epigenome-wide association studies to date have been conducted in blood. However, metabolic syndrome is mediated by a dysregulation of adiposity and therefore it is critical to study adipose tissue in order to understand the effects of this syndrome on epigenomes. To determine if natural variation in DNA methylation was associated with metabolic syndrome traits, we profiled global methylation levels in subcutaneous abdominal adipose tissue. We measured association between 32 clinical traits related to diabetes and obesity in 201 people from the Metabolic Syndrome in Men cohort. We performed epigenome-wide association studies between DNA methylation levels and traits, and identified associations for 13 clinical traits in 21 loci. We prioritized candidate genes in these loci using expression quantitative trait loci, and identified 18 high confidence candidate genes, including known and novel genes associated with diabetes and obesity traits. Using methylation deconvolution, we examined which cell types may be mediating the associations, and concluded that most of the loci we identified were specific to adipocytes. We determined whether the abundance of cell types varies with metabolic traits, and found that macrophages increased in abundance with the severity of metabolic syndrome traits. Finally, we developed a DNA methylation-based biomarker to assess type 2 diabetes risk in adipose tissue. In conclusion, our results demonstrate that profiling DNA methylation in adipose tissue is a powerful tool for understanding the molecular effects of metabolic syndrome on adipose tissue, and can be used in conjunction with traditional genetic analyses to further characterize this disorder.

DNA Methylation Changes More Slowly Than Physiological States in Response to Weight Loss in Genetically Diverse Mouse Strains.

Responses to a high fat, high sucrose (HFHS) diet vary greatly among inbred strains of mice. We sought to examine the epigenetic (DNA methylation) changes underlying these differences as well as variation in weight loss when switched to a low-fat chow diet. We surveyed DNA methylation from livers of 45 inbred mouse strains fed a HFHS diet for 8 weeks using reduced-representation bisulfite sequencing (RRBS). We observed a total of 1,045,665 CpGs of which 83 candidate sites were significantly associated with HFHS diet. Many of these CpGs correlated strongly with gene expression or clinical traits such as body fat percentage and plasma glucose. Five inbred strains were then studied in the context of weight loss to test for evidence of epigenetic "memory." The mice were first fed a HFHS diet for 6 weeks followed by a low-fat chow diet for 4 weeks. Four of the five strains returned to initial levels of body fat while one strain, A/J, retained almost 50% of the fat gained. A total of 36 of the HFHS diet responsive CpGs exhibited evidence of persistent epigenetic modifications following weight normalization, including CpGs near the genes Scd1 and Cdk1. Our study identifies DNA methylation changes in response to a HFHS diet challenge that revert more slowly than overall body fat percentage in weight loss and provides evidence for epigenetic mediated "memory."

SREBP1 Contributes to Resolution of Pro-inflammatory TLR4 Signaling by Reprogramming Fatty Acid Metabolism.

Macrophages play pivotal roles in both the induction and resolution phases of inflammatory processes. Macrophages have been shown to synthesize anti-inflammatory fatty acids in an LXR-dependent manner, but whether the production of these species contributes to the resolution phase of inflammatory responses has not been established. Here, we identify a biphasic program of gene expression that drives production of anti-inflammatory fatty acids 12-24 hr following TLR4 activation and contributes to downregulation of mRNAs encoding pro-inflammatory mediators. Unexpectedly, rather than requiring LXRs, this late program of anti-inflammatory fatty acid biosynthesis is dependent on SREBP1 and results in the uncoupling of NFκB binding from gene activation. In contrast to previously identified roles of SREBP1 in promoting production of IL1ß during the induction phase of inflammation, these studies provide evidence that SREBP1 also contributes to the resolution phase of TLR4-induced gene activation by reprogramming macrophage lipid metabolism.