Faculty Bibliography

The human genome encodes thousands of long non-coding RNAs (lncRNAs), the majority of which are poorly conserved and uncharacterized. Here we identify a primate-specific lncRNA (CHROME), elevated in the plasma and atherosclerotic plaques of individuals with coronary artery disease, that regulates cellular and systemic cholesterol homeostasis. LncRNA CHROME expression is influenced by dietary and cellular cholesterol via the sterol-activated liver X receptor transcription factors, which control genes mediating responses to cholesterol overload. Using gain- and loss-of-function approaches, we show that CHROME promotes cholesterol efflux and HDL biogenesis by curbing the actions of a set of functionally related microRNAs that repress genes in those pathways. CHROME knockdown in human hepatocytes and macrophages increases levels of miR-27b, miR-33a, miR-33b and miR-128, thereby reducing expression of their overlapping target gene networks and associated biologic functions. In particular, cells lacking CHROME show reduced expression of ABCA1, which regulates cholesterol efflux and nascent HDL particle formation. Collectively, our findings identify CHROME as a central component of the non-coding RNA circuitry controlling cholesterol homeostasis in humans.

Macrophages accumulate prominently in the visceral adipose tissue (VAT) of obese humans and high fat diet (HFD) fed mice, and this is linked to insulin resistance and type II diabetes. While the mechanisms regulating macrophage recruitment in obesity have been delineated, the signals directing macrophage persistence in VAT are poorly understood. We previously showed that the neuroimmune guidance cue netrin-1 is expressed in the VAT of obese mice and humans, where it promotes macrophage accumulation. To better understand the source of netrin-1 and its effects on adipose tissue macrophage (ATM) fate and function in obesity, we generated mice with myeloid-specific deletion of netrin-1 (Ntn1^fl/flLysMCre^+/-; Ntn1^Δmac). Interestingly, Ntn1^Δmac mice showed a modest decrease in HFD-induced adiposity and adipocyte size, in the absence of changes in food intake or leptin, that was accompanied by an increase in markers of adipocyte beiging (Prdm16, UCP-1). Using single cell RNA-seq, combined with conventional histological and flow cytometry techniques, we show that myeloid-specific deletion of netrin-1 caused a 50% attrition of ATMs in HFD-fed mice, particularly of the resident macrophage subset, and altered the phenotype of residual ATMs to enhance lipid handling. Pseudotime analysis of single cell transcriptomes showed that in the absence of netrin-1, macrophages in the obese VAT underwent a phenotypic switch with the majority of ATMs activating a program of genes specialized in lipid handling, including fatty acid uptake and intracellular transport, lipid droplet formation and lipolysis, and regulation of lipid localization. Furthermore, Ntn1^Δmac macrophages had reduced expression of genes involved in arachidonic acid metabolism, and targeted LCMS/MS metabololipidomics analysis revealed decreases in proinflammatory eicosanoids (5-HETE, 6-trans LTB₄, TXB₂, PGD₂) in the obese VAT. Collectively, our data show that targeted deletion of netrin-1 in macrophages reprograms the ATM phenotype in obesity, leading to reduced adipose inflammation, and improved lipid handling and metabolic function.

Obesity can lead to type 2 diabetes and is an epidemic. A major contributor to its adverse effects is inflammation of the visceral adipose tissue (VAT). Life-long caloric restriction (CR), in contrast, results in extended lifespan, enhanced glucose tolerance/insulin sensitivity, and other favorable phenotypes. The effects of CR following obesity are incompletely established, but studies show multiple benefits. Many leukocyte types, macrophages predominantly, reside in VAT in homeostatic and pathological states. CR following obesity transiently increases VAT macrophage content prior to resolution of inflammation and obesity, suggesting that macrophage content and phenotype play critical roles. Here, we examined the heterogeneity of VAT leukocytes and the effects of obesity and CR. In general, our single-cell RNA-sequencing data demonstrate that macrophages are the most abundant and diverse subpopulation of leukocytes in VAT. Obesity induced significant transcriptional changes in all 15 leukocyte subpopulations, with many genes showing coordinated changes in expression across the leukocyte subpopulations. Additionally, obese VAT displayed expansion of one major macrophage subpopulation, which, in silico, was enriched in lipid binding and metabolic processes. This subpopulation returned from dominance in obesity to lean proportions after only 2 weeks of CR, although the pattern of gene expression overall remained similar. Surprisingly, CR VAT is dominated by a different macrophage subpopulation, which is absent in lean conditions. This subpopulation is enriched in genes related to phagocytosis and we postulate that its function includes clearance of dead cells, as well as excess lipids, contributing to limiting VAT inflammation and restoration of the homeostatic state.

Abdominal aortic aneurysms (AAA) are characterized by extensive extracellular matrix (ECM) fragmentation and inflammation. However, the mechanisms by which these events are coupled thereby fueling focal vascular damage are undefined. Here we report through single-cell RNA-sequencing of diseased aorta that the neuronal guidance cue netrin-1 can act at the interface of macrophage-driven injury and ECM degradation. Netrin-1 expression peaks in human and murine aneurysmal macrophages. Targeted deletion of netrin-1 in macrophages protects mice from developing AAA. Through its receptor neogenin-1, netrin-1 induces a robust intracellular calcium flux necessary for the transcriptional regulation and persistent catalytic activation of matrix metalloproteinase-3 (MMP3) by vascular smooth muscle cells. Deficiency in MMP3 reduces ECM damage and the susceptibility of mice to develop AAA. Here, we establish netrin-1 as a major signal that mediates the dynamic crosstalk between inflammation and chronic erosion of the ECM in AAA.

Atherosclerosis is characterized by the retention of modified lipoproteins in the arterial wall. These modified lipoproteins activate resident macrophages and the recruitment of monocyte-derived cells, which differentiate into mononuclear phagocytes that ingest the deposited lipoproteins to become "foam cells": a hallmark of this disease. In this Part 2 of a 4-part review series covering the macrophage in cardiovascular disease, we critically review the contributions and relevant pathobiology of monocytes, macrophages, and foam cells as relevant to atherosclerosis. We also review evidence that via various pathways, a failure of the resolution of inflammation is an additional key aspect of this disease process. Finally, we consider the likely role played by genomics and biological networks in controlling the macrophage phenotype in atherosclerosis. Collectively, these data provide substantial insights on the atherosclerotic process, while concurrently offering numerous molecular and genomic candidates that appear to hold great promise for selective targeting as clinical therapies.