Faculty Bibliography

Investigations into the regulatory mechanisms controlling cholesterol homeostasis have proven fruitful in identifying low-density lipoprotein (LDL)-lowering therapies to reduce the risk of atherosclerotic cardiovascular disease. A major advance was the discovery of proprotein convertase subtilisin/kexin type 9 (PCSK9), a secreted protein that binds the LDL receptor (LDLR) on the cell surface and internalizes it for degradation, thereby blunting its ability to take up circulating LDL. The discovery that loss-of-function mutations in PCSK9 lead to lower plasma levels of LDL cholesterol and protection from cardiovascular disease led to the therapeutic development of PCSK9 inhibitors at an unprecedented pace. However, there remain many gaps in our understanding of PCSK9 regulation and biology, including its posttranscriptional control by microRNAs. Using a high-throughput region(3'-UTR) of human microRNA library screen, we identified microRNAs targeting the 3' untranslated region of human PCSK9. The top 35 hits were confirmed by large-format PCSK9 3'-UTR luciferase assays, and 10 microRNAs were then selected for further validation in hepatic cells, including effects on PCSK9 secretion and LDLR cell surface expression. These studies identified seven novel microRNAs that reduce PCSK9 expression, including miR-221-5p, miR-342-5p, miR-363-5p, miR-609, miR-765, and miR-3165. Interestingly, several of these microRNAs were also found to target other genes involved in LDLR regulation and potently upregulate LDLR cell surface expression in hepatic cells. Together, these data enhance our understanding of post-transcriptional regulators of PCSK9 and their potential for therapeutic manipulation of hepatic LDLR expression.

Disruption of systemic homeostasis by either chronic or acute stressors, such as obesity¹ or surgery², alters cancer pathogenesis. Patients with cancer, particularly those with breast cancer, can be at increased risk of cardiovascular disease due to treatment toxicity and changes in lifestyle behaviors^3-5. While elevated risk and incidence of cardiovascular events in breast cancer is well established, whether such events impact cancer pathogenesis is not known. Here we show that myocardial infarction (MI) accelerates breast cancer outgrowth and cancer-specific mortality in mice and humans. In mouse models of breast cancer, MI epigenetically reprogrammed Ly6C^hi monocytes in the bone marrow reservoir to an immunosuppressive phenotype that was maintained at the transcriptional level in monocytes in both the circulation and tumor. In parallel, MI increased circulating Ly6C^hi monocyte levels and recruitment to tumors and depletion of these cells abrogated MI-induced tumor growth. Furthermore, patients with early-stage breast cancer who experienced cardiovascular events after cancer diagnosis had increased risk of recurrence and cancer-specific death. These preclinical and clinical results demonstrate that MI induces alterations in systemic homeostasis, triggering cross-disease communication that accelerates breast cancer.

Rationale: Regression of atherosclerosis is an important clinical goal, however the pathways that mediate the resolution of atherosclerotic inflammation and reversal of plaques are poorly understood. Regulatory T cells (Tregs) have been shown to be atheroprotective, yet the numbers of these immunosuppressive cells decrease with disease progression, and whether they contribute to atherosclerosis regression is not known. Objective: We investigated the roles of Tregs in the resolution of atherosclerotic inflammation, tissue remodeling and plaque contraction during atherosclerosis regression. Methods and Results: Using multiple independent mouse models of atherosclerosis regression, we demonstrate that an increase in plaque Tregs is a common signature of regressing plaques. Single cell RNA-sequencing of plaque immune cells from revealed that Tregs from regressing plaques shared some similarity with splenic Tregs, but were distinct from skin and colon Tregs supporting recent findings of tissue-dependent Treg heterogeneity. Unlike Tregs from progressing plaques that expressed markers of natural Tregs derived from the thymus, Tregs in regressing plaques lacked Nrp1 and Helios expression, suggesting that they are induced in the periphery during lipid lowering therapy. To test whether Tregs are required for resolution of atherosclerotic inflammation and plaque regression, Tregs were depleted using CD25 monoclonal antibody in atherosclerotic mice during apolipoprotein B anti-sense oligonucleotide-mediated lipid lowering. Morphometric analyses revealed that Treg depletion blocked plaque remodeling and contraction, and impaired hallmarks of inflammation resolution including dampening of the Th1 response, alternative activation of macrophages, efferocytosis, and upregulation of specialized pro-resolving lipid mediators. Conclusions: Our data establish essential roles for Tregs in resolving atherosclerotic cardiovascular disease and provide mechanistic insight into the pathways governing plaque remodeling and regression of disease.

MicroRNAs (miRNA) have emerged as important post-transcriptional regulators of metabolic pathways that contribute to cellular and systemic lipoprotein homeostasis. Here, we identify two conserved miRNAs, miR-224, and miR-520d, which target gene networks regulating hepatic expression of the low-density lipoprotein (LDL) receptor (LDLR) and LDL clearance. In silico prediction of miR-224 and miR-520d target gene networks showed that they each repress multiple genes impacting the expression of the LDLR, including the chaperone molecules PCSK9 and IDOL that limit LDLR expression at the cell surface and the rate-limiting enzyme for cholesterol synthesis HMGCR, which is the target of LDL-lowering statin drugs. Using gain- and loss-of-function studies, we tested the role of miR-224 and miR-520d in the regulation of those predicted targets and their impact on LDLR expression. We show that overexpression of miR-224 or miR-520d dose-dependently reduced the activity of PCSK9, IDOL, and HMGCR 3'-untranslated region (3'-UTR)-luciferase reporter constructs and that this repression was abrogated by mutation of the putative miR-224 or miR-520d response elements in the PCSK9, IDOL, and HMGCR 3'-UTRs. Compared to a control miRNA, overexpression of miR-224 or miR-520d in hepatocytes inhibited PCSK9, IDOL, and HMGCR mRNA and protein levels and decreased PCSK9 secretion. Furthermore, miR-224 and miR-520d repression of PCSK9, IDOL, and HMGCR was associated with an increase in LDLR protein levels and cell surface expression, as well as enhanced LDL binding. Notably, the effects of miR-224 and miR-520d were additive to the effects of statins in upregulating LDLR expression. Finally, we show that overexpression of miR-224 in the livers of Ldlr^+/- mice using lipid nanoparticle-mediated delivery resulted in a 15% decrease in plasma levels of LDL cholesterol, compared to a control miRNA. Together, these findings identify roles for miR-224 and miR-520d in the posttranscriptional control of LDLR expression and function.

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.

Fine-tuning of the body's water balance is regulated by vasopressin (AVP), which induces the expression and apical membrane insertion of aquaporin-2 water channels and subsequent water reabsorption in the kidney. Here we demonstrate that silencing of microRNA-132 (miR-132) in mice causes severe weight loss due to acute diuresis coinciding with increased plasma osmolality, reduced renal total and plasma membrane expression of aquaporin-2, and abrogated increase in AVP levels. Infusion with synthetic AVP fully reversed the antagomir-132-induced diuresis, and low-dose intracerebroventricular administration of antagomir-132 similarly caused acute diuresis. Central and intracerebroventricular antagomir-132 injection both decreased hypothalamic AVP mRNA levels. At the molecular level, antagomir-132 increased the in vivo and in vitro mRNA expression of methyl-CpG-binding protein-2 (MECP2), which is a miR-132 target and which blocks AVP gene expression by binding its enhancer region. In line with this, treatment of hypothalamic N6 cells with a high-salt solution increased its miR-132 levels, whereas it attenuated endogenous Mecp2 mRNA levels. In conclusion, we identified miR-132 as a first miRNA regulating the osmotic balance by regulating the hypothalamic AVP gene mRNA expression.

PURPOSE OF REVIEW/OBJECTIVE:Noncoding RNAs have emerged as important regulators of cellular and systemic lipid metabolism. In particular, the enigmatic class of long noncoding RNAs have been shown to play multifaceted roles in controlling transcriptional and posttranscriptional gene regulation. In this review, we discuss recent advances, current challenges and future opportunities in understanding the roles of lncRNAs in the regulation of lipid metabolism during health and disease. RECENT FINDINGS/RESULTS:Despite comprising the majority of the transcriptionally active regions of the human genome, lncRNA functions remain poorly understood, with fewer than 1% of human lncRNAs functionally characterized. Broadly defined as nonprotein coding transcripts greater than 200 nucleotides in length, lncRNAs execute their functions by forming RNA-DNA, RNA-protein, and RNA-RNA interactions that regulate gene expression through diverse mechanisms, including epigenetic remodeling of chromatin, transcriptional activation or repression, posttranscriptional regulation of mRNA, and modulation of protein activity. It is now recognized that in lipid metabolism, just as in other areas of biology, lncRNAs operate to regulate the expression of individual genes and gene networks at multiple different levels. SUMMARY/CONCLUSIONS:The complexity revealed by recent studies showing how lncRNAs can alter systemic and cell-type-specific cholesterol and triglyceride metabolism make it clear that we have entered a new frontier for discovery that is both daunting and exciting.

Chronic kidney disease is associated with progressive renal fibrosis, where perivascular cells give rise to the majority of α-smooth muscle actin (α-SMA) positive myofibroblasts. Here we sought to identify pericytic miRNAs that could serve as a target to decrease myofibroblast formation. Kidney fibrosis was induced in FoxD1-GC;Z/Red-mice by unilateral ureteral obstruction followed by FACS sorting of dsRed-positive FoxD1-derivative cells and miRNA profiling. MiR-132 selectively increased 21-fold during pericyte-to-myofibroblast formation, whereas miR-132 was only 2.5-fold up in total kidney lysates (both in obstructive and ischemia-reperfusion injury). MiR-132 silencing during obstruction decreased collagen deposition (35%) and tubular apoptosis. Immunohistochemistry, Western blot, and qRT-PCR confirmed a similar decrease in interstitial α-SMA(+) cells. Pathway analysis identified a rate-limiting role for miR-132 in myofibroblast proliferation that was confirmed in vitro. Indeed, antagomir-132-treated mice displayed a reduction in the number of proliferating Ki67(+) interstitial myofibroblasts. Interestingly, this was selective for the interstitial compartment and did not impair the reparative proliferation of tubular epithelial cells, as evidenced by an increase in Ki67(+) epithelial cells, as well as increased phospho-RB1, Cyclin-A and decreased RASA1, p21 levels in kidney lysates. Additional pathway and gene expression analyses suggest miR-132 coordinately regulates genes involved in TGF-β signaling (Smad2/Smad3), STAT3/ERK pathways, and cell proliferation (Foxo3/p300). Thus, silencing miR-132 counteracts the progression of renal fibrosis by selectively decreasing myofibroblast proliferation and could potentially serve as a novel antifibrotic therapy.