MicroRNA regulation of cholesterol metabolism

Abstract

MicroRNAs are small noncoding RNAs that regulate gene expression at the posttranscriptional level. Since many microRNAs have multiple mRNA targets, they are uniquely positioned to regulate the expression of several molecules and pathways simultaneously. For example, the multiple stages of cholesterol metabolism are heavily influenced by microRNA activity. Understanding the scope of microRNAs that control this pathway is highly relevant to diseases of perturbed cholesterol metabolism, most notably cardiovascular disease (CVD). Atherosclerosis is a common cause of CVD that involves inflammation and the accumulation of cholesterol-laden cells in the arterial wall. However, several different cell types participate in atherosclerosis, and perturbations in cholesterol homeostasis may have unique effects on the specialized functions of these various cell types. Therefore, our review discusses the current knowledge of microRNA-mediated control of cholesterol homeostasis, followed by speculation as to how these microRNA–mRNA target interactions might have distinctive effects on different cell types that participate in atherosclerosis.

Introduction

Nearly three decades ago, the discovery of microRNAs (miRNAs) as critical posttranscriptional regulators of cellular function has revolutionized our understanding of pathway and network-level control of homeostasis. These ~22 nucleotide (nt)-long single-stranded molecules, frequently coded within introns and transcribed along with their host genes, bear a 6- to 8-nt-long seed sequence that recognizes complementary patterns in the 3′ untranslated region (UTR) of target mRNAs.¹ By binding to this region, miRNAs guide the target mRNA to the RNA-induced silencing complex to ultimately promote mRNA degradation or prevent its translation. Messenger RNAs can contain multiple binding sites for the same miRNA, as well as binding sites for more than one miRNA, and, notably, microRNAs often target multiple mRNAs that participate in the same biological pathway.² Thus, miRNAs offer a way to fine-tune the entire pathway activity. For example, cholesterol homeostasis, both on intracellular and systemic levels, is coordinated by the actions of several microRNAs on processes ranging from cholesterol uptake, synthesis, efflux, and storage to dietary cholesterol absorption, mobilization, and excretion.^3-5 Thus, miRNAs pose as a powerful tool to regulate cholesterol metabolism.

Next, we will briefly summarize major routes and pathways involved in cholesterol metabolism (Fig. 1), and we recommend readers to visit the reviews cited in this section for a detailed discussion of these pathways. After absorption in the gut, dietary cholesterol and fatty acids are esterified and packaged into chylomicrons, which subsequently enter the bloodstream where they are further metabolized by lipoprotein lipase (LPL), which mediates the hydrolysis of triglycerides (TGs) into fatty acids for their absorption in peripheral tissues.^6-9 The chylomicron remnants of this reaction are taken up by the liver, where their components are reassembled with apolipoprotein E (ApoE) into very-low-density-lipoprotein (VLDL) particles.^6,10 These are secreted into the circulation once again, where they interact again with LPL and/or endothelial lipase in capillary beds to promote further lipid absorption.⁶ VLDL remnants, or intermediate-density lipoproteins (IDLs), can circulate and either be taken up by the liver or further hydrolyzed by hepatic lipase to produce IDL remnants, called low-density lipoproteins (LDLs), which now contain a relatively high cholesterol content.⁶ LDL-associated cholesterol (LDL-C) is absorbed by the liver and peripheral tissues. Engagement of ApoB, the main apolipoprotein of LDLs, with the LDL receptor (LDLR), promotes endocytosis of LDL particles.¹¹ The LDLR may subsequently be recycled to the plasma membrane, whereas the LDL particle is delivered from the late endosome to the lysosome, where it is degraded to its constituent lipids, a process that involves the hydrolysis of cholesterol esters.¹¹ Free cholesterol can be transported to the plasma membrane or the endoplasmic reticulum (ER), where it can be esterified by acyl-coenzyme A:cholesterol acyltransferase (ACAT).¹² The resultant cholesteryl esters may either be stored in cytosolic lipid droplets or incorporated as components of lipoproteins in the circulation.^11,13

Figure 1.

Schematic overview of major cholesterol metabolism pathways. After dietary lipid absorption, chylomicrons in the circulation (1) are cleaved by lipoprotein lipase to produce chylomicron remnants (CMRs) (2) and triglycerides, which can be taken up by peripheral tissues (3). CMRs subsequently enter the liver (4) and are repackaged as VLDL particles that are released into the circulation (5). VLDL particles are broken down into IDLs (6) that are further broken down into LDLs by hepatic lipase (7). LDL particles are taken up by peripheral tissues (8a), or cleared by the liver (8b). Efflux of excess cholesterol onto HDL/ApoAI by peripheral tissues occurs via ABCA1/ABCG1 (9). HDL is taken up by the liver (10), and excess cholesterol is then catabolized to bile acids (11) and excreted (12).

Delivery of unesterified cholesterol to the ER also regulates the activity of homeostatic pathways and intracellular cholesterol-sensing mechanisms. One such example is the transcription factor family known as sterol regulatory element–binding proteins (SREBPs).¹⁴ Specifically, SREBP-2 activity transcriptionally drives the expression of genes involved in cholesterol biosynthesis and uptake upon depletion of cellular sterol levels.¹⁴ On the other hand, the accumulation of cholesterol within the cell can also drive its disposal: cholesterol efflux is also transcriptionally regulated by the nuclear liver X receptors (LXRs) in response to elevated cellular levels of cholesterol.¹⁵ LXRs bind to and regulate the expression of not only genes that encode proteins involved in cholesterol efflux but also absorption, transport, excretion, and bile acid conversion.¹⁶ LXR-induced cholesterol export from cells is mediated by ATP-binding cassette (ABC) transporters, such as ABC subfamily A member 1 (ABCA1), ABC subfamily G member 1 (ABCG1), ABCG5, and ABCG8. ABCA1 in the liver mediates cholesterol transport to ApoA-I in the circulation. In macrophages, cholesterol efflux forms nascent high-density lipoprotein (HDL) that serves as an acceptor for cholesterol efflux from ABCG1.¹⁷ Efflux of excess cholesterol from peripheral cells into HDL particles then return to the liver where they are taken up by the scavenger receptor B1 for excretion from the body in a process known as reverse cholesterol transport (RCT).¹⁸ Cholesterol is then targeted for elimination via catabolism to bile acids or direct biliary secretion.

Perturbations in the above processes controlling cholesterol homeostasis are thought to increase the risk and severity of atherosclerotic cardiovascular disease (CVD). For over 20 years, CVD has prevailed as the leading cause of death in the United States, surpassing even the total deaths caused by cancer. Atherosclerosis, a primary cause of CVD-related deaths, is a disease of dyslipidemia and inflammation in which high circulating levels of cholesterol promote vascular lipid retention and plaque development.^19,20 Notably, epidemiological studies have reported an inverse correlation between circulating HDL-C levels and the risk of CVD.²¹ On the other hand, patients with familial hypercholesteremia, a disorder where mutations in Ldlr result in elevated circulating LDL-C, have increased susceptibility to CVD.²² As will be discussed in this review in more detail, perturbations in cholesterol homeostasis on a cellular level can promote phenotypic switching and other pathological behaviors that may contribute to plaque development. Thus, elucidating the various mechanisms that orchestrate the activity of cholesterol-related pathways is imperative in understanding the pathogenic mechanisms of the disease and identifying novel therapeutic targets. In fact, over the past several years, an extensive body of literature has emerged around miRNA-mediated control of these processes, particularly surrounding miRNA-33 (miR-33). In this review, we will discuss the current knowledge about miRNA-mediated control of the aforementioned steps in cholesterol metabolism with a particular emphasis on how this regulation contributes to the pathogenesis of lipid disorders, such as atherosclerosis. The vast majority of the literature on miRNA-mediated regulation of cholesterol homeostasis has focused on their actions in macrophages and hepatocytes. Therefore, our review will be concluded by a discussion of the potential roles of some of these miRNAs in other cell types that participate in atherosclerosis.

miRNA regulation of VLDL/LDL metabolism

MicroR-33 is a family of sterol-sensitive miRNAs that consists of miR-33a and miR-33b, with the latter isoform expressed in humans and nonhuman primates, but not in mice. Located within the intronic regions of SREBF2 (for miR-33a) and SREBF1 (for miR-33b), these miRNAs are cotranscribed with their host genes upon cholesterol depletion or insulin stimulation, respectively.^23-27 Identical seed sequences produce highly similar targeting profiles, although there is a 2-nt difference outside of the seed region.²⁸ The functions of miR-33 were originally identified in a series of seminal studies approximately one decade ago.^23-27 While a vast amount of the literature has focused on how miR-33 regulates HDL-associated cholesterol (HDL-C) metabolism, several studies have investigated the effect of this miRNA on VLDL/LDL metabolism as well. In fact, miR-33 is the most extensively studied candidate when it comes to miRNA regulation of cholesterol homeostasis, and its regulation of lipid homeostasis has been validated across an assortment of animal models and diseases.

Regarding VLDL/LDL metabolism, the effects of miR-33 appear to be context-dependent. Global knockout of miR-33 in chow-fed mice increased plasma LDL/VLDL cholesterol (LDL-C/VLDL-C) levels,^26,29 while only LDL-C levels were changed upon high-fat diet (HFD, 60% kcal from fat) feeding.²⁹ However, changing the genetic background of the mice alters these results: after a western diet (WD, 40% kcal/fat with 1.25% added cholesterol30 or 20% kcal/fat with 0.15% added cholesterol³¹) feeding, VLDL-C and VLDL-TG levels are increased upon miR-33 knockout in Ldlr^−/− mice, but not upon miR-33 knockout in Apoe^−/− mice; this may be due to the fact that Apoe^−/− mice have significantly higher VLDL levels at baseline, which might mask any differences.^30,31 Additionally, other groups have reported that injection of miR-33 inhibitors in mice fed an HFD or a WD did not affect plasma VLDL/LDL-C levels.^24,32 This effect could be due to the fact that the inhibitors may accumulate primarily in some tissues, such as the liver, and, therefore, may not mirror the effects of whole-body knockout.

Additional concerns about the effect of miR-33 inhibitors were raised on the basis of their potential negative long-term side effects. Specifically, Goedeke et al. showed that long-term treatment of mice with miR-33 inhibitors produces potent hepatic steatosis and accumulation of VLDL-associated TG (VLDL-TG) upon HFD (60% kcal/fat).³³ Contrary to this finding, however, long-term miR-33 inhibition in monkeys fed high-carbohydrate (71% kcal/fat), moderate-cholesterol diet decreases VLDL-C and VLDL-TG levels and does not seem to have the same deleterious effects,³⁴ although this could be due to the presence of miR-33b in primates or the different type of diet used. To this end, the recent use of miR-33b knock-in (miR-33b^KI) mice has facilitated a specific analysis of the role of this miR-33 isoform in regulating VLDL/LDL metabolism. miR-33b^KI mice have been described to have decreased VLDL-C and VLDL-TG and a decrease in LDL-C.³⁵ However, whether the use of these humanized mice prevents the deleterious effects of long-term inhibitor treatment remains to be determined.

The mechanisms by which miR-33 may regulate VLDL/LDL metabolism have been approached by Allen and colleagues. They described that miR-33 decreases hepatic VLDL production by targeting N-ethylmaleimide-sensitive factor (Nsf), an ATPase implicated in intracellular vesicular trafficking processes.³⁶ Notably, the effect of miR-33 on VLDL secretion in mice is rescued by the overexpression of Nsf, thus directly linking the miRNA–target interaction to the phenotypic outcome. In fact, since NSF is involved in vesicle trafficking, not exclusively to VLDL, miR-33 manipulations altered circulating levels of several liver-secreted proteins, such as ApoE, ApoAI, and albumin.³⁶

In addition to miR-33, several other miRNAs have been described to regulate VLDL/LDL metabolism, which will be discussed in the remainder of this section (Table 1 and Fig. 2). miR-122 is the most abundant miRNA in the liver, and population-based analyses from the laboratory of Manuel Mayr confirmed that circulating miR-122 levels positively correlate with plasma cholesterol levels in humans, thus implicating it in the regulation of these pathways.³⁷ In fact, genetic deletion of this miRNA decreases circulating levels of LDL, and to a lesser extent, VLDL and HDL.³⁸ In addition, injection of mice and nonhuman primates with antagomirs to pharmacologically inhibit miR-122 reduced total plasma cholesterol levels (in both HDL and LDL fractions).^39-42 However, the effects of miR-122 deletion were paired with significantly increased hepatic steatosis, marked by TG accumulation and infiltration of inflammatory cells, thus casting doubt on the potential safety of sustained miR-122 therapeutics.^38,43 In addition, the full list of direct target genes responsible for this effect still remains to be defined in order to designate it as a potential therapeutic target for the regulation of cholesterol metabolism.

Table 1.

Summary of microRNAs that regulate cholesterol homeostasis

	miRNA	Target	Relevant pathway	Cell types studied
VLDL/LDL metabolism	miR-33	NSF	VLDL production	Hepatocytes
	miR-199	LDLR	Cholesterol uptake	Hepatocytes
		CAV1
		RAB5A
	miR-140	LDLR	Cholesterol uptake	Hepatocytes
	miR-185	LDLR	Cholesterol uptake	Hepatocytes
		KSRP	LDLR stability
	miR-27a/b	LDLR	LDLR activity	Hepatocytes
		LDLRAP1
		LRP6
		Predicted: CD36 and LPL	Oxidized LDL uptake	Macrophages
	miR-148a, miR-128-1, miR-130b, miR-301b	LDLR	Cholesterol uptake	Hepatocytes
	miR-30c	MTP	VLDL production	Hepatocytes
	miR-224, miR-520d	PCSK9 and IDOL	LDLR stability	Hepatocytes
Cholesterol transport and storage	miR-33	NPC1	Cholesterol transport	Macrophages and hepatocytes
		OSBPL6
		NSF		Hepatocytes
		Predicted: CPT1A, CROT, and HADHB	Beta oxidation/lipid droplet accumulation	Hepatocytes
		ATG5, LAMP1, and PRKAA1	Lipid droplet catabolism	Macrophages
	miR-27b	OSBPL6	Cholesterol transport	Macrophages and hepatocytes
		ACAT	Cholesterol esterification	Macrophages
	miR-26	ARL7	Cholesterol transport	Macrophages
Cholesterol efflux and HDL metabolism	miR-33	ABCA1 and ABCG1	Cholesterol efflux	Macrophages and hepatocytes
		PGC-1a, PDK4, and SLC25A5	Mitochondrial biogenesis	Macrophages
	miR-144, miR-27,miR-101	ABCA1	Cholesterol efflux	Macrophages and hepatocytes
	miR-26, miR-19b, miR-302a, miR-20a/b, miR-758	ABCA1	Cholesterol efflux	Macrophages
	miR-34a	ABCA1, ABCG1, and LXRa	Cholesterol efflux	Macrophages
	miR-145	ABCA1	Cholesterol efflux	Macrophages, hepatocytes, and pancreatic islet cells
	miR-148a, miR-128-1, miR-130b, miR-301b, miR-128-2	ABCA1	Cholesterol efflux	Hepatocytes
	miR-106b	ABCA1	Cholesterol efflux	Hepatocytes and neuronal cells
	miR-613	LXRα	Cholesterol efflux	Hepatocytes
	miR-206	LXRa	Cholesterol efflux	Hepatocytes
HDL uptake and cholesterol excretion	miR-33	ABCB11 and ATP8B1	Bile secretion	Hepatocytes
	miR-185, miR-96, miR-223	SR-BI	HDL uptake	Hepatocytes
	miR-125, miR-455	SR-BI	HDL uptake	Steroidogenic cells
Cholesterol biosynthesis	miR-185	SREBP2	Cholesterol biosynthesis	Hepatocytes
	miR-195	HMGCR	Cholesterol biosynthesis	Breast carcinoma cells
	miR-223	HMGCS	Cholesterol biosynthesis	Hepatocytes
	miR-224, miR-520d	HMGCR	Cholesterol biosynthesis	Hepatocytes
	miR-26	INSIG1	SREBP2 activity	Mammary epithelial cells
	miR-130b	INSIG1	SREBP2 activity	Hepatocytes
	miR-96	INSIG2	SREBP2 activity	HeLa and liver cells
	miR-182	FBXW7	SREBP2 activity	HeLa and liver cells

Figure 2.

Regulation of VLDL/LDL metabolism by miRNAs. Schematic model of miRNA regulation of VLDL/LDL homeostasis. Regulation of LDL uptake is managed at the level of the LDL receptor as well as its interacting proteins, such as LDLRAP1 and LRP6. Additionally, miRNA regulation of the LDLR-antagonizing proteins IDOL and PCSK9 can serve to regulate LDL uptake in cells by promoting LDLR degradation. Lastly, VLDL biogenesis, which occurs in the liver, can be inhibited by the actions of miR-30c and miR-33 on MTP and NSF, respectively.

In addition to miR-122, miR-155 and miR-34a also decrease circulating VLDL/LDL levels.^44,45 Macrophages from miR-155^−/− mice have reduced the expression of Hmgcr and Ldlr. LXRα RNA was then confirmed as a miR-155 target, which could in part explain the observed reduction in Ldlr expression.⁴⁴ However, the role of miR-155 in murine atherosclerosis appears to be context-dependent and may differ depending on whether early or late time points are studied.^46-50 Regulation of LDL metabolism via direct targeting of hepatic Ldlr has also been reported by miR-199, miR-140, and miR-148 (Fig. 2); the latter will be discussed in more detail further below. MicroR-140-5p directly targets LDLR and decreases LDL uptake in human hepatocytes.⁵¹ miR-199, on the other hand, has a couple of additional targets involved in LDL internalization that contribute to its repression of receptor-mediated endocytosis in hepatocytes, such as CAV1 and RAB5A.⁵² MicroR-185 is another miRNA that directly targets LDLR; however, its role in regulating LDLR levels is more complex, since it also targets the LDLR-destabilizing protein KH-type splicing regulatory protein (KSRP) in humans, but not in mice.⁵³ While miR-185 antagonism increases plasma LDL-C levels in mice, this likely will not hold true for humans, and studies in nonhuman primates will be required to determine the exact role of miR-185 in atherogenesis.⁵³

The miR-27 family of miRNAs consists of two isoforms: miR-27a and miR-27b. These miRNAs share the same seed sequence and have both been implicated in the regulation of lipid metabolism. An in silico screen conducted by the Sethupathy laboratory pinpointed miR-27 as having a notable number of seed matches in the 3′ UTRs of lipid metabolism-related genes, such as angiopoietinlike 3 (ANGPTL3) and peroxisome proliferator–activated receptor gamma (PPARG). Additionally, mature miR-27b was upregulated in the livers of WD-fed mice, while levels of the primary transcript were unchanged, suggesting that the regulation of miR-27 transcript stability could be a key process in its lipid responsiveness.⁵⁴ In the liver, miR-27a/b regulates several molecules involved in LDL metabolism, such as ApoB, LRP6, LDLRAP1, and LDLR itself (Fig. 2).^55-57 Additionally, miR-27a also increases PCSK9 expression by mechanisms yet to be understood; however, the authors speculate that the existence of a potential miR-27a–binding site in the PCSK9 promoter may account for this effect.^56,57 In vivo injection of miR-27 agomirs or antagomirs in Apoe^−/− mice decreases or increases LDL-C levels, respectively.^58,59 However, other research has shown that miR-27b does not influence plasma cholesterol levels in mice.⁵⁷ This could be due to the different genetic status of the mice (Apoe^−/− versus wild-type), or the mode of miR-27 manipulation (agomir/antagomir injection versus AAV-miR-27 injection).

Aside from its regulation in the liver, miR-27a/b has also been implicated in macrophage cholesterol metabolism. Specifically, miR-27a/b limits the uptake of oxidized LDL (oxLDL) by macrophages, which may be explained by their targeting of LPL and/or regulation of CD36 levels or other scavenger receptors.⁶⁰ More recent work from this group has confirmed that miR-27 antagonism increases inflammation and macrophage cholesterol accumulation in an LPL-dependent manner.⁵⁸ On the basis of its described effects in the liver and macrophages, it seems logical that miR-27 should regulate atherosclerosis development. In fact, in Apoe^−/− mice fed a WD, miR-27 antagomir injection increased aortic root lesion area and lipid deposition in the aortic wall.⁵⁸ However, the cell type–specific role of miR-27 still needs to be established with knockout models, and the exact miR-27 target responsible for the effects on plasma cholesterol still needs to be definitively determined in vivo.

Work from our laboratory identified miR-148a as another novel regulator of LDLR expression using a genome-wide miRNA screen that sought to identify novel posttranscriptional regulators of LDL uptake in hepatocytes.⁶¹ MicroR-148a is upregulated in the livers of WD-fed mice by LXR in an SREBP1c-dependent manner, thus situating miR-148a in a negative feedback loop of cholesterol regulation.⁶¹ Similar findings were confirmed in a sister study published by the Näär laboratory, which reported that miR-128-1, miR-130b, and miR-301b were additionally found to decrease LDLR expression and LDL uptake in hepatocytes (Fig. 2).⁶² In Apoe^−/− mice, miR-128-1 inhibition decreases levels of circulating VLDL-C and LDL-C; however, the in vivo consequence of miR-130b and miR-301b manipulation on LDL-C homeostasis still needs to be established.⁶²

MicroR-30c is a liver-enriched miRNA that targets microsomal triglyceride transfer protein (Mttp), which leads to reduced apoB/apoB100 secretion and reduced plasma VLDL and LDL levels (Fig. 2).⁶³ In addition, miR-30c agonism and antagonism decreases and increases aortic root lesion area and lipid deposition in the aortic wall, respectively.^63-65 Notably, miR-30c mimics also reduced serum cholesterol of obese and diabetic mice, thus revealing its capacity to regulate systemic cholesterol homeostasis in numerous models of dyslipidemia.⁶⁵ Lastly, recent research has implicated miR-224 and miR-520d in the regulation of LDL metabolism as well. These miRNAs indirectly regulate LDLR cell surface expression via targeting PCSK9 and IDOL, two proteins that otherwise antagonize LDLR levels (Fig. 2). In vivo, injection of Ldlr^+/− mice with miR-224 mimic decreases levels of circulating LDL-C.⁶⁶ However, the expression of miR-224 target genes was unchanged in the livers of these mice, suggesting that this effect may be somehow due to the combined actions of other tissues or a previously unidentified target.⁶⁶

miRNA regulation of cholesterol transport, storage, and subcellular localization

In the founding papers of miR-33, NPC1 (Niemann–Pick disease, type C1), a protein involved in transporting cholesterol from the late endosome/lysosome to other subcellular compartments of the cell, was also identified as a miR-33 target in human, but not mouse cells.²³ Later research confirmed that miR-33a*, the passenger strand, targets NPC1 as well.⁶⁷ Other targets of miR-33 related to intracellular cholesterol trafficking include Nsf, as discussed previously,³⁶ and OSBPL6 (oxysterol-binding protein-like 6; Table 1).⁶⁸ OSBPL6 is a cytoplasmic sterol-binding protein involved in cholesterol transport between the endosome and ER, whose expression positively correlates with cellular sterol levels, an effect that is likely orchestrated by this miRNA regulation. Additional analysis of the 3′ UTR of OSBPL6 by Ouimet and colleagues showed a seed match for miR-27b as well.⁶⁸ MicroR-27b was confirmed to regulate OSBPL6 expression in THP-1 macrophages. Thus, by targeting OSBPL6, miR-33 and miR-27b may also indirectly regulate cholesterol trafficking within the cell.⁶⁸ Additional research has shown that miR-27 decreases the ratio of cellular levels of cholesterol esters to free cholesterol via targeting ACAT1, which could have significant implications for foam cell formation and cytotoxicity in atherosclerosis.⁶⁰

In addition to its regulation of cholesterol transport, miR-33 has also been implicated in cholesterol storage and subcellular localization. Namely, miR-33 promotes lipid droplet formation in human hepatocytes likely via its inhibition of fatty acid oxidation,⁶⁹ and it also inhibits autophagy pathways involved in lipid droplet catabolism and bacterial clearance to promote intracellular lipid accumulation and prevent macrophage efferocytosis.^70,71 Additionally, by preventing cholesterol efflux, miR-33 promotes the formation of free cholesterol–rich lipid rafts: in macrophages, this enhances their LPS-induced cytokine production, thus linking metabolic regulation by miR-33 to inflammation;⁷² in fibroblasts, this promotes adaptive fibrosis in mouse models of heart failure.⁷³

ARL7 (ADP ribosylation factor-like 7), an LXR target gene, transports cholesterol to ABCA1 in the plasma membrane for efflux. MicroR-26 has recently been shown to target ARL7 in macrophages stimulated with an LXR agonist and attenuate cholesterol efflux.⁷⁴ LXR transcription factors, often considered master regulators of lipid homeostasis, are activated by cholesterol biosynthetic intermediates and oxidation derivatives. Thus, this recent work situates miR-26 as a downstream regulator of LXR-dependent pathways and cholesterol homeostasis.

miRNA regulation of cholesterol efflux and HDL metabolism

A substantial body of evidence supports a prominent role for miR-33 in the regulation of RCT by targeting the cholesterol efflux transporters ABCA1 and ABCG1 in numerous cell types (Fig. 3). Additionally, the species-specific effects of miR-33 are evident since it does not target ABCG1 in human cells. Functionally, miR-33a and miR-33b decrease cholesterol efflux to ApoA1 (via ABCA1) and HDL (via ABCG1) in mice, but only ApoA1 in humans; this is matched by a regulation of HDL biogenesis in the mouse liver and RCT.^23-27,35,75

Figure 3.

MicroRNA-dependent regulation of cholesterol efflux. Notably, several microRNAs target Abca1, which has an appreciablylong 3′ UTR and effluxes cholesterol to poorly lipidated ApoAI. In addition, miR-34 and miR-33 target Abcg1, which effluxes cholesterol to HDL. Frequently, this regulation of ABC transporter expression influences circulating levels of HDL. In addition to the direct regulation of these transporters, microRNAs, such as miR-613, miR-34, and miR-206, indirectly decrease their expression via repression of LXRα, the major transcriptional driver of ABC transporter expression. Lastly, by targeting mitochondrial SLC25A5, PDK4, and PPARGC1A, miR-33 may impact ATP production and subsequent ABC transporter function.

Given these data, it seems logical that miR-33 should potently regulate circulating HDL-C levels in vivo. For example, miR-33 loss-of-function mice, either with or without knockout of Ldlr or Apoe, have an increase in plasma HDL when fed chow or WD/HFD, while miR-33b^KI mice have decreased plasma HDL-C levels.^28,29,31,76 However, only a transient increase in HDL-C was observed when fed a high-fat, high-cholesterol diet,⁷⁷ and no effect of miR-33 loss-of-function was observed upon WD feeding.^30,78 These differences are likely due to the higher cholesterol content in the diet of the latter two experiments: since miR-33 is downregulated in the liver under high-sterol conditions, the high-cholesterol diet may mask any effects of anti-miR-33 therapy. Additionally, similar experiments conducted in nonhuman primates showed an increase in plasma HDL-C upon miR-33 inhibition.³⁴ In addition to its regulation of cholesterol efflux via direct ABCA1 targeting, miR-33 also indirectly regulates cholesterol efflux by antagonizing PPARGC1A (peroxisome proliferator–activated receptor gamma coactivator 1-alpha), PDK4 (pyruvate dehydrogenase lipoamide kinase isozyme 4), SLC25A5 (ADP/ATP translocase), and multiple complexes in the electron transport chain.⁷⁹ Mechanistically, this regulates cellular oxygen consumption and ATP production, which is thought to be required for proper ABCA1 functionality.⁷⁹ Notwithstanding this evidence, recent work from our laboratory has emphasized that the specific interaction between miR-33 and Abca1 is largely responsible for regulating macrophage cholesterol efflux, and to a lesser extent, circulating HDL-C levels in vivo.⁸⁰ Moving forward, studies focused on the role of miR-33 in other cell types and in cell–cell communication should be pursued, as miR-33 has been reported to be contained in endothelial-derived exosomes that are taken up by macrophages and vascular smooth muscle cells (SMCs) in vitro.⁸¹

In addition to miR-33, several other miRNAs regulate ABCA1 expression (Fig. 3). ABCA1 has an appreciably long 3′ UTR and, therefore, has the capacity to be regulated by several miRNAs. For example, our laboratory and others have shown that by virtue of its targeting of ABCA1, miR-144 decreases cholesterol efflux and plasma HDL levels in the mouse liver and macrophages.^82,83 The precise role of miR-144 in atherosclerosis regulation was later addressed by Hu et al., who showed that the previously observed effects, in combination with elevated plasma cytokine levels in miR-144 mimic–treated mice, increase the severity of atherosclerosis in WD-fed Apoe^−/− mice.⁸⁴

Together with its regulation of ARL7, miR-26 also targets ABCA1 and attenuates cholesterol efflux to ApoAI.⁷⁴ Subsequent studies showed that miR-26 decreases plasma HDL-C levels in Apoe^−/− mice fed a WD.⁸⁵ Interestingly, data from these studies suggest that miR-26 attenuates oxLDL-induced inflammation in human arterial endothelial cells (ECs), thus further elucidating some of the potential cell type–specific roles of miR-26 in atherosclerosis.⁸⁵ However, future studies with genetic deletion of miR-26 will be required to determine the exact cell type–specific contributions of miR-26 to hypercholesterolemia and atherosclerosis progression.

Direct targeting of ABCA1 by miR-27 in macrophages, foam cells, and hepatocytes functionally translates to decreased cholesterol efflux to ApoAI.^55,57,60 Subsequent work from our laboratory was the first to confirm the role of miR-27b in controlling lipid metabolism in vivo; while miR-27b agonism decreases hepatic ABCA1 levels, it does not regulate circulating HDL levels in mice.⁵⁷ The researchers speculate that this effect might be due to the targeting of other lipid-related transcripts by miR-27. However, the exact target interactions that explain this effect still need to be determined.⁵⁷ Regardless, the effect of miR-27b on atherosclerosis progression still needs to be analyzed since silencing of other cholesterol-regulating miRNAs, such as miR-33, can affect lesion development despite no appreciable changes in HDL levels.^30,78 Interestingly, miR-27a directly targets retinoid X receptor (RXR)-α, and may indirectly regulate LXR signaling and cholesterol efflux since LXRs form obligate heterodimers with RXR proteins upon ligand binding.⁵⁵ Lastly, as mentioned previously, miR-27 regulates ANGPTL3, and may, therefore, indirectly regulate endothelial lipase activity and HDL levels; however, this exact relationship still needs to be conclusively determined.⁵⁴

Recent work by Xu et al. has shown that miR-34a expression is upregulated by both lipid overload and inflammatory stimuli in macrophages. Since miR-34a targets ABCG1, ABCA1, and NR1H3 (LXRα to attenuate macrophage cholesterol efflux (Fig. 3), this potentially places macrophage miR-34a in a feed-forward loop that encourages foam cell formation.⁴⁵ In accordance with all of these data, macrophage-specific deletion of miR-34a increases plasma HDL-C and attenuates atherosclerosis.⁴⁵ Interestingly, miR-34a has been shown to have atherogenic effects in other cell types as well: SMC-derived miR-34a promotes vascular calcification, and endothelial-specific miR-34a promotes endothelial apoptosis and atherosclerosis progression in Apoe^−/− mice.^86,87

By targeting ABCA1, miR-145 decreases cholesterol efflux to ApoAI in hepatocytes, macrophages, and pancreatic islets.^88,89 Interestingly, dual overexpression of miR-33 and miR-145 did not synergistically enhance ABCA1 repression.⁸⁸ Global deletion of miR-143/miR-145 in mice attenuates atherosclerosis, an effect that researchers hypothesized to be due in part to its regulation of ABCA1 and cholesterol efflux. The subsequent investigation into the cell type-specific roles of miR-145 surprisingly revealed that while miR-145 mimicry decreases ABCA1 expression in hepatocytes and macrophages, endogenous miR-145 inhibition did not increase ABCA1 levels. Instead, they identified that vascular SMC–secreted miR-145 is taken up by macrophages and that the absence of this cell–cell miRNA transfer in vivo may explain the antiatherosclerotic effect of global miR-145 knockout.⁸⁹ Atheroprotective effects of endothelial-derived miR-145 on vascular SMC activity have also been described—thus highlighting the potentially diverse roles of secreted miR-145 in atherosclerosis protection.⁹⁰

Numerous other miRNAs have been reported to regulate ABCA1 in macrophages or hepatocytes: miR-19b, miR-148a, miR-302a, miR-20a/b, miR-128-1, miR-130b, miR-301b, miR-106b, miR-758, miR-101, and miR-128-2 (Table 1 and Fig. 3). All except for the last four miRNAs have been confirmed to regulate circulating HDL-C levels as well, and miR-106b, miR-19b, miR-302a, and miR-20a/b have all been reported to have proatherogenic effects.^61,62,91-98 However, evidence for potential HDL-C regulation has recently been supported by reports that miR-758, along with miR-33b, is significantly upregulated in human plaques that display posttranscriptional downregulation of ABCA1.⁹⁹ Additionally, miR-223 targets an inhibitory upstream transcriptional regulator of ABCA1, and, therefore, indirectly increases ABCA1 expression.¹⁰⁰

The regulation of the expression of each of these miRNAs is coordinated by numerous stimuli, such as sterol loading or depletion and subsequent LXR-or SREBP2-driven transcription. However, recent work has shown that other noncoding RNAs can regulate the expression of some of the aforementioned miRNAs. Expression of the long noncoding RNA cholesterol homeostasis regulator of miRNA expression (CHROME) is high in both the liver and macrophages and regulates mature ABCA1 transcript levels as well as cholesterol efflux to ApoAI.¹⁰¹ Since nascent ABCA1 transcript levels were unchanged, this suggests a posttranscriptional mechanism by which CHROME regulates ABCA1 levels. In fact, it was identified that CHROME interacts with several miRNAs that have previously been implicated in ABCA1 posttranscriptional regulation: miR-27b, miR-33a/b, and miR-128 (Fig. 3), thus emphasizing that the posttranscriptional control of cholesterol homeostasis involves multifaceted and numerous pathways.¹⁰¹

Lastly, in addition to direct modulation of ABC transporters, the miRNA-mediated regulation of cholesterol efflux can also occur at the level of the LXRs (Fig. 3). Notably, miR-613, which targets NR1H3, has been implicated in a feedback loop of LXR signaling mediated by SREBP-1c in hepatocytes.^102,103 Subsequent studies have, in fact, confirmed that miR-613–mediated targeting of NR1H3 decreases cholesterol efflux to ApoAI and HDL in PPARγ-activated macrophages,¹⁰⁴ and additional work will be required to investigate whether this regulates HDL biogenesis or atherosclerosis progression in vivo. MicroR-206 has also been implicated in the regulation of LXR signaling in numerous cell types: miR-206 directly targets NR1H3 and decreases LXR-induced expression of target genes, such as SREBP1 and FASN in hepatocytes.^105,106 Surprisingly, miR-206 has the opposite effect in macrophages and increases LXRα levels, target gene expression, and cholesterol efflux.¹⁰⁶ However, the reason why the mechanism of action of this miRNA is different in the liver versus macrophages is still unclear.

miRNA regulation of HDL uptake and cholesterol excretion

MicroR-33 also represses bile secretion by targeting ABCB11 and ATP8B1 in hepatocytes (Table 1).¹⁰⁷ ABCB11 facilitates the secretion of bile acids across the apical membrane of hepatocytes, while ATP8B1 promotes lipid packing of the apical membrane to maintain its structure. In addition to its effect on bile acid secretion, miR-33 also represses bile acid synthesis via targeting CYP7A1 in hepatocytes;¹⁰⁸ miR-33’s interaction with these mRNAs, in combination with its effect on ABCA1, compound to increase RCT.

SCARB1 (SR-BI) is an LXR-activated gene that is key in moderating HDL uptake in the liver so that it can be metabolized and excreted. miR-185, miR-96, and miR-223 decrease SCARB1 expression and HDL uptake in hepatocytes in a synergistic fashion.¹⁰⁹ In fact, miR-223 has been validated to target SCARB1 in hepatocytes and human coronary artery ECs and subsequently decreases their uptake of HDL.¹⁰⁰ Other regulators of SCARB1 expression and HDL-C uptake include mir-34a, miR-125, and miR-455.^45,110 MicroR-34a deletion has also been reported to increase ABCG5 and ABCG8 expression, potentially indirectly via its targeting of NR1H3, and this is associated with a decreased bile acid pool size, which may impair free cholesterol excretion pathways.⁴⁵

miRNA regulation of cholesterol biosynthesis

The pioneer work on miRNA antagonism showed that the inhibition of liver-enriched miR-122 in mice decreased the expression of several cholesterol biosynthetic enzymes. However, this effect is indirect, and the true direct target genes and downstream implications of this effect still remain to be examined in further detail.^39,40 MicroR-27a/b has also been implicated in the regulation of Hmgcr expression and other cholesterol biosynthetic enzymes in vitro and in vivo.^54,59 MicroR-27a was also shown to be sterol responsive in mouse hepatocytes, with its expression increased under cholesterol-loaded conditions and decreased under cholesterol-depleted conditions, thereby implicating it in a feedback loop of cholesterol modulation.⁵⁹ Similarly, miR-185 directly targets SREBF2 and subsequently decreases the expression of SREBP-2 target genes in vitro and in vivo (Table 1 and Fig. 4).¹¹¹ Interestingly, miR-185 is directly transcriptionally regulated by SREBP-1c, which situates miR-185 as a bridge between these major regulators of lipid homeostasis. Upstream activation of LXRs by the oxidative transformation of excess cholesterol increases SREBP1-c and, consequently, miR-185 expression, which reciprocally feeds back on SREBP-2 to prevent extraneous cholesterol production.¹¹¹ Lastly, miR-223, miR-195, miR-224, and miR-520d have been implicated in the regulation of cholesterol biosynthesis enzymes as well (Fig. 4).^66,100,112

Figure 4.

Regulation of the cholesterol-sensing SREBP2 pathway by miRNAs. While direct targeting of SREBP2, the master transcriptional regulator of cholesterol biosynthesis, is accomplished by miR-185, several other miRNAs indirectly regulate its activity. For example, miR-26, miR-130b, and miR-96 all target INSIG proteins, which otherwise anchor SREBP2 in the ER membrane in sterol-replete conditions. Additionally, miR-182 targets the ubiquitin ligase FBXW7, which otherwise antagonizes SREBP2 levels.

In addition to direct control of biosynthetic enzymes, regulation of SREBP2 translocation to the nucleus serves as another modality by which cholesterol biosynthesis can be regulated. INSIG1, which anchors SREBP2 in the ER membrane and prevents its nuclear translocation, has also been identified as a direct target of miR-26 and miR-130b (Fig. 4);^62,113 however, whether this interaction can actually determine the capacity for a cell to respond to changes in sterol levels is unknown. In addition, Jeon and colleagues identified INSIG2 (another SREBP-anchoring protein in the ER) as a target of miR-96 and FBXW7 (an SREBP ubiquitin ligase that degrades SREBP2) as a target of miR-182 (Fig. 4).¹¹⁴ Strikingly, antagonism of miR-182 in mice near abolished SREBP2 nuclear translocation in conditions of cholesterol depletion. Conversely, miR-182 transfection increased SREBP2 nuclear translocation, an effect that was inhibited with coexpression of FBXW7.¹¹⁴ This locus was also under transcriptional control of SREBP2, thus identifying a novel SREBP/miRNA axis by which cholesterol homeostasis can be regulated, in addition to the SREBP/miR-33 canon.¹¹⁴

miRNA regulation of cholesterol absorption

The role of intestinal-specific miRNAs in regulating cholesterol homeostatic processes is still a new field of study; however, this section will discuss our current knowledge of these processes. Early studies of the roles for miRNAs in controlling intestinal cell biology and function utilized wild-type and intestinal-specific Dicer1 (one of the key enzymes involved in miRNA biogenesis) knockout mice to identify broad phenotypic changes that occur upon loss of intestinal epithelial miRNAs. Specifically, Dicer1 knockout mice had reduced body weight and increased fecal lipid content after WD feeding, suggesting possible lipid malabsorption.¹¹⁵ Additionally, inducible global deletion of Dicer leads to neutral lipid accumulation in the small intestine as well as a significantly altered proteome; however, it is difficult to definitively say whether this effect is due to intestinal epithelial cell–specific miRNAs or changes in exogenous/circulating miRNAs.¹¹⁶ Specific studies in Caco-2 cells reveal that fatty acid stimulation changes cholesterol-related gene expression, and this effect is altered upon Dicer knockdown. MicroR-30c and miR-192 were identified as miRNAs upregulated by this fatty acid treatment, with many predicted targets involved in cholesterol metabolism.¹¹⁷ While the relevant list of miR-30c/miR-192 targets needs to be verified, this highlights the potential importance of miRNA-mediated regulation of cholesterol homeostasis in the intestine.

Recent research has begun to shine more light on this topic by the use of intestinal gene expression analysis in wild-type and Dicer1 knockout mice following oral lipid gavage. The expression of several transcripts involved in lipid metabolism, such as Acat1 (an enzyme involved in cholesterol esterification) and Orl1 (an oxLDL receptor), was altered.¹¹⁸ MicroR-425-5p, miR-31-5p, miR-99b-5p, miR-200a-5p, and miR-200b-5p were all identified as regulators of the expression of these genes; however, direct miRNA–mRNA targeting interactions still need to be confirmed.¹¹⁸ Additionally, in the intestine, SR-BI is required for proper chylomicron production and is posttranscriptionally regulated by LXR-induced miR-96-5p.¹¹⁹ Lastly, global miR-34a deletion in Apoe^−/− and Ldlr^−/− mice fed a WD decreases plasma TG and cholesterol levels. Mechanistically, this effect is likely due to the downregulation of hepatic CYP7A1 and CYP8B1 to limit bile acid secretion and subsequent lipid absorption.⁴⁵ While to our knowledge, there are limited reports that have investigated whether intestinal-specific loss of any miRNA prevents diet-induced atherosclerosis, it is becoming clear that these molecules may serve as an early intervention therapy to potentially prevent excessive lipid absorption upon WD feeding.

miR-33 regulates other lipid metabolism pathways and atherogenesis

Not surprisingly, owing to its numerous effects on cholesterol metabolism, several groups have studied the role of miR-33 in regulating atherosclerosis progression both in mice and nonhuman primates. However, these effects of miR-33 on atherosclerosis are not only due to its regulation of sterol homeostasis, since its regulation of fatty acid metabolism may also contribute to these phenotypes. For example, miR-33 has also been implicated in mitochondrial function and fatty acid metabolism. As mentioned previously, miR-33 can regulate cholesterol efflux via ATP production.⁷⁹ miR-33 also targets PRKAA1, a subunit of AMPK—a vital cellular energy sensor that promotes fatty acid β-oxidation in the liver and inhibits cholesterol and TG synthesis.¹²⁰ This miR-33/target interaction promotes fatty acid oxidation and increases glycolysis, which can ultimately regulate macrophage differentiation into the M1- and M2-like subtypes.¹²⁰ miR-33 also regulates fatty acid β-oxidation in hepatocytes and macrophages by targeting the enzymes carnitine palmitoyltransferase 1A (CPT1A), carnitine O-octanoyltransferase (CROT), and hydroxyacyl-CoA dehydrogenase trifunctional multienzyme complex subunit beta (HADHB).^25,69 So far, both the miR-33a passenger strand and miR-33b have also been validated to target enzymes involved in β-oxidation.^67,69 In addition to its regulation of fatty acid metabolism, miR-33 has been shown to regulate insulin signaling and glucose metabolism.^69,121 This highlights the potential ways by which miR-33 can potentially regulate obesity and diabetes, which increase the risk of CVD.

Interestingly, miR-33 loss-of-function also results in some of the hallmarks of metabolic syndrome. Upon the discovery that miR-33^−/− mice fed HFD develop weight gain, hepatic steatosis, abnormal glucose levels, and insulin intolerance, SREBP1—a master regulator of lipid homeostasis and lipogenesis—was later confirmed as a miR-33 target and deletion of one copy of Srebf1 ameliorates the miR-33 knockout phenotype.¹²² Complementarily, miR-33b^KI mice have reduced hepatic expression of Abca1 and Srebp1 as well as reduced cholesterol efflux by macrophages.⁷⁵ However, the work from Karunakaran et al. and Goedeke et al. does not validate Srebf1 as a miR-33 target, and they did not see a difference in weight gain in anti-miR-33–treated mice, a result that may be due to the different models used for miR-33 loss-of-function.^32,33 Additionally, while Price et al. recapitulated the metabolic dysfunction in miR-33 knockout mice, the authors speculate that this is likely due to SREBP-1–independent mechanisms.²⁹ In fact, miR-33–mediated effects in the brain may drive this phenotype, as knockout mice exhibited increased food consumption that was eliminated upon cohousing with control mice.²⁹ However, the exact mechanism by which miR-33 regulates food intake still warrants investigation.

Considering all of these metabolic effects of miR-33, the ultimate effect on atherosclerosis progression depends on the mouse model and diet used, while its effect on plaque regression seems to be beneficial. For example, miR-33 antagonism promotes atherosclerosis regression in WD-fed Ldlr^−/− mice.⁷⁶ In another model of atherosclerosis regression, diabetic mice are reported to have reduced regression, while miR-33 inhibition reduced the severity of this diabetic phenotype.¹²³ However, when it comes to atherosclerosis progression, the effects of miR-33 are more nuanced. miR-33 antagonism in Ldlr^−/− mice fed a diet with a 0.3% added cholesterol, but not a diet with a 1.25% added cholesterol, decreases lesion size and macrophage content in the plaque.^77,78 However, these effects may be due to the fact that the diet with higher cholesterol suppresses miR-33 expression in the liver and thus masks the effect of the inhibitors. In addition, miR-33^−/− Apoe^−/− mice fed a WD have reduced atherosclerosis progression compared with Apoe^−/− mice,³¹ and bone marrow (BM) transplantation from miR-33^−/− Ldlr^−/− mice into Ldlr^−/− mice has less atherosclerosis progression compared with mice receiving Ldlr^−/− BM, suggesting that the role of miR-33 in BM-derived cells, such as macrophages, plays a key role in its effects on atherosclerosis.³⁰ Regardless, altogether, a larger body of evidence supports an antiatherogenic role for decreases in whole-body miR-33 levels. Recently, a direct assessment of a specific miR-33/target interaction has highlighted the importance of Abca1 repression in regulating atherosclerosis progression. Remarkably, mutating the endogenous miR-33 binding site in the Abca1 3′ UTR in mice largely resembles the antiatherosclerotic effects of miR-33 knockout.⁸⁰ However, the phenotype in these mice was not as severe, implicating other miR-33 targets in its regulation of atherogenic processes.

Recent research has also begun to implicate miR-33b in atherosclerosis progression as well. In miR-33b^KI mice on an Apoe^−/− background fed a WD, decreased weight gain and large plaque size with an unstable morphology were observed.³⁵ Additionally, mice with only miR-33b had larger plaque size and hepatic TG content than mice with only miR-33a, suggesting that miR-33b may exert a stronger role than miR-33a during atherosclerosis progression.²⁸ In an attempt to decipher the cell type–specific roles of miR-33b in atherosclerosis, BM transplantation from miR-33b^KI mice into Ldlr^−/− mice does not affect plaque size, which might be due to the fact that miR-33b expression in macrophages is considerably lower than miR-33a levels.³⁰ However, another group has reported miR-33b and miR-33a expression to be comparable in macrophages in miR-33b^KI mice, and they subsequently reported that miR-33b^KI BM transplantation into Apoe^−/− mice increased the plaque size compared with mice receiving control BM.²⁸ Ideally, future studies utilizing different diets and models of atherosclerosis will help to validate the roles of these miR-33 isoforms in atherosclerosis.

New horizons: miRNA regulation of cholesterol homeostasis

As evidenced by our review, the vast majority of the literature has focused on the role of miRNAs in regulating cholesterol homeostasis in primarily macrophages and hepatocytes. Granted, these two cell types are essential for regulating whole-body cholesterol metabolism and preventing lipid accumulation in atherosclerotic plaques. However, research has also shown that cholesterol pathways are key for the proper functioning of many other cell types involved in atherosclerosis progression, including SMC, ECs, neutrophils, T cells, and others. For the remainder of this review, we will: (1) highlight some cholesterol pathways that are relevant to the activation and function of these cells; (2) discuss how the miRNA profile changes upon dysfunction of these cells; and (3) how miRNAs discussed in this review may be involved in the dysfunction of these cells during the progression of the lipid-related disease. While this discussion will only touch on a few potentially relevant miRNAs, it aims to highlight how all of the candidates discussed in our review should be imagined within the context of other cell types.

Smooth muscle cells

SMCs, located in the media of the arterial wall, are physiologically crucial for the modulation of vascular tone. In atherosclerosis, SMCs adopt a migratory phenotype and can invade the plaque, where they can contribute to foam cell formation or promote degradation of the local basement membrane and contribute to plaque destabilization.¹²⁴ Extensive links have been made between aberrant cholesterol homeostasis and SMC dysfunction. Notably, in the plaque, SMCs constitute a significant portion of foam cells,¹²⁵ and SMC-derived foam cells have been verified in culture and in vivo through lineage tracing studies.^126,127 In fact, cholesterol loading eventually sparks the adoption of macrophage-like gene expression and function.¹²⁸

miR-145, previously described to regulate Abca1 expression,^88,89 has been implicated in SMC phenotype switching. Mechanistically, cholesterol loading downregulates miR-143/145, which otherwise maintains the expression of the SMC differentiation gene myocardin. The authors speculated that upon miR-143/145 downregulation, KLF4 becomes derepressed and, in turn, decreases myocardin expression; relatedly, oxidized phospholipids have been shown to decrease the expression of SMC differentiation marker genes, such as myocardin, in a KLF4-dependent manner.^129,130 However, whether miR-145 promotes SMC foam cell formation by virtue of its targeting of ABCA1 still needs to be examined in detail, especially since intimal SMC cholesterol loading is associated with decreased ABCA1 expression and efflux to ApoAI. However, this effect on cholesterol efflux is not able to be restored by LXR-induced rescue of ABCA1 expression, indicating that this defect may be due to lack of a required ABCA1 cofactor.¹³¹ It, therefore, seems plausible that miR-33 may also participate in the regulation of this SMC phenotypic switch: (1) direct targeting of ABCA1 could be responsible for decreasing its expression, and (2) regulation of mitochondrial pathways/ATP production may explain why LXR agonism still fails to rescue the effect. MicroR-33 has, in fact, been implicated in SMC function; specifically, miR-33 knockout decreases CCL2 expression in SMCs.¹³² However, experiments regarding whether miR-33 can regulate SMC cholesterol loading are warranted.

Interestingly, the TargetScan analysis of the myocardin 3′ UTR reveals that miR-34a-5p also has a binding site on myocardin that is conserved in humans and chimps. Since miR-34a expression is upregulated in human plaques and is increased by cholesterol loading,⁴⁵ this may situate miR-34a as another hyperlipidemia-induced regulator of SMC plasticity. However, miR-34a also targets KLF4¹³³ and may indirectly increase myocardin expression; regardless, in vitro analyses should readily identify the ultimate effect of cholesterol loading–induced miR-34a on the SMC fate. However, the lack of a conserved binding site in mice precludes the use of definitive lineage-tracing studies.

Notably, cholesterol loading increases the vasoconstrictive activity of the aorta and decreases the SMC–fibronectin adhesion force, which are associated with SMC phenotype switching.¹³⁴ Vascular tone is also potently regulated by endothelial-derived nitric oxide (NO), and thus miRNAs that simultaneously regulate SMC cholesterol homeostasis, as well as NO signaling in SMCs, may prove to be potent targets for the regulation of SMC phenotype switching, atherosclerosis, and other diseases of vascular stiffness, such as hypertension. Notably, endothelial miR-155 is an endogenous regulator of endothelial NO synthase (eNOS) expression and NO production.¹³⁵ In addition, it is also packaged into microvesicles, significantly more so under the context of endothelial inflammation.¹³⁶ Whether endothelial-derived miR-155 is taken up by vascular smooth muscle cells is an interesting concept. By targeting LXR– and potentially promoting intracellular cholesterol accumulation, endothelial-derived miR-155 may regulate SMC cholesterol homeostasis. This could suggest another mechanism by which miR-155 regulates atherosclerosis in inflammatory contexts; in this case, via regulation of vascular tone by controlling endothelial NO production and via regulation of SMC phenotype switching by regulating cholesterol loading. Regardless, in general, since miRNAs dynamically modulate gene expression in response to stimuli, they may prove to be key rheostats in the modulation of cellular cholesterol levels and SMC pathobiology.

Endothelial cells

ECs serve as a primary barrier to subendothelial lipoprotein accumulation. When lipoproteins become oxidized, they induce endothelial inflammation that is greatly responsible for the recruitment of immune cells into the plaque.¹²⁴ The contribution of many metabolic pathways to EC function and dysfunction in atherosclerosis has already been comprehensively reviewed.^137,138 One reason why it is key to understand how cholesterol metabolism in ECs is regulated is exemplified in a study from the Lusis laboratory.¹³⁹ These researchers used two different strains of mice with differing susceptibility to atherosclerosis development. Culturing aortic ECs from each of these mice showed that the susceptible strain showed a much greater extent of endothelial inflammation after oxLDL treatment compared with the atherosclerosis-resistant strain. oxLDL-stimulated endothelial inflammation is one of the earliest detectable events in atherosclerosis, and this study showed that endothelial responses to cholesterol could significantly impact the development of atherosclerotic plaques. Therefore, a thorough understanding of the molecular mechanisms behind the inflammatory and metabolic responses of EC to this stimulus is required. To this end, several bioinformatic reports indicate that the miRNA profile of ECs changes upon inflammation, activation/angiogenesis, senescence, and hypoxia,^140-143 and, therefore, it is imperative to map out which of these molecules may be relevant for endothelial cholesterol homeostasis.

For example, there is strong experimental evidence that LXR signaling and cholesterol efflux via ABC transporters are essential for proper endothelial function. LXR signaling attenuates endothelial inflammation downstream of lysophosphatidylcholine, a component of oxLDL,¹⁴⁴ and also promotes NO production.¹⁴⁵ Not surprisingly, ABC transporters tend to exert similar effects as the LXRs. For example, cholesterol loading via deletion of ABCA1/G1 decreases eNOS activity and NO production, which are otherwise considered atheroprotective, by promoting the caveolin (Cav)-1–eNOS interaction, and this effect can be reversed upon HDL incubation.^146,147 ABC transporters have also been reported to have anti-inflammatory effects in ECs and decrease monocyte adhesion; in fact, EC-specific loss of ABC transporters worsens the atherosclerotic phenotype.^148,149 Additionally, SREBF2 is upregulated by inflammatory stimuli,¹⁵⁰ suggesting that this cholesterol pathway may be important in regulating EC function in atherosclerosis. Therefore, miRNAs that regulate ABCA1 or LXR expression may prove to be important in EC dysfunction in atherosclerosis.

In addition, miR-223, which, as discussed in our review, can regulate ABCA1 and SCARB1 expression, has been reported to be transferred to ECs by HDL and subsequently inhibits ICAM1 expression and endothelial inflammation.¹⁵¹ Notably, SR-BI and Cav-1 act in concert as one of the mechanisms by which ECs transfer lipoproteins to the underlying environment.¹⁵² Thus, changes in the levels of circulating miR-223 may be one of the earliest miRNA effectors of subendothelial lipoprotein retention in atherogenesis, and whether endothelial miR-223 can be antiatherogenic by preventing endothelial lipoprotein uptake should be validated experimentally. Recent research has shown that elevated shear stress (an atheroprotective stimulus) induced by an arteriovenous shunt leads to the upregulation of miR-223 in the vessel wall.¹⁵³ Therefore, low-shear stress, which is associated with atheroprone regions, may lead to downregulation of miR-223 and subsequent perturbations in endothelial lipoprotein transcytosis mechanisms. Furthermore, miR-223 targets tissue factor in ECs.¹⁵⁴ Therefore, miR-223 has the potential to regulate endothelial-associated atherosclerotic processes ranging from initial lipoprotein uptake to thrombogenicity of the plaque. Additionally, as mentioned earlier, miR-199a/b targets Cav-1 in ECs. Since endothelial Cav-1 has recently been shown to be atherogenic and critical in mediating transcytosis of lipoproteins to the subendothelial space,¹⁵⁵ the role of miR-199 in fine-tuning the governing role of ECs in LDL transcytosis also warrants investigation.

Neutrophils and other immune cells

Original descriptions of atherosclerosis did not acknowledge neutrophils as a relevant cell type that contributes to the progression of the disease due to their rare identification within plaques. However, recently improved staining protocols and lineage tracing studies reveal that neutrophils are, in fact, located within lesions.¹⁵⁶ This immune cell may contribute to atherosclerosis progression by producing myeloperoxidase, which may promote LDL oxidation and subsequent endothelial inflammation. They can also promote endothelial apoptosis and contribute to extracellular matrix degradation to destabilize the plaque. In addition, the level of circulating neutrophils positively correlates with atherosclerotic lesion size, thus emphasizing the proatherogenic role of this cell type in CVD.^156,157

Hypercholesteremia promotes neutrophil release from the BM, a process that increases the severity of atherosclerosis, by increasing their expression of CXCR2/CXCL1.¹⁵⁷ The analysis of the 3′ UTR on Target Scan¹⁵⁸ reveals a miR-33-5p–binding site, conserved only in humans and chimps, in the 3′ UTR of CXCR2. However, whether hypercholesteremia-induced miR-33 downregulation contributes in any part to the derepression of CXCR2 and subsequent neutrophilia has not been investigated. Notably, Drechsler et al. report that CXCR2 is also important for neutrophil adhesion to the endothelium,¹⁵⁷ and this would suggest two modalities by which miR-33 may have an antiatherogenic role regarding the contribution of neutrophils specifically to atherosclerosis.

A direct link between cholesterol regulation, as discussed in this review, and neutrophil homeostasis lies in the roles of ApoAI and HDL. Notably, promotion of cholesterol efflux via ABCA1 and ABCG1 to ApoAI and HDL, respectively, decreases Cd11b expression in neutrophils, and infusion of ApoAI reduces inflammatory stimulus–induced leukocyte adhesion to the endothelium as assessed by intravital microscopy.¹⁵⁹ Another role for cholesterol efflux has been implicated in neutrophil biology. Specifically, myeloid-specific loss of ABC transporters increases inflammasome priming and activation, neutrophil recruitment, and neutrophil extracellular trap (NET) formation in the plaques of Ldlr^−/− mice fed a WD; however, concomitant deletion of Nlrp3 was necessary for the loss of these transporters to have an effect on lesion area.¹⁶⁰ Any of the previously discussed miRNAs that regulate cholesterol efflux, most notably miR-33, may, therefore, contribute to neutrophil infiltration into the subendothelial space or NET formation and consequently promote atherosclerosis. For example, miR-155 has been implicated in NET formation by increasing PAD4 levels.¹⁶¹ However, since miR-155 has also been reported to target LXRα,⁴⁴ whether this regulation of ABC transporter expression can promote NETosis warrants investigation, and other miRNAs that may regulate ABCA1/G1 but have not yet been directly implicated in NETosis may be worth examining as well. Importantly, miR-155 is highly expressed in stimulated neutrophils, and feeding with a WD increases the amount of miR-155 present in neutrophil-derived microvesicles.¹⁶² miR-155 from neutrophil-derived microvesicles increases endothelial inflammation via NF-κB signaling and promotes atherosclerosis.¹⁶² The authors claim that this is due to miR-155 targeting of Bcl6, a negative regulator of NF-κB signaling, but LXRα has also been implicated in the regulation of this inflammatory pathway.^16,162 Moreover, extracellular vesicles from SMCs also deliver miR-155 to ECs, suggesting that miR-155 may be involved in atherosclerosis on multiple levels.^162,163 To summarize, the regulation of cholesterol homeostasis by miR-155 may promote NETosis, endothelial inflammation, and atherosclerosis.

miRNA regulation of cholesterol homeostasis may also be important in hematopoietic stem cells, dendritic cells, and T cells. In hematopoietic stem cells, ApoE bound to the extracellular surface promotes cholesterol efflux from ABCA1 and ABCG1.¹⁶⁴ The subsequent accumulation of intracellular cholesterol leads to neutrophilia, monocytosis, and a more rapid progression of atherosclerosis; not surprisingly, LXR stimulation reduces hematopoetic stem cell proliferation.¹⁶⁴ In dendritic cells, ABC transporter deficiency is associated with increased inflammasome priming and caspase 1 cleavage and increased cytokine production in vitro, which is accompanied in vivo with increased T cell activation.¹⁶⁵ Therefore, miRNA regulation of ABC transporters may prove to be key in controlling hematopoiesis and dendritic cell/T cell activation. For instance, the expression of miR-223 is dynamic during different stages of hematopoiesis, and whether its indirect regulation of cholesterol efflux or its regulation of cholesterol biosynthetic enzyme levels can contribute to hematopoiesis or atherosclerosis should be studied. As reviewed recently,¹⁶⁶ several cholesterol-related proteins regulate T cell function and signaling. For example, LXR/ABC transporter–dependent efflux controls lipid raft composition, TCR signaling, and cytokine production,^167-169 while SREBP1a/2-stimulated cholesterol biosynthesis is required for clonal expansion,¹⁷⁰ thus indicating that preventing T cell intracellular cholesterol accumulation may impact atherosclerosis. Studies of the roles of the aforementioned miRNAs in the regulation of T cell activation, different T cell subsets, and their contribution to atherosclerosis are warranted.

Conclusion

miRNAs appear to contribute to the regulation of every step in cholesterol homeostasis, ranging from cholesterol absorption in the gut to bile acid production and cholesterol excretion. Concrete evidence shows that hepatic- and macrophage-derived miRNAs are critical mediators of cholesterol homeostasis and atherosclerosis progression. However, as mentioned earlier in this review, there is ample evidence of miRNA communication between cell types, and changes in the circulating levels of several miRNAs are associated with dyslipidemia, CVD, diabetes, and obesity.^171-174 Moving forward, new research should turn to examine the roles of these miRNAs in other cell types that contribute to atherosclerosis progression, such as SMCs, ECs, and hematopoietic/immune cells. Owing to the abundant connection between cholesterol homeostasis and the functioning of these cell types, any of the miRNAs discussed in our review may serve to be viable regulators of the function and dysfunction of these cells. Accordingly, we provide a discussion for some strong potential candidates, such as miR-33, miR-155, miR-34a, and others. However, the use of cell type–specific knockouts will be crucial in definitively deciphering the pathological effects of these miRNAs in atherosclerosis.