microRNA-33 Regulates Macrophage Autophagy in Atherosclerosis

Abstract

Objective Defective autophagy in macrophages leads to pathologic processes that contribute to atherosclerosis, including impaired cholesterol metabolism and defective efferocytosis. Autophagy promotes the degradation of cytoplasmic components in lysosomes and plays a key role in the catabolism of stored lipids to maintain cellular homeostasis. miR-33 is a post-transcriptional regulator of genes involved in cholesterol homeostasis, yet the complete mechanisms by which miR-33 controls lipid metabolism are unknown. We investigated whether miR-33 targeting of autophagy contributes to its regulation of cholesterol homeostasis and atherogenesis. Approach and Results Using CARS microscopy we show that miR-33 drives lipid droplet accumulation in macrophages, suggesting decreased lipolysis. Inhibition of neutral and lysosomal hydrolysis pathways revealed that miR-33 reduced cholesterol mobilization by a lysosomal-dependent mechanism, implicating repression autophagy. Indeed, we show that miR-33 targets key autophagy regulators and effectors in macrophages to reduce lipid droplet catabolism, an essential process to generate free cholesterol for efflux. Notably, miR-33 regulation of autophagy lies upstream of its known effects on ABCA1-dependent cholesterol efflux, as miR-33 inhibitors fail to increase efflux upon genetic or chemical inhibition of autophagy. Furthermore, we find that miR-33 inhibits apoptotic cell clearance via an autophagy-dependent mechanism. Macrophages treated with anti-miR-33 show increased efferocytosis, lysosomal biogenesis and degradation of apoptotic material. Finally, we show that treating atherosclerotic Ldlr−/− mice with anti-miR-33 restores defective autophagy in macrophage foam cells and plaques, and promotes apoptotic cell clearance to reduce plaque necrosis. Conclusions Collectively, these data provide insight into the mechanisms by which miR-33 regulates cellular cholesterol homeostasis and atherosclerosis.

Genetic Dissection of the Impact of miR-33a and miR-33b during the Progression of Atherosclerosis

“…Additionally, we observe an additional function of miR-33 in regulating the uptake of apoptotic cells by Mθs in vitro , which could possibly also contribute to the changes seen in necrotic core size. Interestingly, another group has very recently reported similar changes in efferocytosis upon inhibition of miR-33, which were attributed to regulation of autophagy by miR-33 (Ouimet et al, 2017). However, neither the specific autophagic genes reported to be targeted by miR-33, nor most other genes related to autophagic function were found to be significantly altered in our RNA-seq analysis.…”

“…Recent work has elucidated the role of miRNAs in the development of numerous metabolic conditions including obesity, Type II diabetes and cardiovascular disease, which are responsible for the majority of chronic illnesses and deaths in the United States and other developed countries. Many factors including insulin signaling, inflammation, autophagy, mitochondrial function, and lipid metabolism are involved in the development of these cardiometabolic diseases, and previous work suggests a role for miR-33 in regulating all of these critical processes (Allen et al, 2012; Davalos et al, 2011; Goedeke et al, 2013; Karunakaran et al, 2015; Marquart et al, 2010; Ouimet et al, 2017; Ouimet et al, 2015; Rayner et al, 2010). …”

“…Additionally, some studies of long-term anti-miR-33 treatment have reported deleterious effects including increased circulating levels of triglycerides (TAGs) and the development of hepatic steatosis (Allen et al, 2014; Goedeke et al, 2014). Most of the beneficial effects of miR-33 on atherosclerosis have been attributed to its ability to increase circulating levels of HDL-C and/or promote Mθ cholesterol efflux (Rayner et al, 2011b; Rayner et al, 2010), although additional functions of miR-33 including regulation of mitochondrial function, autophagy and Mθ activation status may also contribute to these effects (Karunakaran et al, 2015; Ouimet et al, 2017; Ouimet et al, 2015). Importantly, studies performed in monkeys have demonstrated that antagonism of miR-33 can also increase circulating levels of HDL-C in non-human primates (Rayner et al, 2011a; Rottiers et al, 2013).…”

Macrophages

“…miR-33 controls M2 polarization through the alteration of mitochondrial function 18 and key metabolic (AMP-activated protein kinase) 19 and autophagy effectors, 20,21 of high relevance to atherosclerosis and associated cardiometabolic diseases. Long-term inhibition of miR-33 (subcutaneous anti-miR) in C57/Bl6 mice fed a high-fat diet upregulates hepatic ABCA1 (but not SREBP1), promotes whole-body oxidative metabolism and M2 polarization in adipose tissue, and decreases circulating triglyceride levels, although it does not improve insulin resistance.…”

Recent Advances in the Genetics of Atherothrombotic Disease and Its Determinants

“…30 Studies reported in ATVB emphasize the diversity of miRNA species that affect different pathways underlying atherogenesis and vascular biology. For instance, Ouimet et al 31 investigated whether miR-33 contributes to cholesterol homeostasis by targeting autophagy. They showed that miR-33 targets key autophagy regulators and effectors in macrophages to reduce lipid droplet catabolism, which is essential to generate free cholesterol for efflux.…”