Macrophage autophagy is a highly anti-atherogenic process that promotes the catabolism of cytosolic lipid droplets (LDs) to maintain cellular lipid homeostasis. Selective autophagy relies on tags such as ubiquitin and a set of selectivity factors including selective autophagy receptors (SARs) to label specific cargo for degradation. Originally described in yeast cells, “lipophagy” refers to the degradation of LDs by autophagy. Yet, how LDs are targeted for autophagy is poorly defined. Here, we employed mass spectrometry to identify lipophagy factors within the macrophage foam cell LD proteome. In addition to structural proteins (e.g., PLIN2), metabolic enzymes (e.g., ACSL) and neutral lipases (e.g., PNPLA2), we found the association of proteins related to the ubiquitination machinery (e.g., AUP1) and autophagy (e.g., HMGB, YWHA/14-3-3 proteins). The functional role of candidate lipophagy factors (a total of 91) was tested using a custom siRNA array combined with high-content cholesterol efflux assays. We observed that knocking down several of these genes, including Hmgb1, Hmgb2, Hspa5 , and Scarb2 , significantly reduced cholesterol efflux, and SARs SQSTM1/p62, NBR1 and OPTN localized to LDs, suggesting a role for these in lipophagy. Using yeast lipophagy assays, we established a genetic requirement for several candidate lipophagy factors in lipophagy, including HSPA5, UBE2G2 and AUP1. Our study is the first to systematically identify several LD-associated proteins of the lipophagy machinery, a finding with important biological and therapeutic implications. Targeting these to selectively enhance lipophagy to promote cholesterol efflux in foam cells may represent a novel strategy to treat atherosclerosis. Abbreviations: ADGRL3: adhesion G protein-coupled receptor L3; agLDL: aggregated low density lipoprotein; AMPK: AMP-activated protein kinase; APOA1: apolipoprotein A1; ATG: autophagy related; AUP1: AUP1 lipid droplet regulating VLDL assembly factor; BMDM: bone-marrow derived macrophages; BNIP3L: BCL2/adenovirus E1B interacting protein 3-like; BSA: bovine serum albumin; CALCOCO2: calcium binding and coiled-coil domain 2; CIRBP: cold inducible RNA binding protein; COLGALT1: collagen beta(1-O)galactosyltransferase 1; CORO1A: coronin 1A; DMA: deletion mutant array; Faa4: long chain fatty acyl-CoA synthetase; FBS: fetal bovine serum; FUS: fused in sarcoma; HMGB1: high mobility group box 1; HMGB2: high mobility group box 2: HSP90AA1: heat shock protein 90: alpha (cytosolic): class A member 1; HSPA5: heat shock protein family A (Hsp70) member 5; HSPA8: heat shock protein 8; HSPB1: heat shock protein 1; HSPH1: heat shock 105kDa/110kDa protein 1; LDAH: lipid droplet associated hydrolase; LIPA: lysosomal acid lipase A; LIR: LC3-interacting region; MACROH2A1: macroH2A.1 histone; MAP1LC3: microtubule-associated protein 1 light chain 3; MCOLN1: mucolipin 1; NBR1: NBR1, autophagy cargo receptor; NPC2: NPC intracellular cholesterol t...

Rationale: Atherosclerosis is characterized by an accumulation of foam cells within the arterial wall, resulting from excess cholesterol uptake and buildup of cytosolic lipid droplets (LDs). Autophagy promotes LD clearance by freeing stored cholesterol for efflux, a process that has been shown to be atheroprotective. While the role of autophagy in LD catabolism has been studied in macrophage-derived foam cells, this has remained unexplored in vascular smooth muscle cell (VSMC)-derived foam cells that constitute a large fraction of foam cells within atherosclerotic lesions. Objective: We performed a comparative analysis of autophagy flux in lipid-rich aortic intimal populations to determine whether VSMC-derived foam cells metabolize LDs similarly to their macrophage counterparts. Methods and Results: Atherosclerosis was induced in GFP-LC3 transgenic mice by PCSK9 (proprotein convertase subtilisin/kexin type 9)-adeno-associated viral injection and Western diet feeding. Using flow cytometry of aortic digests, we observed a significant increase in dysfunctional autophagy of VSMC-derived foam cells during atherogenesis relative to macrophage-derived foam cells. Using cell culture models of lipid-loaded VSMC and macrophage, we show that autophagy-mediated cholesterol efflux from VSMC foam cells was poor relative to macrophage foam cells, and largely occurs when HDL (high-density lipoprotein) is used as a cholesterol acceptor, as opposed to apoA-1 (apolipoproteinA-1). This was associated with the predominant expression of ABCG1 in VSMC foam cells. Using metformin, an autophagy activator, cholesterol efflux to HDL was significantly increased in VSMC, but not in macrophage, foam cells. Conclusions: These data demonstrate that VSMC and macrophage foam cells perform cholesterol efflux by distinct mechanisms, and that autophagy flux is highly impaired in VSMC foam cells, but can be induced by pharmacological means. Further investigation is warranted into targeting autophagy specifically in VSMC foam cells, the predominant foam cell subtype of advanced atherosclerotic plaques, to promote reverse cholesterol transport and resolution of the atherosclerotic plaque.

A hallmark of sterile and nonsterile inflammation is the increased accumulation of cytoplasmic lipid droplets (LDs) in non-adipose cells. LDs are ubiquitous organelles specialized in neutral lipid storage and hydrolysis. Originating in the ER, LDs are comprised of a core of neutral lipids (cholesterol esters, triglycerides) surrounded by a phospholipid monolayer and several LD-associated proteins. The perilipin (PLIN1-5) family are the most abundant structural proteins present on the surface of LDs. While PLIN1 is primarily expressed in adipocytes, PLIN2 and PLIN3 are ubiquitously expressed. LDs also acquire a host of enzymes and proteins that regulate LD metabolism. Amongst these are neutral lipases and selective lipophagy factors that promote hydrolysis of LD-associated neutral lipid. In addition, LDs physically associate with other organelles such as mitochondria through inter-organelle membrane contact sites that facilitate lipid transport. Beyond serving as a source of energy storage, LDs participate in inflammatory and infectious diseases, regulating both innate and adaptive host immune responses. Here, we review recent studies on the role of LDs in the regulation of immunometabolism.

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Atherosclerosis is characterized by a build-up of foam cells in the arterial wall, resulting from excess cholesterol uptake and accumulation of cytosolic lipid droplets (LDs). Autophagy has been shown to be atheroprotective in part by promoting the catabolism of LDs which liberates free cholesterol for efflux out of foam cells to cholesterol acceptors (ApoA-I or HDL) for removal from the body. Apart from macrophages (MΦ), vascular smooth muscle cells (VSMCs) comprise 50-70% of foam cells in the plaques. Unlike MΦ, the capacity of VSMC foam cells to metabolize cholesterol via autophagy is unknown. Here, we performed a comparative analysis of the autophagic capacity and cholesterol efflux of arterial foam cell subtypes of the atherosclerotic plaque. Atherosclerosis was induced in hypercholesterolemic autophagy reporter mice (GFP-LC3 mice receiving PCSK9-AAV and fed a Western diet). Autophagic flux in aortic digests was assessed by quantifying GFP-LC3 fluorescence after ex vivo treatment with the autophagy inhibitor Bafilomycin A1 or vehicle. MΦ foam cells displayed functional autophagy as shown by an accumulation of GFP-LC3 upon autophagy inhibition. In contrast, VSMC foam cells did not similarly accumulate GFP-LC3 upon bafilomycin treatment, suggesting dysfunction autophagy in these cells. Additionally, immunostaining of late-stage aortic roots showed MΦ, but not VSMC, foam cells induction of the active autophagy marker pATG16L1. Cell culture studies of lipid loaded MΦ and VSMC corroborated this inability for VSMCs to initiate autophagy in vivo . In vitro , MΦ foam cells effluxed cholesterol to ApoA-I (14%) and HDL (50%), whereas VSMC foam cells minimally effluxed cholesterol to HDL (7%) but not apoA-I. However, unlike MΦ foam cells, VSMC efflux was pharmacologically induced by treatment with metformin. Our data therefore demonstrates a lack of functional autophagy in VSMC, as compared to MΦ foam cells, which impairs their ability to perform cholesterol efflux. This autophagy defect in VSMC foam cells can be increased by autophagy activation using metformin, highlighting both the importance of understanding cholesterol metabolism in all foam cell populations and a new avenue to treat atherosclerosis.

Atherosclerosis is characterized by a build-up of foam cells in the arterial wall, resulting from excess cholesterol uptake and accumulation of cytosolic lipid droplets (LDs). Autophagy has been shown to be atheroprotective in part by promoting the catabolism of LDs which liberates free cholesterol for efflux out of foam cells to cholesterol acceptors (ApoA-I or HDL) for removal from the body. Apart from macrophages (MΦ), vascular smooth muscle cells (VSMCs) comprise 50-70% of foam cells in the plaques. Unlike MΦ, the capacity of VSMC foam cells to metabolize cholesterol via autophagy is unknown. Here, w e performed a comparative analysis of the autophagic capacity and cholesterol efflux of arterial foam cell subtypes of the atherosclerotic plaque . Atherosclerosis was induced in hypercholesterolemic autophagy reporter mice (GFP-LC3 mice receiving PCSK9-AAV and fed a Western diet). Autophagic flux in aortic digests was assessed by quantifying GFP-LC3 fluorescence after ex vivo treatment with the autophagy inhibitor Bafilomycin A1 or vehicle. MΦ foam cells displayed functional autophagy as shown by an accumulation of GFP-LC3 upon autophagy inhibition. In contrast, VSMC foam cells did not similarly accumulate GFP-LC3 upon bafilomycin treatment, suggesting dysfunction autophagy in these cells. Additionally, immunostaining of late-stage aortic roots showed MΦ, but not VSMC, foam cells induction of the active autophagy marker pATG16L1. Cell culture studies of lipid loaded MΦ and VSMC corroborated this inability for VSMCs to initiate autophagy in vivo . In vitro , MΦ foam cells effluxed cholesterol to ApoA-I (14%) and HDL (50%), whereas VSMC foam cells minimally effluxed cholesterol to HDL (7%) but not apoA-I. However, unlike MΦ foam cells, VSMC efflux was pharmacologically induced by treatment with metformin. Our data therefore demonstrates a lack of functional autophagy in VSMC, as compared to MΦ foam cells, which impairs their ability to perform cholesterol efflux. This autophagy defect in VSMC foam cells can be increased by autophagy activation using metformin, highlighting both the importance of understanding cholesterol metabolism in all foam cell populations and a new avenue to treat atherosclerosis.

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