Autophagy Is Differentially Regulated in Leukocyte and Nonleukocyte Foam Cells During Atherosclerosis.

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 (microtubule-associated proteins 1A/1B light chain 3) 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 VSMCs and macrophages, 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) was 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.

Expression of murine muscle-enriched A-type lamin-interacting protein (MLIP) is regulated by tissue-specific alternative transcription start sites.

Muscle-enriched lamin-interacting protein (Mlip) is an alternatively spliced gene whose splicing specificity is dictated by tissue type. MLIP is most abundantly expressed in brain, cardiac, and skeletal muscle. In the present study, we systematically mapped the transcriptional start and stop sites of murine Mlip Rapid amplification of cDNA ends (RACE) of Mlip transcripts from the brain, heart, and skeletal muscle revealed two transcriptional start sites (TSSs), exon 1a and exon 1b, and only one transcriptional termination site. RT-PCR analysis of the usage of the two identified TSSs revealed that the heart utilizes only exon 1a for MLIP expression, whereas the brain exclusively uses exon 1b TSS. Loss of Mlip exon 1a in mice resulted in a 7-fold increase in the prevalence of centralized nuclei in muscle fibers with the Mlip exon1a-deficient satellite cells on single fibers exhibiting a significant delay in commitment to a MYOD-positive phenotype. Furthermore, we demonstrate that the A-type lamin-binding domain in MLIP is encoded in exon 1a, indicating that MLIP isoforms generated with exon 1b TSS lack the A-type lamin-binding domain. Collectively these findings suggest that Mlip tissue-specific expression and alternative splicing play a critical role in determining MLIP's functions in mice.

Deletion of MLIP (muscle-enriched A-type lamin-interacting protein) leads to cardiac hyperactivation of Akt/mammalian target of rapamycin (mTOR) and impaired cardiac adaptation.

Aging and diseases generally result from tissue inability to maintain homeostasis through adaptation. The adult heart is particularly vulnerable to disequilibrium in homeostasis because its regenerative abilities are limited. Here, we report that MLIP (muscle enriched A-type lamin-interacting protein), a unique protein of unknown function, is required for proper cardiac adaptation. Mlip(-/-) mice exhibited normal cardiac function despite myocardial metabolic abnormalities and cardiac-specific overactivation of Akt/mTOR pathways. Cardiac-specific MLIP overexpression led to an inhibition of Akt/mTOR, providing evidence of a direct impact of MLIP on these key signaling pathways. Mlip(-/-) hearts showed an impaired capacity to adapt to stress (isoproterenol-induced hypertrophy), likely because of deregulated Akt/mTOR activity. Genome-wide association studies showed a genetic association between Mlip and early response to cardiac stress, supporting the role of MLIP in cardiac adaptation. Together, these results revealed that MLIP is required for normal myocardial adaptation to stress through integrated regulation of the Akt/mTOR pathways.

Phagotrophy in the nitrogen-fixing haptophyte Braarudosphaera bigelowii.

Biological nitrogen fixation provides fixed nitrogen for microbes living in the oligotrophic open ocean. UCYN-A2, the previously known symbiont of Braarudosphaera bigelowii, now believed to be an early-stage B. bigelowii organelle that exchanges fixed nitrogen for fixed carbon, is globally distributed. Indirect evidence suggested that B. bigelowii might be a mixotrophic (phagotrophic) phototrophic flagellate. The goal of this study was to determine if B. bigelowii can graze on bacteria using several independent approaches. The results showed that B. bigelowii grazed on co-occurring bacteria at a rate of 5-7 cells/h/B. bigelowii and that the overall grazing rate was significantly higher at nighttime than at daytime. Bacterial abundance changes, assessed with 16S rRNA gene amplicon sequencing analysis, may have indicated preferential grazing by B. bigelowii on specific bacterial genotypes. In addition, Lysotracker™ staining of B. bigelowii suggested digestive activity inside B. bigelowii. Carbon and nitrogen fixation measurements revealed that the carbon demand of B. bigelowii could not be fulfilled by photosynthesis alone, implying supplementation by heterotrophy. These independent lines of evidence together revealed that B. bigelowii engages in phagotrophy, which, beyond serving as a supplementary source of carbon and energy, may also facilitate the indirect assimilation of inorganic nutrients.

Cell-specific measurements show nitrogen fixation by particle-attached putative non-cyanobacterial diazotrophs in the North Pacific Subtropical Gyre.

Biological nitrogen fixation is a major important source of nitrogen for low-nutrient surface oceanic waters. Nitrogen-fixing (diazotrophic) cyanobacteria are believed to be the primary contributors to this process, but the contribution of non-cyanobacterial diazotrophic organisms in oxygenated surface water, while hypothesized to be important, has yet to be demonstrated. In this study, we used simultaneous 15N-dinitrogen and 13C-bicarbonate incubations combined with nanoscale secondary ion mass spectrometry analysis to screen tens of thousands of mostly particle-associated, cell-like regions of interest collected from the North Pacific Subtropical Gyre. These dual isotope incubations allow us to distinguish between non-cyanobacterial and cyanobacterial nitrogen-fixing microorganisms and to measure putative cell-specific nitrogen fixation rates. With this approach, we detect nitrogen fixation by putative non-cyanobacterial diazotrophs in the oxygenated surface ocean, which are associated with organic-rich particles (<210 µm size fraction) at two out of seven locations sampled. When present, up to 4.1% of the analyzed particles contain at least one active putative non-cyanobacterial diazotroph. The putative non-cyanobacterial diazotroph nitrogen fixation rates (0.76 ± 1.60 fmol N cell-1 d-1) suggest that these organisms are capable of fixing dinitrogen in oxygenated surface water, at least when attached to particles, and may contribute to oceanic nitrogen fixation.

Two co-dominant nitrogen-fixing cyanobacteria demonstrate distinct acclimation and adaptation responses to cope with ocean warming.

The globally dominant N2 -fixing cyanobacteria Trichodesmium and Crocosphaera provide vital nitrogen supplies to subtropical and tropical oceans, but little is known about how they will be affected by long-term ocean warming. We tested their thermal responses using experimental evolution methods during 2 years of selection at optimal (28°C), supra-optimal (32°C) and suboptimal (22°C) temperatures. After several hundred generations under thermal selection, changes in growth parameters, as well as N and C fixation rates, suggested that Trichodesmium did not adapt to the three selection temperature regimes during the 2-year evolution experiment, but could instead rapidly and reversibly acclimate to temperature shifts from 20°C to 34°C. In contrast, over the same timeframe apparent thermal adaptation was observed in Crocosphaera, as evidenced by irreversible phenotypic changes as well as whole-genome sequencing and variant analysis. Especially under stressful warming conditions (34°C), 32°C-selected Crocosphaera cells had an advantage in survival and nitrogen fixation over cell lines selected at 22°C and 28°C. The distinct strategies of phenotypic plasticity versus irreversible adaptation in these two sympatric diazotrophs are both viable ways to maintain fitness despite long-term temperature changes, and so could help to stabilize key ocean nitrogen cycle functions under future warming scenarios.

Identification of novel lipid droplet factors that regulate lipophagy and cholesterol efflux in macrophage foam cells.

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 transporter 2; OPTN: optineurin; P/S: penicillin-streptomycin; PLIN2: perilipin 2; PLIN3: perilipin 3; PNPLA2: patatin like phospholipase domain containing 2; RAB: RAB, member RAS oncogene family; RBBP7, retinoblastoma binding protein 7, chromatin remodeling factor; SAR: selective autophagy receptor; SCARB2: scavenger receptor class B, member 2; SGA: synthetic genetic array; SQSTM1: sequestosome 1; TAX1BP1: Tax1 (human T cell leukemia virus type I) binding protein 1; TFEB: transcription factor EB; TOLLIP: toll interacting protein; UBE2G2: ubiquitin conjugating enzyme E2 G2; UVRAG: UV radiation resistance associated gene; VDAC2: voltage dependent anion channel 2; VIM: vimentin.

Autophagy regulates cholesterol efflux from macrophage foam cells via lysosomal acid lipase.

The lipid droplet (LD) is the major site of cholesterol storage in macrophage foam cells and is a potential therapeutic target for the treatment of atherosclerosis. Cholesterol, stored as cholesteryl esters (CEs), is liberated from this organelle and delivered to cholesterol acceptors. The current paradigm attributes all cytoplasmic CE hydrolysis to the action of neutral CE hydrolases. Here, we demonstrate an important role for lysosomes in LD CE hydrolysis in cholesterol-loaded macrophages, in addition to that mediated by neutral hydrolases. Furthermore, we demonstrate that LDs are delivered to lysosomes via autophagy, where lysosomal acid lipase (LAL) acts to hydrolyze LD CE to generate free cholesterol mainly for ABCA1-dependent efflux; this process is specifically induced upon macrophage cholesterol loading. We conclude that, in macrophage foam cells, lysosomal hydrolysis contributes to the mobilization of LD-associated cholesterol for reverse cholesterol transport.

p38 MAPK signaling regulates recruitment of Ash2L-containing methyltransferase complexes to specific genes during differentiation.

Cell-specific patterns of gene expression are established through the antagonistic functions of trithorax group (TrxG) and Polycomb group (PcG) proteins. Several muscle-specific genes have previously been shown to be epigenetically marked for repression by PcG proteins in muscle progenitor cells. Here we demonstrate that these developmentally regulated genes become epigenetically marked for gene expression (trimethylated on histone H3 Lys4, H3K4me3) during muscle differentiation through specific recruitment of Ash2L-containing methyltransferase complexes. Targeting of Ash2L to specific genes is mediated by the transcriptional regulator Mef2d. Furthermore, this interaction is modulated during differentiation through activation of the p38 MAPK signaling pathway via phosphorylation of Mef2d. Thus, we provide evidence that signaling pathways regulate the targeting of TrxG-mediated epigenetic modifications at specific promoters during cellular differentiation.