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Understanding the derangements in cellular processes that underlie macrophage dysfunction is critical to the development of future therapeutics. The autophagy-lysosome system in macrophages plays critical role in the degradation and recycling of long-lived/damaged intracellular material. Atherosclerotic plaque macrophages develop an impairment in autophagy and lysosomes which is a contributing factor to plaque progression. Thus, one can surmise that harnessing programs of lysosomal repair and lysosomal biogenesis in macrophages can have atheroprotective effects. A class of important regulators of the autophagy-lysosomal system are cytosolic lectins. When the lysosome membrane becomes damaged, Galectin-3 (Gal3) recognizes the damage and recruits autophagic receptors and delivers the cargo to autophagosomes. Additionally, Gal3 may recruit the ESCRT complex and facilitate the repair and restoration of lysosomes. We hypothesized that Gal3 provides a protective role in atherogenic macrophages, by repairing or removing damaged lysosomes, thus preventing the relevant deleterious signaling pathways such as inflammasome activation and apoptosis. We utilized Gal3-KO macrophages and mice to study the role of Gal3 in atherosclerosis. First, we show that Gal3-KO macrophages are highly sensitive to lysosome membrane damaging agents including atherogenic oxLDL and cholesterol crystals, exhibiting decreased autophagy and protein aggregate clearance compared to the WT controls. Furthermore, the recruitment of lysosome repair mechanisms involving the ESCRT complex are blunted. Such blunting of critical repair mechanisms results in increased macrophage apoptosis and inflammasome/IL-1β activation. These findings are also recapitulated in vivo, where bone marrow transplants of atherogenic LDLR-null mice with Gal3-deficient bone marrow results in increased plaque size and altered plaque composition with significant macrophage accumulation, apoptosis, and necrotic core formation, characteristic features of advanced plaques. Overall, our data demonstrate important roles for Gal3 in macrophage lysosome repair mechanisms which are a critical response against atherosclerosis.

The mechanistic target of rapamycin (mTOR) pathway is considered a key regulator of cellular proliferation, survival, metabolism, and degradation. Yet its role in atherosclerosis and in macrophages of the atherosclerotic plaque remains poorly understood. Our previous study has demonstrated macrophage mTORC1 signaling promotes atherogenesis through inhibiting autophagy and increasing apoptosis. Using macrophage-specific Rictor- and mTOR-deficient mice, we now dissect the distinct functions of mTORC2 pathways in atherogenesis. In contrast to the protective effect against atherosclerosis from blocking macrophage mTORC1, macrophage-specific mTORC2-deficient mice have the opposite phenotype with the development of larger and more complex lesions. In cultured macrophages, we show that mTORC2 signaling inhibits the FoxO1 transcription factor leading to suppression of pro-inflammatory pathways, especially the inflammasome/IL-1β response, which also manifest in vivo. In addition, administration of FoxO1 inhibitors efficiently rescued the hyperinflammation caused by mTORC2 deficiency in vitro and in vivo. Interestingly, deletion of macrophage mTOR, which ablates both mTOR-dependent pathways leads to minimal change in plaques reflecting the apparently balanced and opposing roles of its signaling arms. Our data provide the first mechanistic details of macrophage mTOR signaling in atherosclerosis and suggest that therapeutic measures aimed at modulating mTOR need to account for its dichotomous functions.

Defects in the E3 ubiquitin ligase Parkin are sine qua non of Parkinson and related neurodegenerative diseases. Loss of ubiquitination of distinct targets leads to accumulation of cytotoxic protein aggregates, loss of mitophagy, and defects in lipid metabolism. Given the critical role of these processes in plaque macrophages, we set out to define the role of Parkin in atherosclerosis. Pro-atherogenic LDLR-KO mice were transplanted with bone marrow (BM) from wild type and Parkin-KO mice and lesion formation stimulated by 4 months of Western diet feeding. Mice transplanted with wild type BM developed lipid-rich atherosclerotic plaques in both the aortic root and whole aorta replete with foam cell macrophages. In contrast, mice transplanted with Parkin-deficient BM had significantly reduced plaque burden with concomitant reductions in lipid content, foam cell macrophages, and surrogates of plaque complexity including reduced lesional apoptosis and necrotic core formation. The paradoxical atherogenic role for Parkin including development of lipid-laden foam cell macrophages led us to focus on the impact of Parkin in macrophage lipid handling. We find that Parkin mediates the ubiquitination and stabilization of scavenger receptor CD36, a key lipid transporter in macrophages. Parkin-null macrophages had reduced expression of CD36 and concomitant reductions in oxLDL uptake. Further, Parkin deficiency decreased cholesterol efflux in macrophages exacerbating macrophage lipid storage. These results suggest that the non-canonical functions of Parkin in lipid metabolism outweigh its traditional cytoprotective roles in macrophages and mediate its deleterious effects on atherosclerotic progression.

Dysfunction in macrophage lysosomes leading to poor handling of atherogenic lipids, apoptotic cells, and other cytotoxic debris leads to progression of atherosclerotic plaque size and complexity. Stimulation of TFEB, the master transcriptional regulator of autophagy-lysosomal biogenesis, in macrophages has been shown to promote the degradative capacity of plaque macrophages with reductions in atherosclerosis. Thus, there is interest in harnessing this pathway for cardiovascular therapeutics. One of the mechanisms by which TFEB is activated and triggered to translocate to the nucleus is the release of calcium from the lysosomal lumen to the cytosol via specialized channels located at the lysosomal membrane including mucolipin 1, also known as the transient receptor potential cation channel mucolipin 1 (TRPML1). We find that TRPML1 is highly expressed in macrophages and is involved in a feedback loop by which TFEB positively regulates TRPML1 mRNA expression to further support long-term autophagy-lysosomal biogenesis. Utilizing a mammalian TRPML agonist, mucolipin synthetic agonist 1 (ML-SA1), we show that induction of lysosomal calcium release can robustly trigger TFEB nuclear translocation in macrophages. In turn, numerous downstream TFEB transcriptional targets mediating autophagy-lysosomal biogenesis are upregulated resulting in salutary effects including enhanced aggrephagy and clearance of cytotoxic p62-enriched protein aggregates, blunting of inflammasome activation and proinflammatory IL-1β production, and reductions in macrophage apoptosis. Taken together, our data support that stimulation of lysosomal calcium flux can be a novel strategy of enhancing TFEB and macrophage degradative capacity in atherosclerosis.

High-protein intake is common in Western societies and generally considered healthy. However, results from some epidemiological studies suggest elevated protein intake is associated with increased risk for ischemic cardiovascular diseases. In addition, results from studies conducted in mice show that a high protein, compared with a standard Western diet, increases atherosclerosis burden and lesion complexity. The adverse effect of protein ingestion on plaque biology in mice is mediated by amino acid-mammalian target of rapamycin (mTOR)-dependent inhibition of autophagy in macrophages. Here, we evaluate the effect of graded amounts of protein ingestion on this amino acid-mTORC1-autophagy mechanism in human monocytes/macrophages and identify leucine as the key amino acid responsible for activating mTORC1 in macrophages. We describe the presence of a threshold effect of high protein intake on this deleterious signaling pathway wherein protein content greater than about 22% of total energy, which is consumed by nearly 1/4 th of the population in Western societies, acutely activates mTORC1 signaling in monocytes/macrophages. Furthermore, we identify leucine as the critical amino acid modulator and threshold indicator, capable of the dose-dependent mTORC1 activation and downstream functional effects. Finally, by designing specific mouse diets with protein contents mimicking graded levels of protein ingestion in our study participants, we demonstrate the presence of a dietary protein threshold effect in driving atherosclerosis in mouse models. These data demonstrate the potential deleterious impact of excessive protein intake on macrophages and atherosclerotic plaque progression.

Background: Atherosclerosis is characterized by accumulation of modified lipoproteins in intima of aorta leads to the infiltration of monocytes/macrophages. In the atherosclerotic plaque, these macrophages uptake modified lipoproteins and become foam cells. Macrophage 23-kD stress protein (MSP23), a stress-inducible protein, was found for the first time in the macrophages. Recent reports suggested that the shear stress induce MSP23 expression in endothelial cells. However, the role of MSP23 in macrophages has not been studied yet. In this study, we aim to elucidate atheroprotective effect of MSP23 in macrophages. Methods and Results: We show that MSP23 is highly expressed in macrophages among atherogenesis related cells. Double deficiency of MSP23/ApoE mice accelerated plaque formation with increased macrophage infiltrations. Biochemical analyses revealed that MSP23/ApoE double deficient mice exhibited a significant increase in total cholesterol in serum when compared to ApoE deficient mice. Macrophages from MSP23/ApoE double deficient mice expressed higher levels of scavenger receptors and took up more modified lipoproteins compared with ApoE deficient mice, but they expressed low levels of ATP binding cassette protein family A member 1 and were defective in cholesterol efflux. Furthermore, we found that macrophage CE mass was increased in MSP23 deficient macrophages. In bone marrow transplantation experiments, we confirmed that MSP23 plays pivotal atheorprotecitve role in macrophage lineage cells. Conclusions: In this study, we elucidate that MSP23 inhibits atherogenesis by intestinal cholesterol ester hydrolysis and augmenting cholesterol efflux from macrophages resulting in reducing formation of foam cells. Thus, MSP23 appears to be a promising therapeutic target for enhancing cholesterol efflux.