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Defective inflammation resolution is the underlying cause of prevalent chronic inflammatory diseases, such as arthritis, asthma, cancer, and neurodegenerative and cardiovascular diseases. Inflammation resolution is governed by several endogenous factors, including fatty acid-derived specialized proresolving mediators and proteins, such as annexin A1. Specifically, specialized proresolving mediators comprise a family of mediators that include arachidonic acid-derived lipoxins, omega-3 fatty acid eicosapentaenoic acid-derived resolvins, docosahexaenoic acid-derived resolvins, protectins, and maresins. Emerging evidence indicates that imbalances between specialized proresolving mediators and proinflammatory mediators are associated with several prevalent human diseases, including atherosclerosis. Mechanisms that drive this imbalance remain largely unknown and will be discussed in this review. Furthermore, the concept of dysregulated inflammation resolution in atherosclerosis has been known for several decades. Recently, there has been an explosion of new work with regard to the therapeutic application of proresolving ligands in experimental atherosclerosis. Therefore, this review will highlight recent advances in our understanding of how inflammation resolution may become defective in atherosclerosis and the potential for proresolving therapeutics in atherosclerosis. Last, we offer insight for future implications of the field.

Atherosclerosis is initiated by the subendothelial accumulation of apolipoprotein B-containing lipoproteins (apoB LPs), which initiates a sterile inflammatory response dominated by monocyte-macrophages but including all classes of innate and adaptive immune cells. These inflammatory cells, together with proliferating smooth muscle cells and extracellular matrix, promote the formation of subendothelial lesions, or plaques. In the vast majority of cases, these lesions do not cause serious clinical symptoms, which is due in part to a resolution-repair response that limits tissue damage. However, a deadly minority of lesions progress to the point where they can trigger acute lumenal thrombosis, which may then cause unstable angina, myocardial infarction, sudden cardiac death, or stroke. Many of these clinically dangerous lesions have hallmarks of defective inflammation resolution, including defective clearance of dead cells (efferocytosis), necrosis, a defective scar response, and decreased levels of lipid mediators of the resolution response. Efferocytosis is both an effector arm of the resolution response and an inducer of resolution mediators, and thus its defect in advanced atherosclerosis amplifies plaque progression. Pre-clinical causation/treatment studies have demonstrated that "replacement therapy" with exogenously administered resolving mediators can improve lesional efferocytosis and prevent plaque progression. Work in this area has the potential to potentiate the cardiovascular benefits of apoB LP-lowering therapy.

Summary Nonalcoholic steatohepatitis (NASH) is a leading cause of liver disease worldwide. However, the molecular basis of how benign steatosis progresses to NASH is incompletely understood, which has limited the identification of therapeutic targets. Here we show that the transcription regulator TAZ (WWTR1) is markedly higher in hepatocytes in human and murine NASH liver than in normal or steatotic liver. Most importantly, silencing of hepatocyte TAZ in murine models of NASH prevented or reversed hepatic inflammation, hepatocyte death, and fibrosis, but not steatosis. Moreover, hepatocyte-targeted expression of TAZ in a model of steatosis promoted NASH features, including fibrosis. In vitro and in vivo mechanistic studies revealed that a key mechanism linking hepatocyte TAZ to NASH fibrosis is TAZ/TEA domain (TEAD)-mediated induction of Indian hedgehog (Ihh), a secretory factor that activates fibrogenic genes in hepatic stellate cells. In summary, TAZ represents a previously unrecognized factor that contributes to the critical process of steatosis-to-NASH progression.

Positive regulation of hepatic glucose production (HGP) and stimulation of glycogenolysis during fasting are critical processes to prevent hypoglycemia. A key hormone that carries out this function is glucagon, which is secreted by pancreatic α-cells in response to fasting and acts on its receptor on hepatocytes to promote HGP and glycogenolysis (1). However, as often happens, processes that are critical in physiology can promote disease processes in nonphysiological settings. As such, glucagon-induced HGP is a major contributor to excessive HGP and hyperglycemia in obesity and type 2 diabetes (T2D) (2). In this issue of Diabetes , Wang et al. (3) provide evidence that another circulating factor that is increased in fasting and obesity/T2D—prostaglandin F2α (PGF2α)—also promotes HGP in both fasting and obese, insulin-resistant mice. Previous work has elucidated a number of pathways through which activation of the hepatocyte glucagon receptor (GcgR), which is a G-protein–coupled receptor (GPCR), promotes HGP and glycogenolysis in both fasting and obesity. Specifically, adenylyl cyclase/cyclic AMP-mediated protein kinase A (PKA) activation by G proteins of the GcgR controls the transcriptional regulation of HGP and glycogenolysis through at least three distinct but complementary processes (Fig. 1): phosphorylation of CREB, which results in recruitment of its coactivators, CBP and p300; deactivation of a kinase called SIK2; and activation of inositol 3-phosphate receptors (IP3Rs), leading to release of endoplasmic reticulum calcium into the cytosol and subsequent activation of the phosphatase calcineurin and the kinase Ca2+/calmodulin-activated protein kinase IIγ …

Atherosclerotic lesional macrophages express molecules that promote plaque progression, but lack of mechanisms to therapeutically target these molecules represents a major gap in translational cardiovascular research. Here, we tested the efficacy of a small interfering RNA (siRNA) nanoparticle (NP) platform targeting a plaque-destabilizing macrophage molecule—Ca(2+)/calmodulin-dependent protein kinase γ (CaMKIIγ). CaMKIIγ becomes activated in advanced human and mouse plaque macrophages and drives plaque necrosis by suppressing the expression of the efferocytosis receptor MerTK. When macrophage-targeted siCamk2g NPs were administered to Western diet–fed Ldlr(−/−) mice, the atherosclerotic lesions showed decreased CaMKIIγ and increased MerTK expression in macrophages, improved phagocytosis of apoptotic cells (efferocytosis), decreased necrotic core area, and increased fibrous cap thickness—all signs of increased plaque stability—compared with mice treated with control siRNA NPs. These findings demonstrate that atherosclerosis-promoting genes in plaque macrophages can be targeted with siRNA NPs in a preclinical model of advanced atherosclerosis.

Atherosclerosis is a lipid-driven inflammatory disease of the arterial intima in which the balance of pro-inflammatory and inflammation-resolving mechanisms dictates the final clinical outcome. Intimal infiltration and modification of plasma-derived lipoproteins and their uptake mainly by macrophages, with ensuing formation of lipid-filled foam cells, initiate atherosclerotic lesion formation, and deficient efferocytotic removal of apoptotic cells and foam cells sustains lesion progression. Defective efferocytosis, as a sign of inadequate inflammation resolution, leads to accumulation of secondarily necrotic macrophages and foam cells and the formation of an advanced lesion with a necrotic lipid core, indicative of plaque vulnerability. Resolution of inflammation is mediated by specialized pro-resolving lipid mediators derived from omega-3 fatty acids or arachidonic acid and by relevant proteins and signalling gaseous molecules. One of the major effects of inflammation resolution mediators is phenotypic conversion of pro-inflammatory macrophages into macrophages that suppress inflammation and promote healing. In advanced atherosclerotic lesions, the ratio between specialized pro-resolving mediators and pro-inflammatory lipids (in particular leukotrienes) is strikingly low, providing a molecular explanation for the defective inflammation resolution features of these lesions. In this Review, we discuss the mechanisms of the formation of clinically dangerous atherosclerotic lesions and the potential of pro-resolving mediator therapy to inhibit this process.

The mechanisms driving atherothrombotic risk in individuals with JAK2The goal of this study was to assess atherosclerosis and underlying mechanisms in hypercholesterolemic mice with hematopoietic Jak2Irradiated low-density lipoprotein receptor knockout ( LdlrHematopoietic Jak2

Chronic unresolved inflammation plays a causal role in the development of advanced atherosclerosis, but the mechanisms that prevent resolution in atherosclerosis remain unclear. Here, we use targeted mass spectrometry to identify specialized pro-resolving lipid mediators (SPM) in histologically-defined stable and vulnerable regions of human carotid atherosclerotic plaques. The levels of SPMs, particularly resolvin D1 (RvD1), and the ratio of SPMs to pro-inflammatory leukotriene B4 (LTB4), are significantly decreased in the vulnerable regions. SPMs are also decreased in advanced plaques of fat-fed Ldlr−/− mice. Administration of RvD1 to these mice during plaque progression restores the RvD1:LTB4 ratio to that of less advanced lesions and promotes plaque stability, including decreased lesional oxidative stress and necrosis, improved lesional efferocytosis, and thicker fibrous caps. These findings provide molecular support for the concept that defective inflammation resolution contributes to the formation of clinically dangerous plaques and offer a mechanistic rationale for SPM therapy to promote plaque stability., Atherosclerosis progression is linked to inflammatory processes in the blood vessel wall. Here, the authors show that, with the progression of atherosclerosis, the resolution of inflammation is impaired as the result of an imbalance between specialized pro-resolving lipid mediators and leukotrienes.

The acute inflammatory response requires a coordinated resolution program to prevent excessive inflammation, repair collateral damage, and restore tissue homeostasis, and failure of this response contributes to the pathology of numerous chronic inflammatory diseases. Resolution is mediated in part by long-chain fatty acid-derived lipid mediators called specialized proresolving mediators (SPMs). However, how SPMs are regulated during the inflammatory response, and how this process goes awry in inflammatory diseases, are poorly understood. We now show that signaling through the Mer proto-oncogene tyrosine kinase (MerTK) receptor in cultured macrophages and in sterile inflammation in vivo promotes SPM biosynthesis by a mechanism involving an increase in the cytoplasmic:nuclear ratio of a key SPM biosynthetic enzyme, 5-lipoxygenase. This action of MerTK is linked to the resolution of sterile peritonitis and, after ischemia-reperfusion (I/R) injury, to increased circulating SPMs and decreased remote organ inflammation. MerTK is susceptible to ADAM metallopeptidase domain 17 (ADAM17)-mediated cell-surface cleavage under inflammatory conditions, but the functional significance is not known. We show here that SPM biosynthesis is increased and inflammation resolution is improved in a new mouse model in which endogenous MerTK was replaced with a genetically engineered variant that is cleavage-resistant (Mertk(CR)). Mertk(CR) mice also have increased circulating levels of SPMs and less lung injury after I/R. Thus, MerTK cleavage during inflammation limits SPM biosynthesis and the resolution response. These findings contribute to our understanding of how SPM synthesis is regulated during the inflammatory response and suggest new therapeutic avenues to boost resolution in settings where defective resolution promotes disease progression.

Inflammation is an essential protective biological response involving a coordinated cascade of signals between cytokines and immune signaling molecules that facilitate return to tissue homeostasis after acute injury or infection. However, inflammation is not effectively resolved in chronic inflammatory diseases such as atherosclerosis and can lead to tissue damage and exacerbation of the underlying condition. Therapeutics that dampen inflammation and enhance resolution are currently of considerable interest, in particular those that temper inflammation with minimal host collateral damage. Here we present the development and efficacy investigations of controlled-release polymeric nanoparticles incorporating the anti-inflammatory cytokine interleukin 10 (IL-10) for targeted delivery to atherosclerotic plaques. Nanoparticles were nanoengineered via self-assembly of biodegradable polyester polymers by nanoprecipitation using a rapid micromixer chip capable of producing nanoparticles with retained IL-10 bioactivity post-exposure to organic solvent. A systematic combinatorial approach was taken to screen nanoparticles, resulting in an optimal bioactive formulation from in vitro and ex vivo studies. The most potent nanoparticle termed Col-IV IL-10 NP22 significantly tempered acute inflammation in a self-limited peritonitis model and was shown to be more potent than native IL-10. Furthermore, the Col-IV IL-10 nanoparticles prevented vulnerable plaque formation by increasing fibrous cap thickness and decreasing necrotic cores in advanced lesions of high fat-fed LDLr(-/-) mice. These results demonstrate the efficacy and pro-resolving potential of this engineered nanoparticle for controlled delivery of the potent IL-10 cytokine for the treatment of atherosclerosis.

Nonalcoholic steatohepatitis (NASH) has emerged as the leading cause of chronic liver disease worldwide and is rapidly increasing in prevalence owing to the obesity epidemic. Incomplete understanding of the mechanisms of NASH progression, particularly fibrosis, has limited the design of therapeutic strategies. My previous published study indicated that a transcription factor in hepatocytes called TAZ (WWTR1) contributes to steatosis-to-NASH progression. However, the mechanism that induces high TAZ expression in NASH liver remains unknown. The objective of this study is to understand the mechanism of hepatocyte TAZ upregulation in NASH, which may suggest novel therapeutic strategies to prevent NASH and its comorbidities. We hypothesize that hepatic cholesterol upregulates TAZ through PKA-Ca2+-RhoA mediated Hippo pathway inactivation. In vitro, we use liposome to deplete and replete cholesterol in hepatocyte cells and check downstream signaling pathway that includes cholesterol translocation, Ca2+ signaling, RhoA activity, Hippo pathway, and E3 ligase activity. In vivo, we used different concentration cholesterol diet to build the correlation between increased TAZ/liver fibrosis and cholesterol, and AAV8-H1 mediated hepatic-gene silencing to prove the functions of the molecules in the pathway, including AsterB/C, RhoA, and Trcp. My data suggest that hepatic cholesterol accumulation is associated with liver fibrosis and increased TAZ. In addition, cholesterol loading of hepatocytes increases TAZ, and cholesterol depletion decreases TAZ in a proteasome/Trcp-dependent manner. Mechanistically, Hippo pathway kinase-Lats2 inhibits TAZ expression on the post-transcriptional level through TAZ phosphorylation. Moreover, intracellular cholesterol increases cytosolic Ca2+ and activates RhoA signaling, then represses Lats2 activity, so TAZ degradation is inhibited and total protein level increases. Finally, blocking cholesterol translocation from cell membrane to intracellular suppresses the pathway and inhibits TAZ level. We conclude that hepatic cholesterol induces high-level TAZ in NASH through PKA-Ca2+-RhoA mediated Hippo pathway inactivation, and cholesterol transportation plays an important role during this process. These data provide a plausible mechanism underlying liver cholesterol as a risk factor for NASH fibrosis and suggest new therapeutic interventions to block NASH fibrosis and to prevent NASH-associated HCC. Citation Format: Xiaobo Wang, Arun Radhakrishnan, Douglas Covey, Jay Horton, Robert Schwabe, Peter Tontonoz, Kun-Liang Guan, Ira Tabas. Hepatic cholesterol upregulates TAZ in nonalcoholic steatohepatitis [abstract]. In: Proceedings of the AACR Special Conference on the Hippo Pathway: Signaling, Cancer, and Beyond; 2019 May 8-11; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(8_Suppl):Abstract nr B04.

NASH has emerged as the leading cause of chronic liver disease and hepatocellular carcinoma (HCC) worldwide. However, there is a dearth of treatment options, which is due in large part to a poor understanding of NASH pathophysiology, particularly in the conversion of the relatively benign steatosis to clinically important fibrosis and HCC. We hypothesized that the transcription factor TAZ (WWTR1) in hepatocytes (HCs) promotes the progression of steatosis to fibrotic NASH and HCC. We studied liver tissue from humans with NASH and NASH-HCC and used several mouse models, including a dietary model of obesity, insulin resistance, NASH fibrosis, and HCC. NASH mice were treated with various AAV8-H1-shRNAs to silence genes specifically in HCs. The data show that TAZ is markedly increased in HCs in both human and murine NASH liver compared with normal or steatotic liver. In murine models of NASH, silencing of HC TAZ prevents or reverses hepatic inflammation, HC death, and fibrosis. Moreover, HC-targeted expression of TAZ in a mouse model of steatosis promoted NASH features, including fibrosis. Mechanistic studies using cultured cells and molecular-genetic causation studies in mice revealed that a key mechanism linking is TAZ-mediated induction of Indian hedgehog (Ihh), an HC secretory factor that activates fibrogenic genes in hepatic stellate cells (Wang et al., Cell Metabolism). The mechanism of TAZ upregulation in NASH involves a pathway in which excess cholesterol in HCs disrupts the normal proteasomal degradation of TAZ. Prolonged feeding of mice with the NASH diet leads to HCC, with both the tumors and surrounding tissues expressing high levels of TAZ. Silencing TAZ in HCs after advanced NASH has developed eliminates the progression to HCC, and silencing HC TAZ in mice with NASH-HCC shrinks the tumors. The mechanisms of linking TAZ to NASH-HCC likely both niche (surrounding tissue) and oncogenic effects. In conclusion, HC TAZ, whose levels markedly increase during NASH progression via a pathway involving cholesterol enrichment of HCs, plays an essential role in the progression of steatosis to fibrotic NASH and in the progression of NASH to HCC. As such, silencing TAZ in hepatocytes in the setting of early NASH is a potentially powerful new strategy to prevent the progression of NASH to cirrhosis and HCC. Citation Format: Xiaobo Wang, Robert Schwabe, Ira Tabas. The role of hepatocyte TAZ in NASH and NASH-HCC [abstract]. In: Proceedings of the AACR Special Conference on the Hippo Pathway: Signaling, Cancer, and Beyond; 2019 May 8-11; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(8_Suppl):Abstract nr IA23.

Continual efferocytic clearance of apoptotic cells (ACs) by macrophages prevents necrosis and promotes injury resolution. How continual efferocytosis is promoted is not clear. Here, we show that the process is optimized by linking the metabolism of engulfed cargo from initial efferocytic events to subsequent rounds. We found that continual efferocytosis is enhanced by the metabolism of AC-derived arginine and ornithine to putrescine by macrophage arginase 1 (Arg1) and ornithine decarboxylase (ODC). Putrescine augments HuR-mediated stabilization of the mRNA encoding the GTP-exchange factor Dbl, which activates actin-regulating Rac1 to facilitate subsequent rounds of AC internalization. Inhibition of any step along this pathway after first-AC uptake suppresses second-AC internalization, whereas putrescine addition rescues this defect. Mice lacking myeloid Arg1 or ODC have defects in efferocytosis in vivo and in atherosclerosis regression, while treatment with putrescine promotes atherosclerosis resolution. Thus, macrophage metabolism of AC-derived metabolites allows for optimal continual efferocytosis and resolution of injury.

Rationale: The mechanisms driving atherothrombotic risk in individuals with JAK2 V617F ( Jak2 VF ) positive clonal hematopoiesis or myeloproliferative neoplasms are poorly understood. Objective: The goal of this study was to assess atherosclerosis and underlying mechanisms in hypercholesterolemic mice with hematopoietic Jak2 VF expression. Methods and Results: Irradiated low-density lipoprotein receptor knockout ( Ldlr −/− ) mice were transplanted with bone marrow from wild-type or Jak2 VF mice and fed a high-fat high-cholesterol Western diet. Hematopoietic functions and atherosclerosis were characterized. After 7 weeks of Western diet, Jak2 VF mice showed increased atherosclerosis. Early atherosclerotic lesions showed increased neutrophil adhesion and content, correlating with lesion size. After 12 weeks of Western diet, Jak2 VF lesions showed increased complexity, with larger necrotic cores, defective efferocytosis, prominent iron deposition, and costaining of erythrocytes and macrophages, suggesting erythrophagocytosis. Jak2 VF erythrocytes were more susceptible to phagocytosis by wild-type macrophages and showed decreased surface expression of CD47, a “don’t-eat-me” signal. Human JAK2VF erythrocytes were also more susceptible to erythrophagocytosis. Jak2 VF macrophages displayed increased expression and production of proinflammatory cytokines and chemokines, prominent inflammasome activation, increased p38 MAPK (mitogen-activated protein kinase) signaling, and reduced levels of MerTK (c-Mer tyrosine kinase), a key molecule mediating efferocytosis. Increased erythrophagocytosis also suppressed efferocytosis. Conclusions: Hematopoietic Jak2 VF expression promotes early lesion formation and increased complexity in advanced atherosclerosis. In addition to increasing hematopoiesis and neutrophil infiltration in early lesions, Jak2 VF caused cellular defects in erythrocytes and macrophages, leading to increased erythrophagocytosis but defective efferocytosis. These changes promote accumulation of iron in plaques and increased necrotic core formation which, together with exacerbated proinflammatory responses, likely contribute to plaque instability.

A new paradigm for preventing atherosclerotic cardiovascular disease (ASCVD) is needed. The most recent US data show the long‐term decline in cardiovascular deaths has stopped, and has started to increase in the most at‐risk populations.1 Indeed, rising rates of obesity and diabetes mellitus in the setting of suboptimal risk factor control have resulted in a similar number of cardiovascular events occurring in those aged

Inflammation resolution counterbalances excessive inflammation and restores tissue homeostasis after injury. Failure of resolution contributes to the pathology of numerous chronic inflammatory diseases. Resolution is mediated by endogenous specialized proresolving mediators (SPMs), which are derived from long-chain fatty acids by lipoxygenase (LOX) enzymes. 5-LOX plays a critical role in the biosynthesis of two classes of SPMs, lipoxins and resolvins. Cytoplasmic localization of the nonphosphorylated form of 5-LOX is essential for SPM biosynthesis, whereas nuclear localization of phosphorylated 5-LOX promotes proinflammatory leukotriene production. We previously showed that MerTK, an efferocytosis receptor on macrophages, promotes SPM biosynthesis by increasing the abundance of nonphosphorylated, cytoplasmic 5-LOX. Here, we found that activation of MerTK in human macrophages led to ERK-mediated expression of the gene encoding sarcoplasmic/endoplasmic reticulum calcium ATPase 2 (SERCA2), which decreased the cytosolic Ca(2+) concentration and suppressed the activity of calcium/calmodulin-dependent protein kinase II (CaMKII). This in turn reduced the activities of the mitogen-activated protein kinase (MAPK) p38 and the kinase MK2, resulting in the increased abundance of the nonphosphorylated, cytoplasmic form of 5-LOX and enhanced SPM biosynthesis. In a zymosan-induced peritonitis model, an inflammatory setting in which macrophage MerTK activation promotes resolution, inhibition of ERK activation delayed resolution, which was characterized by an increased number of neutrophils and decreased amounts of SPMs in tissue exudates. These findings contribute to our understanding of how MerTK signaling induces 5-LOX-derived SPM biosynthesis and suggest a therapeutic strategy to boost inflammation resolution in settings where defective resolution promotes disease progression.

The mechanisms driving increased athero-thrombotic risk in individuals with JAK2V617F positive clonal hematopoiesis or myeloproliferative neoplasms are poorly understood. This study was to assess atherosclerosis and thrombosis and associated mechanisms in hypercholesterolemic mice with hematopoietic JAK2V617F expression. Irradiated Ldlr -/- mice were transplanted with bone marrow from WT or JAK2V617F mice and fed a high fat high cholesterol diet (WD). After 7 weeks of WD JAK2V617F mice showed increased hematopoiesis, platelet activation, accelerated thrombosis and increased atherosclerosis. Early atherosclerotic lesions showed increased neutrophils, correlating with lesion size and in association with increased rolling and adhesion of neutrophils on endothelial cells in carotid artery. After 12 weeks of WD JAK2V617F lesions were slightly larger than controls but showed additional complexity, with increased necrotic core area, prominent iron deposition and co-staining of erythrocytes and macrophages suggesting erythrophagocytosis. JAK2V617F erythrocytes were more susceptible to phagocytosis by WT macrophages and showed decreased surface expression of CD47, a “don’t eat me” signal. Human JAK2V617F erythrocytes were also more susceptible to erythrophagocytosis. JAK2V617F macrophages displayed increased expression of pro-inflammatory cytokines and chemokines, increased p38 map kinase signaling and increased cleavage and reduced levels of MerTK, a key molecule mediating phagocytosis of apoptotic cells (efferocytosis) in atherosclerotic lesions. Erythrophagocytosis also suppressed efferocytosis. As a result, JAK2V617F lesional efferocytosis was reduced in advanced lesions. In conclusion, JAK2V617F promotes early lesion formation, arterial thrombosis and increased complexity in advanced atherosclerosis. In addition to increasing hematopoiesis and neutrophil infiltration in early lesions, JAK2V617F caused cellular defects in erythrocytes and macrophages, leading to increased erythrophagocytosis but defective efferocytosis. These changes appear to promote accumulation of iron and increased necrotic core formation which, together with exacerbated pro-inflammatory responses, may contribute to plaque instability.

Defective macrophage efferocytosis drives important diseases including atherosclerosis, whereas enhancing efferocytosis has potential therapeutic benefits. A systematic approach to identify regulators of efferocytosis in an unbiased manner holds promise for novel molecular mechanisms and targets. This study establishes an experimental framework using a novel CRISPR knockout screen to systematically map the regulatory networks of macrophage efferocytosis. We infected bone marrow-derived macrophages (BMDM) isolated from Cas9 transgenic mice with a pooled, genome-wide lentiviral library with 78,637 gRNAs, and induced efferocytosis by fluorescently-labeled apoptotic Jurkat cells. We used FACS to isolate BMDM that engulfed multiple apoptotic cells (enriched in gRNAs targeting negative regulators) or did not engulf apoptotic cells (enriched in gRNAs targeting positive regulators), and determined gRNA abundance by deep sequencing. The top-ranked 1,000 genes analyzed by two published methods MAGeCK and PBNPA showed substantial overlap (53% for positive regulators, 36% for negative regulators). We recovered many of the known regulators. e.g., Actr2 , Actr3 and Arpc3 , key components of the Arp2/3 complex regulating actin polymerization, were ranked 2, 8 and 4; Atp6v0b and Atp6v0c , subunits of vacuolar ATPase responsible for acidification of intracellular organelles, were ranked 22 and 27. Cd36 , a well-known efferocytosis receptor, was ranked 5. The top ranked positive regulators were enriched in GO terms “positive regulation of response to stimulus”, “Fcγ receptor signaling pathway” and “phagocytosis” (FDR-adjusted P = 2.19E-14, 1.10E-12 and 4.08E-11, respectively). Individual gRNAs verified a number of top hits from the pooled screen. In summary, we have developed a functional screen that has defined number of genes essential for macrophage efferocytosis. By focusing on a functional phenotype beyond cell viability and growth, we illustrate the versatility of CRISPR screens and provide a general strategy for systematically identifying gene of interest and uncovering novel regulators of complex macrophage functions.

Emerging data suggest that hypercholesterolemia has stimulatory effects on adaptive immunity and that these effects can promote atherosclerosis and perhaps other inflammatory diseases. However, research in this area has relied primarily on inbred strains of mice whose adaptive immune system can differ substantially from that of humans. Moreover, the genetically induced hypercholesterolemia in these models typically results in plasma cholesterol levels that are much higher than those in most humans. To overcome these obstacles, we studied human immune system-reconstituted mice (hu-mice) rendered hypercholesterolemic by treatment with adeno-associated virus 8-proprotein convertase subtilisin/kexin type 9 (AAV8-PCSK9) and a high-fat/high-cholesterol Western-type diet (WD). These mice had a high percentage of human T cells and moderate hypercholesterolemia. Compared with hu-mice that had lower plasma cholesterol, the PCSK9-WD mice developed a T cell-mediated inflammatory response in the lung and liver. Human CD4+ and CD8+ T cells bearing an effector memory phenotype were significantly elevated in the blood, spleen, and lungs of PCSK9-WD hu-mice, whereas splenic and circulating regulatory T cells were reduced. These data show that moderately high plasma cholesterol can disrupt human T cell homeostasis in vivo. This process may not only exacerbate atherosclerosis, but also contribute to T cell-mediated inflammatory diseases in the hypercholesterolemia setting.

Atherosclerosis is characterized by the retention of modified lipoproteins in the arterial wall. These modified lipoproteins activate resident macrophages and the recruitment of monocyte-derived cells, which differentiate into mononuclear phagocytes that ingest the deposited lipoproteins to become "foam cells": a hallmark of this disease. In this Part 2 of a 4-part review series covering the macrophage in cardiovascular disease, we critically review the contributions and relevant pathobiology of monocytes, macrophages, and foam cells as relevant to atherosclerosis. We also review evidence that via various pathways, a failure of the resolution of inflammation is an additional key aspect of this disease process. Finally, we consider the likely role played by genomics and biological networks in controlling the macrophage phenotype in atherosclerosis. Collectively, these data provide substantial insights on the atherosclerotic process, while concurrently offering numerous molecular and genomic candidates that appear to hold great promise for selective targeting as clinical therapies.

Fibrosis is the major determinant of morbidity and mortality in patients with nonalcoholic steatohepatitis (NASH) but has no approved pharmacotherapy in part because of incomplete understanding of its pathogenic mechanisms. Here, we report that hepatocyte Notch activity tracks with disease severity and treatment response in patients with NASH and is similarly increased in a mouse model of diet-induced NASH and liver fibrosis. Hepatocyte-specific Notch loss-of-function mouse models showed attenuated NASH-associated liver fibrosis, demonstrating causality to obesity-induced liver pathology. Conversely, forced activation of hepatocyte Notch induced fibrosis in both chow- and NASH diet–fed mice by increasing Sox9-dependent Osteopontin (Opn) expression and secretion from hepatocytes, which activate resident hepatic stellate cells. In a cross-sectional study, we found that OPN explains the positive correlation between liver Notch activity and fibrosis stage in patients. Further, we developed a Notch inhibitor [Nicastrin antisense oligonucleotide (Ncst ASO)] that reduced fibrosis in NASH diet–fed mice. In summary, these studies demonstrate the pathological role and therapeutic accessibility of the maladaptive hepatocyte Notch response in NASH-associated liver fibrosis.

Summary Regulatory T (Treg) cell responses and apoptotic cell clearance (efferocytosis) represent critical arms of the inflammation resolution response. We sought to determine whether these processes might be linked through Treg-cell-mediated enhancement of efferocytosis. In zymosan-induced peritonitis and lipopolysaccharide-induced lung injury, Treg cells increased early in resolution, and Treg cell depletion decreased efferocytosis. In advanced atherosclerosis, where defective efferocytosis drives disease progression, Treg cell expansion improved efferocytosis. Mechanistic studies revealed the following sequence: (1) Treg cells secreted interleukin-13 (IL-13), which stimulated IL-10 production in macrophages; (2) autocrine-paracrine signaling by IL-10 induced Vav1 in macrophages; and (3) Vav1 activated Rac1 to promote apoptotic cell engulfment. In summary, Treg cells promote macrophage efferocytosis during inflammation resolution via a transcellular signaling pathway that enhances apoptotic cell internalization. These findings suggest an expanded role of Treg cells in inflammation resolution and provide a mechanistic basis for Treg-cell-enhancement strategies for non-resolving inflammatory diseases.

Atherosclerosis is a maladaptive, nonresolving chronic inflammatory disease that occurs at sites of blood flow disturbance. The disease usually remains silent until a breakdown of integrity at the arterial surface triggers the formation of a thrombus. By occluding the lumen, the thrombus or emboli detaching from it elicits ischaemic symptoms that may be life-threatening. Two types of surface damage can cause atherothrombosis: plaque rupture and endothelial erosion. Plaque rupture is thought to be caused by loss of mechanical stability, often due to reduced tensile strength of the collagen cap surrounding the plaque. Therefore, plaques with reduced collagen content are thought to be more vulnerable than those with a thick collagen cap. Endothelial erosion, on the other hand, may occur after injurious insults to the endothelium instigated by metabolic disturbance or immune insults. This review discusses the molecular mechanisms involved in plaque vulnerability and the development of atherothrombosis.

The prevalence of obesity-induced type 2 diabetes (T2D) is increasing worldwide, and new treatment strategies are needed. We recently discovered that obesity activates a previously unknown pathway that promotes both excessive hepatic glucose production (HGP) and defective insulin signaling in hepatocytes, leading to exacerbation of hyperglycemia and insulin resistance in obesity. At the hub of this new pathway is a kinase cascade involving calcium/calmodulin-dependent protein kinase II (CaMKII), p38α mitogen-activated protein kinase (MAPK), and MAPKAPK2/3 (MK2/3). Genetic-based inhibition of these kinases improves metabolism in obese mice. Here, we report that treatment of obese insulin-resistant mice with an allosteric MK2/3 inhibitor, compound (cmpd) 28, ameliorates glucose homeostasis by suppressing excessive HGP and enhancing insulin signaling. The metabolic improvement seen with cmpd 28 is additive with the leading T2D drug, metformin, but it is not additive with dominant-negative MK2, suggesting an on-target mechanism of action. Allosteric MK2/3 inhibitors represent a potentially new approach to T2D that is highly mechanism based, has links to human T2D, and is predicted to avoid certain adverse effects seen with current T2D drugs.

Rationale: Myeloid-derived C/EBP-homologous protein (CHOP), an effector of the endoplasmic reticulum stress–induced unfolded protein response, promotes macrophage apoptosis in advanced atherosclerosis, but the role of CHOP in vascular smooth muscle cells (VSMCs) in atherosclerosis is not known. Objective: To investigate the role of CHOP in SM22α + VSMCs in atherosclerosis. Methods and Results: Chop fl/fl mice were generated and crossed into the Apoe −/− and SM22α-CreKI + backgrounds. SM22α-CreKI causes deletion of floxed genes in adult SMCs. After 12 weeks of Western-type diet feeding, the content of α-actin–positive cells in aortic root lesions was decreased in Chop fl/fl SM22α-CreKI + Apoe −/− versus control Chop fl/fl Apoe −/− mice, and aortic explant–derived VSMCs from the VSMC-CHOP–deficient mice displayed reduced proliferation. Krüppel-like factor 4 (KLF4), a key suppressor of VSMC proliferation, was increased in lesions and aortic VSMCs from Chop fl/fl SM22α-CreKI + Apoe −/− mice, and silencing Klf4 in CHOP-deficient VSMCs restored proliferation. CHOP deficiency in aortic VSMCs increased KLF4 through 2 mechanisms mediated by the endoplasmic reticulum stress effector activating transcription factor 4: transcriptional induction of Klf4 mRNA and decreased proteasomal degradation of KLF4 protein. Conclusions: These findings in SM22α-CHOP–deficient mice imply that CHOP expression in SM22α + VSMCs promotes cell proliferation by downregulating KLF4. The mechanisms involve newly discovered roles of CHOP in the transcriptional and post-translational regulation of KLF4.

Atherosclerosis occurs in the subendothelial space (intima) of medium-sized arteries at regions of disturbed blood flow and is triggered by an interplay between endothelial dysfunction and subendothelial lipoprotein retention. Over time, this process stimulates a nonresolving inflammatory response that can cause intimal destruction, arterial thrombosis, and end-organ ischemia. Recent advances highlight important cell biological atherogenic processes, including mechanotransduction and inflammatory processes in endothelial cells, origins and contributions of lesional macrophages, and origins and phenotypic switching of lesional smooth muscle cells. These advances illustrate how in-depth mechanistic knowledge of the cellular pathobiology of atherosclerosis can lead to new ideas for therapy.

Rationale— Mitochondrial oxidative stress (mitoOS) has been shown to be increased in various cell types in human atherosclerosis and with aging. However, the role of cell type–specific mitoOS in atherosclerosis in the setting of advanced age and the molecular mechanisms remains to be determined in vivo. Objective— The aim of this study was to examine the role of myeloid cell mitoOS in atherosclerosis in aged mice. Approach and Results— Lethally irradiated low-density lipoprotein receptor–deficient mice ( Ldlr −/− ) were reconstituted with bone marrow from either wild-type or mitochondrial catalase (mCAT) mice. mCAT transgenic mice contain ectopically expressed human catalase gene in mitochondria, which reduces mitoOS. Starting at the age of 36 weeks, mice were fed the Western-type diet for 16 weeks. We found that mitoOS in lesional myeloid cells was suppressed in aged mCAT→ Ldlr −/− chimeric mice compared with aged controls, and this led to a significant reduction in aortic root atherosclerotic lesion area despite higher plasma cholesterol levels. Neutrophil extracellular traps (NETs), a proinflammatory extracellular structure that contributes to atherosclerosis progression, were significantly increased in the lesions of aged mice compared with lesions of younger mice. Aged mCAT→ Ldlr −/− mice had less lesional neutrophils and decreased NETs compared with age-matched wild-type→ Ldlr −/− mice, whereas young mCAT→ and wild-type→ Ldlr −/− mice had comparable numbers of neutrophils and similar low levels of lesional NETs. Using cultured neutrophils, we showed that suppression of mitoOS reduced 7-ketocholesterol–induced NET release from neutrophils of aged but not younger mice. Conclusions— MitoOS in lesional myeloid cells enhanced atherosclerosis development in aged mice, and this enhancement was associated with increased lesional NETs. Thus, mitoOS-induced NET formation is a potentially new therapeutic target to prevent atherosclerosis progression during aging.

Atherosclerosis is an arterial disease process characterized by the focal subendothelial accumulation of apolipoprotein-B-containing lipoproteins, immune and vascular wall cells, and extracellular matrix. The lipoproteins acquire features of damage-associated molecular patterns and trigger first an innate immune response, dominated by monocyte-macrophages, and then an adaptive immune response. These inflammatory responses often become chronic and non-resolving and can lead to arterial damage and thrombosis-induced organ infarction. The innate immune response is regulated at various stages, from hematopoiesis to monocyte changes and macrophage activation. The adaptive immune response is regulated primarily by mechanisms that affect the balance between regulatory and effector T cells. Mechanisms related to cellular cholesterol, phenotypic plasticity, metabolism, and aging play key roles in affecting these responses. Herein, we review select topics that shed light on these processes and suggest new treatment strategies.

Atherothrombotic vascular disease is often triggered by a distinct type of atherosclerotic lesion that displays features of impaired inflammation resolution, notably a necrotic core and thinning of a protective fibrous cap that overlies the core. A key cause of plaque necrosis is defective clearance of apoptotic cells, or efferocytosis, by lesional macrophages, but the mechanisms underlying defective efferocytosis and its possible links to impaired resolution in atherosclerosis are incompletely understood. Here, we provide evidence that proteolytic cleavage of the macrophage efferocytosis receptor c-Mer tyrosine kinase (MerTK) reduces efferocytosis and promotes plaque necrosis and defective resolution. In human carotid plaques, MerTK cleavage correlated with plaque necrosis and the presence of ischemic symptoms. Moreover, in fat-fed LDL receptor-deficient (Ldlr-/-) mice whose myeloid cells expressed a cleavage-resistant variant of MerTK, atherosclerotic lesions exhibited higher macrophage MerTK, lower levels of the cleavage product soluble Mer, improved efferocytosis, smaller necrotic cores, thicker fibrous caps, and increased ratio of proresolving versus proinflammatory lipid mediators. These findings provide a plausible molecular-cellular mechanism that contributes to defective efferocytosis, plaque necrosis, and impaired resolution during the progression of atherosclerosis.

Protein therapeutics have gained attention recently for treatment of a myriad of human diseases due to their high potency and unique mechanisms of action. We present the development of a novel polymeric thermosponge nanoparticle for efficient delivery of labile proteins using a solvent-free polymer thermo-expansion mechanism with clinical potential, capable of effectively delivering a range of therapeutic proteins in a sustained manner with no loss of bioactivity, with improved biological half-lives and efficacy in vivo.

Inflammation contributes to many of the characteristics of plaques implicated in the pathogenesis of acute coronary syndromes. Moreover, inflammatory pathways not only regulate the properties of plaques that precipitate acute coronary syndromes but also modulate the clinical consequences of the thrombotic complications of atherosclerosis. This synthesis will provide an update on the fundamental mechanisms of inflammatory responses that govern acute coronary syndromes and also highlight the ongoing balance between proinflammatory mechanisms and endogenous pathways that can promote the resolution of inflammation. An appreciation of the countervailing mechanisms that modulate inflammation in relation to acute coronary syndromes enriches our fundamental understanding of the pathophysiology of this important manifestation of atherosclerosis. In addition, these insights provide glimpses into potential novel therapeutic interventions to forestall this ultimate complication of the disease.

The phagocytosis of apoptotic cells (ACs), or efferocytosis, by DCs is critical for self-tolerance and host defense. Although many efferocytosis-associated receptors have been described in vitro, the functionality of these receptors in vivo has not been explored in depth. Using a spleen efferocytosis assay and targeted genetic deletion in mice, we identified a multiprotein complex — composed of the receptor tyrosine kinase AXL, LDL receptor–related protein–1 (LRP-1), and RAN-binding protein 9 (RANBP9) — that mediates DC efferocytosis and antigen cross-presentation. We found that AXL bound ACs, but required LRP-1 to trigger internalization, in murine CD8α+ DCs and human-derived DCs. AXL and LRP-1 did not interact directly, but relied on RANBP9, which bound both AXL and LRP-1, to form the complex. In a coculture model of antigen presentation, the AXL/LRP-1/RANBP9 complex was used by DCs to cross-present AC-associated antigens to T cells. Furthermore, in a murine model of herpes simplex virus–1 infection, mice lacking DC-specific LRP-1, AXL, or RANBP9 had increased AC accumulation, defective viral antigen-specific CD8+ T cell activation, enhanced viral load, and decreased survival. The discovery of this multiprotein complex that mediates functionally important DC efferocytosis in vivo may have implications for future studies related to host defense and DC-based vaccines.

Rationale: Mitochondrial oxidative stress (mitoOS) has been shown to correlate with the progression of human atherosclerosis. However, definitive cell type–specific causation studies in vivo are lacking, and the molecular mechanisms of potential proatherogenic effects remain to be determined. Objective: Our aims were to assess the importance of macrophage mitoOS in atherogenesis and to explore the underlying molecular mechanisms. Methods and Results: We first validated Western diet–fed Ldlr −/− mice as a model of human mitoOS–atherosclerosis association by showing that non-nuclear oxidative DNA damage, a marker of mitoOS in lesional macrophages, correlates with aortic root lesion development. To investigate the importance of macrophage mitoOS, we used a genetic engineering strategy in which the OS suppressor catalase was ectopically expressed in mitochondria (mCAT) in macrophages. MitoOS in lesional macrophages was successfully suppressed in these mice, and this led to a significant reduction in aortic root lesional area. The mCAT lesions had less monocyte-derived cells, less Ly6c hi monocyte infiltration into lesions, and lower levels of monocyte chemotactic protein-1. The decrease in lesional monocyte chemotactic protein-1 was associated with the suppression of other markers of inflammation and with decreased phosphorylation of RelA (NF-κB p65), indicating decreased activation of the proinflammatory NF-κB pathway. Using models of mitoOS in cultured macrophages, we showed that mCAT suppressed monocyte chemotactic protein-1 expression by decreasing the activation of the IκB-kinase β–RelA NF-κB pathway. Conclusions: MitoOS in lesional macrophages amplifies atherosclerotic lesion development by promoting NF-κB–mediated entry of monocytes and other inflammatory processes. In view of the mitoOS–atherosclerosis link in human atheromata, these findings reveal a potentially new therapeutic target to prevent the progression of atherosclerosis.

Objective— Coronary heart disease is associated with monocytosis. Studies using animal models of monocytosis and atherosclerosis such as ApoE −/− mice have shown bone marrow (BM) hematopoietic stem and multipotential progenitor cell (HSPC) expansion, associated with increased cell surface expression of the common β subunit of the granulocyte macrophage colony–stimulating factor/interleukin-3 receptor (CBS) on HSPCs. ApoE −/− mice also display increased granulocyte macrophage colony–stimulating factor–dependent monocyte production in the spleen. We investigated the role of the CBS in cholesterol-driven HSPC expansion, monocytosis, and atherosclerosis. Approach and Results— Ldlr −/− mice were transplanted with ApoE −/− Cbs −/− or ApoE −/− BM followed by Western-type diet feeding. Compared with ApoE −/− BM–transplanted controls, ApoE −/− Cbs −/− BM–transplanted mice had reduced BM and splenic HSPC proliferation, fewer blood monocytes and neutrophils, and reduced macrophage content and area of early atherosclerotic lesions. More advanced lesions showed diminished macrophage and collagen content; however, lesion size was unchanged, reflecting an increase in necrotic core area, associated with a marked decrease in Abcg1 expression and increased macrophage apoptosis. Compared with wild-type mice, Western-type diet–fed ApoE −/− mice showed increased CBS expression on granulocyte macrophage colony–stimulating factor–producing innate response activator B cells and expansion of this population. ApoE −/− Cbs −/− BM–transplanted Ldlr −/− mice showed a marked decrease in innate response activator B cells compared with ApoE −/− BM–transplanted Ldlr −/− controls. Conclusions— Increased levels of CBS on HSPCs and splenic innate response activator B cells lead to expansion of these populations in ApoE −/− BM–transplanted Ldlr −/− mice, contributing to monocytosis and increased lesional macrophage content. However, in more advanced lesions, the CBS also has a role in atherosclerotic plaque stabilization.

Mitochondrial-derived reactive oxygen species (mtROS) is one of the major sources of cellular ROS, and excessive mtROS is associated with atherosclerosis progression in both human and mouse models. This review aims to summarize the most recent studies showing the existence, the causes and pathological consequences of excessive mtROS in atherosclerosis. Despite numerous association and causation studies demonstrating the importance of mtROS in atherosclerosis progression, the failure of antioxidant therapy in human randomized clinical trials1-3 demands more definitive, cell-type specific investigations. Better mechanistic understanding of mtROS in atherosclerosis may lead to more effective therapeutic strategies.

Objective— Defective autophagy in macrophages leads to pathological 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. microRNA-33 (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 coherent anti-Stokes Raman scattering 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 of 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 (ATP-binding cassette transporter A1)-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.

AXL, a member of the TAM (Tyro3, Axl, MerTK) family of receptors, plays important roles in cell survival, clearance of dead cells (efferocytosis), and suppression of inflammation, which are processes that critically influence atherosclerosis progression. Whereas MerTK deficiency promotes defective efferocytosis, inflammation, and plaque necrosis in advanced murine atherosclerosis, the role of Axl in advanced atherosclerosis progression is not known. Towards this end, bone marrow cells from Axl−/− or wild-type mice were transplanted into lethally irradiated Ldlr−/− mice. These chimeric mice were then fed the Western-type diet (WD) for 17 weeks. We demonstrate that lesional macrophages in WT mice express Axl but that Axl deficiency in bone marrow-derived cells does not affect lesion size, cellularity, necrosis, or inflammatory parameters in advanced atherosclerotic plaques. Moreover, apoptosis of lesional cells was unaffected, and we found no evidence of defective lesional efferocytosis. In contrast to previously reported findings with MerTK deficiency, hematopoietic cell-Axl deficiency in WD-fed Ldlr−/− mice does not affect the progression of advanced atherosclerosis or lesional processes associated with TAM receptor signaling. These findings suggest a heretofore unappreciated TAM receptor hierarchy in advanced atherosclerosis.

Tissue-type plasminogen activator (tPA) initiates fibrinolysis, the primary mechanism that dissolves a thrombus. We have shown that in lean mice the hepatocytes maintain a basal level of circulating tPA that influences fibrinolysis in the occurrence of a vessel injury. As a serine protease, tPA is inhibited by the serpin plasminogen activator inhibitor 1 (PAI1). Both PAI1 and tPA which can be produced by hepatocytes, which is important for regulating energy metabolism and is sensitive to metabolic stress. In obesity, despite an increase in plasma tPA, blood tPA activity and fibrinolysis are reduced primarily due to a larger increase in PAI1. However, the source and regulatory mechanism of this phenomena are unknown. Palmitate treatment of human primary hepatocytes causes an increase in tPA mRNA/protein, reflecting the situation in the obese liver. This increase, however, is overcompensated by a larger increase in PAI1 mRNA, resulting in decreased net tPA activity in the cell culture medium. Silencing PAI1 mRNA with si-SERPINE1 in these cells increased tPA activity in the culture medium. Surprisingly, silencing PAI1 also prevented the increase of tPA mRNA induced by palmitate. Building on this observation, we further treated the cells with active recombinant PAI1 (rPAI1), which induced tPA expression. Next, we fed PAI1fl/flmice with a diet-induced obesity (DIO) diet for three months and silenced their hepatic PAI1 using AAV8-TBG-Cre, which inactivates ~95% floxed gene expression in hepatocytes but not in other cell types of the liver or other tissues. We found that while plasma tPA activity was increased in hepatocyte-PAI1 knockout mice, plasma tPA protein concentration was reduced. In other words, reducing hepatocyte PAI1 in obesity stopped the compensatory increase in liver tPA mRNA and protein. Thus, PAI1 induces tPA, which is likely a compensatory response. To explore the mechanism whereby rPAI1 induces tPA, we considered CREB1, as it is a transcription activator of tPA expression in endothelial cells and is expressed in hepatocytes. We found that CREB1 was activated (phosphorylated) by rPAI1 in human primary hepatocytes and that silencing CREB1 prevented the elevated tPA expression induced by rPAI1. Next, we silenced hepatic CREB1 in obese mice by treating DIO diet-fed CREB1fl/flmice with AAV8-TBG-Cre, which blocked the compensatory increase in tPA and thus worsened the impaired fibrinolysis in obesity. LDL receptor-related protein 1 (LRP1), the major cellular receptor of PAI1, can stimulate CREB1 transcription activity in neurons and adipocytes. As LRP1 is expressed on the surface of hepatocytes, we hypothesized that PAI1 activates CREB1 through LRP1-mediated signaling to increase tPA expression in obesity. Indeed, silencing LRP1 with si-LRP1 stopped the increase of tPA expression induced by rPAI1 in human primary hepatocytes. Interestingly, treating the cells with a mutant rPAI1 lacking the LRP1-interacting heparin-binding domain was unable to induce tPA expression. In summary, hepatocyte tPA mRNA/protein is increased in obesity, but this increase is ultimately overcompensated by a larger increase in PAI1, resulting in decreased plasma tPA activity and fibrinolysis. The markedly increased PAI1, through activating LRP1-CREB1 signaling pathway, drives an increase in hepatic tPA expression as a "compensatory" pathway in obesity. Preventing this compensatory pathway in obesity resulted in the worsening of fibrinolysis. Therapeutic boosting of this compensatory pathway may provide novel strategies to restore effective fibrinolysis in obesity. Disclosures No relevant conflicts of interest to declare.

Atherosclerosis is a chronic inflammatory disease with activation of both the innate and adaptive arms of the immune system. Dendritic cells (DCs) are potent activators of adaptive immunity and have been identified in the normal arterial wall and within atherosclerotic lesions. Recent evidence points to a functional role for DCs in all stages of atherosclerosis because of their myriad functions including lipid uptake, antigen presentation, efferocytosis, and inflammation resolution. Moreover, DC-based vaccination strategies are currently being developed for the treatment of atherosclerosis. This review will focus on the current evidence as well as the proposed roles for DCs in the pathogenesis of atherosclerosis and discuss future therapeutic strategies.

Multiple systemic factors and local stressors in the arterial wall can disturb the functions of endoplasmic reticulum (ER), causing ER stress in endothelial cells (ECs), smooth muscle cells (SMCs), and macrophages during the initiation and progression of atherosclerosis. As a protective response to restore ER homeostasis, the unfolded protein response (UPR) is initiated by three major ER sensors: protein kinase RNA-like ER kinase (PERK), inositol-requiring protein 1α (IRE1α), and activating transcription factor 6 (ATF6). The activation of the various UPR signaling pathways displays a temporal pattern of activation at different stages of the disease. The ATF6 and IRE1α pathways that promote the expression of protein chaperones in ER are activated in ECs in athero-susceptible regions of pre-lesional arteries and before the appearance of foam cells. The PERK pathway that reduces ER protein client load by blocking protein translation is activated in SMCs and macrophages in early lesions. The activation of these UPR signaling pathways aims to cope with the ER stress and plays a pro-survival role in the early stage of atherosclerosis. However, with the progression of atherosclerosis, the extended duration and increased intensity of ER stress in lesions lead to prolonged and enhanced UPR signaling. Under this circumstance, the PERK pathway induces expression of death effectors, and possibly IRE1α activates apoptosis signaling pathways, leading to apoptosis of macrophages and SMCs in advanced lesions. Importantly, UPR-mediated cell death is associated with plaque instability and the clinical progression of atherosclerosis. Moreover, UPR signaling is linked to inflammation and possibly to macrophage differentiation in lesions. Therapeutic approaches targeting the UPR may have promise in the prevention and/or regression of atherosclerosis. However, more progress is needed to fully understand all of the roles of the UPR in atherosclerosis and to harness this information for therapeutic advances.

Objective— Acyl-CoA:cholesterol acyltransferase (ACAT) converts cholesterol to cholesteryl esters in plaque foam cells. Complete deficiency of macrophage ACAT has been shown to increase atherosclerosis in hypercholesterolemic mice because of cytotoxicity from free cholesterol accumulation, whereas we previously showed that partial ACAT inhibition by Fujirebio compound F1394 decreased early atherosclerosis development. In this report, we tested F1394 effects on preestablished, advanced lesions of apolipoprotein-E–deficient mice. Methods and Results— Apolipoprotein-E–deficient mice on Western diet for 14 weeks developed advanced plaques, and were either euthanized (Baseline), or continued on Western diet with or without F1394 and euthanized after 14 more weeks. F1394 was not associated with systemic toxicity. Compared with the baseline group, lesion size progressed in both groups; however, F1394 significantly retarded plaque progression and reduced plaque macrophage, free and esterified cholesterol, and tissue factor contents compared with the untreated group. Apoptosis of plaque cells was not increased, consistent with the decrease in lesional free cholesterol. There was no increase in plaque necrosis and unimpaired efferocytosis (phagocytic clearance of apoptotic cells). The effects of F1394 were independent of changes in plasma cholesterol levels. Conclusion— Partial ACAT inhibition by F1394 lowered plaque cholesterol content and had other antiatherogenic effects in advanced lesions in apolipoprotein-E–deficient mice without overt systemic or plaque toxicity, suggesting the continued potential of ACAT inhibition for the clinical treatment of atherosclerosis, in spite of recent trial data.

The burden of type 2 diabetes and its major complication cardiovascular disease is rapidly increasing worldwide. Understanding the underlying pathogenic mechanisms of these diseases is crucial to develop novel therapeutics. Recent work using genetic and biochemical methods in mouse models and human samples have identified disturbed calcium signalling and endoplasmic reticulum stress as emerging factors involved in the pathogenesis of many metabolic diseases. In this review, we will highlight the specific roles of calcium signalling and endoplasmic reticulum stress response in the development of insulin resistance and atherosclerosis.

Objective: Following myocardial infarction (MI), recruited and resident phagocytes are mobilized at the heart to promote clearance of dying myocardial cells by efferocytosis. This occurs in part through the actions of the macrophage receptor tyrosine kinases, Tyro 3, Axl, and Mertk (TAM), although potential differential contributions of TAMs on recruited vs resident phagocytes, as well as the contribution to clinically-relevant reperfusion, are unknown. We set out to test the significance of recruited vs resident Mertk + phagocytes during ischemia/reperfusion (I/R) and in comparison to Axl + phagocytes. Methods and Results: In contrast to MI, I/R resulted in elevated inflammation and early loss of Mertk on phagocytes in the infarcted tissue with a concomitant increase in serum levels of soluble Mertk in both mice and humans. In mice, Mertk-deficiency during I/R led to decreased cardiac efferocytosis, increased infarct size, and depressed cardiac contractility, newly revealing the importance of efferocytosis during clinically-relevant reperfusion. These endpoints were similarly affected after blocking blood-borne recruited monocytes with CCR2 antagonists, thereby specifically implicating resident Mertk + phagocytes to cardiac repair. We observed the highest expression of Mertk on Ly6C lo MHCII lo cardiac resident macrophages, which resulted in increased efferocytosis-efficiency by these cells compared to other cardiac phagocytes. Shedding of Mertk from Ly6C lo MHCII lo cardiac resident macrophages was observed after I/R and specific inhibition of Mertk cleavage using mice that express a cleavage-resistant Mertk reduced infarct size after I/R, identifying Mertk cleavage as a new therapeutic target after reperfusion injury. Interestingly, Axl was selectively expressed on Ly6C lo MHCII hi cardiac resident macrophages and surprisingly, Axl-deficiency during I/R led to reduced infarct size and improved cardiac contractility, suggesting differential roles between TAMs during cardiac repair. Conclusions: These data newly support the importance of efferocytosis pathways post-I/R and reveal both Axl and specifically Mertk on resident macrophages as key molecular mediators of inflammation resolution during atherothrombosis.

The remarkable plasticity and plethora of biological functions performed by macrophages have enticed scientists to study these cells in relation to atherosclerosis for >50 years, and major discoveries continue to be made today. It is now understood that macrophages play important roles in all stages of atherosclerosis, from initiation of lesions and lesion expansion, to necrosis leading to rupture and the clinical manifestations of atherosclerosis, to resolution and regression of atherosclerotic lesions. Lesional macrophages are derived primarily from blood monocytes, although recent research has shown that lesional macrophage-like cells can also be derived from smooth muscle cells. Lesional macrophages take on different phenotypes depending on their environment and which intracellular signaling pathways are activated. Rather than a few distinct populations of macrophages, the phenotype of the lesional macrophage is more complex and likely changes during the different phases of atherosclerosis and with the extent of lipid and cholesterol loading, activation by a plethora of receptors, and metabolic state of the cells. These different phenotypes allow the macrophage to engulf lipids, dead cells, and other substances perceived as danger signals; efflux cholesterol to high-density lipoprotein; proliferate and migrate; undergo apoptosis and death; and secrete a large number of inflammatory and proresolving molecules. This review article, part of the Compendium on Atherosclerosis , discusses recent advances in our understanding of lesional macrophage phenotype and function in different stages of atherosclerosis. With the increasing understanding of the roles of lesional macrophages, new research areas and treatment strategies are beginning to emerge.

Signaling through MyD88, an adaptor utilized by all TLRs except TLR3, is pro-atherogenic; however, it is unknown whether signaling through TIR-domain-containing adaptor-inducing interferon-β (TRIF), an adaptor used only by TLRs 3 and 4, is relevant to atherosclerosis. We determined that the TRIFLps2 lack-of-function mutation was atheroprotective in hyperlipidemic low density lipoprotein (LDL) receptor knockout (LDLr−/−) mice. LDLr−/− mice were crossed with either TRIFLps2 or TLR3 knockout mice. After feeding an atherogenic diet for 10–15 wks, atherosclerotic lesions in the heart sinus and aorta were quantitated. LDLr−/− mice with TRIFLps2 were significantly protected from atherosclerosis. TRIFLps2 led to a reduction in cytokines secreted from peritoneal macrophages (Mϕ) in response to hyperlipidemia. Moreover, heart sinus valves from hyperlipidemic LDLr−/− TRIFLps2 mice had significantly fewer lesional Mϕ. However, LDLr−/− mice deficient in TLR3 showed some enhancement of disease. Collectively, these data suggest that hyperlipidemia resulting in endogenous activation of the TRIF signaling pathway from TLR4 leads to pro-atherogenic events.

Recent GWAS have identified SNPs at a human chromosom1 locus associated with coronary artery disease risk and LDL cholesterol levels. The SNPs are also associated with altered expression of hepatic sortilin-1 (SORT1), which encodes a protein thought to be involved in apoB trafficking and degradation. Here, we investigated the regulation of Sort1 expression in mouse models of obesity. Sort1 expression was markedly repressed in both genetic (ob/ob) and high-fat diet models of obesity; restoration of hepatic sortilin-1 levels resulted in reduced triglyceride and apoB secretion. Mouse models of obesity also exhibit increased hepatic activity of mammalian target of rapamycin complex 1 (mTORC1) and ER stress, and we found that administration of the mTOR inhibitor rapamycin to ob/ob mice reduced ER stress and increased hepatic sortilin-1 levels. Conversely, genetically increased hepatic mTORC1 activity was associated with repressed Sort1 and increased apoB secretion. Treating WT mice with the ER stressor tunicamycin led to marked repression of hepatic sortilin-1 expression, while administration of the chemical chaperone PBA to ob/ob mice led to amelioration of ER stress, increased sortilin-1 expression, and reduced apoB and triglyceride secretion. Moreover, the ER stress target Atf3 acted at the SORT1 promoter region as a transcriptional repressor, whereas knockdown of Atf3 mRNA in ob/ob mice led to increased hepatic sortilin-1 levels and decreased apoB and triglyceride secretion. Thus, in mouse models of obesity, induction of mTORC1 and ER stress led to repression of hepatic Sort1 and increased VLDL secretion via Atf3. This pathway may contribute to dyslipidemia in metabolic disease.

In the fasted state, increases in circulating glucagon promote hepatic glucose production through induction of the gluconeogenic program. Triggering of the cyclic AMP pathway increases gluconeogenic gene expression via the de-phosphorylation of the CREB co-activator CRTC2 (ref. 1). Glucagon promotes CRTC2 dephosphorylation in part through the protein kinase A (PKA)-mediated inhibition of the CRTC2 kinase SIK2. A number of Ser/Thr phosphatases seem to be capable of dephosphorylating CRTC2 (refs 2, 3), but the mechanisms by which hormonal cues regulate these enzymes remain unclear. Here we show in mice that glucagon stimulates CRTC2 dephosphorylation in hepatocytes by mobilizing intracellular calcium stores and activating the calcium/calmodulin-dependent Ser/Thr-phosphatase calcineurin (also known as PP3CA). Glucagon increased cytosolic calcium concentration through the PKA-mediated phosphorylation of inositol-1,4,5-trisphosphate receptors (InsP(3)Rs), which associate with CRTC2. After their activation, InsP(3)Rs enhanced gluconeogenic gene expression by promoting the calcineurin-mediated dephosphorylation of CRTC2. During feeding, increases in insulin signalling reduced CRTC2 activity via the AKT-mediated inactivation of InsP(3)Rs. InsP(3)R activity was increased in diabetes, leading to upregulation of the gluconeogenic program. As hepatic downregulation of InsP(3)Rs and calcineurin improved circulating glucose levels in insulin resistance, these results demonstrate how interactions between cAMP and calcium pathways at the level of the InsP(3)R modulate hepatic glucose production under fasting conditions and in diabetes.

Summary In advanced atherosclerosis, macrophage apoptosis coupled with defective phagocytic clearance of the apoptotic cells (efferocytosis) promotes plaque necrosis, which precipitates acute atherothrombotic cardiovascular events. Oxidative and endoplasmic reticulum (ER) stress in macrophages are important causes of advanced lesional macrophage apoptosis. We now show that proapoptotic oxidative/ER stress inducers trigger another stress reaction in macrophages, autophagy. Inhibition of autophagy by silencing ATG5 or other autophagy mediators enhances apoptosis and NADPH oxidase-mediated oxidative stress while at the same time rendering the apoptotic cells less well recognized by efferocytes. Most importantly, macrophage ATG5 deficiency in fat-fed Ldlr −/− mice increases apoptosis and oxidative stress in advanced lesional macrophages, promotes plaque necrosis, and worsens lesional efferocytosis. These findings reveal a protective process in oxidatively stressed macrophages relevant to plaque necrosis, suggesting a mechanism-based strategy to therapeutically suppress atherosclerosis progression and its clinical sequelae.

SummaryDefective insulin signaling in hepatocytes is a key factor in type 2 diabetes. In obesity, activation of calcium/calmodulin-dependent protein kinase II (CaMKII) in hepatocytes suppresses ATF6, which triggers a PERK-ATF4-TRB3 pathway that disrupts insulin signaling. Elucidating how CaMKII suppresses ATF6 is therefore essential to understanding this insulin resistance pathway. We show that CaMKII phosphorylates and blocks nuclear translocation of histone deacetylase 4 (HDAC4). As a result, HDAC4-mediated SUMOylation of the corepressor DACH1 is decreased, which protects DACH1 from proteasomal degradation. DACH1, together with nuclear receptor corepressor (NCOR), represses Atf6 transcription, leading to activation of the PERK-TRB3 pathway and defective insulin signaling. DACH1 is increased in the livers of obese mice and humans, and treatment of obese mice with liver-targeted constitutively nuclear HDAC4 or DACH1 small hairpin RNA (shRNA) increases ATF6, improves hepatocyte insulin signaling, and protects against hyperglycemia and hyperinsulinemia. Thus, DACH1-mediated corepression in hepatocytes emerges as an important link between obesity and insulin resistance.