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The clearance of apoptotic cells (ACs) by macrophages (efferocytosis) promotes tissue repair by preventing necrosis and inflammation and by activating pro-resolving pathways, including continual efferocytosis. A key resolution processin vivois efferocytosis-induced macrophage proliferation (EIMP), in which AC-derived nucleotides trigger Myc-mediated macrophage proliferation, thereby increasing the pool of efferocytosis-competent macrophages. Here we show that EIMP requires a second input that is integrated with cellular metabolism, notably, efferocytosis-induced lactate production. While the AC-nucleotide pathway leads to induction ofMycmRNA, lactate signaling is required for the stabilization of Myc protein and subsequent macrophage proliferation. Lactate, via GPR132 and protein kinase A, activates AMP kinase, which increases the NAD+:NADH ratio. This upstream pathway then activates the NAD+-dependent protein deacetylase, SIRT1, which deacetylates Myc to promote its stabilization. Inhibition or silencing of any step along this pathway prevents the increase in Myc protein and proliferation in efferocytosing macrophages despite the presence of the AC-nucleotide/MycmRNA pathway. To test importancein vivo, we transplanted mice with bone marrow cells in which the lactate biosynthetic enzyme lactate dehydrogenase A (LDHA) was knocked down. We then subjected these mice and control bone marrow-transplanted mice to dexamethasone-induced thymocyte apoptosis, a model of high-AC burden. The thymi of the LDHA-knockdown cohort showed reduced macrophage Myc protein expression and proliferation, impaired AC clearance, and increased tissue necrosis. Thus, efferocytosis-induced macrophage proliferation, which is a key process in tissue resolution, requires inputs from two independent efferocytosis-induced processes: a signaling pathway induced by AC-derived nucleotides and a cellular metabolism pathway involving lactate production. These findings illustrate how seemingly distinct pathways in efferocytosing macrophages are integrated to carry out a key process in tissue resolution.

Nanotechnology could improve our understanding of the pathophysiology of atherosclerosis and contribute to the development of novel diagnostic and therapeutic strategies to further reduce the risk of cardiovascular disease. Macrophages have key roles in atherosclerosis progression and, therefore, macrophage-associated pathological processes are important targets for both diagnostic imaging and novel therapies for atherosclerosis. In this Review, we highlight efforts in the past two decades to develop imaging techniques and to therapeutically manipulate macrophages in atherosclerotic plaques with the use of rationally designed nanoparticles. We review the latest progress in nanoparticle-based imaging modalities that can specifically target macrophages. Using novel molecular imaging technology, these modalities enable the identification of advanced atherosclerotic plaques and the assessment of the therapeutic efficacy of medical interventions. Additionally, we provide novel perspectives on how macrophage-targeting nanoparticles can deliver a broad range of therapeutic payloads to atherosclerotic lesions. These nanoparticles can suppress pro-atherogenic macrophage processes, leading to improved resolution of inflammation and stabilization of plaques. Finally, we propose future opportunities for novel diagnostic and therapeutic strategies and provide solutions to challenges in this area for the purpose of accelerating the clinical translation of nanomedicine for the treatment of atherosclerotic vascular disease., In this Review, Tao and colleagues discuss the latest advances in nanoparticle-based imaging and therapeutic approaches targeting macrophages in atherosclerotic plaques, highlight opportunities for novel macrophage-targeting nanomedicines for atherosclerosis diagnosis and treatment, and provide solutions to challenges in this area to accelerate clinical translation., Key points Because macrophages have key roles in atherosclerosis progression, macrophage-mediated pro-atherosclerotic processes are important targets for both diagnostic imaging and novel therapies for atherosclerosis.Nanotechnology is particularly advantageous in its capacity to substantially improve the pharmacokinetic profile and chemical stability of encapsulated diagnostic and therapeutic agents for atherosclerosis management.The rational design of nanoparticle-based imaging agents that can specifically target inflammatory macrophages in atherosclerotic plaques offers diagnostic potential to non-invasively quantify atherosclerosis plaque burden, evaluate the efficacy of medical interventions and serve as surrogate end points.Targeted nanotherapeutics that can modulate plaque macrophage functions by the activation or suppression of specific signalling pathways have shown great promise in preclinical models by improving therapeutic efficacy and reducing off-target and systemic adverse effects.Rapid advances in nanotechnology and bioengineering and an improved understanding of atherosclerotic pathophysiology have accelerated the development of novel nanotherapeutics for atherosclerosis diagnosis and treatment.The success of various nanomedicine-based approaches in preclinical studies of atherosclerosis and their use in human cancer bode well for their future application in the diagnosis and treatment of patients with cardiovascular disease.

Necroptosis contributes to hepatocyte death in nonalcoholic steatohepatitis (NASH), but the fate and roles of necroptotic hepatocytes (necHCs) in NASH remain unknown. We show here that the accumulation of necHCs in human and mouse NASH liver is associated with an up-regulation of the “don’t-eat-me” ligand CD47 on necHCs, but not on apoptotic hepatocytes, and an increase in the CD47 receptor SIRPα on liver macrophages, consistent with impaired macrophage-mediated clearance of necHCs. In vitro, necHC clearance by primary liver macrophages was enhanced by treatment with either anti-CD47 or anti-SIRPα. In a proof-of-concept mouse model of inducible hepatocyte necroptosis, anti-CD47 antibody treatment increased necHC uptake by liver macrophages and inhibited markers of hepatic stellate cell (HSC) activation, which is responsible for liver fibrogenesis. Treatment of two mouse models of diet-induced NASH with anti-CD47, anti-SIRPα, or AAV8-H1-shCD47 to silence CD47 in hepatocytes increased the uptake of necHC by liver macrophages and decreased markers of HSC activation and liver fibrosis. Anti-SIRPα treatment avoided the adverse effect of anemia found in anti-CD47–treated mice. These findings provide evidence that impaired clearance of necHCs by liver macrophages due to CD47-SIRPα up-regulation contributes to fibrotic NASH, and suggest therapeutic blockade of the CD47-SIRPα axis as a strategy to decrease the accumulation of necHCs in NASH liver and dampen the progression of hepatic fibrosis.

Aims The neutrophil–lymphocyte ratio (NLR) is a readily available inflammatory biomarker that may associate with atherosclerosis and predict cardiovascular (CV) events. The aims of this study are to determine whether the NLR predicts incident major adverse cardiovascular events (MACE) and is modified by anti-inflammatory therapy. Methods and results Baseline and on-treatment NLRs were calculated from complete blood counts among 60 087 participants randomized in the CANTOS, JUPITER, SPIRE-1, SPIRE-2, and CIRT trials to receive placebo or canakinumab, rosuvastatin, bococizumab, or methotrexate, respectively, and followed up for MACE. All analyses were performed first in CANTOS, and then externally validated in the other four trials. For the five trials, hazard ratios for major CV events and mortality comparing NLR quartiles were computed using Cox proportional hazards models, and the effect of each randomized intervention on the NLR was evaluated in comparison to placebo. The NLR modestly correlated with interleukin-6, C-reactive protein, and fibrinogen levels but minimally with lipids. In all five randomized trials, baseline NLR predicted incident CV events and death; the per-quartile increase in risk of MACE was 20% in CANTOS [95% confidence interval (CI) 14–25%, P Conclusion The NLR, an easily obtained inflammatory biomarker, independently predicts CV risk and all-cause mortality, and is reduced by interleukin-1β blockade with canakinumab.

Fibrosis contributes to ~45% of deaths in western countries. In chronic liver disease, fibrosis is a major factor determining outcomes, but efficient antifibrotic therapies are lacking. Although platelet-derived growth factor and transforming growth factor–β constitute key fibrogenic mediators, they do not account for the well-established link between cell death and fibrosis in the liver. Here, we hypothesized that damage-associated molecular patterns (DAMPs) may link epithelial cell death to fibrogenesis in the injured liver. DAMP receptor screening identified purinergic receptor P2Y14 among several candidates as highly enriched in hepatic stellate cells (HSCs), the main fibrogenic cell type of the liver. Conversely, P2Y14 ligands uridine 5′-diphosphate (UDP)–glucose and UDP-galactose were enriched in hepatocytes and were released upon different modes of cell death. Accordingly, ligand-receptor interaction analysis that combined proteomic and single-cell RNA sequencing data revealed P2Y14 ligands and P2Y14 receptor as a link between dying cells and HSCs, respectively. Treatment with P2Y14 ligands or coculture with dying hepatocytes promoted HSC activation in a P2Y14-dependent manner. P2Y14 ligands activated extracellular signal–regulated kinase (ERK) and Yes-associated protein (YAP) signaling in HSCs, resulting in ERK-dependent HSC activation. Global and HSC-selective P2Y14 deficiency attenuated liver fibrosis in multiple mouse models of liver injury. Functional expression of P2Y14 was confirmed in healthy and diseased human liver and human HSCs. In conclusion, P2Y14 ligands and their receptor constitute a profibrogenic DAMP pathway that directly links cell death to fibrogenesis.

Cardiovascular disease (CVD) is the leading cause of death in patients with diabetes, and tight glycemic control fails to reduce the risk of developing CVD. Thiazolidinediones (TZDs), a class of peroxisome proliferator–activated receptor γ (PPARγ) agonists, are potent insulin sensitizers with antiatherogenic properties, but their clinical use is limited by side effects. PPARγ deacetylation on two lysine residues (K268 and K293) induces brown remodeling of white adipose tissue and uncouples the adverse effects of TZDs from insulin sensitization. Here we show that PPARγ deacetylation confers antiatherogenic properties and retains the insulin-sensitizing effects of TZD while circumventing its detriments. We generated mice homozygous with mice with deacetylation-mimetic PPARγ mutations K268R/K293R (2KR) on an LDL-receptor knockout (Ldlr−/−) background. 2KR:Ldlr−/− mice showed smaller atherosclerotic lesion areas than Ldlr−/− mice, particularly in aortic arches. With rosiglitazone treatment, 2KR:Ldlr−/− mice demonstrated a residual antiatherogenic response and substantial protection against bone loss and fluid retention. The antiatherosclerotic effect of 2KR was attributed to the protection of endothelium, indicated by improved endothelium-dependent vasorelaxation and repressed expression of proatherogenic factors including inducible nitric oxide synthase, interleukin-6, and NADPH oxidase 2. Therefore, manipulating PPARγ acetylation is a promising therapeutic strategy to control risk of CVD in diabetes treatment.

Nonalcoholic fatty liver disease is the most prevalent liver disease worldwide, affecting 20%-25% of the adult population. In 25% of patients, nonalcoholic fatty liver disease progresses to nonalcoholic steatohepatitis (NASH), which increases the risk for the development of cirrhosis, liver failure, and hepatocellular carcinoma. In patients with NASH, liver fibrosis is the main determinant of mortality. Here, we review how interactions between different liver cells culminate in fibrosis development in NASH, focusing on triggers and consequences of hepatocyte-macrophage-hepatic stellate cell (HSC) crosstalk. We discuss pathways through which stressed and dead hepatocytes instigate the profibrogenic crosstalk with HSC and macrophages, including the reactivation of developmental pathways such as TAZ, Notch, and hedgehog; how clearance of dead cells in NASH via efferocytosis may affect inflammation and fibrogenesis; and insights into HSC and macrophage heterogeneity revealed by single-cell RNA sequencing. Finally, we summarize options to therapeutically interrupt this profibrogenic hepatocyte-macrophage-HSC network in NASH.

sPhagocytic clearance of dying cells, termed efferocytosis, must occur efficiently to maintain homeostasis and prevent disease. Yet, our understanding of this important biological process remains incomplete. To search for novel regulators of efferocytosis, we performed a FACS-based genome-wide CRISPR knockout screen in primary murine macrophages. We identified a novel role for WDFY3 in efferocytosis by macrophages. WDFY3 deficiency in macrophages specifically impaired uptake, not binding, of apoptotic cells due to defective actin depolymerization. We further revealed that WDFY3 directly interacts with GABARAP, thus facilitating LC3 lipidation and subsequent lysosomal acidification to permit the degradation of apoptotic cell components. Although the C-terminus of WDFY3 was sufficient to rescue impaired degradation, full-length WDFY3 is still required for regulating uptake. Finally, WDFY3 is required for efficient efferocytosis in vivo in mice and in primary human macrophages. The work expands our knowledge of the mechanisms of macrophage efferocytosis, and more broadly, provides a general strategy for genome-wide CRISPR screen to interrogate complex functional phenotypes in primary macrophages.HighlightsFunctional readout for pooled genome-wide CRISPR screen in primary macrophages.WDFY3 is discovered as a regulator of macrophage efferocytosis in vitro and in vivo.WDFY3 deficiency led to impaired uptake, as opposed to binding, of apoptotic cells due to defective actin depolymerization.WDFY3 directly interacts with GABARAP, facilitating LC3 lipidation and subsequent lysosomal acidification to permit the degradation of apoptotic cell components.C-terminal WDFY3 is sufficient to regulate the degradation of engulfed apoptotic cells while full-length WDFY is required for regulating uptake.

The clearance of apoptotic cells by professional and non-professional phagocytes — a process termed ‘efferocytosis’ — is essential for the maintenance of tissue homeostasis. Accordingly, defective efferocytosis underlies a growing list of chronic inflammatory diseases. Although much has been learnt about the mechanisms of apoptotic cell recognition and uptake, several key areas remain incompletely understood. This Review focuses on new discoveries related to how phagocytes process the metabolic cargo they receive during apoptotic cell uptake; the links between efferocytosis and the resolution of inflammation in health and disease; and the roles of efferocytosis in host defence. Understanding these aspects of efferocytosis sheds light on key physiological and pathophysiological processes and suggests novel therapeutic strategies for diseases driven by defective efferocytosis and impaired inflammation resolution. Clearing away dead cells — a process known as efferocytosis — is crucial for normal tissue homeostasis and is impaired in several pathological processes. This Review describes new insights into how efferocytes deal with the engulfed dead cell cargo, how efferocytosis supports the resolution of inflammation and how this understanding is informing new therapeutic strategies.

Summary Apoptotic cell clearance by macrophages (efferocytosis) promotes resolution signaling pathways, which can be triggered by molecules derived from the phagolysosomal degradation of apoptotic cells. We show here that nucleotides derived from the hydrolysis of apoptotic cell DNA by phagolysosomal DNase2a activate a DNA−PKcs−mTORC2/Rictor pathway that increases Myc to promote non-inflammatory macrophage proliferation. Efferocytosis-induced proliferation expands the pool of resolving macrophages in vitro and in mice, including zymosan-induced peritonitis, dexamethasone-induced thymocyte apoptosis, and atherosclerosis regression. In the dexamethasone-thymus model, hematopoietic Rictor deletion blocked efferocytosing macrophage proliferation, apoptotic cell clearance, and tissue resolution. In atherosclerosis regression, silencing macrophage Rictor or DNase2a blocked efferocyte proliferation, apoptotic cell clearance, and plaque stabilization. In view of previous work showing that other types of apoptotic cell cargo can promote resolution in individual efferocytosing macrophages, the findings here suggest that signaling-triggered apoptotic cell-derived nucleotides can amplify this benefit by increasing the number of these macrophages.

Efferocytosis, the clearance of apoptotic cells (ACs) by macrophages, is critical for tissue resolution, with defects driving many diseases. Mechanisms of efferocytosis-mediated resolution are incompletely understood. Here, we show that AC-derived methionine regulates resolution through epigenetic repression of the extracellular signal-regulated kinase 1/2 (ERK1/2) phosphatase Dusp4. We focus on two key efferocytosis-induced pro-resolving mediators, prostaglandin E

Non-alcoholic steatohepatitis (NASH) is a leading cause of hepatocellular carcinoma (HCC), but mechanisms linking NASH to eventual tumor formation remain poorly understood. Herein, we investigate the role of TAZ/WWTR1, which is induced in hepatocytes in NASH, in the progression of NASH to HCC.The roles of hepatocyte TAZ and its downstream targets were investigated in diet-induced and genetic models of NASH-HCC using gene-targeting, adeno-associated virus 8 (AAV8)-H1-mediated gene silencing, or AAV8-TBG-mediated gene expression. The biochemical signature of the newly elucidated pathway was probed in liver specimens from humans with NASH-HCC.When hepatocyte-TAZ was silenced in mice with pre-tumor NASH using AAV8-H1-shTaz (short-hairpin Taz), subsequent HCC tumor development was suppressed. In this setting, the tumor-suppressing effect of shTaz was not dependent of TAZ silencing in the tumors themselves and could be dissociated from the NASH-suppressing effects of shTaz. The mechanism linking pre-tumor hepatocyte-TAZ to eventual tumor formation involved TAZ-mediated induction of the NOX2-encoding gene Cybb, which led to NADPH-mediated oxidative DNA damage. As evidence, DNA damage and tumor formation could be suppressed by treatment of pre-tumor NASH mice with AAV8-H1-shCybb; AAV8-TBG-OGG1, encoding the oxidative DNA-repair enzyme 8-oxoguanine glycosylase; or AAV8-TBG-NHEJ1, encoding the dsDNA repair enzyme non-homologous end-joining factor 1. In surrounding non-tumor tissue from human NASH-HCC livers, there were strong correlations between TAZ, NOX2, and oxidative DNA damage.TAZ in pre-tumor NASH-hepatocytes, via induction of Cybb and NOX2-mediated DNA damage, contributes to subsequent HCC tumor development. These findings illustrate how NASH provides a unique window into the early molecular events that can lead to tumor formation and suggest that NASH therapies targeting TAZ might also prevent NASH-HCC.Non-alcoholic steatohepatitis (NASH) is emerging as the leading cause of a type of liver cancer called hepatocellular carcinoma (HCC), but molecular events in pre-tumor NASH hepatocytes leading to HCC remain largely unknown. Our study shows that a protein called TAZ in pre-tumor NASH-hepatocytes promotes damage to the DNA of hepatocytes and thereby contributes to eventual HCC. This study reveals a very early event in HCC that is induced in pre-tumor NASH, and the findings suggest that NASH therapies targeting TAZ might also prevent NASH-HCC.

Aberrant hepatocyte Notch activity is critical to the development of nonalcoholic steatohepatitis (NASH)–induced liver fibrosis, but mechanisms underlying Notch reactivation in developed liver are unclear. Here, we identified that increased expression of the Notch ligand Jagged1 (JAG1) tracked with Notch activation and nonalcoholic fatty liver disease (NAFLD) activity score (NAS) in human liver biopsy specimens and mouse NASH models. The increase in Jag1 was mediated by hepatocyte Toll-like receptor 4 (TLR4)–nuclear factor κB (NF-κB) signaling in pericentral hepatocytes. Hepatocyte-specific Jag1 overexpression exacerbated fibrosis in mice fed a high-fat diet or a NASH-provoking diet rich in palmitate, cholesterol, and sucrose and reversed the protection afforded by hepatocyte-specific TLR4 deletion, whereas hepatocyte-specific Jag1 knockout mice were protected from NASH-induced liver fibrosis. To test therapeutic potential of this biology, we designed a Jag1-directed antisense oligonucleotide (ASO) and a hepatocyte-specific N-acetylgalactosamine (GalNAc)–modified siRNA, both of which reduced NASH diet–induced liver fibrosis in mice. Overall, these data demonstrate that increased hepatocyte Jagged1 is the proximal hit for Notch-induced liver fibrosis in mice and suggest translational potential of Jagged1 inhibitors in patients with NASH.

Macrophage polarization is critical to inflammation and resolution of inflammation. We previously showed that high-mobility group box 1 (HMGB1) can engage receptor for advanced glycation end product (RAGE) to direct monocytes to a proinflammatory phenotype characterized by production of type 1 IFN and proinflammatory cytokines. In contrast, HMGB1 plus C1q form a tetramolecular complex cross-linking RAGE and LAIR-1 and directing monocytes to an antiinflammatory phenotype. Lipid mediators, as well as cytokines, help establish a milieu favoring either inflammation or resolution of inflammation. This study focuses on the induction of lipid mediators by HMGB1 and HMGB1 plus C1q and their regulation of IRF5, a transcription factor critical for the induction and maintenance of proinflammatory macrophages. Here, we show that HMGB1 induces leukotriene production through a RAGE-dependent pathway, while HMGB1 plus C1q induces specialized proresolving lipid mediators lipoxin A4, resolvin D1, and resolvin D2 through a RAGE- and LAIR-1-dependent pathway. Leukotriene exposure contributes to induction of IRF5 in a positive-feedback loop. In contrast, resolvins (at 20 nM) block IRF5 induction and prevent the differentiation of inflammatory macrophages. Finally, we have generated a molecular mimic of HMGB1 plus C1q, which cross-links RAGE and LAIR-1 and polarizes monocytes to an antiinflammatory phenotype. These findings may provide a mechanism to control nonresolving inflammation in many pathologic conditions.

Tissue-type plasminogen activator (tPA) is a major mediator of fibrinolysis and, thereby, prevents excessive coagulation without compromising hemostasis. Studies on tPA regulation have focused on its acute local release by vascular cells in response to injury or other stimuli. However, very little is known about sources, regulation, and fibrinolytic function of noninjury-induced systemic plasma tPA. We explore the role and regulation of hepatocyte-derived tPA as a source of basal plasma tPA activity and as a contributor to fibrinolysis after vascular injury. We show that hepatocyte tPA is downregulated by a pathway in which the corepressor DACH1 represses ATF6, which is an inducer of the tPA gene Plat. Hepatocyte-DACH1–knockout mice show increases in liver Plat, circulating tPA, fibrinolytic activity, bleeding time, and time to thrombosis, which are reversed by silencing hepatocyte Plat. Conversely, hepatocyte-ATF6–knockout mice show decreases in these parameters. The inverse correlation between DACH1 and ATF6/PLAT is conserved in human liver. These findings reveal a regulated pathway in hepatocytes that contributes to basal circulating levels of tPA and to fibrinolysis after vascular injury.

Summary DACH1 abundance is reduced in human malignancies, including breast cancer. Herein DACH1 was detected among multipotent fetal mammary stem cells in the embryo, among mixed lineage precursors, and in adult basal cells and (ERα+) luminal progenitors. Dach1 gene deletion at 6 weeks in transgenic mice reduced ductal branching, reduced the proportion of mammary basal cells (Lin− CD24med CD29high) and reduced abundance of basal cytokeratin 5, whereas DACH1 overexpression induced ductal branching, increased Gata3 and Notch1, and expanded mammosphere formation in LA-7 breast cells. Mammary gland-transforming growth factor β (TGF-β) activity, known to reduce ductal branching and to reduce the basal cell population, increased upon Dach1 deletion, associated with increased SMAD phosphorylation. Association of the scaffold protein Smad anchor for receptor activation with Smad2/3, which facilitates TGF-β activation, was reduced by endogenous DACH1. DACH1 increases basal cells, enhances ductal formation and restrains TGF-β activity in vivo., Highlights • Dach1 is expressed in mammary gland fetal stem cells and adult luminal cells • Dach1 expands mammary gland basal/myoepithelial cells • Dach1 induces post-natal mammary gland ductal formation • Dach1 retrains TGF-β activity in the mammary gland in vivo, DACH1 abundance, is reduced in human malignancies, including breast cancer. In this article, Dr. Pestell and his colleagues showed that DACH1 is expressed in multipotent mammary gland fetal stem cells and both the adult basal and ERα+ luminal cells, promotes mammary gland stem cell and basal/myoepithelial cell expansion, promotes mammary gland ductal branching, and restrains TGF-β action in the pubertal mammary gland.

Macrophages in atherosclerotic lesions promote plaque progression and are an attractive therapeutic target in cardiovascular research. Here we present a protocol for synthesis of small interfering RNA (siRNA) nanoparticles (NP) that target lesional macrophages as a potential treatment for atherosclerosis. Ca

Hepatocellular carcinoma (HCC), the fourth leading cause of cancer mortality, develops almost exclusively in patients with chronic liver disease (CLD) and advanced fibrosis(1,2). Here we interrogated functions of hepatic stellate cells (HSC), the main source of liver fibroblasts(3), during hepatocarcinogenesis. Genetic depletion, activation or inhibition established HSC as tumour-promoting in mouse models of HCC. HSC were enriched in the preneoplastic environment, where they closely interacted with hepatocytes and modulated hepatocarcinogenesis by regulating hepatocyte proliferation and death. Analysis of mouse and human HSC subpopulations and their associated mediators by single cell RNA-sequencing in conjunction with genetic ablation revealed dual functions of HSC in hepatocarcinogenesis. Hepatocyte growth factor, enriched in quiescent and cytokine-producing HSC (cyHSC), protected from hepatocyte death and HCC development. In contrast, type I collagen, enriched in activated myofibroblastic HSC (myHSC), promoted proliferation and tumour development via increased stiffness and TAZ activation in pretumoural hepatocytes and via activation of discoidin domain receptor 1 in established tumours. An increasing HSC dysbalance between cyHSC and myHSC during liver disease progression was associated with elevated HCC risk in patients. In summary, the dynamic shift of HSC subpopulations and their mediators during CLD is associated with a switch from HCC protection to HCC promotion.

Efferocytosis, the process through which apoptotic cells (ACs) are cleared through actin-mediated engulfment by macrophages, prevents secondary necrosis, suppresses inflammation, and promotes resolution. Impaired efferocytosis drives the formation of clinically dangerous necrotic atherosclerotic plaques, the underlying etiology of coronary artery disease (CAD). An intron of the gene encoding PHACTR1 contains rs9349379 (A>G), a common variant associated with CAD. As PHACTR1 is an actin-binding protein, we reasoned that if the rs9349379 risk allele G causes lower PHACTR1 expression in macrophages, it might link the risk allele to CAD via impaired efferocytosis. We show here that rs9349379-G/G was associated with lower levels of PHACTR1 and impaired efferocytosis in human monocyte–derived macrophages and human atherosclerotic lesional macrophages compared with rs9349379-A/A. Silencing PHACTR1 in human and mouse macrophages compromised AC engulfment, and Western diet–fed Ldlr(–/–) mice in which hematopoietic Phactr1 was genetically targeted showed impaired lesional efferocytosis, increased plaque necrosis, and thinner fibrous caps — all signs of vulnerable plaques in humans. Mechanistically, PHACTR1 prevented dephosphorylation of myosin light chain (MLC), which was necessary for AC engulfment. In summary, rs9349379-G lowered PHACTR1, which, by lowering phospho-MLC, compromised efferocytosis. Thus, rs9349379-G may contribute to CAD risk, at least in part, by impairing atherosclerotic lesional macrophage efferocytosis.

Tyro3, AXL, and MerTK (TAM) receptors are activated in macrophages in response to tissue injury and as such have been proposed as therapeutic targets to promote inflammation resolution during sterile wound healing, including myocardial infarction. Although the role of MerTK in cardioprotection is well characterized, the unique role of the other structurally similar TAMs, and particularly AXL, in clinically relevant models of myocardial ischemia/reperfusion infarction (IRI) is comparatively unknown. Utilizing complementary approaches, validated by flow cytometric analysis of human and murine macrophage subsets and conditional genetic loss and gain of function, we uncover a maladaptive role for myeloid AXL during IRI in the heart. Cross signaling between AXL and TLR4 in cardiac macrophages directed a switch to glycolytic metabolism and secretion of proinflammatory IL-1β, leading to increased intramyocardial inflammation, adverse ventricular remodeling, and impaired contractile function. AXL functioned independently of cardioprotective MerTK to reduce the efficacy of cardiac repair, but like MerTK, was proteolytically cleaved. Administration of a selective small molecule AXL inhibitor alone improved cardiac healing, which was further enhanced in combination with blockade of MerTK cleavage. These data support further exploration of macrophage TAM receptors as therapeutic targets for myocardial infarction.

Atherosclerotic vascular disease resulting from unstable plaques is the leading cause of morbidity and mortality in subjects with type 2 diabetes (T2D), and thus a major therapeutic goal is to discover T2D drugs that can also promote atherosclerotic plaque stability. Genetic or pharmacologic inhibition of mitogen-activated protein kinase-activated protein kinase-2 (MK2) in obese mice improves glucose homeostasis and enhances insulin sensitivity. We developed two novel orally active small-molecule inhibitors of MK2, TBX-1 and TBX-2, and tested their effects on metabolism and atherosclerosis in high-fat Western diet (WD)-fed Ldlr-/- mice. Ldlr-/- mice were first fed the WD to allow atherosclerotic lesions to become established, and the mice were then treated with TBX-1 or TBX-2. Both compounds improved glucose metabolism and lowered plasma cholesterol and triglyceride, without an effect on body weight. Most importantly, the compounds decreased lesion area, lessened plaque necrosis, and increased fibrous cap thickness in the aortic root lesions of the mice. Thus, in a preclinical model of high-fat feeding and established atherosclerosis, MK2 inhibitors improved metabolism and also enhanced atherosclerotic plaque stability, suggesting potential for further clinical development to address the epidemic of T2D associated with atherosclerotic vascular disease.

Objective: ODC (ornithine decarboxylase)-dependent putrescine synthesis promotes the successive clearance of apoptotic cells (ACs) by macrophages, contributing to inflammation resolution. However, it remains unknown whether ODC is required for other arms of the resolution program. Approach and Results: RNA sequencing of ODC-deficient macrophages exposed to ACs showed increases in mRNAs associated with heightened inflammation and decreases in mRNAs related to resolution and repair compared with WT (wild type) macrophages. In zymosan peritonitis, myeloid ODC deletion led to delayed clearance of neutrophils and a decrease in the proresolving cytokine, IL (interleukin)-10. Nanoparticle-mediated silencing of macrophage ODC in a model of atherosclerosis regression lowered IL-10 expression, decreased efferocytosis, enhanced necrotic core area, and reduced fibrous cap thickness. Mechanistically, ODC deletion lowered basal expression of MerTK (MER tyrosine-protein kinase)—an AC receptor—via a histone methylation–dependent transcriptional mechanism. Owing to lower basal MerTK, subsequent exposure to ACs resulted in lower MerTK-Erk (extracellular signal-regulated kinase) 1/2–dependent IL-10 production. Putrescine treatment of ODC-deficient macrophages restored the expression of both MerTK and AC-induced IL-10. Conclusions: These findings demonstrate that ODC-dependent putrescine synthesis in macrophages maintains a basal level of MerTK expression needed to optimally resolve inflammation upon subsequent AC exposure. Graphic Abstract: A graphic abstract is available for this article.

With the rapid expansion of the obesity epidemic, nonalcoholic fatty liver disease is now the most common chronic liver disease, with almost 25% global prevalence. Nonalcoholic fatty liver disease ranges in severity from simple steatosis, a benign ‘pre-disease’ state, to the liver injury and inflammation that characterize nonalcoholic steatohepatitis (NASH), which in turn predisposes individuals to liver fibrosis. Fibrosis is the major determinant of clinical outcomes in patients with NASH and is associated with increased risks of cirrhosis and hepatocellular carcinoma. NASH has no approved therapies, and liver fibrosis shows poor response to existing pharmacotherapy, in part due to an incomplete understanding of the underlying pathophysiology. Patient and mouse data have shown that NASH is associated with the activation of developmental pathways: Notch, Hedgehog and Hippo–YAP–TAZ. Although these evolutionarily conserved fundamental signals are known to determine liver morphogenesis during development, new data have shown a coordinated and causal role for these pathways in the liver injury response, which becomes maladaptive during obesity-associated chronic liver disease. In this Review, we discuss the aetiology of this reactivation of developmental pathways and review the cell-autonomous and cell-non-autonomous mechanisms by which developmental pathways influence disease progression. Finally, we discuss the potential prognostic and therapeutic implications of these data for NASH and liver fibrosis. Emerging data have revealed that nonalcoholic steatohepatitis (NASH) and fibrosis are associated with the reactivation of developmental pathways in the liver injury response. This Review describes the role of these pathways in liver development and in the pathogenesis of NASH and fibrosis.

Fibrinolysis is initiated by tissue-type plasminogen activator (tPA) and inhibited by plasminogen activator inhibitor 1 (PAI-1). In obese humans, plasma PAI-1 and tPA proteins are increased, but PAI-1 dominates, leading to reduced fibrinolysis and thrombosis. To understand tPA–PAI-1 regulation in obesity, we focused on hepatocytes, a functionally important source of tPA and PAI-1 that sense obesity-induced metabolic stress. We showed that obese mice, like humans, had reduced fibrinolysis and increased plasma PAI-1 and tPA, due largely to their increased hepatocyte expression. A decrease in the PAI-1 (SERPINE1) gene corepressor Rev-Erbα increased PAI-1, which then increased the tPA gene PLAT via a PAI-1/LRP1/PKA/p-CREB1 pathway. This pathway was partially counterbalanced by increased DACH1, a PLAT-negative regulator. We focused on the PAI-1/PLAT pathway, which mitigates the reduction in fibrinolysis in obesity. Thus, silencing hepatocyte PAI-1, CREB1, or tPA in obese mice lowered plasma tPA and further impaired fibrinolysis. The PAI-1/PLAT pathway was present in primary human hepatocytes, and associations among PAI-1, tPA, and PLAT in livers from obese and lean humans were consistent with these findings. Knowledge of PAI-1 and tPA regulation in hepatocytes in obesity may suggest therapeutic strategies for improving fibrinolysis and lowering the risk of thrombosis in this setting.

Cardiovascular disease (CVD) is the leading cause of death in diabetic patients; however, tight glycemic control fails to lower the risk. The thiazolidinediones (TZDs), a class of PPARg agonists, are potent insulin sensitizers with anti-atherogenic properties, but their clinical utilization is limited by the side effects. PPARg deacetylation on two lysine residues (K268 and K293) induces brown remodeling of white adipose tissue and uncouples TZD’s adverse effects from insulin sensitization. Here we show that PPARg deacetylation confers anti-atherogenic properties and retains the insulin-sensitizing effects of TZD while circumventing its detriments. We generated mice homozygous for deacetylation-mimetic PPARg mutations K268R/K293R (2KR) mice on an LDL-receptor knockout (Ldlr–/–) background. 2KR:Ldlr–/– mice showed reduced atherosclerotic lesions compared to Ldlr–/– mice, particularly in aortic arches. With rosiglitazone treatment, 2KR:Ldlr–/– mice demonstrated a residual anti-atherogenic response and significant protection against bone loss and fluid retention. The anti-atherosclerotic effect of 2KR was attributed to the protection of endothelium, indicated by the improved endothelium-dependent vasorelaxation and repressed expression of pro-atherogenic factors including inducible NO synthase (iNOS), IL-6, and NADPH oxidase 2 (Nox2). Therefore, manipulating PPARg acetylation is a promising therapeutic strategy to control CVD risk in diabetes treatment.

Summary Incomplete understanding of how hepatosteatosis transitions to fibrotic non-alcoholic steatohepatitis (NASH) has limited therapeutic options. Two molecules that are elevated in hepatocytes in human NASH liver are cholesterol, whose mechanistic link to NASH remains incompletely understood, and TAZ, a transcriptional regulator that promotes fibrosis but whose mechanism of increase in NASH is unknown. We now show that increased hepatocyte cholesterol upregulates TAZ and promotes fibrotic NASH. ASTER-B/C-mediated internalization of plasma membrane cholesterol activates soluble adenylyl cyclase (sAC; ADCY10), triggering a calcium-RhoA-mediated pathway that suppresses β-TrCP/proteasome-mediated TAZ degradation. In mice fed with a cholesterol-rich NASH-inducing diet, hepatocyte-specific silencing of ASTER-B/C, sAC, or RhoA decreased TAZ and ameliorated fibrotic NASH. The cholesterol-TAZ pathway is present in primary human hepatocytes, and associations among liver cholesterol, TAZ, and RhoA in human NASH liver are consistent with the pathway. Thus, hepatocyte cholesterol contributes to fibrotic NASH by increasing TAZ, suggesting new targets for therapeutic intervention.

Macrophage immunometabolism, the changes in intracellular metabolic pathways that alter the function of these highly plastic cells, has been the subject of intense interest in the past few years, in part because macrophage immunometabolism plays important roles in atherosclerosis and other inflammatory diseases. In this review article, part of the Compendium on Atherosclerosis , we introduce the concepts of (1) intracellular immunometabolism—the canonical pathways of intrinsic cell activation leading to changes in intracellular metabolism, which in turn alter cellular function; and (2) intercellular immunometabolism—conditions in which intermediates of cellular metabolism are transferred from one cell to another, thereby altering the function of the recipient cell. The recent discovery that the metabolite cargo of dead and dying cells ingested through efferocytosis by macrophages can alter metabolic pathways and downstream function of the efferocyte is markedly changing the way we think about macrophage immunometabolism. Metabolic transitions of macrophages contribute to their functions in all stages of atherosclerosis, from lesion initiation to formation of advanced lesions characterized by necrotic cores, to lesion regression following aggressive lipid lowering. This review article discusses recent advances in our understanding of these different aspects of macrophage immunometabolism in atherosclerosis. With the increasing understanding of the roles of macrophage immunometabolism in atherosclerosis, new exciting concepts and potential targets for intervention are emerging.

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.

Non-resolving inflammation drives the development of clinically dangerous atherosclerotic lesions by promoting sustained plaque inflammation, large necrotic cores, thin fibrous caps, and thrombosis. Resolution of inflammation is not merely a passive return to homeostasis, but rather an active process mediated by specific molecules, including fatty acid-derived specialized pro-resolving mediators (SPMs). In advanced atherosclerosis, there is an imbalance between levels of SPMs and proinflammatory lipid mediators, which results in sustained leukocyte influx into lesions, inflammatory macrophage polarization, and impaired efferocytosis. In animal models of advanced atherosclerosis, restoration of SPMs limits plaque progression by suppressing inflammation, enhancing efferocytosis, and promoting an increase in collagen cap thickness. This Review discusses the roles of non-resolving inflammation in atherosclerosis and highlights the unique therapeutic potential of SPMs in blocking the progression of clinically dangerous plaques.

Obesity-induced metabolic disease involves functional integration among several organs via circulating factors, but little is known about crosstalk between liver and visceral adipose tissue (VAT)1. In obesity, VAT becomes populated with inflammatory adipose tissue macrophages (ATMs)2,3. In obese humans, there is a close correlation between adipose tissue inflammation and insulin resistance4,5, and in obese mice, blocking systemic or ATM inflammation improves insulin sensitivity6–8. However, processes that promote pathological adipose tissue inflammation in obesity are incompletely understood. Here we show that obesity in mice stimulates hepatocytes to synthesize and secrete dipeptidyl peptidase 4 (DPP4), which acts with plasma factor Xa to inflame ATMs. Silencing expression of DPP4 in hepatocytes suppresses inflammation of VAT and insulin resistance; however, a similar effect is not seen with the orally administered DPP4 inhibitor sitagliptin. Inflammation and insulin resistance are also suppressed by silencing expression of caveolin-1 or PAR2 in ATMs; these proteins mediate the actions of DPP4 and factor Xa, respectively. Thus, hepatocyte DPP4 promotes VAT inflammation and insulin resistance in obesity, and targeting this pathway may have metabolic benefits that are distinct from those observed with oral DPP4 inhibitors.

Atherosclerosis is the underlying etiology of cardiovascular disease, the leading cause of death worldwide. Atherosclerosis is a heterogeneous disease in which only a small fraction of lesions lead to heart attack, stroke, or sudden cardiac death. A distinct type of plaque containing large necrotic cores with thin fibrous caps often precipitates these acute events. Here, we show that Ca2+/calmodulin-dependent protein kinase gamma (CaMKII gamma) in macrophages plays a major role in the development of necrotic, thin-capped plaques. Macrophages in necrotic and symptomatic atherosclerotic plaques in humans as well as advanced atherosclerotic lesions in mice demonstrated activation of CaMKII. Western diet-fed LDL receptor-deficient (Ldlr(-/-)) mice with myeloid-specific deletion of CaMKII had smaller necrotic cores with concomitantly thicker collagen caps. These lesions demonstrated evidence of enhanced efferocytosis, which was associated with increased expression of the macrophage efferocytosis receptor MerTK. Mechanistic studies revealed that CaMKII gamma-deficient macrophages and atherosclerotic lesions lacking myeloid CaMKII gamma had increased expression of the transcription factor ATF6. We determined that ATF6 induces liver X receptor-alpha (LXR alpha), an Mertk-inducing transcription factor, and that increased MerTK expression and efferocytosis in CaMKII gamma-deficient macrophages is dependent on LXR alpha. These findings identify a macrophage CaMKII gamma/ATF6/LXR alpha/MerTK pathway as a key factor in the development of necrotic atherosclerotic plaques.