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

RATIONALE: Atherosclerosis is characterized by an accumulation of foam cells within the arterial wall, resulting from excess cholesterol uptake and buildup of cytosolic lipid droplets (LDs). Autophagy promotes LD clearance by freeing stored cholesterol for efflux, a process that has been shown to be atheroprotective. While the role of autophagy in LD catabolism has been studied in macrophage-derived foam cells, this has remained unexplored in vascular smooth muscle cell (VSMC)-derived foam cells that constitute a large fraction of foam cells within atherosclerotic lesions. OBJECTIVE: We performed a comparative analysis of autophagy flux in lipid-rich aortic intimal populations to determine whether VSMC-derived foam cells metabolize LDs similarly to their macrophage counterparts. METHODS AND RESULTS: Atherosclerosis was induced in GFP-LC3 (microtubule-associated proteins 1A/1B light chain 3) transgenic mice by PCSK9 (proprotein convertase subtilisin/kexin type 9)-adeno-associated viral injection and Western diet feeding. Using flow cytometry of aortic digests, we observed a significant increase in dysfunctional autophagy of VSMC-derived foam cells during atherogenesis relative to macrophage-derived foam cells. Using cell culture models of lipid-loaded VSMCs and macrophages, we show that autophagy-mediated cholesterol efflux from VSMC foam cells was poor relative to macrophage foam cells, and largely occurs when HDL (high-density lipoprotein) was used as a cholesterol acceptor, as opposed to apoA-1 (apolipoproteinA-1). This was associated with the predominant expression of ABCG1 in VSMC foam cells. Using metformin, an autophagy activator, cholesterol efflux to HDL was significantly increased in VSMC, but not in macrophage, foam cells. CONCLUSIONS: These data demonstrate that VSMC and macrophage foam cells perform cholesterol efflux by distinct mechanisms, and that autophagy flux is highly impaired in VSMC foam cells, but can be induced by pharmacological means. Further investigation is warranted into targeting autophagy specifically in VSMC foam cells, the predominant foam cell subtype of advanced atherosclerotic plaques, to promote reverse cholesterol transport and resolution of the atherosclerotic plaque.

miR-223 Exerts Translational Control of Proatherogenic Genes in Macrophages.

BACKGROUND: A significant burden of atherosclerotic disease is driven by inflammation. Recently, microRNAs (miRNAs) have emerged as important factors driving and protecting from atherosclerosis. miR-223 regulates cholesterol metabolism and inflammation via targeting both cholesterol biosynthesis pathway and NFkB signaling pathways; however, its role in atherosclerosis has not been investigated. We hypothesize that miR-223 globally regulates core inflammatory pathways in macrophages in response to inflammatory and atherogenic stimuli thus limiting the progression of atherosclerosis. METHODS AND RESULTS: Loss of miR-223 in macrophages decreases Abca1 gene and protein expression as well as cholesterol efflux to apoA1 (Apolipoprotein A1) and enhances proinflammatory gene expression. In contrast, overexpression of miR-223 promotes the efflux of cholesterol and macrophage polarization toward an anti-inflammatory phenotype. These beneficial effects of miR-223 are dependent on its target gene, the transcription factor Sp3. Consistent with the antiatherogenic effects of miR-223 in vitro, mice receiving miR223-/- bone marrow exhibit increased plaque size, lipid content, and circulating inflammatory cytokines (ie, IL-1ß). Deficiency of miR-223 in bone marrow-derived cells also results in an increase in circulating pro-atherogenic cells (total monocytes and neutrophils) compared with control mice. Furthermore, the expression of miR-223 target gene (Sp3) and pro-inflammatory marker (Il-6) are enhanced whereas the expression of Abca1 and anti-inflammatory marker (Retnla) are reduced in aortic arches from mice lacking miR-223 in bone marrow-derived cells. In mice fed a high-cholesterol diet and in humans with unstable carotid atherosclerosis, the expression of miR-223 is increased. To further understand the molecular mechanisms underlying the effect of miR-223 on atherosclerosis in vivo, we characterized global RNA translation profile of macrophages isolated from mice receiving wild-type or miR223-/- bone marrow. Using ribosome profiling, we reveal a notable upregulation of inflammatory signaling and lipid metabolism at the translation level but less significant at the transcription level. Analysis of upregulated genes at the translation level reveal an enrichment of miR-223-binding sites, confirming that miR-223 exerts significant changes in target genes in atherogenic macrophages via altering their translation. CONCLUSIONS: Our study demonstrates that miR-223 can protect against atherosclerosis by acting as a global regulator of RNA translation of cholesterol efflux and inflammation pathways.

RIPK1 Expression Associates With Inflammation in Early Atherosclerosis in Humans and Can Be Therapeutically Silenced to Reduce NF-κB Activation and Atherogenesis in Mice.

BACKGROUND: Chronic activation of the innate immune system drives inflammation and contributes directly to atherosclerosis. We previously showed that macrophages in the atherogenic plaque undergo RIPK3 (receptor-interacting serine/threonine-protein kinase 3)-MLKL (mixed lineage kinase domain-like protein)-dependent programmed necroptosis in response to sterile ligands such as oxidized low-density lipoprotein and damage-associated molecular patterns and that necroptosis is active in advanced atherosclerotic plaques. Upstream of the RIPK3-MLKL necroptotic machinery lies RIPK1 (receptor-interacting serine/threonine-protein kinase 1), which acts as a master switch that controls whether the cell undergoes NF-κB (nuclear factor κ-light-chain-enhancer of activated B cells)-dependent inflammation, caspase-dependent apoptosis, or necroptosis in response to extracellular stimuli. We therefore set out to investigate the role of RIPK1 in the development of atherosclerosis, which is driven largely by NF-κB-dependent inflammation at early stages. We hypothesize that, unlike RIPK3 and MLKL, RIPK1 primarily drives NF-κB-dependent inflammation in early atherogenic lesions, and knocking down RIPK1 will reduce inflammatory cell activation and protect against the progression of atherosclerosis. METHODS: We examined expression of RIPK1 protein and mRNA in both human and mouse atherosclerotic lesions, and used loss-of-function approaches in vitro in macrophages and endothelial cells to measure inflammatory responses. We administered weekly injections of RIPK1 antisense oligonucleotides to Apoe-/- mice fed a cholesterol-rich (Western) diet for 8 weeks. RESULTS: We find that RIPK1 expression is abundant in early-stage atherosclerotic lesions in both humans and mice. Treatment with RIPK1 antisense oligonucleotides led to a reduction in aortic sinus and en face lesion areas (47.2% or 58.8% decrease relative to control, P<0.01) and plasma inflammatory cytokines (IL-1α [interleukin 1α], IL-17A [interleukin 17A], P<0.05) in comparison with controls. RIPK1 knockdown in macrophages decreased inflammatory genes (NF-κB, TNFα [tumor necrosis factor α], IL-1α) and in vivo lipopolysaccharide- and atherogenic diet-induced NF-κB activation. In endothelial cells, knockdown of RIPK1 prevented NF-κB translocation to the nucleus in response to TNFα, where accordingly there was a reduction in gene expression of IL1B, E-selectin, and monocyte attachment. CONCLUSIONS: We identify RIPK1 as a central driver of inflammation in atherosclerosis by its ability to activate the NF-κB pathway and promote inflammatory cytokine release. Given the high levels of RIPK1 expression in human atherosclerotic lesions, our study suggests RIPK1 as a future therapeutic target to reduce residual inflammation in patients at high risk of coronary artery disease.

Loss of MLKL (Mixed Lineage Kinase Domain-Like Protein) Decreases Necrotic Core but Increases Macrophage Lipid Accumulation in Atherosclerosis.

OBJECTIVES: During the advancement of atherosclerosis, plaque cellularity is governed by the influx of monocyte-derived macrophages and their turnover via apoptotic and nonapoptotic forms of cell death. Previous reports have demonstrated that programmed necrosis, or necroptosis, of plaque macrophages contribute to necrotic core formation. Knockdown or inhibition of the necrosome components RIPK1 (receptor-interacting protein kinase 1) and RIPK3 (receptor-interacting protein kinase 3) slow atherogenesis, and activation of the terminal step of necroptosis, MLKL (mixed lineage kinase domain-like protein), has been demonstrated in advanced human atherosclerotic plaques. However, whether MLKL directly contributes to lesion development and necrotic core formation has not been investigated. Approaches and Results: MLKL expression was knocked down in atherogenic Apoe-knockout mice via the administration of antisense oligonucleotides. During atherogenesis, Mlkl knockdown decreased both programmed cell death and the necrotic core in the plaque. However, total lesion area remained unchanged. Furthermore, treatment with the MLKL antisense oligonucleotide unexpectedly reduced circulating cholesterol levels compared with control antisense oligonucleotide but increased the accumulation of lipids within the plaque and in vitro in macrophage foam cells. MLKL colocalized with the late endosome and multivesicular bodies in peritoneal macrophages incubated with atherogenic lipoproteins. Transfection with MLKL antisense oligonucleotide increased lipid localization with the multivesicular bodies, suggesting that upon Mlkl knockdown, lipid trafficking becomes defective leading to enhanced lipid accumulation in macrophages. CONCLUSIONS: These studies confirm the requirement for MLKL as the executioner of necroptosis, and as such a significant contributor to the necrotic core during atherogenesis. We also identified a previously unknown role for MLKL in regulating endosomal trafficking to facilitate lipid handling in macrophages during atherogenesis.

Paradoxical Suppression of Atherosclerosis in the Absence of microRNA-146a.

RATIONALE: Inflammation is a key contributor to atherosclerosis. MicroRNA-146a (miR-146a) has been identified as a critical brake on proinflammatory nuclear factor κ light chain enhancer of activated B cells signaling in several cell types, including endothelial cells and bone marrow (BM)-derived cells. Importantly, miR-146a expression is elevated in human atherosclerotic plaques, and polymorphisms in the miR-146a precursor have been associated with risk of coronary artery disease. OBJECTIVE: To define the role of endogenous miR-146a during atherogenesis. METHODS AND RESULTS: Paradoxically, Ldlr-/- (low-density lipoprotein receptor null) mice deficient in miR-146a develop less atherosclerosis, despite having highly elevated levels of circulating proinflammatory cytokines. In contrast, cytokine levels are normalized in Ldlr-/-;miR-146a-/- mice receiving wild-type BM transplantation, and these mice have enhanced endothelial cell activation and elevated atherosclerotic plaque burden compared with Ldlr-/- mice receiving wild-type BM, demonstrating the atheroprotective role of miR-146a in the endothelium. We find that deficiency of miR-146a in BM-derived cells precipitates defects in hematopoietic stem cell function, contributing to extramedullary hematopoiesis, splenomegaly, BM failure, and decreased levels of circulating proatherogenic cells in mice fed an atherogenic diet. These hematopoietic phenotypes seem to be driven by unrestrained inflammatory signaling that leads to the expansion and eventual exhaustion of hematopoietic cells, and this occurs in the face of lower levels of circulating low-density lipoprotein cholesterol in mice lacking miR-146a in BM-derived cells. Furthermore, we identify sortilin-1(Sort1), a known regulator of circulating low-density lipoprotein levels in humans, as a novel target of miR-146a. CONCLUSIONS: Our study reveals that miR-146a regulates cholesterol metabolism and tempers chronic inflammatory responses to atherogenic diet by restraining proinflammatory signaling in endothelial cells and BM-derived cells.

Extracellular Vesicles Secreted by Atherogenic Macrophages Transfer MicroRNA to Inhibit Cell Migration.

OBJECTIVE: During inflammation, macrophages secrete vesicles carrying RNA, protein, and lipids as a form of extracellular communication. In the vessel wall, extracellular vesicles (EVs) have been shown to be transferred between vascular cells during atherosclerosis; however, the role of macrophage-derived EVs in atherogenesis is not known. Here, we hypothesize that atherogenic macrophages secrete microRNAs (miRNAs) in EVs to mediate cell-cell communication and promote proinflammatory and proatherogenic phenotypes in recipient cells. APPROACH AND RESULTS: We isolated EVs from mouse and human macrophages treated with an atherogenic stimulus (oxidized low-density lipoprotein) and characterized the EV miRNA expression profile. We confirmed the enrichment of miR-146a, miR-128, miR-185, miR-365, and miR-503 in atherogenic EVs compared with controls and demonstrate that these EVs are taken up and transfer exogenous miRNA to naive recipient macrophages. Bioinformatic pathway analysis suggests that atherogenic EV miRNAs are predicted to target genes involved in cell migration and adhesion pathways, and indeed delivery of EVs to naive macrophages reduced macrophage migration both in vitro and in vivo. Inhibition of miR-146a, the most enriched miRNA in atherogenic EVs, reduced the inhibitory effect of EVs on macrophage migratory capacity. EV-mediated delivery of miR-146a repressed the expression of target genes IGF2BP1 (insulin-like growth factor 2 mRNA-binding protein 1) and HuR (human antigen R or ELAV-like RNA-binding protein 1) in recipient cells, and knockdown of IGF2BP1 and HuR using short interfering RNA greatly reduced macrophage migration, highlighting the importance of these EV-miRNA targets in regulating macrophage motility. CONCLUSIONS: EV-derived miRNAs from atherogenic macrophages, in particular miR-146a, may accelerate the development of atherosclerosis by decreasing cell migration and promoting macrophage entrapment in the vessel wall.

IRF2BP2 Reduces Macrophage Inflammation and Susceptibility to Atherosclerosis.

RATIONALE: Inflammation impairs macrophage cholesterol clearance from vascular tissues and promotes atherosclerosis. Inflammatory macrophages suppress expression of the transcription cofactor interferon regulatory factor 2-binding protein 2 (IRF2BP2), and genetic variants near IRF2BP2 associate with ischemic heart disease progression in humans. OBJECTIVES: To test whether IRF2BP2 in macrophages affects atherosclerosis in mice and humans. METHODS AND RESULTS: We generated mice that delete IRF2BP2 in macrophages. IRF2BP2-deficient macrophages worsened atherosclerosis in irradiated low-density lipoprotein receptor null-recipient mice and in apolipoprotein E null mice. IRF2BP2-deficient macrophages were inflammatory and had impaired cholesterol efflux because of their inability to activate the cholesterol transporter ABCA1 in response to cholesterol loading. Their expression of the anti-inflammatory transcription factor Krüppel-like factor 2 was markedly reduced. Promoter studies revealed that IRF2BP2 is required for MEF2-dependent activation of Krüppel-like factor 2. Importantly, restoring Krüppel-like factor 2 in IRF2BP2-deficient macrophages attenuated M1 inflammatory and rescued M2 anti-inflammatory gene activation and improved the cholesterol efflux deficit by restoring ABCA1 activation in response to cholesterol loading. In a cohort of 1066 angiographic cases and 1011 controls, homozygous carriers of a deletion polymorphism (rs3045215) in the 3' untranslated region sequence of human IRF2BP2 mRNA had a higher risk of coronary artery disease (recessive model, odds ratio [95% confidence interval]=1.560 [1.179-2.065], P=1.73E-03) and had lower IRF2BP2 (and Krüppel-like factor 2) protein levels in peripheral blood mononuclear cells. The effect of this deletion polymorphism to suppress protein expression was confirmed in luciferase reporter studies. CONCLUSION: Ablation of IRF2BP2 in macrophages worsens atherosclerosis in mice, and a deletion variant that lowers IRF2BP2 expression predisposes to coronary artery disease in humans.

Macrophage Mitochondrial Energy Status Regulates Cholesterol Efflux and Is Enhanced by Anti-miR33 in Atherosclerosis.

RATIONALE: Therapeutically targeting macrophage reverse cholesterol transport is a promising approach to treat atherosclerosis. Macrophage energy metabolism can significantly influence macrophage phenotype, but how this is controlled in foam cells is not known. Bioinformatic pathway analysis predicts that miR-33 represses a cluster of genes controlling cellular energy metabolism that may be important in macrophage cholesterol efflux. OBJECTIVE: We hypothesized that cellular energy status can influence cholesterol efflux from macrophages, and that miR-33 reduces cholesterol efflux via repression of mitochondrial energy metabolism pathways. METHODS AND RESULTS: In this study, we demonstrated that macrophage cholesterol efflux is regulated by mitochondrial ATP production, and that miR-33 controls a network of genes that synchronize mitochondrial function. Inhibition of mitochondrial ATP synthase markedly reduces macrophage cholesterol efflux capacity, and anti-miR33 required fully functional mitochondria to enhance ABCA1-mediated cholesterol efflux. Specifically, anti-miR33 derepressed the novel target genes PGC-1α, PDK4, and SLC25A25 and boosted mitochondrial respiration and production of ATP. Treatment of atherosclerotic Apoe(-/-) mice with anti-miR33 oligonucleotides reduced aortic sinus lesion area compared with controls, despite no changes in high-density lipoprotein cholesterol or other circulating lipids. Expression of miR-33a/b was markedly increased in human carotid atherosclerotic plaques compared with normal arteries, and there was a concomitant decrease in mitochondrial regulatory genes PGC-1α, SLC25A25, NRF1, and TFAM, suggesting these genes are associated with advanced atherosclerosis in humans. CONCLUSIONS: This study demonstrates that anti-miR33 therapy derepresses genes that enhance mitochondrial respiration and ATP production, which in conjunction with increased ABCA1 expression, works to promote macrophage cholesterol efflux and reduce atherosclerosis.

Therapeutic Inhibition of miR-33 Promotes Fatty Acid Oxidation but Does Not Ameliorate Metabolic Dysfunction in Diet-Induced Obesity.

OBJECTIVE: miR-33 has emerged as an important regulator of lipid homeostasis. Inhibition of miR-33 has been demonstrated as protective against atherosclerosis; however, recent studies in mice suggest that miR-33 inhibition may have adverse effects on lipid and insulin metabolism. Given the therapeutic interest in miR-33 inhibitors for treating atherosclerosis, we sought to test whether pharmacologically inhibiting miR-33 at atheroprotective doses affected metabolic parameters in a mouse model of diet-induced obesity. APPROACH AND RESULTS: High-fat diet (HFD) feeding in conjunction with treatment of male mice with 10 mg/kg control anti-miR or anti-miR33 inhibitors for 20 weeks promoted equivalent weight gain in all groups. miR-33 inhibitors increased plasma total cholesterol and decreased serum triglycerides compared with control anti-miR, but not compared with PBS-treated mice. Metrics of insulin resistance were not altered in anti-miR33-treated mice compared with controls; however, respiratory exchange ratio was decreased in anti-miR33-treated mice. Hepatic expression of miR-33 targets Abca1 and Hadhb were derepressed on miR-33 inhibition. In contrast, protein levels of putative miR-33 target gene SREBP-1 or its downstream targets genes Fasn and Acc were not altered in anti-miR33-treated mice, and hepatic lipid accumulation did not differ between groups. In the adipose tissue, anti-miR33 treatment increased Ampk gene expression and markers of M2 macrophage polarization. CONCLUSIONS: We demonstrate in a mouse model of diet-induced obesity that therapeutic silencing of miR-33 may promote whole-body oxidative metabolism but does not affect metabolic dysregulation. This suggests that pharmacological inhibition of miR-33 at doses known to reduce atherosclerosis may be a safe future therapeutic.

The intracellular redox state is a core determinant of mitochondrial fusion.

Mitochondrial hyperfusion has recently been shown to function as a cellular stress response, providing transient protection against apoptosis and mitophagy. However, the mechanisms that mediate this response remain poorly understood. In this study, we demonstrate that oxidized glutathione (GSSG), the core cellular stress indicator, strongly induces mitochondrial fusion. Biochemical and functional experiments show that GSSG induces the generation of disulphide-mediated mitofusin oligomers, in a process that also requires GTP hydrolysis. Our data outline the molecular events that prime the fusion machinery, providing new insights into the coupling of mitochondrial fusion with the cellular stress response.

Mitochondrial Fragmentation Promotes Inflammation Resolution Responses in Macrophages via Histone Lactylation.

During the inflammatory response, macrophage phenotypes can be broadly classified as pro-inflammatory/classically activated "M1", or pro-resolving/alternatively "M2" macrophages. Although the classification of macrophages is general and assumes there are distinct phenotypes, in reality macrophages exist across a spectrum and must transform from a pro-inflammatory state to a proresolving state following an inflammatory insult. To adapt to changing metabolic needs of the cell, mitochondria undergo fusion and fission, which have important implications for cell fate and function. We hypothesized that mitochondrial fission and fusion directly contribute to macrophage function during the pro-inflammatory and proresolving phases. In the present study, we find that mitochondrial length directly contributes to macrophage phenotype, primarily during the transition from a pro-inflammatory to a proresolving state. Phenocopying the elongated mitochondrial network (by disabling the fission machinery using siRNA) leads to a baseline reduction in the inflammatory marker IL-1ß, but a normal inflammatory response to LPS, similar to control macrophages. In contrast, in macrophages with a phenocopied fragmented phenotype (by disabling the fusion machinery using siRNA) there is a heightened inflammatory response to LPS and increased signaling through the ATF4/c-Jun transcriptional axis compared to control macrophages. Importantly, macrophages with a fragmented mitochondrial phenotype show increased expression of proresolving mediator arginase 1 and increased phagocytic capacity. Promoting mitochondrial fragmentation caused an increase in cellular lactate, and an increase in histone lactylation which caused an increase in arginase 1 expression. These studies demonstrate that a fragmented mitochondrial phenotype is critical for the proresolving response in macrophages and specifically drive epigenetic changes via lactylation of histones following an inflammatory insult.

ATF2 orchestrates macrophage differentiation and activation to promote antibacterial responses.

The differentiation and activation of macrophages are critical regulatory programs that are central to host inflammation and pathogen defense. However, the transcriptional regulatory pathways involved in these programs are not well understood. Herein, we demonstrate that the activity and expression of the transcription factor ATF2 is precisely regulated during primary human monocyte-to-macrophage differentiation and that its activation is linked to M1 polarization and antibacterial responses. Genetic perturbation experiments demonstrated that deletion of ATF2 (THP-ΔATF2) resulted in irregular and abnormal macrophage morphology, whereas macrophages overexpressing ATF2 (THP-ATF2) developed round and pancake-like morphology, resembling classically activated (M1) macrophages. Mechanistically, we show that ATF2 binds to the core promoter of PPM1A, a phosphatase that regulates monocyte-to-macrophage differentiation, to regulate its expression. Functionally, overexpression of ATF2 sensitized macrophages to M1 polarization, resulting in increased production of major histocompatibility complex class II, IL-1ß, and IP-10; improved phagocytic capacity; and enhanced control of the intracellular pathogen Mycobacterium tuberculosis. Gene expression profiling revealed that overexpression of ATF2 reprogramed macrophages to promote antibacterial pathways enriched in chemokine signaling, metabolism, and antigen presentation. Consistent with pathways analysis, metabolic profiling revealed that genetic overexpression or stimuli-induced activation of ATF2 alters the metabolic capacity of macrophages and primes these cells for glycolytic metabolism during M1 polarization or bacterial infection. Our findings reveal that ATF2 plays a central role during macrophage differentiation and M1 polarization to enhance the functional capacities of macrophages.

Virally programmed extracellular vesicles sensitize cancer cells to oncolytic virus and small molecule therapy.

Recent advances in cancer therapeutics clearly demonstrate the need for innovative multiplex therapies that attack the tumour on multiple fronts. Oncolytic or "cancer-killing" viruses (OVs) represent up-and-coming multi-mechanistic immunotherapeutic drugs for the treatment of cancer. In this study, we perform an in-vitro screen based on virus-encoded artificial microRNAs (amiRNAs) and find that a unique amiRNA, herein termed amiR-4, confers a replicative advantage to the VSVΔ51 OV platform. Target validation of amiR-4 reveals ARID1A, a protein involved in chromatin remodelling, as an important player in resistance to OV replication. Virus-directed targeting of ARID1A coupled with small-molecule inhibition of the methyltransferase EZH2 leads to the synthetic lethal killing of both infected and uninfected tumour cells. The bystander killing of uninfected cells is mediated by intercellular transfer of extracellular vesicles carrying amiR-4 cargo. Altogether, our findings establish that OVs can serve as replicating vehicles for amiRNA therapeutics with the potential for combination with small molecule and immune checkpoint inhibitor therapy.

RIPK1 gene variants associate with obesity in humans and can be therapeutically silenced to reduce obesity in mice.

Obesity is a major public health burden worldwide and is characterized by chronic low-grade inflammation driven by the cooperation of the innate immune system and dysregulated metabolism in adipose tissue and other metabolic organs. Receptor-interacting serine/threonine-protein kinase 1 (RIPK1) is a central regulator of inflammatory cell function that coordinates inflammation, apoptosis and necroptosis in response to inflammatory stimuli. Here we show that genetic polymorphisms near the human RIPK1 locus associate with increased RIPK1 gene expression and obesity. We show that one of these single nucleotide polymorphisms is within a binding site for E4BP4 and increases RIPK1 promoter activity and RIPK1 gene expression in adipose tissue. Therapeutic silencing of RIPK1 in vivo in a mouse model of diet-induced obesity dramatically reduces fat mass, total body weight and improves insulin sensitivity, while simultaneously reducing macrophage and promoting invariant natural killer T cell accumulation in adipose tissue. These findings demonstrate that RIPK1 is genetically associated with obesity, and reducing RIPK1 expression is a potential therapeutic approach to target obesity and related diseases.

Publisher Correction: RIPK1 gene variants associate with obesity in humans and can be therapeutically silenced to reduce obesity in mice.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Delivery of MicroRNAs by Chitosan Nanoparticles to Functionally Alter Macrophage Cholesterol Efflux in Vitro and in Vivo.

The prevention and treatment of cardiovascular diseases (CVD) has largely focused on lowering circulating LDL cholesterol, yet a significant burden of atherosclerotic disease remains even when LDL is low. Recently, microRNAs (miRNAs) have emerged as exciting therapeutic targets for cardiovascular disease. miRNAs are small noncoding RNAs that post-transcriptionally regulate gene expression by degradation or translational inhibition of target mRNAs. A number of miRNAs have been found to modulate all stages of atherosclerosis, particularly those that promote the efflux of excess cholesterol from lipid-laden macrophages in the vessel wall to the liver. However, one of the major challenges of miRNA-based therapy is to achieve tissue-specific, efficient, and safe delivery of miRNAs in vivo. We sought to develop chitosan nanoparticles (chNPs) that can deliver functional miRNA mimics to macrophages and to determine if these nanoparticles can alter cholesterol efflux and reverse cholesterol transport in vivo. We developed chNPs with a size range of 150-200 nm via the ionic gelation method using tripolyphosphate (TPP) as a cross-linker. In this method, negatively charged miRNAs were encapsulated in the nanoparticles by ionic interactions with polymeric components. We then optimized the efficiency of intracellular delivery of different formulations of chitosan/TPP/miRNA to mouse macrophages. Using a well-defined miRNA with roles in macrophage cholesterol metabolism, we tested whether chNPs could deliver functional miRNAs to macrophages. We find chNPs can transfer exogenous miR-33 to naïve macrophages and reduce the expression of ABCA1, a potent miR-33 target gene, both in vitro and in vivo, confirming that miRNAs delivered via nanoparticles can escape the endosomal system and function in the RISC complex. Because miR-33 and ABCA1 play a key role in regulating the efflux of cholesterol from macrophages, we also confirmed that macrophages treated with miR-33-loaded chNPs exhibited reduced cholesterol efflux to apolipoprotein A1, further confirming functional delivery of the miRNA. In vivo, mice treated with miR33-chNPs showed decreased reverse cholesterol transport (RCT) to the plasma, liver, and feces. In contrast, when efflux-promoting miRNAs were delivered via chNPs, ABCA1 expression and cholesterol efflux into the RCT pathway were improved. Over all, miRNAs can be efficiently delivered to macrophages via nanoparticles, where they can function to regulate ABCA1 expression and cholesterol efflux, suggesting that these miRNA nanoparticles can be used in vivo to target atherosclerotic lesions.

A mouse monoclonal antibody specific for mouse apoB48 and apoB100 produced by immunizing "apoB39-only" mice with mouse apoB48.

We have generated and characterized a murine monoclonal antibody (mAb) that binds to both mouse apolipoprotein (apo) B48 and apoB100. We immunized "apoB39-only" mice (mice that synthesize a truncated form of apoB, apoB39, but no apoB48 or apoB100) with lipoproteins containing mouse apoB48 and then used splenocytes from the immunized mice to create hybridomas. We identified a hybridoma, 2G11, that secretes a mAb that binds to mouse apoB48 and apoB100 but not to apoB39. Antibody 2G11 also binds apoB48 and apoB100 from rats and hamsters but not from humans. The mAb recognizes mouse apoB equally in very low and low density lipoproteins and was used to quantify apoB in wild-type, apoE-deficient and low-density lipoprotein receptor-deficient mice and in mice treated with an antisense drug that lowers plasma apoB levels. The antibody will be an important reagent for studying mouse models of atherosclerosis. The study also underscores the utility of genetically modified mice for generating mouse mAbs against mouse proteins.

The Keap1-Nrf2 Stress Response Pathway Promotes Mitochondrial Hyperfusion Through Degradation of the Mitochondrial Fission Protein Drp1.

AIMS: Mitochondrial function is coupled to metabolic and survival pathways through both direct signaling cascades and dynamic changes in mitochondrial morphology. For example, a hyperfused mitochondrial reticulum is activated upon cellular stress and is protective against cell death. As part of a genome-wide small inhibitory ribonucleic acid screen, we identified the central redox regulator, Keap1, as a novel regulator of mitochondrial morphology. Here, we aimed to determine the mechanism through which redox signaling and Keap1 mediate changes in mitochondrial morphology. RESULTS: We found that the Nrf2 transcription factor is required for mitochondrial hyperfusion induced by knockdown of Keap1. Nrf2, which is negatively regulated by Keap1, mediates the cell's response to stress by controlling the expression of several hundred genes, including proteasome expression. We next showed that increased proteasome activity, a result of increased Nrf2 activity, is responsible for the degradation of the mitochondrial fission protein Drp1, which occurs in an ubiquitin-independent manner. INNOVATION: Our study described a novel pathway by which Nrf2 activation, known to occur in response to increased oxidative stress, decreases mitochondrial fission and contributes to a hyperfused mitochondrial network. CONCLUSION: This study has identified the Keap1-Nrf2 nexus and modulation of proteasomal activity as novel avenues to inhibit mitochondrial fission. These findings are important, because inhibiting mitochondrial fission is a promising therapeutic approach to restore the balance between fission and fusion, which is attractive for an increasing number of disorders linked to mitochondrial dysfunction. Antioxid. Redox Signal. 27, 1447-1459.

Targeting macrophage necroptosis for therapeutic and diagnostic interventions in atherosclerosis.

Atherosclerosis results from maladaptive inflammation driven primarily by macrophages, whose recruitment and proliferation drive plaque progression. In advanced plaques, macrophage death contributes centrally to the formation of plaque necrosis, which underlies the instability that promotes plaque rupture and myocardial infarction. Hence, targeting macrophage cell death pathways may offer promise for the stabilization of vulnerable plaques. Necroptosis is a recently discovered pathway of programmed cell necrosis regulated by RIP3 and MLKL kinases that, in contrast to apoptosis, induces a proinflammatory state. We show herein that necroptotic cell death is activated in human advanced atherosclerotic plaques and can be targeted in experimental atherosclerosis for both therapeutic and diagnostic interventions. In humans with unstable carotid atherosclerosis, expression of RIP3 and MLKL is increased, and MLKL phosphorylation, a key step in the commitment to necroptosis, is detected in advanced atheromas. Investigation of the molecular mechanisms underlying necroptosis showed that atherogenic forms of low-density lipoprotein increase RIP3 and MLKL transcription and phosphorylation-two critical steps in the execution of necroptosis. Using a radiotracer developed with the necroptosis inhibitor necrostatin-1 (Nec-1), we show that (123)I-Nec-1 localizes specifically to atherosclerotic plaques in Apoe (-/-) mice, and its uptake is tightly correlated to lesion areas by ex vivo nuclear imaging. Furthermore, treatment of Apoe (-/-) mice with established atherosclerosis with Nec-1 reduced lesion size and markers of plaque instability, including necrotic core formation. Collectively, our findings offer molecular insight into the mechanisms of macrophage cell death that drive necrotic core formation in atherosclerosis and suggest that this pathway can be used as both a diagnostic and therapeutic tool for the treatment of unstable atherosclerosis.

Knockdown of glyoxalase 1 mimics diabetic nephropathy in nondiabetic mice.

Differences in susceptibility to diabetic nephropathy (DN) between mouse strains with identical levels of hyperglycemia correlate with renal levels of oxidative stress, shown previously to play a central role in the pathogenesis of DN. Susceptibility to DN appears to be genetically determined, but the critical genes have not yet been identified. Overexpression of the enzyme glyoxalase 1 (Glo1), which prevents posttranslational modification of proteins by the glycolysis-derived α-oxoaldehyde, methylglyoxal (MG), prevents hyperglycemia-induced oxidative stress in cultured cells and model organisms. In this study, we show that in nondiabetic mice, knockdown of Glo1 increases to diabetic levels both MG modification of glomerular proteins and oxidative stress, causing alterations in kidney morphology indistinguishable from those caused by diabetes. We also show that in diabetic mice, Glo1 overexpression completely prevents diabetes-induced increases in MG modification of glomerular proteins, increased oxidative stress, and the development of diabetic kidney pathology, despite unchanged levels of diabetic hyperglycemia. Together, these data indicate that Glo1 activity regulates the sensitivity of the kidney to hyperglycemic-induced renal pathology and that alterations in the rate of MG detoxification are sufficient to determine the glycemic set point at which DN occurs.