The IRG1-itaconate axis protects from cholesterol-induced inflammation and atherosclerosis.

Atherosclerosis is fueled by a failure to resolve lipid-driven inflammation within the vasculature that drives plaque formation. Therapeutic approaches to reverse atherosclerotic inflammation are needed to address the rising global burden of cardiovascular disease (CVD). Recently, metabolites have gained attention for their immunomodulatory properties, including itaconate, which is generated from the tricarboxylic acid-intermediate cis-aconitate by the enzyme Immune Responsive Gene 1 (IRG1/ACOD1). Here, we tested the therapeutic potential of the IRG1-itaconate axis for human atherosclerosis. Using single-cell RNA sequencing (scRNA-seq), we found that IRG1 is up-regulated in human coronary atherosclerotic lesions compared to patient-matched healthy vasculature, and in mouse models of atherosclerosis, where it is primarily expressed by plaque monocytes, macrophages, and neutrophils. Global or hematopoietic Irg1-deficiency in mice increases atherosclerosis burden, plaque macrophage and lipid content, and expression of the proatherosclerotic cytokine interleukin (IL)-1ß. Mechanistically, absence of Irg1 increased macrophage lipid accumulation, and accelerated inflammation via increased neutrophil extracellular trap (NET) formation and NET-priming of the NLRP3-inflammasome in macrophages, resulting in increased IL-1ß release. Conversely, supplementation of the Irg1-itaconate axis using 4-octyl itaconate (4-OI) beneficially remodeled advanced plaques and reduced lesional IL-1ß levels in mice. To investigate the effects of 4-OI in humans, we leveraged an ex vivo systems-immunology approach for CVD drug discovery. Using CyTOF and scRNA-seq of peripheral blood mononuclear cells treated with plasma from CVD patients, we showed that 4-OI attenuates proinflammatory phospho-signaling and mediates anti-inflammatory rewiring of macrophage populations. Our data highlight the relevance of pursuing IRG1-itaconate axis supplementation as a therapeutic approach for atherosclerosis in humans.

Obesity research: Moving from bench to bedside to population.

Globally, obesity is on the rise. Research over the past 20 years has highlighted the far-reaching multisystem complications of obesity, but a better understanding of its complex pathogenesis is needed to identify safe and lasting solutions.

Disruption of the productive encounter complex results in dysregulation of DIAPH1 activity.

The diaphanous-related formin, Diaphanous 1 (DIAPH1), is required for the assembly of Filamentous (F)-actin structures. DIAPH1 is an intracellular effector of the receptor for advanced glycation end products (RAGE) and contributes to RAGE signaling and effects such as increased cell migration upon RAGE stimulation. Mutations in DIAPH1, including those in the basic "RRKR" motif of its autoregulatory domain, diaphanous autoinhibitory domain (DAD), are implicated in hearing loss, macrothrombocytopenia, and cardiovascular diseases. The solution structure of the complex between the N-terminal inhibitory domain, DID, and the C-terminal DAD, resolved by NMR spectroscopy shows only transient interactions between DID and the basic motif of DAD, resembling those found in encounter complexes. Cross-linking studies placed the RRKR motif into the negatively charged cavity of DID. Neutralizing the cavity resulted in a 5-fold decrease in the binding affinity and 4-fold decrease in the association rate constant of DAD for DID, indicating that the RRKR interactions with DID form a productive encounter complex. A DIAPH1 mutant containing a neutralized RRKR binding cavity shows excessive colocalization with actin and is unresponsive to RAGE stimulation. This is the first demonstration of a specific alteration of the surfaces responsible for productive encounter complexation with implications for human pathology.

Experimentally Induced Peripheral Venous Congestion Exacerbates Inflammation, Oxidative Stress, and Neurohormonal and Endothelial Cell Activation in Patients With Systolic Heart Failure.

BACKGROUND: Venous congestion (VC) is a hallmark of symptomatic heart failure (HF) requiring hospitalization; however, its role in the pathogenesis of HF progression remains unclear. We investigated whether peripheral VC exacerbates inflammation, oxidative stress and neurohormonal and endothelial cell (EC) activation in patients with HF with reduced ejection fraction (HFrEF). METHODS AND RESULTS: Two matched groups of patients with HFrEF and with no peripheral VC vs without recent HF hospitalization were studied. We modeled peripheral VC by inflating a cuff around the dominant arm, targeting ∼ 30 mmHg increase in venous pressure (venous stress test [VST]). Blood and ECs were sampled before and after 90 minutes of VST. We studied 44 patients (age 53 ± 12 years, 32% female). Circulating endothelin-1, tumor necrosis factor-α, interleukin-6, isoprostane, angiotensin II (ang-2), angiopoietin-2, vascular cell adhesion molecule-1, and CD146 significantly increased after the VST. Enhanced endothelin-1 and angiopoietin-2 responses to the VST were present in patients with vs without recent hospitalization and were prospectively associated with incident HF-related events; 6698 messenger ribonucleic acid (mRNA probe sets were differentially expressed in ECs after VST. CONCLUSIONS: Experimental VC exacerbates inflammation, oxidative stress, neurohormonal and EC activation and promotes unfavorable transcriptome remodeling in ECs of patients with HFrEF. A distinct biological sensitivity to VC appears to be associated with high risk for HF progression.

Silencing Myeloid Netrin-1 Induces Inflammation Resolution and Plaque Regression.

[Figure: see text].

The receptor for advanced glycation end products and its ligands' expression in OVE26 diabetic sciatic nerve during the development of length-dependent neuropathy.

Type 1 diabetes (T1D) may affect the peripheral nervous system and alter the expression of proteins contributing to inflammation and cellular cytoskeleton dysfunction, in most cases leading to the development of diabetic length-dependent neuropathy (DLDN). In the present study, we performed immunohistochemistry (IHC) to probe the expression of the receptor for advanced glycation end products (RAGE); its key ligands, high-mobility group box 1 (HMGB1), S100 calcium-binding protein B (S100B), and carboxymethyl-lysine (CML - advanced glycation end products (AGE)); and its cytoplasmic tail-binding partner, diaphanous related formin 1 (DIAPH1) and associated molecules, beta-actin (ACTB) and profilin 1 (PFN1) proteins in sciatic nerves harvested from seven-month old FVB/OVE26 mice with genetically-mediated T1D. We found that the amount of RAGE, HMGB1, and S100B proteins was elevated in diabetic vs the non-diabetic groups, while the amount of DIAPH1, ACTB, as well as PFN1 proteins did not differ between these groups. Moreover, our data revealed linear dependence between RAGE and HMGB1 proteins. Interaction criss-cross of selected sets of proteins in the sciatic nerve revealed that there were connected in a singular network. Our results indicate that T1D may alter expression patterns of RAGE axis proteins and thus contribute to DLDN.

Leukocyte Heterogeneity in Adipose Tissue, Including in Obesity.

Adipose tissue (AT) plays a central role in both metabolic health and pathophysiology. Its expansion in obesity results in increased mortality and morbidity, with contributions to cardiovascular disease, diabetes mellitus, fatty liver disease, and cancer. Obesity prevalence is at an all-time high and is projected to be 50% in the United States by 2030. AT is home to a large variety of immune cells, which are critical to maintain normal tissue functions. For example, γδ T cells are fundamental for AT innervation and thermogenesis, and macrophages are required for recycling of lipids released by adipocytes. The expansion of visceral white AT promotes dysregulation of its immune cell composition and likely promotes low-grade chronic inflammation, which has been proposed to be the underlying cause for the complications of obesity. Interestingly, weight loss after obesity alters the AT immune compartment, which may account for the decreased risk of developing these complications. Recent technological advancements that allow molecular investigation on a single-cell level have led to the discovery of previously unappreciated heterogeneity in many organs and tissues. In this review, we will explore the heterogeneity of immune cells within the visceral white AT and their contributions to homeostasis and pathology.

An Eclectic Cast of Cellular Actors Orchestrates Innate Immune Responses in the Mechanisms Driving Obesity and Metabolic Perturbation.

The escalating problem of obesity and its multiple metabolic and cardiovascular complications threatens the health and longevity of humans throughout the world. The cause of obesity and one of its chief complications, insulin resistance, involves the participation of multiple distinct organs and cell types. From the brain to the periphery, cell-intrinsic and intercellular networks converge to stimulate and propagate increases in body mass and adiposity, as well as disturbances of insulin sensitivity. This review focuses on the roles of the cadre of innate immune cells, both those that are resident in metabolic organs and those that are recruited into these organs in response to cues elicited by stressors such as overnutrition and reduced physical activity. Beyond the typical cast of innate immune characters invoked in the mechanisms of metabolic perturbation in these settings, such as neutrophils and monocytes/macrophages, these actors are joined by bone marrow-derived cells, such as eosinophils and mast cells and the intriguing innate lymphoid cells, which are present in the circulation and in metabolic organ depots. Upon high-fat feeding or reduced physical activity, phenotypic modulation of the cast of plastic innate immune cells ensues, leading to the production of mediators that affect inflammation, lipid handling, and metabolic signaling. Furthermore, their consequent interactions with adaptive immune cells, including myriad T-cell and B-cell subsets, compound these complexities. Notably, many of these innate immune cell-elicited signals in overnutrition may be modulated by weight loss, such as that induced by bariatric surgery. Recently, exciting insights into the biology and pathobiology of these cell type-specific niches are being uncovered by state-of-the-art techniques such as single-cell RNA-sequencing. This review considers the evolution of this field of research on innate immunity in obesity and metabolic perturbation, as well as future directions.

Journey to a Receptor for Advanced Glycation End Products Connection in Severe Acute Respiratory Syndrome Coronavirus 2 Infection: With Stops Along the Way in the Lung, Heart, Blood Vessels, and Adipose Tissue.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has affected millions of people worldwide and the pandemic has yet to wane. Despite its associated significant morbidity and mortality, there are no definitive cures and no fully preventative measures to combat SARS-CoV-2. Hence, the urgency to identify the pathobiological mechanisms underlying increased risk for and the severity of SARS-CoV-2 infection is mounting. One contributing factor, the accumulation of damage-associated molecular pattern molecules, is a leading trigger for the activation of nuclear factor-kB and the IRF (interferon regulatory factors), such as IRF7. Activation of these pathways, particularly in the lung and other organs, such as the heart, contributes to a burst of cytokine release, which predisposes to significant tissue damage, loss of function, and mortality. The receptor for advanced glycation end products (RAGE) binds damage-associated molecular patterns is expressed in the lung and heart, and in priming organs, such as the blood vessels (in diabetes) and adipose tissue (in obesity), and transduces the pathological signals emitted by damage-associated molecular patterns. It is proposed that damage-associated molecular pattern-RAGE enrichment in these priming tissues, and in the lungs and heart during active infection, contributes to the widespread tissue damage induced by SARS-CoV-2. Accordingly, the RAGE axis might play seminal roles in and be a target for therapeutic intervention in SARS-CoV-2 infection.

MicroRNA-33 Inhibits Adaptive Thermogenesis and Adipose Tissue Beiging.

[Figure: see text].

The RAGE/DIAPH1 Signaling Axis & Implications for the Pathogenesis of Diabetic Complications.

Increasing evidence links the RAGE (receptor for advanced glycation end products)/DIAPH1 (Diaphanous 1) signaling axis to the pathogenesis of diabetic complications. RAGE is a multi-ligand receptor and through these ligand-receptor interactions, extensive maladaptive effects are exerted on cell types and tissues targeted for dysfunction in hyperglycemia observed in both type 1 and type 2 diabetes. Recent evidence indicates that RAGE ligands, acting as damage-associated molecular patterns molecules, or DAMPs, through RAGE may impact interferon signaling pathways, specifically through upregulation of IRF7 (interferon regulatory factor 7), thereby heralding and evoking pro-inflammatory effects on vulnerable tissues. Although successful targeting of RAGE in the clinical milieu has, to date, not been met with success, recent approaches to target RAGE intracellular signaling may hold promise to fill this critical gap. This review focuses on recent examples of highlights and updates to the pathobiology of RAGE and DIAPH1 in diabetic complications.

AgeR deletion decreases soluble fms-like tyrosine kinase 1 production and improves post-ischemic angiogenesis in uremic mice.

Peripheral arterial disease occurs more frequently and has a worse prognosis in patients with chronic kidney disease (CKD). The receptor for advanced glycation end products (RAGE) is involved in multiple aspects of uremia-associated vasculopathy. Previous data suggest that the RAGE pathway may promote soluble fms-like tyrosine kinase 1 (sFlt1) production, an anti-angiogenic molecule. Thus, we tested the hypothesis that the deletion of AgeR would decrease sFlt1 production and improve post-ischemic revascularization in uremic condition. We used a well-established CKD model (5/6 nephrectomy) in WT and AgeR-/- C57/Bl6 mice. Hindlimb ischemia was induced by femoral artery ligation. Revascularization was evaluated by complementary approaches: ischemic limb retraction, LASCA imagery, and capillary density. The production of sFlt1 was assessed at both RNA and protein levels. After hindlimb ischemia, uremic mice showed slower functional recovery (p < 0.01), decreased reperfusion (p < 0.01), lower capillary density (p = 0.02), and increased circulating sFlt1 levels (p = 0.03). AgeR deletion restored post-ischemic angiogenesis and was protective from sFlt1 increase in uremic mice. These findings show the main role of RAGE in post-ischemic angiogenesis impairment associated with CKD. RAGE may represent a key target for building new therapeutic approaches to improve the outcome of CKD patients with PAD.

Microglia RAGE exacerbates the progression of neurodegeneration within the SOD1G93A murine model of amyotrophic lateral sclerosis in a sex-dependent manner.

BACKGROUND: Burgeoning evidence highlights seminal roles for microglia in the pathogenesis of neurodegenerative diseases including amyotrophic lateral sclerosis (ALS). The receptor for advanced glycation end products (RAGE) binds ligands relevant to ALS that accumulate in the diseased spinal cord and RAGE has been previously implicated in the progression of ALS pathology. METHODS: We generated a novel mouse model to temporally delete Ager from microglia in the murine SOD1G93A model of ALS. Microglia Ager deficient SOD1G93A mice and controls were examined for changes in survival, motor function, gliosis, motor neuron numbers, and transcriptomic analyses of lumbar spinal cord. Furthermore, we examined bulk-RNA-sequencing transcriptomic analyses of human ALS cervical spinal cord. RESULTS: Transcriptomic analysis of human cervical spinal cord reveals a range of AGER expression in ALS patients, which was negatively correlated with age at disease onset and death or tracheostomy. The degree of AGER expression related to differential expression of pathways involved in extracellular matrix, lipid metabolism, and intercellular communication. Microglia display increased RAGE immunoreactivity in the spinal cords of high AGER expressing patients and in the SOD1G93A murine model of ALS vs. respective controls. We demonstrate that microglia Ager deletion at the age of symptomatic onset, day 90, in SOD1G93A mice extends survival in male but not female mice. Critically, many of the pathways identified in human ALS patients that accompanied increased AGER expression were significantly ameliorated by microglia Ager deletion in male SOD1G93A mice. CONCLUSIONS: Our results indicate that microglia RAGE disrupts communications with cell types including astrocytes and neurons, intercellular communication pathways that divert microglia from a homeostatic to an inflammatory and tissue-injurious program. In totality, microglia RAGE contributes to the progression of SOD1G93A murine pathology in male mice and may be relevant in human disease.

AGE/RAGE/DIAPH1 axis is associated with immunometabolic markers and risk of insulin resistance in subcutaneous but not omental adipose tissue in human obesity.

BACKGROUND/OBJECTIVES: The incidence of obesity continues to increase worldwide and while the underlying pathogenesis remains largely unknown, nutrient excess, manifested by "Westernization" of the diet and reduced physical activity have been proposed as key contributing factors. Western-style diets, in addition to higher caloric load, are characterized by excess of advanced glycation end products (AGEs), which have been linked to the pathophysiology of obesity and related cardiometabolic disorders. AGEs can be "trapped" in adipose tissue, even in the absence of diabetes, in part due to higher expression of the receptor for AGEs (RAGE) and/or decreased detoxification by the endogenous glyoxalase (GLO) system, where they may promote insulin resistance. It is unknown whether the expression levels of genes linked to the RAGE axis, including AGER (the gene encoding RAGE), Diaphanous 1 (DIAPH1), the cytoplasmic domain binding partner of RAGE that contributes to RAGE signaling, and GLO1 are differentially regulated by the degree of obesity and/or how these relate to inflammatory and adipocyte markers and their metabolic consequences. SUBJECTS/METHODS: We sought to answer this question by analyzing gene expression patterns of markers of the AGE/RAGE/DIAPH1 signaling axis in abdominal subcutaneous (SAT) and omental (OAT) adipose tissue from obese and morbidly obese subjects. RESULTS: In SAT, but not OAT, expression of AGER was significantly correlated with that of DIAPH1 (n = 16; [Formula: see text], [0.260, 1.177]; q = 0.008) and GLO1 (n = 16; [Formula: see text], [0.364, 1.182]; q = 0.004). Furthermore, in SAT, but not OAT, regression analyses revealed that the expression pattern of genes in the AGE/RAGE/DIAPH1 axis is strongly and positively associated with that of inflammatory and adipogenic markers. Remarkably, particularly in SAT, not OAT, the expression of AGER positively and significantly correlated with HOMA-IR (n = 14; [Formula: see text], [0.338, 1.249]; q = 0.018). CONCLUSIONS: These observations suggest associations of the AGE/RAGE/DIAPH1 axis in the immunometabolic pathophysiology of obesity and insulin resistance, driven, at least in part, through expression and activity of this axis in SAT.

S100A9-RAGE Axis Accelerates Formation of Macrophage-Mediated Extracellular Vesicle Microcalcification in Diabetes Mellitus.

OBJECTIVE: Vascular calcification is a cardiovascular risk factor and accelerated in diabetes mellitus. Previous work has established a role for calcification-prone extracellular vesicles in promoting vascular calcification. However, the mechanisms by which diabetes mellitus provokes cardiovascular events remain incompletely understood. Our goal was to identify that increased S100A9 promotes the release of calcification-prone extracellular vesicles from human macrophages in diabetes mellitus. Approach and Results: Human primary macrophages exposed to high glucose (25 mmol/L) increased S100A9 secretion and the expression of receptor for advanced glycation end products (RAGE) protein. Recombinant S100A9 induced the expression of proinflammatory and osteogenic factors, as well as the number of extracellular vesicles with high calcific potential (alkaline phosphatase activity, P<0.001) in macrophages. Treatment with a RAGE antagonist or silencing with S100A9 siRNA in macrophages abolished these responses, suggesting that stimulation of the S100A9-RAGE axis by hyperglycemia favors a procalcific environment. We further showed that an imbalance between Nrf-2 (nuclear factor 2 erythroid related factor 2) and NF-κB (nuclear factor-κB) pathways contributes to macrophage activation and promotes a procalcific environment. In addition, streptozotocin-induced diabetic Apoe-/-S100a9-/- mice and mice treated with S100a9 siRNA encapsulated in macrophage-targeted lipid nanoparticles showed decreased inflammation and microcalcification in atherosclerotic plaques, as gauged by molecular imaging and comprehensive histological analysis. In human carotid plaques, comparative proteomics in patients with diabetes mellitus and histological analysis showed that the S100A9-RAGE axis associates with osteogenic activity and the formation of microcalcification. CONCLUSIONS: Under hyperglycemic conditions, macrophages release calcific extracellular vesicles through mechanisms involving the S100A9-RAGE axis, thus contributing to the formation of microcalcification within atherosclerotic plaques.

Diabetes and Cardiovascular Complications: The Epidemics Continue.

PURPOSE OF REVIEW: The cardiovascular complications of type 1 and 2 diabetes are major causes of morbidity and mortality. Extensive efforts have been made to maximize glycemic control; this strategy reduces certain manifestations of cardiovascular complications. There are drawbacks, however, as intensive glycemic control does not impart perennial protective benefits, and these efforts are not without potential adverse sequelae, such as hypoglycemic events. RECENT FINDINGS: Here, the authors have focused on updates into key areas under study for mechanisms driving these cardiovascular disorders in diabetes, including roles for epigenetics and gene expression, interferon networks, and mitochondrial dysfunction. Updates on the cardioprotective roles of the new classes of hyperglycemia-targeting therapies, the sodium glucose transport protein 2 inhibitors and the agonists of the glucagon-like peptide 1 receptor system, are reviewed. In summary, insights from ongoing research and the cardioprotective benefits of the newer type 2 diabetes therapies are providing novel areas for therapeutic opportunities in diabetes and CVD.

Multiomics of World Trade Center Particulate Matter-induced Persistent Airway Hyperreactivity. Role of Receptor for Advanced Glycation End Products.

Pulmonary disease after World Trade Center particulate matter (WTC-PM) exposure is associated with dyslipidemia and the receptor for advanced glycation end products (RAGE); however, the mechanisms are not well understood. We used a murine model and a multiomics assessment to understand the role of RAGE in the pulmonary long-term effects of a single high-intensity exposure to WTC-PM. After 1 month, WTC-PM-exposed wild-type (WT) mice had airway hyperreactivity, whereas RAGE-deficient (Ager-/-) mice were protected. PM-exposed WT mice also had histologic evidence of airspace disease, whereas Ager-/- mice remained unchanged. Inflammatory mediators such as G-CSF (granulocyte colony-stimulating factor), IP-10 (IFN-γ-induced protein 10), and KC (keratinocyte chemoattractant) were differentially expressed after WTC-PM exposure. WTC-PM induced α-SMA, DIAPH1 (protein diaphanous homolog 1), RAGE, and significant lung collagen deposition in WT compared with Ager-/- mice. Compared with WT mice with PM exposure, relative expression of phosphorylated to total CREB (cAMP response element-binding protein) and JNK (c-Jun N-terminal kinase) was significantly increased in the lung of PM-exposed Ager-/- mice, whereas Akt (protein kinase B) was decreased. Random forests of the refined lung metabolomic profile classified subjects with 92% accuracy; principal component analysis captured 86.7% of the variance in three components and demonstrated prominent subpathway involvement, including known mediators of lung disease such as vitamin B6 metabolites, sphingolipids, fatty acids, and phosphatidylcholines. Treatment with a partial RAGE antagonist, pioglitazone, yielded similar fold-change expression of metabolites (N6-carboxymethyllysine, 1-methylnicotinamide, N1+N8-acetylspermidine, and succinylcarnitine [C4-DC]) between WT and Ager-/- mice exposed to WTC-PM. RAGE can mediate WTC-PM-induced airway hyperreactivity and warrants further investigation.

Diabetes Mellitus and Cardiovascular Disease.

Cardiovascular disease remains a leading cause of morbidity and mortality in people with types 1 or 2 diabetes mellitus. Although beneficial roles for strict control of hyperglycemia have been suggested, such a strategy is not without liabilities. Specifically, the risk of hypoglycemia and its consequences remain an omnipresent threat with such approaches. The advent of the CVOT (Cardiovascular Outcomes Trials) for new antidiabetes mellitus treatments has uncovered unexpected benefits of cardiovascular protection in some of the new classes of agents, such as the GLP-1 RAs (glucagon-like peptide-1 receptor agonists) and the SGLT-2 (sodium-glucose cotransporter-2) inhibitors. Further, state-of-the-art approaches, such as antibodies to PCKSK9 (proprotein convertase subtilisin-kexin type 9); RNA therapeutics; agents targeting distinct components of the immune/inflammatory response; and novel small molecules that block the actions of RAGE (receptor for advanced glycation end products) signaling, also hold potential as new therapies for diabetes mellitus and cardiovascular disease. Finally, interventions such as weight loss, through bariatric surgery, may hold promise for benefit in diabetes and cardiovascular disease. In this Brief Review, some of the novel approaches and emerging targets for the treatment of diabetes mellitus and cardiovascular disease are discussed. Ultimately, identification of the optimal timing and combinations of such interventions, especially in the context of personalized approaches, together with emerging disease-modifying agents, holds great promise to reduce the burden that diabetes poses to the cardiovascular system.