Oxidative stress is a central mechanism by which the receptor for advanced glycation endproducts (RAGE) mediates its pathological effects. Multiple experimental inquiries in RAGE-expressing cultured cells have demonstrated that ligand-RAGE interaction mediates generation of reactive oxygen species (ROS) and consequent downstream signal transduction and regulation of gene expression. The primary mechanism by which RAGE generates oxidative stress is via activation of NADPH oxidase; amplification mechanisms in the mitochondria may further drive ROS production. Recent studies indicating that the cytoplasmic domain of RAGE binds to the formin mDia1 provide further support for the critical roles of this pathway in oxidative stress; mDia1 was required for activation of rac1 and NADPH oxidase in primary murine aortic smooth muscle cells treated with RAGE ligand S100B. In vivo, in multiple distinct disease models in animals, RAGE action generates oxidative stress and modulates cellular/tissue fate in range of disorders, such as in myocardial ischemia, atherosclerosis, and aneurysm formation. Blockade or genetic deletion of RAGE was shown to be protective in these settings. Indeed, beyond cardiovascular disease, evidence is accruing in human subjects linking levels of RAGE ligands and soluble RAGE to oxidative stress in disorders such as doxorubicin toxicity, acetaminophen toxicity, neurodegeneration, hyperlipidemia, diabetes, preeclampsia, rheumatoid arthritis and pulmonary fibrosis. Blockade of RAGE signal transduction may be a key strategy for the prevention of the deleterious consequences of oxidative stress, particularly in chronic disease.

Rationale The mammalian Diaphanous-related formin, mDia1, governs microtubule and microfilament dynamics while functioning as an effector for Rho small GTP-binding proteins during key cellular processes such as adhesion, cytokinesis, cell polarity and morphogenesis. The cytoplasmic domain of the receptor for advanced glycation endproducts (RAGE) binds to the formin homology 1 (FH1) domain of mDia1; mDia1 is required for RAGE ligand-induced cellular migration in transformed cells. Objective As a key mechanism in vascular remodeling is the induction of smooth muscle cell migration, we tested the role of mDia1 in this process. Methods and Results We report that endothelial denudation injury to the murine femoral artery significantly upregulates mDia1 mRNA transcripts and protein in the injured vessel, particularly in vascular smooth muscle cells within the expanding neointima. Loss of mDia1 expression significantly reduces pathological neointimal expansion consequent to injury. In primary murine aortic smooth muscle cells, mDia1 is required for RAGE ligand-induced membrane translocation of c-Src, which leads to Rac1 activation, redox phosphorylation of AKT/GSK3β and consequent smooth muscle cell migration. Conclusions We conclude that mDia1 integrates oxidative and signal transduction pathways triggered, at least in part by RAGE ligands, therefore regulates pathological neointimal expansion.

Diabetes exacerbates cardiovascular disease, at least in part through suppression of macrophage cholesterol efflux and levels of the cholesterol transporters ATP binding cassette transporter A1 (ABCA1) and ABCG1. The receptor for advanced glycation end products (RAGE) is highly expressed in human and murine diabetic atherosclerotic plaques, particularly in macrophages. We tested the hypothesis that RAGE suppresses macrophage cholesterol efflux and probed the mechanisms by which RAGE downregulates ABCA1 and ABCG1. Macrophage cholesterol efflux to apolipoprotein A1 and HDL and reverse cholesterol transport to plasma, liver, and feces were reduced in diabetic macrophages through RAGE. In vitro, RAGE ligands suppressed ABCG1 and ABCA1 promoter luciferase activity and transcription of ABCG1 and ABCA1 through peroxisome proliferator–activated receptor-γ (PPARG)–responsive promoter elements but not through liver X receptor elements. Plasma levels of HDL were reduced in diabetic mice in a RAGE-dependent manner. Laser capture microdissected CD68+ macrophages from atherosclerotic plaques of Ldlr−/− mice devoid of Ager (RAGE) displayed higher levels of Abca1, Abcg1, and Pparg mRNA transcripts versus Ager-expressing Ldlr−/− mice independently of glycemia or plasma levels of total cholesterol and triglycerides. Antagonism of RAGE may fill an important therapeutic gap in the treatment of diabetic macrovascular complications.

Amyotrophic lateral sclerosis (ALS) is a fatal motor neuron disorder of largely unknown pathogenesis. Recent studies suggest that enhanced oxidative stress and neuroinflammation contribute to the progression of the disease. Mounting evidence implicates the receptor for advanced glycation end-products (RAGE) as a significant contributor to the pathogenesis of certain neurodegenerative diseases and chronic conditions. It is hypothesized that detrimental actions of RAGE are triggered upon binding to its ligands, such as AGEs (advanced glycation end products), S100/calgranulin family members, and High Mobility Group Box-1 (HMGB1) proteins. Here, we examined the expression of RAGE and its ligands in human ALS spinal cord. Tissue samples from age-matched human control and ALS spinal cords were tested for the expression of RAGE, carboxymethyllysine (CML) AGE, S100B, and HMGB1, and intensity of the immunofluorescent and immunoblotting signals was assessed. We found that the expression of both RAGE and its ligands was significantly increased in the spinal cords of ALS patients versus age-matched control subjects. Our study is the first report describing co-expression of both RAGE and its ligands in human ALS spinal cords. These findings suggest that further probing of RAGE as a mechanism of neurodegeneration in human ALS is rational.

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The etiology of amyotrophic lateral sclerosis (ALS), a fatal motor neuron disorder characterized by progressive muscle weakness and spasticity, remains largely unknown. Approximately 5–10% of cases are familial, and of those, 15–20% are associated with mutations in the gene encoding Cu/Zn superoxide dismutase (SOD1). Mutations of the SOD1 gene interrupt cellular homeostasis and contribute to cellular toxicity evoked by the presence of altered SOD1, along with other toxic species, such as advanced glycation end products (AGEs). AGEs trigger activation of their chief cell surface receptor, RAGE (receptor for advanced glycation end products), and induce RAGE-dependent cellular stress and inflammation in neurons, thereby affecting their function and leading to apoptosis. Here, we show for the first time that the expression of RAGE is higher in the SOD1 transgenic mouse model of ALS vs. wild-type mouse spinal cord. We tested whether pharmacological blockade of RAGE may delay the onset and progression of disease in this mouse model. Our findings reveal that treatment of SOD1 transgenic mice with soluble RAGE (sRAGE), a natural competitor of RAGE that sequesters RAGE ligands and blocks their interaction with cell surface RAGE, significantly delays the progression of ALS and prolongs life span compared to vehicle treatment. We demonstrate that in sRAGE-treated SOD1 transgenic animals at the final stage of the disease, a significantly higher number of neurons and lower number of astrocytes is detectable in the spinal cord. We conclude that RAGE antagonism may provide a novel therapeutic strategy for ALS intervention.

Objective Diabetic subjects are at higher risk of ischemic peripheral vascular disease (PVD). We tested the hypothesis that advanced glycation end products (AGEs) and their receptor (RAGE) block neoangiogenesis and blood flow recovery after hind limb ischemia induced by femoral artery ligation (FAL) through modulation of immune/inflammatory mechanisms. Approach and Results Wild type (WT) mice rendered diabetic with streptozotocin and subjected to unilateral FAL displayed increased accumulation and expression of AGEs and RAGE in ischemic muscle. In diabetic WT mice, FAL attenuated neoangiogenesis and impaired blood flow recovery, in parallel with reduced macrophage content in ischemic muscle and suppression of early inflammatory gene expression, including chemokine (C-C motif) ligand 2 (Ccl2) and early growth response gene 1 (Egr1) versus non-diabetic mice. Deletion of Ager or transgenic expression of Glo1 (reduces AGEs) restored adaptive inflammation, neoangiogenesis and blood flow recovery in diabetic mice. In diabetes, deletion of Ager increased circulating Ly6Chi monocytes and augmented macrophage infiltration into ischemic muscle tissue after FAL. In vitro, macrophages grown in high glucose display inflammation that is skewed to expression of tissue damage versus tissue repair gene expression. Further, macrophages grown in high versus low glucose demonstrate blunted macrophage-endothelial cell interactions. In both settings, these adverse effects of high glucose were reversed by Ager deletion in macrophages. Conclusions These findings indicate that RAGE attenuates adaptive inflammation in hind limb ischemia; underscore microenvironment-specific functions for RAGE in inflammation in tissue repair versus damage; and illustrate that AGE/RAGE antagonism may fill a critical gap in diabetic PVD.

We addressed the involvement of the receptor for advanced glycation end products (RAGE) in the impairment of the cellular cholesterol efflux elicited by glycated albumin. Albumin was isolated from type 1 (DM1) and type 2 (DM2) diabetes mellitus (HbA1c > 9%) and non-DM subjects (C). Moreover, albumin was glycated in vitro (AGE-albumin). Macrophages from Ager null and wild-type (WT) mice, or THP-1 transfected with siRNA-AGER, were treated with C, DM1, DM2, non-glycated or AGE-albumin. The cholesterol efflux was reduced in WT cells exposed to DM1 or DM2 albumin as compared to C, and the intracellular lipid content was increased. These events were not observed in Ager null cells, in which the cholesterol efflux and lipid staining were, respectively, higher and lower when compared to WT cells. In WT, Ager, Nox4 and Nfkb1, mRNA increased and Scd1 and Abcg1 diminished after treatment with DM1 and DM2 albumin. In Ager null cells treated with DM-albumin, Nox4, Scd1 and Nfkb1 were reduced and Jak2 and Abcg1 increased. In AGER-silenced THP-1, NOX4 and SCD1 mRNA were reduced and JAK2 and ABCG1 were increased even after treatment with AGE or DM-albumin. RAGE mediates the deleterious effects of AGE-albumin in macrophage cholesterol efflux.