Obesity and diabetes are leading causes of cardiovascular morbidity and mortality. Although extensive strides have been made in the treatments for non-diabetic atherosclerosis and its complications, for patients with diabetes, these therapies provide less benefit for protection from cardiovascular disease (CVD). These considerations spur the concept that diabetes-specific, disease-modifying therapies are essential to identify, especially as the epidemics of obesity and diabetes continue to expand. Hence, as hyperglycemia is a defining feature of diabetes, it is logical to probe the impact of the specific consequences of hyperglycemia on the vessel wall, immune cell perturbation, and endothelial dysfunction—all harbingers to the development of CVD. In this context, high levels of blood glucose stimulate the formation of the irreversible advanced glycation end products, the products of non-enzymatic glycation and oxidation of proteins and lipids. AGEs accumulate in diabetic circulation and tissues and the interaction of AGEs with their chief cellular receptor, receptor for AGE or RAGE, contributes to vascular and immune cell perturbation. The cytoplasmic domain of RAGE lacks endogenous kinase activity; the discovery that this intracellular domain of RAGE binds to the formin, DIAPH1, and that DIAPH1 is essential for RAGE ligand-mediated signal transduction, identifies the specific cellular means by which RAGE functions and highlights a new target for therapeutic interruption of RAGE signaling. In human subjects, prominent signals for RAGE activity include the presence and levels of two forms of soluble RAGE, sRAGE, and endogenous secretory (es) RAGE. Further, genetic studies have revealed single nucleotide polymorphisms (SNPs) of the AGER gene (AGER is the gene encoding RAGE) and DIAPH1, which display associations with CVD. This Review presents current knowledge regarding the roles for RAGE and DIAPH1 in the causes and consequences of diabetes, from obesity to CVD. Studies both from human subjects and animal models are presented to highlight the breadth of evidence linking RAGE and DIAPH1 to the cardiovascular consequences of these metabolic disorders.

Despite advances in lipid-lowering therapies, people with diabetes continue to experience more limited cardiovascular benefits. In diabetes, hyperglycemia sustains inflammation and preempts vascular repair. We tested the hypothesis that the receptor for advanced glycation end-products (RAGE) contributes to these maladaptive processes. We report that transplantation of aortic arches from diabetic, Western diet–fed Ldlr ^—/— mice into diabetic Ager ^—/— ( Ager , the gene encoding RAGE) versus WT diabetic recipient mice accelerated regression of atherosclerosis. RNA-sequencing experiments traced RAGE-dependent mechanisms principally to the recipient macrophages and linked RAGE to interferon signaling. Specifically, deletion of Ager in the regressing diabetic plaques downregulated interferon regulatory factor 7 ( Irf7 ) in macrophages. Immunohistochemistry studies colocalized IRF7 and macrophages in both murine and human atherosclerotic plaques. In bone marrow–derived macrophages (BMDMs), RAGE ligands upregulated expression of Irf7 , and in BMDMs immersed in a cholesterol-rich environment, knockdown of Irf7 triggered a switch from pro- to antiinflammatory gene expression and regulated a host of genes linked to cholesterol efflux and homeostasis. Collectively, this work adds a new dimension to the immunometabolic sphere of perturbations that impair regression of established diabetic atherosclerosis and suggests that targeting RAGE and IRF7 may facilitate vascular repair in diabetes.

The receptor for advanced glycosylation end products (RAGE) is a multiligand receptor involved in diverse cell signaling pathways. Previous studies show that this gene expresses several splice variants in human, mouse, and dog. Alternative splicing (AS) plays an important role in expanding transcriptomic and proteomic diversity, and it has been related to disease. AS is also one of the main evolutionary mechanisms in mammalian genomes. However, limited information is available regarding the AS of RAGE in a wide context of mammalian tissues. In this study, we examined in detail the different RAGE mRNAs generated by AS from six mammals, including two primates (human and monkey), two artiodactyla (cow and pig), and two rodentia (mouse and rat) in 6–18 different tissues including fetal, adult, and tumor. By nested reverse transcription-polymerase chain reaction (RT-PCR) we identified a high number of splice variants including noncoding transcripts and predicted coding ones with different potential protein modifications affecting mainly the transmembrane and ligand-binding domains that could influence their biological function. However, analysis of RNA-seq data enabled detecting only the most abundant splice variants. More than 80% of the detected RT-PCR variants (87 of 101 transcripts) are novel (different exon/intron structure to the previously described ones), and interestingly, 20–60% of the total transcripts (depending on the species) are noncoding ones that present tissue specificity. Our results suggest that RAGE undergoes extensive AS in mammals, with different expression patterns among adult, fetal, and tumor tissues. Moreover, most splice variants seem to be species specific, especially the noncoding variants, with only two (canonical human Tv1-RAGE, and human N-truncated or Tv10-RAGE) conserved among the six different species. This could indicate a special evolution pattern of this gene at mRNA level.

Inter-organelle contact and communication between mitochondria and sarco/endoplasmic reticulum (SR/ER) maintain cellular homeostasis and are profoundly disturbed during tissue ischemia. We tested the hypothesis that the formin Diaphanous-1 (DIAPH1), which regulates actin dynamics, signal transduction and metabolic functions, contributes to these processes. We demonstrate that DIAPH1 interacts directly with Mitofusin-2 (MFN2) to shorten mitochondria-SR/ER distance, thereby enhancing mitochondria-ER contact in cells including cardiomyocytes, endothelial cells and macrophages. Solution structure studies affirm the interaction between the Diaphanous Inhibitory Domain and the cytosolic GTPase domain of MFN2. In male rodent and human cardiomyocytes, DIAPH1-MFN2 interaction regulates mitochondrial turnover, mitophagy, and oxidative stress. Introduction of synthetic linker construct, which shorten the mitochondria-SR/ER distance, mitigated the molecular and functional benefits ofDIAPH1silencing in ischemia. This work establishes fundamental roles for DIAPH1-MFN2 interaction in the regulation of mitochondria-SR/ER contact networks. We propose that targeting pathways that regulate DIAPH1-MFN2 interactions may facilitate recovery from tissue ischemia.

Objective—: Diabetic subjects are at higher risk of ischemic peripheral vascular disease. We tested the hypothesis that advanced glycation end products (AGEs) and their receptor (RAGE) block angiogenesis and blood flow recovery after hindlimb ischemia induced by femoral artery ligation through modulation of immune/inflammatory mechanisms. Approach and Results—: Wild-type mice rendered diabetic with streptozotocin and subjected to unilateral femoral artery ligation displayed increased accumulation and expression of AGEs and RAGE in ischemic muscle. In diabetic wild-type mice, femoral artery ligation attenuated angiogenesis and impaired blood flow recovery, in parallel with reduced macrophage content in ischemic muscle and suppression of early inflammatory gene expression, including Ccl2 (chemokine [C-C motif] ligand-2) and Egr1 (early growth response gene-1) versus nondiabetic mice. Deletion of Ager (gene encoding RAGE) or transgenic expression of Glo1 (reduces AGEs) restored adaptive inflammation, angiogenesis, and blood flow recovery in diabetic mice. In diabetes mellitus, deletion of Ager increased circulating Ly6C ^hi monocytes and augmented macrophage infiltration into ischemic muscle tissue after femoral artery ligation. 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 hindlimb 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 peripheral vascular disease.