Modulation of soluble receptor for advanced glycation end products by angiotensin-converting enzyme-1 inhibition in diabetic nephropathy

Recent studies have identified that first-line renoprotective agents that interrupt the renin-angiotensin system not only reduce BP but also can attenuate advanced glycation end product (AGE) accumulation. This study used in vitro, preclinical, and human approaches to explore the potential effects of these agents on the modulation of the receptor for AGE (RAGE). Bovine aortic endothelial cells that were exposed to the angiotensin-converting enzyme inhibitor (ACEi) ramiprilat in the presence of high glucose demonstrated a significant increase in soluble RAGE (sRAGE) secreted into the medium. In streptozotocin-induced diabetic rats, ramipril treatment (ACEi) at 3 mg/L for 24 wk reduced the accumulation of skin collagen-linked carboxymethyllysine and pentosidine, as well as circulating and renal AGE. Renal gene upregulation of total RAGE (all three splice variants) was observed in ACEi-treated animals. There was a specific increase in the gene expression of the splice variant C-truncated RAGE (sRAGE). There were also increases in sRAGE protein identified within renal cells with ACEi treatment, which showed AGE-binding ability. This was associated with decreases in renal full-length RAGE protein from ACEi-treated rats. Decreases in plasma soluble RAGE that were significantly increased by ACEi treatment were also identified in diabetic rats. Similarly, there was a significant increase in plasma sRAGE in patients who had type 1 diabetes and were treated with the ACEi perindopril. Complexes between sRAGE and carboxymethyllysine were identified in human and rodent diabetic plasma. It is postulated that ACE inhibition reduces the accumulation of AGE in diabetes partly by increasing the production and secretion of sRAGE into plasma.     

Requirement for p38 and p44/p42 mitogen-activated protein kinases in RAGE-mediated nuclear factor-kappaB transcriptional activation and cytokine secretion

Advanced glycation end product (AGE) activation of the signal-transducing receptor for AGE (RAGE) has been linked to a proinflammatory phenotypic change within cells. However, the precise intracellular signaling pathways involved have not been elucidated. We demonstrate here that human serum albumin modified with N(epsilon)-(carboxymethyl)lysine (CML), a major AGE adduct that progressively accumulates with aging, diabetes, and renal failure, induced nuclear factor (NF)-kappaB-driven reporter gene expression in human monocytic THP-1 cells. The NF-kappaB response was blocked with a synthetic peptide corresponding to the putative ligand-binding domain of RAGE, with anti-RAGE antiserum, and by coexpression of truncated receptors lacking the intracellular domain. Signal transduction from RAGE to NF-kappaB involved the generation of reactive oxygen species, since reporter gene expression was blocked with the antioxidant N-acetyl-L-cysteine. CML-modified albumin produced rapid transient activation of tyrosine phosphorylation, extracellular signal-regulated kinase 1 and 2, and p38 mitogen-activated protein kinase (MAPK), but not c-Jun NH(2)-terminal kinase. RAGE-mediated NF-kappaB activation was suppressed by the selective p38 MAPK inhibitor SB203580 and by coexpression of a kinase-dead p38 dominant-negative mutant. Activation of NF-kappaB by CML-modified albumin increased secretion of proinflammatory cytokines (tumor necrosis factor-alpha, interleukin-1beta, and monocyte chemoattractant protein-1) severalfold, and inhibition of p38 MAPK blocked these increases. These results indicate that p38 MAPK activation mediates RAGE-induced NF-kappaB-dependent secretion of proinflammatory cytokines and suggest that accelerated inflammation may be a consequence of cellular activation induced by this receptor.     

PPARgamma ligands increase expression and plasma concentrations of adiponectin, an adipose-derived protein

Insulin resistance and its dreaded consequence, type 2 diabetes, are major causes of atherosclerosis. Adiponectin is an adipose-specific plasma protein that possesses anti-atherogenic properties, such as the suppression of adhesion molecule expression in vascular endothelial cells and cytokine production from macrophages. Plasma adiponectin concentrations are decreased in obese and type 2 diabetic subjects with insulin resistance. A regimen that normalizes or increases the plasma adiponectin might prevent atherosclerosis in patients with insulin resistance. In this study, we demonstrate the inducing effects of thiazolidinediones (TZDs), which are synthetic PPARgamma ligands, on the expression and secretion of adiponectin in humans and rodents in vivo and in vitro. The administration of TZDs significantly increased the plasma adiponectin concentrations in insulin resistant humans and rodents without affecting their body weight. Adiponectin mRNA expression was normalized or increased by TZDs in the adipose tissues of obese mice. In cultured 3T3-L1 adipocytes, TZD derivatives enhanced the mRNA expression and secretion of adiponectin in a dose- and time-dependent manner. Furthermore, these effects were mediated through the activation of the promoter by the TZDs. On the other hand, TNF-alpha, which is produced more in an insulin-resistant condition, dose-dependently reduced the expression of adiponectin in adipocytes by suppressing its promoter activity. TZDs restored this inhibitory effect by TNF-alpha. TZDs might prevent atherosclerotic vascular disease in insulin-resistant patients by inducing the production of adiponectin through direct effect on its promoter and antagonizing the effect of TNF-alpha on the adiponectin promoter.     

A Receptor‐Based Bioadsorbent to Target Advanced Glycation End Products in Chronic Kidney Disease

The accumulation of advanced glycation end products (AGEs) has been reported to be a major contributor to chronic systemic inflammation. AGEs are not efficiently removed by hemodialysis or the kidney of a chronic kidney disease (CKD) patient. The goal of this study was to develop a receptor for AGEs (RAGE)‐based bioadsorbent device that was capable of removing endogenous AGEs from human blood. The extracellular domain of RAGE was immobilized onto agarose beads to generate the bioadsorbent. The efficacy of AGE removal from saline, serum, and whole blood; biological effects of AGE reduction; and hemocompatibility and stability of the bioadsorbent were investigated. The bioadsorbent bound AGE‐modified bovine serum albumin (AGE‐BSA) with a binding capacity of 0.73 ± 0.07 mg AGE‐BSA/mL bioadsorbent. The bioadsorbent significantly reduced the concentration of total AGEs in serum isolated from end‐stage kidney disease patients by 57%. AGE removal resulted in a significant reduction of vascular cell adhesion molecule‐1 expression in human endothelial cells and abolishment of osteoclast formation in osteoclast progenitor cells. A hollow fiber device loaded with bioadsorbent‐reduced endogenous AGEs from recirculated blood to 36% of baseline levels with no significant changes in total protein or albumin concentration. The bioadsorbent maintained AGE‐specific binding capacity after freeze‐drying and storage for 1 year. This approach provides the foundation for further development of soluble RAGE‐based extracorporeal therapies to selectively deplete serum AGEs from human blood and decrease inflammation in patients with diabetes and/or CKD.     

Receptor for advanced glycation end products is involved in impaired angiogenic response in diabetes

Angiogenic response is impaired in diabetes. Here, we examined the involvement of receptor for advanced glycation end products (RAGE) in diabetes-related impairment of angiogenesis in vivo. Angiogenesis was determined in reconstituted basement membrane protein (matrigel) plugs containing vascular endothelial growth factor (VEGF) implanted into nondiabetic or insulin-deficient diabetic wild-type or RAGE(-/-) mice. The total, endothelial, and smooth muscle (or pericytes) cells in the matrigel were significantly decreased in diabetes, with the regulation dependent on RAGE. In the matrigel, proangiogenic VEGF expression was decreased, while antiangiogenic thrombospondin-1 was upregulated in diabetic mice, regardless of the presence of RAGE. In wild-type mice, proliferating cell nuclear antigen (PCNA)-positive cells in the matrigel were significantly less in diabetic than in nondiabetic mice, while the numbers of transferase-mediated dUTP nick-end labeling (TUNEL)-positive cells were significantly higher. This alteration in PCNA- and TUNEL-positive cells in diabetes was not observed in RAGE(-/-) mice. Similarly, the percentage of nuclear factor kappaB-activated cells is enhanced in diabetes, with the regulation dependent on the presence of RAGE. Importantly, adenovirus-mediated overexpression of endogenous secretory RAGE, a decoy receptor for RAGE, restores diabetes-associated impairment of angiogenic response in vivo. Thus, RAGE appears to be involved in impairment of angiogenesis in diabetes, and blockade of RAGE might be a potential therapeutic target.     

Alagebrium reduces glomerular fibrogenesis and inflammation beyond preventing RAGE activation in diabetic apolipoprotein E knockout mice

Advanced glycation end products (AGEs) are important mediators of diabetic nephropathy that act through the receptor for AGEs (RAGE), as well as other mechanisms, to promote renal inflammation and glomerulosclerosis. The relative contribution of RAGE-dependent and RAGE-independent signaling pathways has not been previously studied in vivo. In this study, diabetic RAGE apoE double-knockout (KO) mice with streptozotocin-induced diabetes were treated with the AGE inhibitor, alagebrium (1 mg/kg/day), or the ACE inhibitor, quinapril (30 mg/kg/day), for 20 weeks, and renal parameters were assessed. RAGE deletion attenuated mesangial expansion, glomerular matrix accumulation, and renal oxidative stress associated with 20 weeks of diabetes. By contrast, inflammation and AGE accumulation associated with diabetes was not prevented. However, treatment with alagebrium in diabetic RAGE apoE KO mice reduced renal AGE levels and further reduced glomerular matrix accumulation. In addition, even in the absence of RAGE expression, alagebrium attenuated cortical inflammation, as denoted by the reduced expression of monocyte chemoattractant protein-1, intracellular adhesion molecule-1, and the macrophage marker cluster of differentiation molecule 11b. These novel findings confirm the presence of important RAGE-independent as well as RAGE-dependent signaling pathways that may be activated in the kidney by AGEs. This has important implications for the design of optimal therapeutic strategies for the prevention of diabetic nephropathy.     

Differential effects of advanced glycation end-products on renal tubular cell inflammation

Aim: The authors recently showed that advanced glycation end‐products (AGE) in the form of glycated albumin (GA) upregulated renal tubular expression of interleukin (IL)‐8 and soluble intercellular adhesion molecule‐1 (sICAM‐1), but not other important cytokines known to mediate diabetic nephropathy. This implies that other molecules such as the carbonyl intermediates of AGE or other modified protein lysine‐albumin may participate in diabetic tubular injury. Methods: Human proximal tubular epithelial cells (PTEC) were growth‐arrested and exposed to methylglyoxal (MG), MG‐bovine serum albumin (BSA)‐AGE, carboxymethyllysine (CML)‐BSA, AGE‐BSA or BSA with or without prior addition of rosiglitazone that was previously shown to attenuate the pro‐inflammatory effect of GA alone. Results: MG‐BSA‐AGE and AGE‐BSA upregulated tubular expression of connective tissue growth factor (CTGF), transforming growth factor (TGF)‐β, and vascular endothelial growth factor (VEGF), whereas CML‐BSA stimulated expression of IL‐6, CCL‐2, CTGF, TGF‐β and VEGF. These AGE compounds also activated nuclear factor (NF)‐κB and their effects were attenuated by pre‐incubation with anti‐RAGE antibody. MG and BSA did not affect the expression of any of these molecules. Rosiglitazone did not affect the in vitro biological effects of MG, MG‐BSA‐AGE, AGE‐BSA or CML‐BSA on PTEC. Conclusion: AGE exhibit differential inflammatory and fibrotic effects on PTEC via RAGE activation and NF‐κB signal transduction. Rosiglitazone had no effect on these responses. Further investigations on compounds that nullify the downstream effects of these AGE are warranted.     

Damage to the peritoneal membrane by glucose degradation products is mediated by the receptor for advanced glycation end-products

Peritoneal dialysis is limited by morphologic changes of the peritoneal membrane. Use of peritoneal dialysis fluids (PDF) that contain glucose degradation products (GDP) generates advanced glycation end-products (AGE) within the peritoneal cavity. It is unknown whether peritoneal damage is causally related to AGE-receptor for AGE (RAGE) interaction. The effects of PDF were compared with different amounts of GDP on morphologic changes of the peritoneal membrane in 48 wild-type (WT) and 48 RAGE-deficient mice. PDF (1 ml) were instilled twice daily over a period of 12 wk. Groups with eight animals each received no manipulation (sham); sham instillation (sham i.p.); or filter-sterilized, glucose-free, conventional low GDP- or high GDP PDF. In vitro (generation of AGE fluorescence in PDF) and in vivo (immunohistochemistry for carboxymethyllysine), a GDP-dependent increase of AGE formation occurred. Inflammation and neoangiogenesis were augmented in WT mice that were treated with high GDP accompanied by upregulation of CD3+ T cells, increased NF-kappaB binding activity, increased lectin, and vascular endothelial growth factor expression. Furthermore, pronounced submesothelial fibrosis was found with increased expression of TGF-beta1. Exposure to low GDP resulted in only mild inflammation and neoangiogenesis (compared with sham i.p.) and no fibrosis in WT mice. The findings in WT contrasted with those in RAGE-deficient mice, which showed no increased inflammation (CD3+ T cells and NF-kappaB binding activity), neoangiogenesis (by lectin and vascular endothelial growth factor expression), or fibrosis (expression of TGF-beta1) after long-term exposure to GDP-containing PDF. Peritoneal damage by GDP in PDF is dependent at least in part on AGE-RAGE interaction.     

Inhibition of the interaction of AGE-RAGE prevents hyperglycemia-induced fibrosis of the peritoneal membrane

The peritoneal membrane of long-term peritoneal dialysis patients is characterized by a loss of ultrafiltration capacity, associated morphologically with submesothelial fibrosis and neoangiogenesis. Exposure to high glucose concentrations in peritoneal dialysate and the resultant advanced glycation end-products (AGE) accumulation have been implicated in the development of these changes, but their exact pathophysiological role is unknown. We examined the effect of the interaction of AGE with one of their receptors (i.e., RAGE) on the function and structure of the peritoneum exposed to high ambient glucose concentrations. Streptozotocin-induced diabetic rats and control rats were treated during 6 wk with either neutralizing monoclonal anti-RAGE antibodies or control antibodies. The expression of RAGE was strongly enhanced in the peritoneal membrane of the diabetic animals. The diabetic peritonea were characterized by an elevated transport of small solutes, lower ultrafiltration rates, a higher vascular density, and an upregulation of endothelial nitric oxide synthase expression. These parameters were unaffected by treatment with anti-RAGE antibodies. In contrast, anti-RAGE but not control antibodies prevented upregulation of TGF-beta, development of submesothelial fibrosis, and fibronectin accumulation in the peritoneum of diabetic animals. In conclusion, binding of AGE to RAGE increases the expression of TGF-beta and contributes to the development of submesothelial fibrosis. Neoangiogenesis and the resultant loss of ultrafiltration capacity are mediated by different pathogenetic pathways.     

Glycation, glycoxidation and diabetes mellitus

Advanced glycation end-products (AGEs) result from a reaction between carbohydrates and the free amino groups of proteins, lipids, and DNA. Non enzymatic glycation, glycoxidation with glucose auto-oxidation and the polyol pathway are involved in glycated protein formation. AGEs also named glycotoxins are found in excess in pathological situations such as diabetes mellitus, renal failure, and aging or after absorption of food containing glycated products. Three major pathophysiological mechanisms are described to explain AGE toxicity, first AGEs can accumulate in the vessel wall and in collagen of different tissues; second in situ glycation is possible; third, AGEs bind to cell receptors inducing deleterious consequences. AGE receptor RAGE is a multiligand member of the immunoglobulin superfamily of cell surface molecules. AGE-receptor interaction can alter, macrophage, endothelial cell, mesangial and mesothelial cell functions and can induce inflammation. Oxidant stress, vascular hyperpermeability, vascular cell adhesion molecule-1 (VCAM-1) overexpression and monocytes chemotactic Protein-1 (MCP-1) production have been observed after cell activation by AGEs. AGEs appear to be involved in the genesis of diabetic macro but also microangiopathy such as retinopathy and glomerulosclerosis. New drugs are tested to prevent or break the AGE-protein cross-linkage, or to control the AGE-receptor interaction and their consequences. Dietary treatment, strict glycemic control and preservation of renal function remain the best approach for preventing AGE formation and limiting their deleterious effects.     

Receptors for advanced glycation end-products (AGE)--expression by endothelial cells in non-diabetic uraemic patients

BACKGROUND: Cellular actions of advanced glycation end-products (AGE) aremediated by a receptor for AGE (RAGE), a novel integral membraneprotein. Immunohistochemical studies show only low-level RAGEantigen expression in endothelial cells. DESIGN: It was the purpose of the study to compare expression of RAGEantigen by endothelial cells in non-diabetic uraemic patients(n=8) with non-uraemic controls (n=11). Samples of arterialtissue were obtained at the time of renal transplantation (inuraemic patients) and abdominal surgery (in controls). RAGEantigen was visualized using guinea-pig anti-RAGE IgG and PAPtechnique. RESULTS: Marked staining for RAGE was noted in endothelial cells, botharterial endothelium and endothelium of vasa vasorum of normoglycaemicuraemic patients, but was not demonstrable in endothelial cellsof large arteries and only faintly expressed in vasa vasorumof non-uraemic individuals. CONCLUSION: Normal endothelial cells do not constitually express RAGE antigen;in contrast it is expressed by arterial and capillary endothelialcells of uraemic patients. The observation is of note in viewof the putative role of AGE of causing atherosclerotic and non-atheroscleroticvascular lesions.     

Molecular mechanisms of AGE/RAGE-mediated fibrosis in the diabetic heart

Chronic hyperglycemia is one of the main characteristics of diabetes. Persistent exposure to elevated glucose levels has been recognized as one of the major causal factors of diabetic complications. In pathologies, like type 2 diabetes mellitus(T2DM), mechanical and biochemical stimuli activate profibrotic signaling cascades resulting in myocardial fibrosis and subsequent impaired cardiac performance due to ventricular stiffness. High levels of glucose nonenzymatically react with long-lived proteins, such as collagen, to form advanced glycation end products(AGEs). AGE-modified collagen increase matrix stiffness making it resistant to hydrolytic turnover, resulting in an accumulation of extracellular matrix(ECM) proteins. AGEs account for many of the diabetic cardiovascular complications through their engagement of the receptor for AGE(RAGE). AGE/RAGE activation stimulates the secretion of numerous profibrotic growth factors, promotes increased collagen deposition leading to tissue fibrosis, as well as increased RAGE expression. To date, the AGE/RAGE cascade is not fully understood. In this review, we willdiscuss one of the major fibrotic signaling pathways, the AGE/RAGE signaling cascade, as well as propose an alternate pathway via Rap1 a that may offer insight into cardiovascular ECM remodeling in T2 DM. In a series of studies, we demonstrate a role for Rap1 a in the regulation of fibrosis and myofibroblast differentiation in isolated diabetic and non-diabetic fibroblasts. While these studies are still in a preliminary stage, inhibiting Rap1 a protein expression appears to down-regulate the molecular switch used to activate the ζ isotype of protein kinase C thereby promote AGE/RAGE-mediated fibrosis.     

Expression of receptors for advanced glycosylated end-products in renal disease

BACKGROUND: Advanced glycation endproducts (AGEs) are believed to mediatelong-term complications in diabetes mellitus. In this contextwe studied the expression of the receptor for AGEs (RAGE) inthe kidney of patients with a variety of different renal diseases. METHODS: RAGE was detected by immunocytochemistry in renal biopsies.We compared the staining for RAGE in nine patients with diabeticnephropathy, 20 with inflammatory and/or immune complex and10 with non-inflammatory renal diseases. Normal renal tissuefrom seven patients with tumour nephrectomies served as controls. RESULTS: In controls the only cells expressing RAGE constitutively wereinterstitial cells and vascular smooth muscle cells (6/7), whiledistal tubular cells were rarely positive (1/7). Endothelialcells of arteries/arterioles, glomerular endothelial cells,podocytes, and capsular epithelial cells were consistently negative. In diabetic nephropathy, inflammatory and/or immune complex,and non-inflammatory renal diseases, all cell types mentionedabove became positive for RAGE. Whilst the distribution of RAGEin the tissue was quite similar, staining intensity in inflammatoryand/or immune complex diseases was higher than in diabetic nephropathy. CONCLUSION: RAGE induction in the kidney is not specific for diabetic nephropathyand occurs in other types of renal diseases as well.     

Hyperglycemia and Liver Ischemia Reperfusion Injury: A Role for the Advanced Glycation Endproduct and Its Receptor Pathway

Although pretransplant diabetes is a risk factor for mortality post–liver transplant, the underlying mechanism has not been fully defined. In a murine liver partial warm ischemia model, we addressed the question of how diabetes/hyperglycemia impacted tissue inflammatory injuries against ischemia reperfusion (IR), focusing on the advanced glycation endproduct (AGE) and its receptor (RAGE) pathway. Our results showed that hepatocellular injury was exacerbated in streptozotocin‐induced diabetic mice against IR, in association with hyper‐inflammatory immune activation in livers. Serum levels of AGEs, but not HMGB1, were increased in diabetic mice in response to liver IR. Both RAGE antagonist peptides and small interfering RNA alleviated liver injuries and inhibited inflammatory immune activation against IR in diabetic, but not normal, mice. Kupffer cells (KCs)/macrophages, but not hepatocytes, from diabetic mice expressed significantly higher levels of RAGE, leading to their hyper‐inflammatory responsiveness to both TLR ligands and AGEs. In vitro, hyperglycemia increased macrophage RAGE expression and enhanced their TLR responses. Our results demonstrated that activation of the AGE–RAGE signaling pathway in KCs was responsible for hyper‐inflammatory immune responses and exacerbated hepatocellular injuries in diabetic/hyperglycemic hosts against liver IR.     

Endogenous secretory receptor for advanced glycation end-products and cardiovascular disease in end-stage renal disease.

The receptor for advanced glycation end-products (RAGE) is involved in microvascular and macrovascular complications in diabetes. The expression of RAGE is up-regulated in atherosclerotic plaques of diabetic animals, and the augmentation of atherosclerosis in diabetic mice is inhibited by the competition of RAGE. An endogenous secretory RAGE (esRAGE) was identified as a novel splice variant carrying all of the extracellular domains, but devoid of the transmembrane and intracytoplasmic domains. The esRAGE is released from the cells, to bind advanced glycation end-products, and this is capable of neutralizing the actions of advanced glycation end-products on endothelial cells in culture. The adenoviral overexpression of esRAGE restores the impairment of vascular dysfunction in diabetes, suggesting that esRAGE may be an important inhibitor of RAGE signaling in vivo, and may be useful for the prevention of diabetic vascular complications. In 203 age-matched and sex-matched type 2 diabetic and 134 nondiabetic subjects, plasma esRAGE levels were inversely associated with carotid or femoral atherosclerosis. In patients with end-stage renal disease (ESRD), plasma esRAGE levels are higher than in those without ESRD. In a cohort of 206 patients (including 171 nondiabetic patients) with chronic renal failure who were followed for a median of 111 months, the cumulative incidence of cardiovascular death by Kaplan-Meier estimation was significantly higher in subjects in the lowest tertile of plasma esRAGE than in those in the middle or highest tertile. Compared with the lowest tertile of plasma esRAGE, the hazards ratios for the highest and middle tertile were 0.40 (95% confidence interval, 0.18 to 0.89) and 0.26 (95% confidence interval, 0.10 to 0.66), respectively. The higher risk at the lower level of esRAGE was still significant even after adjustment for body mass index, hypertension, dyslipidemia, and vascular complications, and was confounded only by age and diabetes. Thus, we postulate that plasma esRAGE is a potential protective factor and a novel biomarker against the occurrence of cardiovascular disease in ESRD.     

RAGE and modulation of ischemic injury in the diabetic myocardium

OBJECTIVE—Subjects with diabetes experience an increased risk of myocardial infarction and cardiac failure compared with nondiabetic age-matched individuals. The receptor for advanced glycation end products (RAGE) is upregulated in diabetic tissues. In this study, we tested the hypothesis that RAGE affected ischemia/reperfusion (I/R) injury in the diabetic myocardium. In diabetic rat hearts, expression of RAGE and its ligands was enhanced and localized particularly to both endothelial cells and mononuclear phagocytes.RESEARCH DESIGN AND METHODS—To specifically dissect the impact of RAGE, homozygous RAGE-null mice and transgenic (Tg) mice expressing cytoplasmic domain-deleted RAGE (DN RAGE), in which RAGE-dependent signal transduction was deficient in endothelial cells or mononuclear phagocytes, were rendered diabetic with streptozotocin. Isolated perfused hearts were subjected to I/R.RESULTS—Diabetic RAGE-null mice were significantly protected from the adverse impact of I/R injury in the heart, as indicated by decreased release of LDH and lower glycoxidation products carboxymethyl-lysine (CML) and pentosidine, improved functional recovery, and increased ATP. In diabetic Tg mice expressing DN RAGE in endothelial cells or mononuclear phagocytes, markers of ischemic injury and CML were significantly reduced, and levels of ATP were increased in heart tissue compared with littermate diabetic controls. Furthermore, key markers of apoptosis, caspase-3 activity and cytochrome c release, were reduced in the hearts of diabetic RAGE-modified mice compared with wild-type diabetic littermates in I/R.CONCLUSIONS—These findings demonstrate novel and key roles for RAGE in I/R injury in the diabetic heart.Cardiac complications remain a leading cause of morbidity and mortality in subjects with diabetes (1–3). Although many factors contribute to depressed cardiac function in diabetes, innate disturbances within the diabetic heart contribute importantly to progressive dysfunction, which often leads to irreversible failure and death (3). Alterations in substrate metabolism and increased levels of oxygen free radicals have been observed in diabetic tissues. Inflammatory cytokines may exert direct negative inotropic effects on cardiac myocytes and contribute to aberrant remodeling in the failed heart (4–8). The pathophysiology of diabetes-associated cardiac complications is complex and involves a host of factors linked to metabolic and immune/inflammatory cell activation.The accumulation of late-stage glycoxidation adducts of proteins, termed advanced glycation end products (AGEs), occurs in diabetic tissues. AGEs modify long-lived molecules in the blood vessel wall and structural tissues of the heart considerably earlier than symptomatic cardiac dysfunction occurs (9). A major way in which AGEs exert their cellular effects is by ligation of the multiligand receptor for AGE (RAGE) (10–13).We tested the role of RAGE in rodent models of type 1 diabetes, and we show that pharmacological blockade of ligand-RAGE interaction or genetic modulation of RAGE suppresses ischemia/reperfusion (I/R) injury in the isolated perfused heart, at least in part secondary to critical contributions evoked from RAGE-expressing endothelial cells and mononuclear phagocytes in the diabetic heart.