High-molecular group box 1-protein, S100/calgranulins, advanced glycation end products (AGEs), amyloid-beta peptides and the family of beta-sheet fibrils contribute to a number of inflammatory conditions by promoting cellular dysfunction and breaking immune tolerance. The receptor of AGE (RAGE) is a multiligand receptor of the immunoglobulin superfamily of cell surface molecules that acts as a pattern recognition receptor. Besides binding ligands actively participating in inflammation and immune responses, RAGE serves as an endothelial adhesion receptor for leukocyte integrins and promotes leukocyte recruitment and extravasation of infiltrating cells. Engagement of RAGE subsequently converts transient cellular stimulation into sustained cellular dysfunction driven by long-term activation of the proinflammatory nuclear factor-kappaB. Deletion of RAGE and pharmacological interventions targeting interruption of RAGE-ligand interaction suppresses inflammation and dampens tissue damage in experimental models of inflammatory disorders, thus delineating RAGE as a potential therapeutic target in inflammation.

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.

Astrocytes and microglia associate to amyloid plaques, a pathological hallmark of Alzheimer disease. Microglia are activated by and can phagocytose beta-amyloid (Abeta). Scavenger receptors (SRs) are among the receptors mediating the uptake of fibrillar Abeta in vitro. However, little is known about the function of the astrocytes surrounding the plaques or the nature of their interaction with Abeta. It is unknown whether glial cells bind to nonfibrillar Abeta and if binding of astrocytes to Abeta depends on the same Scavenger receptors described for microglia. We determined the binding of glia to Abeta by an adhesion assay and evaluated the presence of scavenger receptors in glial cells by immunocytochemistry, immunohistochemistry of brain sections, and immunoblot. We found that astrocytes and microglia from neonatal rats adhered in a concentration-dependent manner to surfaces coated with fibrillar Abeta or nonfibrillar Abeta. Fucoidan and poly(I), known ligands for SR-type A, inhibited adhesion of microglia and astrocytes to Abeta and also inhibited Abeta phagocytosis. In contrast, a ligand for SR-type B like low density lipoprotein, did not compete glial adhesion to Abeta. Microglia presented immunodetectable SR-BI, SR-AI/AII, RAGE, and SR-MARCO (macrophage receptor with collagenous structure, a member of the SR-A family). Astrocytes presented SR-BI and SR-MARCO. To our knowledge, this is the first description of the presence of SR-MARCO in astrocytes. Our results indicate that both microglia and astrocytes adhere to fibrillar and nonfibrillar Abeta. Adhesion was mediated by a fucoidan-sensitive receptor. We propose that SR-MARCO could be the Scavenger receptor responsible for the adhesion of astrocytes and microglia to Abeta.

Amphoterin, a major form of HMG (high mobility group) 1 proteins, is highly expressed in immature and malignant cells. A role in cell motility is suggested by the ability of amphoterin to promote neurite extension through RAGE (receptor of advanced glycation end products), an immunoglobulin superfamily member that communicates with the GTPases Cdc42 and Rac. We show here that cell contact with the laminin matrix induces accumulation of both amphoterin mRNA and protein close to the plasma membrane, which is accompanied by extracellular export of amphoterin. A role for amphoterin in extracellular matrix-dependent cell regulation is further suggested by the finding that specific decrease of amphoterin mRNA and protein, using antisense oligonucleotides transfected into cells, inhibits cell migration to laminin in a transfilter assay whereas the oligonucleotides in the culture medium have no effect. Moreover, affinity-purified anti-amphoterin antibodies inhibit cell migration to laminin, supporting an extracellular role for the endogenous amphoterin in cell motility. The finding that amphoterin expression is more pronounced in cells with a motile phenotype as compared to cells of dense cultures, is consistent with the results of the cell migration assays. Our results strongly suggest that amphoterin is a key player in the migration of immature and transformed cells.

OBJECTIVE

Advanced glycation end-products (AGE) may cause vascular stiffening by forming crosslinks through the collagen molecule or by interaction with their cellular transductional receptor (RAGE). A secreted isoform of RAGE, termed soluble RAGE (sRAGE), may contribute to the removal/detoxification of AGE by acting as a decoy. Here we studied the plasma sRAGE levels in hypertensive and normotensive human subjects. We also investigated the relationship between blood pressure parameters and plasma sRAGE concentrations.

METHODS

A cross-sectional case-control study.

METHODS

The outpatient clinic of a university teaching hospital. Participants were 147 never-treated patients with essential hypertension (87 men and 60 women, aged 50 +/- 10 years) and 177 normotensive controls (118 men and 59 women, aged 49 +/- 10 years).

METHODS

Plasma sRAGE levels determined by enzyme-linked immunosorbent assay, systolic blood pressure (SBP), diastolic blood pressure, pulse pressure (PP) and mean arterial pressure.

RESULTS

The plasma concentration of sRAGE [median (interquartile range)] was 1206 (879-1658) pg/ml in hypertensive subjects and 1359 (999-2198) pg/ml in normotensive controls (P = 0.002). Simple correlation analysis revealed that log-transformed sRAGE levels were inversely correlated with SBP (r = -0.11; P < 0.001) and PP (r = -0.23; P < 0.001). Forward-selection multiple regression analysis revealed that log-transformed sRAGE levels were determined more strongly by PP (F = 3.127, P < 0.001).

CONCLUSIONS

Plasma sRAGE levels are decreased in patients with essential hypertension and are inversely related to PP. Our results raise the possibility that sRAGE may play a role in arterial stiffening and its complications.

Alcohol-induced neuroinflammation is mediated by proinflammatory cytokines, including IL-1β. IL-1β production requires caspase-1 activation by inflammasomes-multiprotein complexes that are assembled in response to danger signals. We hypothesized that alcohol-induced inflammasome activation contributes to increased IL-1β in the brain. WT and TLR4-, NLRP3-, and ASC-deficient (KO) mice received an ethanol-containing or isocaloric control diet for 5 weeks, and some received the rIL-1ra, anakinra, or saline treatment. Inflammasome activation, proinflammatory cytokines, endotoxin, and HMGB1 were measured in the cerebellum. Expression of inflammasome components (NLRP1, NLRP3, ASC) and proinflammatory cytokines (TNF-α, MCP-1) was increased in brains of alcohol-fed compared with control mice. Increased caspase-1 activity and IL-1β protein in ethanol-fed mice indicated inflammasome activation. TLR4 deficiency protected from TNF-α, MCP-1, and attenuated alcohol-induced IL-1β increases. The TLR4 ligand, LPS, was not increased in the cerebellum. However, we found up-regulation of acetylated and phosphorylated HMGB1 and increased expression of the HMGB1 receptors (TLR2, TLR4, TLR9, RAGE) in alcohol-fed mice. NLRP3- or ASC-deficient mice were protected from caspase-1 activation and alcohol-induced IL-1β increase in the brain. Furthermore, in vivo treatment with rIL-1ra prevented alcohol-induced inflammasome activation and IL-1β, TNF-α, and acetylated HMGB1 increases in the cerebellum. Conversely, intracranial IL-1β administration induced TNF-α and MCP-1 in the cerebellum. In conclusion, alcohol up-regulates and activates the NLRP3/ASC inflammasome, leading to caspase-1 activation and IL-1β increase in the cerebellum. IL-1β amplifies neuroinflammation, and disruption of IL-1/IL-1R signaling prevents alcohol-induced inflammasome activation and neuroinflammation. Increased levels of acetylated and phosphorylated HMGB1 may contribute to alcoholic neuroinflammation.

Advanced glycation endproducts (AGEs) and a receptor for AGEs (RAGE) have been linked in the pathogenesis of diabetic nephropathy. RAGE is usually localized to podocytes and is increased in diabetes. RAGE activation increases reactive oxygen species production, which mediates hyperglycemia-induced podocyte apoptosis in early diabetic nephropathy. Here, we examined the interaction of AGE and RAGE on podocyte apoptosis. When we exposed murine cultured podocytes to bovine serum albumin (BSA) that was modified by AGEs or to carboxymethyl-lysine BSA, more apoptosis was found when compared with unmodified BSA. Similarly, more podocytes underwent detachment and apoptosis when cultured on AGE-modified collagen IV than on native collagen IV. AGEs isolated from sera of patients with chronic kidney disease also caused apoptosis of podocytes. Apoptosis was diminished by small interference RNA (siRNA) for RAGE in podocytes exposed to AGE-BSA, but not to AGE-modified collagen IV. Both AGE- and carboxymethyl-lysine modified-BSA activated p38MAP kinase and inhibition of this kinase reduced the apoptotic effect of AGE-BSA. Exposure to AGE-BSA was associated with Akt dephosphorylation and FOXO4 transcriptional activation leading to an increase in the expression of an effector protein of apoptosis, Bim. siRNA for FOXO4 abolished AGE-BSA-induced apoptosis of podocytes. Our study suggests that an AGE-RAGE interaction contributes to podocyte apoptosis by activation of the FOXO4 transcription factor.

A host of diabetes-related insults to the central nervous system (CNS) have been clearly documented in type-1 and -2 diabetic patients as well as experimental animal models. These host of neurological disorders encompass hemodynamic impairments (e.g., stroke), vascular dementia, cognitive deficits (mild to moderate), as well as a number of neurochemical, electrophysiological and behavioral alterations. The underlying causes of diabetes-induced CNS complications are multifactorial and are relatively little understood although it is now evident that blood-brain barrier (BBB) damage plays a significant role in diabetes-dependent CNS disorders. Changes in plasma glucose levels (hyper- or hypoglycemia) have been associated with altered BBB transport functions (e.g., glucose, insulin, choline, amino acids, etc.), integrity (tight junction disruption), and oxidative stress in the CNS microcapillaries. Last two implicating a potential causal role for upregulation and activation of the receptor for advanced glycation end products (RAGE). This type I membrane-protein also transports amyloid-beta (Aβ) from the blood into the brain across the BBB thus, establishing a link between type 2 diabetes mellitus (T2DM) and Alzheimer's disease (AD, also referred to as "type 3 diabetes"). Hyperglycemia has been associated with progression of cerebral ischemia and the consequent enhancement of secondary brain injury. Difficulty in detecting vascular impairments in the large, heterogeneous brain microvascular bed and dissecting out the impact of hyper- and hypoglycemia in vivo has led to controversial results especially with regard to the effects of diabetes on BBB. In this article, we review the major findings and current knowledge with regard to the impact of diabetes on BBB integrity and function as well as specific brain microvascular effects of hyper- and hypoglycemia.

OBJECTIVE

In diabetes, vascular dysfunction is characterized by impaired endothelial function due to increased oxidative stress. Empagliflozin, as a selective sodium-glucose co-transporter 2 inhibitor (SGLT2i), offers a novel approach for the treatment of type 2 diabetes by enhancing urinary glucose excretion. The aim of the present study was to test whether treatment with empagliflozin improves endothelial dysfunction in type I diabetic rats via reduction of glucotoxicity and associated vascular oxidative stress.

METHODS

Type I diabetes in Wistar rats was induced by an intravenous injection of streptozotocin (60 mg/kg). One week after injection empagliflozin (10 and 30 mg/kg/d) was administered via drinking water for 7 weeks. Vascular function was assessed by isometric tension recording, oxidative stress parameters by chemiluminescence and fluorescence techniques, protein expression by Western blot, mRNA expression by RT-PCR, and islet function by insulin ELISA in serum and immunohistochemical staining of pancreatic tissue. Advanced glycation end products (AGE) signaling was assessed by dot blot analysis and mRNA expression of the AGE-receptor (RAGE).

RESULTS

Treatment with empagliflozin reduced blood glucose levels, normalized endothelial function (aortic rings) and reduced oxidative stress in aortic vessels (dihydroethidium staining) and in blood (phorbol ester/zymosan A-stimulated chemiluminescence) of diabetic rats. Additionally, the pro-inflammatory phenotype and glucotoxicity (AGE/RAGE signaling) in diabetic animals was reversed by SGLT2i therapy.

CONCLUSIONS

Empagliflozin improves hyperglycemia and prevents the development of endothelial dysfunction, reduces oxidative stress and improves the metabolic situation in type 1 diabetic rats. These preclinical observations illustrate the therapeutic potential of this new class of antidiabetic drugs.

RAGE (receptor for advanced glycation end-products) is a multiligand receptor of the immunoglobulin superfamily involved in inflammation, diabetes, atherosclerosis, nephropathy, neurodegeneration, and cancer. Advanced glycation end-products, high mobility group box-1 (amphoterin), β-amyloid fibrils, certain S100 proteins, and DNA and RNA are RAGE ligands. Upon RAGE ligation, adaptor proteins (i.e., diaphanous-1, TIRAP, MyD88 and/or other as yet unidentified adaptors) associate with RAGE cytoplasmic domain resulting in signaling. However, RAGE activation may not be restricted to pathological statuses, the receptor being involved in tissue homeostasis and regeneration/repair upon acute injury, and in resolution of inflammation. RAGE effects are strongly dependent on the cell type and the context, which may condition therapeutic strategies aimed at reducing RAGE signaling.

We present the draft genome for the Rickettsia endosymbiont of Ixodes scapularis (REIS), a symbiont of the deer tick vector of Lyme disease in North America. Among Rickettsia species (Alphaproteobacteria: Rickettsiales), REIS has the largest genome sequenced to date (>2 Mb) and contains 2,309 genes across the chromosome and four plasmids (pREIS1 to pREIS4). The most remarkable finding within the REIS genome is the extraordinary proliferation of mobile genetic elements (MGEs), which contributes to a limited synteny with other Rickettsia genomes. In particular, an integrative conjugative element named RAGE (for Rickettsiales amplified genetic element), previously identified in scrub typhus rickettsiae (Orientia tsutsugamushi) genomes, is present on both the REIS chromosome and plasmids. Unlike the pseudogene-laden RAGEs of O. tsutsugamushi, REIS encodes nine conserved RAGEs that include F-like type IV secretion systems similar to that of the tra genes encoded in the Rickettsia bellii and R. massiliae genomes. An unparalleled abundance of encoded transposases (>650) relative to genome size, together with the RAGEs and other MGEs, comprise ~35% of the total genome, making REIS one of the most plastic and repetitive bacterial genomes sequenced to date. We present evidence that conserved rickettsial genes associated with an intracellular lifestyle were acquired via MGEs, especially the RAGE, through a continuum of genomic invasions. Robust phylogeny estimation suggests REIS is ancestral to the virulent spotted fever group of rickettsiae. As REIS is not known to invade vertebrate cells and has no known pathogenic effects on I. scapularis, its genome sequence provides insight on the origin of mechanisms of rickettsial pathogenicity.

Nonenzymatic protein glycation results in the formation of advanced glycation end products (AGEs) that are implicated in the pathology of diabetes, chronic inflammation, Alzheimer's disease, and cancer. AGEs mediate their effects primarily through a receptor-dependent pathway in which AGEs bind to a specific cell surface associated receptor, the Receptor for AGEs (RAGE). N(ɛ)-carboxy-methyl-lysine (CML) and N(ɛ)-carboxy-ethyl-lysine (CEL), constitute two of the major AGE structures found in tissue and blood plasma, and are physiological ligands of RAGE. The solution structure of a CEL-containing peptide-RAGE V domain complex reveals that the carboxyethyl moiety fits inside a positively charged cavity of the V domain. Peptide backbone atoms make specific contacts with the V domain. The geometry of the bound CEL peptide is compatible with many CML (CEL)-modified sites found in plasma proteins. The structure explains how such patterned ligands as CML (CEL)-proteins bind to RAGE and contribute to RAGE signaling.

BACKGROUND

Diabetes and periodontitis are complex chronic diseases with an established bidirectional relationship. There is long-established evidence that hyperglycaemia in diabetes is associated with adverse periodontal outcomes. However, given the ubiquity of periodontal diseases and the emerging global diabetes epidemic, the complications of which contribute to significant morbidity and premature mortality, it is timely to review the role of periodontitis in diabetes.

OBJECTIVE

To report the epidemiological evidence from cross-sectional, prospective and intervention studies for the impact of periodontal disease on diabetes incidence, control and complications and to identify potential underpinning mechanisms.

BACKGROUND

Over the last 20 years, consistent and robust evidence has emerged that severe periodontitis adversely affects glycaemic control in diabetes and glycaemia in non-diabetes subjects. In diabetes patients, there is a direct and dose-dependent relationship between periodontitis severity and diabetes complications. Emerging evidence supports an increased risk for diabetes onset in patients with severe periodontitis. Biological mechanisms: Type 2 diabetes is preceded by systemic inflammation, leading to reduced pancreatic b-cell function, apoptosis and insulin resistance.Increasing evidence supports elevated systemic inflammation (acute-phase and oxidative stress biomarkers) resulting from the entry of periodontal organisms and their virulence factors into the circulation, providing biological plausibility for the effects of periodontitis on diabetes. AGE (Advanced Glycation Endproducts)-RAGE (Receptor for AGEs) interactions and oxidative-stress-mediated pathways provide plausible mechanistic links in the diabetes to periodontitis direction.

METHODS

Randomized controlled trials (RCTs) consistently demonstrate that mechanical periodontal therapy associates with approximately a 0.4% reduction in HbA1C at 3 months, a clinical impact equivalent to adding a second drug to a pharmacological regime for diabetes. RCTs are needed with larger numbers of subjects and longer term follow-up, and if results are substantiated, adjunctive periodontal therapies subsequently need to be evaluated. There is no current evidence to support adjunctive use of antimicrobials for periodontal management of diabetes patients.

CONCLUSIONS

Given the current evidence, it is timely to provide guidelines for periodontal care in diabetes patients for medical and dental professionals and recommendations for patients/the public.

Caspase-11, a cytosolic endotoxin (lipopolysaccharide: LPS) receptor, mediates pyroptosis, a lytic form of cell death. Caspase-11-dependent pyroptosis mediates lethality in endotoxemia, but it is unclear how LPS is delivered into the cytosol for the activation of caspase-11. Here we discovered that hepatocyte-released high mobility group box 1 (HMGB1) was required for caspase-11-dependent pyroptosis and lethality in endotoxemia and bacterial sepsis. Mechanistically, hepatocyte-released HMGB1 bound LPS and targeted its internalization into the lysosomes of macrophages and endothelial cells via the receptor for advanced glycation end-products (RAGE). Subsequently, HMGB1 permeabilized the phospholipid bilayer in the acidic environment of lysosomes. This resulted in LPS leakage into the cytosol and caspase-11 activation. Depletion of hepatocyte HMGB1, inhibition of hepatocyte HMGB1 release, neutralizing extracellular HMGB1, or RAGE deficiency prevented caspase-11-dependent pyroptosis and death in endotoxemia and bacterial sepsis. These findings indicate that HMGB1 interacts with LPS to mediate caspase-11-dependent pyroptosis in lethal sepsis.

Effects of APOE genotype on age-related slopes of cortical thinning was estimated by measuring the thickness of the cerebral cortex on a point-by-point basis across the cortical mantle in 96 healthy non-demented volunteers aged 48-75 years. Fifty nine were APOE epsilon 4- (no epsilon 4 allele) and 37 were epsilon 4+ (1 or 2 epsilon 4 alleles). The genotype groups had similar age, sex and IQ. Two T(1)-weighted MP-RAGE sequences were averaged for each participant to yield images with high signal-to-noise ratio, and quantified using semi-automated analysis tools. epsilon 4 carriers had thicker cortex than non-carriers in several frontal and temporal areas in both hemispheres, but showed a steeper age-related decline in adjacent areas. Upon comparison of the epsilon 4-specific age-related thinning with previously published patterns of thinning in normal aging and Alzheimer's disease (AD), we conclude that APOE epsilon 4 may function to accelerate thinning in areas found to decline in aging (medial prefrontal and pericentral cortex), but also to initiate thinning in areas associated with AD and amyloid-beta aggregation (occipitotemporal and basal temporal cortex).

In this study we show that embryonic neurite growth-promoting protein amphoterin binds to carboxylated N -glycans previously identified on mammalian endothelial cells. Since amphoterin is a ligand for the receptor for advanced glycation end products (RAGE), and the ligand-binding V-domain of the receptor contains two potential N -glycosylation sites, we hypothesized that N -glycans on RAGE may mediate its interactions with amphoterin. In support of this, anti-carboxylate antibody mAbGB3.1 immunoprecipitates bovine RAGE, and PNGase F treatment reduces its molecular mass by 4.5 kDa, suggesting that the native receptor is a glycoprotein. The binding potential of amphoterin to RAGE decreases significantly in presence of soluble carboxylated glycans or when the receptor is deglycosylated. Oligosaccharide analysis shows that RAGE contains complex type anionic N -glycans with non-sialic acid carboxylate groups, but not the HNK-1 (3-sulfoglucuronyl beta1-3 galactoside) epitope. Consistent with the functional localization of RAGE and amphoterin at the leading edges of developing neurons, mAbGB3.1 stains axons and growth cones of mouse embryonic cortical neurons, and inhibits neurite outgrowth on amphoterin matrix. The carboxylated glycans themselves promote neurite outgrowth in embryonic neurons and RAGE-transfected neuroblastoma cells. This outgrowth requires full-length, signalling-competent RAGE, as cells expressing cytoplasmic domain-deleted RAGE are unresponsive. These results indicate that carboxylated N -glycans on RAGE play an important functional role in amphoterin-RAGE-mediated signalling.

Advanced glycation end products (AGEs) and their receptors are strongly implicated in the development of diabetes complications. When stimulated by AGEs, the receptors for AGEs (RAGEs) induce inflammation and are thought to fuel disease progression. Soluble circulating RAGE (sRAGE) may counteract the detrimental effects of RAGE. We measured sRAGE in stored plasma from a random sample of 1,201 participants in the Atherosclerosis Risk in Communities (ARIC) Study who were aged 47-68 years, had normal kidney function, and had no history of cardiovascular disease. In cross-sectional analyses, black race, male sex, higher BMI, and higher C-reactive protein were independently associated with low sRAGE. The racial difference was striking, with blacks approximately three times more likely to have low sRAGE compared with whites even after adjustment. During ~18 years of follow-up, there were 192 incident coronary heart disease events, 53 ischemic strokes, 213 deaths, and 253 cases of diabetes (among the 1,057 persons without diabetes at baseline). In multivariable Cox models comparing risk in the first quartile with that in the fourth quartile of baseline sRAGE, low levels of sRAGE were significantly associated with risk of diabetes (hazard ratio 1.64 [95% CI 1.10-2.44]), coronary heart disease (1.82 [1.17-2.84]), and mortality (1.72 [1.11-2.64]) but not ischemic stroke (0.78 [0.34-1.79]). In conclusion, we found that low levels of sRAGE were a marker of future chronic disease risk and mortality in the community and may represent an inflammatory state. Racial differences in sRAGE deserve further examination.

The receptor for advanced glycation end products (RAGE) is a pattern recognition receptor (PRR) that interacts with diverse endogenous ligands. Ligation of RAGE triggers a series of cellular signaling events, including the activation of transcription factor NF-kappaB, leading to the production of pro-inflammatory cytokines, and causing inflammation. While acute inflammation serves to resolve pathogen infection and stresses, which promote tissue repair, persistent inflammation results in maladaptive tissue remodeling and damage. RAGE signaling has been implicated in multiple detrimental human illnesses including diabetes, atherosclerosis, arthritis, and Alzheimer's disease. In addition, prolonged inflammation often serves as the precursor for arterial remodeling that underlies the exponential increase of age-associated arterial diseases. Despite the significant progress and exciting discoveries in RAGE research, little is known on the biochemistry of RAGE and the signaling mechanism of RAGE remains poorly defined. The biological impact of RAGE signaling in clinical situations and aging-associated diseases also remains to be fully realized. This review attempts to provide a comprehensive summary on both recent findings and missing pieces of the RAGE puzzle.

The clinical relevance of the receptor for advanced glycation end products (RAGE) and amphoterin expression was examined in 119 non-diabetic patients with colorectal carcinoma. Expression of RAGE was examined by immunohistochemistry and that of amphoterin by in situ hybridization. RAGE expression was observed in 55% (64/119) of the cases. RAGE positivity in Dukes' B, C and D cases was 19, 81 and 100%, respectively (p<0.0001). Amphoterin was expressed in most cases regardless of tumor stage. Survival analysis of Dukes' B and C cases showed a significantly poorer prognosis for patients with co-expression of RAGE and amphoterin than for patients without co-expression. The results suggest that co-expression of RAGE and amphoterin is closely associated with invasion and metastasis of colorectal cancer.

Microvascular accumulation and neuronal overproduction of amyloid-beta peptide (Abeta) are pathologic features of Alzheimer's disease (AD). In this study, we examined the receptor for advanced glycation endproducts (RAGE), a multi-ligand receptor found in both neurons and cerebral microvascular endothelia that binds Abeta. RAGE expression was assessed in aged controls (n = 6), patients with early AD-like pathology (n = 6), and severe, Braak V-VI AD (n = 6). Human hippocampi were stained with a specific polyclonal antibody directed against RAGE (Research Diagnostics, Flanders, NJ). Immunoreactivity was localized in both neurons and cerebral endothelial cells. Quantitative image-analyses were performed on grayscale images to assess the total surface area of endothelial RAGE immunoreaction product in cross sections of cerebral microvessels (5-20 microm). Confocal images were acquired for confirmation of RAGE immunoreactivity in both microvessels and neurons by coupling RAGE with CD-31 and neurofilament, respectively. A significant increase in endothelial RAGE immunoreactivity was found in severe Braak V-VI AD patients when compared to aged controls (p < 0.001), and when compared to patients with early AD pathology (p = 0.0125). In addition, a significant increase in endothelial RAGE immunoreactivity was witnessed when comparing aged controls having no reported AD pathology with patients having early AD-like pathology (p = 0.038). Our data suggest that microvascular RAGE levels increase in conjunction with the onset of AD, and continue to increase linearly as a function of AD pathologic severity (p < 0.0001).

Interactions between advanced glycation end products (AGEs) and the receptor for AGE (RAGE) are implicated in the vascular complications in diabetes. We have identified eight novel polymorphisms, of which the -1420 (GGT)n, -1393 G/T, -1390 G/T, and -1202 G/A were in the overlapping PBX2 3' untranslated region (UTR), and the -429 T/C (66.5% TT, 33.5% TC/CC), -407 to -345 deletion (99% I, 1% I/D, 0% D), -374 T/A (66.4% TT, 33.6% TA/AA), and +20 T/A were in the RAGE promoter. To evaluate the effects on transcriptional activity, we measured chloramphenicol acetyl transferase (CAT) reporter gene expression, driven by variants of the -738 to +49 RAGE gene fragment containing the four polymorphisms identified close to the transcriptional start site. The -429 C, -374 A, and 63-bp deletion alleles resulted in a mean increase of CAT expression of twofold (P < 0.0001), threefold (P < 0.001), and fourfold (P < 0.05), respectively, with the -374 T and A alleles yielding highly differential binding of nuclear protein extract from both monocyte- and hepatocyte-derived cell lines. The prevalence of the functional polymorphisms were investigated in subjects with type 2 diabetes (106 with and 109 without retinopathy), with the -429 C allele showing an increase in the retinopathy group (P < 0.05). These data suggest that the polymorphisms involved in differences in RAGE gene regulation may influence the pathogenesis of diabetic vascular complications.

OBJECTIVE

Dysregulation of inflammatory adipokines by the adipose tissue plays an important role in obesity-associated insulin resistance. Pathways leading to this dysregulation remain largely unknown. We hypothesized that the receptor for advanced glycation end products (RAGE) and the ligand N(ε)-(carboxymethyl)lysine (CML) are increased in adipose tissue and, moreover, that activation of the CML-RAGE axis plays an important role in obesity-associated inflammation and insulin resistance.

RESULTS

In this study, we observed a strong CML accumulation and increased expression of RAGE in adipose tissue in obesity. We confirmed in cultured human preadipocytes that adipogenesis is associated with increased levels of CML and RAGE. Moreover, CML induced a dysregulation of inflammatory adipokines in adipocytes via a RAGE-dependent pathway. To test the role of RAGE in obesity-associated inflammation further, we constructed an obese mouse model that is deficient for RAGE (ie, RAGE(-/-)/Leptr(Db-/-) mice). RAGE(-/-)/Leptr(Db-/-) mice displayed an improved inflammatory profile and glucose homeostasis when compared with RAGE(+/+)/Leptr(Db-/-) mice. In addition, CML was trapped in adipose tissue in RAGE(+/+)/Leptr(Db-/-) mice but not in RAGE(-/-)/Leptr(Db-/-). RAGE-mediated trapping in adipose tissue provides a mechanism underlying CML accumulation in adipose tissue and explaining decreased CML plasma levels in obese subjects. Decreased CML plasma levels in obese individuals were strongly associated with insulin resistance.

CONCLUSIONS

RAGE-mediated CML accumulation in adipose tissue and the activation of the CML-RAGE axis are important mechanisms involved in the dysregulation of adipokines in obesity, thereby contributing to the development of obesity-associated insulin resistance.

S100A12 is a member of the S100 family of EF-hand calcium-binding proteins. Human S100A12 is predominantly expressed and secreted by neutrophil granulocytes and, therefore, has been assigned to the S100 protein subfamily of calgranulins or myeloid-related proteins. Intracellular S100A12 exists as an anti-parallel homodimer and upon calcium-dependent activation interacts with target proteins to regulate cellular functions. Extracellular S100A12 exists majorily as homodimer and hexamer, respectively, and shows cytokine-like characteristics. It is part of the innate immune response and linked to certain autoimmune reactions. Human S100A12 is markedly overexpressed in inflammatory compartments, and elevated serum levels of S100A12 are found in patients suffering from various inflammatory, neurodegenerative, metabolic, and neoplastic disorders. In this regard, interaction of calcium-activated S100A12 with the multiligand receptor for advanced glycation endproducts (RAGE) and its soluble form (sRAGE) plays a central pathogenetic role. Recent clinical evidence suggests a high potential of S100A12 as a sensitive and specific diagnostic marker of localized inflammatory processes.

BACKGROUND

Despite recent modifications, the clinical definition of the acute respiratory distress syndrome (ARDS) remains non-specific, leading to under-diagnosis and under-treatment. This study was designed to test the hypothesis that a biomarker panel would be useful for biologic confirmation of the clinical diagnosis of ARDS in patients at risk of developing ARDS due to severe sepsis.

METHODS

This was a retrospective case control study of 100 patients with severe sepsis and no evidence of ARDS compared to 100 patients with severe sepsis and evidence of ARDS on at least two of their first four ICU days. A panel that included 11 biomarkers of inflammation, fibroblast activation, proteolytic injury, endothelial injury, and lung epithelial injury was measured in plasma from the morning of ICU day two. A backward elimination model building strategy on 1,000 bootstrapped data was used to select the best performing biomarkers for further consideration in a logistic regression model for diagnosis of ARDS.

RESULTS

Using the five best-performing biomarkers (surfactant protein-D (SP-D), receptor for advanced glycation end-products (RAGE), interleukin-8 (IL-8), club cell secretory protein (CC-16), and interleukin-6 (IL-6)) the area under the receiver operator characteristic curve (AUC) was 0.75 (95% CI: 0.7 to 0.84) for the diagnosis of ARDS. The AUC improved to 0.82 (95% CI: 0.77 to 0.90) for diagnosis of severe ARDS, defined as ARDS present on all four of the first four ICU days.

CONCLUSIONS

Abnormal levels of five plasma biomarkers including three biomarkers generated by lung epithelium (SP-D, RAGE, CC-16) provided excellent discrimination for diagnosis of ARDS in patients with severe sepsis. Altered levels of plasma biomarkers may be useful biologic confirmation of the diagnosis of ARDS in patients with sepsis, and also potentially for selecting patients for clinical trials that are designed to reduce lung epithelial injury.