Toll-like receptor 4 (TLR4) activation in neuron and astrocytes by High Mobility Group Box 1 (HMGB1) protein is a key mechanism of seizure generation. HMGB1 also activates the Receptor for Advanced Glycation Endproducts (RAGE), but it was unknown whether RAGE activation contributes to seizures or to HMGB1 proictogenic effects. We found that acute EEG seizures induced by 7ng intrahippocampal kainic acid (KA) were significantly reduced in Rage-/- mice relative to wild type (Wt) mice. The proictogenic effect of HMGB1 was decreased in Rage-/- mice, but less so, than in Tlr4-/- mice. In a mouse mesial temporal lobe epilepsy (mTLE) model, status epilepticus induced by 200ng intrahippocampal KA and the onset of the spontaneous epileptic activity were similar in Rage-/-, Tlr4-/- and Wt mice. However, the number of hippocampal paroxysmal episodes and their duration were both decreased in epileptic Rage-/- and Tlr4-/- mice vs Wt mice. All strains of epileptic mice displayed similar cognitive deficits in the novel object recognition test vs the corresponding control mice. CA1 neuronal cell loss was increased in epileptic Rage-/- vs epileptic Wt mice, while granule cell dispersion and doublecortin (DCX)-positive neurons were similarly affected. Notably, DCX neurons were preserved in epileptic Tlr4-/- mice. We did not find compensatory changes in HMGB1-related inflammatory signaling nor in glutamate receptor subunits in Rage-/- and Tlr4-/- naïve mice, except for ~20% NR2B subunit reduction in Rage-/- mice. RAGE was induced in neurons, astrocytes and microvessels in human and experimental mTLE hippocampi. We conclude that RAGE contributes to hyperexcitability underlying acute and chronic seizures, as well as to the proictogenic effects of HMGB1. RAGE and TLR4 play different roles in the neuropathologic sequelae developing after status epilepticus. These findings reveal new molecular mechanisms underlying seizures, cell loss and neurogenesis which involve inflammatory pathways upregulated in human epilepsy.

BACKGROUND

Adaptation to hypoxia and consequent pro-inflammatory gene expression of prostate and breast carcinomas have been implicated in the progression toward cancer malignant phenotype. Only partial data are available for the human tumor glioblastoma multiforme (GBM). The aim of our study was to analyze the hypoxic and pro-inflammatory microenvironment in GBMs and to demonstrate that in a stem/progenitor cell line derived from human glioblastoma (GBM-SCs), hypoxia activates a coordinated inflammatory response, evidencing an invasive and migratory phenotype.

METHODS

From each of 10 human solid glioblastomas, clinically and histopathologically characterized, we obtained three surgical samples taken from the center and the periphery of the tumor, and from adjacent host normal tissue. Molecular and morphological analyses were carried out using quantitative real-time PCR and western blot (WB). GBM stem and differentiated cells were incubated under hypoxic conditions and analyzed for pro-inflammatory gene expression and for invasive/migratory behavior.

RESULTS

A panel of selected representative pro-inflammatory genes (RAGE and P2X7R, COX2, NOS2 and, PTX3) were analyzed, comparing tumor, peritumor and host normal tissues. Tumors containing leukocyte infiltrates (as assessed using CD45 immunohistochemistry) were excluded. Selected genes were overexpressed in the central regions of the tumors (i.e. in the more hypoxic areas), less expressed in peripheral regions, and poorly expressed or absent in adjacent normal host tissues. Western blot analysis confirmed that the corresponding pro-inflammatory proteins were also differently expressed. Hypoxic stem cell lines showed a clear time-dependent activation of the entire panel of pro-inflammatory genes as compared to differentiated tumor cells. Biological assays showed that invasive and migratory behavior was strengthened by hypoxia only in GBM stem cells.

CONCLUSIONS

In human solid glioblastoma we have observed a coordinated overexpression of a panel of pro-inflammatory genes as compared to host normal tissue. We have also evidenced a similar pattern of overexpressed genes in GBM-SCs after hypoxic treatment, showing also a gain of invasive and migratory function that was lost when these stem cells differentiated. We suggest that, as has been previously described for prostatic and mammary carcinoma, in human glioblastoma acquisition of a proinflammatory phenotype may be relevant for malignant progression.

The pattern-recognition receptor (PRR) family includes Toll-like receptors (TLRs), nucleotide-binding oligomerization domain (NOD) -like receptors (NLRs), RIG-I-like receptors (RLRs), C-type lectin receptors (CLRs) and the receptor for advanced glycation end products (RAGE). They recognize various microbial signatures or host-derived danger signals and trigger an immune response. Eosinophils are multifunctional leucocytes involved in the pathogenesis of several inflammatory processes, including parasitic helminth infection, allergic diseases, tissue injury and tumour immunity. Human eosinophils express several PRRs, including TLR1-5, TLR7, TLR9, NOD1, NOD2, Dectin-1 and RAGE. Receptor stimulation induces survival, oxidative burst, activation of the adhesion system and release of cytokines (interleukin-1β, interleukin-6, tumour necrosis factor-α and granulocyte-macrophage colony-stimulating factor), chemokines (interleukin-8 and growth-related oncogene-α) and cytotoxic granule proteins (eosinophil cationic protein, eosinophil-derived neurotoxin, eosinophil peroxidase and major basic protein). It is also evident that eosinophils play an immunomodulatory role by interacting with surrounding cells. The presence of a broad range of PRRs in eosinophils indicates that they are not only involved in defence against parasitic helminths, but also against bacteria, viruses and fungi. From a clinical perspective, eosinophilic PRRs seem to be involved in both allergic and malignant diseases by causing exacerbations and affecting tumour growth, respectively.

Diabetic vascular complication is a leading cause of acquired blindness, end-stage renal failure, a variety of neuropathies and accelerated atherosclerosis, which could account for disabilities and high mortality rates in patients with diabetes. Chronic hyperglycemia is essentially involved in the development and progression of diabetic micro- and macroangiopathy. Among various metabolic derangements implicated in the pathogenesis of diabetic vascular complication, advanced glycation end product (AGE) hypothesis is most compatible with the theory of 'hyperglycemic memory'. In this review, we discuss the molecular mechanisms of diabetic vascular complication, specially focusing on AGEs and their receptor (RAGE) system. Several types of AGE inhibitors and their therapeutic implications in this devastating disorder are also discussed here.

Advanced glycation end products (AGEs) accumulate with age and at an accelerated rate in diabetes. AGEs bind cell-surface receptors including the receptor for advanced glycation end products (RAGE). The dependence of RAGE binding on specific biochemical characteristics of AGEs is currently unknown. Using standardized procedures and a variety of AGE measures, the present study aimed to characterize the AGEs that bind to RAGE and their formation kinetics in vitro. To produce AGEs with varying RAGE binding affinity, bovine serum albumin (BSA) AGEs were prepared with 0.5M glucose, fructose, or ribose at times of incubation from 0 to 12 weeks or for up to 3 days with glycolaldehyde or glyoxylic acid. The AGE-BSAs were characterized for RAGE binding affinity, fluorescence, absorbance, carbonyl content, reactive free amine content, molecular weight, pentosidine content, and N-epsilon-carboxymethyl lysine content. Ribose-AGEs bound RAGE with high affinity within 1 week of incubation in contrast to glucose- and fructose-AGE, which required 12 and 6 weeks, respectively, to generate equivalent RAGE ligands (IC50=0.66, 0.93, and 1.7 microM, respectively). Over time, all of the measured AGE characteristics increased. However, only free amine content robustly correlated with RAGE binding affinity. In addition, detailed protocols for the generation of AGEs that reproducibly bind RAGE with high affinity were developed, which will allow for further study of the RAGE-AGE interaction.

OBJECTIVE

Peripheral neuropathy associated with type 2 diabetes (DPN) is not widely modeled. We describe unique features of DPN in type 2 diabetic Zucker diabetic fatty (ZDF) rats.

METHODS

We evaluated the structural, electrophysiological, behavioral, and molecular features of DPN in ZDF rats and littermates over 4 months of hyperglycemia. The status of insulin signaling transduction molecules that might be interrupted in type 2 diabetes and selected survival-, stress-, and pain-related molecules was emphasized in dorsal root ganglia (DRG) sensory neurons.

RESULTS

ZDF rats developed slowing of motor sciatic-tibial and sensory sciatic digital conduction velocity and selective mechanical allodynia with preserved thermal algesia. Diabetic sural axons, preserved in number, developed atrophy, but there was loss of large-calibre dermal and small-calibre epidermal axons. In diabetic rats, insulin signal transduction pathways in lumbar DRGs were preserved or had trends toward upregulation: mRNA levels of insulin receptor beta-subunit (IRbeta), insulin receptor substrate (IRS)-1, and IRS-2. The numbers of neurons expressing IRbeta protein were also preserved. There were trends toward early rises of mRNA levels of heat shock protein 27 (HSP27), the alpha2delta1 calcium channel subunit, and phosphatidylinositol 3-kinase in diabetes. Others were unchanged, including nuclear factor-kappaB (NF-kappaB; p50/p105) and receptor for advanced glycosylation endproducts (RAGE) as was the proportion of neurons expressing HSP27, NF-kappaB, and RAGE protein.

CONCLUSIONS

ZDF type 2 diabetic rats develop a distal degenerative sensory neuropathy accompanied by a selective long-term pain syndrome. Neuronal insulin signal transduction molecules are preserved.

HMGB1 (high mobility group box 1 protein) is a nuclear protein that can also act as an extracellular trigger of inflammation, proliferation and migration, mainly through RAGE (the receptor for advanced glycation end products); HMGB1-RAGE interactions have been found to be important in a number of cancers. We investigated whether HMGB1 is an autocrine factor in human glioma cells. Western blots showed HMGB1 and RAGE expression in human malignant glioma cell lines. HMGB1 induced a dose-dependent increase in cell proliferation, which was found to be RAGE-mediated and involved the MAPK/ERK pathway. Moreover, in a wounding model, it induced a significant increase in cell migration, and RAGE-dependent activation of Rac1 was crucial in giving the tumour cells a motile phenotype. The fact that blocking DNA replication with anti-mitotic agents did not reduce the distance migrated suggests the independence of the proliferative and migratory effects. We also found that glioma cells contain HMGB1 predominantly in the nucleus, and cannot secrete it constitutively or upon stimulation; however, necrotic glioma cells can release HMGB1 after it has translocated from the nucleus to cytosol. These findings provide the first evidence supporting the existence of HMGB1/RAGE signalling pathways in human glioblastoma cells, and suggest that HMGB1 may play an important role in the relationship between necrosis and malignancy in glioma tumours by acting as an autocrine factor that is capable of promoting the growth and migration of tumour cells.

BACKGROUND

Human S100A12 is a member of the S100 family of EF-hand calcium-modulated proteins that are associated with many diseases including cancer, chronic inflammation and neurological disorders. S100A12 is an important factor in host/parasite defenses and in the inflammatory response. Like several other S100 proteins, it binds zinc and copper in addition to calcium. Mechanisms of zinc regulation have been proposed for a number of S100 proteins e.g. S100B, S100A2, S100A7, S100A8/9. The interaction of S100 proteins with their targets is strongly dependent on cellular microenvironment.

RESULTS

The aim of the study was to explore the factors that influence S100A12 oligomerization and target interaction. A comprehensive series of biochemical and biophysical experiments indicated that changes in the concentration of calcium and zinc led to changes in the oligomeric state of S100A12. Surface plasmon resonance confirmed that the presence of both calcium and zinc is essential for the interaction of S100A12 with one of its extracellular targets, RAGE--the Receptor for Advanced Glycation End products. By using a single-molecule approach we have shown that the presence of zinc in tissue culture medium favors both the oligomerization of exogenous S100A12 protein and its interaction with targets on the cell surface.

CONCLUSIONS

We have shown that oligomerization and target recognition by S100A12 is regulated by both zinc and calcium. Our present work highlighted the potential role of calcium-binding S100 proteins in zinc metabolism and, in particular, the role of S100A12 in the cross talk between zinc and calcium in cell signaling.

Cognitive decline in chronic diabetic patients is a less investigated topic. Diabetes and obesity are among the modifiable risk factors for Alzheimer's disease (AD), the most common form of dementia. Studies have identified several overlapping neurodegenerative mechanisms, including oxidative stress, mitochondrial dysfunction, and inflammation that are observed in these disorders. Advanced glycation end products generated by chronic hyperglycemia and their receptor RAGE provide critical links between diabetes and AD. Peripheral inflammation observed in obesity leads to insulin resistance and type 2 diabetes. Although the brain is an immune-privileged organ, cross-talks between peripheral and central inflammation have been reported. Damage to the blood brain barrier (BBB) as seen with aging can lead to infiltration of immune cells into the brain, leading to the exacerbation of central inflammation. Neuroinflammation, which has emerged as an important cause of cognitive dysfunction, could provide a central mechanism for aging-associated ailments. To further add to these injuries, adult neurogenesis that provides neuronal plasticity is also impaired in the diabetic brain. This review discusses these molecular mechanisms that link obesity, diabetes and AD. This article is part of a Special Issue entitled: Oxidative Stress and Mitochondrial Quality in Diabetes/Obesity and Critical Illness Spectrum of Diseases - edited by P. Hemachandra Reddy.

Interactions between danger-associated molecular patterns (DAMP) and pathogen-associated molecular patterns (PAMP) and pattern recognition receptors such as Toll-like receptors (TLRs) are critical for the regulation of the inflammatory process via activation of nuclear factor-κB (NF-κB) and cytokine secretion. In this report, we investigated the capacity of lipopolysaccharide (LPS) -free S100A9 (DAMP) protein to activate human and mouse cells compared with lipoprotein-free LPS (PAMP). First, we showed that LPS and S100A9 were able to increase NF-κB activity followed by increased cytokine and nitric oxide (NO) secretion both in human THP-1 cells and in mouse bone marrow-derived dendritic cells. Surprisingly, although S100A9 triggered a weaker cytokine response than LPS, we found that S100A9 more potently induced IκBα degradation and hence NF-κB activation. Both the S100A9-induced response and the LPS-induced response were completely absent in TLR4 knockout mice, whereas it was only slightly affected in RAGE knockout mice. Also, we showed that LPS and S100A9 NF-κB induction were strongly reduced in the presence of specific inhibitors of TLR-signalling. Chloroquine reduced S100A9 but not LPS signalling, indicating that S100A9 may need to be internalized to be fully active as a TLR4 inducer. This was confirmed using A488-labelled S100A9 that was internalized in THP-1 cells, showing a raise in fluorescence after 30 min at 37°. Chloroquine treatment significantly reduced the fluorescence. In summary, our data indicate that both human and mouse S100A9 are TLR4 agonists. Importantly, S100A9 induced stronger NF-κB activation albeit weaker cytokine secretion than LPS, suggesting that S100A9 and LPS activated NF-κB in a qualitatively distinct manner.

OBJECTIVE

Women with polycystic ovary syndrome (PCOS) carry a number of cardiovascular risk factors and are considered to be at increased risk for atherosclerosis. Elevated concentrations of advanced glycation end-products (AGE), which exert their effects through interaction with specific receptors (RAGE), have been implicated in the cellular and tissue damage during atherosclerotic processes.

METHODS

We investigated serum AGE levels in 29 young women with PCOS as well as the expression of their receptor, RAGE, in circulating monocytes and compared them levels with 22 healthy control women.

RESULTS

Women with PCOS had higher levels of serum AGE proteins compared to healthy individuals (9.81 +/- 0.16 vs. 5.11 +/- 0.16, P < 0.0001), and increased RAGE expression was observed in monocytes of PCOS women compared to controls (30.91 +/- 10.11 vs. 7.97 +/- 2.61, P < 0.02). A positive correlation was observed between AGE proteins and testosterone (T) levels (r = 0.73, P < 0.0001). The correlation between AGE proteins and T levels remained high (partial correlation coefficient = 0.61, P = 0.0001) after controlling for body mass index (BMI), insulin levels and the area under the curve for glucose (AUCGLU) during an oral glucose tolerance test (OGTT). A positive correlation was also observed between AGE proteins and the free androgen index (FAI) (r = 0.58, P < 0.0001), waist-to-hip ratio (WHR) (r = 0.31, P < 0.02), insulin (r = 0.46, P < 0.001), homeostasis model assessment (HOMA) (r = 0.47, P < 0.0001), AUCGLU (r = 0.52, P < 0.002) and RAGE (r = 0.59, P < 0.01). A negative correlation was observed between AGE proteins and glucose/insulin ratio (GLU/INS) (r = -0.35, P < 0.01), and the quantitative insulin sensitivity check index (QUICKI) (r =-0.50, P < 0.01). In multiple regression analysis T was the only independent predictor of AGE levels (P < 0.0001, b = 0.044) between BMI, insulin, SHBG and AUCGLU (adjusted R2 = 0.59, F = 44.41, P < 0.0001).

CONCLUSIONS

These data clearly demonstrate, for the first time, that PCOS women without overt hyperglycaemia have increased AGE levels and elevated RAGE expression when compared with controls.

The calcium-binding protein S100A12 causes inflammation through interaction with the multiligand receptor for advanced glycation end products (RAGE). Blocking of S100A12 showed promising therapeutic effects in mice. We investigated 31 individuals with Kawasaki disease, and recorded an association between expression of S100A12 and activity of Kawasaki disease. Serum concentrations of S100A12 decreased quickly in 28 patients who responded to treatment with gammaglobulin (from 463 microg/L [SD 316] to 184 microg/L [147] within 24 h, p<0.0001). Since the interaction of S100A12 with multiligand receptors has a key role in inflammatory responses, this protein could serve as a novel target for future therapeutic interventions in inflammatory disorders.

OBJECTIVE

Receptor for advanced glycation end products (AGEs) (RAGE) plays a central role in the process of plaque rupture in diabetic patients. Recently, it has been reported that RAGE may be downregulated by improving glycemic control. In contrast, despite being well known that RAGE may be induced in human vessels in a glucose-independent fashion, also by myeloperoxidase (MPO)-dependent AGE generation, no data exist regarding the possibility of a pharmacological modulation of glucose-independent RAGE generation. Thus, the aim of this study was to characterize the effect of simvastatin on the expression of RAGE and RAGE-dependent plaque-destabilizing genes in human atherosclerotic plaques.

RESULTS

Seventy type 2 diabetic patients with asymptomatic carotid artery stenosis (>70%) were randomized to American Heart Association (AHA) step 1 diet plus simvastatin (40 mg/d) or AHA step 1 diet alone for 4 months before endarterectomy. Plaque expression of MPO, AGEs, RAGE, NF-kappaB, COX-2, mPGES-1, matrix metalloproteinase (MMP)-2 and MMP-9, lipid and oxidized LDL (oxLDL) content, procollagen 1, and interstitial collagen was analyzed by immunohistochemistry and Western blot; zymography was used to detect MMP activity. Plaques from the simvastatin group had less (P<0.0001) immunoreactivity for MPO, AGEs, RAGE, p65, COX-2, mPGES-1, MMP-2, and MMP-9, lipids and oxLDL; reduced (P<0.0001) gelatinolytic activity; increased (P<0.0001) procollagen 1 and collagen content; and fewer (P<0.0001) macrophages, T-lymphocytes, and HLA-DR+ cells. Of interest, RAGE inhibition by simvastatin, observed not only in plaque sections but also in plaque-derived macrophages, was reverted by addition of AGEs in vitro.

CONCLUSIONS

This study supports the hypothesis that simvastatin inhibits plaque RAGE expression by decreasing MPO-dependent AGE generation. This effect in turn might contribute to plaque stabilization by inhibiting the biosynthesis of PGE2-dependent MMPs, responsible for plaque rupture.

Advanced glycation end products (AGEs) are believed to play an important role in the development of diabetic complications. AGEs are increased in experimental diabetes and treatment with the inhibitor of advanced glycation end products, aminoguanidine, has been shown to attenuate the level of these products in tissues undergoing complications. Recently, an AGE-binding protein has been isolated from bovine lung endothelial cells and termed the receptor for advanced glycated end products (RAGE). The present study sought to determine the distribution of AGE and RAGE in tissues susceptible to the long-term complications of diabetes including the kidney, eye, nerve, arteries as well as in a tissue resistant to such complications, the lung. Using polyclonal antisera both AGE and RAGE were found to co-localize in the renal glomerulus. AGE staining was clearly increased with age and was further increased by diabetes. Aminoguanidine treatment reduced AGE accumulation in the kidney. Co-localisation of AGE and RAGE was demonstrated in the inner plexiform layer and the inner limiting membrane of the retina and in nerve bundles from mesenteric arteries. In the aorta, both AGE and RAGE were found in the intima, media and adventitia. Medial staining was increased in diabetes and was reduced by aminoguanidine treatment. A similar pattern was observed for RAGE in the aorta. In the lung, RAGE was found widely distributed throughout the lung whereas the distribution of AGE staining was more limited, primarily localising to macrophages. The co-localisation of AGEs and RAGE in sites of diabetic microvascular injury suggests that this ligand-receptor interaction may represent an important mechanism in the genesis of diabetic complications.

Sepsis still represents an important clinical and economic challenge for intensive care units. Severe complications like multi-organ failure with high mortality and the lack of specific diagnostic tools continue to hamper the development of improved therapies for sepsis. Fundamental questions regarding the cellular pathogenesis of experimental and clinical sepsis remain unresolved. According to experimental data, inhibiting macrophage migration inhibitory factor, high-mobility group box protein 1 (HMGB1), and complement factor C5a and inhibiting the TREM-1 (triggering receptor expressed on myeloid cells 1) signaling pathway and apoptosis represent promising new therapeutic options. In addition, we have demonstrated that blocking the signal transduction pathway of receptor of advanced glycation endproducts (RAGE), a new inflammation-perpetuating receptor and a member of the immunoglobulin superfamily, increases survival in experimental sepsis. The activation of RAGE by advanced glycation end-products, S100, and HMGB1 initiates nuclear factor kappa B and mitogen-activated protein kinase pathways. Importantly, the survival rate of RAGE knockout mice was more than fourfold that of wild-type mice in a septic shock model of cecal ligation and puncture (CLP). Additionally, the application of soluble RAGE, an extracellular decoy for RAGE ligands, improves survival in mice after CLP, suggesting that RAGE is a central player in perpetuating the innate immune response. Understanding the basic signal transduction events triggered by this multi-ligand receptor may offer new diagnostic and therapeutic options in patients with sepsis.

HMGB1 is a member of the high-mobility group protein superfamily that has been widely studied as nuclear proteins that bind DNA, stabilize nucleosomes and facilitate gene transcription. A series of recent discoveries revealed a cytokine activity of HMGB1, that when secreted into the extracellular milieu, mediates downstream inflammatory responses in endotoxemia, arthritis and sepsis. HMGB1 is properly defined as a cytokine because it stimulates proinflammatory responses in monocytes/macrophages, is produced during inflammatory responses in vivo in standardized models of systemic and local inflammation, mediates delayed endotoxin lethality, and is required for the full expression of inflammation in animal models of endotoxemia, sepsis and arthritis. HMGB1 is either actively secreted by monocytes/macrophages or passively released from necrotic cells from any tissue. These pathways are central for the biology of HMGB1 as a cytokine, since they provide key mechanisms that integrate the inflammatory response to infectious and non-infectious cell injuries. Receptor signal transduction of HMGB1 occurs in part through the receptor for advanced glycation end-products (RAGE) expressed on monocytes/macrophages, endothelial cells, neurons and smooth-muscle cells. HMGB1 is a late-acting cytokine, because it first appears in the extracellular milieu 8-12 h after the initial macrophage response to proinflammatory stimuli. Knowledge of the cytokine role of HMGB1 has implications for understanding downstream cytokine cascades, regulation of delayed innate immune responses and targeting treatment towards these processes. Effectiveness of delayed treatment with HMGB1 blockade up to 24 h after induction of experimental sepsis offers a unique window of opportunities to allow rescue from lethal sepsis.

Receptor for advanced glycation end-products (RAGE) is known to be involved in microvascular complications in diabetes. RAGE is also profoundly associated with macrovascular complications in diabetes through regulation of atherogenesis, angiogenic response, vascular injury, and inflammatory response. The potential significance of RAGE in the pathogenesis of cardiovascular disease appears not to be confined solely to nondiabetic rather than diabetic conditions. Numerous truncated forms of RAGE have recently been described, and the C-terminally truncated soluble form of RAGE has received much attention. Soluble RAGE consists of several forms, including endogenous secretory RAGE (esRAGE), which is a spliced variant of RAGE, and a shedded form derived from cell-surface RAGE. These heterogeneous forms of soluble RAGE, which carry all of the extracellular domains but are devoid of the transmembrane and intracytoplasmic domains, bind ligands including AGEs and can antagonize RAGE signaling in vitro and in vivo. ELISA systems have been developed to measure plasma esRAGE and total soluble RAGE, and the pathophysiological roles of soluble RAGE have begun to be unveiled clinically. In this review, we summarize recent findings regarding pathophysiological roles in cardiovascular disease of RAGE and soluble RAGE and discuss their potential usefulness as therapeutic targets and biomarkers for the disease.

OBJECTIVE

Receptor for advanced glycation end products (RAGE) is a cell surface molecule that binds a variety of ligands, including high mobility group box chromosomal protein 1 (HMGB-1), a potent proinflammatory cytokine. RAGE-ligand interaction leads to an inflammatory response. A truncated form of the receptor, soluble RAGE (sRAGE), has been suggested to function as a decoy abrogating cellular activation, but its endogenous activity is not fully understood. We undertook this study to assess the properties of sRAGE in vivo and in vitro and to analyze the role of sRAGE in HMGB-1-induced arthritis.

METHODS

Mice were injected intraarticularly with HMGB-1 and treated systemically with sRAGE prior to histologic joint evaluation. All animals were subjected to peritoneal lavage to assess the local effect of sRAGE treatment. For in vitro studies, mouse splenocytes were incubated with sRAGE followed by assessment of NF-kappaB activation and cytokine production. The chemotactic properties of sRAGE were investigated using in vitro migration assay.

RESULTS

Soluble RAGE was determined to have proinflammatory properties since it gave rise to production of interleukin-6, tumor necrosis factor alpha, and macrophage inflammatory protein 2. This effect was triggered by interaction with leukocyte beta2 integrin Mac-1 and was mediated via NF-kappaB. Systemic treatment with sRAGE significantly down-regulated HMGB-1-triggered arthritis, but the observed effect was due to a deviation of the inflammatory response from the joint to the peritoneal cavity rather than a genuine antiinflammatory effect. Apart from its proinflammatory properties, sRAGE was proven to act as a chemotactic stimulus for neutrophils.

CONCLUSIONS

We conclude that sRAGE interacts with Mac-1, thereby acting as an important proinflammatory and chemotactic molecule.

Activation of the receptor for advanced glycation endproducts (RAGE) by its multiple ligands can trigger diverse signaling pathways with injurious or pro-survival consequences. In this study, we show that Rage mRNA and protein levels were stimulated in the mouse brain after experimental stroke and systemic hypoxia. In both cases, RAGE expression was primarily associated with neurons. Activation of RAGE-dependent pathway(s) post-ischemia appears to have a neuroprotective role because mice genetically deficient for RAGE exhibited increased infarct size 24 h after injury. Up-regulation of RAGE expression was also observed in primary neurons subjected to hypoxia or oxygen-glucose deprivation, an in vitro model of ischemia. Treatment of neurons with low concentrations of S100B decreased neuronal death after oxygen-glucose deprivation, and this effect was abolished by a neutralizing antibody against RAGE. Conversely, high concentrations of exogenous S100B had a cytotoxic effect that seems to be RAGE-independent. As an important novel finding, we demonstrate that hypoxic stimulation of RAGE expression is mediated by the transcription factor hypoxia-inducible factor-1. This conclusion is supported by the finding that HIF-1alpha down-regulation by Cre-mediated excision drastically decreased RAGE induction by hypoxia or desferrioxamine. In addition, we showed that the mouse RAGE promoter region contains at least one functional HIF-1 binding site, located upstream of the proposed transcription start site. A luciferase reporter construct containing this RAGE promoter fragment was activated by hypoxia, and mutation at the potential HIF-1 binding site decreased hypoxia-dependent promoter activation. Specific binding of HIF-1 to this putative HRE in hypoxic cells was detected by chromatin immunoprecipitation assay.

OBJECTIVE

The high-mobility group box-1 protein (HMGB1) serves as the prototypic damage-associated molecular pattern molecule, interacting with a variety of defined pattern recognition receptors in the microenvironment of damaged or necrotic tissue. As regulatory T cells (T(reg)) play a crucial role in autoimmune diseases and tumor immune escape, the previously unexamined role of HMGB1 on the function of T(reg) is of great interest.

METHODS

Human CD4(+)CD25(+)CD127(-) T(reg) and CD4(+)CD25(-)CD127(+) conventional T cells (T(con)) were phenotypically analyzed for their constitutive as well as HMGB1-modulated expression of Toll-like receptors (TLR) and the receptor for advanced glycation end products (RAGE). Furthermore, the influence of recombinant and complexed HMGB1 from necrotic cell supernatant on the function of T(reg) and T(con) was investigated.

RESULTS

T(reg) express significantly higher levels of RAGE on the cell surface than T(con), while levels of TLR4 are similar. HMGB1 modulates T(reg) biology by inducing migration and prolonging survival. Furthermore, HMGB1 enhances IL-10 release and T(reg) suppressive capacity in a RAGE-dependent manner. In addition, HMGB1 directly suppresses IFNγ release of T(con) and inhibits their proliferation via TLR4.

CONCLUSIONS

HMGB1 directly enhances immune inhibitory functions of T(reg) via RAGE-mediated mechanisms and limits the number and activity of T(con). HMGB1 effects on T(reg) may alter immune reactivity in the setting of chronic inflammatory states such as cancer.

We reported that RAGE (receptor for advanced glycation end products), a multiligand receptor of the immunoglobulin superfamily expressed in myoblasts, when activated by its ligand amphoterin (HMGB1), stimulates rat L6 myoblast differentiation via a Cdc42-Rac-MKK6-p38 mitogen-activated protein kinase pathway, and that RAGE expression in skeletal muscle tissue is developmentally regulated. We show here that inhibition of RAGE function via overexpression of a signaling deficient RAGE mutant (RAGE delta cyto) results in increased myoblast proliferation, migration, and invasiveness, and decreased apoptosis and adhesiveness, whereas myoblasts overexpressing RAGE behave the opposite, compared with mock-transfected myoblasts. These effects are accompanied by a decreased induction of the proliferation inhibitor, p21(Waf1), and increased induction of cyclin D1 and extent of Rb, ERK1/2, and JNK phosphorylation in L6/RAGE delta cyto myoblasts, the opposite occurring in L6/RAGE myoblasts. Neutralization of culture medium amphoterin negates effects of RAGE activation, suggesting that amphoterin is the RAGE ligand involved in RAGE-dependent effects in myoblasts. Finally, mice injected with L6/RAGE delta cyto myoblasts develop tumors as opposed to mice injected with L6/RAGE or L6/mock myoblasts that do not. Thus, the amphoterin/RAGE pair stimulates myoblast differentiation by the combined effect of stimulation of differentiation and inhibition of proliferation, and deregulation of RAGE expression in myoblasts might contribute to their neoplastic transformation.

Chronic hyperglycemia promotes insulin resistance at least in part by increasing the formation of advanced glycation end products (AGEs). We have previously shown that in L6 myotubes human glycated albumin (HGA) induces insulin resistance by activating protein kinase Calpha (PKCalpha). Here we show that HGA-induced PKCalpha activation is mediated by Src. Coprecipitation experiments showed that Src interacts with both the receptor for AGE (RAGE) and PKCalpha in HGA-treated L6 cells. A direct interaction of PKCalpha with Src and insulin receptor substrate-1 (IRS-1) has also been detected. In addition, silencing of IRS-1 expression abolished HGA-induced RAGE-PKCalpha co-precipitation. AGEs were able to induce insulin resistance also in vivo, as insulin tolerance tests revealed a significant impairment of insulin sensitivity in C57/BL6 mice fed a high AGEs diet (HAD). In tibialis muscle of HAD-fed mice, insulin-induced glucose uptake and protein kinase B phosphorylation were reduced. This was paralleled by a 2.5-fold increase in PKCalpha activity. Similarly to in vitro observations, Src phosphorylation was increased in tibialis muscle of HAD-fed mice, and co-precipitation experiments showed that Src interacts with both RAGE and PKCalpha. These results indicate that AGEs impairment of insulin action in the muscle might be mediated by the formation of a multimolecular complex including RAGE/IRS-1/Src and PKCalpha.

OBJECTIVE

To examine safety, tolerability, and efficacy of PF-04494700, an inhibitor of the receptor for advanced glycation end products (RAGE), in mild to moderate Alzheimer disease (AD).

METHODS

Double-blind, placebo-controlled trial at 40 academic centers (United States). Subjects with AD and Mini-Mental State Examination score 14-26 were randomized to PF-04494700 60 mg/day × 6 days, then 20 mg daily (high dose); 15 mg/day × 6 days, then 5 mg daily (low dose); or placebo, for 18 months. Clinical and laboratory measures were used to evaluate safety and tolerability. The primary efficacy measure was the Alzheimer's Disease Assessment Scale-cognitive (ADAS-cog). Secondary measures assessed clinical stage, function, behavior, MRI, and CSF biomarkers.

RESULTS

A total of 399 subjects were randomized. In a prespecified interim analysis, when 50% of subjects had completed the 6-month visit, the high dose was associated with confusion, falls, and greater ADAS-cog decline and was discontinued. A second prespecified analysis compared low-dose and placebo groups for futility and safety approximately 12 months after all subjects were randomized. This analysis met criteria for futility, and treatment was discontinued. There were no safety concerns in the low-dose group. Analyses including post-futility data showed decreased decline on the ADAS-cog in the low-dose group at month 18. Other clinical and biomarker measures showed no differences between low-dose treatment and placebo.

CONCLUSIONS

PF-04494700 at 20 mg/d was associated with increased adverse events and cognitive decline. At 5 mg/d, PF-04494700 had a good safety profile. A potential benefit for this low dose on the ADAS-cog is not conclusive, because of high dropout and discontinuation rates subsequent to the interim analyses.

METHODS

This study provides Class I evidence that in patients with AD high-dose PF-04494700 increased cognitive decline at 6 months and Class IV evidence that low-dose PF-04494700 slowed cognitive decline at 18 months.

Advanced Glycation End products (AGEs) are the products of nonenzymatic glycation and oxidation of proteins and lipids. Formation of AGEs is increased in response to hyperglycaemia, reactive oxygen species and ageing. AGEs are proinflammatory and can modify the extracellular matrix. RAGE (Receptor for Advanced Glycation End Products) mediates some of the effects of AGEs.

METHODS

Formalin-fixed lung tissue from patients who had lobectomy for bronchial carcinoma was used to investigate the presence of AGEs and RAGE. Subjects were divided into those with COPD and controls. Immunostaining for AGEs and RAGE was performed and the intensity of staining measured.

RESULTS

Subjects with COPD and controls were similar in age and smoking history but FEV(1)% predicted was lower for COPD than controls. Intensity of staining for AGEs was greater in the airways (p = 0.025) and alveolar walls (p = 0.004) in COPD. Intensity of staining for RAGE was also significantly increased in alveolar walls (p = 0.03) but not the airways. FEV(1)% predicted was correlated with the intensity of staining for AGEs in the airways and alveoli.

CONCLUSIONS

The increased staining for both AGEs and RAGE in COPD lung raises the possibility that the RAGE-AGEs interaction may have a role in the pathogenesis of COPD.