Role of the receptor for advanced glycation endproducts (RAGE) in retinal vasodegenerative pathology during diabetes in mice.

AIMS/HYPOTHESIS: The receptor for AGEs (RAGE) is linked to proinflammatory pathology in a range of tissues. The objective of this study was to assess the potential modulatory role of RAGE in diabetic retinopathy. METHODS: Diabetes was induced in wild-type (WT) and Rage (-/-) mice (also known as Ager (-/-) mice) using streptozotocin while non-diabetic control mice received saline. For all groups, blood glucose, HbA1c and retinal levels of methylglyoxal (MG) were evaluated up to 24 weeks post diabetes induction. After mice were killed, retinal glia and microglial activation, vasopermeability, leucostasis and degenerative microvasculature changes were determined. RESULTS: Retinal expression of RAGE in WT diabetic mice was increased after 12 weeks (p < 0.01) but not after 24 weeks. Rage (-/-) mice showed comparable diabetes but accumulated less MG and this corresponded to enhanced activity of the MG-detoxifying enzyme glyoxalase I in their retina when compared with WT mice. Diabetic Rage (-/-) mice showed significantly less vasopermeability, leucostasis and microglial activation (p < 0.05-0.001). Rage (-/-) mice were also protected against diabetes-related retinal acellular capillary formation (p < 0.001) but not against pericyte loss. CONCLUSIONS/INTERPRETATION: Rage (-/-) in diabetic mice is protective against many retinopathic lesions, especially those related to innate immune responses. Inhibition of RAGE could be a therapeutic option to prevent diabetic retinopathy.

Methylglyoxal as a new biomarker in patients with septic shock: an observational clinical study.

INTRODUCTION: The role of reactive carbonyl species, such as methylglyoxal (MG), has been overlooked within the context of the sepsis syndrome. The aims of this study were to assess the impact of MG formation in different inflammatory settings and to evaluate its use for early diagnosis as well as prognosis of the sepsis syndrome. METHODS: In total, 120 patients in three groups were enrolled in this observational clinical pilot study. The three groups included patients with septic shock (n = 60), postoperative controls (n = 30), and healthy volunteers (n = 30). Plasma samples from patients with septic shock were collected at sepsis onset and after 24 hours and 4, 7, 14, and 28 days. Plasma samples from postoperative controls were collected prior to surgery, immediately following the end of the surgical procedure as well as 24 hours later and from healthy volunteers once. Plasma levels of MG were determined by high-performance liquid chromatography. Additionally, plasma levels of procalcitonin, C-reactive protein, soluble CD14 subtype, and interleukin-6 were determined. RESULTS: Patients with septic shock showed significantly higher plasma levels of MG at all measured times, compared with postoperative controls. MG was found to identify patients with septic shock more effectively-area under the curve (AUC): 0.993-than procalcitonin (AUC: 0.844), C-reactive protein (AUC: 0.791), soluble CD14 subtype (AUC: 0.832), and interleukin-6 (AUC: 0.898) as assessed by receiver operating characteristic (ROC) analysis. Moreover, plasma levels of MG in non-survivors were significantly higher than in survivors (sepsis onset: *P = 0.018 for 90-day survival; **P = 0.008 for 28-day survival). Plasma levels of MG proved to be an early predictor for survival in patients with septic shock (sepsis onset: ROC-AUC 0.710 for 28-day survival; ROC-AUC 0.686 for 90-day survival). CONCLUSIONS: MG was identified as a marker for monitoring the onset, development, and remission of sepsis and was found to be more useful than routine diagnostic markers. Further studies are required to determine the extent of MG modification in sepsis and whether targeting this pathway could be therapeutically beneficial to the patient. TRIAL REGISTRATION: German Clinical Trials Register DRKS00000505. Registered 8 November 2010.

RAGE regulates immune cell infiltration and angiogenesis in choroidal neovascularization.

PURPOSE: RAGE regulates pro-inflammatory responses in diverse cells and tissues. This study has investigated if RAGE plays a role in immune cell mobilization and choroidal neovascular pathology that is associated with the neovascular form of age-related macular degeneration (nvAMD). METHODS: RAGE null (RAGE-/-) mice and age-matched wild type (WT) control mice underwent laser photocoagulation to generate choroidal neovascularization (CNV) lesions which were then analyzed for morphology, S100B immunoreactivity and inflammatory cell infiltration. The chemotactic ability of bone marrow derived macrophages (BMDMs) towards S100B was investigated. RESULTS: RAGE expression was significantly increased in the retina during CNV of WT mice (p<0.001). RAGE-/- mice exhibited significantly reduced CNV lesion size when compared to WT controls (p<0.05). S100B mRNA was upregulated in the lasered WT retina but not RAGE-/- retina and S100B immunoreactivity was present within CNV lesions although levels were less when RAGE-/- mice were compared to WT controls. Activated microglia in lesions were considerably less abundant in RAGE-/- mice when compared to WT counterparts (p<0.001). A dose dependent chemotactic migration was observed in BMDMs from WT mice (p<0.05-0.01) but this was not apparent in cells isolated from RAGE-/- mice. CONCLUSIONS: RAGE-S100B interactions appear to play an important role in CNV lesion formation by regulating pro-inflammatory and angiogenic responses. This study highlights the role of RAGE in inflammation-mediated outer retinal pathology.

CD166/ALCAM mediates proinflammatory effects of S100B in delayed type hypersensitivity.

Promiscuity of pattern recognition receptors, such as receptor for advanced glycation end products (RAGE), allows for a complex regulatory network controlling inflammation. Scavenging of RAGE ligands by soluble RAGE treatment is effective in reducing delayed-type hypersensitivity (DTH), even in RAGE(-/-) mice by 50% (p < 0.001). This has led to the hypothesis that molecules scavenged by soluble RAGE bind to receptors other than RAGE. This study identifies CD166/ALCAM (ALCAM) as a close structural and functional homolog of RAGE, and it shows that binding of S100B to CD166/ALCAM induces dose- and time-dependent expression of members of the NF-κB family in wild type (WT) and RAGE(-/-) mouse endothelial cells. Blocking CD166/ALCAM expression using small interfering RNA completely inhibited S100B-induced NF-κB activation in RAGE(-/-), but not in WT cells. The in vivo significance of these observations was demonstrated by attenuation of DTH in WT and RAGE(-/-) animals pretreated with CD166/ALCAM small interfering RNA by 50% and 40%, respectively (p < 0.001). Experiments in ALCAM(-/-) animals displayed an only slight reduction of 16% in DTH, explained by compensatory reciprocal upregulation of RAGE in animals devoid of CD166/ALCAM, and vice versa. Consistently, ALCAM(-/-) mice, but not WT mice treated with RAGE small interfering RNA show a 35% reduction in DTH, and ALCAM(-/-) RAGE(-/-) double-knockout mice show a 27% reduction in DTH reaction. Thus, S100B is a proinflammatory cytokine bridging RAGE and CD166/ALCAM downstream effector mechanisms, both being compensatory upregulated after genetic deletion of its counterpart.

RAGE-mediated interstitial fibrosis in neonatal obstructive nephropathy is independent of NF-κB activation.

Urinary tract obstruction during nephron development causes tubular apoptosis, tubular atrophy, and interstitial fibrosis. Leukocyte recruitment is critical in the development of obstructive nephropathy leading to interstitial inflammation and renal fibrosis. RAGE, the receptor of advanced glycation end products, is implicated in chronic inflammation and has been recently identified as a novel receptor for the ß2-integrin Mac-1, cooperating with ICAM-1 and thereby directly mediating leukocyte recruitment in vivo. Here, we studied the role of RAGE and ICAM-1 in a model of unilateral ureteral obstruction in neonatal mice. Interestingly, the number of infiltrating leukocytes was independent of RAGE and ICAM-1 in the ureteral obstructed neonatal kidney. By contrast, galectin-3, a marker for profibrogenic M2 macrophages, was strongly reduced in ureteral obstructed RAGE and RAGE-Icam1 knockout mice. Snail expression and loss of E-cadherin but not NF-κB activation were attenuated in both knockout models. Epithelial cell cycle arrest at G2/M, which mediates kidney fibrosis, and transforming growth factor-ß expression were reduced in ureteral obstructed RAGE knockout mice. Thus, RAGE and ICAM-1 promote renal fibrosis in the developing kidney upon ureteral obstruction. Combined RAGE- and ICAM-1-blocking strategies may prove beneficial in neonatal obstructive nephropathy.

The receptor for advanced glycation end products is dispensable in a mouse model of oral and esophageal carcinogenesis.

Aberrant expression of the receptor for advanced glycation end products (RAGE) and its ligands, such as S100/Calgranulins, has been demonstrated in squamous cell carcinomas of the upper aerodigestive tract. However, the question whether RAGE signaling is causally linked with neoplastic transformation of keratinocytes in mucosal epithelia has not been addressed so far. We used the well-established mouse model of 4-nitroquinoline-1-oxide (4-NQO) induced tumorigenesis to investigate tumor development in control and RAGE-deficient (Rage(-/-)) animals. Although 4-NQO induced lesions of the tongue and the esophagus showed strong induction of the RAGE ligands S100a8 and S100a9, we did not observe any significant difference in tumor incidence or multiplicity between control and Rage(-/-) mice. Furthermore, detailed analysis of tumor sections by histological and immunohistochemical staining revealed no difference in either the size or histological architecture of dysplastic lesions, tumor cell proliferation, or the number of inflammatory immune cells in the tumor microenvironment. Finally, we detected induced transcript and protein levels of the Toll-like receptor 4 (Tlr4) in 4-NQO induced lesions, suggesting that signaling via the S100-Tlr4 axis may compensate for the lack of RAGE in early stages of tumor development. Our data demonstrate that RAGE is dispensable in the onset of genotoxic induced oral and esophageal squamous cell carcinoma and provide evidence for an alternative pathway of S100-Calgranulin signaling via Tlr4.

Serum soluble receptor for advanced glycation end products (sRAGE) is independently associated with cigarette smoking in non-diabetic healthy subjects.

This study was designed to explore the relationship between serum levels of soluble receptor for advanced glycation end products (sRAGE) and cigarette smoking in non-diabetic healthy subjects. A total of 98 non-diabetic, otherwise healthy male subjects were recruited. A fasting blood sample and medical history including detail history of cigarette smoking was collected. The serum sRAGE levels were found significantly higher (p=0.002) in cigarette smokers (1475±422 pg/ml, n=45) compared with non-smokers (1165±350 pg/ml, n=53). Moreover, among the cigarette smokers, serum sRAGE levels were found significantly correlated with number of cigarettes smoked per day (r=0.60, p<0.001). In bivariate analysis in the total population, sRAGE positively correlated with smoking habit (r=0.37, p=0.002) and negatively correlated with systolic (r=-0.32, p=0.01) and diastolic blood pressure (r=-0.36, p=0.003). However, in stepwise multivariate linear regression model, sRAGE showed a significant independent association with smoking habit (b=0.32, p=0.007, R2=0.23). In conclusion, this study for the first time shows a significant elevation of serum sRAGE in cigarette smokers compared with non-smokers, a strong correlation between sRAGE and number of cigarettes smoked per day and an independent association of sRAGE with smoking habit in non-diabetic healthy subjects.

Receptor for advanced glycation endproducts (RAGE) is a key regulator of oval cell activation and inflammation-associated liver carcinogenesis in mice.

UNLABELLED: The receptor for advanced glycation endproducts (RAGE) is a multiligand receptor and member of the immunoglobulin superfamily. RAGE is mainly involved in tissue damage and chronic inflammatory disorders, sustaining the inflammatory response upon engagement with damage-associated molecular pattern molecules (DAMPs) such as S100 proteins and high-mobility group box 1 (HMGB1). Enhanced expression of RAGE and its ligands has been demonstrated in distinct tumors and several studies support its crucial role in tumor progression and metastasis by still unknown mechanisms. Here we show that RAGE supports hepatocellular carcinoma (HCC) formation in the Mdr2(-/-) mouse model, a prototype model of inflammation-driven HCC formation, which mimics the human pathology. Mdr2(-/-) Rage(-/-) (dKO) mice developed smaller and fewer HCCs than Mdr2(-/-) mice. Interestingly, although in preneoplastic Mdr2(-/-) livers RAGE ablation did not affect the onset of inflammation, premalignant dKO livers showed reduced liver damage and fibrosis, in association with decreased oval cell activation. Oval cells expressed high RAGE levels and displayed reduced proliferation upon RAGE silencing. Moreover, stimulation of oval cells with HMGB1 promoted an ERK1/2-Cyclin D1-dependent oval cell proliferation in vitro. Finally, genetic and pharmacologic blockade of RAGE signaling impaired oval cell activation in an independent mouse model of oval cell activation, the choline deficient ethionine-supplemented dietary regime. CONCLUSION: Our data identified a novel function of RAGE in regulating oval cell activation and tumor development in inflammation-associated liver carcinogenesis.

Endothelial progenitor cells in regeneration after acute lung injury: do they play a role?

Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are common disorders in patients requiring critical care. The clinical management of these disorders is difficult and unrewarding, and thus they are among the most common causes of death in intensive care units. The activation and damage of pulmonary endothelium comprise the hallmark of ALI/ARDS. Therefore, the recruitment of circulating endothelial progenitor cells (EPCs) to these lesions may exert a beneficial effect on the clinical course of ALI/ARDS. Consequently, cell-based therapies using stem cells to regenerate lung tissue have emerged as potential novel treatment strategies. Although initial studies suggested implantations of exogenously administered bone marrow-derived progenitor cells into damaged vessel walls, recent evidence indicates that this is rather a rare occurrence with uncertain physiologic significance. In the past few years, different populations of progenitor cells were identified, with different functional capacities. This review (1) highlights the different populations of EPCs identified or administered in different models of ALI/ARDS, (2) reports on whether beneficial effects of EPCs could be demonstrated, and (3) puts the conflicting results of different studies into perspective.

Targeting activation of specific NF-κB subunits prevents stress-dependent atherothrombotic gene expression.

Psychosocial stress has been shown to be a contributing factor in the development of atherosclerosis. Although the underlying mechanisms have not been elucidated entirely, it has been shown previously that the transcription factor nuclear factor-κB (NF-κB) is an important component of stress-activated signaling pathway. In this study, we aimed to decipher the mechanisms of stress-induced NF-κB-mediated gene expression, using an in vitro and in vivo model of psychosocial stress. Induction of stress led to NF-κB-dependent expression of proinflammatory (tissue factor, intracellular adhesive molecule 1 [ICAM-1]) and protective genes (manganese superoxide dismutase [MnSOD]) via p50, p65 or cRel. Selective inhibition of the different subunits and the respective kinases showed that inhibition of cRel leads to the reduction of atherosclerotic lesions in apolipoprotein(-/-) (ApoE(-/-)) mice via suppression of proinflammatory gene expression. This observation may therefore provide a possible explanation for ineffectiveness of antioxidant therapies and suggests that selective targeting of cRel activation may provide a novel approach for the treatment of stress-related inflammatory vascular disease.

Cellular infiltrates and NFκB subunit c-Rel signaling in kidney allografts of patients with clinical operational tolerance.

BACKGROUND: Nuclear factor kappa B (NFκB) plays a potential role in tolerance by orchestrating onset and resolution of inflammation and regulatory T cell differentiation through subunit c-Rel. We characterized cellular infiltrates and expression of NFκB1, c-Rel and its upstream regulators phosphatidylinositol 3-kinase/RAC-alpha serine/threonine kinase, in allograft biopsies from patients with spontaneous clinical operational tolerance (COT). METHODS: Paraffin-fixed kidney allograft biopsies from 40 patients with COT (n=4), interstitial rejection (IR; n=12), borderline changes (BC; n=12), and long-term allograft function without rejection (NR; n=12) were used in the study. Cellular infiltrates and immunohistochemical expression of key proteins of the NFκB pathway were evaluated in the cortical tubulointerstitium and in cellular infiltrates using digital image analysis software. Results were given as mean±SEM. RESULTS: Biopsies from patients with COT exhibited a comparable amount of cellular infiltrate to IR, BC, and NR (COT, 191±81; IR, 291±62; BC, 178±45; and NR, 210±42 cells/mm) but a significantly higher proportion of forkhead box P3-positive cells (COT, 11%±1.7%; IR, 3.5%±0.70%; BC, 3.4%±0.57%; and NR, 3.7%±0.78% of infiltrating cells; P=0.02). c-Rel expression in cellular infiltrates was significantly elevated in IR, BC, and NR when analyzing the number of positive cells per mm (P=0.02) and positive cells per infiltrating cells (P=0.04). In contrast, tubular PI3K and c-Rel expression were significantly higher in IR and BC but not in NR compared with COT (P=0.03 and P=0.006, respectively). With RAC-alpha serine-threonine kinase, similar tendencies were observed (P=0.2). CONCLUSIONS: Allografts from COT patients show significant cellular infiltrates but a distinct expression of proteins involved in the NFκB pathway and a higher proportion of forkhead box P3-positive cells.

RAGE-dependent activation of gene expression of superoxide dismutase and vanins by AGE-rich extracts in mice cardiac tissue and murine cardiac fibroblasts.

Advanced glycation end products (AGEs) are stable compounds formed from initial Maillard reaction products. They are considered as markers for ageing and often associated with age-related, degenerative diseases. Bread crust represents an established model for nutritional compounds rich in AGEs and is able to induce antioxidative defense genes such as superoxide dismutases and vanins in cardiac cells. The aim of this study was to investigate to what extend the receptor for AGEs (RAGE) contributes to this response. Signal transduction in response to bread crust extract was analysed in cardiac fibroblasts derived from C57/B6-NCrl (RAGE +/+) and the corresponding RAGE-knock out C57/B6-NCrl mouse strain (RAGE -/-). Activation of superoxide dismutases in animals was then analysed upon bread crust feeding in these two mice strains. Cardiac fibroblasts from RAGE -/- mice did not express RAGE, but the expression of AGER-1 and AGER-3 was up-regulated, whereas the expression of SR-B1 was down-regulated. RAGE -/- cells were less sensitive to BCE in terms of MAP-kinase phosphorylation and NF-κB reporter gene activation. Bread crust extract induced mRNA levels of MnSOD and Vnn-1 were also reduced in RAGE -/- cells, whereas Vnn-3 mRNA accumulation seemed to be RAGE receptor independent. In bread crust feeding experiments, RAGE -/- mice did not exhibit an activation of MnSOD-mRNA and -protein accumulation as observed for the RAGE +/+ animals. In conclusion, RAGE was clearly a major factor for the induction of antioxidant defense signals derived from bread crust in cardiac fibroblast and mice. Nevertheless higher doses of bread crust extract could overcome the RAGE dependency in cell cultures, indicating that additional mechanisms are involved in BCE-mediated activation of SOD and vanin expression.

Methylglyoxal activates nociceptors through transient receptor potential channel A1 (TRPA1): a possible mechanism of metabolic neuropathies.

Neuropathic pain can develop as an agonizing sequela of diabetes mellitus and chronic uremia. A chemical link between both conditions of altered metabolism is the highly reactive compound methylglyoxal (MG), which accumulates in all cells, in particular neurons, and leaks into plasma as an index of the severity of the disorder. The electrophilic structure of this cytotoxic ketoaldehyde suggests TRPA1, a receptor channel deeply involved in inflammatory and neuropathic pain, as a molecular target. We demonstrate that extracellularly applied MG accesses specific intracellular binding sites of TRPA1, activating inward currents and calcium influx in transfected cells and sensory neurons, slowing conduction velocity in unmyelinated peripheral nerve fibers, and stimulating release of proinflammatory neuropeptides from and action potential firing in cutaneous nociceptors. Using a model peptide of the N terminus of human TRPA1, we demonstrate the formation of disulfide bonds based on MG-induced modification of cysteines as a novel mechanism. In conclusion, MG is proposed to be a candidate metabolite that causes neuropathic pain in metabolic disorders and thus is a promising target for medicinal chemistry.

Identical LDL-cholesterol lowering but non-identical effects on NF-κB activity: High dose simvastatin vs combination therapy with ezetimibe.

OBJECTIVE: Lowering LDL-cholesterol by statins has been proven to be associated with reduction of proinflammatory regulators e.g. activation of the transcription factor NF-κB. To our knowledge, anti-inflammatory potential of newer cholesterol lowering agents such as ezetimibe is less intensively studied. Therefore we analyzed the effects of equipotent LDL-lowering therapy with simvastatin alone compared to a combination with ezetimibe on NF-κB activation in peripheral blood mononuclear cells (PBMCs) of patients with type 2 diabetes. METHODS: Thirty-one patients with type 2 diabetes were included in a double-blind, randomized trial receiving either 80 mg simvastatin (sim80; n = 10) or a combination of 10 mg simvastatin and 10 mg ezetimibe (sim10eze10; n = 11) or placebo (n = 9) for eight weeks. NF-κB binding activity and inflammatory markers (IL-6, hsCRP) were analyzed at baseline and after eight weeks of treatment. NF-κB binding activity was analyzed by electrophoretic mobility shift assay. IL-6 and hsCRP were measured by ELISA. RESULTS: After eight weeks of treatment LDL-cholesterol was lowered to the same extent in both treatment groups (p = 0.40) but not in placebo. However, patients taking sim80 showed a significant reduction of mononuclear NF-κB binding activity compared to baseline (p = 0.009) while no effect was observed in the sim10eze10 group (p = 0.79). Similar differences in anti-inflammatory effects were also observed when analyzing hsCRP (sim80: p = 0.03; sim10eze10: p = 0.40) and IL-6 levels (sim80: p = 0.15; sim10eze10: p = 0.95). CONCLUSION: High dose simvastatin therapy reduces proinflammatory transcription factor NF-κB binding activity and hsCRP levels, while combination of low dose simvastatin with ezetimibe resulting in a similar LDL-reduction does not affect these inflammatory markers.

L-arginine and asymmetric dimethylarginine are early predictors for survival in septic patients with acute liver failure.

Dysfunctions of the L-arginine (L-arg)/nitric-oxide (NO) pathway are suspected to be important for the pathogenesis of multiple organ dysfunction syndrome (MODS) in septic shock. Therefore plasma concentrations of L-arg and asymmetric dimethylarginine (ADMA) were measured in 60 patients with septic shock, 30 surgical patients and 30 healthy volunteers using enzyme linked immunosorbent assay (ELISA) kits. Plasma samples from patients with septic shock were collected at sepsis onset, and 24 h, 4 d, 7 d, 14 d and 28 d later. Samples from surgical patients were collected prior to surgery, immediately after the end of the surgical procedure as well as 24 h later and from healthy volunteers once. In comparison to healthy volunteers and surgical patients, individuals with septic shock showed significantly increased levels of ADMA, as well as a decrease in the ratio of L-arg and ADMA at all timepoints. In septic patients with an acute liver failure (ALF), plasma levels of ADMA and L-arg were significantly increased in comparison to septic patients with an intact hepatic function. In summary it can be stated, that bioavailability of NO is reduced in septic shock. Moreover, measurements of ADMA and L-arg appear to be early predictors for survival in patients with sepsis-associated ALF.

S100A8/A9 aggravates post-ischemic heart failure through activation of RAGE-dependent NF-κB signaling.

The extracellular heterodimeric protein S100A8/A9 activates the innate immune system through activation of the receptor of advanced glycation end products (RAGE) and Toll-like receptors. As activation of RAGE has recently been associated with sustained myocardial inflammation and heart failure (HF) we studied the role of S100A8/A9 in the development of post-ischemic HF. Hypoxia led to sustained induction of S100A8/A9 accompanied by increased nuclear factor (NF-)κB binding activity and increased expression of pro-inflammatory cytokines in cardiac fibroblasts and macrophages. Knockdown of either S100A8/A9 or RAGE rescued the induction of pro-inflammatory cytokines and NF-κB activation after hypoxia. In a murine model of post-ischemic HF both cardiac RNA and protein levels of S100A8/A9 were elevated as soon as 30 min after hypoxia with sustained activation up to 28 days after ischemic injury. Treatment with recombinant S100A8/A9 resulted in reduced cardiac performance following ischemia/reperfusion. Chimera experiments after bone marrow transplantation demonstrated the importance of RAGE expression on immune cells for their recruitment to the injured myocardium aggravating post-ischemic heart failure. Signaling studies in isolated ventricles indicated that MAP kinases JNK, ERK1/2 as well as NF-κB mediate signals downstream of S100A8/A9-RAGE in post-ischemic heart failure. Interestingly, cardiac performance was not affected by administration of S100A8/A9 in RAGE(-/-)-mice, which demonstrated significantly improved cardiac recovery compared to WT-mice. Our study provides evidence that sustained activation of S100A8/A9 critically contributes to the development of post-ischemic HF driving the progressive course of HF through activation of RAGE.

Receptor for advanced glycation endproducts (RAGE) deficiency protects against MPTP toxicity.

Parkinson's disease (PD) is a common neurodegenerative disorder of unknown pathogenesis characterized by the loss of nigrostriatal dopaminergic neurons. Oxidative stress, microglial activation and inflammatory responses seem to contribute to the pathogenesis. The receptor for advanced glycation endproducts (RAGE) is a multiligand receptor of the immunoglobulin superfamily of cell surface molecules. The formation of advanced glycation end products (AGEs), the first ligand of RAGE identified, requires a complex series of reactions including nonenzymatic glycation and free radical reactions involving superoxide-radicals and hydrogen peroxide. Binding of RAGE ligands results in activation of nuclear factor-kappaB (NF-κB). We show that RAGE ablation protected nigral dopaminergic neurons against cell death induced by the neurotoxin MPTP that mimics most features of PD. In RAGE-deficient mice the translocation of the NF-κB subunit p65 to the nucleus, in dopaminergic neurons and glial cells was inhibited suggesting that RAGE involves the activation of NF-κB. The mRNA level of S100, one of the ligands of RAGE, was increased after MPTP treatment. The dopaminergic neurons treated with MPP(+) and S100 protein showed increased levels of apoptotic cell death, which was attenuated in RAGE-deficient mice. Our results suggest that activation of RAGE contributes to MPTP/MPP(+)-induced death of dopaminergic neurons that may be mediated by NF-κB activation.

HMGB1 is associated with atherosclerotic plaque composition and burden in patients with stable coronary artery disease.

OBJECTIVES: The role of inflammation in atherosclerosis is widely appreciated. High mobility group box 1 (HMGB1), an injury-associated molecular pattern molecule acting as a mediator of inflammation, has recently been implicated in the development of atherosclerosis. In this study, we sought to investigate the association of plasma HMGB1 with coronary plaque composition in patients with suspected or known coronary artery disease (CAD). DESIGN: HMGB1, high sensitive troponin T (hsTnT) and high sensitive C-reactive protein (hsCRP) were determined in 152 consecutive patients with suspected or known stable CAD who underwent clinically indicated 256-slice coronary computed tomography angiography (CCTA). Using CCTA, we assessed 1) coronary calcification, 2) non-calcified plaque burden and 3) the presence of vascular remodeling in areas of non-calcified plaques. RESULTS: Using univariate analysis, hsCRP, hsTnT and HMGB1 as well as age, and atherogenic risk factors were associated with non-calcified plaque burden (r = 0.21, p = 0.009; r = 0.48, p<0.001 and r = 0.34, p<0.001, respectively). By multivariate analysis, hsTnT and HMGB1 remained independent predictors of the non-calcified plaque burden (r = 0.48, p<0.01 and r = 0.34, p<0.001, respectively), whereas a non-significant trend was noticed for hs-CRP (r = 0.21, p = 0.07). By combining hsTnT and HMGB1, a high positive predictive value for the presence of non-calcified and remodeled plaque (96% and 77%, respectively) was noted in patients within the upper tertiles for both biomarkers, which surpassed the positive predictive value of each marker separately. CONCLUSIONS: In addition to hs-TnT, a well-established cardiovascular risk marker, HMGB1 is independently associated with non-calcified plaque burden in patients with stable CAD, while the predictive value of hs-CRP is lower. Complementary value was observed for hs-TnT and HMGB1 for the prediction of complex coronary plaque.

Nuclear factor erythroid-derived 2 (Nfe2) regulates JunD DNA-binding activity via acetylation: a novel mechanism regulating trophoblast differentiation.

We recently demonstrated that the bZip transcription factor nuclear factor erythroid-derived 2 (Nfe2) represses protein acetylation and expression of the transcription factor glial cell missing 1 (Gcm1) in trophoblast cells, preventing excess syncytiotrophoblast formation and permitting normal placental vascularization and embryonic growth. However, the Gcm1 promoter lacks a Nfe2-binding site and hence the mechanisms linking Nfe2 and Gcm1 expression remained unknown. Here we show that Nfe2 represses JunD DNA-binding activity to the Gcm1 promoter during syncytiotrophoblast differentiation. Interventional studies using knockdown and knockin approaches show that enhanced JunD DNA-binding activity is required for increased expression of Gcm1 and syncytiotrophoblast formation as well as impaired placental vascularization and reduced growth of Nfe2(-/-) embryos. Induction of Gcm1 expression requires binding of JunD to the -1441 site within the Gcm1 promoter, which is distinct from the -1314 site previously shown to induce Gcm1 expression by other bZip transcription factors. Nfe2 modulates JunD binding to the Gcm1 promoter via acetylation, as reducing JunD acetylation using the histone acetyltransferase inhibitor curcumin reverses the increased JunD DNA-binding activity observed in the absence of Nfe2. This identifies a novel mechanism through which bZip transcription factors interact. Within the placenta this interaction regulates Gcm1 expression, syncytiotrophoblast formation, placental vascularization, and embryonic growth.

RAGE and ICAM-1 differentially control leukocyte recruitment during acute inflammation in a stimulus-dependent manner.

BACKGROUND: The receptor for advanced glycation endproducts, RAGE, is involved in the pathogenesis of many inflammatory conditions, which is mostly related to its strong activation of NF-κB but also due to its function as ligand for the ß2-integrin Mac-1. To further dissect the stimulus-dependent role of RAGE on leukocyte recruitment during inflammation, we investigated ß2-integrin-dependent leukocyte adhesion in RAGE-/- and Icam1-/- mice in different cremaster muscle models of inflammation using intravital microscopy. RESULTS: We demonstrate that RAGE, but not ICAM-1 substantially contributes to N-formyl-methionyl-leucyl-phenylalanine (fMLP)-induced leukocyte adhesion in TNF-α-pretreated cremaster muscle venules in a Mac-1-dependent manner. In contrast, fMLP-stimulated leukocyte adhesion in unstimulated cremaster muscle venules is independent of RAGE, but dependent on ICAM-1 and its interaction with LFA-1. Furthermore, chemokine CXCL1-stimulated leukocyte adhesion in surgically prepared cremaster muscle venules was independent of RAGE but strongly dependent on ICAM-1 and LFA-1 suggesting a differential and stimulus-dependent regulation of leukocyte adhesion during inflammation in vivo. CONCLUSION: Our results demonstrate that RAGE and ICAM-1 differentially regulate leukocyte adhesion in vivo in a stimulus-dependent manner.