OBJECTIVE

Mitochondrial transcription factor A (TFAM) is normally bound to and remains associated with mitochondrial DNA (mtDNA) when released from damaged cells. We hypothesized that TFAM, bound to mtDNA (or equivalent CpG-enriched DNA), amplifies TNFα release from TLR9-expressing plasmacytoid dendritic cells (pDCs) by engaging RAGE.

METHODS

Murine Flt3 ligand-expanded splenocytes obtained from C57BL/6 mice were treated with recombinant human TFAM, alone or in combination with CpG-enriched DNA with subsequent TNFα release measured by ELISA. The role of RAGE was determined by pre-treatment with soluble RAGE or heparin or by employing matching RAGE (-/-) splenocytes. TLR9 signaling was evaluated using a specific TLR9-blocking oligonucleotide and by inhibiting endosomal processing, PI3K and NF-κB. Additional studies examined whether heparin sulfate moieties or endothelin converting enzyme-1 (ECE-1)-dependent recycling of endosomal receptors were required for TFAM and CpG DNA recognition.

RESULTS

TFAM augmented splenocyte TNFα release in response to CpGA DNA, which was strongly dependent upon pDCs and regulated by RAGE and TLR9 receptors. Putative TLR9 signaling pathways, including endosomal acidification and signaling through PI3K and NF-κB, were essential for splenocyte TNFα release in response to TFAM+CpGA DNA. Interestingly, TNFα release depended upon endothelin converting enzyme (ECE)-1, which cleaves and presumably activates TLR9 within endosomes. Recognition of the TFAM-CpGA DNA complex was dependent upon heparin sulfate moieties, and recombinant TFAM Box 1 and Box 2 proteins were equivalent in terms of augmenting TNFα release.

CONCLUSIONS

TFAM promoted TNFα release in a splenocyte culture model representing complex cell-cell interactions in vivo with pDCs playing a critical role. To our knowledge, this study is the first to incriminate ECE-1-dependent endosomal cleavage of TLR9 as a critical step in the signaling pathway leading to TNFα release. These findings, and others reported herein, significantly advance our understanding of sterile immune responses triggered by mitochondrial danger signals.

Airway mucus hyperproduction is a common feature of chronic airway diseases such as severe asthma, chronic obstructive pulmonary disease and cystic fibrosis, which are closely associated with neutrophilic airway inflammation. S100A8, S100A9 and S100A12 are highly abundant proteins released by neutrophils and have been identified as important biomarkers in many inflammatory diseases. Herein, we report a new role for S100A8, S100A9 and S100A12 for producing MUC5AC, a major mucin protein in the respiratory tract. All three S100 proteins induced MUC5AC mRNA and the protein in normal human bronchial epithelial cells as well as NCI-H292 lung carcinoma cells in a dose-dependent manner. A Toll-like receptor 4 (TLR4) inhibitor almost completely abolished MUC5AC expression by all three S100 proteins, while neutralization of the receptor for advanced glycation end-products (RAGE) inhibited only S100A12-mediated production of MUC5AC. The S100 protein-mediated production of MUC5AC was inhibited by the pharmacological agents that block prominent signalling molecules for MUC5AC expression, such as mitogen-activated protein kinases, nuclear factor-κB (NF-κB) and epidermal growth factor receptor. S100A8, S100A9 and S100A12 equally elicited both phosphorylation of extracellular signal-regulated kinase (ERK) and nuclear translocation of NF-κB/degradation of cytosolic IκB with similar kinetics through TLR4. In contrast, S100A12 preferentially activated the ERK pathway rather than the NF-κB pathway through RAGE. Collectively, these data reveal the capacity of these three S100 proteins to induce MUC5AC production in airway epithelial cells, suggesting that they all serve as key mediators linking neutrophil-dominant airway inflammation to mucin hyperproduction.

High mobility group box 1 (HMGB1) is a nuclear protein that can bind to DNA and act as a co-factor for gene transcription. When released into extracellular fluid, it plays a proinflammatory role by acting as a damage-associated molecular pattern molecule (DAMP) (also known as an alarmin) to initiate innate immune responses by activating multiple cell surface receptors such as the receptor for advanced glycation end-products (RAGE) and toll-like receptors (TLRs), TLR2, TLR4 or TLR9. This proinflammatory role is now considered to be important in the pathogenesis of a wide range of kidney diseases whether they result from hemodynamic changes, renal tubular epithelial cell apoptosis, kidney tissue fibrosis or inflammation. This review summarizes our current understanding of the role of HMGB1 in kidney diseases and how the HMGB1-mediated signaling pathway may constitute a new strategy for the treatment of kidney diseases.

OBJECTIVE

Advanced glycation end-products (AGEs) levels are high in western diets and contribute to tissue injury via activation of RAGE (receptor for AGEs) and generation of reactive oxygen species (ROS). Here, we determined if high dietary AGE intake worsens progression of non-alcoholic fatty liver disease (NAFLD).

METHODS

Male Sprague Dawley rats were fed a methionine choline deficient (MCD) diet for 6 weeks before 6 weeks of a high AGE MCD diet through baking. They were compared with animals on MCD diet or a methionine choline replete (MCR) diet alone for 12 weeks. Hepatic ROS, triglycerides, biochemistry, picro-sirius morphometry, hepatic mRNA expression and immunohistochemistry were determined. Primary hepatic stellate cells (HSCs) from both MCR and MCD animals were exposed to AGEs. ROS, proliferation and mRNA expression were determined.

RESULTS

The high AGE MCD diet increased hepatic AGE content and elevated triglycerides, NADPH dependent superoxide production, HNE adducts, steatosis, steatohepatitis (CD43, IL-6, TNF-α) and fibrosis (α-SMA, CTGF, COL1A, picrosirius) compared to MCD alone. In HSCs, AGEs significantly increased ROS production, bromodeoxyuridine proliferation and MCP-1, IL-6, α-SMA, and RAGE expression in HSCs from MCD but not MCR animals. These effects were abrogated by RAGE or NADPH oxidase blockade.

CONCLUSIONS

In the MCD model of NAFLD, high dietary AGEs increases hepatic AGE content and exacerbates liver injury, inflammation, and liver fibrosis via oxidative stress and RAGE dependent profibrotic effects of AGEs on activated HSCs. This suggests that pharmacological and dietary strategies targeting the AGE/RAGE pathway could slow the progression of NAFLD.

Diabetic angiopathy including micro- and macroangiopathy is concerned with high rate of morbidity and mortality in patients with long-standing diabetes. Receptor for advanced glycation end products (RAGE) and its ligands have been considered as important pathogenic triggers for the progression of the vascular injuries in diabetes. The deleterious link between RAGE and diabetic angiopathy has been demonstrated in animal studies. Preventive and therapeutic strategies focusing on RAGE and its ligand axis may be of great importance in relieving diabetic vascular complications and reducing the burden of disease.

Immune and bone cells are functionally coupled by pro-inflammatory cytokine intercellular signaling networks common to both tissues and their crosstalk may contribute to the etiologies of some immune-associated bone pathologies. For example, the receptor activator of NF-kappaB ligand (RANKL)/osteoprotegerin (OPG)/receptor activator of NF-kappaB (RANK) signaling axis plays a critical role in dendritic cell (DC) function as well as bone remodeling. The expression of RANKL by immune cells may contribute to bone loss in periodontitis, arthritis, and multiple myeloma. A recent discovery reveals that DCs release the chromatin protein high mobility group box 1 (HMGB1) as a potent immunomodulatory cytokine mediating the interaction between DCs and T-cells, via HMGB1 binding to the membrane receptor for advanced glycation end products (RAGE). To determine whether osteoblasts or osteoclasts express and/or release HMGB1 into the bone microenvironment, we analyzed tissue, cells, and culture media for the presence of this molecule. Our immunohistochemical and immunocytochemical analyses demonstrate HMGB1 expression in primary osteoblasts and osteoclasts and that both cells express RAGE. HMGB1 is recoverable in the media of primary osteoblast cultures and cultures of isolated osteoclast precursors and osteoclasts. Parathyroid hormone (PTH), a regulator of bone remodeling, attenuates HMGB1 release in cultures of primary osteoblasts and MC3T3-E1 osteoblast-like cells but augments this release in the rat osteosarcoma cell line UMR 106-01, both responses primarily via activation of adenylyl cyclase. PTH-induced HMGB1 discharge by UMR cells exhibits similar release kinetics as reported for activated macrophages. These data confirm the presence of the HMGB1/RAGE signaling axis in bone.

Advanced glycation endproducts (AGEs) and the receptor for AGEs (RAGE) have been linked to the pathogenesis of diabetic complications, such as retinopathy, neuropathy, and nephropathy. AGEs may induce β-cell dysfunction and apoptosis, another complication of diabetes. However, the role of AGE-RAGE interaction in AGE-induced pancreatic β-cell failure has not been fully elucidated. In this study, we investigated whether AGE-RAGE interaction could mediate β-cell failure. We explored the potential mechanisms in insulin secreting (INS-1) cells from a pancreatic β-cell line, as well as primary rat islets. We found that glycated serum (GS) induced apoptosis in pancreatic β-cells in a dose- and time-dependent manner. Treatment with GS increased RAGE protein production in cultured INS-1 cells. GS treatment also decreased bcl-2 gene expression, followed by mitochondrial swelling, increased cytochrome c release, and caspase activation. RAGE antibody and knockdown of RAGE reversed the β-cell apoptosis and bcl-2 expression. Inhibition of RAGE prevented AGE-induced pancreatic β-cell apoptosis, but could not restore the function of glucose stimulated insulin secretion (GSIS) in rat islets. In summary, the results of the present study demonstrate that AGEs are integrally involved in RAGE-mediated apoptosis and impaired GSIS dysfunction in pancreatic β-cells. Inhibition of RAGE can effectively protect β-cells against AGE-induced apoptosis, but cannot reverse islet dysfunction in GSIS.

Altered expression of chondroitin sulfate (CS) and heparan sulfate (HS) at the surfaces of tumor cells plays a key role in malignant transformation and tumor metastasis. Previously we demonstrated that a Lewis lung carcinoma (LLC)-derived tumor cell line with high metastatic potential had a higher proportion of E-disaccharide units, GlcUA-GalNAc(4,6-O-disulfate), in CS chains than low metastatic LLC cells and that such CS chains are involved in the metastatic process. The metastasis was markedly inhibited by the pre-administration of CS-E from squid cartilage rich in E units or by preincubation with a phage display antibody specific for CS-E. However, the molecular mechanism of the inhibition remains to be investigated. In this study the receptor molecule for CS chains containing E-disaccharides expressed on LLC cells was revealed to be receptor for advanced glycation end products (RAGE), which is a member of the immunoglobulin superfamily predominantly expressed in the lung. Interestingly, RAGE bound strongly to not only E-disaccharide, but also HS-expressing LLC cells. Furthermore, the colonization of the lungs by LLC cells was effectively inhibited by the blocking of CS or HS chains at the tumor cell surface with an anti-RAGE antibody through intravenous injections in a dose-dependent manner. These results provide the clear evidence that RAGE is at least one of the critical receptors for CS and HS chains expressed at the tumor cell surface and involved in experimental lung metastasis and that CS/HS and RAGE are potential molecular targets in the treatment of pulmonary metastasis.

Neurovascular dysfunction is an important component of Alzheimer's disease, leading to reduced clearance across the blood-brain barrier and accumulation of neurotoxic β-amyloid (Aβ) peptides in the brain. It has been shown that the ABC transport protein P-glycoprotein (P-gp, ABCB1) is involved in the export of Aβ from the brain into the blood. To determine whether Aβ influences the expression of key Aβ transporters, we studied the effects of 1-day subcutaneous Aβ1-40 and Aβ1-42 administration via Alzet mini-osmotic pumps on P-gp, BCRP, LRP1, and RAGE expression in the brain of 90-day-old male FVB mice. Our results demonstrate significantly reduced P-gp, LRP1, and RAGE mRNA expression in mice treated with Aβ1-42 compared to controls, while BCRP expression was not affected. The expression of the four proteins was unchanged in mice treated with Aβ1-40 or reverse-sequence peptides. These findings indicate that, in addition to the age-related decrease of P-gp expression, Aβ1-42 itself downregulates the expression of P-gp and other Aβ-transporters, which could exacerbate the intracerebral accumulation of Aβ and thereby accelerate neurodegeneration in Alzheimer's disease and cerebral β-amyloid angiopathy.

The receptor for advanced glycation end products (RAGE) plays a crucial role in several disease processes including diabetes, inflammation, and cancer. Compared with apoptosis ("programmed cell death"), autophagy is a genetically programmed, evolutionarily conserved cell survival process that degrades long-lived cellular proteins and organelles ("programmed cell survival"). Recently we reported that RAGE is an important regulator of oxidative stress in pancreatic cancer cells. Upregulation of RAGE expression by the nuclear factor (NF)-κB pathway decreases reactive oxygen species (ROS)-induced oxidative injury. In contrast, suppression of RAGE expression increases pancreatic tumor cell sensitivity to oxidative injury. Furthermore, RAGE is a positive regulator of autophagy, and negative regulator of apoptosis during oxidative stress. These findings provide insight into how crosstalk between apoptosis and autophagy is mediated via ROS signaling with a process involving RAGE.

OBJECTIVE

To review the evidence for the molecular and cellular processes that may potentially link periodontal disease and diabetes. The pathogenic roles of cytokines and metabolic molecules (e.g. glucose, lipids) are explored and the role of periodontal bacteria is also addressed. Paradigms for bidirectional relationships between periodontitis and diabetes are discussed and opportunities for elaborating these models are considered.

METHODS

Database searches were performed using MeSH terms, keywords, and title words. Studies were evaluated and summarized in a narrative review.

RESULTS

Periodontal microbiota appears unaltered by diabetes and there is little evidence that it may influence glycaemic control. Small-scale clinical studies and experiments in animal models suggest that IL-1b, TNF-a, IL-6, OPG and RANKL may mediate periodontitis in diabetes. The AGE-RAGE axis is likely an important pathway of tissue destruction and impaired repair in diabetes-associated periodontitis. A role for locally activated pro-inflammatory factors in the periodontium, which subsequently impact on diabetes, remains speculative.

CONCLUSIONS

There is substantial information on potential mechanistic pathways which support a close association between diabetes and periodontitis, but there is a real need for longitudinal clinical studies using larger patient groups, integrated with studies of animal models and cells/tissues in vitro.

OBJECTIVE

Knowledge of the role of advanced glycation end products (AGE), their receptor (RAGE), and the receptor's soluble form (sRAGE), in heart failure (HF) is very limited. We evaluated the clinical role of the AGE-RAGE system in HF and in particular any association it might have with ischaemic aetiology.

RESULTS

We measured fluorescent AGE, glycated albumin and sRAGE in 103 patients with chronic HF. We showed that sRAGE but not AGE was related to ischaemic aetiology (1638.3 ± 207.4 ischaemic vs. 1065.1 ± 94.2 pg/mL non-ischaemic group; P =0.016) independent of age, sex, diabetes, renal function, clinical severity, or other variables (OR: 1.091; 95% CI (confidence interval): 1.032-1.153; P =0.007). Moreover, sRAGE was directly associated with extent of coronary disease (OR for three vessel disease compared with non-coronary lesions: 1.186; 95% CI: 1.065-1.322; P =0.002). We also found a correlation between sRAGE and severity of HF, which increased with New York Heart Association (NYHA) class (741.9 ± 88.9 pg/mL in class 1, 1195.9 ± 113.2 pg/mL in class II, and 1724.8 ± 245.7 pg/mL in class III (P < 0.05)) and brain natriuretic peptide (BNP) levels (667.4 ± 68.0 vs. 1344.5 ± 126.0 pg/mL for BNP < and ≥400 pg/mL, respectively).

CONCLUSIONS

sRAGE is an indicator of chronic heart failure severity and an independent marker of coronary artery disease and its severity in patients with CHF.

OBJECTIVE

Skeletal muscle myopathy is a common diabetes complication. One possible cause of myopathy is myocyte failure to repair contraction-generated plasma membrane injuries. Here, we test the hypothesis that diabetes induces a repair defect in skeletal muscle myocytes.

METHODS

Myocytes in intact muscle from type 1 (INS2(Akita+/-)) and type 2 (db/db) diabetic mice were injured with a laser and dye uptake imaged confocally to test repair efficiency. Membrane repair defects were also assessed in diabetic mice after downhill running, which induces myocyte plasma membrane disruption injuries in vivo. A cell culture model was used to investigate the role of advanced glycation end products (AGEs) and the receptor for AGE (RAGE) in development of this repair defect.

RESULTS

Diabetic myocytes displayed significantly more dye influx after laser injury than controls, indicating a repair deficiency. Downhill running also resulted in a higher level of repair failure in diabetic mice. This repair defect was mimicked in cultured cells by prolonged exposure to high glucose. Inhibition of the formation of AGE eliminated this glucose-induced repair defect. However, a repair defect could be induced, in the absence of high glucose, by enhancing AGE binding to RAGE, or simply by increasing cell exposure to AGE.

CONCLUSIONS

Because one consequence of repair failure is rapid cell death (via necrosis), our demonstration that repair fails in diabetes suggests a new mechanism by which myopathy develops in diabetes.

Interaction of advanced glycation end products (AGE) with AGE-receptors induces several cellular phenomena relating potentially to diabetic complications. Five AGE-receptors identified so far are RAGE (receptor for AGE), 80 K-H, OST-48, galectin-3, and SR-A (macrophage scavenger receptor type I and II). Since SR-A belongs to the class A scavenger receptor family and the scavenger receptor collectively represents a family of multiligand lipoprotein receptors, it is possible that CD36 belonging to the class B scavenger receptor family (SR-B) can recognize AGE-proteins as a ligand. This was tested in the present study at the cellular level using CHO (Chinese hamster ovary) cells overexpressing human CD36 (CHO-CD36 cells). 125I-AGE-BSA (bovine serum albumin) was endocytosed in a dose-dependent fashion and underwent lysosomal degradation by CHO-CD36 but not wild-type CHO cells. Endocytic uptake of 125I-AGE-BSA by these cells was inhibited 50% by oxidized LDL (Ox-LDL) and 60% by FA6-152, an anti-CD36 antibody inhibiting cellular binding of Ox-LDL. Our results indicate that CD36 expressed by these cells mediates endocytic uptake and subsequent intracellular degradation of AGE-proteins. Because CD36 is one of the major Ox-LDL receptors and is upregulated in macrophage- and smooth muscle cell-derived foam cells in human atherosclerotic lesions, the present results suggest that, like Ox-LDL, AGE-proteins generated in situ are recognized by CD36, which might contribute to the pathogenesis of diabetic macrovascular complications.

OBJECTIVE

Increased lipid peroxidation and inflammation are major factors in the pathogenesis of nonalcoholic fatty liver disease (NAFLD). A lipoxidation product that could play a role in the induction of hepatic inflammation is N(ε)-(carboxymethyl)lysine (CML). The aim of the present study was to investigate the relationship between steatosis and CML and to study the role of CML in hepatic inflammation.

METHODS

We included 74 obese individuals, which were categorized into 3 groups according to the grade of hepatic steatosis. CML accumulation in liver biopsies was assessed by immunohistochemistry and plasma CML levels were measured by mass spectrometry. Plasma CML levels were also determined in the hepatic artery, portal, and hepatic vein of 22 individuals, and CML fluxes across the liver were calculated. Hepatocyte cell lines were used to study CML formation during intracellular lipid accumulation and the effect of CML on pro-inflammatory cytokine expression. Gene expression levels of the inflammatory markers were determined in liver biopsies of the obese individuals.

RESULTS

CML accumulation was significantly associated with the grade of hepatic steatosis, the grade of hepatic inflammation, and gene expression levels of inflammatory markers PAI-1, IL-8, and CRP. Analysis of CML fluxes showed no release/uptake of CML by the liver. Lipid accumulation in hepatocytes, induced by incubation with fatty acids, was associated with increased CML formation and expression of the receptor for advanced glycation endproducts (RAGE), PAI-1, IL-8, IL-6, and CRP. Pyridoxamine and aminoguanidine inhibited the endogenous CML formation and the increased RAGE, PAI-1, IL-8, IL-6, and CRP expression. Incubation of hepatocytes with CML-albumin increased the expression of RAGE, PAI-1, and IL-6, which was inhibited by an antibody against RAGE.

CONCLUSIONS

Accumulation of CML and a CML-upregulated RAGE-dependent inflammatory response in steatotic livers may play an important role in hepatic steatosis and in the pathogenesis of NAFLD.

P73 antisense RNA 1 T (non-protein coding), also known as TP73-AS1, is a long non-coding RNA (lncRNA) which is involved in cell proliferation and the development of tumors. However, the exact effects and molecular mechanisms of TP73-AS1 in hepatocellular carcinoma (HCC) progression are still unknown. The present study is aimed to investigate the detailed functions and the mechanism of TP73-AS1 in regulation of HCC cell proliferation.

TP73-AS1 expression in HCC tissues and cell lines was determined using real-time PCR assays; the correlation of TP73-AS1 expression with clinicopathological features of HCC was analyzed. The functions of TP73-AS1 in regulation of HCC cell proliferation was evaluated using MTT and BrdU assays. The candidate upstream miRNAs of HMGB1 were screened using miRcode, miRWalk, miRanda and Target scan, verified using real-time PCR assays. The interaction between TP73-AS1 and miR-200a was confirmed using Luciferase report gene assays. The proten levels of HMGB1 signaling-related factors in response to co-processing TP73-AS1 knockdown and miR-200a inhibition were determined using Western blot assays and ELISA. Further, miR-200a, HMGB1 mRNA and RAGE mRNA and their correlations in HCC tissues were determined.

TP73-AS1 was upregulated in HCC tissues and cell lines. High TP73-AS1 expression was correlated with worse clinicopathological features, poorer prognosis and shorter survival. Knockdown of TP73-AS1 inhibited the HCC proliferation and the expression levels of HMGB1, RAGE and NF-κB in HCC cells. By using online tools, we screened out several candidate upstream miRNAs of HMGB1, among which miR-200a overexpression inhibited HMGB1 mRNA expression the most significantly. By using luciferase assays, we confirmed that miR-200a could directly bind to TP73-AS1 and the 3'UTR of HMGB1; TP73-AS1 competed with HMGB1 for miR-200a binding. MiR-200a inhibition could up-regulate HMGB1, RAGE, NF-κB expression as well as NF-κB regulated cytokines levels, which could be partially restored by si-TP73-AS1. In HCC tissues, miR-200a was down-regulated while HMGB1 and RAGE were up-regulated; TP73-AS1 was inversely correlated with miR-200a, while positively correlated with HMGB1 and RAGE, respectively.

Our data indicated that TP73-AS1 might be an oncogenic lncRNA that promoted proliferation of HCC and could be regarded as a therapeutic target in human HCC.

The advanced glycosylation end product (AGE)-binding proteins identified so far include the components of the AGE-receptor complex p60, p90 and galectin-3, receptor for advanced glycosylation end products (RAGE), and the macrophage scavenger receptor types I and II. Galectin-3 interacts with beta-galactoside residues of several cell surface and matrix glycoproteins through the carbohydrate recognition domain and is also capable of peptide-peptide associations mediated by its N-terminus domain. These structural properties enable galectin-3 to exert multiple functions, including the modulation of cell adhesion, the control of cell cycle, and the mRNA splicing activity. Moreover, in macrophages, astrocytes, and endothelial cells, galectin-3 has been shown to exhibit a high-affinity binding for AGEs; the lack of a transmembrane anchor sequence or signal peptide suggests that it associates with other AGE-receptor components rather than playing an independent role as AGE-receptor. In tissues that are targets of diabetic vascular complications, such as the mesangium and the endothelium, galectin-3 is not expressed or only weakly expressed under basal conditions, at variance with p90 and p60 but becomes detectable with aging and is induced or up-regulated by the diabetic milieu, which only slightly affects the expression of p90 or p60. This (over)expression of galectin-3 may in turn modulate AGE-receptor-mediated events by modifying the function of the AGE-receptor complex, which could play a role in the pathogenesis of target tissue injury. Up-regulated galectin-3 expression may also exert direct effects on tissue remodeling, independently of AGE ligands, by virtue of its adhesive and growth regulating properties.

Advanced glycation end-products (AGEs), senescent macroprotein derivatives formed at an accelerated rate in diabetes, are closely associated with vascular calcification in humans. In this study, the hypothesis that AGEs enhance calcification in cultured vascular smooth muscle cells (VSMCs) was tested. Using real-time polymerase chain reaction (PCR) and specific protein assays, it was demonstrated that rat aortic VSMCs incubated with AGEs exhibited an increased expression of the AGE receptor (RAGE) and typical bone proteins, such as osteopontin and alkaline phosphatase. Incubation with AGEs also enhanced calcium accumulation in VSMCs in time- and dose-dependent manners. These AGEs-mediated changes in VSMCs were partially attenuated by a neutralizing antibody to RAGE. The results suggest that AGEs that accumulate in diabetes could elicit the osteoblastic differentiation of VSMCs, thereby contributing to vascular calcification via the RAGE pathway. Interruption of the AGE-RAGE interaction might be a promising target for therapeutic intervention to prevent diabetic vascular calcification.

Accumulating evidence indicates that abnormal deposition of amyloid-β (Aβ) peptide in the brain is responsible for endothelial cell damage and consequently leads to blood-brain barrier (BBB) leakage. However, the mechanisms underlying BBB disruption are not well described. We employed an monolayer BBB model comprising bEnd.3 cell and found that BBB leakage was induced by treatment with Aβ(1-42), and the levels of tight junction (TJ) scaffold proteins (ZO-1, Claudin-5, and Occludin) were decreased. Through comparisons of the effects of the different components of Aβ(1-42), including monomer (Aβ(1-42)-Mono), oligomer (Aβ(1-42)-Oligo), and fibril (Aβ(1-42)-Fibril), our data confirmed that Aβ(1-42)-Oligo is likely to be the most important damage factor that results in TJ damage and BBB leakage in Alzheimer's disease. We found that the incubation of bEnd.3 cells with Aβ(1-42) significantly up-regulated the level of receptor for advanced glycation end-products (RAGE). Co-incubation of a polyclonal antibody to RAGE and Aβ(1-42)-Oligo in bEnd.3 cells blocked RAGE suppression of Aβ(1-42)-Oligo-induced alterations in TJ scaffold proteins and reversed Aβ(1-42)-Oligo-induced up-regulation of RAGE, matrix metalloproteinase (MMP)-2, and MMP-9. Furthermore, we found that these effects induced by Aβ(1-42)-Oligo treatment were effectively suppressed by knockdown of RAGE using small interfering RNA (siRNA) transfection. We also found that GM 6001, a broad-spectrum MMP inhibitor, partially reversed the Aβ(1-42)-Oligo-induced inhibitor effects in bEnd.3 cells. Thus, these results suggested that RAGE played an important role in Aβ-induced BBB leakage and alterations of TJ scaffold proteins, through a mechanism that involved up-regulation of MMP-2 and MMP-9.

Low-grade inflammation (LGI) is a central phenomenon in the genesis of obesity and insulin-resistance characterized by IL-6 in human serum. Whereas this LGI was initially thought to be mainly attributed to macrophage activation, it is now known that pre-adipocytes and adipocytes secrete several adipokines including IL-6 and participate to LGI and associated pathologies. In macrophages, HMGB1 is a nuclear yet secreted protein and acts as a cytokine to drive the production of inflammatory molecules through RAGE and TLR2/4. In this paper we tested the secretion of HMGB1 and the auto- and paracrine contribution to fat inflammation using the human preadipocyte cell line SW872 as a model. We showed that 1) human SW872 secreted actively HMGB1, 2) IL-6 production was positively linked to high levels of secreted HMGB1, 3) recombinant HMGB1 boosted IL-6 expression and this effect was mediated by the receptor RAGE and did not involve TLR2 or TLR4. These results suggest that HMGB1 is a major adipokine contributing to LGI implementation and maintenance, and can be considered as a target to develop news therapeutics in LGI associated pathologies such as obesity and type II diabetes.

OBJECTIVE

This cross-sectional study aimed at evaluating the possible association of serum levels of glycated albumin (GA) and endogenous secretory RAGE (esRAGE) with angiographically significant coronary artery disease (CAD) in patients with type 2 diabetes mellitus (T2DM) and non-diabetic patients.

METHODS

Serum levels of GA, esRAGE, and inflammatory markers were measured in 1320 patients undergoing coronary angiography from three large districts in Shanghai. Patients were divided into four groups based on the presence/absence of T2DM and of significant CAD (diameter stenosis>>or=70%).

RESULTS

Serum levels of GA, high-sensitivity C-reactive protein (hsCRP), tumor necrosis factor (TNF)-alpha and interleukin (IL)-6 were significantly higher and, in contrast, serum esRAGE levels were lower in patients with both T2DM and significant CAD than in all other groups (all P<0.01), whereas there were no significant differences in GA and esRAGE levels between non-diabetic patients with CAD and those without. Serum GA and esRAGE levels correlated with angiographic CAD severity (P=0.031 and P=0.001), extent (both P<0.001) and number of diseased coronary arteries (both P<0.001) in diabetic CAD patients. At multivariable regression analysis, male gender, age, hypertension, cigarette smoking, HDL-C, lipoprotein (a), GA, esRAGE, hsCRP and TNF-alpha were all independent determinants of significant CAD in diabetic patients. Moreover, male gender, age, hypertension, lipoprotein (a), GA and esRAGE remained independently associated with three-vessel disease.

CONCLUSIONS

Patients with T2DM and CAD feature elevated serum GA and decreased serum esRAGE levels. Here serum GA and esRAGE levels are associated with angiographic extent and severity of CAD.

The receptor for advanced glycation end products (RAGE) is a signaling receptor protein of the immunoglobulin superfamily implicated in multiple pathologies. It binds a diverse repertoire of ligands, but the structural basis for the interaction of different ligands is not well understood. We earlier showed that carboxylated glycans on the V-domain of RAGE promote the binding of HMGB1 and S100A8/A9. Here we study the role of these glycans on the binding and intracellular signaling mediated by another RAGE ligand, S100A12. S100A12 binds carboxylated glycans, and a subpopulation of RAGE enriched for carboxylated glycans shows more than 10-fold higher binding potential for S100A12 than total RAGE. When expressed in mammalian cells, RAGE is modified by complex glycans predominantly at the first glycosylation site (N25IT) that retains S100A12 binding. Glycosylation of RAGE and maximum binding sites for S100A12 on RAGE are also cell type dependent. Carboxylated glycan-enriched population of RAGE forms higher order multimeric complexes with S100A12, and this ability to multimerize is reduced upon deglycosylation or by using non-glycosylated sRAGE expressed in E. coli. mAbGB3.1, an antibody against carboxylated glycans, blocks S100A12-mediated NF-kappaB signaling in HeLa cells expressing full-length RAGE. These results demonstrate that carboxylated N-glycans on RAGE enhance binding potential and promote receptor clustering and subsequent signaling events following oligomeric S100A12 binding.

Psoriasis is a common complex genetic disease characterized by hyperplasia and inflammation in the skin; however, the relative contributions of epidermal cells and the immune system to disease pathogenesis remain unclear. Linkage studies have defined a psoriasis susceptibility locus (PSORS4) on 1q21, the epidermal differentiation complex, which includes genes for small S100 calcium-binding proteins. These proteins are involved in extracellular and intracellular signaling during epithelial host defense, linking innate and adaptive immunity. Inflammation-prone psoriatic skin constitutively expresses elevated concentrations of S100A7 (psoriasin) and S100A15 (koebnerisin) in the epidermis. Here, we report that genetically modified mice expressing elevated amounts of doxycycline-regulated mS100a7a15 in skin keratinocytes demonstrated an exaggerated inflammatory response when challenged by exogenous stimuli such as abrasion (Koebner phenomenon). This immune response was characterized by immune cell infiltration and elevated concentrations of T helper 1 (T(H)1) and T(H)17 proinflammatory cytokines, which have been linked to the pathogenesis of psoriasis and were further amplified upon challenge. Both inflammation priming and amplification required mS100a7a15 binding to the receptor of advanced glycation end products (RAGE). mS100a7a15 potentiated inflammation by acting directly as a chemoattractant for leukocytes, further increasing the number of inflammatory cells infiltrating the skin. This study provides a pathogenetic psoriasis model using a psoriasis candidate gene to link the epidermis and innate immune system in inflammation priming, highlighting the S100A7A15-RAGE axis as a potential therapeutic target.

The debate over the efficacy of bioaugmentation rages on, with research continuing to demonstrate that its advantages for soil bioremediation are difficult to predict; however, when it works, the results are often very encouraging. The difficulties arise from, among others, the diversity of the microorganisms used, environmental heterogeneity, and variations in the influence of critical parameters (e.g. humidity, microbial predation and "bioavailability') which, unfortunately, are not even always identified.