OBJECTIVE

C-reactive protein (CRP), an acute-phase reactant produced mainly by the liver, is elevated in diabetes, thus contributing to the development and progression of atherosclerosis. However, the molecular mechanism underlying the elevation of CRP in diabetes is not fully understood. Since a crosstalk between AGE and angiotensin II (Ang II) has been proposed in the pathogenesis of accelerated atherosclerosis in diabetes, we examined here whether and how telmisartan, a unique Ang II type 1 receptor blocker (ARB) with peroxisome proliferator-activated receptor-gamma (PPAR-gamma)-modulating activity, could inhibit AGE-induced CRP expression in a human hepatoma cell line, Hep3B cells.

METHODS

Protein levels of the receptor for AGE (RAGE) were analysed by western blots. Gene expression was analysed by quantitative real-time RT-PCR. CRP released into the medium was measured with ELISA. Intracellular formation of reactive oxygen species (ROS) was measured using the fluorescent probe CM-H(2)DCFDA.

RESULTS

Telmisartan, but not candesartan, another ARB, downregulated RAGE mRNA levels in a dose-dependent manner. Telmisartan decreased basal as well as AGE-induced RAGE protein expression in Hep3B cells. Furthermore, telmisartan dose-dependently inhibited AGE-induced ROS generation and subsequent CRP gene and protein induction in Hep3B cells. GW9662, an inhibitor of PPAR-gamma, blocked the inhibitory effects of telmisartan on RAGE expression and its downstream signalling in Hep3B cells.

CONCLUSIONS

Our present study indicates a unique beneficial aspect of telmisartan: it may work as an anti-inflammatory agent against AGE by suppressing RAGE expression via PPAR-gamma activation in the liver and may play a protective role in vascular injury in diabetes.

Expression of the paired-box 7 (PAX7) transcription factor is regulated during both myoblast proliferation and differentiation: high levels of PAX7 compromise myogenic differentiation because of excess and prolonged proliferation, whereas low levels of PAX7 result in precocious differentiation. We showed that myogenin repressed Pax7 transcription in differentiating myoblasts by binding to specific recognition sites in the Pax7 promoter, and that high-mobility group box 1 (HMGB1)-receptor for advanced glycation end-products (RAGE) signaling was required for myogenin induction and myogenin-dependent repression of Pax7 transcription. In addition, PAX7 negatively and myogenin positively regulated RAGE expression. RAGE, a multiligand receptor of the immunoglobulin superfamily, was not expressed in adult skeletal muscles, and was transiently expressed in activated, proliferating and differentiating satellite cells (SCs) in injured muscles. Compared with wild-type muscles, Rage(-/-) muscles exhibited increased numbers of basal SCs that were further increased in injured Rage(-/-) muscles following elevated myoblast asymmetric division; complete regeneration of injured Rage(-/-) muscles was found to be delayed by ~1 week. Thus, RAGE signaling physiologically repressed Pax7 transcription in SCs by upregulating myogenin, thereby accelerating muscle regeneration and limiting SC self-renewal.

OBJECTIVE

High-mobility group protein B1 (Hmgb1) is released from necrotic cells and induces an inflammatory response. Although Hmgb1 has been implicated in ischemia/reperfusion (IR) injury of the brain, its role in IR injury of the retina remains unclear. Here, the authors provide evidence that Hmgb1 contributes to retinal damage after IR.

METHODS

Retinal IR injury was induced by unilateral elevation of intraocular pressure and the level of Hmgb1 in vitreous humor was analyzed 24 hours after reperfusion. To test the functional significance of Hmgb1 release, ischemic or normal retinas were treated with the neutralizing anti-Hmgb1 antibody or recombinant Hmgb1 protein respectively. To elucidate in which cell type Hmgb1 exerts its effect, primary retinal ganglion cell (RGC) cultures and glia RGC cocultures were treated with Hmgb1. To clarify the downstream signaling pathways involved in Hmgb1-induced effects in the ischemic retina, receptor for advanced glycation end products (Rage)-deficient mice (RageKO) were used.

RESULTS

Hmgb1 is accumulated in the vitreous humor 24 hours after IR. Inhibition of Hmgb1 activity with neutralizing antibody significantly decreased retinal damage after IR, whereas treatment of retinas or retinal cells with Hmgb1 induced a loss of RGCs. The analysis of RageKO versus wild-type mice showed significantly reduced expression of proinflammatory genes 24 hours after reperfusion and significantly increased survival of ganglion cell layer neurons 7 days after IR injury.

CONCLUSIONS

These results suggest that an increased level of Hmgb1 and signaling via the Rage contribute to neurotoxicity after retinal IR injury.

HIV-1-infected brains are characterized by increased amyloid deposition. To study the influence of HIV-1 on amyloid beta (Abeta) homeostasis at the blood-brain barrier (BBB) level, we employed a model of brain microvascular endothelial cells exposed to HIV-1 in the presence or absence of Abeta. HIV-1 markedly increased endogenous Abeta levels and elevated accumulation of exogenous Abeta. Simvastatin, the HMG-CoA reductase inhibitor, blocked these effects. We next evaluated the effects of HIV-1 and/or simvastatin on expression of the receptor for lipoprotein related protein (LRP1) and the receptor for advanced glycation end products (RAGE), known to regulate Abeta transport across the BBB. LRP1 expression was not affected by HIV-1; however, it was increased by simvastatin. Importantly, simvastatin attenuated HIV-1-induced RAGE expression. These results suggest that HIV-1 may directly contribute to Abeta accumulation at the BBB level. In addition, statins may protect against increased Abeta levels associated with HIV-1 infection in the brain.

The high mobility group 1B protein (HMGB1) mediates chronic inflammatory responses in endothelial cells, which play a critical role in atherosclerosis. However, the underlying mechanism is unknown. The goal of our study was to identify the effects of HMGB1 on the RAGE-induced inflammatory response in endothelial cells and test the possible involvement of the endoplasmic reticulum stress pathway. Our results showed that incubation of endothelial cells with HMGB1 (0.01-1 μg/ml) for 24h induced a dose-dependent activation of endoplasmic reticulum stress transducers, as assessed by PERK and IRE1 protein expression. Moreover, HMGB1 also promoted nuclear translocation of ATF6. HMGB1-mediated ICAM-1 and P-selectin production was dramatically suppressed by PERK siRNA or IRE1 siRNA. However, non-targeting siRNA had no such effects. HMGB1-induced increases in ICAM-1 and P-selectin expression were also inhibited by a specific eIF2α inhibitor (salubrinal) and a specific JNK inhibitor (SP600125). Importantly, a blocking antibody specifically targeted against RAGE (anti-RAGE antibody) decreased ICAM-1, P-selectin and endoplasmic reticulum stress molecule (PERK, eIF2α, IRE1 and JNK) protein expression levels. Collectively, these novel findings suggest that HMGB1 promotes an inflammatory response by inducing the expression of ICAM-1 and P-selectin via RAGE-mediated stimulation of the endoplasmic reticulum stress pathway.

Deposition of cross-linked insoluble protein aggregates such as amyloid plaques is characteristic for Alzheimer's disease. Microglial activation by these extracullar deposits has been proposed to play a crucial role in functional degeneration as well as cell death of neurones. A sugar-derived post-translational modification of long-lived proteins, advanced glycation endproducts (AGEs), activate specific signal transduction pathways, resulting in the up-regulation of various pro-inflammatory signals such as cytokines [interleukin-6 (IL-6), tumour necrosis factor-alpha (TNF-alpha)] and inducible nitric oxide synthase (iNOS). Our goal was to study AGE-activated signal transduction pathways involved in the induction of pro-inflammatory effectors in the murine microglial cell line N-11. Chicken egg albumin-AGE (CEA-AGE), used as model AGE, induces nitric oxide (NO), TNF-alpha and IL-6 production. The AGE receptor, RAGE, and the transcription factor, nuclear factor kappa B (NF-kappaB), appear to be involved in all pathways, since a neutralizing RAGE antibody and a peptide inhibiting NF-kappaB translocation down-regulated NO, TNF-alpha and IL-6 production. NO and TNF-alpha, but not IL-6 production appear to be regulated independently, since NOS inhibitors did not decrease TNF-alpha secretion and a neutralizing TNF-alpha antibody did not reduce NO production, while employment of NOS inhibitors reduced significantly the secretion of IL-6. Inhibition of the MAP-kinase-kinase (MEK) and phosphatidylinositol 3-kinase (PI3K) pathway, but not that of mitogen-activated protein kinase-p38 (MAPK-p38), reduced NO, TNF-alpha and IL-6 significantly, suggesting that simultaneous activation of the first two pathways is necessary for the AGE-induced induction of these pro-inflammatory stimuli.

Lipid mediators such as prostaglandin E2 (PGE2) play a central role during atherogenesis as a consequence of inflammation. PGE2 is produced from phospholipids by a cascade of enzymatic reactions involving phospholipase A2 (PLA2), cyclooxygenase (COX), and prostaglandin E synthase (PGES). It is released by several cell types, including vascular smooth muscle cells (VSMCs). Recent work has shown that the secretory PLA2-IIA (sPLA2-IIA), the most abundant isoform of secreted PLA2 in VSMCs, acts as a potent cytokine and activates VSMCs through a positive feedback loop. High mobility group protein 1 (HMGB1), also known as amphoterin, is a ubiquitous protein that plays various roles in the nucleus. HMGB1 is released by necrotic cells and by immune cells in response to various inflammatory mediators and acts as a potent proinflammatory cytokine. The present study investigates the role of HMGB1 in the activation of sPLA2-IIA expression and PGE2 production in VSMCs. Recombinant HMGB1 slightly activated the sPLA2-IIA, COX-2, and mPGES-1 genes but dramatically stimulated these genes in VSMCs that had been incubated with the proinflammatory cytokine IL-1beta for 24 h. This effect was accompanied by significantly increased PGE2 release. Induction of the three known receptors of HMGB1, namely RAGE, TLR-2, and TLR-4, by IL-1beta suggests that proinflammatory cytokines sensitize VSMCs to HMGB1. This provides new insights into the role of HMGB1 in VSMCs, suggesting it may be essential for the progression of atherosclerosis.

Our seminal discovery of high mobility group box 1 (HMGB1) as a late mediator of lethal systemic inflammation has prompted a new field of investigation for the development of experimental therapeutics. We previously reported that a major Danshen ingredient, tanshinone IIA sodium sulfonate (TSN-SS), selectively inhibited endotoxin-induced HMGB1 release and conferred protection against lethal endotoxemia and sepsis. To investigate the underlying mechanisms by which TSN-SS effectively inhibits HMGB1 release, we examined whether TSN-SS stimulates HMGB1 uptake by macrophages and whether genetic depletion of HMGB1 receptors [e.g., toll-like receptors (TLR)2, TLR4, or the receptor for advanced glycation end product (RAGE)] or pharmacological inhibition of endocytosis impairs TSN-SS-facilitated HMGB1 cellular uptake. TSN-SS stimulated internalization of exogenous HMGB1 protein into macrophage cytoplasmic vesicles that subsequently co-localized with microtubule-associated protein light chain 3 (LC3)-positive punctate structures (likely amphisomes). Meanwhile, it time-dependently elevated cellular levels of internalized HMGB1, leading to elevated LC3-II production and aggregation. Although genetic depletion of TLR2, TLR4, and/or RAGE did not impair TSN-SS-mediated HMGB1 uptake, specific inhibitors of the clathrin- and caveolin-dependent endocytosis significantly impaired TSN-SS-mediated HMGB1 uptake. Co-treatment with a lysosomal inhibitor, bafilomycin A1, led to enhanced accumulation of endogenous LC3-II and internalized exogenous HMGB1 in TSN-SS/rHMGB1-treated macrophages. Taken together, these findings suggest that TSN-SS may facilitate HMGB1 endocytic uptake, and subsequently delivered it to LC3-positive vacuoles (possibly amphisomes) for degradation via a lysosome-dependent pathway.

BACKGROUND

High-mobility group box 1 protein (HMGB1) belonging to endogenous danger signals prolongs eosinophil survival and acts as a chemoattractant.

OBJECTIVE

The authors evaluated the role of HMGB1 in the pathogenesis of asthma characterized by eosinophilic airway inflammation.

METHODS

Firstly, HMGB1 expressions in induced sputum obtained from human asthmatics were determined. This was followed by an evaluation of the role of HMGB1 in a murine model of asthma using anti-HMGB1 antibodies. Then the effect of HMGB1 on the receptor of advanced glycation end products (RAGE) expressions on CD11b-CD11c(+) cells isolated from a murine model of asthma were measured to elucidate the mechanisms involved.

RESULTS

Sputum HMGB1 expressions were markedly higher in asthmatics than in normal controls, and were positively correlated with sputum eosinophilia and sputum TNF-α, IL-5 and IL-13 expressions. In a murine model of asthma, HMGB1 expressions in lung tissue and HMGB1 levels in bronchoalveolar lavage fluid were significantly elevated and eosinophilic airway inflammation, non-specific airway hyperresponsiveness, and pathological changes were attenuated by blocking HMGB1 activity. Furthermore, we found that enhanced RAGE expressions on CD11b-CD11c(+) also significantly decreased when HMGB1 activity was blocked.

CONCLUSIONS

Our findings suggest that HMGB1 plays a key role in the pathogenesis of clinical and experimental asthma characterized by eosinophilic airway inflammation.

Autophagy is a double-edged sword in tumorigenesis and plays an important role in the resistance of cancer cells to chemotherapy. S100A8 is a member of the S100 calcium-binding protein family and plays an important role in the drug resistance of leukemia cells, with the mechanisms largely unknown. Here we report that S100A8 contributes to drug resistance in leukemia by promoting autophagy. S100A8 level was elevated in drug resistance leukemia cell lines relative to the nondrug resistant cell lines. Adriamycin and vincristine increased S100A8 in human leukemia cells, accompanied with upregulation of autophagy. RNA interference-mediated knockdown of S100A8 restored the chemosensitivity of leukemia cells, while overexpression of S100A8 enhanced drug resistance and increased autophagy. S100A8 physically interacted with the autophagy regulator BECN1 and was required for the formation of the BECN1-PI3KC3 complex. In addition, interaction between S100A8 and BECN1 relied upon the autophagic complex ULK1-mAtg13. Furthermore, we discovered that exogenous S100A8 induced autophagy, and RAGE was involved in exogenous S100A8-regulated autophagy. Our data demonstrated that S100A8 is involved in the development of chemoresistance in leukemia cells by regulating autophagy, and suggest that S100A8 may be a novel target for improving leukemia therapy.

Amphoterin, the major product of the high mobility group-1 gene, is a ligand associated with cancer invasion and metastasis through activation of the receptor for advanced glycation end products (RAGE). Expression of amphoterin and RAGE was examined in prostatectomy specimens from 40 patients with pT3 prostate cancer (18 non-metastatic and 22 metastatic) preoperatively treated with lutenizing hormone-releasing hormone (LH-RH) agonist. Amphoterin expression was detected in tumor cells of 6 (27%) metastatic and 0 non-metastatic cases (p<0.0001). Amphoterin was also detected in prostatic stromal cells of 14 (63%) metastatic cases and 2 (11%) non-metastatic cases (p=0.0010). RAGE production was detected in cancer cells of 16 (73%) metastatic and 6 (33%) non-metastatic cases (p=0.0244). A total of 2 (22%) non-metastatic and 16 (73%) metastatic cases showed co-expression of amphoterin and RAGE in tumor cells or in tumor cells and stromal cells (p=0.0001). The in vitro invasive capacity of PC-3, a prostatic cancer cell line that co-expressed amphoterin and RAGE, was suppressed by treatment with amphoterin antisense S-oligodeoxynucleotide (ODN). Primary cultured human prostatic stromal cells secreted no amphoterin; however, amphoterin secretion was induced by androgen deprivation. The conditioned medium of human prostatic stromal cells deprived of androgen recovered the in vitro invasive capacity of PC-3 cells suppressed by amphoterin antisense S-ODN. These results suggest that androgen deprivation provides a paracrine interaction between cancer and stromal cells through the RAGE-amphoterin system in advanced prostate cancer.

BACKGROUND

It is well known that diabetes mellitus is a major risk factor for vascular diseases such as atherosclerosis and restenosis after angioplasty. It has become clear that advanced glycation end products (AGE) and their receptor (RAGE) are implicated in vascular diseases, especially in diabetes mellitus. Nevertheless, the mechanisms by which diabetes mellitus is often associated with vascular diseases remain unclear.

RESULTS

To study the role of endogenous cytokines such as tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 in the development of vascular diseases and in the expression of RAGE, we used semapimod, a pharmacological inhibitor of cytokine production, and examined its effect on neointimal formation in the femoral artery of obese Zucker (OZ) rats. We also used an adenovirus construct expressing a dominant negative mutant of the receptor for TNF-alpha (AdTNFRDeltaC) to block the action of endogenous TNF-alpha. Semapimod significantly suppressed neointimal formation and RAGE expression in OZ rats compared with untreated OZ rats. This inhibitory effect of semapimod on neointimal formation was overcome by infection of an adenovirus expressing RAGE into the femoral artery of OZ rats. Furthermore, AdTNFRDeltaC infection significantly suppressed neointimal formation and RAGE expression in the femoral artery of OZ rats.

CONCLUSIONS

These results suggest that endogenous cytokines, especially TNF-alpha, were implicated in neointimal formation in OZ rats and that RAGE was a mediator of the effect of these cytokines on neointimal formation.

Glycation reactions resulting in the generation and accumulation of advanced glycation endproducts (AGEs) are potential mechanisms by which bone protein may be altered in vivo. AGEs accumulate in the bone increasingly with age come into close contact with osteoblasts or osteoclasts. The direct effect of AGEs on bone cells has not been thoroughly investigated. This study aimed to examine whether glycated bovine serum albumin (AGE - BSA) as an AGE modulate the mRNA expression of various genes in primary human osteoblast cultures. The following parameters were included: RAGE (receptor for AGEs), alkaline phosphatase, osteocalcin, osterix and RANKL (receptor activator of nuclear factor-kappaB ligand). Primary human osteoblast cultures were obtained from bone specimens of six patients with osteoarthrosis. Human osteoblasts were treated in AGE - BSA or control-BSA (non-glycated BSA) containing medium (5 mg/ml each) over a time course of seven days. After RT-PCR the mRNA expression was measured by real-time PCR. Related to control - BSA exposure, the mRNA expression of RAGE, RANKL and osterix increased during AGE - BSA treament. For alkaline phosphatase and osteocalcin a tendency of down-regulation was found. In summary, the study presents evidence that advanced glycation end products accumulated in bone alter osteoblasts by activation the AGE - RAGE pathway (RAGE mRNA up-regulation), inducing enhanced osteoclastogenesis (RANKL mRNA up-regulation) and impaired matrix mineralization (down-regulation of alkaline phosphatase and osteocalcin mRNA). Thus, AGEs may play a functional role in the development of bone diseases (e.g. osteoporosis).

At the concentrations normally found in the brain extracellular space the glial-derived protein, S100B, protects neurons against neurotoxic agents by interacting with the receptor for advanced glycation end products (RAGE). It is known that at relatively high concentrations S100B is neurotoxic causing neuronal death via excessive stimulation of RAGE. S100B is detected within senile plaques in Alzheimer's disease, where its role is unknown. The present study was undertaken to evaluate a putative neuroprotective role of S100B against Abeta amyloid-induced neurotoxicity. We treated LAN-5 neuroblastoma cultures with toxic amounts of Abeta25-35 amyloid peptide. Our results show that at nanomolar concentrations S100B protects cells against Abeta-mediated cytotoxicity, as assessed by 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and terminal deoxynucleotidyl transferase-mediated dUTP-fluorescein isothiocyanate nick end-labeling (TUNEL) experiments, by countering the Abeta-mediated decrease in the expression of the anti-apoptotic factor Bcl-2. This effect depends on S100B binding to RAGE because S100B is unable to contrast Abeta-mediated neurotoxicity in neurons overexpressing a signaling-deficient RAGE mutant lacking the cytosolic and transducing domain. Our data suggest that at nanomolar doses S100B counteracts Abeta peptide neurotoxicity in a RAGE-mediated manner. However, at micromolar doses S100B is toxic to LAN-5 cells and its toxicity adds to that of the Abeta peptide, suggesting that additional molecular mechanisms may be involved in the neurotoxic process.

BACKGROUND

The proinflammatory cytokine S100A12 (also known as EN-RAGE) is associated with cardiovascular morbidity and mortality in hemodialysis patients. In the current study, we tested the hypothesis that S100A12 expressed in vascular smooth muscle in nonatherosclerosis-prone C57BL/6J mice on normal rodent chow diet, but exposed to the metabolic changes of chronic kidney disease (CKD), would develop vascular disease resembling that observed in patients with CKD.

METHODS

CKD was induced in S100A12 transgenic mice and wild-type littermate mice not expressing human S100A12 by surgical ligation of the ureters. The aorta was analyzed after 7 weeks of elevated BUN (blood urea nitrogen), and cultured aortic smooth muscle cells were studied.

RESULTS

We found enhanced vascular medial calcification in S100A12tg mice subjected to CKD. Vascular calcification was mediated, at least in part, by activation of the receptor for S100A12, RAGE (receptor for advanced glycation endproducts), and by enhanced oxidative stress, since inhibition of NADPH-oxidase Nox1 and limited access of S100A12 to RAGE attenuated the calcification and gene expression of osteoblastic genes in cultured vascular smooth muscle cells.

CONCLUSIONS

S100A12 augments CKD-triggered osteogenesis in murine vasculature, reminiscent of features associated with enhanced vascular calcification in patients with chronic and end-stage kidney disease.

Angiotensin II (Ang II), a vasoactive peptide that is also considered a growth factor, has been implicated in both normal and diabetic cellular proliferation. We recently found that activation of janus kinase 2 (JAK2) is essential for the Ang II-induced proliferation of vascular smooth muscle cells (VSMCs) and that high glucose augments Ang II-induced proliferation of VSMCs by increasing signal transduction through activation of JAK2. Here, we demonstrate that S100B, a ligand for the receptor of advanced glycation end products (RAGEs), augmented both Ang II-induced tyrosine phosphorylation of JAK2 and cell proliferation in VSMCs in a receptor-dependent manner. We also found that S100B-RAGE interaction triggered intracellular generation of reactive oxygen species (ROS), VSMC proliferation, and JAK2 tyrosine phosphorylation via activation of phospholipase D (PLD)2. These results provide direct evidence for linkages between PLD2, ROS production, and S100B-RAGE-induced enhancement of Ang II-induced cell proliferation and activation of JAK2 in VSMCs.

Accumulating evidence has shown a strong relationship between Alzheimer's disease (AD), cerebral amyloid angiopathy (CAA), and cerebrovascular disease. Cognitive impairment in AD patients can result from cortical microinfarcts associated with CAA, as well as the synaptic and neuronal disturbances caused by cerebral accumulations of β-amyloid (Aβ) and tau proteins. The pathophysiology of AD may lead to a toxic chain of events consisting of Aβ overproduction, impaired Aβ clearance, and brain ischemia. Insufficient removal of Aβ leads to development of CAA and plays a crucial role in sporadic AD cases, implicating promotion of Aβ clearance as an important therapeutic strategy. Aβ is mainly eliminated by three mechanisms: (1) enzymatic/glial degradation, (2) transcytotic delivery, and (3) perivascular drainage (3-"d" mechanisms). Enzymatic degradation may be facilitated by activation of Aβ-degrading enzymes such as neprilysin, angiotensin-converting enzyme, and insulin-degrading enzyme. Transcytotic delivery can be promoted by inhibition of the receptor for advanced glycation end products (RAGE), which mediates transcytotic influx of circulating Aβ into brain. Successful use of the RAGE inhibitor TTP488 in Phase II testing has led to a Phase III clinical trial for AD patients. The perivascular drainage system seems to be driven by motive force generated by cerebral arterial pulsations, suggesting that vasoactive drugs can facilitate Aβ clearance. One of the drugs promoting this system is cilostazol, a selective inhibitor of type 3 phosphodiesterase. The clearance of fluorescent soluble Aβ tracers was significantly enhanced in cilostazol-treated CAA model mice. Given that the balance between Aβ synthesis and clearance determines brain Aβ accumulation, and that Aβ is cleared by several pathways stated above, multi-drugs combination therapy could provide a mainstream cure for sporadic AD.

Engagement of the receptor for advanced glycation end products (RAGE) by its signal transduction ligands is implicated in the development and progression of atherosclerosis. TNFalpha, a proinflammatory cytokine, is a potent inducer of RAGE expression in endothelial cells. In the present study, we demonstrate that reactive oxygen species (ROS) generated by TNFalpha stimulated human umbilical vein endothelial cells (HUVECs) induce RAGE expression. The complex III of mitochondrial respiratory chain appears to be the primary source of ROS. The gp91phox subunit of NADPH oxidase appears to be the source of ROS that induces TNFalpha-dependent mitochondrial ROS generation and subsequent RAGE expression. We also demonstrate that the ROS-mediated RAGE induction occurs via activation of NF-kappaB, a proinflammatory transcription factor. Thus, stimulation of HUVECs by TNFalpha evokes the following sequence of events: stimulation of NADPH oxidase ->> generation of ROS ->> activation of the mitochondrial respiratory chain ->> stimulation of NF-kappaB activity ->> induction of RAGE expression.

It is generally believed that some inflammatory antigens can recognize Toll-like receptors on synovial fibroblasts (SFs) and then activate downstream signals, leading to the formation of RASFs and inducing rheumatoid arthritis (RA). The objective of the current work was to study on the hypothesis that outer PAMP (LPS) binds to the inner DAMP (HMGB1) and becomes a complex that recognizes TLRs/RAGE on SFs, thus initiating a signaling cascade that leads to the secretion of inflammatory cytokines and chemokines, production of tissue-destructive enzymes, and formation of RASFs, finally resulting in RA. Osteoarthritis synovial fibroblasts (OASFs) were co-cultured with HMGB1-LPS complex in vitro for five generations to induce the transformation of human SFs to RA-like SFs (tOASFs). Then, changes of tOASFs in cell cycle and apoptosis-autophagy balance were investigated in vitro, and the pathogenicity of tOASFs was evaluated in a SCID mouse model in vivo. In vitro cell cycle analysis showed more tOASFs passing through the G1/S checkpoint and moving to S or G2 phase. Flow cytometry and confocal microscopy showed that apoptosis was reduced and autophagy was enhanced significantly in tOASFs as compared with those in OASFs. The expression of certain receptors and adhesion molecules in tOASFs was upregulated. In vivo experiments showed that tOASFs attached to, invaded, and degraded the co-implanted cartilage. In addition, histochemistry showed excessive proliferation of tOASFs and the expression of matrix metalloproteinases (MMPs). Based on the above findings, we conclude that HMGB1-LPS complex could promote the formation of RASFs.

RAGE, a receptor for advanced glycation endproducts, is an immunoglobulin-like cell surface receptor that is often described as a pattern recognition receptor due to the structural heterogeneity of its ligand. RAGE is an important cellular cofactor for amyloid beta-peptide (Abeta)-mediated cellular perturbation relevant to the pathogenesis of Alzheimer's disease (AD). The interaction of RAGE with Abeta in neurons, microglia, and vascular cells accelerates and amplifies deleterious effects on neuronal and synaptic function. RAGE-dependent signaling contributes to Abeta-mediated amyloid pathology and cognitive dysfunction observed in the AD mouse model. Blockade of RAGE significantly attenuates neuronal and synaptic injury. In this review, we summarize the role of RAGE in the pathogenesis of AD, specifically in Abeta-induced cellular perturbation.

OBJECTIVE

Receptor for advanced glycation end products (RAGE) serves as a pattern recognition receptor for several endogenous ligands that are potent inducers of inflammation. By activating endothelial cells and leukocytes, RAGE augments recruitment of leukocytes to sites of inflammation, which is a key process, especially in vasculitis. Soluble RAGE (sRAGE) acts as a naturally occurring inhibitor of RAGE by neutralizing proinflammatory ligands, e.g., S100A12. This neutrophil-derived protein has been reported to be associated with Kawasaki disease (KD) and to provoke proinflammatory responses. The aim of this study was to investigate circulating sRAGE in an acute inflammatory disorder and to compare these data directly with concentrations of the proinflammatory RAGE ligand S100A12.

METHODS

Serum concentrations of sRAGE and S100A12 were analyzed by specific enzyme-linked immunosorbent assays in 50 children with KD, and additionally in 39 patients with juvenile idiopathic arthritis (JIA). In 28 of the patients with KD, levels were analyzed longitudinally over the course of the disease.

RESULTS

Patients with KD and those with systemic-onset JIA had decreased levels of sRAGE during active disease, especially those patients with KD who were more severely affected and not responding to treatment. In addition, the level of sRAGE correlated negatively with the level of proinflammatory S100A12. After intravenous immunoglobulin (IVIG) therapy in patients with KD, the S100A12:sRAGE ratio was significantly different between responders and nonresponders.

CONCLUSIONS

Inverse regulation of both sRAGE and its proinflammatory ligand S100A12 seems to be a relevant molecular mechanism promoting systemic inflammation. Calculating the S100A12:sRAGE ratio might help to detect patients with KD who are at risk of being unresponsive to IVIG therapy.

During normal aging and amyloid beta-peptide (Abeta) disorders such as Alzheimer's disease (AD), one finds increased deposition of Abeta and activated monocytes/microglial cells in the brain. Our previous studies show that Abeta interaction with a monolayer of normal human brain microvascular endothelial cells results in increased adherence and transmigration of monocytes. Relatively little is known of the role of Abeta accumulated in the AD brain in mediating trafficking of peripheral blood monocytes (PBM) across the blood-brain barrier (BBB) and concomitant accumulation of monocytes/microglia in the AD brain. In this study, we showed that interaction of Abeta(1--40) with apical surface of monolayer of brain endothelial cells (BEC), derived either from normal or AD individuals, resulted in increased transendothelial migration of monocytic cells (HL-60 and THP-1) and PBM. However, transmigration of monocytes across the BEC monolayer cultivated in a Transwell chamber was increased 2.5-fold when Abeta was added to the basolateral side of AD compared with normal individual BEC. The Abeta-induced transmigration of monocytes was inhibited in both normal and AD-BEC by antibodies to the putative Abeta receptor, receptor for advanced glycation end products (RAGE), and to the endothelial cell junction molecule, platelet-endothelial cell adhesion molecule-1 (PECAM-1). We conclude that interaction of Abeta with the basolateral surface of AD-BEC induces cellular signaling, promoting transmigration of monocytes from the apical to basolateral direction. We suggest that Abeta in the AD brain parenchyma or cerebrovasculature initiates cellular signaling that induces PBM to transmigrate across the BBB and accumulate in the brain.

HIF1α and NFkB are two transcription factors very frequently activated in tumors and involved in tumor growth, progression, and resistance to chemotherapy. In fact, HIF1α and NFkB together regulate transcription of over a thousand genes that, in turn, control vital cellular processes such as adaptation to the hypoxia, metabolic reprograming, inflammatory reparative response, extracellular matrix digestion, migration and invasion, adhesion, etc. Because of this wide involvement they could control in an integrated manner the origin of the malignant phenotype. Interestingly, hypoxia and inflammation have been sequentially bridged in tumors by the discovery that alarmin receptors genes such as RAGE, P2X7, and some TLRs, are activated by HIF1α; and that, in turn, alarmin receptors strongly activate NFkB and proinflammatory gene expression, evidencing all the hallmarks of the malignant phenotype. Recently, a large number of drugs have been identified that inhibit one or both transcription factors with promising results in terms of controlling tumor progression. In addition, many of these molecules are natural compounds or off-label drugs already used to cure other pathologies. Some of them are undergoing clinical trials and soon they will be used alone or in combination with standard anti-tumoral agents to achieve a better treatment of tumors with reduction of metastasis formation and, more importantly, with a net increase in survival. This review highlights the central role of HIF1α activated in hypoxic regions of the tumor, of NFkB activation and proinflammatory gene expression in transformed cells to understand their progression toward malignancy. Different molecules and strategies to inhibit these transcription factors will be reviewed. Finally, the central role of a new class of deacetylases called Sirtuins in regulating HIF1α and NFkB activity will be outlined.