Gray matter abnormalities have been found in anorexia nervosa (AN) in several brain regions. However, little is known about white matter abnormalities under the condition of AN. To comprehensively assess the microstructural integrity of white matter pathways in women with anorexia nervosa, we performed voxel-based Diffusion Tensor Imaging (DTI). 21 women with AN according to DSM-IV criteria (9 of them recovered) and 20 female age-matched healthy control subjects were enrolled in the study. The patients had a mean body mass index of 17.2 kg/m(2) (controls: 19.6 kg/m(2)). High resolution T1 images (MP-RAGE) and DTI were performed on a 3 T Siemens-scanner. Images were pre-processed and analyzed using a modified protocol for DTI in SPM2. Fractional anisotropy (FA) maps were compared using t-tests (p < 0.05, corrected). Compared with controls, AN patients showed bilateral reductions of FA maps in the posterior thalamic radiation which includes the optic radiation, and the left mediodorsal thalamus. Our study is limited by the small sample size and its cross-sectional design. A longitudinal design with the same individuals assessed when acutely ill and recovered is warranted for future studies. For the first time, the findings of our DTI study identified disturbances of associational and commissural fibers in the bilateral occipitotemporal white matter. The results help narrowing the prevailing biological models of AN by suggesting that body image distortion is related to microstructural alterations of white matter tracts connecting the extrastriate visual cortex with other brain regions involved in body perception.

RAGE (receptor for advanced glycation end products) is a multi-ligand receptor that belongs to the immunoglobulin superfamily of transmembrane proteins. RAGE binds AGEs (advanced glycation end products), HMGB1 (high-mobility group box-1; also designated as amphoterin), members of the S100 protein family, glycosaminoglycans and amyloid β peptides. Recent studies using tools of structural biology have started to unravel common molecular patterns in the diverse set of ligands recognized by RAGE. The distal Ig domain (V1 domain) of RAGE has a positively charged patch, the geometry of which fits to anionic surfaces displayed at least in a proportion of RAGE ligands. Association of RAGE to itself, to HSPGs (heparan sulfate proteoglycans), and to Toll-like receptors in the cell membrane plays a key role in cell signaling initiated by RAGE ligation. Ligation of RAGE activates cell signaling pathways that regulate migration of several cell types. Furthermore, RAGE ligation has profound effects on the transcriptional profile of cells. RAGE signaling has been mainly studied as a pathogenetic factor of several diseases, where acute or chronic inflammation plays a role. Recent studies have suggested a physiological role for RAGE in normal lung function and in neuronal signaling.

BACKGROUND

Reduction in the deposition of amyloid β (Aβ) has been the primary target for Alzheimer's disease (AD) therapeutics recently, but in clinical trials this approach has generally been unsuccessful. A common feature of AD pathology is a complex inflammatory component that could be a target for treatment. One feature of this inflammation has been the involvement of the receptor for advanced glycation endproducts (RAGE), whose ligands include advanced glycation-endproduct-modified proteins as well as lipids and Aβ, which are found at elevated levels in AD brains.

METHODS

In this article, the authors describe the key features of RAGE and how it could have a role in AD pathogenesis. They also summarize experimental animal and clinical data that demonstrate the therapeutic effect of RAGE inhibition and consider what these findings mean for human disease.

CONCLUSIONS

RAGE has multiple ligands, including Aβ, that are increased in AD brains. Inhibiting RAGE-ligand interactions without activating receptor signaling can reduce multiple pathological pathways relevant for AD. Several RAGE inhibitors and modulators are now being tested as therapeutics for AD. Recent Phase II studies have established the good safety and tolerability of TTP448 with some evidence of positive benefit at lower dose. This suggests that further studies are required.

Alzheimer's disease (AD), one of the most dreaded neurodegenerative disorders, is characterized by cortical and cerebrovascular amyloid β peptide (Aβ) deposits, neurofibrillary tangles, chronic inflammation, and neuronal loss. Increased bone fracture rates and reduced bone density are commonly observed in patients with AD, suggesting one or more common denominators between both disorders. However, very few studies are available that have addressed this issue. Here, we present evidence for a function of amyloid precursor protein (APP) and Aβ in regulating osteoclast (OC) differentiation in vitro and in vivo. Tg2576 mice, which express the Swedish mutation of APP (APPswe) under the control of a prion promoter, exhibit biphasic effects on OC activation, with an increase of OCs in younger mice (< 4 months old), but a decrease in older Tg2576 mice >> 4 months old). The increase of OCs in young Tg2576 mice appears to be mediated by Aβ oligomers and receptor for advanced glycation end products (RAGE) expression in bone marrow macrophages (BMMs). However, the decrease of OC formation and activity in older Tg2576 mice may be due to the increase of soluble rage (sRAGE) in aged Tg2576 mice, an inhibitor of RANKL-induced osteoclastogenesis. These results suggest an unexpected function of APPswe/Aβ, reveal a mechanism underlying altered bone remodeling in AD patients, and implicate APP/Aβ and RAGE as common denominators for both AD and osteoporosis.

Apoptotic cells trigger immune tolerance in engulfing phagocytes. This poorly understood process is believed to contribute to the severe immunosuppression and increased susceptibility to nosocomial infections observed in critically ill sepsis patients. Extracellular high mobility group box 1 (HMGB1) is an important mediator of both sepsis lethality and the induction of immune tolerance by apoptotic cells. We have found that HMGB1 is sensitive to processing by caspase-1, resulting in the production of a fragment within its N-terminal DNA-binding domain (the A-box) that signals through the receptor for advanced glycation end products (RAGE) to reverse apoptosis-induced tolerance. In a two-hit mouse model of sepsis, we show that tolerance to a secondary infection and its associated mortality were effectively reversed by active immunization with dendritic cells treated with HMGB1 or the A-box fragment, but not a noncleavable form of HMGB1. These findings represent a novel link between caspase-1 and HMGB1, with potential therapeutic implications in infectious and inflammatory diseases.

HMGB1 is an architectural chromatin-binding protein that can be released actively by activated cells or passively by dying cells and can serve as a DAMP molecule to drive the pathogenesis of inflammatory and angiogenic diseases. Through TLR4 and RAGE signaling pathways, HMGB1 could regulate vascular growth in vivo and in vitro through diverse mechanisms, including induction of proangiogenic cytokine release and activation of ECs, macrophages, EPCs, and mesoangioblasts, all of which could contribute to vessel formation. Accordingly, HMGB1 plays a significant role in many angiogenesis-related conditions, such as tumors, PDR, wound-healing, and ischemia-induced angiogenesis. In this review, we focus on the regulatory role of HMGB1 in angiogenesis and recent progress in therapeutic strategies targeting HMGB1.

An increase in the interaction between advanced glycation end-products (AGEs) and their receptor RAGE is believed to contribute to the pathogenesis of chronic complications of Diabetes mellitus, which can include bone alterations such as osteopenia. We have recently found that extracellular AGEs can directly regulate the growth and development of rat osteosarcoma UMR106 cells, and of mouse calvaria-derived MC3T3E1 osteoblasts throughout their successive developmental stages (proliferation, differentiation and mineralisation), possibly by the recognition of AGEs moieties by specific osteoblastic receptors which are present in both cell lines. In the present study we examined the possible expression of RAGE by UMR106 and MC3T3E1 osteoblastic cells, by immunoblot analysis. We also investigated whether short-, medium- or long-term exposure of osteoblasts to extracellular AGEs, could modify their affinity constant and maximal binding for AGEs (by 125I-AGE-BSA binding experiments), their expression of RAGE (by immunoblot analysis) and the activation status of the osteoblastic ERK 1/2 signal transduction mechanism (by immunoblot analysis for ERK and P-ERK). Our results show that both osteoblastic cell lines express readily detectable levels of RAGE. Short-term exposure of phenotypically mature osteoblastic UMR106 cells to AGEs decrease the cellular density of AGE-binding sites while increasing the affinity of these sites for AGEs. This culture condition also dose-dependently increased the expression of RAGE and the activation of ERK. In proliferating MC3T3E1 pre-osteoblasts, 24-72 h exposure to AGEs did not modify expression of RAGE, ERK activation or the cellular density of AGE-binding sites. However, it did change the affinity of these binding sites forAGEs, with both higher- and lower-affinity sites now being apparent. Medium-term ( 1 week) incubation of differentiated MC3T3E1 osteoblasts with AGEs, induced a simultaneous increase in RAGE expression and in the relative amount of P-ERK. Mineralising MC3T3E1 cultures grown for 3 weeks in the presence of extracellular AGEs showed a decrease both in RAGE and P-ERK expression. These results indicate that, in phenotypically mature osteoblastic cells, changes in ERK activation closely follow the AGEs-induced regulation of RAGE expression. Thus, the AGEs-induced biological effects that we have observed previously in osteoblasts, could be mediated by RAGE in the later stages of development, and mediated by other AGE receptors in the earlier pre-osteoblastic stage.

BACKGROUND

Self injurious behaviour (SIB), the deliberate, repetitive infliction of self harm, is present in a wide variety of neuropsychiatric disorders, including Tourette syndrome (TS). Although SIB occurs in up to 60% of individuals with TS, and can cause significant clinical impairment and distress, little is known about its aetiology.

OBJECTIVE

This study examined the relationship between SIB and other behavioural features that commonly co-occur with TS in nearly 300 subjects with TS participating in three genetic studies. SIB, obsessions, compulsions, tic severity, attention deficit hyperactivity disorder related impulsivity, risk taking behaviours, and rages were systematically assessed in all subjects.

METHODS

Using logistic regression, a best fit model was determined for both mild to moderate SIB and severe SIB.

RESULTS

Mild/moderate SIB in TS was correlated with the presence of obsessive and compulsive symptoms such as the presence of aggressive obsessions or violent or aggressive compulsions, and with the presence of obsessive-compulsive disorder and overall number of obsessions. Severe SIB in TS was correlated with variables related to affect or impulse dysregulation; in particular, with the presence of episodic rages and risk taking behaviours. Both mild/moderate and severe SIB were also correlated with tic severity.

CONCLUSIONS

This study suggests that mild/moderate and severe SIB in TS may represent different phenomena, which has implications for clinical management of these symptoms.

Uric acid (UA) is a risk factor for endothelial dysfunction, a process in which inflammation may play an important role. UA increases high mobility group box chromosomal protein 1 (HMGB1) expression and extracellular release in endothelial cells. HMGB1 is an inflammatory cytokine that interacts with the receptor for advanced glycation end products (RAGE), inducing an oxidative stress and inflammatory response, which leads to endothelial dysfunction. In this study, human umbilical vein endothelial cells (HUVECs) were incubated with a high concentration of UA (20 mg/dL) after which endothelial function and the expression of HMGB1, RAGE, nuclear factor kappa B (NF-κB), inflammatory cytokines, and adhesion molecules were evaluated. UA inhibited endothelial nitric oxide synthase (eNOS) expression and nitric oxide (NO) production in HUVECs, increased intracellular HMGB1 expression and extracellular HMGB1 secretion, and upregulated RAGE expression. UA also activated NF-κB and increased the level of inflammatory cytokines. Blocking RAGE significantly suppressed the upregulation of RAGE and HMGB1 and prevented the increase in DNA binding activity of NF-κB and the levels of inflammatory cytokines. It also blocked the decrease in eNOS expression and NO production induced by UA. Our results suggest that high concentrations of UA cause endothelial dysfunction via the HMGB1/RAGE signaling pathway.

The aim of the present study was to investigate in vivo the effect of high-mobility group box 1 protein (HMGB1) on activity of regulatory T cells (Tregs) and the influence on T-cell-mediated immunity after thermal injury. Male Wistar rats were randomly divided into four groups as follows: sham burn group, burn group, burn with ethyl pyruvate treatment group, and burn with antireceptor for advanced glycation end products (RAGE) antibody treatment group, and they were killed on postburn days 1, 3, 5, and 7, respectively, with eight animals at each time point. Magnetic cell sorting microbeads were used to isolate splenic Tregs and a column of nylon wool to obtain T cells. Phenotypes, including cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4), forkhead/winged helix transcription factor p3 (Foxp3), RAGE, and IL-2Ralpha, were analyzed by flow cytometry. Levels of HMGB1, IL-10, IL-2, IL-4 and interferon gamma were determined by enzyme-linked immunosorbent assay kits, and real-time reverse transcription-polymerase chain reaction was performed to detect mRNA expression of IL-10, IL-2, and IL-2Ralpha. Serum HMGB1 levels were significantly elevated during postburn days 1 to 7. In the burn group, CTLA-4 and Foxp3 expression levels of Tregs were strongly enhanced in comparison to the sham-injured group, and the capacity of Tregs to produce IL-10 was markedly increased. Administration of ethyl pyruvate to inhibit HMGB1 or anti-RAGE antibody could significantly decrease expression levels of CTLA-4, Foxp3 on Tregs, and IL-10 production after burns. Simultaneously, proliferative activity and expression levels of IL-2 and IL-2Ralpha of T cell were restored. The excessively released HMGB1 might stimulate CD4+CD25+Treg activity via binding RAGE on the surface of Tregs and trigger a shift of T(H)1 to T(H)2 with suppression of T-lymphocyte immune function after burn injury.

The word "Anna" was spoken by 12 female and 11 male subjects with six different emotional expressions: "rage/hot anger," "despair/lamentation," "contempt/disgust," "joyful surprise," "voluptuous enjoyment/sensual satisfaction," and "affection/tenderness." In an acoustical analysis, 94 parameters were extracted from the speech samples and broken down by correlation analysis to 15 parameters entering subsequent statistical tests. The results show that each emotion can be characterized by a specific acoustic profile, differentiating that emotion significantly from all others. If aversive emotions are tested against hedonistic emotions as a group, it turns out that the best indicator of aversiveness is the ratio of peak frequency (frequency with the highest amplitude) to fundamental frequency, followed by the peak frequency, the percentage of time segments with nonharmonic structure ("noise"), frequency range within single time segments, and time of the maximum of the peak frequency within the utterance. Only the last parameter, however, codes aversiveness independent of the loudness of an utterance.

A nuclear protein, high mobility group box 1 (HMGB1), is released passively by necrotic cells and actively by macrophages/monocytes in response to exogenous and endogenous inflammatory stimuli. After binding to the receptor for advanced glycation end products (RAGE), or Toll-like receptor 4 (TLR4), HMGB1 activates macrophages/monocytes to express proinflammatory cytokines, chemokines, and adhesion molecules. Pharmacological suppression of its activities or release is protective against lethal endotoxemia and sepsis, establishing HMGB1 as a critical mediator of lethal systemic inflammation. In light of observations that many viruses (e.g., West Nile virus, Salmon anemia virus) can induce passive HMGB1 release, we propose a potential pathogenic role of HMGB1 in viral infectious diseases.

OBJECTIVE

To assess the involvement of the AGE-specific receptor (AGER, also known as RAGE) axis and nuclear factor kappa-B (NFKB, also known as NF-kappaB) activation in the development of lacrimal gland and tear film dysfunction in diabetes, the present study evaluated: (1) lacrimal gland and tear film alterations in diabetic rats; and (2) the expression of AGE, AGER and NFKB in ocular tissues of normoglycaemic and diabetic rats.

METHODS

Diabetes was induced in male Wistar rats with intravenous streptozotocin. Tear secretion parameters were measured and NFKB expression was evaluated in lacrimal glands of control and diabetic rats by western blot. Immunohistochemistry with confocal microscopy was used to assess AGE, AGER and NFKB expression in lacrimal glands of both groups.

RESULTS

Lacrimal gland weight and tear film volume were lower in diabetic than in control rats (p=0.01 and 0.02, respectively). IL1B and TNF concentrations in tears were higher in diabetic than in control rats (p=0.007 and 0.02, respectively). NFKB protein was identified in rat cornea, conjunctiva and lacrimal glands. AGE, AGER and NFKB expression were greater in lacrimal glands of diabetic than in those of control rats.

CONCLUSIONS

Diabetes induces significant alterations in rat lacrimal gland structure and secretion. The higher expression of AGE, AGER and NFKB in lacrimal glands of diabetic rats suggests that these factors are involved in signalling and in subsequent inflammatory alterations related to dry eye in diabetes mellitus.

High-mobility group box 1 (HMGB1) protein is a nuclear DNA-binding protein, which functions as an alarmin when released from cells. Recent studies implicate extracellular HMGB1 in the pathogenesis of systemic lupus erythematosus (SLE), a prototypical autoimmune disease characterized by the formation of multiple autoantibodies, especially those directed against nucleosomes and double-stranded (ds)DNA. Elevated concentrations of HMGB1 are observed in sera as well as in skin lesions of patients with lupus. Of importance, serum HMGB1 and anti-HMGB1 autoantibody levels correlate with disease activity. In the blood of patients with SLE, HMGB1 is complexed with nucleosomes, at least partially. Moreover, HMGB1-nucleosome complexes from apoptotic cells activate antigen-presenting cells. Injection of HMGB1-nucleosome complexes into nonautoimmune mice results in the formation of autoantibodies against dsDNA and histones in a Toll-like receptor (TLR) 2-dependent manner. Additionally, HMGB1, as a part of DNA-anti-DNA immune complexes, can interact with receptor for advanced glycation end products (RAGE) on the surface of plasmacytoid dendritic cells and B cells leading to TLR9-dependent interferon (IFN)α release and activation of autoreactive B cells, respectively. HMGB1 attached to neutrophil extracellular traps may contribute to IFNα production by facilitating the recognition of self-nucleic acids. Furthermore, HMGB1, complexed with DNA and pathogenic anti-DNA autoantibodies, activates its receptors, TLR2, TLR4 and RAGE, and may thereby be involved in anti-DNA autoantibody-induced kidney damage in lupus nephritis. Collectively, these findings suggest that HMGB1 is a potential marker of disease activity and, because of its probable involvement in the pathogenesis, a novel therapeutic target in SLE.

Non-enzymatic modification of proteins by reducing sugars, a process that is also known as Maillard reaction, leads to the formation of advanced glycation end products (AGEs) in vivo. It is now well established that formation and accumulation of AGEs progress during normal aging, and at an extremely accelerated rate under diabetes, thus being implicated in various types of AGE-related disorders such as diabetic vascular complications, neurodegenerative diseases and cancers. Further, there is accumulating evidence that AGEs and their receptor RAGE interaction elicits oxidative stress generation and subsequently alters gene expression in various types of cells. In addition, digested food-derived AGEs are found to play an important role in the pathogenesis of the AGE-related disorders as well. Indeed, restriction of diet-derived AGEs not only blocks the progression of atherosclerosis and renal injury, but also improves insulin resistance in animal models. AGE-poor diets reduce serum levels of inflammatory biomarkers in patients with diabetes or chronic renal failure. These observations suggest that the restriction of food-derived AGEs or the inhibition of absorption of dietary AGEs may be a novel target for therapeutic intervention in the AGE-related disorders. In this paper, we review the pathological role of food-derived AGEs in various types of disorders and discuss the potential utility of oral adsorbent that inhibits the absorption of AGEs in these devastating diseases.

The aim of this study was the evaluation of the normal sellar anatomy in vitro and in vivo with high-field MRI and its application in the diagnosis of sellar pathologies in comparison to standard MRI. All high-field MR images were obtained using a 3T Bruker Medspec 30/80 Scanner with a head birdcage transmit/receive coil and an actively shielded gradient system with a maximum gradient strength of 45 mT/m. Firstly an in vitro study of the sella turcica was performed to depict normal pituitary and sellar anatomy at high field. After a pilot-study this sequence-protocol was established: A RARE sequence (TR/TE = 7790/19 ms; matrix size, 512 x 512; RARE factor = 8, FOV, 200 mm) was used for T2-weighted coronal, axial and sagittal images. A 3D gradient echo sequence with magnetization-preparation (MP-RAGE, TR/TE/TI = 33.5/7.6/800 ms, matrix size, 512 x 512; FOV, 200 mm, effective slice thickness, 1.88 mm; 3 averages) was used for acquisition of T1-weighted pre- and post-contrast images. Between January 2002 and March 200458 patients were enrolled in this study. Seven patients were examined for suspected microadenoma and in 51 patients 3T MRI was used to obtain additional information about the sellar lesion already known to be present from standard MRI. In 21 cases the accuracy of the imaging findings was assessed afterwards by comparison with intraoperative findings. The infiltration of the medial cavernous sinus wall was suspected on standard MRI on 15 sides (47%), on high-field MRI on 9 sides (28%) and could be verified by intraoperative findings on 6 sides (19%). Accordingly, sensitivity to infiltration was 83% for 3T and 67% for standard MRI. Specificity was 84% for 3T and 58% for standard MRI. Moreover, high-field MRI revealed microadenomas in 7 patients with a median diameter of 4mm (range 2-9 mm). The segments of the cranial nerves were seen as mean 4 hypointense spots (range 2-5 spots) on high-field MRI in contrast to 3 spots (range 0-4 spots) on standard MRI. This difference was considerably significant (P < 0.001, Wilcoxon rank sum test). The histopathological results revealed pituitary adenoma in 16 patients and non-adenomatous sellar pathologies such as Rathke's cleft cyst, sarcoidosis, meningeoma and metastasis in 5 patients. High-field MRI is superior to standard MRI for the prediction of invasion of adjacent structures in patients with pituitary adenomas and improves surgical planning of sellar lesion.

Diabetes, is a metabolic disorder characterised by chronic hyperglycaemia, hypertension, dyslipidaemia, microalbuminuria and inflammation. Moreover, there are a number of complications associated with this condition including retinopathy, neuropathy and nephropathy. Diabetic nephropathy, is the major cause of end-stage renal disease in Western societies affecting a substantial proportion (25-40%) of patients with diabetes. Advanced glycation end products (AGEs) have been identified as important modulators of the development and progression of diabetic nephropathy, through both receptor dependant and independent interactions. AGEs elicit their receptor mediated effects via their engagement with numerous receptors and binding proteins which are broadly thought to be either inflammatory (RAGE and AGE-R2) or clearance receptors (AGE-R1, AGE-R3, CD36, Scr-II, FEEL-1 and FEEL-2). Modulation of AGE receptor expression is an important potential therapeutic approach worth consideration as a treatment for diabetic nephropathy and likely applicable to other vascular complications.

OBJECTIVE

The receptor for advanced glycation end products (RAGE) is expressed at high levels in the lung, particularly in type 1 alveolar cells, and has been shown to amplify injury triggered by acute stress. Previous studies suggest serum concentrations of soluble RAGE increase during pulmonary reperfusion injury after transplantation. RAGE blockade has been shown to suppress hepatic and cardiac ischemia and reperfusion injury in mice. Thus we tested the hypothesis that RAGE mediates tissue-injury mechanisms in ischemia and reperfusion injury in the lung.

METHODS

C57BL/6 mice were subjected to 30 minutes of pulmonary ischemia by clamping the left hilum, followed by 60 minutes of reperfusion. Lung function was assessed by means of blood gas analysis, and capillary leak was assessed by injecting fluorescein isothiocyanate-labeled albumin and comparing fluorescence in bronchial lavage fluid with that in serum. Histologic analysis of the lung was performed by a pathologist naive to the experimental conditions.

RESULTS

In animals subjected to RAGE blockade, significant increases in Po(2) (108 vs 73 mm Hg, P = .0094) and more than 3-fold decrease in capillary leak Relative Fluorescent Units (RFU, 6.12 vs 1.75; P = .001) were observed. Histologic examination revealed significant injury reduction in soluble RAGE-treated animals versus control animals. RAGE knockout mice exhibited a protected phenotype when exposed to pulmonary ischemia and reperfusion. Additionally, interleukin 8 production and nuclear factor kappaB activation were increased in control mice.

CONCLUSIONS

Abrogation of RAGE signaling attenuates pulmonary ischemia and reperfusion injury. This study suggests that RAGE might play a central role in pulmonary reperfusion injury and in transplantation and that blockade of RAGE might offer a potential target to abrogate pulmonary reperfusion injury in clinical transplantation.

OBJECTIVE

To investigate the effect of glyceraldehyde-derived advanced glycation end-products (Glycer-AGEs) on hepatocellular carcinoma (HCC) cells.

METHODS

Two HCC cell lines (Hep3B and HepG2 cells) and human umbilical vein endothelial cells (HUVEC) were used. Cell viability was determined using the WST-8 assay. Western blotting, enzyme linked immunosorbent assay, and real-time reverse transcription-polymerase chain reactions were used to detect protein and mRNA. Angiogenesis was evaluated by assessing the proliferation, migration, and tube formation of HUVEC.

RESULTS

The receptor for AGEs (RAGE) protein was detected in Hep3B and HepG2 cells. HepG2 cells were not affected by the addition of Glycer-AGEs. Glycer-AGEs markedly increased vascular endothelial growth factor (VEGF) mRNA and protein expression, which is one of the most potent angiogenic factors. Compared with the control unglycated bovine serum albumin (BSA) treatment, VEGF mRNA expression levels induced by the Glycer-AGEs treatment were 1.00 ± 0.10 vs 1.92 ± 0.09 (P < 0.01). Similarly, protein expression levels induced by the Glycer-AGEs treatment were 1.63 ± 0.04 ng/mL vs 2.28 ± 0.17 ng/mL for the 24 h treatment and 3.36 ± 0.10 ng/mL vs 4.79 ± 0.31 ng/mL for the 48 h treatment, respectively (P < 0.01). Furthermore, compared with the effect of the control unglycated BSA-treated conditioned medium, the Glycer-AGEs-treated conditioned medium significantly increased the proliferation, migration, and tube formation of HUVEC, with values of 122.4% ± 9.0% vs 144.5% ± 11.3% for cell viability, 4.29 ± 1.53 vs 6.78 ± 1.84 for migration indices, and 71.0 ± 7.5 vs 112.4 ± 8.0 for the number of branching points, respectively (P < 0.01).

CONCLUSIONS

These results suggest that Glycer-AGEs-RAGE signaling enhances the angiogenic potential of HCC cells by upregulating VEGF expression.

OBJECTIVE

Vascular remodeling diseases (VRD) are mainly characterized by inflammation and a vascular smooth muscle cells (VSMCs) proproliferative and anti-apoptotic phenotype. Recently, the activation of the advanced glycation endproducts receptor (RAGE) has been shown to promote VSMC proliferation and resistance to apoptosis in VRD in a signal transducer and activator of transcription (STAT)3-dependant manner. Interestingly, we previously described in both cancer and VRD that the sustainability of this proproliferative and antiapoptotic phenotype requires activation of the transcription factor NFAT (nuclear factor of activated T-cells). In cancer, NFAT activation is dependent of the oncoprotein provirus integration site for Moloney murine leukemia virus (Pim1), which is regulated by STAT3 and activated in VRD. Therefore, we hypothesized that RAGE/STAT3 activation in VSMC activates Pim1, promoting NFAT and thus VSMC proliferation and resistance to apoptosis. Methods/Results- In vitro, freshly isolated human carotid VSMCs exposed to RAGE activator Nε-(carboxymethyl)lysine (CML) for 48 hours had (1) activated STAT3 (increased P-STAT3/STAT3 ratio and P-STAT3 nuclear translocation); (2) increased STAT3-dependent Pim1 expression resulting in NFATc1 activation; and (3) increased Pim1/NFAT-dependent VSMC proliferation (PCNA, Ki67) and resistance to mitochondrial-dependent apoptosis (TMRM, Annexin V, TUNEL). Similarly to RAGE inhibition (small interfering RNA [siRNA]), Pim1, STAT3 and NFATc1 inhibition (siRNA) reversed these abnormalities in human carotid VSMC. Moreover, carotid artery VSMCs isolated from Pim1 knockout mice were resistant to CML-induced VSMC proliferation and resistance to apoptosis. In vivo, RAGE inhibition decreases STAT3/Pim1/NFAT activation, reversing vascular remodeling in the rat carotid artery-injured model.

CONCLUSIONS

RAGE activation accounts for many features of VRD including VSMC proliferation and resistance to apoptosis by the activation of STAT3/Pim1/NFAT axis. Molecules aimed to inhibit RAGE could be of a great therapeutic interest for the treatment of VRD.

Heart failure is an increasingly prevalent disorder with considerable morbidity and mortality. Although many causal mechanisms such as inherited cardiomyopathies, ischemic cardiomyopathy, or muscular overload are easily identified in clinical practice, the events that determine the progression of cardiac injury to heart failure and adverse ventricular remodeling are still unclear. Yet there is compelling evidence that inflammatory mechanisms contribute to the progression of heart failure. High-mobility group box-1 (HMGB1) is a newly recognized potent innate "danger signal" that is released by necrotic cells and by activated immune cells. HMGB1 signals via the receptor for advanced glycation end-product (RAGE) and members of the toll-like receptor (TLR) family. We have demonstrated an important role for HMGB1 and RAGE in the pathogenesis of early- and late-phase complications following ischemia/reperfusion (I/R) injury of the heart. In addition, enhanced postmyocardial infarction remodeling in type 1 diabetes mellitus was partially mediated by HMGB1 activation. We propose that the interaction of HMGB1 and RAGE is a key component initiating and sustaining the inflammatory response in inflammatory cardiomyopathy eventually leading to heart failure. Thus HMGB1-antagonizing gene therapy represents a new therapeutic strategy.

BACKGROUND

The receptor for advanced glycation end-products (RAGE) is highly expressed in the lung, where it is believed to have a homeostatic role. Reduced plasma levels of soluble RAGE (sRAGE) have been reported in patients with chronic obstructive pulmonary disease (COPD). The aim of the present study was to evaluate the association of plasma sRAGE levels with a longitudinal decline of lung function. We have also measured plasma levels of high mobility group box 1 (HMGB1), a RAGE ligand which has been associated with chronic inflammatory diseases including COPD.

METHODS

Baseline plasma concentrations of sRAGE and HMGB1 were measured in non-smokers (n = 32), smokers without COPD (n = 212), and smokers with COPD (n = 51), and the associations of the plasma sRAGE and HMGB1 levels with longitudinal declines of lung function during a 4-year follow-up period were analysed.

RESULTS

The plasma levels of sRAGE were significantly lower in smokers without COPD and in smokers with COPD, as compared to those of non-smokers. Plasma sRAGE levels positively correlated with FVC and FEV1 and inversely correlated with BMI and pack-years. Lower sRAGE levels were associated with greater declines of FEV1/FVC over 4 years in all participants. Moreover, multivariate regression analysis indicated that the baseline plasma sRAGE concentration was an independent predictor of FEV1/FVC decline in all groups. A subgroup analysis showed that decreased sRAGE levels are significantly associated with a more rapid decline of FEV1/FVC in smokers with COPD. There was no significant correlation between plasma HMGB1 levels and longitudinal decline of lung function.

CONCLUSIONS

Lower plasma concentrations of sRAGE were associated with greater progression of airflow limitations over time, especially in smokers with COPD, suggesting that RAGE might have a protective role in the lung.

OBJECTIVE

Severe injury to the liver, such as that induced by toxic doses of acetaminophen, triggers a cascade of events leading to hepatocyte death. It is hypothesized that activation of the receptor for advanced glycation end products (RAGE) might contribute to acetaminophen-induced liver toxicity by virtue of its ability to generate reactive oxygen species, at least in part via nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, and thereby activate downstream signaling pathways leading to cellular injury.

METHODS

A model was employed in which toxic doses of acetaminophen (1125 mg/kg) were administered to C57BL/6 mice. To block RAGE, mice received murine soluble (s) RAGE, the extracellular ligand binding domain of the receptor that acts as a decoy to interrupt ligand-RAGE signaling.

RESULTS

Animals treated with sRAGE displayed increased survival compared with vehicle treatment, and markedly decreased hepatic necrosis. Consistent with an important role for RAGE-triggered oxidant stress in acetaminophen-induced injury, a significant reduction of nitrotyrosine protein adducts was observed in hepatic tissue in sRAGE-treated versus vehicle-treated mice receiving acetaminophen, in parallel with significantly increased levels of glutathione. In addition, pro-regenerative cytokines tumor necrosis factor-alpha and interleukin-6 were increased in sRAGE-treated versus vehicle-treated mice.

CONCLUSIONS

These findings implicate RAGE-dependent mechanisms in acetaminophen-induced liver damage and suggest that blockade of this pathway may impart beneficial effects in toxin-induced liver injury.

OBJECTIVE

Polymyositis and dermatomyositis are characterised by muscle weakness and fatigue even in patients with normal muscle histology via unresolved pathogenic mechanisms. In this study, we investigated the mechanisms by which high mobility group box protein 1 (HMGB1) acts to accelerate muscle fatigue development.

METHODS

Intact single fibres were dissociated from flexor digitorum brevis (FDB) of wild type, receptor for advanced glycation endproduct (RAGE) knockout and toll like receptor 4 (TLR4) knockout mice and cultured in the absence or presence of recombinant HMGB1. A decrease in sarcoplasmic reticulum Ca(2+) release during a series of 300 tetanic contractions, which reflects the development of muscle fatigue, was determined by measuring myoplasmic free tetanic Ca(2+). TLR4 and major histocompatibility complex (MHC)-class I expression in mouse FDB fibres were investigated by immunofluorescence and confocal microscopy. Immunohistochemistry was used to investigate TLR4, MHC-class I and myosin heavy chain expression in muscle fibres of patients.

RESULTS

Our results demonstrate that TLR4 is expressed in human and mouse skeletal muscle fibres, and coexpressed with MHC-class I in muscle fibres of patients with myositis. Furthermore, we show that HMGB1 acts via TLR4 but not RAGE to accelerate muscle fatigue and to induce MHC-class I expression in vitro. In order to bind and signal via TLR4, HMGB1 must have a reduced cysteine 106 and a disulphide linkage between cysteine 23 and 45.

CONCLUSIONS

The HMGB1-TLR4 pathway may play an important role in causing muscle fatigue in patients with polymyositis or dermatomyositis and thus is a potential novel target for future therapy.