Levels Of Receptor For Advanced Glycation End-products Research Articles

Hippocampal RAGE immunoreactivity in early and advanced Alzheimer's disease

Microvascular accumulation and neuronal overproduction of amyloid-β peptide (Aβ) are pathologic features of Alzheimer's disease (AD). In this study, we examined the receptor for advanced glycation endproducts (RAGE), a multi-ligand receptor found in both neurons and cerebral microvascular endothelia that binds Aβ. RAGE expression was assessed in aged controls ( n = 6), patients with early AD-like pathology ( n = 6), and severe, Braak V–VI AD ( n = 6). Human hippocampi were stained with a specific polyclonal antibody directed against RAGE (Research Diagnostics, Flanders, NJ). Immunoreactivity was localized in both neurons and cerebral endothelial cells. Quantitative image-analyses were performed on grayscale images to assess the total surface area of endothelial RAGE immunoreaction product in cross sections of cerebral microvessels (5–20 µm). Confocal images were acquired for confirmation of RAGE immunoreactivity in both microvessels and neurons by coupling RAGE with CD-31 and neurofilament, respectively. A significant increase in endothelial RAGE immunoreactivity was found in severe Braak V–VI AD patients when compared to aged controls ( p < 0.001), and when compared to patients with early AD pathology ( p = 0.0125). In addition, a significant increase in endothelial RAGE immunoreactivity was witnessed when comparing aged controls having no reported AD pathology with patients having early AD-like pathology ( p = 0.038). Our data suggest that microvascular RAGE levels increase in conjunction with the onset of AD, and continue to increase linearly as a function of AD pathologic severity ( p < 0.0001).

The Pro12Ala polymorphism of peroxisome proliferator-activated receptor-γ2 gene is associated with plasma levels of soluble RAGE (Receptor for Advanced Glycation Endproducts) and the presence of peripheral arterial disease

Objectives Recent evidences suggest that the activation of peroxisome proliferator-activated receptor (PPAR)-γ2, which plays an important role in vascular homeostasis, also regulates the expression of the Receptor for Advanced Glycation End products (RAGE). In turn, low levels of soluble RAGE (sRAGE) have recently emerged as a valuable biomarker of vascular inflammation. The potential alterations in sRAGE concentrations in peripheral arterial disease (PAD), however, have not been yet investigated. The aim of the present study was to clarify whether the Pro12Ala polymorphism of the PPAR-γ2 gene is related to plasma sRAGE levels and the presence of PAD in nondiabetic Italian individuals. Design and methods A total of 201 patients with PAD and 201 PAD-free control subjects were investigated. Genotyping of the Pro12Ala polymorphism of the PPAR-γ2 gene was performed by means of PCR-RFLPs. Plasma sRAGE levels were determined by ELISA. Results Subjects carrying at least one Ala12 allele of the PPAR-γ2 gene had lower sRAGE levels (all p values < 0.001). The prevalence rate of the Ala12 allele was significantly higher in PAD patients (14.0%) than in controls (8.0%, p = 0.009). In multivariate logistic regression analysis after adjustment for potential confounders, the Ala12 allele was significantly and independently associated with the risk of PAD (OR = 1.57, 95% CI = 1.11–2.65, p = 0.021). Conclusions Our data indicate that the Ala12 allele of the PPAR-γ2 gene is associated with lower levels of the soluble decoy receptor sRAGE and the presence of PAD.

Ovariectomy increases neuronal amyloid-β binding alcohol dehydrogenase level in the mouse hippocampus

Ovarian hormone decline after menopause may influence cognitive performance and increase the risk for Alzheimer's disease (AD) in women. Amyloid-β peptide (Aβ) has been proposed to be the primary cause of AD. In this study, we examined whether ovariectomy (OVX) could affect the levels of cofactors Aβ-binding alcohol dehydrogenase (ABAD) and receptor for advanced glycation endproducts (RAGE), which have been reported to potentiate Aβ-mediated neuronal perturbation, in mouse hippocampus, correlating with estrogen and Aβ levels. Female ICR mice were randomly divided into ovariectomized or sham-operated groups, and biochemical analyses were carried out at 5 weeks after the operation. OVX for 5 weeks significantly decreased hippocampal 17β-estradiol level, while it tended to reduce the hormone level in serum, compared with the sham-operated control. In contrast, OVX did not affect hippocampal Aβ 1-40 level, although it significantly increased serum Aβ 1-40 level. Furthermore, we demonstrated that OVX increased hippocampal ABAD level in neurons, but not astrocytes, while it did not affect RAGE level. These findings suggest that the expression of neuronal ABAD depends on estrogen level in the hippocampus and the increase in serum Aβ and hippocampal ABAD induced by ovarian hormone decline may be associated with pre-stage of memory deficit in postmenopausal women and Aβ-mediated AD pathology.

A Novel Function of the Receptor for Advanced Glycation End-Products (RAGE) in Association with Tumorigenesis and Tumor Differentiation of HCC

BackgroundThe expression of the receptor for advanced glycation end products (RAGE) has an impact on the mechanisms giving rise to characteristic features of various cancer cells. The purpose of this study was to elucidate the clinicopathological relevance of the level of RAGE expression in patients with hepatocellular carcinoma (HCC) and to explore the effect of RAGE expression on the characteristic features of HCC.MethodsThe expression of RAGE was assessed in paired cancer and noncancerous tissues with HCC, using reverse-transcription polymerase chain reaction (RT-PCR), and immunohistochemistry. The quantitative RT-PCR data were analyzed in association with the clinicopathological factors of the patients with HCC. In in vitro experiments, the survival of RAGE-transfected Cos7 and mock-transfected Cos7 cells was compared under hypoxic conditions. In addition, after reducing RAGE levels in RAGE-transfected Cos7 cells by siRNA, similar experiments were performed.ResultsThe expression of RAGE mRNA was lower in normal liver than in hepatitis and highest in HCC. Furthermore, in HCC, it was high in well- and moderately differentiated tumors but declined as tumors dedifferentiated to poorly differentiated HCC. Furthermore, HCC lines resistant to hypoxia were found to have higher levels of RAGE expression, and RAGE transfectant also showed significantly prolonged survival under hypoxia.ConclusionsOur results suggest that HCC during the early stage of tumorigenesis with less blood supply may acquire resistance to stringent hypoxic milieu by hypoxia-induced RAGE expression.

Poster 238: RAGE, Inflammation, and Temporomandibular Joint Disorders

Temporomandibular Joint Disorders (TMDs) are a common cause of facial pain. Although the etiology of TMDs is yet to be fully elucidated, histologic and arthroscopic studies strongly suggest that the inflammatory process is similar to that seen in osteoarthritis of other synovial joints. The Receptor for Advanced Glycation Endproducts (RAGE) is a multiligand receptor that is present on cells of the monocyte/macrophage lineage, vascular cells, and many tumor cells. RAGE is expressed at low levels in most normal tissues and is upregulated where its ligands are present. This receptor binds multiple ligands including a number of Advanced Glycation Endproducts (AGEs), S100/calgranulins, proinflammatory cytokines, MAC-1, a leukocyte beta-2 integrin receptor, and amphoterin, also known as HMGB1, a DNA binding protein released during cell necrosis, resulting in activation of a number of critical cell signaling pathways as well as the generation of reactive oxygen species. It appears that RAGE is emerging as a key inflammatory mediator. In order to investigate the role that RAGE and its ligands play in TMD inflammation we have utilized a transgenic murine model that overexpressses human Tumor Necrosis Factor-alpha (tg hTNF-alpha), resulting in progressive inflammation and destruction of the synovial joints. Furthermore, to test the impact of genetic deletion of RAGE these transgenic animals were breed into the RAGE null background. Animals were sacrificed at 3, 5, and 11 months of age. The temporomandibular joints (TMJ) and knee joints were examined by H & E staining, immunocytochemistry, western blot analysis for RAGE and S100b protein, and ELISA for Interleukin-1 beta (IL-1 bet&cjs0744;a and AGEs). Age and sex-matched C57BL animals and RAGE null animals served as controls. Analysis was by ANOVA and the Tukey-Kramer poc-hoc test was performed for all pair-wise comparisons. Significance was set at p < 0.05. Transgenic animals showed progressive inflammation and joint destruction with increased age, although there were significant differences between the TMJ and knee joints. At 11 months knee joints showed a large amount of destruction and synovitis. While the TMJ had some synovitis, there was only minimal articular destruction. Histological examination showed a more aggressive disease pattern in affected females compared to males in both TMJ and knee joints. RAGE staining correlated with disease progression although there was greater intensity in the knee joints. This was confirmed by western blot analysis. A significant increase in S100b levels was found in tg hTNF-alpha mice compared to control C57B6 mice. Transgenic hTNF-alpha/RAGE null mice had baseline levels of both RAGE and S100b. TNF-alpha mice expressed significantly higher levels of AGEs than control mice. Interestingly, AGE levels were significantly increased in many of the tg hTNF-alpha/RAGE null samples compared with TNF-alpha mice. IL-1 beta ELISA results demonstrated that TNF-alpha affected samples had greater levels of IL-1 beta compared with control mice. However, TNF-alpha/RAGE null samples were comparable to controls. The tg hTNF-alpha mouse is a useful model of TMD inflammation. Affected animals showed significant inflammatory changes with more severe disease observed in the knee joints when compared to the TMJs. Furthermore, RAGE upregulation is critical in the arthritic progression as insertion of the RAGE null background into the TNF mouse decreases a number of inflammatory mediators to baseline levels. RAGE blockage may have therapeutic value in the treatment of TMDs and inflammatory arthridities.

Plasma Receptor for Advanced Glycation End-products Predicts Duration of ICU Stay and Mechanical Ventilation in Patients After Lung Transplantation

Primary graft dysfunction, formerly termed reperfusion pulmonary edema, is the leading cause of short-term complications after lung transplantation. New evidence shows that alveolar type I epithelial cells play an active role in alveolar fluid transport and are therefore presumed to be critical in the absorption of pulmonary edema. We tested the potential relevance of a novel marker of alveolar type I cell injury, the receptor for advanced glycation end-products (RAGE), to short-term outcomes of lung transplantation. The study was a prospective, observational cohort study of 20 patients undergoing single lung, bilateral lung or combined heart-lung transplantation. Plasma biomarkers were measured 4 hours after allograft reperfusion. Higher plasma RAGE levels were associated with a longer duration of mechanical ventilation and longer intensive care unit length of stay, in contrast to markers of alveolar type II cell injury, endothelial injury and acute inflammation. Specifically, for every doubling in plasma RAGE levels, the duration of mechanical ventilation increased on average by 26 hours, adjusting for ischemia time (95% confidence interval [CI] 7.4 to 44.7 hours, p = 0.01). Likewise, for every doubling of plasma RAGE levels, intensive care unit length of stay increased on average by 1.8 days, again adjusting for ischemia time (95% CI 0.13 to 3.45 days p = 0.04). In contrast, the clinical diagnosis of primary graft dysfunction was not as predictive of these short-term outcomes. Higher levels of plasma RAGE measured shortly after reperfusion predicted poor short-term outcomes from lung transplantation. Elevated plasma RAGE levels may have both pathogenetic and prognostic value in patients after lung transplantation.

Receptor for Advanced Glycation End-Products Is a Marker of Type I Cell Injury in Acute Lung Injury

Receptor for advanced glycation end-products (RAGE) is one of the alveolar type I cell-associated proteins in the lung. To test the hypothesis that RAGE is a marker of alveolar epithelial type I cell injury. Rats were instilled intratracheally with 10 mg/kg lipopolysaccharide or hydrochloric acid. RAGE levels were measured in the bronchoalveolar lavage (BAL) and serum in the rats and in the pulmonary edema fluid and plasma from patients with acute lung injury (ALI; n = 22) and hydrostatic pulmonary edema (n = 11). In the rat lung injury studies, RAGE was released into the BAL and serum as a single soluble isoform sized approximately 48 kD. The elevated levels of RAGE in the BAL correlated well with the severity of experimentally induced lung injury. In the human studies, the RAGE level in the pulmonary edema fluid was significantly higher than the plasma level (p < 0.0001). The median edema fluid/plasma ratio of RAGE levels was 105 (interquartile range, 55-243). The RAGE levels in the pulmonary edema fluid from patients with ALI were higher than the levels from patients with hydrostatic pulmonary edema (p < 0.05), and the plasma RAGE level in patients with ALI were significantly higher than the healthy volunteers (p < 0.001) or patients with hydrostatic pulmonary edema (p < 0.05). RAGE is a marker of type I alveolar epithelial cell injury based on experimental studies in rats and in patients with ALI.

Upregulation of RAGE and its ligands in proliferative retinal disease

We sought to study the presence of the receptor for advanced glycation endproducts (RAGE) and its ligands, advanced glycation endproducts (AGEs), S100/calgranulins and amphoterin (high mobility group box 1 protein; HMGB1), in the vitreous cavity and epiretinal membranes (ERMs) of eyes of patients with proliferative diabetic retinopathy (PDR) and proliferative vitreoretinopathy (PVR). Undiluted vitreous specimens were collected from 30 eyes of 30 patients undergoing pars plana vitrectomy for repair of retinal detachment (RD) secondary to PDR ( n=15) or PVR ( n=15). The vitreous samples obtained from 10 eyes undergoing macular hole repair were used as controls. Epiretinal membranes were obtained from eight eyes with PDR and from 10 eyes with PVR. The levels of AGEs in the vitreous were measured using ELISA. The vitreous levels of soluble RAGE (sRAGE), S100/calgranulins and amphoterin were measured using Western blot analyses. The localization of RAGE and its ligands in ERMs was determined with immunohistochemistry. The vitreous levels of sRAGE were significantly increased in both PDR and PVR ( p≤0.05) compared to control vitreous. In both PDR and PVR, the vitreous levels of AGEs ( p≤0.01), S100/calgranulins ( p≤0.05), and amphoterin ( p≤0.01) were also elevated compared to control eyes. Expression of RAGE was detected in six of eight ERMs from eyes with PDR and eight of 10 ERMs from eyes with PVR. Many cells expressing RAGE also expressed vimentin, suggesting a glial cell origin. Ligands for RAGE were also detected in ERMs, with AGEs detected in five eyes with PDR and eight eyes with PVR. Similarly, S100 and amphoterin ERM expression was observed in six eyes with PDR; these ligands were also expressed in ERMs from eyes with PVR (8 and 7 cases, respectively). We conclude that RAGE and its ligands are increased in the vitreous cavity of eyes with PDR and PVR and are present in ERMs of eyes with these proliferative retinal disorders. These findings suggest a role for the proinflammatory RAGE axis in the pathogenesis of proliferative retinal diseases.

Colon Cancer Cell-Derived High Mobility Group 1/Amphoterin Induces Growth Inhibition and Apoptosis in Macrophages

High mobility group (HMGB)1/amphoterin is a multifunctional cytokine involved in invasion and metastasis of cancer and in inflammation. To investigate HMGB1/amphoterin effects on macrophages, U937 human monocytic leukemia cells and rat peritoneal and human alveolar macrophages were examined. U937 cells expressed low levels of an HMGB1/amphoterin receptor, receptor for advanced glycation end-products (RAGE), whereas RAGE production was induced in differentiated phorbol 12-myristate 13-acetate (PMA)-U937 cells. Treatment with cultured medium of HMGB1/amphoterin-secreting WiDr human colon cancer cells showed growth inhibition of both U937 and PMA-U937 cells and apoptosis in PMA-U937 cells. The number of PMA-U937 cells was markedly decreased by co-culture with WiDr cells exposed to HMGB1/amphoterin sense S-oligodeoxynucleotide (ODN) in spheroids or monolayers. In contrast, PMA-U937 cells co-cultured with WiDr cells exposed to HMGB1/amphoterin anti-sense S-ODN were preserved in number. PMA-U937 cells exposed to RAGE anti-sense S-ODN were insensitive to WiDr-cultured medium. Recombinant human HMGB1/amphoterin induced growth inhibition in thioglycollate-induced rat peritoneal macrophages, PMA-U937 cells, and human alveolar macrophages, an effect that was abrogated by absorption with anti-HMGB1 antibody. Phosphorylation of JNK and Rac1 was induced in PMA-U937 cells treated with HMGB1/amphoterin. These results suggest that HMGB1/amphoterin induces growth inhibition and apoptosis in macrophages through RAGE intracellular signaling pathway.