Overexpression of the receptor for advanced glycation end-products in the auditory cortex of rats with noise-induced hearing loss.

BACKGROUND: The receptor for advanced glycation end-products (RAGE) is involved in neuroinflammation. This study investigated the changes in RAGE expression following noise-induced hearing loss. METHODS: Three-week-old female Sprague-Dawley rats were exposed to 115 dB SPL white noise for 4 h daily for 3 d (noise group, n = 16). In parallel, age and sex-matched control rats were raised under standard conditions without noise exposure (control group, n = 16). After 2 h (noise immediate, n = 8) and 4 wk (noise 4-week, n = 8) of noise exposure, the auditory cortex was harvested and cytoplasmic and nuclear fractions were isolated. The gene expression levels of tumor necrosis factor alpha (TNF-α), interleukin 6 (IL6), interleukin 1 beta (IL1ß), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), and RAGE were evaluated using real-time reverse transcription polymerase chain reaction. The protein expression levels of nuclear RAGE and cytosolic RAGE were evaluated using western blotting. Additionally, matrix metalloproteinase 9 (MMP9) was pharmacologically inhibited in the noise immediate group, and then nuclear and cytosolic RAGE expression levels were evaluated. RESULTS: The noise immediate and noise 4-week groups exhibited increased auditory thresholds at 4, 8, 16, and 32 kHz frequencies. The genes encoding the pro-inflammatory cytokines TNF-α, IL6, IL1ß, and NF- κB were increased 3.74, 1.63, 6.42, and 6.23-fold in the noise immediate group, respectively (P = 0.047, 0.043, 0.044, and 0.041). RAGE mRNA expression was elevated 1.42-fold in the noise 4-week group (P = 0.032). Cytosolic RAGE expression was increased 1.76 and 6.99-fold in the noise immediate and noise 4-week groups, respectively (P = 0.04 and 0.03). Nuclear RAGE expression was comparable between the noise and control groups. matrix metalloproteinase 9 (MMP9) inhibition reduced cytosolic RAGE expression in the noise immediate group (P = 0.004). CONCLUSIONS: Noise exposure increased the expression of cytosolic RAGE in the auditory cortex and upregulated pro-inflammatory genes, but this response could be alleviated by MMP9 inhibition.

Receptor for advanced glycation end products up-regulation in cerebral endothelial cells mediates cerebrovascular-related amyloid ß accumulation after Porphyromonas gingivalis infection.

Cerebrovascular-related amyloidogenesis is found in over 80% of Alzheimer's disease (AD) cases, and amyloid ß (Aß) generation is increased in the peripheral macrophages during infection of Porphyromonas gingivalis (P. gingivalis), a causal bacterium for periodontitis. In this study, we focused on receptor for advanced glycation end products (RAGE), the key molecule involves in Aß influx after P. gingivalis infection to test our hypothesis that Aß transportation from periphery into the brain, known as "Aß influx," is enhanced by P. gingivalis infection. Using cultured hCMEC/D3 cell line, in comparison to uninfected cells, directly infection with P. gingivalis (multiplicity of infection, MOI = 5) significantly increased a time-dependent RAGE expression resulting in a dramatic increase in Aß influx in the hCMEC/D3 cells; the P. gingivalis-up-regulated RAGE expression was significantly decreased by NF-κB and Cathepsin B (CatB)-specific inhibitors, and the P.gingivalis-increased IκBα degradation was significantly decreased by CatB-specific inhibitor. Furthermore, the P. gingivalis-increased Aß influx was significantly reduced by RAGE-specific inhibitor. Using 15-month-old mice (C57BL/6JJmsSlc, female), in comparison to non-infection mice, systemic P. gingivalis infection for three consecutive weeks (1 × 108  CFU/mouse, every 3 days, intraperitoneally) significantly increased the RAGE expression in the CD31-positive endothelial cells and the Aß loads around the CD31-positive cells in the mice's brains. The RAGE expression in the CD31-positive cells was positively correlated with the Aß loads. These observations demonstrate that the up-regulated RAGE expression in cerebral endothelial cells mediates the Aß influx after P. gingivalis infection, and CatB plays a critical role in regulating the NF-κB/RAGE expression. Cover Image for this issue: https://doi.org/10.1111/jnc.15073.

Inhibiting HMGB1-RAGE axis prevents pro-inflammatory macrophages/microglia polarization and affords neuroprotection after spinal cord injury.

BACKGROUND: Spinal cord injury (SCI) favors a persistent pro-inflammatory macrophages/microglia-mediated response with only a transient appearance of anti-inflammatory phenotype of immune cells. However, the mechanisms controlling this special sterile inflammation after SCI are still not fully elucidated. It is known that damage-associated molecular patterns (DAMPs) released from necrotic cells after injury can trigger severe inflammation. High mobility group box 1(HMGB1), a ubiquitously expressed DNA binding protein, is an identified DAMP, and our previous study demonstrated that reactive astrocytes could undergo necroptosis and release HMGB1 after SCI in mice. The present study aimed to explore the effects and the possible mechanism of HMGB1on macrophages/microglia polarization, as well as the neuroprotective effects by HMGB1 inhibition after SCI. METHODS: In this study, the expression and the concentration of HMGB1 was determined by qRT-PCR, ELISA, and immunohistochemistry. Glycyrrhizin was applied to inhibit HMGB1, while FPS-ZM1 to suppress receptor for advanced glycation end products (RAGE). The polarization of macrophages/microglia in vitro and in vivo was detected by qRT-PCR, immunostaining, and western blot. The lesion area was detected by GFAP staining, while neuronal survival was examined by Nissl staining. Luxol fast blue (LFB) staining, DAB staining, and western blot were adopted to evaluate the myelin loss. Basso-Beattie-Bresnahan (BBB) scoring and rump-height Index (RHI) assay was applied to evaluate locomotor functional recovery. RESULTS: Our data showed that HMGB1 can be elevated and released from necroptotic astrocytes and HMGB1 could induce pro-inflammatory microglia through the RAGE-nuclear factor-kappa B (NF-κB) pathway. We further demonstrated that inhibiting HMGB1 or RAGE effectively decreased the numbers of detrimental pro-inflammatory macrophages/microglia while increased anti-inflammatory cells after SCI. Furthermore, our data showed that inhibiting HMGB1 or RAGE significantly decreased neuronal loss and demyelination, and improved functional recovery after SCI. CONCLUSIONS: The data implicated that HMGB1-RAGE axis contributed to the dominant pro-inflammatory macrophages/microglia-mediated pro-inflammatory response, and inhibiting this pathway afforded neuroprotection for SCI. Thus, therapies designed to modulate immune microenvironment based on this cascade might be a prospective treatment for SCI.

AGER1 downregulation associates with fibrosis in nonalcoholic steatohepatitis and type 2 diabetes.

Type 2 diabetes is clinically associated with progressive necroinflammation and fibrosis in nonalcoholic steatohepatitis (NASH). Advanced glycation end-products (AGEs) accumulate during prolonged hyperglycemia, but the mechanistic pathways that lead to accelerated liver fibrosis have not been well defined. In this study, we show that the AGEs clearance receptor AGER1 was downregulated in patients with NASH and diabetes and in our NASH models, whereas the proinflammatory receptor RAGE was induced. These findings were associated with necroinflammatory, fibrogenic, and pro-oxidant activity via the NADPH oxidase 4. Inhibition of AGEs or RAGE deletion in hepatocytes in vivo reversed these effects. We demonstrate that dysregulation of NRF2 by neddylation of cullin 3 was linked to AGER1 downregulation and that induction of NRF2 using an adeno-associated virus-mediated approach in hepatocytes in vivo reversed AGER1 downregulation, lowered the level of AGEs, and improved proinflammatory and fibrogenic responses in mice on a high AGEs diet. In patients with NASH and diabetes or insulin resistance, low AGER1 levels were associated with hepatocyte ballooning degeneration and ductular reaction. Collectively, prolonged exposure to AGEs in the liver promotes an AGER1/RAGE imbalance and consequent redox, inflammatory, and fibrogenic activity in NASH.

Effect of AGER on the biological behavior of non­small cell lung cancer H1299 cells.

Advanced glycosylation end-product specific receptor (AGER) is a multi-ligand cell surface receptor abnormally expressed in lung cancer, and is a member of the immunoglobulin superfamily. Therefore, this study aimed to explore the effect of AGER on the biological behavior of non­small cell lung cancer (NSCLC) H1299 cell line. A microarray­based gene expression profiling analysis of the GSE27262 dataset from the Gene Expression Omnibus (GEO) database was conducted to identify differentially expressed genes, which were verified using The Cancer Genome Atlas (TCGA) database. The expression of AGER in the normal human lung BEAS­2B cell line and NSCLC H1299 cell line was examined using reverse transcription­quantitative PCR. Lentiviral interference and overexpression vectors of AGER were constructed and transfected into H1299 cells using Lipofectamine®. AGER expression and biological properties, including cell viability, apoptosis, migration and invasion abilities, in H1299 cells were investigated using MTT, flow cytometry, wound healing and Transwell assays. AGER was expressed at a low level in NSCLC tissues and H1299 cells (P<0.05). Compared with control cells, AGER overexpression cells displayed decreased cell viability, proliferation, migration and invasion abilities, and significantly increased levels of apoptosis. Furthermore, AGER overexpression increased the expression of Bax and decreased the expression of Bcl­2 in H1299 cells (P<0.05), and AGER knockdown displayed the opposite effects on H1299 cells. Therefore, AGER overexpression decreased the proliferation, invasion and migration abilities of H1299 cells, and increased apoptosis. The present study suggested that AGER might serve as a potential molecular marker for NSCLC.

AGER expression and alternative splicing in bronchial biopsies of smokers and never smokers.

Cigarette smoking is one of the major risk factors for the development of chronic obstructive pulmonary disease (COPD). Evidence is accumulating that Receptor for Advanced Glycation-End products (RAGE)-signaling is a key pathway in the pathophysiology of COPD. To date, it is unknown how smoking affects RAGE expression. In the current study, we investigated the effect of smoking on AGER, the gene encoding RAGE, expression and on alternative splicing of AGER. To this end, we conducted RNA-Seq on bronchial biopsies for asymptomatic smokers (n = 36) and never smokers (n = 40). Total AGER gene expression was accessed using DESeq2, while alternative splicing was investigated by measuring the number of specific split reads spanning exon-exon junctions and the total split reads. One of the major isoforms of RAGE is endogenous soluble (es) RAGE, an anti-inflammatory decoy receptor, making up for approximately 10% of the total amount of soluble (s)RAGE. We found that smokers show decreased total gene expression of AGER in bronchial biopsies, while the relative abundance of the esRAGE isoform is increased. Furthermore, no difference in the serum levels of total sRAGE were observed between smokers and non-smokers. Our data indicates that smoking initiates a protective anti-inflammatory mechanism with decreased expression of the pro-inflammatory gene AGER and increased relative abundance of the anti-inflammatory isoform esRAGE.

Expression of soluble receptor for advanced glycation end products is associated with disease severity in congenital diaphragmatic hernia.

Pulmonary hypertension (PH) and lung hypoplasia are major contributors to morbidity and mortality in newborns with congenital diaphragmatic hernia (CDH). The soluble receptor for advanced glycation end products (sRAGE) is a marker of endothelial function and might be associated with disease severity in CDH newborns. In a cohort of 30 CDH newborns and 20 healthy control newborns, sRAGE concentration was measured at birth and at 6 h, 12 h, 24 h, 48 h, and 7-10 days. In healthy newborns, sRAGE was significantly higher at birth and at 48 h compared with CDH newborns (both P < 0.001). Among CDH newborns, sRAGE was significantly lower at birth (P = 0.033) and at 7-10 days (P = 0.035) in patients receiving extracorporeal membrane oxygenation (ECMO) compared with patients not receiving ECMO. In contrast, CDH newborns receiving ECMO had significantly higher values at 6 h (P = 0.001), 12 h (P = 0.004), and 48 h (0.032). Additionally, sRAGE correlated significantly with PH severity, intensity and duration of mechanical ventilation, and prenatally assessed markers of CDH severity (lung size, liver herniation). The probability to receive ECMO therapy was five times higher in CDH newborns with sRAGE concentrations below the calculated cutoff of 650 pg/ml at birth (P = 0.002) and nine times higher in CDH newborns with sRAGE concentrations above the cutoff of 3,500 pg/ml at 6 h (P = 0.001). These findings suggest a potential involvement of sRAGE in the pathophysiology of CDH and may act as a therapeutic target in future treatment approaches.

HMGB1/RAGE Mediates UVB-Induced Secretory Inflammatory Response and Resistance to Apoptosis in Human Melanocytes.

The major modifiable risk factor in melanomagenesis is UV exposure and mutagenesis of melanocytes. Other UV-induced events that contribute to early tumorigenesis are poorly understood. Herein we show that the repeated exposure of human primary melanocytes to UVB results in a sustained senescence response, increases in expression of signal transducer and activator of transcription 1, MX1, OAS2, and IRF7 proteins of up to 75-fold, and resistance to subsequent UVB-induced apoptosis. In the setting of UVB-induced DNA damage, we detected time-dependent increases in the release of damage-associated molecular patterns such as high-mobility group box 1 (HMGB1). After intermittent UVB exposure, melanocytes treated with the JAK inhibitor ruxolitinib reduced expression of HMGB1 and MX1 as well as activation of JAK1 (pJAK1) and signal transducer and activator of transcription 1 (pSTAT1). In addition, melanocytes expressing small hairpin RNA selective for the HMGB1 receptor, receptor for advanced glycosylation end product (RAGE), exhibited decreased expression of both HMGB1 and MX1 after UVB exposure. The response of small hairpin RAGE-infected cells to human recombinant HMGB1 was blunted with decreased MX1 expression and JAK activation. Finally, depletion of receptor for advanced glycosylation end product decreased UVB-induced resistance to apoptosis (P < 0.05). These findings highlight a cell autonomous response to UV damage, contribute to their resistance to apoptosis and cell death, and may have implications for early stages of melanoma development.

Intermittent Hypoxia Enhances THP-1 Monocyte Adhesion and Chemotaxis and Promotes M1 Macrophage Polarization via RAGE.

Intermittent hypoxia (IH) that resulted from obstructive sleep apnea (OSA) has been found to be a risk factor of coronary artery disease. IH and the receptor for advanced glycation end products (RAGE) expression are known to activate monocyte/macrophage and associated with atherosclerosis development, while their effects on monocyte adhesion, chemotaxis to the endothelium, and macrophage polarization remain unknown. In the present study, RAGE in THP-1 monocytes was inhibited by shRNA lentiviral particles, followed by exposure to IH. Cell adhesion assay, transwell migration assay, and macrophage polarization assays were performed to study the effects of IH and RAGE. The mRNA and protein expression levels were investigated by RT/real-time PCR and western blot analysis, respectively. We found that IH increased RAGE expression and activated NF-кB signalling in THP-1 monocytes. The results also revealed that IH enhanced the MCP-1-mediated THP-1 monocyte adhesion and chemotaxis and promoted macrophage polarization toward a proinflammatory phenotype, which was mediated by RAGE activity. Additionally, inhibition of chemokine receptor type 2 (CCR2) suppressed the IH-induced monocyte adhesion and chemotaxis. These results demonstrated a potential role of monocyte adhesion, chemotaxis, and macrophage polarization in the development cardiovascular diseases induced by IH and identified that RAGE could be a promising therapeutic target to prevent atherosclerosis in patients with OSA.

Receptor for Advanced Glycation End Products (RAGE) is Expressed Predominantly in Medium Spiny Neurons of tgHD Rat Striatum.

Receptor for advanced glycation end products (RAGE) is a multi-ligand receptor involved in the pathology of several progressive neurodegenerative disorders including Huntington's disease (HD). We previously showed that the expression of RAGE and its colocalization with ligands were increased in the striatum of HD patients, increasing with grade severity, and that the pattern of RAGE expression coincided with the medio-lateral pattern of neurodegeneration. However, the exact role of RAGE in HD remains elusive. In order to address the necessity for a direct functional study, we aimed to characterize the pattern of RAGE expression in the transgenic rat model of HD (tgHD rats). Our results showed that RAGE expression was expanded laterally in tgHD rat caudate-putamen (CPu) compared to wildtype littermates, but the expression was unchanged by disease severity. The rostro-caudal location did not affect RAGE expression. RAGE was predominantly expressed in the medium spiny neurons (MSN) where it colocalized most extensively with N-carboxymethyllysine (CML), which largely contradicts with observations from human HD brains. Overall, the tgHD rat model only partially recapitulated the pattern in striatal RAGE expression in human brains, raising a question about its reliability as an animal model for future functional studies.

Inhibition of GLO1 in Glioblastoma Multiforme Increases DNA-AGEs, Stimulates RAGE Expression, and Inhibits Brain Tumor Growth in Orthotopic Mouse Models.

Cancers that exhibit the Warburg effect may elevate expression of glyoxylase 1 (GLO1) to detoxify the toxic glycolytic byproduct methylglyoxal (MG) and inhibit the formation of pro-apoptotic advanced glycation endproducts (AGEs). Inhibition of GLO1 in cancers that up-regulate glycolysis has been proposed as a therapeutic targeting strategy, but this approach has not been evaluated for glioblastoma multiforme (GBM), the most aggressive and difficult to treat malignancy of the brain. Elevated GLO1 expression in GBM was established in patient tumors and cell lines using bioinformatics tools and biochemical approaches. GLO1 inhibition in GBM cell lines and in an orthotopic xenograft GBM mouse model was examined using both small molecule and short hairpin RNA (shRNA) approaches. Inhibition of GLO1 with S-(p-bromobenzyl) glutathione dicyclopentyl ester (p-BrBzGSH(Cp)2) increased levels of the DNA-AGE N²-1-(carboxyethyl)-2'-deoxyguanosine (CEdG), a surrogate biomarker for nuclear MG exposure; substantially elevated expression of the immunoglobulin-like receptor for AGEs (RAGE); and induced apoptosis in GBM cell lines. Targeting GLO1 with shRNA similarly increased CEdG levels and RAGE expression, and was cytotoxic to glioma cells. Mice bearing orthotopic GBM xenografts treated systemically with p-BrBzGSH(Cp)2 exhibited tumor regression without significant off-target effects suggesting that GLO1 inhibition may have value in the therapeutic management of these drug-resistant tumors.

Chronic Arsenic Exposure Increases Aß(1-42) Production and Receptor for Advanced Glycation End Products Expression in Rat Brain.

Chronic arsenic exposure during development is associated with alterations of chemical transmission and demyelination, which result in cognitive deficits and peripheral neuropathies. At the cellular level, arsenic toxicity involves increased generation of reactive species that induce severe cellular alterations such as DNA fragmentation, apoptosis, and lipid peroxidation. It has been proposed that arsenic-associated neurodegeneration could evolve to Alzheimer disease in later life.1,2 In this study, the effects of chronic exposure to inorganic arsenic (3 ppm by drinking water) in Wistar rats on the production and elimination of Amyloid-ß (Aß) were evaluated. Male Wistar rats were exposed to 3 ppm of arsenic in drinking water from fetal development until 4 months of age. After behavioral deficits induced by arsenic exposure through contextual fear conditioning were verified, the brains were collected for the determination of total arsenic by inductively coupled plasma-mass spectrometry, the levels of amyloid precursor protein and receptor for advanced glycation end products (RAGE) by Western blot analysis as well as their transcript levels by RT-qPCR, Aß(1-42) estimation by ELISA assay and the enzymatic activity of ß-secretase (BACE1). Our results demonstrate that chronic arsenic exposure induces behavioral deficits accompanied of higher levels of soluble and membranal RAGE and the increase of Aß(1-42) cleaved. In addition, BACE1 enzymatic activity was increased, while immunoblot assays showed no differences in the low-density lipoprotein receptor-related protein 1 (LRP1) receptor among groups. These results provide evidence of the effects of arsenic exposure on the production of Aß(1-42) and cerebral amyloid clearance through RAGE in an in vivo model that displays behavioral alterations. This work supports the hypothesis that early exposure to metals may contribute to neurodegeneration associated with amyloid accumulation.

Adiponectin controls the apoptosis and the expression of tight junction proteins in brain endothelial cells through AdipoR1 under beta amyloid toxicity.

Alzheimer's disease (AD) is the most common neurodegenerative disease, characterized by excessive beta amyloid (Aß) deposition in brain, leading to blood-brain barrier (BBB) disruption. The mechanisms of BBB disruption in AD are still unclear, despite considerable research. The adipokine adiponectin is known to regulate various metabolic functions and reduce inflammation. Though adiponectin receptors have been reported in the brain, its role in the central nervous system has not been fully characterized. In the present study, we investigate whether adiponectin contributes to the tight junction integrity and cell death of brain endothelial cells under Aß-induced toxicity conditions. We measured the expression of adiponectin receptors (AdipoR1 and AdipoR2) and the alteration of tight junction proteins in in vivo 5xFAD mouse brain. Moreover, we examined the production of reactive oxygen species (ROS) and the loss of tight junction proteins such as Claudin 5, ZO-1, and inflammatory signaling in in vitro brain endothelial cells (bEnd.3 cells) under Aß toxicity. Our results showed that Acrp30 (a globular form of adiponectin) reduces the expression of proinflammatory cytokines and the expression of RAGE as Aß transporters into brain. Moreover, we found that Acrp 30 attenuated the apoptosis and the tight junction disruption through AdipoR1-mediated NF-κB pathway in Aß-exposed bEnd.3 cells. Thus, we suggest that adiponectin is an attractive therapeutic target for treating BBB breakdown in AD brain.

Receptor for advanced glycation end products is overexpressed in psoriatic plaques independent of disease severity.

BACKGROUND: Enhanced expression and excitation of the receptor for advanced glycation end products is considered to play a role in the regulation of many pro-inflammatory genes involved in the pathogenesis of psoriasis. AIM: We investigated the expression of receptor for advanced glycation end product in various cell types, in lesional and peri-lesional skin of patients with psoriasis, and its correlation with disease severity. METHODS: Paraffin-embedded punch biopsy tissue taken from psoriatic plaques and peri-lesional normal appearing skin tissue of twenty patients with psoriasis, and normal skin samples of eleven healthy participants, were enrolled in the study. The sections were stained immunohistochemically with anti-receptor for advanced glycation end product antibody. The intensity of receptor for advanced glycation end product expression was assessed semi-quantitatively on epidermal cells, microvascular endothelium, dermal fibroblasts and inflammatory cells. They were graded as follows: 0 (no staining), 1 (weak), 2 (moderate) and 3 (strong) intensity. RESULTS: Receptor for advanced glycation end product expression on epidermis, microvascular endothelium, inflammatory cells and fibroblasts in the psoriatic plaques was more intense than perilesional and normal tissue (all P < 0.05). It did not correlate with disease severity. LIMITATIONS: The main limitation of our study is that this was a semi-quantitative assessment, detected immunohistochemically in skin biopsies. CONCLUSION: Receptor for advanced glycation end product expression may have an important role in psoriasis pathogenesis, independent of disease severity.

Increased expression of damage-associated molecular patterns (DAMPs) in osteoarthritis of human knee joint compared to hip joint.

Osteoarthritis (OA) is a degenerative disease characterized by the destruction of cartilage. The greatest risk factors for the development of OA include age and obesity. Recent studies suggest the role of inflammation in the pathogenesis of OA. The two most common locations for OA to occur are in the knee and hip joints. The knee joint experiences more mechanical stress, cartilage degeneration, and inflammation than the hip joint. This could contribute to the increased incidence of OA in the knee joint. Damage-associated molecular patterns (DAMPs), including high-mobility group box-1, receptor for advanced glycation end products, and alarmins (S100A8 and S100A9), are released in the joint in response to stress-mediated chondrocyte and cartilage damage. This facilitates increased cartilage degradation and inflammation in the joint. Studies have documented the role of DAMPs in the pathogenesis of OA; however, the comparison of DAMPs and its influence on OA has not been discussed. In this study, we compared the DAMPs between OA knee and hip joints and found a significant difference in the levels of DAMPs expressed in the knee joint compared to the hip joint. The increased levels of DAMPs suggest a difference in the underlying pathogenesis of OA in the knee and the hip and highlights DAMPs as potential therapeutic targets for OA in the future.

Expression of the receptor for advanced glycation end products in acquired reactive perforating collagenosis.

BACKGROUND: Acquired reactive perforating collagenosis (ARPC) is a rare skin disorder characterized by transepidermal elimination of dermal collagen. There is little data regarding the pathogenesis of ARPC. The receptor for advanced glycation end products (RAGE) is a multiligand transmembrane receptor that plays an important role in inflammatory responses and may be involved in the pathogenesis of ARPC. AIM: To explore the expression of RAGE in ARPC. METHODS: Paraffin-embedded punch biopsy specimens of 41 patients with ARPC and of 11 healthy controls with normal skin were obtained from the Department Of Pathology. Clinical data of all patients were reviewed from the medical files. All specimens were stained immunohistochemically with antibody to RAGE (Anti-RAGE). The intensity of RAGE expression was assessed semi-quantitatively on epidermal cells, microvascular endothelium, dermal fibroblasts and inflammatory cells and graded as 0 (no staining), 1 (weak), 2 (moderate) and 3 (strong). The patients were divided into diabetic and nondiabetic groups for analysis. RESULTS: RAGE expression in microvascular endothelium, inflammatory cells and fibroblasts of patients with ARPC was more intense than normal tissues of healthy participants (P values are 0.005, 0.017 and P > 0.05). LIMITATIONS: Our method of assessment of RAGE expression was semi-quantitative. CONCLUSION: We observed an overexpression of RAGE in lesional samples of patients with ARPC which was independent of the presence of diabetes.

The effects of physical exercise on nonmotor symptoms and on neuroimmune RAGE network in experimental parkinsonism.

Parkinson's disease (PD) prodromal stages comprise neuropsychiatric perturbations that critically compromise a patient's quality of life. These nonmotor symptoms (NMS) are associated with exacerbated innate immunity, a hallmark of overt PD. Physical exercise (PE) has the potential to improve neuropsychiatric deficits and to modulate immune network including receptor for advanced glycation end products (RAGE) and Toll-like receptors (TLRs) in distinct pathological settings. Accordingly, the present study aimed to test the hypothesis that PE 1) alleviates PD NMS and 2) modulates neuroimmune RAGE network in experimental PD. Adult Wistar rats subjected to long-term mild treadmill were administered intranasally with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and probed for PD NMS before the onset of motor abnormalities. Twelve days after MPTP, neuroimmune RAGE network transcriptomics (real-time quantitative PCR) was analyzed in frontal cortex, hippocampus, and striatum. Untrained MPTP animals displayed habit-learning and motivational deficits without gross motor impairments (cued version of water-maze, splash, and open-field tests, respectively). A suppression of RAGE and neuroimmune-related genes was observed in frontal cortex on chemical and physical stressors (untrained MPTP: RAGE, TLR5 and -7, and p22 NADPH oxidase; saline-trained animals: RAGE, TLR1 and -5 to -11, TNF-α, IL-1ß, and p22 NADPH oxidase), suggesting the recruitment of compensatory mechanisms to restrain innate inflammation. Notably, trained MPTP animals displayed normal cognitive/motivational performances. Additionally, these animals showed normal RAGE expression and neuroprotective PD-related DJ-1 gene upregulation in frontal cortex when compared with untrained MPTP animals. These findings corroborate PE efficacy in improving PD NMS and newly identify RAGE network as a neural substrate for exercise intervention. Additional research is warranted to unveil functional consequences of PE-induced modulation of RAGE/DJ-1 transcriptomics in PD premotor stages.NEW & NOTEWORTHY This study newly shows that physical exercise (PE) corrects nonmotor symptoms of the intranasal 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model of experimental parkinsonism. Additionally, we show that suppression of neuroimmune receptor for advanced glycation end products (RAGE) network occurs in frontal cortex on chemical (MPTP) and physical (PE) interventions. Finally, PE normalizes frontal cortical RAGE transcriptomics and upregulates the neuroprotective DJ-1 gene in the intranasal MPTP model of experimental parkinsonism.

HMGB1 attenuates TGF-ß-induced epithelial-mesenchymal transition of FaDu hypopharyngeal carcinoma cells through regulation of RAGE expression.

Abnormal expression of high-mobility group box-1 (HMGB1) protein occurs in many tumors and is closely associated with tumor invasion and metastasis. However, a role for HMGB1 in epithelial-mesenchymal transition (EMT) in hypopharyngeal carcinoma has not been previously reported. We cultured cells of the hypopharyngeal carcinoma cell line FaDu in vitro and then treated them with 5 ng/ml TGF-ß1 for 48 h to induce EMT. Vimentin, Snail, and HMGB1 expression patterns were then detected using immunofluorescence staining; HMGB1 mRNA and protein expression were verified by RT-PCR and western blot analyses. HMGB1 was then silenced in FaDu cells using RNAi, followed by detection of Vimentin, Snail, and HMGB1 expressions by immunofluorescence staining. The mRNA expression levels of Vimentin, Snail, HMGB1, and E-cadherin were verified by RT-PCR, while protein expression of HMGB1 and receptor for advanced glycation end products (RAGE) were detected by western blot analysis. The biological behavior of FaDu cells was observed before and after HMGB1 silencing using wound healing and cell invasion assays. Following culture with 5 ng/ml TGF-ß1 for 48 h, the morphology of FaDu cells changed from a regular cobblestone-like appearance into a spindle-like shape. Expression levels of Vimentin, Snail, and HMGB1 were upregulated at both mRNA and protein levels as determined by RT-PCR, immunofluorescence, and western blotting. After HMGB1 silencing, mRNA expression levels of the epithelial cell marker E-cadherin were upregulated. Meanwhile, expression levels of the mesenchymal markers Vimentin and Snail were decreased. Western blotting revealed that HMGB1 and RAGE were downregulated. RNAi-mediated inhibition of HMGB1 expression decreased the capacities of FaDu cells for invasion and metastasis as determined by wound healing and cell invasion assays. HMGB1 is essential for maintaining the interstitial cell phenotype in TGF-ß1-induced EMT of FaDu cells, and silencing HMGB1 greatly inhibits the invasive and metastatic ability of these cells.

Lung adenocarcinoma expressing receptor for advanced glycation end-products with primary systemic AL amyloidosis: a case report and literature review.

BACKGROUND: Receptor for advanced glycation end-products (RAGE), a receptor for amyloids, is constitutively expressed in lungs and generally observed to be downregulated in lung cancer tissues. However, increasing levels of RAGE or serum amyloids is associated with poor outcome in lung cancer patients. We report a rare case of primary systemic amyloid light-chain (AL) amyloidosis in biopsy-proven multiple organs with early-stage non-small cell lung cancer (NSCLC) that displayed strong staining for RAGE in the tumour tissue. Interestingly, compared with randomly selected lung cancer biopsy samples, including all representative histological subtypes of NSCLC and small-cell lung cancer, only the NSCLC in the present case showed strong expression for RAGE that can bind amyloids. CASE PRESENTATION: A 71-year-old woman was admitted to our hospital for comprehensive investigation of nephrotic syndrome. Computed tomography showed a small nodule in the right upper lung lobe with hilar mediastinal lymph node enlargement. Pathological examination of transbronchial biopsy samples of the nodule yielded a diagnosis of lung adenocarcinoma. Furthermore, the pathological detection of amyloid deposition in biopsy samples of a subcarinal lymph node, gastric and duodenal mucosa, cardiac muscle, and bone marrow led to a diagnosis of primary systemic AL amyloidosis with nephrotic syndrome and cardiomyopathy. In addition, RAGE was detected in lung tumour tissues surrounded by normal lung tissues with amyloid deposition. CONCLUSION: The RAGE positivity of the lung cancer cells in this case suggests an interaction between amyloid-containing tissues and RAGE-expressing cancer cells. Lung adenocarcinoma with RAGE expression may be a complication of underlying amyloidosis.

All-Trans Retinoic Acid supplementation prevents cardiac fibrosis and cytokines induced by Methylglyoxal.

Methylglyoxal (MG), a metabolic intermediate of glycolysis is a precursor for endogeneous production of advanced glycation end-products. The increased production of MG have negative influence over the structure and function of different biomolecules and thus plays an important role in the pathogenesis of diabetic cardiac complications. Retinoic acid (RA), an active metabolite of vitamin A, has a major role in preventing cardiac remodeling and ventricular fibrosis. Hence, the objective of the present study was to determine whether rats administered with all-trans retinoic acid (RA) could attenuate MG induced pathological effects. Wistar rats were divided into 4 groups. Group 1 rats were kept as control; Group 2 rats were administrated with MG (75 mg/kg/day) for 8 weeks. Group 3 rats were given RA (Orally, 1.0 mg/kg/day) along with MG; Group 4 rats received RA alone. Cardiac antioxidant status, induction of fibrosis, AGE receptor (RAGE) and cytokines expression was evaluated in the heart tissues. Administration of MG led to depletion of antioxidant enzymes, induction of fibrosis (p < 0.001), up-regulated expression of RAGE (3.5 fold), TGF-ß (4.4 fold), SMAD2 (3.7 fold), SMAD3 (6.0 fold), IL-6 (4.3 fold) and TNF-α (5.5 fold) in the heart tissues compared to control rats. Moreover, the exogenous administration of MG caused significant (p < 0.001) increase in the circulating CML levels. Whereas, RA treatment prevented the induction of fibrosis and restored the levels of cytokines and RAGE expression. Methylglyoxal-induced fibrosis can lead to pathological effects in the heart tissues. RA attenuates the effects of MG in the heart, suggesting that it can be of added value to usual diabetic therapy.