Dysregulation of Receptor for Advanced Glycation End Products (RAGE) Expression as a Biomarker of Keratoconus

Background. Because of the implications of Receptor for Advanced Glycation End Products (RAGE) in keratoconus (KC), we describe a differential expression of RAGE transcripts and proteins in corneal tissues and tears of KC and healthy patients. Methods. Using a case-controlled study, corneal epitheliums and tears of KC and healthy subjects were obtained during corneal collagen cross-linking and photorefractive keratectomy (PKR) and during usual consultations. Quantitative reverse transcription (RT-qPCR) and Western-Blot were performed to analyze RAGE transcripts and proteins’ expression in corneal tissues and tears. Results. One hundred and six patients were included in this study. The characteristics of the patients were as follows: 56 KC (25 corneal epithelium and 31 tears) and 50 control subjects (25 corneal epithelium and 25 tears). Transcripts of RAGE, HMGB1, and S100 family ligands were quantified by RT-qPCR, identifying a significantly higher expression of RAGE and HMGB1 in the healthy group than in the KC group ( p = 0.03 and 0.04, respectively). Western Blot showed a significantly higher fl-RAGE expression in KC corneal epithelium than control ( p < 0.001 ) and lower s-RAGE expression in KC tears than control ( p = 0.04 ). Conclusions. Linked with the inflammatory process occurring in KC pathophysiology, we propose for the first time that the RAGE expression (total and truncated forms of receptor and ligands) in KC corneal tissues and tear samples provides viable biomarkers.

Reduced receptor for advanced glycation end products is associated with α-SMA expression in patients with idiopathic pulmonary fibrosis and mice

Background Idiopathic pulmonary fibrosis (IPF) is a chronic and progressive interstitial lung disease. Despite alveolar epithelial cells is crucial role in lung, its contribution and the associated biomarker remain unknown in the pathogenesis of IPF. Recently, environmental factors including stone dust, silica and cigarette smoking were found as risk factors involved in IPF. Receptor for advanced glycation end products (RAGE) is a member of the immunoglobulin super family of cell surface receptors. It has been shown that interaction between RAGE and its ligands on immune cells mediates cellular migration and regulation of pro-inflammation. RAGE is highly expressed in the lung, in particular, alveolar epithelial cells. Therefore, we determined whether RAGE expression is associated with fibrosis-associated genes in patients with IPF and mice. Results When bleomycin (BLM) was intratracheally administered to C57BL/6 mice for 1, 2 weeks, macrophage and neutrophils were significantly increased. The fibrotic nodule formed and accumulation of collagen was determined after BLM injection in H&E- and Masson’s trichrome staining. Levels of elastin, Col1a1 and fibronectin were increased in quantitative real-time PCR and protein levels of α-SMA was increased in western blot analysis. In the lung tissues of 1 mg/kg BLM-induced mice, RAGE expression was gradually decreased in 1- and 2 weeks in immunohistochemistry and western blot analysis, and 3 mg/kg of BLM-induced mice exhibited decreased RAGE levels while α-SMA expression was increased. We next determined RAGE expression in the lungs of IPF patients using immunohistochemistry. As a result, RAGE expression was decreased, while α-SMA expression was increased compared with non-IPF subjects. Conclusions Our findings suggest that reduced RAGE was associated with increased fibrotic genes in BLM-induced mice and patients with IPF. Therefore, RAGE could be applied with a biomarker for prognosis and diagnosis in the pathogenesis of IPF.

Sitagliptin attenuates arterial calcification by downregulating oxidative stress-induced receptor for advanced glycation end products in LDLR knockout mice

Diabetes is a complex disease characterized by hyperglycemia, dyslipidemia, and insulin resistance. Plasma advanced glycation end products (AGEs) activated the receptor for advanced glycation end products (RAGE) and the activation of RAGE is implicated to be the pathogenesis of type 2 diabetic mellitus (T2DM) patient vascular complications. Sitagliptin, a dipeptidyl peptidase-4 (DPP4) inhibitor, is a new oral hypoglycemic agent for the treatment of T2DM. However, the beneficial effects on vascular calcification remain unclear. In this study, we used a high-fat diet (HFD)-fed low-density lipoprotein receptor deficiency (LDLR−/−) mice model to investigate the potential effects of sitagliptin on HFD-induced arterial calcification. Mice were randomly divided into 3 groups: (1) normal diet group, (2) HFD group and (3) HFD + sitagliptin group. After 24 weeks treatment, we collected the blood for chemistry parameters and DPP4 activity measurement, and harvested the aorta to evaluate calcification using immunohistochemistry and calcium content. To determine the effects of sitagliptin, tumor necrosis factor (TNF)-α combined with S100A12 was used to induce oxidative stress, activation of nicotinamide adenine dinucleotide phosphate (NADPH), up-regulation of bone markers and RAGE expression, and cell calcium deposition on human aortic smooth muscle cells (HASMCs). We found that sitagliptin effectively blunted the HFD-induced artery calcification and significantly lowered the levels of fasting serum glucose, triglyceride (TG), nitrotyrosine and TNF-α, decreased the calcium deposits, and reduced arterial calcification. In an in-vitro study, both S100A12 and TNF-α stimulated RAGE expression and cellular calcium deposits in HASMCs. The potency of S100A12 on HASMCs was amplified by the presence of TNF-α. Sitagliptin and Apocynin (APO), an NADPH oxidase inhibitor, inhibited the TNF-α + S100A12-induced NADPH oxidase and nuclear factor (NF)-κB activation, cellular oxidative stress, RAGE expression, osteo transcription factors expression and calcium deposition. In addition, treatment with sitagliptin, knockdown of RAGE or TNF-α receptor blunted the TNF-α + S100A12-induced RAGE expression. Our findings suggest that sitagliptin may suppress the initiation and progression of arterial calcification by inhibiting the activation of NADPH oxidase and NF-κB, followed by decreasing the expression of RAGE.

Xiaoyaosan Exerts Antidepressant Effect by Downregulating RAGE Expression in Cingulate Gyrus of Depressive-Like Mice

Xiaoyaosan (XYS), as a classic Chinese medicine compound, has been proven to have antidepressant effect in many studies, but its mechanism has not been clarified. In our previous studies, we found that chronic stress can induce depressive-like behavior and lead to emotion-related cingulate gyrus (Cg) dysfunction, as well as the decrease of neurotrophic factors and the increase of inflammatory-related proteins. Therefore, we speculated that XYS may play an antidepressant role by regulating the inflammation-related receptor of advanced glycation protein end product (RAGE) to affect the functional connectivity (FC) signal of the Cg and improve the depressive-like behavior. In order to verify this hypothesis, we analyzed the FC and RAGE expression in the Cg of depressive-like mice induced by chronic unpredictable mild stress (CUMS) and verified it with RAGE knockout mice. At the same time, we detected the effect of XYS on the depressive-like behavior, expression of RAGE, and the FC of the Cg of mice. The results showed that the FC of the Cg of depressive-like mice induced by CUMS was weakened, and the expression of RAGE was upregulated. The antidepressant effect of XYS is similar to that of fluoxetine hydrochloride, which can significantly reduce the depressive-like behavior of mice and inhibit the expression of the RAGE protein and mRNA in the Cg, and increase the FC of the Cg in mice. In conclusion, XYS may play an antidepressant role by downregulating the expression of RAGE in the Cg of depressive-like mice induced by CUMS, thereby affecting the functional signal and improving the depressive-like behavior.

Effect of High Cholesterol Regulation of LRP1 and RAGE on Aβ Transport Across the Blood-brain Barrier in Alzheimer’s Disease

Background: High cholesterol aggravates the risk development of Alzheimer's disease (AD). AD is closely related to the transport impairment of amyloid-β (Aβ) in the blood-brain barrier. It is unclear whether high cholesterol affects the risk of cognitive impairment in AD by affecting Aβ transport. The purpose of the study is to investigate whether high cholesterol regulates Aβ transport through low-density lipoprotein receptor-related protein 1 (LRP1) and receptor for advanced glycation end products (RAGE) in the risk development of AD. Methods: We established high cholesterol AD mice model. The learning and memory functions were evaluated by Morris water maze (MWM). Cerebral microvascular endothelial cells were isolated, cultured, and observed. The expression levels of LRP1 and RAGE of endothelial cells and their effect on Aβ transport in vivo were observed. The expression level of LRP1 and RAGE was detected in cultured microvessels after using Wnt inhibitor DKK-1 and β-catenin inhibitor XAV-939. Results: Hypercholesterolemia exacerbated spatial learning and memory impairment. Hypercholes- terolemia increased serum Aβ40 level, while serum Aβ42 level did not change significantly. Hyper- cholesterolemia decreased LRP1 expression and increased RAGE expression in cerebral microvascular endothelial cells. Hypercholesterolemia increased brain apoptosis in AD mice. In in vitro experiment, high cholesterol decreased LRP1 expression and increased RAGE expression, increased Aβ40 expression in cerebral microvascular endothelial cells. High cholesterol regulated the expressions of LRP1 and RAGE and transcriptional activity of LRP1 and RAGE promoters by the Wnt/β-catenin signaling pathway. Conclusion : High cholesterol decreased LRP1 expression and increased RAGE expression in cerebral microvascular endothelial cells, which led to Aβ transport disorder in the blood-brain barrier. Increased Aβ deposition in the brain aggravated apoptosis in the brain, resulting to cognitive impairment of AD mice.

Copper, Iron, Selenium and Lipo-Glycemic Dysmetabolism in Alzheimer’s Disease

The aim of the present review is to discuss traditional hypotheses on the etiopathogenesis of Alzheimer’s disease (AD), as well as the role of metabolic-syndrome-related mechanisms in AD development with a special focus on advanced glycation end-products (AGEs) and their role in metal-induced neurodegeneration in AD. Persistent hyperglycemia along with oxidative stress results in increased protein glycation and formation of AGEs. The latter were shown to possess a wide spectrum of neurotoxic effects including increased Aβ generation and aggregation. In addition, AGE binding to receptor for AGE (RAGE) induces a variety of pathways contributing to neuroinflammation. The existing data also demonstrate that AGE toxicity seems to mediate the involvement of copper (Cu) and potentially other metals in AD pathogenesis. Specifically, Cu promotes AGE formation, AGE-Aβ cross-linking and up-regulation of RAGE expression. Moreover, Aβ glycation was shown to increase prooxidant effects of Cu through Fenton chemistry. Given the role of AGE and RAGE, as well as metal toxicity in AD pathogenesis, it is proposed that metal chelation and/or incretins may slow down oxidative damage. In addition, selenium (Se) compounds seem to attenuate the intracellular toxicity of the deranged tau and Aβ, as well as inhibiting AGE accumulation and metal-induced neurotoxicity.

Deletion of RAGE fails to prevent hepatosteatosis in obese mice due to impairment of other AGEs receptors and detoxifying systems

Advanced glycation endproducts (AGEs) are involved in several diseases, including NAFLD and NASH. RAGE is the main receptor mediating the pro-inflammatory signalling induced by AGEs. Therefore, targeting of RAGE has been proposed for prevention of chronic inflammatory diseases. However, the role of RAGE in the development of NAFLD and NASH remains poorly understood. We thus aimed to analyse the effect of obesity on AGEs accumulation, AGE-receptors and AGE-detoxification, and whether the absence of RAGE might improve hepatosteatosis and inflammation, by comparing the liver of lean control, obese (LeptrDb−/−) and obese RAGE-deficient (RAGE−/− LeptrDb−/−) mice. Obesity induced AGEs accumulation and RAGE expression with hepatosteatosis and inflammation in LeptrDb−/−, compared to lean controls. Despite the genetic deletion of RAGE in the LeptrDb−/− mice, high levels of intrahepatic AGEs were maintained accompanied by decreased expression of the protective AGE-receptor-1, impaired AGE-detoxifying system glyoxalase-1, and increased expression of the alternative AGE-receptor galectin-3. We also found sustained hepatosteatosis and inflammation as determined by persistent activation of the lipogenic SREBP1c and proinflammatory NLRP3 signalling pathways. Thus, RAGE targeting is not effective in the prevention of NAFLD in conditions of obesity, likely due to the direct liver specific crosstalk of RAGE with other AGE-receptors and AGE-detoxifying systems.

Hypercholesterolemia and 27-Hydroxycholesterol Increase S100A8 and RAGE Expression in the Brain: a Link Between Cholesterol, Alarmins, and Neurodegeneration

Alterations in cholesterol metabolism in the brain have a major role in the physiology of Alzheimer’s disease (AD). Oxysterols are cholesterol metabolites with multiple implications in memory functions and in neurodegeneration. Previous studies have shown detrimental effects of cholesterol metabolites in neurons, but its effect in glial cells is unknown. We used a high-fat/high-cholesterol diet in mice to study the effects of hypercholesterolemia over the alarmin S100A8 cascade in the hippocampus. Using CYP27Tg, a transgenic mouse model, we show that the hypercholesterolemia influence on the brain is mediated by the excess of 27-hydroxycholesterol (27-OH), a cholesterol metabolite. We also employed an acute model of 27-OH intraventricular injection in the brain to study RAGE and S100A8 response. We used primary cultures of neurons and astrocytes to study the effect of high levels of 27-OH over the S100A8 alarmin cascade. We report that a high-fat/high-cholesterol diet leads to an increase in S100A8 production in the brain. In CYP27Tg, we report an increase of S100A8 and its receptor RAGE in the hippocampus under elevated 27-OH in the brain. Using siRNA, we found that 27-OH upregulation of RAGE in astrocytes and neurons is mediated by the nuclear receptor RXRγ. Silencing RXRγ in neurons prevented 27-OH-mediated upregulation of RAGE. These results show that S100A8 alarmin and RAGE respond to high levels of 27-OH in the brain in both neurons and astrocytes through RXRγ. Our study supports the notion that 27-OH mediates detrimental effects of hypercholesterolemia to the brain via alarmin signaling.

RAGE Differentially Altered in vitro Responses in Vascular Smooth Muscle Cells and Adventitial Fibroblasts in Diabetes-Induced Vascular Calcification

The Advanced Glycation End-Products (AGE)/Receptor for AGEs (RAGE) signaling pathway exacerbates diabetes-mediated vascular calcification (VC) in vascular smooth muscle cells (VSMCs). Other cell types are involved in VC, such as adventitial fibroblasts (AFBs). We hope to elucidate some of the mechanisms responsible for differential signaling in diabetes-mediated VC with this work. This work utilizes RAGE knockout animals and in vitro calcification to measure calcification and protein responses. Our calcification data revealed that VSMCs calcification was AGE/RAGE dependent, yet AFBs calcification was not an AGE-mediated RAGE response. Protein expression data showed VSMCs lost their phenotype marker, α-smooth muscle actin, and had a higher RAGE expression over non-diabetics. RAGE knockout (RKO) VSMCs did not show changes in phenotype markers. P38 MAPK, a downstream RAGE-associated signaling molecule, had significantly increased activation with calcification in both diabetic and diabetic RKO VSMCs. AFBs showed a loss in myofibroblast marker, α-SMA, due to calcification treatment. RAGE expression decreased in calcified diabetic AFBs, and P38 MAPK activation significantly increased in diabetic and diabetic RKO AFBs. These findings point to potentially an alternate receptor mediating the calcification response in the absence of RAGE. Overall, VSMCs and AFBs respond differently to calcification and the application of AGEs.

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 (IL1β), 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, IL1β, 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.