RAGE Deficiency Research Articles

Macrophage RAGE deficiency prevents myocardial fibrosis by repressing autophagy-mediated macrophage alternative activation.

Myocardial fibrosis (MF) is the characteristic pathological feature of various cardiovascular diseases that lead to heart failure (HF) or even fatal outcomes. Alternatively, activated macrophages are involved in the development of fibrosis and tissue remodeling. Although the receptor for advanced glycation end products (RAGE) is involved in MF, its potential role in regulating macrophage function in cardiac fibrosis has not been fully investigated. We aimed to determine the role of macrophage RAGE in transverse aortic constriction (TAC)-induced MF. In this study, we found that RAGE expression was markedly increased in the infiltrated alternatively activated macrophages within mice hearts after TAC. RAGE knockout mice showed less infiltration of alternatively activated macrophages and attenuated cardiac hypertrophy and fibrosis compared to the wild-type mice. Our data suggest that mice with macrophage-specific genetic deletion of RAGE were protected from interstitial fibrosis and cardiac dysfunction when subjected to pressure overload, which led to a decreased proportion of alternatively activated macrophages in heart tissues. Our invitro experiments demonstrated that RAGE deficiency inhibited the differentiation into alternatively activated macrophages by suppressing autophagy activation. In the co-culture system, invitro polarization of RAW264.7 macrophages toward an alternatively activated phenotype stimulated the expression of α-smooth muscle actin and collagen in cardiac fibroblasts. However, the knockdown of RAGE and inhibition of autophagy in macrophages showed reduced fibroblast-to-myofibroblast transition (FMT). Collectively, our results suggest that RAGE plays an important role in the recruitment and activation of alternatively activated macrophages by regulating autophagy, which contributes to MF. Thus, blockage of RAGE signaling may be an attractive therapeutic target for the treatment of hypertensive heart disease.

Pathogenic Role of RAGE in Tau Transmission and Memory Deficits

BackgroundIn tauopathies, brain regions with tau accumulation strongly correlate with clinical symptoms, and spreading of misfolded tau along neural network leads to disease progression. However, the underlying mechanisms by which tau proteins enter neurons during pathological propagation remain unclear. MethodsTo identify membrane receptors responsible for neuronal propagation of tau oligomers, we established a cell-based tau uptake assay and screened complementary DNA expression library. Tau uptake and propagation were analyzed in vitro and in vivo using a microfluidic device and stereotactic injection. The cognitive function of mice was assessed using behavioral tests. ResultsFrom a genome-wide cell-based functional screening, RAGE (receptor for advanced glycation end products) was isolated to stimulate the cellular uptake of tau oligomers. Rage deficiency reduced neuronal uptake of pathological tau prepared from rTg4510 mouse brains or cerebrospinal fluid from patients with Alzheimer’s disease and slowed tau propagation between neurons cultured in a 3-chamber microfluidic device. RAGE levels were increased in the brains of rTg4510 mice and tau oligomer-treated neurons. Rage knockout decreased tau transmission in the brains of nontransgenic mice after injection with Alzheimer’s disease patient-derived tau and ameliorated memory loss after injection with GFP-P301L-tau-AAV. Treatment of RAGE antagonist FPS-ZM1 blocked transsynaptic tau propagation and inflammatory responses and alleviated cognitive impairment in rTg4510 mice. ConclusionsThese results suggest that in neurons and microglia, RAGE binds to pathological tau and facilitates neuronal tau pathology progression and behavioral deficits in tauopathies.

RAGE deficiency ameliorates autoimmune hepatitis involving inhibition of IL-6 production via suppressing protein Arid5a in mice.

Activation of T cells and pro-inflammatory cytokines are essential for human autoimmune hepatitis. RAGE is one of the receptors for the inflammatory alarm molecule high mobility group box 1 (HMGB1), and it is involved in autoimmune hepatitis. However, the molecular mechanism of RAGE in the context of autoimmune hepatitis remains elusive. This study aimed to identify the function and mechanism of RAGE in autoimmune hepatitis. The role and underlying mechanisms of RAGE signaling-driven immune inflammatory response in ConA-induced experimental hepatitis were examined using the RAGE-deficient mice. We found that the RAGE deficiency protected the mouse from liver inflammatory injury caused by the ConA challenge. mRNA expression of VCAM-1, IL-6, and TNF-α within the livers is markedly decreased in RAGE-deficient mice compared to wild-type mice. In parallel, RAGE deficiency leads to reduced levels of the serum pro-inflammatory cytokines IL-6 and TNF-α as compared with wild-type control mice. RAGE-deficient mice exhibit increased hepatic NK cells and decreased CD4+ T cells compared with wild-type control mice. Notably, in vivo blockade of IL-6 in wild-type mice significantly protected mice from ConA-induced hepatic injury. Furthermore, RAGE deficiency impaired IL-6 production and was associated with decreased expression of Arid5a in liver tissues, a half-life IL-6 mRNA regulator. RAGE signaling is important in regulating the development of autoimmune hepatitis. Immune regulation of RAGE may represent a novel therapeutic strategy to prevent immune-mediated liver injury.

RAGE promotes dysregulation of iron and lipid metabolism in alcoholic liver disease

Alcoholic liver disease (ALD) is associated with hepatic inflammatory activation and iron overload. The receptor for advanced glycation end products (RAGE) is an important metabolic mediator during the development of ALD. The aim of this study was to determine the effect of RAGE on iron homeostasis in ALD. We found increased circulating transferrin, hepcidin and ferritin in ALD patients and positively correlated with RAGE level. RAGE knockout (RAGE−/−) and wild-type mice were subjected to chronic alcoholic feeding for 6 weeks to induce ALD, and RAGE inhibitor, iron chelator or lipid peroxidation inhibitor were administered. We showed that chronic alcohol administration triggered hepatic steatosis, inflammation, and oxidative stress, which were eliminated by deficiency or inhibition of RAGE. Surprisingly, pathways of hepatic iron metabolism were significantly altered, including increased iron uptake (Tf/TfR) and storage (Ferritin), as well as decreased iron export (FPN1/Hepcidin). In vitro experiments confirmed that RAGE had different effects on the mechanism of iron metabolism of hepatocytes and macrophages respectively. In conclusion, our data revealed preclinical evidence for RAGE inhibition as an effective intervention for alleviating alcohol-induced liver injury.

RAGE displays sex-specific differences in obesity-induced adipose tissue insulin resistance

BackgroundThe receptor for advanced glycation end products (RAGE) plays an important role in obesity-associated insulin sensitivity. We have also previously reported that RAGE deficiency improved insulin resistance in obesity-induced adipose tissue. The current study was aimed to elucidate the sex-specific mechanism of RAGE deficiency in adipose tissue metabolic regulation and systemic glucose homeostasis.MethodsRAGE-deficient (RAGE−/−) mice were fed a high-fat diet (HFD) and subjected to glucose and insulin tolerance tests. Subcutaneous adipose tissue (sAT) was collected, and macrophage polarization was assessed by quantitative real-time PCR. Immunoblotting was performed to evaluate the insulin signaling in adipose tissues.ResultsUnder HFD feeding conditions, body weight and adipocyte size of female RAGE deficient (RAGE−/−) were markedly lower than that of male mice. Female RAGE−/− mice showed significantly improved glucose and insulin tolerance compared to male RAGE−/− mice, accompanied with increased M2 macrophages polarization. Expressions of genes involved in anti-oxidant and browning were up-regulated in adipose tissues of female RAGE−/− mice. Moreover, insulin-induced AKT phosphorylation was significantly elevated in adipose tissue in female RAGE−/− mice compared to male RAGE−/− mice.ConclusionsOur findings suggest that RAGE-mediated adipose tissue insulin resistance is sex-specific, which is associated with different expression of genes involved in anti-oxidant and browning and insulin-induced AKT phosphorylation.

Receptor of Advanced Glycation End Products Deficiency Attenuates Cisplatin-Induced Acute Nephrotoxicity by Inhibiting Apoptosis, Inflammation and Restoring Fatty Acid Oxidation.

Cisplatin is a widely used and potent anti-neoplastic agent, but severe and inescapable side effects in multiple normal tissues and organs limit its application, especially nephrotoxicity. Molecular mechanisms of cisplatin nephrotoxicity involve mitochondrial damage, oxidative stress, endoplasmic reticulum stress, inflammation, apoptosis, necroptosis, etc. Receptor of advanced glycation end products (RAGE) is a multiligand pattern recognition receptor, engaged in inflammatory signaling and mitochondrial homeostasis. Whether inhibition of RAGE alleviates cisplatin-induced nephropathy has not been investigated. Here, we revealed that RAGE deficiency attenuates cisplatin-induced acute nephrotoxicity, as evidenced by reduced apoptosis, inflammation, lipid accumulation, restored mitochondrial homeostasis and fatty acid oxidation in renal tubular epithelial cells (TECs). In vitro studies showed that, the RAGE-specific inhibitor FPS-ZM1 attenuated the cisplatin-induced decrease of cell viability and fatty acid oxidation in the normal rat renal TEC line NRK-52E cells. Taken together, RAGE knockout mitigated cisplatin-induced acute nephrotoxicity by inhibiting apoptosis, inflammation, and restoring fatty acid oxidation in TECs, suggesting that RAGE inhibition could be a therapeutic option for cisplatin-induced acute nephrotoxicity.

Targeting adaptor protein SLP76 of RAGE as a therapeutic approach for lethal sepsis

Accumulating evidence shows that RAGE has an important function in the pathogenesis of sepsis. However, the mechanisms by which RAGE transduces signals to downstream kinase cascades during septic shock are not clear. Here, we identify SLP76 as a binding partner for the cytosolic tail of RAGE both in vitro and in vivo and demonstrate that SLP76 binds RAGE through its sterile α motif (SAM) to mediate downstream signaling. Genetic deficiency of RAGE or SLP76 reduces AGE-induced phosphorylation of p38 MAPK, ERK1/2 and IKKα/β, as well as cytokine release. Delivery of the SAM domain into macrophages via the TAT cell-penetrating peptide blocks proinflammatory cytokine production. Furthermore, administration of TAT-SAM attenuates inflammatory cytokine release and tissue damage in mice subjected to cecal ligation and puncture (CLP) and protects these mice from the lethality of sepsis. These findings reveal an important function for SLP76 in RAGE-mediated pro-inflammatory signaling and shed light on the development of SLP76-targeted therapeutics for sepsis.

RAGE and TLR4 differentially regulate airway hyperresponsiveness: Implications for COPD.

The receptor for advanced glycation end products (RAGE) and Toll-like receptor 4 (TLR4) is implicated in COPD. Although these receptors share common ligands and signalling pathways, it is not known whether they act in concert to drive pathological processes in COPD. We examined the impact of RAGE and/or TLR4 gene deficiency in a mouse model of COPD and also determined whether expression of these receptors correlates with airway neutrophilia and airway hyperresponsiveness (AHR) in COPD patients. We measured airway inflammation and AHR in wild-type, RAGE-/- , TLR4-/- and TLR4-/- RAGE-/- mice following acute exposure to cigarette smoke (CS). We also examined the impact of smoking status on AGER (encodes RAGE) and TLR4 bronchial gene expression in patients with and without COPD. Finally, we determined whether expression of these receptors correlates with airway neutrophilia and AHR in COPD patients. RAGE-/- mice were protected against CS-induced neutrophilia and AHR. In contrast, TLR4-/- mice were not protected against CS-induced neutrophilia and had more severe CS-induced AHR. TLR4-/- RAGE-/- mice were not protected against CS-induced neutrophilia but were partially protected against CS-induced mediator release and AHR. Current smoking was associated with significantly lower AGER and TLR4 expression irrespective of COPD status, possibly reflecting negative feedback regulation. However, consistent with preclinical findings, AGER expression correlated with higher sputum neutrophil counts and more severe AHR in COPD patients. TLR4 expression did not correlate with neutrophilic inflammation or AHR. Inhibition of RAGE but not TLR4 signalling may protect against airway neutrophilia and AHR in COPD.

Relevance of Receptor for Advanced Glycation end Products (RAGE) in Murine Antibody-Mediated Autoimmune Diseases.

It is incompletely understood how self-antigens become targets of humoral immunity in antibody-mediated autoimmune diseases. In this context, alarmins are discussed as an important level of regulation. Alarmins are recognized by various receptors, such as receptor for advanced glycation end products (RAGE). As RAGE is upregulated under inflammatory conditions, strongly binds nucleic acids and mediates pro-inflammatory responses upon alarmin recognition, our aim was to examine its contribution to immune complex-mediated autoimmune diseases. This question was addressed employing RAGE−/− animals in murine models of pristane-induced lupus, collagen-induced, and serum-transfer arthritis. Autoantibodies were assessed by enzyme-linked immunosorbent assay, renal disease by quantification of proteinuria and histology, arthritis by scoring joint inflammation. The associated immune status was determined by flow cytometry. In both disease entities, we detected tendentiously decreased autoantibody levels in RAGE−/− mice, however no differences in clinical outcome. In accordance with autoantibody levels, a subgroup of the RAGE−/− animals showed a decrease in plasma cells, and germinal center B cells and an increase in follicular B cells. Based on our results, we suggest that RAGE deficiency alone does not significantly affect antibody-mediated autoimmunity. RAGE may rather exert its effects along with other receptors linking environmental factors to auto-reactive immune responses.

RAGE deficiency does not affect non-alcoholic steatohepatitis and atherosclerosis in Western type diet-fed Ldlr\u2212/\u2212 mice

Non-alcoholic fatty liver disease is a spectrum of liver diseases ranging from steatosis only to non-alcoholic steatohepatitis (NASH). The latter is characterized by hepatic inflammation, which increases the risk of cardiovascular disease. It is poorly understood which factors contribute to the onset of hepatic inflammation characterizing the progression from steatosis to NASH. Previously, we demonstrated increased advanced glycation endproducts (AGEs) in the livers of NASH patients. We hypothesise that AGEs play a key role in NASH development by activating their proinflammatory receptor, RAGE. RAGE-deficient mice and wildtype littermates, both on Ldlr−/− background, were fed a Western type diet (WTD) for 3 or 12 weeks. Flow cytometry, histology, gene expression and AGE measurements were performed to evaluate the effects of RAGE deficiency. RAGE-deficient mice displayed reduced weight gain and visceral fat expansion compared to control mice. No difference in adipose tissue inflammation was observed between groups. RAGE deficiency did not affect WTD-induced monocytosis, circulating lipids or hepatic steatosis. WTD-induced hepatic neutrophil and macrophage accumulation and atherosclerotic plaque development was comparable between control and RAGE-deficient mice. No difference in AGE levels was observed. RAGE does not seem to play a major role in the development of NASH or atherosclerosis in a hyperlipidemic mouse model.

Blockade of HMGB1 Attenuates Diabetic Nephropathy in Mice

Activation of TLR2 or TLR4 by endogenous ligands such as high mobility group box 1 (HMGB1) may mediate inflammation causing diabetic kidney injury. We determined whether blockade of HMGB1 signaling by: (1) supra-physiological production of endogenous secretory Receptor for Advanced Glycation End-products (esRAGE), a receptor for HMGB1; (2) administration of HMGB1 A Box, a specific competitive antagonist, would inhibit development of streptozotocin induced diabetic nephropathy (DN). Wild-type diabetic mice developed albuminuria, glomerular injuries, interstitial fibrosis and renal inflammation. Using an adeno-associated virus vector, systemic over-expression of esRAGE afforded significant protection from all parameters. No protection was achieved by a control vector which expressed human serum albumin. Administration of A Box was similarly protective against development of DN. To determine the mechanism(s) of protection, we found that whilst deficiency of TLR2, TLR4 or RAGE afforded partial protection from development of DN, over-expression of esRAGE provided additional protection in TLR2−/−, modest protection against podocyte damage only in TLR4−/− and no protection in RAGE−/− diabetic mice, suggesting the protection provided by esRAGE was primarily through interruption of RAGE and TLR4 pathways. We conclude that strategies to block the interaction between HMGB1 and its receptors may be effective in preventing the development of DN.

Rage induces hepatocellular carcinoma proliferation and sorafenib resistance by modulating autophagy

The receptor for advanced glycation end products (Rage) is involved in the development of various tumors and acts as an oncogenic protein. Rage is overexpressed in tumors including hepatocellular carcinoma (HCC). However, the molecular mechanism of Rage in HCC progression and sorafenib resistance remains unclear. In this study, enhanced Rage expression is highly associated proliferation and contributes to sorafenib resistance. Rage deficiency contributed to autophagy induction through activating AMPK/mTOR signaling pathway, which is important for sorafenib response. Moreover, the interactions between Rage and Rage ligands such as high mobility group box 1 (HMGB1) and s100a4 positively increased Rage expression. Our data indicate that Rage may be a potential target for therapeutic intervention in HCC and biomarker for sorafenib resistance.

Lack of Receptor for Advanced Glycation End Products Leads to Less Severe Staphylococcal Skin Infection but More Skin Abscesses and Prolonged Wound Healing.

Lack of receptor for advanced glycation end products (RAGE) ameliorates several infections including Staphylococcus aureus pneumonia. We sought to investigate the role of RAGE in staphylococcal skin infection in mice. Wild-type (WT) and RAGE deficient (RAGE-/-) mice were subcutaneously inoculated with S. aureus SH1000 strain in abscess-forming dose or necrotic dose. Clinical signs of dermatitis, along with histopathological changes, were compared between the groups. The skin lesion size was smaller in RAGE-/- mice. Infected RAGE-/- mice expressed lower proinflammatory cytokines in local skins compared to control mice. Low dose of bacteria caused more abscess formation in RAGE-/- mice compared to skin necrosis that was more often observed in WT mice. As a result of more abscess formation, the wound healing was prolonged in RAGE-/- mice. Importantly, RAGE-/- mice had lower bacterial loads in the skin than controls, which is correlated with higher local levels of myeloperoxidase before skin infection. In vitro, enhanced phagocytic capacity of neutrophils and macrophages obtained from RAGE-/- mice compared to control mice was observed. RAGE deficiency up-regulates phagocytic capacity of phagocytes, resulting in lower bacterial burden in local skin and milder skin lesions in mice with staphylococcal skin infection.

Advanced glycation end products receptor RAGE controls myocardial dysfunction and oxidative stress in high-fat fed mice by sustaining mitochondrial dynamics and autophagy-lysosome pathway

Oxidative stress and mitochondrial dysfunction are recognized as major contributors of cardiovascular damage in diabetes and high fat diet (HFD) fed mice. Blockade of receptor for advanced glycation end products (RAGE) attenuates vascular oxidative stress and development of atherosclerosis. We tested whether HFD-induced myocardial dysfunction would be reversed in RAGE deficiency mice, in association with changes in oxidative stress damage, mitochondrial respiration, mitochondrial fission and autophagy-lysosomal pathway. Cardiac antioxidant capacity was upregulated in RAGE−/− mice under normal diet as evidenced by increased superoxide dismutase and sirtuin mRNA expressions. Mitochondrial fragmentation and mitochondrial fission protein Drp1 and Fis1 expressions were increased in RAGE−/− mice. Autophagy-related protein expressions and cathepsin-L activity were increased in RAGE−/− mice suggesting sustained autophagy-lysosomal flux. HFD induced mitochondrial respiration defects, cardiac contractile dysfunction, disrupted mitochondrial dynamics and autophagy inhibition, which were partially prevented in RAGE−/− mice. Our results suggest that cardioprotection against HFD in RAGE−/− mice include reactivation of autophagy, as inhibition of autophagic flux by chloroquine fully abrogated beneficial myocardial effects and its stimulation by rapamycin improved myocardial function in HFD wild type mice. As mitochondrial fission is necessary to mitophagy, increased fragmentation of mitochondrial network in HFD RAGE−/− mice may have facilitated removal of damaged mitochondria leading to better mitochondrial quality control. In conclusion, modulation of RAGE pathway may improve mitochondrial damage and myocardial dysfunction in HFD mice. Attenuation of cardiac oxidative stress and maintenance of healthy mitochondria population ensuring adequate energy supply may be involved in myocardial protection against HFD.

Abstract 456: Rage Suppresses Angiogenesis and Blood Flow Recovery After Hind Limb Ischemia in Diabetic Mice Through Modulation of Macrophage Inflammation and Macrophage-endothelial Interactions

Peripheral vascular disease is a condition characterized by atherosclerotic narrowed arteries distal to the aorta which triggers an acute or critical limb ischemia. Development of ischemic PVD has been considered one of the principal complications of diabetes, leading to amputation of digits and limbs. Advanced glycation end products (AGE) ligands and their receptor (RAGE) have been implicated in multiple key mechanisms underlying diabetes and diabetic complications, including hypoxia and ischemia/reperfusion injury. We tested the hypothesis that vascular recovery after hind limb ischemia would be rescued by deficiency of RAGE, at least in part through modulation of macrophage dysfunction. Wild type (WT) and Ager deficient mice were rendered diabetic with streptozotocin, and subjected to unilateral hind limb ischemia. Previous results showed an increased accumulation and expression of AGEs and RAGE in ischemic muscle, especially in diabetic WT mice. Attenuated angiogenesis and impaired blood flow recovery were also observed, in parallel with reduced early inflammatory macrophage infiltration into ischemic muscle in the WT diabetic mice. We performed flow cytometry to analyze circulating monocyte subsets: pro-inflammatory Ly6G/C hi and anti-inflammatory Ly6G/C lo . Work by others reported higher levels of monocytes in diabetes in the baseline state without injury; our data indicate that the increase is mostly attributed to the pro-inflammatory Ly6G/C hi population, being significantly lower in the Ager -/- diabetic mice. After seven days of ischemia to the unilateral hind limb, lower levels of circulating pro-inflammatory Ly6G/C hi monocytes were found in both WT and Ager deficient diabetic mice. After four weeks of injury, the pro-inflammatory monocytes levels were significantly recovered to baseline levels in Ager -/- mice, whereas WT mice failed to reacquire their baseline levels. In vitro studies using murine endothelial cells and murine macrophages revealed that RAGE suppressed macrophage-endothelial cell interaction, particularly in diabetes-relevant concentrations of D-glucose. These data suggest unique ischemia-dependent mechanisms in hind limb ischemia through RAGE down-regulation of the early adaptive immune response.

RAGE-mediated extracellular matrix proteins accumulation exacerbates HySu-induced pulmonary hypertension.

Extracellular matrix (ECM) proteins accumulation contributes to the progression of pulmonary arterial hypertension (PAH), a rare and fatal cardiovascular condition defined by high pulmonary arterial pressure, whether primary, idiopathic, or secondary to other causes. The receptor for advanced glycation end products (RAGE) is constitutively expressed in the lungs and plays an important role in ECM deposition. Nonetheless, the mechanisms by which RAGE mediates ECM deposition/formation in pulmonary arteries and its roles in PAH progression remain unclear. Expression of RAGE and its activating ligands, S100/calgranulins and high mobility group box 1 (HMGB1), were increased in both human and mouse pulmonary arterial smooth muscle cells (PASMCs) under hypoxic conditions and were also strikingly upregulated in pulmonary arteries in hypoxia plus SU5416 (HySu)-induced PAH in mice. RAGE deletion alleviated pulmonary arterial pressure and restrained extracellular matrix accumulation in pulmonary arteries in HySu-induced PAH murine model. Moreover, blocking RAGE activity with a neutralizing antibody in human PASMCs, or RAGE deficiency in mouse PASMCs exposed to hypoxia, suppressed the expression of fibrotic proteins by reducing TGF-β1 expression. RAGE reconstitution in deficient mouse PASMCs restored hypoxia-stimulated TGF-β1 production via ERK1/2 and p38 MAPK pathway activation and subsequently increased ECM protein expression. Interestingly, HMGB1 acting on RAGE, not toll-like receptor 4 (TLR4), induced ECM deposition in PASMCs. Finally, in both idiopathic PAH patients and HySu-induced PAH mice, soluble RAGE (sRAGE) levels in serum were significantly elevated compared to those in controls. Activation of RAGE facilitates the development of hypoxia-induced pulmonary hypertension by increase of ECM deposition in pulmonary arteries. Our results indicate that sRAGE may be a potential biomarker for PAH diagnosis and disease severity, and that RAGE may be a promising target for PAH treatment.

RAGE deficiency attenuates the protective effect of Lidocaine against sepsis-induced acute lung injury.

Lidocaine (Lido) is reported to suppress inflammatory responses and exhibit a therapeutic effect in models of cecal ligation and puncture (CLP)-induced acute lung injury (ALI). The receptor for advanced glycation end product (RAGE) exerts pro-inflammatory effects by enhancing pro-inflammatory cytokine production. However, the precise mechanism by which Lido confers protection against ALI is not clear. ALI was induced in RAGE WT and RAGE knockout (KO) rats using cecal ligation and puncture (CLP) operations for 24h. The results showed that Lido significantly inhibited CLP-induced lung inflammation and histopathological lung injury. Furthermore, Lido significantly reduced CLP-induced upregulation of HMGB1 and RAGE expression and activation of the NF-κB and MAPK signaling pathways. With the use of RAGE KO rats, we demonstrate here that RAGE deficiency attenuates the protective effect of Lido against CLP-induced lung inflammatory cell infiltration and histopathological lung injury. These results suggest that RAGE deficiency attenuates the protective effect of Lido against CLP-induced ALI by attenuating the pro-inflammatory cytokines production.

RAGE deficiency alleviates aortic valve calcification in ApoE−/− mice via the inhibition of endoplasmic reticulum stress

Receptor for advanced glycation end products (RAGE) and endoplasmic reticulum (ER) stress have been shown to be involved in calcific aortic valve disease (CAVD). However, the association between RAGE and ER stress remains unknown in the pathogenesis of CAVD. The current study aims to test the hypothesis that RAGE deficiency alleviates aortic valve calcification via the inhibition of ER stress. Up-regulation of RAGE and ER stress markers in calcified human aortic valves were confirmed by immunoblotting. Aortic valve calcification was evaluated in atherosclerotic prone ApoE−/− mice or in mice with dual deficiencies of ApoE and RAGE (ApoE−/−RAGE−/−) fed with high cholesterol diet for 24weeks. Echocardiography and histological examination show that genetic deficiency of RAGE attenuates aortic valve calcification in ApoE−/− mice. Meanwhile, RAGE deficiency inhibited the osteogenic signaling and ER stress activation as well as suppressed macrophage infiltration in vivo. Cultured human aortic valve interstitial cells (AVICs) were treated with high molecular group box 1 protein (HMGB1) as in vitro model. We found that HMGB1 induced osteoblastic differentiation and calcification through RAGE/ER stress. Furthermore, Sox9 up-regulation and intranuclear translocation mediated the pro-osteogenic effect of HMGB1 on AVICs. RAGE or ER stress knockdown reduced the up-regulation of monocyte chemoattractant protein-1 (MCP-1) and tumor necrosis factor-α (TNF-α) in human AVICs exposed to HMGB1.These novel findings demonstrate that RAGE deficiency protects against aortic valve calcification in high cholesterol diet-fed ApoE−/− mice via inhibition of ER stress. HMGB1 induces AVIC osteoblastic differentiation and calcification through RAGE/ER stress/Sox9 pathway.

RAGE Deficiency Impairs Bacterial Clearance in Murine Staphylococcal Sepsis, but Has No Significant Impact on Staphylococcal Septic Arthritis.

BackgroundSeptic arthritis is a serious joint disease often caused by Staphylococcus aureus (S. aureus). Receptor for Advanced Glycation End products (RAGE) has an important role in several infections. We sought to investigate the role of RAGE in staphylococcal septic arthritis and sepsis in mice.MethodsWild-type (WT) and RAGE deficient (RAGE-/-) mice were intra-articularly or intravenously inoculated with an arthritic or septic dose of S. aureus LS-1 strain. Clinical arthritis, weight development and mortality were monitored for 14 days. Serum levels of cytokines, kidney bacterial loads as well as micro-CT and histopathology of the joints were assessed.ResultsRAGE-/- mice with septic arthritis had significantly lower IL-17A and higher bone mineral density (BMD) compared to the control group. However, no significant differences between the groups were observed regarding the weight loss, the severity and frequency of arthritis, and bacterial loads in the kidneys. In mice with sepsis, the overall mortality rate was similar in RAGE-/- (39%) and in WT mice (45%). However, RAGE-/- mice with sepsis had significantly higher bacterial load in their kidneys compared to the WT controls. In line with data from hematogenous S. aureus arthritis, RAGE deficiency had no impact on arthritis severity in local joint infection.ConclusionsOur results indicate that lack of RAGE has no significant impact on septic arthritis. However, RAGE-/- mice had significantly higher BMD compared to WT mice, which coincided with lower IL-17A in RAGE-/- mice. In sepsis, RAGE deficiency impairs bacterial kidney clearance.

RAGE: A novel target for breast cancer growth and metastasis.

Metastasis is a major cause of mortality in Breast Cancer (BC) patients in part due to lack of clinically established targeted therapies. Among the different types of BC, triple negative BC (TNBC) (ER-, PR-, HER2-) has been associated the most with poor prognosis and survival due to early metastasis to other organs and a lack of clinically established targeted therapies. Hence, elucidating novel mechanisms that regulate metastasis would lead to the development of targeted therapies and new treatments for TNBC and metastatic breast cancers. It is now well accepted that solid tumors, including those in the breast, have an inflammatory microenvironment. Receptor for advanced glycation end products (RAGE) is a member of the immunoglobulin superfamily of cell surface molecules which has been associated with chronic inflammation, which in turn enhances the progression of various cancers [1]. We have shown that RAGE is expressed in a panel of aggressive BC cell lines and TNBC tissues [3]. High RAGE expression was also observed in lymph node and distant metastases patient samples. In addition, we observed that high RAGE expression was associated with poor prognosis in breast cancer [3]. RAGE has also been shown to play important role in various cancers including pancreatic cancer. Under hypoxic environment, RAGE was shown to interact directly to mutant KRAS and upregulate HIF1α that leads to the development of pancreatic cancer [2]. We have shown that RAGE deficiency inhibits the growth of murine breast cancer tumor cells [3]. RAGEdeficient mice have shown less chemically-induced inflammation [1]. Furthermore, we have shown that RAGE neutralizing antibody treatment significantly inhibited breast cancer metastasis in an intracardial mouse model [3]. RAGE is a multi-ligand receptor and binds to several inflammatory ligands such as advanced glycation end products (AGE), high mobility group box 1 peptide (HMGB-1), amyloid-β peptide and the S100 family of proteins. Our mechanistic investigation has revealed that RAGE mediates its functional effects in breast cancer by binding to S100A7 [3]. S100A7 is a small molecular weight calcium-binding protein [4]. Although a number of putative functions have been proposed for S100A7, its biological role, particularly in BC, remains to be defined [4]. Phylogenetic analyses have shown the mouse ancestor mS100a7a15 to be most related to human S100A7 [5]. It has been shown that mS100a7a15 is up-regulated during carcinogen-induced mammary tumorigenesis. However, the direct functional role of mS100a7a15 in disease progression is not well-characterized. We have shown that mS100a7a15 overexpression induced hyperplasia in mammary glands of these transgenic mice [5]. Upon binding to ligands, RAGE activates its downstream signaling mechanisms that augment and maintain chronic inflammatory conditions [1]. We have also shown that S100A7 enhances NF-kB activation and its nuclear translocation in TNBC cells. These features of RAGE make it an ideal candidate for therapeutic strategies against inflammation induced cancers. The link between inflammation and cancer has been postulated for a long time because of the presence of inflammatory cells in the tumor microenvironment, including myeloid cells and tumor associated macrophages (TAMs) [6]. These cells have been shown to significantly enhance tumor growth and the metastasis of various tumors and are key inhibitors of anti-tumor immunity [6]. In addition, collaborative interactions of tumor cells with TAMs have been associated with poor prognosis in BC [6]. TAMs have also been shown to play an important role in enhancing metastasis and drug resistance [6]. Therefore, inhibition of TAM accumulation and recruitment is a promising strategy for enhancing the effectiveness of immune-based therapies against tumors [6]. We and others have shown that RAGE is also expressed on immune cells [3, 5, 7]. RAGE expression was detected in different cell types present within the tumor microenvironment including macrophages. In addition, RAGE has been shown to be present in the microglia in the gliomas. Chen et al (2014) have shown that RAGE signaling in glioma TAMs regulates angiogenesis most likely by MMP9 [8]. We also showed in breast cancer mouse model that blocking of RAGE signaling significantly inhibited tumor growth, metastasis and angiogenesis through inhibition of MMP9+ TAMs [3]. In addition, RAGE also binds to S100A8/A9 to recruit myeloid derived suppressor cells (MDSCs) and thereby enhance cancer growth and metastasis [7]. These studies suggest that RAGE could enhance tumor growth and metastasis through regulating tumor microenvironment. Overall, our results indicate that RAGE could be used as a novel therapeutic target and biomarker for TNBCs. Furthermore RAGE/S100A7 signaling through paracrine and endocrine mechanisms in epithelial as well as TAMs plays an important role in linking inflammation to breast cancer development and metastasis (Fig. ​(Fig.1).1). In addition, RAGE also binds to S100A8/A9 to recruit myeloid derived suppressor cells (MDSCs) and thereby enhance breast cancer growth and metastasis. Figure 1 Role of S100A7/RAGE-signaling axis in breast cancer growth and metastasis