Role Of Receptor For Advanced Glycation End-products Research Articles

RAGE is a critical factor of sex-based differences in age-induced kidney damage.

Introduction: Advanced glycation end products (AGEs) are a heterogeneous group of molecules with potential pathophysiological effects on the kidneys. Fibrosis together with the accumulation of AGEs has been investigated for its contribution to age-related decline in renal function. AGEs mediate their effects in large parts through their interactions with the receptor for AGEs (RAGE). RAGE is a transmembrane protein that belongs to the immunoglobulin superfamily and has the ability to interact with multiple pro-inflammatory/pro-oxidative ligands. The role of RAGE in aging kidneys has not been fully characterized, especially for sex-based differences. Methods: Therefore, we analyzed constitutive RAGE knockout (KO) mice in an age- and sex-dependent manner. Paraffin-embedded kidney sections were used for histological analysis and protein expression of fibrosis and damage markers. RNA expression analysis from the kidney cortex was done by qPCR for AGE receptors, kidney damage, and early inflammation/fibrosis factors. FACS analysis was used for immune cell profiling of the kidneys. Results: Histological analysis revealed enhanced infiltration of immune cells (positive for B220) in aged (>70weeks old) KO mice in both sexes. FACS analysis revealed a similar pattern of enhanced B-1a cells in aged KO mice. There was an age-based increase in pro-fibrotic and pro-inflammatory markers (IL-6, TNF, TGF-β1, and SNAIL1) in KO male mice that presumably contributed to renal fibrosis and renal damage (glomerular and tubular). In fact, in KO mice, there was an age-dependent increase in renal damage (assessed by NGAL and KIM1) that was accompanied by increased fibrosis (assessed by CTGF). This effect was more pronounced in male KO mice than in the female KO mice. In contrast to the KO animals, no significant increase in damage markers was detectable in wild-type animals at the age examined (>70weeks old). Moreover, there is an age-based increase in AGEs and scavenger receptor MSR-A2 in the kidneys. Discussion: Our data suggest that the loss of the clearance receptor RAGE in male animals further accelerates age-dependent renal damage; this could be in part due to an increase in AGEs load during aging and the absence of protective female hormones. By contrast, in females, RAGE expression seems to play only a minor role when compared to tissue pathology.

Insulin/IGF Axis and the Receptor for Advanced Glycation End Products: Role in Meta-inflammation and Potential in Cancer Therapy.

In metabolic conditions such as obesity and diabetes, which are associated with deregulated signaling of the insulin/insulin-like growth factor system (IIGFs), inflammation plays a dominant role. In cancer, IIGFs is implicated in disease progression, particularly during obesity and diabetes; however, further mediators may act in concert with IIGFs to trigger meta-inflammation. The receptor for advanced glycation end-products (RAGE) and its ligands bridge together metabolism and inflammation in obesity, diabetes, and cancer. Herein, we summarize the main mechanisms of meta-inflammation in malignancies associated with obesity and diabetes; we provide our readers with the most recent understanding and conceptual advances on the role of RAGE at the crossroad between impaired metabolism and inflammation, toward disease aggressiveness. We inform on the potential hubs of cross-communications driven by aberrant RAGE axis and dysfunctional IIGFs in the tumor microenvironment. Furthermore, we offer a rationalized view on the opportunity to terminate meta-inflammation via targeting RAGE pathway, and on the possibility to shut its molecular connections with IIGFs, toward a better control of diabetes- and obesity-associated cancers.

Role of RAGE in the Pathogenesis of Neurological Disorders.

This review reflects upon our own as well as other investigators' studies on the role of receptor for advanced glycation end-products (RAGE), bringing up the latest information on RAGE in physiology and pathology of the nervous system. Over the last ten years, major progress has been made in uncovering many of RAGE-ligand interactions and signaling pathways in nervous tissue; however, the translation of these discoveries into clinical practice has not come to fruition yet. This is likely, in part to be the result of our incomplete understanding of this crucial signaling pathway. Clinical trials examining the therapeutic efficacy of blocking RAGE-external ligand interactions by genetically engineered soluble RAGE or an endogenous RAGE antagonist, has not stood up to its promise; however, other trials with different blocking agents are being considered with hope for therapeutic success in diseases of the nervous system.

Dual Nature of RAGE in Host Reaction and Nurturing the Mother-Infant Bond.

Non-enzymatic glycation is an unavoidable reaction that occurs across biological taxa. The final products of this irreversible reaction are called advanced glycation end-products (AGEs). The endogenously formed AGEs are known to be bioactive and detrimental to human health. Additionally, exogenous food-derived AGEs are debated to contribute to the development of aging and various diseases. Receptor for AGEs (RAGE) is widely known to elicit biological reactions. The binding of RAGE to other ligands (e.g., high mobility group box 1, S100 proteins, lipopolysaccharides, and amyloid-β) can result in pathological processes via the activation of intracellular RAGE signaling pathways, including inflammation, diabetes, aging, cancer growth, and metastasis. RAGE is now recognized as a pattern-recognition receptor. All mammals have RAGE homologs; however, other vertebrates, such as birds, amphibians, fish, and reptiles, do not have RAGE at the genomic level. This evidence from an evolutionary perspective allows us to understand why mammals require RAGE. In this review, we provide an overview of the scientific knowledge about the role of RAGE in physiological and pathological processes. In particular, we focus on (1) RAGE biology, (2) the role of RAGE in physiological and pathophysiological processes, (3) RAGE isoforms, including full-length membrane-bound RAGE (mRAGE), and the soluble forms of RAGE (sRAGE), which comprise endogenous secretory RAGE (esRAGE) and an ectodomain-shed form of RAGE, and (4) oxytocin transporters in the brain and intestine, which are important for maternal bonding and social behaviors.

1190-P: Diabetes Activates Pancreatic Stellate Cells through RAGE Signaling in Pancreatic Ductal Adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive carcinoma with poor prognosis. Its poor prognosis is ascribed to the extensive fibrotic stroma. Pancreatic stellate cells (PSCs) are major contributors to PDAC fibrosis. Type 2 diabetes (T2D) is an established risk factor for PDAC. The common mechanistic link might be the accumulation of advanced glycation end-products (AGEs) and the signaling through receptor for advanced glycation end-products (RAGE), the receptor for AGEs. Nevertheless, the role of RAGE in PSCs activation was not evaluated in PDAC with T2D. First, PSCs were isolated from male obese T2D model, db/db (db) and db/m (C) mice at 12 weeks of age. mRNA expression of the makers for PSCs activation like TGF-β1, αSMA and collagen type1A1 were significantly increased in PSCs isolated from db compared to C (p<0.01, respectively). To evaluate the implication of RAGE, PSCs isolated from C57BL/6 (WT) and RAGE deleted mice (RKO) were stimulated with 12.5-100 μg/ml AGEs. AGEs increased mRNA expression of TGF-β1, αSMA and collagen type1A1 in WT-PSCs with dose dependent manner (p<0.01, respectively) despite no increase in mRNA expressions in RKO-PSCs. Furthermore, PSCs were isolated from high fat diet induced obese WT and RKO for 8 weeks. The mRNA expressions of TGF-β1, αSMA and collagen type1A1 were comparable between two groups. We evaluated surgically resected human PDAC subjects with T2D (PDT2D, 33 cases) and without T2D (PDNT2D, 83cases). αSMA-labeled PSCs significantly surrounded the ducts with dysplasia in PDT2D compared to PDNT2D (p<0.05). The nuclear expression of NF-κB in stromal cells, which was downstream molecules of RAGE, was significantly higher in PDT2D than PDNT2D (p<0.05). Collectively, RAGE signaling was activated in PSCs in T2D, which could be implicated in severe stromal fibrosis in PDAC. Inhibition of RAGE signaling in PSCs may link to a new therapy, in which the stromal fibrosis and cytokine production are reduced in PDAC with T2D. Disclosure C. Uchida: None. H. Mizukami: None. K. Kudo: None. Y. Yamamoto: None. Funding Japan Society for the Promotion of Science (JP17K17579)

A Role for RAGE in DNA Double Strand Breaks (DSBs) Detected in Pathological Placentas and Trophoblast Cells.

Impaired DNA damage responses are associated with several diseases, including pregnancy complications. Recent research identified an ATM-kinase dependent function for the nuclear isoform of the receptor for advanced glycation end-products (RAGE) during double strand break (DSB)-repair. RAGE contributes to end-resectioning of broken DNA sites by binding with the MRE11-Rad50-Nbs1 (MRN) complex. Placental research is limited regarding the impact of genomic instability and the mechanism for potential repair. We tested the hypothesis regarding the involvement of RAGE during the repair of placental DNA-DSBs. We first identified that the pregnancy complications of PE and preterm labor (PTL) experience loss of genomic integrity and an in vitro trophoblast cell model was used to characterize trophoblast DSBs. Colocalized immunofluorescence of γ-H2AX and RAGE support the potential involvement of RAGE in cellular responses to DNA-DSBs. Immunoblotting for both molecules in PE and PTL placenta samples and in trophoblast cells validated a connection. Co-immunoprecipitation studies revealed interactions between RAGE and pATM and MRE11 during DNA-DSBs. Reduced cellular invasion confirmed the role of genomic instability in trophoblastic function. Collectively, these experiments identified genomic instability in pregnancy complications, the impact of defective DNA on trophoblast function, and a possible RAGE-mediated mechanism during DNA-DSB repair.

Downregulation of RAGE Inhibits Cell Proliferation and Induces Apoptosis via Regulation of PI3K/AKT Pathway in Cervical Squamous Cell Carcinoma.

AimThe receptor for advanced glycation endproducts (RAGE) expression has been reported to be implicated with cancer development. In this study, the role of RAGE in the regulation of cervical squamous cancer cell proliferation, apoptosis and the mechanism of RAGE involved in the biological behaviors were explored.MethodsThe RAGE expression was overexpressed or downregulated by lentivirus transfection. The effect of RAGE expression on cell proliferation was explored by CCK-8, MTT, and BrdU assay, and the effect of RAGE on tumor development was confirmed by the xenograft mouse model along with the immunohistochemistry stain of proliferating cell nuclear antigen (PCNA). Apoptosis was investigated by flow cytometry and TUNEL assay. Western blotting was performed to investigate the expression of possible proteins, including Bax, Bcl-2, PI3K, p-PI3K, AKT, and p-AKT.ResultsOverexpression of RAGE promoted proliferation of cervical squamous cancer cell and increased PCNA expression. In the meantime, RAGE overexpression inhibited cell apoptosis along with a decrease of Bax/Bcl-2 ratio, and induction of PI3K/AKT activation. The in vivo results showed that overexpression of RAGE enhanced tumor growth. Conversely, knockdown of RAGE exhibited opposed effects on cervical cancer cells and xenograft mouse model. Furthermore, RAGE inhibitor FPS-ZM1 effectively inhibited SiHa cell viability and PCNA expression, and increased cell apoptosis and Bax/Bcl-2 ratio. Moreover, PI3K inhibitor LY294002 effectively inhibited activation of PI3K and AKT, and further repressed RAGE overexpression-induced cell proliferation and apoptosis inhibition.ConclusionRAGE promotes the growth ability of cervical squamous cell carcinoma by inducing PCNA expression and inhibiting cell apoptosis via inactivation of the PI3K/AKT pathway.

Abstract P3-01-11: Targeting RAGE inhibits breast cancer invasion and metastasis

Abstract Background: Breast cancer (BC) is treatable with early detection, but once metastasis occurs, there is no cure. Therefore, it is of upmost importance to find targetable biomarkers that are involved in modulating the multi-step metastatic process. The Receptor for Advanced Glycation Endproducts (RAGE) and its ligands are an inflammatory pathway that are involved in modulating breast cancer progression and metastasis. We recently demonstrated that genetic or pharmacological interruption of RAGE signaling affected tumor progression and metastasis. However, no studies have dissected the role of RAGE in tumor growth from the metastatic cascade. Here, we show for the first time in multiple metastatic breast cancer models that targeting RAGE impairs BC metastasis. Methods: We tested the anti-metastatic effect of RAGE in vitro using the RAGE inhibitor FPS-ZM1 or RAGE shRNA knockdown in cell invasion, proliferation, migration and sphere formation assays (with 4T1, Py8119 and E0771 mouse BC cells). For in vivo assays we used orthotopic BC models (4T1/BALB/c and E0771 & Py8119/C57BL6) and experimental metastasis assays (tail vein injection of 4T1/BALB/c and Py8119/C57BL6). To target RAGE, we used genetic (shRNA in 4T1 cells and RAGE knockout in C57BL6 mice) and pharmacological approaches (I.P. injection of FPS-ZM1). To investigate the synergistic effects of RAGE inhibition on progression and metastasis, we tested combination therapy of low-dose doxorubicin and FPS-ZM1. Results: Inhibition of RAGE with FPS-ZM1 (1-5uM) impaired tumor cell invasion of 4T1, E0771 and Py8119 cells. Similarly, in spheroid assays, treatment of cells with FPS-ZM1 resulted in fewer and smaller colonies. However, FPS-ZM1 treatment did not affect cell proliferation. In vivo studies revealed that FPS-ZM1 (1mg/kg) has a modest effect on tumor growth in 4T1/BALB/c injected mice but displayed a dramatic inhibitory effect on metastasis to the lungs. In experimental metastasis assays, tail-vein injection of 4T1 cells in BALB/c mice demonstrated that FPS-ZM1 treatment strongly impaired metastatic disease in mice compared to controls. To dissect the genetic role of RAGE in the tumor versus host, we test the effect of RAGE knockdown in 4T1 cells in experimental metastasis assays. RAGE shRNA impaired metastasis to the lungs, albeit to a lower degree seen with the RAGE inhibitor. To test the role of the host, we injected Py8119 cells into wild-type and RAGE knockout (RKO) mice. RKO mice displayed fewer metastatic burden compared to wild-type mice. Finally, in our combination treatment experiments, treatment of 4T1-injected BALB/c mice with Doxorubicin and FPS-ZM1 (alone and in combination), demonstrated that drug combination was more effective in inhibiting lung metastasis than either reagent alone. We are currently assessing how RAGE mechanistically drives these metastatic changes. Conclusion: Our data strongly suggests RAGE plays an important role in breast cancer metastasis, with less of an effect on tumor growth. Ongoing studies in our lab are testing which stage of the metastatic cascade RAGE is involved in, and the underlying mechanisms driving these processes. In conclusion, the use of RAGE inhibitors could represent a novel therapeutic approach for metastatic breast cancer. Citation Format: Gyong Ha Hwang, Melinda Magna, Barbara Mera, Toni Yeasky, Taekyoung Kwak, Lucas Outcault, Masaru Takabatake, Thuy-Mai Le, Marc E. Lippman, Barry I. Hudson. Targeting RAGE inhibits breast cancer invasion and metastasis [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-01-11.

Abstract 628: Diabetes-Mediated Vascular Calcification is RAGE-Dependent

Type II diabetes mellitus (DM) is characterized by chronic hyperglycemia, and medial vascular calcification is a common cardiovascular complication of DM. This leads to aortic stiffening, which can leave patients at an increased risk for heart attack or stroke. Advanced Glycation End-Products (AGEs)/Receptor for AGEs (RAGE) signaling cascade has been implicated as a potentiator of diabetes-mediated vascular calcification, but it is not well understood. AGE/RAGE signaling influences both cellular and systemic responses to increase bone matrix proteins in hyperglycemic and calcification conditions and has also been shown to increase oxidative stress by promoting diabetes-mediated vascular calcification. This causes a phenotypic switch of vascular smooth muscle cells (VSMCs) to osteoblast-like cells and the hypothesized activation of adventitial fibroblasts (AFBs) to a myofibroblast phenotype. The purpose of this research is to understand AGE/RAGE mediated vascular calcification as a complication of diabetes. Calcification was induced for 7 days in primary mouse VSMCs and AFBs of non-diabetic, diabetic, non-diabetic RAGE knockout (RKO), and diabetic RKO, and then treated with AGEs to activate RAGE. Alizarin Red S staining was utilized to visualize calcification. Intracellular calcium levels were quantified and normalized to cell number (DAPI). Pronounced calcification was observed in non-diabetic VSMCs and the loss of RAGE resulted in decreased calcification in the non-diabetic RKO VSMCs. AFBs were exposed to the same experimental conditions as the VSMCs and calcification was increased in the diabetic AFBs while calcification was significantly decreased in the diabetic RKO AFBs. These data demonstrated that diabetes-mediated vascular calcification was RAGE-dependent in both cell types. Literature has cited the VSMC as the primary mediator for vascular calcification, but we have shown that the AFBs in the outer layer of the aorta have the ability to calcify and this is mediated by RAGE signaling, which elucidates a role for RAGE in diabetes-mediated vascular calcification. Understanding the role of AGE/RAGE signaling in diabetes-mediated vascular calcification will allow for possible targets for pharmacological intervention.

Extracorporal Shock Wave Therapy Enhances Receptor for Advanced Glycated End-Product-Dependent Flap Survival and Angiogenesis.

Loss of skin flaps due to deteriorated wound healing is a crucial clinical issue. Extracorporal shock wave therapy (ESWT) promotes flap healing by inducing angiogenesis and suppressing inflammation. The receptor for advanced glycation end-products (RAGEs) was identified to play a pivotal role in wound healing. However, to date, the role of RAGE in skin flaps and its interference with ESWT are unknown. Caudally pedicled musculocutanous skin flaps in RAGE and wt mice were treated with low-dose extracorporal shock waves (s-RAGE, s-wt) and analyzed for flap survival, histomorphologic studies, and immunohistochemistry during a 10-day period. Animals without ESWT served in each genotype as a control group (c-RAGE, c-wt). Statistical analysis was carried out by repeated-measures analysis of variance. Flap necrosis was significantly reduced after ESWT in wt animals but increased in RAGE-deficient animals. Morphometric differences between the 4 groups were identified and showed a delayed wound healing with dysregulated inflammatory cells and deteriorated angiogenesis in RAGE animals. Furthermore, spatial and temporal differences were observed. The RAGE controls inflammation and angiogenesis in flap healing. The protective effects of ESWT are dependent on intact RAGE signaling, which enables temporary targeted infiltration of immune cells and neoangiogenesis.

22016 ATVB Plenary Lecture: Receptor for Advanced Glycation Endproducts and Implications for the Pathogenesis and Treatment of Cardiometabolic Disorders: Spotlight on the Macrophage.

The receptor for advanced glycation endproducts (RAGE) interacts with a unique repertoire of ligands that form and collect in the tissues and circulation in diabetes mellitus, aging, inflammation, renal failure, and obesity. RAGE is expressed on multiple cell types linked to tissue perturbation in these settings. This brief review focuses on the role of RAGE in monocytes/macrophages and how RAGE ligand engagement on these cells mediates seminal changes in monocyte/macrophage migration, oxidative stress, cholesterol efflux, and pro- versus anti-inflammatory cues that signal to tissue damage. Studies using mice devoid of Ager (gene encoding RAGE) or pharmacological antagonists of RAGE are protective in animal models of diabetes mellitus, atherosclerosis, and high-fat diet-induced obesity, in least in part through key roles in monocytes/macrophages. RAGE signal transduction requires the interaction of RAGE cytoplasmic domain with the formin, DIAPH1 (diaphanous 1) and novel antagonists of this interaction show significant promise in attenuation of the maladaptive effects of RAGE ligands in cellular and in vivo models. Finally, this brief review discusses evidence for RAGE axis perturbation in human monocytes/macrophages and how tracing RAGE activity in these cells may identify target engagement biomarkers of RAGE antagonism for future clinical trials.

Abstract P3-06-01: Therapeutic targeting of RAGE in the tumor and tumor microenvironment inhibits breast progression and metastasis

Abstract Background: The Receptor for Advanced-Glycation End-products (RAGE) is highly expressed in various cancers and its expression is correlated with poorer outcomes in breast cancer. We have previously implicated RAGE in breast cancer, but whether RAGE drives breast cancer progression and metastasis either through tumor cell intrinsic effects, non-tumor cells of the tumor microenvironment, or both, is not fully understood. More importantly, studies are lacking that target RAGE therapeutically in cancer, and may therefore represent a novel treatment for breast cancer metastasis. Methods: Using multiple human and murine breast cancer models we dissected the tumor intrinsic versus tumor microenvironment role of RAGE in metastasis. RAGE was targeted in tumor cells using multiple shRNAs, in non-tumor cells by global gene knockout in mice, and both by therapeutically targeting with the novel RAGE inhibitor FPS-ZM1. In vivo orthotopic models included the NSG (NOD-SCID-gamma) xenograft mouse model (with MDA-MB-231 cells; herein 231), BALBc (4T-1 and 67NR), and C57BL6 wild-type and RAGE knockout (RAGE -/-) mice (with MMTV-PyMT spontaneous breast cancer derived AT-3 cells). Results: We first tested how RAGE impacts tumor cell intrinsic mechanisms using either RAGE shRNAs or FPS-ZM1 in 231, 4175 (231 isogenic highly metastatic cells) and 4T-1 cells. RAGE shRNA and FPS-ZM1 both decreased RAGE MAP-kinase signaling, transwell invasion and soft agar colony formation, without affecting proliferation. In vivo, RAGE shRNA knockdown in 231 cells did not affect tumor growth, but inhibited metastasis to lung and liver. RAGE shRNA knockdown in 4175 cells, decreased orthotopic tumor growth, and reduced tumor angiogenesis and tumor recruitment of leukocyte / macrophages. Furthermore, RAGE shRNA knockdown dramatically decreased metastasis of 4175 cells to lung and liver in a time and sized matched manner compared to shRNA controls. Similarly, RAGE knockdown in 4T-1 cells reduced cell invasion and colony formation, and inhibited lung metastasis from the orthotopic site in BALBc immunocompetent mice. To test the non-tumor cell microenvironment role of RAGE, we performed syngeneic studies with orthotopically injected AT-3 cells in RAGE +/+ and RAGE -/- C57BL6 mice. RAGE -/- mice displayed striking impairment of tumor cell growth compared to RAGE +/+ mice, along with decreased MAP-kinase signaling, tumor angiogenesis and inflammatory cell recruitment. Finally, to test the combined inhibition of RAGE in both tumor cell intrinsic and non-tumor cells of the microenvironment, we performed in vivo treatment of 4175 tumors with FPS-ZM1 (1mg/kg, twice per week). Compared to vehicle, FPS-ZM1 inhibited primary tumor growth, inhibited tumor angiogenesis and inflammatory cell recruitment, and most importantly prevented metastasis to lung and liver. Conclusion: These data clearly demonstrate a role for RAGE in breast cancer progression and metastasis through distinct effects in the tumor cell and non-tumor cells of the tumor microenvironment. Furthermore, our data from drug inhibitor studies highlight the combined targeting of RAGE in the tumor and tumor microenvironment, and as a viable therapeutic means for breast and other metastatic cancers. Citation Format: Kwak T, Drews-Elger K, Ergonul A, Braley A, Hwang GH, El-Ashry D, Slingerland JM, Lippman ME, Hudson BI. Therapeutic targeting of RAGE in the tumor and tumor microenvironment inhibits breast progression and metastasis [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P3-06-01.

Deletion of the Formin, Drf1, is Protective Against Renal Damage in a Murine Model of Diabetes

Our studies have shown the cytoplasmic domain of the receptor for advanced glycation endproducts (RAGE) binds to the formin molecule, diaphanous‐1 (mDia1). mDia1 is a member of the formin family of intracellular molecules involved in cellular migration which act as effectors of Rho GTPase signaling. In RAGE‐expressing cells devoid of Drf1 (gene encoding mDia1), incubation with RAGE ligands failed to generate reactive oxygen species or activate key signaling cellular stress pathways. Here, we sought to determine if mDia1 plays key roles in a murine model of diabetic nephropathy (DN). Our preliminary data reveal that mDia1 expression is increased in human and murine diabetic podocytes and parietal epithelial cells in a diffuse and global pattern compared to age‐matched controls. Furthermore, expression patterns of mDia1 are highly analogous to those of RAGE. While the role of RAGE has been shown to play a key role in the development of DN, the potential contribution of mDia1 has yet to be elucidated. Therefore, we tested the hypothesis that mDia1 contributes to the development and progression of diabetic renal disease in a murine model.Male wild‐type and Drf1‐null (WT, Drf1KO; all in the C57BL/6 background) mice were rendered diabetic at 6 weeks of age with streptozotocin and sacrificed after 3 or 6 months of diabetes. Kidneys were harvested and processed for histology and gene expression and stained with periodic‐acid Schiff (PAS) and semi‐quantitative scoring was used to determine the degree of mesangial sclerosis by average findings in >100 glomeruli/mouse (scale 0–3+; 0=absent, 1=mild, 2=moderate, 3=severe). Glomerular basement membrane (GBM) thickness and podocyte foot process effacement (FPE) were measured by ultrastructural analysis (>8 glomeruli/mouse). Gene expression of inflammatory markers (transforming growth factor beta; Tgfb, interleukin 6; Il6, and monocyte chemoattractant protein 1; Ccl2) were assessed by quantitative real time PCR using RNA prepared from whole kidney cortex and normalized to beta‐actin.This preliminary study suggests that deletion of Drf1 in mice results in a substantial protection against indices of DN by reducing podocyte effacement, inflammation and fibrosis in type 1 DM. Histological and microscopic observations show that mDiaKO mice have a reduced GBM, less podocyte FPE and a decrease in mesangial sclerosis compared to WT mice after 6 months of diabetes. Genotype WT Drf1KO ND (n=5) DM (n=5) ND (n=5) DM (n=5) GBM (nm) 217.3±14.1 288.5±17.46^ 228.3±16.5 246.0±23.2* FPE [%] 9.6±3.6 29.0±6.8^ 9.8±2.3* 13.0±4.7* MS 0.4±0.4 2.0±0.4^ 0.0±0.0* 1.3±0.6*# p<0.05 vs. WT ND, p<0.05 vs WT DM, p<0.05 vs Drf1KO ND. Values shown are Mean±SEM. Ratio of target gene fold‐change normalized to beta‐actin. Gene expression of inflammatory markers in diabetes is downregulated with deletion of mDia1 compared to WT after both 3 and 6 months. Condition 3 months 6 months WTND Drf1KOND WTDM Drf1KODM WTND Drf1KOND WTDM Drf1KODM n=3 n=3 n=3 n=3 n=3 n=3 n=5 n=3 Tgfb 1 1.02 2.67^# 1.09* 1 0.52ab 7.32ac 0.82abc Il6 1 0.96 1.49^# 1.21.^*# 1 0.52ab 2.13ac 0.73abc Ccl2 1 1.01 1.43^# 1.17^*# 1 0.75ab 1.82ac 1.12abc p<0.05 vs. WT ND 3 months, p<0.05 vs WT DM 3 months, p<0.05 vs Drf1KO ND 3 months, p<0.05 vs WT ND 6 months, p<0.05 vs WT DM 6 months, p<0.05 vs Drf1KO ND 6 months. Values shown are Mean±SEM.

Absence of RAGE in an animal experimental model of Duchenne muscular dystrophy results in reduced muscle necrosis and inflammation

Duchenne muscular dystrophy (DMD) is a lethal X-linked neuromuscular disorder characterized by progressive muscle degeneration due to lack of dystrophin, a protein essential for the integrity of sarcolemma during contraction. Chronic inflammation is a hallmark of muscles in DMD subjects, and contributes to progressive muscle wasting. RAGE (receptor for advanced glycation end-products) is a multiligand receptor of the immunoglobulin superfamily involved in physiological and pathological processes including inflammation and myogenesis [1]. While absent in healthy adult muscle tissue, RAGE is expressed in regenerating myofibers during muscle regeneration [2,3], in dystrophic muscles and activated immune cells. To have information about the role of RAGE in the pathophysiology of DMD we generated a double mutant mouse lacking dystrophin and RAGE (mdx/Ager–/– mouse) by cross-breeding dystrophic (mdx) mice with RAGE-null (Ager-/-) mice. Comparison of Quadriceps femoris of mdx and mdx/Ager–/– mice at different ages (i.e., 2, 3, 4 and 5 weeks, and 6 and 12 months of age) showed that the absence of RAGE in dystrophic mice did not affect the onset of the pathology. However, compared with age-matched mdx mice, muscles of 5 week- and 6 and 12 month-old mdx/Ager–/– mice showed i) significantly reduced numbers of necrotic myofibers, ii) a shift towards higher values of the cross-sectional areas (CSA) of myofibers, which was also evident in regenerating (centrally-nucleated) myofibers, and iii) reduced areas of immune cell infiltrate. The expression of MAC3, a marker of activated macrophages, was strongly reduced in muscles of mdx/Ager–/– mice compared with mdx mice. Moreover, muscles of mdx/Ager–/– mice exhibited significantly reduced PAX7+ve and myogenin+ve cell numbers, suggesting a reduced recruitment of muscle precursor cells and more efficient regeneration in dystrophic mice lacking RAGE. Our results suggest that RAGE may sustain inflammatory and degenerative processes in dystrophic muscles, and the inhibition of its expression/activity might represent a potential therapeutic approach in DMD patients.This work was supported by grants from MIUR 2012N8YJC3, AFM-Telethon 16812 and Fondazione CRP 2015.0325.021.

Advanced glycation end-products induced VEGF production and inflammatory responses in human synoviocytes via RAGE-NF-κB pathway activation.

Aging and diabetes are known to be the major cause to affect the progression of osteoarthritis (OA). Advanced glycation end products (AGEs) have been observed to accumulate in various organs especially in joint tissue and do damage to the joint tissue during aging and diabetes. Synovial angiogenesis and inflammation are observed across the full range of OA severity. The signaling pathway of AGEs on vascular endothelial growth factor (VEGF) production and inflammatory responses in synoviocytes are still unclear. Here, we investigated the role of receptor for AGEs (RAGE) and the signaling pathway involved in AGEs-induced VEGF production and inflammatory responses in human synoviocytes. Human synoviocytes were cultured and treated with AGEs (25-100 µg/ml). AGEs significantly induced the protein expressions of cyclooxygenase-2 (COX-2) and VEGF and the productions of prostaglandin-E2 (PGE2), VEGF, interleukin-6 (IL-6), and metalloproteinase-13 (MMP-13) in human synoviocytes in a dose-dependent manner. Moreover, AGEs markedly activated the phosphorylations of IκB kinase (IKK)α/β, IκBα, and nuclear factor (NF)-κB-p65 proteins in human synoviocytes in a time-dependent manner. Treatment with neutralizing antibody for RAGE statistically significantly decreased the AGEs-induced increase in COX-2, VEGF, PGE2, IL-6, and MMP13 and AGEs-activated NF-κB pathway activation. Taken together, these findings indicate that AGEs are capable of inducing VEGF production and inflammatory responses via RAGE-NF-κB pathway activation in human synoviocytes. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:791-800, 2016.

Abstract 5292: Determination of the receptor for advanced glycation end-products in breast cancer patients previous chemotherapy

Abstract The receptor for advanced glycation end-products (RAGE) is a newly recognized factor regulating cancer cell invasion and metastasis. RAGE has recently been recognized as a cancer-associated protein responsible for cancer progression and metastasis in gastrointestinal and epithelial cancers. The aim was to examine the role of RAGE in breast cancer previous to chemotherapy treatment. Methods: A retrospective study in surgical specimens obtained from 68 consecutive patients who underwent surgery for stage II-III breast cancer at the Oncology Hospital at National Medical Center SXXI, between 2008 and 2009. RAGE expression was examined by immunohistochemistry (IHQ) and PCR in 68 patients with stage II or III breast cancer previous chemotherapy treatment. RAGE expression was compared with clinicopathological parameters including clinical stage, invasive depth, nodal metastasis, disease recurrence and disease-free survival. The positive expression rate of RAGE was defined as 50% or more of stain in IHQ. Results: In all 68 samples no RAGE membrane expression was found significantly. Just one specimen was 50% positive in tumor IHQ and no clinicopathological correlation was found. In PCR the results were more significant. Our findings shows suggestive no evidence for the presence of RAGE in membrane, but possibly RAGE gene expression could had relationship in the progression of breast cancer in stages II and III. Citation Format: Jorge A. Guadarrama-Orozco, Erika B. Ruiz-García, Hector A. Maldonado-Martinez, Lilia P. Barron-Rodríguez, Fernando Mainero-Ratchelous, Horacio Astudillo-delaVega. Determination of the receptor for advanced glycation end-products in breast cancer patients previous chemotherapy. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5292. doi:10.1158/1538-7445.AM2015-5292

Abstract 805: Subcellular distribution of RAGE affects its functions in melanoma growth and progression

Abstract The receptor for advanced glycation end-products (RAGE) is known to act as a central driver of tumorigenesis by sustaining a chronic inflammatory tumor microenvironment. Until to date, RAGE has been exclusively described as a cell surface receptor being activated upon engagement with its various extra-cellular ligands, e.g. S100B, S100A8/A9, HMGB1, and others. This study aimed at elucidating the functional role of RAGE and its isoforms depending on their subcellular distribution in the context of melanoma development, growth and progression. Therefore, various in vitro models using melanoma cells or melanocytes and melanoma mouse models as well as tissue-microarrays representing human specimens of malignant melanoma and benign nevi were applied. The expression analyses revealed an overexpression of RAGE in melanoma cells compared to melanocytes/nevocytes. Moreover, RAGE protein was found to be localized primarily in the nucleus of melanocytes/nevocytes whereas a predominant cell surface/cytoplasmic localization of RAGE is observed in melanoma cells. Nuclear translocation of RAGE depends on the nuclear transport machinery and site-directed mutagenesis of predicted DNA binding sites within the RAGE protein was applied in order to study its functional role in the nucleus. Furthermore, knockdown of RAGE indicated its central function in apoptosis induction. In conclusion, RAGE and its isoforms are overexpressed and aberrantly localized in malignant melanoma cells compared to melanocytes/nevocytes. Our data point towards a novel tumor-protective function of RAGE in melanoma development depending on its nuclear localization. Citation Format: Maike Reith, Wolf-Henning Gebhardt, Kathrin Tarnanidis, Nikolaus B. Wagner, Kristian Ikenberg, Coretta Kehrel, Jochen Utikal, Christoffer Gebhardt. Subcellular distribution of RAGE affects its functions in melanoma growth and progression. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 805. doi:10.1158/1538-7445.AM2015-805

Deletion of mDia1 is Protective Against Renal Damage in a Murine Model of Diabetes

Our studies have shown the cytoplasmic domain of the receptor for advanced glycation endproducts (RAGE) binds to the formin molecule, diaphanous-1 (mDia1). mDia1 is a member of the formin family of intracellular molecules involved in cellular migration which act as effectors of Rho GTPase signaling. In RAGE-expressing cells devoid of mDia1, incubation with RAGE ligands failed to generate reactive oxygen species or activate key signaling cellular stress pathways. Our preliminary data reveal that mDia1 expression is increased in human and murine diabetic kidneys and expression patterns of mDia1 are highly analogous to RAGE expression patterns. In the diabetic glomerulus, RAGE and mDia1 are highly expressed in podocytes. While the role of RAGE has been shown to play a key role in the development of DN, the potential contribution of mDia1 has yet to be elucidated. Therefore, we tested the hypothesis that mDia1 contributes to the development of diabetic renal disease in a murine model. This preliminary study suggests that deletion of mDia1 in mice results in a substantial protection against indices of renal disease by reducing podocyte effacement, inflammation and fibrosis in type 1 DM.

Identification of a functional interaction of HMGB1 with Receptor for Advanced Glycation End-products in a model of neuropathic pain.

Recent studies indicate that the release of high mobility group box 1 (HMGB1) following nerve injury may play a central role in the pathogenesis of neuropathic pain. HMGB1 is known to influence cellular responses within the nervous system via two distinct receptor families; the Receptor for Advanced Glycation End-products (RAGE) and Toll-like receptors (TLRs). The degree to which HMGB1 activates a receptor is thought to be dependent upon the oxidative state of the ligand, resulting in the functional isoforms of all-thiol HMGB1 (at-HMGB1) acting through RAGE, and disufide HMGB1 (ds-HMGB1) interacting with TLR4. Though it is known that dorsal root ganglia (DRG) sensory neurons exposed to HMGB1 and TLR4 agonists can influence excitation, the degree to which at-HMGB1 signaling through neuronal RAGE contributes to neuropathic pain is unknown. Here we demonstrate that at-HMGB1 activation of nociceptive neurons is dependent on RAGE and not TLR4. To distinguish the possible role of RAGE on neuropathic pain, we characterized the changes in RAGE mRNA expression up to one month after tibial nerve injury (TNI). RAGE mRNA expression in lumbar dorsal root ganglion (DRG) is substantially increased by post-injury day (PID) 28 when compared with sham injured rodents. Protein expression at PID28 confirms this injury-induced event in the DRG. Moreover, a single exposure to monoclonal antibody to RAGE (RAGE Ab) failed to abrogate pain behavior at PID 7, 14 and 21. However, RAGE Ab administration produced reversal of mechanical hyperalgesia on PID28. Thus, at-HMGB1 activation through RAGE may be responsible for sensory neuron sensitization and mechanical hyperalgesia associated with chronic neuropathic pain states.

Knockdown of RAGE inhibits growth and invasion of gastric cancer cells

The receptor for advanced glycation end-products (RAGE) is an oncogenic trans-membranous receptor, which is overexpressed in multiple human cancers. However, the role of RAGE in gastric cancer is still elusive. In this study, we investigated the expression and molecular mechanisms of RAGE in gastric cancer cells. Forty cases of gastric cancer and corresponding adjacent non-cancerous tissues (ANCT) were collected, and the expression of RAGE was assessed using immunohistochemistry (IHC) in biopsy samples. Furthermore, RAGE signaling was blocked by constructed recombinant small hairpin RNA lentiviral vector (Lv-shRAGE) used to transfect into human gastric cancer SGC-7901 cells. The expression of AKT, proliferating cell nuclear antigen (PCNA) and matrix metallopeptidase-2 (MMP-2) was detected by Real-time PCR and Western blot assays. Cell proliferative activities and invasive capability were respectively determined by MTT and Transwell assays. Cell apoptosis and cycle distribution were analyzed by flow cytometry. As a consequence, RAGE was found highly expressed in cancer tissues compared with the ANCT (70.0% vs 45.0%, P=0.039), and correlated with lymph node metastases (P=0.026). Knockdown of RAGE reduced cell proliferation and invasion of gastric cancer with decreased expression of AKT, PCNA and MMP-2, and induced cell apoptosis and cycle arrest. Altogether, upregulation of RAGE expression is associated with lymph node metastases of gastric cancer, and blockade of RAGE signaling suppresses growth and invasion of gastric cancer cells through AKT pathway, suggesting that RAGE may represent a potential therapeutic target for this aggressive malignancy.