Regulation of Receptor for Advanced Glycation End Products (RAGE) Ectodomain Shedding and Its Role in Cell Function.

The receptor for advanced glycation end products (RAGE) is a multiligand transmembrane receptor that can undergo proteolysis at the cell surface to release a soluble ectodomain. Here we observed that ectodomain shedding of RAGE is critical for its role in regulating signaling and cellular function. Ectodomain shedding of both human and mouse RAGE was dependent on ADAM10 activity and induced with chemical activators of shedding (ionomycin, phorbol 12-myristate 13-acetate, and 4-aminophenylmercuric acetate) and endogenous stimuli (serum and RAGE ligands). Ectopic expression of the splice variant of RAGE (RAGE splice variant 4), which is resistant to ectodomain shedding, inhibited RAGE ligand dependent cell signaling, actin cytoskeleton reorganization, cell spreading, and cell migration. We found that blockade of RAGE ligand signaling with soluble RAGE or inhibitors of MAPK or PI3K blocked RAGE-dependent cell migration but did not affect RAGE splice variant 4 cell migration. We finally demonstrated that RAGE function is dependent on secretase activity as ADAM10 and γ-secretase inhibitors blocked RAGE ligand-mediated cell migration. Together, our data suggest that proteolysis of RAGE is critical to mediate signaling and cell function and may therefore emerge as a novel therapeutic target for RAGE-dependent disease states.

Vascular and inflammatory stresses mediate atherosclerosis via RAGE and its ligands in apoE-/- mice.

Endothelial dysfunction is a key triggering event in atherosclerosis. Following the entry of lipoproteins into the vessel wall, their rapid modification results in the generation of advanced glycation endproduct epitopes and subsequent infiltration of inflammatory cells. These inflammatory cells release receptor for advanced glycation endproduct (RAGE) ligands, specifically S100/calgranulins and high-mobility group box 1, which sustain vascular injury. Here, we demonstrate critical roles for RAGE and its ligands in vascular inflammation, endothelial dysfunction, and atherosclerotic plaque development in a mouse model of atherosclerosis, apoE-/- mice. Experiments in primary aortic endothelial cells isolated from mice and in cultured human aortic endothelial cells revealed the central role of JNK signaling in transducing the impact of RAGE ligands on inflammation. These data highlight unifying mechanisms whereby endothelial RAGE and its ligands mediate vascular and inflammatory stresses that culminate in atherosclerosis in the vulnerable vessel wall.

Mice deficient in PKCbeta and apolipoprotein E display decreased atherosclerosis.

Endothelial activation is a central initiating event in atheroma formation. Evidence from our laboratory and others has demonstrated links between activation of early growth response-1 (Egr-1) and atherosclerosis and also has demonstrated that activated protein kinase C (PKC) betaII is a critical upstream regulator of Egr-1 in response to vascular stress. We tested the role of PKCbeta in regulating key events linked to atherosclerosis and show that the aortas of apoE(-/-) mice display an age-dependent increase in PKCbetaII antigen in membranous fractions vs. C57BL/6 animals with a approximately 2-fold increase at age 6 wk and a approximately 4.5-fold increase at age 24 wk. Consistent with important roles for PKCbeta in atherosclerosis, a significant decrease in atherosclerotic lesion area was evident in PKCbeta(-/-)/apoE(-/-) vs. apoE(-/-) mice by approximately 5-fold, in parallel with significantly reduced vascular transcripts for Egr-1 and matrix metalloproteinase (MMP)-2 antigen and activity vs. apoE(-/-) mice. Significant reduction in atherosclerosis of approximately 2-fold was observed in apoE(-/-) mice fed ruboxistaurin chow (PKCbeta inhibitor) vs. vehicle. In primary murine and human aortic endothelial cells, the PKCbeta-JNK mitogen-activated protein kinase pathway importantly contributes to oxLDL-mediated induction of MMP2 expression. Blockade of PKCbeta may be beneficial in mitigating endothelial perturbation and atherosclerosis.

Noninvasive monitoring the biology of atherosclerotic plaque development with radiolabeled annexin V and matrix metalloproteinase inhibitor in spontaneous atherosclerotic mice.

OBJECTIVES: To compare the ability of (99m)Tc-labeled broad-based matrix metalloproteinase inhibitor (RP805) (MPI) and (99m)Tc-annexin V to identify more advanced atherosclerotic disease in apolipoprotein E-null (apoE(-/-)) mice. BACKGROUND: Both MMP expression and apoptotic cell death occur in both early and in advanced atherosclerotic plaques. METHODS: Eight 6-9-week-old apoE(-/-) mice, 10 apoE(-/-) mice at 20 weeks, and 12 apoE(-/-) at 40 weeks were injected with both tracers in alternating sequence separated by 48 h, underwent planar imaging and were killed. Radiotracer uptake was quantified from the scans as percent whole body and from tissue as percent injected dose per gram (%ID/g). Quantitative immunohistopathology of the aorta and carotids for macrophages, MMPs, and caspase was performed. RESULTS: At 6 weeks, mice showed no tracer uptake in the chest or neck and had minimal lesion. At 20 weeks, uptake of annexin V as %ID was borderline higher than MPI (1.10 ± .48% vs .77 ± .31%, P = .09), between 20 and 40 weeks aortic lesion area increased from 37.4 ± 12.0% to 46.2 ± 7.4% and at 40 weeks MPI was significantly greater than annexin V uptake (1.11 ± .66% vs .70 ± .16%, P = .05). On histology there were greater increases in % MMP-2 and -9 than % caspase positive cells. Carotid uptake of MPI was greater than annexin V at both 20 and 40 weeks (1.25 ± .48% vs .78 ± .25%, P = .02 and 3.70 ± 1.45% vs 2.25 ± .66%, P = .005). The carotid lesion area at 40 weeks was 74 ± 9% with greater % cells positive for MMP's than caspase. %ID/g annexin V correlated significantly with % macrophages and with caspase-3 positive cells and %ID/g MPI correlated significantly with % macrophages and with MMP-2 and -9 positive cells. CONCLUSIONS: In apoE(-/-) mice, MMP expression is greater than apoptosis as the disease progresses and MPI may be a better imaging agent for more advanced disease.

Identification, classification, and expression of RAGE gene splice variants.

The receptor for advanced glycation end-products (RAGE) is a single-transmembrane, multiligand receptor of the immunoglobulin superfamily. RAGE up-regulation is implicated in numerous pathological states including vascular disease, diabetes, cancer, and neurodegeneration. The understanding of the regulation of RAGE is important in both disease pathogenesis and normal homeostasis. Here, we demonstrate the characterization and identification of human RAGE splice variants by analysis of RAGE cDNA from tissue and cells. We identified a vast range of splice forms that lead to changes in the protein coding region of RAGE, which we have classified according to the Human Gene Nomenclature Committee (HGNC). These resulted in protein changes in the ligand-binding domain of RAGE or the removal of the transmembrane domain and cytosolic tail. Analysis of splice variants for premature termination codons reveals approximately 50% of identified variants are targeted to the nonsense-mediated mRNA decay pathway. Expression analysis revealed the RAGE_v1 variant to be the primary secreted soluble isoform of RAGE. Taken together, identification of functional splice variants of RAGE underscores the biological diversity of the RAGE gene and will aid in the understanding of the gene in the normal and pathological state.

A clinical comparison of slow- and rapid-escalation treprostinil dosing regimens in patients with pulmonary hypertension.

BACKGROUND AND OBJECTIVE: Subcutaneous treprostinil is an effective treatment for pulmonary arterial hypertension (PAH). A previous pivotal study indicated that infusion site pain was dose dependent and resulted in suboptimal dose escalation by week 12 and a reduced clinical benefit. We hypothesized that a rapid-escalation treprostinil dosing regimen would be as safe and effective as a slow-escalation dosing regimen. METHODS: Twenty-three patients received treprostinil to treat PH of various aetiologies and were randomized into two groups. Group 1 (11 patients: seven females and four males, aged 51.7 +/- 15.4 years) received a slow-escalation regimen, and group 2 (12 patients: ten females and two males, aged 51.3 +/- 16.7 years) were exposed to rapid dose escalation. The dose escalation, exercise capacity (a 6-minute walk test [6WT] or a shuttle walk test [SWT]), WHO classification, blood pressure, heart rate, respiration rate, baseline haemodynamics and adverse events were followed up for 12 weeks. RESULTS: Baseline haemodynamics did not differ significantly between the treatment groups. At follow-up, the treprostinil dose reached 12.9 +/- 2.7 ng/kg/min in group 1 and 20.3 +/- 5.8 ng/kg/min in group 2 (p < 0.01). The patients' WHO classification improved significantly (p < 0.05), with no difference between the groups. Improvement of exercise capacity was greater in group 2 (6WT and SWT, p < 0.05). Infusion site pain occurred in 81.8% of group 1 and in 58.3% of group 2 (p < 0.05) patients. Other adverse events and changes in the heart rate, respiration rate and blood pressure were similar in both groups. CONCLUSION: The rapid-dosing regimen is as safe and effective as the slow-escalation regimen and may be associated with even better clinical outcomes. Infusion site pain is not dose dependent.

Receptor for advanced-glycation end products: key modulator of myocardial ischemic injury.

BACKGROUND: The beneficial effects of reperfusion therapies have been limited by the amount of ischemic damage that occurs before reperfusion. To enable development of interventions to reduce cell injury, our research has focused on understanding mechanisms involved in cardiac cell death after ischemia/reperfusion (I/R) injury. In this context, our laboratory has been investigating the role of the receptor for advanced-glycation end products (RAGE) in myocardial I/R injury. METHODS AND RESULTS: In this study we tested the hypothesis that RAGE is a key modulator of I/R injury in the myocardium. In ischemic rat hearts, expression of RAGE and its ligands was significantly enhanced. Pretreatment of rats with sRAGE, a decoy soluble part of RAGE receptor, reduced ischemic injury and improved functional recovery of myocardium. To specifically dissect the impact of RAGE, hearts from homozygous RAGE-null mice were isolated, perfused, and subjected to I/R. RAGE-null mice were strikingly protected from the adverse impact of I/R injury in the heart, as indicated by decreased release of LDH, improved functional recovery, and increased adenosine triphosphate (ATP). In rats and mice, activation of the RAGE axis was associated with increases in inducible nitric oxide synthase expression and levels of nitric oxide, cyclic guanosine monophosphate (cGMP), and nitrotyrosine. CONCLUSIONS: These findings demonstrate novel and key roles for RAGE in I/R injury in the heart. The findings also demonstrate that the interaction of RAGE with advanced-glycation end products affects myocardial energy metabolism and function during I/R.

Infection with a periodontal pathogen increases mononuclear cell adhesion to human aortic endothelial cells.

BACKGROUND: As a link between periodontal infections and an increased risk for vascular disease has been demonstrated, we assessed the ability of the Gram-negative periodontal pathogen Porphyromonas gingivalis to modulate properties of endothelial cells linked to inflammation and proatherogenic pathways. METHODS AND RESULTS: Primary human aortic endothelial cells (HAEC) were infected with either P. gingivalis strain 381 or its non-invasive fimbriae-deficient mutant, DPG3, and incubated with U-937 monocytes, or Jurkat T cells. P. gingivalis-infected HAEC demonstrated significantly increased adhesion of immune cells compared to non-infected cells or those infected with DPG3. Heat-killed bacteria had no effect on mononuclear cell adhesion and P. gingivalis LPS had only a minimal effect. P. gingivalis infection significantly increased HAEC expression of VCAM-1, ICAM-1 and E-selectin, and enhanced production of IL-6, IL-8 and MCP-1. CONCLUSION: These data demonstrate that live invasive P. gingivalis 381 elicits a pro-atherogenic response in HAEC.

Central role of PKCbeta in neointimal expansion triggered by acute arterial injury.

We tested the hypothesis that PKCbeta contributes to vascular smooth muscle cell (SMC) migration and proliferation; processes central to the pathogenesis of restenosis consequent to vascular injury. Homozygous PKCbeta null (-/-) mice or wild-type mice fed the PKCbeta inhibitor, ruboxistaurin, displayed significantly decreased neointimal expansion in response to acute femoral artery endothelial denudation injury compared with controls. In vivo and in vitro analyses demonstrated that PKCbetaII is critically linked to SMC activation, at least in part via regulation of ERK1/2 MAP kinase and early growth response-1. These data highlight novel roles for PKCbeta in the SMC response to acute arterial injury and suggest that blockade of PKCbeta may represent a therapeutic strategy to limit restenosis.

RAGE modulates vascular inflammation and atherosclerosis in a murine model of type 2 diabetes.

Previous studies demonstrated that induction of diabetes with streptozotocin (stz) accelerated atherosclerosis in hyperlipidemic apo E null (-/-) mice. Blockade of the Receptor for Advanced Glycation Endproducts (RAGE) in those animals suppressed acceleration of atherosclerotic lesion area, in a manner independent of changes in levels of glucose, insulin or lipids. In the present studies, we extended these concepts to a murine model of type 2 diabetes, and bred apo E -/- mice into the db/db background. Db/db mice are a model of obesity and insulin resistance-mediated hyperglycemia. Compared to apo E -/- m/db (non-diabetic) mice, apo E -/- db/db (diabetic) mice displayed accelerated atherosclerosis at the aortic sinus. Consistent with an important role for RAGE in this process, administration of soluble (s) RAGE, the extracellular ligand-binding domain of RAGE, resulted in significantly reduced atherosclerotic lesion area in a glycemia- and lipid-independent manner. In parallel, apo E -/- db/db mice displayed RAGE-dependent enhanced expression of Vascular Cell Adhesion Molecule-1, tissue factor and matrix metalloproteinase (MMP)-9 antigen/activity in aortae compared to non-diabetic animals. In addition, consistent with the premise that upregulation of RAGE ligands and RAGE occurs even in the non-diabetic, hyperlipidemic state, albeit to lesser degrees than in diabetes, administration of sRAGE to apo E -/- m/db mice resulted in decreased atherosclerotic lesion area at the aortic sinus. Taken together, these findings establish a new murine model for the study of atherosclerosis in type 2 diabetes and highlight important roles for RAGE in proatherogenic mechanisms in hyperglycemia triggered by insulin resistance.

Early growth response-1 promotes atherogenesis: mice deficient in early growth response-1 and apolipoprotein E display decreased atherosclerosis and vascular inflammation.

Early growth response-1 (Egr-1) regulates expression of proinflammatory and procoagulant genes in acute cell stress. Experimental evidence suggested that Egr-1 transcripts were upregulated in human atherosclerotic plaques versus adjacent unaffected tissue. To test the impact of Egr-1 in chronic vascular stress, we examined its role in a murine model of atherosclerosis. Real-time PCR analysis of aortae retrieved from apoE-/- mice demonstrated increased Egr-1 transcripts in an age-dependent manner, compared with aortae retrieved from C57BL/6 control animals. Therefore, homozygous Egr-1-/- mice were bred into the apoE-/- background. Homozygous double-knockout mice (Egr-1-/-/apoE-/-) in the C57BL/6 background were maintained on normal chow diet. At age 14 and 24 weeks, atherosclerotic lesion area and complexity at the aortic root were strikingly decreased in mice deficient in both Egr-1 and apoE compared with mice deficient in apoE alone. In parallel, transcripts for genes regulating the inflammatory/prothrombotic response were diminished in Egr-1-/-/apoE-/- aortae versus apoE-/-. In vitro, oxidized low-density lipoprotein (OxLDL), a key factor inciting atherogenic mechanisms in the vasculature, upregulated Egr-1 expression in monocytes via the MEK-ERK1/2 pathway. We conclude that Egr-1 broadly regulates expression of molecules critically linked to atherogenesis and lesion progression.

(124)I PET/CT in Patients with Differentiated Thyroid Cancer: Clinical and Quantitative Image Analysis.

BACKGROUND: Although radioactive iodine (RAI) imaging/therapy is one of the earliest applications of theranostics, there remain a number of unresolved clinical questions as to the optimization of diagnostic techniques/protocols and improvements in patient-specific treatment planning strategies. The objectives of this study were to determine the imaging characteristics and clinical feasibility of (124)I positron emission tomography/computed tomography (PET/CT) for the determination of extent of disease and evaluation of RAI kinetics in its physiologic and neoplastic distribution in patients with differentiated thyroid cancer (DTC). METHODS: The study was designed as a prospective phase II diagnostic trial of patients with confirmed DTC. Following adequate preparation, patients received 2 mCi (124)I in liquid form and sequential whole-body PET/CT imaging was performed at five time points (2-4 h, 24 ± 6 h, 48 ± 6 h, 72 ± 6 h, and 96 ± 6 h post-administration). All patients who had (124)I imaging subsequently underwent RAI treatment with (131)I, with administered activities ranging from 100 to 300 mCi. Post-treatment scans were obtained 5-7 days after RAI treatment. A by-patient and by-lesion analysis of the (124)I images was performed and compared with the post-treatment (131)I scans as well as F-18 FDG PET/CT images. Quantitative image analysis was also performed to determine the total functional volume (mL), activity per functional volume (µCi/mL), and cumulated activity (µCi/h) for remnants, salivary glands, and nodal metastases. RESULTS: Fifteen patients (6 women; Mage = 57 years; range 29-91 years) were enrolled into the study. Forty-six distinct lesions were identified in these 15 patients on (124)I PET/CT images, with a sensitivity of 92.5%. In addition, (124)I identified 22.5% more foci of RAI-avid lesions compared with the planar (131)I post-treatment scans. This study demonstrates different kinetic profiles for normal thyroid remnants (peaked at 24 h with mono-exponential clearance), salivary glands (peaked at 4 h with bi-exponential clearance), and metastatic lesions (protracted retention), as well as individual variations in functional volumes and thus cumulated activities. CONCLUSIONS: (124)I PET/CT is a valuable clinical imaging tool/agent, both in determining the extent of disease in the setting of metastatic DTC and in the functional volumetric and kinetic evaluation of target lesions.

The RAGE Gly82Ser polymorphism is not associated with cardiovascular disease in the Framingham offspring study.

The receptor for advanced glycation end-products (RAGE) is expressed to enhance degrees in human atherosclerotic plaques and co-localizes with inflammatory and pro-oxidant mediators in the vulnerable regions of the plaque. Previous studies highlighted a number of variants in the gene encoding the receptor, including a Gly to Ser substitution at amino acid 82 within the ligand-binding domain of RAGE. The Ser82 allele enhanced ligand-binding affinity and increased ligand-stimulated generation of inflammatory mediators in transfected cells and human monocytes compared to the common RAGE Gly82 allele. Thus it was logical to test the hypothesis that increased prevalence of the Gly82Ser polymorphism was associated with cardiovascular events in the Framingham offspring study (n=1632). Our analyses revealed that the Gly82Ser RAGE polymorphism did not demonstrate any association with the incidence of cardiovascular disease in diabetic or non-diabetic subjects (Gly82 96%, Ser82 4%). Analysis of specific manifestations of cardiovascular disease, including coronary heart disease (CHD), cardiovascular disease (CVD), myocardial infarction (MI) and ischemic disease (ISD) revealed no association with RAGE genotype. Further studies are required on other more prevalent genetic variants of RAGE and cardiovascular disease.

An analysis of characteristics of subjects examined for incretin effects on pancreatic pathology.

A recent autopsy analysis asserted that incretin drugs have the potential of increasing the risk for pancreatic cancer and for pancreatic neuroendocrine tumors. We examined the Network for Pancreatic Organ Donors with Diabetes (nPOD) database from which that analysis was derived. Our findings raise important questions about the comparability of the two groups of diabetes patients used for the analysis. Our review of the data available on the nPOD Web site and our reading of the earlier article lead us to the conclusion that the data, and the implications of the data, as expressed by the authors of the autopsy analysis are vastly overstated and are a misrepresentation of the information available.

Alternatively spliced RAGEv1 inhibits tumorigenesis through suppression of JNK signaling.

Receptor for advanced glycation end products (RAGE) and its ligands are overexpressed in multiple cancers. RAGE has been implicated in tumorigenesis and metastasis, but little is known of the mechanisms involved. In this study, we define a specific functional role for an alternate splice variant termed RAGE splice variant 1 (RAGEv1), which encodes a soluble endogenous form of the receptor that inhibits tumorigenesis. RAGEv1 was downregulated in lung, prostate, and brain tumors relative to control matched tissues. Overexpressing RAGEv1 in tumor cells altered RAGE ligand stimulation of several novel classes of genes that are critical in tumorigenesis and metastasis. Additionally, RAGEv1 inhibited tumor formation, cell invasion, and angiogenesis induced by RAGE ligand signaling. Analysis of signal transduction pathways underlying these effects revealed marked suppression of c-jun-NH(2)-kinase (JNK) pathway signaling, and JNK inhibition suppressed signaling through the RAGE pathway. Tumors expressing RAGEv1 were significantly smaller than wild-type tumors and displayed prominently reduced activation of JNK. Our results identify RAGEv1 as a novel suppressor, the study of which may offer new cancer therapeutic directions.

Interaction of the RAGE cytoplasmic domain with diaphanous-1 is required for ligand-stimulated cellular migration through activation of Rac1 and Cdc42.

Cellular migration is a fundamental process linked to diverse pathological states such as diabetes and its complications, atherosclerosis, inflammation, and cancer. The receptor for advanced glycation end products (RAGE) is a multiligand cell surface macromolecule which binds distinct ligands that accumulate in these settings. RAGE-ligand interaction evokes central changes in key biological properties of cells, including proliferation, generation of inflammatory mediators, and migration. Although RAGE-dependent signal transduction is critically dependent on its short cytoplasmic domain, to date the proximate mechanism by which this RAGE domain engages and stimulates cytoplasmic signaling pathways has yet to be identified. Here we show that the RAGE cytoplasmic domain interacts with Diaphanous-1 (Dia-1) both in vitro and in vivo. We employed the human RAGE cytoplasmic domain as "bait" in the yeast two-hybrid assay and identified the formin homology (FH1) domain of Dia-1 as a potential binding partner of this RAGE domain. Immunoprecipitation studies revealed that the RAGE cytoplasmic domain interacts with the FH1 domain of Dia-1. Down-regulation of Dia-1 expression by RNA interference blocks RAGE-mediated activation of Rac-1 and Cdc42 and, in parallel, RAGE ligand-stimulated cellular migration. Taken together, these findings indicate that the interaction of the RAGE cytoplasmic domain with Dia-1 is required to transduce extracellular environmental cues evoked by binding of RAGE ligands to their cell surface receptor, a chief consequence of which is Rac-1 and Cdc42 activation and cellular migration. Because RAGE and Dia-1 are implicated in the regulation of inflammatory, vascular, and transformed cell migration, these findings highlight this interaction as a novel target for therapeutic intervention in inflammation, atherosclerosis, diabetes, and cancer.

Protein kinase C beta/early growth response-1 pathway: a key player in ischemia, atherosclerosis, and restenosis.

Atherosclerosis, restenosis, and the consequences of ischemia are the major causes of morbidity and mortality worldwide. Elucidation of key contributing pathways in animal models of ischemia-reperfusion injury, atherosclerosis, and restenosis consequent to vascular injury may lead to great interest in determining if blocking these pathways could prevent vascular disease in human subjects. This review details the evidence that the protein kinase C (PKC) beta/early growth response-1 axis plays a central role in the response to both acute and chronic vascular stresses in animal models and also indicates the clinical implications of a specific inhibitor of PKCbeta, ruboxistaurin (LY333531).