Faculty Bibliography

BACKGROUND: Receptor for advanced glycated end product (RAGE) expression is a prominent feature of atherosclerosis. We have previously shown in apoE null mice uptake of a radiolabeled anti-RAGE antibody in atherosclerotic plaque and now evaluate RAGE-directed imaging to identify advanced plaques in a large animal model. METHODS: Nine hyperlipidemic (HL) pigs were injected with 603.1 +/- 129.5 MBq of (99m)Tc-anti-RAGE F(ab')2, and after 6 h (blood pool clearance), they underwent single-photon emission computed tomography/computed tomography (SPECT/CT) imaging of the neck, thorax, and hind limbs. Two HL pigs received (99m)Tc non-immune IgG F(ab')2, and three farm pigs were injected with (99m)Tc-anti-RAGE F(ab')2. After imaging, the pigs were euthanized. The aorta from the root to bifurcation was dissected, and the innominates, proximal carotids, and coronaries were dissected and counted, stained for H&E and RAGE, and AHA-classified. RESULTS: On pathology, 24% of the arterial segments showed AHA class III or IV lesions, and these lesions were confined almost exclusively to coronaries and carotids with % stenosis from 15% to 65%. Scatter plots of %ID/g for class III/IV vs. I/II lesions showed almost complete separation. Focal vascular uptake of tracer visualized on SPECT scans corresponded to class III/IV lesions in the coronary and carotid vessels. In addition, uptake in the hind limbs was noted in the HL pigs and corresponded to RAGE staining of small arteries in the muscle sections. Correlations for the vascular lesions were r = 0.747, P = 0.001 for %ID vs. %ID/g and r = 0.83, P = 0.002 for %ID/g vs. % RAGE staining. CONCLUSIONS: Uptake of radiolabeled anti-RAGE antibody in coronary and carotid fibroatheroma and in the small arteries of the hind limbs in a relevant large animal model of atherosclerosis supports the important role of RAGE in atherosclerosis and peripheral artery disease as a target for imaging and treatment.

INTRODUCTION: Intestinal fibrosis is a common and severe complication of inflammatory bowel disease (IBD) characterized by excessive deposition of extracellular matrix components (ECM) and for which efficient and well-tolerated therapies are currently lacking. Inflamed colonic mucosa of patients with active IBD show a significant increase in expression of the receptor for advanced glycation end products (RAGE), a member of the immunoglobulin superfamily of cell surface receptors, able to regulate chronic inflammation by activating the NF-kB pathway and inducing inflammatory and oxidative stress. In addition, a growing body of evidences in kidney, liver and lung fibrosis shows how the increased myofibroblast activation and numbers and the consequent ECM accumulation are regulated by RAGE. All these data may place this receptor among the most innovative and promising targets for new antifibrotic therapies in IBD AIMS & METHODS: We propose to investigate the involvement of RAGE in the development of the DSS-induced intestinal fibrosis in mice. Chronic colitis and fibrosis were induced in C57BL/6 wild type (WT) and RAGE null mice by administration of 2.5% (w/v) dextran sulfate sodium (DSS) in drinking water for 5 days followed by 7 days of water, for three cycles. Three days after the last cycle of treatment, the entire colon was rapidly excised and scored for the assessment of macroscopic lesion, including dilation, thickness and adhesion, on a 0-3 scale by an investigator naive to the experimental conditions. The sum of the scores of colonic lesions was expressed as total macroscopic score. Tissue specimens, collected from distal colon, were subject to Hematoxylin/Eosin staining, to assess the degree of inflammation, and Picrosirius red staining was performed to assess collagen deposition. Thus, a total microscopic score was calculated evaluating presence of ulceration, inflammatory degree, depth of lesions and fibrotic degree. mRNA expression of the main profibrotic mediator, Tgf-beta1, and the expression of ECM components, mainly collagen types I-III (Col1A1 gene) and fibronectin (Fn-1 gene), were evaluated by quantitative RT-PCR RESULTS: Compared to WT mice, DSS-treated C57/Bl6 RAGE null mice showed a significant 29% decrease of the colon weight/length ratio (p<0.0001), an indicator of wall thickening. A total macroscopic score of 6 +/- 0.92 was assessed in colons recovered from DSS-treated WT mice, but in mice devoid of RAGE, the appearance of the macroscopic lesions was significantly reduced, 1.43 +/- 0.54 (p<0.0001, n=15). DSS-treated RAGE null mice showed also a significant 49% decrease of total microscopic score compared to WT mice. mRNA Tgf-beta1 expression was significantly increased 3.4 fold by the DSS administration in WT mice colon, whereas it was unchanged in RAGE null mice compared to mice receiving only tap water. Col1A1 and Fn-1 genes were upregulated in DSS-treated WT mice (5.52 folds, p= 0.137 and 53 folds, p= 0.0016, respectively). Lack of RAGE decreased 3.17 folds (p= 0.0341) Col1A1 expression and totally prevents the Fn-1 upregulation induced by DSS treatment CONCLUSION: The potential profibrotic role of RAGE in IBD could both shed light into the complex and dynamic fibrogenic processes in IBD and pave the way for new anti-fibrotic agents and approaches in this disease

RATIONALE: A major conceptual challenge in sarcoidosis is to understand how fibrosis occurs in an environment dominated by the expression of Th1 cytokines such as interferon-gamma, which is known to inhibit collagen synthesis. RAGE and its ligands are histologically co-localized to areas of sarcoidosis granulomatous, and a RAGE polymorphism is associated with increased risk of developing sarcoidosis. We recently identified that histological expression of the RAGE ligand serum amyloid A (SAA) was correlated with collagen deposition around sarcoidosis granulomas (AJRCCM 2010, 181:360). We explored the effects of RAGE stimulation by SAA on lung inflammation and fibrosis using a model of mKatG-induced granulomatous inflammation. METHODS: Wild-type (C57wt) RAGE-deficient (RAGEko) C57BL/6 mice were sensitized to recombinant mKatG, then re-exposed to mKatG-coated sepharose beads via orotracheal instillation, with or without SAA administration on days 0 and 7, and evaluated at 2wks. Inflammation and collagen deposition were quantified by digital microscopy and reported as group median values. Steady state whole-lung mRNA expression of cytokine and fibrosis-related genes was assessed by real-time PCR and reported as fold-change in SAA-treated vs untreated mice. RESULTS: In C57wt, granuloma area around mKatG-coated beads was 48% greater in SAA-treated animals vs. mKatG-beads alone (76907 vs 51582, p<0.0004), and this was associated with higher whole-lung mRNA expression of IFNg (1.27-fold), TNF (3.00-fold), and IL10 (3.25-fold) in SAA treated vs untreated C57wt (p<0.05, all comparisons). In RAGEko, the effect of SAA on granuloma area was attenuated (12% increase; 52576 vs 46710, p=ns) and failed to up-regulate IFNg (0.85-fold, p=ns), TNF (0.95-fold, p=ns), or IL10 (0.71-fold, p<0.05) expression. SAA administration resulted in 36% greater collagen deposition around mKatG granulomas identified by Sirius Red staining in C57wt (48 vs 35.5, p<0.03) and higher mRNA expression of type-1 collagen (2.32-fold), type-3 collagen (1.33-fold), and the collagen molecular chaperone Hsp47 (1.68-fold) (p<0.05, all comparisons). Consistent with the known effects of other RAGE ligands, SAA also up-regulated the expression of RAGE (1.59-fold, p<0.05). In contrast to C57wt, SAA administration did not significantly alter granuloma collagen content (16.5 vs 31) nor up-regulate mRNA expression of type-1 collagen (1.12-fold) or Hsp47 (1.14-fold) in RAGEko (p=ns, all comparisons). SAA administration was associated with reduced type-3 collagen expression (0.62-fold, p<0.05) in RAGEko. CONCLUSION: These results suggest that local inflammatory and fibrotic responses in experimental granulomatous lung inflammation are mediated, in part, through RAGE stimulation and support the therapeutic use of strategies that block this pathway in the management of sarcoidosis

OBJECTIVE: To compare bariatric surgery versus intensive medical weight management (MWM) in patients with type 2 diabetes mellitus (T2DM) who do not meet current National Institutes of Health criteria for bariatric surgery and to assess whether the soluble form of receptor for advanced glycation end products (sRAGE) is a biomarker to identify patients most likely to benefit from surgery. BACKGROUND: There are few studies comparing surgery to MWM for patients with T2DM and BMI less than 35. METHODS: Fifty-seven patients with T2DM and BMI 30 to 35, who otherwise met the criteria for bariatric surgery were randomized to MWM versus surgery (bypass, sleeve or band, based on patient preference). The primary outcomes assessed at 6 months were change in homeostatic model of insulin resistance (HOMA-IR) and diabetes remission. Secondary outcomes included changes in HbA1c, weight, and sRAGE. RESULTS: The surgery group had improved HOMA-IR (-4.6 vs +1.6; P = 0.0004) and higher diabetes remission (65% vs 0%, P < 0.0001) than the MWM group at 6 months. Compared to MWM, the surgery group had lower HbA1c (6.2 vs 7.8, P = 0.002), lower fasting glucose (99.5 vs 157; P = 0.0068), and fewer T2DM medication requirements (20% vs 88%; P < 0.0001) at 6 months. The surgery group lost more weight (7. vs 1.0 BMI decrease, P < 0.0001). Higher baseline sRAGE was associated with better weight loss outcomes (r = -0.641; P = 0.046). There were no mortalities. CONCLUSIONS: Surgery was very effective short-term in patients with T2DM and BMI 30 to 35. Baseline sRAGE may predict patients most likely to benefit from surgery. These findings need to be confirmed with larger studies.ClinicalTrials.gov ID: NCT01423877.

BACKGROUND: The receptor for advanced glycation end products (RAGE) shares common ligands and signaling pathways with TLR4, a key mediator of house dust mite (Dermatophagoides pteronyssinus) (HDM) sensitization. We hypothesized that RAGE and its ligand high-mobility group box-1 (HMGB1) cooperate with TLR4 to mediate HDM sensitization. OBJECTIVES: To determine the requirement for HMGB1 and RAGE, and their relationship with TLR4, in airway sensitization. METHODS: TLR4(-/-), RAGE(-/-), and RAGE-TLR4(-/-) mice were intranasally exposed to HDM or cockroach (Blatella germanica) extracts, and features of allergic inflammation were measured during the sensitization or challenge phase. Anti-HMGB1 antibody and the IL-1 receptor antagonist Anakinra were used to inhibit HMGB1 and the IL-1 receptor, respectively. RESULTS: The magnitude of allergic airway inflammation in response to either HDM or cockroach sensitization and/or challenge was significantly reduced in the absence of RAGE but not further diminished in the absence of both RAGE and TLR4. HDM sensitization induced the release of HMGB1 from the airway epithelium in a biphasic manner, which corresponded to the sequential activation of TLR4 then RAGE. Release of HMGB1 in response to cockroach sensitization also was RAGE dependent. Significantly, HMGB1 release occurred downstream of TLR4-induced IL-1alpha, and upstream of IL-25 and IL-33 production. Adoptive transfer of HDM-pulsed RAGE(+/+)dendritic cells to RAGE(-/-) mice recapitulated the allergic responses after HDM challenge. Immunoneutralization of HMGB1 attenuated HDM-induced allergic airway inflammation. CONCLUSION: The HMGB1-RAGE axis mediates allergic airway sensitization and airway inflammation. Activation of this axis in response to different allergens acts to amplify the allergic inflammatory response, which exposes it as an attractive target for therapeutic intervention.

Objective. Receptor for advanced glycated endproducts (RAGE) plays an important role in atherogenesis in diabetes. We imaged RAGE to investigate the effect of glucose control to suppress RAGE and reduce atherosclerosis in apolipoprotein E null (apoE(-/-)) diabetic mice. Methods and Results. Thirty-three apoE(-/-) mice received streptozotocin and 6 weeks later 15 began treatment with insulin implants. Blood glucose measurements during study averaged: 140 +/- 23 mg/dL (treated) and 354 +/- 14 mg/dL (untreated). After 15 wk 30 mice were injected with (99m)Tc-anti-RAGE F(ab')2, 3 with (99m)Tc-nonimmune IgG F(ab')2, and all with CT contrast agent and underwent SPECT/CT imaging. At necropsy, the proximal aorta was weighed, counted, and sectioned and the % injected dose per gram (%ID/g) was calculated. From the merged SPECT/CT scans, tracer uptake localized to arteries was lower in the treated mice: 3.15 +/- 1.82 x 10(-3) versus 8.69 +/- 4.58 x 10(-3)%ID (P = 0.001). Percent cross-sectional lesion area was smaller in the treated (14.3 +/- 7.8% versus 29.5 +/- 10.9%) (P = 0.03). RAGE uptake on scans (%ID) correlated with quantitative RAGE staining in the atheroma and with %ID/g (R = 0.6887; P = 0.01). Lesion size as percent cross-sectional area was smaller in the treated (14.3 +/- 7.8% versus 29.5 +/- 10.9%) (P = 0.03). RAGE uptake on scans (%ID) correlated with quantitative RAGE staining in the atheroma and with %ID/g (R = 0.6887; P = 0.01). Conclusions. These results support the importance of suppressing RAGE to reduce atherosclerotic complications of diabetes and value of molecular imaging to assess treatment effect.