Faculty Bibliography

OBJECTIVE: Cardiovascular disease is the leading cause of death in patients with end-stage renal disease. Serum amyloid A (SAA) is an acute phase protein and a binding partner for the multiligand receptor for advanced glycation end products (RAGE). We investigated the role of the interaction between SAA and RAGE in uremia-related atherogenesis. APPROACH AND RESULTS: We used a mouse model of uremic vasculopathy, induced by 5 of 6 nephrectomy in the Apoe(-/-) background. Sham-operated mice were used as controls. Primary cultures of Ager(+/+) and Ager(-/-) vascular smooth muscle cells (VSMCs) were stimulated with recombinant SAA, S100B, or vehicle alone. Relevance to human disease was assessed with human VSMCs. The surface area of atherosclerotic lesions at the aortic roots was larger in uremic Apoe(-/-) than in sham-operated Apoe(-/-) mice (P<0.001). Furthermore, atherosclerotic lesions displayed intense immunostaining for RAGE and SAA, with a pattern similar to that of alpha-SMA. Ager transcript levels in the aorta were 6x higher in uremic animals than in controls (P<0.0001). Serum SAA concentrations were higher in uremic mice, not only after 4 weeks of uremia but also at 8 and 12 weeks of uremia, than in sham-operated animals. We investigated the functional role of RAGE in uremia-induced atherosclerosis further, in animals lacking RAGE. We found that the induction of uremia in Apoe(-/-) Ager(-/-) mice did not accelerate atherosclerosis. In vitro, the stimulation of Ager(+/+) but not of Ager(-/-) VSMCs with SAA or S100B significantly induced the production of reactive oxygen species, the phosphorylation of AKT and mitogen-activated protein kinase-extracellular signal-regulated kinases and cell migration. Reactive oxygen species inhibition with N-acetyl cysteine significantly inhibited both the phosphorylation of AKT and the migration of VSMCs. Similar results were obtained for human VSMCs, except that the phosphorylation of mitogen-activated protein kinase-extracellular signal-regulated kinases, rather than of AKT, was subject to specific redox-regulation by SAA and S100B. Furthermore, human aortic atherosclerotic sections were positively stained for RAGE and SAA. CONCLUSIONS: Uremia upregulates SAA and RAGE expression in the aortic wall and in atherosclerotic lesions in mice. Ager(-/-) animals are protected against the uremia-induced acceleration of atherosclerosis. SAA modulates the functions of murine and human VSMCs in vitro in a RAGE-dependent manner. This study, therefore, identifies SAA as a potential new uremic toxin involved in uremia-related atherosclerosis through interaction with RAGE.

Introduction Brown adipose tissue (BAT) is one of the primary tissues responsible for adaptive non-shivering thermogenesis, mediated by mitochondria uncoupling protein-1 (Ucp1), and contributes to energy expenditure. BAT has been shown to play a role in adiposity, insulin resistance and hyperlipidaemia. We and others^1-2 have shown that genetic deficiency of RAGE (receptor for advanced glycation end products) prevented the effects of a high-fat diet (HFD) on energy expenditure, weight gain, adipose tissue inflammation, and insulin resistance. The aim of this study is to compare and quantitate the BAT activity and BAT volume in RAGE null (Ager ^-/-) and wild type (WT) mice in response to HFD and low-fat diets (LFD) using in vivo PET imaging and ex vivo autoradiography (ARG) studies with [¹⁸F]FDG. Since BAT is not readily visible with [¹⁸F] FDG at room temperature based on our previous BAT imaging study³, all comparative studies were conducted under identical mild- cold stimulation conditions with [¹⁸F]FDG. Methods PET/CT imaging (Siemens, Inveon) of 4 groups of male C57/BL6 mice were compared (Ager ^-/--LFD, Ager ^-/--HFD, WT-LFD and WT-HFD). Before each scan, all mice were fasted for at least 4 h and then exposed to a cold-pack (0- 4degreeC) for 30 min. For each study, a dynamic PET scan was carried out for 1 h, followed by a CT scan (6 min) for the purpose of attenuation correction and co-registration. SUVmean (mean standard uptake values), SUVR (ratio of SUV BAT/muscle), and %ID/g for the interscapular BAT were determined. Mice were dissected after the scan to obtain the BAT tissue for further quantification of BAT via ex-vivo autoradiography (ARG) study. Results Preliminary results are summarized here: 1) For LFD mice, the uptake was significantly higher in WT-LFD (SUVR: 11.7) than that in Ager ^-/--LFD (SUVR: 2.63); 2) The SUVR were similar for both WT-HFD and Ager ^-/--HFD (2.53 and 2.44, respectively) (Fig.1). 3) Among Ager ^-/- types, the uptake was similar in both Ager ^-/--HFD (SUVR: 2.44) and Ager ^-/--LFD (SUVR: 2.63), and the %ID/g values were similar in both as well (Fig.1). These findings corroborate our previous findings on Ucp1²; that is, (1) Ucp1-mRNA transcripts in BAT are almost halved in Ager ^-/--LFD than in WTLFD; (2) in HFD, Ucp1-mRNA transcripts did not differ in BAT between WT and Ager ^-/- mice; (3) a bigger change was observed in Ucp1 between WT-LFD and WT-HFD, while a smaller change was seen between Ager ^-/--LFD and Ager ^-/- -HFD. Results from ex vivo ARG studies were consistent with the PET imaging data. Conclusion In WT mice, BAT activity was significantly reduced after HFD as compared to LFD; however, BAT activity was not affected by HFD in Ager ^-/- mice. The consistent findings between the current in vivo PET imaging study of BAT and our previous study of Ucp1 further support our hypothesis that RAGE may contribute to altered energy expenditure, and provide a protective effect against HFD by Ager deletion. [TABLE PRESENTED]

Objective: In diabetes, hyperglycemia causes the accumulation of advanced glycation end products (AGEs) that trigger reactive oxygen species (ROS) generation through binding the receptor for AGEs (RAGE). Because exogenous growth factors have had little success in enhancing chronic wound healing, we investigated whether hyperglycemia-induced AGEs interfere with cellular responses to extracellular signals. We used stromal cell-derived factor-1 (SDF-1), an angiogenic chemokine also known to promote stem cell recruitment in skin wounds. Approach: Human leukemia-60 (HL-60) cells and mouse peripheral blood mononuclear cells (PBMCs), which express the SDF-1 receptor CXCR-4, were incubated for 24 h in medium supplemented with 25 mM d-glucose. Soluble RAGE (sRAGE) was used to block RAGE activation. Response to SDF-1 was measured in cellular migration and ROS assays. A diabetic murine excisional wound model measured SDF-1 liposome and sRAGE activity in vivo. Results: Hyperglycemia led to significant accumulation of AGEs, decreased SDF-1-directed migration, and elevated baseline ROS levels; it suppressed the ROS spike normally triggered by SDF-1. sRAGE decreased the ROS baseline and restored both the SDF-1-mediated spike and cell migration. Topically applied sRAGE alone promoted healing and enhanced the effect of exogenous SDF-1 on diabetic murine wounds. Innovation: While there is interest in using growth factors to improve wound healing, this strategy is largely ineffective in diabetic wounds. We show that sRAGE may restore signaling, thus potentiating the effect of exogenously applied growth factors. Conclusion: Blocking RAGE with sRAGE restores SDF-1-mediated cellular responses in hyperglycemic environments and may potentiate the effectiveness of SDF-1 applied in vivo.

SCOPE: Nvarepsilon -Carboxymethyl-lysine (CML) is a prominent advanced glycation end-product which is not only found in vivo but also in food. It is known that a percentage of the dietary CML (dCML) is absorbed into the circulation and only partly excreted in the urine. Several studies have tried to measure how much dCML remains in tissues. However obstacles to interpreting the data have been found. METHODS AND RESULTS: A new protocol which discriminates dCML from native CML (nCML) has been developed. Three CML isotopes with different mass-to-charge ratios were used: nCML Nepsilon -carboxymethyl-L-lysine, dCML Nepsilon -[13 C]carboxy[13 C]methyl-L-lysine and internal standard Nepsilon -carboxymethyl-L-[4,4,5,5-2 H4 ]lysine. Wild-type (n = 7) and RAGE-/- (n = 8) mice were fed for 30 days with either a control, or a BSA-bound dCML-enriched diet. Organs were analyzed for nCML and dCML using liquid chromatography-tandem mass spectrometry. Mice exposed to dCML showed an accumulation in all tissues tested except fat. The rate of deposition was high (81-320 mugdCML /g dry matter) in kidneys, intestine, and lungs and low (<5 mug/g) in heart, muscle, and liver. This accumulation was not RAGE dependent. CONCLUSION: The kidney is not the only organ affected by the accumulation of dCML. Its high accumulation in other tissues and organs may also, however, have important physiological consequences.

Background: The receptor for advanced glycation products (RAGE) is a member of the immunoglobulin superfamily able to regulate chronic inflammation. The prolonged AGE exposition in kidney, liver, and lung induces up regulation of alphaSMA, the main marker of myofibroblasts activation, resulting in an accumulation of extracellular matrix components (ECM) and consequent fibrosis. At intestinal level fibrosis is a common and severe compliance of inflammatory bowel disease (IBD), and inflamed colonic mucosa of patients with active IBD shows a significant increase of AGE and RAGE. Our aim was to determine the role of RAGE in intestinal fibrosis. Methods: Fibrosis was induced in C57BL/6 wild-type (WT) and RAGE-/- mice by for 3 cycles of 2,5% (w/v) dextran sulfate sodium (DSS) administration for 6 weeks, macroscopic (including dilation, thickness, and adhesion) and microscopic lesions (inflammation by Hematoxylin/Eosin and collagen deposition by Picrosirius red staining) were scored for all colonic specimens. mRNA expression of the main profibrotic mediator, TGF-beta (Tgf-beta1 gene), alphaSMA (ACTA-1 gene), and the expression of ECM components, mainly collagen types I-III (Col1A1 gene) and fibronectin (FN-1 gene), was evaluated by quantitative RT-polymerase chain reaction (PCR). Moreover, RAGE gene expression levels were evaluated in TGF-beta-stimulated intestinal fibroblasts and epithelial cells, as well as in human primary intestinal fibroblasts isolated from IBD patients Results: Compared with WT mice, DSS-treated C57/Bl6 RAGE- /- mice showed a lower colon weight/length ratio (29%, p < 0.0001), an indicator of wall thickening. In RAGE-/-mice, the macroscopic score was significantly reduced compared with WT mice (6 +/- 0.92 vs 1.43 +/- 0.54, p < 0.0001). DSS-treated RAGE-/- mice showed less then 50% (p < 0.001) of total microscopic score valued in WT mice. DSS administration also induced significant increase of Tgf-beta1, ACTA-1, Col1A1, and FN-1 gene expression in WT mice colon. DSS-treated RAGE-/- mice showed lower expression of Col1A1 (3.17 fold, p < 0.01), Tgf-beta1 (2 fold, p < 0.01), ACTA-1 (1.2 fold, p < 0.05) than WT mice, whereas FN-1 expression was totally prevented. In addition, myofibroblasts deriving by TGF-beta-treated intestinal fibroblasts and epithelial cells showed enhanced mRNA RAGE expression by 1.5 fold (p < 0.01) and 2.3 fold (p < 0.01), respectively. Additionally, in primary human intestinal fibroblasts, obtained by UC patients, a significant increase by 3.8 fold (p < 0.05) of RAGE gene expression was observed. Conclusions: The potential profibrotic role of RAGE in the development of intestinal fibrosis could shed light into the complex and dynamic fibrogenic processes in IBD

The weak oligomerization exhibited by many transmembrane receptors has a profound effect on signal transduction. The phenomenon is difficult to characterize structurally due to the large sizes of and transient interactions between monomers. The receptor for advanced glycation end products (RAGE), a signaling molecule central to the induction and perpetuation of inflammatory responses, is a weak constitutive oligomer. The RAGE domain interaction surfaces that mediate homo-dimerization were identified by combining segmental isotopic labeling of extracellular soluble RAGE (sRAGE) and nuclear magnetic resonance spectroscopy with chemical cross-linking and mass spectrometry. Molecular modeling suggests that two sRAGE monomers orient head to head forming an asymmetric dimer with the C termini directed toward the cell membrane. Ligand-induced association of RAGE homo-dimers on the cell surface increases the molecular dimension of the receptor, recruiting Diaphanous 1 (DIAPH1) and activating signaling pathways.

Acetylation of proteins as a post-translational modification is gaining rapid acceptance as a cellular control mechanism on par with other protein modification mechanisms such as phosphorylation and ubiquitination. Through genetic manipulations and evolving proteomic technologies, identification and consequences of transcription factor acetylation is beginning to emerge. In this review, we summarize the field and discuss newly unfolding mechanisms and consequences of transcription factor acetylation in normal and stressed hearts. This article is part of a Special Issue entitled: The role of post-translational protein modifications on heart and vascular metabolism edited by Jason R.B. Dyck & Jan F.C. Glatz.

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. View this table: In this window In a new window Table 1 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. (Table Presented)