RECEPTOR FOR ADVANCED GLYCATION END-PRODUCTS: EXPRESSION AND SIGNALING

The NF-B transcription factor family plays a central role in many aspects of the immune response, and activation of this family of transcription factors has been shown to trigger many disease processes. Thus, the ability to modulate NF-kB activity may be an attractive way to treat these diseases. We used an in vitro cell-based assay to test potential NF-kB inhibitors by measuring their effect on IL-1-induced expression of the NF-B dependent intracellular adhesion molecule-1 (ICAM-1, CD54). To develop the cell-based system we sorted IL-1b-responsive U373 human astrocytoma cells to obtain a population of cells with minimum background expression and maximum induced expression of CD54 following stimulation with IL-1. We tested ethyl pyruvate, a novel anti-inflammatory drug candidate, and the ability of related compounds to block activation of NF-kB activity by measuring the expression of CD54 on U373 cells exposed to IL-1. 4-hydroxyphenylpyruvic acid was the best inhibitor of CD54 upregulation. We further tested the compounds using the mouse macrophage-like RAW 264.7 cell line which produce a variety of cytokines and nitric oxide (NO•) following exposure to lipopolysaccharide (LPS) in an NF-kB-dependent manner. The drugs downregulated LPS-induced IL-6 production, iNOS upregulation, and NO• production following the same efficacy trend observed in the primary screening using CD54 expression in U373 cells. These studies show the ease of using an endogenous reporter gene (i.e., CD54) and FACS analysis to rapidly characterize the relative efficacy of pharmacologic inhibitors. A second completely unrelated topic of the dissertation dealt with the receptor for advanced glycation end-products (RAGE). RAGE is thought to be important in a variety of pathological conditions, including diabetes, sepsis, atherosclerosis, renal diseases, hypertension and Alzheimer's disease. However, RAGE proximal signaling events are still unclear. We were able to establish that original RAGE, sequenced from bovine lung, is only present in the lung. This observation was based on antibody specificity, Northern blotting and N-glycosylation analysis. One of the antibodies that we used (H-300, Santa Cruz, CA) was very selective for lung RAGE and not cross-react with other RAGE isoforms. Only lung RAGE had a transcript size of 1.4 kb as determined by Northern blot and only lung RAGE was N-glycosylated. Non-lung tissues and cell lines appeared to express their own unique RAGE isoforms. Non-lung derived cell lines were permissive for lung RAGE isoform expression but lung derived cell lines were not. Interestingly, all transfected cell lines (of lung and non-lung origin) expressed RAGE mRNA transcripts. In addition, we established that previously described endogenous soluble RAGE (esRAGE) does not contain any of the canonical RAGE epitopes, but includes sequence encoded in intron 9. RAGE knockout mice lose esRAGE isoform along with the canonical one confirming that esRAGE originates from the RAGE gene. Signaling studies with pro-inflammatory stimuli in mouse lung slices of wild-type and knockout mice revealed the importance of RAGE in LPS and IL-1-induced inflammatory response, but not when reported RAGE ligands, including AGEs, HMGB1 and S100B, were applied

Small interfering RNA mediated Poly (ADP-ribose) Polymerase-1 inhibition upregulates the heat shock response in a murine fibroblast cell line

Poly (ADP-ribose) polymerase-1 (PARP-1) is a highly conserved multifunctional enzyme, and its catalytic activity is stimulated by DNA breaks. The activation of PARP-1 and subsequent depletion of nicotinamide adenine dinucleotide (NAD+) and adenosine triphosphate (ATP) contributes to significant cytotoxicity in inflammation of various etiologies. On the contrary, induction of heat shock response and production of heat shock protein 70 (HSP-70) is a cytoprotective defense mechanism in inflammation. Recent data suggests that PARP-1 modulates the expression of a number of cellular proteins at the transcriptional level. In this study, small interfering RNA (siRNA) mediated PARP-1 knockdown in murine wild-type fibroblasts augmented heat shock response as compared to untreated cells (as evaluated by quantitative analysis of HSP-70 mRNA and HSP-70 protein expression). These events were associated with increased DNA binding of the heat shock factor-1 (HSF-1), the major transcription factor of the heat shock response. Co-immunoprecipitation experiments in nuclear extracts of the wild type cells demonstrated that PARP-1directly interacted with HSF-1. These data demonstrate that, in wild type fibroblasts, PARP-1 plays a pivotal role in modulating the heat shock response both through direct interaction with HSF-1 and poly (ADP-ribosylation)

P-hydroxyphenylpyruvate, an intermediate of the Phe/Tyr catabolism, improves mitochondrial oxidative metabolism under stressing conditions and prolongs survival in rats subjected to profound hemorrhagic shock

The aim of this study was to test the effect of a small volume administration of p-hydroxyphenylpyruvate (pHPP) in a rat model of profound hemorrhagic shock and to assess a possible metabolic mechanism of action of the compound. The results obtained show that hemorrhaged rats treated with 2-4% of the estimated blood volume of pHPP survived significantly longer (p<0.001) than rats treated with vehicle. In vitro analysis on cultured EA.hy 926 cells demonstrated that pHPP improved cell growth rate and promoted cell survival under stressing conditions. Moreover, pHPP stimulated mitochondria-related respiration under ATP-synthesizing conditions and exhibited antioxidant activity toward mitochondria-generated reactive oxygen species. The compound effects reported in the in vitro and in vivo analyses were obtained in the same millimolar concentration range. These data disclose pHPP as an efficient energetic substrates-supplier to the mitochondrial respiratory chain as well as an antioxidant supporting the view that the compound warrants further evaluation as a therapeutic agent. © 2014 Cotoia et al

Lack of the Receptor for Advanced Glycation End-Products Attenuates E. coli Pneumonia in Mice

Background: The receptor for advanced glycation end-products (RAGE) has been suggested to modulate lung injury in models of acute pulmonary inflammation. To study this further, model systems utilizing wild type and RAGE knockout (KO) mice were used to determine the role of RAGE signaling in lipopolysaccharide (LPS) and E. coli induced acute pulmonary inflammation. The effect of intraperitoneal (i.p.) and intratracheal (i.t.) administration of mouse soluble RAGE on E. coli injury was also investigated. Methodology/Principal Findings: C57BL/6 wild type and RAGE KO mice received an i.t. instillation of LPS, E. coli, or vehicle control. Some groups also received i.p. or i.t. administration of mouse soluble RAGE. After 24 hours, the role of RAGE expression on inflammation was assessed by comparing responses in wild type and RAGE KO. RAGE protein levels decreased in wild type lung homogenates after treatment with either LPS or bacteria. In addition, soluble RAGE and HMGB1 increased in the BALF after E. coli instillation. RAGE KO mice challenged with LPS had the same degree of inflammation as wild type mice. However, when challenged with E. coli, RAGE KO mice had significantly less inflammation when compared to wild type mice. Most cytokine levels were lower in the BALF of RAGE KO mice compared to wild type mice after E. coli injury, while only monocyte chemotactic protein-1, MCP-1, was lower after LPS challenge. Neither i.p. nor i.t. administration of mouse soluble RAGE attenuated the severity of E. coli injury in wild type mice. Conclusions/Significance: Lack of RAGE in the lung does not protect against LPS induced acute pulmonary inflammation, but attenuates injury following live E. coli challenge. These findings suggest that RAGE mediates responses to E. coli-associated pathogen-associated molecular pattern molecules other than LPS or other bacterial specific signaling responses. Soluble RAGE treatment had no effect on inflammation. © 2011 Ramsgaard et al

Guided Bone Regeneration with Ammoniomethacrylate-Based Barrier Membranes in a Radial Defect Model

Large bone defects pose an unsolved challenge for orthopedic surgeons. Our group has previously reported the construction of a barrier membrane made of ammoniomethacrylate copolymer USP (AMCA), which supports the adhesion, proliferation, and osteoblastic differentiation of human mesenchymal stem cells (hMSCs). In this study, we report the use of AMCA membranes to seclude critical segmental defect (~1.0 cm) created in the middle third of rabbit radius and test the efficiency of bone regeneration. Bone regeneration was assessed by radiography, biweekly for 8 weeks. The results were verified by histology and micro-CT at the end of the follow-up. The AMCA membranes were found superior to no treatment in terms of new bone formation in the defect, bone volume, callus surface area normalized to total volume, and the number of bone trabeculae, after eight weeks. Additional factors were then assessed, and these included the addition of simvastatin to the membrane, coating the membrane with human MSC, and a combination of those. The addition of simvastatin to the membranes demonstrated a stronger effect at a similar radiological follow-up. We conclude that AMCA barrier membranes per se and simvastatin delivered in a controlled manner improve bone regeneration outcome

Para-hydroxyphenylpyruvate inhibits the pro-inflammatory stimulation of macrophage preventing LPS-mediated nitro-oxidative unbalance and immunometabolic shift

Targeting metabolism is emerging as a promising therapeutic strategy for modulation of the immune response in human diseases. In the presented study we used the lipopolysaccharide (LPS)-mediated activation of RAW 264.7 macrophage-like cell line as a model to investigate changes in the metabolic phenotype and to test the effect of p-hydroxyphenylpyruvate (pHPP) on it. pHPP is an intermediate of the PHE/TYR catabolic pathway, selected as analogue of the ethyl pyruvate (EP), which proved to exhibit antioxidant and anti-inflammatory activities. The results obtained show that LPS-priming of RAW 264.7 cell line to the activated M1 state resulted in up-regulation of the inducible nitric oxide synthase (iNOS) expression and consequently of NO production and in release of the pro-inflammatory cytokine IL-6. All these effects were prevented dose dependently by mM concentrations of pHPP more efficiently than EP. Respirometric and metabolic flux analysis of LPS-treated RAW 264.7 cells unveiled a marked metabolic shift consisting in downregulation of the mitochondrial oxidative phosphorylation and upregulation of aerobic glycolysis respectively. The observed respiratory failure in LPS-treated cells was accompanied with inhibition of the respiratory chain complexes I and IV and enhanced production of reactive oxygen species. Inhibition of the respiratory activity was also observed following incubation of human neonatal fibroblasts (NHDF-neo) with sera from septic patients. pHPP prevented all the observed metabolic alteration caused by LPS on RAW 264.7 or by septic sera on NHDF-neo. Moreover, we provide evidence that pHPP is an efficient reductant of cytochrome c. On the basis of the presented results a working model, linking pathogen-associated molecular patterns (PAMPs)-mediated immune response to mitochondrial oxidative metabolism, is put forward along with suggestions for its therapeutic control

Para-hydroxyphenylpyruvate inhibits the pro-inflammatory stimulation of macrophage preventing LPS-mediated nitro-oxidative unbalance and immunometabolic shift

Targeting metabolism is emerging as a promising therapeutic strategy for modulation of the immune response in human diseases. In the presented study we used the lipopolysaccharide (LPS)-mediated activation of RAW 264.7 macrophage-like cell line as a model to investigate changes in the metabolic phenotype and to test the effect of p-hydroxyphenylpyruvate (pHPP) on it. pHPP is an intermediate of the PHE/TYR catabolic pathway, selected as analogue of the ethyl pyruvate (EP), which proved to exhibit antioxidant and anti-inflammatory activities. The results obtained show that LPS-priming of RAW 264.7 cell line to the activated M1 state resulted in up-regulation of the inducible nitric oxide synthase (iNOS) expression and consequently of NO production and in release of the pro-inflammatory cytokine IL-6. All these effects were prevented dose dependently by mM concentrations of pHPP more efficiently than EP. Respirometric and metabolic flux analysis of LPS-treated RAW 264.7 cells unveiled a marked metabolic shift consisting in downregulation of the mitochondrial oxidative phosphorylation and upregulation of aerobic glycolysis respectively. The observed respiratory failure in LPS-treated cells was accompanied with inhibition of the respiratory chain complexes I and IV and enhanced production of reactive oxygen species. Inhibition of the respiratory activity was also observed following incubation of human neonatal fibroblasts (NHDF-neo) with sera from septic patients. pHPP prevented all the observed metabolic alteration caused by LPS on RAW 264.7 or by septic sera on NHDF-neo. Moreover, we provide evidence that pHPP is an efficient reductant of cytochrome c. On the basis of the presented results a working model, linking pathogen-associated molecular patterns (PAMPs)-mediated immune response to mitochondrial oxidative metabolism, is put forward along with suggestions for its therapeutic control

Effect of pHPP on cell growth and cell viability under stressing conditions.

<p>EA.hy 926 cells (10⁴ in 100 µl of complete DMEM +10% FBS) were left to adhere to the bottom of the impedentiometric microplate wells in a controlled environment. Impedance was recorded continuously every 5 min and expressed as cell index (CI). (<b>A</b>) Once the CI reached a steady value, the medium was substituted with DMEM containing dissolved pHPP at the indicated concentrations and the impedance recorded for three days. The time scale has been split to distinguish early from late phases and the CI scale enlarged in the left part of the diagram. The values shown are means of 2 independent experiments. (<b>B</b>) Cells were incubated in complete DMEM for 5 h and then the medium substituted with PBS without or with the indicated concentrations of pHPP and the CI recorded for the following 5 h. The values shown are means of 2 independent experiments. (<b>C</b>) Cells were incubated with serum- and glucose-free DMEM (DMEM -S, -G) without or with 10 mM pHPP±20 mM DCA and CI recorded for the following 23 h. Subsequently, the medium was changed to PBS supplemented without or with the same compounds as before. The traces were normalized to the CI values reached before the medium change; each time point is the average ± SEM of 3 independent experiments. The right panel depicts representative phase contrast micrographs of the cells at the end of the experiment; the dark connected circles are the micro-electrodes at the bottom of the impedentiometric wells.</p

Confocal microscopy analysis of the effect of pHPP on production of mitochondrial superoxide anion in cultured cells.

<p>EA.hy 926 cells were grown on coverslips in complete DMEM +10% FBS (<b>A</b>) or without FBS (<b>B</b>) (for 4 hours) either in the absence and in the presence of 10 mM pHPP followed by addition of the mt-O₂^.− fluorescent probe MitoSox (0.5 µM, 15 min incubation). The pictures are optical fields under each condition as imaged by laser scanning confocal microscopy and are representative of two independent experiments yielding similar outcomes. White bar: 50 µm. The insets show selected enlargements rendered to highlight the intracellular compartmentalization of the fluorescent signal. (<b>C</b>) Histogram showing the quantitative analysis of the intracellular fluorescent signal along with the statistical significance of the differences; bars indicate mean values ± SD from 5 randomly selected optical fields/experiment taken under each condition (each optical field contained ≈20 cells). See under Materials and Methods for further details.</p

Respirometric analysis of EA.hy 926 cells.

<p>Intact EA.hy 926 cells were assayed by high-resolution respirometry in the buffer described in Materials and Methods supplemented with the indicated concentrations of pHPP. Panel <b>A</b> depicts OCR measured under resting phosphorylating conditions (OCR_R), in the presence of FoF1-ATP synthase inhibitor oligomycin (OCR_O), or in the presence of the protonophoric uncoupler FCCP (OCR_U). The bar-values were corrected for the residual OCR measured in the presence of the respiratory chain blocker rotenone (shown in the inset) and are means ± SEM of 4–6 independent experiments under each condition. Panel <b>B</b> shows OCR linked to ATP synthesis obtained from the values shown in (A) subtracting OCR_O from the OCR_R. Panel <b>C</b> shows the respiratory control ratios (RCR) obtained from the values shown in (A) dividing OCR_U by the OCR_O. *, P<0.05; **, P<0.01 (vs untreated). (<b>D</b>) OCR_U was measured as described in (A); the inhibitors of the tyrosine catabolism and of glycolysis (DCA and 2-DG, respectively) were tested at the indicated concentrations on the respiratory activity assessed in the absence or in the presence of 2.5 mM pHPP. The bar values are percentages of the OCR_U measured in control untreated cells and are the means ± SEM of 4–5 independent experiments under each condition. *, P<0.05, **, P<0.01 (vs inhibitor-untreated).</p