1 Novartis Institutes for BioMedical Research, Cambridge, Massachusetts
2 Department of Molecular Genetics, Microbiology and Immunology, University of Medicine and Dentistry of New Jersey, Piscataway, New Jersey
3 PTC Therapeutics, South Plainfield, New Jersey
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ABSTRACT |
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Advanced glycation end products (AGEs) are a heterogeneous group of irreversibly bound, complex structures that form nonenzymatically when reducing sugars react with free amino groups on macromolecules (rev. in 1). AGEs are highly reactive and continue to react with nearby amino groups to produce both intra- and intermolecular crosslinks (2). The formation of AGEs has been found to occur in aging and at an accelerated rate in diabetic patients (rev. in 3). The deposition of these covalent adducts on various macromolecules has been reported to contribute to the development of the complications of aging and diabetes through both direct chemical- (covalent crosslink formation) and cell surface receptormediated pathways (4).
The most characterized AGE binding protein is the receptor for AGEs (RAGE). RAGE, a 45-kDa protein belonging to the immunoglobulin superfamily, is present on the cell surface of a variety of cells, including endothelial cells, mononuclear phagocytes, and hepatocytes (5,6). RAGE is a multiligand receptor that has also been shown to bind to several proteins in the S100 family including S100A12 (EN-RAGE) and S100b (7,8). S100b and S100A12 are calcium binding proteins with inflammatory properties (rev. in 9). Activation of RAGE by its various ligands reportedly induces a variety of proinflammatory and procoagulant cellular responses, resulting from the activation of nuclear factor-B (NF-
B) (10), including the expression of vascular cell adhesion molecule-1 (VCAM-1), tumor necrosis factor-
(TNF-
), interleukin (IL)-6, and tissue factor (TF) (7,1114).
Chronic infusion of model AGEs into normal/healthy animals has been reported to elicit pathologies similar to those observed in diabetes. For example, several studies reported that injection of healthy mice with 6 mg/day of model AGEs for 4 weeks resulted in an increase in the expression of several genes implicated in diabetic nephropathy, including TGF-ß, type IV collagen, and laminin (1517). Another group reported an increase in vascular permeability and defective vasodilatory responses in rats and rabbits injected with model AGEs for 4 weeks (18). Administration of model AGEs into healthy animals was also reported to increase VCAM-1 and ICAM-1 expression, intimal proliferation, and lipid deposits, all of which are implicated in atherosclerosis (19,20). Only a few studies have examined the effects of acute administration of model AGEs. Stern and colleagues (10,13) reported that within hours of infusion of various amounts of model AGEs (0.11.0 mg/mouse), increases in liver IL-6 mRNA, lung heme oxygenase mRNA, lung staining for VCAM-1, NF-B activation in liver, and tissue TBARS were observed.
Previously, we have found that RAGE binding AGEs can be created reproducibly using the reducing sugarsglucose, fructose, or ribose (21). Interestingly, we also found that those AGE preparations, which were essentially endotoxin free (0.2 ng/mg protein), were incapable of inducing VCAM-1 or TNF-
secretion regardless of RAGE binding affinity (22). Therefore, our previous findings suggested that RAGE binding affinity does not correlate with cellular activation. Furthermore, our results suggested that AGE proteins may not be general drivers of proinflammatory cellular responses. The objective of the current study was to clarify the role of AGEs in cell activation through gene expression profiling using both in vitro and in vivo model systems. Changes in gene expression of cultured endothelial cells (ECs) treated with either AGEs or S100b were studied. As positive controls, ECs were also treated with the known inflammatory triggers TNF-
or lipopolysaccharide/endotoxin (LPS). The effects of AGEs were studied in vivo using healthy mice exposed to two different treatment conditions: 1) intravenous injection of a single dose of model AGEs (
10 mg/mouse) or 2) four intraperitoneal injections of model AGEs (10 mg · mouse-1 · day-1). In both cases, the liver was extracted for gene expression profiling. The liver was chosen to study the effects of AGEs in vivo, because of its well-characterized responsiveness to inflammatory stimuli, especially with respect to the acute-phase response.
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RESEARCH DESIGN AND METHODS |
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Preparation of ribose-derived model AGEs.
Ribose-derived model AGEs (Rib BSA) were prepared with 500 mmol/l ribose (6-week incubation) and characterized as described previously (21). Endotoxin levels were measured by Associates of Cape Cod (Falmouth, MA) using the gel-clot method and were found to be <0.2 ng/mg AGE-BSA. Control BSA (Ctrl BSA) used in these experiments was the same endotoxin-tested BSA used as starting material for AGE-BSA preparations; however, the BSA was kept frozen until needed. As needed, the BSA was thawed and diluted using dialysis buffer to the same concentration as the stock Rib BSA (48.9 mg/ml). After dialysis, the final protein concentration was determined using the BCA assay. As reported previously, the half-maximal inhibition concentration (IC50) for Rib BSA in a cell-free human soluble RAGE (hsRAGE) binding assay was 0.11 µmol/l (21). In contrast, Ctrl BSA showed no detectable binding affinity for hsRAGE (21).
Preparation of S100b.
The Hans Kocher lab (Novartis Pharmaceuticals, Basel, Switzerland) generously provided recombinant human S100b. Endotoxin levels were determined to be 2.5 ng/mg protein. Using the cell-free hsRAGE assay reported previously, the IC50 for S100b was 0.24 µmol/l (21).
Cell culture.
Human microvascular ECs (HMEC-4) were obtained from Dr. Edwin Ades (Centers for Disease Control and Prevention, Atlanta, GA). HMEC-4 cells were derived from human foreskin and immortalized by constitutive expression of the T-antigen of SV40 virus (23). Monolayers were propagated in growth medium (MCDB131, supplemented with 10% heat-inactivated FBS, 2 mmol/l L-glutamine, 10 ng/ml epidermal growth factor, 1 µg/ml hydrocortisone [HC], and 1% antibiotic-antimicotic in 5% CO2 at 37°C). The cells were grown to confluence in T-175 flasks (5 x 106 cells per flask in 20 ml medium). Cells were passaged once a week following mild trypsinization with 0.05% Trypsin-EDTA at 37°C for 5 min. HMEC-4 cells were used at passage 22. When 80% confluent, cells were treated for 18 h at 37°C with 0.5 mg/ml Ctrl BSA, 0.5 mg/ml Rib BSA, or 0.2 mg/ml S100b diluted in growth medium, except the FBS, which was used to 5% (three flasks per treatment group).
Administration of model AGEs to mice.
C57BL/6J mice were obtained from The Jackson Laboratory at 46 weeks of age and were allowed to acclimate for at least 1 week before use. The mice were 610 weeks of age (25 g) at the time of the experiment. Model AGEs were injected intravenously in a volume of 10 ml/kg or intraperitoneally at a volume of 50 ml/kg. Mice were administered with a single intravenous injection of 400 mg/kg Ctrl BSA or Rib BSA (
10 mg/mouse) or of 4 mg/kg (
0.1 mg/mouse) of LPS (three mice/group). After 24 h, mice were killed by CO2 asphyxiation and the liver was removed for RNA isolation. In a separate experiment, mice were injected with 400 mg/kg i.p. of Ctrl BSA or Rib BSA daily for 4 days (five mice/group). On day 5, mice were killed and the liver was removed for RNA isolation. The use and care of laboratory animals at the Novartis Institutes for Biomedical Research through institutional policy complies with or exceeds all requirements mandated by the Animal Welfare Act and state and local laws governing the use of animals in research.
RNA extraction for microarray analysis.
Total RNA was isolated from cultured cells and murine liver tissue using TRIzol reagent according to the manufacturers instructions. The total RNA was further purified using the clean-up protocol in the Qiagen RNeasy kit according to the manufacturers instructions. Final RNA concentrations were determined spectrophotometrically at 260 nm. Quality of the total RNA (300 ng/lane) was determined by subjecting the samples to 1% agarose gel electrophoresis. RNA integrity was confirmed by ribosomal 18S and 28S RNA ethidium bromide staining.
Microarray analysis.
Purified total RNA was used to synthesize double-stranded cDNA using Superscript Choice System. The cDNA was then transcribed in vitro using Enzo BioArray high-yield transcript labeling kit to form biotin-labeled cRNA. The labeled cRNA was fragmented and hybridized to the microarray for 16 h at 45°C. The array was washed and stained using the GeneChip Fluidics station. For HMEC-4 cells, cRNA was hybridized to Affymetrix hg U133A chips. For the murine tissuederived cRNA, the Affymetrix MG U74Av2 chips were utilized. The array was scanned and the data were captured using the Affymetrix GeneChip Laboratory Information Management System (LIMS). The Affymetrix GeneChip MAS4.0 software was used to generate the average difference calls (AvgDiff).
For each experiment, pairwise comparison of replicates showed that there were no outliers and that the twofold difference could be considered significant. Therefore, data were filtered using the following criteria: fold change twofold or greater with (Students t test P < 0.05) and mean AvgDiff values 200. Note: some of the probes recognize multiple genes within a family; therefore, the gene sequence recognized by the probe is either identical to the sequence provided under the listed gene accession number or similar to that gene sequence.
Clustering.
Hierarchical clustering to generate an experimental tree was performed using GeneSpring software and the default settings (measure similarity by standard correlation with a separation ratio of 0.5 and a minimum distance of 0.001). Experiment trees were generated using two different lists of genes. The first list identified genes that differed in expression between mice treated with a single bolus of either Rib BSA (10 mg/mouse) or Ctrl BSA (10 mg/mouse). Selection criteria included a mean average difference of at least 200 (a twofold difference between the two treatment groups; P < 0.05 Welsh T-test, unequal variance, no additional Bonferroni corrections). The second list identified genes that differed in expression between mice treated with LPS versus Ctrl BSA using the same selection criteria described above. Of note, another group of mice was injected with a lower dose of Rib BSA (0.3 mg/mouse) and no significant changes in gene expression were observed compared with Ctrl BSA treatment (data not shown).
Data analysis.
Statistical analysis was performed in Excel (Microsoft, Redmond, WA). Triplicate experiments were analyzed unless otherwise noted. Experiment tree graphs were created in GeneSpring (Silicon Genetics, Redwood City, CA).
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RESULTS |
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Table 6 lists selected genes upregulated in the liver from mice treated with a single bolus of model AGE compared with mice treated with a single bolus of Ctrl BSA. The list further demonstrates that although some genes upregulated by Rib BSA are also upregulated by LPS, the overall expression patterns differed. For example, of the 43 genes that changed at least 10-fold after LPS treatment, only 4 of those genes were also upregulated by Rib BSA (serum amyloid A1, serum amyloid A3, monocyte chemotactic protein-1, and MARCO). In contrast, both M and P lysozyme were upregulated by Rib BSA, but not by LPS. No genes were found significantly downregulated in the liver.
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DISCUSSION |
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Gene expression profiling of S100b-treated endothelial cells confirmed S100b as the mediator of inflammation as previously reported (7) and, therefore, also confirmed the validity of our in vitro model system. In our studies, S100b triggered an immune response defined mainly by expression of chemokines, adhesion molecules, and genes involved in antigen presentation, which included MHC class I and II alleles and several proteasome subunits (summarized in Fig. 1). In addition, increased expression of those gene classes is dependent on activation of the NF-B pathway (29), confirming previous reports that NF-
B is a central transcription factor in the cellular response to S100b (7). Taken together, the changes in gene expression observed after S100b treatment described an activated endothelium.
Although S100b treatment induced some similar gene expression changes compared with TNF- or LPS, the overall pattern of gene expression varies greatly (compare Tables 3 and 5). Thus, the effects on gene expression observed when HMEC-4 cells were treated with S100b are unlikely due to contaminating LPS. In addition, the gene expression changes observed after treatment of HMEC-4 cells with TNF-
or LPS further validated our in vitro model system, showing that the HMEC-4 cells are responsive to proinflammatory triggers.
Exposure of healthy animals to high doses of model AGEs triggered a modest immune response in liver tissue defined mainly as a macrophage-based clearance/detoxification response. Overall, mice injected with model AGEs failed to display gene expression changes indicative of a strong induction of the NF-B pathway. At least six of the nine genes that increased in expression after a single intravenous administration of model AGEs are associated with macrophage activation and differentiation (monocyte chemotactic protein-1, M and P lysozymes, MARCO, CD5L, and thymosin) (30) (Table 6, Fig. 1). These results were confirmed by a second experiment measuring gene expression changes in livers from mice treated for 4 days with high doses of model AGEs. These mice also showed an increase in a large number of the genes associated with macrophage activation or differentiation, including lysozyme P and M, MARCO, CD5L, TYRO, CD68, properdin factor, and complement C1qB (30). This suggests that the majority of the cellular responses that followed exposure to exogenous AGEs were derived from the macrophage-derived Kupffer cells. The upregulation of lysozyme is interesting, because lysozyme has been reported to bind AGEs and improve renal excretion of AGEs (31). In addition, a 2.8-fold increase in VCAM-1 was observed in liver tissue from mice treated for 4 days with model AGEs (total amount of intraperitoneally injected AGE: 40 mg/mouse). In these same mice, a two- to fourfold increase in soluble VCAM-1 was measured by enzyme-linked immunosorbent assay (data not shown). Stern and Schmidt (11) reported that healthy mice injected with a single bolus 0.50 mg/mouse of model AGE showed a two- to threefold increase in VCAM-1 expression in the lung according to immunohistological staining. We did not see an increase in liver VCAM-1 mRNA after a single injection of 10 mg/mouse (a 20 times larger dose).
Our results do not show that AGEs trigger a strong inflammatory response. Previously, animals injected with a single dose of exogenous AGEs have been reported to increase the expression of a variety of inflammatory mediators, including liver IL-6 and heme oxygenase (10,13). However, our experiments showed no evidence of an increase in IL-6 expression. Furthermore, if IL-6 expression was induced in our study, then a significant increase in the expression of acute-phase proteins such as C-reactive protein and fibrinogen would have been observed. Although we feel our data accurately reflect the effects of AGEs in vivo, there are several differences between this study and previously published studies, including 1) strain of mouse (SJL vs. C57BL/6), 2) dose of model AGE (0.5 vs. 10 or 40 mg/mouse), and 3) likely the composition of the AGE preparations (4) time point (6 vs. 24 h or 5 days). In addition, a longer-term study using AGE-modified mouse serum albumin might result in induction of inflammatory mediators. Although many of the known AGE structures that have been shown to form under in vitro conditions have also been found in vivo (3235), model AGEs may not accurately reflect the chemical composition of AGEs formed in vivo.
The present study is one of the first to look at AGE-induced effects on gene expression using this oligonucleotide array technology. Ideally all of the genes observed to change should be confirmed by additional techniques, such as Northern blot or RT-PCR. In the HMEC-4 cell system, we have confirmed, using a cell-based enzyme-linked immunosorbent assay, that the VCAM-1 gene expression changes elicited by TNF-, LPS, and S100b described herein reflect a change in protein levels as well (22). In the animal studies, the increase in mRNA expression of P lysozyme in mice injected with model AGEs was confirmed by Northern blot analysis (data not shown).
Exposure of healthy animals to high doses of model AGEs triggered a modest immune response in the liver tissue defined mainly as a macrophage-based clearance/detoxification response. The significance of these changes is unclear. Future work will be required to decipher whether these effects reflect specific AGE-mediated cellular responses or these effects may actually be an artifact resulting from injection of high concentrations of modified proteins. For example, although Ctrl BSA is used for comparison, the ribose-modified protein may be denatured and elicit nonspecific effects. Unlike previous reports, the observed immune response in AGE-treated mice did not entail expression of high levels of inflammatory mediators, although very modest changes in the inflammatory mediators VCAM-1 and monocyte chemotactic protein-1 were noted in mice exposed to model AGEs. Overall, our data do not convincingly demonstrate that model AGEs are signaling molecules, despite previous work showing that the AGEs bind to RAGE with high affinity (21).
Although our work suggests that AGEs do not trigger a significant inflammatory immune response, accumulation of AGEs on macromolecules is known to adversely affect both the functional properties and clearance of these molecules. The resulting biomechanical changes to these molecules have been shown to contribute to the pathology of several disease states, including atherosclerosis and diabetic complications (3638). Thus, the biomechanical effects of AGEs may prove to be more detrimental in vivo than the proposed cell-surface receptor-mediated pathways.
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ACKNOWLEDGMENTS |
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Address correspondence and reprint requests to Thomas E. Hughes, Novartis Institutes for BioMedical Research, 100 Technology Square, Bldg. 601/Rm. 5155, Cambridge, MA 02139. E-mail: thomase.hughes{at}pharma.novartis.com
Received for publication August 1, 2003 and accepted in revised form November 12, 2003
AGE, advanced glycation end product; Ctrl BSA, BSA incubated in the absence of modifying agent; EC, endothelial cell; HC, hydrocortisone; HMEC, human microvascular EC; hsRAGE, human soluble RAGE; ICAM-1, intercellular adhesion molecule-1; IB
, inhibitor of nuclear factor-
B; IL, interleukin; LPS, lipopolysaccharide/endotoxin; MHC, major histocompatibility complex; RAGE, receptor for AGE; Rib BSA, BSA incubated with ribose; TF, tissue factor; TGF-ß, transforming growth factor-ß; TNF-
, tumor necrosis factor-
;VCAM-1, vascular cell adhesion molecule-1
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REFERENCES |
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