Activation of the receptor for advanced glycation end products (RAGE) reportedly triggers a variety of proinflammatory responses. However, our previous work revealed that RAGE-binding AGEs free of endotoxin were incapable of inducing vascular cell adhesion molecule-1 (VCAM-1) or tumor necrosis factor-α (TNF-α) expression. Thus, the objective of this study was to clarify the role of AGEs in cell activation through gene expression profiling using both in vitro and in vivo model systems. Endothelial cells treated with AGE-BSA, previously shown to bind RAGE with high affinity, did not show gene expression changes indicative of an inflammatory response. In contrast, the alternate RAGE ligand, S100b, triggered an increase in endothelial mRNA expression of a variety of immune-related genes. 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 or 2) four intraperitoneal injections of model AGEs (once per day). In both cases, the liver was extracted for gene expression profiling. Both of the short-term AGE treatments resulted in a moderate increase in liver mRNA levels for genes involved in macrophage-based clearance/detoxification of foreign agents. Our findings using AGEs with strong RAGE-binding properties indicate that AGEs may not uniformly play a role in cellular activation.

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 receptor–mediated 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-κB (NF-κB) (10), including the expression of vascular cell adhesion molecule-1 (VCAM-1), tumor necrosis factor-α (TNF-α), 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-β, 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 (0.1–1.0 mg/mouse), increases in liver IL-6 mRNA, lung heme oxygenase mRNA, lung staining for VCAM-1, NF-κB activation in liver, and tissue TBARS were observed.

Previously, we have found that RAGE binding AGEs can be created reproducibly using the reducing sugars—glucose, 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-α 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-α 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.

Bovine albumin (Fraction V, sterile filtered, endotoxin tested), d(−) ribose, sodium phosphate monobasic, sodium phosphate dibasic, sodium hydroxide, recombinant human epidermal growth factor, hydrocortisone, gelatin, recombinant human TNF-α, and lipopolysaccharide from E. coli 0111:B4 were obtained from Sigma (St. Louis, MO). PBS (10×) was purchased from Roche Diagnostics Corporation (Mannheim, Germany). Endotoxin-free distilled water, sterile PBS without calcium and magnesium, MCDB 131 media, heat-inactivated FBS, l-glutamine, antibiotic/antimicotic, 0.05% trypsin/0.53 mmol/l EDTA, penicillin and streptomycin, TRIzol reagent, and Superscript II Choice System were purchased from Gibco BRL/Life Technologies (Gaithersburg, MD). Sterilization filters (Express filter; 0.22 μm; 250 ml) were obtained from Millipore (Bedford, MA). Bicinchoninic acid (BCA) protein assay kit was purchased from Pierce (Rockford, IL). T-175 Falcon flasks were purchased from Fisher Scientific (Pittsburgh, PA). RNeasy kits were obtained from Qiagen (Valencia, CA). A BioArray High Yield DNA Transcript kit was purchased from ENZO Diagnostics (Farmingdale, NY).

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 × 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 4–6 weeks of age and were allowed to acclimate for at least 1 week before use. The mice were 6–10 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 manufacturer’s instructions. The total RNA was further purified using the clean-up protocol in the Qiagen RNeasy kit according to the manufacturer’s 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 tissue–derived 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 (Student’s 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).

Isolation of genes regulated in ECs by model AGE-BSAs.

To identify genes regulated in the endothelium after exposure to AGE-BSAs with high RAGE binding affinity, HMEC-4 cells were treated for 18 h at 37°C with 0.5 mg/ml Rib BSA or Ctrl BSA. Total RNA was isolated from the treated cells and used for microarray analysis. As shown in Tables 1 and 2, analysis identified only five genes that were significantly upregulated and only four genes were significantly downregulated. The responses were very modest, with no gene increasing by more than threefold. Overall, the genes found to be regulated did not comprise the expected proinflammatory mRNA phenotype. In fact, the genes found to be upregulated showed no obvious expression pattern. However, several genes that have been suggested to be involved in the control cell proliferation were downregulated after treatment with Rib BSA wk6, including inhibitors of DNA binding-1, -2, and -3 (Table 2).

S100b regulated gene expression in endothelial cells.

When HMEC-4 cells were treated for 18 h at 37°C with S100b, 44 genes were significantly upregulated and 10 were significantly downregulated as assessed by microarray analysis (Tables 3 and 4). Many of the genes upregulated were indicative of an activated endothelium, including genes encoding a number of chemokines and adhesion molecules and genes encoding proteins involved in antigen presentation, including expression of a variety of major histocompatibility complex (MHC) class I and II alleles and subunits of the proteasome (Table 3).

For comparison, HMEC-4 cells were also treated with two known inflammatory triggers—TNF-α (20 ng/ml) or LPS (200 ng/ml) for 4 h at 37°C. As expected, numerous proinflammatory genes were differentially regulated (84 genes after TNF-α treatment; 165 genes after LPS treatment). Table 5 shows the top 35 genes that were upregulated in both TNF-α and LPS treatments. HMEC-4 cells treated with TNF-α or LPS resulted in a strong inflammatory cellular response, which included an increase in the expression of several cytokines/chemokines, adhesion molecules, transcription factors/regulators, and proteins involved in apoptosis to name a few.

Gene expression profiles from livers of mice injected with exogenous model AGEs.

While the effects observed in cultured cells are often indicative of what happens in vivo, the cultured cell model system is limited because it is unable to account for interactions between different cell types that occur within an animal. Therefore, the effects of acute administration of exogenous AGEs to healthy mice were evaluated. Healthy C57BL/6 mice were intravenously injected with either a single bolus of Rib BSA or Ctrl BSA (10 mg/mouse). After 24 h, the livers were removed and total RNA was isolated. Another group of healthy mice was intraperitoneally injected for 4 days with either Rib BSA or Ctrl BSA (10 mg · mouse−1 · day−1). On the 5th day, the livers were removed and total RNA was isolated. The preparations used in this study were essentially endotoxin free (≤0.2 ng/mg AGE-BSA) according to the gel-clot method. However, to be sure the genes differentially regulated in animals injected with Rib BSA were not due to trace amounts of endotoxin, the expression pattern in AGE-treated animals was compared with animals injected with endotoxin. Using GeneSpring software, cluster analysis illustrated the samples from LPS-treated mice did not cluster with the model AGE–treated mice (data not shown). These data suggest that the biological responses induced by model AGEs are not similar to the responses elicited by LPS; therefore, the biological activity of the model AGEs used in this study is not likely a result of endotoxin contamination.

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.

In mice treated with model AGEs for 4 days (10 mg/mouse i.p.), 26 genes were upregulated in the liver at least twofold, and no genes were significantly downregulated. Genes with at least a 2.5-fold upregulation are listed in Table 7. LPS was not included as a control in this experiment. A number of genes that had been identified after a single injection of model AGEs were also elevated after four injections of model AGEs, including M and P lysozyme and the macrophage scavenger receptors MARCO and CD5L.

To gain a better understanding of the role RAGE ligands play in cellular activation, we evaluated the effects of model AGEs or recombinant S100b on EC gene expression using microarray technology. AGE treatment of HMEC-4 cells did not induce an inflammatory mRNA phenotype as predicted by the literature (see below). However, treatment with S100b induced an mRNA phenotype of activated endothelium (Tables 3 and 4, Fig. 1). In addition, treatment of ECs with known inflammatory triggers TNF-α or LPS induced a strong inflammatory immune response defined by an increase in the expression of genes, including cytokines, cytokine receptors, chemokines, MMP-1, and various cell adhesion molecules (Table 5, Fig. 1). These data suggest that RAGE binding AGEs may not be general drivers of inflammation. In contrast, this work confirmed S100b as mediator of inflammation either by activating RAGE or through other pathways yet to be elucidated. Future work will be required to determine whether the various reported RAGE ligands activate different cellular responses.

Numerous studies have been published showing cells exposed to AGEs resulted in significant alterations in the expression of many genes, including IL-1β (24), TNF-α (12), IL-6 (13), platelet-derived growth factor (25), insulin-like growth factor-1 (IGF-1) (26), thrombomodulin, VCAM-1 (11), and TF (14,27,28). Of these genes, all were present on the chip; however, the majority were considered below detection level. The only exception was platelet-derived growth factor, which displayed a low level of expression that did not differ between Ctrl BSA and Rib BSA treatments. Treating cells with AGE-BSAs for multiple time periods may provide a more complete picture. For example, longer/chronic exposure of cells might result in changes in the above-mentioned genes. However, treatment of HMEC-4 cells up to 72 h failed to induce VCAM-1 as assessed by enzyme-linked immunosorbent assay (22). In addition, total RNA extracted from HMEC-4 cells treated for 4 h with endotoxin-free AGE-BSA also did not show an increase in proinflammatory gene expression (data not shown).

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-κB pathway (29), confirming previous reports that NF-κB 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-α 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-α 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-κB 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-α, 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.

FIG. 1.

Summary of gene expression changes induced in cultured endothelial cells treated with AGE-BSA, S100b, TNF-α, or LPS.

FIG. 1.

Summary of gene expression changes induced in cultured endothelial cells treated with AGE-BSA, S100b, TNF-α, or LPS.

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TABLE 1

Upregulated genes in HMEC-4 cells after treatment with Rib BSA

Gene nameANFold changePotential function
Immunoglobulin lambda chain VJ region (IGL) AF043584 2.8 Role unclear 
Bone morphogenetic protein 4 D30751 2.3 Cell proliferation, differentiation, and apoptosis 
ICAM2 NM_000873 2.0 Leukocyte adhesion 
N2,N2-dimethylguanosine tRNA methyltransferase AF196479 2.0 Methylation guanosine of tRNAs 
Similar to bone morphogenetic protein 7 (osteogenic protein 1) BC004248 2.0 Possibly member of TGF-β superfamily 
Gene nameANFold changePotential function
Immunoglobulin lambda chain VJ region (IGL) AF043584 2.8 Role unclear 
Bone morphogenetic protein 4 D30751 2.3 Cell proliferation, differentiation, and apoptosis 
ICAM2 NM_000873 2.0 Leukocyte adhesion 
N2,N2-dimethylguanosine tRNA methyltransferase AF196479 2.0 Methylation guanosine of tRNAs 
Similar to bone morphogenetic protein 7 (osteogenic protein 1) BC004248 2.0 Possibly member of TGF-β superfamily 

Fold change for all listed genes statistically significant; P < 0.05 (Student’s t test; model AGE-treated vs. control BSA untreated; unpaired assume unequal variance in both populations). AN is the nucleotide accession number for each gene.

TABLE 2

Downregulated genes in HMEC-4 cells after treatment with Rib BSA

Gene nameANFold changePotential function
Inhibitor of DNA binding 2 NM_002166 −4.1 Inhibitor of bHLH transcription factors 
Inhibitor of DNA binding 1 D13889 −2.9 Inhibitor of bHLH transcription factors 
Retinol dehydrogenase 11 NM_016026 −2.2 Role unknown 
Inhibitor of DNA binding 3 NM_002167 −2.1 Inhibitor or bHLH transcription factors 
Gene nameANFold changePotential function
Inhibitor of DNA binding 2 NM_002166 −4.1 Inhibitor of bHLH transcription factors 
Inhibitor of DNA binding 1 D13889 −2.9 Inhibitor of bHLH transcription factors 
Retinol dehydrogenase 11 NM_016026 −2.2 Role unknown 
Inhibitor of DNA binding 3 NM_002167 −2.1 Inhibitor or bHLH transcription factors 

Fold change for all listed genes statistically significant; P < 0.05 (Student’s t test; model AGE-treated vs. control BSA untreated; unpaired assume unequal variance in both populations). AN is the nucleotide accession number for each gene.

TABLE 3

Upregulated genes in HMEC-4 cells after treatment with S100b

Gene nameANFold changePotential function
Cytokines/chemokines    
 Monocyte chemotactic protein (MCP-1) NM_002982 44.6 Monocyte/basophil chemotactant 
 Chemokine CXC ligand 2 (GRO2 oncogene) NM_002089 10.2 Polymorphonuclear leukocyte chemotactant 
 Small inducible cytokine A5 (RANTES) NM_002985 3.9 Monocytes/memory T-cell/eosinophil chemotactant 
 Pre-B-cell colony enhancing factor (PBEF) NM_005746 2.4 B-cell precursor maturation 
Membrane proteins    
 HLA-B, allele A∗2711 NM_005514 3.9 Antigen presentation 
 Interferon induced transmembrane protein 1 (IFITM1) NM_003641 3.8 Implicated in cell growth inhibition 
 HLA class I heavy chain (HLA-Cw∗1701) NM_002117 3.7 Antigen presentation 
 Phospholipid scramblase 3 (PLSCR3) NM_020360 3.3 Cell activation or injury 
 HLA-B39 NM_005514 3.0 Antigen presentation 
 Vascular cell adhesion molecule 1 (VCAM1) NM_080682 2.9 Monocyte and lymphocyte adhesion molecule 
 HLA-Cw1 M12679 2.9 Antigen presentation 
 MHC class I-C, clone MGC:11039 BC004489.1 2.9 Antigen presentation 
 MHC class I HLA B71 L07950.1 2.8 Antigen presentation 
 HLA-G2.1 M90684.1 2.7 Antigen presentation 
 MHC, class I, HLA-J M80469 2.6 Non-function pseudogene 
 Highly similar to HLA-B and -C NG_002397 2.5 Antigen presentation 
 Similar to HLA-F, α chain AW514210 2.5 Antigen presentation 
 Tissue specific transplantation antigen P35B (TSTA3) NM_003313 2.4 Leukocyte adhesion 
 Transferrin receptor (p90, CD71) BC001188 2.3 Iron transport 
 HLA-G2.2 M90685.1 2.3 Antigen presentation 
 Similar to MHC, class I, HLA-A11 AA573862 2.3 Antigen presentation 
 Mpv17 transgene NM_002437 2.0 ROS metabolism 
 Nicotinamide N-methyltransferase (NNMT) NM_006169 2.0 N-methylation of nicotinamide and other pyridines 
Proteases    
 Proteasome subunit, β type 8 (PSMB8) NM_148919 2.7 Protein degradation 
 Proteasome activator subunit 2 NM_002818 2.1 Protein degradation 
 Proteasome subunit, β type, 10 (PSMB10) NM_002801 2.0 Protein degradation 
Enzymes    
 Highly similar to aldolase A AK026577 2.3 Similar to enzyme that converts fructose-1,6-bisphosphate to glyceraldehyde 3-phosphate 
 Aldolase A NM_000034 2.1 Glycolysis 
 Peptidylprolyl isomerase F (cyclophilin F) NM_005729 2.1 Protein folding 
Mitochondrial proteins    
 Superoxide dismutase 2, mitochondrial NM_000636 4.7 Catalyzes conversion of superoxide radicals to molecular oxygen 
 Mitochondrial ribosome protein L4 NM_015956 2.3 Component of mitochondrial ribosome 
 Death-associated protein 3 NM_004632 2.2 Inducer of apoptosis 
Secreted proteins    
 Pentaxin-related gene NM_002852 2.7 Acute-phase response 
 Midkine NM_002391 2.2 Heparin binding growth factor 
Transcription factors    
 CCAAT enhancer binding protein (CEBP) δ NM_005195 2.6 Regulates expression of various acute-phase proteins and cytokines 
 Nuclear factor of κ light polypeptide gene enhancer in B-cells inhibitor, α (IκBα) NM_020529 2.1 Inhibits NF-κB from entering the nucleus 
 CAAT enhancer binding protein (CEBP), β NM_005194 2.0 Regulates expression of various acute-phase proteins and cytokines 
 RNA binding motif protein 6 (RBM6) NM_005777 2.0 Tumor suppressor 
Hypothetical proteins    
 Natural killer cell transcript 4 (NK4) NM_004221 21.6 Role unknown 
 Interferon-stimulated protein, 15 kDa (ISG15) NM_005101 6.2 Role unknown 
 Interferon, α-inducible protein (clone IFI-6-16) (G1P3) NM_002038 5.2 Role unknown 
 KIAA0090 protein NM_015047 2.9 Role unknown 
 MGC5627 protein NM_024096 2.3 Role unknown 
 Hypothetical protein LOC57333 BC013436 2.0 Role unknown 
Gene nameANFold changePotential function
Cytokines/chemokines    
 Monocyte chemotactic protein (MCP-1) NM_002982 44.6 Monocyte/basophil chemotactant 
 Chemokine CXC ligand 2 (GRO2 oncogene) NM_002089 10.2 Polymorphonuclear leukocyte chemotactant 
 Small inducible cytokine A5 (RANTES) NM_002985 3.9 Monocytes/memory T-cell/eosinophil chemotactant 
 Pre-B-cell colony enhancing factor (PBEF) NM_005746 2.4 B-cell precursor maturation 
Membrane proteins    
 HLA-B, allele A∗2711 NM_005514 3.9 Antigen presentation 
 Interferon induced transmembrane protein 1 (IFITM1) NM_003641 3.8 Implicated in cell growth inhibition 
 HLA class I heavy chain (HLA-Cw∗1701) NM_002117 3.7 Antigen presentation 
 Phospholipid scramblase 3 (PLSCR3) NM_020360 3.3 Cell activation or injury 
 HLA-B39 NM_005514 3.0 Antigen presentation 
 Vascular cell adhesion molecule 1 (VCAM1) NM_080682 2.9 Monocyte and lymphocyte adhesion molecule 
 HLA-Cw1 M12679 2.9 Antigen presentation 
 MHC class I-C, clone MGC:11039 BC004489.1 2.9 Antigen presentation 
 MHC class I HLA B71 L07950.1 2.8 Antigen presentation 
 HLA-G2.1 M90684.1 2.7 Antigen presentation 
 MHC, class I, HLA-J M80469 2.6 Non-function pseudogene 
 Highly similar to HLA-B and -C NG_002397 2.5 Antigen presentation 
 Similar to HLA-F, α chain AW514210 2.5 Antigen presentation 
 Tissue specific transplantation antigen P35B (TSTA3) NM_003313 2.4 Leukocyte adhesion 
 Transferrin receptor (p90, CD71) BC001188 2.3 Iron transport 
 HLA-G2.2 M90685.1 2.3 Antigen presentation 
 Similar to MHC, class I, HLA-A11 AA573862 2.3 Antigen presentation 
 Mpv17 transgene NM_002437 2.0 ROS metabolism 
 Nicotinamide N-methyltransferase (NNMT) NM_006169 2.0 N-methylation of nicotinamide and other pyridines 
Proteases    
 Proteasome subunit, β type 8 (PSMB8) NM_148919 2.7 Protein degradation 
 Proteasome activator subunit 2 NM_002818 2.1 Protein degradation 
 Proteasome subunit, β type, 10 (PSMB10) NM_002801 2.0 Protein degradation 
Enzymes    
 Highly similar to aldolase A AK026577 2.3 Similar to enzyme that converts fructose-1,6-bisphosphate to glyceraldehyde 3-phosphate 
 Aldolase A NM_000034 2.1 Glycolysis 
 Peptidylprolyl isomerase F (cyclophilin F) NM_005729 2.1 Protein folding 
Mitochondrial proteins    
 Superoxide dismutase 2, mitochondrial NM_000636 4.7 Catalyzes conversion of superoxide radicals to molecular oxygen 
 Mitochondrial ribosome protein L4 NM_015956 2.3 Component of mitochondrial ribosome 
 Death-associated protein 3 NM_004632 2.2 Inducer of apoptosis 
Secreted proteins    
 Pentaxin-related gene NM_002852 2.7 Acute-phase response 
 Midkine NM_002391 2.2 Heparin binding growth factor 
Transcription factors    
 CCAAT enhancer binding protein (CEBP) δ NM_005195 2.6 Regulates expression of various acute-phase proteins and cytokines 
 Nuclear factor of κ light polypeptide gene enhancer in B-cells inhibitor, α (IκBα) NM_020529 2.1 Inhibits NF-κB from entering the nucleus 
 CAAT enhancer binding protein (CEBP), β NM_005194 2.0 Regulates expression of various acute-phase proteins and cytokines 
 RNA binding motif protein 6 (RBM6) NM_005777 2.0 Tumor suppressor 
Hypothetical proteins    
 Natural killer cell transcript 4 (NK4) NM_004221 21.6 Role unknown 
 Interferon-stimulated protein, 15 kDa (ISG15) NM_005101 6.2 Role unknown 
 Interferon, α-inducible protein (clone IFI-6-16) (G1P3) NM_002038 5.2 Role unknown 
 KIAA0090 protein NM_015047 2.9 Role unknown 
 MGC5627 protein NM_024096 2.3 Role unknown 
 Hypothetical protein LOC57333 BC013436 2.0 Role unknown 

Fold change for all listed genes statistically significant; P < 0.05 (Student’s t test; S100b-treated vs. media control–untreated; unpaired assume unequal variance in both populations). AN is the nucleotide accession number for each gene.

TABLE 4

Downregulated genes in HMEC-4 cells after treatment with S100b

Gene nameANFold changePotential function
Stress response proteins    
 Metallothionein 1E M10942 −2.3 Binds toxic metals and scavenges free radicals 
 Similar to metallothionein 1E AL031602 −2.6 See function of metallothionein 1E 
 Selenoprotein W, 1 (SEPW1) NM_003009 −2.1 Antioxidant 
Enzymes    
 RNA polymerase II (DNA directed) polypeptide A NM_000937 −2.7 Largest subunit of RNA polymerase II (220 kD) 
 Short-chain dehydrogenase reductase 1 (SDR1) NM_004753 −2.5 Regeneration of retinol (Vit. A) from retinal 
 Highly similar to transglutaminase 2 BC003551 −2.2 99% identical to transglutaminase 2, a protein cross-linking enzyme 
 Ubiquitin protein ligase E3A NM_000462 −2.0 Protein ubiquitination 
Mitochondrial proteins    
 Glutaminase C AF158555 −2.4 Converts l-glutamine to l-glutamate 
Cytoskeletal-associated protein    
 Caldesmon 1 (CALD1) NM_033157 −2.0 Actomyosin regulatory protein 
Other proteins    
 RNA binding protein BRUNOL3 U69546 −2.0 Translation repression 
 Clone 24775 AF052169 −2.1 Role unknown 
Gene nameANFold changePotential function
Stress response proteins    
 Metallothionein 1E M10942 −2.3 Binds toxic metals and scavenges free radicals 
 Similar to metallothionein 1E AL031602 −2.6 See function of metallothionein 1E 
 Selenoprotein W, 1 (SEPW1) NM_003009 −2.1 Antioxidant 
Enzymes    
 RNA polymerase II (DNA directed) polypeptide A NM_000937 −2.7 Largest subunit of RNA polymerase II (220 kD) 
 Short-chain dehydrogenase reductase 1 (SDR1) NM_004753 −2.5 Regeneration of retinol (Vit. A) from retinal 
 Highly similar to transglutaminase 2 BC003551 −2.2 99% identical to transglutaminase 2, a protein cross-linking enzyme 
 Ubiquitin protein ligase E3A NM_000462 −2.0 Protein ubiquitination 
Mitochondrial proteins    
 Glutaminase C AF158555 −2.4 Converts l-glutamine to l-glutamate 
Cytoskeletal-associated protein    
 Caldesmon 1 (CALD1) NM_033157 −2.0 Actomyosin regulatory protein 
Other proteins    
 RNA binding protein BRUNOL3 U69546 −2.0 Translation repression 
 Clone 24775 AF052169 −2.1 Role unknown 

Fold change for all listed genes statistically significant; P < 0.05 (Student’s t test; S100b-treated vs. media control–untreated; unpaired assume unequal variance in both populations). AN is the nucleotide accession number for each gene.

TABLE 5

Upregulated genes in HMEC-4 cells after treatment with LPS or TNF-α

Gene nameAN+LPS+TNF-αPotential function
Cytokines/chemokines     
 Interleukin 6 NM_000600 106.9 21.1 Inflammatory cytokine 
 Chemokine CC ligand 20 NM_004591 57.3 33.4 Lymphocyte chemotactant 
 Interleukin 8 M28130 50.3 33.9 Chemokine of CXC motif 
 Chemokine CXC ligand 2 (GRO2) NM_002089 49.1 27.5 Polymorphonuclear leukocyte chemotactant 
 Chemokine CXC ligand 1 (GRO1) NM_001511 45.7 28.9 Polymorphonuclear leukocyte chemotactant 
 Monocyte chemotactic protein (MCP-1) NM_002982 28.5 24.1 Monocyte/basophil chemotactant 
 Chemokine CXC ligand 3 (GRO 3) NM_002090 21.4 6.1 Polymorphonuclear leukocyte chemotactant 
 Chemokine CC ligand 5 NM_002985 10.0 3.1 Monocyte/memory T-cells/eosinophil chemotactant 
Cytokine receptors     
 Interleukin 7 receptor M29696 15.0 7.7 Component of IL-7 receptor complex that directly binds IL7 
 Interleukin 15 receptor, alpha U31628 5.8 5.0 Component of IL-15 receptor complex 
Adhesion molecules     
 VCAM-1 NM_080682 90.6 148.0 Monocyte and lymphocyte adhesion molecule 
 ICAM-1 NM_000201 43.5 88.1 Monocyte and lymphocyte adhesion molecule 
 Ninjurin 1 U91512 9.1 7.5 Implicated in cell adhesion 
 Endothelial cell–specific molecule 1 NM_007036 7.3 8.4 Antagonizes ICAM-1 for LFA1 binding 
 TNF-α–induced protein 6 NM_007115 7.8 17.3 Implicated in leukocyte adhesion; related to CD44 
Transcription factors/regulators     
 NF-κB p49/p100 subunit NM_002502 18.1 14.0 Regulates expression of a variety of proinflammatory genes 
 NF-κB p105 (precursor to p50) subunit M58603 11.9 6.2 Regulates expression of a variety of proinflammatory genes 
 IκBα NM_020529 5.4 4.8 Inhibitor of NF-κB 
 TNF-α–induced protein 3 (A20) NM_006290 14.8 22.1 Inhibitor of NF-κB 
 Interferon regulatory factor 1 NM_002198 15.5 9.5 Transcription of IFN alpha and beta 
Enzymes     
 GTP cyclohydrolase 1 (dopa-responsive dystonia) NM_000161 17.6 15.4 Synthesis of aromatic side chains in phe, tyr, trp 
 Superoxide dismutase 2, mitochondrial NM_000636 17.9 36.4 Conversion of superoxide radicals to molecular oxygen 
 MMP 1 (interstitial collagenase) M13509 3.8 2.5 Degradation interstitial collagens, types I, II, and III 
ECM molecules     
 Tenascin C (hexabrachion) NM_002160 11.9 9.4 Inhibitor of chemotaxis of polymorphonuclear leukocytes and monocytes 
Proteins involved in apoptosis     
 Caspase-like apoptosis regulatory protein 2 (clarp) AF005775 7.0 6.4 Positively regulates caspase 8 
 Phorbol-12-myristate-13-acetate-induced protein 1 (NOXA) NM_021127 5.5 3.0 Promoter of apoptosis 
 Apoptosis inhibitor 1 (baculoviral IAP repeat-containing 2) U45878 16.5 28.7 Regulator of apoptosis, interacts with TRAF 1&2 
Proteins involved in cell growth     
 Jun B; proto-oncogene M29039 10.3 6.7 Promoter of cell growth 
 MAD; mothers against decapentaplegic homolog 3 NM_005902 8.6 5.3 Imparts growth inhibitory effects of TGF-β 
Proteins with unknown function     
 Interferon, alpha-inducible protein (clone IFI-15K) NM_005101 17.7 6.5 Role unknown 
 Interferon stimulated gene 20 kDa NM_002201 13.5 5.9 Nuclear protein 
 Hypothetical protein FLJ90005 W27419 7.0 6.3 Role unknown 
 Interferon-induced protein with tetratricopeptide repeats 1 (IFI-56K) M24594 38.5 5.0 Implicated in translation; interacts with initiation factor eIF-3 
 Interferon-induced protein with tetratricopeptide repeats 2 (IFI-54K) NM_001547 35.6 4.7 Role unknown 
 TNF-α–induced protein 2 M92357 22.1 17.7 Implicated as retinoic acid targeted gene; potential oncogene 
Gene nameAN+LPS+TNF-αPotential function
Cytokines/chemokines     
 Interleukin 6 NM_000600 106.9 21.1 Inflammatory cytokine 
 Chemokine CC ligand 20 NM_004591 57.3 33.4 Lymphocyte chemotactant 
 Interleukin 8 M28130 50.3 33.9 Chemokine of CXC motif 
 Chemokine CXC ligand 2 (GRO2) NM_002089 49.1 27.5 Polymorphonuclear leukocyte chemotactant 
 Chemokine CXC ligand 1 (GRO1) NM_001511 45.7 28.9 Polymorphonuclear leukocyte chemotactant 
 Monocyte chemotactic protein (MCP-1) NM_002982 28.5 24.1 Monocyte/basophil chemotactant 
 Chemokine CXC ligand 3 (GRO 3) NM_002090 21.4 6.1 Polymorphonuclear leukocyte chemotactant 
 Chemokine CC ligand 5 NM_002985 10.0 3.1 Monocyte/memory T-cells/eosinophil chemotactant 
Cytokine receptors     
 Interleukin 7 receptor M29696 15.0 7.7 Component of IL-7 receptor complex that directly binds IL7 
 Interleukin 15 receptor, alpha U31628 5.8 5.0 Component of IL-15 receptor complex 
Adhesion molecules     
 VCAM-1 NM_080682 90.6 148.0 Monocyte and lymphocyte adhesion molecule 
 ICAM-1 NM_000201 43.5 88.1 Monocyte and lymphocyte adhesion molecule 
 Ninjurin 1 U91512 9.1 7.5 Implicated in cell adhesion 
 Endothelial cell–specific molecule 1 NM_007036 7.3 8.4 Antagonizes ICAM-1 for LFA1 binding 
 TNF-α–induced protein 6 NM_007115 7.8 17.3 Implicated in leukocyte adhesion; related to CD44 
Transcription factors/regulators     
 NF-κB p49/p100 subunit NM_002502 18.1 14.0 Regulates expression of a variety of proinflammatory genes 
 NF-κB p105 (precursor to p50) subunit M58603 11.9 6.2 Regulates expression of a variety of proinflammatory genes 
 IκBα NM_020529 5.4 4.8 Inhibitor of NF-κB 
 TNF-α–induced protein 3 (A20) NM_006290 14.8 22.1 Inhibitor of NF-κB 
 Interferon regulatory factor 1 NM_002198 15.5 9.5 Transcription of IFN alpha and beta 
Enzymes     
 GTP cyclohydrolase 1 (dopa-responsive dystonia) NM_000161 17.6 15.4 Synthesis of aromatic side chains in phe, tyr, trp 
 Superoxide dismutase 2, mitochondrial NM_000636 17.9 36.4 Conversion of superoxide radicals to molecular oxygen 
 MMP 1 (interstitial collagenase) M13509 3.8 2.5 Degradation interstitial collagens, types I, II, and III 
ECM molecules     
 Tenascin C (hexabrachion) NM_002160 11.9 9.4 Inhibitor of chemotaxis of polymorphonuclear leukocytes and monocytes 
Proteins involved in apoptosis     
 Caspase-like apoptosis regulatory protein 2 (clarp) AF005775 7.0 6.4 Positively regulates caspase 8 
 Phorbol-12-myristate-13-acetate-induced protein 1 (NOXA) NM_021127 5.5 3.0 Promoter of apoptosis 
 Apoptosis inhibitor 1 (baculoviral IAP repeat-containing 2) U45878 16.5 28.7 Regulator of apoptosis, interacts with TRAF 1&2 
Proteins involved in cell growth     
 Jun B; proto-oncogene M29039 10.3 6.7 Promoter of cell growth 
 MAD; mothers against decapentaplegic homolog 3 NM_005902 8.6 5.3 Imparts growth inhibitory effects of TGF-β 
Proteins with unknown function     
 Interferon, alpha-inducible protein (clone IFI-15K) NM_005101 17.7 6.5 Role unknown 
 Interferon stimulated gene 20 kDa NM_002201 13.5 5.9 Nuclear protein 
 Hypothetical protein FLJ90005 W27419 7.0 6.3 Role unknown 
 Interferon-induced protein with tetratricopeptide repeats 1 (IFI-56K) M24594 38.5 5.0 Implicated in translation; interacts with initiation factor eIF-3 
 Interferon-induced protein with tetratricopeptide repeats 2 (IFI-54K) NM_001547 35.6 4.7 Role unknown 
 TNF-α–induced protein 2 M92357 22.1 17.7 Implicated as retinoic acid targeted gene; potential oncogene 

Fold change for all listed genes statistically significant; P < 0.05 (Student’s t test; LPS vs. Ctrl BSA or TNF-α vs. Ctrl BSA; unpaired assume unequal variance in both populations). AN is the nucleotide accession number for each gene.

TABLE 6

Genes upregulated by a single intravenous administration of Rib BSA in mouse liver

Gene nameAN+RibBSA+LPSPotential function
Monocyte chemotactic protein (MCP-1) M19681 12* 31* Monocyte/basophil chemotactant 
Lysozyme P structural X51547 11* Absent Antibacterial enzyme 
MARCO U18424 10* 10* Scavenger receptor; binds oxidized LDL 
Serum amyloid A3 X03505 9.1* 105* Acute-phase protein 
Lysozyme M M21050 3.5* 0.5 Antibacterial enzyme 
CD5L NM_005894 3.4* Absent Scavenger receptor of cysteine-rich family 
Serum amyloid A1 M13521 3.3* 46.5* Acute-phase protein 
Prothymosin β 4 U38967 3.1* 1.0 Migration of macrophages and other cell types 
Procollagen type IV M15832 2.4* 4.0* Extracellular matrix molecule 
Gene nameAN+RibBSA+LPSPotential function
Monocyte chemotactic protein (MCP-1) M19681 12* 31* Monocyte/basophil chemotactant 
Lysozyme P structural X51547 11* Absent Antibacterial enzyme 
MARCO U18424 10* 10* Scavenger receptor; binds oxidized LDL 
Serum amyloid A3 X03505 9.1* 105* Acute-phase protein 
Lysozyme M M21050 3.5* 0.5 Antibacterial enzyme 
CD5L NM_005894 3.4* Absent Scavenger receptor of cysteine-rich family 
Serum amyloid A1 M13521 3.3* 46.5* Acute-phase protein 
Prothymosin β 4 U38967 3.1* 1.0 Migration of macrophages and other cell types 
Procollagen type IV M15832 2.4* 4.0* Extracellular matrix molecule 
*

P < 0.05 (Student’s t test; model AGE-treated or LPS-treated vs. control BSA untreated; unpaired assume unequal variance in both populations). AN is the nucleotide accession number for each gene.

TABLE 7

Genes upregulated in mouse liver after 4 injections of Rib BSA

Gene nameAN+ Rib BSAPotential function
UI-M-AL0-abv-e-12-0-UI.s1 AI838080 6.4 EST; role unknown 
Lysozyme P X51547 4.3 Antibacterial enzyme 
Ribonucleotide reductase M2 subunit NM_009104 3.8 Cell-cycle regulated rate-limiting DNA synthesis enzyme 
MARCO U18424 3.7 Scavenger receptor; binds oxidized LDL 
Viral envelope–like protein (G7e) U69488 3.6 Lymphoid expressed gene 
Lysozyme M M21050 3.5 Antibacterial enzyme 
Adipose fatty acid binding protein (422) gene M20497 3.2 Involved in cellular fatty acid uptake 
Retinoic acid-inducible E3 protein U29539 3.1 Role unknown 
Ly-6 alloantigen (Ly-6E.1) X04653 3.0 T-cell activation 
UI-M-BH1-amo-d-08-0-UI.s1 AW048937 2.9 EST; role unknown 
CD5L NM_005894 2.9 Scavenger receptor of cysteine-rich family 
VCAM-1 NM_011693 2.8 Mediates adhesion of monocytes and lymphocytes 
EGF-like module containing, mucin-like, hormone receptor-like sequence 1 XM_128711 2.8 Role unknown 
Mitogen-responsive 96 kDa phosphoprotein p96 U18869 2.7 Role unknown 
TYRO protein tyrosine kinase binding protein (DAP12) NM_011662 2.7 NK cell activation 
CD68 antigen NM_009853 2.5 Specific for monocyte/macrophage cells 
Properdin factor, complement XM_135820 2.5 Complement protein 
Complement C1q B chain NM_009777 2.5 Complement protein 
UI-M-BH1-alf-e-03-0-UI.s1 AW046124 2.5 EST; role unknown 
Gene nameAN+ Rib BSAPotential function
UI-M-AL0-abv-e-12-0-UI.s1 AI838080 6.4 EST; role unknown 
Lysozyme P X51547 4.3 Antibacterial enzyme 
Ribonucleotide reductase M2 subunit NM_009104 3.8 Cell-cycle regulated rate-limiting DNA synthesis enzyme 
MARCO U18424 3.7 Scavenger receptor; binds oxidized LDL 
Viral envelope–like protein (G7e) U69488 3.6 Lymphoid expressed gene 
Lysozyme M M21050 3.5 Antibacterial enzyme 
Adipose fatty acid binding protein (422) gene M20497 3.2 Involved in cellular fatty acid uptake 
Retinoic acid-inducible E3 protein U29539 3.1 Role unknown 
Ly-6 alloantigen (Ly-6E.1) X04653 3.0 T-cell activation 
UI-M-BH1-amo-d-08-0-UI.s1 AW048937 2.9 EST; role unknown 
CD5L NM_005894 2.9 Scavenger receptor of cysteine-rich family 
VCAM-1 NM_011693 2.8 Mediates adhesion of monocytes and lymphocytes 
EGF-like module containing, mucin-like, hormone receptor-like sequence 1 XM_128711 2.8 Role unknown 
Mitogen-responsive 96 kDa phosphoprotein p96 U18869 2.7 Role unknown 
TYRO protein tyrosine kinase binding protein (DAP12) NM_011662 2.7 NK cell activation 
CD68 antigen NM_009853 2.5 Specific for monocyte/macrophage cells 
Properdin factor, complement XM_135820 2.5 Complement protein 
Complement C1q B chain NM_009777 2.5 Complement protein 
UI-M-BH1-alf-e-03-0-UI.s1 AW046124 2.5 EST; role unknown 

Fold change for all listed genes statistically significant; P < 0.05 (Student’s t test; model AGE-treated vs. control BSA untreated; unpaired assume unequal variance in both populations). AN is the nucleotide accession number for each gene.

We thank Marlene Dressman for her contributions in analyzing the gene expression changes observed in mice following a single administration of model AGEs. We thank Shari Caplan for generously providing probes for Northern blot analysis. We would also like to thank Arco Jeng’s lab for their expertise in Northern blot analysis. Helpful discussions from John Rediske are kindly acknowledged.

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