Divergent Pathways of Expression Are Activated by the RAGE Ligands S100b and AGE-BSA Jessica V. Valencia,1,2 Manisha Mone,1 Jin Zhang,1 Marla Weetall,3 Frank P. Buxton,1 and Thomas E. Hughes1

Activation of the receptor for advanced glycation end The formation of AGEs has been found to occur in aging products (RAGE) reportedly triggers a variety of proin- and at an accelerated rate in diabetic patients (rev. in 3). flammatory responses. However, our previous work re- The deposition of these covalent adducts on various vealed that RAGE-binding AGEs free of endotoxin were macromolecules has been reported to contribute to the incapable of inducing vascular -1 development of the complications of aging and diabetes (VCAM-1) or tumor necrosis factor-␣ (TNF-␣) expres- through both direct chemical- (covalent crosslink forma- sion. Thus, the objective of this study was to clarify the tion) and cell surface receptor–mediated pathways (4). role of AGEs in cell activation through profiling using both in vitro and in vivo model systems. The most characterized AGE binding is the Endothelial cells treated with AGE-BSA, previously receptor for AGEs (RAGE). RAGE, a 45-kDa protein shown to bind RAGE with high affinity, did not show belonging to the immunoglobulin superfamily, is present gene expression changes indicative of an inflammatory on the cell surface of a variety of cells, including endothe- response. In contrast, the alternate RAGE ligand, lial cells, mononuclear phagocytes, and hepatocytes (5,6). S100b, triggered an increase in endothelial mRNA ex- RAGE is a multiligand receptor that has also been shown pression of a variety of immune-related . The to bind to several in the S100 family including effects of AGEs were studied in vivo using healthy mice S100A12 (EN-RAGE) and S100b (7,8). S100b and S100A12 exposed to two different treatment conditions: 1) intra- venous injection of a single dose of model AGEs or 2) are calcium binding proteins with inflammatory properties four intraperitoneal injections of model AGEs (once per (rev. in 9). Activation of RAGE by its various ligands day). In both cases, the liver was extracted for gene reportedly induces a variety of proinflammatory and pro- expression profiling. Both of the short-term AGE treat- coagulant cellular responses, resulting from the activation ments resulted in a moderate increase in liver mRNA of nuclear factor-␬B (NF-␬B) (10), including the expres- levels for genes involved in -based clear- sion of vascular cell adhesion molecule-1 (VCAM-1), tumor ance/detoxification of foreign agents. Our findings using necrosis factor-␣ (TNF-␣), interleukin (IL)-6, and tissue AGEs with strong RAGE-binding properties indicate factor (TF) (7,11–14). that AGEs may not uniformly play a role in cellular activation. Diabetes 53:743–751, 2004 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 dvanced glycation end products (AGEs) are a model AGEs for 4 weeks resulted in an increase in the heterogeneous group of irreversibly bound, expression of several genes implicated in diabetic ne- ␤ complex structures that form nonenzymatically phropathy, including TGF- , type IV collagen, and laminin when reducing sugars react with free amino (15–17). Another group reported an increase in vascular A permeability and defective vasodilatory responses in rats groups on macromolecules (rev. in 1). AGEs are highly reactive and continue to react with nearby amino groups and rabbits injected with model AGEs for 4 weeks (18). to produce both intra- and intermolecular crosslinks (2). 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 From the 1Novartis Institutes for BioMedical Research, Cambridge, Massa- implicated in atherosclerosis (19,20). Only a few studies chusetts; the 2Department of Molecular Genetics, Microbiology and Immunol- ogy, University of Medicine and Dentistry of New Jersey, Piscataway, New have examined the effects of acute administration of Jersey; and 3PTC Therapeutics, South Plainfield, New Jersey. model AGEs. Stern and colleagues (10,13) reported that Address correspondence and reprint requests to Thomas E. Hughes, Novar- tis Institutes for BioMedical Research, 100 Technology Square, Bldg. 601/Rm. within hours of infusion of various amounts of model 5155, Cambridge, MA 02139. E-mail: [email protected]. AGEs (0.1–1.0 mg/mouse), increases in liver IL-6 mRNA, Received for publication 1 August 2003 and accepted in revised form 12 heme oxygenase mRNA, lung staining for VCAM-1, November 2003. AGE, advanced glycation end product; Ctrl BSA, BSA incubated in the NF-␬B activation in liver, and tissue TBARS were absence of modifying agent; EC, endothelial cell; HC, hydrocortisone; HMEC, observed. human microvascular EC; hsRAGE, human soluble RAGE; ICAM-1, intercel- lular adhesion molecule-1; I␬B␣, inhibitor of nuclear factor-␬B; IL, interleukin; Previously, we have found that RAGE binding AGEs can LPS, lipopolysaccharide/endotoxin; MHC, major histocompatibility complex; be created reproducibly using the reducing sugars—glu- RAGE, receptor for AGE; Rib BSA, BSA incubated with ribose; TF, tissue cose, fructose, or ribose (21). Interestingly, we also found factor; TGF-␤, transforming growth factor-␤; TNF-␣, tumor necrosis factor-␣; VCAM-1, vascular cell adhesion molecule-1. that those AGE preparations, which were essentially en- © 2004 by the American Diabetes Association. dotoxin free (Յ0.2 ng/mg protein), were incapable of

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inducing VCAM-1 or TNF-␣ secretion regardless of RAGE Administration of model AGEs to mice. C57BL/6J mice were obtained binding affinity (22). Therefore, our previous findings 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 suggested that RAGE binding affinity does not correlate (ϳ25 g) at the time of the experiment. Model AGEs were injected intrave- with cellular activation. Furthermore, our results sug- nously in a volume of 10 ml/kg or intraperitoneally at a volume of 50 ml/kg. gested that AGE proteins may not be general drivers of Mice were administered with a single intravenous injection of 400 mg/kg Ctrl proinflammatory cellular responses. The objective of the BSA or Rib BSA (ϳ10 mg/mouse) or of 4 mg/kg (ϳ0.1 mg/mouse) of LPS (three mice/group). After 24 h, mice were killed by CO2 asphyxiation and the current study was to clarify the role of AGEs in cell liver was removed for RNA isolation. In a separate experiment, mice were activation through gene expression profiling using both in injected with 400 mg/kg i.p. of Ctrl BSA or Rib BSA daily for 4 days (five vitro and in vivo model systems. Changes in gene expres- mice/group). On day 5, mice were killed and the liver was removed for RNA sion of cultured endothelial cells (ECs) treated with either isolation. The use and care of laboratory animals at the Novartis Institutes for AGEs or S100b were studied. As positive controls, ECs Biomedical Research through institutional policy complies with or exceeds all requirements mandated by the Animal Welfare Act and state and local laws were also treated with the known inflammatory triggers governing the use of animals in research. TNF-␣ or lipopolysaccharide/endotoxin (LPS). The effects RNA extraction for microarray analysis. Total RNA was isolated from of AGEs were studied in vivo using healthy mice exposed cultured cells and murine liver tissue using TRIzol reagent according to the to two different treatment conditions: 1) intravenous in- manufacturer’s instructions. The total RNA was further purified using the ϳ clean-up protocol in the Qiagen RNeasy kit according to the manufacturer’s jection of a single dose of model AGEs ( 10 mg/mouse) or instructions. Final RNA concentrations were determined spectrophotometri- 2) four intraperitoneal injections of model AGEs (10 mg ⅐ cally at 260 nm. Quality of the total RNA (300 ng/lane) was determined by Ϫ1 Ϫ1 mouse ⅐ day ). In both cases, the liver was extracted subjecting the samples to 1% agarose gel electrophoresis. RNA integrity was for gene expression profiling. The liver was chosen to confirmed by ribosomal 18S and 28S RNA ethidium bromide staining. study the effects of AGEs in vivo, because of its well- Microarray analysis. Purified total RNA was used to synthesize double- stranded cDNA using Superscript Choice System. The cDNA was then characterized responsiveness to inflammatory stimuli, es- transcribed in vitro using Enzo BioArray high-yield transcript labeling kit to pecially with respect to the acute-phase response. 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 RESEARCH DESIGN AND METHODS Affymetrix hg U133A chips. For the murine tissue–derived cRNA, the Bovine albumin (Fraction V, sterile filtered, endotoxin tested), D(Ϫ) ribose, Affymetrix MG U74Av2 chips were utilized. The array was scanned and the sodium phosphate monobasic, sodium phosphate dibasic, sodium hydroxide, data were captured using the Affymetrix GeneChip Laboratory Information recombinant human epidermal growth factor, hydrocortisone, gelatin, recom- Management System (LIMS). The Affymetrix GeneChip MAS4.0 software was binant human TNF-␣, and lipopolysaccharide from E. coli 0111:B4 were used to generate the average difference calls (AvgDiff). obtained from Sigma (St. Louis, MO). PBS (10ϫ) was purchased from Roche For each experiment, pairwise comparison of replicates showed that there Diagnostics Corporation (Mannheim, Germany). Endotoxin-free distilled wa- were no outliers and that the twofold difference could be considered ter, sterile PBS without calcium and magnesium, MCDB 131 media, heat- significant. Therefore, data were filtered using the following criteria: fold inactivated FBS, L-glutamine, /antimicotic, 0.05% trypsin/0.53 mmol/l change twofold or greater with (Student’s t test P Ͻ 0.05) and mean AvgDiff EDTA, penicillin and streptomycin, TRIzol reagent, and Superscript II Choice values Յ200. Note: some of the probes recognize multiple genes within a System were purchased from Gibco BRL/Life Technologies (Gaithersburg, family; therefore, the gene sequence recognized by the probe is either MD). Sterilization filters (Express filter; 0.22 ␮m; 250 ml) were obtained from identical to the sequence provided under the listed gene accession number or Millipore (Bedford, MA). Bicinchoninic acid (BCA) protein assay kit was similar to that gene sequence. purchased from Pierce (Rockford, IL). T-175 Falcon flasks were purchased Clustering. Hierarchical clustering to generate an experimental tree was from Fisher Scientific (Pittsburgh, PA). RNeasy kits were obtained from performed using GeneSpring software and the default settings (measure Qiagen (Valencia, CA). A BioArray High Yield DNA Transcript kit was similarity by standard correlation with a separation ratio of 0.5 and a minimum purchased from ENZO Diagnostics (Farmingdale, NY). distance of 0.001). Experiment trees were generated using two different lists Preparation of ribose-derived model AGEs. Ribose-derived model AGEs of genes. The first list identified genes that differed in expression between (Rib BSA) were prepared with 500 mmol/l ribose (6-week incubation) and mice treated with a single bolus of either Rib BSA (10 mg/mouse) or Ctrl BSA characterized as described previously (21). Endotoxin levels were measured (10 mg/mouse). Selection criteria included a mean average difference of at by Associates of Cape Cod (Falmouth, MA) using the gel-clot method and least 200 (a twofold difference between the two treatment groups; P Ͻ 0.05 were found to be Ͻ0.2 ng/mg AGE-BSA. Control BSA (Ctrl BSA) used in these Welsh T-test, unequal variance, no additional Bonferroni corrections). The experiments was the same endotoxin-tested BSA used as starting material for second list identified genes that differed in expression between mice treated AGE-BSA preparations; however, the BSA was kept frozen until needed. As with LPS versus Ctrl BSA using the same selection criteria described above. Of needed, the BSA was thawed and diluted using dialysis buffer to the same note, another group of mice was injected with a lower dose of Rib BSA (0.3 concentration as the stock Rib BSA (48.9 mg/ml). After dialysis, the final mg/mouse) and no significant changes in gene expression were observed protein concentration was determined using the BCA assay. As reported compared with Ctrl BSA treatment (data not shown).

previously, the half-maximal inhibition concentration (IC50) for Rib BSA in a Data analysis. Statistical analysis was performed in Excel (Microsoft, cell-free human soluble RAGE (hsRAGE) binding assay was 0.11 ␮mol/l (21). Redmond, WA). Triplicate experiments were analyzed unless otherwise noted. In contrast, Ctrl BSA showed no detectable binding affinity for hsRAGE (21). Experiment tree graphs were created in GeneSpring (Silicon Genetics, Red- Preparation of S100b. The Hans Kocher lab (Novartis Pharmaceuticals, wood City, CA). Basel, Switzerland) generously provided recombinant human S100b. Endo- toxin 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 mol/l (21). RESULTS Cell culture. Human microvascular ECs (HMEC-4) were obtained from Dr. Edwin Ades (Centers for Disease Control and Prevention, Atlanta, GA). Isolation of genes regulated in ECs by model AGE- HMEC-4 cells were derived from human foreskin and immortalized by BSAs. To identify genes regulated in the endothelium after constitutive expression of the T-antigen of SV40 virus (23). Monolayers were exposure to AGE-BSAs with high RAGE binding affinity, propagated in growth medium (MCDB131, supplemented with 10% heat- HMEC-4 cells were treated for 18 h at 37°C with 0.5 mg/ml inactivated FBS, 2 mmol/l L-glutamine, 10 ng/ml epidermal growth factor, 1 ␮ Rib BSA or Ctrl BSA. Total RNA was isolated from the g/ml hydrocortisone [HC], and 1% antibiotic-antimicotic in 5% CO2 at 37°C). The cells were grown to confluence in T-175 flasks (5 ϫ 106 cells per flask in treated cells and used for microarray analysis. As shown in 20 ml medium). Cells were passaged once a week following mild trypsiniza- Tables 1 and 2, analysis identified only five genes that were tion with 0.05% Trypsin-EDTA at 37°C for 5 min. HMEC-4 cells were used at significantly upregulated and only four genes were signif- passage 22. When ϳ80% 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 icantly downregulated. The responses were very modest, medium, except the FBS, which was used to 5% (three flasks per treatment with no gene increasing by more than threefold. Overall, group). the genes found to be regulated did not comprise the

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TABLE 1 Upregulated genes in HMEC-4 cells after treatment with Rib BSA Fold Gene name AN change Potential 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-␤ superfamily Fold change for all listed genes statistically significant; P Ͻ 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. expected proinflammatory mRNA phenotype. In fact, the injected with either a single bolus of Rib BSA or Ctrl BSA genes found to be upregulated showed no obvious expres- (10 mg/mouse). After 24 h, the livers were removed and sion pattern. However, several genes that have been total RNA was isolated. Another group of healthy mice suggested to be involved in the control cell proliferation was intraperitoneally injected for 4 days with either Rib were downregulated after treatment with Rib BSA wk6, BSA or Ctrl BSA (10 mg ⅐ mouseϪ1 ⅐ dayϪ1). On the 5th day, including inhibitors of DNA binding-1, -2, and -3 (Table 2). the livers were removed and total RNA was isolated. The S100b regulated gene expression in endothelial cells. preparations used in this study were essentially endotoxin When HMEC-4 cells were treated for 18 h at 37°C with free (Յ0.2 ng/mg AGE-BSA) according to the gel-clot S100b, 44 genes were significantly upregulated and 10 method. However, to be sure the genes differentially were significantly downregulated as assessed by microar- regulated in animals injected with Rib BSA were not due to ray analysis (Tables 3 and 4). Many of the genes upregu- trace amounts of endotoxin, the expression pattern in lated were indicative of an activated endothelium, AGE-treated animals was compared with animals injected including genes encoding a number of chemokines and with endotoxin. Using GeneSpring software, cluster anal- adhesion molecules and genes encoding proteins involved ysis illustrated the samples from LPS-treated mice did not in antigen presentation, including expression of a variety cluster with the model AGE–treated mice (data not of major histocompatibility complex (MHC) class I and II shown). These data suggest that the biological responses alleles and subunits of the proteasome (Table 3). induced by model AGEs are not similar to the responses For comparison, HMEC-4 cells were also treated with elicited by LPS; therefore, the biological activity of the two known inflammatory triggers—TNF-␣ (20 ng/ml) or model AGEs used in this study is not likely a result of LPS (200 ng/ml) for4hat37°C. As expected, numerous endotoxin contamination. proinflammatory genes were differentially regulated (84 Table 6 lists selected genes upregulated in the liver from genes after TNF-␣ treatment; 165 genes after LPS treat- mice treated with a single bolus of model AGE compared ment). Table 5 shows the top 35 genes that were upregu- with mice treated with a single bolus of Ctrl BSA. The list lated in both TNF-␣ and LPS treatments. HMEC-4 cells further demonstrates that although some genes upregu- treated with TNF-␣ or LPS resulted in a strong inflamma- lated by Rib BSA are also upregulated by LPS, the overall tory cellular response, which included an increase in the expression patterns differed. For example, of the 43 genes expression of several /chemokines, adhesion that changed at least 10-fold after LPS treatment, only 4 of molecules, transcription factors/regulators, and proteins those genes were also upregulated by Rib BSA (serum involved in apoptosis to name a few. amyloid A1, serum amyloid A3, chemotactic Gene expression profiles from livers of mice injected protein-1, and MARCO). In contrast, both M and P ly- with exogenous model AGEs. While the effects observed sozyme were upregulated by Rib BSA, but not by LPS. No in cultured cells are often indicative of what happens in genes were found significantly downregulated in the liver. vivo, the cultured cell model system is limited because it is In mice treated with model AGEs for 4 days (10 mg/ unable to account for interactions between different cell mouse i.p.), 26 genes were upregulated in the liver at least types that occur within an animal. Therefore, the effects of twofold, and no genes were significantly downregulated. acute administration of exogenous AGEs to healthy mice Genes with at least a 2.5-fold upregulation are listed in were evaluated. Healthy C57BL/6 mice were intravenously Table 7. LPS was not included as a control in this

TABLE 2 Downregulated genes in HMEC-4 cells after treatment with Rib BSA Fold Gene name AN change Potential function Inhibitor of DNA binding 2 NM_002166 Ϫ4.1 Inhibitor of bHLH transcription factors Inhibitor of DNA binding 1 D13889 Ϫ2.9 Inhibitor of bHLH transcription factors Retinol dehydrogenase 11 NM_016026 Ϫ2.2 Role unknown Inhibitor of DNA binding 3 NM_002167 Ϫ2.1 Inhibitor or bHLH transcription factors Fold change for all listed genes statistically significant; P Ͻ 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.

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TABLE 3 Upregulated genes in HMEC-4 cells after treatment with S100b Fold Gene name AN change Potential 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 A5 (RANTES) NM_002985 3.9 /memory T-cell/eosinophil chemotactant Pre-B-cell colony enhancing factor (PBEF) NM_005746 2.4 B-cell precursor maturation Membrane proteins NM_005514 3.9 Antigen presentation 2711ءHLA-B, allele A Interferon induced transmembrane protein 1 (IFITM1) NM_003641 3.8 Implicated in cell growth inhibition NM_002117 3.7 Antigen presentation (1701ءHLA class I heavy chain (HLA-Cw 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, ␣ 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, ␤ type 8 (PSMB8) NM_148919 2.7 Protein degradation Proteasome activator subunit 2 NM_002818 2.1 Protein degradation Proteasome subunit, ␤ 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) ␦ NM_005195 2.6 Regulates expression of various acute-phase proteins and cytokines Nuclear factor of ␬ light polypeptide gene enhancer NM_020529 2.1 Inhibits NF-␬B from entering the nucleus in B-cells inhibitor, ␣ (I␬B␣) CAAT enhancer binding protein (CEBP), ␤ 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, ␣-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 Ͻ 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.

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TABLE 4 Downregulated genes in HMEC-4 cells after treatment with S100b Fold Gene name AN change Potential function Stress response proteins Metallothionein 1E M10942 Ϫ2.3 Binds toxic metals and scavenges free radicals Similar to metallothionein 1E AL031602 Ϫ2.6 See function of metallothionein 1E Selenoprotein W, 1 (SEPW1) NM_003009 Ϫ2.1 Antioxidant Enzymes RNA polymerase II (DNA directed) polypeptide A NM_000937 Ϫ2.7 Largest subunit of RNA polymerase II (220 kD) Short-chain dehydrogenase reductase 1 (SDR1) NM_004753 Ϫ2.5 Regeneration of retinol (Vit. A) from retinal Highly similar to transglutaminase 2 BC003551 Ϫ2.2 99% identical to transglutaminase 2, a protein cross-linking enzyme Ubiquitin protein ligase E3A NM_000462 Ϫ2.0 Protein ubiquitination Mitochondrial proteins Glutaminase C AF158555 Ϫ2.4 Converts L-glutamine to L-glutamate Cytoskeletal-associated protein Caldesmon 1 (CALD1) NM_033157 Ϫ2.0 Actomyosin regulatory protein Other proteins RNA binding protein BRUNOL3 U69546 Ϫ2.0 Translation repression Clone 24775 AF052169 Ϫ2.1 Role unknown Fold change for all listed genes statistically significant; P Ͻ 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. experiment. A number of genes that had been identified example, longer/chronic exposure of cells might result in after a single injection of model AGEs were also elevated changes in the above-mentioned genes. However, treat- after four injections of model AGEs, including M and P ment of HMEC-4 cells up to 72 h failed to induce VCAM-1 lysozyme and the macrophage scavenger receptors as assessed by enzyme-linked immunosorbent assay (22). MARCO and CD5L. In addition, total RNA extracted from HMEC-4 cells treated for 4 h with endotoxin-free AGE-BSA also did not DISCUSSION show an increase in proinflammatory gene expression To gain a better understanding of the role RAGE ligands (data not shown). play in cellular activation, we evaluated the effects of Gene expression profiling of S100b-treated endothelial model AGEs or recombinant S100b on EC gene expression cells confirmed S100b as the mediator of inflammation as using microarray technology. AGE treatment of HMEC-4 previously reported (7) and, therefore, also confirmed the cells did not induce an inflammatory mRNA phenotype as validity of our in vitro model system. In our studies, S100b predicted by the literature (see below). However, treat- triggered an immune response defined mainly by expres- ment with S100b induced an mRNA phenotype of activated sion of chemokines, adhesion molecules, and genes in- endothelium (Tables 3 and 4, Fig. 1). In addition, treatment volved in antigen presentation, which included MHC class of ECs with known inflammatory triggers TNF-␣ or LPS I and II alleles and several proteasome subunits (summa- induced a strong inflammatory immune response defined rized in Fig. 1). In addition, increased expression of those by an increase in the expression of genes, including cyto- gene classes is dependent on activation of the NF-␬B kines, cytokine receptors, chemokines, MMP-1, and vari- pathway (29), confirming previous reports that NF-␬Bisa ous cell adhesion molecules (Table 5, Fig. 1). These data central transcription factor in the cellular response to suggest that RAGE binding AGEs may not be general S100b (7). Taken together, the changes in gene expression drivers of inflammation. In contrast, this work confirmed observed after S100b treatment described an activated S100b as mediator of inflammation either by activating endothelium. RAGE or through other pathways yet to be elucidated. Although S100b treatment induced some similar gene Future work will be required to determine whether the expression changes compared with TNF-␣ or LPS, the various reported RAGE ligands activate different cellular overall pattern of gene expression varies greatly (compare responses. Tables 3 and 5). Thus, the effects on gene expression Numerous studies have been published showing cells observed when HMEC-4 cells were treated with S100b are exposed to AGEs resulted in significant alterations in the unlikely due to contaminating LPS. In addition, the gene expression of many genes, including IL-1␤ (24), TNF-␣ expression changes observed after treatment of HMEC-4 (12), IL-6 (13), platelet-derived growth factor (25), insulin- cells with TNF-␣ or LPS further validated our in vitro like growth factor-1 (IGF-1) (26), thrombomodulin, model system, showing that the HMEC-4 cells are respon- VCAM-1 (11), and TF (14,27,28). Of these genes, all were sive to proinflammatory triggers. present on the chip; however, the majority were consid- Exposure of healthy animals to high doses of model ered below detection level. The only exception was plate- AGEs triggered a modest immune response in liver tissue let-derived growth factor, which displayed a low level of defined mainly as a macrophage-based clearance/detoxifi- expression that did not differ between Ctrl BSA and Rib cation response. Overall, mice injected with model AGEs BSA treatments. Treating cells with AGE-BSAs for multi- failed to display gene expression changes indicative of a ple time periods may provide a more complete picture. For strong induction of the NF-␬B pathway. At least six of the

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TABLE 5 Upregulated genes in HMEC-4 cells after treatment with LPS or TNF-␣ Gene name AN ϩLPS ϩTNF-␣ Potential 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 NM_002982 28.5 24.1 Monocyte/basophil chemotactant (MCP-1) 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-␣–induced protein 6 NM_007115 7.8 17.3 Implicated in leukocyte adhesion; related to CD44 Transcription factors/regulators NF-␬B p49/p100 subunit NM_002502 18.1 14.0 Regulates expression of a variety of proinflammatory genes NF-␬B p105 (precursor to p50) M58603 11.9 6.2 Regulates expression of a variety of proinflammatory subunit genes I␬B␣ NM_020529 5.4 4.8 Inhibitor of NF-␬B TNF-␣–induced protein 3 (A20) NM_006290 14.8 22.1 Inhibitor of NF-␬B Interferon regulatory factor 1 NM_002198 15.5 9.5 Transcription of IFN alpha and beta Enzymes GTP cyclohydrolase 1 (dopa- NM_000161 17.6 15.4 Synthesis of aromatic side chains in phe, tyr, trp responsive dystonia) Superoxide dismutase 2, mito- NM_000636 17.9 36.4 Conversion of superoxide radicals to molecular chondrial 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 of polymorphonuclear leuko- cytes and monocytes Proteins involved in apoptosis Caspase-like apoptosis regulatory AF005775 7.0 6.4 Positively regulates caspase 8 protein 2 (clarp) Phorbol-12-myristate-13-acetate-in- NM_021127 5.5 3.0 Promoter of apoptosis duced protein 1 (NOXA) Apoptosis inhibitor 1 (baculoviral U45878 16.5 28.7 Regulator of apoptosis, interacts with TRAF 1&2 IAP repeat-containing 2) Proteins involved in cell growth Jun B; proto-oncogene M29039 10.3 6.7 Promoter of cell growth MAD; mothers against decapentaple- NM_005902 8.6 5.3 Imparts growth inhibitory effects of TGF-␤ gic homolog 3 Proteins with unknown function Interferon, alpha-inducible protein NM_005101 17.7 6.5 Role unknown (clone IFI-15K) 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 tetra- M24594 38.5 5.0 Implicated in translation; interacts with initiation tricopeptide repeats 1 (IFI-56K) factor eIF-3 Interferon-induced protein with tetra- NM_001547 35.6 4.7 Role unknown tricopeptide repeats 2 (IFI-54K) TNF-␣–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 Ͻ 0.05 (Student’s t test; LPS vs. Ctrl BSA or TNF-␣ vs. Ctrl BSA; unpaired assume unequal variance in both populations). AN is the nucleotide accession number for each gene. nine genes that increased in expression after a single chemotactic protein-1, M and P lysozymes, MARCO, CD5L, intravenous administration of model AGEs are associated and thymosin) (30) (Table 6, Fig. 1). These results were with macrophage activation and differentiation (monocyte confirmed by a second experiment measuring gene expres-

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TABLE 6 Genes upregulated by a single intravenous administration of Rib BSA in mouse liver Gene name AN ϩRibBSA ϩLPS Potential 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 ␤ 4 U38967 3.1* 1.0 Migration of and other cell types Procollagen type IV M15832 2.4* 4.0* Extracellular matrix molecule *P Ͻ 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. sion changes in livers from mice treated for 4 days with immunohistological staining. We did not see an increase in high doses of model AGEs. These mice also showed an liver VCAM-1 mRNA after a single injection of 10 mg/ increase in a large number of the genes associated with mouse (a 20 times larger dose). macrophage activation or differentiation, including ly- Our results do not show that AGEs trigger a strong sozyme P and M, MARCO, CD5L, TYRO, CD68, properdin inflammatory response. Previously, animals injected with factor, and complement C1qB (30). This suggests that the a single dose of exogenous AGEs have been reported to majority of the cellular responses that followed exposure increase the expression of a variety of inflammatory me- to exogenous AGEs were derived from the macrophage- diators, including liver IL-6 and heme oxygenase (10,13). derived Kupffer cells. The upregulation of lysozyme is However, our experiments showed no evidence of an interesting, because lysozyme has been reported to bind increase in IL-6 expression. Furthermore, if IL-6 expres- AGEs and improve renal excretion of AGEs (31). In sion was induced in our study, then a significant increase addition, a 2.8-fold increase in VCAM-1 was observed in in the expression of acute-phase proteins such as C-reac- liver tissue from mice treated for 4 days with model AGEs tive protein and fibrinogen would have been observed. (total amount of intraperitoneally injected AGE: 40 mg/ Although we feel our data accurately reflect the effects of mouse). In these same mice, a two- to fourfold increase in AGEs in vivo, there are several differences between this soluble VCAM-1 was measured by enzyme-linked immu- study and previously published studies, including 1) strain nosorbent assay (data not shown). Stern and Schmidt (11) of mouse (SJL vs. C57BL/6), 2) dose of model AGE (0.5 vs. reported that healthy mice injected with a single bolus 0.50 10 or 40 mg/mouse), and 3) likely the composition of the mg/mouse of model AGE showed a two- to threefold AGE preparations (4) time point (6 vs. 24 h or 5 days). In increase in VCAM-1 expression in the lung according to addition, a longer-term study using AGE-modified mouse

TABLE 7 Genes upregulated in mouse liver after 4 injections of Rib BSA Gene name AN ϩ Rib BSA Potential 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 Ͻ 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.

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FIG. 1. Summary of gene expression changes induced in cultured endothelial cells treated with AGE-BSA, S100b, TNF-␣, or LPS. serum albumin might result in induction of inflammatory Although our work suggests that AGEs do not trigger a mediators. Although many of the known AGE structures significant inflammatory immune response, accumulation that have been shown to form under in vitro conditions of AGEs on macromolecules is known to adversely affect have also been found in vivo (32–35), model AGEs may not both the functional properties and clearance of these mol- accurately reflect the chemical composition of AGEs ecules. The resulting biomechanical changes to these mol- formed in vivo. ecules have been shown to contribute to the pathology of The present study is one of the first to look at AGE- several disease states, including atherosclerosis and dia- induced effects on gene expression using this oligonucle- betic complications (36–38). Thus, the biomechanical effects otide array technology. Ideally all of the genes observed to of AGEs may prove to be more detrimental in vivo than the change should be confirmed by additional techniques, proposed cell-surface receptor-mediated pathways. such as Northern blot or RT-PCR. In the HMEC-4 cell system, we have confirmed, using a cell-based enzyme- ACKNOWLEDGMENTS linked immunosorbent assay, that the VCAM-1 gene ex- We thank Marlene Dressman for her contributions in pression changes elicited by TNF-␣, LPS, and S100b analyzing the gene expression changes observed in mice described herein reflect a change in protein levels as well following a single administration of model AGEs. We (22). In the animal studies, the increase in mRNA expres- thank Shari Caplan for generously providing probes for sion of P lysozyme in mice injected with model AGEs was Northern blot analysis. We would also like to thank Arco confirmed by Northern blot analysis (data not shown). Jeng’s lab for their expertise in Northern blot analysis. Exposure of healthy animals to high doses of model Helpful discussions from John Rediske are kindly ac- AGEs triggered a modest immune response in the liver knowledged. tissue defined mainly as a macrophage-based clearance/ detoxification response. The significance of these changes is unclear. Future work will be required to decipher REFERENCES whether these effects reflect specific AGE-mediated cellu- 1. Ulrich P, Cerami A: Protein glycation, diabetes, and aging. Recent Prog lar responses or these effects may actually be an artifact Horm Res 56:1–21, 2001 2. Makita Z, Bucala R, Rayfield EJ, Friedman EA, Kaufman AM, Korbet SM, resulting from injection of high concentrations of modified Barth RH, Winston JA, Fuh H, Manogue KR, Vlassara H: Reactive glyco- proteins. For example, although Ctrl BSA is used for sylation endproducts in diabetic uraemia and treatment of renal failure. comparison, the ribose-modified protein may be denatured Lancet 343:1519–1522, 1994 and elicit nonspecific effects. Unlike previous reports, the 3. Bierhaus A, Hofmann MA, Ziegler R, Nawroth PP: AGEs and their observed immune response in AGE-treated mice did not interaction with AGE-receptors in vascular disease and diabetes mellitus. I. The AGE concept. Cardiovasc Res 37:586–600, 1998 entail expression of high levels of inflammatory mediators, 4. Vlassara H, Palace MR: Diabetes and advanced glycation endproducts. although very modest changes in the inflammatory medi- J Intern Med 251:87–101, 2002 ators VCAM-1 and monocyte chemotactic protein-1 were 5. Neeper M, Schmidt AM, Brett J, Yan SD, Wang F, Pan, YCE, Elliston K, noted in mice exposed to model AGEs. Overall, our data Stern D, Shaw A: Cloning and expression of a cell surface receptor for advanced glycosylation end products of proteins. J Biol Chem 267:14998– do not convincingly demonstrate that model AGEs are 15004, 1992 signaling molecules, despite previous work showing that 6. Brett J, Schmidt AM, Yan SD, Zou S, Weidman E, Pinsky D, Neeper M, the AGEs bind to RAGE with high affinity (21). Shaw A, Migheli A, Stern D: Survey of the distribution of a newly

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