Divergent Pathways of Gene Expression Are Activated by the RAGE Ligands S100b and AGE-BSA

Divergent Pathways of Gene 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 cell adhesion molecule-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 gene expression
profiling using both in vitro and in vivo model systems.                               The most characterized AGE binding protein 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 genes. The                                  to bind to several proteins 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 macrophage-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
                                                                                    model AGEs for 4 weeks resulted in an increase in the

           dvanced glycation end products (AGEs) are a
           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
groups on macromolecules (rev. in 1). AGEs are highly                               permeability and defective vasodilatory responses in rats
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: thomase.hughes@pharma.novartis.com.              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           lung 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 (ⱕ0.2 ng/mg protein), were incapable of
DIABETES, VOL. 53, MARCH 2004                                                                                                                 743

inducing VCAM-1 or TNF-␣ secretion regardless of RAGE                             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
binding affinity (22). Therefore, our previous findings                           acclimate for at least 1 week before use. The mice were 6 –10 weeks of age
suggested that RAGE binding affinity does not correlate                           (⬃25 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 (⬃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
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
                                                                                  Biomedical Research through institutional policy complies with or exceeds all
AGEs or S100b were studied. As positive controls, ECs                             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
                                                                                  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 䡠                         cally at 260 nm. Quality of the total RNA (300 ng/lane) was determined by
mouse⫺1 䡠 day⫺1). 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.
                                                                                  Microarray analysis. Purified total RNA was used to synthesize double-
study the effects of AGEs in vivo, because of its well-                           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(⫺) 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 (10⫻) 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, antibiotic/antimicotic, 0.05% trypsin/0.53 mmol/l   change twofold or greater with (Student’s t test P ⬍ 0.05) and mean AvgDiff
EDTA, penicillin and streptomycin, TRIzol reagent, and Superscript II Choice      values ⱕ200. 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 ⬍ 0.05
were found to be ⬍0.2 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-
inactivated FBS, 2 mmol/l L-glutamine, 10 ng/ml epidermal growth factor, 1
                                                                                  HMEC-4 cells were treated for 18 h at 37°C with 0.5 mg/ml
␮g/ml hydrocortisone [HC], and 1% antibiotic-antimicotic in 5% CO2 at 37°C).      Rib BSA or Ctrl BSA. Total RNA was isolated from the
The cells were grown to confluence in T-175 flasks (5 ⫻ 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 ⬃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
                                                                                  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
744                                                                                                                          DIABETES, VOL. 53, MARCH 2004

Upregulated genes in HMEC-4 cells after treatment with Rib BSA
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 ⬍ 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 䡠 mouse⫺1 䡠 day⫺1). 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 (ⱕ0.2 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) for 4 h at 37°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 cytokines/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, monocyte 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
Downregulated genes in HMEC-4 cells after treatment with Rib BSA
Gene name                                         AN                      change                              Potential 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.

DIABETES, VOL. 53, MARCH 2004                                                                                                            745

Upregulated genes in HMEC-4 cells after treatment with S100b
Gene name                                                            AN         change                      Potential function
  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 Aⴱ2711                                           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-Cwⴱ1701)                          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, ␣ 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
  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
  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 ⬍ 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.

746                                                                                                               DIABETES, VOL. 53, MARCH 2004

Downregulated genes in HMEC-4 cells after treatment with S100b
Gene name                                                           AN           change                       Potential 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
  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.

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-␬B is a
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
DIABETES, VOL. 53, MARCH 2004                                                                                                                  747

Upregulated genes in HMEC-4 cells after treatment with LPS or TNF-␣
Gene name                                          AN         ⫹LPS      ⫹TNF-␣                          Potential function
  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
  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
  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
  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 chemotaxis 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
Fold change for all listed genes statistically significant; P ⬍ 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-
748                                                                                                            DIABETES, VOL. 53, MARCH 2004

Genes upregulated by a single intravenous administration of Rib BSA in mouse liver
Gene name                                          AN          ⫹RibBSA          ⫹LPS                     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 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.

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

Genes upregulated in mouse liver after 4 injections of Rib BSA
Gene name                                               AN        ⫹ 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 ⬍ 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.

DIABETES, VOL. 53, MARCH 2004                                                                                                            749

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-
pression changes elicited by TNF-␣, LPS, and S100b                    We thank Marlene Dressman for her contributions in
described herein reflect a change in protein levels as well           analyzing the gene expression changes observed in mice
                                                                      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
tissue defined mainly as a macrophage-based clearance/
detoxification response. The significance of these changes
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DIABETES, VOL. 53, MARCH 2004                                                                                                                                  751
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