Divergent Pathways of Gene Expression Are Activated by the RAGE Ligands S100b and AGE-BSA
←
→
Page content transcription
If your browser does not render page correctly, please read the page content below
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
A
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; IB␣, 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 743DIVERGENT mRNA PHENOTYPES INDUCED BY RAGE LIGANDS
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 2004J.V. VALENCIA AND ASSOCIATES
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 ⬍ 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
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 ⫺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 745DIVERGENT mRNA PHENOTYPES INDUCED BY RAGE LIGANDS
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 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
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, ␣ (IB␣)
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 2004J.V. VALENCIA AND ASSOCIATES
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 ⫺2.3 Binds toxic metals and scavenges free radicals
Similar to metallothionein 1E AL031602 ⫺2.6 See function of metallothionein 1E
Selenoprotein W, 1 (SEPW1) NM_003009 ⫺2.1 Antioxidant
Enzymes
RNA polymerase II (DNA directed) polypeptide A NM_000937 ⫺2.7 Largest subunit of RNA polymerase II (220 kD)
Short-chain dehydrogenase reductase 1 (SDR1) NM_004753 ⫺2.5 Regeneration of retinol (Vit. A) from retinal
Highly similar to transglutaminase 2 BC003551 ⫺2.2 99% identical to transglutaminase 2, a protein
cross-linking enzyme
Ubiquitin protein ligase E3A NM_000462 ⫺2.0 Protein ubiquitination
Mitochondrial proteins
Glutaminase C AF158555 ⫺2.4 Converts L-glutamine to L-glutamate
Cytoskeletal-associated protein
Caldesmon 1 (CALD1) NM_033157 ⫺2.0 Actomyosin regulatory protein
Other proteins
RNA binding protein BRUNOL3 U69546 ⫺2.0 Translation repression
Clone 24775 AF052169 ⫺2.1 Role unknown
Fold change for all listed genes statistically significant; P ⬍ 0.05 (Student’s t test; S100b-treated vs. media control– untreated; unpaired assume
unequal variance in both populations). AN is the nucleotide accession number for each gene.
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 747DIVERGENT mRNA PHENOTYPES INDUCED BY RAGE LIGANDS
TABLE 5
Upregulated genes in HMEC-4 cells after treatment with LPS or TNF-␣
Gene name AN ⫹LPS ⫹TNF-␣ 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
IB␣ 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 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
oncogene
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 2004J.V. VALENCIA AND ASSOCIATES TABLE 6 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 TABLE 7 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
DIVERGENT mRNA PHENOTYPES INDUCED BY RAGE LIGANDS
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
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
interaction with AGE-receptors in vascular disease and diabetes mellitus.
observed immune response in AGE-treated mice did not 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
750 DIABETES, VOL. 53, MARCH 2004J.V. VALENCIA AND ASSOCIATES
characterized receptor for advanced glycation end products in tissues. Hughes TE: Advanced glycation endproduct ligands for the receptor for
Am J Pathol 143:1699 –1712, 1993 advanced glycation endproducts: biochemical characterization and forma-
7. Hofmann MA, Drury S, Fu C, Qu W, Taguchi A, Lu Y, Avila C, Bierhaus A, tion kinetics. Anal Biochem 324:68 –78, 2004
Neurath MF, Beach D, McClary J, Morser J, Stern D, Schmidt AM: RAGE 22. Valencia JV, Hughes TE: AGEs do not induce cell surface VCAM-1 on
mediates a novel proinflammatory axis: a central cell surface receptor for endothelial cells (Abstract). Diabetes 51 (Suppl. 2):A502, 2002
S100/calgranulin polypeptides. Cell 97:889 –901, 1999 23. Ades EW, Candal FJ, Swerlick RA, George VG, Summers S, Bosse DC,
8. Bucciarelli LG, Wendt T, Rong L, Lalla E, Hofmann MA, Goova MT, Lawley TJ: HMEC-1: establishment of an immortalized human microvas-
Taguchi A, Yan SF, Yan SD, Stern DM, Schmidt AM: RAGE is a multiligand cular endothelial cell line. J Invest Dermatol 99:683– 690, 1992
receptor of the immunoglobulin superfamily: implications for homeostasis 24. Vlassara H, Brownlee M, Manogue KR, Dinarello CA, Pasagian A: Cachec-
and chronic disease. Cell Mol Life Sci 59:1117–1128, 2002 tin/TNF and IL-1 induced by glucose-modified proteins: role in normal
9. Donato R: S100: a multigenic family of calcium-modulated proteins of the tissue remodeling. Science 240:1546 –1548, 1988
EF-hand type with intracellular and extracellular functional roles. Int 25. Kirstein M, Brett J, Radoff S, Ogawa S, Stern D, Vlassara H: Advanced
J Biochem Cell Biol 33:637– 668, 2001 protein glycosylation induces transendothelial human monocyte chemo-
10. Yan SD, Schmidt AM, Anderson GM, Zhang J, Brett J, Zou YS, Pinsky D, taxis and secretion of platelet-derived growth factor: role in vascular
Stern D: Enhanced cellular oxidant stress by the interaction of advanced disease of diabetes and aging. Proc Natl Acad Sci U S A 87:9010 –9018, 1990
glycation end products with their receptors/binding proteins. J Biol Chem 26. Kirstein M, Aston C, Hintz R, Vlassara H: Receptor-specific induction of
269:9889 –9897, 1994
insulin-like growth factor I in human monocytes by advanced glycosylation
11. Schmidt AM, Hori O, Chen JX, Li JF, Crandall J, Zhang J, Cao R, Yan SD,
end product-modified proteins. J Clin Invest 90:439 – 446, 1992
Brett J, Stern D: Advanced glycation endproducts interacting with their
27. Esposito C, Gerlach H, Brett J, Stern D, Vlassara H: Endothelial receptor-
endothelial receptor induce expression of vascular cell adhesion mole-
mediated binding of glucose-modified albumin is associated with increased
cule-1 (VCAM-1) in cultured human endothelial cells and in mice: a
monolayer permeability and modulation of cell surface coagulant proper-
potential mechanism for the accelerated vasculopathy of diabetes. J Clin
ties. J Exp Med 170:1387–1407, 1989
Invest 96:1395–1403, 1995
12. Miyata T, Hori O, Zhang J, Yan SD, Ferran L, Iida Y, Schmidt AM: The 28. Yamagishi S, Fujimori H, Yonekura H, Yamamoto Y, Yamamoto H: Ad-
receptor for advanced glycation end products (RAGE) is a central media- vanced glycation endproducts inhibit prostacyclin production and induce
tor of the interaction of AGE-beta2microglobulin with human mononu- plasminogen activator inhibitor-1 in human microvascular endothelial
clear phagocytes via an oxidant-sensitive pathway: implications for the cells. Diabetologia 41:1435–1441, 1998
pathogenesis of dialysis-related amyloidosis. J Clin Invest 98:1088 –1094, 29. Pahl HL: Activators and target genes of Rel/NF-kB transcription factors.
1996 Oncogene 18:6853– 6866, 1999
13. Schmidt AM, Hasu M, Popov D, Zhang JH, Chen J, Yan SD, Brett J, Cao R, 30. Greaves DR, Gordon S: Macrophage-specific gene expression: current
Costache G, Stern D: Receptor for advanced glycation end products paradigms and future challenges. Int J Hematol 76:6 –15, 2002
(AGEs) has a central role in vessel wall interactions and gene activation in 31. Zheng F, Cai W, Mitsuhashi T, Vlassara H: Lysozyme enhances renal
response to circulating AGE proteins. Proc Natl Acad Sci U S A 91:8807– excretion of advanced glycation endproducts in vivo and suppresses
8811, 1994 adverse age-mediated cellular effects in vitro: a potential AGE sequestra-
14. Bierhaus A, Illmer T, Kasper M, Luther T, Quehenberger P, Nawroth PP: tion therapy for diabetic nephropathy? Mol Med 7:737–747, 2001
Advanced glycation end product (AGE)-mediated induction of tissue 32. Makita Z, Vlassara H, Cerami A, Bucala R: Immunochemical detection of
factor in cultured endothelial cells is dependent on RAGE. Circulation advanced glycosylation end products in vivo. J Biol Chem 267:5133–5138,
96:2262–2271, 1997 1992
15. Striker LJ, Striker GE: Administration of AGEs in vivo induces extracellu- 33. Reddy S, Bichler J, Wells-Knecht KJ, Thorpe SR, Baynes JWN: Epsilon-
lar matrix gene expression. Nephrol Dial Transplant 11 (Suppl. 5):62– 65, (carboxymethyl)lysine is a dominant advanced glycation end product
1996 (AGE) antigen in tissue proteins. Biochemistry 34:10872–10878, 1995
16. Yang CW, Vlassara H, Striker GE, Striker LJ: Administration of AGEs in 34. Shamsi FA, Partal A, Sady C, Glomb MA, Nagaraj RH: Immunological
vivo induces genes implicated in diabetic glomerulosclerosis. Kidney Int evidence for methylglyoxal-derived modifications in vivo: determination of
47 (Suppl. 49):S55–S58, 1995 antigenic epitopes. J Biol Chem 273:6928 – 6936, 1998
17. Yang CW, Vlassara H, Peten EP, He CJ, Striker GE, Striker LJ: Advanced 35. Al-Abed Y, Bucala R: Structure of a synthetic glucose derived advanced
glycation end products up-regulate gene expression found in diabetic glycation end product that is immunologically cross-reactive with its
glomerular disease. Proc Natl Acad Sci U S A 91:9436 –9440, 1994 naturally occurring counterparts. Bioconjug Chem 11:39 – 45, 2000
18. Vlassara H, Fuh H, Makita Z, Krungkrai S, Cerami A, Bucala R: Exogenous 36. Sims TJ, Rasmussen LM, Oxlund H, Bailey AJ: The role of glycation
advanced glycosylation end products induce complex vascular dysfunc- cross-links in diabetic vascular stiffening. Diabetologia 39:946 –51, 1996
tion in normal animals: a model for diabetic and aging complications. Proc 37. Bucala R, Makita Z, Vega G, Grundy S, Koschinsky T, Cerami A, Vlassara
Natl Acad Sci U S A 89:12043–12047, 1992 H: Modification of low density lipoprotein by advanced glycation end
19. Vlassara H, Fuh H, Donnelly T, Cybulsky M: Advanced glycation endprod- products contributes to the dyslipidemia of diabetes and renal insuffi-
ucts promote adhesion molecule (VCAM-1, ICAM-1) expression and ath- ciency. Proc Natl Acad Sci U S A 91:9441–9445, 1994
eroma formation in normal rabbits. Mol Med 1:447– 456, 1995 38. Duraisamy Y, Slevin M, Smith N, Bailey J, Zweit J, Smith C, Ahmed N,
20. Crauwels HM, Herman AG, Bult H: Local application of advanced glycation Gaffney J: Effect of glycation on basic fibroblast growth factor induced
end products and intimal hyperplasia in the rabbit collared carotid artery. angiogenesis and activation of associated signal transduction pathways in
Cardiovasc Res 47:173–182, 2000 vascular endothelial cells: possible relevance to wound healing in diabetes.
21. Valencia JV, Weldon SC, Quinn D, Kiers GH, DeGroot J, TeKoppele JM, Angiogenesis 4:277–288, 2001
DIABETES, VOL. 53, MARCH 2004 751You can also read
