THE JOURNAL OF BIOLOGICAL CHEMISTRY
1208712095, 2005
2005 by The American Society for Biochemistry and Molecular Biology, Inc.
Advanced Glycation End Products Enhance Expression of
Pro-apoptotic Genes and Stimulate Fibroblast Apoptosis through
Cytoplasmic and Mitochondrial Pathways*
Received for publication, June 7, 2004, and in revised form, November 29, 2004
Published, JBC Papers in Press, December 6, 2004, DOI 10.1074/jbc.M406313200
Zoubin Alikhani, Mani Alikhani, Coy M. Boyd, Kiyoko Nagao, Philip C. Trackman,
and Dana T. Graves
From the Department of Periodontology and Oral Biology, Boston University School of Dental Medicine,
Boston, Massachusetts 02118
Both aging and diabetes are characterized by the for-
mation of advanced glycation end products (AGEs).
Both exhibit other similarities including deficits in
wound healing that are associated with higher rates of
fibroblast apoptosis. In order to investigate a potential
mechanism for enhanced fibroblast apoptosis in diabe-
tes and aged individuals, experiments were carried out
to determine whether the predominant advanced glyca-
tion end product in skin, N--(carboxymethyl) lysine
(CML)-collagen, could induce fibroblast apoptosis. In
vivo experiments established that CML-collagen but not
unmodified collagen induced fibroblast apoptosis and
that apoptosis was dependent upon caspase-3, -8, and -9
activity. In vitro experiments demonstrated that CML-
collagen but not control collagen induced a time- and
dose-dependent increase in fibroblast apoptosis. By use
of blocking antibodies, apoptosis was shown to be medi-
ated through receptor for AGE signaling. AGE-induced
apoptosis was largely dependent on the effector
caspase, caspase-3, which was activated through both
cytoplasmic (caspase-8-dependent) and mitochondrial
(caspase-9) pathways. CML-collagen had a global effect
of enhancing mRNA levels of pro-apoptotic genes that
included several classes of molecules including ligands,
receptors, adaptor molecules, mitochondrial proteins,
and others. However, the pattern of expression was not
identical to the pattern of apoptotic genes induced by
tumor necrosis factor .
Advanced glycation end products (AGEs)1 result from non-
enzymatic reactions of carbohydrates and oxidized lipids with
proteins (1, 2). Lysine side chains condense with aldehyde or
ketone structures to form a reversible Schiffs base. This struc-
* This work was supported by National Institutes of Health Grants
DE07559, DE11254, AR49920, and DE14066. The costs of publication of
this article were defrayed in part by the payment of page charges. This
article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Periodon-
tology and Oral Biology, Boston University School of Dental Medicine,
700 Albany St., Boston, MA 02118. Tel.: 617-638-8547; Fax: 617-638-
4924; E-mail: dgraves@bu.edu.
1
The abbreviations used are: AGE, advanced glycation end product;
RAGE, receptor for advanced glycation end product; CML, N--(car-
boxymethyl) lysine; TNF, tumor necrosis factor; PBS, phosphate-buff-
ered saline; TUNEL, terminal deoxynucleotidyl transferase-mediated
nick end labeling; AFC, 7-amino-4-trifluoromethyl coumarin; ELISA,
enzyme-linked immunosorbent assay; EMSA, electrophoretic mobility
shift analysis; RT, reverse transcription; NF-B, nuclear factor B; Z,
benzyloxycarbonyl; fmk, fluoromethyl ketone; GAPDH, glyceraldehyde-
3-phosphate dehydrogenase.
This paper is available on line at http://www.jbc.org
Vol. 280, No. 13, Issue of April 1, pp.
Printed in U.S.A.
ture isomerizes to a more reactive ketoamine (Amadori prod-
uct), which then may undergo additional rearrangement, de-
hydration, condensation, and oxidation reactions. These
structures can result in protein cross-links and in a variety of
protein modifications that collectively are known as advanced
glycation end products (3). N--(Carboxymethyl) lysine (CML)
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structure is a prevalent AGE and is generated principally by
oxidative cleavage of Amadori intermediates (3).
AGEs form in tissues and accumulate during aging. AGE
formation occurs throughout life, and AGEs are found at sig-
nificantly higher levels in aged individuals (4, 5). Their forma-
tion is considered to be one of the major etiologic factors in the
pathophysiology of aging (6 8). AGEs have also been studied
in relation to development of many age-related chronic diseases
including cataract formation, neurodegenerative disorders
such as Alzheimer disease, osteoarthritis and changes observed
in myocardial dysfunction (9 14).
AGE formation is enhanced by hyperglycemia and has been
linked to many of the complications of diabetes. By functionally
blocking AGE activity or by applying AGEs in vivo, it has been
shown that AGEs contribute to several pathologic processes.
These include diabetes-associated cataract formation, nephrop-
athy, retinopathy, neuropathy, and periodontal disease (15
20). AGEs also impair osseous wound healing (21).
Originally it was thought that most of the effects of AGEs
were the result of cross-linking of matrix and other proteins (3).
Although cross-linking is physiologically important, glycation
can alter proteins so that they acquire signaling properties,
which they do not otherwise possess. Several AGE cell surface
receptors/binding proteins have been identified. The most thor-
oughly studied is receptor for advanced glycation end products
(RAGE). RAGE is a multi-ligand member of the immunoglob-
ulin superfamily of cell surface receptors that binds many
ligands including those of the S100/calgranulin family (22).
Other receptors/binding proteins include macrophage scav-
enger receptor types I and II, oligosaccharyl transferase-48
(AGE-R1), 80K-H phosphoprotein (AGE-R2), and galectin-3
(AGE-R3). These are expressed on a wide range of cells includ-
ing smooth muscle cells, monocytes, macrophages, endothelial
cells, podocytes, astrocytes, microglia, and fibroblasts. Most of
the biologic activities associated with AGEs have been shown to
be transduced by RAGE, whereas the scavenger receptors are
thought to regulate removal of AGEs (23, 24). AGEs can mod-
ulate inflammatory events by stimulating production of reac-
tive oxygen species, chemotaxis, activation of monocytes/
macrophages, and stimulation of interleukin-1 and TNF
production (2530).
It is well known that AGEs form in the skin as part of the
aging process and as a result of diabetes (3133). Accumulation
12087
12088 AGEs Stimulate Fibroblast Apoptosis

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FIG. 1. AGE stimulates fibroblast apoptosis in vivo. A, histologic sections at the site of injection in the mouse scalp. Top panel, TUNEL
staining of specimens 24 h after injection with unmodified collagen; bottom panel, TUNEL staining 24 h after injection of CML-collagen. Large
arrow points to apoptotic cell, whereas small arrows point toward normal cells counterstained with nuclear fast red. B, quantitative analysis of
fibroblast apoptosis after CML-collagen injection. The number of fibroblastic TUNEL-positive apoptotic cells was counted in specimens 24 h after
injection of CML-collagen, unmodified collagen, or the control, zero time point. Each value represents the mean of six specimens S.E. The
experiment was performed twice with similar results.

of AGEs in skin has been related to tissue stiffening and lack of
elasticity during aging (5, 31, 32). However, AGEs in connec-
tive tissue can have detrimental effects through cell signaling.
For example, RAGE activation in dermal fibroblasts reduces
collagen synthesis and matrix production (34). AGEs also con-
tribute to impaired diabetic wound healing, in part through
interfering with formation of an extracellular matrix (35).
One mechanism through which AGEs may affect pathologic
processes is by enhanced apoptosis as supported by in vitro
studies. AGEs are pro-apoptotic for cultured retinal pericytes,
corneal endothelial cells, neuronal cells, and renal mesangial
cells (36 38). The mechanisms by which AGEs lead to apo-
ptosis are not well understood. There are reports suggesting
that AGEs may enhance apoptosis indirectly through increas-
ing oxidative stress or via induced expression of pro-apoptotic
cytokines (36, 38, 39). Interestingly, enhanced apoptosis of
these cells is also associated with diabetic complications such
as retinopathy, neuropathy, nephropathy, and accelerated vas-
culopathy (38 40). Enhanced apoptosis has been linked to
many of the detrimental effects of aging, which is also associ-
ated with AGE accumulation (41). Because fibroblasts play
important roles in the maintenance and healing of dermal
connective tissue, the accumulation of AGEs in skin may have
a detrimental effect, in part, through promoting fibroblast apo-
ptosis. To address this issue, we investigated the apoptotic
effect of AGEs on fibroblasts and the mechanisms through
which AGEs induce apoptosis in these cells.
MATERIALS AND METHODS
CML-collagenCML-collagen was prepared by chemical modifica-
tion of acid-soluble bovine skin collagen (Sigma), as described previ-
ously (21, 42). Briefly, 50 mg of collagen was dissolved in 25 ml of 1 mM
HCl freshly made in sterile water and incubated at 37 C with occa-
sional mixing. Sterile PBS (25 ml) was added, followed by sodium
cyanoborohydride (1.42 g) and glyoxylic acid (0.715 g). Control collagen
was prepared at the same time, except that no glyoxylic acid was added.
All samples were then incubated at 37 C for 24 h. AGE collagen and
control collagen were then exhaustively dialyzed against distilled wa-
ter. In dose-response experiments where higher concentrations of CML-
collagen were used, AGE and control collagen was dialyzed against
PBS. Both CML-collagen and control collagen were soluble at the con-
centrations stored and tested. In total, 3 8% of lysine residues in
CML-collagen were converted to CML, as determined by the trinitro-
benzenesulfonic acid assay (43). The percentage of modification of col-
lagen that we have generated is 10-fold less than the amount used in a
recent report to assess CML binding and activation of NF-B (42) and
only a small amount higher than that reported for the skin of aged or
diabetic individuals (31). CML-collagen was highly reactive on Western
blots with anti-CML monoclonal antibody 6D12 (Wako, Richmond, VA),
whereas control collagen was not reactive. The amount of endotoxin
contamination was measured by Pyrochrome Limulus Amebocyte Ly-
sate assay (Associates of Cape Cod, Inc., Woods Hole, MA) and found to
be low (0.094 0.01 ng/mg lipopolysaccharide in control collagen and
0.087 0.01 ng/mg lipopolysaccharide in CML-collagen).
AnimalsCD1 mice were purchased from Charles River Laborato-
ries, (Waltham, MA). These are outbred mice that were selected be-
cause they do not exhibit strain-associated responses, as has been
reported in some cases where a particular strain of mouse has been
studied (45, 46). All procedures involving mice were approved by the
Boston University Medical Center Institutional Animal Care and Use
Committee. Mice were anesthetized with injection of ketamine (80
mg/kg) and xylazine (10 mg/kg) in sterile PBS. CML-collagen or unmod-
ified collagen was injected into the loose connective tissue adjacent to
calvarial bone at a point on the midline of the skull located between the
ears. Injection at this anatomic site can be reproducibly achieved. For
each data point, there were six mice (n 6). We undertook preliminary
experiments to identify a dose for CML-collagen that gave a moderate
number of apoptotic cells. On this basis, 100 g of CML-collagen or an
equal amount of unmodified collagen was injected. Mice were eutha-
nized 24 h after injection. In addition to CML-collagen or unmodified
collagen alone, some animals were treated by intraperitoneal injection
of caspase-3, -8, or -9 inhibitor (1 mg/kg) 1 h before CML-collagen
injection and locally (25 g) at the time of CML-collagen injection. The
caspase-3 inhibitor Z-DEVD-fmk, the caspase-8 inhibitor Z-IETD-fmk,
and the caspase-9 inhibitor Z-LEHD-fmk were purchased from R&D
Systems (Minneapolis, MN). Control mice received CML-collagen or
 AGEs Stimulate Fibroblast Apoptosis
FIG. 2. AGE stimulates fibroblast apoptosis in vitro. A, primary
human adult dermal fibroblasts were incubated with CML-collagen or
unmodified collagen (200 g/ml) for 1, 3, 6, and 24 h. Apoptosis was
determined by ELISA. The experiment was performed twice with sim-
ilar results. B, primary human adult dermal fibroblasts were incubated
with CML-collagen or unmodified collagen (0 800 g/ml) for 24 h, and
apoptosis was measured by ELISA. Each value represents the mean of
five replicates S.E. The experiment was performed three times with
similar results.
unmodified collagen containing 2% Me2SO (Sigma-Aldrich).
Preparation of Histologic SectionsAnimals were euthanized by de-
capitation, and their heads were fixed for 72 h in cold 4% paraformal-
dehyde and decalcified by incubation with cold Immunocal (Decal Corp.,
Congers, NY) for 12 days with solution changed daily. Paraffin-em-
bedded sagittal sections were prepared at a thickness of 5 6 m.
TUNEL Assay and Quantitative Histologic AnalysisApoptotic cells
were detected by an in situ TUNEL assay by means of a TACS 2
TdT-Blue Label kit purchased from Trevigen (Gaithersburg, MD), fol-
lowing the manufacturers instructions. Sections were counterstained
with nuclear fast red. The number of fibroblastic apoptotic cells was
determined in six specimens per group by counting the number of
TUNEL-positive cells that had the characteristic microscopic appear-
ance of fibroblasts. Counts and measurements were confirmed by re-
analysis of all the specimens by an independent examiner. The intra-
and inter-examiner variations were 10%. Students t test was used to
determine significant differences between the experimental and control
groups at the p 0.05 level.
Caspase ActivityCaspase-3, -8, and -9 activities from in vitro and in
vivo experiments were assayed with fluorometric kits purchased from
R&D Systems. Briefly, after sacrifice at the indicated time points,
murine scalps were immediately dissected from the calvaria and frozen
in liquid nitrogen. Frozen tissues were pulverized, and lysates were
prepared using cell lysis buffer provided by R&D Systems. After cen-
trifugation, total protein was quantitated using a BCA protein assay kit
(Pierce). Caspase-3 activity was detected by using the specific caspase-3
fluorogenic substrate, DEVD peptide conjugated to 7-amino-4-triflu-
oromethyl coumarin (AFC). Caspase-8 activity was detected by using
the specific caspase-8 fluorogenic substrate, IETD-AFC. Caspase-9 ac-
tivity was detected by using the specific caspase-9 fluorogenic sub-
strate, LEHD-AFC. Measurements were made on a fluorescent micro-
plate reader using filters for excitation (400 nm) and detection of
emitted light (505 nm). In some assays, recombinant caspase-3 enzyme
(R&D Systems) was used as a positive control. Buffers without cell
lysate and cell lysate without substrate were used as negative controls.
The same caspase-3, -8, and-9 inhibitors were used in both in vivo and
12089
FIG. 3. CML-collagen-induced apoptosis is mediated through
RAGE. Primary human adult dermal fibroblasts were incubated with
CML-collagen (200 g/ml) in the presence or absence of anti-RAGE
polyclonal antibody specific for the extracellular domain of RAGE (10
g/l) or non-immune serum (10 g/l) for 24 h. In some cases, cells
were incubated with antiserum or non-immune serum alone. The ex-
tent of apoptosis was determined by ELISA. Each value represents the
mean of five replicates S.E. The experiment was performed three
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times with similar results.
FIG. 4. Caspase activity is stimulated by CML-collagen in fi-
broblasts in vitro. Caspase-3,-8, and -9 activities were measured by
fluorometric assays in lysates from cultured primary human adult
dermal fibroblasts incubated with CML-collagen or unmodified collagen
(200 g/ml) for 24 h. Each value represents the mean of five replicates
S.E. The experiment was performed four times with similar results.
in vitro studies. In addition, the pan-caspase (general caspase) inhibitor
Z-VAD-fmk was purchased from R&D Systems for in vitro assays. For
each group, there were six specimens (n 6).
Cell CulturePrimary human adult dermal fibroblasts were pur-
chased from Cambrex (Walkersville, MD). Cells were propagated and
maintained in Dulbeccos modified Eagles medium (Cambrex) supple-
mented with 10% fetal bovine serum, gentamicin (100 g/ml), and
amphotericin B (100 ng/ml) at 37 C in a humidified atmosphere of 5%
CO2. Experiments with CML-collagen were performed in culture me-
dium supplemented with 0.5% fetal bovine serum. Assays were per-
formed when the cultures reached 75 85% confluence. In most exper-
iments, 200 g/ml CML-collagen or unmodified control collage was
used, which is equivalent to 2 M. Apoptosis of fibroblasts was deter-
mined by an ELISA technique measuring histone-associated DNA frag-
ments (Roche Applied Science), following the manufacturers instruc-
tions. In these studies, 20,000 fibroblasts/cm2 were treated with CML-
collagen (200 g/ml) or unmodified collagen (200 g/ml) for 24 h.
Apoptosis was determined by ELISA, and the cell numbers were as-
sessed in corresponding wells to normalize apoptosis measurements.
For caspase activity measurements, the same technique was used as
described above for in vivo studies. In some cases, cells were treated
with caspase-3, caspase-8, caspase-9, caspase-8 caspase-9, or pan-
caspase inhibitors (50 M) at the time of CML-collagen stimulation. The
dose of each inhibitor was selected based on our previous results (47).
12090 AGEs Stimulate Fibroblast Apoptosis

FIG. 5. Caspase inhibitors prevent

AGE-stimulated fibroblast apoptosis
and caspase-3 activity in vitro. Pri-
mary human adult dermal fibroblasts
were incubated with unmodified or CML-
collagen (200 g/ml) with or without
caspase inhibitors (50 M) for 24 h as de-
scribed under Materials and Methods.
A, the extent of apoptosis was determined
by ELISA and performed twice with sim-
ilar results. Each value represents the
mean of five replicates S.E. B,
caspase-3 activity was measured using a
fluorometric assay in lysates from cell cul-
tures obtained after 24 h of stimulation
with CML-collagen (200 g/ml) with or
without caspase-8 or -9 inhibitors as de-

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scribed under Materials and Methods.
The experiment was performed three
times with similar results. Each value
represents the mean of five replicates
S.E.

Control cells were incubated in assay medium supplemented with ve-
hicle alone (2% Me2SO) in the culture media.
Inhibition of RAGEAntibodies to the extracellular domain of
RAGE have been shown to inhibit binding of CML modified proteins to
RAGE and have been used to assess the impact of RAGE activation of
cellular events (42, 48). To study the role of RAGE, fibroblast cultures
were incubated with CML-collagen or unmodified collagen (200 g/ml)
in the presence or absence of anti-RAGE polyclonal antibodies specific
for the extracellular domain of RAGE (10 g/l) or non-immune serum
(10 g/l) for 24 h (Biocompare, San Francisco, CA). As controls, cells
were incubated with 10 g/ml RAGE antiserum or non-immune serum
without AGE stimulation. The extent of apoptosis was determined by
ELISA. Statistical difference between samples was determined by one-
way analysis of variance followed by Tukeys multiple comparison tests.
EMSAFibroblast cultures were incubated in assay medium for 1 h
with 200 g/ml CML-collagen or unmodified collagen in the presence or
absence of a specific NF-B inhibitor, SN50 (100 g/ml) (Biomol, Plym-
outh Meeting, PA). Nuclear proteins were extracted using protein ex-
traction kit (Pierce) following the manufacturers instructions. Concen-
trations of nuclear proteins were measured by using BCA protein assay
kit (Pierce). Interaction between NF-B in the protein extract and DNA
probe was investigated using EMSA kit from Panomics (Redwood City,
CA) following the manufacturers instruction.
MicroarrayFibroblast cell cultures were exposed to CML-collagen
(200 g/ml) or TNF- (20 ng/ml) for 6 h. TNF- was purchased from
R&D Systems. Control cells were exposed to either unmodified collagen
(200 g/ml) or vehicle alone (PBS). RNA was extracted using the
RNeasy Mini Kit (Qiagen, Valencia, CA). Human Apoptosis GEArray Q
series kit that includes 96 key genes involved in apoptosis was pur-
chased from SuperArray, Inc. (Bethesda, MD). Total RNA was used as
the template for 32P-labeled cDNA probe synthesis following the man-
ufacturers instructions. After overnight hybridization with labeled
probe, the GEArray membranes were exposed using a PhosphorImager.
The resulting digital image was converted to a raw data file using
Scanalyze software (www.microarray.org/software.html). GEArray An-
alyzer software (SuperArray, Inc.) was used for data analysis. The
microarray was performed twice with similar results, and the mean
values of the two experiments are presented for each gene. The microar-
ray for TNF- stimulation was performed simultaneously with the AGE
stimulation array.
RT-PCRFive g of total RNA was used to produce cDNA using a
First Strand cDNA Synthesis Kit (SuperArray, Inc.). PCR was per-
formed on the synthesized cDNA using the SingleGene PCR Kit pur-
chased from SuperArray, Inc. following the manufacturers protocol.
Specific primers were used for each gene, and GAPDH was used as
internal control. This kit has the advantage that GAPDH and the gene of
interest are amplified in the same tube and for the same number of cycles.
Based on a pilot study, 24 and 28 cycles were chosen to compare the gene
expression. After electrophoresis on a 2% agarose gel containing 0.5 g/ml
ethidium bromide, bands were visualized on a UV-box. The density of
each band was measured and compared using Quantity One software
(Bio-Rad). The optical density of each band was normalized by the value
of the GAPDH in the same lane. Two separate RT-PCRs were performed
with similar results. Students t test was used to determine significant
differences between the experimental and control groups.
RESULTS
In Vivo Induction of Apoptosis by CML-collagenTo study
the effect of AGEs on fibroblast apoptosis in vivo, 100 g of
CML-collagen or control collagen was injected in the scalp of
CD1 mice. Histologic sections were examined for fibroblast
apoptosis by their characteristic appearance in the TUNEL
assay (Fig. 1). Fibroblast apoptosis could be detected in the
CML-collagen-treated group. In comparison, there was virtu-
ally no induction of fibroblast apoptosis in mice treated with
unmodified collagen compared with the zero time point (Fig.
1A). Quantitative analysis indicated that CML-collagen stim-
ulated a 4-fold increase in apoptotic cells compared with un-
 AGEs Stimulate Fibroblast Apoptosis
modified collagen (Fig. 1B) (p 0.05). When expressed as the
percentage of TUNEL-positive fibroblasts, the levels were
0.85 0.05 in the CML-collagen-inoculated group and 0.21
0.01 (p 0.05) for control collagen-treated group.
In Vitro Induction of Apoptosis by CML-collagenIn vitro
studies were carried out to measure the direct effect of AGEs on
fibroblast apoptosis under well-defined conditions. Apoptosis of
human adult primary skin fibroblasts was measured by the
relative amount of histone-associated DNA fragments in the
cytoplasm as determined by ELISA. A time course experiment
determined that AGE-induced apoptosis was not detected be-
fore 6 h (Fig. 2A). At the 6 h time point, a 1.7-fold increase was
noted, whereas at 24 h, a 3-fold increase in apoptosis was
detected. The increase at 6 and 24 h was statistically signifi-
cant (p 0.05). CML-collagen induced a dose-dependent in-
crease in fibroblast apoptosis, whereas control collagen did not
(Fig. 2B). At 200 g/ml, CML-collagen compared with unmod-
ified collagen induced a 3-fold increase in apoptosis, which was
statistically significant (p 0.05). Saturating levels were
reached at 400 g/ml CML-collagen with a 5-fold increase in
apoptosis (p 0.05). In subsequent studies, a concentration of
200 g/ml CML-collagen or control collagen and a time point of
24 h were used unless stated otherwise.
Role of RAGE in Induction of Apoptosis by CML-collagen
To determine the significance of RAGE activation in the apop-
totic response to AGEs, human adult dermal fibroblasts were
incubated with CML-collagen (200 g/ml) in the presence or
absence of anti-RAGE polyclonal antibodies specific for the
extracellular domain of RAGE (10 g/ml) or non-immune se-
rum (10 g/ml) for 24 h (Fig. 3). CML-collagen stimulated a
significant increase in fibroblast apoptosis (p 0.05). However,
anti-RAGE antibody was able to completely block the apoptotic
effects of AGE so that there was no difference between fibro-
blasts incubated with control collagen and cells incubated with
CML-collagen plus RAGE antibody. Matched pre-immune se-
rum had no effect on CML-collagen-induced apoptosis, demon-
strating specificity of the antibody. As additional controls, cells
incubated with RAGE antibody or pre-immune serum in the
absence of CML-collagen had no direct effect on apoptosis. The
results obtained are consistent with functional studies in which
RAGE antibody inhibited activation of cells by CML-albumin
(data not shown).
In Vitro Effects of CML-collagen on Activation of Caspases-3,
-8, and -9 To further investigate mechanisms of AGE-induced
fibroblast apoptosis, the activation of initiator and executioner
caspases was measured in adult human fibroblasts after stim-
ulation by CML-collagen or unmodified collagen (Fig. 4). CML-
collagen stimulated a 5-fold increase in caspase-3, a 4.3-fold
increase in caspase-8, and a 3.2-fold increase in caspase-9
activity compared with cells treated with unmodified collagen
alone. Each was statistically significant (p 0.05).
Effects of Different Caspase Inhibitors on Apoptosis in
VitroTo investigate the effect of caspase-3, caspase-8, and
caspase-9 activity on AGE-induced apoptosis in human dermal
fibroblasts, cells were incubated with a pan-caspase and
caspase-3, -8, or -9 inhibitors at the time of CML-collagen
stimulation (Fig. 5A). The concentration of inhibitors used was
50 M, based on previous studies that demonstrated that this
concentration inhibits virtually all activity in a highly specific
manner (49 52). The pan-caspase inhibitor reduced apoptosis
compared with CML-collagen alone by 92%. Caspase-3 inhibi-
tor reduced the apoptotic effect of CML-collagen by 85%, a level
similar to that of the pan-caspase inhibitor. Caspase-8 inhibi-
tor alone and caspase-9 inhibitor alone significantly reduced
apoptosis by 65% and 32%, respectively (p 0.05). However,
when caspase-8 and -9 inhibitors were combined, the result
12091
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FIG. 6. AGEs stimulate fibroblast apoptosis through an NF-B-
independent mechanism. A, primary adult human dermal fibro-
blasts were incubated for 24 h with 200 g/ml CML-collagen or unmod-
ified collagen in the presence or absence of NF-B inhibitor SN50 (100
g/ml). Nuclear extracts were isolated and incubated with biotinylated
NF-B probe or probe plus excess unlabeled probe. EMSA was carried
out as described under Materials and Methods. The arrow points to
the NF-B band in lane 2. The experiment was performed four times
with similar results. B, primary adult human fibroblasts were incu-
bated with CML-collagen (200 g/ml) with or without NF-B inhibitor
SN50 (100 g/ml) or unmodified collagen (200 g/ml). Apoptosis was
measured 24 h later by ELISA. Each value represents the mean of five
replicates S.E. The experiment was performed three times with
similar results.
was similar to that of the caspase-3 inhibitor (p 0.05). Taken
together, these results suggest that AGEs function primarily
through caspase-3, whose induction can be accounted for by
activation from both caspases-8 and-9. The latter is demon-
strated by (p 0.05), establishing that caspase-3 activity is
significantly reduced in the presence of caspase-8 and -9 inhib-
itors (p 0.05) (Fig. 5B).
Role of NF-B in AGE-stimulated ApoptosisTo investigate
the effect of AGE on activation of NF-B in fibroblasts, adult
human dermal fibroblasts were incubated with CML-collagen
or unmodified collagen for 1 h. Using EMSA, it was shown that
CML-collagen but not unmodified collagen induced NF-B ac-
tivation (Fig. 6A). This effect could be blocked completely by an
NF- B inhibitor (SN50, 100 g/ml) (p 0.05). To investigate
the role of NF-B activation in AGE- induced apoptosis, adult
human dermal fibroblasts were treated with CML-collagen and
SN50 simultaneously, and apoptosis was measured by ELISA.
Inhibition of NF-B did not suppress fibroblast apoptosis (Fig.
6B). In fact, the level of apoptosis was higher in the presence of
SN50 inhibitor (p 0.05).
In Vitro Effect of CML-collagen on Expression of Apoptotic
Genes and Comparison with TNFExperiments with microar-
rays were carried out to study the impact of AGE on expression
of apoptosis-related genes. For the purpose of this analysis, a
minimum 2-fold change in the level of expression was used as
a threshold (Tables I and II). In addition, microarrays were also
carried out under identical conditions to compare AGE modu-
lation of apoptotic genes with that of TNF-. As shown in Table
12092 AGEs Stimulate Fibroblast Apoptosis
TABLE I
Fold change in apoptotic gene expression by AGE or TNF stimulation in vitro
Comparison of apoptotic gene expression in fibroblasts in vitro stimulated by CML-collagen or TNF-. Primary human dermal fibroblasts were
incubated with 200 g/ml CML-collagen or 20 ng/ml TNF-. Cells exposed to unmodified collagen or PBS were used as controls. Total RNA was
isolated 6 h after stimulation and subjected to microarray analysis. Left panels show genes whose expression is up-regulated 2-fold by both AGE
and TNF stimulation. Right panels show genes that were differentially regulated by AGE or TNF, i.e. AGE or TNF but not both induced a 2-fold
change in mRNA level.
Increased 2-fold by both AGE and TNF Regulated differently by AGE and TNF
Gene AGE TNF AGE/TNF ratio Gene AGE TNF AGE/TNF ratio
Ligands Ligands
LT- 5.01 6.41 0.78 CD27L 1.35 3.56 0.38
TNF 3.74 5.84 0.64 CD40L 1.33 2.51 0.53
OX40L 3.56 2.51 1.42 Lt 1.10 2.2 0.50
TNFSF9 3.03 5.18 0.58 TRAIL 1.04 3.54 0.29
APRIL 2.85 3.38 0.84 TNFSF12 0.84 2.12 0.40
CD30L 2.27 2.01 1.13 FasL 0.82 3.11 0.26
Receptors Receptors
TNFRSF9 7.67 3.12 2.46 TRAIL-R3 1.69 2.13 0.79
Fas 4.49 6.19 0.73 DR3 1.24 2.63 0.47
CD27 2.61 3.87 0.67 TNFRSF14 1.18 2.75 0.43
LTR 2.61 2.80 0.93 TNFR1 1.58 5.01 0.32
CD40 2.11 2.23 0.95 TNFR2 1.54 2.54 0.61
OX40 0.78 3.12 0.25
Mitochondrial Mitochondrial
Bfl-1 5.84 4.72 1.24 Blk 1.47 3.23 0.46
MCL-1 4.36 4.29 1.02 Bok/Mtd 1.25 2.08 0.60

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Bax 2.15 2.45 0.88 Bak 0.83 2.55 0.33
Nip-3 2.11 3.40 0.62 Bad 0.60 3.45 0.17
Caspases Caspases
Caspase-9 3.13 2.92 1.07 None
Caspase-8 3.07 3.50 0.88
Caspase-6 2.68 3.02 0.89
Caspase-3 2.52 3.12 0.81
Caspase-1 2.48 2.51 0.99
Caspase-2 2.19 3.52 0.62
Caspase-7 2.16 3.21 0.67
Caspase-4 2.05 2.16 0.95
Adaptors Adaptors
TRAF2 6.52 3.52 1.85 Flash 1.54 2.58 0.60
CASPER 5.64 7.16 0.79 Apaf-1 1.46 3.45 0.42
TRAF1 4.45 8.34 0.53 DAP-kinase2 1.41 2.07 0.68
RIP 2.57 3.35 0.77 MyD88 1.19 2.03 0.59
I-TRAF 2.50 3.74 0.67 CRADD 0.83 3.24 0.26
FADD 2.23 3.27 0.68 TRAF6 0.71 2.11 0.34
Others Others
GADD45 8.00 7.81 1.02 XIAP 1.43 2.12 0.67
P53 6.58 5.42 1.21 CIDE-A 1.32 2.12 0.62
Cardiac/Rip2 3.65 4.53 0.81 RPA 1.02 2.11 0.48
Chk2(RAD53) 3.52 3.50 1.01 Hus1 0.94 2.12 0.44
Mdm2 2.52 2.47 1.02 P63 0.87 0.49 1.78
IAP-2 2.30 2.35 0.98
BCL-10 2.25 3.06 0.74

TABLE II (CD27L, CD40L, Lt, TRAIL, TNFSF12, and FasL) were in-
Apoptotic gene expression decreased at least 2-fold by AGE duced more than 2-fold by TNF- stimulation, but not by AGE.
or TNF stimulation
From the TNF receptor family of the genes, Fas, CD27, LTR,
Microarrays were carried out as described in Table I. The fold change
in apoptotic gene expression modulated by AGE or TNF is shown. Only CD40, and TNFRSF9 mRNA levels increased 27-fold in re-
one gene, Bcl-2, was down-regulated by both AGE and TNF stimulation. sponse to both AGE and TNF-. However, other members of
Gene AGE TNF AGE/TNF ratio
this family exhibited increased expression by TNF- but not
AGE stimulation (TRAIL-R3, DR3, TNFRSF14, TNFR1,
Mitochondrial TNFR2, and OX40). Of the mitochondrial proteins that regu-
Bcl-2 0.45 0.45 1.0
Adaptors late apoptosis, AGE and TNF both increased Bax, Blf-1, and
TRAF3/CRAF1 0.31 1.27 0.24 Nip-3, which are pro-apoptotic members of the Bcl-2 family,
TRAF4 0.09 2.39 0.04 and MCL-1, which is an anti-apoptotic member of the Bcl-2
TRAF5 0.45 1.54 0.29 family. In addition, TNF- enhanced the pro-apoptotic proteins
Bcl-x 1.38 0.25 5.52
Others
Blk, Bak, and Bad, whereas AGE had no significant effect on
Nop30 0.45 0.74 0.61 expression of these genes. The caspase family showed signifi-
ATM 0.38 1.10 0.35 cant increases in expression after both stimuli. Initiator
caspases-2, -8, and -9 and effector caspases -3, -6, and -7
showed a 23-fold increase by both AGE and TNF. From the
I, CML-collagen compared with unmodified collagen induced a adaptor family, TRAF2, CASPER, TRAF1, RIP, I-TRAF, and
25-fold increase in the expression of six members of the TNF FADD increased between 2- and 8-fold in response to both
ligand superfamily (LT-, TNF-, OX40L, TNFSF9, APRIL, stimuli. The genes of six adaptor molecules (Flash, Apaf1,
and CD30L). The same six ligands had enhanced mRNA levels DAP-kinase, myD88, CRADD, and TRAF6) increased more
stimulated by TNF. Another six TNF superfamily ligands than 2-fold in response to TNF but not to AGE. P53, a cell cycle
 AGEs Stimulate Fibroblast Apoptosis 12093
TABLE III
Apoptotic gene expression not increased or decreased 2-fold by both AGE and TNF stimulation
Microarrays were carried out as described in Table I. Genes that did not exhibit a 2-fold change in expression by both AGE and TNF stimulation
are shown. Each value presented is the fold induction compared to unmodified protein (for AGE) or buffer alone (for TNF) and represents the mean
of two separate microarrays, which gave virtually identical results.
TNF ligand family TNF receptor family Bcl-2 family Caspases Adaptor Other

TNFSF14 TRAIL-R, Trail receptor Biml, HRK, Bcl-w, Caspase-10, caspase-5, Bar and Trip CIDE-B, DFF40, DFFA, Chk1,
(DR5), TRAIL-R4, and Bik caspase-14, and IAP-1, Nod/CARD4, Apollon/
and CD30 caspase-13 Bruce, survivin, and NAIP/
BIRC1

TABLE IV
Apoptotic gene expression modulated by CML-collagen or TNF- in
fibroblasts in vitro, as measured by RT-PCR
Primary human dermal fibroblasts were incubated with 200 g/ml
CML-collagen or modified collagen or 20 ng/ml TNF- or vehicle alone
(sterile PBS). Total RNA was isolated 6 h after stimulation and sub-
jected to RT-PCR as described under Materials and Methods. Each
lane represents the gene of interest and an internal standard, GAPDH,
amplified for 24 or 28 cycles. Each value represents the mean fold
increase of each experimental group compared with the respective
control obtained in two separate experiments.
CML-collagen/control TNF/control
Gene

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24 cycles 28 cycles 24 cycles 28 cycles
P53 3.43 3.72 3.15 3.33
TNF SF 9 2.8 3.06 2.8 2.7
Bax 2.23 2.02 2.24 2.13
TRAIL 1.5 1.01 2.58 2.74
Bcl-2 0.43 0.49 0.42 0.44

cycles to establish that differences obtained were not an

FIG. 7. Comparison of AGE- and TNF-stimulated expression of
apoptotic genes in vitro as measured by RT-PCR. Expression of
selected apoptotic genes was measured by RT-PCR of total RNA ex-
tracted from fibroblast cultures stimulated with CML-collagen or un-
modified collagen (200 g/ml) for 6 h. Other cells were stimulated with
TNF (20 ng/ml) or PBS alone, and the extracted RNA was used for the
same purpose. RT-PCR was carried out using primer pairs from Super-
Array in which an internal standard, GAPDH, is run for the same
number of cycles as the gene of interest. The amplicon of each gene is
shown after 24 and 28 cycles of PCR. The experiment was performed
twice with similar results.
regulator that is strongly pro-apoptotic, increased almost 6-fold
in both groups.
In Table II, molecules down-regulated more than 2-fold by
AGE and/or TNF stimulation are shown. Expression of the
anti-apoptotic gene Bcl-2 was reduced more than 2-fold by both
AGE and TNF. The pro-apoptotic adaptor molecule TRAF4
mRNA levels decreased almost 10-fold in response to AGE
while increasing 2-fold after TNF stimulation. Adaptor mole-
cules TRAF3 and TRAF5 decreased in response to AGE but did
not change more than 2-fold after TNF- stimulation. The
mRNA level of the anti-apoptotic gene Bcl-x was diminished
4-fold by TNF but not by AGE. Two other pro-apoptotic genes,
Nop30 and ATM, were down-regulated by AGE but not by TNF.
A list of other apoptotic genes whose expression was not en-
hanced or diminished more than 2-fold is shown in Table III.
To confirm the results of the microarray, RT-PCR using
specific primers for five selected genes of interest was exam-
ined (Fig. 7). Amplification was performed over 24 and 28
artifact of a particular number of amplifications. Densitomet-
ric analysis of the resulting bands indicates that the RT-PCR
results confirmed the data obtained for the microarray very
well (Table IV). In every case, the RT-PCR results exhibited
the same 2-fold change or lack of 2-fold change in cells stim-
ulated by CML-collagen or TNF- compared with the respec-
tive control.
In Vivo Effects of CML-collagen on Activation of Different
CaspasesTo determine whether AGEs also induce caspase
activity in vivo, experiments were performed in which CML-
collagen and unmodified collagen were injected into the scalp,
and the activation of caspases was measured (Fig. 8A). CML-
collagen (100 g) increased caspase-3 activity 2.5-fold, in-
creased caspase-8 activity 2.4-fold, and increased caspase-9
activity 2.2-fold compared with unmodified collagen (p 0.05).
To study the functional role of caspase-8 and -9 in AGE-
induced apoptosis, mice received injection with CML-collagen
with or without specific caspase inhibitors (Fig. 8B). Caspase-8
inhibitor decreased AGE-induced fibroblast apoptosis in vivo
77%, whereas caspase-9 inhibitor decreased it by 43% (p
0.05). These results agree well with in vitro data.
DISCUSSION
To study the apoptotic effect of AGE stimulation on fibro-
blasts and also to investigate mechanisms through which
AGEs induce apoptosis in these cells, CML-collagen was in-
jected subcutaneously in the scalps of mice. The direct effects of
AGE stimulation on fibroblasts were examined in culture. The
results establish that AGEs significantly induce apoptosis in
fibroblasts in vivo and in vitro and that it is largely dependent
upon caspase-8 and -9 activation of caspase-3. To our knowl-
edge, these studies are the first to characterize the caspase
pathways through which AGEs induce apoptosis in vitro and in
vivo. Moreover, AGE stimulation has a predominant effect of
enhancing expression of pro-apoptotic genes.
In order to identify the mechanisms through which AGE-
induced apoptosis was signaled, we identified the receptor in-
12094 AGEs Stimulate Fibroblast Apoptosis
FIG. 8. AGEs induce caspase activity in connective tissue in
vivo. A, caspase activity was measured using fluorometric assays in
lysates from tissue obtained after injection of CML-collagen or unmod-
ified collagen in the scalps of mice. The experiment was performed twice
with similar results. B, the number of TUNEL-positive apoptotic fibro-
blasts was quantified 24 h after injection of the scalp with CML-
collagen with or without caspase-8 or -9 inhibitors. After 24 h, the
extent of apoptosis was measured by TUNEL staining of histologic
sections. Each value represents the mean of specimens from six mice
S.E. The experiment was performed three times with similar results.
volved and the downstream events. By use of a blocking anti-
body, it was shown that virtually all of the apoptotic effect of
AGEs on fibroblast apoptosis was mediated through the RAGE.
Previously, it was shown that RAGE activation reduces colla-
gen production in fibroblasts (34). In addition, it has been
reported that blocking RAGE significantly enhances wound
healing in diabetes (35). As discussed below, enhanced fibro-
blast apoptosis represents one of the potential mechanisms
through which AGEs could impair the healing process.
By using different caspase inhibitors in vivo and in vitro, we
established that AGEs activate caspase-8, which signals apo-
ptosis through the cytosolic pathway, and, to a lesser extent,
caspase-9, which signals through the mitochondrial pathway.
The functional importance of caspase activation in fibroblast
apoptosis was shown by use of specific inhibitors. Consistent
with the degree of activation, inhibition of caspase-8 in vitro
had a greater impact than inhibition of caspase-9. However,
caspase-8 and -9 inhibitors together were more potent than
either inhibitor alone in reducing AGE-induced apoptosis, fur-
ther supporting the concept that AGE signaling involves both
caspase-8 and -9 pathways. Inhibitor studies also showed that
both of these pathways stimulated activation of the effector
caspase, caspase-3. The involvement of caspase-3 in AGE-stim-
ulated fibroblast apoptosis was shown by enhanced caspase-3
activity and through inhibition of apoptosis with a specific
caspase-3 inhibitor. The observation that caspase-3 inhibitor
had an effect similar to that of a pan-caspase inhibitor suggests
that AGE stimulation operates principally through this effector
caspase. These results are in contrast to TNF, which induces
fibroblast apoptosis almost exclusively through the cytosolic,
caspase-8-dependent pathway (47).
To date, there have been relatively few reports examining
the effect of AGEs on gene expression and no reports examining
global induction of apoptotic genes. We demonstrated that
AGEs induced a full range of apoptotic genes involving ligands,
receptors, adaptor molecules, mitochondrial proteins, and var-
ious other molecules that participate in apoptosis. Although
the net effect was to induce expression of pro-apoptotic genes,
there were instances in which the expression of some was
down-regulated, for example, the adapter molecules TRAF3,
TRAF4, and TRAF5. For those genes whose expression has
previously been reported to be induced by AGEs, we observed
similar results, even though the cell type was different. For
example, AGEs have been reported to down-regulate Bcl-2 in
retinal pigment epithelial cell and pericytes (39, 53) and to
up-regulate bax mRNA levels in mesangial cells and retinal
neurons (54, 55). Similar down-regulation of bcl-2 and up-
regulation of bax were found in AGE-stimulated fibroblasts.
The modulation of apoptotic gene expression was also com-
pared between AGE and TNF- stimulation under identical
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conditions. Whereas TNF induced a pattern that was over-
whelmingly pro-apoptotic, including the inhibition of several
anti-apoptotic genes, AGE stimulated a somewhat different
pattern as discussed above. This would suggest that there are
differences in the pathways through which AGE and TNF
affect mRNA levels of apoptotic genes. A potential mechanism
by which apoptosis through AGE or TNF could differ is through
differences in stimulation of anti-apoptotic mechanisms. Al-
though both AGEs and TNF have been shown to activate
NF-B (56, 57), it has also been reported that there are differ-
ences with AGEs exhibiting a more prolonged induction (58).
Thus, AGEs and TNF may have differences in the pathways
that lead to activation of caspase activity or may have differ-
ences in activation of NF-B, thereby exhibiting differences in
anti-apoptotic processes.
Advanced glycation end products are present at much higher
levels in individuals with diabetes and in aged individuals.
With increasing age, there is a reduction in fibroblast prolifer-
ation and life span, an increase in their apoptosis, and a di-
minished rate of dermal wound healing (59, 60). Fibroblasts
play a leading role in wound healing, particularly in the skin.
To participate in wound repair, they must migrate into the
wounded area, proliferate, and form new matrix. Because ad-
equate healing requires a sufficient number of cells to repair
wounds, factors that cause enhanced apoptosis could impair
healing, as suggested by recent reports (44, 61). Thus, the
formation of advanced glycation end products represents one of
the potential mechanisms through which wound healing in
aged and diabetic individuals may be diminished.
AcknowledgmentWe thank Alicia Ruff for help in preparing the
manuscript.
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Advanced Glycation End Products Enhance Expression of Pro-apoptotic Genes and
Stimulate Fibroblast Apoptosis through Cytoplasmic and Mitochondrial Pathways
Zoubin Alikhani, Mani Alikhani, Coy M. Boyd, Kiyoko Nagao, Philip C. Trackman and
Dana T. Graves
J. Biol. Chem. 2005, 280:12087-12095.
doi: 10.1074/jbc.M406313200 originally published online December 6, 2004

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