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Department of Pediatrics, Oregon Health Sciences University (S.M.T., S.D.C., J.T., H.-S.K., Y.O., R.G.R.), Portland, Oregon 97201; and Cardiorenal Cell Biology, Scios, Inc. (M.M.C., A.H.J.), Sunnyvale, California 94086
Address all correspondence and requests for reprints to: Dr. Stephen M. Twigg, Department of Pediatrics, NRC-5, Mark O. Hatfield Research Center, Oregon Health Sciences University, 3181 SW Sam Jackson Park Road, Portland Oregon 97201. E-mail: twiggs{at}ohsu.edu
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Pathological hallmarks in most tissues where diabetes complications occur include expansion of extracellular matrix (ECM) and angiogenesis (1). The ECM expansion has been proposed to be due to a combination of increased ECM production (14) (15) and biochemically modified matrix, with a reduction in ECM breakdown (16). Connective tissue growth factor (CTGF), also known as insulin-like growth factor (IGF)-binding protein-related protein-2 (IGFBP-rP2) (17), is a potent inducer of ECM in fibroblasts (18, 19) and a potent angiogenic factor (20, 21). A potential role for CTGF in fibrotic disease states is increasingly being described (22, 23, 24), suggesting that CTGF may be a mediator of ECM expansion and fibrosis in diabetes. The aim of this study was to determine whether CTGF is up-regulated by AGE and subsequently to explore the cellular mechanism(s) that might be responsible for this effect.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Intact carboxyl-terminal flag-tagged IGFBP-rP2 (CTGF), used as a
standard in the Western immunoblots and in the CTGF enzyme-linked
immunosorbent assay (ELISA), was purified from a baculovirus expression
system, and pure IGFBP-rP2 (CTGF) protein was quantitated using
Coomassie-stained gels with BSA as standard, all as previously
described (29). The approximately 14-kDa fragment of
IGFBP-rP2 (CTGF), described in Fig. 7A
, was purified from a highly
proteolyzed preparation of pure IGFBP-rP2 (CTGF) protein, using
late-harvested SF-9 insect cell lysates. After initial purification of
this preparation by means of flag protein-Sepharose affinity
chromatography (29), the fragment was separated from any
remaining intact IGFBP-rP2 (CTGF) using a high performance
size-fractionation gel permeation column (HR-75, Pharmacia Biotech, Piscataway, NJ) with PBS as buffer, with fast
performance liquid chromatography, eluting at 0.5 ml/min with 0.5-ml
fractions. Pure, approximately 14-kDa fragment was confirmed by
Coomassie staining and Western immunoblot using IGFBP-rP2 (CTGF)
primary antibody (not shown).
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The AGE content in the preparations was assessed by means of
fluorescence, SDS-PAGE analysis, and ELISA. The fluorescence content,
measured with a fluorescence spectrometer at 390 nm emission after 450
nm excitation in relative fluorescence units per mg BSA, was 11.2
± 2.5 for control BSA, 67.1 ± 12.5 for AGE BSA from
glycolaldehyde, 52.3 ± 6.3 for AGE BSA from glucose, and 9.7
± 1.2 for aminoguanidine added to BSA and glucose (termed
aminoguanidine BSA) (31). By SDS-PAGE analysis under
reducing conditions, followed by Coomassie staining, the AGE BSA
produced from D-glucose and glycolaldehyde was shown to
have high Mr species consistent with the
intermolecular cross-linking ability of AGE, as previously described
(26). In contrast, the control BSA and aminoguanidine BSA
preparations did not have these high Mr forms
(data not shown). By competitive ELISA (32), performed by
Dr. P. Foiles (Alteon, Inc., Ramsey, NJ), using a synthetic
N-
-carboxymethyl lysine (CML) analog as the standard, the
CML content of the preparations (picomoles of CML per µg BSA ±
95% confidence interval) was 82 ± 8.5 for AGE BSA from
glycolaldehyde and 13 ± 1.4 for AGE BSA from glucose and was
undetectable (<1) for control BSA and also undetectable when
aminoguanidine at 100 mM was coincubated with BSA
and glucose. Unless otherwise indicated in the text, the data described
refer to the use of AGE BSA synthesized from glucose.
Cell culture
Primary cell cultures of nonfetal human dermal fibroblasts,
CRL-2097 and CRL-1474, were purchased from American Type Culture Collection (Manassas, VA). Cells were maintained in MEM
supplemented with 10% FBS and were used in these studies between
passages 4 and 12. The human primary cultures of dermal fibroblasts,
designated A35 (derived from the forearm of a 70-yr-old male) and A305
(newborn foreskin fibroblasts), were gifts from Dr. S. Goldstein,
Memorial Veteran’s Hospital (Little Rock, AR). These cells were
maintained in DMEM with 15% FBS.
Cell treatment
After trypsinization, cells were grown in 12-well plates for 5
days in their respective media with FBS until they were confluent. For
experiments requiring the use of blocking antibodies to RAGE and AGE,
cells were grown in 24-well plates under the same conditions. Cells
were then incubated in their respective serum-free medium for 16 h
and then treated with additions on day 0 under serum-free conditions,
using fresh media. Unless otherwise indicated in the text, the
conditioned media were not changed after adding the treatments. When
cells were transiently treated for 8 h with reagents, they were
washed with PBS, and fresh serum-free medium with 0.05% BSA was added.
Cell lysates and conditioned media were harvested up to 3 days after
treatments. For experiments involving the use of blocking antibodies or
antioxidants, cells were preincubated with the antibody or reagent for
2 h under serum-free conditions before adding AGE or control BSA
directly to the medium.
Total RNA isolation and analysis by quantitative real-time
RT-PCR
Total RNA was isolated from duplicate wells using the RNeasy
Minikit from QIAGEN (Valencia, CA) and was then analyzed
by quantitative real-time PCR using an ABI Prism 7700 Sequence
Detection System (PE Applied Biosystems, Foster City, CA).
This system is based on the ability of the 5'-nuclease activity of
Taq polymerase to cleave a nonextendable dual labeled
fluorogenic hybridization probe during the extension phase of PCR. The
following sequence-specific primers and probes for human CTGF,
IGFBP-rP1, IGFBP-3, and 18S ribosomal RNA were designed using Primer
Express Software 1.0 (PE Applied Biosystems): for CTGF:
forward, 5'-GAGGAAAACATTAAGAAGGGCAAA-3'; reverse,
5'-CGGCACAGGTCTTGATGA-3'; and probe,
5'-6FAM-TTTGAGCTTTCTGGCTGCACCAGTGT-TAMRA-3'; for IGFBP-rP1: forward,
5'-GCGGAAAATGGCAGACAATT-3'; reverse, 5'-CTTGAGGGTTTGGGTTTCCA-3';
and probe, 5'-6FAM-TTCGCTCCATGATGCGTTATCTGGG-TAMRA-3'; for IGFBP-3:
forward, 5'-AAGGTGGGTAGGCACGTTGTAG-3'; reverse,
5'-ATATCAAAACCCGAATCCACTTTACT-3'; and probe,
5'-6FAM-CAAAGCAATGTCTAGTCCCGGTATGTCCAA-TAMRA-3'; for 18S: forward,
5'-CGGCTACCACATCCAAGGAA-3'; reverse, 5'-GCTGGAATTACCGCGGCT-3'; and
probe, 5'-6FAM-TGCTGGCACCAGACTTGCCCTC-TAMRA-3'. Primers were used at a
concentration of 200 nM and probes at 100
nM in each reaction. Multiscribe reverse
transcriptase and AmpliTaq Gold polymerase (PE Applied Biosystems) were used in all RT-PCR reactions. Each RNA sample
was analyzed in triplicate. Relative quantitation of 18S ribosomal RNA
and human CTGF, IGFBP-rP1, and IGFBP-3 messenger RNAs (mRNAs) was
calculated using the comparative threshold cycle number for each sample
fitted to a five-point standard curve (ABI prism 7700 User Bulletin 2,
PE Applied Biosystems). The standard curve was constructed
using a serial dilution of total RNA extracted from human cardiac
fibroblasts that had been treated with TGFß1 at 1 ng/ml for 24
h. Expression levels were normalized to 18S ribosomal RNA and related
to relevant controls, as indicated in the text.
Preparation of conditioned media and cell lysates
Cell lysate samples were harvested after treatment, by washing
cells with PBS, then adding 100 µl cold RIPA lysis buffer [20
mM Tris (pH 8.0), 150 mM NaCl, 1% Nonidet
P-40, 0.5% sodium deoxycholate, and 0.1% SDS] plus a protease
inhibitor cocktail (Roche Molecular Biochemicals,
Mannheim, Germany) directly to each well. Plates were rocked for 15 min
at 4 C, and lysates were collected and centrifuged at 10,000 x
g for 10 min at 4 C. The supernatants from duplicate wells
within each experiment were pooled and stored at -20 C until analysis.
The total protein concentration was determined for each sample by use
of the DC Protein Assay reagent (Bio-Rad Laboratories, Inc., Hercules, CA). Then 20 µg total protein were loaded per
lane for SDS-PAGE analysis, and 5 µg total protein were added to each
ELISA well for CTGF quantitation.
Western immunoblot analysis
Conditioned medium samples were separated on 15% nonreducing
SDS-PAGE. Proteins were electrotransferred onto nitrocellulose, and
membranes were blocked with 5% nonfat dry milk/TBS with 0.1%
(vol/vol) Tween 20 for 1 h at 22 C, then incubated in IGFBP-rP2
(CTGF) antiserum at 1:1000 dilution at 4 C overnight. After incubation
of membranes with a horseradish peroxidase (HRP)-labeled secondary
antibody for 1 h at 22 C, immunoreactive proteins were detected by
use of enhanced chemiluminescence (NEN Life Science Products, Boston, MA).
CTGF (IGFBP-rP2) ELISA
The anti-IGFBP-rP2 (CTGF) antibody (8800) (25) was
biotinylated by incubating protein A affinity-purified 8800 (0.8 µg)
with 150 µg sulfo-NHS-LC biotin (Pierce Chemical Co.,
Rockford, IL) for 2 h at 22 C, followed by separation from
unreacted biotin through a size-fractionation and desalting column with
PBS as buffer according to the manufacturer’s instructions.
Affinity-purified 8800 antibody (600 ng/well) in 10 mM
sodium carbonate, pH 9.6, was adsorbed to 96-well immunoplates
(Nalge Nunc International, Rochester, NY) by a 20-h
incubation at 4 C. The unbound antibody was removed, and the wells were
blocked by incubation with PBS and 0.1% (vol/vol) Triton X-100 (buffer
A) containing 10 g/liter BSA for 2 h at 37 C, then washed four
times with buffer A. Purified intact recombinant human (rh) IGFBP-rP2
(CTGF) in buffer A and 1 g/liter BSA was used to generate standard
curves. Standard and samples (100 µl/well) were incubated in
duplicate at 4 C for 20 h. The plate was washed, then incubated
with biotinylated IGFBP-rP2 (CTGF) antibody (80 ng/well) for 20 h
at 4 C. After washing, the plate was incubated with streptavidin-HRP
(1:500) for 30 min at 22 C, followed by substrate [0.1 g/liter
3,3',5,5'-tetramethylbenzidine in 0.2 M sodium acetate (pH
6) containing 0.06% (wt/wt)
H2O2] for 30 min at 22 C.
The reaction was stopped by the addition of 2 M
H2SO4, and the absorbance
was measured at 450 nm using a microplate reader. The interassay
coefficient of variation was 8.1% for the middle concentration (10
ng/well) of rhIGFBP-rP2 (CTGF) standard used. No cross-reactivity was
detected with 1 µg/well purified rhIGFBP-3, rhIGFBP-rP1 (mac 25), or
rhIGFBP-rP3 (Nov H; not shown).
CTGF (IGFBP-rP2) cell association assay
To determine whether increases in rhIGFBP-rP2 (CTGF) in the
whole cell lysates after AGE treatment are due to increases in
rhIGFBP-rP2 (CTGF) at a cell-associated site either on the cell surface
or in the extracellular matrix, rather than intracellularly, 2097
fibroblasts in confluent monolayer were treated under serum-free
conditions with AGE or control BSA (each at 100 µg/ml) for 3 days in
replicates of four. After washing twice with PBS at 4 C, biotinylated
rhIGFBP-rP2 (CTGF) antibody (800 ng/well) together with
streptavidin-HRP (1:500) was added for 2 h at 22 C in PBS and
0.1% BSA. In some wells the streptavidin-HRP (1:500) was added in the
absence of primary antibody to determine nonspecific binding and
endogenous cellular peroxidase activity. After two further (gentle) PBS
washes, developing substrate was added, and absorbance was read as
described for the IGFBP-rP2 (CTGF) ELISA above.
Densitometric analysis
To quantify the relative induction of CTGF after Western
immunoblots, densitometric measurement was performed using GS-700
Imaging Densitometer with Mutli-Analyst Software (Bio-Rad Laboratories, Inc.).
Statistical analysis
Results are expressed as the mean ± SD or the
mean ± SEM as indicated in the text. All data were
pooled from three or four independent experiments, each performed in
triplicate. Differences between groups were assessed using Student’s
two-tailed paired t test in Excel 98 (Microsoft Corp., Redmond, WA). P < 0.05 was
considered statistically significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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), and a
progressive increase occurred over the 3-day time course of the study
(Fig. 1B). In contrast, no change in CTGF mRNA over time was seen with
the same concentration of control BSA (Fig. 1, A and B). These results
were confirmed by Northern analysis (not shown). Transient treatment of
cells with AGE for 8 h, followed by washing of cells with PBS and
replacement with fresh serum-free medium, also caused a progressive
increase in CTGF mRNA over subsequent days (Fig. 1C), with a clear
persistence of the effect for at least 72 h after AGE
addition.
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).
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).
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). IGFBP-3 is the predominant IGFBP present in human fibroblast
conditioned medium (33), and its mRNA also was not
increased by AGE (Fig. 3B). In comparison with the lack of effect of
AGE treatment, TGFß1 treatment modestly up-regulated IGFBP-rP1,
and possibly IGFBP-3 mRNA, at 48 h (Fig. 3B), as previously
described (33, 34). Also consistent with previous
observations in cultured human fibroblasts (35), a
pronounced up-regulation of CTGF mRNA occurred after TGFß1 addition
(Fig. 3B).
Formation of products of nonenzymatic glycosylation was inhibited by
the dihydrazine compound, aminoguanidine (13). When cells
were treated with BSA that had been coincubated for 10 weeks with both
glucose and aminoguanidine as described in Materials and
Methods, no increase in CTGF mRNA was observed compared with
control BSA treatment alone or with serum-free medium without any
addition (Fig. 4A). This result
confirms that the active component in the AGE reagent used is a
product of nonenzymatic glycosylation.
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). As this antibody is
specific to AGE and it does not bind to amadori products
(26), which are early products of nonenzymatic
glycosylation, these results show that AGE is the active component in
the synthesized reagent responsible for increasing CTGF in these
studies.
AGE may bind to and activate one or more of the defined cell surface
receptors for AGE (8). The AGE receptor subtype, termed
RAGE, has recently been shown to be present on the surface of human
fibroblasts (27), and in some cell systems the induction
of growth factors by AGE has been shown to be mediated by RAGE
(36). When cells were preincubated with a blocking
antibody of RAGE activation by AGE ligand, the induction of CTGF mRNA
by AGE was attenuated by the anti-RAGE IgG by 64.1%, on the average
(Fig. 4B). Higher concentrations of anti-RAGE IgG did not have any
additional effect (not shown). In contrast, the AGE effect was not
significantly inhibited by normal rabbit serum IgG (Fig. 4B). These
results show that RAGE is at least partly mediating the AGE induction
of CTGF mRNA in the fibroblasts.
As reactive oxygen (RO) species are commonly generated in cells after
activation of AGE receptors by its ligand (9, 37), an
effect of inhibiting RO species formation during AGE treatment was
studied. Preincubation of the fibroblasts with the antioxidants
dimethylsulfoxide or N-acetyl cysteine, however, did not
inhibit the increases in CTGF mRNA (Fig. 4C). These results imply that
RO species are unlikely to play a role in the observed AGE effect on
CTGF.
A potential role for autocrine TGFß1 in CTGF mRNA induction by AGE
was then examined. TGFß1 is a potent inducer of CTGF gene expression
in this cell system (Fig. 3A), and in addition, AGE may induce TGFß1
mRNA and protein in some cells (13). Induction of CTGF
mRNA by rhTGFß1 added to the cultured fibroblasts was fully inhibited
by a TGFß1-neutralizing antibody at 24 h (Fig. 5A) and 48 h (Fig. 5B). In contrast,
when the same antibody was added under the same conditions in parallel
wells, no significant inhibition of the CTGF mRNA increase induced by
AGE occurred (Fig. 5, A and B), indicating that the effect of AGE is
TGFß1 independent in this cell system.
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). A progressive increase in intact
CTGF as well as previously described lower Mr
immunoreactive forms, at approximately 14 and 20 kDa (38),
occurred (Fig. 6A). High Mr immunoreactive
material (>80 kDa) was also more prominent in the AGE-treated medium,
which may include CTGF covalently cross-linked by AGE. All of these
increases were more marked using AGE BSA synthesized from
glycolaldehyde compared with AGE BSA synthesized from glucose (not
shown). Using densitometric analysis from three independent
experiments, the intact CTGF in the medium was increased by AGE
compared with control BSA treatment (mean ± SEM) by
3.6 ± 1.1-fold on day 1, 8.0 ± 1.3-fold on day 2, and
14.5 ± 3.1-fold on day 3 (P < 0.05 for all days
of AGE treatment compared with control BSA treatment).
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). There was no CTGF
fragment or high Mr immunoreactive material
observed in the lysates (not shown). Densitometric analysis of CTGF
from lysates from four independent experiments (mean ±
SEM) gave the following results for fold change with AGE
treatment compared with control BSA treatment: 0.89 ± 0.33 on day
1, 0.97 ± 0.17 on day 2, and 1.53 ± 0.25 on day 3
(P < 0.05 for day 3 only for AGE compared with control
BSA on the respective day).
Considering that the increase in CTGF in whole cell lysates on day 3
after AGE treatment was relatively modest, changes in cell lysate CTGF
were further determined by a CTGF ELISA, as described in
Materials and Methods. This assay can measure endogenous
intact CTGF, which is present in whole cell lysates, but due to a lack
of parallelism with the intact rhCTGF used as the standard, it cannot
be used to accurately measure the 14-kDa CTGF fragment (Fig. 7A), which is present
in the fibroblast-conditioned media (Fig. 6A). Consistent with the
Western immunoblots of cell lysates (Fig. 6B), the ELISA also showed
that AGE treatment reproducibly increased CTGF in the fibroblast whole
cell lysates on day 3 (Fig. 7B). Thus, in contrast to the increases in
CTGF protein observed in the conditioned media, there was no increase
in CTGF protein in the first 2 days after AGE treatment in the whole
cell lysates compared with control, and there was only a modest and
delayed increase in CTGF in the lysates, which was much less striking
than the increases in CTGF protein observed in the conditioned media
(Fig. 6A).
To determine whether the increase in CTGF in the whole cell lysates
seen by day 3 of AGE treatment was accessible to the extracellular
environment, a cell association assay for CTGF was performed, as
described in Materials and Methods. This assay uses binding
of a biotinylated CTGF primary antibody to endogenous CTGF protein,
followed by antibody detection using a streptavidin-HRP system. As no
plasma membrane-permeabilizing agents were used in the protocol, the
specific signal detected by the CTGF primary antibody was due to CTGF
present on the cell surface or in the extracellular matrix, rather than
CTGF present in an intracellular compartment. As shown in Fig. 7C, at
72 h AGE at 100 µg/ml specifically increased the absorbance
signal compared with control (P < 0.05 for analysis of
combined data from four independent experiments). In parallel wells,
under the same conditions of confluent cell monolayers in serum-free
media, cell number determined by hemocytometer counting and trypan blue
exclusion was not changed by AGE treatment compared with control BSA
(not shown). Thus, these results indicate that at 72 h, AGE
treatment increases cell-associated CTGF compared with BSA control
treatment alone.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The up-regulation of CTGF by AGE appears to be specific for CTGF and is generalizable to skin fibroblasts from differing sources and passage number. In each of the four fibroblast cell lines studied, AGE up-regulated CTGF. In the cell line most extensively studied, CRL-2097, CTGF was regulated by AGE in early passages (passage 4) and also at later passages (passage 12). In contrast to effects on CTGF, the two other members of the IGFBP superfamily that were studied, IGFBP-3 and IGFBP-rP1, were not up-regulated by AGE. Further studies will be required to determine whether AGE affects other members of the CCN (CTGF, Cyrbl, Nov) family.
The concentrations of AGE BSA used in these experiments approximate those used in vitro in other studies exploring biological effects of AGE on cells (13, 27). Although there is no universal standard method for measuring specific AGE components at this time, and the AGE antibodies used in assays measuring AGE differ (39), the AGE BSA concentrations studied are in the broad range for AGE concentrations found in diabetic serum (40).
Few AGE components have been defined biochemically to date, and the specific end-product(s) that might be mediating the effect on CTGF was not identified in this work. AGE adducts existing in diabetic tissues that have been shown to signal through AGE receptors include mainly CML (28) and imidazoline-based products (41). Considering that CML adduct is a ligand for RAGE (28), that our AGE reagent contained CML, and that at least part of the AGE effect on CTGF has been shown to be mediated through RAGE, it is plausible that CML adducts are one of the AGE components operative in this study. In the current work, when AGE BSA synthesized from glycolaldehyde was studied in experiments where AGE BSA synthesized from glucose was also used in treatments, each at the same AGE concentration of 100 µg/ml BSA with mRNA measurements over 3 sequential days, the induction of CTGF mRNA by these reagents did not differ (data not shown). As these two AGE reagents contain differing amounts of CML (as described in Materials and Methods), the CML adduct cannot be the only explanation for the observed AGE effect on CTGF mRNA in these AGE preparations. Further experiments with pure CML and other pure AGE adducts, when available, will be required to address this issue.
A number of subtypes of cell surface receptor bind AGE specifically and are responsible for mediating multiple cellular effects of AGE (42). These receptors exist in four main classes: RAGE, AGE-R1, AGE-R2, and AGE-R3 (8). In the diabetic environment, the increased AGE present is hypothesized to bind and activate AGE receptors, and in some studies, the induction of growth factors by AGE has been shown to be mediated by AGE receptors, including RAGE (43). Our studies indicate that RAGE is responsible for mediating at least part of the effect of AGE on CTGF mRNA. The possibility that other AGE receptors might also contribute to these effects is not excluded by this work.
A role for growth factors in contributing to chronic diabetes-related end-organ complications, particularly vascular endothelial growth factor (VEGF), TGFß1, IGF-I, and platelet-derived growth factor, is under increasing evaluation, and a potential role for CTGF in chronic diabetic complications is emerging. CTGF is a potent profibrotic agent (18, 44), which is reflected in its ability to induce ECM components and increase fibroblast DNA synthesis (18) and to promote angiogenesis (20, 21). CTGF mRNA levels are up-regulated in many chronic disease states where fibrosis is prominent (22, 23, 24). Two separate studies involving renal mesangial cells and differing diabetic rat models recently reported that CTGF gene expression (45) as well as protein (46) are increased in mesangial cells after exposure to high glucose and in vivo in diabetic rat kidneys. Immunohistochemical studies of kidney tissue in human end-stage renal disease showed increased CTGF protein in diabetic kidneys as well as other nephropathies (47), and CTGF mRNA is markedly increased in advanced atheromatous lesions (48).
This is the first report of CTGF induction by advanced
glycosylation end products, and it provides a potentially critical
linkage among AGE, growth factors, and fibrosis. AGE induction of
growth factors and cytokines has been described for VEGF, TGFß1,
IGF-I and platelet-derived growth factor, TNF
, IL-1ß, and IL-6
(3) mainly in various endothelial and mesenchymal cultured
cells and in some cases by AGE administration in vivo
(15). CTGF appears to fit well into this group of
proinflammatory and/or profibrotic proteins.
The striking persistent effect over 3 days of AGE on CTGF mRNA even after transient treatment suggests that regulation of CTGF by the AGE reagent tested is complex and may involve multiple interrelated intracellular signals. The cellular mechanism of AGE induction of CTGF mRNA was not defined in this study. RO species were not implicated, because antioxidants were ineffective in inhibiting AGE induction of CTGF. TGFß1 is a known potent inducer of CTGF gene expression, and CTGF is implicated as a downstream mediator of TGFß1 effects (49), particularly in fibrosis (44). We were unable to show, however, that TGFß1 is a mediator in the AGE induction of CTGF. In the current work, both the early time course of initial induction of CTGF mRNA by AGE at 8 h as well as the inability of TGFß1-neutralizing antibodies to inhibit AGE induction of CTGF suggest that AGE is operating through mechanisms that are independent of TGFß1. Although studies involving the use of exogenously added neutralizing antibodies have potential limitations in assessing the role of endogenous protein bioactivity, that total TGFß1 measurements in conditioned media measured by TGFß1 ELISA (Promega Corp., Madison, WI) in these cells were not increased by AGE compared with control BSA treatment (data not shown) is also supportive that TGFß1 is not a mediator of AGE induction of CTGF in this work. These results contrast with studies describing TGFß-dependent effects of glucose on CTGF up-regulation in human mesangial cells (45, 46), but are consistent with other studies showing that various reagents can potently up-regulate CTGF mRNA independently of TGFß1 (50).
In human fibroblast primary cultures, CTGF exists at very low levels in conditioned medium and is often present in low Mr fragment forms, which may also have bioactivity (38). That intact CTGF was readily detectable in the medium after AGE treatment may be partly related to posttranslational modification of CTGF, with cross-linking of CTGF protein by AGE into a high Mr immunoreactive form and redistribution of CTGF from a cell-associated site into the conditioned medium. In addition to the CTGF increases in the conditioned medium and consistent with the progressive increase in CTGF mRNA after AGE treatment, AGE caused increases in intact CTGF in whole cell lysates at 72 h. Further analysis showed that the CTGF increase in the lysates included protein that was cell associated and in a site accessible to the extracellular environment. To what extent the bioactivity of CTGF protein is affected by its presence in the medium compared with a cell-associated site is an important issue for future study of CTGF bioactivity.
There is a rationale to potentially link AGE effects and diabetic complications with the induction of CTGF in skin and, by association, with pathology in other tissues. A feature commonly present in human diabetes, even in late childhood and adolescence, is skin thickening and contracture (51), termed diabetic sclerosis. This process affects mainly the distal extremities and is characterized by expansion of extracellular matrix, fibroblast proliferation, and angiogenesis (52). The presence of overt diabetic sclerosis of skin is correlated with the presence and future development of end-organ complications, particularly diabetic nephropathy and retinopathy (53). AGE products are increased in human diabetic skin (6), and the levels of AGE in skin also correlate positively with the presence of diabetic microvascular kidney and eye disease (5, 6). That the ability of CTGF to induce fibrosis has been well characterized in skin (18, 44) makes skin fibroblasts a relevant cell model for the current study.
Clearly, in vivo and longer term studies are required to substantiate a more definitive role for induction of CTGF by AGE in potentially mediating diabetic fibrotic complications in skin and other organs. Activation of receptors for AGE, particularly RAGE (54), has also been implicated in the pathogenesis of fibrosis that develops in chronic diseases other than diabetes (42, 54, 55). That AGE up-regulates CTGF in nontransformed human fibroblasts suggests that CTGF may be a factor mediating the observed AGE and RAGE effects, which is a hypothesis that requires further testing.
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Acknowledgments |
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Footnotes |
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Received October 3, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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vß3, promotes
endothelial cell survival, and induces angiogenesis in vivo. Mol Cell
Biol 19:2958–2966)-(Carboxymethyl)lysine adducts of proteins are ligands
for receptor for advanced glycation end products that activate cell
signaling pathways and modulate gene expression. J Biol Chem 274:31740–31749This article has been cited by other articles:
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 E. J. Kuiper, J. M. Hughes, R. J. Van Geest, I. M. C. Vogels, R. Goldschmeding, C. J. F. Van Noorden, R. O. Schlingemann, and I. Klaassen Effect of VEGF-A on Expression of Profibrotic Growth Factor and Extracellular Matrix Genes in the Retina Invest. Ophthalmol. Vis. Sci., September 1, 2007; 48(9): 4267 - 4276. [Abstract] [Full Text] [PDF]
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R. G. Dean, L. C. Balding, R. Candido, W. C. Burns, Z. Cao, S. M. Twigg, and L. M. Burrell Connective Tissue Growth Factor and Cardiac Fibrosis after Myocardial Infarction J. Histochem. Cytochem., October 1, 2005; 53(10): 1245 - 1256. [Abstract] [Full Text] [PDF]
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S. V. McLennan, X. Y. Wang, V. Moreno, D. K. Yue, and S. M. Twigg Connective Tissue Growth Factor Mediates High Glucose Effects on Matrix Degradation through Tissue Inhibitor of Matrix Metalloproteinase Type 1: Implications for Diabetic Nephropathy Endocrinology, December 1, 2004; 145(12): 5646 - 5655. [Abstract] [Full Text] [PDF]
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 P. Roestenberg, F. A. van Nieuwenhoven, L. Wieten, P. Boer, T. Diekman, A. M. Tiller, W. M. Wiersinga, N. Oliver, W. Usinger, S. Weitz, et al. Connective Tissue Growth Factor Is Increased in Plasma of Type 1 Diabetic Patients With Nephropathy Diabetes Care, May 1, 2004; 27(5): 1164 - 1170. [Abstract] [Full Text] [PDF]
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D. R. Hinton, C. Spee, S. He, S. Weitz, W. Usinger, L. LaBree, N. Oliver, and J. I. Lim Accumulation of NH2-Terminal Fragment of Connective Tissue Growth Factor in the Vitreous of Patients With Proliferative Diabetic Retinopathy Diabetes Care, March 1, 2004; 27(3): 758 - 764. [Abstract] [Full Text] [PDF]
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C. Tikellis, M. E. Cooper, Stephen. M. Twigg, W. C. Burns, and M. Tolcos Connective Tissue Growth Factor Is Up-Regulated in the Diabetic Retina: Amelioration by Angiotensin-Converting Enzyme Inhibition Endocrinology, February 1, 2004; 145(2): 860 - 866. [Abstract] [Full Text] [PDF]
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 S. Lam, R. N. van der Geest, N. A.M. Verhagen, F. A. van Nieuwenhoven, I. E. Blom, J. Aten, R. Goldschmeding, M. R. Daha, and C. van Kooten Connective Tissue Growth Factor and IGF-I Are Produced by Human Renal Fibroblasts and Cooperate in the Induction of Collagen Production by High Glucose Diabetes, December 1, 2003; 52(12): 2975 - 2983. [Abstract] [Full Text] [PDF]
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 D. Foell, D. Kane, B. Bresnihan, T. Vogl, W. Nacken, C. Sorg, O. FitzGerald, and J. Roth Expression of the pro-inflammatory protein S100A12 (EN-RAGE) in rheumatoid and psoriatic arthritis Rheumatology, November 1, 2003; 42(11): 1383 - 1389. [Abstract] [Full Text] [PDF]
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J. M. Forbes, M. E. Cooper, M. D. Oldfield, and M. C. Thomas Role of Advanced Glycation End Products in Diabetic Nephropathy J. Am. Soc. Nephrol., August 1, 2003; 14(90003): S254 - 258. [Abstract] [Full Text] [PDF]
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R. Asleh, S. Marsh, M. Shilkrut, O. Binah, J. Guetta, F. Lejbkowicz, B. Enav, N. Shehadeh, Y. Kanter, O. Lache, et al. Genetically Determined Heterogeneity in Hemoglobin Scavenging and Susceptibility to Diabetic Cardiovascular Disease Circ. Res., June 13, 2003; 92(11): 1193 - 1200. [Abstract] [Full Text] [PDF]
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D. A. Kass Getting Better Without AGE: New Insights Into the Diabetic Heart Circ. Res., April 18, 2003; 92(7): 704 - 706. [Full Text] [PDF]
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S. M. Twigg, Z. Cao, S. V. MCLennan, W. C. Burns, G. Brammar, J. M. Forbes, and M. E. Cooper Renal Connective Tissue Growth Factor Induction in Experimental Diabetes Is Prevented by Aminoguanidine Endocrinology, December 1, 2002; 143(12): 4907 - 4915. [Abstract] [Full Text] [PDF]
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S. M. Twigg, A. H. Joly, M. M. Chen, J. Tsubaki, H.-S. Kim, V. Hwa, Y. Oh, and R. G. Rosenfeld Connective Tissue Growth Factor/IGF-Binding Protein-Related Protein-2 Is a Mediator in the Induction of Fibronectin by Advanced Glycosylation End-Products in Human Dermal Fibroblasts Endocrinology, April 1, 2002; 143(4): 1260 - 1269. [Abstract] [Full Text] [PDF]
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)
compared with the curves generated with a 14-kDa fragment of CTGF
(IGFBP-rP2; 
), endogenous intact CTGF present in
2097 cell-conditioned medium (
), and 2097 cell whole cell lysates
(
). B, Results of analysis of CTGF in the whole cell lysates by CTGF
ELISA after treatment up to 72 h with AGE or control BSA, each at
100 µg/ml. Data are the mean ± 1 SD of four
independent experiments. *, P < 0.05
vs. control BSA on day 3. C, Results showing
cell-associated CTGF after AGE treatment compared with control BSA (100
µg/ml) at 72 h, using biotinylated anti-CTGF IgG primary
antibody followed by HRP-labeled secondary antibody or the
secondary antibody alone, as described in Materials and
Methods. Data are the mean ± 1 SD of four
independent experiments. *, P < 0.05
vs. all other groups.
