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Department of Cell Biology and Anatomy, University of Miami School of Medicine, Miami, Florida 33136 USA;
FibroGen, Inc., South San Francisco, California 94080, USA; and
* Cleveland Clinic Florida, Ft. Lauderdale, Florida 33309, USA
2Correspondence: Department of Cell Biology and Anatomy, University of Miami School of Medicine (R-124), 1600 NW 10th Ave., Miami, FL 33136, USA. E-mail: ggrotend{at}mednet.med.miami.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: fibrosis • wound repair • granulation tissue • cholera toxin • transgenic
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Peptide factors appear to orchestrate the
repair cascade not only by controlling platelet function, leukotaxis,
cytokine synthesis, and angiogenesis, but also by directing the
progression of fibroblast phenotypes that ultimately results in the
formation of mature scar tissue by regulating the ability of
fibroblasts to proliferate and to quantitatively and qualitatively
change their extracellular matrix component production profiles. One of
the primary regulatory factors involved in initiating the wound healing
cascade is transforming growth factor beta
(TGF-ß).3
Although TGF-ß is involved in many diverse physiological processes
including inflammation, neoplastic progression, cell cycle regulation,
and development, its central role in the wound healing cascade has been
confirmed by numerous in vitro and in vivo
studies. In vitro, the ability of TGF-ß to stimulate the
proliferation and extracellular matrix component synthesis of cultured
fibroblasts is well documented (2
3
4
5
6
7)
. Similarly,
injection of TGF-ß into the subcutis of neonatal mice induces the
formation of granulation tissue (4
, 8)
and TGF-ß or
TGF-ß mRNA has been detected at sites of normal wound repair as well
as in the lesions of numerous fibrotic disorders (9
10
11
12)
.
Previous studies have indicated that the TGF-ß stimulation of
extracellular matrix synthesis is not shared by other growth factors
such as fibroblast growth factor or platelet-derived growth factor
(6
, 13)
. The observation that protein synthesis is
required for TGF-ß to induce extracellular matrix protein gene
expression indicates that some other intermediate factor or factors may
be involved in these signaling pathways (6)
.
One protein with demonstrated potential as a downstream mediator for
TGF-ß signaling in fibroblastic cells is the cysteine-rich peptide,
connective tissue growth factor (CTGF) (14)
. CTGF is not
only mitogenic and chemotactic for fibroblasts (15)
, but
also stimulates the synthesis of at least two extracellular matrix
components: type I collagen and fibronectin (8)
.
Fibroblast CTGF gene expression is strongly induced by TGF-ß but not
by other growth factors (16)
. Protein synthesis is not
required for TGF-ß induction of CTGF gene expression (16
, 17)
, and a novel TGF-ß response element has been identified in
the CTGF promoter (17)
. This novel TGF-ß response
element shares partial sequence homology with the consensus sequence
for the cAMP response element, and its activation by TGF-ß is
inhibited by cAMP analogs and agents elevating intracellular cAMP
(14
, 18)
. Coordinate expression of TGF-ß and CTGF mRNAs
has been observed during wound repair (16)
, and CTGF
transcripts are induced in skin fibroblasts at the site of intradermal
injection of TGF-ß in neonatal mice (8)
. Moreover, CTGF
injection sites develop granulation tissue histologically similar to
that induced by TGF-ß injection (8)
. Thus, CTGF mimics
many of the fibroblast-activating/matrix-forming activities of TGF-ß
both in vitro and in vivo; CTGF gene expression
is directly stimulated by TGF-ß, strongly suggesting that CTGF may
function as a downstream mediator of TGF-ß action on fibroblasts.
Two of our recent in vitro studies support this hypothesis,
demonstrating that CTGF acts as a downstream mediator of one TGF-ß
regulated fibroblast function: TGF-ß-induced, anchorage-independent
proliferation of a continuous line of cultured normal rat kidney (NRK)
fibroblasts (18
, 19)
. We investigated whether CTGF is also
essential for TGF-ß-induced fibroblast collagen synthesis in
vitro and if agents that block CTGF synthesis or action can
inhibit TGF-ß-induced granulation tissue formation in
vivo.
Our results confirm that CTGF by itself is a potent inducer of cultured fibroblast collagen synthesis and demonstrate that blocking CTGF action with an anti-CTGF antibody or blocking CTGF synthesis with an antisense CTGF gene or agents elevating cAMP prevent TGF-ß-induced collagen synthesis in NRK and human foreskin fibroblast cultures. Using in vivo studies, we found that cAMP-elevating agents can effectively block TGF-ß induction of a CTGF promoter transgene in neonatal murine dermis as well as selectively reduce TGF-ß-induced granulation tissue collagen formation in wound chambers subcutaneously (s.c.) implanted in rats. However, inhibition of fibroblast influx/proliferation may also contribute to the decreased collagen content of granulation tissue, as cAMP elevation significantly decreased the number of fibroblastic cells present at TGF-ß injected dermal sites in neonatal mice. Collectively, these data demonstrate that CTGF mediates TGF-ß-induced collagen synthesis and suggest that CTGF may be a useful target for antifibrotic therapies, as anti-CTGF antibodies and cAMP-elevating agents both have the potential to act as antifibrotic therapeutics by inhibiting CTGF action or TGF-ß-induced CTGF gene expression.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Recombinant human
TGF-ß1 and TGF-ß2 and
CTX were obtained from Gibco BRL (Gaithersburg, Md.) and 8-Br-cAMP and
8-Br-cGMP from Sigma Chemical (St. Louis, Mo.). Chicken anti-human
rCTGF was raised against recombinant human CTGF as described previously
(19)
.
Goat anti-CTGF immunoglobulin G production and purification
Goat anti-human CTGF antibodies were prepared using a standard
IACUC approved protocol where the animals were challenged initially
(day 1) with 20 µg of pure recombinant human CTGF suspended in 150
µg of Freund's complete adjuvant, followed by subsequent challenges
of 20 µg CTGF suspended in 100 µg of Freund's incomplete adjuvant
on days 28, 35, 42, 49, 56, 77, 84, etc. The animals were bled on days
42, 49, 56, 63, 91, 98, 105, etc. Anti-CTGF titers were initially
screened using Western blot assays and the highest titers of serum were
pooled. The total immunoglobulin G (IgG) fraction from the serum was
isolated using protein G Sepharose; this material was further purified
using CTGF-Affi-Gel 10 as an affinity matrix to yield CTGF-specific
IgG.
Cell cultures
NRK fibroblasts, clone NRK-49F, a continuous line of cultured
normal rat kidney fibroblasts (20)
, were obtained from
American Type Culture Collection (ATCC; Rockville, Md.) and from Dr. R.
Assoian, University of Pennsylvania. Human foreskin fibroblasts were
established from explant cultures. Cell cultures were maintained in
Dulbecco's modified Eagle media (DMEM) containing 2.5% fetal bovine
serum (FBS) and 2.5% Nu-Serum I (NS) (Collaborative Biomedical,
Bedford, Mass.) and passaged prior to confluence. Numerous
investigators (2
3
4
5
6
7)
have shown that both NRK and foreskin
fibroblasts are sensitive target cells for TGF-ß regulation of
fibroblast proliferation and collagen synthesis. NRK fibroblasts stably
transfected with an antisense CTGF gene (NRK-ASCTGF) were transfected
and cloned in our laboratory as described previously (18)
.
Assay of fibroblast collagen synthesis
Growth-arrested monolayers of NRK or foreskin fibroblasts were
prepared by seeding 10,000 cells/well in 48-well plates and allowing
fibroblasts to grow to confluence in 5 to 7 days in DMEM + 2.5%
FBS/NS. Fibroblast monolayers were then serum starved in DMEM
containing 25 mM HEPES and ITS premix (Collaborative Biomedical) for 1
to 8 days before initiating assays for the effect of CTGF and other
biological agents on collagen synthesis. To evaluate the effect of CTGF
and other biological agents on collagen synthesis, biological agents
along with 10 ng/ml of insulin-like growth factor II and 50 µg/ml
ascorbic acid were added to the starved culture media; collagen
synthesis during the terminal 24 h of a 48 h treatment period
was assessed by measuring 3H-proline
incorporation into pepsin-resistant, salt-precipitated
extracellular/cell surface-associated collagen using a quantitative
assay (21)
we have used extensively (22
23
24
25)
.
Each experimental condition was done in duplicate or triplicate wells;
results are expressed as cpm/well ± SE, as the tested
reagents caused only minor changes in the cell numbers of these
growth-arrested cultures over the 2 day assay period. When comparing
cell types with differing confluent cell densities, results are
expressed as cpm/103 cells after counting
trypsinized cell monolayers. Moreover, the length of growth arrest had
no quantitative effect on responses to any test reagent except CTGF,
whose collagen synthesis stimulatory activity was most notable in
cultures arrested for the longest time periods. We previously noted a
similar effect for CTGF induction of fibroblast DNA synthesis
(15)
.
Western blot analysis
Western blots of supernatant media CTGF were performed as
described previously (8)
. Briefly, cell culture
supernatant media were subject to sodium dodecyl sulfate-polyacrylamide
gel electrophoresis (SDS-PAGE) on 12% acrylamide gels and transferred
to nitrocellulose filters by electroblotting. Blots were incubated with
2 µg/ml chicken anti-CTGF antibodies and then with
peroxidase-conjugated affinity-purified rabbit anti-chicken IgY (1:1000
dilution, Organon Teknika-Cappel, West Chester, Pa.). Antigens were
detected with a chemiluminescent substrate system (ECL, Amersham,
Arlington Heights, Ill.).
RNA isolation and Northern blotting
Total cellular RNA was extracted from cell monolayers by a
single-step method of RNA isolation (Trizol Reagent, Gibco BRL) and
Northern blot analysis was performed largely as described previously
(8
, 25)
. Hybridization signals were compared using a
scanner and quantitated based on a standard area of pixels after
normalizing for 18s rRNA controls. Double-stranded cDNA fragments used
for probes were labeled by random priming with digoxigenin-modified
dUTP and hybridization signals were detected with an anti-digoxigenin
antibody coupled to alkaline phosphatase and a chemiluminescent
substrate detection system (Boehringer Mannheim, Indianapolis, Ind.).
The CTGF probe was derived from a 1.1 kb human cDNA fragment that
encompassed the open reading frame of the CTGF transcript. The
pro
1(I)collagen probe was a 1.5 kb fragment at the 3' end of the
open reading frame cleaved from plasmid Hf677 (ATCC).
CTGF promoter/LacZ constructs and transgenic mice development
A 965 bp SacI/XhoI fragment containing the
CTGF promoter was cloned into a pSV-ß-galactosidase plasmid (Promega,
Madison Wis.). The final construct, PRM1, contained the CTGF promoter,
the bacterial ß-galactosidase gene, and an SV40 polyadenylation
signal, and was digested with SmaI/BamHI and
separated from the plasmid by agarose gel electrophoresis prior to
injection into pronuclei. The final 897 bp CTGF promoter sequence was
composed of bases -823 to +74 relative to the transcription start site
and is identical to the P0 fragment of the human CTGF promoter
described previously that exhibits a strong induction by TGF-ß
(17)
.
The PRM1 DNA fragment were submitted to the University of Miami
Transgene Facility, and pronuclear injections and breeding were
performed routinely (26)
. Briefly, ova were obtained from
superovulated C57BL/6J females mated to SJL males. Transgenic mice were
generated by introducing 1.5 ng/ml of linearized DNA into one of the
pronuclei of recently fertilized mouse ova. Zygotes were transferred
into the oviducts of pseudopregnant B6SJL mice. Tail genomic DNA
was obtained using prescribed techniques (27)
, and
Southern blotting was performed routinely using a probe consisting
of a 3 kb HindIII fragment of the original plasmid. Two
positive founder mice were separated and each line was bred to
homozygosity. The resulting PRM1 transgenic lines were named
TgN(CTGGLRp)1Grg and TgN(CTGGLRp)2Grg according to the
standardized nomenclature for transgenic animals (28)
.
Although both lines expressed detectable levels of lacZ at sites of
endogenous CTGF expression in the embryos, the TgN1Grg line exhibited a
more intense signal and was used for the studies reported here.
In vivo CTGF promoter/LacZ transgene induction
studies
One-day-old transgenic neonatal mice were injected once daily
for 3 days with 800 ng TGF-ß in a 20 µl saline vehicle in the
dermis at the nape of the neck and simultaneously with 800 ng TGF-ß
and 100 ng of cholera toxin in the dermis at a site on the top line
over the lumbar vertebrae. Transgenic neonatal mice were assayed for
ß-galactosidase activity as described previously, with X-gal staining
producing a grossly visible blue stain in the cells and tissues
expressing lacZ (29)
. Briefly, embryos and neonates were
fixed for 1 h in paraformaldehyde, then washed three times for 30
min each in phosphate-buffered saline containing 2 mM
MgCl2, 4 mM potassium ferrocyanide, 5 mM
potassium ferricyanide, 0.2% Nonidet P-40, and 0.1% deoxycholate.
After X-gal staining, the neonates were washed in phosphate-buffered
saline and soaked in formalin for 24 h and 70% ethanol for
24 h.
Wound chamber studies
Wound chamber studies were performed using the previously
described Schilling-Hunt wound chamber model (30
31
32
33)
.
Briefly, a 1 cm longitudinal incision was made on the middle of the
dorsum of anesthetized and shaved male Sprague-Dawley rats (300 g).
After blunt scissor dissection beneath the panniculus carnosus on both
sides of the incision, wound chambers (1x3 cm), with medical grade
silicone elastomer plugs on each end, were implanted into the two
dorsal s.c. pockets. The chambers used in these experiments were
composed of high-density nylon mesh, which does not induce any foreign
body reaction. Animals were randomized into eight treatment groups;
beginning on day 0, six chambers per treatment group were injected
daily with either saline/1% bovine serum albumin control vehicle, CTX
(0.1 and 1.0 mg), or 8-Br-cAMP (10 and 100 mg) with or without 1 µg
of TGF-ß2, which was injected every other day
starting on day 1. This schedule was maintained for 14 days, on which
day the animals were killed and the chambers removed by dissection. All
tissue was carefully removed from the outside of the chamber and the
chamber contents were solubilized by pepsin digestion. Aliquots of the
pepsin digests were then subjected to SDS-PAGE and Coomassie staining,
and compared by densitometry to Coomassie stained collagen standards to
biochemically quantitate chamber collagen content. Results were
averaged for each treatment group and expressed as mg of
collagen/chamber ± SE.
s.c. Administration of factors to mice
Neonatal NIH Swiss mice were injected in the nape of the neck
daily for 3 days with 20 µl total volume of either saline control,
800 ng TGF-ß, or 400 ng CTGF with or without the addition of 10, 50,
or 100 ng of cholera toxin to the injection mixture (4
, 8)
. At least three mice were used for every injection set.
Animals were killed 24 h after the last injection and large
biopsies of the area containing and surrounding the injection site were
removed. Sections were prepared for histopathologic examination after
routine formalin fixation, processing, and staining with either
Mayer's hematoxylin and eosin or tissue-specific Giemsa.
Immunohistochemical staining with rabbit anti-mouse CD45 (leukocyte
common antigen) was performed routinely on additional 5 µM sections
to further delineate monocytes from fibroblasts.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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1(I)collagen mRNA expression and type I collagen protein
synthesis in NRK fibroblasts when RNA expression is assessed by
Northern blotting and collagen synthesis by PAGE analysis after
[35S]methionine biosynthetic labeling
(8)
. However, such data are not quantitative and we have
now analyzed the effect of CTGF on NRK fibroblast synthesis using a
quantitative microwell assay (21)
. The dose-response curve
presented in Fig. 1
demonstrates that CTGF can stimulate large increases in collagen
synthesis over a range of low concentrations from 0.04 to 1 ng/ml, with
maximal activity observed at only 1 ng/ml (27 pM) of CTGF. Higher CTGF
concentrations of up to 25 ng/ml did not further stimulate collagen
synthesis (data not shown). Although we have not investigated here the
individual collagen types whose synthesis is stimulated by CTGF, our
previous report indicates that type I collagen, the major collagen type
produced by NRK fibroblasts, is stimulated by CTGF treatment
(8)
. Whether CTGF stimulates the synthesis of other
collagen types and their contribution to the collagen levels measured
by our quantitative microwell assay remain to be investigated. Since
the levels of collagen synthesis induced by 1 ng/ml or less of CTGF
were comparable to that induced by 5 ng/ml TGF-ß (Fig. 1)
and CTGF
gene expression is known to be induced by TGF-ß (16
, 17)
, we wanted to determine whether CTGF was required for
TGF-ß stimulation of fibroblast collagen synthesis. To do this, we
examined TGF-ß-induced collagen synthesis in NRK fibroblasts stably
transfected with an antisense CTGF gene (NRK-ASCTGF), which effectively
blocks TGF-ß-activated CTGF gene expression (18)
. As
shown in Fig. 1
, the collagen synthesis of these NRK-ASCTGF fibroblasts
was only minimally stimulated by TGF-ß treatment, suggesting that
CTGF synthesis is essential for TGF-ß induction of collagen
synthesis.
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We also prepared an anti-CTGF-specific neutralizing antibody by
immunizing goats with purified recombinant human CTGF and investigated
whether anti-CTGF IgG could block TGF-ß-induced fibroblast collagen
synthesis. Using a similar strategy we previously demonstrated that an
anti-CTGF antibody raised in chickens blocks the TGF-ß-induced,
anchorage-independent growth of NRK fibroblasts (18)
.
Because our chicken anti-CTGF exhibited only limited neutralizing
activity, requiring 500 µg/ml of IgY for 50% neutralization of NRK
fibroblast proliferation, we developed the goat anti-CTGF antibody
described here, which has similar properties to the chicken anti-CTGF
IgY but is 10-fold more potent, causing a 50% neutralization of
TGF-ß-induced, anchorage-independent growth of NRK fibroblasts at
only 50 µg/ml (data not shown). As shown in Fig. 2
, 50 µg/ml of this affinity-purified goat anti-CTGF IgG almost totally
neutralized the collagen synthesis stimulatory activity of 25 ng/ml of
CTGF in NRK fibroblast cultures. An equivalent concentration of
purified nonimmune goat IgG had no neutralizing activity, and the goat
anti-CTGF IgG had little effect on NRK fibroblast basal collagen
synthesis. When the goat anti-CTGF IgG was added to the media of NRK
fibroblast cultures stimulated with 5 ng/ml of TGF-ß, the anti-CTGF
blocked the TGF-ß-induced collagen synthesis in a
concentration-dependent manner, causing approximately a 50%
neutralization at 10 µg/ml and a complete (100%) inhibition at 50
µg/ml (Fig. 3
A). Similar results were obtained when goat anti-CTGF was
added to TGF-ß stimulated human foreskin fibroblast cultures (Fig. 3B
). These data demonstrate that CTGF mediates
TGF-ß-induced fibroblast collagen synthesis.
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Elevation of intracellular cAMP blocks TGF-ß-induced collagen
synthesis by cultured NRK and human foreskin fibroblasts
We previously reported that elevation of intracellular cAMP levels
can block the DNA synthesis needed for cell proliferation in
TGF-ß-stimulated NRK fibroblasts by suppressing CTGF gene expression
(19)
. To determine what effect suppressing CTGF gene
expression by elevating levels of cAMP has on TGF-ß-induced collagen
synthesis, we examined growth-arrested cultures of NRK and foreskin
fibroblasts. As detailed in Fig. 4
A, 100 ng/ml of the adenylyl cyclase activator, cholera toxin
(CTX), and a membrane-permeable analog of cAMP, 8-Br-cAMP (1 mM),
inhibited TGF-ß (5 ng/ml) -induced collagen synthesis in NRK
fibroblast cultures by 71% and 76%, respectively. This inhibitory
effect appeared to be selective for agents elevating cAMP as 1 mM
8-Br-cGMP did not decrease TGF-ß stimulation of collagen synthesis.
Moreover, CTX and cAMP had no effect on basal collagen synthesis,
suggesting that only TGF-ß-induced collagen synthesis is subject to
cAMP-mediated down-regulation. These results were confirmed in several
additional experiments with NRK fibroblasts as well as with two
separate strains of human foreskin fibroblasts (Fig. 4B
).
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To determine whether levels of CTGF production correlated with
fibroblast collagen synthesis, as we have previously found in NRK
fibroblast proliferation studies (19)
, we compared
foreskin fibroblast pro1(I)collagen and CTGF mRNA levels by
Northern blotting and assayed supernatant media for CTGF peptides by
Western blotting. The data presented in Fig. 5
for one strain of foreskin fibroblasts show that after 24 h of
treatment, TGF-ß-induced increases in the 4.8 and 5.8 kb mRNA
transcripts for pro1(I)collagen. These increases were paralleled by
enhanced levels of the 2.4 kb CTGF mRNA transcript and immunoreactive
38 kDa CTGF peptides. Moreover, 8-Br-cAMP almost completely inhibited
the TGF-ß induction of CTGF peptides and mRNA transcripts and caused
a 50–60% decrease in pro1(I)collagen mRNA transcripts. Taken
together, these data indicate that elevated cAMP levels selectively
inhibit TGF-ß-induced fibroblast collagen synthesis via
transcriptional mechanisms, depressing CTGF production. Consequently,
TGF-ß-induced fibroblast collagen synthesis appears to be dependent
on the direct action of increased levels of newly synthesized CTGF.
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In our previously reported studies investigating NRK fibroblast
mitogenesis, we were able to confirm the requirement for CTGF in
TGF-ß-stimulated mitogenesis by overcoming the cAMP or CTX blockade
of TGF-ß-induced proliferation by the addition of exogenous CTGF to
the cultured cells (19)
. Accordingly, we tested the
ability of CTGF to restore collagen synthesis in the TGF-ß-activated
cell cultures that had been treated with either CTX or 8-Br-cAMP.
Surprisingly, we could not restore the collagen synthesis, even with
CTGF concentrations as high as 25 ng/ml (Fig. 6
), although CTGF alone in concentrations of 1 ng/ml can clearly induce
maximal collagen synthesis (Fig. 1)
. This apparent paradox was
investigated by assaying the effect elevated intracellular cAMP levels
had on collagen synthesis induced by CTGF itself. The data reported in
Fig. 6
indicate that in addition to inhibiting collagen synthesis by
blocking TGF-ß-induced CTGF gene expression, elevated intracellular
cAMP levels also directly suppress CTGF-induced collagen synthesis, as
both CTX and 8-Br-cAMP treatment blocked NRK fibroblast collagen
synthesis stimulated by treatment with CTGF itself. Thus, cAMP appears
to influence TGF-ß stimulation of collagen production in two ways:
1) by inhibiting CTGF synthesis and 2) by
blocking the CTGF signaling pathway that regulates collagen synthesis.
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CTX inhibits expression of a CTGF promoter/lacZ reporter transgene
in vivo
Because the adenylyl cyclase activator CTX was capable of
significantly inhibiting the induction of the CTGF gene by TGF-ß
in vitro, we wanted to determine whether it could be used
in vivo to block induction of the CTGF gene. For this
purpose, we used a line of transgenic mice we constructed that contains
a transgene composed of the human CTGF promoter controlling the
expression of a bacterial ß-galactosidase (lacZ) reporter gene. When
stained with X-gal, these transgenic mice produce a grossly visible
blue stain in the cells and tissues expressing lacZ. Using in
situ hybridization techniques, we have demonstrated that s.c.
injection of TGF-ß in the nape of the neck of neonatal mice induced
CTGF transcripts in the fibroblasts, forming granulation tissue at the
site of injection (8)
. We repeated this study with our
CTGF promoter/lacZ transgenic mouse line. Neonatal transgenic mice were
injected as described previously (4
, 8)
with TGF-ß (800
ng) alone or TGF-ß (800 ng) and CTX (100 ng) daily for 3 days.
Individual mice were injected with the two mixtures at sites separated
by 1 cm so that each individual could serve as an internal control. The
mice were killed 6 h after the last injection and stained with
X-gal to detect expression of the transgene. In the region of the
TGF-ß injected skin, a circumscribed area of blue stain was grossly
visible through the epidermis, and the dermis and superficial subcutis
exhibited blue staining granulation tissue (Fig. 7
A–C). In contrast, the site injected with both TGF-ß and
CTX lacked any detectable blue staining (Fig. 7A
).
Saline-injected transgenic controls did not produce granulation tissue
at the site, and the dermis and subcutis did not stain (data not
shown). These results indicate that CTX can effectively inhibit CTGF
expression in vivo, as predicted from our cell culture
experiments, and suggest that our transgenic mice may be a convenient
model with which to study regulation of CTGF gene expression in
vivo.
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CTX and 8-Br-cAMP selectively block TGF-ß-induced collagen
deposition in vivo
To quantitatively evaluate the effects of CTX and cAMP analogs on
TGF-ß stimulation of extracellular matrix formation in
vivo, we used the Schilling-Hunt wound chamber model (30
, 31)
. We and others have used this model to evaluate the ability
of various growth factors, including TGF-ß, to influence the ongoing
wound repair process that occurs in response to s.c. implantation of
the wound chambers (16
, 32
, 33)
. Because the chambers are
all of uniform size and shape, the tissue formed within the chamber can
be harvested precisely and quantitative measurements can be made on the
contents. The results of these experiments are illustrated in
Fig. 8
. As expected, TGF-ß injection stimulated a quantitative increase of
42% in the amount of collagen present within harvested chambers
compared to control saline-injected chambers. Coinjection of either 1
µg of CTX or 10 µg or 100 µg of 8-Br-cAMP with TGF-ß completely
blocked the increase in collagen deposition induced by TGF-ß,
reducing collagen deposition to levels comparable to or less than
saline-injected controls. The collagen levels in chambers treated with
either 0.1 or 1 µg of CTX without TGF-ß were relatively unaffected
and exhibited no more than an 18% reduction from saline-injected
control chambers. These results demonstrated that elevation of in
vivo fibroblast cAMP levels totally inhibits the wound chamber
collagen deposition induced by exogenous TGF-ß. Elevation of cAMP
appeared to only partially suppress the collagen deposition that occurs
in the absence of exogenous TGF-ß. This suggests that the collagen
deposition occurring in untreated wound chambers is only partially
induced by endogenous TGF-ß and is consistent with previous
observations that wounds in rats treated with a neutralizing antibody
to TGF-ß heal effectively, but have a partially reduced collagen
content and a less scar-like appearance (34)
. This ability
of cAMP elevating agents to selectively inhibit only the collagen
synthesis induced by TGF-ß suggests such agents may have
potential as antifibrotic therapeutics, since they would effectively
inhibit TGF-ß-induced, CTGF-mediated collagen synthesis while
sparing both basal and TGF-ß-independent induced collagen synthesis.
|
CTX blocks granulation tissue fibroblast population increases
induced by TGF-ß in vivo
Although the wound chamber studies provide an accurate assay of
in vivo collagen synthesis, they do not assess the
contribution fibroblast influx/proliferation has on granulation tissue
collagen formation. In contrast, neonatal mice injection studies do
provide an accurate means of assessing the recruitment of fibroblasts
during granulation tissue formation but do not allow for practical
quantitative biochemical analysis of matrix components due to
difficulties in obtaining uniform samples. Since our previous studies
indicate that both TGF-ß and CTGF stimulate a fibroblast-rich
granulation tissue when injected into the dermis of neonatal mice, we
wanted to determine whether CTX could inhibit this effect of TGF-ß.
The formation of granulation tissue in this model of early wound repair
involves both new matrix protein synthesis and a simultaneous increase
in the matrix fibroblast population by both chemotactic recruitment and
subsequent proliferation of fibroblasts at the site, all of which are
demonstrated biological activities of CTGF (4
, 8
, 15)
. The
results of our study are presented as hematoxylin- and eosin-stained
sections in Fig. 9
and as quantitated data counting the number of fibroblasts and
inflammatory neutrophils present in Fig. 10
. Monocytes were differentiated from fibroblasts by immunohistochemistry
for CD45 (leukocyte common antigen) and the number of monocytes was
found to be similar in all injection samples, with counts averaging
1–3 per high-power field. As shown, intradermal injection of 800 ng of
TGF-ß induced accumulation of a loose extracellular matrix and the
presence of a significant fibroblast population without substantial
inflammatory cell infiltration (Fig. 9A
, Fig. 10
). When 100
ng of CTX was injected, a different response, consisting of
neutrophilic inflammation with edema and few fibroblasts, was observed
(Fig. 9B
, Fig. 10
). Since CTX is a bacterially derived
protein used as a vaccine adjuvant due to its leukotactic and
lymphokine-inducing activities, the leukocytic infiltrate was expected
(35)
. At a dose of 100 ng of CTX, the neonates showed no
adverse systemic clinical signs, had full stomachs at necropsy, and
were similar in size to noninjected littermates. When 800 ng TGF-ß
and 100 ng CTX were coinjected, the markedly increased fibroblast
population induced by TGF-ß was greatly reduced and the histological
appearance more closely resembled that injected with CTX alone (i.e., a
neutrophilic infiltrate but few fibroblasts) (Fig. 9C
, Fig. 10
). We also injected doses of 10 ng and 50 ng of CTX with 800 ng of
TGF-ß and obtained a relative dose response in the number of
fibroblasts present at the site (data not shown).
|
|
To determine whether CTX was acting exclusively by inhibiting CTGF gene
expression or was also blocking CTGF activity, we coinjected 400 ng of
CTGF with CTX. The resulting granulation tissue (Fig. 9D
,
Fig. 10
) contained the expected neutrophilic infiltrate and an enhanced
number of fibroblasts comparable to sites injected with CTGF alone
(8)
. This indicates that CTX does not directly
block the ability of CTGF to increase fibroblast population numbers
during the formation of granulation tissue. Accordingly, when CTGF was
coinjected with TGF-ß and CTX, it prevented CTX from down-regulating
TGF-ß-stimulated fibroblast population numbers by replacing the
endogenous CTGF whose synthesis was blocked by CTX injection (Fig. 9
E, Fig. 10
). Thus, it appears that cAMP-elevating
agents inhibit TGF-ß-induced chemotactic and mitogenic responses that
lead to increased in vivo granulation tissue fibroblast
populations solely by blocking CTGF gene expression. In contrast, the
synthesis of matrix components such as collagen is inhibited by the
blockade of both CTGF synthesis and CTGF action.
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
, 11)
. Nonetheless,
many believe that TGF-ß plays a major role in initiating these
complex biological processes, as TGF-ß stimulates both fibroblast
proliferation and the synthesis and secretion of several extracellular
matrix components (1
2
3
4
5
6
7
8
9
10
11
12)
. Consequently, there has been an
intense effort to identify agents that selectively block the biological
actions of TGF-ß, much of which has focused on TGF-ß signaling
pathways. One signaling molecule selectively induced in connective
tissue cells by TGF-ß is the 38 kDa cysteine-rich peptide, CTGF
(16
, 17)
. CTGF mimics many of TGF-ß actions on
connective tissue cells, acting as a fibroblast chemoattractant,
mitogen, and inducer of extracellular matrix component synthesis
(8
, 15
, 18)
. In previous studies with cultured
fibroblasts, we demonstrated that the blocking of CTGF action with
anti-CTGF antibodies or blocking CTGF gene expression with an antisense
CTGF gene or with agents elevating cAMP inhibits TGF-ß-induced
fibroblast proliferation (18
, 19)
.
In the studies reported here, we examined whether blockade of CTGF
action or synthesis could also inhibit TGF-ß-induced fibroblast
collagen synthesis. Our results demonstrate that similar to cell
proliferation, blockade of CTGF action with neutralizing antibodies or
inhibition of CTGF synthesis by gene repression significantly reduces
fibroblast collagen synthesis induced by TGF-ß. Specific goat
anti-CTGF antibodies reduced collagen protein production induced by
TGF-ß by 85–100%, demonstrating that in the fibroblast cultures we
have studied, TGF-ß induces collagen synthesis exclusively via a
CTGF-dependent pathway. Whether TGF-ß induces collagen synthesis in
all fibroblasts or other mesenchyme-derived cells, such as chondrocytes
and vascular smooth muscle cells, by CTGF-dependent pathways remains to
be investigated. We also found that NRK fibroblasts stably transfected
with an antisense CTGF gene were virtually unresponsive to TGF-ß
induction of collagen synthesis and that agents that block CTGF gene
induction by TGF-ß, such as CTX and 8-Br-cAMP, reduced TGF-ß
induction of pro1(I)collagen mRNA transcripts by 50–60% and
reduced collagen (primarily type I) protein production by 70–80%.
These data are consistent with previous studies demonstrating that
TGF-ß stimulation of collagen gene expression requires protein
synthesis (6)
. The current work demonstrates that CTGF is
one of the proteins that must be synthesized for TGF-ß up-regulation
of type I collagen gene expression. Whether CTGF mediates
TGF-ß-induced expression of other collagen types or other
TGF-ß-regulated fibroblast genes is currently unknown. However, some
TGF-ß-inducible effects on fibroblasts appear to be CTGF independent,
as CTX and 8-Br-cAMP do not block TGF-ß-induced changes in NRK and
NIH/3T3 fibroblast morphology (19)
. Such CTGF-independent
actions may be mediated by other TGF-ß signaling pathways, such as
Smads, which have recently been implicated in TGF-ß activation of the
human type VII collagen gene (36)
. Nevertheless, elevation
of intracellular cAMP levels has been reported to down-regulate the
expression of several additional TGF-ß inducible genes, including
c-sis, c-myc, collagenase, procathepsin L,
laminin, and plasminogen activator inhibitor (13
, 37
38
39
40
41)
.
Thus, sensitivity to cAMP appears to be a common feature for many
TGF-ß-regulated genes. Whether the expression of any of these genes
is regulated via a CTGF-dependent signaling pathway,
however, remains to be investigated.
Even though this paper appears to be the first to demonstrate that cAMP
inhibits TGF-ß-induced collagen synthesis in fibroblasts, several
studies have shown that elevation of intracellular cAMP levels with
phosphodiesterase inhibitors such as pentoxifylline inhibit fibroblast
growth and collagen synthesis induced by serum and/or
fibroblast-activating cytokines (23
, 24
, 42)
. Similarly,
prostaglandin E2, a known inducer of intracellular cAMP, has long been
known to inhibit serum-driven fibroblast collagen synthesis
(43)
. Since pentoxifylline inhibition of fibroblast
collagen synthesis in the presence of serum has generally been reported
to be greater than in fibroblasts deprived of serum stimulation for
48 h, one potential mechanism consistent with our observations is
that pentoxifylline-elevated cAMP levels may be inhibiting collagen
synthesis by blocking the induction of CTGF synthesis by TGF-ß
contained in serum. This may be the case, as we found that 8-Br-cAMP
inhibits the collagen synthesis of NRK fibroblasts cultured in 5% FBS
(data not shown). Moreover, pentoxifylline has been reported to inhibit
collagen synthesis in animal models of hepatic fibrosis thought to be
dependent on TGF-ß for development, demonstrating that in addition to
8-Br-cAMP and CTX, other agents that elevate intracellular levels of
cAMP in connective tissue cells can also function as effective in
vivo inhibitors of fibrotic tissue formation (44)
.
The decrease in collagen synthesis caused by pentoxifylline has been
linked to the down-regulation of the transcription factor nuclear
factor 1 (25)
, which has been reported to be required for
TGF-ß-induced activation of the type I and III procollagen promoters
(45
, 46)
. The role of CTGF, whether any, in regulating
nuclear factor l expression is currently unknown.
In previous studies where we identified elevated intracellular cAMP as
an important repressor of TGF-ß-induced, CTGF gene expression, we
were able to overcome the blockade of TGF-ß-induced cell
proliferation by cAMP or CTX with the addition of exogenous CTGF to the
NRK fibroblast cultures (19)
. In contrast to these
observations, our current studies revealed that addition of CTGF to NRK
fibroblast activated with TGF-ß in the presence of cAMP or CTX did
not restore collagen production because elevated intracellular cAMP
levels also blocked collagen synthesis directly induced by CTGF itself.
Combined, our data suggest that there are cAMP-sensitive differences in
the signaling pathways from the CTGF receptor for cell proliferation
vs. collagen synthesis, as schematically illustrated in Fig. 11
. The nature of the signaling pathways distal to the CTGF receptor that
regulate collagen synthesis and are affected by cAMP remain to be
determined. However, they likely involve complex indirect interactions,
since no collagen gene promoters are known to contain the cAMP
responsive element (47)
.
|
Our animal study findings parallel our tissue culture observations and suggest that cAMP-elevating agents may have therapeutic potential as agents selectively inhibiting CTGF-induced fibrosis. Intradermal coinjection of CTX with TGF-ß suppressed TGF-ß activation of a human CTGF promoter/lacZ reporter transgene in transgenic mice and both 8-Br-cAMP and CTX blocked TGF-ß-induced collagen deposition in a wound chamber model of fibrosis in rats. CTX also reduced granulation tissue fibroblast population increases induced by intradermal injections of TGF-ß in neonatal mice, but not increases induced by CTGF or TGF-ß combined with CTGF. Thus, blockade of in vivo CTGF synthesis reduces TGF-ß-induced granulation tissue formation by inhibiting both collagen synthesis and fibroblast accumulation. Whereas the use of two separate in vivo wound repair models was necessary to accurately quantitate granulation tissue collagen content and fibroblast population numbers, their use also allowed us to examine the effects of elevated cAMP levels on granulation tissue formation induced by both short- and long-term TGF-ß treatment. The short-term treatment of three daily TGF-ß intradermal injections used in our neonatal mice studies causes the formation of a loose, fibroblast-rich granulation tissue closely resembling that seen in early normal wound repair. In contrast, the long-term treatment schedule of TGF-ß injections every other day for 14 days used in our wound chamber studies induces wound chambers filled with large quantities of a dense extracellular matrix rich in organized collagen, more closely resembling the matrix observed in tissue derived from sites of fibrotic disease. Our results demonstrate that elevation of cAMP levels blocks granulation tissue formation and matrix collagen accumulation regardless of whether tissue fibroblast exposure to TGF-ß is acute, as occurs in normal wound repair, or is chronic, as likely occurs in fibrotic diseases, provided that cAMP elevating agents are coadministered with TGF-ß.
The progression of wound repair from inflammation to early granulation
tissue formation through active matrix synthesis to remodeled mature
scar involves a cascade of cytokines, matrix components, and other
factors operating in a specific temporal sequence to drive fibroblasts
and other wound site cell populations through a series of phenotypic
changes (1)
. Collectively, the current and previously
reported data strongly suggest that after initial release of TGF-ß by
activated platelets and infiltrating monocytes, newly synthesized CTGF
stimulates a fibroblast chemotactic response and subsequent
proliferation of fibroblasts, resulting in a markedly increased wound
site fibroblast population. This expanded fibroblast population can
then be stimulated by paracrine/autocrine CTGF to produce enhanced
quantities of collagen and possibly other matrix components. That
TGF-ß synthesis precedes CTGF expression during wound repair has been
aptly demonstrated in wound chamber studies in rats (16)
.
Though TGF-ß was found to be maximally expressed 3 days after
wounding, CTGF peak expression did not occur until days 6–9 and
persisted through day 15, well into the active matrix synthesis phase
of wound repair. Thus, it appears that the prolonged expression of CTGF
and its long time course of action allow wound site fibroblasts to
continue to accumulate, proliferate and synthesize enhanced quantities
of collagen or other matrix components for a considerable time after a
relatively brief exposure to TGF-ß. Thus, TGF-ß/CTGF-mediated
tissue formation after injury may function in many ways like a
developmental process during embryogenesis. That is, brief exposure of
responsive cells in an appropriate environment to an inducer is
sufficient to initiate a complex biological response, such as limb
formation during embryogenesis or, in this case, connective tissue
formation after activation with TGF-ß.
The inappropriate or over expression of tissue-associated TGF-ß has
long been linked with the development of the pathological fibrosis
characteristic of numerous fibrotic disorders (11
, 12)
.
More recently the presence of CTGF expression, often along with
TGF-ß, has also been noted in tissue sampled from several diseases in
which fibrosis represents the major or an accompanying pathology. These
diseases include scleroderma (48)
, keloids and other
localized skin fibrotic diseases (49)
, atherosclerosis
(50)
, glomerulosclerosis (51)
, pulmonary
fibrosis (52)
, inflammatory bowel disease
(53)
, and mammary tumors associated with the development
of a fibrous stroma (54)
. The elucidation of downstream
mediators of TGF-ß, like CTGF in pathways of granulation tissue
formation and fibrosis may provide therapeutic targets for diminishing
these fibrotic effects in a more selective manner. Our results indicate
that agents that elevate intracellular cAMP have the potential to be
the first generation of therapeutics effectively targeting one of these
downstream mediators (i.e., CTGF). Our observations from cell culture
systems and animal models indicate that basal in vitro
collagen synthesis and that portion of induced in vivo
collagen synthesis occurring independent of TGF-ß activation are both
insensitive to down-regulation by cAMP elevating agents. These results
are both surprising and unexpected. They suggest that distinct
molecular mechanisms may regulate basal/TGF-ß-independent vs.
TGF-ß/CTGF-induced collagen gene expression and subsequent
collagen synthesis. Consequently, it may be possible in the future to
selectively inhibit the collagen synthesis induced by TGF-ß at tissue
sites of pathological fibrosis with therapeutic agents designed to
block the synthesis or action of CTGF while sparing basal collagen
synthesis and that induced independently of TGF-ß.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
3 Abbreviations: CTGF, connective tissue growth
factor; CTX, cholera toxin; DMEM, Dulbecco's modified Eagle media;
FBS, fetal bovine serum; Ig, immunoglobulin; NRK, normal rat kidney;
NS, Nu-Serum I; s.c., subcutaneously; SDS-PAGE, sodium dodecyl
sulfate-polyacrylamide gel electrophoresis; TGF, transforming growth
factor.
Received for publication February 18, 1999. Accepted for publication April 17, 1999.
|
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A. LEASK and D. J. ABRAHAM TGF-{beta} signaling and the fibrotic response FASEB J, May 1, 2004; 18(7): 816 - 827. [Abstract] [Full Text] [PDF]
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Q. Garrett, P. T. Khaw, T. D. Blalock, G. S. Schultz, G. R. Grotendorst, and J. T. Daniels Involvement of CTGF in TGF-{beta}1-Stimulation of Myofibroblast Differentiation and Collagen Matrix Contraction in the Presence of Mechanical Stress Invest. Ophthalmol. Vis. Sci., April 1, 2004; 45(4): 1109 - 1116. [Abstract] [Full Text] [PDF]
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M. KJAeR Role of Extracellular Matrix in Adaptation of Tendon and Skeletal Muscle to Mechanical Loading Physiol Rev, April 1, 2004; 84(2): 649 - 698. [Abstract] [Full Text] [PDF]
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G. R. GROTENDORST, H. RAHMANIE, and M. R. DUNCAN Combinatorial signaling pathways determine fibroblast proliferation and myofibroblast differentiation FASEB J, March 1, 2004; 18(3): 469 - 479. [Abstract] [Full Text] [PDF]
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M. Bohm, M. Raghunath, C. Sunderkotter, M. Schiller, S. Stander, T. Brzoska, T. Cauvet, H. B. Schioth, T. Schwarz, and T. A. Luger Collagen Metabolism Is a Novel Target of the Neuropeptide {alpha}-Melanocyte-stimulating Hormone J. Biol. Chem., February 20, 2004; 279(8): 6959 - 6966. [Abstract] [Full Text] [PDF]
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D. W. Esson, A. Neelakantan, S. A. Iyer, T. D. Blalock, L. Balasubramanian, G. R. Grotendorst, G. S. Schultz, and M. B. Sherwood Expression of Connective Tissue Growth Factor after Glaucoma Filtration Surgery in a Rabbit Model Invest. Ophthalmol. Vis. Sci., February 1, 2004; 45(2): 485 - 491. [Abstract] [Full Text] [PDF]
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S. Yamashita, A. Maeshima, I. Kojima, and Y. Nojima Activin A Is a Potent Activator of Renal Interstitial Fibroblasts J. Am. Soc. Nephrol., January 1, 2004; 15(1): 91 - 101. [Abstract] [Full Text] [PDF]
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E. Ogawa, W. M. Elliott, F. Hughes, T. J. Eichholtz, J. C. Hogg, and S. Hayashi Latent adenoviral infection induces production of growth factors relevant to airway remodeling in COPD Am J Physiol Lung Cell Mol Physiol, January 1, 2004; 286(1): L189 - L197. [Abstract] [Full Text] [PDF]
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 K. Heinemeier, H. Langberg, J. L. Olesen, and M. Kjaer Role of TGF-{beta}1 in relation to exercise-induced type I collagen synthesis in human tendinous tissue J Appl Physiol, December 1, 2003; 95(6): 2390 - 2397. [Abstract] [Full Text]
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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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 J. T. Daniels, G. S. Schultz, T. D. Blalock, Q. Garrett, G. R. Grotendorst, N. M. Dean, and P. T. Khaw Mediation of Transforming Growth Factor-{beta}1-Stimulated Matrix Contraction by Fibroblasts: A Role for Connective Tissue Growth Factor in Contractile Scarring Am. J. Pathol., November 1, 2003; 163(5): 2043 - 2052. [Abstract] [Full Text] [PDF]
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A. Holmes, D. J. Abraham, Y. Chen, C. Denton, X. Shi-wen, C. M. Black, and A. Leask Constitutive Connective Tissue Growth Factor Expression in Scleroderma Fibroblasts Is Dependent on Sp1 J. Biol. Chem., October 24, 2003; 278(43): 41728 - 41733. [Abstract] [Full Text] [PDF]
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P. Bonniaud, P. J. Margetts, M. Kolb, T. Haberberger, M. Kelly, J. Robertson, and J. Gauldie Adenoviral Gene Transfer of Connective Tissue Growth Factor in the Lung Induces Transient Fibrosis Am. J. Respir. Crit. Care Med., October 1, 2003; 168(7): 770 - 778. [Abstract] [Full Text] [PDF]
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A Ivarsen, T Laurberg, and T Moller-Pedersen Characterisation of corneal fibrotic wound repair at the LASIK flap margin Br. J. Ophthalmol., October 1, 2003; 87(10): 1272 - 1278. [Abstract] [Full Text] [PDF]
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M. Ruperez, O. Lorenzo, L. M. Blanco-Colio, V. Esteban, J. Egido, and M. Ruiz-Ortega Connective Tissue Growth Factor Is a Mediator of Angiotensin II-Induced Fibrosis Circulation, September 23, 2003; 108(12): 1499 - 1505. [Abstract] [Full Text] [PDF]
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T. D. Blalock, M. R. Duncan, J. C. Varela, M. H. Goldstein, S. S. Tuli, G. R. Grotendorst, and G. S. Schultz Connective Tissue Growth Factor Expression and Action in Human Corneal Fibroblast Cultures and Rat Corneas after Photorefractive Keratectomy Invest. Ophthalmol. Vis. Sci., May 1, 2003; 44(5): 1879 - 1887. [Abstract] [Full Text] [PDF]
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R. M. Mason and N. A. Wahab Extracellular Matrix Metabolism in Diabetic Nephropathy J. Am. Soc. Nephrol., May 1, 2003; 14(5): 1358 - 1373. [Abstract] [Full Text] [PDF]
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 K.-Y. Hung, J.-W. Huang, C.-T. Chen, P.-H. Lee, and T.-J. Tsai Pentoxifylline modulates intracellular signalling of TGF-{beta} in cultured human peritoneal mesothelial cells: implications for prevention of encapsulating peritoneal sclerosis Nephrol. Dial. Transplant., April 1, 2003; 18(4): 670 - 676. [Abstract] [Full Text] [PDF]
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L. L. Soon, T.-A. Yie, A. Shvarts, A. J. Levine, F. Su, and K.-M. Tchou-Wong Overexpression of WISP-1 Down-regulated Motility and Invasion of Lung Cancer Cells through Inhibition of Rac Activation J. Biol. Chem., March 21, 2003; 278(13): 11465 - 11470. [Abstract] [Full Text] [PDF]
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 C. R. Harlow, M. Rae, L. Davidson, P. C. Trackman, and S. G. Hillier Lysyl Oxidase Gene Expression and Enzyme Activity in the Rat Ovary: Regulation by Follicle-Stimulating Hormone, Androgen, and Transforming Growth Factor-{beta} Superfamily Members in Vitro Endocrinology, January 1, 2003; 144(1): 154 - 162. [Abstract] [Full Text] [PDF]
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J. K. Burgess, P. R. A. Johnson, Q. Ge, W. W. Au, M. H. Poniris, B. E. McParland, G. King, M. Roth, and J. L. Black Expression of Connective Tissue Growth Factor in Asthmatic Airway Smooth Muscle Cells Am. J. Respir. Crit. Care Med., January 1, 2003; 167(1): 71 - 77. [Abstract] [Full Text] [PDF]
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J. Li, P. Lothar Schwimmbeck, C. Tschope, S. Leschka, L. Husmann, S. Rutschow, F. Reichenbach, M. Noutsias, U. Kobalz, W. Poller, et al. Collagen degradation in a murine myocarditis model: relevance of matrix metalloproteinase in association with inflammatory induction Cardiovasc Res, November 1, 2002; 56(2): 235 - 247. [Abstract] [Full Text] [PDF]
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E. Gore-Hyer, D. Shegogue, M. Markiewicz, S. Lo, D. Hazen-Martin, E. L. Greene, G. Grotendorst, and M. Trojanowska TGF-beta and CTGF have overlapping and distinct fibrogenic effects on human renal cells Am J Physiol Renal Physiol, October 1, 2002; 283(4): F707 - F716. [Abstract] [Full Text] [PDF]
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C. R. Harlow, L. Davidson, K. H. Burns, C. Yan, M. M. Matzuk, and S. G. Hillier FSH and TGF-{beta} Superfamily Members Regulate Granulosa Cell Connective Tissue Growth Factor Gene Expression in Vitro and in Vivo Endocrinology, September 1, 2002; 143(9): 3316 - 3325. [Abstract] [Full Text] [PDF]
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R. G. Crystal, P. B. Bitterman, B. Mossman, M. I. Schwarz, D. Sheppard, L. Almasy, H. A. Chapman, S. L. Friedman, T. E. King Jr., L. A. Leinwand, et al. Future Research Directions in Idiopathic Pulmonary Fibrosis: Summary of a National Heart, Lung, and Blood Institute Working Group Am. J. Respir. Crit. Care Med., July 15, 2002; 166(2): 236 - 246. [Abstract] [Full Text] [PDF]
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H. Matsuoka, T. Arai, M. Mori, S. Goya, H. Kida, H. Morishita, H. Fujiwara, I. Tachibana, T. Osaki, and S. Hayashi A p38 MAPK inhibitor, FR-167653, ameliorates murine bleomycin-induced pulmonary fibrosis Am J Physiol Lung Cell Mol Physiol, July 1, 2002; 283(1): L103 - L112. [Abstract] [Full Text] [PDF]
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 Q H Song, V E Klepeis, M A Nugent, and V Trinkaus-Randall TGF-{beta}1 regulates TGF-{beta}1 and FGF-2 mRNA expression during fibroblast wound healing Mol. Pathol., June 1, 2002; 55(3): 164 - 176. [Abstract] [Full Text] [PDF]
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E E-D A Moussad, M A E Rageh, A K Wilson, R D Geisert, and D R Brigstock Temporal and spatial expression of connective tissue growth factor (CCN2; CTGF) and transforming growth factor {beta} type 1 (TGF-{beta}1) at the utero-placental interface during early pregnancy in the pig Mol. Pathol., June 1, 2002; 55(3): 186 - 192. [Abstract] [Full Text] [PDF]
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H. Yokoi, M. Mukoyama, A. Sugawara, K. Mori, T. Nagae, H. Makino, T. Suganami, K. Yahata, Y. Fujinaga, I. Tanaka, et al. Role of connective tissue growth factor in fibronectin expression and tubulointerstitial fibrosis Am J Physiol Renal Physiol, May 1, 2002; 282(5): F933 - F942. [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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 J. Liu, V.-M. Kosma, T. Vanttinen, C. Hyden-Granskog, and R. Voutilainen Gonadotrophins inhibit the expression of insulin-like growth factor binding protein-related protein-2 mRNA in cultured human granulosa-luteal cells Mol. Hum. Reprod., February 1, 2002; 8(2): 136 - 141. [Abstract] [Full Text] [PDF]
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J. M. Davidson Smad about Elastin Regulation Am. J. Respir. Cell Mol. Biol., February 1, 2002; 26(2): 164 - 166. [Full Text] [PDF]
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 C Raetsch, J D Jia, G Boigk, M Bauer, E G Hahn, E-O Riecken, and D Schuppan Pentoxifylline downregulates profibrogenic cytokines and procollagen I expression in rat secondary biliary fibrosis Gut, February 1, 2002; 50(2): 241 - 247. [Abstract] [Full Text] [PDF]
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J. M. Mason, H.-P. Xu, S. K. Rao, A. Leask, M. Barcia, J. Shan, R. Stephenson, and S. Tabibzadeh Lefty Contributes to the Remodeling of Extracellular Matrix by Inhibition of Connective Tissue Growth Factor and Collagen mRNA Expression and Increased Proteolytic Activity in a Fibrosarcoma Model J. Biol. Chem., January 4, 2002; 277(1): 407 - 415. [Abstract] [Full Text]
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C.-C. Chen, F.-E Mo, and L. F. Lau The Angiogenic Factor Cyr61 Activates a Genetic Program for Wound Healing in Human Skin Fibroblasts J. Biol. Chem., December 7, 2001; 276(50): 47329 - 47337. [Abstract] [Full Text] [PDF]
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P. A. Folger, D. Zekaria, G. Grotendorst, and S. K. Masur Transforming Growth Factor-{beta}-Stimulated Connective Tissue Growth Factor Expression during Corneal Myofibroblast Differentiation Invest. Ophthalmol. Vis. Sci., October 1, 2001; 42(11): 2534 - 2541. [Abstract] [Full Text] [PDF]
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J. HEUSINGER-RIBEIRO, M. EBERLEIN, N. A. WAHAB, and M. GOPPELT-STRUEBE Expression of Connective Tissue Growth Factor in Human Renal Fibroblasts: Regulatory Roles of RhoA and cAMP J. Am. Soc. Nephrol., September 1, 2001; 12(9): 1853 - 1861. [Abstract] [Full Text] [PDF]
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I. E. Blom, A. J. van Dijk, L. Wieten, K. Duran, Y. Ito, L. Kleij, M. deNichilo, T. J. Rabelink, J. J. Weening, J. Aten, et al. In vitro evidence for differential involvement of CTGF, TGF{beta}, and PDGF-BB in mesangial response to injury Nephrol. Dial. Transplant., June 1, 2001; 16(6): 1139 - 1148. [Abstract] [Full Text] [PDF]
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A Leask, S Sa, A Holmes, X Shiwen, C M Black, and D J Abraham The control of ccn2 (ctgf) gene expression in normal and scleroderma fibroblasts Mol. Pathol., June 1, 2001; 54(3): 180 - 183. [Abstract] [Full Text]
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S. M. Twigg, M. M. Chen, A. H. Joly, S. D. Chakrapani, J. Tsubaki, H.-S. Kim, Y. Oh, and R. G. Rosenfeld Advanced Glycosylation End Products Up-Regulate Connective Tissue Growth Factor (Insulin-Like Growth Factor-Binding Protein-Related Protein 2) in Human Fibroblasts: A Potential Mechanism for Expansion of Extracellular Matrix in Diabetes Mellitus Endocrinology, May 1, 2001; 142(5): 1760 - 1769. [Abstract] [Full Text]
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 D. G. Peters, A. B. Kassam, E. Feingold, E. Heidrich-O'Hare, H. Yonas, R. E. Ferrell, and A. Brufsky Molecular Anatomy of an Intracranial Aneurysm : Coordinated Expression of Genes Involved in Wound Healing and Tissue Remodeling Stroke, April 1, 2001; 32(4): 1036 - 1042. [Abstract] [Full Text] [PDF]
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N. Sedlaczek, J.-D. Jia, M. Bauer, H. Herbst, M. Ruehl, E. G. Hahn, and D. Schuppan Proliferating Bile Duct Epithelial Cells Are a Major Source of Connective Tissue Growth Factor in Rat Biliary Fibrosis Am. J. Pathol., April 1, 2001; 158(4): 1239 - 1244. [Abstract] [Full Text] [PDF]
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R. B. Slee, S. G. Hillier, P. Largue, C. R. Harlow, G. Miele, and M. Clinton Differentiation-Dependent Expression of Connective Tissue Growth Factor and Lysyl Oxidase Messenger Ribonucleic Acids in Rat Granulosa Cells Endocrinology, March 1, 2001; 142(3): 1082 - 1089. [Abstract] [Full Text] [PDF]
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Y. ITO, R. GOLDSCHMEDING, R. J. BENDE, N. CLAESSEN, M. A. CHAND, L. KLEIJ, T. J. RABELINK, J. J. WEENING, and J. ATEN Kinetics of Connective Tissue Growth Factor Expression during Experimental Proliferative Glomerulonephritis J. Am. Soc. Nephrol., March 1, 2001; 12(3): 472 - 484. [Abstract] [Full Text]
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G. R. Grotendorst, L. F. Lau, and B. Perbal CCN Proteins Are Distinct from, and Should Not Be Considered Members of, the Insulin-Like Growth Factor-Binding Protein Superfamily J. Clin. Endocrinol. Metab., February 1, 2001; 86(2): 944 - 945. [Full Text]
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M. Löhr, C. Schmidt, J. Ringel, M. Kluth, P. Müller, H. Nizze, and R. Jesnowski Transforming Growth Factor-{beta}1 Induces Desmoplasia in an Experimental Model of Human Pancreatic Carcinoma Cancer Res., January 1, 2001; 61(2): 550 - 555. [Abstract] [Full Text]
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P. D. Ellis, Q. Chen, P. J. Barker, J. C. Metcalfe, and P. R. Kemp Nov Gene Encodes Adhesion Factor for Vascular Smooth Muscle Cells and Is Dynamically Regulated in Response to Vascular Injury Arterioscler. Thromb. Vasc. Biol., August 1, 2000; 20(8): 1912 - 1919. [Abstract] [Full Text] [PDF]
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 K. S. Frazier, A. Paredes, P. Dube, and E. Styer Connective Tissue Growth Factor Expression in the Rat Remnant Kidney Model and Association with Tubular Epithelial Cells Undergoing Transdifferentiation Vet. Pathol., July 1, 2000; 37(4): 328 - 335. [Abstract] [Full Text] [PDF]
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G. R. Grotendorst, L. F. Lau, and B. Perbal CCN Proteins Are Distinct from and Should Not Be Considered Members of the Insulin-Like Growth Factor-Binding Protein Superfamily Endocrinology, June 1, 2000; 141(6): 2254 - 2256. [Full Text] [PDF]
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A. Hahn, J. Heusinger-Ribeiro, T. Lanz, S. Zenkel, and M. Goppelt-Struebe Induction of Connective Tissue Growth Factor by Activation of Heptahelical Receptors. MODULATION BY Rho PROTEINS AND THE ACTIN CYTOSKELETON J. Biol. Chem., November 22, 2000; 275(48): 37429 - 37435. [Abstract] [Full Text] [PDF]
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R. C. Chambers, P. Leoni, O. P. Blanc-Brude, D. E. Wembridge, and G. J. Laurent Thrombin Is a Potent Inducer of Connective Tissue Growth Factor Production via Proteolytic Activation of Protease-activated Receptor-1 J. Biol. Chem., November 3, 2000; 275(45): 35584 - 35591. [Abstract] [Full Text] [PDF]
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C.-C. Chen, N. Chen, and L. F. Lau The Angiogenic Factors Cyr61 and Connective Tissue Growth Factor Induce Adhesive Signaling in Primary Human Skin Fibroblasts J. Biol. Chem., March 23, 2001; 276(13): 10443 - 10452. [Abstract] [Full Text] [PDF]
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A. Holmes, D. J. Abraham, S. Sa, X. Shiwen, C. M. Black, and A. Leask CTGF and SMADs, Maintenance of Scleroderma Phenotype Is Independent of SMAD Signaling J. Biol. Chem., March 30, 2001; 276(14): 10594 - 10601. [Abstract] [Full Text] [PDF]
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L. Ulloa and S. Tabibzadeh Lefty Inhibits Receptor-regulated Smad Phosphorylation Induced by the Activated Transforming Growth Factor-beta Receptor J. Biol. Chem., June 8, 2001; 276(24): 21397 - 21404. [Abstract] [Full Text] [PDF]
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S. C. FRANCIS, M. K. RAIZADA, A. A. MANGI, L. G. MELO, V. J. DZAU, P. R. VALE, J. M. ISNER, D. W. LOSORDO, J. CHAO, M. J. KATOVICH, et al. Genetic targeting for cardiovascular therapeutics: are we near the summit or just beginning the climb? Physiol Genomics, December 21, 2001; 7(2): 79 - 94. [Abstract] [Full Text] [PDF]
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