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Hypoxia-induced increase in the production of extracellular matrix proteins in systemic sclerosis.

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ARTHRITIS & RHEUMATISM
Vol. 56, No. 12, December 2007, pp 4203–4215
DOI 10.1002/art.23074
© 2007, American College of Rheumatology
Hypoxia-Induced Increase in the Production of
Extracellular Matrix Proteins in Systemic Sclerosis
Jörg H. W. Distler,1 Astrid Jüngel,2 Margarita Pileckyte,3 Jochen Zwerina,4 Beat A. Michel,2
Renate E. Gay,2 Otylia Kowal-Bielecka,5 Marco Matucci-Cerinic,6 Georg Schett,4
Hugo H. Marti,7 Steffen Gay,2 and Oliver Distler2
Objective. Insufficient angiogenesis with tissue
ischemia and accumulation of extracellular matrix are
hallmarks of systemic sclerosis (SSc). Based on the
severely decreased oxygen levels in the skin of patients
with SSc, we aimed to investigate the role of hypoxia in
the pathogenesis of SSc.
Methods. Subtractive hybridization was used to
compare gene expression in dermal fibroblasts under
hypoxic and normoxic conditions. Dermal fibroblasts
were further characterized by exposure to different
concentrations of oxygen and for different time periods
as well as by interference with hypoxia-inducible factor
1␣ (HIF-1␣). The systemic normobaric hypoxia model
in mice was used for in vivo analyses.
Results. Several extracellular matrix proteins and
genes involved in extracellular matrix turnover, such as
thrombospondin 1, pro␣2(I) collagen, fibronectin 1,
insulin-like growth factor binding protein 3, and transforming growth factor ␤–induced protein, were induced
by hypoxia in SSc and healthy dermal fibroblasts. The
induction of these genes was time- and dose-dependent.
Experiments with HIF-1␣–knockout mouse embryonic
fibroblasts, deferoxamine/cobalt ions as chemical stabilizers of HIF-1␣, and HIF-1␣ small interfering RNA
consistently showed that extracellular matrix genes are
induced in dermal fibroblasts by HIF-1␣–dependent, as
well as HIF-1␣–independent, mechanisms. Using the
systemic normobaric hypoxia mouse model, we demonstrated that dermal hypoxia leads to the induction of the
identified extracellular matrix genes in vivo after both
short exposure and prolonged exposure to hypoxia.
Conclusion. These data show that hypoxia contributes directly to the progression of fibrosis in patients with SSc by increasing the release of major
extracellular matrix proteins. Targeting of hypoxia
pathways might therefore be of therapeutic value in
patients with SSc.
The cellular signaling pathways involved in the
response to hypoxia have been elucidated in detail
during the last several years (1). Many downstream
effects of hypoxia are mediated via stabilization of the
transcription factor hypoxia-inducible factor 1␣ (HIF1␣) (2). Under normoxic conditions, hydroxylation and
acetylation of the oxygen-dependent domain of HIF-1␣
promote binding of the von Hippel-Lindau tumor suppressor protein (pVHL) and rapid degradation in proteasomes. With decreasing levels of oxygen, hydroxylation and acetylation of HIF-1␣ do not occur due to the
lack of molecular oxygen, pVHL does not bind, and the
HIF-1␣ protein is stabilized. After translocation into the
nucleus, HIF-1␣ binds with its dimerization partner
HIF-1␤/aryl hydrocarbon receptor nuclear translocator
(ARNT) to defined hypoxia-responsive elements in regulatory regions of target genes, such as vascular endothelial growth factor (VEGF), and increases their transcription. While HIF-1␣ is an important mediator of
1
Jörg H. W. Distler, MD: Center of Experimental Rheumatology and Zurich Center of Integrative Human Physiology, University
Hospital Zurich, Zurich, Switzerland, and University of Erlangen–
Nuremberg, Erlangen, Germany; 2Astrid Jüngel, PhD, Beat A. Michel,
MD, Renate E. Gay, MD, Steffen Gay, MD, Oliver Distler, MD:
Center of Experimental Rheumatology and Zurich Center of Integrative Human Physiology, University Hospital Zurich, Zurich, Switzerland; 3Margarita Pileckyte, MD, PhD: Kaunas Medical University
Hospital, Kaunas, Lithuania; 4Jochen Zwerina, MD, Georg Schett,
MD: University of Erlangen–Nuremberg, Erlangen, Germany; 5Otylia
Kowal-Bielecka, MD: Medical University of Bialystok, Bialystok,
Poland; 6Marco Matucci-Cerinic, MD: University of Florence, Florence, Italy; 7Hugo H. Marti, MD: University of Heidelberg, Heidelberg, Germany.
Address correspondence and reprint requests to Oliver Distler, MD, Center of Experimental Rheumatology and Zurich Center of
Integrative Human Physiology, University Hospital Zurich, Gloriastrasse 25, CH-8091 Zurich, Switzerland. E-mail: Oliver.Distler@
usz.ch.
Submitted for publication April 22, 2007; accepted in revised
form August 13, 2007.
4203
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DISTLER ET AL
hypoxia signaling, HIF-1␣–independent mechanisms,
such as messenger RNA (mRNA) stabilization and
increased transcription by other HIF family members,
also contribute to the cellular responses to hypoxia (1,2).
Systemic sclerosis (SSc) is a chronic fibrotic disorder of unknown cause that affects the skin and a
variety of internal organs (3). The hallmark of SSc is an
excessive accumulation of extracellular matrix proteins,
which are released by activated interstitial fibroblasts.
Extracellular matrix proteins that are increased in the
skin include collagens, fibronectin, glycosaminoglycans,
and thrombospondin (4). The resulting progressive fibrosis of the skin and involved organs is a major cause of
morbidity and mortality in SSc patients (5). The mechanisms leading to the activation of interstitial fibroblasts
are incompletely understood, despite their central role
in the pathogenesis of SSc.
Abnormalities of the microvascular system are
another key feature of SSc. Endothelial cell damage is
among the earliest changes in the disease and results in
disorganization of the capillary architecture and loss of
capillaries. The reduction of capillary density is associated with an insufficient formation of new vessels via
angiogenesis (6). Together with the increased distance
to blood vessels caused by the accumulation of extracellular matrix proteins, the decreased capillary density
reduces the supply of oxygen and leads to tissue hypoxia.
We recently showed that patients with SSc have severely
reduced levels of oxygen in fibrotic skin as compared
with the skin of healthy volunteers (7). However, the
molecular effects of hypoxia in SSc and their role in the
fibrotic process in vivo have not been analyzed. Thus, the
aim of the present study was to identify and characterize
genes regulated by hypoxia, using subtractive hybridization as a screening technique, and to address the induction of these genes in a systemic normobaric hypoxia
model in mice.
MATERIALS AND METHODS
Patients and fibroblast cultures. SSc and control fibroblasts were derived from biopsies of affected skin obtained
from SSc patients or normal skin obtained from healthy
volunteers. Fibroblasts were obtained from biopsy samples by
enzymatic digestion, as described previously (8). All SSc
patients fulfilled the criteria for SSc as suggested by LeRoy and
Medsger (9). Fibroblasts from passages 3–8 were used for the
experiments. All patients and controls signed a consent form
that had been approved by the local Institutional Review
Boards. Mouse embryonic fibroblasts (MEFs) from HIF1␣⫹/⫹ (wild-type) and HIF-1␣–/– (knockout) mice were kindly
provided by R. Johnson (10).
Culture conditions and induction of hypoxia. Fibroblasts were grown in Dulbecco’s modified Eagle’s medium
(Life Technologies, Basel, Switzerland) as described elsewhere
(8). For exposure to hypoxia, fibroblasts were transferred into
an incubator (Forma Scientific, Illkirch, France) and exposed
to a humidified atmosphere containing 5% CO2 and between
1% and 16% O2 volume/volume (hypoxia) as indicated below
(7). For controls, cells were cultured under the same conditions except that the atmosphere contained 20% O2 v/v
(normoxia). For all experiments, cells were used when they
reached 30–50% confluence. In another set of experiments,
SSc and normal dermal fibroblasts were grown to 50% confluence in 12-well plates. Deferoxamine mesylate and cobalt
chloride (both from Sigma, Deisenhofen, Germany), dissolved
in distilled water, were added to the medium for 12 hours at a
final concentration of 100 ␮g/ml.
Western blotting. Cultured cells were removed from
the incubator and rinsed immediately with ice-cold phosphate
buffered saline. Extraction of nuclear proteins and transfer
onto nitrocellulose membranes was performed according to
standard protocols (11). HIF-1␣ protein was detected using
monoclonal mouse anti–HIF-1␣ mgc3 antibodies (12), as
described elsewhere (7).
Suppressive subtractive hybridization. SSc fibroblasts
exposed to hypoxic or normoxic conditions for 24 hours were
used for these experiments. Suppressive subtractive hybridization was performed using the PCR-Select system (Clontech,
Palo Alto, CA). The oxygen concentration under the hypoxic
condition (1% O2) is equivalent to a PO2 value of 7 mm Hg,
which is close to the 10% percentile measured in the fibrotic
skin of SSc patients (6).
Isolation of total RNA was performed with TRIzol LS
reagent (Gibco BRL, Basel, Switzerland). After analysis of the
RNA quality on agarose gel, 1 ␮g of total RNA from SSc
dermal fibroblasts exposed to hypoxia for 24 hours or from
normoxic controls were reverse transcribed into hypoxic and
normoxic complementary DNA (cDNA) pools using the
SMART cDNA synthesis kit, which ensures full-length transcription of mRNA (Clontech).
Suppressive subtractive hybridization was then performed. Briefly, cDNA from an SSc fibroblast culture exposed
to hypoxic conditions for 24 hours was used as tester. After
creation of blunt-ended fragments by digestion with Rsa I,
tester cDNA was separated into 2 pools, which were ligated to
2 different adaptors. The cDNA pool from normoxic fibroblasts digested with Rsa I, but not ligated to any adaptor, was
used as a driver. Hybridization of tester cDNA from hypoxic
fibroblasts with an excess of driver cDNA from normoxic
controls leads to equalization and enrichment of differentially
expressed sequences among tester cDNA molecules, since only
cDNA molecules with 2 different adaptors at both ends are
amplified exponentially, whereas cDNA molecules present in
both hypoxic and normoxic fibroblasts are not amplified. After
2 hybridization steps, hypoxia-induced sequences were further
amplified by nested polymerase chain reaction (PCR) using
primers against sequences of the 2 different adaptors.
For creation of a subtractive cDNA library, the PCR
mixture enriched for differentially expressed genes was digested with Rsa I to cleave the adaptors from the cDNA
strands. After purification with the High Pure PCR Product
Purification kit (Boehringer, Mannheim, Germany), cDNA
HYPOXIA-INDUCED EXTRACELLULAR MATRIX PROTEINS IN SSc
fragments were ligated into the pPCR-Script Amp SK(⫹)
vector (Stratagene, Basel, Switzerland) with T4-ligase at 16°C
for 10 hours. Vectors were then amplified in Epicurian Coli
XL10-Gold ultracompetent cells (Stratagene). Plasmid DNA
was prepared with the Concert High Purity Plasmid Miniprep
system (Gibco BRL). Isolated plasmids containing sequences
from the library of hypoxia-induced genes were analyzed by
automatic dideoxy-sequencing (Microsynth, Balgach, Switzerland). For each gene, the homology with published sequences
was analyzed by searching GenBank databases.
From the cDNA library of hypoxia-induced genes
obtained by subtractive hybridization, 58 clones were randomly
selected, sequenced, and identified using National Center for
Biotechnology Information BLAST databases. Because similar
to other differential expression screening techniques, suppression subtractive hybridization produces false-positive results,
the differential expression of the identified genes was confirmed and quantified by real-time PCR using SYBR Green.
By using this strategy, the induction of 48% of the identified
genes (28 clones) could be confirmed. This percentage is well
within the range of the percentages from other studies using
suppressive subtractive hybridization and confirms the validity
of the experimental approach (13).
Quantitative real-time PCR. TRIzol LS reagent was
used for RNA isolation from tissues and cultured cells. Tissue
samples were homogenized with the Dispergierstation T8.10
(IKA Labortechnik, Wohlen, Switzerland). Tissue samples
from the mouse experiments were homogenized with the
Dispergierstation T8.10, and TRIzol LS reagent was used for
RNA isolation of tissues and cultured cells. For quantification
of mRNA, SYBR Green real-time PCR was performed using
the ABI Prism 7700 Sequence Detection system (PE Applied
Biosystems, Rotkreuz, Switzerland) as described previously
(14). Specific primer pairs for each gene were designed with
Primer Express software (PE Applied Biosystems). (A table of
primers used in these experiments can be obtained by contacting the authors.) Samples without enzyme in the reverse
transcription were used as a control (non–reverse transcriptase
control) to exclude genomic contamination. Nonspecific signals caused by primer dimers were excluded by dissociation
curve analysis, by analysis of the reaction products on agarose
gels, and by use of no-template controls.
For quantification of VEGF mRNA, TaqMan realtime PCR was performed with the TaqMan probe and primer
sequences for VEGF previously described (7). To normalize
for the amounts of loaded cDNA, ␤-actin was used as an
endogenous control. After confirmation that the amplification
efficiency of the genes of interest and the endogenous control
␤-actin was equal, differences were calculated according to the
threshold cycle (Ct) and the comparative Ct method for
relative quantification. All measurements were performed in
duplicate.
Transfection of human dermal fibroblasts with small
interfering RNAs (siRNA) against HIF-1␣ and HIF-2␣. Three
distinct predesigned siRNA against HIF-1␣ and HIF-2␣, as
well as control siRNA, were purchased from Ambion (Huntingdon, UK). Transfection of cells was performed by nucleofection using an Amaxa system (Amaxa, Cologne, Germany) as
described previously (6). Eight hours after transfection, the
medium was changed, and the cells were either exposed to
hypoxia (1% oxygen) or were cultured under normoxic condi-
4205
tions as described previously (6). After 48 hours, RNA was
isolated and analyzed by real-time PCR as described above.
Inhibition of transforming growth factor ␤ (TGF␤)
signaling. To investigate the contribution of TGF␤ signaling to
the induction of extracellular matrix proteins by hypoxia,
human dermal fibroblasts were cultured in the presence of a
neutralizing mouse anti-human TGF␤ antibody (R&D Systems, Wiesbaden, Germany). The median neutralization dose
of the neutralizing TGF␤ antibody in the presence of 0.25
ng/ml of TGF␤ was 30 ng/ml. Concentrations of the neutralizing TGF␤ antibody from 20 ng/ml to 500 ng/ml were used for
our experiments. Cells incubated with isotype mouse antihuman antibodies (R&D Systems) at the same concentrations
were used as controls. After 48 hours under either hypoxic or
normoxic conditions, the expression of extracellular matrix
proteins was analyzed.
Collagen protein measurements. Collagen protein was
measured with the Sircol collagen assay as described elsewhere
(8). This assay detects protein from types I–XIV collagen using
a quantitative Sirius Red binding method.
Animals. All experiments were performed according to
protocols approved by the local Animal Research Ethics
Committee. Female C57BL/6 mice ages 4–6 weeks (n ⫽ 4
animals per group) were exposed to systemic normobaric
hypoxia by substitution of oxygen with nitrogen in a closed
Persplex chamber using a Digamix 2M 302/a-F pump (H.
Wösthoff Messtechnik, Bochum, Germany) at a flow rate of 37
liters/minute (11). Mice were allowed to adapt to hypoxia over
a period of 1 hour, with gradually decreased inspiratory O2
fractions, from 21% to 6%, and were then maintained at a
fraction of inspired oxygen (FiO2) level of 6% for 24 or 48
hours. The animals were killed immediately after exposure to
hypoxia.
Stereotactic microdissection of the dermis. The dermis
was separated from mouse skin specimens by stereotactic
microdissection as described previously (15). Briefly, freshly
frozen skin specimens from mice exposed to hypoxia and to
normoxia (controls) were cut into 15-␮m sections and placed
on glass slides. Using a histologic stereomicroscope (Stemi
DV4; Zeiss, Oberkochen, Germany) at 40⫻ magnification,
10–15 sections from each animal were microdissected by
removing the epidermal and subcutaneous parts of the tissue.
The remaining dermis was scraped from the slide and immediately placed on dry ice to prevent degradation of RNA.
Statistical analysis. Data are expressed as the mean ⫾
SEM. Wilcoxon’s signed rank test for related samples and the
Mann-Whitney test for unrelated samples were used for statistical analyses. P values less than 0.05 were considered
statistically significant.
RESULTS
Expression of HIF-1␣ and VEGF in dermal
fibroblast cultures. We first compared the induction of
HIF-1␣ by immunoblotting of cultured dermal fibroblasts from SSc patients and healthy controls after
exposure to hypoxic conditions. While no expression of
HIF-1␣ protein was observed in the normoxic controls, a
strong and stable expression of HIF-1␣ was found in
4206
DISTLER ET AL
Table 1. Extracellular matrix proteins and genes involved in extracellular matrix regulation in fibroblasts from SSc patients and normal controls*
SSc fibroblasts
Sequence identity
Accession
number
Thrombospondin 1
Pro␣2(I) collagen
Fibronectin 1
Microfibrillar-associated protein 4
TGF␤-induced protein
IGFBP-3
XM_031616
NM_000089
XM_030549
XM_045044
XM_038210
XM_038123
Up-regulated
cultures
6
6
6
5
6
6
of
of
of
of
of
of
6
6
6
6
6
6
Mean fold
induction
3.30 ⫾ 0.93†
2.10 ⫾ 0.27†
1.46 ⫾ 0.27†
2.38 ⫾ 0.54
2.22 ⫾ 0.38†
6.26 ⫾ 1.08†
Normal fibroblasts
Up-regulated
cultures
3
5
5
4
5
5
of
of
of
of
of
of
5
5
5
5
5
5
Mean fold
induction
2.70 ⫾ 0.45
1.97 ⫾ 0.35†
2.13 ⫾ 0.41†
2.63 ⫾ 0.86
2.24 ⫾ 0.53†
7.48 ⫾ 0.88†
* Hypoxia-induced genes that were identified by suppressive subtractive hybridization were confirmed in additional samples by real-time polymerase
chain reaction analysis. SSc ⫽ systemic sclerosis; TGF␤ ⫽ transforming growth factor ␤; IGFBP-3 ⫽ insulin-like growth factor binding protein 3.
† P ⬍ 0.05 for dermal fibroblasts cultured under hypoxic conditions compared with normoxic control fibroblasts.
dermal SSc and normal fibroblasts cultured under hypoxic conditions in 1% oxygen. Consistent with recent
observations (6), we found no differences in the levels of
HIF-1␣ protein between SSc and normal fibroblasts
after 24 hours of hypoxia (data not shown).
Because VEGF is one of the best-characterized
downstream targets of HIF-1␣ and is further increased
by hypoxia due to mRNA stabilization, we next analyzed
the levels of VEGF mRNA by TaqMan real-time PCR.
Consistent with the results of the HIF-1␣ Western blot
analysis, a mean ⫾ SEM 2.0 ⫾ 0.1–fold induction of
VEGF mRNA was observed in SSc fibroblasts exposed
to hypoxia as compared with normoxic controls (P ⬍
0.05). Again, there was no significant difference between
SSc and normal fibroblasts (2.1 ⫾ 0.2–fold upregulation). Together, these results show that hypoxia
can be sufficiently induced in dermal fibroblasts under
these experimental conditions, with no differences in the
expression of HIF-1␣ and VEGF between SSc and
normal dermal fibroblasts.
Hypoxia-induced genes in cultured dermal fibroblasts. We next aimed to identify downstream targets of
hypoxia signaling in dermal fibroblasts using suppression
subtractive hybridization. The differential expression of
genes identified by subtractive hybridization was confirmed in additional cultures of fibroblasts from SSc
patients and was compared with that in normal fibroblasts. The identified and confirmed genes were grouped
according to their main biologic function.
One group consisted of genes known to inhibit
cell proliferation and included basic leucine transcription factor, B cell translocation factor, cyclin T1, and
peripheral myelin protein 22/growth arrest–specific 3.
Furthermore, induction of enzymes with functions in
metabolic pathways, such as triosephosphate isomerase
1 (TPI-1), phosphoglycerate kinase 1 (PGK-1), and
NADH dehydrogenase B8 subunit, was observed.
Among these enzymes, TPI-1 and PGK-1 are well
characterized as hypoxia-driven HIF-1␣ target genes (1),
which further confirms that the experimental setting
used for the differential screening approach successfully
activated cellular hypoxia pathways in the dermal fibroblasts. Six clones of the hypoxia-induced cDNA library
were homologous to recently identified gene sequences,
for which biologic functions have not yet been established. Moreover, a number of genes with different
biologic functions, such as Toll-like receptor 4 and
cadherin 13, were found to be induced by hypoxia. (A
table showing the genes identified in these experiments
can be obtained by contacting the authors.) Consistent
with the findings of the HIF-1␣ and VEGF analyses,
hypoxia induced the majority of the identified genes to a
similar extent in SSc and normal fibroblasts.
Hypoxia-induced extracellular matrix proteins.
The most interesting group of genes was identified from
the hypoxia-induced cDNA library encoded for extracellular matrix proteins and for genes that are involved in
extracellular matrix regulation (Table 1). Genes significantly induced by hypoxia in SSc fibroblasts included
fibronectin 1 (1.46 ⫾ 0.27–fold induction compared with
normoxic controls; P ⬍ 0.05), thrombospondin 1 (3.30 ⫾
0.93–fold induction; P ⬍ 0.05), pro␣2(I) collagen
(COL1A2) (2.10 ⫾ 0.27–fold induction; P ⬍ 0.05),
insulin-like growth factor binding protein 3 (IGFBP-3)
(6.26 ⫾ 1.08–fold induction; P ⬍ 0.05) and TGF␤induced protein (TGF␤i) (2.22 ⫾ 0.38–fold induction;
P ⬍ 0.05). Similar to the other genes, the extracellular
matrix proteins were induced in SSc fibroblasts and
normal fibroblasts to a similar extent by hypoxia (Table
1). In additional experiments, another extracellular matrix protein, cartilage oligomeric matrix protein (COMP;
thrombospondin 5), was induced by hypoxia (2.8 ⫾
0.4–fold induction; P ⬍ 0.05). We also confirmed these
results on the protein level by showing a significant
HYPOXIA-INDUCED EXTRACELLULAR MATRIX PROTEINS IN SSc
4207
Figure 1. Time course of the up-regulation of extracellular matrix proteins and related genes induced by exposure to hypoxia for 12–48 hours.
Prolonged exposure to hypoxia resulted in an even stronger induction of mRNA for A, thrombospondin 1, B, fibronectin 1, C, pro␣2(I) collagen (col
1A2), D, transforming growth factor ␤–induced protein (TGF␤i), and E, insulin-like growth factor binding protein 3 (IGFBP-3) in cultured dermal
fibroblasts from patients with systemic sclerosis (SSc; n ⫽ 5) and normal controls (n ⫽ 5) as compared with normoxic controls (defined as a value
of 1). Values are the mean and SEM. ⴱ ⫽ P ⬍ 0.05 for dermal fibroblasts cultured under hypoxic conditions (1% oxygen) compared with the same
fibroblasts cultured under normoxic conditions (20% oxygen) for the same time period.
induction of collagens after exposure to hypoxia using
the Sircol collagen assay (data not shown).
Further increases in the production of extracellular matrix proteins after prolonged exposure to hypoxia. In vivo, chronic hypoxia is present in the dermis of
patients with SSc because of reduced capillary density
and the accumulation of extracellular matrix proteins
(6). Thus, the exposure of fibroblasts to hypoxia for 24
hours, as used for the suppressive subtractive hybridization experiments, might not fully reflect the situation in
vivo. To address this issue, SSc and normal dermal
fibroblast cultures were exposed to hypoxic conditions
for prolonged times and were compared with control
cultures exposed to normoxic conditions.
Notably, after prolonged exposure to hypoxia,
most of the extracellular matrix proteins and related
genes were significantly increased to levels higher than
those achieved after shorter exposure times (Figure 1).
After 12 hours, there was only minor up-regulation, but
after 24 hours of hypoxia, up-regulation of all genes
reached statistical significance as compared with controls. The levels of fibronectin 1, thrombospondin 1,
4208
DISTLER ET AL
Figure 2. Oxygen concentration–dependent induction of extracellular matrix proteins and related genes in dermal fibroblasts. Expression of A,
fibronectin 1, B, thrombospondin 1, C, pro␣2(I) collagen (col 1A2), D, transforming growth factor ␤–induced protein (TGF␤i), and E, insulin-like
growth factor binding protein 3 (IGFBP-3) in cultured dermal fibroblasts from patients with systemic sclerosis (SSc; n ⫽ 5) and normal controls (NH;
n ⫽ 5) was analyzed by real-time polymerase chain reaction after 48 hours of exposure to 20%, 16%, 11%, 8.5%, 6%, or 1% oxygen. Values are
the mean and SEM. ⴱ ⫽ P ⬍ 0.05 for dermal fibroblasts cultured under hypoxic conditions (as indicated) compared with the same fibroblasts cultured
under normoxic conditions (20% oxygen) for the same time period.
COL1A2, and IGFBP-3 increased further after 36 hours
in a time-dependent manner, and the degree of induction by hypoxia was even more pronounced after 48
hours (Figure 1). The induction of TGF␤i reached its
maximum between 24 hours and 36 hours, with a
constant up-regulation at later time points. In contrast,
the induction of COMP decreased from a mean ⫾ SEM
of 2.8 ⫾ 0.4–fold after 12 hours to 1.3 ⫾ 0.1–fold after 96
hours. Again, we did not observe significant differences
between SSc and normal fibroblasts for any of these
extracellular matrix proteins. These data suggest that
prolonged exposure to hypoxia at levels similar to those
in vivo has even more profound effects on the induction
of most extracellular matrix proteins.
Correlation of the induction of extracellular matrix proteins with the levels of oxygen. To further
examine the functional association between hypoxia and
extracellular matrix proteins, dermal fibroblasts from
HYPOXIA-INDUCED EXTRACELLULAR MATRIX PROTEINS IN SSc
SSc patients and normal controls were exposed to different oxygen concentrations, ranging from 21% to 1%.
The expression of IGFBP-3 did not differ at oxygen
concentrations between 16% and 11% as compared with
SSc fibroblasts under normoxic conditions. However, at
lower oxygen levels, there was a significant
concentration-dependent increase in the levels of
IGFBP-3 (Figure 2E). At 8.5% oxygen, IGFBP-3 levels
were up-regulated by 1.9 ⫾ 0.1–fold and increased
further to 2.8 ⫾ 0.4–fold at 6% oxygen. The greatest
induction of IGFBP-3, with an increase of 6.3 ⫾ 1.1–
fold, was measured at 1% oxygen (Figure 2E), which is
consistent with the reduced oxygen levels measured in
vivo in the dermis of patients with SSc (6). Similar results
were obtained with normal fibroblasts. Analogous to the
results for IGFBP-3, significant oxygen concentration–
dependent induction was also observed for fibronectin 1,
thrombospondin 1, COL1A2, and TGF␤i (Figures 2A–
D). These data further confirm the functional correlation between hypoxia and extracellular matrix proteins
by showing a concentration-dependent induction, with
the strongest effects at reduced oxygen concentrations
equivalent to those present in vivo in patients with SSc
(6).
Role of HIF-1␣ in hypoxia-mediated induction of
extracellular matrix proteins. Based on the dose- and
time-dependent induction of several extracellular matrix
proteins and related genes by hypoxia, we hypothesized
that targeting of the major hypoxia transcription factor
HIF-1 might be a valuable approach to inhibiting the
hypoxia-mediated accumulation of extracellular matrix
proteins.
To test this hypothesis, embryonic fibroblasts
from HIF-1␣–/– and HIF-1␣⫹/⫹ mice were cultured
under hypoxic and normoxic conditions. A strong, HIF1␣–dependent regulation of TGF␤i was observed, with a
mean ⫾ SEM reduction of 85 ⫾ 8% in MEFs from
HIF-1␣–/– mice, as compared with TGF␤i expression in
MEFs from HIF-1␣⫹/⫹ mice, under hypoxic conditions
(Figure 3). This reduced expression in HIF-1␣–/– MEFs
was in the range of the reduced expression of PGK-1
(94 ⫾ 3% reduction in HIF-1␣–/– MEFs), a well-defined
HIF-1␣ target that was used as a positive control in these
experiments (Figure 3).
In contrast, the mean induction of IGFBP-3 and
fibronectin decreased by only 35 ⫾ 12% and 47 ⫾ 10%,
respectively, in MEFs from HIF-1␣–/– mice compared
with those from HIF-1␣⫹/⫹ mice under hypoxic conditions, suggesting that the transcriptional activation of
these genes is only partially mediated by HIF-1␣, but is
also mediated via HIF-1␣–independent pathways (Fig-
4209
Figure 3. Role of the transcription factor hypoxia-inducible factor 1␣
(HIF-1␣) in the induction of extracellular matrix components by
hypoxia. The contribution of HIF-1␣ to the up-regulation of fibronectin 1, insulin-like growth factor binding protein 3 (IGFBP-3), and
transforming growth factor ␤–induced protein (TGF␤i) was analyzed
using mouse embryonic fibroblasts (MEFs) from HIF-1␣⫹/⫹ (wildtype [WT]) and HIF-1␣–/– (knockout) mice. Phosphoglycerate kinase 1
(PGK-1), an oxygen-sensitive gene known to be HIF-1␣–dependent,
was used as a positive control. Mean induction by hypoxia in MEFs
from WT mice was defined as 100%, and the mean induction in MEFs
from HIF-1␣–/– mice is shown relative to this value. Values are the
mean and SEM of 4 independent experiments.
ure 3). The expression of Col1a2 was below the level of
detection in mouse embryonic fibroblasts, and thrombospondin 1 was not induced by hypoxia in the mouse
cell line.
To confirm these results in a human system and
with adult cells, human dermal fibroblasts were stimulated with deferoxamine mesylate (DFX) and cobalt ions
(Co2⫹). DFX and Co2⫹ mimic hypoxia by preventing the
degradation of HIF-1␣ protein under normoxic conditions (16). In addition to the genes mentioned above,
COL1A2 and thrombospondin 1 were induced by DFX
and Co2⫹ in human dermal fibroblasts, confirming the
induction of these genes by hypoxia and suggesting at
least partial regulation by HIF-1␣ (Figure 4A).
The results with the chemical stabilizers of
HIF-1␣ were confirmed by knockdown experiments
using specific siRNA against HIF-1␣. PGK-1 and
VEGF, two well-established HIF-1–dependent genes,
were used as positive controls in our analysis of the
efficacy of the suppression of HIF-1␣ by the siRNA. The
induction of PGK-1 by hypoxia was reduced to 40 ⫾ 8%,
and the induction of VEGF was reduced to 26 ⫾ 8%
Figure 4. Hypoxia-inducible factor 1␣ (HIF-1␣)–dependent induction of extracellular matrix genes induced by hypoxia in human dermal fibroblasts. A, Induction of extracellular
matrix proteins by deferoxamine mesylate (DFx) and cobalt chloride (CoCl2) in fibroblasts from patients with systemic sclerosis (SSc). Similar to hypoxic conditions, the HIF-1␣
stabilizers DFx and CoCl2 induced extracellular matrix proteins and related genes in dermal fibroblasts. Thrombospondin 1, pro␣2(I) collagen (col 1A2), and transforming growth
factor ␤–induced protein (TGF␤i) were induced to a similar extent as under hypoxic conditions. In contrast, fibronectin 1 was only slightly induced and insulin-like growth factor
binding protein 3 (IGFBP-3) was induced to a much lesser extent than under hypoxic conditions, suggesting that HIF-1␣–independent mechanisms are involved. B–F, Modulation
of the induction of thrombospondin 1 (B), pro␣2(I) collagen (C), TGF␤i (D), IGFBP-3 (E), and fibronectin 1 (F) in SSc fibroblasts by 3 different small interfering RNAs (siRNA)
against HIF-1␣. Consistent with the results obtained with the chemical stabilizers of HIF-1␣, siRNA against HIF-1␣ strongly reduced the induction of thrombospondin, pro␣2(I)
collagen, and TGF␤i by hypoxia, whereas the expression of fibronectin 1 and IGFBP-3 was not significantly reduced. Values are the mean and SEM.
4210
DISTLER ET AL
HYPOXIA-INDUCED EXTRACELLULAR MATRIX PROTEINS IN SSc
4211
Figure 5. Transforming growth factor ␤ (TGF␤)–dependent induction of extracellular matrix proteins by hypoxia in human dermal fibroblasts. To
study the role of TGF␤ in the induction of extracellular matrix genes by hypoxia, dermal fibroblasts from patients with systemic sclerosis were
incubated with neutralizing antibodies against TGF␤ (aTGFb AB) at 20 ng/ml, 100 ng/ml, or 500 ng/ml and cultured for 48 hours under hypoxic or
normoxic conditions. Neutralizing antibodies against TGF␤ reduced the induction of A, pro␣2(I) collagen, B, fibronectin 1, C, thrombospondin 1,
and D, transforming growth factor ␤–induced protein under both hypoxic and normoxic conditions. The reductions were more pronounced in
hypoxic fibroblasts. These results suggest that TGF␤ plays a major role in the induction of extracellular matrix proteins by hypoxia. Values are the
mean and SEM.
(data not shown); these findings confirm that HIF-1␣
was efficiently suppressed in our system.
The 3 siRNA against HIF-1␣ strongly suppressed
the induction of COL1A2, thrombospondin 1, and
TGF␤i, to 26 ⫾ 12%, 32 ⫾ 7%, and 56 ⫾ 14%, as
compared with normoxic control siRNA, suggesting
HIF-1␣–dependent regulation of these genes (Figures
4B–D). Consistent with the results with MEFs from
HIF-1␣–/– mice, the induction of IGFBP-3 and fibronectin 1 were only slightly reduced (Figure 4E and F),
suggesting that HIF-1␣ does not play a major role in the
induction of these genes under hypoxic conditions.
In contrast to HIF-1␣, transfection of human
dermal fibroblasts with siRNA against HIF-2␣ did not
reduce the induction of COL1A2, thrombospondin 1,
fibronectin 1, IGFBP-3, or TGF␤i (Figures 4B–F).
These findings are evidence against an important role of
HIF-2␣ in the induction of these extracellular matrix
genes in dermal fibroblasts from SSc patients and
healthy controls.
These data show on different experimental levels
that the effects of hypoxia on the induction of extracellular matrix genes are mediated by both HIF-1␣–
dependent and HIF-1␣–independent pathways. Thus,
targeting of the HIF-1␣ system could be a strategy by
which to inhibit the hypoxia-driven synthesis of extracel-
4212
lular matrix proteins, but it might not completely block
the hypoxia-mediated effects on the activation of dermal
fibroblasts. Moreover, inhibition of HIF-2␣ seems to be
inefficient to prevent the induction of extracellular matrix proteins under hypoxic conditions.
Dependence of extracellular matrix protein induction on TGF␤. TGF␤ is a major stimulus for the
induction of extracellular matrix proteins in SSc fibroblasts. In addition, hypoxia has recently been shown to
induce the expression of connective tissue growth factor
in SSc fibroblasts (17). To investigate whether the
up-regulation of extracellular matrix proteins is mediated by TGF␤-dependent pathways, we incubated SSc
fibroblasts under hypoxic and normoxic conditions with
neutralizing antibodies against TGF␤. Neutralizing antibodies against TGF␤ completely abrogated the induction of COL1A2, fibronectin 1, thrombospondin 1, and
TGF␤i under hypoxic conditions (Figures 5A–D). In
fibroblasts subjected to normoxic conditions, neutralizing antibodies against TGF␤ also reduced the production of extracellular matrix proteins. However, the inhibitory effects were not as pronounced as those seen
with fibroblasts exposed to hypoxic conditions, suggesting that TGF␤ plays a key role in the induction of
extracellular matrix proteins by hypoxia.
Induction of extracellular matrix proteins in the
dermis in an in vivo mouse model of hypoxia. We next
aimed to confirm the in vitro effects of hypoxia on
dermal fibroblasts in vivo by using a mouse model of
systemic normobaric hypoxia. This mouse model has
been validated for the induction of systemic hypoxia in
various tissues (11), but it is unknown whether it induces
cellular hypoxia in the skin. Thus, we first analyzed the
expression of the hypoxia-driven genes VEGF and
PGK-1 in RNA extracts from dermal specimens from
the back of mice subjected to hypoxic conditions. In fact,
VEGF as well as PGK-1 mRNA increased significantly
after exposure to hypoxia, by 2.0 ⫾ 0.4–fold and 2.3 ⫾
0.4–fold, respectively, as compared with mice maintained under normoxic conditions (P ⬍ 0.05) (data not
shown). The induction of VEGF and PGK-1 after
exposure to hypoxia was also found in dermal RNA
extracts from the skin of the ear (2.1 ⫾ 0.4–fold and
2.4 ⫾ 0.1–fold induction, respectively; P ⬍ 0.05) (data
not shown). The induction of these 2 established markers of hypoxia at different sites confirmed that hypoxiainduced pathways were activated in the mouse skin.
We next analyzed the expression of the identified
hypoxia-induced extracellular matrix proteins and related genes in total RNA extracts from the dermis of
hypoxia-treated mice. After 24 hours of hypoxia, all
DISTLER ET AL
Figure 6. Synthesis of extracellular matrix proteins and related genes
in the dermis of hypoxia-treated mice. The expression of the hypoxiainduced genes identified by subtractive hybridization in cultured
dermal fibroblasts from the dermis of mice treated with hypoxia for 24
hours and 48 hours was analyzed by real-time polymerase chain
reaction using SYBR Green. Values are the mean and SEM of 4 mice
per experimental condition. col 1A2 ⫽ pro␣2(I) collagen; TGFbi ⫽
transforming growth factor ␤–induced protein; IGFBP-3 ⫽ insulin-like
growth factor binding protein 3.
genes identified by subtractive hybridization were induced in the dermis, as compared with control mice
maintained under normoxic conditions (Figure 6). For
example, hypoxic mice produced 3.0 ⫾ 0.2–fold more
IGFBP-3 and 2.1 ⫾ 0.2–fold more TGF␤i than did
normoxic controls in dermis samples obtained from their
back. Similar results were found in studies of dermis
samples from the ears of hypoxia-treated mice.
Since prolonged exposure to hypoxia resulted in a
further up-regulation of these genes in vitro, we speculated that this might also be true for the situation in vivo.
Indeed, a stronger induction of fibronectin 1, thrombospondin 1, and Col1a2 was detected in the dermis of
mice exposed to hypoxia for 48 hours (Figure 6). Again,
similar results were obtained for dermis samples from
the back and ears of hypoxia-treated mice.
Together, these data confirm in an in vivo model
that hypoxia in the skin results in an increased production of several extracellular matrix proteins and related
genes. This induction by hypoxia is time-dependent, with
stronger effects after prolonged exposure to hypoxia, as
was seen in the skin of patients with SSc.
HYPOXIA-INDUCED EXTRACELLULAR MATRIX PROTEINS IN SSc
DISCUSSION
Severe tissue hypoxia and activated interstitial
fibroblasts are characteristic features of systemic sclerosis (SSc). The present study was performed with the
objective of identifying molecular pathways that are
induced by hypoxia and that contribute to the chronic
activation of dermal fibroblasts in SSc. The most intriguing finding was the induction of extracellular matrix
proteins in dermal fibroblasts. The up-regulation of
extracellular matrix proteins by hypoxia demonstrates
that hypoxia directly contributes to the development and
progression of fibrosis in this disease and therefore
provides a novel link between the vascular changes and
fibrosis in SSc.
The extracellular matrix proteins we identified
(fibronectin 1, thrombospondin 1, and collagens) are
produced in excessive amounts by SSc fibroblasts and
form a major part of the fibrotic material in SSc skin (4).
TGF␤i is an extracellular matrix protein that is expressed at high levels in fibrotic lesions, such as arteriosclerotic plaques (18), and in zones of thickened extracellular matrix in the bladder (19). While its role in SSc
has not yet been addressed, TGF␤i has been demonstrated to bind to types I, II, and IV collagen (20) and to
promote the attachment and spreading of fibroblasts,
activities similar to those of fibronectin 1 (21).
Another hypoxia-induced protein identified was
IGFBP-3, which is involved in the regulation of extracellular matrix proteins. IGFBP-3 binds to extracellular
matrix components, protects IGF-1 from degradation,
and modulates the effects of IGF-1 on target cells (22).
The ligand IGF-1 is profibrotic through its stimulation of
the synthesis of collagen and down-regulation of the
production of collagenases in fibroblasts. Moreover,
IGFBP-3 is overexpressed in fibroblasts from the fibrotic
skin of patients with SSc and directly induces the synthesis of fibronectin in lung fibroblasts (22,23).
The findings of our differential expression
screening analysis of hypoxia-induced genes have a
direct impact on virtually all in vitro studies dealing with
the pathogenesis of SSc. Hypoxia has a wide range of
molecular effects on cells through both HIF-1␣–
dependent as well as HIF-1␣–independent mechanisms
(1). This is underlined by our results in dermal fibroblasts, which identified hypoxia-induced molecules with
a role in such diverse biologic processes as angiogenesis,
proliferation, cellular metabolic pathways, and extracellular matrix accumulation. Cell culture studies performed under normoxic conditions at 20% oxygen
clearly do not resemble the hypoxic environment present
4213
in vivo in SSc tissues (7). Thus, the interference with
multiple hypoxia-driven pathways is overlooked with
normoxic culture conditions and has not been taken into
account in the majority of in vitro experiments in the
past.
While the hypoxic stimulus is only present in the
skin of patients with SSc, the hypoxia-induced expression profile was not different between normal and SSc
dermal fibroblasts in our experimental setting. These
results suggest that the induction of extracellular matrix
proteins by chronic hypoxia represents a common biologic reaction pattern rather than disease-specific effects. However, since the oxygen levels are severely
reduced in the skin of SSc patients as compared with
healthy controls (7), the activation of dermal fibroblasts
by hypoxia is operative only in SSc, but not in healthy
controls.
Although it was beyond the scope of the present
study to analyze the effects of hypoxia in other organ
systems and disease settings, the induction of extracellular matrix proteins by hypoxia might also play a role in
other fibrotic diseases associated with vascular abnormalities. Indeed, consistent with our data, chronic hypoxia, for example, has been shown to increase matrix
components in renal tubular interstitial cells, fibroblasts,
and hepatic stellate cells in vitro (24–26). However, an
up-regulation of extracellular matrix proteins and the
role of HIF-1␣ in this process have not yet been
investigated.
Short-term hypoxia such as that which occurs in
patients with vasospasms due to primary Raynaud’s
phenomenon is not sufficient to induce fibroblast activation. Exposure of dermal fibroblasts to hypoxia for less
than 6 hours has no effects on the stabilization of
HIF-1␣ protein (7), and in the present study, this did not
induce the production of extracellular matrix proteins.
This observation is important, because it explains why
patients with primary Raynaud’s phenomenon do not
progress to a state of fibroblast activation with increased
extracellular matrix protein deposition despite their
short-term hypoxia caused by vasospasm, a situation
opposite that in SSc patients with chronic hypoxia
caused by fibrosis and capillary reduction.
The induction of major extracellular matrix proteins by hypoxia raises the question of whether hypoxia
signaling pathways could be a target for therapeutic
approaches. In this regard, it should be emphasized that
the hypoxia-induced release of extracellular matrix proteins worsens the hypoxia of interstitial fibroblasts by
further increasing the distance to blood vessels. This
finally results in a vicious circle of extracellular matrix
4214
DISTLER ET AL
accumulation and hypoxia. Consistent with previous
studies, we demonstrated that the induction of extracellular matrix proteins is dependent on TGF␤ (25). The
stimulation of downstream effects of TGF␤ seems not to
depend on the induction of TGF␤, because we did not
observe a consistently increased synthesis of TGF␤
mRNA and protein under hypoxic conditions (data not
shown). Alternative explanations include increased activation of latent TGF␤ complexes or optimized signaling
of TGF␤.
Our experiments with MEFs from HIF-1␣–/–
mice and with siRNA against HIF-1␣ suggest that
targeting of the HIF-1␣ system could be a strategy for
inhibiting, at least in part, the hypoxia-driven synthesis
of extracellular matrix proteins. Along this line, Kung
and coworkers (27) identified a small-molecule inhibitor
that blocks the interaction of HIF-1 with the transcriptional coactivator p300, thereby attenuating hypoxiainducible transcription in vitro and in vivo. Unfortunately, this compound proved to be too toxic to allow
further development for its use in humans (28). A
number of drugs, however, have been identified that
indirectly down-regulate HIF-1. This includes inhibitors
of histone deacetylases such as trichostatin A, which are
in clinical trials in patients with cancers and have
recently been shown to reduce the release of collagen
from SSc dermal fibroblasts in vitro (29,30). Whether
this also holds true in vivo and whether histone deacetylase inhibitors could be a potential therapy for patients
with SSc remains to be analyzed.
In conclusion, the present study is the first to
show that hypoxia induces multiple extracellular matrix
proteins in dermal fibroblasts in vitro as well as in
systemic normobaric hypoxia in vivo. The induction of
extracellular matrix proteins by hypoxia was dose- and
time-dependent, with higher inductions under conditions of chronic hypoxia and under low oxygen concentrations as are present in the skin of SSc patients.
Analysis with MEFs from HIF-1␣–/– mice indicated that
the induction of extracellular matrix genes by hypoxia is
at least partly driven by HIF-1␣. Considering that progressive fibrosis is a major cause of morbidity and
mortality in SSc patients, targeting of hypoxia pathways
such as HIF-1 might be a promising novel approach for
the treatment of the late fibrotic stages of SSc.
AUTHOR CONTRIBUTIONS
Drs. J. H. W. Distler and O. Distler had full access to all of the
data in the study and take responsibility for the integrity of the data
and the accuracy of the data analysis.
Study design. J. H. W. Distler, Schett, S. Gay, O. Distler.
Acquisition of data. J. H. W. Distler, Jüngel, Pileckyte, KowalBielecka, Marti, O. Distler.
Analysis and interpretation of data. J. H. W. Distler, Jüngel, Michel,
R. E. Gay, Marti, S. Gay, O. Distler.
Manuscript preparation. J. H. W. Distler, Jüngel, Zwerina, R. E. Gay,
Matucci-Cerinic, S. Gay, O. Distler.
Statistical analysis. J. H. W. Distler, O. Distler.
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