triclostatina a para esclerodermia

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ARTHRITIS & RHEUMATISM

Vol. 56, No. 8, August 2007, pp 2755–2764

DOI 10.1002/art.22759

© 2007, American College of Rheumatology

Trichostatin A Prevents the Accumulation of Extracellular

Matrix in a Mouse Model of Bleomycin-Induced Skin Fibrosis

Lars C. Huber,1 Jorg H. W. Distler,2 Falk Moritz,1 Hossein Hemmatazad,1 Thomas Hauser,3

Beat A. Michel,1 Renate E. Gay,1 Marco Matucci-Cerinic,4 Steffen Gay,1

Oliver Distler,1 and Astrid Jungel1

Objective. Tissue fibrosis is a hallmark compro-

mising feature of many disorders. In this study, we

investigated the antifibrogenic effects of the histone

deacetylase inhibitor trichostatin A (TSA) on cytokine-

driven fibrotic responses in vitro and in vivo. Methods. Skin fibroblasts from patients with sys-

temic sclerosis (SSc) and normal healthy control sub-

jects were stimulated with profibrotic cytokines in com-

bination with TSA. Human Col1(I) and fibronectin

were measured using real-time polymerase chain reac-

tion, and levels of soluble collagen were estimated using

the SirCol collagen assay. Electromobilty shift assay

and confocal fluorescence microscopy were used to

investigate the intracellular distribution of Smad tran-

scription factors. For in vivo analysis, skin fibrosis was

quantified by histologic assessment of mouse skin tissue

in a model of bleomycin-induced fibrosis.

Results. Reductions in the cytokine-induced tran-scription of Col1(I) and fibronectin were observed in

both normal and SSc skin fibroblasts following the

addition of TSA. Similarly, the expression of total

collagen protein in TSA-stimulated SSc skin fibroblasts

was reduced to basal levels. The mechanism of action of

TSA included inhibition of the nuclear translocation

and DNA binding of profibrotic Smad transcription

factors. Western blot analysis revealed an up-regulation

of the cell cycle inhibitor p21 by TSA, leading to reduced

proliferation of fibroblasts. In addition, in bleomycin-induced fibrosis in mice, TSA prevented dermal accu-

mulation of extracellular matrix in vivo.

Conclusion. These findings provide novel insights

into the epigenetic regulation of fibrosis. TSA and

similar inhibitory compounds appear to represent early

therapeutic strategies for achieving reversal of the

cytokine-driven induction of matrix synthesis that leads

to fibrosis.

Systemic sclerosis (SSc) is characterized by severefibrosis of the skin and various internal organs. SSc skin

fibroblasts show distinct features of a dysregulated phe-notype in vitro and in vivo, leading ultimately to theproduction of high levels of collagen and other proteinsof the extracellular matrix as well as a reduced expres-

sion of matrix-degrading enzymes. However, the mech-anisms responsible for the activation of fibroblasts, andthus the development of chronic and progressive fibroticdisease, remain unclear (for review, see ref. 1).

Several profibrotic cytokines have been shown tobe strongly associated with the pathogenesis of SSc,including transforming growth factor (TGF) (2),interleukin-4 (IL-4) (3), and platelet-derived growth

factor (PDGF) (4). Moreover, alterations in the down-stream signaling pathways of TGF have been observed,in particular, an increase in the expression of phosphor-

ylated Smad transcription factors (5,6). These cellularalterations have been reported to be stable over multiplegenerations of SSc fibroblasts in vitro. Genetic studies,in contrast, could not identify clear associations be-tween specific mutations of the genome and the

Supported in part by the Swiss National Science Foundation(SNF 3200B0-103691).

1Lars C. Huber, MD, Falk Moritz, MD, Hossein Hem-matazad, MD, Beat A. Michel, MD, Renate E. Gay, MD, Steffen Gay,MD, Oliver Distler, MD, Astrid Jungel, PhD: University HospitalZurich, and Zurich Center for Integrative Human Physiology, Zurich,Switzerland; 2Jorg H. W. Distler, MD: University of Erlangen-Nuremberg, Erlangen, Germany, and University Hospital Zurich,Zurich, Switzerland; 3Thomas Hauser, MD, University Hospital Zu-rich, Zurich, Switzerland; 4Marco Matucci-Cerinic, MD, PhD: Univer-sity of Florence, Florence, Italy.

Address correspondence and reprint requests to Lars C.Huber, MD, Center of Experimental Rheumatism, University HospitalZurich, Gloriastrasse 25, Zurich 8091, Switzerland. E-mail:[email protected].

Submitted for publication December 21, 2006; accepted inrevised form April 20, 2007.

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excessive accumulation of extracellular matrix (7). Theprofibrotic phenotype of SSc fibroblasts could thereforebe imprinted by a combination of epigenetic alterations,such as methylation and acetylation, as has been recentlysuggested (8).

Epigenetic modifications comprise heritable al-terations in the DNA itself without any changes in thenucleotide sequence. Unlike alterations of the genome,epigenetic changes are reversible and offer the potentialopportunity to reverse the epigenetic pattern throughtherapeutic strategies (9). Modifications of histones, inparticular, histone (de)acetylation, are among the prin-cipal mechanisms that have been described as epigeneticchanges.

In normal resting cells, DNA is highly organized within nucleosomes, in an octomeric core unit of chro-matin and in DNA-binding nucleoproteins (histones)

(10). Thereby, the dynamic packaging of chromatinregulates the extent of gene transcription. Histone hy-peracetylation is generally associated with a loosenedstate of chromatin and increased rates of gene transcrip-tion. Conversely, deacetylation of histones causes tighter

wrapping of the DNA around the nucleosome andprevents transcription factors and RNA polymerase IIfrom binding (11). Targeted deacetylation is induced byhistone deacetylases (HDACs), which are multisubunitenzyme complexes that remove the acetyl group fromthe histones via a charge-relay system, using zinc ionsas prosthetic groups (for review, see ref. 12). HDACinhibitors function by dislodging the zinc ion, thus

turning off the charge-relay system. The most potentreversible HDAC inhibitor known is trichostatin A(TSA), which fits exactly into the catalytic site of theHDAC enzymes (13).

During recent years, TSA has been used inclinical trials as a novel therapeutic strategy for variouscancers, and these studies have shown that TSA inducescell cycle arrest, cell differentiation, and apoptotic celldeath (14–16). Of interest, TSA was also postulated as alead compound in the development of antifibrogenicdrugs (17), and was tested as a promising therapeuticagent in hepatic fibrosis (18) and in the prevention of

cutaneous radiation syndrome (19).Fibrotic diseases comprise a wide array of clinical

entities. In this regard, novel molecules blocking the ex-cessive accumulation of extracellular matrix are of greatinterest in the development of therapeutic strategies. Inthe present study, we investigated the antifibrogeniceffects of TSA, both in human SSc skin fibroblasts in

vitro and in an animal model of skin fibrosis in vivo.

PATIENTS AND METHODS

Patients and fibroblast cultures. Normal and SScfibroblast cultures were prepared from biopsy specimens that were obtained from the skin of patients with SSc (n 6) andhealthy control subjects (n 3) at the University of Florence.

Biopsy samples from the SSc patients were from areas of affected skin. All patients fulfilled the criteria for SSc assuggested by LeRoy et al (20), and all subjects signed a consentform that was approved by the institutional review board of theUniversity of Florence.

After enzymatic digestion of the skin biopsy specimens with dispase II (Boehringer-Mannheim, Rotkreuz, Switzer-land), cells were cultured in Dulbecco’s modified Eagle’smedium (DMEM) containing 10% heat-inactivated fetal calf serum (FCS), 25 m M HEPES, 100 units/ml penicillin, 100g/ml streptomycin, 2 m M L-glutamine, and 2.5 g/ml ampho-tericin B (all from Gibco BRL, Basel, Switzerland). Fibroblastsfrom passages 4–8 were used for these experiments. Cells weretreated with TSA (Sigma, St. Louis, MO) in a concentration of 2 M (21). A stock solution of TSA was prepared in ethanol

and stored at 80°C. The final concentration of ethanol in themedium was 0.06%.

Real-time reverse transcription–polymerase chain re-action (RT-PCR). Total RNA was isolated from SSc fibroblastsusing the RNeasy kit (Qiagen, Basel, Switzerland) according tothe manufacturer’s instructions, which included DNase treat-ment. To generate complementary DNA (cDNA), total RNA(300–500 ng) was subjected to RT using Moloney murineleukemia virus reverse transcriptase (2.5 units/l), randomhexamers (2.5 M ), dNTPs (2 m M each), and RNase inhibitor(1 unit/l) (all from Applied Biosystems, Rotkreuz, Switzer-land). The RT reaction was performed in a total volume of 20 l in a GeneAmp PCR cycler (Applied Biosystems) at 25°Cfor 10 minutes, followed by 30 minutes at 48°C and then 5minutes at 95°C. The cDNA was stored at 20°C until

analyzed further. Samples without enzyme were used as neg-ative controls in the RT reaction, to exclude genomic contam-ination. Quantification of specific messenger RNA (mRNA) was performed by TaqMan single-reporter and SYBR Greenreal-time PCRs, using the ABI Prism 7700 Sequence DetectionSystem (Applied Biosystems) as described previously (22).

SYBR Green real-time PCR was performed for humanCol1(I) (forward primer 5-TCAAGAGAAGGCTCACGA-TGG-3, reverse primer 5-TCACGGTCACGAACCACATT-3) and human fibronectin 1 (forward primer 5-TTCTAAG- ATTTGGTTTGGGATCAAT-3, reverse primer 5-TCTTG-GTTGGCTGCATATGC-3). To confirm specific amplifica-tion by the SYBR Green PCR, a dissociation curve analysis was performed for each primer pair, and both non-RT negativecontrols and water controls were used for these analyses. Theamounts of loaded cDNA were normalized using a predevel-oped 18S ribosomal RNA control kit (Applied Biosystems) asan endogenous control.

Differential gene expression was calculated with thethreshold cycle (Ct), and relative quantification was calculated with the comparative Ct method. Only samples with a differ-ence of at least 4 cycles between the signals in cDNA samplesand negative controls (corresponding to a 24 [16-fold] differ-ence in expression) were considered for the calculations. Allexperiments were performed at least in duplicate.

2756 HUBER ET AL



Measurement of collagen. Total soluble collagen in cellculture supernatants was quantified using the SirCol collagenassay (Biocolor, Belfast, Northern Ireland). For these experi-ments, confluent cells were incubated for 24 hours with 40 lDMEM/5% FCS per cm2 of culture dish surface. One milliliterof Sirius red dye, an anionic dye that reacts specifically with

basic side-chain groups of collagens under assay conditions, was added to 400 l supernatant, followed by incubation undergentle rotation for 30 minutes at room temperature. Aftercentrifugation at 12,000 g for 10 minutes, the collagen-bounddye was redissolved with 1 ml of 0.5 M NaOH, and theabsorbance was measured at 540 nm in an MRX enzyme-linked immunosorbent assay reader (Dynex Technologies,Chantilly, VA).

Microtiter tetrazolium (MTS) assay. SSc and normaldermal fibroblasts were incubated with TSA (2 M ) in 96-wellplates for 48–72 hours. Cell proliferation was analyzed using anMTS assay (CellTiter96 AQueous Cell Proliferation Assay;Promega, Wallisellen, Switzerland) according to the manufac-turer’s instructions. For this analysis, an MRX microplatereader (Dynex Technologies) was used, at a test wavelength of

490 nm. Untreated fibroblasts were used as controls. Western blot analysis for p21. Induction of the cell

cycle inhibitor p21WAF1/Cip1 by TSA was analyzed by Westernblotting. SSc skin fibroblasts (2 106 /well) were incubated inthe absence or presence of TSA (2 M ) for 48 hours and lysedin Laemmli buffer. Protein extracts from SSc fibroblasts wereanalyzed by electrophoresis on 10% sodium dodecyl sulfate–polyacrylamide gels, and transferred onto a nitrocellulosemembrane. The membrane was blocked for 1 hour with Trisbuffered saline (TBS) containing 0.1% Tween 20 (TBST) and5% dehydrated skim milk. Blots were then incubated overnightat 4°C in the presence of monoclonal antibodies againstp21WAF1/Cip1 (Cell Signaling Technology, Beverly, MA) or-tubulin (Sigma). The blots were washed 3 times with TBSTfor 15 minutes each, and incubated with horseradish

peroxidase–conjugated goat anti-mouse IgG secondary anti-bodies at room temperature for 1 hour. Signals were detected with enhanced chemiluminescence Western blot detectionreagents (Amersham Bioscience, Freiburg, Germany) andexposed to radiographic films (SuperRX; Fujifilm MedicalSystems, Stamford, CT).

Electrophoretic mobility shift assay (EMSA). SSc skinfibroblasts were cultured to confluence in cell culture flasks(225 cm2) and incubated with medium only or with TSA(2 M ) for 48 hours. TGF (5 ng/ml; R&D Systems, Abing-don, UK) was then added for 60 minutes. Cells were collectedby scratching in ice-cold phosphate buffered saline (PBS).Nuclear extracts were prepared according to the protocoldescribed by Andrews and Faller (23). The concentration of nuclear protein was determined using the bicinchoninic acidprotein assay reagent kit (Pierce, Rockford, IL), with normal-ization for the amount of protein within each experiment.

Nonradioactive EMSA was performed using an EMSAkit (Panomics, Redwood City, CA) according to the manufac-turer’s instructions. Three micrograms of nuclear protein wasincubated for 30 minutes at 21°C with biotinylated oligonucleo-tides containing the Smad3/4 binding site (Panomics). As anegative control, the binding reaction was performed in thepresence of an excess of unlabeled double-stranded oligo-nucleotide. The samples were electrophoretically separated

(120V for 1.5–2 hours) in a nondenaturing polyacrylamide gel(6% with 2.5% glycerol) and blotted (300 mA for 30–40minutes) on a Biodyne B (0.45 m) positively charged nylonmembrane (Pall, Basel, Switzerland). The transfer buffer con-tained 20% methanol, 0.27 M Tris, and 2 M glycine.

After transfer, the membrane was ultraviolet (UV)–

crosslinked at 254 nm for 3 minutes, using a Stratalinker UVcrosslinker (Stratagene, La Jolla, CA). Biotin was labeled with alkaline phosphatase–conjugated streptavidin (1:1,000;Dako, Glostrup, Denmark), and streptavidin was detected with CDP-Star substrate (Applied Biosystems) according tothe manufacturer’s instructions. Chemiluminescence signals were visualized by exposing the membrane to an Agfa CurixOrtho HT-A film (Agfa-Gevaert, Kontich, Belgium) for 2minutes (24).

Confocal microscopy for Smad3. For confocal fluores-cence microscopy, SSc skin fibroblasts (5,000/well) were grownovernight in 24-well plates containing round coverslips (Hecht Assistant, Altnau, Switzerland). Fresh medium with 10% FCS,containing TGF (5 ng/ml) and/or TSA (2 M ), was thenadded. After incubation at 37°C for 1–2 hours, SSc skin

fibroblasts were fixed with methanol and the nuclei werestained with DAPI.

The intracellular localization of Smad transcriptionfactors was investigated by confocal fluorescence microscopy.Cells were incubated with polyclonal goat antibodies againstSmad3 (Santa Cruz Biotechnology, Santa Cruz, CA) or withcontrol antibodies, followed by incubation with fluoresceinisothiocyanate (FITC)–labeled secondary rabbit anti-goat an-tibodies (Dako). Confocal fluorescence microscopy was per-formed using a Leica SP2 inverted microscope (Leica, Wetzlar,Germany).

Bleomycin-induced dermal fibrosis. Skin fibrosis wasinduced in 6–8-week-old, pathogen-free female C3H/HeJ mice(Sankyo, Tokyo, Japan) by local injection of bleomycin for24–28 days (25,26). One hundred microliters of bleomycin

dissolved in PBS at a concentration of 0.5 mg/ml was admin-istered every other day by subcutaneous (SC) injection indefined areas of the upper back. SC injection of 100 l PBS was used as the control. Two subgroups of mice were treated with bleomycin followed by TSA in different concentrations.TSA was dissolved in PBS/1% DMSO and administered everyother day by intraperitoneal (IP) injection in a total volume of 0.1 ml. The concentrations of TSA were 0.5 g/gm/day forlow-dose therapy and 1 g/gm/day for high-dose therapy. After4 weeks, the animals were killed by CO2 asphyxiation. Theinjected skin was removed and processed for histologic analy-sis. Experiments were performed in 2 independent series.

Histologic analysis. The injected sections of skin werefixed in 4% formalin and embedded in paraffin. Five-micrometer sections were stained with hematoxylin and eosinfor the determination of dermal thickness. For analysis of connective tissue, Masson’s trichrome staining was performed.

Dermal thickness of the injected sections was analyzed with an Imager1 microscope (Carl-Zeiss, Jena, Germany) at200-fold magnification, by measuring the distance betweenthe epidermal–dermal junction and the dermal–subcutaneousfat junction at sites of induration in 3 consecutive skin sectionsfrom each animal. In each series of experiments, the dermalthickness was calculated as the fold increase compared withthat in controls. The analysis was performed by 2 independent

TRICHOSTATIN A IN SKIN FIBROSIS 2757



examiners (AJ and LCH) who were blinded to the treatmentgroups.

Statistical analysis. Results are expressed as themean SEM. Statistical analysis was performed using Graph-Pad Prism software, version 4.03 (GraphPad Software, SanDiego, CA). For analysis between different groups, Wilcoxon’s

test was used. P values less than or equal to 0.05 wereconsidered significant.

RESULTS

Effects of TSA on the expression of profibrotic

molecules at the mRNA and protein levels. TSA wasdescribed as a lead compound in the development of antifibrogenic drugs for hepatic fibrosis (17). In thiscontext, we investigated the antifibrogenic effects of TSA by exposing human SSc skin fibroblasts to TGF (5ng/ml) alone or in combination with different concen-trations of TSA. As shown in Figure 1, levels of mRNA

for both Col1(I) and fibronectin were strongly inducedby TGF in SSc skin fibroblasts, as expected. In partic-ular, following treatment with TGF, mRNA forCol1(I) was induced a mean SEM 2.7 0.5 fold andmRNA for fibronectin was induced 5.7 1.3 fold.

These effects were abolished when TSA (2 M ) was added simultaneously with TGF to cultures of SScskin fibroblasts. In this regard, addition of TSA reducedthe TGF-stimulated production of Col1(I) mRNAfrom a mean SEM 2.7 0.5 fold to 0.9 0.1 fold ( P

0.05) and the TGF-stimulated induction of fibronectinmRNA from 5.7 1.3 fold to 2.4 1.3 fold ( P 0.05)(n 4 for each) (Figure 1). In experiments with skinfibroblasts from healthy controls, TGF induced theproduction of Col1(I) mRNA a mean SEM 2.3 0.8fold and the production of fibronectin mRNA 3.5 0.6

fold, whereas the addition of TSA reduced the amountof TGF-induced mRNA production to 1.1 0.5 foldfor Col1(I) and to 2.4 1.3 fold for fibronectin(Figure 1).

We next tested the effects of TSA on skin fibro-blasts at the protein level, by analyzing the supernatantso f TGF- and TSA-treated SSc skin fibroblasts(Figure 2). Similar to the data obtained from the assess-ments of mRNA, TGF (5 ng/ml) strongly increased thede novo synthesis of collagen from a mean SEM190 29 pg/ml to 455 43 pg/ml. The combination of TGF and TSA down-regulated the synthesis of colla-gen to 170 55 pg/ml ( P 0.05), which was below the

basal levels found in cultures of unstimulated cells. Addition of TSA to unstimulated SSc skin fibroblastsreduced the synthesis of collagen from 190 29 pg/ml to115 49 pg/ml. These results clearly indicate that TSAinhibits the synthesis of collagen by SSc skin fibroblasts.

To investigate whether TSA also blocks the ac-tion of other profibrotic cytokines, we exposed SSc skinfibroblasts to IL-4 (5 ng/ml) or PDGF (20 ng/ml) alone

Figure 1. Effects of trichostatin A (TSA) on the transcription of

extracellular matrix genes. Systemic sclerosis (SSc) and normal dermalfibroblasts were stimulated with transforming growth factor (TGF)

(5 ng/ml) (solid bars) or with TGF (5 ng/ml) plus TSA (2 M )

(shaded bars), and compared with unstimulated fibroblasts (open bar).Results are the mean and SEM fold change in levels of mRNA for

Col1(I) and fibronectin relative to that in unstimulated cells, from 4

independent experiments; values are normalized to the expression of 18S ribosomal RNA. P 0.05 versus SSc cells treated with TGF

alone.

Figure 2. Inhibitory effects of TSA on the release of collagen protein

in SSc dermal fibroblasts. TGF, interleukin-4 (IL-4), and platelet-

derived growth factor (PDGF) up-regulated the de novo synthesis of collagen by SSc fibroblasts. When TSA (2 M ) was added to the

cultures, both the basal and cytokine-induced production of collagen

were inhibited. Results are the mean and SEM from 4 independentexperiments. See Figure 1 for other definitions.

2758 HUBER ET AL



or in combination with TSA (2 M ) for 48 hours. Thestimulatory effects of IL-4 and PDGF on SSc skinfibroblasts were similar to the effects observed withTGF. The collagen production by SSc fibroblasts wasincreased to 288 73 pg/ml following stimulation with

IL-4, and increased to 235 16 pg/ml following stimu-lation with PDGF. Strikingly, addition of TSA (2 M )reversed both the IL-4–induced collagen synthesis (to103 25 pg/ml) and the PDGF-stimulated collagensynthesis (to 103 22 pg/ml), representing a return tonormal levels, suggesting that TSA functions as aninhibitor of the final common pathway in the productionof extracellular matrix proteins.

Kinetics of TSA. We found that the observedeffects of TSA were both dose- and time-dependent.Untreated cells and TGF-stimulated cells were ex-posed to TSA in concentrations of 100 n M , 500 n M ,1 M , and 2 M . Quantification of collagen protein

within 48 hours after incubation showed that TSA actedin a clear dose-dependent manner, with the highestreduction in collagen synthesis observed following addi-tion of 2 M TSA and the lowest reduction observed

with 100 n M TSA. These doses are within the physio-logic range of concentrations of TSA that have beenapplied in vitro in other studies (18,27).

Similarly, TSA acted in a time-dependent man-ner. Protein levels were measured following a singleaddition of TSA and TGF to the cultures, with thelevels determined at 48 hours, 96 hours, and 144 hoursafter incubation. The maximal effects of TSA were

observed after incubation for 48 hours (results notshown). These results confirm that TSA functions as areversible HDAC inhibitor.

Previous studies have shown that TSA might leadto inhibition of the cell cycle as well as apoptotic celldeath (28,29). With the doses of TSA used in the presentstudy, no increase in the rate of apoptosis could beobserved, as determined by annexin V/propidium iodidedouble-staining and fluorescence-activated cell sorteranalysis (results not shown). However, an effect of TSA

was observed on the cell cycle. When SSc skin fibroblasts were exposed to TSA (2 M ), proliferation of fibroblasts was reduced by 29 3% after 72 hours, as measured by

MTS assay. To investigate the molecular mechanisms of this TSA-induced cell cycle inhibition, Western blottingfor the cell cycle inhibitor p21 was performed. WhenSSc skin fibroblasts were analyzed after exposure toTSA for 48 hours, p21 was clearly induced (results notshown).

Levels of Smad transcription factors. The down-stream signaling action of TGF is mainly mediated by

Smad transcription factors. To determine whether TSAalters the expression pattern of these molecules, EMSA

was performed. SSc skin fibroblasts were exposed toTGF, alone or in combination with TSA, for 2 hours.

As shown in Figure 3, TGF strongly induced theDNA-binding fraction of the profibrotic Smad3/4. Thiseffect, however, was almost completely abolished when

the cells were coincubated with TSA.These findings were also confirmed on the mor-

phologic level, by confocal fluorescence microscopy of skin fibroblasts using FITC-labeled anti-Smad3 anti-bodies (Figure 4). In untreated control SSc fibroblasts(Figure 4a), green fluorescence within the nuclei, repre-senting the presence of Smad3 molecules, could beobserved only sporadically. After exposure of SSc skin

Figure 3. Representative results from electrophoretic mobility shift

assay, demonstrating the inhibition of nuclear Smad3/4 by TSA in SSc

dermal fibroblasts. Lane 1, Positive control; lane 2, free probe withoutnuclear extracts; lane 3, nuclear extract from unstimulated SSc fibro-

blasts; lane 4, nuclear extract from TGF-stimulated SSc fibroblasts;

lane 5, nuclear extract from unstimulated SSc fibroblasts treated withTSA; lane 6, nuclear extract from TGF-stimulated SSc fibroblasts

treated with TSA; lane 7, nuclear extract from unstimulated SSc

fibroblasts with an excess of unlabeled cold probe; control lanes were

ran on a separate gel. Signals for Smad3/4–DNA complexes provideevidence of a strong activation of Smad3/4 by TGF (5 ng/ml) (lane 4).

This activation could be inhibited when TSA (2 M ) was added (lane

6). See Figure 1 for definitions.




fibroblasts to TSA alone (Figure 4b), a similar pattern of scant Smad3 distribution was seen. When TGF wasadded, however, Smad3 molecules were observed to bedistributed over the cytoplasm and within the nucleus(Figure 4c). The addition of TSA completely abolishedthis TGF-induced nuclear translocation (Figure 4d).

Effects of TSA on bleomycin-induced dermalfibrosis in vivo. Following our in vitro studies, we testedthe effects of TSA in vivo using a previously describedanimal model of bleomycin-induced skin fibrosis (25).No obvious toxic effects were observed in TSA-treatedmice.

Repeated SC application of bleomycin causedmarked thickening of the mouse skin at the sites of

injection. When analyzed histologically, the skin frombleomycin-exposed animals showed clear signs of tissuefibrosis, including the accumulation of extracellular ma-trix within the dermis and the adjacent subcutis, thusleading to replacement of subcutaneous fat tissue withfibrillar collagen bundles (Figures 5a and b).

When TSA was administered by IP injection afterSC injections of bleomycin, the fibrotic effects of bleo-mycin were clearly reduced, as shown in Figures 5c andd. These observations were quantified by the measure-ment of dermal thickness. In particular, injection of bleomycin induced an increase in dermal thickness by amean SEM 71 14% as compared with that inuntreated control mice ( P 0.001). Injections of TSA

Figure 4. Representative images from confocal fluorescence microscopy of human SSc dermal fibroblasts. SScfibroblasts were cultured on chamber slides with TSA and/or TGF and stained with DAPI for localization of nuclei

(red) and fluorescein isothiocyanate–labeled anti-Smad3 antibodies (green). Intranuclear Smad3 molecules could beobserved only sporadically in untreated SSc fibroblasts (a) and in TSA-treated SSc fibroblasts (b). Stimulation of thefibroblasts with TGF resulted in cytoplasmic and nuclear distribution of Smad3 molecules (c). Simultaneous

application of TGF in conjunction with TSA completely abolished this TGF-induced nuclear translocation in SSc

fibroblasts (d). See Figure 1 for definitions.

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prevented thickening of the skin. At a TSA dosage of 0.5 g/gm/day, the level of bleomycin-induced dermalfibrosis was significantly reduced ( P 0.003), to 16 4% of that in PBS-treated control animals. Similar levelsof dermal thickness were observed when TSA wasapplied IP at a higher dosage (Figure 6), indicating

that therapeutic effects could be achieved even withsubmicromolar doses of TSA.

Moreover, morphologic analysis of the mouseskin with Masson’s trichrome staining, used for detec-tion of collagen fibers on paraffin-embedded tissuesections, further revealed the dense accumulation of

Figure 5. Prevention of the accumulation of extracellular matrix by trichostatin A (TSA) in experimental dermal fibrosis. Skin fibrosis was induced

in mice by repeated subcutaneous injections of bleomycin (b–d). Groups of mice were either left untreated (b) or treated with intraperitoneal

injections of TSA (c and d) at a concentration of 0.5 g/gm/day (c) or 1.0 g/gm/day (d). Phosphate buffered saline–treated mice served as controls

(a). Injections of TSA prevented an increase in dermal thickness, as analyzed by computer-assisted measurement of the dermo–epidermal distance

(red bars), and also prevented the accumulation of dense collagen bundles (arrows in b) that would replace subcutaneous fat tissue (asterisks in a

and b versus c and d). Representative tissue sections in a–d were examined at the same magnification (original magnification 200).




collagen fibers induced by repeated bleomycin injec-tions, whereas TSA partly prevented this process. Inparticular, dermal collagen bundles were less denselypacked, and the subcutaneous fat was still present.These data are consistent with the results obtained in

vitro and confirm the role of TSA as a promising leadcompound in the development of antifibrogenic drugs.

DISCUSSION

Fibrosis is a clinical hallmark of many disorders,and excessive accumulation of extracellular matrix

within internal organs ultimately leads to severe compli-cations, such as respiratory distress, liver failure, anddeath. At the moment, no effective treatment is knownto inhibit fibrotic processes. In the context of SSc,numerous studies have identified a profibrotic pheno-type of fibroblasts in which the cells are stimulated byelevated levels of various cytokines at the early stages of the disease (4,30), ultimately leading to the developmentand progression of tissue fibrosis.

Of interest, the profibrotic cellular features char-

acterizing this phenotype remain unchanged during celldivision (8). In addition to genetic mutations and clonalselection, which have been suggested as possible mech-anisms, there has been growing interest in epigeneticmodifications. Most of the evidence regarding the roleof epigenetic alterations in disease has been gained fromthe use of epigenetic drugs, such as HDAC inhibitors.

In the present study, we tested the effect of TSA,

the strongest HDAC inhibitor currently known (12), oncollagen production by skin fibroblasts, using in vitroand in vivo experiments with SSc skin fibroblasts and amouse model of bleomycin-induced skin fibrosis. Theaddition of TSA normalized the levels of mRNA tran-

scripts for Col1(I) (procollagen) and fibronectin, as well as the levels of collagen protein in cytokine-stimulated SSc skin fibroblasts. Consistent with the datafrom other studies, our findings indicate the involvementof epigenetic histone modifications in the expression of fibrogenic molecules. The use of HDAC inhibitors pre-

vents the removal of acetyl groups from core histones,and a state of histone hyperacteylation is usually associ-ated with increased rates of gene transcription.

Since TSA inhibited the growth factor–inducedexpression of profibrotic molecules in our experi-ments, several possible mechanisms of action can bediscussed. One possible mechanism is the induction of genes essential for the suppression of collagen expres-sion. Conversely, TSA or gene products induced by TSAmight interfere with the signaling cascade involved intissue fibrosis. Furthermore, TSA might affect the pro-duction of cellular products indirectly by, for example,inducing cell cycle arrest, apoptosis, or cell death. Bind-ing of TGF to the serine/theronine kinase receptor(TGFRII) activates TGFRI and initiates a down-stream signaling cascade through Smad transcriptionfactors. In particular, Smad proteins become activatedby phosphorylation. Phosphorylated Smad2 and Smad3then form a complex with Smad4, resulting in a hetero-

meric structure that translocates to the nucleus (6,31).Together with other DNA-binding factors, the Smadcomplexes regulate the expression of several profibroticgenes. Smad6 and Smad7, in contrast, have been linkedto antifibrotic activities.

Of the factors investigated in the present study,the DNA-binding activities of Smad3 and Smad4 wereclearly affected, and nuclear translocation was inhibitedby TSA. Whether this effect is attributable to epigeneticmodifications of histones or whether TSA directly inhib-its the phosphorylation of Smads has to be addressed infurther studies. Moreover, TSA appears to have otherfunctional properties in addition to the inhibition of

histone deacetylation. Recent reports have described anovel, histone acetylation–independent mechanism by

which HDAC inhibitors cause dephosphorylation of intracellular proteins, which challenges the view of therole of TSA as a sole HDAC inhibitor (32).

Collagen repressors, such FLI1 and p53, were notinvestigated in the present study, but previous findingssuggest that FLI1 is affected by the use of epigenetic

Figure 6. Change in dermal thickness in groups of mice with

bleomycin-induced skin fibrosis treated with TSA at either 1 g/gm/

day (TSA high) or 0.5 g/gm/day (TSA low) or left untreated, ascompared with a control group of phosphate buffered saline (PBS)–

treated mice. Results are the mean and SEM fold change in dermal

thickness relative to controls.

P

0.05. See Figure 5 for otherdefinitions.

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drugs (8). Alternatively, the fact that TSA provokes cellcycle arrest could be another explanation for the re-duced production of collagen proteins in SSc skin fibro-blasts. Several studies performed in cancer cells haves ho wn t ha t T SA i nd uc es t he e xp re ss io n o f

the cyclin-dependent kinase inhibitor p21, thus leadingto cell cycle inhibition in melanoma and glioma cells(28,33). Consistent with these findings, TSA stronglyinduced the expression of p21 in SSc skin fibroblasts,and the population of proliferating cells was clearlyreduced. Taken together, the findings from our experi-ments indicate that TSA prevents the accumulation of extracellular matrix by several mechanisms, includingepigenetic alterations of the expression pattern of Smadtranscription factors, and interference with regulation of the cell cycle.

Our data are consistent with the results from

previous studies in which the antifibrotic properties of TSA have been demonstrated (18,27). However, ourstudy amends the data obtained in vitro, with experi-ments performed in vivo using a common animal modelof skin fibrosis. In the bleomycin-induced fibrosis mousemodel, micromolecular concentrations of TSA pre-

vented the development of skin fibrosis without theoccurrence of obvious toxic side effects. After applica-tion of 0.5 g/gm of TSA per day, the bleomycin-inducedcollagen bundles within the dermis and the replacementof subcutaneous fat tissue were reduced to levels com-parable with those in the skin of untreated animals.However, increasing the applied concentration of TSA

to 1.0 g/gm/day did not result in further improvement.These findings indicate that the therapeutic effects of TSA could be achieved even with doses in the submi-cromolar range.

In conclusion, we have shown that the HDACinhibitor TSA has potent antifibrogenic effects on SScskin fibroblasts in vitro. With respect to the pivotal roleof profibrotic cytokines such as TGF, PDGF, and IL-4in the pathogenesis of fibrotic diseases, we were able toshow that TSA abrogates the stimulating effects of thesefactors on extracellular matrix production. Moreover, ina mouse model of bleomycin-induced fibrosis, TSA

prevented the development of skin fibrosis as quantifiedby changes to the dermal thickness within the bleomycininjection sites. With regard to the strong antifibrogenicactivities of TSA, we propose that TSA and relatedinhibitory compounds should be considered for use asearly pharmacologic strategies to conquer the develop-ment and progression of fibrosis in SSc and othercytokine-driven fibrotic entities.

ACKNOWLEDGMENTS

We thank all staff members of the laboratory of electron microscopy at the University of Zurich for theirexcellent support, in particular, Dr. M. Hoechli for providingassistance with the confocal microscopy.

AUTHOR CONTRIBUTIONS

Dr. Huber had full access to all of the data in the study andtakes responsibility for the integrity of the data and the accuracy of thedata analysis.Study design. Huber, Distler, Hauser, R. E. Gay, Matucci-Cerinic, S.Gay, Distler, Jungel.

Acquisition of data. Huber, Moritz, Hemmatazad, Hauser, Jungel. Analysis and interpretation of data. Huber, Distler, S. Gay, Jungel.Manuscript preparation. Huber, Moritz, Michel, R. E. Gay, S. Gay,Dist ler, Jungel.Statistical analysis. Huber.

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triclostatina a para esclerodermia

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