DD tissue specimens were obtained from four adult patients undergoing fasciectomy for DD. Patients who underwent carpal tunnel release and showed no evidence of DD contributed the control tissue from normal palmar fascia (n = 3) or carpal ligament (n = 1). All DD tissues used were from primary releases. The tissue was separated macroscopically in nodules and cords. Only nodules were used in this study. For details on how samples were prepared, see the Additional files.
Clinical sample preparation
After excision, the nodule was divided in three portions. One portion was placed in 10% formalin and further processed for immunohistochemistry. The second was immediately placed into liquid nitrogen for protein extraction. The third portion was used for primary cell culture. All of the patients underwent excision independently of this study and had not undergone previous surgery on their hands. Oral consent for removal of the tissue and storage in the tissue bank for research purposes was obtained from the patients. Individual consent for this specific project was waived by the local ethics committee because the research was performed on 'waste' material, which was stored in a coded fashion.
Recombinant human TGF-β3 (OSI Pharmaceuticals Inc., Melville, NY, USA) and recombinant human BMP6 (Creative BioMolecules, Hopkinton MA, USA) were generously provided by K Iwata and K Sampath, respectively. SB-431542 compound, which targets ALK4, ALK5 and ALK7, was purchased from Tocris Bioscience, Ellisville, Missouri, USA. The PD98059 compound, which targets mitogen-activated protein kinase kinase 1 (MEK1), was purchased from Cell Signaling Technology (Danvers, MA, USA). The vascular endothelial growth factor (VEGF) receptor inhibitor PTK787/ZK222584, the epidermal growth factor (EGF) receptor inhibitor PKI166 and the PDGF receptor inhibitor STI571 (also known as imatinib mesylate) were kindly provided by Novartis, Amsterdam, The Netherlands. The protein kinase C activator 12-O-tetradecanoyl-phorbol-13-acetate (TPA) was obtained from Sigma (Sigma Chemical Co., St. Louis, MO, USA).
To obtain primary cells, tissues were minced under sterile conditions into pieces that measured approximately 1 × 1 × 2 mm3. Ten to twenty pieces were placed as explants into the wells of six-well plates and stored in 37°C incubators in 5% CO2. Primary cells from passages 3 through 6 were used for the experiments. All of the cells were subcultured in DMEM containing 4.5 g/L glucose (Gibco, Breda, The Netherlands) supplemented with 10% foetal bovine serum (FBS) (Integro, Zaandam, The Netherlands), 100 IU/mL penicillin and 100 IU/mL streptomycin (Invitrogen, Breda, The Netherlands).
RNA isolation and quantitative real-time PCR
Total RNA was extracted by using the RNeasy Kit (Qiagen, Venlo, The Netherlands) according to the manufacturer's instructions. Reverse transcriptase PCR was performed using the RevertAid H Minus First Strand cDNA Synthesis Kit (Fermentas, St. Leon-Rot, Germany) according to the manufacturer's instructions. All of the samples were plated in duplicate, and TaqMan PCR reactions were performed using the StepOnePlus Real-Time PCR System (Applied Biosystems, Carlsbad, California, USA). Lack of DNA contamination was verified and gene expression levels were determined using the comparative ΔCt method with glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as the reference.
Quantitative PCR primers
Human TGF-β1 through TGF-β3, α-SMA, PAI-1, c-myc, COl1A2, fibronectin, Smad1 through Smad3, CTGF, PDGF-A, PDGF-B and GAPDH gene expression was analysed using the following forward and reverse primers: TGF-β1, 5'-CTCTCCGACCTGCCACAGA-3' and 5'-AACCTAGATGGGCGCGATCT-3'; TGF-β2, 5'-CCGCCCACTTTCTACAGACCC-3' and 5'-GCGCTGGGTGGGAGATGTTAA-3'; TGF-β3, 5'-CTGGCCCTGCTGAACTTTG-3' and 5'-AAGGTGGTGCAAGTGGACAGA-3'; α-SMA, 5'-CACCTTCCAGCAGATGTGGAT-3' and 5'-AAGCATTTGCGGTGGACAAT-3'; PAI-1, 5'-TCTTTGGTGAAGGGTCTGCT-3' and 5'-CTGGGTTTCTCCTCCTGT TG-3'; c-myc, 5'-CGTCTCCACACATCAGCACAA-3' and 5'-CACTGTCCAACTTGACCCTCTTG-3'; COl1A2, 5'-GATGTTGAACTTGTTGCTGAGG-3' and 5'-TCTTTCCCCATTCATTTGTCTT-3'; fibronectin, 5'-GAGGCCACCATCACTGGTT-3' and 5'-AGTGCGATGACATAGATGGTGTA-3'; Smad1, 5'-TGAACCATGGATTTGAGACAGT-3' and 5'-CTGGCGGTGGTATTCTGC-3'; Smad2, 5'-CGAAAAGGATTGCCACATGTT-3' and 5'-TTGAGTTCATGATGACTGTGAAGATC-3'; Smad3, 5'-CGGTCAACCAGGGCTTTG-3' and 5'-CAGCCTTTGACGAAGCTCATG-3'; CTGF, 5'-TTGCGAAGCTGACCTGGAAGAGAA-3' and 5'-AGCTCGGTATGTCTTCATGCTGGT-3'; PDGF-A, 5'-CCTCACATCCGTGTCCTCTT-3' and 5'-ACACGAGCAGTGTCAAGTGC-3'; PDGF-B, 5'-TGCTGTTGAGGTGGCTGTAG-3' and 5'-GAAAATGCAGGGTGGAGGTA-3'; TGF-α, 5'-TAACCACGAGACCCTCAACC-3' and 5'-CCCAAGCCTTAGCTGTCTTG-3'; and GAPDH, 5'-ATCACTGCCACCCAGAAGAC-3' and 5'-ATGAGGTCCACCACCCTGTT-3'.
MTS-based proliferation assay
Cells were seeded into 96-well plates at 7 × 103 cells/well and treated the next day with the indicated inhibitors or DMSO as a control. Increases in the number of viable cells after culture were measured daily for 4 days using an 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS)-based proliferation assay according to the manufacturer's instructions (CellTiter 96 AQueous One Solution Cell Proliferation Assay System; Promega, Leiden, The Netherlands) and using the measured absorbance at 490 nm on day 0 as the reference.
Tissue lysate preparation and Western blot analysis
For tissue lysates, biopsies were frozen in liquid nitrogen and pulverised using a mortar. Thereafter, the triturated tissues were incubated in ice-cold lysis buffer (150 mmol NaCl, 20 mmol Tris·HCl, pH 7.5, 1% Nonidet P-40, 5 mmol sodium ethylenediaminetetraacetic acid (EDTA) and one Complete Protease Inhibitor Cocktail Tablet (Roche, Woerden, The Netherlands) per 50 mL of solution) for 30 minutes. Prior to centrifugation at 4°C for 15 minutes at 14 × 103 rpm, the samples underwent extensive vortexing and sonification. The total protein content of the supernatant was determined using the DC Protein Assay (Bio-Rad Laboratories, Veenendaal, The Netherlands)). Equal amounts of total protein (100 μg/μL) were loaded onto a 10% gel, followed by SDS-PAGE and Western blot analysis. For cell-based assays, cells were plated onto six-well plates at a density of 4 × 105 cells/well, stimulated with the indicated reagents and directly lysed in sample buffer (250 mmol Tris·HCl, pH 6.8, 8% SDS, 40% glycerol, 5% β-mercaptoethanol, and bromophenol blue) after 18 hours. Antibodies specifically targeting Smad1 (Zymed, San Francisco, CA, USA), Smad2/3 (BD Transduction Laboratories, Breda, The Netherlands), phosphorylated ERK1/2 (P-ERK1/2; Cell Signaling Technology), PAI-1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), Col1α 2 (Col1A2; SouthernBiotech, Birmingham, Alabama, USA), α-SMA (Sigma Chemical Co.), fibronectin/ED-A (Abcam, Cambridge, MA, USA) and c-myc (Santa Cruz Biotechnology) were purchased. Antibodies targeting phosphorylated Smad1 and Smad2 (P-Smad1 and P-Smad2, respectively) were described previously . P-Smad3 was obtained from E Leof (Mayo Clinic, Rochester, MN, USA), and P-ERK1/2 antibodies were a gift from WH Moolenaar (Netherlands Cancer Institute, Amsterdam, The Netherlands). Equal loading was confirmed using an anti-β-actin antibody (Sigma Chemical Co.). Quantitative Western blot analysis was performed using secondary goat anti-rabbit IRDye 680 and goat anti-mouse IRDye 800 CW with the Odyssey Scanner (LI-COR Biosciences, Lincoln, Nebraska USA) according to the manufacturer's instructions.
For immunofluorescence staining, cells were grown on coverslips overnight. Cells were fixed with ice-cold methanol for 30 minutes, washed twice with PBS, quenched with 20 mmol NH4Cl, and permeabilised with 0.1% Triton X-100 the following day. Cells were then incubated in blocking solution (PBS containing 3.0% BSA) for 45 minutes followed by incubation for 1 hour with anti-α-SMA antibody (Sigma Chemical Co.) diluted 1:100 in blocking solution. After washing, the labelled secondary antibody Alexa Fluor 488 goat anti-mouse immunoglobulin G (IgG) (Invitrogen) was used. Nuclei were stained using Hoechst 33258 (Invitrogen) according to the manufacturer's instructions. Specimens were visualised by using an Olympus IX51 inverted microscope at ×100 magnification using the cellF Soft Imaging System (Olympus, Zoeterwoude, The Netherlands).
Cells were cultured overnight on coverslips. The next day fixation in acetone followed by staining for α-SMA (α-SMA/1, M851, 1A4 clone; Dako, Carpinteria, CA, USA) at 1:500 dilution was performed for 60 minutes. Endogenous peroxidase was quenched with 0.1% natriumazide/0.3% hydrogen peroxide in PBS. After post-antibody blocking, goat poly-horseradish peroxidase (HRP) anti-mouse IgG (Immunologic, Duiven, The Netherlands) was added for 30 minutes. The colouring reaction was developed with 3-amino-9-ethylcarbazole (AEC), and counterstaining was performed with H & E.
Paraffin-embedded tissue samples of 5-μm thickness were sequentially cut. Before blocking endogenous peroxidase activity with 1% hydrogen peroxide (Merck, Amsterdam, The Netherlands) in 2% PBS, sections were deparaffinised and rehydrated using xylene and a descending alcohol series. Blocking was performed with the following sequence: 2.5% periodic acid, 0.02% sodium borohydride and Protein Block (Dako).
Detection of TGF-β3: After deparaffinisation, antigen retrieval was performed in citrate buffer. Blocking was done with Protein Block (Dako) for 20 minutes. TGF-β3 antibody (Abcam) was applied overnight in a humid chamber at 4°C. Slides were rinsed in PBS, after which biotinylated link antibody was added (LSAB2 System; Dako) for 60 minutes. After slides were washed in PBS, streptavidin conjugate (LSAB2 System) was applied for 60 minutes.
Detection of P-Smad2: Prior to the application of Protein Block for 20 minutes, sections were pretreated with proteinase K (2 μg/mL in PBS) at 37°C for 30 minutes. P-Smad2 (Ser465/467; Cell Signaling Technology) was added overnight in a humid chamber at 4°C. Slides were rinsed in PBS, after which biotinylated link antibody was added (LSAB2 System) for 60 minutes. After PBS washing, streptavidin conjugate (LSAB2 System) was applied for 60 minutes.
Detection of α-SMA: After quenching endogenous peroxidase activity with 0.3% H2O2 in methanol, slides were heated in Tris-EDTA for 10 minutes at 100°C for antigen retrieval. The α-SMA antibody (α-SMA/1, M851, 1A4 clone; Dako, Glostrup, Denmark) was applied for 60 minutes followed by post-antibody blocking (Immunologic) for 15 minutes. After rinsing, goat poly-HRP against mouse IgG (Immunologic) was added for 30 minutes followed by PBS washing. All colouring reactions were developed by using 3,3'-diaminobenzidine (Sigma Chemical Co.) followed by counterstaining with H & E. Unlabelled samples were scored by an independent pathologist. Scoring was rated as follows: no staining (-) (except for staining in blood vessel walls), weak staining (+), moderate staining (++) and intense staining (+++).
Detection of P-ERK1/2: Before blocking endogenous peroxidase activity with 40% methanol and 1% H2O2 (Merck) in PBS, sections were deparaffinised and rehydrated using xylene and a descending alcohol series. Antigen retrieval using proteinase K (2.5 μL in 100 mmol Tris, pH 9.0, and 50 mmol EDTA, pH 8.0) for 10 minutes at 37°C was followed by three washes with 0.1 mol Tris-buffered saline (pH 7.4) containing 0.02% Tween 20 (TBST). Thereafter slides were incubated in 0.5% (Boehringer, Ingelheim, Germany) blocking reagent (BMP) in TBST for 60 minutes at 37°C. Subsequently, the P-ERK1/2 antibody (1:100; Cell Signaling Technology) diluted in 0.5% BMP/TBST was applied overnight at 4°C. Next, a species-specific biotinylated anti-IgG antibody (1:600 dilution in 0.5% BMP/TBST) was applied, followed by 45 minutes at 37°C. Incubation with streptavidin-HRP (1:200 dilution in 0.5% BMP/TBST) for 30 minutes at 37°C preceded and followed an amplification step using biotinyl-tyramide. Staining was carried out using AEC (Sigma Chemical Co., Zwijndrecht, The Netherlands) and Mayer's haematoxylin (Merck) according to the manufacturers' instructions. A water-based mounting solution was applied, and staining was visualised by using an Olympus IX51 inverted microscope equipped with the cellF Soft Imaging System (Olympus). Unlabelled samples were scored by an independent researcher.
Fibroblast-populated collagen lattice contraction assay
Three-dimensional fibroblast-populated collagen lattice (FPCL) contraction assays were carried out with primary cell cultures from passages 4 through 6. The assay was performed as described previously by others, with some modifications [30–32]. The collagen lattices were prepared by mixing a neutralising solution of COL1 (eight parts 3 mg/mL COL1 (PurCol®, Daiichi Sankyo, Parsippany, New Jersey, USA) one part 10 × α-MEM (Invitrogen) and one part HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) buffer, pH 9.0). Final collagen and cell concentrations were adjusted to 2 mg/mL and 86 × 103 cells/mL using PBS, respectively. The cell-collagen mixture was aliquoted into PBS + 2% BSA-pretreated 24-well culture dishes (0.5 mL/well) and left to polymerise for 1 hour at 37°C. In each well, to the top of the polymerised lattice, we added 0.5 mL/well of DMEM containing 10% FBS. After 2 days of incubation at 37°C, the attached FPCLs were mechanically released from the sides of the culture plates, and fresh media supplemented with 0.5% FBS and the indicated substances were added. Images were obtained at various time points over a 5-day period using the Odyssey Scanner (LI-COR Biosciences). Collagen lattice areas were measured using the corresponding Odyssey 2.1 software.
Values are expressed as means ± standard error of the mean. For statistical comparisons of two samples, an unpaired, a two-tailed Student's t-test with distinction of equal and unequal variances in a group (Levene's test) was used to determine the significance of differences between means. In addition, a nonparametric Mann-Whitney U test under the null hypothesis that the distributions of both groups (control versus Dupuytren-derived fibroblasts) were equal was performed for the data set shown in Figure 2C. All of the relevant comparisons were considered to be significantly different at P < 0.05. Experiments were performed at least three times, and representative results are shown.