Dupuytren's disease (DD) is a common and heritable fibrosis of the palmar fascia that typically manifests as permanent finger contractures. The molecular interactions that induce the development of hyper-contractile fibroblasts, or myofibroblasts, in DD are poorly understood. We have identified IGF2 and IGFBP6, encoding insulin-like growth factor (IGF)-II and IGF binding protein (IGFBP)-6 respectively, as reciprocally dysregulated genes and proteins in primary cells derived from contracture tissues (DD cells). Recombinant IGFBP-6 inhibited the proliferation of DD cells, patient-matched control (PF) cells and normal palmar fascia (CT) cells. Co-treatments with IGF-II, a high affinity IGFBP-6 ligand, were unable to rescue these effects. A non-IGF-II binding analog of IGFBP-6 also inhibited cellular proliferation, implicating IGF-II-independent roles for IGFBP-6 in this process. IGF-II enhanced the proliferation of CT cells, but not DD or PF cells, and significantly enhanced DD and PF cell contractility in stressed collagen lattices. While IGFBP-6 treatment did not affect cellular contractility, it abrogated the IGF-II-induced contractility of DD and PF cells in stressed collagen lattices. IGF-II also significantly increased the contraction of DD cells in relaxed lattices, however this effect was not evident in relaxed collagen lattices containing PF cells. The disparate effects of IGF-II on DD and PF cells in relaxed and stressed contraction models suggest that IGF-II can enhance lattice contractility through more than one mechanism. This is the first report to implicate IGFBP-6 as a suppressor of cellular proliferation and IGF-II as an inducer of cellular contractility in this connective tissue disease.

BackgroundDupuytren's contracture (DC) is a fibroproliferative disorder characterized by the progressive development of a scar-like collagen-rich cord that affects the palmar fascia of the hand and leads to digital flexion contractures. DC is most commonly treated by surgical resection of the diseased tissue, but has a high reported recurrence rate ranging from 27% to 80%. We sought to determine if the transcriptomic profiles of fibroblasts derived from DC-affected palmar fascia, adjacent phenotypically normal palmar fascia, and non-DC palmar fascial tissues might provide mechanistic clues to understanding the puzzle of disease predisposition and recurrence in DC.MethodsTo achieve this, total RNA was obtained from fibroblasts derived from primary DC-affected palmar fascia, patient-matched unaffected palmar fascia, and palmar fascia from non-DC patients undergoing carpal tunnel release (6 patients in each group). These cells were grown on a type-1 collagen substrate (to better mimic their in vivo environments). Microarray analyses were subsequently performed using Illumina BeadChip arrays to compare the transcriptomic profiles of these three cell populations. Data were analyzed using Significance Analysis of Microarrays (SAM v3.02), hierarchical clustering, concordance mapping and Venn diagram.ResultsWe found that the transcriptomic profiles of DC-disease fibroblasts and fibroblasts from unaffected fascia of DC patients exhibited a much greater overlap than fibroblasts derived from the palmar fascia of patients undergoing carpal tunnel release. Quantitative real time RT-PCR confirmed the differential expression of select genes validating the microarray data analyses. These data are consistent with the hypothesis that predisposition and recurrence in DC may stem, at least in part, from intrinsic similarities in the basal gene expression of diseased and phenotypically unaffected palmar fascia fibroblasts. These data also demonstrate that a collagen-rich environment differentially alters gene expression in these cells. In addition, Ingenuity pathway analysis of the specific biological pathways that differentiate DC-derived cells from carpal tunnel-derived cells has identified the potential involvement of microRNAs in this fibroproliferative disorder.ConclusionsThese data show that the transcriptomic profiles of DC-disease fibroblasts and fibroblasts from unaffected palmar fascia in DC patients are highly similar, and differ significantly from the transcriptomic profiles of fibroblasts from the palmar fascia of patients undergoing carpal tunnel release.

IGF2 expression and β-catenin levels are increased in Frozen Shoulder Syndrome Abstract Purpose: Frozen Shoulder Syndrome is a brosis of the shoulder joint capsule that is clinically associated with Dupuytren's disease, a brosis of the palmar fascia. Little is known about any commonalities in the pathophysiology of these connective tissue broses. β-catenin, a protein that transactivates gene expression, and levels of IGF2 mRNA, encoding insulin-like growth factor-II, are elevated in Dupuytren's disease. e aim of this study was to determine if correlating changes in β-catenin levels and IGF2 expression are evident in Frozen Shoulder Syndrome.Methods: Tissue from patients with Frozen Shoulder Syndrome and rotator cu tear were obtained during shoulder arthroscopies. Total protein extracts were prepared from tissue aliquots and β-catenin immunoreactivity was assessed by Western immunoblotting. In parallel, primary broblasts were derived from these tissues and assessed for IGF2 expression by quantitative PCR.Results: β-catenin levels were signi cantly increased in Frozen Shoulder Syndrome relative to rotator cu tear when assessed by Western immunoblotting analyses. IGF2 mRNA levels were signi cantly increased in primary broblasts derived from frozen shoulder syndrome tissues relative to broblasts derived from rotator cu tissues. Conclusions:As in Dupuytren's disease, β-catenin levels and IGF2 expression are elevated in Frozen Shoulder Syndrome. ese ndings support the hypothesis that these connective tissue broses share a common pathophysiology. Materials and Methods Tissue collectionTissue sections were collected with approval of the Human Subjects Research Ethics Board (HSREB) at Western University from surgical specimens of patients undergoing shoulder arthroscopy for the treatment of either FSS or subacromial decompression for RCT. An arthroscopic punch was used to obtain tissue specimens from the rotator cu interval immediately adjacent to the antero-superior arthroscopic portal from patients with FSS and RCT. Representative samples of these tissues were removed at the time of surgery and immediately transported to the laboratory. e tissues were either snap frozen in liquid nitrogen for total protein extraction or processed for primary broblast derivation. Western immunoblottingTotal protein extracts were prepared from snap frozen tissue using modi ed RIPA bu er. Tissue lysate (25 μg) was subjected to Western blot analysis and β-catenin levels were assessed using an anti-β-catenin monoclonal antibody (clone 14, Transduction Laboratories, Lexington, KY). β-actin levels were assessed in parallel using an anti-β-actin antibody (Sigma, St Louis, MO) to normalize for variability in total protein loading. Antibody speci c bands were visualized using enhanced chemiluminescence (ECL) and Kodak XLS lm. Densitometry analysis was carried out using Scion Image so ware (Scion Corporation, Beta 4.0.2, Frederick, MD). Normalized measurements of β-catenin were plotted as the sample mean (β-catenin /actin) ratio ± standard error...

Dupuytren's contracture (DC) is a fibroproliferative disorder of unknown etiology characterized by a scar-like contracture that develops in the palm and/or digits. We have previously reported that the eta subunit of the chaperonin containing T-complex polypeptide (CCT-eta) is increased in fibrotic wound healing, and is essential for the accumulation of α-smooth muscle actin (α-SMA) in fibroblasts. The purpose of this study was to determine if CCT-eta is similarly implicated in the aberrant fibrosis seen in DC and to investigate the role of CCT-eta in the behavior of myo/fibroblasts in DC. Fibroblasts were obtained from DC-affected palmar fascia, from adjacent phenotypically normal palmar fascia in the same DC patients (PF), and from non-DC palmar fascial tissues in patients undergoing carpal tunnel (CT) release. Inherent contractility in these three populations was examined using fibroblast-populated collagen lattices (FPCLs) and by cell traction force microscopy. Expression of CCT-eta and α-SMA protein was determined by Western blot. The effect of CCT-eta inhibition on the contractility of DC cells was determined by deploying an siRNA versus CCT-eta. DC cells were significantly more contractile than both matching palmar fascial (PF) cells and CT cells in both assays, with PF cells demonstrating an intermediate contractility in the FPCL assay. Whereas α-SMA protein was significantly increased only in DC cells compared to PF and CT cells, CCT-eta protein was significantly increased in both PF and DC cells compared to CT cells. siRNA-mediated depletion of CCT-eta inhibited the accumulation of both CCT-eta and α-SMA protein in DC cells, and also significantly decreased the contractility of treated DC cells. These observations suggest that increased expression of CCT-eta appears to be a marker for latent and active disease in these patients and to be essential for the increased contractility exhibited by these fibroblasts.