Free radicals and Dupuytren's contracture.

The concentration of substrate expressed as hypoxanthine capable of reacting with xanthine oxidase to release superoxide free radicals (O2-) was measured in control and Dupuytren''s contracture palmar fascia. In Dupuytren''s contracture palmar fascia the concentration of hypoxanthine was six times that of control and was greatest in "nodular" areas. Xanthine oxidase activity was also detected in Dupuytren''s contracture palmar fascia. These results suggest a greater potential for hypoxanthine-xanthine oxidase generated oxygen free radical formation in Dupuytren''s contracture than in control palmar fascia. Production of free radicals may be an important factor in the pathogenesis of Dupuytren''s contracture. The benefit of allopurinol in the management of Dupuytren''s contracture and other fibrotic conditions may thus be explained, as allopurinol binds to xanthine oxidase and prevents release of free radicals.     

Identification of Histological Patterns in Clinically Affected and Unaffected Palm Regions in Dupuytren's Disease

Dupuytren''s disease is a fibro-proliferative disease characterized by a disorder of the extracellular matrix (ECM) and high myofibroblast proliferation. However, studies failed to determine if the whole palm fascia is affected by the disease. The objective of this study was to analyze several components of the extracellular matrix of three types of tissues—Dupuytren''s diseased contracture cords (DDC), palmar fascia clinically unaffected by Dupuytren''s disease contracture (NPF), and normal forehand fascia (NFF). Histological analysis, quantification of cells recultured from each type of tissue, mRNA microarrays and immunohistochemistry for smooth muscle actin (SMA), fibrillar ECM components and non-fibrillar ECM components were carried out. The results showed that DDC samples had abundant fibrosis with reticular fibers and few elastic fibers, high cell proliferation and myofibroblasts, laminin and glycoproteins, whereas NFF did not show any of these findings. Interestingly, NPF tissues had more cells showing myofibroblasts differentiation and more collagen and reticular fibers, laminin and glycoproteins than NFF, although at lower level than DDC, with similar elastic fibers than DDC. Immunohistochemical expression of decorin was high in DDC, whereas versican was highly expressed NFF, with no differences for aggrecan. Cluster analysis revealed that the global expression profile of NPF was very similar to DDC, and reculturing methods showed that cells corresponding to DDC tissues proliferated more actively than NPF, and NPF more actively than NFF. All these results suggest that NPF tissues may be affected, and that a modification of the therapeutic approach used for the treatment of Dupuytren''s disease should be considered.     

Genome-Wide Analysis Using Exon Arrays Demonstrates an Important Role for Expression of Extra-Cellular Matrix,Fibrotic Control and Tissue Remodelling Genes in Dupuytren's Disease

Dupuytren''s disease (DD) is a classic example of pathological fibrosis which results in a debilitating disorder affecting a large sector of the human population. It is characterized by excessive local proliferation of fibroblasts and over-production of collagen and other components of extracellular matrix (ECM) in the palmar fascia. The fibrosis progressively results in contracture of elements between the palmar fascia and skin causing flexion deformity or clawing of the fingers and a severe reduction in hand function. While much is known about the pathogenesis and surgical treatment of DD, little is known about the factors that cause its onset and progression, despite many years of research. Gene expression patterns in DD patients now offers the potential to identify genes that direct the pathogenesis of DD. In this study we used primary cultures of fibroblasts derived from excisional biopsies of fibrotic tissue from DD patients to compare the gene expression profiles on a genome-wide basis with normal control fibroblasts. Our investigations have identified genes that may be involved with DD pathogenesis including some which are directly relevant to fibrosis. In particular, these include significantly reduced expression levels of three matrix metallopeptidases (MMP1, MMP3, MMP16), follistatin, and STAT1, and significantly increased expression levels of fibroblast growth factors (FGF9, FGF11), a number of collagen genes and other ECM genes in DD patient samples. Many of these gene products are known to be involved in fibrosis, tumour formation and in the normal processes of tissue remodelling. In addition, alternative splicing was identified in some DD associated genes. These highly sensitive genomic investigations provide new insight into the molecular mechanisms that may underpin the development and progression of DD.     

Post‐transcriptional regulation of α‐smooth muscle actin determines the contractile phenotype of Dupuytren's nodular cells

The objective was to study Dupuytren's myofibroblast cells in constrained collagen matrices in order to more closely emulate their in vivo environment and, to correlate their contractility with α‐smooth muscle actin (α‐SMA) expression and determine if dermal fibroblasts regulate Dupuytren's myofibroblast phenotype. Isotonic and isometric force contraction by cells isolated from Dupuytren's nodules, palmar and non‐palmar skin fibroblasts was measured in collagen matrices. The effect of co‐culturing nodule cells with dermal fibroblasts on isometric contraction was examined. Isometric contraction was correlated with levels of α‐SMA mRNA by pcr and protein by Western blotting, and α‐SMA distribution assessed by immunofluorescence. Dupuytren's nodule cells exhibited similar levels of isotonic contraction to both palmar and non‐palmar dermal fibroblasts. However, nodule cells generated high levels of isometric force (mean: 3.5 dynes/h), which continued to increase over 24 h to a maximum of 173 dynes. In contrast, dermal fibroblasts initially exhibited low levels of contraction (mean: 0.5 dynes/h) and reached tensional homeostasis on average after 15 h (range: 4–20 h), with a maximum force of 52 dynes. Although all three cell types had similar α‐SMA mRNA levels, increased levels of α‐SMA protein were observed in nodule cells compared to dermal fibroblasts. α‐SMA localised to stress fibres in 35% (range: 26–50%) of nodule cells compared to only 3% (range:0–6%) of dermal fibroblasts. Co‐cultures of Dupuytren's cells and dermal fibroblasts showed no contractile differences. The contractile phenotype of Dupuytren's myofibroblasts is determined by increased α‐SMA protein distributed in stress fibres, not by cellular mRNA levels. Dupuytren's cell contractility is not influenced by dermal fibroblasts. J. Cell. Physiol. 224: 681–690, 2010. © 2010 Wiley‐Liss, Inc.     

IGF-II and IGFBP-6 regulate cellular contractility and proliferation in Dupuytren's disease

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.     

Dupuytren's Contracture—Etiology and Principles of Treatment

Although Dupuytren''s contracture was first described more than 300 years ago, the cause is not yet certainly known. It is only agreed that the disease process affecting the palmar fascia is most commonly found in older-aged males of Caucasian descent.Conservative or non-surgical treatment is usually ineffective. Surgical treatment includes fasciotomy, partial fasciectomy and total fasciectomy. Fasciotomy is indicated for localized disease or for patients with medical conditions which preclude more extensive operation. Subtotal fasciectomy with or without skin graft is the most commonly employed procedure. Total palmar fasciectomy is seldom indicated. Improved function of the hand can be anticipated after adequate surgical intervention.     

Increased CCT-eta expression is a marker of latent and active disease and a modulator of fibroblast contractility in Dupuytren’s contracture

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.     

Mechanical and migratory properties of normal,scar, and Dupuytren's fibroblasts

Mechanical properties of myofibroblasts play a key role in Dupuytren's disease. Here, we used atomic force microscopy to measure the viscoelastic properties of 3 different types of human primary fibroblasts derived from a same patient: normal and scar dermal fibroblasts and palmar fascial fibroblasts from Dupuytren's nodules. Different stiffness hydrogels (soft ~1 kPa and stiff ~ 50 kPa) were used as cell culture matrix to mimic the mechanical properties of the natural tissues, and atomic force microscopy step response force curves were used to discriminate between elastic and viscous properties of cells. Since transforming growth factor‐β1 (TGF‐β1) is known to induce expression of α–smooth muscle actin positive stress fibers in myofibroblasts, we investigated the behavior of these fibroblasts before and after applying TGF‐β1. Finally, we performed an in vitro cell motility test, the wound healing or scratch assay, to evaluate the migratory properties of these fibroblasts. We found that (1) Dupuytren's fibroblasts are stiffer than normal and scar fibroblasts, the elastic modulus E ranging from 4.4, 2.1, to 1.8 kPa, for Dupuytren's, normal and scar fibroblasts, respectively; (2) TGF‐β1 enhances the level of α–smooth muscle actin expression and thus cell stiffness in Dupuytren's fibroblasts (E, ~6.2 kPa); (3) matrix stiffness influences cell mechanical properties most prominently in Dupuytren's fibroblasts; and (4) Dupuytren's fibroblasts migrate slower than the other fibroblasts by a factor of 3. Taking together, our results showed that mechanical and migratory properties of fibroblasts might help to discriminate between different pathological conditions, helping to identify and recognize specific cell phenotypes.     

Cell behavior and cell-matrix interactions of human palmar aponeurotic cells in vitro

The present investigation has been performed to better characterize, in vitro, normal aponeurotic cells in comparison with dermal fibroblasts and with cells derived from Dupuytren's affected aponeuroses. Cells were cultured in monolayer and/or into three-dimensional collagen gels. Cell structure, adhesion, and spreading capability on different substrates, as well as integrin expression were investigated by light and electron microscopy and by flow cytometry. Cell-matrix interactions were also analyzed by gel retraction experiments in the presence, or absence, of RGD peptides and anti-integrin antibodies. Normal aponeurotic cells, compared with dermal fibroblasts, exhibited in vitro peculiar structural features, which were substantially maintained in Dupuytren's aponeurotic cells, irrespective of the substrate they were grown on. By contrast, the aponeurotic cell behavior was different in normal and diseased cells, these latter approaching that of dermal fibroblasts. Normal aponeurotic cells, in fact, were characterized by low efficiency in retracting the collagen gel, low α₂, α₁, and α₅ integrin subunit expression and low adhesion properties onto collagen and fibronectin, whereas cells isolated from the aponeuroses of Dupuytren's patients exhibited higher capability of retracting the collagen gel, increased adhesion properties toward collagen and fibronectin, and higher levels of integrin expression. No differences were observed between dermal fibroblasts from Dupuytren's patients or from normal subjects. These in vitro results are consistent with those previously obtained in situ, suggesting that palmar aponeurotic cells have a peculiar phenotype and that changes in cell-matrix interactions occur in Dupuytren's contracture. Moreover, by comparing data obtained from the retracted fibrotic cords and the still clinically unaffected aponeuroses of the same patients, it may be noted that Dupuytren's disease is not only confined to the clinically involved branches, but includes the whole aponeurosis of the affected hand. J. Cell. Physiol. 173:415–422, 1997. © 1997 Wiley-Liss, Inc.     

Dupuytren's disease.

Dupuytren''s disease is an extremely common malady, affecting as many as 3% of the general population. Presenting features are variable and include simple asymptomatic palmar nodules or refractory contractures of the interphalangeal joints. Substantial associations with knuckle pads, plantar nodules, and Peyronie''s disease are noteworthy. Although a strong familial tendency is present, the precise pathologic mechanism is unknown. Treatment is frequently unnecessary, but when indicated it includes a variety of surgical alternatives. An appropriately timed referral to a surgical specialist before irreversible contracture of the interphalangeal joints can prevent a permanent loss of function. When surgical intervention is not elected, careful and regular follow-up is necessary to detect early joint contracture.     

Periostin differentially induces proliferation, contraction and apoptosis of primary Dupuytren's disease and adjacent palmar fascia cells

Dupuytren's disease, (DD), is a fibroproliferative condition of the palmar fascia in the hand, typically resulting in permanent contracture of one or more fingers. This fibromatosis is similar to scarring and other fibroses in displaying excess collagen secretion and contractile myofibroblast differentiation. In this report we expand on previous data demonstrating that POSTN mRNA, which encodes the extra-cellular matrix protein periostin, is up-regulated in Dupuytren's disease cord tissue relative to phenotypically normal palmar fascia. We demonstrate that the protein product of POSTN, periostin, is abundant in Dupuytren's disease cord tissue while little or no periostin immunoreactivity is evident in patient-matched control tissues. The relevance of periostin up-regulation in DD was assessed in primary cultures of cells derived from diseased and phenotypically unaffected palmar fascia from the same patients. These cells were grown in type-1 collagen-enriched culture conditions with or without periostin addition to more closely replicate the in vivo environment. Periostin was found to differentially regulate the apoptosis, proliferation, α smooth muscle actin expression and stressed Fibroblast Populated Collagen Lattice contraction of these cell types. We hypothesize that periostin, secreted by disease cord myofibroblasts into the extra-cellular matrix, promotes the transition of resident fibroblasts in the palmar fascia toward a myofibroblast phenotype, thereby promoting disease progression.     

Dupuytren's Contracture of the Fingers—A Simplified Approach to Surgical Treatment

Hematoma formation, delay in healing, pain, stiffened finger joints are complications that sometimes follow classical surgical approaches to Dupuytren''s contracture. A new surgical approach to the disease that can correct the contractures without the attendant morbidity is urgently needed. By treating Dupuytren''s as any other scar contracture (division of the contracting soft tissue at its point of maximal tension and interposing normal free full-thickness skin) postoperative morbidity can be greatly decreased. Full return of function was achieved within 21 days following operation in 85 percent of the cases in which finger contractures were present before operation. Contracture release of 100 joints was done by this means, with loss of only one graft and without recurrence of the disease. This technique offers simple surgical control of progressive and recurrent Dupuytren''s contracture of the fingers with minimal postoperative morbidity.     

Improving identification of differentially expressed genes by integrative analysis of Affymetrix and Illumina arrays

Together with the widely used Affymetrix microarrays, the recently introduced Illumina platform has become a cost-effective alternative for genome-wide studies. To efficiently use data from both array platforms, there is a pressing need for methods that allow systematic integration of multiple datasets, especially when the number of samples is small. To address these needs, we introduce a meta-analytic procedure for combining Affymetrix and Illumina data in the context of detecting differentially expressed genes between the platforms. We first investigate the effect of different expression change estimation procedures within the platforms on the agreement of the most differentially expressed genes. Using the best estimation methods, we then show the benefits of the integrative analysis in producing reproducible results across bootstrap samples. In particular, we demonstrate its biological relevance in identifying small but consistent changes during T helper 2 cell differentiation.     

Platelet derived growth factor BB is a ligand for dermatan sulfate chain(s) of small matrix proteoglycans from normal and fibrosis affected fascia

Structural requirements of the short isoform of platelet derived growth factor BB (PDGF-BB) to bind dermatan sulfate (DS)/chondroitin sulfate (CS) are unknown. Meanwhile the interaction may be important for tissue repair and fibrosis which involve both high activity of PDGF-BB and matrix accumulation of DS. We examined by the solid phase assay the growth factor binding to DS chains of small proteoglycans from various fasciae as well as to standard CSs. Before the assay a structural analysis of DSs and CSs was accomplished involving the evaluation of their epimerization and/or sulfation patterns. In addition, in vivo acceptors for PDGF-BB in fibrosis affected fascia were detected. PDGF-BB binding sites on DSs/CSs are located in long chain sections with the same type of hexuronate isomer however without any apparent preference to glucuronate or iduronate residues. Alternatively, the interaction seems to involve two shorter DS chain sections assembling disaccharides with the same type of hexuronate isomer which are separated by disaccharide(s) with another hexuronate one. Moreover, DS/CS affinity to the growth factor most probably depends on an accumulation of di-2,4-O-sulfated disaccharides in binding site while the presence of 6-O-sulfated N-acetyl-galactosamine residues rather attenuates the binding. All examined fascia DSs and standard CSs showed significant PDGF-BB binding capability with the highest affinity found for normal palmar fascia decorin DS. In fibrosis affected palmar fascia DS/CS proteoglycans are able to form with PDGF-BB supramolecular complexes also including other matrix components such as type III collagen and fibronectin which bind the growth factor covalently. Our results suggest that DS chains of fascia matrix small PGs may regulate PDGF-BB availability leading to restriction of fibrosis associated with Dupuytren's disease or to control of normal fascia repair.