Responsiveness of Dupuytren's disease fibroblasts to 5 alpha-dihydrotestosterone

PURPOSE: We recently showed that androgen receptors are expressed in Dupuytren's contracture. The aim of the present work was to test the responsiveness of Dupuytren's fibroblasts to 5 alpha-dihydrotestosterone (5 alpha-DHT), the active form of testosterone. RESULTS: Cultured palmar fascia cells from 10 patients with Dupuytren's contracture and 4 normal subjects were exposed to 5 alpha-DHT (10 or 100 ng/mL) for 1, 3, 7, and 15 days. Their phenotype was analyzed immunohistochemically for alpha-smooth muscle actin and androgen receptor expression and proliferation rates were studied. RESULTS: At 15 days the higher concentration of 5 alpha-DHT induced an increase in Dupuytren's fibroblast proliferation, whereas anti-alpha-smooth muscle actin exhibited the strongest expression. At the same time point androgen receptor expression decreased with the lower concentration and disappeared altogether with the higher dose of 5 alpha-DHT. CONCLUSIONS:The palmar fascia is a target tissue for androgen action via androgen receptors. Further studies are required to determine whether control of androgen receptor may control the evolution of Dupuytren's disease.     

Chromosomal abnormalities in Dupuytren's contracture and carpal tunnel syndrome.

The cytogenetics of cell cultures derived from Dupuytren's tissue, adjacent palmar fascia and palmar skin from patients undergoing fasciectomy have been examined and the results compared to cell cultures established from palmar fascia, flexor retinaculum and palmar skin of patients undergoing carpal tunnel decompression. Chromosomal abnormalities were detected in cell cultures from Dupuytren's tissue in eight of the nine patients studied. Clones of cells trisomic for chromosome 8 were found in five of the nine patients. Trisomy 8 was also present in two of five flexor retinaculum cultures from carpal tunnel syndrome cases. These findings in both Dupuytren's contracture and carpal tunnel syndrome suggest the presence of chromosomal instability in the palmar fascia. The significance of the chromosomal abnormalities is however unclear, but they indicate a possible common pathway in the onset of pathological fibrosis.     

The collagen changes of Dupuytren's contracture

In Dupuytren's contracture there is an increase in the ratio of type III to type I collagen. The objective of this study was to determine if fibroblasts from patients with Dupuytren's contracture have an intrinsic aberration in collagen production or whether local factors govern the collagen changes in Dupuytren's contracture. Using a new collagen micro-method, we found that fibroblasts cultured from palmar fascia affected by Dupuytren's contracture produced similar collagen to fibroblasts derived from the palmar fascia of age- and sex-matched patients with carpal tunnel syndrome. Furthermore, the collagen changes of Dupuytren's contracture could be reproduced in all cell lines by increasing fibroblast density. At high fibroblast density, type I collagen production was inhibited: a finding that could account for the increased types III/I collagen ratio in Dupuytren's contracture. These results suggest that a genetic defect in collagen production is unlikely and that the important phenomenon is an increase in fibroblast density.     

An investigation into the role of inflammatory cells in Dupuytren's disease

An immunohistochemical study was performed on nodules excised from the palmar fascia of patients with Dupuytren's contracture. In cellular nodules, antibodies to actin (used as a marker for myofibroblasts), desmin, vimentin, Mac 387 (a macrophage marker) and leucocyte common antigen were used. A correlation was demonstrated between the numbers of macrophages and the presence of myofibroblasts. The presence of myofibroblasts is generally considered to indicate the active stage of the disease. Inflammatory cells other than macrophages were largely absent from the nodules, although lymphocytes were frequent in the tissue around the nodules. Microvascular changes were prominent in the nodules and pericyte proliferation was observed around occluded capillaries. Release of growth factors from macrophages may be important in Dupuytren's contracture, as is the case in other fibrotic diseases. The possible role of macrophages in the aetiology of Dupuytren's disease is discussed.     

Enzymes of glucose metabolism in palmar fascia and Dupuytren's contracture.

Several enzymes participating in glucose metabolism and some of the acid hydrolases were assayed in palmar fascia and Dupuytren's contracture with fluorometric microanalytical methods. The enzyme activities of glucose metabolism were lower in normal palmar fascia than in dermis. The fascia of Dupuytren's contracture exhibited a general increase in the enzyme activities of glucose catabolism. Little alteration was found in alanine aminotransferase and UDP-glucose dehydrogenase activity in the lesion. Lysosomal hydrolytic enzyme activities were increased five to ten times in Dupuytren's tissue. The dermis overlying Dupuytren's contracture exhibited an increase in the enzyme activities of glucose catabolism, but to a lesser degree than did the fascia of the lesion. The epidermis of involved palmar skin displayed normal enzyme activities.     

The effect of 5-fluorouracil on Dupuytren fibroblast proliferation and differentiation

INTRODUCTION: Dupuytren's disease is a proliferative disease with contractile properties, prone to recur after surgery. Intra-operatively applied 5-fluorouracil has been used to avoid scar problems in the eye after glaucoma filtration surgery and was therefore investigated as a means to inhibit proliferation and myofibroblast differentiation in Dupuytren fibroblasts in vitro. METHOD: Primary cell lines were obtained by explants from Dupuytren's tissue (n = 6), non-diseased palmar fascia from patients with Dupuytren's disease (n = 3) and carpal ligament from patients undergoing carpal tunnel release (n = 3). The effect of 5-fluorouracil on proliferation was assessed by cell counting. Myofibroblast differentiation, an intergral part of Dupuytren's contracture, was investigated by staining for alpha smooth muscle actin, a marker for contractile cells, using immunohisto-chemical methods. RESULTS: A single exposure to 5-fluorouracil caused a sustained inhibition of proliferation in Dupuytren's and non-diseased fascia cultures, whilst the effect on carpal ligament cultures was transient. Untreated Dupuytren's fibroblasts exhibited the highest myofibroblast differentiation, whilst differentiation in non-diseased fascia cultures was shown to be proportional to cell density and virtually non-existent in carpal ligament cultures. After 5-fluorouracil exposure, the differentiation was significantly reduced in Dupuytren's fibroblasts cultures, reduced at high cell densities in non-diseased fascia and unchanged in carpal ligament cell cultures. DISCUSSION: 5-fluorouracil inhibits both proliferation and myofibroblast differentiation in Dupuytren's cell cultures and may have a potential use as an adjuvant treatment to Dupuytren surgery in order to reduce the rate of recurrence and contracture.     

Gene expression analysis of Dupuytren's disease: the role of TGF-beta2

Dupuytren's disease is characterised by nodular fibroblastic proliferation of the palmar fascia leading to contracture of the hand. Transforming growth factor beta (TGF-beta) is thought to play a role in its pathogenesis. We performed a cDNA microarray analysis of Dupuytren's diseased cord tissue with an emphasis on TGF-beta isoforms. Normal-appearing transverse ligament of the palmar fascia from adjacent to the diseased cord and palmar fascia from patients undergoing carpal tunnel release were used as controls. TGF-beta gene expression was confirmed by quantitative real-time polymerase chain reaction. Over 20 unique genes were found to be significantly up-regulated, including several previously reported genes. A dominant increase in TGF-beta2 expression was seen in the cord tissue, whereas TGF-beta1 and TGF-beta3 were found not to be significantly up-regulated. Quantitative real-time polymerase chain reaction confirmed these findings. This gene expression profile allows for further experiments that may eventually lead to gene therapy to block the development and progression of Dupuytren's disease clinically.     

The myofibroblast in Dupuytren's contracture.

Dupuytren's contracture nodules, but not cords, contain myofibroblasts. These cells, which combine many electron microscopic, physiologic, and immunohistochemical characteristics of fibroblasts and smooth muscle cells, are probably the active force of contraction. Prominent myofibroblasts and intracellular microtubules correlate with increased likelihood of clinical recurrence after surgery. Tissue culture of cells derived from Dupuytren's contracture myofibroblasts show consistently slower cell replication than from fibroblasts and show persistence of electron microscopic characteristics in early passages. Research in Dupuytren's contracture myofibroblasts has been done on human tissue and so has clinical correlation. Myofibroblast presence may help to predict recurrence of disease and suggests that palmar skin should be excised when adherent to disease nodules. The theory of myofibroblasts helps explain why the open technique often succeeds, and why full thickness skin grafts inhibit recurrent contracture.     

Cellular structure and biology of Dupuytren's disease

Numerous studies support the idea that the myofibroblast is a key cell responsible for the tissue contraction in Dupuytren's disease. In vitro models have been developed to study the underlying cellular basis of myofibroblast differentiation and contraction. Studies suggest that the growth factor TGF-beta 1 combined with mechanical stress can promote the differentiation of fibroblasts into myofibroblasts. Agonists, such as LPA and thrombin, can promote the contraction of myofibroblasts through specific intracellular signaling pathways that regulate levels of phosphorylated myosin light chain. Agents that can affect these intracellular signaling pathways hold promise as a means to decrease contraction of the myofibroblast and of the palmar fascia in Dupuytren's disease. Finally, the recent finding that IFN-gamma can suppress both the differentiation of the myofibroblast and the generation of contractile force, together with preliminary clinical results using IFN-gamma, suggest the potential use of IFN-gamma for nonsurgical therapy of Dupuytren's disease. Future studies into the cellular basis of tissue contraction should provide alternative methods to improve management of Dupuytren's contracture.     

The effect of steroids on Dupuytren's disease: role of programmed cell death

This study compared the rates of proliferation and apoptosis of cells within nodules of Dupuytren's disease and nodules from patients that had been injected preoperatively with steroid (Depo-Medrone). It also compared the effects of steroids in apoptosis in cultured Dupuytren's cells and control fibroblasts from palmar fascia and fascia lata. Steroids reduced the rate of fibroblast proliferation and increased the rate of apoptosis of both fibroblasts and inflammatory cells in Dupuytren's tissue. Steroids also produced apoptosis of cultured Dupuytren's cells but not of palmar fascia and fascia lata cells.     

Gelatinase A activity in Dupuytren's disease

PURPOSE: Dupuytren's contracture is a fibroproliferative disorder of the hand characterized by an abnormal myofibroblast and fibroblast proliferation and extracellular matrix deposition leading to retraction and deformation of the palm. Recent studies have shown that molecules of extracellular matrix may coordinate morphogenesis, cell differentiation, and most importantly, fibrogenesis in tissue. Gelatinase A (MMP-2) is a member of the matrix metalloproteinase family of proteolytic enzymes that contribute to remodeling the extracellular matrix by degrading its components. The aim of this study was to determine the level of MMP-2 activation in the palmar fascia of patients with Dupuytren's contracture with reference to the clinical stages of disease progression and recurrence of the contracture after surgery. METHODS: The level of relative MMP-2 activation, expressed by the active to latent MMP-2 ratio, was investigated with use of zymography and computerized densitometry in 16 normal and 71 pathologic tissues characterizing different clinical stages of the disease progression. RESULTS: We found that the level of MMP-2 activation was significantly elevated in the palmar fascias with Dupuytren's contracture compared with normal tissues. We did not find statistically significant differences between groups with different stages of the disease progression. We also did not find a relation between a high level of MMP-2 activation and the recurrence in the area of surgically treated Dupuytren's contracture. CONCLUSIONS: The differences in MMP-2 activation between contractured and normal fascia suggest a participation of this enzyme in the promotion of Dupuytren's disease. We did not find a relationship, however, between the level of MMP-2 activation and the secondary contracture.     

Dupuytren's contracture: morphological and biochemical changes in palmar aponeurosis

The palmar aponeurosis removed from ten patients with Dupuytren's contracture was studied using morphological and biochemical approaches. The histological characteristic of Dupuytren's contracture is the presence of numerous nodules among the lamellar structures of the collagen fibres. In the nodules, there are many active fibroblasts which are surrounded by immature fibres and metachromatic substances demonstrated by toluidine blue staining. Ultrastructurally, the active fibroblasts have the characteristics of myofibroblasts, as previously reported by Dr. Gabbiani. We found that some fibroblasts have intracellular collagen fibrils in the cytoplasm. When assayed by Siegel and Martin's method, lysyl oxidase activity of the palmar aponeurosis was significantly higher in Dupuytren's contracture than in normal hands. Biochemical studies such as electrophoretic analysis of mucopolysaccharides, determination of uronic acid and collagen contents were undertaken to compare the aponeurosis of Dupuytren's contracture with normal cases. The uronic acid contents were higher in Dupuytren's contracture than in the controls. However, no difference between the two groups was found in the collagen contents and in the composition of the mucopolysaccharides. These characteristic features; existence of myofibroblasts and intracellular collagen fibrils and increase in the activity of lysyl oxidase probably play a significant role in the establishment of flexion contracture of the fingers in Dupuytren's contracture.     

Dupuytren's contracture: an update of biomolecular aspects and therapeutic perspectives

The so-called fibrogenic cytokines, able to induce the growth of fibroblasts and their differentiation into myofibroblasts and to stimulate their production of extracellular matrix, are involved in the genesis of Dupuytren's contracture. Although many studies have been made of biomolecular aspects of palmar fibromatosis, practical applications from them are still far from imminent because of the real difficulty of blocking their action in vivo, even in a chronic, progressive lesion such as Dupuytren's disease. Consequently, surgical excision of the palmar fascia still remains the treatment of choice.     

Dupuytren's disease: comparative growth dynamics and morphology between cultured myofibroblasts (nodule) and fibroblasts (cord)

The excised palmar fascia of 11 patients with Dupuytren's disease was separated clinically into nodules and cords. Myofibroblasts were seen by light and electron microscopy in each of the nodules, but the cords generally lacked myofibroblasts. Only one cord specimen had microscopic features that were intermediate between nodule and cord. Electron microscopy demonstrated that in vivo differences between myofibroblasts from nodules and fibroblasts from cords and control skin samples could be preserved in vitro. Growth studies showed slower growth of cultured myofibroblasts (mean +/- SD generation time 68.7 +/- 15 h) than cord-derived fibroblasts (mean +/- SD generation time 51.5 +/- 0.9 h). These data suggest that the life cycle of the myofibroblasts from Dupuytren's disease nodules differs from that of fibroblasts found in cordlike tissues. These myofibroblasts have biological characteristics nearly identical to those of myofibroblasts found in other contracting tissues, such as granulating wounds and breast cancer.     

The different characteristics of Dupuytren's disease fibroblasts derived from either nodule or cord: expression of alpha-smooth muscle actin and the response to stimulation by TGF-beta1

Mechanisms behind the onset and progression of Dupuytren's disease are poorly understood. Both myofibroblasts and transforming growth factor beta 1 (TGF-beta(1)) have been implicated. We studied fibroblast cultures derived from nodules or cords of Dupuytren's contracture tissue to determine the proportion of myofibroblasts present in comparison with flexor retinaculum fibroblast cultures. We identified myofibroblasts by immunohistochemical staining for alpha-SMA. We then investigated the effects of TGF-beta(1) stimulation on these fibroblasts. Basal myofibroblast/fibroblast proportions were 9.7% in nodule cell cultures, 2.7% in cord cell cultures and only 1.3% in flexor retinaculum cell cultures. Nodule and cord myofibroblast proportions increased to 25.4% and 24.2%, respectively, in response to TGF-beta(1) treatment. Flexor retinaculum cell cultures showed no response to TGF-beta(1) stimulation. Fibroblasts cultured from specific regions of Dupuytren's tissue retain myofibroblast features in culture. TGF-beta(1) stimulation causes an increased myofibroblast phenotype to similar levels in both nodule and cord, suggesting that previously quiescent cord fibroblasts can be reactivated to become myofibroblasts by TGF-beta(1). This could be an underlying reason for high recurrence rates seen after surgery or progression following injury.     

The cytoskeleton and extracellular matrix of the Dupuytren's disease "myofibroblast": an immunofluorescence study of a nonmuscle cell type

The cytoskeleton and the extracellular matrix of myofibroblasts in nodules from Dupuytren's diseased palmar fascia were examined by indirect immunofluorescence. Primary antibodies used as probes of these tissue compartments were directed against (1) smooth muscle myosin, (2) nonmuscle myosin--components of the cytoplasmic contractile apparatus in smooth muscle and nonmuscle cells, respectively, (3) laminin, and (4) fibronectin--extracellular glycoproteins mediating cell-matrix attachment in smooth muscle and nonmuscle fibroblastic cells, respectively. The Dupuytren's nodular cells stained for nonmuscle myosin and fibronectin but not for smooth muscle myosin or laminin; this indicated that, at the level of biochemical differentiation, these cells are a nonmuscle type. Staining for fibronectin between nodular cells was dramatically increased over that seen between fibroblasts of the normal palmar fascia. Because of the non-muscle nature of the distinctive contractile cell type of the Dupuytren's nodule, we suggest that the term myofibroblast should be considered a misnomer when applied to this pathogenic cell type.     

Langerhans cells in Dupuytren's contracture

We have examined biopsies of Dupuytren's contracture palmar fascia, overlying subcutis and skin, and have correlated the distribution of gross macroscopic changes in the hand, mapped pre- and intraoperatively, with light microscopic immunohistochemical findings. We report increased numbers of S100 positive Langerhans cells (an epidermal cell of dendritic lineage) and CD45 positive cells, both in "nodules" and at dermo-epidermal junctions, in the biopsied tissues. This suggests that Langerhans cells migrate from the epidermis into Dupuytren's contracture tissue, possibly in response to local changes in levels of inflammatory cytokines within the tissue. Our findings, together with other reports of increased numbers of dermal dendrocytes and inflammatory cells in Dupuytren's contracture tissue, lend circumstantial support to the "extrinsic theory" of the pathogenesis of Dupuytren's contracture. However, the earliest stages of the disease process have not been defined, and therefore the events which ultimately produce fibrosis in the palmar fascial complex in susceptible individuals could begin in the skin and/or within deeper tissues, especially where there is dysregulation of the immune system.     

Prostaglandins influence myofibroblast contractility in Dupuytren''s disease

This study investigated if the vasoactive prostaglandins, PGE2, and PGF2 alpha, were identifiable in association with nodular myofibroblasts of patients with Dupuytren's disease. Immunocytochemic studies, using antibodies specific for these prostaglandins, have confirmed their association with myofibroblasts. Radioimmunoassay was used to quantitate the prostaglandins. Our results indicate a significant increase of both prostaglandins, especially PGF2 alpha, in Dupuytren's palmar fascia when compared with control fascia. These endogenous prostaglandins may influence the contractile behavior of myofibroblasts in Dupuytren's disease to contribute to the pathobiology of this disorder.     

Regulation of proliferation and platelet-derived growth factor expression in palmar fibromatosis (Dupuytren contracture) by mechanical strain

Palmar fibromatosis (Dupuytren contracture) causes fibrosis of specific palmar fascial bands. These bands are subjected to repetitive mechanical strain in situ. Primary cell cultures were derived from (a) palmar fibromatosis from eight patients, (b) uninvolved palmar fascia (Skoog's fibers) from four of these patients and (c) normal palmar fascia from four additional patients. The cells were plated onto collagen-coated membranes either subjected to cyclic strain (25% maximal strain at 1 Hz) or without strain. Bromodeoxy uridine incorporation showed an increase in proliferation in all cultures subjected to strain. This increase was highest for palmar fibromatosis (10 to 40% nuclear incorporation, p = 0.02). Skoog's fibers and fascia from the normal individuals showed a trend (not significant) toward increase with strain (8 to 25%, p = 0.15 for Skoog's fibers, and 8 to 15%, p = 0.45 for normal fascia). Cyclic strain increased the expression of platelet derived growth factor-A relative to glyceraldehyde-3-phosphate dehydrogenase in palmar fibromatosis (2.2 to 3.5, p = 0.05) and Skoog's fibers (0.8 to 2.0, p = 0.04). The expression of platelet-derived growth factor-B relative to glyceraldehyde-3-phosphate dehydrogenase was enhanced by cyclic strain only in the fibromatosis tissue (0.7 to 2.1, p = 0.04). The normal fascia did not express platelet-derived growth factor. Platelet-derived growth factor neutralizing antibody decreased bromodeoxyuridine incorporation in fibromatosis cultures subjected to cyclic strain to near levels for those grown in the absence of strain (38 to 16%, p = 0.05). Conditioned medium from fibromatosis cells grown under stain showed a trend toward increased proliferation in additional fibromatosis cultures compared with conditioned medium from fibromatosis cells grown without strain (9 to 15% nuclear incorporation, p = 0.20). The observed palmar fibromatosis contracture can be partially explained on the basis of the cell's response to cyclic strain, which may be mediated by plateletderived growth factor.