Our aims were to describe the distribution of alpha-smooth muscle actin (SMA)-containing cells in
Dupuytren's tissue in vivo and to determine the effects of selected agents in regulating the expression of SMA in Dupuytren's cells in vitro.
In selected hypercellular zones of Dupuytren's nodules up to 40% of the cells contained SMA, as shown by immunohistochemistry. A lower percentage (20%) of SMA-containing cells was found in regions of lower cellularity. A notable finding was that treatment in vitro of Dupuytren's cells with platelet-derived growth factor significantly reduced the content of SMA. Cells from the same patients showed a significant increase in expression of SMA in response to treatment with transforming growth factor, which confirmed recent findings. In addition, interferon-gamma, which has been previously used as a treatment for Dupuytren's disease in a clinical study, had no reproducible effect on the expression of this actin isoform. Our findings are of significance for the conservative management of contractures.
J Bone Joint Surg [Br] 2003;85-B:448-55.
Received 4 February 2002: Accepted after revision 14 June 2002
Dupuytren's contracture, or palmar fibromatosis, is one of several fibromatoses characterised by nodular or distributed aggregates of immature fibroblasts dispersed in a dense collagen matrix.1 As a result of the proliferation and action of myofibroblasts in the palmar fascia, patients with Dupuytren's disease may have progressive and irreversible flexion contractures of the phalangeal joints. Myofibroblasts were first implicated as being responsible for contracture in Dupuytren's disease on the basis of their ultrastructural identification in transmission electron-microscopic studies.2 Later investigations showed the expression of ax-smooth muscle actin (SMA) in cells from tissue explants.3 It was also shown that myofibroblasts were capable of generating a contractile force.3,4
Historically, surgery has been the main method of treatment of Dupuytren's disease, but the growing understanding of the role of cytokines in the aetiology of the disease suggests that non-surgical alternatives could be developed. Modification of the disease may result from regulation of the contractile cell phenotype by cytokines. A recent study5 has shown the effectiveness of using pharmacological agents to reduce in vitro the contraction of Dupuytren's cells stimulated by lysophosphatidic acid.
Luck6 described three phases through which Dupuytren's diseased tissue is believed to progress: proliferative, involutional, and residual. The relative abundance and distribution of myofibroblasts in Dupuytren's tissue vary according to the phase of the disease. During the proliferative stage, when fascial fibroplasia causes a nodular lesion, myofibroblasts appear and contribute extensively to the nodule. The involutional phase is marked by an alignment of the cells with the lines of stress within the tissue, and the myofibroblasts show a similar pattern. In the residual phase, the nodule is replaced by scar tissue and myofibroblasts can no longer be identified. They appear to have been replaced by mature fibroblasts.7 During the progression of the disease, SMA is also expressed transiently. It appears and increases during the proliferative phase, remains during the involutional phase, and disappears during the residual phase.8
One objective of our study was to evaluate immunohistochemically the distribution of SMA-expressing cells in Dupuytren's tissue in order to determine the number of these cells in the various cellular regions which comprise Dupuytren's nodule and to establish the prevalence of this actin isoform in situ by direct examination of tissue samples. A second objective was to determine the effects of the following cytokines on the expression of SMA in Dupuytren's cells in vitro by Western blot analysis: transforming growth factor (TGF)-beta1, platelet-derived growth factor (PDGF)-BB, and interferon (IFN)-gamma. Recent immunofluorescence studies have shown that TGF-beta1 can increase the expression of SMA in cells isolated from Dupuytren's tissue9 and that IFN-gamma can block the TGF-(31 stimulation of SMA.10 Previous studies11,12 have shown a reduction of SMA in fibroblasts from other sources treated with PDGFBB by Western blot analysis.
A recent study13 has used injections of purified clostrid ial collagenase to disrupt the Dupuytren's nodule in the nonsurgical management of the disease. While the results are promising there is a concern about the incidence of recurrence of the contracture. A direct comparison of the effects of selected growth factors on the expression of SMA in Dupuytren's cells could be instructive in providing adjunctive therapy for the enzymic treatment of the disease and to reduce the incidence of recurrence. The value of our study extends beyond Dupuytren's disease. The development of a non-surgical treatment for Dupuytren's disease may also be implicated in the management of other contractures.
Materials and Methods
Tissue specimens were obtained from 13 patients undergoing subtotal fasciectomy for Dupuytren's contracture (Table I). The indication for surgery was failure of conservative management. The excised tissue was obtained for study under a protocol which was approved by the Institutional Review Board. Two patients had recurrence of the disease. Samples from eight patients were allocated for SMA immunohistochemistry and specimens from the remaining five were designated for cell culture and Western blot analysis of the effects of selected cytokines on the expression of SMA.
Immunohistochemistry. The specimens were rinsed with phosphate-buffered saline solution (PBS) and fixed in formalin for seven days before being processed in the TissueTek Vacuum Filtration Processor. The samples were embedded in paraffin. Microtomed sections, 7 (mu)m thick, were mounted on to glass microscope slides. Several sections were fixed on each slide. The slides were stained with a monoclonal antibody for SMA.
In preparation for immunohistochemistry, the slides were first deparaffinised in xylene and then rehydrated in alcohol and water baths. The sections were digested for one hour with 0.1% trypsin (Sigma Chemical Co, St Louis, Missouri). After digestion, the slides were rinsed with PBS before 3% H^sub 2^O^sub 2^ was added for ten minutes to quench endogenous peroxidase. They were rinsed with PBS and incubated with 30% goat serum (#G9023; Sigma Chemical Co) for ten minutes to block non-specific sites. The primary antibody, mouse monoclonal anti-smooth muscle actin antibody (Cat A2547, clone 1A4, Sigma Chemical Co), was applied to all but one section on each slide. The last section was treated with mouse serum at the same protein concentration as that of the monoclonal antibody solution and served as a negative control. The slides remained incubated with these solutions for two hours and then were rinsed with PBS. They were then incubated with the secondary antibody, biotinylated goat anti-mouse immunoglobulin (#B7151, Sigma Chemical Co) for two minutes before being rinsed again with PBS. Extra Avidin-conjugated peroxidase was added for 20 minutes and the slides were rinsed further with PBS followed by distilled water. The labelling was developed by incubation with the substrate reagent (00-2007, Zymed) for ten minutes. The slides were then washed in distilled water before being placed in a bath of the counterstain, Mayer's haematoxylin, for 20 minutes. Coverslips were applied to the slides with warmed glycerol gelatin.
Immunohistochemical evaluation. The stained slides were examined using a Vanox AH-2 microscope (Olympus, Tokyo, Japan) by normal and polarised light. Four histological zones were identified on the basis of their cellularity. Vascular. These zones comprised a unique category of cellularity since vascular smooth muscle always stained positive for SMA. This zone was not included in the evaluation of expression of SMA in myofibroblasts because of the confounding presence of a large number of vascular cells. Vascular zones were considered to be areas which contained one or more blood vessels.
Hypercellular. These contained more than 100 cells per mm^sup 2^.
Moderately cellular. These had between 50 and 99 cells per mm^sup 2^.
Hypocellular. These contained fewer than 50 cells per mm^sup 2^. The percentage of the section represented by each of these four zones was estimated.
Cell morphology was determined according to nuclear aspect ratio. The cells were categorised as either round, oval, or elongated according to the following criteria: 1) round, length:width = 1; 2) oval, 5.
Elongated cells were also analysed for crimp. Cells were considered to be crimped if they had at least one fold along their length, the period of which was generally comparable to that of the crimp of the adjacent collagen matrix. The percentage of cells with each of the three morphologies was determined in each of the four zones.
We evaluated only immunohistochemically prepared slides on which the negative control section displayed no noticeable presence of the chromogen. The smooth muscle cells in the vascular zones were used as a positive control. Cells were considered to be positive for smooth muscle actin only if they stained with the same chromogen intensity which was found in the vascular smooth muscle. The percentage of SMA-positive cells was determined by dividing the number of positively-stained cells by the total number of cells in the zone and rounding the results to the nearest 5% (for values greater than 5%). The percentage of the SMA-containing cells among those which were crimped was also determined. Examination of the biopsied tissues from three patients (cases 2, 4 and 7) showed notable differences in selected regions, and these regions were evaluated separately.
Cell culture. The tissue samples were placed in PBS until they were prepared for cell culture within three hours of removal. After rinsing the tissue samples thoroughly with PBS the fat and soft tissues surrounding the fibrotic lesion were removed. Each sample was then divided into 1 x 1 x 2 mm sections. These were used as explants and placed into two-dimensional six-well culture plates containing 2 ml of Dulbecco's modified medium (D-MEM F12) supplemented with 10% heat-inactivated fetal bovine serum (Australian Certified; Hyclone, Logan, Utah) and 1% antibiotic-antimycotic solution prepared with 10000 U/ml of penicillin G sodium, 10000 (mu)g/ml of streptomycin sulphate, and 25 (mu)g/ ml of amphotericin B in 0.85% saline (100X; Gibco BRL, Grand Island, New York). The culture plates were placed in an incubator at 37 deg C in an atmosphere of 5% CO2 and 95% air. The media were changed three times a week. The explants were observed daily for signs of outgrowth. When the fibroblasts from the explants achieved confluence, the cell number was estimated using a haemocytometer, and the cells were subcultured into four 25 ml culture flasks. The amount of prepared medium added increased to 5 ml per flask and the media continued to be changed three times a week. When the cells in the flasks from the first or second passage were 75% confluent, they were treated with the selected cytokines (see below).
Cytokine treatment. The growth factors and doses which we used included: TGF-beta1, 1 ng/ml (Sigma Chemical Co); PDGF-BB, 10 ng/ml (BioMimetic Pharmaceuticals Inc, Franklin, Tennessee), and interferon-gamma, 1000 U/ml (Sigma Chemical Co). These doses were based on previous studies performed on human gingival fibroblasts,11 cells isolated from torn human rotator cuffs12 and on smooth muscle cells.14 One or two 25 ml flasks were prepared for each agent and the untreated control group. The flasks were incubated with the agent and 5 ml of prepared medium for four days. The control cultures received only the culture medium. The cells were then removed from the flasks by digestion with trypsin. They were centrifuged, resuspended in PBS and counted. The solution was then centrifuged again, the PBS removed, and lysis buffer added at 0.5 ml per 2 million cells and placed in the freezer until a Western blot analysis was performed.
Smooth muscle actin Western blot analysis. Tubes containing the lysis buffer and cells from the tissue digest and cell cultures were microcentrifuged and the supernatant removed. Protein extracted from smooth muscle cells (human aorta) served as the positive control. To perform the protein assay, 0.15 (mu)g/pl of bovine serum albumin was used in amounts of 10, 20, 40 and 60 (mu)l, with 200 pl of dye. dd H20 was added to bring the total amount to 1000 (mu)l and a standard curve plotted. Then 20 (mu)l of supernatant from each sample was added to 780 (mu)l of ddH2O and 200 (mu) of dye. The mixture was incubated for ten minutes at room temperature and readings were taken at a wavelength of 595 nm.
The following amounts of protein were analysed: 5 pg of the smooth muscle cell control, 10 (mu)g of specimens 9 to 12 and 15 (mu)g of specimen 13. Stacking and resolving gets were prepared for gel electrophoresis. The protein standard and sample were prepared with sample buffer, heated in boiling water for five minutes, and loaded into wells of the gel. The gel was run in IX running buffer (Tris/Glycine/SDS) for 1.5 hours at 110 volts.
The blot was transferred by soaking the gel, membrane, filter paper, and sponge in the transfer buffer for ten minutes. After loading the system into the holder, it was run for one hour at 100 volts. After removal, the membranes were washed in the transfer buffer for five minutes and placed in 5% dry milk overnight. They were then incubated with the primary anti-SMA antibody for two hours and rinsed three times in transfer buffer for ten minutes each. This was followed by application of the secondary antibody and a luminol-based chemiluminescent detection system. Films were digitised for densitometric analysis using NIH Image processing and analysis software (National Institutes of Health, Bethesda, Maryland). The results were reported as a percentage of the densitometric readings of the positive smooth muscle cell control.
Histological examination revealed wide variation in the composition of the tissue within individual histological sections and among samples from different patients (Table 11). The regions stratified on the basis of cell number density as hypercellular, moderately cellular, and hypocellular, generally had histological features which coincided with the three stages of progression of the disease, namely proliferative, involutional, and residual.
The specimens were generally of low vascularity with the vascular area representing 5% or less of the section, except in two cases where it reached 15% (Figs la and b). Two specimens (1 and 4b) had tissue with few cells. Most of the histological sections comprised hypercellular and moderately cellular material. The relative amounts of these two tissues varied among samples. The greatest amount of hypercellular material in a section was 75% (specimen 6). Cell morphology. The cells found throughout the regions of high and low cellularity had an appearance consistent with fibroblasts, some plump (rounded) and others elongated (Fig. 1c). The hypervascular regions were generally populated by round or oval cells while the areas of low cell number density contained elongated cells (Table II). The rounded cells did not appear to be transverse views of elongated cells because the diameter of the nucleus was greater than the thickness of the nucleus of the elongated cells. These plump cells were more likely to be fibroblasts with abundant cytoplasm, immature fibroblasts or undifferentiated mesenchymal cells. In some sections rounded cells in a hypercellular material blended into a hypocellular fibrocollagenous matrix with elongated and crimped cells (Fig. lc). Elongated cells with crimp (Fig. Id) were only found in the moderately cellular and hypocellular regions (Table II).
The fibrocollagenous regions in areas of low cellularity showed a crimp with a peak-to-peak spacing (period) of 10 to 20 gm (Fig 1d) which was prominent in polarised light microscopy (Fig. 1e). The crimped region often occupied a large percentage of the tissue area. Many of the elongated cells in zones of crimped collagen showed the same period of the crimp along their length (Table II; Fig. 1d).
SMA immunohistochemistry. Virtually all the cells in some regions of the tissues stained positive for SMA (Table II; Fig. 1c). There was, however, a wide variability in the percentage of SMA-positive cells in the regions with various cellularities, with the greatest percentages in the hypercellular and moderately cellular zones (Table II). There was difficulty, however, in definitively identifying SMA-staining cells in the hypocellular regions because of the very narrow profile of many of the elongated cells, as shown by some of the cells in Figure I d. Therefore there may have been a greater percentage of SMA-expressing cells in this zone than had been recorded. There was, however, a marked difference in the percentage of SMA-staining cells in the hypercellular, moderately cellular, and hypocellular zones (Fig. 2). One-factor analysis of variance (ANOVA) showed a significant effect of zone on the percentage of SMAexpressing cells (p = 0.007).
In some specimens (2a and 7b) most of the elongated/ crimped cells contained SMA (Table II; Fig. 1d). There was, however, no systematic finding in this regard since many crimped cells did not stain for SMA in other samples.
SMA Western blot analysis and the effects of selected cytokines. In four of the five specimens allocated for Western blot analysis cytoplasmic protein extracted from cells immediately after isolation from the tissue digests showed the presence of SMA (Fig. 3). The most prominent band in the film was at the 42kDa location coincident with the SMA extracted from the smooth muscle cell controls. In each of these cases, however, an unexplained second band was also present which did not appear in the cytoplasmic protein extracted from the cultured cells (Fig. 3). SMA was detectable in varying amounts in the cells from the three patients (specimens 9. 10 and II) evaluated after the first passage and in both specimens (12 and 13) analysed after passage 2.
A consistent finding was the decrease in the SMA content of cells treated with PDGF-BB and an increase in those treated with TGF-beta1 (Fig. 3). Treatment with IFN-gamma had a variable effect in these cells. Comparison of the relative amounts of SMA in the untreated and treated Dupuytren's cells was performed using densitometry of the Western blot films. The intensity of a band of SMA from an untreated or treated Dupuytren cell sample was divided by the intensity of the SMA band from a sample of the smooth muscle cell control on the same gel. In this way the amount of SMA in the Dupuytren's cells was expressed as a percentage of that found in smooth muscle cell controls. This method of comparison was used for the samples from four patients (9 to 12) for which the same amount of cytoplasmic protein (10 pg) extracted from the Dupuytren's samples and control smooth muscle cells was analysed (Fig. 4). The 10 (mu)g sample of cytoplasmic protein from the Dupuytren's cells contained more than 50% of the content of SMA found in 5 (mu)g of protein from the smooth muscle cell control. The difference of a factor of two in the amount of protein analysed suggested that the cells from the Dupuytren's specimens had approximately 25% of the SMA content of smooth muscle cells. Treatment with PDGF-BB resulted in a reduction of 65% in the SMA content of the cells (Fig. 4), and treatment with TGF-1 an increase of 3.4-fold in the content of SMA.
One-factor ANOVA showed that the effect of treatment with cytokine on the content of SMA was statistically significant (p = 0.0006, Fig. 4). Two-tailed, paired Student's ttests showed that the PDGF-BB-induced decrease and TGFbeta1-induced increase in expression of SMA were statistically significant (p = 0.028 and p = 0.049, respectively). There was no statistically significant effect of IFN- (p = 0.2) on the content of SMA of the cells.
The effect of treatment with PDGF-BB on cell proliferation was marked (Fig. 5). There was a ten-fold increase in cell number after the four-day treatment. This compared with an approximate twofold increase in the untreated control cells. One-factor ANOVA showed a significant effect of treatment with cytokine on the increase in cell number (p
The variability of the cellularity of Dupuytren's nodules was clearly in evidence in our study, within sections from the same nodule. SMA-expressing cells were found in regions with different cellularity, with the percentage of SMA-containing cells decreasing with decreasing cellularity of the region. As has been previously found,8 the percentage of SMA-containing cells was elevated in the proliferative phase. SMA-expressing cells all but disappeared during the residual phases which was characterised by a low cellnumber density. The zones with uniform crimp of the fibrocollagenous matrix were of interest. The fact that the constituent SMA-containing cells in these regions showed the same crimp suggests that they have a role in producing the buckling force which may have been responsible for creating this architectural feature.
Immunohistochemistry may thus be of value in demonstrating the percentage of SMA-containing cells and give an indication of their state of contraction based on their buckled appearance. It is not clear, however, that such a study of biopsied material could be of value in providing a guide to the prognosis of the disease in a particular patient. This is due in part to the variability of the histological composition of the tissue comprising the lesion and to the known transience of SMA expression.15 Thus while it is known that SMA-expressing connective-tissue cells can stay in a contracted state indefinitely in much the same way that smooth muscle cells maintain tone,16 in some cases there may be a down-regulation of SMA expression or apoptosis of the SMA-containing cells.17
The demonstration of the prevalence of SMA-containing cells in Dupuytren's tissues in our study further focuses attention on the regulation of the expression of this isoform of actin as a means of controlling the disease. A notable finding was the significant decrease in SMA in PDGF-BBtreated Dupuytren's cells. A comparable result had been previously obtained in a study using human gingival fibroblasts.11 PDGF-alpha and PDGF-beta along with TGF-beta have been found to be elevated in Dupuytren's nodules, compared with healthy tissues.18 PDGF is produced in the diseased tissue19 and it appears to have a role in the proliferative stage of this disease.20 It binds to cell-membrane receptors on the myofibroblasts during both the proliferative and involutional stages of Dupuytren's.20 Furthermore, PDGF has been shown to have dose-dependent mitogenic effects.21 Other effects attributed to PDGF include increased synthesis of type-III collagen,22 the reorganisation of actin filaments23 and the stimulation of the production of arachidonic acid which can be converted to prostaglandin.23 It is possible that the effect of PDGF in down-regulating expression of SMA may be a homeostatic mechanism to counter the up-regulating effect of TGF-P. Alternatively, it may be that the rapid cell proliferation stimulated by PDGF precludes the expression of this particular actin isoform. These findings warrant further study including a dose-response analysis.
Our finding of a significant increase in expression of SMA by treatment with TGF-beta1 of Dupuytren's cells in vitro, coincided with recent immunofluorescence studies which have shown that TGF-beta1 increased the percentage of SMA-containing cells in Dupuytren's tissue.24 The expression of TGF-beta in Dupuytren's tissue has been considered to be important because of its multiple effects which could promote the processes underlying the pathogenesis of the disease: mitogenesis,25 chemotaxis26 and the synthesis of collagen and fibronectin.27 TGF-beta can also induce expression of SMA,28 suggesting that stimulation of fibroblasts by TGF-beta may induce their differentiation into myofibroblasts. Furthermore, in a released collagen lattice contraction assay, TGF-beta1 has been shown to increase the force of contraction generated by myofibroblasts obtained from Dupuytren's tissue.9 It would be interesting in future studies to determine if PDGF-BB could reduce or prevent the stimulation by TGFbeta of the expression of SMA in Dupuytren's cells.
A cytokine produced by helper T lymphocytes, IFN-gamma, is believed to suppress the differentiation of myofibroblasts. Treatment with IFN-gamma has been shown to decrease expression of SMA in cultured fibroblasts29 and in addition, to block effectively changes in the expression of SMA and the formation of fibronectin fibrils and fibronexus which are normally incurred by exposure to TGF-beta1.9 Furthermore, IFN-gamma can reduce the contractile force caused by Dupuytren's myofibroblasts treated with TGF-beta1. A pilot study to determine the effects in vivo of the treatment of Dupuytren's disease with IFN-gamma has shown that this can decrease the amount of SMA expressed in the myofibroblasts, decrease the size of the nodule, and decrease symptoms.30 In contrast to these previous findings our study did not reveal a meaningful effect of IFN-gamma in reducing the expression of SMA in Dupuytren's cells. Additional work will be necessary to resolve the differences in response to IFN-gamma found in this and previous studies.
Our study may provide a useful basis for the comparison of the relative effects of selected cytokines on the expression of SMA in Dupuytren's disease. This could be helpful in identifying novel strategies for the non-surgical treatment of this often debilitating problem. Moreover, our findings could provide insights in new approaches to the management of other fibrotic contractures such as joint contractures.
The authors are grateful for the assistance of Sandra Zaptaka-Taylor with the histology and help with the initial Western blots from Xiu-Ying Zhang MD. The efforts of Mark Koris, MD, in providing specimens are also appreciated. This study was supported in part by the Harvard Centre for Craniofacial Tissue Engineering. Brigham Orthopaedic Foundation and the US Department of Veterans Affairs. The PDGF was kindly provided by BioMimetic Pharmaceuticals, Inc. Franklin. Tennessee.
No benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article
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H. B. Hindman, R. Marty-Roix, J-B. Tang, J. B. Jupiter, B. P. Simmons, M. Spector
From Harvard Medical School, and the VA Boston Healthcre System, Boston, USA
H. B. Hindman, BA
R. Marty-Roix, BS
B. P. Simmons, MD
M. Spector, PhD*
Department of Orthopaedic Surgery, Brigham and Women's Hospital, 75 Francis Street, Boston, Massachusetts 02115, USA.
*VA Boston Healthcare System, 150 South Huntington Avenue, Boston, Massachusetts OZ 130, USA.
J-B. Tang, MD
Department of Orthopaedics, Nantong Medical College, Jiangsu, China.
J. B. Jupiter, MD
Department of Orthopaedic Surgery, Massachusetts General Hospital, 15 Parkman Street, Boston, Massachusetts 02114, USA.
Correspondence should be sent to Dr M. Spector.
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