Dupuytren's disease: physiologic changes in nodule and cord fibroblasts through aging in vitro

The pathogenesis of the fibrotic disease Dupuytren's contracture remains unclear. The disease process includes two structurally distinct fibrotic elements, the nodule and the cord. It has been proposed that as the disease progresses, nodules develop into cords. To corroborate that hypothesis, the authors took advantage of cultured fibroblast differences found between gap junction intercellular communication and fibroblast-populated collagen lattice contraction. Paired fibroblast cell lines of nodules and cords derived from four patients with Dupuytren's disease were maintained in culture for at least eight passages. The presence of gap junction intercellular communication in nodule- and cord-derived fibroblasts was documented and reported as a coupling index. The contraction of free-floating nodule- or cord-derived collagen lattices was also documented and reported. Early passage (passage 4) cord-derived fibroblasts showed a significant increase in coupling index compared with passage 4 nodule-derived fibroblasts (4.0 +/- 0.4 versus 2.5 +/- 0.3, respectively), where p < or = 0.01. However, late passage (passage 8) nodule- and cord-derived fibroblasts were equivalent in their coupling index (4.1 +/- 0.4 versus 4.4 +/- 0.4, respectively). Early passage nodule-derived fibroblast-populated collagen lattices contracted by 64 percent, whereas late passage nodule-derived lattices showed less contraction, at only 40 percent. Early and late passage cord-derived lattices contracted 46 and 37 percent, respectively. All nodule- and cord-derived cell lines were statistically equivalent at lattice contraction by passage 8. These in vitro studies support the hypothesis that fibroblasts derived from Dupuytren's contracture nodules change their phenotype after undergoing repeated cell passage, acquiring a cord-like fibroblast phenotype. Dupuytren's nodules represent the early, active form of fibrosis in which cells are more proliferative, better at fibroblast-populated collagen lattice contraction, and display less gap junction intercellular communication. The speculation is that alterations in gap junction intercellular communication may be involved in the progression of Dupuytren's nodules to cords as the disease progresses.     

N-acetyl-L-cysteine abrogates fibrogenic properties of fibroblasts isolated from Dupuytren's disease by blunting TGF-beta signalling

Dupuytren's disease, a benign fibroproliferative disorder of the palmar fascia, represents an ideal model to study tissue fibrosis. Transforming growth factor-beta1 (TGF-beta1) and its downstream Smad signalling system is well established as a key player during fibrogenesis. Thus, targeting this basic pathomechanism seems suitable to establish new treatment strategies. One such promising treatment involves the substance N-acetyl-L-cysteine (NAC), shown to have antifibrotic properties in hepatic stellate cells and rat fibroblasts. In order to investigate antifibrotic effects of N-acetyl-L-cysteine (NAC), fibroblasts were isolated from surgically resected fibrotic palmar tissues (Dupuytren fibroblasts, DF) and exposed to different concentrations of NAC and recombinant TGF-beta1. Fibroblasts isolated from tendon pulleys served as controls (control fibroblasts, CF). Smad signalling was investigated by a Smad binding element driven reporter gene analysis. Both cell types express TGF-beta1, indicating autocrine signalling in DF and CF. This was confirmed by comparing reporter gene activity from LacZ and Smad7 adenovirus infected cells. NAC treatment resulted in abrogation of Smad mediated signalling comparable to ectopically overexpressed Smad7, even when the cells were stimulated with recombinant TGF-beta1 or ectopically expressed a constitutively active TGF-beta receptor type I. Additionally, NAC dose-dependently decreased expression of three major indicators of impaired fibrotic matrix turnover, namely alpha-smooth muscle actin (alpha-SMA), alpha 1 type I procollagen (Col1A1), and plasminogen activator inhibitor-type I (PAI-1). Our results suggest that TGF-beta signalling and subsequent expression of fibrogenesis related proteins in Dupuytren's disease is abrogated by NAC thus providing a basis for a therapeutic strategy in Dupuytren's disease and other fibroproliferative disorders.     

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.     

Epidermal growth factor in Dupuytren's disease

The aim of this article was to show the participation of epidermal growth factor (EGF) in the pathogenesis of Dupuytren's disease (palmar contracture). The concentration of EGF in specimens obtained from 68 patients with Dupuytren's contracture and 14 controls was examined immunochemically with the use of enzyme-linked immunosorbent assay. The determined EGF concentration in pathologic aponeurosis with symptoms of Dupuytren's disease (median, 6.29 ng/g; range, 1.67 to 63.09 ng/g) showed significantly different values (p = 0.036) in comparison with the control group (median, 10.1 ng/g; range, 5.13 to 39.81 ng/g). The changes in EGF concentration were shown in tested groups of pathologic tissues that were formed according to the clinical stage of disease progression. The significantly lower concentration than that seen in the control group characterizes tissues with first and third degrees of palmar contracture progression (p = 0.025 and p = 0.018, respectively). In the group of patients with second-degree disease progression, the EGF level increased transiently. Nevertheless, in comparison with the other groups, the difference was not significant. The group with the fourth degree of the disease showed EGF concentrations that resembled the control values. The authors conclude that significant differences in levels of EGF concentration between contractured and normal fasciae may suggest the participation of this cytokine in the pathogenesis of Dupuytren's disease.     

Dupuytren's disease metabolite analyses reveals alterations following initial short-term fibroblast culturing

Dupuytren's disease (DD) is an ill-defined fibroproliferative disorder affecting the palm of the hand, resulting in progressive and irreversible digital contracture. In view of the abnormal gene dysregulation found in DD, and its potential effect on metabolites at a functional level, we chose to examine the metabolic profile involved in DD. Using Fourier transform infrared (FT-IR) spectroscopy to generate metabolic fingerprints of cultured cells, we compared the profiles of DD cords and nodules (1) against the unaffected transverse palmar fascia (internal control), (2) against carpal ligamentous fascia (external control), and (3) against fibroblasts from fat surrounding the nodule and skin overlying the nodule (environmental control). We also determined the effects of serial passaging of the cells on DD fingerprints. Subsequently, gas chromatography-mass spectrometry (GC-MS) was employed for metabolic profiling in order to identify metabolites characteristic of the DD tissue phenotypes. We developed a robust metabolomic analysis procedure of DD using cultured fibroblasts derived from DD tissues. Our carefully controlled culture conditions, combined with assessment of metabolic phenotypes by FT-IR and GC-MS, enabled us to demonstrate metabolic differences between DD and unaffected transverse palmar fascia and between DD and healthy control tissue. In early passage (0-3) the metabolic differences were clear, but cells from subsequent passages (4-6) started to lose this distinction between diseased and non-diseased origin. The dysregulated metabolites we identified were leucine, phenylalanine, lysine, cysteine, aspartic acid, glycerol-3-phosphate and the vitamin precursor to coenzyme A. Early passage DD cells exhibit a clear metabolic profile, in which central metabolic pathways appear to be involved. Experimental conditions have been identified in which these DD data are reproducible. The experimental reproducibility will be useful in DD diagnostics and for DD systems biology.     

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.     

In vitro study of novel collagenase (XIAFLEX®) on Dupuytren's disease fibroblasts displays unique drug related properties

Dupuytren's disease (DD) is a benign, fibroproliferative disease of the palmar fascia, with excessive extracellular matrix (ECM) deposition and over-production of cytokines and growth factors, resulting in digital fixed flexion contractures limiting hand function and patient quality of life. Surgical fasciectomy is the gold standard treatment but is invasive and has associated morbidity without limiting disease recurrence. Injectable Collagenase Clostridium histolyticum (CCH)--Xiaflex?--is a novel, nonsurgical option with clinically proven in vivo reduction of DD contractures but with limited in vitro data demonstrating its cellular and molecular effects. The aim of this study was to delineate the effects of CCH on primary fibroblasts isolated from DD and non-DD anatomical sites (using RTCA, LDH, WST-1, FACS, qRT-PCR, ELISA and In-Cell Quantitative Western Blotting) to compare the efficacy of varying concentrations of Xiaflex? against a reagent grade Collagenase, Collagenase A. Results demonstrated that DD nodule and cord fibroblasts had greater proliferation than those from fat and skin. Xiaflex? exposure resulted in dose- and time-dependent inhibition of cellular spreading, attachment and proliferation, with cellular recovery after enzyme removal. Unlike Collagenase A, Xiaflex? did not cause apoptosis. Collagen expression patterns were significantly (p<0.05) different in DD fibroblasts across anatomical sites - the highest levels of collagen I and III were detected in DD nodule, with DD cord and fat fibroblasts demonstrating a smaller increase in both collagen expression relative to DD skin. Xiaflex? significantly (p<0.05) down-regulated ECM components, cytokines and growth factors in a dose-dependent manner. An in vitro scratch wound assay model demonstrated that, at low concentrations, Xiaflex? enabled a faster fibroblast reparatory migration into the wound, whereas, at high concentrations, this process was significantly (p<0.05) inhibited. This is the first report elucidating potential mechanisms of action of Xiaflex? on Dupuytren fibroblasts, offering a greater insight and a better understanding of its effect in DD.     

The contractile properties and responses to tensional loading of Dupuytren's disease--derived fibroblasts are altered: a cause of the contracture?

Dupuytren's disease causes disability because of the development of finger flexion deformities, with distinct nodule and cord formation. This results in physical shortening of the diseased fascial tissue through a combination of cell-mediated contraction and matrix remodeling. It is this fixed tissue fabric shortening that prevents finger extension. In this experimental study, the relative contractile properties of Dupuytren nodule- and cord-derived fibroblasts were quantified in a culture force monitor model, in comparison with normal carpal ligament fibroblasts. Nine nodule, 10 cord, and four carpal ligament fibroblast cell lines were studied; each cell line was derived from a separate patient. The contractile forces generated by nodule and cord fibroblasts were significantly greater than the force generated by carpal ligament fibroblasts. There were also significant differences between nodule- and cord-derived fibroblasts, with the nodule cells demonstrating the greatest contractile force generation. The contraction profiles of both cord and nodule Dupuytren fibroblasts demonstrated delays in the attainment of tensional homeostasis, with an absence of a plateau phase by 20 hours. After the contraction phase, cell-seeded constructs were subjected to a series of four uniaxial mechanical overloads and cellular responses were monitored during each subsequent 30-minute period. Dupuytren nodule and cord fibroblast responses were significantly altered, compared with carpal ligament fibroblasts, exhibiting an increased and opposite response. Dupuytren fibroblasts, particularly nodule fibroblasts, exhibited increased force generation and a delay in reaching tensional homeostasis. The data suggest that these cells have an inherently higher basal tension and contractile ability. This results in increased shortening of the matrix, and the delay in reaching tensional homeostasis might exacerbate this response. These results represent a theoretical framework regarding the fundamental processes involved in the pathogenesis and progression of clinical flexion deformities in Dupuytren disease.     

Dupuytren's disease: history, diagnosis, and treatment

LEARNING OBJECTIVES: After studying this article, the participant should be able to: 1. Describe the clinical features of the disease. 2. Describe the pathoanatomical structures in Dupuytren's disease. 3. Outline the various factors associated with Dupuytren's disease. 4. Describe the modalities for surgical and nonsurgical treatment of the condition. 5. Outline recent biomolecular knowledge about the basis of Dupuytren's disease. SUMMARY: Dupuytren's disease is characterized by nodule formation and contracture of the palmar fascia, resulting in flexion deformity of the fingers and loss of hand function. The authors review the historical background, clinical features, and current therapy of Dupuytren's disease; preview treatment innovations; and present molecular data related to Dupuytren's disease. These new findings may improve screening for Dupuytren's disease and provide a better understanding of the disease's pathogenesis.     

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.     

Construction of transgenic pea lines with modified expression of diamine oxidase and modified nodulation responses with exogenous putrescine

Diamine oxidase (DAO) might influence pea nodule development either by regulating the peroxide-driven cross-linking of glycoproteins in the infection thread matrix or by modifying the metabolism of diamines and polyamines in host cells. Transformed lines of pea (Pisum sativum) with the coding sequence for DAO (PSAO-1) in sense orientation behind a tissue-specific promoter (pENOD12A) showed strong co-suppression of DAO activity in extracts from nodules and epicotyls, whereas the antisense constructs were relatively unaffected. No difference in nodule number was observed between transformed lines and controls, suggesting that DAO does not normally have an essential role in nodule initiation. However, lines showing co-suppression of DAO were less sensitive to the inhibitory effects of exogenous putrescine and less active in the cross-linking of matrix glycoprotein, indicating that putrescine-derived products of DAO activity could retard nodule development. Inoculation of co-suppressed lines with Rhizobium strain B661 (a lipopolysaccharide-defective mutant) resulted in more extreme impairment of nodule development and nitrogen fixation capacity, relative to lines with normal levels of DAO, which suggests that DAO may serve to reduce the endogenous level of inhibitory diamines or polyamines in nodules under physiological stress. We conclude that the most critical role of DAO in pea nodule development is apparently in the regulation of diamine levels in host tissues.     

MMP-14 and MMP-2 are key metalloproteases in Dupuytren's disease fibroblast-mediated contraction

Dupuytren's disease (DD) is a common fibrotic condition of the palmar fascia, leading to deposition of collagen-rich cords and progressive flexion of the fingers. The molecular mechanisms underlying the disease are poorly understood. We have previously shown altered expression of extracellular matrix-degrading proteases (matrix metalloproteases, MMPs, and 'a disintegrin and metalloprotease domain with thrombospondin motifs', ADAMTS, proteases) in palmar fascia from DD patients compared to control and shown that the expression of a sub-set of these genes correlates with post-operative outcome. In the current study we used an in vitro model of collagen contraction to identify the specific proteases which mediate this effect. We measured the expression of all MMPs, ADAMTSs and their inhibitors in fibroblasts derived from the palmar fascia of DD patients, both in monolayer culture and in the fibroblast-populated collagen lattice (FPCL) model of cell-mediated contraction. Key proteases, previously identified in our tissue studies, were expressed in vitro and regulated by tension in the FPCL, including MMP1, 2, 3, 13 and 14. Knockdown of MMP2 and MMP14 (but not MMP1, 3 and 13) inhibited cell-mediated contraction, and knockdown of MMP14 inhibited proMMP-2 activation. Interestingly, whilst collagen is degraded during the FPCL assay, this is not altered upon knockdown of any of the proteases examined. We conclude that MMP-14 (via its ability to activate proMMP-2) and MMP-2 are key proteases in collagen contraction mediated by fibroblasts in DD patients. These proteases may be drug targets or act as biomarkers for disease progression.     

Dermatan sulfate remodeling associated with advanced Dupuytren's contracture

Dermatan sulfate (DS) widespread as a component of extracellular matrix proteoglycans, is characterized by great bio-reactivity and remarkable structural heterogeneity due to distinct degrees of sulfation and glucuronosyl epimerization and different polymerization degrees. However, DS metabolism under various biological conditions is poorly known. Dupuytren's contracture is a benign fibromatosis leading to complex remodeling of the palmar fascia structure and properties. However, it remains unclear whether the disease affects the structure of DS, which is the major tissue glycosaminoglycan. Thus the aim of the study was to examine the structure of the total DS in Dupuytren's fascia. DS chains were extracted from 5 samples of normal fascia and 7 specimens of Dupuytren's tissue by papain digestion followed by fractionation with cetylpyridinium chloride. Then, DS structure analysis was performed comprising the evaluation of its molecular masses and sensitivity to hyaluronidase and chondroitinase B. Dupuytren's contracture is associated with significant remodeling of DS chain structure revealed by (1) a distinct profile of chain molecular masses characterized by the appearance of long size components as well as the increase in the content of small size chains; (2) a different glucuronosyl epimerization pattern connected with the enhanced content of glucuronate disaccharide blocks; (3) chain oversulfation. These structural alterations in total DS may modify the GAG interactions especially affecting collagen fibrillogenesis and growth factor availability. Thus, Dupuytren's contracture associated DS remodeling may promote the phenomena typical for advanced disease: apoptosis and reduction in cell number as well as the appearance of dense pseudotendinous collagen matrix.     

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.     

Differences in calcium accumulation between human plantar and palmar aponeuroses

To elucidate the characteristics of calcium accumulation of human plantar and palmar aponeuroses, the authors determined the calcium content of human plantar and palmar aponeuroses by atomic absorption flame emission spectrophotometry. The subjects consisted of 9 men and 14 women, ranging in age from 61 to 93 yr. In the plantar aponeurosis, the calcium content was significantly higher in the anterior and posterior parts than in the middle part. It is known that pressure distribution under the sole of a foot is higher in the anterior and posterior parts than in the middle part. The present study suggests that the accumulation of calcium in the plantar aponeurosis is related with the pressure distribution under the sole of a foot. The calcium content increased progressively with aging in the anterior part of the plantar aponeurosis, but not in the middle and posterior parts. Regarding the palmar aponeurosis, the calcium content was significantly higher in the anterior and posterior parts in comparison with the middle part. It was found that the calcium content increased progressively with aging in the posterior part of the palmar aponeurosis, whereas it did not increase significantly with aging in the anterior and middle parts. Regarding the relationship between the calcium content of the aponeuroses and the bone mineral density, a significant correlation was found between the calcium content in the anterior part of the palmar aponeurosis and the bone mineral density of the scaphoid bone.     

SV40-transformed human cells fail to grow in zinc concentrations which permit normal human fibroblast proliferation

Zinc is a metal known to be required for normal growth of both cells and organisms. When normal and SV₄₀-transformed human tumor cells are plated and grown in medium containg zinc sulfate, a significant fraction of the transformed cells fail to grow at zinc concentrations which are relatively nontoxic to the normal fibroblasts. Although cultured cells respond to certain metallic ions by incresing their metallothionein content, no difference in the ability of normal and transformed cells to produce metallothionein in response to zinc exposure could be detected, thus ruling out this mechanism as a basic for the differing abilities of the cells to grwo in zinc in vitro. These results suggest that zinc may be capable of differntially regulating the growth of normal and SV_40? transformed human fibroblasts.     

Alterations in the extracellular matrix proteoglycan profile in Dupuytren's contracture affect the palmar fascia

Dupuytren's disease is a palmar fibromatosis associated with changes in fibroblast activity that also affect the metabolism of extracellular matrix components. In contrast to disease connected alterations in collagen and non-collagenous glycoproteins (mainly fibronectin), the metabolism of proteoglycans, being glycosaminoglycan modified glycoproteins, is poorly understood. Thus, the aim of the present study was the characterization of matrix proteoglycans (PGs) derived from normal fascia and Dupuytren's fascia. Extracted and purified PGs (particularly small PGs) were analysed for content, molecular mass, immunoreactivity and glycosaminoglycan chain structure. The matrix of normal fascia mainly contains decorin [small dermatan sulfate (DS) PG] with biglycan (another small DSPG) and large chondroitin sulfate(CS)/DSPG representing minor components. Dupuytren's disease is associated with the remodeling of matrix PG composition. The most prominent alteration is an accumulation of biglycan frequently bearing DS chains with higher molecular masses. Moreover, the amount of large CS/DSPG is increased. In contrast, decorin displays changes affecting mainly DS chain structure reflected in (i) an increase in some chain molecular masses, (ii) an enhanced content of iduronate disaccharide clusters, and (iii) oversulfation of disaccharide repeats. The PG alterations observed in Dupuytren's fascia may influence the matrix properties and contribute to disease progression.     

Cell distribution and antigenic properties of mammalian sarcolectins

Sarcolectins are present in a great variety of tissues from mammalian origin. Such substances were observed to be secreted from cultures of human embryonic fibroblasts, human osteosarcoma and rat Rous sarcoma transformed cells and could be extracted from TG 180 Crocker Sarcoma or normal human placenta. All sarcolectins tested here, were comparable by their physicochemical properties to those previously reported in hamster or human sarcomas. Indeed, they are proteins or glycoproteins, resistant to pepsin and migrate in SDS-PAGE in the 65 kDa area. They agglutinate cells with an affinity for simple sugars and degrade previously established interferon-induced antiviral resistance. Considering the hamster sarcolectin as reference in this comparative study, both differences and similarities in the antigenic properties of mouse, rat and human sarcolectin variants were demonstrated. An indirect immunofluorescence assay showed that sarcolectins were specifically labelled on the cell surface but not detected in the cytoplasm after methanol or acetone permeabilization of the membrane. By electron microscopy, using immunoperoxidase labelling, sarcolectins can be localized on the surface of normal, transformed, human or rat cells. Only limited segments of normal cell membranes were labelled, while transformed cells were frequently stained on their whole surface. Other known extracellular proteins, such as fibronectin and collagen, did not share common antigenic determinants with sarcolectins.     

Human fibroblasts from cancer patients: lifespan and transformed phenotype in vitro and role of mesenchyme in vivo

Human fibroblasts cultured in vitro can exhibit a different potential number of population doublings. In normal donors, the average number of population doublings is inversely related to the donor's age. An increased growth potential was detected in skin fibroblasts from breast cancer patients, independently of the donor's age. These cells responded in an abnormal way to 3 biological parameters: (1) colony formation in semisolid medium; (2) colony formation on monolayers of normal human epithelial cells; and (3) increase of saturation densities in overcrowded culture conditions. A third of these cultures, as well as skin fibroblasts from other cancer patients, at the plateau phase of growth exhibited a significant percentage of cells still synthesizing DNA. Exposure to overcrowding, limited in time, caused the selection of a cell subset which displayed new biological, biochemical and functional properties commonly found in transformed cells. The abnormal in vitro behavior of skin fibroblasts from breast cancer patients does not seem to be associated with the expression of oncofetal membrane markers (4F2, IL2 receptor) while the fibroblasts from patients with the adenomatosis of the colon and rectum (ACR) syndrome expressed the 4F2 antigen. This is the first time that the IL2 receptor is found on non-hematopoietic cells. Fibroblastic cells with abnormal characteristics, which may also present a decreased efficiency in organizing a primitive fibrin matrix, could represent in vivo an anarchistic milieu, favoring disturbed epithelial-stromal interactions and the emergence of the less structured tumor stromatic tissue.