The role of an MMP inhibitor in the regulation of mechanical tension by Dupuytren's disease fibroblasts.

Mechanical tension and contracture are two related facets of tissue biology. This study assessed the effect of ilomastat, a broad-spectrum matrix metalloprotease (MMP) inhibitor, on generation of tension by Dupuytren's disease fibroblasts. Nodule and cord-derived fibroblasts were isolated from five patients with Dupuytren's disease; flexor retinaculum acted as the control. A culture force monitor (CFM) provided an in vitro model of tissue organization to assess development of mechanical tension, lattice contraction and spatial remodelling by fibroblasts. Responses to ilomastat were compared to treatment with a control peptide. Nodule and cord-derived fibroblasts exhibited a two-fold increase in tension compared with flexor retinaculum. Ilomastat significantly inhibited development of tension by nodule and cord but not flexor retinaculum derived fibroblasts at 100 microM. These results imply that MMP activity mediates regulation of tensile strength by Dupuytren's disease fibroblasts and may be an important therapeutic target in patients with Dupuytren's disease.

Evaluation of Permacol as a cultured skin equivalent.

Skin loss following severe burn requires prompt wound closure to avoid such complications as fluid and electrolyte imbalance, infection, immune suppression, and pain. In clinical situations in which insufficient donor skin is available, the development of cultured skin equivalents (dermal matrices seeded with keratinocytes and fibroblasts) may provide a useful alternative. The aim of this study was to assess the suitability of a porcine-derived dermal collagen matrix (Permacol) to function as a cultured skin equivalent in supporting the growth of keratinocytes in vitro and providing cover to full thickness wounds in the BALB C/nude mouse model. A histological comparison was against Glycerol treated-Ethylene Oxide Sterilised Porcine Dermis (Gly-EO Dermis) which has successfully been used as a cultured skin equivalent in previous studies. Both Gly-EO Dermis and to a lesser extent Permacol were able to support the growth of cultured keratinocytes following a 16-day period of cell culture, however, this study was only able to demonstrate the presence of an epidermal layer on Gly-EO dermis 2 weeks after grafting onto full-thickness wounds in the BALB C/nude mouse model.

Current prospects for adult stem cell-based therapies in ocular repair and regeneration.

Recent advances in stem cell biology have led to the exploration of stem cell-based therapies to treat a wide range of human diseases. In the ophthalmic field, much hope has been placed on the potential use of these cells to restore sight, particularly in those conditions in which other established treatments have failed and in which visual function has been irreversibly damaged by disease or injury. At present, there are many limitations for the immediate use of embryonic stem cells to treat ocular disease, and as more evidence emerges that adult stem cells are present in the adult human eye, it is clear that these cells may have advantages to develop into feasible therapeutic treatments without the problems associated with embryonic research and immune rejection. Here we discuss the current prospects for the application of various adult ocular stem cells to human therapies for restoration of vision.

Changes in collagen metabolism in response to endothelin-1: evidence for fibroblast heterogeneity.

Endothelin-1 (Et-1) is a 21-amino acid peptide primarily synthesized by endothelial cells. It was originally classified as a potent vasoconstrictor but recent evidence suggests that it also possesses a wide variety of non-vascular actions. It stimulates fibroblast and smooth muscle cell proliferation and it has been shown to stimulate fibroblast collagen metabolism. However, studies on its ability to regulate collagen production remain incomplete, and its effect on post-translational processing of procollagen has not been studied. This report details the effect of Et-1 on the rates of procollagen synthesis and degradation in two fibroblast cell lines; human foetal lung (HFL-1) and whole foetal rat fibroblasts (Rat 2). Fibroblast cultures were incubated for 24 hr in the presence or absence of Et-1 before procollagen metabolism was determined by measuring hydroxyproline. Non-collagen metabolism was also determined in these cultures from the uptake of tritiated phenylalanine. Et-1 stimulated procollagen synthesis in HFL-1 fibroblasts and reduced synthesis in Rat 2 cells. The response was dose dependent with the greatest effect at 1.10(-6) M Et-1 for both cell types (155 +/- 6% of control (mean +/- SD, n = 6, P < 0.01) and 61 +/- 4% of control (n = 4, P < 0.01) for HFL-1 and Rat 2 fibroblasts, respectively). Non-collagen protein synthesis was increased to 148 +/- 5% of control (P < 0.05) at 1.10(-6) M Et-1. Non-collagen protein synthesis remained unaffected in the HFL-1 fibroblast cultures. Procollagen degradation, expressed as a proportion of total procollagen synthesis, was decreased in HFL-1 fibroblasts (control, 29 +/- 2%; Et-1, 1.10(-6) M; 21 +/- 2%; P < 0.01), and increased in Rat 2 fibroblasts (control 42 +/- 1%; Et-1, 1.10(-6) M; 49 +/- 1%; P < 0.01). Blocking of the EtA receptor for Et-1, using the receptor antagonist-BQ123, abolished the effect of Et-1 on procollagen metabolism in both cell types. These results suggest that different populations of fibroblasts exhibit heterogeneous responses to Et-1. It is concluded that Et-1 may play an important role in the extent and distribution of fibrosis seen in diseases associated with the overproduction of Et-1.

Insulin-like growth factor I is a major fibroblast mitogen produced by primary cultures of human airway epithelial cells.

1. The ability of airway epithelial cells to produce insulin-like growth factor I may be important in the pathogenesis of subepithelial fibrosis observed in the airways of patients with asthma. We determined whether human airway epithelial cells are capable of producing polypeptide mediators that could induce fibroblast proliferative activity, in particular insulin-like growth factor I. 2. We examined 12 primary cultures of human airway epithelial cells grown to confluence on collagen gel-coated dishes. Using a colorimetric assay based on the uptake and subsequent release of Methylene Blue, increased proliferation of human fetal lung fibroblasts was detected in conditioned media from airway epithelial cells. The median stimulation of fibroblast proliferation was 49.9% (range 25.6-113.3%) above control values (observed at 1:2 dilution of media). 3. A neutralizing antiserum to insulin-like growth factor I partly inhibited fibroblast proliferation induced by epithelial cell conditioned media by 52.2% (49.9-109%; n = 5). 4. Radioimmunoassay for insulin-like growth factor I in conditioned media demonstrated a median concentration of 54.1 ng/ml (32.4-96.8 ng/ml). 5. Insulin-like growth factor I mRNA was detected in epithelial cell monolayers by Northern blot analysis using an insulin-like growth factor I cDNA probe. 6. The insulin-like growth factor I gene is expressed in cultured human airway epithelial cells, which also secrete insulin-like growth factor I protein. Insulin-like growth factor I also accounts for the major mitogenic activity for fibroblasts of cultured human epithelial cell conditioned media. Insulin-like growth factor I may function in a paracrine manner to modulate fibroblast behaviour and may be involved in airway processes, such as those occurring in asthma.

Role of thrombin in pulmonary fibrosis.

Pulmonary fibrosis commonly develops in systemic sclerosis. We assessed the role of thrombin in promoting fibroblast proliferation in the lungs in this disorder. Bronchoalveolar lavage fluid (BALF) thrombin concentrations were higher in ten patients with systemic sclerosis than in 12 healthy controls (14.6 vs 3.6 nmol/L, p < 0.02), but values in patients with cryptogenic fibrosing alveolitis (n = 10) or sarcoidosis (n = 10) were not increased. BALF from all patients induced fibroblast proliferation. This proliferation was attenuated by thrombin inhibitors for BALF from systemic sclerosis patients only. We suggest thrombin contributes to lung fibroblast proliferation in this disorder.

Interleukin-8. Differential expression in lone fibrosing alveolitis and systemic sclerosis.

Fibrosing alveolitis may occur alone (CFA) or in association with systemic sclerosis (FASSc). FASSc was recently shown to have a prognostic advantage over CFA. Because interleukin-8 (IL-8) is likely to be a major determinant of neutrophil alveolitis, we evaluated IL-8 expression in patients with CFA and FASSc and compared it with that in normal individuals and sarcoidosis and systemic sclerosis patients without pulmonary involvement (SSc no FA). IL-8 protein in bronchoalveolar lavage fluid (BALF) was assessed by immunoassay, and IL-8 mRNA expression was assessed using Northern analysis and reverse transcription-polymerase chain reaction (RT-PCR) and in situ hybridization of lung parenchyma. Compared with normal subjects, IL-8 concentration was significantly greater in both CFA (p < 0.001) and FASSc groups (p < 0.05) but no different in sarcoidosis. The IL-8 concentration in CFA was higher than in FASSc (p < 0.01) and was related to BAL % neutrophils (rs = 0.48, p < 0.01). IL-8 mRNA expression evaluated by Northern analysis was seen only in patients with CFA and FASSc and was related to BAL % neutrophils (rs = 0.63, p < 0.01). We suggest that IL-8 is a key factor in the pathogenesis of fibrosing alveolitis and that the poorer prognosis of CFA compared with FASSc is related to higher levels of IL-8 within the lower respiratory tract.

Activation of peripheral blood mononuclear cells in bronchoalveolar lavage fluid from patients with sarcoidosis: visualisation of single cell activation products.

BACKGROUND: Interstitial lung diseases are characterised by the recruitment of mononuclear cells to disease sites where maturation occurs and activation products, including lysozyme (LZM), are released. Analysis of in vitro cell culture supernatants for activation products masks the functional heterogeneity of cell populations. It is therefore necessary to examine the secretion of activation products by single cells to assess whether the activation of newly recruited mononuclear phagocytes at the sites of disease in the lung is uniform and controlled by the local microenvironment. METHODS: The reverse haemolytic plaque assay was used to evaluate, at a single cell level, the ability of bronchoalveolar lavage (BAL) fluid from seven patients with sarcoidosis to activate Ficoll-Hypaque-separated peripheral blood mononuclear cells by comparison with BAL fluid from six normal volunteers and nine patients with systemic sclerosis. Monolayers of peripheral blood mononuclear cells and sheep red blood cells were cultured either alone or in the presence of 20% (v/v) BAL fluid with a polyclonal anti-LZM antibody. LZM/anti-LZM complexes bound to red blood cells surrounding the secreting cells were disclosed following complement lysis of red blood cells and quantification of plaque dimensions using microscopy and image analysis. RESULTS: Bronchoalveolar lavage fluid from all the patients with sarcoidosis increased LZM secretion by peripheral blood mononuclear cells compared with unstimulated mononuclear cells. By contrast, BAL fluid from the other individuals had no effect on LZM secretion. CONCLUSIONS: Single cells activated by BAL fluid can be evaluated by the reverse haemolytic plaque assay. BAL fluid from patients with sarcoidosis, but not from patients with systemic sclerosis or normal individuals, contains components capable of activating mononuclear phagocytes to secrete lysozyme.

Increased levels of endothelin-1 in bronchoalveolar lavage fluid from patients with systemic sclerosis contribute to fibroblast mitogenic activity in vitro.

Pulmonary fibrosis is a major cause of morbidity and mortality in patients with systemic sclerosis (SSc). The pathogenesis of this condition is poorly understood, but one of the earliest pathologic features is endothelial and epithelial cell injury with subsequent regeneration. Endothelial and epithelial cells can release several mediators, including endothelin-1 (ET-1). In this study, we investigated the levels of ET-1 in bronchoalveolar lavage fluid (BALF) from patients with SSc and assessed the contribution of ET-1 to the fibroblast mitogenic activity induced by these fluids. A total of 26 patients were evaluated and divided into those with evidence of pulmonary fibrosis, assessed by thin-section computed tomography (group I, n = 16), and those with a normal scan (group II, n = 10). BALF from both groups of patients stimulated fibroblast proliferation. Values expressed as median (range) percentage increase above media controls were 25.5% (5.0 to 47.8%) and 27.6% (10.9 to 51.6%) for groups I and II, respectively (P < 0.02 in both cases). Mitogenic activity was inhibited by about 40% in the presence of either a neutralizing antibody to ET-1 or two synthetic ET-1 receptor ligands. Levels of ET-1 in BALF, expressed as medians (range) were 2.90 ng/mg albumin (0.68 to 5.75) in patients with SSc and 1.23 ng/mg albumin (0.84 to 2.0) in control patients (P < 0.02). Furthermore, ET-1 levels in BALF from patients in group II (3.83 ng/mg albumin, range 1.76 to 5.75) were elevated compared with those in group I (2.62 ng/mg albumin, range 0.68 to 3.81; P < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Insulin-like growth factor-I is partially responsible for fibroblast proliferation induced by bronchoalveolar lavage fluid from patients with systemic sclerosis.

1. Interstitial lung disease is a common complication of systemic sclerosis. The mechanism by which excess collagen is deposited in the lung is poorly understood, but is thought to involve release of mediators which activate lung fibroblasts. In this study we investigated and partially characterized the fibroblast proliferative activity of bronchoalveolar lavage fluid from 29 patients with systemic sclerosis, 19 with and 10 without evidence of lung disease assessed by thin-section computed tomography. 2. Bronchoalveolar lavage fluid from both groups of patients stimulated fibroblast proliferation compared with control subjects: systemic sclerosis with normal computed tomography, 27.7 (range 10.5-57.9)% above control; systemic sclerosis with abnormal computed tomography, 26.7 (range 5.0-47.8)% above control, P < 0.02 in both cases. 3. The activity was reduced by about one-third by neutralizing antibodies to insulin-like growth factor-1 but not platelet-derived growth factor. Levels of insulin-like growth factor-1 of bronchoalveolar fluid were increased in patients with systemic sclerosis [2.10 (range 1.10-3.48) ng/ml of bronchoalveolar lavage fluid] compared with controls [1.45 (range 1.10-2.05) ng/ml; P < 0.01]. When patients were subdivided into those with abnormal computed tomography [2.10 (range 1.20-3.48) ng/ml] and those with normal computed tomography [1.85 (range 1.10-2.90) ng/ml] only the values for the group with evidence of lung disease were increased compared with control subjects (P < 0.02). Platelet-derived growth factor could not be detected in bronchoalveolar lavage fluid from any group. Fractionation of bronchoalveolar lavage fluid demonstrated activity in several fractions consistent with the molecular masses of insulin-like growth factor-1 associated with binding proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

Growth factor activity in the lung during compensatory growth after pneumonectomy: evidence of a role for IGF-1.

Unilateral pneumonectomy in rats causes compensatory growth of the remaining lung. During this growth, there are large increases in the cell numbers and in the rates of collagen and non-collagen protein production. We examined possible mechanisms by which these changes might occur. Assessment of the effect of bronchoalveolar lavage (BAL) fluid on fibroblasts in vitro demonstrated the presence of stimulatory activity for fibroblast replication in control animals. This activity was greatly increased two and six days postpneumonectomy (115 +/- 26% and 75 +/- 18% above control values, respectively), but had returned to normal by 14 days. Preliminary characterization suggests that the activity is heat labile and consists of at least two moieties with apparent molecular weights of 5-15 kD and 70-220 kD. The activity was partially blocked by antibodies to insulin-like growth factor-1 (IGF-1), and levels of IGF-1 were increased by about 100% (p less than 0.001) two days after pneumonectomy compared with control values. Examination of BAL cells demonstrated an early influx of leucocytes into the remaining lung of pneumonectomized rats. At two days, about 25% of the lavageable cells were neutrophils, but macrophages were the predominant cell type at all times. The extravascular albumin space of the lung increased by about 65% (p less than 0.01), six days after pneumonectomy. The influx of circulatory proteins and cells are potential sources of the increased mitogenic activity observed in the lung.

The effect of transforming growth factor beta on rates of procollagen synthesis and degradation in vitro.

Transforming growth factor beta (TGF beta) is known to stimulate procollagen production and steady-state levels of procollagen mRNAs, but its ability to affect post-translational processing of procollagen has been little studied. This paper demonstrates the application of recently developed ultrasensitive methods for measuring hydroxyproline to assess rates of procollagen synthesis and degradation in vitro with and without TGF beta. Foetal rat fibroblasts synthesized 8.63 +/- 0.21 pmol hydroxyproline/micrograms DNA per h, which corresponds to approx. 40 molecules of procollagen/cell per s. Addition of TGF beta to cultures increased total amounts of procollagen synthesized and degraded by 112% and 82%, respectively, but there was a significant decrease in the proportion of procollagen degraded (control, 38.0 +/- 1.1%; TGF beta, 32.3 +/- 0.9%; P less than 0.005). This study demonstrates a novel mechanism which may contribute to the TGF beta-induced increase in procollagen production by fibroblasts.