Inhibition of neointimal hyperplasia in a rabbit vein graft model following non-viral transfection with human iNOS cDNA.

Vein graft failure caused by neointimal hyperplasia (IH) after coronary artery bypass grafting with saphenous veins is a major clinical problem. The lack of safe and efficient vectors for vascular gene transfer has significantly hindered progress in this field. We have developed a Receptor-Targeted Nanocomplex (RTN) vector system for this purpose and assessed its therapeutic efficacy in a rabbit vein graft model of bypass grafting. Adventitial delivery of ß-Galactosidase showed widespread transfection throughout the vein wall on day 7, estimated at about 10% of cells in the adventitia and media. Vein grafts were then transfected with a plasmid encoding inducible nitric oxide synthase (iNOS) and engrafted into the carotid artery. Fluorescent immunohistochemistry analysis of samples from rabbits killed at 7 days after surgery showed that mostly endothelial cells and macrophages were transfected. Morphometric analysis of vein graft samples from the 28-day groups showed approximately a 50% reduction of neointimal thickness and 64% reduction of neointimal area in the iNOS-treated group compared with the surgery control groups. This study demonstrates efficacy of iNOS gene delivery by the RTN formulation in reducing IH in the rabbit model of vein graft disease.

Regulation of matrix turnover: fibroblasts, forces, factors and fibrosis.

Fibroblasts are multifunctional cells that are responsible for matrix homoeostasis, continuously synthesizing and degrading a diverse group of extracellular molecules and their receptors. Rates of turnover of matrix molecules and the proteases that degrade them are normally under the control of diverse chemical and mechanical cues, with cytokines, growth factors, proteases, lipid mediators and mechanical forces playing roles. The maintenance of this homoeostasis is vital to the preservation of normal tissue function and is clearly lost in chronic diseases of the joints, skin and internal organs where destruction and excessive deposition is seen. Current research is focusing on defining the key pathways of activation either in resident fibroblasts, matrix-producing cells derived from circulating fibrocytes, or from transdifferentiation of resident cells. The common downstream signalling pathways are also being defined, as well as the gene interactions leading to altered cell phenotype. The present article reviews these findings and our current concepts of the key molecular events leading to tissue damage and excessive matrix deposition in tissue fibrosis. These studies are leading to an appreciation of the complexity of events with multiple pathways involved, but, as the facts emerge, we are finding promising new ways to treat fibrosis and halt the inexorable progression that is a feature of so many fibrotic and remodelling disorders.

Procollagen type I gene expression and cell proliferation are increased in lipodermatosclerosis.

BACKGROUND: Lipodermatosclerosis (LDS) is characterized by a hardening and hyperpigmentation of lower leg skin as a consequence of chronic venous insufficiency. The degree of skin hardening or fibrosis associated with LDS is proposed to relate directly to skin breakdown and venous ulcer formation as well as to a subsequent delay in ulcer healing. OBJECTIVES: To determine whether elevated procollagen type I gene expression and increased cell proliferation are responsible for the fibrotic changes associated with LDS. METHODS: Skin biopsies were obtained from the legs of patients with varying degrees of chronic venous disease and were assessed for procollagen gene expression by in-situ hybridization and for cell proliferation by immunolocalization of proliferating cell nuclear antigen. RESULTS: The number of cells expressing procollagen type I mRNA (COL1A1) was significantly higher in the dermis of LDS-affected skin compared with samples from the other patient groups. In addition, there was a significant increase in the number of dermal fibroblasts undergoing proliferation in both LDS samples and skin samples prior to LDS changes compared with control samples. However, there was no significant difference in level of inflammation in biopsy samples between patient classes. CONCLUSIONS: These results suggest that enhanced cell proliferation and procollagen gene expression are both involved in LDS development. Furthermore, fibrotic changes may occur in the absence of, or subsequent to, any significant inflammatory response, indicating that additional profibrotic factors produced in the skin as a consequence of chronic venous insufficiency may play a role in LDS formation.

Attenuation of bleomycin induced pulmonary fibrosis in mice using the heme oxygenase inhibitor Zn-deuteroporphyrin IX-2,4-bisethylene glycol.

BACKGROUND: Pulmonary fibrosis is associated with a poor prognosis. The pathogenesis of fibrotic lung disorders remains unclear, but the extent of tissue damage due to the persistent presence of oxidants or proteases is believed to be important. The heme degrading enzyme heme oxygenase (HO) has been found to be expressed in experimental fibrosis, and generation of free iron and carbon monoxide (CO) by HO has been implicated in oxidant induced lung damage. A study was undertaken to examine the effects of the HO inhibitor Zn-deuteroporphyrin-IX-2,4-bisethylene glycol (Zndtp) on the development of pulmonary fibrosis in the bleomycin model of lung injury and repair. METHODS: Zndtp (10 micro mol/kg) was administered subcutaneously twice daily to mice 1 week following the intratracheal instillation of 0.025 U bleomycin. Animals were killed 10 or 21 days after bleomycin instillation and indices of lung damage and fibrosis were evaluated. RESULTS: Bleomycin treatment induced pulmonary cytotoxicity, increased levels of active transforming growth factor beta (TGF-beta), enhanced lung collagen accumulation, and decreased glutathione content. Zndtp administration significantly attenuated these indices. CONCLUSIONS: Administration of Zndtp in the bleomycin model resulted in appreciable alveolar cytoprotection and amelioration of pulmonary fibrosis. This molecule and its analogues may warrant further consideration in the treatment of acute lung injury and fibrotic lung disorders.

Pulmonary gene therapy. Realistic hope for the future, or false dawn in the promised land?

Pulmonary gene therapy offers the hope of treatment for conditions such as cystic fibrosis, lung cancer, pulmonary fibrosis and acute respiratory distress syndrome for which current therapy is inadequate. Although initial clinical trials in cystic fibrosis and non-small cell lung cancer have shown promise the results have not been as good as might have been anticipated. However, clinical improvement has been demonstrated in conditions such as haemophilia [82], cardiovascular disease [83], head and neck cancer [84] and X-linked severe combined immunodeficiency disease [85]. The lack of success of pulmonary gene therapy is due, in part on the physical barriers to transfection perfected by the lung to prevent toxicity from inhaled particles, and partly due to the poor transfection efficiency of non-viral systems, and the immunogenicity of viral systems, of gene transfer. The LID vector goes some way to addressing the problems associated with current gene delivery strategies. With continued improvements in the properties of both viral and non-viral gene delivery systems leading to improved transfection efficiency with reduced toxicity, as well as the development of strategies aimed at reducing the physical barriers to pulmonary transfection, and targeting gene delivery systems to the site of injury, it is likely that pulmonary gene therapy will be used successfully to ameliorate a number of devastating pulmonary conditions.

Short course dexamethasone treatment following injury inhibits bleomycin induced fibrosis in rats.

BACKGROUND: Corticosteroids are routinely used in patients with pulmonary fibrosis. The timing for initiation of treatment is likely to be crucial for corticosteroids to exert an antifibrotic effect. Experimental studies in animals have examined the effect of corticosteroid treatment starting before or at the time of lung injury. However, this is not representative of the human condition as treatment only begins after disease has been established. We examined the effect of a short course corticosteroid treatment starting 3 days after bleomycin induced lung injury on the development of pulmonary fibrosis. METHODS: Bleomycin (1.5 mg/kg) was instilled intratracheally into rats to induce pulmonary fibrosis. The effect of a 3-day course of dexamethasone (0.5 mg/kg) initiated 3 days after bleomycin induced lung injury on cell proliferation and collagen deposition was examined by analysing bronchoalveolar lavage (BAL) fluid and lung tissue. RESULTS: Treating bleomycin exposed animals after injury with dexamethasone for 3 days inhibited lung collagen deposition compared with animals exposed to bleomycin without dexamethasone treatment (15.2 (2.2) mg collagen/lung v 22.5 (2.1) mg/lung; p<0.05). Dexamethasone treatment reduced pulmonary parenchymal cell proliferation in bleomycin exposed rats but did not influence BAL fluid mitogenic activity for lung fibroblasts or alter the BAL fluid levels of the fibrogenic mediators transforming growth factor-beta(1), platelet derived growth factor-AB, and thrombin. CONCLUSIONS: A 3 day course of dexamethasone treatment initiated 3 days after bleomycin induced lung injury reduces lung cell proliferation and collagen deposition by mechanisms other than through reduction of transforming growth factor-beta(1), platelet derived growth factor-AB, and thrombin levels in BAL fluid. We propose that an early short course treatment with dexamethasone may be useful in inhibiting pulmonary fibrosis.

Formation of LID vector complexes in water alters physicochemical properties and enhances pulmonary gene expression in vivo.

There is currently an urgent need to develop efficient gene-delivery systems for the lung that are free of inflammatory effects. The LID vector is a synthetic gene delivery system, comprised of lipofectin (L), an integrin-targeting peptide (I) and DNA (D) that has previously been shown to have high transfection efficiency in the lung. We have assessed the effect of alternative methods of complex preparation on structural features of the complex, levels and duration of reporter gene expression and the host response to the LID vector. We have demonstrated that making the complex in water affects the structure of the LID complexes making them smaller and more stable with a more cationic surface charge than complexes prepared in phosphate-buffered saline (PBS). When the LID vector was constituted in water and instilled intratracheally into the lungs of mice there was a 10-fold increase in luciferase activity compared with preparation in PBS. Furthermore, luciferase activity was still evident 1 week following vector instillation. This enhancement may be because of altered complex structure, although effects of the hypotonic vector solution on the lung cannot be excluded. The inflammatory effects of instilling the LID vector in water were minimal, even after three administrations of the LID vector, with only mild alterations in cytokine and broncho-alveolar lavage fluid (BALF) cell profiles. These results demonstrate that the LID vector can generate high, and prolonged, levels of gene expression in the lung from small quantities of DNA and that careful attention to synthetic polyplex structure may be important to optimize efficiency of gene expression in vivo.

Direct thrombin inhibition reduces lung collagen, accumulation, and connective tissue growth factor mRNA levels in bleomycin-induced pulmonary fibrosis.

Dramatic activation of the coagulation cascade has been extensively documented for pulmonary fibrosis associated with acute and chronic lung injury. In addition to its role in hemostasis, thrombin exerts profibrotic effects via activation of the major thrombin receptor, protease-activated receptor-1. In this study, we examined the effect of the direct thrombin inhibitor, UK-156406 on fibroblast responses in vitro and on bleomycin-induced pulmonary fibrosis in rats. UK-156406 significantly inhibited thrombin-induced fibroblast proliferation, procollagen production, and connective tissue growth factor (CTGF) mRNA levels when used at equimolar concentration to the protease. Thrombin levels in bronchoalveolar lavage fluid and expression of thrombin and protease-activated receptor-1 in lung tissue were increased after intratracheal instillation of bleomycin. The characteristic doubling in lung collagen in bleomycin-treated animals (38.4 +/- 2.0 mg versus 17.1 +/- 1.4 mg, P < 0.01) was preceded by significant elevations in alpha1(I) procollagen and CTGF mRNA levels (3.0 +/- 0.4-fold and 6.3 +/- 0.4-fold respectively, (P < 0.01), and total inflammatory cell number. UK-156406, administered at an anticoagulant dose, attenuated lung collagen accumulation in response to bleomycin by 35 +/- 12% (P < 0.05), inhibited alpha1(I) procollagen and CTGF mRNA levels by 50% and 35%, respectively (P < 0.05), but had no effect on inflammatory cell recruitment. This is the first report showing that direct thrombin inhibition abrogates lung collagen accumulation in bleomycin-induced pulmonary fibrosis.

Localisation of transforming growth factor beta1 and beta3 mRNA transcripts in normal and fibrotic human lung.

BACKGROUND: Transforming growth factor beta1 is implicated in the pathogenesis of lung fibrosis. It promotes extracellular matrix accumulation by increasing procollagen synthesis and reducing degradation. TGFbeta1 gene and protein expression increase in experimental lung fibrosis, and TGFbeta1 antibodies attenuate fibrosis in mice. The role of other TGFbeta isoforms is unclear. This study aimed to localise TGFbeta1 and TGFbeta3 gene expression in fibrotic human lung and compare it with that in normal human lung. METHODS: Lung tissue from patients with cryptogenic fibrosing alveolitis and fibrosis associated with systemic sclerosis was examined by in situ hybridisation. Macroscopically normal lung from carcinoma resections was used as control tissue. Digoxigenin labelled riboprobes were synthesised from TGFbeta isoform specific cDNA templates. RESULTS: The digoxigenin labelled riboprobes were sensitive and permitted precise cellular localisation of mRNA transcripts. TGFbeta1 and TGFbeta3 mRNA transcripts were widespread in normal lung and localised to alveolar macrophages and bronchiolar epithelium. TGFbeta1 but not TGFbeta3 mRNA was detected in mesenchymal and endothelial cells. In fibrotic lung tissue mRNA transcripts for both isoforms were also detected in metaplastic type II cells. TGFbeta1 gene expression was enhanced in some patients. TGFbeta3 was expressed in fibrotic lung but was not consistently altered compared with controls. CONCLUSION: TGFbeta1 mRNA transcripts were localised in normal and fibrotic human lung and TGFbeta3 gene expression in human lung fibrosis was shown for the first time. The results suggest that TGFbeta1 may play the predominant role in pathogenesis. It is suggested that TGFbeta1 should be the primary target of anticytokine treatments for pulmonary fibrosis.

Cyclooxygenase-2 deficiency results in a loss of the anti-proliferative response to transforming growth factor-beta in human fibrotic lung fibroblasts and promotes bleomycin-induced pulmonary fibrosis in mice.

Prostaglandin E(2) (PGE(2)) inhibits fibroblast proliferation and collagen production. Its synthesis by fibroblasts is induced by profibrotic mediators including transforming growth factor (TGF)-beta(1). However, in patients with pulmonary fibrosis, PGE(2) levels are decreased. In this study we examined the effect of TGF-beta(1) on PGE(2) synthesis, proliferation, collagen production, and cyclooxygenase (COX) mRNA levels in fibroblasts derived from fibrotic and nonfibrotic human lung. In addition, we examined the effect of bleomycin-induced pulmonary fibrosis in COX-2-deficient mice. We demonstrate that basal and TGF-beta(1)-induced PGE(2) synthesis is limited in fibroblasts from fibrotic lung. Functionally, this correlates with a loss of the anti-proliferative response to TGF-beta(1). This failure to induce PGE(2) synthesis is because of an inability to up-regulate COX-2 mRNA levels in these fibroblasts. Furthermore, mice deficient in COX-2 exhibit an enhanced response to bleomycin. We conclude that a decreased capacity to up-regulate COX-2 expression and COX-2-derived PGE(2) synthesis in the presence of increasing levels of profibrotic mediators such as TGF-beta(1) may lead to unopposed fibroblast proliferation and collagen synthesis and contribute to the pathogenesis of pulmonary fibrosis.

Fibroproliferation occurs early in the acute respiratory distress syndrome and impacts on outcome.

The fibroproliferative phase of acute respiratory distress syndrome (ARDS) has traditionally been regarded as a late event but recent studies that suggest increased lung collagen turnover within 24 h of diagnosis challenge this view. We hypothesized that fibroproliferation is initiated early in ARDS, characterized by the presence of fibroblast growth factor activity in the lung and would relate to clinical outcome. Patients fulfilling American/European Consensus Committee criteria for ARDS and control patients ventilated for non-ARDS respiratory failure underwent bronchoalveolar lavage (BAL) and serum sampling within 24 h of diagnosis and again at 7 d. The ability of BAL fluid (BALF) to stimulate human lung fibroblast proliferation in vitro was examined in relation to concentrations of N-terminal peptide for type III procollagen (N-PCP-III) in BALF/serum and clinical indices. At 24 h, ARDS lavage fluid demonstrated potent mitogenic activity with a median value equivalent to 70% (range 31-164) of the response to serum, and was significantly higher than control lavage (32% of serum response, range 11-42; p < 0.05). At 24 h, serum N-PCP-III concentrations were elevated in the ARDS group compared with control patients (2.8 U/ml; range 0.6-14.8 versus 1.1 U/ml; range 0.4-3.7, p < 0.0001) as were BALF N-PCP-III concentrations (2.9 U/ml; range 0. 6-11.4 versus 0.46 U/ ml; range 0.00-1.63, p < 0.01). In addition, BALF N-PCP-III concentrations at 24 h were significantly elevated in nonsurvivors of ARDS compared with survivors (p < 0.05). At 7 d, the mitogenic activity remained elevated in the ARDS group compared with control (p < 0.05) and was also significantly higher in ARDS nonsurvivors compared with survivors (67%; range 45-120 versus 31%; range 16-64, p < 0.05). These data are consistent with the hypothesis that fibroproliferation is an early response to lung injury and an important therapeutic target.

Increased collagen production in fibroblasts cultured from irradiated skin and effect of TGF beta(1)- clinical study.

Fibrosis in normal tissues is a common and dose-limiting late complication of radiotherapy at many cancer sites, but its pathogenesis is poorly understood. We undertook a controlled study of the effect of irradiation on the collagen production of fibroblasts cultured from skin biopsies taken from patients undergoing radiotherapy treatment. Eight weeks after a single 8 Gy fraction using 300 kV X-rays, five patients treated at the Royal Marsden Hospital underwent biopsy of the irradiated site and of the contralateral, unirradiated body site. Fibroblasts from irradiated and control, unirradiated sites were cultured in vitro, and collagen production rates were measured during a 48-hour incubation under standardized conditions and in the presence and absence of transforming growth factor beta(1)(TGF beta(1)), 1 ng/ml, using HPLC. Collagen production was elevated in cells cultured from irradiated skin; median collagen production rates 61.16 pmoles hydroxyproline/10(5)cells/hour in irradiated cells, 39.78 pmoles hydroxyproline/10(5)cells/hour in unirradiated cells, P = 0.016 (Mann-Whitney U-test). In fibroblasts from unirradiated sites, collagen production rates were increased by the addition of TGF beta(1); however, in three of the cell lines cultured from irradiated sites this effect of TGF beta(1)on collagen production was not observed.

Angiotensin II is mitogenic for human lung fibroblasts via activation of the type 1 receptor.

The expression of renin-angiotensin system components and the elevation of angiotensin-converting enzyme (ACE) in a number of fibrotic lung diseases suggests angiotensin II (AII) could play a role in the pathogenesis of pulmonary fibrosis. However, the effect of AII on lung fibroblasts has not previously been assessed and the mechanisms by which AII induces cell proliferation in mesenchymal cells are not fully understood. We have examined the ability of AII to stimulate fetal and adult human lung fibroblast proliferation in vitro. In particular, we have assessed the receptor subtypes involved and the possible autocrine role of transforming growth factor beta (TGF-beta) and platelet-derived growth factor (PDGF), two recognized fibroblast mitogens. Angiotensin type 1 (AT1), but not type 2, receptors were identified on fetal and adult human lung fibroblasts by immunocytochemistry. AII (1 microM) increased DNA synthesis (determined by [(3)H]thymidine incorporation) in fetal and adult cells by 211 +/- 18% and 150 +/- 14%, respectively (p < 0.01), and was inhibited by a specific AT1 receptor antagonist, Losartan (74 +/- 14%). A proliferative response to AII was confirmed by direct cell counts. Subsequently, fibroblasts were incubated with neutralizing antibodies to TGF-beta and PDGF. Anti-TGF-beta antibodies inhibited AII-induced DNA synthesis by 73 +/- 13%. However, no effect was seen with anti-PDGF antibodies. In conclusion, we have shown that angiotensin II induces human lung fibroblast proliferation in vitro via activation of the AT1 receptor and involves the autocrine action of TGF-beta.

An integrin-targeted non-viral vector for pulmonary gene therapy.

Gene therapy offers potential for the treatment of severe respiratory diseases. However, the vectors which are currently available have drawbacks limiting their therapeutic application. Here we report on an integrin-targeted, non-viral gene delivery system for pulmonary gene transfer. We demonstrate that this vector can deliver the lacZ reporter gene to the lung, transfecting bronchial epithelium and parenchymal cells with similar efficiency to an adenoviral vector and with greater efficiency than a cationic liposome. In addition, vector administration can be repeated leading to further gene expression without inducing inflammation. The advantages of this novel gene delivery system provide considerable potential for targeted gene therapy in vivo. Gene Therapy (2000) 7, 393-400.

Mast cell tryptase stimulates human lung fibroblast proliferation via protease-activated receptor-2.

Mast cells play a potentially important role in fibroproliferative diseases, releasing mediators including tryptase that are capable of stimulating fibroblast proliferation and procollagen synthesis. The mechanism by which tryptase stimulates fibroblast proliferation is unclear, although recent studies suggest it can activate protease-activated receptor (PAR)-2. We therefore investigated the role of PAR-2 in tryptase-induced proliferation of human fetal lung and adult lung parenchymal and airway fibroblasts and, for comparative purposes, adult dermal fibroblasts. Tryptase (0.7-70 mU/ml) induced concentration-dependent increases in proliferation of all fibroblasts studied. Antipain, bis(5-amidino-2-benzimidazolyl)methane, and benzamidine inhibited tryptase-induced fibroblast proliferation, demonstrating that proteolytic activity is required for the proliferative effects of tryptase. RT-PCR demonstrated the presence of PAR-2 mRNA, and immunohistochemical staining localized PAR-2 to the cell surface of lung fibroblasts. In addition, specific PAR-2 activating peptides, SLIGKV and SLIGRL, mimicked the proliferative effects of tryptase. In contrast, human dermal fibroblasts only weakly stained with the PAR-2 antibody, PAR-2 mRNA was almost undetectable, and fibroblasts did not respond to PAR-2 activating peptides. These results suggest that tryptase induces lung, but not dermal, fibroblast proliferation via activation of PAR-2 and are consistent with the hypothesis that the release of tryptase from activated mast cells may play an important role in the fibroproliferative response observed in asthma, chronic obstructive pulmonary disease, and patients with pulmonary fibrosis.

Fibroblast mitogens in bronchoalveolar lavage (BAL) fluid from asbestos-exposed subjects with and without clinical evidence of asbestosis: no evidence for the role of PDGF, TNF-alpha, IGF-1, or IL-1 beta.

Asbestosis is a fibrotic lung disease resulting from inhalation of asbestos fibres. Its pathogenesis is poorly understood but probably involves stimulation of fibroblast proliferation and collagen production by mediators released from inflammatory and resident lung cells. In vitro studies have implicate PDGF, TNF-alpha, IGF-1, TGF-beta, and IL-1 in asbestosis, but the role of these mediators in vivo is not known. This study aimed to characterize mediators in bronchoalveolar lavage (BAL) fluid from patients exposed to asbestos with (n = 24) or without (n = 34) asbestosis, compared with ten normal subjects. Human lung fibroblasts were exposed to serial dilutions of BAL fluids and the effects on fibroblast proliferation were assessed. The median mitogenic activity of BAL fluid from asbestos-exposed (17 per cent above medium control, range 3-44 per cent) and asbestosis (14 per cent, range 2-60 per cent) groups was higher than that of BAL fluid from controls (10 per cent, range 2-20 per cent; P < 0.01 and P < 0.05, respectively), but there was no significant difference between the patient groups. The mitogenic activity of BAL fluids was not reduced by incubation with neutralizing antibodies to PDGF-AA, PDGF-AB, PDGF-BB, TNF-alpha, IGF-1, and IL-1 beta. We conclude that BAL fluids from patients exposed to asbestos contain mitogens for human lung fibroblasts, but that PDGF, TNF-alpha, IGF-1, or IL-1 beta do not contribute to this activity.

A novel transforming growth factor beta2 antisense transcript in mammalian lung.

Transforming growth factor (TGF) beta2 gene expression was examined in murine, rat and human lung by in situ hybridization with riboprobes. Hybridization signal was observed in a variety of cells with the sense probe, and Northern-blot analysis with this probe demonstrated the presence of a novel 3.5 kb transcript. This first report suggesting the existance of a natural TGFbeta2 antisense transcript raises the possibility that such a transcript may play a role in regulating TGFbeta2 production.

Thrombin stimulates fibroblast procollagen production via proteolytic activation of protease-activated receptor 1.

Thrombin is a multifunctional serine protease that has a crucial role in blood coagulation. It is also a potent mesenchymal cell mitogen and chemoattractant and might therefore have an important role in the recruitment and local proliferation of mesenchymal cells at sites of tissue injury. We hypothesized that thrombin might also affect the deposition of connective tissue proteins at these sites by directly stimulating fibroblast procollagen production. To address this hypothesis, the effect of thrombin on procollagen production and gene expression by human foetal lung fibroblasts was assessed over 48 h. Thrombin stimulated procollagen production at concentrations of 1 nM and above, with maximal increases of between 60% and 117% at 10 nM thrombin. These effects of thrombin were, at least in part, due to increased steady-state levels of alpha1(I) procollagen mRNA. They could furthermore be reproduced with thrombin receptor-activating peptides for the protease-activated receptor 1 (PAR-1) and were completely abolished when thrombin was rendered proteolytically inactive with the specific inhibitors d-Phe-Pro-ArgCH2Cl and hirudin, indicating that thrombin is mediating these effects via the proteolytic activation of PAR-1. These results suggest that thrombin might influence the deposition of connective tissue proteins during normal wound healing and the development of tissue fibrosis by stimulating fibroblast procollagen production.

Increased endothelin-1 and its localization during the development of bleomycin-induced pulmonary fibrosis in rats.

Endothelin-1 (Et-1) has been implicated in the pathogenesis of pulmonary fibrosis with increased levels in the lung tissue of patients with pulmonary fibrosis and profibrotic effects in vitro. In this study we have investigated the temporal changes in lung Et-1 levels and immunohistochemical localization in relation to collagen deposition during the development of bleomycin-induced pulmonary fibrosis in rats. Lung Et-1 content doubled by 3 d following the intratracheal instillation of bleomycin, and continued to increase up to 7 d when values were about threefold greater than controls. Thereafter, the values for bleomycin-treated animals remained constant up to 21 d. There was no change in collagen content at 3 d but after 7 d there was a 25% increase and by 21 d levels were almost double those of the controls. In normal lung, Et-1 was predominantly associated with epithelial cells of conducting and nonconducting airways. Following bleomycin administration, intense staining of macrophages and conducting airway and alveolar epithelial cells was observed with marked staining of perivascular, peribronchiolar, and alveolar septal connective tissue, as well as the venular and arterial intima and media. These results demonstrate elevation of Et-1 levels prior to an increase in collagen content which, along with its localization within developing fibrotic lesions, provides further evidence of a profibrotic role for Et-1 in the pathogenesis of pulmonary fibrosis.

Thrombin stimulates smooth muscle cell procollagen synthesis and mRNA levels via a PAR-1 mediated mechanism.

Thrombin is a serine protease involved in haemostasis which exerts a number of cellular effects, including stimulating mesenchymal cell migration, proliferation, and has been implicated both in normal wound healing and pathological conditions associated with hyperproliferation of smooth muscle cells such as atherosclerosis and restenosis. We hypothesize that thrombin, in addition to its proliferative effects, may also influence the deposition of matrix proteins at sites of vascular injury by directly stimulating smooth muscle cell procollagen production. 10 nM thrombin significantly stimulated rat aortic smooth muscle cell procollagen production by 34 +/- 3% compared to media control cells over a 48 h incubation period, and increased steady state alpha1(I) procollagen mRNA levels by up to 104 +/- 22%. These effects are mediated via interaction of thrombin with the PAR-1 receptor since TRAP (Thrombin Receptor Activating Peptide) stimulated procollagen production by 23 +/- 0.5%. In addition, conditioned medium from thrombin-treated cells stimulated procollagen production by 30 +/- 3% suggesting that thrombin is acting via the production and/or release of an autocrine mediator. These data suggest a novel role for thrombin in vascular wound healing and the development of pathological conditions associated with increased connective tissue deposition.