Intrinsic defence capacity and therapeutic potential of natriuretic peptides in pulmonary hypertension associated with lung fibrosis.

BACKGROUND AND PURPOSE: Idiopathic pulmonary fibrosis (IPF) is a progressive fibro-proliferative disorder refractory to current therapy commonly complicated by the development of pulmonary hypertension (PH); the associated morbidity and mortality are substantial. Natriuretic peptides possess vasodilator and anti-fibrotic actions, and pharmacological augmentation of their bioactivity ameliorates renal and myocardial fibrosis. Here, we investigated whether natriuretic peptides possess an intrinsic cytoprotective function preventing the development of pulmonary fibrosis and associated PH, and whether therapeutics targeting natriuretic peptide signalling demonstrate efficacy in this life-threatening disorder. EXPERIMENTAL APPROACH: Pulmonary haemodynamics, right ventricular function and markers of lung fibrosis were determined in wild-type (WT) and natriuretic peptide receptor (NPR)-A knockout (KO) mice exposed to bleomycin (1 mg·kg(-1) ). Human myofibroblast differentiation was studied in vitro. KEY RESULTS: Exacerbated cardiac, vascular and fibrotic pathology was observed in NPR-A KO animals, compared with WT mice, exposed to bleomycin. Treatment with a drug combination that raised circulating natriuretic peptide levels (ecadotril) and potentiated natriuretic peptide-dependent signalling (sildenafil) reduced indices of disease progression, whether administered prophylactically or to animals with established lung disease. This positive pharmacodynamic effect was diminished in NPR-A KO mice. Atrial natriuretic peptide and sildenafil synergistically reduced TGFß-induced human myofibroblast differentiation, a key driver of remodelling in IPF patients. CONCLUSIONS AND IMPLICATIONS: These data highlight an endogenous host-defence capacity of natriuretic peptides in lung fibrosis and PH. A combination of ecadotril and sildenafil reversed the pulmonary haemodynamic aberrations and remodelling that characterize the disease, advocating therapeutic manipulation of natriuretic peptide bioactivity in patients with IPF.

Is global climate change influencing the overwintering distribution of weakfish Cynoscion regalis?

The pattern of stable isotope signatures in a sub-sample of 67 juvenile weakfish Cynoscion regalis, captured at the mouth of the Christina River, 113 km upstream of the mouth of Delaware Bay (U.S.A) in the autumn of 2000, suggested that they resided at the location since recruitment. The possibility that young C. regalis departed from the generally characteristic life-history pattern of marine migrants at this latitude, i.e. emigrating offshore with the adults in autumn was bolstered by the collection of 69 individuals during the winters of 2000-2006 from the travelling screens of a power plant located at river kilometre 88 including an 118 mm total length juvenile captured in mid-February 2006.

Procoagulant signalling mechanisms in lung inflammation and fibrosis: novel opportunities for pharmacological intervention?

There is compelling evidence that uncontrolled activation of the coagulation cascade following lung injury contributes to the development of lung inflammation and fibrosis in acute lung injury/acute respiratory distress syndrome (ALI/ARDS) and fibrotic lung disease. This article reviews our current understanding of the mechanisms leading to the activation of the coagulation cascade in response to lung injury and the evidence that excessive procoagulant activity is of pathophysiological significance in these disease settings. Current evidence suggests that the tissue factor-dependent extrinsic pathway is the predominant mechanism by which the coagulation cascade is locally activated in the lungs of patients with ALI/ARDS and pulmonary fibrosis. Whilst, fibrin deposition might contribute to the pathophysiology of ALI/ARDS following systemic insult; current evidence suggests that the cellular effects mediated via activation of proteinase-activated receptors (PARs) may be of particular importance in influencing inflammatory and fibroproliferative responses in experimental models involving direct injury to the lung. In this regard, studies in PAR(1) knockout mice have shown that this receptor plays a major role in orchestrating the interplay between coagulation, inflammation and lung fibrosis. This review will focus on our current understanding of excessive procoagulant signalling in acute and chronic lung injury and will highlight the novel opportunities that this may present for therapeutic intervention.

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.

B[a]P-DNA binding in early life-stages of Atlantic tomcod: population differences and chromium modulation.

Atlantic tomcod (Microgadus tomcod) from the Hudson River (HR) are resistant at the molecular and organismic levels to the effects of exposure to dioxin-like aromatic hydrocarbon (AH) compounds, but much less so to benzo[a]pyrene (B[a]P). The aims of this study were to determine in early life-stages of tomcod exposed to B[a]P: (1) if DNA binding levels differed between fish from the HR and Miramichi River (MR), and (2) if co-exposure to chromium could modulate this genotoxic effect. After exposure to [(3)H]B[a]P alone, DNA-bound radioactivity was 5-10-fold higher in embryos and larvae of MR than HR descent. Co-exposure to chromium modulated DNA binding levels in offspring of both populations. In MR embryos, co-exposure to chromium inhibited B[a]P uptake. These results demonstrated resistance to the genotoxic effects of B[a]P in early life stages of HR tomcod at an ecologically important endpoint and suggest the ability of chromium to modulate AH-induced genotoxicity.

The rapamycin analogue SDZ RAD attenuates bleomycin-induced pulmonary fibrosis in rats.

Pulmonary fibrosis is characterized by excessive deposition of extracellular matrix proteins within the pulmonary interstitium. The new macrolide immunosuppressant SDZ RAD, a rapamycin analogue, inhibits growth-factor dependent proliferation of mesenchymal cells and might therefore be of therapeutic interest for the treatment of fibrotic lung disease. In this study the effect of SDZ RAD on lung-collagen accumulation in the bleomycin model of pulmonary fibrosis in rats was investigated. SDZ RAD (2.5 mg x kg(-1) x day(-1)) or drug vehicle were administered orally by daily gavage. Successful dosing was confirmed by measuring splenic weight. Total lung-collagen content was measured by high-performance liquid chromatographic quantitation of hydroxyproline. In animals given bleomycin and drug vehicle, total lung collagen was increased by 182+/-11% (mean+/-SEM) compared with saline controls at 14 days (p<0.001). The increase in lung-collagen accumulation was reduced by 75+/-12% (p<0.01) in animals given SDZ RAD and was accompanied by a concomitant 56+/-6% (p<0.001) reduction in lung weight. SDZ RAD is currently in clinical trials for the prevention of solid organ graft rejection, another condition characterized by excessive extracellular matrix production. The authors propose that SDZ RAD warrants evaluation as a novel therapeutic agent for fibrotic lung disease.

Coagulation cascade proteases and tissue fibrosis.

Fibrotic disorders of the liver, kidney and lung are associated with excessive deposition of extracellular matrix proteins and ongoing coagulation-cascade activity. In addition to their critical roles in blood coagulation, thrombin and the immediate upstream coagulation proteases, Factors Xa and VIIa, influence numerous cellular responses that may play critical roles in subsequent inflammatory and tissue repair processes in vascular and extravascular compartments. The cellular effects of these proteases are mediated via proteolytic activation of a novel family of cell-surface receptors, the protease-activated receptors (PAR-1, -2, -3 and -4). Although thrombin is capable of activating PAR-1, -3 and -4, there is accumulating in vitro evidence that the profibrotic effects of thrombin are predominantly mediated via PAR-1. Factor Xa is capable of activating PAR-1 and PAR-2, but its mitogenic effects for fibroblasts are similarly mediated via PAR-1. These proteases do not exert their profibrotic effects directly, but act via the induction of potent fibrogenic mediators, such as platelet-derived growth factor and connective tissue growth factor. In vivo studies using proteolytic inhibitors, PAR-1 antagonists and PAR-1-deficient mice have provided evidence that coagulation proteases play a key role in tissue inflammation and in a number of vascular pathologies associated with hyperproliferation of smooth muscle cells. More recently, coagulation proteases have also been shown to play a role in the pathogenesis of fibrosis but the relative contribution of their cellular versus their procoagulant effects awaits urgent evaluation in vivo. These studies will be informative in determining the potential application of PAR-1 antagonists as antifibrotic agents.

Role of thrombin and its major cellular receptor, protease-activated receptor-1, in pulmonary fibrosis.

Pulmonary fibrosis is the end stage of a heterogeneous group of disorders and is characterized by the excessive deposition of extracellular matrix proteins within the pulmonary interstitium. There is increasing evidence from a number of studies that activation of the coagulation cascade, with the resultant generation of coagulation proteases, plays a central role in fibrotic lung disease that is associated with acute and chronic lung injury. Consistent with this finding, levels of thrombin are increased in bronchoalveolar lavage fluid from patients and in animal models of this disorder. In addition to its classical role in blood coagulation, thrombin exerts a number of proinflammatory and profibrotic cellular effects in vitro that are critically important in tissue repair processes. These cellular effects are predominantly mediated via proteolytic activation of the major thrombin receptor protease-activated receptor-1 (PAR-1). This has led us to hypothesize that the procoagulant and the downstream cellular effects of thrombin, which are initiated following receptor activation, may be important in promoting tissue fibrosis in vivo. To examine this hypothesis, we assessed the effect of a direct thrombin inhibitor in bleomycin-induced pulmonary fibrosis in rats. Immunohistochemical studies showed that expression of thrombin and PAR-1 in lung tissue increased dramatically after intratracheal instillation of bleomycin, compared with saline-treated animals. After bleomycin instillation, there was a doubling in the amount of lung collagen after 14 days, which was preceded by elevations in alpha(1)(I) procollagen and connective tissue growth factor (CTGF) mRNA levels. However, when bleomycin-treated animals concurrently received a continuous infusion of a direct thrombin inhibitor at an anticoagulant dose, lung collagen accumulation in response to bleomycin was attenuated by up to 40%. Furthermore, alpha(1)(I) procollagen and CTGF mRNA levels were also significantly reduced in these animals. These findings confirm that thrombin is a key mediator in the pathogenesis of this condition and suggest that the cellular effects of thrombin may be critically important in promoting lung collagen accumulation in this experimental model of pulmonary fibrosis. Targeting the profibrotic effects of coagulation proteases warrants further evaluation as a potential therapeutic strategy for fibrotic lung disease.

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.

Factor Xa is a fibroblast mitogen via binding to effector-cell protease receptor-1 and autocrine release of PDGF.

The coagulation cascade protease thrombin is a fibroblast mitogen, but the proliferative potential of other coagulation proteases is not known. In this study we show that factor Xa stimulated human fetal lung fibroblast DNA synthesis in a concentration-dependent manner from 1 nM onward with a fourfold increase at 200 nM. The mitogenic effect of factor Xa was confirmed using a colorimetric proliferation assay and direct cell counting. Factor Xa and thrombin had equivalent potencies, and their stimulatory effects followed a similar time course. Comparable results were also obtained with primary human adult fibroblasts derived from lung, kidney, heart, skin, and liver. Factor VIIa also stimulated fibroblast proliferation, but only at concentrations >10 nM, whereas factor IXa had no effect. To begin to address the mechanism by which factor Xa is acting, we show that human fibroblasts express effector-cell protease receptor-1 and that blocking antibodies to this receptor and the catalytic site of factor Xa inhibited its mitogenic effect. Furthermore, factor Xa upregulated platelet-derived growth factor-A (PDGF-A) mRNA expression, whereas PDGF-B could not be detected, and a blocking antibody to PDGF inhibited the mitogenic effect of factor Xa. We conclude that factor Xa acts as a fibroblast mitogen via binding to effector-cell protease receptor-1 and the autocrine release of PDGF.

Alpha-1-antitrypsin stimulates fibroblast proliferation and procollagen production and activates classical MAP kinase signalling pathways.

Connective tissue formation at sites of tissue repair is regulated by matrix protein synthesis and degradation, which in turn is controlled by the balance between proteases and antiproteases. Recent evidence has suggested that antiproteases may also exert direct effects on cell function, including influencing cell migration and proliferation. The antiprotease, alpha1-antitrypsin, is the major circulating serine protease inhibitor which protects tissues from neutrophil elastase attack. Its deficiency is associated with the destruction of connective tissue in the lung and the development of emphysema, whereas accumulation of mutant alpha1-antitrypsin within hepatocytes often leads to liver fibrosis. In this study, we report that alpha1antitrypsin, at physiologically relevant concentrations, promotes fibroblast proliferation, with maximal stimulatory effects of 118 +/- 2% (n=6, P < 0.02) above media controls for cells exposed to 60 microM. We further show that alpha1antitrypsin also stimulates fibroblast procollagen production, independently of its effects on cell proliferation, with values maximally increased by 34 +/- 3% (n = 6, P < 0.01) above media controls at 30 microM. Finally, mechanistic studies to examine the mechanism by which alpha1-antitrypsin acts, showed that alpha1-antitrypsin induced the rapid activation of p42MAPK and p44MAPK (also known as ERK1/2) and that the specific MEK1 inhibitor PD98059 totally blocked alpha1-antitrypsin's mitogenic effects. These results support the hypothesis that alpha1-antitrypsin may play a role in influencing tissue repair in vivo by directly stimulating fibroblast proliferation and extracellular matrix production via classical mitogen-activated signalling pathways.

Thrombin is a potent inducer of connective tissue growth factor production via proteolytic activation of protease-activated receptor-1.

The coagulation protease thrombin plays a critical role in hemostasis and exerts pro-inflammatory and pro-fibrotic effects via proteolytic activation of the major thrombin receptor, protease-activated receptor-1 (PAR-1). Connective tissue growth factor (CTGF) is a novel fibroblast mitogen and also promotes extracellular matrix protein production. It is selectively induced by transforming growth factor beta (TGF-beta) and is thought to be the autocrine agent responsible for mediating its pro-fibrotic effects. CTGF is up-regulated during tissue repair and in fibrotic conditions associated with activation of the coagulation cascade. We therefore hypothesized that coagulation proteases promote the production of CTGF by cells at sites of tissue injury. To begin to address this hypothesis, we assessed the effect of coagulation proteases on fibroblast CTGF expression in vitro, and we show that thrombin, at physiological concentrations, up-regulated CTGF mRNA levels 5-fold relative to base line (p < 0.01) in fetal fibroblasts and 7-fold in primary adult fibroblasts (p < 0.01). These effects were cycloheximide-insensitive and were not blocked with a pan-specific TGF-beta-neutralizing antibody. They were further paralleled by a concomitant increase in CTGF protein production and could be mimicked with selective PAR-1 agonists. In addition, fibroblasts derived from PAR-1 knockout mice were unresponsive to thrombin but responded normally to TGF-beta(1). Finally, factor Xa, which is responsible for activating prothrombin during blood coagulation, exerted similar stimulatory effects. We propose that coagulation proteases and PAR-1 may play a role in promoting connective tissue formation during normal tissue repair and the development of fibrosis by up-regulating fibroblast CTGF expression.

Cadmium inhibits proteoglycan and procollagen production by cultured human lung fibroblasts.

Chronic inhalation of cadmium at the workplace or in cigarette smoke is associated with emphysema, a disease characterized by extensive disruption of lung connective tissue. We have previously shown that cadmium, at noncytotoxic doses, inhibits fibroblast procollagen production in vitro, with maximal inhibitory effects of 69 +/- 6% (P < 0.01) at 30 µM cadmium chloride (CdCl2). In this paper we show that at similar doses, cadmium also inhibits proteoglycan synthesis, with values reduced by between 36 +/- 4% (P < 0.01) and 42 +/- 6% (P < 0.01) for proteoglycans secreted into the culture media and associated with the cell layer, respectively. The greatest inhibition was obtained for the major matrix-associated proteoglycans, versican, decorin, and the large heparan sulfate proteoglycans, with synthesis values reduced by between 60 and 70%. Biglycan and other heparan sulfate proteoglycans were also affected, with synthesis values reduced by between 25 and 45%. In contrast, total protein synthesis was unaffected. Furthermore, effects of cadmium at the protein level were mirrored by reduction in messenger RNA levels for alpha1(I) procollagen, versican, and decorin. These data support the hypothesis that cadmium may play an important role in the pathogenesis of emphysema associated with chronic inhalation of cadmium fumes by inhibiting the production of connective tissue proteins.

Thrombin.

Thrombin is a multifunctional serine protease which plays a central role in haemostasis by regulating platelet aggregation and blood coagulation. It is formed from its precursor prothrombin following tissue injury and converts fibrinogen to fibrin in the final step of the clotting cascade. It also promotes numerous cellular effects including chemotaxis, proliferation, extracellular matrix turnover and release of cytokines. These actions of thrombin on cells have been implicated in tissue repair processes and in the pathogenesis of inflammatory and fibroproliferative disorders such as pulmonary fibrosis and atherosclerosis. Thrombin mediates its cellular effects by proteolytically activating cell surface receptors. Presently, two such receptors have been described and their roles in regulation of these functions are currently being investigated. The discovery of multiple thrombin receptors creates the possibility of selective receptor blockade of specific thrombin mediated events. New drugs with these actions should add to our current repertoire of thrombin inhibitors used to treat thrombotic diseases.

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.