Robin J. McAnulty

Common Promoter Variant in Cyclooxygenase-2 Represses Gene Expression Evidence of Role in Acute-Phase Inflammatory Response

Objective—Cyclooxygenase (COX)-2 is a key regulatory enzyme in the synthesis of prostanoids associated with trauma and inflammation. We investigated the COX-2 gene for functional variants that may influence susceptibility to disease. Methods and Results—The promoter of COX-2 was screened for variants in healthy subjects by use of polymerase chain reaction-based methods. Promoter activity was investigated by using reporter expression experiments in human lung fibroblasts. Patients undergoing coronary artery bypass graft surgery, with measurements of plasma markers linked to COX-2 activity, were genotyped for association studies. A common COX-2 promoter variant, −765G>C, was found and shown to be carried by >25% of a group of healthy UK subjects. The −765C allele had significantly lower promoter activity compared with −765G, basally (28±3% lower, P <0.005) and in serum-stimulated cells (31±2% lower, P <0.005). In patients subjected to coronary artery bypass graft surgery, the magnitude of rise in levels of C-reactive protein (CRP) was strongly genotype dependent. Compared with −765G homozygotes, patients carrying the −765C allele had significantly lower plasma CRP levels at 1 to 4 days after surgery (14% lower at the peak of CRP levels on day 3, P <0.05 for all time points). Conclusions—For several acute and chronic inflammatory diseases, −765G>C may influence the variability of response observed.

Cyclooxygenase-2 Deficiency Results in a Loss of the Anti-Proliferative Response to Transforming Growth Factor-β 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.

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 1 (I) procollagen and CTGF mRNA levels (3.0 ± 0.4-fold and 6.3 ± 0.4-fold respectively, ( P P 1 (I) procollagen and CTGF mRNA levels by 50% and 35%, respectively ( P

Lysophosphatidic Acid Induces αvβ6 Integrin-Mediated TGF-β Activation via the LPA2 Receptor and the Small G Protein Gαq

Activation of latent transforming growth factor beta (TGF-beta) by alphavbeta6 integrin is critical in the pathogenesis of lung injury and fibrosis. We have previously demonstrated that the stimulation of protease activated receptor 1 promotes alphavbeta6 integrin-mediated TGF-beta activation via RhoA, which is known to modulate cell contraction. However, whether other G protein-coupled receptors can also induce alphavbeta6 integrin-mediated TGF-beta activation is unknown; in addition, the alphavbeta6 integrin signaling pathway has not yet been fully characterized. In this study, we show that lysophosphatidic acid (LPA) induces alphavbeta6-mediated TGF-beta activation in human epithelial cells via both RhoA and Rho kinase. Furthermore, we demonstrate that LPA-induced alphavbeta6 integrin-mediated TGF-beta activity is mediated via the LPA2 receptor, which signals via G alpha(q). Finally, we show that the expression levels of both the LPA2 receptor and alphavbeta6 integrin are up-regulated and are spatially and temporally associated following bleomycin-induced lung injury. Furthermore, both the LPA2 receptor and alphavbeta6 integrin are up-regulated in the overlying epithelial areas of fibrosis in patients with usual interstitial pneumonia. These studies demonstrate that LPA induces alphavbeta6 integrin-mediated TGF-beta activation in epithelial cells via LPA2, G alpha(q), RhoA, and Rho kinase, and that this pathway might be clinically relevant to the development of lung injury and fibrosis.

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.

A simplified method for quantitation of the relative amounts of type I and type III collagen in small tissue samples

Abstract A method is described for the quantitation of the relative amounts of types I and III collagens in rabbit lung tissue. This involved (i) repeated homogenization in the presence of 2% sodium dodecyl sulfate and the production of an acetone dried powder, (ii) reaction with cyanogen bromide, (iii) polyacrylamide gel electrophoresis, and (iv) densitometric scanning of proteins stained by Coomassie blue R-250. Several features of this procedure were shown to offer advantages over methods previously employed. First, the sodium dodecyl sulfate solution was shown to remove the bulk of noncollagen proteins leaving an insoluble residue which could then be reacted with cyanogen bromide without further purification. Second, cyanogen bromide was shown to solubilize essentially all of the collagen in the residue leaving an insoluble pellet with an amino acid analysis similar to elastin. Finally, to facilitate accurate quantitation, types I and III collagen standards were included with each gel so that a standard curve of protein versus staining density could be constructed. This method is assessed to be simpler and more accurate than those employed previously for the quantitation of collagens and can be applied to small tissue samples (

Diminished Prostaglandin E2 Contributes to the Apoptosis Paradox in Idiopathic Pulmonary Fibrosis

RATIONALE Patients with idiopathic pulmonary fibrosis (IPF), a progressive disease with a dismal prognosis, exhibit an unexplained disparity of increased alveolar epithelial cell (AEC) apoptosis but reduced fibroblast apoptosis. OBJECTIVES To examine whether the failure of patients with IPF to up-regulate cyclooxygenase (COX)-2, and thus the antifibrotic mediator prostaglandin (PG)E(2), accounts for this imbalance. METHODS Fibroblasts and primary type II AECs were isolated from control and fibrotic human lung tissue. The effects of COX-2 inhibition and exogenous PGE(2) on fibroblast and AEC sensitivity to Fas ligand (FasL)-induced apoptosis were assessed. MEASUREMENTS AND MAIN RESULTS IPF lung fibroblasts are resistant to FasL-induced apoptosis compared with control lung fibroblasts. Inhibition of COX-2 in control lung fibroblasts resulted in an apoptosis-resistant phenotype. Administration of PGE(2) almost doubled the rate of FasL-induced apoptosis in fibrotic lung fibroblasts compared with FasL alone. Conversely, in primary fibrotic lung type II AECs, PGE(2) protected against FasL-induced apoptosis. In human control and, to a greater extent, fibrotic lung fibroblasts, PGE(2) inhibits the phosphorylation of Akt, suggesting that regulation of this prosurvival protein kinase is an important mechanism by which PGE(2) modulates cellular apoptotic responses. CONCLUSIONS The observation that PGE(2) deficiency results in increased AEC but reduced fibroblast sensitivity to apoptosis provides a novel pathogenic insight into the mechanisms driving persistent fibroproliferation in IPF.

Severity of Lung Injury in Cyclooxygenase-2- Deficient Mice Is Dependent on Reduced Prostaglandin E 2 Production

Levels of prostaglandin E(2) (PGE(2)), a potent inhibitor of fibroblast function, are decreased in the lungs of patients with pulmonary fibrosis, which has been shown to be because of limited expression of cyclooxygenase-2 (COX-2). To further investigate the relative importance of COX-2 and PGE(2) in the development of fibrosis we have used a selective COX-2 inhibitor and COX-2-deficient ((-/-) and (+/-)) mice in studies of bleomycin-induced lung fibrosis. We demonstrate in wild-type mice that bleomycin-induced lung PGE(2) production is predominantly COX-2 mediated. Furthermore, COX-2(+/-) mice show limited induction of PGE(2) and an enhanced fibrotic response with increased lung collagen content compared with wild-type mice after bleomycin injury (P < 0.001). In contrast, COX-2(-/-) mice show increased levels of lung PGE(2), compared with wild-type mice after injury (P < 0.05), because of compensatory up-regulation of COX-1, which appears to be associated with macrophage/monocytes but not fibroblasts derived from these mice. COX-2(-/-) mice show an enhanced and persistent inflammatory response to bleomycin, however the fibrotic response to injury was unaltered compared with wild-type animals. These data provide further direct evidence for the importance of up-regulating COX-2 and PGE(2) expression in protecting against the development of fibrosis after lung injury.

The pathogenesis of pulmonary fibrosis: Is there a fibrosis gene?

Interstitial fibrosis is seen in the lung in response to a variety of insults, and often appears stereotypical in terms of its clinical and pathological features. However, exposure to a known aetiological factor does not always lead to fibrosis. For example in bleomycin-induced pulmonary fibrosis, a wide variation in response is seen both in humans and in animal models, which is not completely accounted for by known risk factors. These observations and the existence of a number of familial forms of lung fibrosis suggest a genetic predisposition. Current hypotheses concerning the pathogenesis of pulmonary fibrosis propose an initial stage involving the influx of inflammatory cells into the interstitium. These cells, together with activated resident cells are then thought to release polypeptide mediators that stimulate the fibroblast proliferation and matrix protein synthesis typical of these disorders. Genetic influences could have an important role in regulating a number of these events, altering the immunological response to injury or modulating collagen metabolism in the lung. However, despite recent advances in molecular genetic techniques, there have been few human studies to date. Most have concentrated on genetic loci with a high degree of polymorphism such as the human leucocyte antigen (HLA) system and yield conflicting results. Others offer tantalising but as yet, incomplete insights into the mechanisms involved. Defining the genetic abnormalities underlying both the familial forms of pulmonary fibrosis and the variations seen in response to lung injury should enhance our understanding of the pathogenic processes and help to focus research in this area.

Fibroblasts Isolated from Normal Lungs and Those with Idiopathic Pulmonary Fibrosis Differ in Interleukin-6/gp130-Mediated Cell Signaling and Proliferation

Interleukin (IL)-6 and IL-11 are elevated in a variety of lung conditions and may impact on repair mechanisms in chronic inflammatory disorders. However, the mechanisms by which these cytokines influence fibroblast proliferation in normal and disease states have not been previously addressed. We examined the effect of these cytokines on proliferation and cell-cycle kinetics of primary human lung fibroblasts obtained from normal patients and patients with idiopathic pulmonary fibrosis (IPF). IL-6 inhibited the proliferation of normal fibroblasts due to the sustained phosphorylation of STAT-3 and production of the cyclin-dependent kinase inhibitor p19(INK4D). In contrast IL-6 was mitogenic for IPF fibroblasts due to the sustained activation of MAPK, which in turn inhibited the production of p27(Kip1), allowing activation of cyclin D(1) and hyperphosphorylation of retinoblastoma protein. IL-11 was mitogenic for both normal and IPF fibroblasts. These results provide strong evidence for a fundamental abnormality in a cytokine-signaling pathway, as opposed to alterations in cytokine production, in the pathogenesis of IPF.