Meprin ß contributes to collagen deposition in lung fibrosis.

Lung fibrosis is a severe disease characterized by epithelial cell injury, inflammation and collagen deposition. The metalloproteases meprinα and meprinß have been shown to enhance collagen maturation and inflammatory cell infiltration via cleavage of cell-cell contact molecules; therefore we hypothesized that meprins could play a role in lung fibrosis. An exhaustive characterization of bleomycin-treated meprinα, meprinß and the double meprinsαß knock-out (KO) with respective wt-littermates was performed by using several different methods. We observed no difference in lung function parameters and no change in inflammatory cells infiltrating the lung between wt and all meprins KO mice after 14 days bleomycin. No difference in epithelial integrity as assessed by e-cadherin protein level was detected in bleomycin-treated lungs. However, morphological analysis in the bleomycin-treated mice revealed decrease collagen deposition and tissue density in meprinß KO, but not in meprinα and meprinαß KO mice. This finding was accompanied by localization of meprinß to epithelial cells in regions with immature collagen in mice. Similarly, in human IPF lungs meprinß was mostly localized in epithelium. These findings suggest that local environment triggers meprinß expression to support collagen maturation. In conclusion, our data demonstrate the in vivo relevance of meprinß in collagen deposition in lung fibrosis.

The low density lipoprotein receptor-related protein (LRP) 1 and its function in lung diseases.

The low density lipoprotein receptor-related protein (LRP) 1 is a ubiquitously expressed, versatile cell surface transmembrane receptor involved in embryonic development and adult tissue homeostasis. LRP1 binds and endocytoses a broad spectrum of over 40 ligands identified thus far, including lipoproteins, extracellular matrix proteins, proteases and protease/inhibitor complexes and growth factors. Interactions with other membrane receptors and intracellular adaptors/scaffolding proteins allow LRP1 to modulate cell migration, survival, proliferation and (trans) differentiation. Because LRP1 displays a wide-range of interactions and activities, its expression and function is temporally and spatially tightly controlled. It is not, therefore, surprising that deregulation of LRP1 production and/or activity is observed in several diseases. In this review, we will systematically examine the evidence for the role of LRP1 in human pathologies placing special emphasis on LRP1-mediated pathogenesis of the lung.

Maggot excretion products from the blowfly Lucilia sericata contain contact phase/intrinsic pathway-like proteases with procoagulant functions.

For centuries, maggots have been used for the treatment of wounds by a variety of ancient cultures, as part of their traditional medicine. With increasing appearance of antimicrobial resistance and in association with diabetic ulcers, maggot therapy was revisited in the 1980s. Three mechanisms by which sterile maggots of the green bottle fly Lucilia sericata may improve healing of chronic wounds have been proposed: Biosurgical debridement, disinfecting properties, and stimulation of the wound healing process. However, the influence of maggot excretion products (MEP) on blood coagulation as part of the wound healing process has not been studied in detail. Here, we demonstrate that specific MEP-derived serine proteases from Lucilia sericata induce clotting of human plasma and whole blood, particularly by activating contact phase proteins factor XII and kininogen as well as factor IX, thereby providing kallikrein-bypassing and factor XIa-like activities, both in plasma and in isolated systems. In plasma samples deficient in contact phase proteins, MEP restored full clotting activity, whereas in plasma deficient in either factor VII, IX, X or II no effect was seen. The observed procoagulant/intrinsic pathway-like activity was mediated by (chymo-) trypsin-like proteases in total MEP, which were significantly blocked by C1-esterase inhibitor or other contact phase-specific protease inhibitors. No significant influence of MEP on platelet activation or fibrinolysis was noted. Together, MEP provides contact phase bypassing procoagulant activity and thereby induces blood clotting in the context of wound healing. Further characterisation of the active serine protease(s) may offer new perspectives for biosurgical treatment of chronic wounds.

Hypoxia- or PDGF-BB-dependent paxillin tyrosine phosphorylation in pulmonary hypertension is reversed by HIF-1α depletion or imatinib treatment.

Chronic exposure to hypoxia induces a pronounced remodelling of the pulmonary vasculature leading to pulmonary hypertension (PH). The remodelling process also entails increased proliferation and decreased apoptosis of pulmonary arterial smooth muscle cells (PASMC), processes regulated by the cytoskeletal protein paxillin. In this study, we aimed to examine the molecular mechanisms leading to deregulation of paxillin in PH. We detected a time-dependent increase in paxillin tyrosine 31 (Y31) and 118 (Y118) phosphorylation following hypoxic exposure (1 % O2) or platelet-derived growth factor (PDGF)-BB stimulation of primary human PASMC. In addition, both, hypoxia- and PDGF-BB increased the nuclear localisation of phospho-paxillin Y31 as indicated by immunofluorescence staining in human PASMC. Elevated paxillin tyrosine phosphorylation in human PASMC was attenuated by hypoxia-inducible factor (HIF)-1α depletion or by treatment with the PDGF-BB receptor antagonist, imatinib. Moreover, we observed elevated paxillin Y31 and Y118 phosphorylation in the pulmonary vasculature of chronic hypoxic mice (21 days, 10 % O2) which was reversible by imatinib-treatment. PDGF-BB-dependent PASMC proliferation was regulated via the paxillin-Erk1/2-cyclin D1 pathway. In conclusion, we suggest paxillin up-regulation and phosphorylation as an important mechanism of vascular remodelling underlying pulmonary hypertension.

Characterization of dendritic cells in testicular draining lymph nodes in a rat model of experimental autoimmune orchitis.

The maturation state of dendritic cells (DC) is regarded as a control point for the induction of peripheral tolerance or autoimmunity. Experimental autoimmune orchitis (EAO) serves as a model to investigate inflammatory-based testicular impairment, which ranks as a significant cause of male infertility. This work aimed to determine whether DC enrichment occurs organotypically in testicular draining lymph nodes (TLN) compared with LN draining the site of immunization (ILN) and thus contributes to the pathogenesis of autoimmune orchitis. In this regard, we quantified and characterized the DC from TLN and ILN in rats with EAO. Flow cytometric analysis showed a significant increase in the percentage of DC (OX62+) only in TLN from EAO rats compared with normal (N) and adjuvant control (C) groups. The number of DC from ILN and TLN expressing CD80, CD86 and major histocompatibility complex (MHC) II was comparable among N, C and experimental (E) groups at 30 and 50 days after the first immunization. However, TLN DC from EAO rats (50 days) showed an increase in mean fluorescence intensity for MHC II compared with N, C and E groups (30 days). The mRNA expression level of IL-10 and IL-12p35 was significantly upregulated in enriched DC fraction from TLN in EAO rats with no significant changes observed in ILN DC. The expression of IL-23p19 mRNA remained unchanged. Functional data, using proliferation assays showed that EAO-DC from TLN, but not from ILN, significantly enhanced the proliferation of naïve T cells compared with C-DC. In summary, our data suggest that the DC in TLN from orchitis rats are mature, present antigens to T cells and stimulate an autoimmune response against testicular antigens, thus causing immunological infertility.

Role of coagulation and fibrinolysis in lung and renal fibrosis.

Elevated procoagulant and suppressed fibrinolytic activities are regularly encountered in different forms of clinical and experimental fibrosis of the lungs and the kidneys. Although primarily serving to provide a provisional matrix of repair largely consisting of fibrin and fibronectin, the involved procoagulant serine proteases and protease inhibitors may also exert distinct cellular downstream signaling events modifying the fibrotic response. In this review, evidence for an impaired regulation of coagulation and fibrinolysis factors in clinical and experimental lung and renal fibrosis is provided and the role of PAR (protease activated receptor) induced profibrotic and HGF (hepatocyte growth factor) elicited antifibrotic cellular events is worked out. In view of experiments obtained in animal models of lung and renal fibrosis, the potential therapeutic usefulness of anticoagulant or profibrinolytic strategies is discussed.

Cellular origin of pro-coagulant and (anti)-fibrinolytic factors in bleomycin-injured lungs.

Excessive pro-coagulant and decreased fibrinolytic activities in the alveolar compartment have been repeatedly documented for inflammatory and fibrotic lung diseases. The current authors determined the contribution of different resident lung cells to the altered local production of coagulation- and fibrinolysis-system components in bleomycin-injured mouse lungs via cell-specific and quantitative assessment of mRNA levels of various pro-coagulant and (anti)-fibrinolytic factors. Laser-assisted microdissection technology was used to sample specific cell populations in combination with subsequent mRNA analysis by real-time quantitative reverse transcriptase-PCR. Additionally, western blot analysis, immunohistochemistry and activity assays were performed. Following bleomycin challenge, the strongest induction of tissue factor and plasminogen activator inhibitor (PAI)-1 mRNA expression was observed in alveolar macrophages (approximately 250- and 60-fold induction, respectively). These factors were also upregulated in alveolar type II cells, but to an approximately six-fold lesser extent. In contrast, PAI-2 expression was induced exclusively in alveolar macrophages. A slight increase of urokinase-type plasminogen activator (uPA) expression was also observed in alveolar macrophages (two-fold induction), but uPA activity was reduced due to a disproportionate increase of PAI production. Alveolar macrophages and, to a lesser extent, alveolar type II cells are the main sources of locally produced pro-coagulant and anti-fibrinolytic factors in bleomycin-injured lungs.

Surfactant protein C mutations in sporadic forms of idiopathic interstitial pneumonias.

Interstitial pneumonias have recently been associated with mutations in the gene encoding surfactant protein C (SFTPC). In particular, SFTPC mutations have been reported in a number of familial forms of pulmonary fibrosis and in infants with interstitial lung diseases. The present study searched for SFTPC mutations in adult patients with sporadic idiopathic interstitial pneumonia. In total, 35 adult patients with sporadic idiopathic interstitial pneumonia and 50 healthy subjects were investigated for SFTPC mutations by direct DNA sequencing. Of the patients with sporadic idiopathic interstitial pneumonia, 25 suffered from idiopathic pulmonary fibrosis and 10 patients from nonspecific interstitial pneumonia. Only two frequent nonsynonymous variants, T138N and S186N, were detected. Allele frequencies of both variations as well as of other identified noncoding alterations did not differ significantly between the diverse patient groups and control subjects. In conclusion, mutations in the gene encoding surfactant protein C are not common in sporadic cases of idiopathic pulmonary fibrosis and nonspecific interstitial pneumonia, suggesting that the mutated gene does not play an important role in the pathogenesis of these forms of idiopathic interstitial pneumonia.

Pro- and antifibrinolytic properties of human pulmonary microvascular versus artery endothelial cells: impact of endotoxin and tumor necrosis factor-alpha.

OBJECTIVE: Microvascular thrombosis is a common feature of acute inflammatory lung injury, as occurs in sepsis and acute respiratory distress syndrome, but the underlying pathomechanisms are presently not fully understood. DESIGN: Experimental. SETTING: University laboratory. SUBJECTS: Lung endothelial cells. INTERVENTIONS: We characterized the expression of tissue-type and urokinase-type plasminogen activator (t-PA and u-PA) as well as plasminogen activator inhibitor (PAI)-1 and PAI-2 in human endothelial cells (EC) from the microvascular pulmonary circulation (HMVEC-L) and compared it with that of EC from pulmonary artery (HPAEC) and umbilical vein (HUVEC) under baseline conditions and upon stimulation with either tumor necrosis factor-alpha or lipopolysaccharide. MEASUREMENTS AND MAIN RESULTS: Real-time reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay were employed for quantification of messenger RNA and protein concentrations. Under baseline conditions, comparable PAI-1 expression was noted in all EC. HPAEC were characterized by significantly higher baseline expression of t-PA and PAI-2 compared with HUVEC and HMVEC-L. In contrast, u-PA messenger RNA concentrations were found to be significantly higher in nonstimulated HMVEC-L compared with HUVEC and HPAEC. In all EC, stimulation with tumor necrosis factor-alpha and lipopolysaccharide increased the expression of PAI-1, PAI-2, and u-PA and decreased t-PA expression. The changes in messenger RNA content were reflected by corresponding changes in the protein concentrations. CONCLUSIONS: High baseline u-PA expression is a prominent feature of human lung microvascular EC, whereas pulmonary artery EC are characterized by high t-PA concentrations. Microbial and inflammatory challenge provokes up-regulation of PAI-1 and PAI-2 and down-regulation of t-PA in both macro- and microvascular pulmonary EC, which may favor local fibrin deposition.