Cellular Microenvironment Stiffness Regulates Eicosanoid Production and Signaling Pathways.

Pathological changes in the biomechanical environment are implicated in the progression of idiopathic pulmonary fibrosis (IPF). Stiffened matrix augments fibroblast proliferation and differentiation and activates TGF-ß1 (transforming growth factor-ß1). Stiffened matrix impairs the synthesis of the antifibrogenic lipid mediator prostaglandin E2 (PGE2) and reduces the expression of the rate-limiting prostanoid biosynthetic enzyme cyclooxygenase-2 (COX-2). We now show that prostaglandin E synthase (PTGES), the final enzyme in the PGE2 biosynthetic pathway, is expressed at lower levels in the lungs of patients with IPF. We also show substantial induction of COX-2, PTGES, prostaglandin E receptor 4 (EP4), and cytosolic phospholipase A2 (cPLA2) expression in human lung fibroblasts cultured in soft collagen hydrogels or in spheroids compared with conventional culture on stiff plastic culture plates. Induction of COX-2, cPLA2, and PTGES expression in spheroid cultures was moderately inhibited by the p38 mitogen-activated protein kinase inhibitor SB203580. The induction of prostanoid biosynthetic enzyme expression was accompanied by an increase in PGE2 levels only in non-IPF-derived fibroblast spheroids. Our study reveals an extensive dysregulation of prostanoid biosynthesis and signaling pathways in IPF-derived fibroblasts, which are only partially abrogated by culture in soft microenvironments.

Casein Kinase 1δ/ε Inhibitor, PF670462 Attenuates the Fibrogenic Effects of Transforming Growth Factor-ß in Pulmonary Fibrosis.

Transforming growth factor-beta (TGF-ß) is a major mediator of fibrotic diseases, including idiopathic pulmonary fibrosis (IPF). However, therapeutic global inhibition of TGF-ß is limited by unwanted immunosuppression and mitral valve defects. We performed an extensive literature search to uncover a little-known connection between TGF-ß signaling and casein kinase (CK) activity. We have examined the abundance of CK1 delta and epsilon (CK1δ/ε) in lung tissue from IPF patients and non-diseased controls, and investigated whether inhibition of CK1δ/ε with PF670462 inhibits pulmonary fibrosis. CK1δ/ε levels in lung tissue from IPF patients and non-diseased controls were assessed by immunohistochemistry. Anti-fibrotic effects of the CK1δ/ε inhibitor PF670462 were assessed in pre-clinical models, including acute and chronic bleomycin mouse models and in vitro experiments on spheroids made from primary human lung fibroblast cells from IPF and control donors, and human A549 alveolar-like adenocarcinoma-derived epithelial cells. Increased expression of CK1δ and ε in IPF lungs compared to non-diseased controls was accompanied by increased levels of the product, phospho-period 2. In vitro, PF670462 prevented TGF-ß-induced epithelial-mesenchymal transition. The stiffness of IPF-derived spheroids was reduced by PF670462 and TGF-ß-induced fibrogenic gene expression was inhibited. The CK1δ/ε inhibitor PF670462 administered systemically or locally by inhalation prevented both acute and chronic bleomycin-induced pulmonary fibrosis in mice. PF670462 administered in a 'therapeutic' regimen (day 7 onward) prevented bleomycin-induced lung collagen accumulation. Elevated expression and activity of CK1 δ and ε in IPF and anti-fibrogenic effects of the dual CK1δ/ε inhibitor, PF670462, support CK1δ/ε as novel therapeutic targets for IPF.

Annexin A2 contributes to lung injury and fibrosis by augmenting factor Xa fibrogenic activity.

In lung injury and disease, including idiopathic pulmonary fibrosis (IPF), extravascular factor X is converted into factor Xa (FXa), a coagulant protease with fibrogenic actions. Extracellular annexin A2 binds to FXa, augmenting activation of the protease-activated receptor-1 (PAR-1). In this study, the contribution of annexin A2 in lung injury and fibrosis was investigated. Annexin A2 immunoreactivity was observed in regions of fibrosis, including those associated with fibroblasts in lung tissue of IPF patients. Furthermore, annexin A2 was detected in the conditioned media and an EGTA membrane wash of human lung fibroblast (LF) cultures. Incubation with human plasma (5% vol/vol) or purified FXa (15-50 nM) evoked fibrogenic responses in LF cultures, with FXa increasing interleukin-6 (IL-6) production and cell number by 270 and 46%, respectively (P < 0.05, n = 5-8). The fibrogenic actions of plasma or FXa were attenuated by the selective FXa inhibitor apixaban (10 µM, or antibodies raised against annexin A2 or PAR-1 (2 µg/ml). FXa-stimulated LFs from IPF patients (n = 6) produced twice as much IL-6 as controls (n = 10) (P < 0.05), corresponding with increased levels of extracellular annexin A2. Annexin A2 gene deletion in mice reduced bleomycin-induced increases in bronchoalveolar lavage fluid (BALF) IL-6 levels and cell number (*P < 0.05; n = 4-12). Lung fibrogenic gene expression and dry weight were reduced by annexin A2 gene deletion, but lung levels of collagen were not. Our data suggest that annexin A2 contributes to lung injury and fibrotic disease by mediating the fibrogenic actions of FXa. Extracellular annexin A2 is a potential target for the treatment of IPF.

The Coagulant Factor Xa Induces Protease-Activated Receptor-1 and Annexin A2-Dependent Airway Smooth Muscle Cytokine Production and Cell Proliferation.

During asthma exacerbation, plasma circulating coagulant factor X (FX) enters the inflamed airways and is activated (FXa). FXa may have an important role in asthma, being involved in thrombin activation and an agonist of protease-activated receptor-1 (PAR-1). Extracellular annexin A2 and integrins are also implicated in PAR-1 signaling. In this study, the potential role of PAR-1 in mediating the effects of FXa on human airway smooth muscle (ASM) cell cytokine production and proliferation was investigated. FXa (5-50 nM), but not FX, stimulated increases in ASM IL-6 production and cell number after 24- and 48-hour incubation, respectively (P < 0.05; n = 5). FXa (15 nM) also stimulated increases in the levels of mRNA for cytokines (IL-6), cell cycle-related protein (cyclin D1), and proremodeling proteins (FGF-2, PDGF-B, CTGF, SM22, and PAI-1) after 3-hour incubation (P < 0.05; n = 4). The actions of FXa were insensitive to inhibition by hirudin (1 U/ml), a selective thrombin inhibitor, but were attenuated by SCH79797 (100 nM), a PAR-1 antagonist, or Cpd 22 (1 µM), an inhibitor of integrin-linked kinase. The selective targeting of PAR-1, annexin A2, or ß1-integrin by small interfering RNA and/or by functional blocking antibodies also attenuated FXa-evoked responses. In contrast, the targeting of annexin A2 did not inhibit thrombin-stimulated ASM function. In airway biopsies of patients with asthma, FXa and annexin A2 were detected in the ASM bundle by immunohistochemistry. These findings establish FXa as a potentially important asthma mediator, stimulating ASM function through actions requiring PAR-1 and annexin A2 and involving integrin coactivation.

A meta-analysis of reverse breech extraction to deliver a deeply impacted head during cesarean delivery.

BACKGROUND: During cesarean delivery, extracting a deeply impacted head is a real challenge for obstetricians. OBJECTIVES: To compare selected maternal and fetal outcome indicators of the "pull" (reverse breech extraction) and "push" methods for impacted fetal head extraction during cesarean delivery. SEARCH STRATEGY: A computer-based search of the Medline, Cochrane library, and HINARI databases. SELECTION CRITERIA: Studies that compared the maternal and perinatal outcomes of the push and pull methods for impacted fetal head extraction during cesarean delivery were included. DATA COLLECTION AND ANALYSIS: A meta-analysis of Mantel-Haenszel odds ratios and standardized mean differences from 11 randomized comparative and retrospective cohort studies was performed. MAIN RESULTS: In the pooled analysis, the risk of uterine incision extension was more than 8 times higher with the push method than with the pull method. Blood loss and operation time were also increased with the push method, and this method was associated with more perinatal deaths and admissions to the neonatal intensive care unit. The risk of wound infection was not significantly different between the 2 methods. CONCLUSIONS: The present meta-analysis demonstrated marked reductions in uterine incision extension, blood loss, and operation time with reverse breech extraction.