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Sepsis is a systemic inflammatory response that requires effective macrophage metabolic functions to resolve ongoing inflammation. Previous work showed that the mechanosensitive cation channel, transient receptor potential vanilloid 4 (TRPV4), mediates macrophage phagocytosis and cytokine production in response to lung infection. Here, we show that TRPV4 regulates glycolysis in a stiffness-dependent manner by augmenting macrophage glucose uptake by GLUT1. In addition, TRPV4 is required for LPS-induced phagolysosome maturation in a GLUT1-dependent manner. In a cecal slurry mouse model of sepsis, TRPV4 regulates sepsis-induced glycolysis as measured by BAL fluid (BALF) lactate and sepsis-induced lung injury as measured by BALF total protein and lung compliance. TRPV4 is necessary for bacterial clearance in the peritoneum to limit sepsis-induced lung injury. It is interesting that BALF lactate is increased in patients with sepsis compared with healthy control participants, supporting the relevance of lung cell glycolysis to human sepsis. These data show that macrophage TRPV4 is required for glucose uptake through GLUT1 for effective phagolysosome maturation to limit sepsis-induced lung injury. Our work presents TRPV4 as a potential target to protect the lung from injury in sepsis. [ABSTRACT FROM AUTHOR]

The urokinase-type plasminogen activator receptor (uPAR) is a glycosylphosphatidylinositol-linked membrane protein with no cytosolic domain that localizes to lipid raft microdomains. Our laboratory and others have documented that lung fibroblasts from patients with idiopathic pulmonary fibrosis (IPF) exhibit a hypermotile phenotype. This study was undertaken to elucidate the molecular mechanism whereby uPAR ligation with its cognate ligand, urokinase, induces a motile phenotype in human lung fibroblasts. We found that uPAR ligation with the urokinase receptor binding domain (amino-terminal fragment) leads to enhanced migration of fibroblasts on fibronectin in a protease-independent, lipid raft-dependent manner. Ligation of uPAR with the amino-terminal fragment recruited α5β1 integrin and the acylated form of the Src family kinase, Fyn, to lipid rafts. The biological consequences of this translocation were an increase in fibroblast motility and a switch of the integrin-initiated signal pathway for migration away from the lipid raft-independent focal adhesion kinase pathway and toward a lipid raft-dependent caveolin-Fyn-Shc pathway. Furthermore, an integrin homologous peptide as well as an antibody that competes with β1 for uPAR binding have the ability to block this effect. In addition, its relative insensitivity to cholesterol depletion suggests that the interactions of α5β1 integrin and uPAR drive the translocation of α5β1 integrin-acylated Fyn signaling complexes into lipid rafts upon uPAR ligation through protein-protein interactions. This signal switch is a novel pathway leading to the hypermotile phenotype of IPF patient-derived fibroblasts, seen with uPAR ligation. This uPAR dependent, fibrotic matrix-selective, and profibrotic fibroblast phenotype may be amenable to targeted therapeutics designed to ameliorate IPF.

Idiopathic pulmonary fibrosis (IPF) is a fatal fibrotic lung disorder with no effective medical treatments available. The generation of myofibroblasts, which are critical for fibrogenesis, requires both a mechanical signal and activated TGF-β; however, it is not clear how fibroblasts sense and transmit the mechanical signal(s) that promote differentiation into myofibroblasts. As transient receptor potential vanilloid 4 (TRPV4) channels are activated in response to changes in plasma membrane stretch/matrix stiffness, we investigated whether TRPV4 contributes to generation of myofibroblasts and/or experimental lung fibrosis. We determined that TRPV4 activity is upregulated in lung fibroblasts derived from patients with IPF. Moreover, TRPV4-deficient mice were protected from fibrosis. Furthermore, genetic ablation or pharmacological inhibition of TRPV4 function abrogated myofibroblast differentiation, which was restored by TRPV4 reintroduction. TRPV4 channel activity was elevated when cells were plated on matrices of increasing stiffness or on fibrotic lung tissue, and matrix stiffness-dependent myofibroblast differentiation was reduced in response to TRVP4 inhibition. TRPV4 activity modulated TGF-β1-dependent actions in a SMAD-independent manner, enhanced actomyosin remodeling, and increased nuclear translocation of the α-SMA transcription coactivator (MRTF-A). Together, these data indicate that TRPV4 activity mediates pulmonary fibrogenesis and suggest that manipulation of TRPV4 channel activity has potential as a therapeutic approach for fibrotic diseases., Introduction Idiopathic pulmonary fibrosis (IPF) is a scarring disorder of the lungs without any effective medical treatment available. As such, the roughly 100,000 patients in the USA with IPF have [...]

Fibroblasts from patients with pulmonary fibrosis express higher levels of the receptor for urokinase, and the extent of fibrosis in some animal models exhibits a dependence on the urokinase receptor. Recent observations have identified the urokinase receptor as a trans-interacting receptor with consequences on signaling and cell responses that vary depending on its interacting partner, the relative levels of expression, and the state of cellular transformation. We undertook this study to define the urokinase-type plasminogen activator cellular receptor (u-PAR)-integrin interactions and to determine the functional consequences of such interactions on normal human lung fibroblast attachment and migration. u-PAR colocalizes in lammelipodia/filopodia with relevant integrins that mediate fibroblast attachment and spreading on the provisional matrix proteins vitronectin, fibronectin, and collagens. Inhibitory antibody studies have revealed that human lung fibroblasts utilize [[alpha].sub.v][[beta].sub.5] to attach to vitronectin, predominantly [[alpha].sub.5][[beta].sub.1] (and [[alpha].sub.v][[beta].sub.3]) to attach to fibronectin, and [[alpha].sub.1][[beta].sub.1], [[alpha].sub.2][[beta].sub.1], and [[alpha].sub.3][[beta].sub.1] to attach to collagen. Blocking studies with [alpha]-integrin subunit decoy peptides and u-PAR neutralizing antibodies indicate that u-PAR modulates the integrin-mediated attachment to purified provisional matrix proteins, to anti-integrin antibodies, or to fibroproliferative lesions from fibrotic lungs. Furthermore, these decoy peptides blunt fibroblast spreading and migration. We show that u-PAR can interact with multiple [alpha]-integrins but with a preference for [[alpha].sub.3]. Taken together, these data demonstrate that u-PAR may interact with multiple integrins in normal human lung fibroblasts thereby promoting attachment, spreading, and migration. Modulation of fibroblast invasion would be expected to lead to amelioration of fibroproliferative diseases of the lung. pulmonary; fibrosis; plasmin

A double-blind, randomized, placebo-controlled trial was conducted to determine the effects of oral cyclophosphamide on lung function and health-related symptoms in patients with evidence of active alveolitis and scleroderma-related interstitial lung disease. It was found that one year of oral cyclophosphamide in patients with symptomatic scleroderma-related interstitial lung disease had a significant but modest beneficial effect on lung function, dyspnea, thickening of the skin, and the health-related quality of life.

Transforming growth factor (TGF)-[beta] plays an important role in tissue fibrogenesis. We previously demonstrated that reduced glutathione (GSH) supplementation blocked collagen accumulation induced by TGF-[beta] in NIH-3T3 cells. In the present study, we show that supplementation of GSH restores the collagen degradation rate in TGF-[beta]-treated NIH-3T3 cells. Restoration of collagen degradation by GSH is associated with a reduction of type I plasminogen activator inhibitor (PAI)-I expression/activity as well as recovery of the activities of cell/extracellular matrix-associated tissue-type plasminogen activator and plasmin. Furthermore, we find that NIH-3T3 cells constitutively express plasminogen mRNA and possess plasmin activity. Blockade of cell surface binding of plasminogen/plasminogen activation with tranexamic acid (TXA) or inhibition of plasmin activity with aprotinin significantly reduces the basal level of collagen degradation both in the presence or absence of exogenous plasminogen. Most importantly, addition of TXA or active PAI-1 almost completely eliminates the restorative effects of GSH on collagen degradation in TGF-[beta] treated cells. Together, our results suggest that the major mechanism by which GSH restores collagen degradation in TGF-[beta]-treated cells is through blocking PAI-1 expression, leading to increased PA/plasmin activity and consequent proteolytic degradation of collagens. This study provides mechanistic evidence for GSH's putative therapeutic effect in the treatment of fibrotic disorders. transforming growth factor-[beta]; plasminogen activation

Transforming growth factor-[beta] (TGF-[beta]) is a potent fibrogenic cytokine. The molecular mechanism underlying TGF-[beta] fibrogenesis, however, has not been completely elucidated. In this study, we showed that TGF[beta] decreased the intracellular GSH content in murine embryo fibroblasts (NIH 3T3), which was followed by an increase in collagen I mRNA content and collagen protein production. Prevention of GSH depletion with N-acetylcysteine (NAC), GSH, or GSH ester abrogated TGF-[beta]-stimulated collagen production, whereas a decrease in intracellular GSH content with L-buthionine-S,R-sulfoximine, an inhibitor of de novo GSH synthesis, enhanced TGF-[beta]-stimulated collagen production. These results suggest that GSH depletion induced by TGF-[beta] may mediate TGF-[beta]-stimulated collagen production. In addition, we showed that TGF-[beta] stimulated superoxide production and increased release of [H.sub.2][O.sub.2] from the cells, whereas GSH ester decreased basal and TGF-[beta] + glucose oxidase-stimulated [H.sub.2][O.sub.2] release. H202, exogenously added or continuously generated by glucose oxidase, enhanced TGF-[beta]-stimulated collagen production, whereas suppression of superoxide production by diphenyliodonium, an NAD(P)H oxidase inhibitor, blocked TGF-[beta]-stimulated collagen production. These data further suggest that reactive oxygen species are involved in TGF-[beta]-stimulated collagen production and that the effect of GSH depletion on TGF-[beta]-stimulated collagen production may be mediated by facilitating reactive oxygen species signaling. glutathione ester; [[sup.3]H]proline incorporation; collagen I mRNA; reactive oxygen species

The alveolar fibrinolytic system is altered in acute lung injury (ALI). Levels of the fibrinolytic protease inhibitor, plasminogen activator inhibitor-1 (PAI-1), are too low in bronchoalveolar lavage to address its prognostic significance. This study was performed to assess whether PAI-1 antigen in undiluted pulmonary edema fluid levels can identify patients with ALI and predict their outcome. PAI-1 antigen levels in both plasma and edema fluid were higher in ALI compared with hydrostatic edema, and edema fluid PAI-1 values identified those with ALI with high sensitivity and specificity. Both the high plasma and edema fluid PAI-1 antigen values were associated with a higher mortality rate and fewer days of unassisted ventilation in patients with ALI. Differences in PAI-1 activity were concordant with levels of PAI-1 antigen. Although the fibrin-derived alveolar D-dimer levels were strikingly similar in both groups, ALI patients had a higher relative proportion of D-monomer. In conclusion, PAI-1 levels in edema fluid and plasma identify those with ALI that have a poor prognosis. The data indicate that fibrin turnover in early ALI is a consequence of a rapid fibrinogen influx and fractional fibrinolytic inhibition. flbrinolysis; prognosis

Methoxyflurane-anesthesized mice with bleomycim-induced lung injury were analyzed to determine the biological activity of fibrinogen in fibroproliferative reactions. Biochemical analysis of bleomycin-induced acute alveolitis indicated the accumulation of fibrin in fibroproliferative lesions. Fibrin accumulation in these regions indicate that the heme protein is cross-linked with fibrils that colocalize with lesional collagens.