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The coagulation system is central to the pathophysiology of acute lung injury. We have previously demonstrated that the anticoagulant activated protein C (APC) prevents increased endothelial permeability in response to edemagenic agonists in endothefial cells and that this protection is dependent on the endothelial protein C receptor (EPCR). We currently investigate the effect of APC in a mouse model of ventilator-induced lung injury (VILI). C57BL/6J mice received spontaneous ventilation (control) or mechanical ventilation (MV) with high ([HV.sub.T]; 20 ml/kg) or low ([LV.sub.T]; 7 ml/kg) tidal volumes for 2 h and were pretreated with APC or vehicle via jugular vein 1 h before MV. In separate experiments, mice were ventilated for 4 h and received APC 30 and 150 min after starting MV. Indices of capillary leakage included bronchoalveolar lavage (BAL) total protein and Evans blue dye (EBD) assay. Changes in pulmonary EPCR protein and Rho-associated kinase (ROCK) were assessed using SDS-PAGE. Thrombin generation was measured via plasma thrombin-antithrombin complexes. HVT induced pulmonary capillary leakage, as evidenced by significant increases in BAL protein and EBD extravasation, without significantly increasing thrombin production. [HV.sub.T] also caused significant decreases in pulmonary, membrane-bound EPCR protein levels and increases in pulmonary ROCK-1. APC treatment significantly decreased pulmonary leakage induced by MV when given either before or after initiation of MV. Protection from capillary leakage was associated with restoration of EPCR protein expression and attenuation of ROCK-1 expression. In addition, mice overexpressing EPCR on the pulmonary endothelium were protected from [HV.sub.T]-mediated injury. Finally, gene microarray analysis demonstrated that APC significantly altered the expression of genes relevant to vascular permeability at the ontology (e.g., blood vessel development) and specific gene (e.g., MAPK-associated kinase 2 and integrin-[[beta].sub.6]) levels. These findings indicate that APC is barrier-protective in VILI and that EPCR is a critical participant in APC-mediated protection. endothelial protein C receptor; mechanical stress; vascular barrier function; ventilator-induced lung injury

To investigate the potential role of the local expression of alternative complement factor B (hBf) in human sepsis, we examined the induction of Bf gene expression in human peripheral blood monocytes (PBMCs) from patients with septic shock and the mechanisms of hBf gene regulation by tumor necrosis factor (TNF)-[alpha], interferon (IFN)-[gamma], and lipopolysaccharide (LPS) in human monocytes. PBMCs from septic shock patients showed increased hBf mRNA expression when compared with control patients. Costimulation with TNF-[alpha] and IFN-[gamma] or stimulation with LPS demonstrated a time-and dose-dependent induction of hBf mRNA expression in human PBMCs. A region of the hBf promoter between -735 and + 128 bp was found to mediate IFN-[gamma], TNF-[alpha], and LPS responsiveness as well as the synergistic effect of IFN-[gamma]/TNF-[alpha] on hBf promoter activity. Site-directed mutagenesis of a IFN-[gamma]-activation site (GAS) cis element (-90 to -82 bp) abrogated IFN-[gamma] responsiveness. Mutagenesis of a nuclear factor (NF)-[kappa]B cis element at -466 to -456 bp abrogated TNF-[alpha] and LPS responsiveness of the Bf promoter. Thus hBf gene expression is induced in PBMCs from septic shock patients, and the induction of hBf by IFN-[gamma] TNF-[alpha], and LPS is through GAS and NF-[kappa]B cis-binding sites on the hBf promoter. Furthermore, activated protein C (APC) inhibited LPS-stimulated hBf promoter activity and protein expression in human monocytes suggesting that the beneficial effect of APC therapy in sepsis may in part be due to inhibition of complement induction and/or activation via the alternative pathway. septic shock; monocytes/macrophages; complement factor B; cytokines

Crystalline silica has long been well established as a fibrogenic agent, and recent evidence has implicated it as a potential human carcinogen. However, the mechanisms of silica-induced disease development and progression are not well understood. Our previo us studies demonstrated that crystalline silica is able to activate activator protein-1 (AP-1) through mitogen-activated protein kinase (MAPK) pathways. The present study investigates the possible involvement of protein kinase C (PKC) in silica-induced activation of the MAPK/AP-1 signal transduction pathway. Treatment of mouse epidermal cells (JB6 cell line) with freshly fractured silica stimulated translocation of PKC[alpha] and PKC [member of] from the cytosol to the membrane and activated AP-1 transcription activity. Pretreatment of cells with PKC inhibitors, including RO-32-0432, calphostin C, and bisindolylmaleimide I, inhibited silica-induced AP-1 activation and phosphorylation of ERKs and p38 kinase. These inhibitory effects by PKC inhibitors were dose dependent. Furthermore, overexpression of dominant negative mutant (DNM) of PKC [alpha] or PKC [member of] markedly blocked AP-1 activation as well as phosphorylation of ERKs and p38 kinase induced by freshly fractured silica. These results demonstrate that PKC [alpha] and PKC [member of] are essential in silica-induced AP-1 activation through the MAP kinase (ERKs and p38 kinases) pathway. protein kinase C; mitogen-activated protein kinase; activator protein-1; silica

Mechanical forces regulate lung maturation in the fetus by promoting type II epithelial differentiation. However, the cell surface receptors that transduce these mechanical cues into cellular responses remain largely unknown. When distal lung type II epithelial cells isolated from embryonic day 19 rat fetuses were cultured on flexible plates coated with laminin, fibronectin, vitronectin, collagen, or elastin and exposed to a level of mechanical strain (5%) similar to that observed in utero, transmembrane signaling responses were induced under all conditions, as measured by ERK activation. However, mechanical stress maximally increased expression of the type II cell differentiation marker surfactant protein C when cells were cultured on laminin substrates. Strain-induced alveolar epithelial differentiation was inhibited by interfering with cell binding to laminin using soluble laminin peptides (IKVIV or YIGSR) or blocking antibodies against integrin [[beta].sub.1], [[alpha].sub3], or [[alpha].sub.6]. Additional studies were carried out with substrates coated directly with different nonactivating anti-integrin antibodies. Blocking integrin [[beta].sub.1] and [[alpha].sub.6] binding sites inhibited both cell adhesion and differentiation, whereas inhibition of [[alpha].sub.3] prevented differentiation without altering cell attachment. These data demonstrate that various integrins contribute to mechanical control of type II lung epithelial cell differentiation on laminin substrates. However, they may act via distinct mechanisms, including some that are independent of their cell anchoring role. laminin; surfactant protein C

Surfactant protein B (SP-B) is required for function of newborn and adult lung, and partial deficiency has been associated with susceptibility to lung injury. In the present study, transgenic mice were produced in which expression of SP-B in type II epithelial cells was conditionally regulated. Concentrations of SP-B were maintained at 60-70% of that normally present in control. Immunostaining for SP-B demonstrated cellular heterogeneity in expression of the protein. In subsets of type II cells in which SP-B staining was decreased, immunostaining for pro-SP-C was increased and lamellar body ultrastructure was disrupted, consistent with focal SP-B deficiency. Fluorescence-activated cell sorting analyses of freshly isolated type II cells identified a population of cells with low SP-B content and a smaller population with increased SP-B content. confirming nonuniform expression of the SP-B transgene. Focal air space enlargement, without cellular infiltration or inflammation, was observed. Pressure-volume curves indicated that maximal tidal volume was unchanged: however, hysteresis was modestly altered and residual volumes were significantly decreased in the SP-B-deficient mice. Chronic, nonuniform SP-B deficiency perturbed pulmonary function and caused air space enlargement. transgenic mice; surfactant protein B; surfactant protein C

A study was conducted to determine if recombinant human soluble thrombomodulin can stem the increase of rat pulmonary vascular permeability induced by endotoxin through protein C activation. Results show that recombinant human soluble thrombomodulin treated with monoclonal antibody F2H5 cannot inhibit endotoxin-induced coagulation. Results also indicate that endotoxin-induced intravascular coagulation is inhibited by recombinant human soluble thrombomodulin through thrombin binding.

Angiogenesis and morphogenesis of murine fetal distal lung in an allograft model. Am J Physiol Lung Cell Mol Physiol 278: L1000-L1007, 2000.--Neovascularization is crucial to lung morphogenesis; however, factors determining vessel growth and formation are poorly understood. The goal of our study was to develop an allograft model that would include maturation of the distal lung, thereby ultimately allowing us to study alveolar development, including microvascular formation. We transplanted 14-day gestational age embryonic mouse lung primordia subcutaneously into the back of nude mice for 3.5-14 days. Lung morphogenesis and neovascularization were evaluated by light microscopy, in situ hybridization, and immunohistochemical techniques. Embryonic 14-day gestational age control lungs had immature structural features consistent with pseudoglandular stage of lung development. In contrast, 14 days after subcutaneous transplantation of a 14-day gestational age lung, the allograft underwent significant structural morphogenesis and neovascularization. This was demonstrated by continued neovascularization and cellular differentiation, resulting in mature alveoli similar to those noted in the 2-day postnatal neonatal lung. Confirmation of maturation of the allograft was provided by progressive type II epithelial cell differentiation as evidenced by enhanced local expression of mRNA for surfactant protein C and a threefold (P [is less than] 0.008) increase in vessel formation as determined by immunocytochemical detection of platelet endothelial cell adhesion molecule-1 expression. Using the tyrosine kinase Flk-1 receptor (flk-1) LacZ transgene embryos, we determined that the neovascularization within the allograft was from the committed embryonic lung endothelium. Therefore, we have developed a defined murine allograft model that can be used to study distal lung development, including neovascularization. The model may be useful when used in conjunction with an altered genetic background (knockout or knock in) of the allograft and has the further decided advantage of bypassing placental barriers for introduction of pharmacological agents or DNA directly into the lung itself. pulmonary microvasculature; lung development; embryo; vasculogenesis; surfactant protein C; platelet endothelial cell adhesion molecule-1

Russo, Scott J., Wenjing Wang, Catherine A. Lomax, and Michael F. Beers. Structural requirements for intracellular targeting of SP-C proprotein. Am. J. Physiol. 277 (Lung Cell. Mol. Physiol. 21): L1034-L1044, 1999.--Rat surfactant protein (SP) C is synthesized as a 194-amino acid proprotein that is proteolytically processed to a 35-amino acid mature form in subcellular compartments distal to the medial Golgi compartment. To identify domains of SP-C proprotein (proSP-C) necessary for endoplasmic reticulum translocation and for targeting to cytosolic processing compartments, we characterized expression patterns of heterologous SP-C fusion proteins in A549 lung epithelial cells and in the rat pheochromocytoma cell line PC-12. cDNA constructs were produced; these constructs encoded fusion proteins consisting of enhanced green fluorescent protein (EGFP) and wild-type proSP-C (EGFP/[SP-C.sup.1-194]), mature SP-C (EGFP/[SP-C.sup.24-59]), or progressive deletions of the [NH.sub.2]- or COOH-terminal flanking domains. By fluorescence microscopy, EGFP/[SP-C.sup.1-194] transfected into A549 cells was translocated and expressed in acidic cytoplasmic vesicles. By deletional analysis, a functional signal peptide was mapped to the domain [Phe.sup.24] to [His.sup.59], whereas a motif for targeting to cytosolic vesicular compartments was localized to the [NH.sub.2] flanking domain [Met.sup.10] to [Gln.sup.23]. Truncations of the distal COOH terminus were retained in the endoplasmic reticulum/Golgi compartment; however, the COOH flanking region alone was insufficient for targeting. In PC-12 cells, EGFP/[SP-C.sup.1-194] was expressed in peripheral cytosolic vesicles, whereas EGFP/[SP-C.sup.24-194] and EGFP/[SP-C.sup.24-59] were each translocated but not targeted. We conclude that two domains in the proSP-C sequence are required for targeting: mature SP-C ([Phe.sup.24] to [Leu.sup.58]) contains a functional signal sequence active in epithelial and nonepithelial cells, whereas [Met.sup.10] to [Gln.sup.23], but not the COOH flanking peptide, is required for targeting to late vesicular compartments. surfactant protein C; propeptide targeting; A549 cells; green fluorescent protein