Alveolar Epithelial Cells Express Both Plasminogen Activator and Tissue Factor

Alveolar Epithelial Cells Express Both Plasminogen Activator and Tissue Factor

3 Hirai KI, Witschi HP. Cote MC. Electron microscopy of butylated hydroxytoluene-induced lung damage. Exp Mol Patbol1978; 27:295-308 4 Adamson IYR, Bo...

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3 Hirai KI, Witschi HP. Cote MC. Electron microscopy of butylated hydroxytoluene-induced lung damage. Exp Mol Patbol1978; 27:295-308 4 Adamson IYR, Bowden DH, Cote MC, Witschi HP. Lung injury induced by butylated hydroxytoluene: cytodynamic and biochemical studies in mice. Lab Invest 1977; 36:26-32 5 Haschek WM. Witschi HP. Pulmonary fibrosis: a possible mechanism. Toxicol Appl Pbannacol1979; 51:475-87 5a Witschi HP, Hoschek WM, Klein-Szanto AJP, Holdc:inen PJ. Potentiation of diffuse lung damage by oxygen: determining variables. Am Rev Respir Dis 1981; 123:98-103 6 Haschek WM, Reiser KM , Klein-Szanto AJP, Kehrer JP. Smith LH, Last JA, et al. Potentiation of butylated bydroxytolueneinduced acute lung damage by oxygen: celllcinetics and collagen metaboUsm. Am Rev Respir Dis 1983; 127:28-34 7 Haschek WM, Meyer KR , Ullrich RL, Witschi HP. Potentiation of chemically induced lung fibrosis by thorax irradiation. lnt J Radiat Oncol Bioi Phys 1980; 6:449-55 8 Brody AR, Soler P, Basset F. Haschek WM, Wit.scbi HP. Epithelial-mesenchymal associations of cells in human pulmonary fibrosis and in BHT-oxygen-induced fibrosis in mice. Exp Lung Res 1981; 2:207-20 9 Halddnen PJ, Morse CC, Martin FM, Dalbey WE, Haschek WM, Witschi HP. Potentiating effects of oxygen in lungs damaged by methylcyclopentadienyl manganese tricarbonyl, cadmium chloride, oleic acid, and antitumor drugs. Toxicol Appl Pbannacol1983; 67:55-09 10 Cooper JAD Jr, White DA, Mathey RA . Drug-induced pulmonary disease. Part I. Cytotoxic drugs. Am Rev Respir Dis 1986; 133:321-40 11 Tryka AF, Skomlk WA, Codleslci JJ, Brain JD. Potentiation of bleomycin-induced lung injury by exposure to 70% oxygen. Am Rev Respir Dis 1982; 126:1074-79 12 Tryka AF, Codleslci Jj. Skomik WA , Brain JD. Progressive pulmonary fibrosis in hamsters. Exp Lung Res 1983; 5:155-70 13 Tryka AF, Wltschi HP, Lindenschmidt RC. Progressive pulmonary fibrosis in rats: abiochemical, celllcinetic, and morphologic analysis. Exp Mol Pathol 1985; 43:348-58 14 Halddnen PJ, Whiteley Jw. Witschi HP. Hyperoxia but not thoracic x-irradiation potentiates bleomycin· and cyclophosphamide-induced lung damage in mice. Am Rev Respir Dis 1982;

126:281-85 15 Halddnen PJ, Haschek WM. Pulmonary toxicity of methylcyclopentadienyl manganese tricarbonyl: noncillated bronchiolar epitheUal (Clara) cell necrosis and alveolar damage in the mouse, rat and hamster. Toxicol Appl Pharmacol1982; 65:11-22 16 Haschek WM, Boyd MR. Haldcinen PJ, Owenby C, Witschi HP. Acute inhalation toxicity of 3-methylfuran in the mouse: pathology, cell lcinetics, and respiratory rate effects. Toxicol Appl Pbannacol1984; 72:124-33 17 Kehrer JP, Klein-Szanto AJP. Thurston DE. Liodenschmidt RC , Witschl HP. QS,S,-trimethyl phosphorodithioate-induced lung damage in rats and mice. Toxicol Appl Pharmacol 1986; 84:48092 18 Lindenscbmidt RC, Tryka AF, Godfrey CA, Frome EL, Witschi HP. Intratracheal versus intravenous administration ofbleomycin in mice: acute effects. Toxiool Appl Pbannacol1986; 85:69-77 19 Martin FM, Witschi HP. Cadmium-induced lung injury: cell lcinetics and long-term effects. Toxiool Appl Pharmacol 1985; 80:215-27 20 Sendelbach LE, Witschi HP, Tryka AF. Acute pulmonary toxicity of beryllium sulfate inhalation in rats and mice: celllcinetics and histopathology. Toxicol Appl Pharmacol 1986; 85:248-56 21 Witschi HP, Godfrey C, Frome E, Lindenschmidt RC. Pulmonary toxicity of cytostatic drugs: cell lcinetics. Fundam Appl Toxicol1987; 8:253-62 22 Haschek WM, Klein-Szanto AJP, Last JA, Reiser KM, Witschi

HP. Long•term morphologic and biochemical features of exper· ime ntally induced lung fibrosis in the mouse. Lab Invest 1982;

46:43S-49 23 Morse CC, Sigler C. Lock S, Haldcinen PJ, Hascbek WM , Witschl HP. Pulmonary toxicity of cyclophosphamide: a 1 year study. Exp Mol Pathol1985; 42:2SHO 24 Travis EL, Bucci L, Fang MZ. Residual damage in mouse lungs at long intervals after cyclophosphamide treatment. Cancer Res 1990; 50:2139-45 25 Sendelbach LE, Tryka AF, Witschi HP. Progressive lung injury over a one-year period after a single inhalation exposure to berylllum sulfate. Am Rev Respir Dis 1989; 139:1003-00 26 Terzaghi M, Nettesheim P, Williams ML. Repopulation of denuded tracheal grafts with normal, preneoplastic and neoplastic epithelial cell populations. Cancer Res 1978; 38:4546-53 27 Adamson I YR. Young L, Bowden DH. Relationship of alveolar epitheUal injury and repair to the induction of pulmonary fibrosis. Am J Pathol1988; 130:377-83 28 Reiser KM , Last JA. Early cellular events in pulmonary fibrosis. Exp Lung Res 1986; 1X:331·55 29 Kelley J. Cytolcines of the lung. Am Rev Respir Dis 1990; 141:765-88 30 Hirai KJ, Yamauchi M, Witsc.hi HP, Cote MC. Disintegration of lung peroxisomes during differentiation of type II cells to type I cells in butylated hydroxytoluene-administered mice. Exp Mol Pathol1983; 39:129-38 31 Lanir A, Karem D , Gershon D. Pulmonary oxygen toxicity: lack of tolerance in mice of various ages. Biochem Biophys Res Commun 1981; 101:1193-99 32 Mansour H , Brun-Pascaud M, Coogerot-Pocidalo MA, Pocidalo JJ. Protection of normobaric oxygen toxicity of the lung by inducers of cytochrome 1450 linked mono-oxygenases. CR Acad Sci Paris 1986; 302:247-49 33 Mansour H , Levacher M, Awulay-Dupuis E, Moreau J, Marquetty C, Coogerot-Pocidalo MA. Genetic differences in response to pulmonary cytochrome 1450 inducers and oxygen toxicity. JAppl Pbysiol1988; 64:1376-80 34 Baker RR, Holm BA. Panus PC, Matalon S. Development of oxygen tolerance in rabbits with no increase in antioxidant enzymes. J Appl Physiol1989; 66:1679-84 35 Margaretten N, Tryka AF, Witschi HP. Oxygen tolerance in mice following exposure to butylated bydroxytoluene. Toxicol Appl Pharmacol1988; 96:147-58 36 Schuller HM, Becker KL, Witschi HP. An animal model for neuroendocrine lung cancer. Carcinogenesis 1988; 9:293-96 37 Schuller HM, Witschl HP, Nylen E, Joshi PA, Correa A, Becker KL. Pathobiology of lung tumors induced in hamsters by 4(methylnitrosamino)-1-{3-pyridyl)-1-butanone and the modulating effect ofhyperoxia. Cancer Res 1990; 50:196(}.65

Alveolar Epithelial Cells Express Both Plasminogen Activator and Tissue Factor* Potential Role In Repair of Lung Injury Bruce C. Marsholl, M.D.; Bl1lnt R. Brown, M .D.; Mark A Rothstein, M.D.; N. V. &o, M.D.; john R. HoldtU, M. D.; and George M. Rodgers, M.D ., Ph .D.

•From the Pulmonary Division, Department of Medicine, University of Utah Health Sciences Center, Salt Lake City. Work was supported in part by a Veterans Administration Associat.e lnvestisator Award and a NIH Clinical Investigator Award (B.C.M.) and a ~terans Administration Research Advisory Crant and Merit Review Crant (C. M.R.). Reprint 1'1lqtutlt&: Dr: Marsholl, Pulmonary Division, 4F240, Unlverlity of Utah, Salt lAke City 84132 CHEST I 99 I 3 I MARCH, 1991 I Supplement

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he formation of fibrin is a part of the normal tissue repair process. Fibrin not only serves as a hemostatic barrier and limiting factor in the exudative process, but also provides a matrix for the tissue repair that follows injury. The plasmin/plasminogen activator system is a key component in the fibrinolysis that accompanies tissue repair. 1 The adult respiratory distress syndrome (ARDS) is a common medical problem associated with a high mortality. lntraalveolar fibrin deposition is a prominent feature of the early phase of this syndrome.• Recent studies suggest that the procoagulantlfibrinolytic balance is altered in the early phase of ARDS such that fibrin deposition is favored. 3•4 The extensive remodeling process that occurs in survivors leads to the eventual return to near-normal lung anatomy and physiologic function in most individuals.u The specific cell types that contribute to the procoagulantl fibrinolytic balance in the alveolar space are not known. The alveolar macrophage has been recognized as a potentially relevant cell in that it has the capacity to express procoagulants, plasminogen activator, and plasminogen activator inhibitors! Since alveolar epithelial cells play a central role in the repair process following acute lung injury, we hypothesized that they might also be a source of procoagulant and/ or fibrinolytic components. To test this hypothesis we studied rat alveolar epithelial cells in vitro, isolated by enzyme dissociation followed by differential adherence to IgG-coated plates.' We recently reporte
Table 1-AWeol.ar Epithelial CeU Procoagulont Actioity• Pmooagulant Activity (mU!..,.g Protein)

Source of Clotting Factors

729 817 40

Pooled normal plasma Factor VIII-deficient plasma Factor VII-deficient plasma Factor X-deficient plasma

<30

•Alveolar epithelial celllysates were incubated with either pooled normal plasma or factor-deficient plasma and CaCJ. and reCalcification clotting times were measured. Clotting times we re con· verted to tissue factor activity, with data expressed per j.Lg prote in. Each value represents the mean of 3 assays.

cal distribution of tissue factor in normal human tissues suggests that it serves as a protective "envelope" ready to initiate coagulation if vascular integrity is compromised . In the lung, alveolar epithelial cells were one site with significant staining for the tissue factor antigen. 13 This observation, coupled with the finding of substantial tissue factor activity in these cells, suggests that tissue factor is a physiologically relevant product of the alveolar epithelium. The regulation of tissue factor by intravascular cells such as monocytes and endothelial cells has received considerable attention.•us However, little is known about regulation of epithelial cell tissue factor activity. We observed that PMA treatment induces downregulation of alveolar epithelial cell tissue factor activity (Fig 1). Current studies are aimed at elucidating the mechanisms of this regulation. We speculate that downregulation of epithelial cell tissue factor activity may be an important component of the repair process in that it alters the procoagulantlfibrinolytic balance to favor

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Cell Lyaate FIGURE 1. Alveolar epithelial cell tissue factor activity: downregu· lation by PMA. After 3 d in culture, confluent monolayers we re washed with PBS and incubated with serum-free medium. After 24 h the serum-free medium was replaced with medium containing 0 or 50 ng/ml of PMA. The cell lysates were harvested in Trisbuffered saline 24 h later and assayed for tissue factor activity by recalcification assay (mean± SE, n 4).

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33rd Annuat lhomas L Petty Aspen Lung Conference

fibrin resorption. The alveolar epithelium should be considered an important contributor to alveolar fibrin deposition and subsequent resorption that typifies acute lung injury. Alveolar epithelial ceUs express key components of both the coagulation and fibrinolytic cascade: tissue factor and u-PA, respectively. Further, with respect to PMA, these components appear to be coordinately regulated. Further investigation of the modulation of these alveolar epithelial cell products may provide new insights into the alveolar cell response to acute lung injury and suggest potential strategies to favorably manipulate this response. REFERENCES

1 Saksela 0 , Riflcin DB. Cell associated plasminogen activation: regulation and physiological functions. Annu Rev Cell Biol1988; 4:93-126 2 Pratt PC, Vollmer RT, Shelburne JD, Crapo JD. Pulmonary morphology in a multihospital collaborative extracorporeal membrane oxygenation project. Am J Pathol1979; 95:191-214 3 Idell S, James KK, Levin EG, Schwartz BS, Manchandra N, Maunder RJ, et al. Local abnormalities in coagulation and fibrinolytic pathways predispose to alveolar fibrin deposition in the adult respiratory distress syndrome. J Clin Invest 1989; 84:695-705 4 Bertozzi P. Astedt B. Zenzius L, Lynch K, LeMaire F, Zapol W, Chapman HA. Depressed bronchoalveolar urokinase activity in patients with adult respiratory distress syndrome. N Engl J Med 1990; 322:89().97 5 Elliott CG, Morris AH, Cengiz ZM . Pulmonary function and exercise gas exchange in survivors or adult respiratory distress syndrome. Am Rev Respir Dis 1981; 123:492-95 6 Alberts WM , Priest CR, Moser KM. Outlook for survivors of ARDS. Chest 1983:84:272-74 7 Chapman HA, Bertozzi P. Reilly JJ. Role of enzymes mediating thrombosis and thrombolysis in lung disease. Chest 1988; 93:1256-63 8 Dobbs LC, Gonzalez R, Williams MC. An improved method for isolating type 2 cells in high yield and purity. Am Rev Respir Dis 1986; 134:141-45 9 Marshall BC, Sageser DS, Rao NV. Emi M, Hoidal JR. Alveolar epithelial cell plasminogen activator: characterization and regulation. J Bioi Chern 1990; 265:8198-204 10 Surprenant YM, Zuckerman SH. Novel microliter assay for the quantitation of procoagulant activity on adherent monocytes, macrophages, and endothelial cells. Thromb Res 1989; 53:33946

11 Nemerson Y. Tissue factor and hemostasis. Blood 1988; 71:1-8 12 Dvorak HF, Senger DR, Dvorak AM, Harvey VS , McDonagh J. Regulation or extravascular coagulation by microvascular permeability. Science 1985; 227:1059-61 13 Drake TA, Morrissey JH, Edgington TS. Selective cellular expression of tissue factor in human tissues. Am J Pathol 1989; 134:1087-97 14 Schwartz BS, Bradshaw JD. Differential regulation of tissue factor and plasminogen activator inhibitor by human mononuclear cells. Blood 1989; 74:1644-50 15 Scarpati EM, Sadler JE. Regulation of endothelial cell coagulant properties, modulation of tissue factor, plasminogen activator inhibitors, and thrombomodulin by PMA and tumor necrosis factor. J Bioi Chern 1989; 264:20705-13

Hypertrophic Alveolar "TYpe II Cells Isolated after Silica-Induced Lung Injury Are Progressing Through the Cell Cycle and Maintain a Commitment to DNA Synthesis in Primary Culture* Ralph) lbnos. M.D.; and Robert J M~Uon, M.D.

,l cute lung injury frequency results in damage to type I

1"1 cells and disruption of the alveolar epithelial lining. Epithelial repair occurs when alveolar type II cells proliferate and differentiate into type I cells, restoring the integrity of the alveolar epithelium. Alveolar type II cell hyperplasia and hypertrophy occur after silica-induced lung injury and are a common response in the course of alveolar epithelial repair. We performed cell cycle analysis of elutriated type II cells isolated after silica instillation in the rat to determine if hypertrophic type II cells were progressing through the cell cycle and were proliferating cells. Alveolar type II cells were isolated from rats 14 d after intratracheal instillation of silica (100 mglkg) and separated into groups of increasing cell size by centrifugal elutriation. The cells were fixed in 70% ethanol and incubated with propidium iodide and RNase prior to analysis on an Epics ftow cytometer. The percentage of cells in the GVM (proliferative) phase of the cell cycle increased with increasing type II cell size: 3.3 ± 0. 7% in the smallest cells and 24.5±5.2% in the largest cells. To determine the proliferative potential of silica type II cells in primary culture and to correlate alveolar type II cell size with the level of in vitro DNA synthesis, we studied DNA synthesis in type II cells isolated after silica instillation. Alveolar type II cells were isolated from rats 1, 2, 3, and 4 wk after intratracheal instillation of silica (100 mglkg), cultured in Dulbecco's modified eagle medium supplemented with 10% fetal bovine serum, and labeled with tritiated thymidine from d 1 to d 3in culture. DNA synthesis was determined by ('H) thymidine incorporation and autoradiographic labeling index. The level of thymidine incorporation increased progressively from 22.3 ± 5.4 X 1()1 dpml well 7 d after silica instillation to 34.4 ± 5.0x1()l dpm!weU at 28 d . The plating efficiency and type II cell purity were the same at all time points after silica instillation. To determine if there were differences in the proliferative potential of normotrophic and hypertrophic silica type II cells, type II cells isolated 14 d after silica instillation were separated into groups of increasing cell size by centrifugal elutriation. The plating efficiency and alveolar type II cell purity (<88%) were the same in all groups ofelutriated cells. The hypertrophic type II cells had a higher level of thymidine incorporation (22.0 ± 2.8 x 1()1 dpmlwell) than the normotrophic type II cells (11.1±0.7 X 1()1 dpmlwell, p<0.01). Each group of elutriated type II cells was capable of responding to the same level ofstimulated DNA synthesis in the presence of insulin, epidermal growth factor, and cholera toxin. The autoradiographic labeling index increased *From the National Jewish Center, University of Colorado Health Sciences Center, Denver. CHEST I 99 I 3 I MARCH, 1991 I Supplement

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