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Hydroxylation of Collagen by Lungs of Rats Administered Bleomycin
Hydroxylation of Collagen by Lungs of Rats Administered Bleomycin* JA
l.DSt, Ph.D.; J E. Gerrletl, Ph.D.; L. G. Arrn.rtrong;
T. R. Gdzleichtc-; and K. M. HeUer; M.D.
I
ntratracheal instillation of a single dose of bleomycin sulfate into rats or hamsters produces acute pulmonary fibrosis. In this model, edema, inflammation, and cellular influx are followed by increased synthesis and deposition of lung collagen in a centriacinar distribution within 4 wk. The bleomycin model of acute fibrosis is especially useful for investigating alterations in collagen metabolism that occur prior to detectable deposition of excess lung collagen, such as increased rate of synthesis of collagen, increased synthesis of type I collagen relative to type III, and increased content of hydroxylysine and hydroxylysine-derived cross-links as compared with normal lung collagen. All of these early changes, which may be observed within 1 wk of bleomycin administration, parallel alterations in collagen synthesis and deposition observed in human lung fibrosis. ~ investigated the hydroxylation of lysine in newly synthesized lung collagen in an in mtro culture system prepared from rats administered bleomycin. Collagen synthesized by tissue minces from lungs of rats administered 1 unit of bleomycin by intratracheal instillation 1 or 2 wk earlier contained relatively more hydroxylysine than collagen made by lungs from saline-instilled control animals. Most if not all of the relative increase in lysine hydroxylation could be localized to the «1 chain of type I collagen. Lung homogenates from bleomycin-treated rats showed increased activity of lysyl hydroxylase (EC 1.14.11.4), the enzyme catalyzing the conversion of collagen-bound lysine to hydroxylysine. Thus, the increased hydroxylation of lysine and of lysinederived cross-links previously observed in collagen of diseased human lungs and in animal models of lung fibrosis is reflected in an in oUro system. This finding should further facilitate studies of the mechanisms underlying altered collagen metabolism in pulmonary fibrosis. •From the Department of Internal Medicine, School of Medicine, and the CalifOrnia Primate Research Center, University of California, Davis.
Molecular Interactions of Isolated Rat ~ II Pneumocytes with Components of Extracellular Matrices In vitro* Phalp L. Sannu, Ph.D.; &rry ~ Ph.D.;
and Kenneth B. Adler; Ph.D.
•From the [)epartment of Anatomy, Physiological Sciences, and
Radiology, CoDege of ~terinary Medicine, North Carolina State
University, Raleigh. Supported by US Public Health Service grants HL 36982, HL 37636, and CA 41359 and by grants from the North Carolina State University College of ~terinary Medicine and the Life Sciences Division of 3M Corporation, St Paul, MN. Dr Adler is an Established Investigator of the American Heart Association. &print ~m: Dr: Sannu, APR-NCSU CVM 4700 HUl.tborough SIYM, llakigh, NC 27606 70S
T
he type II pneumocyte of the pulmonary alveolus is a multifunctional cell capable not only of synthesizing and secreting the lipoproteins of surfactant, but also of acting as the progenitor cell of the alveolar epithelium. The mechanisms that control these critical processes are poorly understood, but likely involve interaction of insoluble extracellular matrices (ECM), principally of the basement membrane, and soluble factors, such as hormones and growth factors. The alveolar basement membrane is known to be composed of type IV collagen,' laminin,' and possibly fibronectin during inflammatory episodes.• Heparan sulfate proteoglycan3 and chondroitin sulfate proteoglycan• have recently been localized to this region by immunologic techniques; this localization is in agreement with earlier reportsu on the cytochemistry of its anionic sites. These sites were found to be asymmetrically arranged within the alveolar basement membrane, with the majority located in the lamina raraextema.u In addition, the anionic sites were quantitatively different between the basement membrane microdomains of type I and type II pneumocytes.• We hypothesized that these quantitative differences might reflect differences in composition of ECM molecules that could influence the phenotypic expression, proliferative capacity, and specific functions of type II cells in vivo. To address this problem, we exposed isolated rat type II cells to different ECM substrates and monitored their proliferative activity and biosynthesis of ECM components in vitro. MATERIALS AND METHODS
Type II cells were isolated from Fisher 344 VAF rats by the panning method of Dobbs et al,• seeded on culture slide chambers (Nunc, Napier, IL) or standard T·llasks, and cultured in Dulbecco's modified eagle medium containing 10% Fetal bovine serum at 37"C and 10% carbon dioxide. Chamber and Bask culture surfaces were coated with either fibronectin (Gibco, Grand Island, NY), laminin (Collaborative Research. Bedford, MA). type IV collagen (Mouse Collaborative Research, Bedford, MA), chondroitin sulfate (Sigma, St Louis), dermatan sulfate (Sigma, St Louis), or beparan sulfate (Sigma, St Louis) at 0.3 (high concentration) or 0.03 (low concentration) ~&'mm•. For proliFerative indices, cultures were pulsed with 10 11>M 5-bromo-2'-deoxyuridine (BrdU) at 24, 72, and 120 h In culture. Cultures were terminated at 48, 96, or 144 h and fixed with methanol. Cells pulsed 24 h with BrdU were treated lOr immunologic detection or BrdU according to the recommendations oF the supplier (Boehringer Mannheim, Indianapolis). Random microscopic fields were counted at 40 x , and the number or BrdUpositive cells was compared with the total number of type II cells counted to yield a proliFerative index. For matrix biosynthesis, cells were continuously labeled For 24 h with either 100 ~Ci/ml L·[>oS]-cysteine or 200 ~Ci/ml [""S)-sulfate after 72 h In culture. lmmunopreclpitation or ECM products was performed according to the method of Frenette et al,•• with the use of specl.6c polyclonal antibodies to laminin, type IV collagen, and fibronectin. Normal rabbit serum was used as a control. Products were separated oo 5% SDS polyacrylamide gels under standard reducing and non-reducing conditions and the radioactive bands were imaged on Kodalc x-ray film. RESULTS
After 48 h in culture, type II cells cultured on uncoated plastic dishes 'and pulsed 24 h with BrdU had a labeling index of3.5-4.5%. The labeling indices of cells cultured on laminin, high concentrations offibronectin, type IV collagen, 33n:l Annual Thomas L Petty Aspen Lung ~
l.DSt, Ph.D.; J E. Gerrletl, Ph.D.; L. G. Arrn.rtrong;
T. R. Gdzleichtc-; and K. M. HeUer; M.D.
I
ntratracheal instillation of a single dose of bleomycin sulfate into rats or hamsters produces acute pulmonary fibrosis. In this model, edema, inflammation, and cellular influx are followed by increased synthesis and deposition of lung collagen in a centriacinar distribution within 4 wk. The bleomycin model of acute fibrosis is especially useful for investigating alterations in collagen metabolism that occur prior to detectable deposition of excess lung collagen, such as increased rate of synthesis of collagen, increased synthesis of type I collagen relative to type III, and increased content of hydroxylysine and hydroxylysine-derived cross-links as compared with normal lung collagen. All of these early changes, which may be observed within 1 wk of bleomycin administration, parallel alterations in collagen synthesis and deposition observed in human lung fibrosis. ~ investigated the hydroxylation of lysine in newly synthesized lung collagen in an in mtro culture system prepared from rats administered bleomycin. Collagen synthesized by tissue minces from lungs of rats administered 1 unit of bleomycin by intratracheal instillation 1 or 2 wk earlier contained relatively more hydroxylysine than collagen made by lungs from saline-instilled control animals. Most if not all of the relative increase in lysine hydroxylation could be localized to the «1 chain of type I collagen. Lung homogenates from bleomycin-treated rats showed increased activity of lysyl hydroxylase (EC 1.14.11.4), the enzyme catalyzing the conversion of collagen-bound lysine to hydroxylysine. Thus, the increased hydroxylation of lysine and of lysinederived cross-links previously observed in collagen of diseased human lungs and in animal models of lung fibrosis is reflected in an in oUro system. This finding should further facilitate studies of the mechanisms underlying altered collagen metabolism in pulmonary fibrosis. •From the Department of Internal Medicine, School of Medicine, and the CalifOrnia Primate Research Center, University of California, Davis.
Molecular Interactions of Isolated Rat ~ II Pneumocytes with Components of Extracellular Matrices In vitro* Phalp L. Sannu, Ph.D.; &rry ~ Ph.D.;
and Kenneth B. Adler; Ph.D.
•From the [)epartment of Anatomy, Physiological Sciences, and
Radiology, CoDege of ~terinary Medicine, North Carolina State
University, Raleigh. Supported by US Public Health Service grants HL 36982, HL 37636, and CA 41359 and by grants from the North Carolina State University College of ~terinary Medicine and the Life Sciences Division of 3M Corporation, St Paul, MN. Dr Adler is an Established Investigator of the American Heart Association. &print ~m: Dr: Sannu, APR-NCSU CVM 4700 HUl.tborough SIYM, llakigh, NC 27606 70S
T
he type II pneumocyte of the pulmonary alveolus is a multifunctional cell capable not only of synthesizing and secreting the lipoproteins of surfactant, but also of acting as the progenitor cell of the alveolar epithelium. The mechanisms that control these critical processes are poorly understood, but likely involve interaction of insoluble extracellular matrices (ECM), principally of the basement membrane, and soluble factors, such as hormones and growth factors. The alveolar basement membrane is known to be composed of type IV collagen,' laminin,' and possibly fibronectin during inflammatory episodes.• Heparan sulfate proteoglycan3 and chondroitin sulfate proteoglycan• have recently been localized to this region by immunologic techniques; this localization is in agreement with earlier reportsu on the cytochemistry of its anionic sites. These sites were found to be asymmetrically arranged within the alveolar basement membrane, with the majority located in the lamina raraextema.u In addition, the anionic sites were quantitatively different between the basement membrane microdomains of type I and type II pneumocytes.• We hypothesized that these quantitative differences might reflect differences in composition of ECM molecules that could influence the phenotypic expression, proliferative capacity, and specific functions of type II cells in vivo. To address this problem, we exposed isolated rat type II cells to different ECM substrates and monitored their proliferative activity and biosynthesis of ECM components in vitro. MATERIALS AND METHODS
Type II cells were isolated from Fisher 344 VAF rats by the panning method of Dobbs et al,• seeded on culture slide chambers (Nunc, Napier, IL) or standard T·llasks, and cultured in Dulbecco's modified eagle medium containing 10% Fetal bovine serum at 37"C and 10% carbon dioxide. Chamber and Bask culture surfaces were coated with either fibronectin (Gibco, Grand Island, NY), laminin (Collaborative Research. Bedford, MA). type IV collagen (Mouse Collaborative Research, Bedford, MA), chondroitin sulfate (Sigma, St Louis), dermatan sulfate (Sigma, St Louis), or beparan sulfate (Sigma, St Louis) at 0.3 (high concentration) or 0.03 (low concentration) ~&'mm•. For proliFerative indices, cultures were pulsed with 10 11>M 5-bromo-2'-deoxyuridine (BrdU) at 24, 72, and 120 h In culture. Cultures were terminated at 48, 96, or 144 h and fixed with methanol. Cells pulsed 24 h with BrdU were treated lOr immunologic detection or BrdU according to the recommendations oF the supplier (Boehringer Mannheim, Indianapolis). Random microscopic fields were counted at 40 x , and the number or BrdUpositive cells was compared with the total number of type II cells counted to yield a proliFerative index. For matrix biosynthesis, cells were continuously labeled For 24 h with either 100 ~Ci/ml L·[>oS]-cysteine or 200 ~Ci/ml [""S)-sulfate after 72 h In culture. lmmunopreclpitation or ECM products was performed according to the method of Frenette et al,•• with the use of specl.6c polyclonal antibodies to laminin, type IV collagen, and fibronectin. Normal rabbit serum was used as a control. Products were separated oo 5% SDS polyacrylamide gels under standard reducing and non-reducing conditions and the radioactive bands were imaged on Kodalc x-ray film. RESULTS
After 48 h in culture, type II cells cultured on uncoated plastic dishes 'and pulsed 24 h with BrdU had a labeling index of3.5-4.5%. The labeling indices of cells cultured on laminin, high concentrations offibronectin, type IV collagen, 33n:l Annual Thomas L Petty Aspen Lung ~