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65 The plasminogen system in pulmonary fibrosis
SESSION VII: Physiology and Disease
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66
THE PLASMINOGEN SYSTEM IN PULMONARY FIBROSIS.
REGULATION OF PLASMINOGEN ACTIVATORS AND MATRIX METALLOPROTEINASES DURING DEVELOPMENT AND REGRESSION OF THE CORPUS LUTEUM IN THE MOUSE
Carmell ~M. Swaisgood, Esther French, Victoria Ploplis. Center for Thrombosis and Vascular Biology, Department of Molecular Cardiology, The Cleveland Clinic Foundation, Cleveland, OH 44195.
Kui Liu, Anna-Carin Hiigglund and Tor Ny Department of Medical Biochemistry and Biophysics, University of Ume~, S901 87, Sweden
Acute and chronic pulmonarydiseases are characterized by impaired fibrinolytic activity within the lung. The fibrinolytie system consists of the zymogen plasminogen (Pig); tissue and urokinase plasminogen activators; plasminogen activator inttibitors, PAI-1 and PAI-2; plasmin, the enzyme responsible for the degradation of fibrin; and plasmin inhibitor, a 2 anti-plasmin. The development of transgenic mice deficient for components of the plasminogen system have indicated that fibrinolysis is compromised in plasminogen null animals. Therefore, we examined the effect of bleomycin, an agent that induces the development of pulmonary fibrosis, in mice deficient for plasminogen (Pig), tissue plasminogen activator (t-PA), and urokinase plasminogen activator (u-PA). Results indicate that Pig, t-PA, and u-PA deficient mice treated with 0.075 U of bleomycin increased the content of collagen in lung tissue relative to wild type, as assayed by hydroxyproline and observed in lung sections stained with Mason Trichrome, two weeks after intratracheal administration. This increase in collagen was due to bleomycin since saline-treated animals showed equivalent collagen levels between the different genotypes. Interestingly, all u-PA 4- (n=3), and two t-PA-t- (n=3) mice treated with bleomycin died before the completion of the experiment. On the other hand, saline treatment did not cause any adverse effect on mice survival. Lungs from u-PA v-and t-PA v-animals assayed post-mortem revealed an increase in collagen content (72 #g and 68 #g hydroxyproline/lung,respectively) relative to wild type (50 #g hydroxyproline/lu ng). The t-PA 4-mice treated with bleomycin that survived the two weeks showed a more striking increase in collagen content (135 #g hydroxyprolinedlung) relative to wild type. Recently it has been reported that bleomycin also induces an increase of collagen in the lung of transgenic mice overexpressing PAI-1. Since collagen content has been correlated to the severity of pulmonary fibrosis, we suggest that impaired expression of the components of the plasminogen system may contribute to the development and progression of this and other acute and chronic pulmonary diseases.
Proteolysis mediated by plasminogen activators (PAs) and matrix metalloproteinases (MMPs) has been associated with various physiological processes in many organs including the ovary. Afrer ovulation, the ruptured follicle converts to a corpus luteum (CL) in a process that involves active tissue remodeling and angiogenesis. The CL is a transient hormone secreting organ that produces progesterone which is required for the maintenance of pregnancy. If no implantation occurs, the CL undergoes regression during which it first loses the ability to produce progesterone and then is structurally degenerated. By using a pseudopregnant mouse modal, we have studied and localized the expression of pretenses and protease irdaibitors during different developmental stages of CL by Northern blot and in situ hybridization analysis. During formation of CL tPA, uPA and PAI-I as well as stromelysin-3, membrane type MMP 0VlT1-MMP) and tissue inhibitor of metalloproteinases 1 (TIMP-I) were expressed but with varying expression pattern. In contrast only uPA was expressed during the functional luteal phase. During the late luteal regression the expression oftPA, uPA, stromelysin-3, MT1-MMP and TIMP-1 increased again, which correlated with an increased apoptosis as determined by in situ 3'-end labeling. Our data demonstrate that pretenses and protease inhibitors are coordinately and temporally expressed during formation and regression of corpus luteum. The tumor related metailoproteinases strumdysin3 and MTI-MMP exhibit normal physiological expressions during this processes. The expression pattern indicate that uPA may play a role during the functional phase. Studies with gene deficient mice that are in progress will reveal the functional role of proteases during the developmental cycle of CL.
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68
PHYSIOLOGICAL CONSEQUENSES OF PLASMINOGEN DEFICIENCY ON FERTILITY AND OVULATION EFFICIENCY
IMPAIRMENT OF POST-LACTATIONAL INVOLUTION OF THE MAMMARY GLAND 1N MICE DEFICIENT FOR THE PLASMINOGEN GENE. S.F. Biorn I, L.R. Lurid, 1~ Bugge:, I.d. Christensen t, R. Osterby 3, d. Degen", Z Werb~ and I~ Dane 1. ~The Finsen Laboratory, DK-2100 Copenhagen; 2Children's Hosp. Res. Found., Cincinnatti, Ohio; 3Electron Microscopy Lab., Aarhus; 3Lab. Pad. and Env. Health, Univ. California, San Francisco.
IN¥, A., q.,eenardsson,G., IHfigghind,A-C., tHiigglrf, P., 2Ploplis,VA., 3Carmeliet,P and ~Ny,T. 1Departmentof Medical Biochemistryand Biophysics,Ume~ University,Umeh, Sweden. 2Center for Thrombosis Vascular Biology,The ClevelandClinic Fotmdation, Cleveland, Ohio, USA. 3Center for Transgene Technology and Gene therapy, Vlaams IntemniversitairInstituut veer Bioteelmologie,KU Leuven,Belgium. The plasminogen activator (PA) system together with the matrix metalloproteinases (MMPs) are thought to play an important role in the degradation of the fonieular wall at the time of ovulation. Consequently, the ovulation efficiency is reduced by 26% in a gnnadotropin induced ovulation model using mice lacking both tPA and UPA gene functions. To further investigate the involvement of the PA-system in the ovulatory process, we have studied the physiological consequences of plasminogen (pig) deficiency on the fertility and ovulation efficiency in adult normally cycling female mice. Our results indicate that fertility and physiological ovulation, measured as pups/litter and oocytes/mouse respectively, is similar for pig-deficient and wild-type mice. However, fewer pigdeficient mice ovulated or delivered pups compared with the wild-type controls. This is most likely due to the impaired health of the adult pig-deficient females. To avoid studying potentially sick mice, we used gonadotropin induced ovulation in young immature female mice as a model system. 25-day-old female mice were treated with pregnant mare's serum gonadotropin (PMSG) and human chorionie gonadotropin (hCG) to induce ovulation. The mice were killed about 10 h after ovulation and the number of oocytes in the oviduct was recorded. By using this model, we observed a 17% significant reduction (,o=0.023) in ovulation efficiency in the pig-deficient females. However, we also observed that the pig-deficient mice had significantly lower body weight than wild-type controls, which could partly be responsible for the difference in ovulation efficiency. Our results suggests that there are redundant mechanisms that generate the proteolytic activity required for follicular wall degradation and that plasmin, together with other pretenses, is required for normal ovulation Future studies using pretense deficient mice will clarify putative redundant mechanism between the PA- and MMP-systems.
We have studied the morphological characteristics of post-lactational mammary gland involution day five in wildtype (pig+/+, n=13), hetero- (pig+/-, n=9) and homozygntenus (pig-/-, n=13) pinsminogen deficient mice. Only animals that had succeded to nourish 7 pups for 10 days were studied thereby excluding about 28 % of the pig -/- mice. The glandular weights (rag) (means and SD: pig+/+ 109 _+ 37; pig+/- 179 _+ 26; pig-/- 217 _+ 52) were different among the groups (p< 0.0001), the increase detectable in pig+/- vs. pig+/+ mice (p=0.01). Morphometric analysis was carried out using standard stereolngical methods. The volume fi'actiuns (%) of lipid (median: pig+/+ 49.7; pig+/- 45.6; pig-/- 34.4) were different among all of the groups (p:=0.0002) and significantly reduced compared with the pig+/- vs. plg+/+ mice (p=0.023). Alveoli were regressed e x e r t for some areas of nun-collapsed alveoli in pig-/- mice, the volume fractions (median: plg+/+LS; pig+/- 4.3; pig-/-4.7) differed (p<0.0011) and was lower in the pig+/- vs. plg+/+ mice at the 0.01 level. Larger duets surrounded by connective tissue differed (median: pig+/+ 2.3; pig+/- 6.4; pig-/- 6.4) among the groups (p=0.0056) significant for pig+/- vs. pig+/+ (p= 0.0488). Finally the volume fractions of the lymph node differed (median: pig+/+3.2; pig+/-2.7; pig-/- 17.3) (p= 0.0015) with a dramatic increase in the homozygoteous mice: pig+/- vs. plg-/mice (p=0.0050). Total volume of alvenli (ram3) were different (median: pig+/+ 1.0; pig+/- 8.6; pig-/- 8.9) (p=0.0001), the increase observed comparing pig+/+ vs. pig+/- (p=0.0011). Likewise total volume of large duets (median: pig+/+ 3. I; pig+/- 10.9; pig-/- 14.8) differed among the groups (p=0.0005) and significantly so for pig+/- vs. pig+/+ (p=0.0194). Connective tissue and big vessels (median: pig+/+ 16.8; pig+/- 25.9; pig-/- 40.5) differed among the groups (p=0.008), with significant increase between pig+/+ vs. pig+/- (p=0.030). The lymph node increased in pig-/- vs. pig+/- (p=0.0326). The data demonstrate altered mammary gland involution in mice deficient for the plasminogen gene indicating a functional role of the plasminogen activation system during thns process.
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66
THE PLASMINOGEN SYSTEM IN PULMONARY FIBROSIS.
REGULATION OF PLASMINOGEN ACTIVATORS AND MATRIX METALLOPROTEINASES DURING DEVELOPMENT AND REGRESSION OF THE CORPUS LUTEUM IN THE MOUSE
Carmell ~M. Swaisgood, Esther French, Victoria Ploplis. Center for Thrombosis and Vascular Biology, Department of Molecular Cardiology, The Cleveland Clinic Foundation, Cleveland, OH 44195.
Kui Liu, Anna-Carin Hiigglund and Tor Ny Department of Medical Biochemistry and Biophysics, University of Ume~, S901 87, Sweden
Acute and chronic pulmonarydiseases are characterized by impaired fibrinolytic activity within the lung. The fibrinolytie system consists of the zymogen plasminogen (Pig); tissue and urokinase plasminogen activators; plasminogen activator inttibitors, PAI-1 and PAI-2; plasmin, the enzyme responsible for the degradation of fibrin; and plasmin inhibitor, a 2 anti-plasmin. The development of transgenic mice deficient for components of the plasminogen system have indicated that fibrinolysis is compromised in plasminogen null animals. Therefore, we examined the effect of bleomycin, an agent that induces the development of pulmonary fibrosis, in mice deficient for plasminogen (Pig), tissue plasminogen activator (t-PA), and urokinase plasminogen activator (u-PA). Results indicate that Pig, t-PA, and u-PA deficient mice treated with 0.075 U of bleomycin increased the content of collagen in lung tissue relative to wild type, as assayed by hydroxyproline and observed in lung sections stained with Mason Trichrome, two weeks after intratracheal administration. This increase in collagen was due to bleomycin since saline-treated animals showed equivalent collagen levels between the different genotypes. Interestingly, all u-PA 4- (n=3), and two t-PA-t- (n=3) mice treated with bleomycin died before the completion of the experiment. On the other hand, saline treatment did not cause any adverse effect on mice survival. Lungs from u-PA v-and t-PA v-animals assayed post-mortem revealed an increase in collagen content (72 #g and 68 #g hydroxyproline/lung,respectively) relative to wild type (50 #g hydroxyproline/lu ng). The t-PA 4-mice treated with bleomycin that survived the two weeks showed a more striking increase in collagen content (135 #g hydroxyprolinedlung) relative to wild type. Recently it has been reported that bleomycin also induces an increase of collagen in the lung of transgenic mice overexpressing PAI-1. Since collagen content has been correlated to the severity of pulmonary fibrosis, we suggest that impaired expression of the components of the plasminogen system may contribute to the development and progression of this and other acute and chronic pulmonary diseases.
Proteolysis mediated by plasminogen activators (PAs) and matrix metalloproteinases (MMPs) has been associated with various physiological processes in many organs including the ovary. Afrer ovulation, the ruptured follicle converts to a corpus luteum (CL) in a process that involves active tissue remodeling and angiogenesis. The CL is a transient hormone secreting organ that produces progesterone which is required for the maintenance of pregnancy. If no implantation occurs, the CL undergoes regression during which it first loses the ability to produce progesterone and then is structurally degenerated. By using a pseudopregnant mouse modal, we have studied and localized the expression of pretenses and protease irdaibitors during different developmental stages of CL by Northern blot and in situ hybridization analysis. During formation of CL tPA, uPA and PAI-I as well as stromelysin-3, membrane type MMP 0VlT1-MMP) and tissue inhibitor of metalloproteinases 1 (TIMP-I) were expressed but with varying expression pattern. In contrast only uPA was expressed during the functional luteal phase. During the late luteal regression the expression oftPA, uPA, stromelysin-3, MT1-MMP and TIMP-1 increased again, which correlated with an increased apoptosis as determined by in situ 3'-end labeling. Our data demonstrate that pretenses and protease inhibitors are coordinately and temporally expressed during formation and regression of corpus luteum. The tumor related metailoproteinases strumdysin3 and MTI-MMP exhibit normal physiological expressions during this processes. The expression pattern indicate that uPA may play a role during the functional phase. Studies with gene deficient mice that are in progress will reveal the functional role of proteases during the developmental cycle of CL.
67
68
PHYSIOLOGICAL CONSEQUENSES OF PLASMINOGEN DEFICIENCY ON FERTILITY AND OVULATION EFFICIENCY
IMPAIRMENT OF POST-LACTATIONAL INVOLUTION OF THE MAMMARY GLAND 1N MICE DEFICIENT FOR THE PLASMINOGEN GENE. S.F. Biorn I, L.R. Lurid, 1~ Bugge:, I.d. Christensen t, R. Osterby 3, d. Degen", Z Werb~ and I~ Dane 1. ~The Finsen Laboratory, DK-2100 Copenhagen; 2Children's Hosp. Res. Found., Cincinnatti, Ohio; 3Electron Microscopy Lab., Aarhus; 3Lab. Pad. and Env. Health, Univ. California, San Francisco.
IN¥, A., q.,eenardsson,G., IHfigghind,A-C., tHiigglrf, P., 2Ploplis,VA., 3Carmeliet,P and ~Ny,T. 1Departmentof Medical Biochemistryand Biophysics,Ume~ University,Umeh, Sweden. 2Center for Thrombosis Vascular Biology,The ClevelandClinic Fotmdation, Cleveland, Ohio, USA. 3Center for Transgene Technology and Gene therapy, Vlaams IntemniversitairInstituut veer Bioteelmologie,KU Leuven,Belgium. The plasminogen activator (PA) system together with the matrix metalloproteinases (MMPs) are thought to play an important role in the degradation of the fonieular wall at the time of ovulation. Consequently, the ovulation efficiency is reduced by 26% in a gnnadotropin induced ovulation model using mice lacking both tPA and UPA gene functions. To further investigate the involvement of the PA-system in the ovulatory process, we have studied the physiological consequences of plasminogen (pig) deficiency on the fertility and ovulation efficiency in adult normally cycling female mice. Our results indicate that fertility and physiological ovulation, measured as pups/litter and oocytes/mouse respectively, is similar for pig-deficient and wild-type mice. However, fewer pigdeficient mice ovulated or delivered pups compared with the wild-type controls. This is most likely due to the impaired health of the adult pig-deficient females. To avoid studying potentially sick mice, we used gonadotropin induced ovulation in young immature female mice as a model system. 25-day-old female mice were treated with pregnant mare's serum gonadotropin (PMSG) and human chorionie gonadotropin (hCG) to induce ovulation. The mice were killed about 10 h after ovulation and the number of oocytes in the oviduct was recorded. By using this model, we observed a 17% significant reduction (,o=0.023) in ovulation efficiency in the pig-deficient females. However, we also observed that the pig-deficient mice had significantly lower body weight than wild-type controls, which could partly be responsible for the difference in ovulation efficiency. Our results suggests that there are redundant mechanisms that generate the proteolytic activity required for follicular wall degradation and that plasmin, together with other pretenses, is required for normal ovulation Future studies using pretense deficient mice will clarify putative redundant mechanism between the PA- and MMP-systems.
We have studied the morphological characteristics of post-lactational mammary gland involution day five in wildtype (pig+/+, n=13), hetero- (pig+/-, n=9) and homozygntenus (pig-/-, n=13) pinsminogen deficient mice. Only animals that had succeded to nourish 7 pups for 10 days were studied thereby excluding about 28 % of the pig -/- mice. The glandular weights (rag) (means and SD: pig+/+ 109 _+ 37; pig+/- 179 _+ 26; pig-/- 217 _+ 52) were different among the groups (p< 0.0001), the increase detectable in pig+/- vs. pig+/+ mice (p=0.01). Morphometric analysis was carried out using standard stereolngical methods. The volume fi'actiuns (%) of lipid (median: pig+/+ 49.7; pig+/- 45.6; pig-/- 34.4) were different among all of the groups (p:=0.0002) and significantly reduced compared with the pig+/- vs. plg+/+ mice (p=0.023). Alveoli were regressed e x e r t for some areas of nun-collapsed alveoli in pig-/- mice, the volume fractions (median: plg+/+LS; pig+/- 4.3; pig-/-4.7) differed (p<0.0011) and was lower in the pig+/- vs. plg+/+ mice at the 0.01 level. Larger duets surrounded by connective tissue differed (median: pig+/+ 2.3; pig+/- 6.4; pig-/- 6.4) among the groups (p=0.0056) significant for pig+/- vs. pig+/+ (p= 0.0488). Finally the volume fractions of the lymph node differed (median: pig+/+3.2; pig+/-2.7; pig-/- 17.3) (p= 0.0015) with a dramatic increase in the homozygoteous mice: pig+/- vs. plg-/mice (p=0.0050). Total volume of alvenli (ram3) were different (median: pig+/+ 1.0; pig+/- 8.6; pig-/- 8.9) (p=0.0001), the increase observed comparing pig+/+ vs. pig+/- (p=0.0011). Likewise total volume of large duets (median: pig+/+ 3. I; pig+/- 10.9; pig-/- 14.8) differed among the groups (p=0.0005) and significantly so for pig+/- vs. pig+/+ (p=0.0194). Connective tissue and big vessels (median: pig+/+ 16.8; pig+/- 25.9; pig-/- 40.5) differed among the groups (p=0.008), with significant increase between pig+/+ vs. pig+/- (p=0.030). The lymph node increased in pig-/- vs. pig+/- (p=0.0326). The data demonstrate altered mammary gland involution in mice deficient for the plasminogen gene indicating a functional role of the plasminogen activation system during thns process.
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