Plasminogen Activators Direct Reorganization of the Liver Lobule after Acute Injury
2001; Elsevier BV; Volume: 158; Issue: 3 Linguagem: Inglês
10.1016/s0002-9440(10)64039-4
ISSN1525-2191
AutoresJorge A. Bezerra, Angela R. Currier, Héctor Melín‐Aldana, Gregg Sabla, Thomas Bugge, Keith W. Kombrinck, Jay L. Degen,
Tópico(s)Trace Elements in Health
ResumoTissue repair requires an adequate cellular proliferation coordinated with the timely proteolysis of matrix elements. Based on the properties of plasminogen activators in liver cell proliferation and tissue proteolysis, we explored the regulatory role of tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA) in liver repair. Using carbon tetrachloride (CCl4) intoxication as a model of acute liver injury, we found that tPA-deficient mice displayed a mild defect in hepatic repair, whereas livers of uPA-deficient mice had a more substantial delay in repair, with injury of centrilobular hepatocytes persisting up to 14 days after CCl4. Notably, functional cooperativity between plasminogen activators was strongly inferred from the profound reparative defect in livers of mice lacking tPA and uPA simultaneously, with persistence of centrilobular injury as far out as 35 days. The defective repair was not because of increased susceptibility of experimental mice to the toxin or to inadequate cellular proliferation. Instead, lack of plasminogen activators led to the accumulation of fibrin and fibronectin within injured areas and poor removal of necrotic cells. These data demonstrate that tPA and uPA play a critical role in hepatic repair via proteolysis of matrix elements and clearance of cellular debris from the field of injury. Tissue repair requires an adequate cellular proliferation coordinated with the timely proteolysis of matrix elements. Based on the properties of plasminogen activators in liver cell proliferation and tissue proteolysis, we explored the regulatory role of tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA) in liver repair. Using carbon tetrachloride (CCl4) intoxication as a model of acute liver injury, we found that tPA-deficient mice displayed a mild defect in hepatic repair, whereas livers of uPA-deficient mice had a more substantial delay in repair, with injury of centrilobular hepatocytes persisting up to 14 days after CCl4. Notably, functional cooperativity between plasminogen activators was strongly inferred from the profound reparative defect in livers of mice lacking tPA and uPA simultaneously, with persistence of centrilobular injury as far out as 35 days. The defective repair was not because of increased susceptibility of experimental mice to the toxin or to inadequate cellular proliferation. Instead, lack of plasminogen activators led to the accumulation of fibrin and fibronectin within injured areas and poor removal of necrotic cells. These data demonstrate that tPA and uPA play a critical role in hepatic repair via proteolysis of matrix elements and clearance of cellular debris from the field of injury. Cellular proliferation and reorganization of the extracellular matrix are central features of tissue repair and remodeling of the liver and extra-hepatic systems. After an acute injury, liver cells undergo well-synchronized rounds of proliferation that are terminated when the original liver mass is restored.1Higgins GM Anderson RM Experimental pathology of the liver. I. Restoration of the liver of the white rat following partial surgical removal.Arch Pathol. 1931; 12: 126-202Google Scholar, 2Grisham JW A morphologic study of deoxyribonucleic acid synthesis and cell proliferation in regenerating rat liver; autoradiography with thymidine-H3.Cancer Res. 1962; 22: 842-849PubMed Google Scholar This proliferative response is regulated by growth-related genes, occurs coordinately with glucose homeostasis, and is essential for survival of the organism.3Cressman DE Greenbaum LE DeAngelis RA Ciliberto G Furth EE Poli V Taub R Liver failure and defective hepatocyte regeneration in interleukin-6-deficient mice.Science. 1996; 274: 1379-1383Crossref PubMed Scopus (1343) Google Scholar, 4Greenbaum LE Li W Cressman DE Peng Y Ciliberto G Poli V Taub R CCAAT enhancer-binding protein beta is required for normal hepatocyte proliferation in mice after partial hepatectomy.J Clin Invest. 1998; 102: 996-1007Crossref PubMed Scopus (242) Google Scholar, 5Rai RM Lee FY Rosen A Yang SQ Lin HZ Koteish A Liew FY Zaragoza C Lowenstein C Diehl AM Impaired liver regeneration in inducible nitric oxide synthase deficient mice.Proc Natl Acad Sci USA. 1998; 95: 13829-13834Crossref PubMed Scopus (222) Google Scholar, 6Yamada Y Kirillova I Peschon JJ Fausto N Initiation of liver growth by tumor necrosis factor: deficient liver regeneration in mice lacking type I tumor necrosis factor receptor.Proc Natl Acad Sci USA. 1997; 94: 1441-1446Crossref PubMed Scopus (851) Google Scholar Concomitant with these processes, remodeling of extracellular matrix components by specialized systems of proteases must occur to clear necrotic debris by cellular scavengers and to form a suitable scaffold for newly formed cells. Although the control of matrix remodeling is not fully understood, transforming growth factor-β1 seems to be an important mediator of matrix production,7Friedman SL Cytokines and fibrogenesis.Semin Liver Dis. 1999; 19: 129-140Crossref PubMed Scopus (353) Google Scholar, 8Hellerbrand C Stefanovic B Giordano F Burchardt ER Brenner DA The role of TGFbeta1 in initiating hepatic stellate cell activation in vivo.J Hepatol. 1999; 30: 77-87Abstract Full Text Full Text PDF PubMed Scopus (327) Google Scholar, 9Qi Z Atsuchi N Ooshima A Takeshita A Ueno H Blockade of type beta transforming growth factor signaling prevents liver fibrosis and dysfunction in the rat.Proc Natl Acad Sci USA. 1999; 96: 2345-2349Crossref PubMed Scopus (298) Google Scholar whereas plasminogen plays a key role in the proteolytic clearance of matrix components and necrotic cells in vivo.10Bezerra JA Bugge TH Melin-Aldana H Sabla G Kombrinck KW Witte DP Degen JL Plasminogen deficiency leads to impaired remodeling after a toxic injury to the liver.Proc Natl Acad Sci USA. 1999; 96: 15143-15148Crossref PubMed Scopus (117) Google Scholar In plasminogen-deficient mice, the reparative response of the liver after a toxic injury was recently shown to be severely impaired, and these animals are unable to successfully restore lobular architecture throughout prolonged periods.10Bezerra JA Bugge TH Melin-Aldana H Sabla G Kombrinck KW Witte DP Degen JL Plasminogen deficiency leads to impaired remodeling after a toxic injury to the liver.Proc Natl Acad Sci USA. 1999; 96: 15143-15148Crossref PubMed Scopus (117) Google Scholar The conversion of the inactive proenzyme plasminogen to the active serine protease, plasmin, by either urokinase-type plasminogen activator (uPA) or tissue-type plasminogen activator (tPA), is critical to the maintenance of hemostasis. Both plasminogen- and plasminogen activator-deficient mice experience widespread microvascular thrombosis, progressive organ damage, and delayed wound healing.11Bugge TH Flick MJ Daugherty CC Degen JL Plasminogen deficiency causes severe thrombosis but is compatible with development and reproduction.Genes Dev. 1995; 9: 794-807Crossref PubMed Scopus (381) Google Scholar, 12Carmeliet P Schoonjans L Kieckens L Ream B Degen J Bronson R De Vos R van den Oord JJ Collen D Mulligan RC Physiological consequences of loss of plasminogen activator gene function in mice.Nature. 1994; 368: 419-424Crossref PubMed Scopus (928) Google Scholar, 13Drew AF Kaufman AH Kombrinck KW Danton MJ Daugherty CC Degen JL Bugge TH Ligneous conjunctivitis in plasminogen-deficient mice.Blood. 1998; 91: 1616-1624Crossref PubMed Google Scholar, 14Ploplis VA Carmeliet P Vazirzadeh S Van Vlaenderen I Moons L Plow EF Collen D Effects of disruption of the plasminogen gene on thrombosis, growth, and health in mice.Circulation. 1995; 92: 2585-2593Crossref PubMed Scopus (313) Google Scholar, 15Romer J Bugge TH Pyke C Lund LR Flick MJ Degen JL Dano K Impaired wound healing in mice with a disrupted plasminogen gene.Nat Med. 1996; 2: 287-292Crossref PubMed Scopus (485) Google Scholar These adverse pleiotropic effects seem to result from the multisystem accumulation of fibrin, as indicated by the general correction of the spontaneous phenotypic abnormalities observed in plasminogen-deficient mice when a loss of fibrinogen is genetically superimposed.16Bugge TH Kombrinck KW Flick MJ Daugherty CC Danton MJ Degen JL Loss of fibrinogen rescues mice from the pleiotropic effects of plasminogen deficiency.Cell. 1996; 87: 709-719Abstract Full Text Full Text PDF PubMed Scopus (341) Google Scholar However, a number of observations support a broader role for the plasminogen activation system outside fibrinolysis,17Chen ZL Strickland S Neuronal death in the hippocampus is promoted by plasmin-catalyzed degradation of laminin.Cell. 1997; 91: 917-925Abstract Full Text Full Text PDF PubMed Scopus (544) Google Scholar, 18Brunner G Gabrilove J Rifkin DB Wilson EL Phospholipase C release of basic fibroblast growth factor from human bone marrow cultures as a biologically active complex with a phosphatidylinositol-anchored heparan sulfate proteoglycan.J Cell Biol. 1991; 114: 1275-1283Crossref PubMed Scopus (132) Google Scholar, 19Odekon LE Blasi F Rifkin DB Requirement for receptor-bound urokinase in plasmin-dependent cellular conversion of latent TGF-beta to TGF-beta.J Cell Physiol. 1994; 158: 398-407Crossref PubMed Scopus (174) Google Scholar particularly in the hepatic microenvironment.20Kim TH Mars WM Stolz DB Petersen BE Michalopoulos GK Extracellular matrix remodeling at the early stages of liver regeneration in the rat.Hepatology. 1997; 26: 896-904Crossref PubMed Scopus (173) Google Scholar, 21Liu ML Mars WM Zarnegar R Michalopoulos GK Collagenase pretreatment and the mitogenic effects of hepatocyte growth factor and transforming growth factor-alpha in adult rat liver.Hepatology. 1994; 19: 1521-1527Crossref PubMed Scopus (111) Google Scholar, 22Mars WM Liu ML Kitson RP Goldfarb RH Gabauer MK Michalopoulos GK Immediate early detection of urokinase receptor after partial hepatectomy and its implications for initiation of liver regeneration.Hepatology. 1995; 21: 1695-1701PubMed Google Scholar, 23Mars WM Kim TH Stolz DB Liu ML Michalopoulos GK Presence of urokinase in serum-free primary rat hepatocyte cultures and its role in activating hepatocyte growth factor.Cancer Res. 1996; 56: 2837-2843PubMed Google Scholar, 24Roselli HT Su M Washington K Kerins DM Vaughan DE Russell WE Liver regeneration is transiently impaired in urokinase-deficient mice.Am J Physiol. 1998; 275: G1472-G1479PubMed Google Scholar, 25Michalopoulos GK DeFrances MC Liver regeneration.Science. 1997; 276: 60-66Crossref PubMed Scopus (2956) Google Scholar Perhaps the most persuasive of these observations is that plasminogen-deficient mice are unable to reorganize hepatic matrix and/or remove necrotic hepatocytes from sites of acute liver injury, regardless of the presence or absence of circulating fibrinogen.10Bezerra JA Bugge TH Melin-Aldana H Sabla G Kombrinck KW Witte DP Degen JL Plasminogen deficiency leads to impaired remodeling after a toxic injury to the liver.Proc Natl Acad Sci USA. 1999; 96: 15143-15148Crossref PubMed Scopus (117) Google Scholar Interestingly, poor removal of necrotic cells has also been reported in the heart of mice lacking uPA; necrotic cardiomyocytes within ischemic heart tissue persist as mummified ghosts, and are not readily replaced by fibrotic scar tissue in uPA-deficient mice.26Heymans S Luttun A Nuyens D Theilmeier G Creemers E Moons L Dyspersin GD Cleutjens JP Shipley M Angellilo A Levi M Nube O Baker A Keshet E Lupu F Herbert JM Smits JF Shapiro SD Baes M Borgers M Collen D Daemen MJAP Carmeliet P Inhibition of plasminogen activators or matrix metalloproteinases prevents cardiac rupture but impairs therapeutic angiogenesis and causes cardiac failure.Nat Med. 1999; 5: 1135-1142Crossref PubMed Scopus (720) Google Scholar Together, these studies suggest that plasminogen- and uPA-mediated clearance of necrotic cells may be central to tissue repair. In the current studies, we explored the role of uPA and its functional homologue tPA in liver cell proliferation, matrix remodeling, and clearance of necrotic cells in vivo. Using a well-established model of acute liver injury in mice genetically engineered to lack tPA (tPAo), uPA (uPAo), or both (tPAo/uPAo),12Carmeliet P Schoonjans L Kieckens L Ream B Degen J Bronson R De Vos R van den Oord JJ Collen D Mulligan RC Physiological consequences of loss of plasminogen activator gene function in mice.Nature. 1994; 368: 419-424Crossref PubMed Scopus (928) Google Scholar we report that although cellular proliferation is not affected by the lack of plasminogen activators, repair is severely impaired. Mice lacking each plasminogen activator individually have a mild (tPAo mice) to moderate (uPAo mice) impairment in repair, whereas the combined loss of uPA and tPA results in severe defect in lobular reorganization with matrix accumulation and poor clearance of necrotic hepatocytes. Mice with a targeted disruption of the genes coding for tPA (tPAo) or uPA (uPAo) were of a mixed 129/C57BL/6 genetic background.12Carmeliet P Schoonjans L Kieckens L Ream B Degen J Bronson R De Vos R van den Oord JJ Collen D Mulligan RC Physiological consequences of loss of plasminogen activator gene function in mice.Nature. 1994; 368: 419-424Crossref PubMed Scopus (928) Google Scholar To generate mice with simultaneous deficiency of tPA and uPA (tPAo/uPAo), we bred double heterozygous mice, and offspring were genotyped by polymerase chain reaction using specific primers that identify endogenous and targeted alleles for the tPA and uPA genes using ear biopsy DNA as template.12Carmeliet P Schoonjans L Kieckens L Ream B Degen J Bronson R De Vos R van den Oord JJ Collen D Mulligan RC Physiological consequences of loss of plasminogen activator gene function in mice.Nature. 1994; 368: 419-424Crossref PubMed Scopus (928) Google Scholar, 27Bugge TH Flick MJ Danton MJ Daugherty CC Romer J Dano K Carmeliet P Collen D Degen JL Urokinase-type plasminogen activator is effective in fibrin clearance in the absence of its receptor or tissue-type plasminogen activator.Proc Natl Acad Sci USA. 1996; 93: 5899-5904Crossref PubMed Scopus (239) Google Scholar All experimental challenges were performed in 2- to 6-month-old littermates. All mice were housed together, fed laboratory chow ad libitum, and were kept in the same room under supervision by the same investigator throughout the study period. Animal protocols were approved by the Institutional Animal Care and Use Committee of the Children’s Hospital Research Foundation (Cincinnati, OH). Gene-targeted mice and control littermates were injected intraperitoneally with 0.5 ml of carbon tetrachloride (CCl4) (Aldrich Chemical Inc., Milwaukee, WI) per kg of body weight as a 25% solution in corn oil.28Paulsen JE The time-course of mouse liver regeneration after carbon tetrachloride injury is influenced by circadian rhythms.Chronobiol Int. 1990; 7: 271-275Crossref PubMed Scopus (6) Google Scholar, 29Yazigi NA Carrick TL Bucuvalas JC Schmidt CS Balistreri WF Bezerra JA Expansion of transplanted hepatocytes during liver regeneration.Transplantation. 1997; 64: 816-820Crossref PubMed Scopus (24) Google Scholar Mice were examined daily and sacrificed 2 to 35 days after CCl4 treatment, as previously described.10Bezerra JA Bugge TH Melin-Aldana H Sabla G Kombrinck KW Witte DP Degen JL Plasminogen deficiency leads to impaired remodeling after a toxic injury to the liver.Proc Natl Acad Sci USA. 1999; 96: 15143-15148Crossref PubMed Scopus (117) Google Scholar In brief, mice were weighed, anesthetized, and blood samples were collected from the inferior vena cava. The liver was removed, blot dried, and weighed; samples were obtained from lobes, embedded in paraffin, sectioned, and stained with hematoxylin and eosin for histological analysis. The remainder of the liver was immediately frozen in liquid nitrogen. Biochemical markers of liver function and injury were determined in plasma within 4 hours of collection by an automated enzymatic assay using the Vistros Chemistry Systems 950 (Johnson & Johnson, Rochester, NY).29Yazigi NA Carrick TL Bucuvalas JC Schmidt CS Balistreri WF Bezerra JA Expansion of transplanted hepatocytes during liver regeneration.Transplantation. 1997; 64: 816-820Crossref PubMed Scopus (24) Google Scholar The proliferative response after CCl4injection was measured by the incorporation of bromodeoxyuridine (BrdU), which was administered intraperitoneally to all mice 2 hours before sacrifice.10Bezerra JA Bugge TH Melin-Aldana H Sabla G Kombrinck KW Witte DP Degen JL Plasminogen deficiency leads to impaired remodeling after a toxic injury to the liver.Proc Natl Acad Sci USA. 1999; 96: 15143-15148Crossref PubMed Scopus (117) Google Scholar BrdU-labeled hepatocytes were identified on 4-μm sections of paraffin-embedded liver samples according to instructions provided in the Cell Proliferation kit (Amersham, Life Science, Arlington Heights, IL).29Yazigi NA Carrick TL Bucuvalas JC Schmidt CS Balistreri WF Bezerra JA Expansion of transplanted hepatocytes during liver regeneration.Transplantation. 1997; 64: 816-820Crossref PubMed Scopus (24) Google Scholar For each liver sample, hepatocyte labeling index (% of hepatocytes incorporating BrdU) was calculated by counting BrdU-labeled and -unlabeled hepatocytes in 10 high-power fields (∼100 hepatocyte nuclei/field) by an investigator unaware of animal genotype. Hepatocyte proliferation was then expressed as the mean (±SD) for all mice in each group, and statistical significance was determined using unpaired t-test, with a significance level of P < 0.05. Liver extracts were prepared from frozen liver samples homogenized in phosphate-buffered saline containing 1% Nonidet P-40, 0.5% deoxycholate, 0.1% sodium dodecyl sulfate, and 10% protease inhibitor cocktail (Sigma Chemical Co., St. Louis, MO). Homogenates were centrifuged at 12,000 × g, for 15 minutes, at 4°C, and protein concentration was determined in the supernatant using the Bradford method-based Bio-Rad assay (Bio-Rad Lab., Inc., Hercules, CA). One hundred μg of homogenate protein were analyzed by zymography using polyacrylamide gels cast with casein and plasminogen as described previously.11Bugge TH Flick MJ Daugherty CC Degen JL Plasminogen deficiency causes severe thrombosis but is compatible with development and reproduction.Genes Dev. 1995; 9: 794-807Crossref PubMed Scopus (381) Google Scholar, 30Heckel JL Sandgren EP Degen JL Palmiter RD Brinster RL Neonatal bleeding in transgenic mice expressing urokinase-type plasminogen activator.Cell. 1990; 62: 447-456Abstract Full Text PDF PubMed Scopus (192) Google Scholar Immunohistochemical detection of fibrinogen and fibronectin (two components of the provisional matrix of wound fields) was performed in liver sections using a rabbit anti-fibrinogen antiserum,10Bezerra JA Bugge TH Melin-Aldana H Sabla G Kombrinck KW Witte DP Degen JL Plasminogen deficiency leads to impaired remodeling after a toxic injury to the liver.Proc Natl Acad Sci USA. 1999; 96: 15143-15148Crossref PubMed Scopus (117) Google Scholar rabbit anti-fibronectin IgG (Sigma Chemical Co.), and the Vectastain ABC-AP detection system (Vector Laboratories, Burlingame, CA); omission of primary antibodies was used in at least one section for each experiment as a negative control.15Romer J Bugge TH Pyke C Lund LR Flick MJ Degen JL Dano K Impaired wound healing in mice with a disrupted plasminogen gene.Nat Med. 1996; 2: 287-292Crossref PubMed Scopus (485) Google Scholar Fast Red TR/naphthol AS-MX (Sigma Chemical Co.) was used to detect alkaline phosphatase activity in situ.10Bezerra JA Bugge TH Melin-Aldana H Sabla G Kombrinck KW Witte DP Degen JL Plasminogen deficiency leads to impaired remodeling after a toxic injury to the liver.Proc Natl Acad Sci USA. 1999; 96: 15143-15148Crossref PubMed Scopus (117) Google Scholar CCl4 is a specific hepatotoxin that destroys a subset of hepatocytes proximal to the central vein that enzymatically convert CCl4 to the free radical CCl3 and other highly-reactive species.31Recknagel RO Glende Jr, EA Dolak JA Waller RL Mechanisms of carbon tetrachloride toxicity.Pharmacol Ther. 1989; 43: 139-154Crossref PubMed Scopus (1183) Google Scholar To explore the role of plasminogen activators in liver repair after an acute toxic injury, mice with single and combined deficits in tPA and uPA were challenged with a single dose of CCl4. Control (tPA+/uPA+) and plasminogen activator-deficient animals displayed a similar susceptibility to CCl4-induced liver damage. Plasma alanine aminotransferase (ALT, an indicator of hepatocyte injury) levels increased >80-fold within 2 days of the initial challenge and returned to near baseline by 7 days, regardless of the genotype (Figure 1). Therefore, deficiency of plasminogen activators did not seem to affect either the initial extent of liver damage or the short-lived toxicity of CCl4. Consistent with the ALT data, visual inspection of livers 2 days after CCl4 revealed a similar diffuse pale/lacy appearance in mice of all four genotypes (Figure 2). The gross appearance of the livers of mice expressing both plasminogen activators (tPA+/uPA+) or expressing solely uPA (ie, tPAo) normalized by 7 days. In contrast, the diffuse pale/lacy appearance persisted in the livers of both uPAo and tPAo/uPAo mice, with only marginal improvement in the former at 14 days. To further define the long-term outcome of hepatic repair, we further carried the study to 35 days. At this time, the livers of tPA+/uPA+, tPAo, and uPAo mice invariably displayed a normal appearance, whereas the livers of tPAo/uPAo mice remained grossly abnormal, and similar to the appearance that had been seen 2 days after CCl4 (data not shown).Figure 2Delayed restoration of normal liver appearance after toxic injury in mice lacking plasminogen activators. Visual inspection of livers from mice lacking plasminogen activators and control littermates shows an indistinguishable pale/lacy appearance in all livers 2 days after CCl4. Although livers of tPA+/uPA+and tPAo mice restore normal appearance at 7 days, uPAo livers appear mildly abnormal at 14 days, and tPAo/uPAo livers display a pronounced pale/lacy appearance at both time points.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To determine the microscopic basis for the abnormal appearance after CCl4 in mice lacking plasminogen activators, we performed histological analyses of paraffin-embedded liver sections. Two days after injury, all livers (n = 7 for each genotype) had widespread liquefaction necrosis in centrilobular hepatocytes regardless of the genotype, compromising 30 to 50% of the entire lobule (Figure 3). There was minimal inflammation and intact cellular components in the unaffected areas of the liver lobule; venular, sinusoidal endothelial, and nonparenchymal cells were well preserved. Systematic analysis of liver samples at 7 to 35 days (n = 3 to 4 for each genotype) showed that the centrilobular injury of tPA+/uPA+ mice resolved at 7 days, whereas tPAo livers had minimal residual necrotic hepatocytes with inflammatory infiltrate in the centrilobular region in three of four mice. At 14 and 35 days, liver sections of tPA+/uPA+ mice and tPAo littermates were normal and indistinguishable. Normalization of centrilobular injury was delayed in uPAo mice, with near resolution seen 14 to 35 days after the initial challenge (Figure 3). These data suggest that in the absence of tPA, uPA can independently promote the reparative response after CCl4 injury and nearly normalize hepatic histology within 7 days. In contrast, tPA cannot support efficient repair in the absence of uPA, as demonstrated by a persistent centrilobular injury at 14 days in uPAo livers. Nevertheless, tPA remains an important physiological adjunct in view of the profound delay in normalization of lobular appearance of livers lacking both tPA and uPA; these livers display persistent centrilobular injury as far out as 35 days after a single dose of CCl4 (Figure 3). Plasminogen activators have been previously linked to hepatocyte proliferation,20Kim TH Mars WM Stolz DB Petersen BE Michalopoulos GK Extracellular matrix remodeling at the early stages of liver regeneration in the rat.Hepatology. 1997; 26: 896-904Crossref PubMed Scopus (173) Google Scholar, 21Liu ML Mars WM Zarnegar R Michalopoulos GK Collagenase pretreatment and the mitogenic effects of hepatocyte growth factor and transforming growth factor-alpha in adult rat liver.Hepatology. 1994; 19: 1521-1527Crossref PubMed Scopus (111) Google Scholar, 22Mars WM Liu ML Kitson RP Goldfarb RH Gabauer MK Michalopoulos GK Immediate early detection of urokinase receptor after partial hepatectomy and its implications for initiation of liver regeneration.Hepatology. 1995; 21: 1695-1701PubMed Google Scholar, 23Mars WM Kim TH Stolz DB Liu ML Michalopoulos GK Presence of urokinase in serum-free primary rat hepatocyte cultures and its role in activating hepatocyte growth factor.Cancer Res. 1996; 56: 2837-2843PubMed Google Scholar, 24Roselli HT Su M Washington K Kerins DM Vaughan DE Russell WE Liver regeneration is transiently impaired in urokinase-deficient mice.Am J Physiol. 1998; 275: G1472-G1479PubMed Google Scholar, 25Michalopoulos GK DeFrances MC Liver regeneration.Science. 1997; 276: 60-66Crossref PubMed Scopus (2956) Google Scholar and one potential mechanism underlying the defective repair of tPAo/uPAo livers is impaired proliferative response. Using BrdU incorporation by hepatocytes as an indicator of proliferation, we found that mice lacking plasminogen activators mounted a proliferative response that was similar to control animals in regards to localization of BrdU-labeled cells within the hepatic lobule and degree of proliferation (Figure 4). BrdU-labeled hepatocytes were uniformly dispersed along noninjured areas of the liver lobule in an indistinguishable manner in both targeted and nontargeted littermates (Figure 4A). Analysis of the number of BrdU-labeled hepatocytes 2 days after CCl4 showed a >10-fold increase greater than baseline levels in all livers regardless of the genotype. Although the increase tended to be more pronounced in the livers of tPAo/uPAo mice, this finding did not reach statistical significance (Figure 4B). Interestingly, the number of BrdU-labeled hepatocytes returned to near baseline levels in nontargeted littermates as well as in tPAo/uPAo mice despite the persistence of pronounced centrilobular tissue damage at 7 and 14 days. This apparent dissociation between proliferative response and hepatic repair probably results from the restoration of normal cellular mass in mice lacking plasminogen activators leading to normalization of hepatic function. The maintenance of plasma albumin at normal levels at both time points in control and plasminogen activator-deficient mice is consistent with a prompt restoration of liver function (data not shown). Interestingly, the coexistence of an adequate proliferative response and the inability to clear necrotic cells in uPAo and tPAo/uPAo livers resulted in an increase in liver mass in these mice to 47 to 49% greater than control and tPAo littermates at 14 days (Figure 5).Figure 5Increased liver mass in mice lacking plasminogen activators after CCl4. Expression of liver weight as a percentage of body weight shows an increase in liver mass in all mice 2 days after CCl4, followed by a gradual decrease in control and tPAo littermates between 7 to 14 days. In contrast, this decrease is not observed in mice lacking uPA or both tPA/uPA, which display a greater liver mass at 14 days (mean ± SD; P < 0.01 for uPAo and tPAo/uPAoversus tPAo and tPA+/uPA+ at 14 days; control = tPA+/uPA+).View Large Image Figure ViewerDownload Hi-res image Download (PPT) Increased uPA activity and plasminogen activation in liver remnants after partial hepatectomy have been suggested to play a regulatory role in liver regeneration.20Kim TH Mars WM Stolz DB Petersen BE Michalopoulos GK Extracellular matrix remodeling at the early stages of liver regeneration in the rat.Hepatology. 1997; 26: 896-904Crossref PubMed Scopus (173) Google Scholar, 22Mars WM Liu ML Kitson RP Goldfarb RH Gabauer MK Michalopoulos GK Immediate early detection of urokinase receptor after partial hepatectomy and its implications for initiation of liver regeneration.Hepatology. 1995; 21: 1695-1701PubMed Google Scholar Therefore, we used zymography to define the hepatic activity of plasminogen activators after a toxic injury and to explore whether the loss of an individual plasminogen activator species is accompanied by a compensatory increase in the level of the remaining plasminogen activator in injured livers. In tPA+/uPA+ mice, liver injury resulted in a rapid and substantial increase in the activity of hepatic plasminogen activators, primarily uPA (Figure 6). The activity of plasminogen activators progressively returned to basal levels throughout the 14-day study period, seemingly in parallel with the microscopic resolution of centrilobular damage. A similar pattern of transiently increased activity of hepatic uPA was seen in tPAo mice after CCl4 administration, although the decrease in uPA activity appeared delayed. Interestingly, the transient change in hepatic uPA activity coincided with the timely resolution of the centrilobular injury of tPAo livers, suggesting that uPA-mediated proteolysis efficiently directs hepatic repair. In uPAo mice, hepatic tPA activity was markedly increased at 7 and 14 days; however, based on the slo
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