Potential conflict of interest: Nothing to report.
Recent studies demonstrating a role for plasminogen activator inhibitor (PAI)-1 in cholestatic liver disease in mice suggested that tissue-type plasminogen activator (tPA) or urokinase plasminogen activator (uPA) might be important after biliary tract obstruction. We now demonstrate that blocking tPA exacerbates liver injury after bile duct ligation (BDL). tPA deficient mice have increased bile infarcts, increased TUNEL positive cells, increased neutrophil infiltration, reduced hepatocyte proliferation and reduced ductular reaction 72 hours after BDL compared to wild type mice. In addition, the protective and proliferative effects of plasminogen activator inhibitor 1 (PAI-1) deficiency after BDL are dramatically blocked by the tPA inhibitor tPA-STOP. One potential mechanism for these effects is that both tPA deficiency and tPA-STOP reduce hepatocyte growth factor (HGF) activation and c-Met phosphorylation in the liver after BDL. In support of this hypothesis, HGF treatment reverses the effects of tPA deficiency in mice. Furthermore, preferential tPA activation in areas of injury after BDL might occur because fibrin accumulates in bile infarcts and activates tPA. Conclusion: tPA inactivation accelerates liver injury after BDL and reduces HGF activation. These data suggest that strategies to increase HGF activation might be protective in liver diseases with biliary tract obstruction even without increased HGF production. (HEPATOLOGY 2007;45:1527–1537.)
Cirrhosis occurs after many types of liver injury including biliary obstruction, which can be studied using surgical bile duct ligation (BDL). BDL predictably induces hepatocyte cell death, neutrophil infiltration, bile duct proliferation, stellate cell activation, and fibrosis.1–3 Preventing or repairing this injury requires restoration of injured parenchymal cells, reconstruction of damaged extracellular matrix, and clearance of cellular debris.4–6 Complex mechanisms control these processes and require coordinated interactions between cytokines, proteases, and inflammatory cells in a well defined temporal and spatial fashion.
Recent studies demonstrate that plasminogen activator inhibitor-1 (PAI-1) exacerbates cholestatic liver disease.7, 8 PAI-1 blocks the proteolytic activity of tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA),9 proteases that convert plasminogen to the active protease plasmin that critically controls fibrinolysis. In addition, tPA, uPA, and plasmin have a variety of other substrates. For example, tPA and uPA can activate hepatocyte growth factor (HGF) by proteolysis.10, 11 HGF is important for survival and proliferation of hepatocytes12–14 and cholangiocytes15–17 and reduces hepatic fibrosis via c-Met activation.12, 18 Thus, tPA or uPA could reduce liver injury by increasing HGF activation. In contrast, plasmin is a key activator of transforming growth factor β 1 (TGFβ1), an important pro-fibrogenic cytokine and a negative regulator of hepatocellular proliferation.19–22 For this reason, plasmin activation could potentially exacerbate hepatic injury. Plasmin and uPA also activate matrix metalloproteinases that control tissue remodeling and might reduce fibrosis.23, 24 Thus, the plasmin protease system could influence liver injury and repair through many distinct molecular mechanisms.
We recently demonstrated increased tPA activation in PAI-1−/− mice after BDL that correlated with reduced liver injury, but no change in uPA activity suggesting that tPA was the major mediator of PAI-1 deficiency induced hepatic protection.7 To test this hypothesis, we performed BDL in tPA−/− mice. These studies confirm that tPA influences many aspects of liver injury and repair after BDL. Specifically, tPA−/− mice have larger bile infarcts, increased neutrophil infiltration, and less hepatocyte and cholangiocyte proliferation after BDL than WT mice. Furthermore, in PAI-1−/− mice, the tPA inhibitor tPA-STOP prevents the protective and proliferative effects of PAI-1 deficiency after BDL. In addition, both tPA deficiency and tPA-STOP reduce hepatic HGF and c-Met activation after BDL suggesting that tPA activity is essential for HGF activation in this setting. In support of this hypothesis, the detrimental effects of tPA deficiency can be overcome by administering active mature HGF. Thus, we now provide substantial evidence that tPA critically regulates liver injury and repair after extrahepatic bile duct obstruction. Moreover, we provide a mechanism for preferential tPA activation because the tPA activator fibrin rapidly accumulates in the bile infarcts after BDL.
BDL or sham surgery was performed as described7 on 8-10 week old (22-26 gram) male C57BL/6J WT (#0664), homozygous C57BL/6J PAI-1−/− (#2507) and tPA−/− (#2508) mice from The Jackson Laboratory (Bar Harbor, ME). To inhibit tPA in PAI-1−/− mice, we infused tPA-STOP (#544, American Diagnostic Inc., Stamford, CT)25, 26 or vehicle control (H2O) continuously for 6 days (2 nmol/hr, 0.5 μl/hr) by osmotic mini-pump (#1007D, Alza, Newark, DE),27 starting 72 hours before BDL. tPA-STOP selectively inhibits tPA (Ki = 0.035 μM for tPA, Ki = 3.4 μM for uPA),28 and also has activity against trypsin (Ki = 0.096 μM) and factor Xa (Ki = 0.021 μM). For some studies, tPA−/− mice were given active recombinant mouse HGF (0.1 mg/Kg, R&D Sys Inc, Minneapolis, MN) or PBS via tail vein injection daily for 6 days starting 72 hours before BDL. Use of mice was approved by Washington University's Animal Care Committee.
tPA, uPA and Plasmin Activity.
Casein and plasminogen zymography were performed as described.7 Plasminogen activator specific bands were verified by their absence in gels lacking plasminogen.
10 randomly selected 1 mm2 hematoxylin and eosin stained 4 μm paraffin sections were analyzed per mouse. Infarct areas were measured using NIH imageJ 1.30 (NIH, Bethesda, MD).7 Microscopic evaluations were performed blinded and the same liver regions were studied to minimize potential effects of intra-hepatic variability.
ApopTag Plus In Situ Apoptosis Fluorescein Detection Kit (S71111, Chemicon, Temecula, CA) was used according to manufacturer's instructions. Hoechst 33342 (1 μg/ml, Molecular Probes, Eugene, OR) was used for nuclear staining. TUNEL-positive cells in 30 random microscopic fields (100×) per mouse were counted.
Serum total and direct bilirubin, gamma-glutamyl transpeptidase (GGT), alanine aminotransferase (ALT) and bile acids were measured using commercial assays according to the manufacturer's instructions (#2692, #960, #0930, Stanbio, Boerne, TX; #BQ092A, Bio-Quant, San Diego, CA).
Deparaffinized and rehydrated sections were incubated for 30 minutes with a freshly prepared solution of 4% pararosaniline (0.1 ml, Sigma) plus 4 % sodium nitrate (0.1 ml, Sigma), plus 0.1 M phosphate buffer (pH = 6.3, 30 ml) to which 1% naphthol AS-D chloroacetate (1 ml, Sigma) was added.29, 30 Extravasated and sinusoidal neutrophils identified by red color and nuclear morphology were counted in 20 randomly selected high-power fields (HPF, 400×) per mouse.
Cxcl1 and Cxcl2.
mRNA levels were determined by quantitative real-time PCR (qRT-PCR) as previously described.7 CXCL1 protein was measured in liver homogenates by enzyme linked immunoassay using a Quantikine kit (#MKC00B, R&D Sys. Inc., Minneapolis, MN).
Hepatocyte and Cholangiocyte Proliferation.
Proliferation was measured by bromodeoxyuridine (BrdU) incorporation.7 Bile ducts were categorized by the number of circumferential cholangiocytes (small ≤ 10 cells; medium 10-20 cells; large > 20 cells).15 Ductular reaction was assessed by immunohistochemistry using rabbit polyclonal anti-cow cytokeratin antibody (wide spectrum screening (WSS), 1:200, DakoCytomation, Carpinteria, CA) that recognizes a variety of keratins, but specifically labels bile duct epithelial cells (BDEC) in the liver.7
Fibrin deposition was assessed after staining with goat polyclonal antibody against fibrinogen (1:200, F8512, Sigma, St Louis, MO),5 Alexa594- secondary antibody (Molecular Probes) and Hoechst 33342 nuclear staining. Adjacent sections were stained with hematoxylin and eosin to localize fibrin. Immunoreactive areas were determined in 10 randomly selected non-overlapping 0.16 mm2 (200×) fields/mouse using NIH imageJ 1.30.
Liver homogenate immunoblotting used goat anti-human α-HGF antibody (1:100, N-17, Santa Cruz Biotech. Inc., Santa Cruz, CA) as described.7 Band optical density was determined using NIH ImageJ 1.30.
c-Met was immunoprecipitated from liver homogenates and protein immunoblot analysis used anti-phosphotyrosine antibody (1:1000, clone 4G10, Upstate, Waltham, MA) or anti-c-Met antibody.7
Experimental results were derived from 4-6 BDL mice and reported as mean ± SEM. Student t-test or Mann-Whitney rank sum test were used with P value < 0.05 considered statistically significant.
tPA Deficiency Eliminates tPA Activity, But Does Not Alter uPA or Plasmin Activity After BDL.
BDL induces reproducible hepatic injury including areas of feathery hepatocyte degeneration caused by bile acid cytotoxicity (bile infarcts). To test the hypothesis that tPA critically influences bile infarct formation, BDL was performed on WT and tPA−/− mice. To confirm tPA deficiency and determine if compensatory changes in uPA or plasmin activity occur in the tPA−/− mice, casein/plasminogen zymography was performed on liver homogenates 72 hours after BDL. As expected, tPA activity was absent in tPA−/− mice (Fig. 1). uPA and plasmin activity were similar in tPA−/− and WT mice suggesting that total hepatic plasmin activity does not depend on tPA after BDL and that compensatory changes in uPA activity do not occur in tPA−/− mice (Fig. 1A,B).
tPA Deficiency Increases Bile Infarct Formation and Hepatocyte Cell Death After BDL.
Because PAI-1−/− mice have increased tPA activity and fewer bile infarcts than WT after BDL,7, 8 we hypothesized that tPA deficiency might increase bile infarct size in this model. To test this hypothesis, bile infarcts were analyzed 72 hours after BDL. Abundant bile infarcts were observed in WT mice, but tPA−/− animals had dramatically more bile infarcts (Fig. 2A,B). Quantitative analysis confirmed 83% and 94% increases in bile infarct number and area respectively in tPA−/− versus WT mice (Fig. 2E,G). TUNEL positive nuclei were also increased in tPA−/− compared to WT mouse liver after BDL (TUNEL positive nuclei per 100× field: WT 30 +/− 5, tPA−/− 45 +/− 5, P < 0.01). To determine directly if protection caused by PAI-1 deficiency could be blocked by inhibiting tPA, PAI-1−/− mice were infused with tPA-STOP, a selective tPA inhibitor with 100-fold higher Ki for uPA than for tPA. tPA-STOP markedly increased bile infarct formation in PAI-1−/− mice (Fig. 2C,D,F,H), similar to the effects of tPA deficiency. In agreement with histological evidence of liver injury, tPA deficiency increased serum ALT and GGT after BDL (Fig. 3A,C). As previously reported,7 ALT levels were similar in WT and PAI-1−/− mice, suggesting that PAI-1 deficiency more effectively prevents cell death than ALT release, but tPA-STOP increased both serum ALT and GGT in PAI-1−/− compared to control animals (Fig. 3B,D). In contrast, direct and total bilirubin levels, and total serum bile acid levels are comparable in WT, tPA−/−, and PAI-1−/− mice treated with tPA-STOP or vehicle, confirming complete biliary tract obstruction in all animals (Fig. 3E–H). Together these data provide strong evidence that tPA dramatically reduces bile infarct formation and hepatocyte cell death after BDL and suggest that protective effects of PAI-1 deficiency are mediated by increased tPA activation.
tPA Deficiency Increases Neutrophil Infiltration into Bile Infarcts After BDL.
Neutrophil extravasation into the liver is an important mechanisms of injury and repair after many types of injury including BDL.29, 31–34 To determine if tPA influences hepatic neutrophil accumulation, naphthol AS-D chloroacetate esterase stained sections obtained 72 hours after BDL in WT and tPA−/− mice were analyzed. These studies demonstrate marked neutrophil accumulation in bile infarcts in both WT and tPA−/− animals (Fig. 4A,B), but tPA−/− mice had more neutrophil accumulation than WT animals (Fig. 4E), correlating with increased size and number of bile infarcts in tPA−/− mice (Fig. 2). As previously reported, PAI-1−/− mice had fewer infiltrating neutrophils than WT,7 but this effect was reversed by tPA-STOP (Fig. 4C,D,F). Because neutrophils significantly contribute to liver injury after BDL,29, 34 we next determined if increased neutrophil accumulation in tPA−/− mice correlated with increased production of neutrophil chemotactic factor mRNAs using quantitative real time PCR. Cxcl1 and Cxcl2 mRNA were markedly elevated in tPA−/− compared to WT mice after BDL (Cxcl1: 5.4 +/− 1.0 fold increase, P = 0.007; Cxcl2: 3.3 +/− 0.7 fold increase, P = 0.009 and Fig. 4G). Similarly tPA-STOP increased Cxcl1 mRNA levels in PAI-1−/− mice (Cxcl1: 5.1 +/− 1.1 fold increase, P = 0.025, Fig. 4H), but for unclear reasons, tPA-STOP did not alter Cxcl2 mRNA levels (P = 0.7). Cxcl1 protein was also increased in tPA−/− mouse liver (P < 0.001 versus WT) and reduced in PAI-1−/− mouse liver after BDL (P < 0.001 versus WT), but this effect was modest compared to the change in mRNA (Fig. 4I,J). Overall, these data demonstrate that tPA reduces neutrophil infiltration into the liver and that tPA deficiency increases the production of neutrophil chemotactic factors after BDL.
tPA Deficiency Reduces Hepatocyte Proliferation After BDL.
In response to hepatocyte injury, hepatocytes proliferate after BDL.2, 7 While it might be anticipated that increased hepatic injury would lead to increased hepatocyte proliferation, we hypothesized that increased injury in tPA−/− mice resulted from reduced HGF activation and that tPA−/− mice would therefore have both increased cell death and reduced hepatocyte proliferation compared to WT animals. To test this hypothesis, hepatocyte proliferation rates were determined by BrdU incorporation 72 hours and one week after BDL in tPA−/− and WT mice. tPA−/− mice had markedly reduced hepatocyte proliferation compared to WT after BDL (Fig. 5A,C). In PAI-1−/− mice, tPA-STOP also reduced hepatocyte proliferation compared to control animals 72 hours after BDL (Fig. 5B). Since hepatocyte proliferation is likely to be important for liver repair and long term survival in these mice, Kaplan-Meier survival analysis was performed on WT and tPA−/− mice after BDL (Fig. 5D). These analyses confirm higher mortality rates in tPA−/− versus WT mice after BDL (P = 0.035). Together, these data strongly suggest that tPA is an important mediator of hepatocyte proliferation after biliary tract obstruction.
tPA Deficiency Reduces Bile Duct Epithelial Cell Proliferation (BDEC) After BDL.
Biliary tract obstruction causes markedly increased BDEC proliferation. To determine if BDEC proliferation depends on tPA, BrdU incorporation into BDEC was evaluated 72 hours after BDL. tPA deficiency reduced the BDEC proliferation in small and medium bile ducts after BDL compared to WT mice (Fig. 6G,I). tPA-STOP largely prevented the increase in BDEC proliferation in PAI-1−/− compared to WT mice after BDL (Fig. 6H,J). These differences in BDEC proliferation correlated well with differences in ductular reaction observed after cytokeratin immunostaining (Fig. 6A–F), strongly suggesting that tPA is an important mediator of BDEC proliferation after biliary tract obstruction.
Fibrin Accumulates in Bile Infarcts After BDL.
The results above suggest that uPA cannot substitute for tPA in the liver injury and repair processes evaluated. Indeed, our prior analyses demonstrated increased tPA, but not uPA, activity in PAI-1−/− mice after BDL.7 One possible explanation for the importance of tPA in these processes would be selective tPA activation. Because tPA activity increases dramatically when tPA binds fibrin,35, 36 we hypothesized that selective tPA activation could occur if fibrin was deposited in the liver after BDL. Indeed, there was minimal hepatic fibrin deposited in the absence of injury (data not shown), but marked fibrin accumulation in bile infarcts in WT, tPA−/−, and PAI-1−/− mice after BDL (Fig. 7A–H). Quantitative image analysis confirmed significantly larger areas of hepatic fibrin immunoreactivity in tPA−/− than WT mice (Fig. 7I). Similarly tPA-STOP increased the area of fibrin deposition in PAI-1−/− versus control mice (Fig. 7J). This selective fibrin accumulation in bile infarcts provides a reasonable explanation for localized tPA activation in areas of liver injury.
tPA Is Critical for HGF Activation After BDL.
HGF is produced as an inactive precursor that requires proteolytic cleavage for activation. Although HGF can be activated by both tPA and uPA,10 we previously hypothesized that HGF activation after BDL is primarily mediated by tPA.7 To test this hypothesis, HGF activation was evaluated by protein immunoblot. Prior analyses demonstrated that both mature and cleaved α-HGF levels are increased in WT mice after BDL compared to sham surgery.7 In current study, both tPA deficiency and tPA-STOP markedly reduce HGF activation after BDL (Fig. 8A–D). Furthermore reduced HGF activation correlated with reduced c-Met activation in tPA−/− mice and in tPA-STOP treated PAI-1−/− mice (Fig. 8E–H). Thus, tPA activity appears essential for efficient HGF activation after BDL.
Active HGF Reverses Detrimental Effects of tPA Deficiency After BDL.
Although we hypothesized that reduced HGF activation adequately explains increased cell death and reduced proliferative repair in tPA−/− versus WT mice after BDL, tPA could be required for more than HGF processing. To test the hypothesis that inadequate HGF activation causes increased liver injury in tPA−/− mice, active HGF protein was administered to tPA−/− mice via tail vein injection. Remarkably HGF administration to tPA−/− mice reduced bile infarct number and area (Fig. 9A,B), reduced the number of TUNEL+ cells (Fig. 10C,D,F), reduced neutrophil extravasation (Fig. 9C), reduced Cxcl1 protein accumulation (Cxcl1 protein levels in tPA−/− mice injected with PBS: 3.4 +/− 0.5 pg/100 μg protein; Cxcl1 protein levels in tPA−/− injected with HGF: 1.9 +/− 0.2 pg/100 μg protein, P = 0.02), and increased hepatocyte proliferation after BDL (Fig. 9D) to levels comparable to those found in WT mice. These findings make it unlikely that tPA deficiency has detrimental effects on the liver after BDL that cannot be reversed by increased active HGF availability.
Since biliary tract obstruction leads to progressive hepatic injury that can result in cirrhosis, delineating molecular mechanisms that determine the severity of cholestatic injury might provide new opportunities to slow disease progression for biliary obstructive disorders. Several observations recently suggested that PAI-1, tPA, uPA and plasminogen influence liver injury. In particular, our analysis of biliary tract obstruction in PAI-1 deficient mice demonstrated that PAI-1 increases the severity of hepatic injury, reduces hepatocyte and cholangiocyte proliferation, increases neutrophil infiltration7 and increases fibrosis (unpublished data) after BDL. We also found that tPA activity was increased in PAI-1−/− mice after BDL, but uPA and plasmin activity were comparable to WT. Bergheim et al.8 demonstrated similar effects of PAI-1 deficiency on liver injury and fibrosis, but reported increases in both tPA and uPA activity in PAI-1−/− mice. This raised questions about whether PAI-1−/− effects on liver injury were mediated by tPA, uPA or both. We now provide evidence that many effects of PAI-1 deficiency depend on tPA, and not uPA.
tPA is a protease that has important functions in fibrinolysis, extracellular matrix remodeling and cytokine processing. To perform these functions, there are both plasmin dependent and plasmin independent roles for tPA. In fibrinolysis, for example, the major action of tPA is conversion of plasminogen to the active protease plasmin.37 Plasmin not only causes fibrinolysis, but also induces matrix metalloproteinases and activates cytokines such as TGFβ.19, 24, 38 tPA also has important plasmin-independent functions in endothelial cell proliferation,39 microglial activation,40 mossy fiber outgrowth stimulation,40 and neuronal death after ischemia.41 Furthermore, tPA directly activates several growth factors, including HGF10, 11 and platelet derived growth factor (PDGF-CC) by proteolysis.42
We now demonstrate that tPA deficiency increases the extent of bile infarcts after BDL. The hypothesis that increased bile infarct size is at least partially caused by increased parenchymal cell damage in tPA−/− mice is supported by increased ALT and GGT serum levels in the mutant animals. Furthermore, there are increased TUNEL positive cells in tPA−/− compared to WT mice after BDL demonstrating an increase in the number of cells with nuclear DNA fragmentation. While controversy exists over the significance of TUNEL staining and the role of necrosis versus apoptosis after BDL,3, 43 our data collectively demonstrate that tPA deficiency increases liver injury and cell death after BDL.
Once the liver is injured, repair mechanisms are induced that include increased hepatocyte and cholangiocyte proliferation. While it might be anticipated that increased cell death would lead to a more robust proliferative repair response, tPA−/− mice have less hepatocyte and cholangiocyte proliferation than WT after BDL. Furthermore, tPA−/− mice have higher mortality rates than WT animals in this setting. Together, these findings suggest that tPA is essential for the normal mechanisms that promote hepatocellular survival and proliferation during biliary tract obstruction. Reduced HGF activation in tPA−/− compared to WT mice after BDL could explain each of these observations since HGF is a survival factor for hepatocytes and mitogen for both hepatocytes and cholangiocytes. Indeed, we now demonstrate that HGF activation and c-Met phosphorylation in tPA−/− mouse liver are markedly reduced after BDL compared to WT. Finally, although not conclusive, the observation that many of the detrimental effects of tPA deficiency can be prevented by exogenous administration of active HGF suggests that tPA may be primarily important for HGF activation in this setting. Lending further credence to this argument is a recent manuscript demonstrating effects of HGF blocking antibodies after BDL that resemble our results in tPA−/− mice.44
In conjunction with increased hepatocellular injury, tPA−/− mice have more neutrophil extravasation than WT after BDL. The precise mechanism for increased neutrophil infiltration is not yet clear. tPA, for example, could reduce neutrophil infiltration by enhancing the release of chemoattractants from the extracellular matrix, similar to the described effect of PAI-1 inhibition on the human protein IL-8.45 Alternatively, increased immobilized fibrin(ogen) might encourage neutrophil infiltration in tPA−/− mice, because fibrin(ogen) is bound with high affinity by αMβ2 integrin expressed on neutrophils.46 HGF also reduces expression of ICAM-1 in the liver after BDL44 and could reduce neutrophil infiltration by this mechanism.34 Current data, however, demonstrate a markedly reduced abundance of mRNA for the murine neutrophil chemoattractants Cxcl1 and Cxcl2 in WT compared to tPA−/− mice after BDL and a smaller reduction in Cxcl1 protein. These observations suggest significant effects on the transcription or stability of these mRNA that are independent of effects on protein localization or fibrinogen accumulation. In any case, increased neutrophil recruitment may significantly exacerbate liver injury in tPA−/− mice creating a vicious cycle that involves tissue damage by the neutrophils and increased production of neutrophil chemotactic factors in response to injury.29, 34
Why can't uPA substitute for tPA in this setting? One reasonable explanation for this observation is that additional mechanisms restrict spatial and temporal activation of these potent proteases. For example, fibrin accumulates in areas of liver injury after BDL, and binding of tPA to fibrin markedly stimulates tPA activity.47 uPA also requires additional factors like binding to uPAR for optimal activity,48, 49 and therefore might have a different spatial and temporal activity pattern than tPA even though liver homogenates have comparable enzymatic activity for both proteases after BDL. While the role of uPA after BDL remains uncertain,50 our data demonstrate that uPA cannot substitute for tPA in this setting. Furthermore, the ability to reverse the effects of PAI-1 deficiency on BDL-induced liver injury using tPA-STOP provides strong suggestive but not conclusive evidence that the protective effects of PAI-1 deficiency are primarily mediated by increased tPA, and not uPA, activity.
The observation that tPA and uPA have distinct functions after BDL is consistent with previous data demonstrating that uPA cannot substitute for tPA in other injury models. For example, after hyperoxia, tPA−/− mice have more severely injured lungs and greater mortality than WT animals.51 Similarly, tPA−/− mice develop more bleomycin-induced lung fibrosis than either WT or uPA−/− animals.52 Furthermore, in a model of crescentic glomerulonephritis, injury is exacerbated in tPA−/− mice, but not in uPA−/− animals.53 In contrast, after single-dose carbon tetrachloride (CCl4)-induced liver injury, tPA−/− mice had a milder defect in liver repair than uPA−/− mice.54 In combination with our data, these studies clearly demonstrate distinct roles for uPA and tPA in injury and repair processes that depend on the precise nature of the underlying injury.
In summary, we demonstrate that tPA protects the liver during and after BDL. At least part of this protection occurs because tPA is required for HGF activation following BDL. These observations suggest that strategies to increase HGF activation will protect the liver from injury during cholestatic liver disease.
We thank Dr. Phillip Tarr, Dr. Louis Muglia, and Dr. David Rudnick for critical comments on the manuscript.