The regulation by fibrinogen and fibrin of tissue plasminogen activator kinetics and inhibition by plasminogen activator inhibitor 1

BACKGROUND: Tissue plasminogen activator (tPA) is unusual in the coagulation and fibrinolysis cascades in that it is produced as an active single-chain enzyme (sctPA) rather than a zymogen. Two chain tPA (tctPA) is produced by plasmin but there are conflicting reports in the literature on the behaviour of sc- and tctPA and little work on inhibition by the specific inhibitor plasminogen activator inhibitor-1 (PAI-1) under physiological conditions. OBJECTIVES: To perform a systematic study on the kinetics of sctPA and tctPA as plasminogen activators and targets for PAI-1. METHODS: Detailed kinetic studies were performed in solution and in the presence of template stimulators, fibrinogen and fibrin, including native fibrin and partially digested fibrin. Numerical simulation techniques were utilized to cope with the challenges of investigating kinetics of activation and inhibition in the presence of fibrin(ogen). RESULTS: Enzyme efficiency (k(cat)/K(m)) was higher for tctPA than sctPA in solution with chromogenic substrate (3-fold) and plasminogen (7-fold) but in the presence of templates, such as fibrinogen and native or cleaved fibrin, the difference disappeared. sctPA was more susceptible to PAI-1 in buffer solution and in the presence of fibrinogen; however, in the presence of fibrin, PAI-1 inhibited more slowly and there was no difference between sc and tctPA. CONCLUSIONS: Fibrinogen and fibrin modulate the activity of tPA differently in regard to their activation of plasminogen and inhibition by PAI-1. Fibrinogen and fibrin stimulate tPA activity against plasminogen but fibrin protects tPA from PAI-1 to promote fibrinolysis.     

Treatment of bleomycin-induced pulmonary fibrosis by transfer of urokinase-type plasminogen activator genes.

During acute and chronic inflammatory lung diseases, the normal fibrinolytic activity in the alveolar space is inhibited by increased levels of plasminogen activator inhibitor 1 (PAI-1). Transgenic mice having increased fibrinolytic activity due to genetic deficiency of PAI-1 develop less fibrosis after bleomycin-induced lung inflammation. These observations led us to hypothesize that pulmonary fibrosis could be limited through enhancement of alveolar fibrinolytic activity by adenovirus-mediated transfer of the urokinase-type plasminogen activator (uPA) gene to the lung. To investigate this hypothesis, 0.075 U of bleomycin was introduced intratracheally into mice. Twenty-one days later, the mice were treated intratracheally with phosphate-buffered saline (PBS), a control adenovirus, or adenoviruses containing murine or human uPA cDNAs. On day 28, the mice were sacrificed, and lung fibrosis was quantitated by measuring hydroxyproline content. As expected, bleomycin caused a doubling in lung hydroxyproline to 345.6+/-28.2 microg/lung (SEM) compared with mice receiving PBS (170.2+/-4.0 microg/lung). Treatment of the bleomycin-injured mice with the control adenovirus on day 21 had no impact on lung fibrosis (338.4+/-17.2 microg/lung). Importantly, the human uPA adenovirus significantly reduced (p<0.05) lung hydroxyproline (281.2+/-22.8 microg/lung), thus attenuating by 38% the bleomycin-induced increase in lung collagen. The improvement in bleomycin-induced lung fibrosis resulting from treatment with the human uPA adenovirus further supports the importance of the fibrinolytic system during inflammatory lung injury and repair.     

Thrombin upregulates production of plasminogen activator inhibitor type 1 in human peritoneal mesothelial cells.

OBJECTIVE: To determine the influence of thrombin, which is generated intraperitoneally during peritoneal dialysis, on the synthesis of fibrinolytic system components in human peritoneal mesothelial cells (HMC). METHODS: Confluently grown HMC, isolated from the omental tissue, were used in the experiments. Conditioned media were obtained by incubating cells with serum-free M199 containing the appropriate concentration of the test compound. Tissue type plasminogen activator (tPA) and plasminogen activator inhibitor type 1 (PAI-1) antigen concentrations were measured by ELISA. Northern blot analysis was conducted for mRNA expression experiments. To test thrombin specificity, we used the thrombin inhibitor hirudin. The protein kinase C (PKC) inhibitor Ro 31-8220 was inserted to examine whether the effect of thrombin depends on PKC activity. RESULTS: Thrombin increased PAI-1 antigen in the conditioned media of HMC in a time- and concentration-dependent manner. After 24 hours incubation, PAI-1 levels increased from 350+/-30 ng/10(5) cells in control conditions to 620+/-30 ng/10(5) cells in HMC exposed to 5 U/mL thrombin (n = 8, p < 0.05). In contrast, there was no effect of thrombin on tPA antigen levels. An increase of PAI-1 mRNA expression was also observed by Northern blot hybridization. Hirudin (10 U/mL) inhibited the thrombin-induced increase in PAI-1 synthesis. In addition, a complete inhibition of the stimulating effect of thrombin on PAI-1 synthesis was obtained by blocking PKC activity with Ro 31-8220 (3 micromol/L). CONCLUSIONS: Thrombin increases PAI-1 synthesis in HMC via a PKC-dependent mechanism.Thereby the synthesis of tPA is not affected. Thus, thrombin may not only promote fibrin formation in the peritoneal cavity, but may also inhibit fibrin degradation by release of free PAI-1 from HMC.     

Bleomycin-induced pulmonary fibrosis in transgenic mice that either lack or overexpress the murine plasminogen activator inhibitor-1 gene.

Impaired fibrinolytic activity within the lung is a common manifestation of acute and chronic inflammatory lung diseases. Because the fibrinolytic system is active during repair processes that restore injured tissues to normal, reduced fibrinolytic activity may contribute to the subsequent development of pulmonary fibrosis. To examine the relationship between the fibrinolytic system and pulmonary fibrosis, lung inflammation was induced by bleomycin in transgenic mice that either overexpressed or were completely deficient in murine plasminogen activator inhibitor-1 (PAI-1). 2 wk after 0.075 U of bleomycin, the lungs of transgenic mice overexpressing PAI-1 contained significantly more hydroxyproline (118 +/- 8 micrograms) than littermate controls (70.5 +/- 8 micrograms, P < 0.005). 3 wk after administration of a higher dose of bleomycin (0.15 U), the lung hydroxyproline content of mice completely deficient in PAI-1 (49 +/- 8 micrograms) was not significantly different (P = 0.63) than that of control animals receiving saline (37 +/- 1 micrograms), while hydroxyproline content was significantly increased in heterozygote (77 +/- 12 micrograms, P = 0.06) and wild-type (124 +/- 19 micrograms, P < 0.001) littermates. These data demonstrate a direct correlation between the genetically determined level of PAI-1 expression and the extent of collagen accumulation that follows inflammatory lung injury. These results strongly support the hypothesis that alterations in fibrinolytic activity influence the extent of pulmonary fibrosis that occurs after inflammatory injury.     

Development of a new test for the global fibrinolytic capacity in whole blood.

BACKGROUND: The development of global tests for the fibrinolytic capacity in blood is hampered by the low base-line fibrinolytic activity in blood, by the involvement of both plasmatic components and blood cells in the fibrinolytic system and by the loss of fibrinolytic activity as a result of the action of plasminogen activator inhibitor-1 (PAI-1). OBJECTIVE: To develop a new test for the global fibrinolytic capacity (GFC) of whole blood samples. METHODS AND RESULTS: Collection of blood in thrombin increased the subsequent generation of fibrin degradation products. This was ascribed to rapid clot formation and concomitant reduction of in vitro neutralization of tissue-type plasminogen activator (tPA) by PAI-1. On the basis of this observation, the following test was designed: blood samples were collected in thrombin with and without aprotinin and clots were incubated for 3 h at 37 degrees C. The GFC was assessed from the difference between the fibrin degradation products in the two sera. The assay was applied to blood samples from patients and healthy subjects. Other hemostasis parameters were determined in plasma samples taken simultaneously. The GFC varied considerably (normal range 0.13-13.6 microg mL(-1)); physical exercise strongly increased the GFC. Statistically significant correlations were found with tPA activity, PAI-1 activity and fibrinogen level. A mixture of antibodies against tPA and urokinase-type plasminogen activator (uPA) completely inhibited the GFC. An inhibitor of activated thrombin-activatable fibrinolysis inhibitor (TAFI) accelerated fibrinolysis 8-fold. CONCLUSION: The new test represents a global assessment of the main fibrinolytic factors in plasma and potentially those associated with blood cells.     

uPA、uPAR、PAI-1血浆含量与卵巢恶性肿瘤的相关性研究

目的：探讨尿激酶型纤溶酶原激活物（uPA）及其受体（uPAR）和抑制剂（PAI-1）血浆含量与卵巢恶性肿瘤之间的关系。方法：收集52例卵巢恶性肿瘤患者血液标本,以30例健康人作对照,用ELISA法分别检测uPA、uPAR和PAI-1的含量。结果：uPA、uPAR在卵巢恶性肿瘤各期之间均有极显著性差异（P〈0．01）,PAI-1在卵巢恶性肿瘤FIGO Ⅰ～Ⅲ期的含量逐渐升高,但在FIG0Ⅳ期时显著下降（P〈0．05）。患者组uPA、uPAR、PAI-1均较对照组升高,差异极显著（P〈0．01）。结论uPA、uPAR在卵巢恶性肿瘤患者可作为预后的判断指标,PAI-1与卵巢恶性肿瘤的分期有一定的相关性。     

The glycocalyx protects erythrocyte-bound tissue-type plasminogen activator from enzymatic inhibition

Coupling tissue-type plasminogen activator (tPA) to carrier red blood cells (RBC) prolongs its intravascular life span and permits its use for thromboprophylaxis. Here, we studied the susceptibility of RBC/tPA to PA inhibitors including plasminogen activator inhibitor-1 (PAI-1) that constrain its activity and may reduce the duration of its effect. Despite lesser spatial and diffusional limitations, soluble tPA was far less effective than RBC/tPA in dissolving clots formed in vitro from blood of wild-type (WT) mice (40 versus 80% lysis at equal doses of tPA). Furthermore, after i.v. injection, soluble tPA lost activity faster in transgenic mice expressing a high level of PAI-1 than in WT mice, whereas the activity of RBC/tPA was unaffected. PAI-1 inactivated soluble tPA at equimolar ratios in vitro, but it had no effect on the amidolytic or fibrinolytic activity of RBC/tPA. RBC/tPA was also more resistant than soluble tPA to in vitro inhibition by other serpins (alpha2-macroglobulin and alpha1-antitrypsin) and pathologically high levels of glucose. However, coupling to RBC did not protect a truncated tPA mutant, Retavase, from plasma inhibitors. Chemical removal of the RBC glycocalyx negated tPA protection from inhibitors: tPA coupled to glycocalyx-stripped RBC bound twice as much 125I-PAI-1 as did tPA coupled to naive RBC, and susceptibility of the bound tPA to inhibition by PAI-1 was restored. Thus, the RBC glycocalyx protects RBC-coupled tPA against inhibition. Resistance to high levels of inhibitors in vivo contributes to the potential utility of RBC/tPA for thromboprophylaxis.     

Regulation of local availability of active tissue-type plasminogen activator in vivo in man.

Free, biologically active tissue-type plasminogen activator (tPA) is the main initiator of intravascular fibrinolysis, but little is known about the regulation of active tPA on the organ level. The aim was to investigate if the local availability of active tPA on the organ level depends on the local release rate of tPA or the arterial input of tPA and plasminogen activator inhibitor type 1 (PAI-1). Also, we wanted to evaluate if plasma levels predict capacity for endothelial release of fibrinolytic proteins. Invasive perfused-forearm studies were performed in 96 healthy subjects. Local release rates of fibrinolytic proteins were assessed at baseline and during endothelial stimulation. Stimulation by methacholine and desmopressin induced a 6- and 12-fold increase in total tPA release rates, respectively. With increasing local release rates of tPA a gradually closer correlation emerged between the total tPA secretion and the forearm output of active tPA (from r = 0.102, ns to r = 0.85, P < 0.0001). Forearm availability of active tPA was not related to arterial input of either tPA or PAI-1. Release rates and plasma levels of tPA were not correlated. Baseline release rates of active tPA increased to noon. The major determinant for the local availability of active tPA is the capacity of the endothelium to release tPA rather than the arterial input of PAI-1 or tPA. Despite a molar excess of PAI-1, the majority of tPA released during stimulation does not undergo local inactivation. The capacity to release tPA locally cannot be predicted from its plasma concentration.     

Plasminogen activator inhibitor-1 in acute hyperoxic mouse lung injury.

Hyperoxia-induced lung disease is associated with prominent intraalveolar fibrin deposition. Fibrin turnover is tightly regulated by the concerted action of proteases and antiproteases, and inhibition of plasmin-mediated proteolysis could account for fibrin accumulation in lung alveoli. We show here that lungs of mice exposed to hyperoxia overproduce plasminogen activator inhibitor-1 (PAI-1), and that PAI-1 upregulation impairs fibrinolytic activity in the alveolar compartment. To explore whether increased PAI-1 production is a causal or only a correlative event for impaired intraalveolar fibrinolysis and the development of hyaline membrane disease, we studied mice genetically deficient in PAI-1. We found that these mice fail to develop intraalveolar fibrin deposits in response to hyperoxia and that they are more resistant to the lethal effects of hyperoxic stress. These observations provide clear and novel evidence for the pathogenic contribution of PAI-1 in the development of hyaline membrane disease. They identify PAI-1 as a major deleterious mediator of hyperoxic lung injury.     

四种烤瓷冠基底金属浸提液干预人牙龈成纤维细胞尿纤溶酶原激活剂和Ⅰ型纤溶酶原激活物抑制剂的表达

背景:修复材料的生物相容性是决定临床修复效果的重要因素之一。目的:通过比较4种金属浸提液对人牙龈成纤维细胞尿纤溶酶原激活剂和Ⅰ型纤溶酶原激活物抑制剂表达的差异探索其生物相容性。方法:选用活体牙龈,原代培养人牙龈成纤维细胞,采用镍铬、钴铬、纯钛及金钯合金4种金属浸提液进行干预,以未进行干预的细胞作为对照。结果与结论:ELISA检测结果显示镍铬和钴铬合金浸提液干预后细胞尿纤溶酶原激活剂表达增加;免疫荧光实验结合电镜观察发现镍铬和钴铬合金浸提液干预的细胞胞质荧光染色深,分布均匀,遍布整个细胞,提示细胞Ⅰ型纤溶酶原激活物抑制剂表达增加。而纯钛和金钯合金浸提液对细胞尿纤溶酶原激活剂和Ⅰ型纤溶酶原激活物抑制剂的表达无影响。说明纯钛和金钯合金的生物相容性优于镍铬和钴铬合金。     

Neointima formation and thrombosis after vascular injury in transgenic mice overexpressing plasminogen activator inhibitor-1 (PAI-1)

Summary.  The controversial role of plasminogen activator inhibitor-1 (PAI-1) in neointima formation and restenosis was studied with the use of a vascular injury model in transgenic mice overexpressing murine PAI-1 (PAI-1 Tg) and in wild-type (WT) controls. Despite the high circulating PAI-1 levels in the PAI-1 Tg mice (52 ± 9.8 ng mL⁻¹ vs. 0.76 ± 0.17 ng mL⁻¹ in WT mice), no significant fibrin deposition was observed in non-injured femoral arteries of 8- to 12-week-old mice. Two weeks after severe electric injury, extensive and comparable fibrin deposition was observed in both genotypes, despite a significantly reduced in situ fibrinolytic activity in arterial sections of the PAI-1 Tg mice. The neointimal and medial areas were similar in WT and PAI-1 Tg mice, resulting in comparable intima/media ratios (e.g. 0.94 ± 0.25 and 1.04 ± 0.17 at the center of the injury). Nuclear cell counts in cross-sectional areas of the neointima of the injured region were also comparable in arteries from WT and PAI-1 Tg mice (224 ± 63, 233 ± 20), and the distribution pattern of α-actin-positive smooth muscle cells was similar. These findings indicate that in a vascular injury model that induces extensive and persistent fibrin deposition in femoral arteries of mice, overexpression of PAI-1 does not affect neointima formation.     

Interaction between plasminogen activator inhibitor type 1 (PAI-1) bound to fibrin and either tissue-type plasminogen activator (t-PA) or urokinase-type plasminogen activator (u-PA). Binding of t-PA/PAI-1 complexes to fibrin mediated by both the finger and the kringle-2 domain of t-PA.

Plasminogen activation is catalyzed both by tissue-type-(t-PA) and by urokinase-type plasminogen activator (u-PA). This reaction is controlled by plasminogen activator inhibitor type 1 (PAI-1) that is either present in plasma or bound to fibrin, present in a thrombus. We studied the mechanism of in vitro inhibition of both t-PA and u-PA activity by PAI-1 bound to fibrin. It is shown that activation of latent PAI-1 unmasks a specific fibrin-binding site that is distinct from its reactive site. This reactive site of activated PAI-1 bound to fibrin is fully exposed to form complexes with t-PA and u-PA, that are unable to activate plasminogen. Upon complex formation with either one of the plasminogen activators, PAI-1 apparently undergoes a conformational change and loses its affinity for fibrin. Consequently, complexes of u-PA and PAI-1 dissociate from the fibrin matrix and are encountered in the fluid phase. In contrast, t-PA/PAI-1 complexes remain bound to fibrin. By employing recombinant t-PA deletion-mutant proteins, that precisely lack domains involved in fibrin binding, we demonstrate that binding of t-PA/PAI-1 complexes is mediated by both the "finger" (F) and the "kringle-2" (K2) domain of t-PA. A model is proposed that explains inhibition of the fibrinolytic process, at the level of plasminogen activation by t-PA, directed by PAI-1 bound to fibrin. An implication of the proposed model is that t-PA/PAI-1 complexes and free t-PA compete for the same binding sites on fibrin.     

纤溶酶原激活物及其抑制剂-1的血浆含量测定在急性肺血栓栓塞症中的意义

目的探讨在急性肺血栓栓塞症(APE)发病中的组织型纤溶酶原激活物(tPA)及其抑制剂-1(PAI-1)的血浆含量、作用及其该病诊断中的意义。方法,对44例APE患者和56例健康正常对照者应用酶联免疫吸附双抗体夹心法(ELISA法)定量测定血浆tPA和PAI-1抗原水平。结果与正常对照组(tPA含量为11．05ng／ml和PAI-1含量为61．31ng／m1)相比,APE组的IPA含量(33．88ng／ml)和PAI-1含量(111．50ng／ml)较高,两组间差异有显著性。在急性肺血栓栓塞症的疾病诊断中,tPA和PAI-1的血浆含量合理诊断截断点分别为21．7ng／ml和79．4ng／ml.结论急性肺血栓栓塞症的发病是由于PAI-1抗原产生和释放增多,而非tPA抗原释放或产生不足所致．tPA和PAI-1抗原血浆含量测定在APE的疾病诊断中具有要意义。     

Upregulation of fibrinolysis by adenovirus-mediated transfer of urokinase-type plasminogen activator genes to lung cells in vitro and in vivo

Impaired fibrinolytic activity within the lungs is a common manifestation of acute and chronic inflammatory lung diseases. Our previous work using transgenic mice showed that upregulation of fibrinolysis reduced pulmonary fibrosis following bleomycin-induced inflammatory lung injury. As a strategy to accelerate fibrinolysis, we generated recombinant adenoviruses containing human and mouse urokinase-type plasminogen activator (uPA) cDNAs. Both vectors induced the expression of functional uPA in human lung-derived epithelial A549 cells. A single intratracheal instillation of these uPA-containing adenoviruses into mouse lungs resulted in increased plasminogen activator activity in bronchoalveolar lavage fluid for at least 2 weeks. Plasma-derived fibrin-rich matrices overlaid on A549 cells infected with these uPA vectors were lysed efficiently in a dose-dependent fashion. Similarly, fibrin matrices formed within intact lungs that had been infected with these uPA-containing adenoviruses were also lysed more rapidly compared with noninfected and control virus-infected lungs. These results indicate that adenovirus-mediated transduction of uPA successfully upregulates fibrinolysis in vitro and in vivo. These uPA vectors can be readily used for testing the role of the fibrinolytic system in animal models of lung fibrosis, with particular attention to their therapeutic potential.     

Plasminogen and Plasminogen Activators Protect against Renal Injury in Crescentic Glomerulonephritis

The plasminogen/plasmin system has the potential to affect the outcome of inflammatory diseases by regulating accumulation of fibrin and other matrix proteins. In human and experimental crescentic glomerulonephritis (GN), fibrin is an important mediator of glomerular injury and renal impairment. Glomerular deposition of matrix proteins is a feature of progressive disease. To study the role of plasminogen and plasminogen activators in the development of inflammatory glomerular injury, GN was induced in mice in which the genes for these proteins had been disrupted by homologous recombination. Deficiency of plasminogen or combined deficiency of tissue type plasminogen activator (tPA) and urokinase type plasminogen activator (uPA) was associated with severe functional and histological exacerbation of glomerular injury. Deficiency of tPA, the predominant plasminogen activator expressed in glomeruli, also exacerbated disease. uPA deficiency reduced glomerular macrophage infiltration and did not significantly exacerbate disease. uPA receptor deficiency did not effect the expression of GN. These studies demonstrate that plasminogen plays an important role in protecting the glomerulus from acute inflammatory injury and that tPA is the major protective plasminogen activator.     

Measurement of different forms of tissue plasminogen activator in plasma

BACKGROUND: We evaluated assays to measure both total tissue plasminogen activator (tPA) and the three principle forms of tPA in plasma: active tPA, tPA complexed with plasminogen activator inhibitor type 1 (PAI-1), and tPA complexed with C1-inhibitor. METHODS: Active tPA was measured by use of an indirect amidolytic assay and immunofunctional assays. tPA/PAI-1, tPA/C1-inhibitor, and total tPA antigen were measured by use of microtiter plates coated with anti-tPA antibodies and, respectively, anti-PAI-1, anti-C1-inhibitor, and anti-tPA antibodies conjugated to peroxidase. RESULTS: The immunofunctional tPA assay detected 1 U/L (0.001 U/mL) tPA and recovered 108% +/- 12% of active tPA added to samples containing high (mean, 60 000 IU/L) PAI-1 activities vs a detection limit of 10 U/L (0.01 U/mL) and 13% +/- 25% recovery for the indirect amidolytic tPA activity assay. For measurement of tPA/PAI-1 complex, polyclonal anti-PAI-1 conjugates recovered 112% +/- 20% of the expected tPA/PAI-1 vs recovery of only 38% +/- 16% when monoclonal anti-PAI-1 conjugates were used. Of three methods tested, two total tPA antigen assays correlated well (r(2) = 0.85) and showed recoveries near 100%, whereas the third method showed lower correlations, higher intercepts, and falsely high recovery. A single anti-tPA capture antibody that performed the best in the individual assay evaluations was used to measure the different forms of tPA in 22 samples with a range of tPA and PAI-1 values. The sum of the molar concentrations of active tPA, tPA/PAI-1, and tPA/C1-inhibitor using the optimized methods was equal to 94% +/- 7% of measured total tPA. CONCLUSION: Optimized assays based on a single anti-tPA capture antibody can be used to accurately measure the major forms of tPA in plasma.     

Critical role of plasminogen activator inhibitor-1 in cholestatic liver injury and fibrosis

Plasminogen activator inhibitor-1 (PAI-1) is an acute phase protein known to correlate with hepatic fibrosis. However, whether or not PAI-1 plays a causal role in this disease process had not been directly tested. Therefore, wild-type or PAI-1 knockout (PAI-1(-/-)) mice underwent bile duct ligation. Mice were sacrificed either 3 or 14 days after surgery for assessment of early (i.e., inflammation) and late (i.e., fibrosis) changes caused by bile duct ligation. Liver injury was determined by histopathology and plasma enzymes. Accumulation of extracellular matrix was evaluated by Sirius red staining and by measuring hydroxyproline content. Hepatic expression of PAI-1 was increased approximately 9-fold by bile duct ligation in wild-type mice. Furthermore, early liver injury and inflammation due to bile duct ligation was significantly blunted in PAI-1(-/-) mice in comparison with wild-type mice. Although PAI-1(-/-) mice were significantly protected against the accumulation of extracellular matrix caused by bile duct ligation, increases in expression of indices of stellate cell activation and collagen synthesis caused by bile duct ligation were not attenuated. Protection did, however, correlate with an elevation in hepatic activities of plasminogen activator and matrix metalloprotease activities. In contrast, the increase in tissue inhibitor of metalloproteases-1 protein, a major inhibitor of matrix metalloproteases, caused by bile duct ligation was not altered in PAI-1(-/-) mice compared with the wild-type strain. The increase in hepatic activity of urokinase-type plasminogen activator was also accompanied by more activation of the hepatocyte growth factor receptor c-Met. Taken together, these data suggest that PAI-1 plays a causal role in mediating fibrosis during cholestasis.