Introduction

Chronic obstruction of the common bile duct may cause hepatic fibrosis and secondary biliary cirrhosis. Thus, chronic cholestatic liver diseases are a leading indication for liver transplantation in adult and pediatric patients^1,2. Among the most prominent cholestatic disorders are primary biliary cirrhosis, secondary biliary cirrhosis, primary sclerosing cholangitis, and extrahepatic biliary atresia³. Even when the underlying cause is treated or removed, it is usually thought that end-stage cirrhosis is an irreversible process⁴. Secondary biliary cirrhosis (SBC) onset is a consequence of congenital defects like biliary atresia, which is a devastating infant disease. Also, SBC is the result of biliary obstruction after iatrogenic cholecystectomies. Laparoscopic cholecystectomy remains the only treatment for symptomatic gallbladder stones and is the most prominent source of biliary injuries. Unfortunately, in some cases the surgery is complicated by vasculobiliary injury. Iatrogenic injury of the biliary tract represents a complex problem for surgeon and patient⁵. Reconstruction consists in performing a biliodigestive anastomosis (BDA), but it represents a surgical challenge. On the other hand, patients with primary biliary cirrhosis have experienced some relief when treated with ursodiol⁶ or methotrexate⁷. However, the controversy remains regarding whether they are effective in reversing fibrosis⁸. It is clear, then, that development of new strategies to reverse cholestasis-induced hepatic damage and the concomitant fibrosis is attractive. Recently, Zhong et al. have reported that viral gene delivery of superoxide dismutase attenuates experimental cholestasis-induced liver fibrosis in the rat⁹. Nonetheless, the nature of the experimental design would preclude the generalized utility of this treatment in patients with biliary obstruction. Our experience in the field has rendered information that one single injection of an adenoviral vector bearing a modified cDNA coding for a nonsecreted form of human urokinase-plasminogen activator (AdHuPA) efficiently reverts CCl₄-induced liver cirrhosis, which resembles human alcoholic cirrhosis¹⁰.

Here, we have combined a surgical procedure to accomplish biliary decompression with AdHuPA-targeted liver delivery to improve the outcome of experimental SBC. Contrary to data obtained from cirrhotic animals treated with BDA plus AdGFP gene delivery, the combined therapy using AdHuPA instead resulted in enhanced liver fibrosis reversion, disappearance of collagen-making cells, and stimulation of hepatocyte cell regeneration. Livers from AdHuPA-injected animals experienced clear-cut expression of human corresponding protein detected by immunohistochemistry. This cascade of events resulted in up-regulated expression of specific collagen-degrading enzymes (metalloproteinases) like MMP-3, MMP-9, and MMP-2. We believe these latter enzymes as accountable for enhanced hepatic fibrosis reversion. Biochemical parameters, specifically bilirubin determinations, as well as functional measurements (ascites, gastric varices) were also down-regulated with a tendency to normalize.

Results and Discussion

Application of gene therapy strategies in cooperation with standard medical practices seem promising and plausible therapeutic measures for a number of pathophysiologic conditions. Thus, in vivo surgical resection plus adjuvant gene therapy protocols have been devised for the treatment of experimental mammary and prostate cancer¹⁶. Along the same rationale, an enhanced therapeutic effect of herpes simplex virus thymidine kinase gene plus ganciclovir (gene therapy approach) in combination with ionizing radiation for mouse prostate cancer has been reported¹⁷. A clinical study using this combined strategy is now ongoing. In the field of chronic hepatic diseases, the development of the Kasai portoenterostomy procedure improved the prognosis for children with biliary atresia^18,19. However, progressive hepatic fibrosis, portal hypertension, and eventual liver failure are common; 70 to 80% of patients eventually require liver transplantation, accounting for 50–60% of all children who undergo liver transplantation^18,19. Therefore, a need for different therapeutic approaches to alleviate cholestatic disorders resulting in liver cirrhosis becomes evident. Ursodeoxycholic acid is currently the most promising therapy for chronic cholestatic liver diseases; however, it cannot prevent fibrosis^20,21. Recently, Zhong et al.⁹ demonstrated that gene delivery of mitochondrial Mn-superoxide dismutase (Mn-SOD) to bile-duct-ligated rats blocks formation of oxygen radicals and production of toxic cytokines, thereby minimizing liver injury caused by cholestasis. However, their study has a major strategic difference with ours. They had to administer the adenoviral vector containing Mn-SOD 3 days before rat bile-duct ligation took place. Potential translational application of such a strategy to the clinical scenario would be difficult.

Here, we used a biliary common duct ligation (BDL) rat model resembling biliary cirrhosis in humans^22,23. In this BDL model, extrahepatic cholestasis due to prolonged obstruction of bile flow led to anatomic destruction of the biliary tree and produced a mortality rate of 20–30% at 4 weeks of ligature. In the surviving animals, biochemical and morphological changes with extensive proliferation of bile ducts, enlarged portal tracts, inflammation, necrosis, and formation of periportal fibrosis with pericentral collagen deposition were evident in 4-week BDL cirrhotic animals (n = 6) deprived of gene therapy treatment (Fig. 1B). We sacrificed animals 10 days after 4-week BD ligation. On the other hand, a matching lot of cirrhotic rats (n = 6) otherwise treated with one single injection of 6 10¹¹ viral particles (vp)/kg clinical grade AdHuPA adenoviral vector showed a slight histologic improvement and a significant 25.8% fibrosis index reduction (P 0.05) (Fig. 1A). It is important to make a note of this, since our previous work in rats with CCl₄-induced cirrhosis treated with AdHuPA¹⁰ demonstrated that overexpression of corresponding hepatic human uPA correlated with induction of MMP-2 and almost complete resolution of periportal and centrilobular fibrosis (85%) compared to a progressive fibrosis in controls. The differences found in this communication regarding percentage of fibrosis reversion may be due to the harshness of the fibrotic process in the BDL model and the different extracellular matrix proteins shaping the scar.

Figure 1.

Histologic analysis of fibrosis in bile duct-ligated (BDL) rats treated with either AdHuPA or AdGFP adenoviral vector. Liver tissue was processed as described under Materials and Methods. (A) Fibrosis index was determined in control rats without bile duct ligation (basal), 4 weeks after BDL, and 7 days later. After this liver biopsy was taken, BDA rats were divided into groups and injected via the iliac vein with either 6 10¹¹ vp/kg AdHuPA (n = 6) or 6 10¹¹ vp/kg control adenoviral vector AdGFP (n = 6). Rats were kept under surveillance for 10 more days and then sacrificed. (B) Both animal groups were maintained for 4 weeks after BDL without bile drainage.

Full figure and legend (181K)

In light of these results, we thought that combination of surgical procedures with AdHuPA gene therapy strategy would result in an enhanced therapeutic effect resulting in relief of the cholestatic disorder, induction of fibrosis reversion, and hepatic cell regeneration.

Therefore, we designed experiments shown in Fig. 2 to evaluate such a combined treatment. Animals showed dramatic elevation of liver enzymes AST, ALT, alkaline phosphatase (AP), and total and direct bilirubins following BDL. We obtained liver biopsies from each rat before BDL and at every step described after ligation. At the end of 2 (Group 1) or 4 (Group 2) weeks all rats had increased liver fibrosis along with histologic and clinical features already described¹⁵. Then, we subjected all animals in both groups to BDA to induce drainage of the clogged bile. This surgical procedure resembles those performed in humans to ameliorate bile obstruction by discontinuation of the harmful agent and reestablishment of bile flow. We left the rats in this stage for 7 days. Hepatic fibrosis index continued to be elevated to similar extents in all animals. Then, we injected 7 2-week BDL cirrhotic rats via the iliac vein with 6 10¹¹ vp/kg AdHuPA and 7 control rats with 6 10¹¹ vp/kg irrelevant adenoviral vector AdGFP. These 14 rats constituted Group 1. Similarly, we treated 5 4-week BDL cirrhotic rats with 6 10¹¹ vp/kg AdHuPA and 5 parallel rats with 6 10¹¹ vp/kg irrelevant adenoviral vector AdGFP. These 10 animals constituted Group 2. We kept all rats under surveillance for 10 more days, monitoring for overall health status, and then sacrificed them. We observed an important and significant 56.9% decrease in fibrosis ratio in Group 1 and 42.9% in Group 2, in rats injected with AdHuPA, but observed no decrease in percentage fibrosis index in animals injected with AdGFP (P < 0.001). Moreover, we have included data from animals without any viral transduction nor surgical intervention as control. These data reflect BDL animals ligated for 2 weeks, plus 7 days (with no biliodigestive anastomosis), plus 10 days, which equals the time span in animals injected with adenovector. Similarly, Fig. 2 shows in the lower right corner BDL animals ligated for 4 weeks without biliodigestive anastomosis and no Ad injection and followed up to 17 days until sacrifice. These last controls enable us to suggest the specific effect of AdHuPA, but not AdGFP, on induction of collagenous extracellular matrix protein degradation reflected by a statistically significant decreased fibrosis index.

Figure 2.

Positive coadjuvant effect of bile drainage plus AdHuPA adenoviral vector. We compared fibrosis index between AdHuPA and AdGFP adenovirus vector injection and between groups of 2 weeks (top) and 4 weeks (bottom) after BDL. Bile drainage itself improved the fibrosis percentage observed, though a synergistic improvement took place after AdHuPA adenovirus vector injection. Liver fibrosis index decreased 56.9% in cirrhotic rats ligated for 2 weeks (P < 0.05) compared with 42.9% in animals with BDL for 4 weeks. A numerical figure of bile-duct-ligated cirrhotic animals that did not receive any adenovector treatment is in the right-hand side. This figure reflects BDL animals ligated for 3 weeks, plus 7 days (with no biliodigestive anastomosis), plus 10 days, which equals the time span in animals injected with adenovector. Similarly, in the lower right are shown BDL animals ligated for 4 weeks without biliodigestive anastomosis and no Ad injection and followed up to 17 days until sacrifice.

Full figure and legend (490K)

Liver fibrosis index was determined by two different pathologists blinded to the study.

Finally, expression of human uPA (see later in Fig. 5) in cirrhotic livers led to an important resolution of fibrosis and regeneration of functional hepatocytes as determined by immunohistochemical reaction with anti-PCNA antibody (Fig. 3). Multiple mechanisms may account for the induction of hepatocyte regeneration and matrix degradation by activation of the metalloproteinase cascade may lead to remodeling of the distorted architecture and angiogenesis and may free up space for hepatocyte expansion. A potential mechanism for the degradation of fibrotic tissue observed with AdHuPA is through the disengagement of latent metalloproteinase complexes with TIMPs rendering active collagen-degrading enzymes (Armendariz-Borunda, unpublished observations).

Figure 5.

Liver tissue sections from BDL cirrhotic animals that were ligated for 4 weeks, underwent bile drainage, and then were injected with either AdHuPA or AdGFP. Tissue sections were incubated with corresponding antibodies. The right shows data of positive area for each antibody.

Full figure and legend (494K)

Figure 3.

Liver sections were incubated overnight at room temperature with mouse monoclonal antibody against -smooth muscle actin diluted 1:50 in PBS. Activated collagen-making cells (hepatic stellate cells) are observed in brown color. HSC activation decreased dramatically in rats treated with AdHuPA (bottom). Hepatocyte regeneration was measured by immunohistochemistry using anti-PCNA antibody. Arrowheads show specific staining. Animals treated with AdGFP (top) showed less cell proliferation compared with animals treated with AdHuPA. Ascites as well gastric varices (GV) improved importantly in rats injected with AdHuPA but not in AdGFP rats. nd, not determined.

Full figure and legend (285K)

Hepatic stellate cell (HSC) activation reflects the capacity of these cells to synthesize an increased amount of collagenous proteins. It is clear that we found the specific marker -smooth muscle actin (-SMA) in considerably smaller amounts in liver sections from rats treated with AdHuPA. Also, we could observe an augmented number of mitotic figures (PCNA) indicating an active liver cell regeneration as opposed to AdGFP-treated animals. Liver tissue slides stained with hematoxylin and eosin are included in Fig. 3C to demonstrate this mitotic index. Ascites as well gastric varices improved importantly in rats treated with HuPA as opposed to rats treated with irrelevant AdGFP (Fig. 3).

Cirrhotic animals showed dramatic elevation of liver enzymes AST, ALT, AP (data not shown), and total and direct bilirubins followed BDL (Fig. 4). One week after BDA and immediately before adenoviral application, rats showed a significant improvement in biochemical liver enzymes (P < 0.05), though the most striking beneficial effect was noted in total bilirubin, specifically direct bilirubin, indicating a clear relief in cholestatic disorder. It is important to point out that we observed this in both groups of animals (2-week BDL and 4-week BDL) injected with the therapeutic adenovector. However, this effect was statistically significant only in Group 1. These results are relevant since it is well established that the relationship of serum bilirubin concentrations to survival is inversely proportional in most of both adult and pediatric patients with cholestatic disorders who progress to the need for liver transplantation²⁴. Higher serum bilirubin values were also clearly associated with much poorer survival in another study²⁵. It is clear, then, that in patients with biliary cirrhosis, elevated serum bilirubin values are an independent predictor of poor prognosis^26,27 and bilirubin is one of the most significant variables in the Mayo mathematical model of survival in patients with biliary cirrhosis²⁴.

Figure 4.

Total and direct bilirubin improvement.

Full figure and legend (165K)

In trying to elucidate the mechanisms of action of human uPA protein in the degradation of collagenous extracellular matrix proteins we performed immunohistochemical staining with specific antibodies against human urokinase-plasminogen activator and metalloproteinases like MMP-3, MMP-9, and MMP-2.

Fig. 5 clearly depicts the specific immunostaining in liver from cirrhotic, drained animals injected with either AdHuPA or AdGFP. Livers from AdHuPA-treated animals underwent a cascade of events resulting in specific detection uPA, MMP-3, MMP-9, and MMP-2.

Although we did not run a kinetics curve at different times post-Ad injection, our reasoning, and according to results shown in other systems, is that uPA activates rat pro-MMP-3 in situ, which in turn triggers the initial event of collagen degradation, and also active MMP-3 would induce activation of pro-MMP-9 and MMP-2, degrading in turn collagenous and gelatinous remaining material.

We did not search for MMP-1 action in our system. However, it is possible that initial breakdown of the major constituent of the fibrous scar in these cirrhotic livers, namely collagen type I, is achieved by MMP-1 and/or MMP-3 and then continued by MMP-9 and MMP-2. It has been recently been shown in other systems that degradation of collagen I clearly occurs during recovery from liver fibrosis, and this may not simply promote or facilitate the hepatocyte regenerative response but is also associated with a diminution in hepatic stellate cell number and a decrease in collagen I and -SMA expression^28,29. These data correlate with our findings.

The results found in this rodent model of cirrhosis are encouraging and warrant the further development of coadjuvant approaches to heal human secondary biliary cirrhosis.

Materials and Methods

Animals and induction of secondary biliary cirrhosis

Wistar female rats were housed in the animal facility of the University of Guadalajara and all animal studies were conducted in accordance with the principles and procedures outlined in the National Institutes of Health's Guide for the Care and Use of Laboratory Animals. Rats weighing 200–250 g were fed a standard rat chow diet. Two groups of animals were used: rats ligated for 2 weeks and then subjected to BDA plus either AdGFP (control animals) or AdHuPA constituted Group 1. Animals ligated for 4 weeks and identically managed as before represented Group 2. Thus, animals included in Group 1 (n = 14) underwent BDL for 2 weeks, followed by BDA. Rats were left at this stage for 7 days. Then, rats (n = 7) were injected via the iliac vein with 6 10¹¹ vp/kg AdHuPA along with 7 rats injected with 6 10¹¹ vp/kg irrelevant adenoviral vector AdGFP used as control. Rats were kept under careful surveillance for 10 days, monitoring for overall health status, and then sacrificed. The procedure involved rats anesthetized at every step with intramuscular dehydrobenzoperidol (400 g/kg) and ketamine (400 l/kg) and from which liver biopsies and serum functional hepatic tests were determined at every step. Animals underwent a 2-cm upper-midline abdominal incision below the xifoides appendix, and the suprapancreatic common bile duct was identified and double ligated with 5-O silk (Ethicon; Johnson and Johnson, Mexico City, Mexico) and transected between the ligatures. Subsequently, the abdominal wall was closed in two layers with continuous 5-O silk. BDA was carried out with internal silastic stent (Medicate Grade Tubing 602-205.40; Dow Corning, Midland, MI) between dilated bile duct and duodenum. That procedure was intended to drain the clogged bile and to reestablish bile flow. Group 2 was composed of 10 cirrhotic animals that underwent BDL, BDA was done 4 weeks later, and immediately 5 rats were injected via the iliac vein with 6 10¹¹ vp/kg AdHuPA along with 5 rats injected with 6 10¹¹ vp/kg irrelevant adenoviral vector AdGFP. The subsequent procedures and tests were performed identically as for Group 1. Rats were kept under careful surveillance for 10 more days, monitored for overall health status, and sacrificed.

Adenovirus vectors

Cloning of the adenoviral plasmid pAd.PGK-NC-huPA has been described before¹¹ and essentially it is a first-generation adenoviral vector bearing a modified cDNA coding for nonsecreted human urokinase plasminogen activator (AdHuPA). The preparations of this Ad vector were monitored for endotoxin and mycoplasm contaminants and were titered as previously described¹². The rationale for using this vector resided in the advantage of the nonsecreted uPA, which does not cause hypocoagulation and spontaneous bleeding, which represent a main drawback in cirrhotic animals.

The AdGFP vector used here (irrelevant adenovirus) is an E1- and E3-deleted replication-defective adenovirus vector previously described¹². The vectors were produced under good laboratory practice conditions. The vectors were titered and characterized as described^12,13 and had a vector particle to infection unit ratio of 30.

Biochemical assays

Blood (1.5 ml) was drawn from animals when each surgical procedure was performed and serum transaminases, ALT, AST, alkaline phosphatase, and total and direct bilirubins were determined in an automated Sincron-7 analyzer at the Hospital Civil of Guadalajara. Five animals were studied in each group at the indicated steps: basal, 2 or 4 weeks after BDL, 7 days after BDA, and 10 days after 6 10¹¹ vp/kg AdHuPA or AdGFP adenovirus vector injection by iliac vein. Results are expressed as means SD.

Histological examination of liver sections

Liver biopsies were taken at every step during the experimental design. That is, a piece of liver was obtained from each rat at basal time, either at 14 or at 28 days after BDL (for Groups 1 and 2, respectively), and then at 7 days after BDA and 10 days after adenoviral vector injection. The liver was fixed by immersion in 10% paraformaldehyde diluted in phosphate-buffered saline (PBS), dehydrated in graded ethylic alcohol, and embedded in paraffin. Sections 5 m thick were stained with hematoxylin/eosin to identify mitotic figures reflecting liver cell regeneration and Masson trichrome to determine the percentage of liver tissue affected by fibrosis, and characteristic proliferation of bile ducts was determined using a computer-assisted automated image analyzer (Image-Pro Plus 4.0) by analyzing 20 random fields per slide and calculating the ratio of connective tissue to the whole area of the liver. Parallel liver slides were stained in 0.1% Picrosirius red solution. Sirius red is a specific dye that has an affinity for collagenous proteins. Then, tissue sections were counterstained with fast green dye, which has an affinity for noncollagenous protein^14,15.

Immunohistochemistry of liver sections

Another set of liver sections was mounted in silane-covered slides and deparaffinized, and the endogenous activity of peroxidase was quenched with 3.0% H₂O₂ in absolute methanol. Liver sections were incubated overnight at room temperature with mouse monoclonal antibodies against PCNA, to determine cell proliferation, and -smooth muscle actin, to search for hepatic stellate cell activation (Boehringer Mannheim, Germany), diluted 1/20 and 1/50, respectively, in PBS and with goat polyclonal antibodies against human uPA (Chemicon International, USA) diluted 1/400 in PBS. We also used goat polyclonal IgG antibodies against rat collagen-degrading enzymes or metalloproteinases MMP-2, -3, and -9 (Santa Cruz Biotechnology, USA) diluted 1/300 in PBS. For this staining, we used liver slides from animals of 4-week BDL plus drainage and 10 days after injection of adenovirus with HuPA or GFP gene. We included a negative control without primary antibody in each slide.

Bound antibodies were detected with peroxidase-labeled rabbit polyclonal antibodies against mouse or goat immunoglobulins and diaminobenzidine and counterstained with hematoxylin. Histopathology was interpreted by two independent board-certified pathologists who were blinded to the study.

Ascites was considered severe (+++) when ascitic fluid comprised >5% of body weight; moderate (+ or ++) when <5% of body weight. We also determined gravity of gastric varices.

Statistical analyses

Results are expressed as means and SD. Student's t test and Mann–Whitney U test were used. P < 0.05 was considered to indicate significant differences between groups.

References

1. Starzl, T. E., Demetris, A. J. and Van Thiel, D. (1989). Liver transplantation (1). N. Engl. J. Med.. 321: 1014–1022. | PubMed | ChemPort |
2. Whitington, P. F. and Balistreri, W. F. (1991). Liver transplantation in pediatrics: Indications, contraindications, and pretransplant management. J. Pediatr.. 118: 169–177. | PubMed | ChemPort |
3. Poupon, R., Chazouilleres, O. and Poupon, R. E. (2000). Chronic cholestatic diseases. J. Hepatol.. 32: (Suppl. 1): 129–140. | Article | PubMed | ISI | ChemPort |
4. Friedman, S. L. (1993). The cellular basis of hepatic fibrosis: Mechanisms and treatment strategies. N. Engl. J. Med.. 328: 1828–1835. | Article | PubMed | ISI | ChemPort |
5. Rossi, R. L. and Tsao, J. L. (1994). Biliary reconstruction. Surg. Clin. North Am.. 74: 825–844. | PubMed | ChemPort |
6. Poupon, R. E., Balkau, B., Eschwege, E. and Poupon, R. (1991). A multicenter, controlled trial of ursodiol for the treatment of primary biliary cirrhosis. UDCA-PBC Study Group. N. Engl. J. Med.. 324: 1548–1554. | PubMed | ChemPort |
7. Kaplan, M. M., DeLellis, R. A. and Wolfe, H. J. (1997). Sustained biochemical and histologic remission of primary biliary cirrhosis in response to medical treatment. Ann. Intern. Med.. 126: 682–688. | PubMed | ChemPort |
8. Zimmerman, H., Reichen, J., Zimmermann, A., Sagesser, H., Thenisch, B. and Hoflin, F. (1992). Reversibility of secondary biliary fibrosis by biliodigestive anastomosis in the rat. Gastroenterology. 103: 579–589. | PubMed |
9. Zhong, Z., Froh, M., Wheeler, M. D., Smutney, O., Lehmann, T. G. and Thurman, R. G. (2002). Viral gene delivery of superoxide dismutase attenuates experimental cholestasis-induced liver fibrosis in the rat. Gene Ther.. 9: 183–191. | Article | PubMed | ISI | ChemPort |
10. Salgado, S., et al. (2000). Liver cirrhosis is reverted by urokinase-type plasminogen activator gene therapy. Mol. Ther.. 2: 545–551. | Article | PubMed | ChemPort |
11. Lieber, A., Peeters, M. J., Gown, A., Perkins, J. and Kay, M. A. (1995). A modified urokinase plasminogen activator induces liver regeneration without bleeding. Hum. Gene Ther.. 6: 1029–1037. | PubMed | ISI | ChemPort |
12. Nyberg-Hoffman, C., Shabram, P., Li, W., Giroux, D. and Aguilar-Cordova, E. (1997). Sensitivity and reproducibility in adenoviral infectious titer determination. Nat. Med.. 3: 808–811. | Article | PubMed | ChemPort |
13. García-Bañuelos, J., Siller-López, F., Miranda, A., Aguilar, L. K., Aguilar-Córdova, E. and Armendáriz-Borunda, J. (2002). Cirrhotic rat livers with extensive fibrosis can be safely transduced with clinical-grade adenoviral vectors: Evidence of cirrhosis reversion. Gene Ther.. 9: 127–134. | Article | PubMed | ChemPort |
14. Lopez-De Leon, A. and Rojkind, M. (1985). A simple micromethod for collagen and total protein determination in formalin-fixed paraffin-embedded section. J. Histochem. Cytochem.. 33: 737–743. | PubMed | ChemPort |
15. Bueno, M. R., Daneri, A. and Armendáriz-Borunda, J. (2000). Cholestasis-induced fibrosis is reduced by interferon alpha-2a and is associated with elevated liver metalloprotease activity. J. Hepatol.. 33: 915–925. | Article | PubMed | ChemPort |
16. Sukin, S. W., et al. (2001). In vivo surgical resection plus adjuvant gene therapy in the treatment of mammary and prostate cancer. Mol. Ther.. 3: 500–506. | Article | PubMed | ChemPort |
17. Chhikara, M., et al. (2001). Enhanced therapeutic effect of HSV-tk + GCV gene therapy and ionizing radiation for prostate cancer. Mol. Ther.. 3: 536–542. | Article | PubMed | ISI | ChemPort |
18. Kasai, M. (1974). Treatment of biliary atresia with special reference to hepatic porto-enterostomy and its modifications. Prog. Pediatr. Surg.. 6: 5–52. | PubMed | ChemPort |
19. Karrer, F. M., et al. (1996). Long-term results with the Kasai operation for biliary atresia. Arch. Surg.. 131: 493–496. | PubMed | ChemPort |
20. Stiehl, A., et al. (1990). Ursodeoxycholic acid-induced changes of plasma and urinary bile acids in patients with primary biliary cirrhosis. Hepatology. 12: 492–497. | PubMed | ChemPort |
21. Skulina, D., et al. (1999). The influence of ursodeoxycholic acid on some biochemical, immunologic and histopathologic parameters in patients with primary biliary cirrhosis. Przegl Lek.. 56: 201–204. | PubMed | ChemPort |
22. Murr, M., Gigot, J. F., Nagorney, D. M., Harmsen, W. S., Ilstrup, D. M. and Farnell, M. B. (1999). Long-term results of biliary reconstruction after laparoscopic bile duct injuries. Arch. Surg.. 134: 604–610. | PubMed | ChemPort |
23. Pickleman, J., Marsan, R. and Borge, M. (2000). Portoenterostomy: An old treatment for a new disease. Arch. Surg.. 135: 811–817. | PubMed | ChemPort |
24. Dickson, E. R., Grambsch, P. M., Fleming, T. R., Fisher, L. D. and Langoworthy, A. (1989). Prognosis in primary biliary cirrhosis: Model for decision making. Hepatology. 10: 1–7. | PubMed | ISI | ChemPort |
25. Miga, D., Sokol, R. J., Mackenzie, T., Narkewicz, M. R., Smith, D. and Karrer, F. M. (2001). Survival after first esophageal variceal hemorrhage in patients with biliary atresia. J. Pediatr.. 139: 291–296. | PubMed | ChemPort |
26. Bonnand, A. M., Heathcote, E. J., Lindor, K. D. and Poupon, R. E. (1999). Clinical significance of serum bilirubin levels under ursodeoxycholic acid therapy in patients with primary biliary cirrhosis. Hepatology. 29: 39–43. | Article | PubMed | ChemPort |
27. Shapiro, J. M., Smith, H. and Schaffner, F. (1979). Serum bilirubin: a prognostic factor in primary biliary cirrhosis. Gut. 20: 137–140. | PubMed | ChemPort |
28. Issa, R., et al. (2003). Mutation in collagen-I that confers resistance to the action of collagenase results in failure of recovery from CCl₄-induced in liver fibrosis, persistence of activated hepatic stellate cells, and diminished hepatocyte regeneration. FASEB J.. 17: 47–49. | PubMed | ChemPort |
29. Iimuro, Y., et al. (2003). Delivery of matrix metalloproteinase-1 attenuates established liver fibrosis in the rat. Gastroenterology. 124: 445–458. | Article | PubMed | ISI | ChemPort |

Acknowledgements

The authors greatly appreciate the support of personnel at the CUCS Animal Facilities, especially the help of Dr. Pedro Díaz. The authors are also indebted to Mario Cárdenas and Rosa Lina Torres-Rodríguez for their invaluable technical help and Ing. Rogelio Troyo for his support in carrying statistic analysis. This work was supported in part by a Grant from CONACyT, No. 28832-M, to J.A.B. and by PIHCSA Medica.