Correction of liver disease following transplantation of normal rat hepatocytes into Long-Evans Cinnamon rats modeling Wilson's disease.

To establish the efficacy of cell therapy in Wilson's disease, we used the Long-Evans Cinnamon (LEC) rat model with atp7b gene mutation and copper toxicosis. Several groups of LEC rats were established, including animals pretreated with retrorsine to exacerbate copper toxicosis and inhibit proliferation in native hepatocytes followed by partial hepatectomy to promote liver repopulation. Hepatocytes from normal, syngeneic LEA rats were transplanted intrasplenically. Animal survival, biliary copper excretion, and hepatic copper were determined. The magnitude of liver repopulation was demonstrated by measuring serum ceruloplasmin and hepatic atp7b mRNA. Long-term survival in LEC rats treated with retrorsine, partial hepatectomy, and cell transplantation was up to 90%, whereas fewer than 10% of animals pretreated with retrorsine, without cell therapy, survived, P < 0.001. Liver repopulation occurred gradually after cell transplantation, ranging from <25% at 6 weeks, 26 to 40% at 4 months, and 74 to 100% at 6 months or beyond. Liver repopulation restored biliary copper excretion capacity and lowered liver copper levels. Remarkably, liver histology was completely normal in LEC rats with extensive liver repopulation, compared with widespread megalocytosis, apoptosis, oval cell proliferation, and cholangiofibrosis in untreated animals. These data indicate that liver repopulation with functionally intact cells can reverse pathophysiological perturbations and cure Wilson's disease.

KATP channels regulate mitogenically induced proliferation in primary rat hepatocytes and human liver cell lines. Implications for liver growth control and potential therapeutic targeting.

To determine whether K(ATP) channels control liver growth, we used primary rat hepatocytes and several human cancer cell lines for assays. K(ATP) channel openers (minoxidil, cromakalim, and pinacidil) increased cellular DNA synthesis, whereas K(ATP) channel blockers (quinidine and glibenclamide) attenuated DNA synthesis. The channel inhibitor glibenclamide decreased the clonogenicity of HepG2 cells without inducing cytotoxicity or apoptosis. To demonstrate the specificity of drugs for K(+) channels, whole-cell patch-clamp recordings were made. Hepatocytes revealed K(+) currents with K(ATP) channel properties. These K(+) currents were augmented by minoxidil and pinacidil and attenuated by glibenclamide as well as tetraethylammonium, in agreement with established responses of K(ATP) channels. Reverse transcription of total cellular RNA followed by polymerase chain reaction showed expression of K(ATP) channel-specific subunits in rat hepatocytes and human liver cell lines. Calcium fluxes were unperturbed in glibenclamide-treated HepG2 cells and primary rat hepatocytes following induction with ATP and hepatocyte growth factor, respectively, suggesting that the effect of K(ATP) channel activity upon hepatocyte proliferation was not simply due to indirect modulation of intracellular calcium. The regulation of mitogen-related hepatocyte proliferation by K(ATP) channels advances our insights into liver growth control. The findings have implications in mechanisms concerning liver development, regeneration, and oncogenesis.

Biliary copper excretion capacity in intact animals: correlation between ATP7B function, hepatic mass, and biliary copper excretion.

Copper toxicosis can occur in the absence of biliary copper excretion. To demonstrate whether biliary copper excretion capacity is correlated with hepatic mass and ATP7B function, we undertook studies in intact animals. Copper-histidine was injected intrasplenically after baseline bile collection, followed by measurement of copper excretion in Long-Evans Cinnamon (LEC) rats lacking atp7b function and in normal, syngeneic Long-Evans Agouti (LEA) rats. The basal biliary copper excretion was very low in LEC rats compared with LEA rats, 8+/-4 and 37+/-18 ng copper/min, respectively; p<0.05. After addition of copper, copper excretion increased significantly (by two- to five-fold) in LEA rats during the 30 minute study period, whereas LEC rats showed only a slight and transient increase in copper excretion. After one-third and two-thirds partial hepatectomy immediately before copper loading, copper excretion decreased progressively. The studies indicate that biliary copper excretion depends on hepatocyte mass and ATP7B gene function. Analysis of copper excretion with our non-radioactive method will facilitate testing of novel therapies and pathophysiological mechanisms in copper toxicity.

Integration and proliferation of transplanted cells in hepatic parenchyma following D-galactosamine-induced acute injury in F344 rats.

To determine whether liver repopulation with cell transplantation could be of therapeutic value in acute hepatic failure, it is necessary to establish the fate of transplanted hepatocytes. This study used dipeptidyl peptidase IV-deficient F344 rats as recipients to analyse the engraftment and proliferation of transplanted hepatocytes. Syngeneic hepatocytes were transplanted intrasplenically 24-30 h after induction of liver injury by D-galactosamine (GalN). Portosystemic shunting was analysed with 99m-Tc-labelled albumin microspheres. GalN-treated rats showed characteristic hepatic necrosis, inflammation, gamma-glutamyl transpeptidase activation, and regenerative activity, without increased portosystemic shunting (>99% 99m-Tc activity was in the liver in normal and GalN-treated rats). Transplanted cells entered hepatic sinusoids promptly and were observed in liver plates at 48 h. The number of transplanted cells increased in GalN-treated rats by approximately seven-fold (range two- to 12-fold), along with evidence for DNA synthesis between 3 and 14 days after cell transplantation and greater prevalence of larger transplanted cell clusters. These findings indicate that the liver can be safely repopulated in animals with acute liver failure, although the time required for regenesis of plasma membrane structures and proliferation in transplanted hepatocytes will need to be considered in developing therapeutic strategies.

Position-specific gene expression in the liver lobule is directed by the microenvironment and not by the previous cell differentiation state.

Mechanisms directing position-specific liver gene regulation are incompletely understood. To establish whether this aspect of hepatic gene expression is an inveterate phenomenon, we used transplanted hepatocytes as reporters in dipeptidyl peptidase IV-deficient F344 rats. After integration in liver parenchyma, the position of transplanted cells was shifted from periportal to perivenous areas by targeted hepatic ablations with carbon tetrachloride. In controls, transplanted cells showed greater glucose-6-phosphatase and lesser glycogen content in periportal areas. This pattern was reversed when transplanted cells shifted from periportal to perivenous areas. Transplanted hepatocytes in perivenous areas exhibited inducible cytochrome P450 activity, which was deficient in periportal hepatocytes. Moreover, cytochrome P450 activity was rapidly extinguished in activated hepatocytes when these cells were transplanted into the nonpermissive liver of suckling rat pups. In cells isolated from the normal F344 rat liver, cytochrome P450 inducibility was originally greater in perivenous hepatocytes; however, periportal cells rapidly acquired this facility in culture conditions. These findings indicate that the liver microenvironment exerts supremacy over prior differentiation state of cells in directing position-specific gene expression. Therefore, persistence of specialized hepatocellular function will require interactions with regulatory signals and substrate availability, which bears upon further analysis of liver gene regulation, including in progenitor and/or stem cells.