The inability of transplanted cells to proliferate in the normal liver hampers cell therapy. We considered that oxidative hepatic DNA damage would impair the survival of native cells and promote proliferation in transplanted cells. Dipeptidyl peptidase-deficient F344 rats were preconditioned with whole liver radiation and warm ischemia-reperfusion followed by intrasplenic transplantation of syngeneic F344 rat hepatocytes. The preconditioning was well tolerated, although serum aminotransferase levels rose transiently and hepatic injury was observed histologically, along with decreased catalase activity and 8-hydroxy adducts of guanine, indicating oxidative DNA damage. Transplanted cells did not proliferate in the liver over 3 months in control animals and animals preconditioned with ischemia-reperfusion alone. Animals treated with radiation alone showed some transplanted cell proliferation. In contrast, the liver of animals preconditioned with radiation plus ischemia-reperfusion was replaced virtually completely over 3 months. Transplanted cells integrated in the liver parenchyma and liver architecture were preserved normally. These findings offer a paradigm for repopulating the liver with transplanted cells. Progressive loss of cells experiencing oxidative DNA damage after radiation and ischemia-reperfusion injury could be of significance for epithelial renewal in additional organs.oxidative damage ͉ hepatocyte L iver repopulation with transplanted cells is of considerable interest for cell and gene therapy (1). Transplanted hepatocytes integrate in the liver parenchyma, function normally, and survive life-long (2-4). However, transplanted cells do not proliferate in the normal adult liver, whereas specific therapies require a significant transplanted cell mass. Proliferation in transplanted cells depends on whether native cells are at survival͞proliferation disadvantages, as suggested by animal studies using exogenous toxins or natural disease, e.g., fumaryl acetoacetate hydroxylase (FAH) mice (hereditary tyrosinemia type-1), Long-Evans Cinnamon (LEC) rats (Wilson's disease), P-glycoprotein-2 (Pgy-2) mutant mice (progressive familial intrahepatic cholestasis), etc. (5-12). Initial clinical studies in familial hypercholesterolemia (FH) or Crigler-Najjar syndrome substantiated these principles (13,14).Genotoxic liver injury is a potent stimulus for transplanted cell proliferation. Rats exposed to retrorsine, a pyrrolizidine alkaloid, or whole liver radiation (RT), which produce DNA adducts and oxidative injury, respectively, lead to extensive transplanted cell proliferation in conjunction with two-thirds partial hepatectomy (PH) (15, 16). Although PH induces hepatic DNA synthesis, its additional effects include oxidative DNA damage, senescence-type changes, including p21 expression, polyploidy, attenuated proliferation capacity, and hepatocyte apoptosis (17-19). Both retrorsine and RT increase PH-induced hepatic polyploidy and apoptosis (20,21). Moreover, the thyroid hormone T3, which regulates PH-induced polyplo...

To determine whether disruption of the hepatic sinusoidal endothelium will facilitate engraftment of transplanted cells, we treated Fischer 344 (F344) rats lacking dipeptidyl peptidase IV (DPPIV) activity with cyclophosphamide (CP). Electron microscopy showed endothelial injury within 6 hours following CP, and, after 24 and 48 hours, the endothelium was disrupted in most hepatic sinusoids. CP did not affect Kupffer cell function. Similarly, CP had no obvious effects on hepatocytes. Intrasplenic transplantation of F344 rat hepatocytes followed by their localization with DPPIV histochemistry showed 3-to 5-fold increases in the number of transplanted cells in CP-treated animals. Transplanted cells integrated in the liver parenchyma more rapidly in CPtreated animals, and hybrid bile canaliculi developed even 1 day after cell transplantation, which was not observed in control animals. To demonstrate whether improved cell engraftment translated into superior liver repopulation, recipient animals were conditioned with retrorsine and two-thirds partial hepatectomy (PH), which induces transplanted cell proliferation. CP treatment of these animals before cell transplantation significantly increased the number and size of transplanted cell foci. In conclusion, disruption of the hepatic sinusoidal endothelium was associated with accelerated entry and integration of transplanted cells in the liver parenchyma. These results provide insights into hepatocyte engraftment in the liver and will help in optimizing liver-directed cell therapy. ( T ransplanted hepatocytes integrate in the liver, survive throughout the life of animals, and express genes normally. 1-3 However, several days are required for transplanted cells to integrate in the liver parenchyma. 4 Moreover, transplanted cells do not proliferate significantly in the normal adult liver, 3 although, when proliferation and/or survival of native and transplanted cells are dissociated, transplanted hepatocytes can repopulate the liver extensively. [5][6][7][8][9][10] The possibility of therapeutic liver repopulation has been tested in several animal models with liver failure, genetic diseases, and metabolic deficiency states. [11][12][13][14] The swiftness with which transplanted cells engraft could be critical for liver repopulation. After injection into the portal system, transplanted cells lodge in hepatic sinusoids because of differences in the dimensions of these cells and sinusoids. 15 This is associated with changes in native hepatocytes, such as gamma-glutamyl transpeptidase activation and disruption of gap junctions. 16 The sinusoidal endothelium is disrupted 16 to 20 hours after transplantation, which provides transplanted cells access to the space of Disse. 4 Transplanted cells then translocate into the liver plate, with reconstitution of gap junctions and bile canaliculi over 3 to 7 days. However, delays in cell engraftment affect transplanted cell proliferation. In rats subjected to two-thirds partial hepatectomy (PH), which induces hepatic DNA synthesis within ...

Protein expression patterns were analyzed in a rat model of hepatic neoplasia to detect changes reflecting biological mechanism or potential therapeutic targets. The rat resistant hepatocyte model of carcinogenesis was studied, with a focus on the earliest preneoplastic lesion visible in the liver, the preneoplastic hyperplastic nodule. Expression differences were shown by two-dimensional polyacrylamide gel electrophoresis and image analysis. Polypeptide masses were measured by peptide mass fingerprinting using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF) and their sequences were obtained by tandem mass spectrometry. Alterations in expression of cytoskeletal and functional proteins were demonstrated, consistent with biological changes known to occur in the preneoplastic cells. Of particular interest was the differential expression of a serine protease inhibitor (serpin) with a role implicated in angiogenesis. Serpin, implicated in the inhibition of angiogenesis, is present in normal liver but has greatly reduced expression at the preneoplastic stage of liver cancer development. Immunofluorescence microscopy with antibodies to this serpin, kallistatin, supports the proteomic identification. Immunofluorescence microscopy with antibodies to the blood vessel marker von Willebrand factor provides evidence for neovascularization in the liver containing multiple preneoplastic nodules. These observations suggest that at an early stage of liver carcinogenesis reduction or loss of angiogenesis inhibitors may contribute to initiation of neoangiogenesis. A number of other identified proteins known to be associated with hepatomas are also present at early-stage neoplasia.