Search

Background Hepatocyte transplantation has been proposed as an alternative to orthotopic liver transplantation to treat metabolic liver diseases. This approach requires preconditioning of the host liver to enhance engraftment of transplanted hepatocytes. Different methods are currently used in preclinical models: partial hepatectomy, portal ligature or embolization, and radiotherapy or chemotherapeutic drugs. However, these methods carry high risks of complications and are problematic for use in clinical practice. Here, we developed an innovative method called volumetric (distal, partial, and random) portal embolization (VPE), which preserves total liver volume. Methods Embolization was performed in the portal trunk of C57BL6 adult mice with polyester microspheres, to ensure a bilateral and distal distribution. The repartition of microspheres was studied by angiographic and histological analyses. Liver regeneration was evaluated by Ki67 labeling. Optimal conditions for VPE were determined, and the resulting regeneration was compared with that after partial hepatectomy (70%). Labeled adult hepatocytes were then transplanted, and engraftment was compared between embolized (n = 19) and nonembolized mice (n = 8). Engraftment was assessed in vivo and histologically by tracking labeled cells at day 5. Results The best volumetric embolization conditions, which resulted in the regeneration of 5% of total liver, were 8 × 10 ten-micron microspheres infused with a 29 G needle directly into the portal trunk at 3.3 μL/s. In these conditions, transplanted hepatocytes engraftment was significantly higher than that in control conditions (3 vs 0.65%). Conclusions The VPE is a new, minimally invasive, and efficient technique to prepare the host liver for cell transplantation.

Innovative cell-based therapies are considered as alternatives to liver transplantation. Recent progress in lentivirus-mediated hepatocyte transduction has renewed interest in cell therapy for the treatment of inherited liver diseases. However, hepatocyte transplantation is still hampered by inefficient hepatocyte engraftment. We previously showed that partial portal vein embolization (PVE) improved hepatocyte engraftment in a nonhuman primate model. We developed here an ex vivo approach based on PVE and lentiviral-mediated transduction of hepatocytes from normal (New Zealand White, NZW) and Watanabe heritable hyperlipidemic (WHHL) rabbits: the large animal model of familial hypercholesterolemia type IIa (FH). FH is a life-threatening human inherited autosomal disease caused by a mutation in the low-density lipoprotein receptor (LDLR) gene, which leads to severe hyperc-holesterolemia and premature coronary heart disease. Rabbit hepatocytes were isolated from the resected left liver lobe, and the portal branches of the median lobes were embolized with Histoacryl® glue under radiologic guidance. NZW and WHHL hepatocytes were each labeled with Hoechst dye or transduced with lentivirus expressing GFP under the control of a liver-specific promoter (mTTR, a modified murine transthyretin promoter) and were then immediately transplanted back into donor animals. In our conditions, 65–70% of the NZW and WHHL hepatocytes were transduced. Liver repopulation after transplantation with the Hoechst-labeled hepatocytes was 3.5 ± 2%. It was 1.4 ± 0.6% after transplantation with either the transduced NZW hepatocytes or the transduced WHHL hepatocytes, which was close to that obtained with Hoechst-labeled cells, given the mean transduction efficacy. Transgene expression persisted for at least 8 weeks posttransplantation. Transduction of WHHL hepatocytes with an LDLR-encoding vector resulted in phenotypic correction in vitro as assessed by internalization of fluorescent LDL ligands. In conclusion, our results have applications for the treatment of inherited metabolic liver diseases, such as FH, by transplantation of lentivirally transduced hepatocytes.

International audience; Heterozygous mutations in JAGGED1, encoding a single-pass transmembrane ligand for the Notch receptors, cause Alagille syndrome (AGS), a polymalformative disorder affecting the liver, heart, eyes and skeleton and characterized by a peculiar facies. Most of the JAGGED1 mutations generate premature termination codons, and as a result, two pathogenic mechanisms causing AGS have been proposed: haploinsufficiency or a dominant-negative effect of putative truncated proteins. To determine whether missense or protein-truncating mutations in JAGGED1 can lead to the synthesis and function of abnormal proteins, we performed cell culture experiments. We showed that human JAGGED1 undergoes a metalloprotease-dependent cleavage resulting in the shedding of its extracellular domain and that this domain seems able to fulfill a biological function in vitro, probably by antagonizing Notch signaling. Moreover, the soluble form of JAGGED1 was able to compete with the transmembrane ligand. Mutant proteins with missense or nonsense mutations were synthesized and gave rise to a chord-like phenotype and a migration defect when expressed by stably transfected cells. These chord-like structures were similar to the phenotype exhibited by fibroblasts isolated from a fetus with a protein-truncating mutation. Results obtained from Notch signaling inhibition and Notch reporter assays showed that this chord-like phenotype, exhibited by mutant JAGGED1 transfectants, may result from an inhibitory effect on Notch signaling. Altogether, our results favor a dominant-negative mechanism of some JAGGED1 mutations in AGS.

Les recepteurs des gonadotrophines et de la TSH appartiennent a un sous-groupe de recepteurs couples aux proteines G qui presentent des particularites dans leur trafic cellulaire : les recepteurs de la TSH et de la FSH ont une distribution cellulaire polarisee basolaterale au niveau des cellules thyroidiennes et des cellules de Sertoli respectivement. Par contre, le recepteur de la LH est exprime de facon circonferentielle au niveau des cellules cibles gonadiques. Pour comprendre cette difference de propriete nous avons exprime ces recepteurs dans un modele de cellules epitheliales polarisees : les cellules MDCK.Nous avons ainsi montre que les trois recepteurs adoptent dans ces cellules une localisation basolaterale. Ils contiennent donc tous un signal de localisation basolaterale. Les recepteurs des gonadotrophines subissent de plus une transcytose partielle qui n’est pas observee pour le recepteur de la TSH. Nous avons montre que les proteines G heterotrimeriques jouent un role dans ce mecanisme de transcytose. Cet effet ne necessite pas une activation de l’adenylate cyclase et implique une population intracellulaire de proteines G differentes de celle qui intervient dans la transduction du signal.Nous avons ensuite identifie la nature du signal d’adressage basolateral du recepteur de la FSH par mutagenese dirigee. Nous avons montre de facon inattendue qu’il est localise dans la partie distale du domaine intracellulaire qui diverge entre les trois recepteurs. Il comprend 14 acides amines et est essentiellement dependant pour son activite d’une tyrosine et d’une leucine. Nous avons montre que ce signal est independant du signal d’endocytose du recepteur. Ce signal est autonome et transferable a une autre proteine membranaire apicale, le recepteur du NGF qu’il convertit en partie en une proteine basolaterale.Il s’agit du premier signal d’adressage basolateral caracterise pour un recepteur couple aux proteines G et plus generalement pour un recepteur hormonal.

In many organs the vascular endothelium forms a barrier which impedes the free diffusion of large molecules. The mechanism by which protein hormones are transported through the endothelial cells to reach their target cells is unknown. We have examined the transport of human chorionic gonadotropin (hCG) in rat testicular microvasculature by electron microscopy and by analysing the transfer of radiolabeled hormone and antibodies. Surprisingly, we have observed that the same receptor molecule which is present in target Leydig cells is also involved in transcytosis through the endothelial cells. The hormone was internalized by coated pits and vesicles on the luminal side of the endothelium. It was then localized in the endosomal compartment and subsequently appeared to be delivered by smooth vesicles into the subendothelial space. Moreover, anti-LH/hCG receptor antibodies were efficiently transported via the same system and delivered into the interstitial space. If generalized, these observations may define a new level of modulation of hormone action and may be of importance for drug targeting into the numerous organs which are responsive to the various protein hormones.

International audience; Transplantation of hepatocytes, whether genetically modified or not, has become an alternative to orthotopic liver transplantation for the treatment of patients with metabolic disease. However, more than ten years after the first clinical trial of ex vivo gene therapy to treat patients with Familial Hypercholesterolemia, there are still a number of impediments to these approaches. Numerous animal models are still being developed on the one hand to improve hepatocyte integration within hepatic parenchyma and function, and on the other hand to develop vectors that drive long-term transgene expression in situ. These include large animal models such as non-human primates, which have recently led to significant progress in hepatocyte transplantation. Simultaneous development of lentiviral vectors from different lentivirus species has permitted the transfer of genes into mitotically-quiescent primary cells including differentiated hepatocytes. Particularly third generation vectors derived from HIV-1 lentivirus are the most widely used and have significantly improved the safety and efficiency of these vectors. Given the shortage of organs and problems related to immunosuppression on one hand, and recent progresses in hepatocyte transduction and transplantation on the other hand, ex vivo approach is becoming a real alternative to allogeneic hepatocyte transplantation. We review the present progresses and limits of the ex vivo liver gene therapy approach in different animal models, emphasizing clinically relevant procedures.

The poor hepatocyte engraftment efficiency and the low level of their expansion in the host liver are a major limitation to cell therapy for the treatment of life-threatening liver diseases. Many rodent models have shown that liver repopulation via transplanted hepatocytes occurs only when liver growth capacity is impaired for an extended period of time. However, these models are not transposable to the clinics and to date there is no safe method to achieve this result in a clinical setting.Therefore, it is necessary to define on large animal models strategies that provide to transplanted hepatocytes sufficient proliferation stimuli to induce their division and that could permit a direct extrapolation to humans. Such procedures should be transposable to patients. We have defined a protocol of liver partial portal branch embolisation and shown that it induces the proliferation of transplanted hepatocytes in non-human primates (Macaca mulatta). This animal model is also appropriate to evaluate the lentiviral-mediated ex vivo gene therapy approach, since simian hepatocytes are efficiently transduced by HIV-1-derived lentivirus vectors.

International audience; The feasibility of ex vivo gene therapy as an alternative to liver transplantation for the treatment of liver metabolic diseases needs to be analyzed in large animal models. This approach requires appropriate gene transfer vectors and effective hepatocyte engraftment. Lentiviral vectors have the ability to transduce nondividing differentiated cells, such as hepatocytes, and portal vein occlusion increases hepatocyte engraftment. We investigated whether reversible portal vein embolization combined with ex vivo lentivirus-mediated gene transfer is an effective approach for successful hepatocyte engraftment in nonhuman primates and whether the transgene remains expressed in the long term in transplanted hepatocytes in situ. Simian hepatocytes were isolated after left lobe resection, and the left and right anterior portal branches of animals were embolized with absorbable material. Isolated hepatocytes were labeled with Hoechst dye or transduced in suspension with lentiviruses expressing green fluorescent protein under the control of the human apolipoprotein A-II promoter and transplanted via the inferior mesenteric vein. The whole procedure was well tolerated. The embolized liver was revascularized within 2 weeks. The volume of nonembolized liver increased from 38.7% +/- 0.8% before embolization to 55.9% +/- 1% after embolization and hepatocytes significantly proliferated (10.5% +/- 0.4% on day 3 after embolization). Liver repopulation after transplantation with Hoechst-labeled hepatocytes was 7.4% +/- 1.2%. Liver repopulation was 2.1% +/- 0.2% with transduced hepatocytes, a proportion similar to that obtained with Hoechst-labeled cells, given that the mean transduction efficacy of simian hepatocyte population was 34%. Transgene expression persisted at 16 weeks after transplantation. Conclusion: We have developed a new approach to improve hepatocyte engraftment and to express a transgene in the long term in nonhuman primates. This strategy could be suitable for clinical applications. (HEPATOLOGY 2009.).

The poor hepatocyte engraftment efficiency and the low level of their expansion in the host liver are a major limitation to cell therapy for the treatment of life-threatening liver diseases. Many rodent models have shown that liver repopulation via transplanted hepatocytes occurs only when liver growth capacity is impaired for an extended period of time. However, these models are not transposable to the clinics and to date there is no safe method to achieve this result in a clinical setting.Therefore, it is necessary to define on large animal models strategies that provide to transplanted hepatocytes sufficient proliferation stimuli to induce their division and that could permit a direct extrapolation to humans. Such procedures should be transposable to patients. We have defined a protocol of liver partial portal branch embolisation and shown that it induces the proliferation of transplanted hepatocytes in non-human primates (Macaca mulatta). This animal model is also appropriate to evaluate the lentiviral-mediated ex vivo gene therapy approach, since simian hepatocytes are efficiently transduced by HIV-1-derived lentivirus vectors.

International audience; BACKGROUND: Hepatocyte transplantation could be an alternative to whole liver transplantation for the treatment of metabolic liver diseases. However, the results of clinical investigations suggest that the number of engrafted hepatocytes was insufficient to correct metabolic disorders. This may partly result from a lack of proliferation of transplanted hepatocytes. In rodents, portal ligation enhances hepatocyte engraftment after transplantation. We investigated the effects of partial portal ligation and embolization on engraftment and proliferation of transplanted hepatocytes in primates. METHODS: Hepatocyte autotransplantation was performed in Macaca monkeys. The left lateral lobe was resected for hepatocyte isolation. The first group of monkeys underwent surgical ligation of the left and right anterior portal branches; in the second group, the same portal territories were obstructed by embolization with biological glue. To evaluate the proportion of cell engraftment hepatocytes were Hoechst-labeled and transplanted via the portal vein. Cell proliferation was measured by BrdU incorporation. RESULTS: Hepatocyte proliferation was induced by both procedures but it was significantly higher after partial portal embolization (23.5% and 11.2% of dividing hepatocytes on days 3 and 7) than after ligation (3% and 0.8%). Hepatocytes engrafted more efficiently after embolization than after ligation. They proliferated and participated to liver regeneration representing 10% of the liver mass on day seven and their number remained constant on day 15. CONCLUSIONS: These data suggest that partial portal embolization of the recipient liver improves engraftment of transplanted hepatocytes in a primate preclinical model providing a new strategy for hepatocyte transplantation.

International audience; BACKGROUND/AIMS: Lentivirus-mediated ex vivo gene therapy is becoming a promising approach for the treatment of liver metabolic disorders. However, the feasibility of this approach needs to be studied in large animal models. The purpose of this study was to evaluate the efficacy of ex vivo gene transfer into Macaca hepatocytes with two different HIV-1 derived lentiviral vectors. METHODS: A self-inactivating lentivector was constructed to express GFP under the control of the hepatic apolipoprotein A-II promoter. Freshly isolated and thawed hepatocytes were transduced in suspension with lentiviral vectors expressing the GFP gene under the control of a ubiquitous promoter (EF1-alpha) and the apolipoprotein A-II promoter. Transduced thawed hepatocytes were transplanted into the spleen of newborn mice, and livers analyzed 4 and 12 weeks after transplantation. RESULTS: We show that lentivectors are efficient in transducing hepatocytes in suspension either freshly isolated or cryopreserved. We also show that thawed and transduced hepatocytes engrafted and participated in liver growth after transplantation into newborn mice and that the apolipoprotein A-II promoter is functional. CONCLUSIONS: Our data show that transplantation of transduced hepatocytes into monkeys should allow to evaluate the fate of transplanted cells and transgene expression in a pre-clinical model of ex vivo gene therapy.

The follicle-stimulating hormone receptor (FSHR) is physiologically localized in the basolateral compartment of the membrane of Sertoli cells. This localization is also observed when the receptor is experimentally expressed in Madin-Darby canine kidney cells. We thus used in vitro mutagenesis and transfection into these polarized cells to delineate the basolateral localization signal of the receptor. The signal was localized in the C-terminal tail of the intracellular domain (amino acids 678–691) at a marked distance of the membrane. Mutation of individual amino acids highlighted the importance of Tyr684 and Leu689. The 14-amino acid sequence was grafted onto the p75 neurotrophin receptor and redirected this apical protein to the basolateral cell membrane compartment. Deletion of amino acids 677–695 did not modify the internalization of the FSHR, showing that the basolateral localization signal of the FSHR is not colinear with its internalization signal.

Monoclonal anti-receptor antibodies were used to study the cellular traffic of the hCG/LH receptor by immunoelectron microscopy. The LHR38 antibody was shown to bind to the extracellular domain of the receptor but not to interfere with hormone binding, adenylate cyclase activation or with the rate of internalization of the receptor. Pig Leydig cells and a permanent L-cell line expressing the LH receptor were used for the study. Incubation with LHR38-gold complexes showed the LH receptors to be randomly distributed over the cell surface including the clathrin coated pits. The LH receptors were internalized via a route including coated pits, coated vesicles and multivesicular bodies to lysosomes. This route is different from that observed for beta-adrenergic, muscarinic, and yeast mating factor receptors and considered previously as possibly general for G-protein-coupled receptors. The use of [125I]LHR38 allowed precise measurement of the rate of internalization, showing the existence of a constitutive pathway which was increased 11-fold by hormone administration. Double labeling experiments suggested that the hormone (hCG-Au15nm) and the receptor (labeled with LHR38-Au5nm) have similar routes of endocytosis, both of them being degraded in lysosomes. Studies of the reappearance of LHR38-Au5nm on the surface of the cells and the use of monensin indicated that only a very small proportion of the receptor molecules were recycled to the cell surface. The distribution and the intracellular pathways of LH receptors are very similar in Leydig cells and transfected L-cells. This opens the possibility of using the latter to study, by in vitro mutagenesis, the molecular mechanisms involved in the cellular traffic of LH receptors.

Over 90 mouse monoclonal antibodies have been raised against rabbit and human uterine progesterone receptor (PR). These antibodies, because of their specificity, are powerful tools with which to examine the localization, structure, and function of PR. A selection of 22 well characterized mABs was made to test their ability to give the best immunocytochemical staining of PR in various species. Comparative analysis of the antibodies led to the following conclusions. Li 417 (and, to a lesser extent, Let 126) was the best monoclonal in humans; Let 126 and Mi 60 were the most sensitive monoclonals in guinea pigs, rabbits, and monkeys. In sheep, sows, cows and mares as well as in rats and chickens Let 81 or Let 548 gave the best results (Let 126 was also effective in sows and mares, while Li 169 was also effective in sheep and cows). Two antibodies (Li 169 and Let 548) cross-reacted with PR in all of the species tested, including mammals and birds, and appeared to recognize two highly conserved antigenic sites. Remarkably, these conserved sequences are located in the highly variable N-terminal part of the receptor; they may, thus, be related to the still poorly understood function of this domain of the receptor.

Five monoclonal antibodies were used for the immunocytochemical study of mammalian progesterone receptor (PR). Initial studies were aimed at defining the optimal experimental conditions for the detection of the receptor, with special emphasis on techniques likely to be used in clinical determinations and in immunoelectron microscopic localization. Specific immunoperoxidase staining was observed either in fixed, frozen sections or in sections of paraffin-embedded tissue. The latter method allowed a better preservation of cellular structures. Among the eight fixatives tested, glutaraldehyde, picric-acid formaldehyde, and paraformaldehyde proved satisfactory. Both indirect immunoperoxidase and the indirect antibody peroxidase-antiperoxidase methods could be used. In immature rabbits or castrated guinea pigs primed by estrogen, i.e. in conditions where its ligand was absent (or present in very low concentration), the PR was confined to the nucleus of immunoreactive cells. This was the case for all the cell types of the endometrium and the myometrium, for the immunostained cells of the oviduct, cervix, vagina, pituitary gland, and for the very weakly stained cells in the liver. No staining was observed in nontarget tissues for progesterone, such as diaphragm, spleen, and small intestine. Nuclear staining was also absent when various control antibodies replaced anti-PR antibodies. This result thus generalizes the observations made on the estrogen receptor, showing that there is no translocation of the receptor from cytoplasm to nucleus under the influence of the hormone. Moreover, a marked heterogeneity in immunostaining was observed among cells of the same type in several tissues, suggesting that there could be large differences in the hormonal sensitivity of individual cells. Cellular distribution of PR immunoreactivity was studied in the uterus, cervix, oviduct, and pituitary gland of rabbits and in the uterus and vagina of guinea pigs. A labeling was observed in all the cell types of the uterus (luminal and glandular epithelium, stroma, and muscularis). In the cervix, nuclear immunostaining was observed in the connective tissue of the lamina propria and in some epithelial and muscle cells. In the vagina, PR immunoreactivity was seen in the basal layers of the stratified squamous epithelium, in the connective tissue of the lamina propria, and in the smooth muscle. In the oviduct, the luminal epithelium, the connective tissue, and the muscularis were stained. In the pituitary gland, selective nuclear labeling was observed in a few scattered cells.

The progesterone receptor has been localized in the rabbit uterus by immunocytochemistry at the electron microscopic level, using monoclonal antibodies and the protein A-gold technique. The progesterone receptor in uterine stromal cells was mainly localized in the nucleus; however, a small fraction of antigen was present in the cytoplasm, where it was associated with the rough endoplasmic reticulum and with free ribosomes. The plasma membrane was not labeled. In the nucleus, the receptor was always associated with condensed chromatin or areas surrounding condensed chromatin, whereas the nuceolus was not labeled. In the chromatin, receptor distribution varied according to the hormonal state: in the absence of progesterone, the receptor was randomly scattered over the clumps of condensed chromatin; after administration of the progestin R5020, it was mainly detected in the border regions between condensed chromatin and nucleoplasm and, to a lesser extent, over dispersed chromatin in the nucleoplasm. These areas have been shown to be the most active sites of gene transcription.

Modifications of uterine blood flow are implicated in many important aspects of reproductive physiology and in several of their pathological disorders. These modifications are hormonally regulated but remain poorly understood, and various complex mechanisms have been proposed. The aim of this study was to investigate the presence and some characteristics of estrogen receptors (ER) and progesterone receptors (PR) in uterine blood vessels. Using monoclonal antibodies and immunocytochemistry we observed the presence of ER and PR in muscle cells (tunica media) of uterine arteries of rabbits and women. ER or PR immunoreactivity was not detected in the endothelium of uterine arteries nor in uterine capillaries or veins. Staining for both receptors was also present in arterial walls from the fallopian tube (isthmus and ampulla) and vagina but not in arteries of nonreproductive tissues (intestinal, renal, hepatic, femoral, and pulmonary arteries, aorta). PR immuno-staining was increased by estrogen in all cell types of the rabbit uterus, but the doses necessary to provoke an intense nuclear staining in uterine arteries were higher than those required for observing strong labeling in glandular, stromal, or myometrial cells. These results suggest that, contrary to many hypotheses previously put forward, sex steroid hormones may regulate uterine blood flow through a direct effect on uterine arterial walls.