The difference between the number of patients needing transplantation for chronic liver disease and the number of organ donors is growing, drawing attention to the urgent requirement for novel therapies. Chronic liver injury is commonly caused by viral hepatitis, alcohol consumption, obesity and metabolic disorders. Prolonged liver injury leads to fibrosis, hepatic scarring and eventually cirrhosis. This project is based on previous studies demonstrating the therapeutic effects of bone marrow-derived macrophages (BMDM) in a murine model of liver fibrosis. BMDM facilitated fibrosis regression and improved liver regeneration. Pro-resolution macrophages exhibited increased expression of MMPs, growth factors and phagocytosis-related genes. However, macrophages derived from bone marrow are inherently heterogeneous and difficult to genetically manipulate. To overcome this limitation, our laboratory has established a protocol whereby pure populations of macrophages can be produced in significant numbers from murine embryonic stem cells (ESC) in vitro, providing an essentially limitless source of macrophages. The first goal of this project was to compare macrophages derived from ESCs (ESDM) with classical BMDM. ESDM displayed characteristic macrophage morphology, could be activated and responded to different cytokines in vitro, and were functionally phagocytic. However, they displayed some differences in their gene expression profile, and were found to be less phagocytic than BMDM. We then assessed whether ESDM could be used in the treatment of a murine model of hepatic injury induced by carbon tetrachloride administration. ESDM therapy helped in the regression of liver fibrosis, down-regulated the number of fibrogenic myofibroblasts, and activated liver progenitor cells. However, a higher number of ESDM compared to BMDMs were required to exert that effect. To assess whether ESDM may be similar to yolk sac derived tissue-resident macrophages, rather than monocyte-derived, we compared their behaviour in a Kupffer cell repopulation assay. Macrophages were depleted using liposomal clodronate treatment then animals were transplanted with either ESDM or BMDM. We demonstrated that ESDM were more efficient than BMDM at repopulating the Kupffer cell compartment and reversing the effects of liposomal clodronate treatment in mice. It is well known that macrophages are very difficult to genetically modify. So our strategy was to genetically modify ESC and then differentiate them to macrophages that carry the modification. By genetically modifying ESCs, we attempted to produce pro-fibrolytic ESDM that over-express MMP12 which is a member of the matrix metalloproteinase family of genes that mainly degrades elastin, an extracellular matrix component. We initially employed a Tet-On 3G expression system to create an ESC line where MMP12 could be expressed in an inducible manner in differentiated macrophages. However, although this inducible strategy functioned in undifferentiated ESCs we could not induce the expression of MMP12 in differentiated macrophages. In an attempt to overcome possible gene-silencing issues, we designed and constructed an expression strategy such that Mmp12 was expressed specifically in macrophages. The ESC line was built such that Mmp12 expression would be driven by the promoter of macrophage colony stimulating factor-1 receptor gene (Csf-1r or c-fms). Using the CRISPR/Cas9 strategy, we successfully targeted the Mmp12 cDNA to the Csf-1r locus but ESDM that were differentiated from targeted ESC lines did not express Mmp12. Thus, despite having adopted two independent strategies, we have failed to generate genetically modified macrophages. As a first step to translate the therapeutic effects of macrophages into the clinical setting, we optimized a feeder- and serum-free protocol to efficiently generate macrophages from human induced pluripotent stem cells.

Les travaux antérieurs du laboratoire ont montré que la Reptine, une AAA+ ATPase, est surexprimée dans le carcinome hépatocellulaire où elle est nécessaire à la prolifération et la survie cellulaire. Il est connu que la Reptine joue un rôle crucial dans la stabilité de la kinase mTOR, mais son rôle physiopathologique in vivo reste inconnu. Les objectifs de ma thèse étaient d’étudier le rôle de la Reptine dans le métabolisme et la régénération hépatique grâce à un nouveau modèle murin d’invalidation hépato-spécifique de la Reptine (Reptin LKO). Nous avons montré que la Reptine régule la stabilité de la protéine mTOR in vivo, via son activité ATPase. De manière inattendue, la délétion ou l’inhibition pharmacologique de la Reptine induisent une inhibition de l’activité mTORC1 et une augmentation de l’activité mTORC2, associées à une inhibition de la lipogenèse et de la production de glucose hépatique. La délétion de la Reptine supprime complètement les phénotypes pathologiques associés au syndrome métabolique induit par un régime riche en graisses. Ainsi, l’inhibition de l’ATPase Reptine pourrait représenter une nouvelle stratégie thérapeutique pour le syndrome métabolique. Dans le modèle Reptin LKO, nous avons observé une perte progressive de l’invalidation de la Reptine associée à un phénomène de régénération hépatique. Nos résultats préliminaires suggèrent que la Reptine est nécessaire à la survie des hépatocytes et est requise pour la prolifération des hépatocytes durant la régénération hépatique après hépatectomie partielle. Pour conclure, l’ensemble de nos résultats suggèrent que la Reptine joue un rôle crucial dans l’homéostasie glucido-lipidique du foie, ainsi que dans la prolifération et la survie des hépatocytes. / Previous studies of the laboratory have shown that Reptin, an AAA+ ATPase, is overexpressed in hepatocellular carcinoma where it is necessary for proliferation and cell survival. It is known that Reptin plays a critical role in the stabilization of the mTOR kinase, but its pathophysiological role in vivo remains unknown. The objectives of my thesis were to study the role of Reptin in liver metabolism and regeneration using a new hepato-specific Reptin knock-out murine model (Reptin LKO). We have shown that hepatic Reptin maintains mTOR protein level in vivo through its ATPase activity. Unexpectedly, loss or pharmacological inhibition of Reptin induces an inhibition of mTORC1 activity and an increase of mTORC2 activity, associated with inhibition of lipogenesis and hepatic glucose production. The deletion of Reptin completely rescued pathological phenotypes associated with the metabolic syndrome induced by a high fat diet. Thus, inhibition of Reptin ATPase could represent a new therapeutic perspective for the metabolic syndrome. In Reptin LKO model, we have observed a progressive loss of Reptin invalidation associated with a liver regeneration phenomenon. Our preliminary data suggest that Reptin is necessary for hepatocyte survival and is required for hepatocyte proliferation during liver regeneration after partial hepatectomy. To conclude, altogether our results suggest that Reptin plays a crucial role in glucose and lipid metabolism in the liver, and in hepatocyte proliferation and survival.

Thesis (Ph. D.)--University of Washington, 2001. / Vita. Includes bibliographical references (leaves 81-96).

Liver possesses the capacity to restore its tissue mass and attain optimal volume in response to physical, infectious and toxic injury. The extraordinary ability of liver to regenerate is the effect of cross-talk between growth factors, cytokines, matrix components and many other factors. Adult liver normally has a very low level of hepatocyte cell division. However, most hepatocytes will rapidly proliferate in response to liver mass reduction. In this study the differences in gene expression in rat liver at 1, 2, 4 and 6 hours after the partial hepatectomy were analyzed using cDNA microarrays. These time points correspond to quiescent liver and early G1 phase in this system. The experiments showed that 309 genes had significantly altered expression for at least one of the analyzed time-points. Differentially expressed genes were clustered according to their temporal expression profiles. PAINT analysis identified several TF binding sites (~20) enriched in specific individual expression patterns. We have obtained time series data on the activity of nine transcription factors (NF-κB, HNF-1, CREB, C/EBP-α, C/EBP-β, AP-2α, PAX-6, GATA-1, STAT-3) that were implicated in our analysis. An early transient activation of NF-κB, HNF-1 and C/EBP-β was observed. By contrast, C/EBP-α rapidly declined. Chromatin immunoprecipitation (ChIP) assay was performed to examine the binding of NF-κB to the promoter regions of Sod2, Mt1a and Cebpb. We see increased NF-κB binding to both Sod2 and... [to full text] / Infekcijos, toksinų ar fizinių veiksnių pažeistos kepenys gali gana greitai atstatyti prarastą audinio masę. Mitozė vyksta tik labai nedidelėje suaugusio organizmo kepenų ląstelių dalyje. Tačiau sumažėjus kepenų masei hepatocitai ima sparčiai proliferuoti. Kepenų gebėjimą regeneruoti lemia sudėtinga augimo veiksnių, citokinų ir kitų ląstelės veiklą reguliuojančių junginių sąveika. Šiame darbe buvo tiriami genų raiškos pokyčiai žiurkės kepenų ląstelėse pradiniame kepenų regeneracijos tarpsnyje. Genų raiškai nustatyti buvo panaudotos DNR mikrogardelės. Buvo nustatyti 309 genai, kurių raiška patikimai pakinta per pirmąsias 6 valandas po dalinės hepatektomijos operacijos. Nustatytieji 309 genai buvo suskirstyti į šešias grupes pagal laikinius raiškos pokyčius. PAINT programos pagalba buvo nustatyti 22 transkripcijos veiksniai, galimai lemiantys laikinius genų raiškos pokyčius po dalinės hepatektomijos operacijos. Devynių transkripcijos veiksnių (NF-κB, GATA-1, HNF-1, C/EBP-α ir C/EBP-β, STAT-3, PAX-6, AP-2α ) aktyvumo pokyčiai buvo įvertinti eksperimentiškai. Nustatyta, kad aktyvių NF-κB, HNF-1 ir C/EBP-β kiekis hepatocitų branduoliuose staigiai padidėja po dalinės hepatektomijos operacijos. Priešingai, aktyvios C/EBP-α formos kiekis sumažėja. Chromatino imunoprecipitacijos (ChIP) metodu buvo tiriamas NF-κB jungimasis prie Sod2, Mt1a ir Cebpb promotorių sričių praėjus 1 valandai po hepatektomijos. Regeneruojančių kepenų mėginiuose rasta daugiau NF-κB prisijungusio prie Sod2... [toliau žr. visą tekstą]

Infekcijos, toksinų ar fizinių veiksnių pažeistos kepenys gali gana greitai atstatyti prarastą audinio masę. Mitozė vyksta tik labai nedidelėje suaugusio organizmo kepenų ląstelių dalyje. Tačiau sumažėjus kepenų masei hepatocitai ima sparčiai proliferuoti. Kepenų gebėjimą regeneruoti lemia sudėtinga augimo veiksnių, citokinų ir kitų ląstelės veiklą reguliuojančių junginių sąveika. Šiame darbe buvo tiriami genų raiškos pokyčiai žiurkės kepenų ląstelėse pradiniame kepenų regeneracijos tarpsnyje. Genų raiškai nustatyti buvo panaudotos DNR mikrogardelės. Buvo nustatyti 309 genai, kurių raiška patikimai pakinta per pirmąsias 6 valandas po dalinės hepatektomijos operacijos. Nustatytieji 309 genai buvo suskirstyti į šešias grupes pagal laikinius raiškos pokyčius. PAINT programos pagalba buvo nustatyti 22 transkripcijos veiksniai, galimai lemiantys laikinius genų raiškos pokyčius po dalinės hepatektomijos operacijos. Devynių transkripcijos veiksnių (NF-κB, GATA-1, HNF-1, C/EBP-α ir C/EBP-β, STAT-3, PAX-6, AP-2α ) aktyvumo pokyčiai buvo įvertinti eksperimentiškai. Nustatyta, kad aktyvių NF-κB, HNF-1 ir C/EBP-β kiekis hepatocitų branduoliuose staigiai padidėja po dalinės hepatektomijos operacijos. Priešingai, aktyvios C/EBP-α formos kiekis sumažėja. Chromatino imunoprecipitacijos (ChIP) metodu buvo tiriamas NF-κB jungimasis prie Sod2, Mt1a ir Cebpb promotorių sričių praėjus 1 valandai po hepatektomijos. Regeneruojančių kepenų mėginiuose rasta daugiau NF-κB prisijungusio prie Sod2... [toliau žr. visą tekstą] / Liver possesses the capacity to restore its tissue mass and attain optimal volume in response to physical, infectious and toxic injury. The extraordinary ability of liver to regenerate is the effect of cross-talk between growth factors, cytokines, matrix components and many other factors. Adult liver normally has a very low level of hepatocyte cell division. However, most hepatocytes will rapidly proliferate in response to liver mass reduction. In this study the differences in gene expression in rat liver at 1, 2, 4 and 6 hours after the partial hepatectomy were analyzed using cDNA microarrays. These time points correspond to quiescent liver and early G1 phase in this system. The experiments showed that 309 genes had significantly altered expression for at least one of the analyzed time-points. Differentially expressed genes were clustered according to their temporal expression profiles. PAINT analysis identified several TF binding sites (~20) enriched in specific individual expression patterns. We have obtained time series data on the activity of nine transcription factors (NF-κB, HNF-1, CREB, C/EBP-α, C/EBP-β, AP-2α, PAX-6, GATA-1, STAT-3) that were implicated in our analysis. An early transient activation of NF-κB, HNF-1 and C/EBP-β was observed. By contrast, C/EBP-α rapidly declined. Chromatin immunoprecipitation (ChIP) assay was performed to examine the binding of NF-κB to the promoter regions of Sod2, Mt1a and Cebpb. We see increased NF-κB binding to both Sod2 and... [to full text]

No description available.

The proprotein convertase subtilisin/kexin type 9, PCSK9, belongs to the proprotein convertase (PC) family. Human mutations in the gene encoding PCSK9 lead to either familial hyper- or hypocholesterolemia, resulting from a gain or loss of function, respectively. Mice lacking PCSK9 are viable and show a 42% decrease in plasma cholesterol levels. The enzyme triggers the degradation of the low density lipoprotein receptor (LDLR) through a partially unknown mechanism. / PCSK9 is very abundant in the liver and intestine during development and adulthood. Hepatocytes have a capacity to reproduce themselves and, upon injury, can repopulate the liver. For a better understanding of the role of PCSK9 in the liver, partial hepatectomy was performed on Pcsk9 +/+, Pcsk9+/- and Pcsk9-/- mice. The absence of PCSK9 resulted in defective liver regeneration, while wild type (WT) and heterozygous mice had no phenotype. Regeneration defects could be prevented by a high cholesterol diet. PCSK9 deficiency, by contributing to maintaining low circulating cholesterol levels may thus hamper liver regeneration. This knowledge is critical for the analysis of future PCSK9 inhibitors expected to be developed in the near future. / Key words. Proprotein convertase subtilisin/kexin 9 (PCSK9), a familial hyper- or hypocholesterolemia, low density lipoprotein receptor, knockout mouse model, partial hepatectomy.

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 4 uppsatser.

Introduction: The reason for the inability of performing the liver resection for colorectal carcinoma metastasis is usually insufficient remnant liver parenchyma after liver resection (future liver remnant volume - FLRV). The current standard method of increasing FLRV is the embolization of the branch of portal vein (portal vein embolization - PVE) on the side of the tumor, and then suspended after hypertrophy of the non-embolised lobe liver resection. Unfortunately, there are some patients who do not increase liver volume despite perfectly executed PVE. Besides that, FLRV occurs during the time necessary for hypertrophy progression of metastatic disease. Therefore, we are trying to find the appropriate way to encourage the growth of remaining liver parenchyma and accelerate hypertrophy of the contralateral liver lobe. From our previous experience (IGA MZ NS 10240), it is possible to be optimistic that there hope is the way of hematopoietic progenitor cells (HPC - adult stem cells) after previous PVE to non-embolised branches of the portal vein. These cells do not only accelerate liver regeneration, but are also able to improve its function (function of the liver) which is especially important for patients after neoadjuvant chemotherapy (steatohepatitis or steatofibrosis), and for patients with...

Avec près de 350 millions de personnes chroniquement infectées, et malgré l’existence de vaccins efficaces, le virus de l’Hépatite B (VHB) reste un problème majeur de santé publique. Parmi les protéines virales, la protéine régulatrice HBx possèdent des activités qui pourraient être particulièrement impliquées dans le développement de CHC. Au cours de ce travail, nous nous sommes intéressés aux différences biologiques entre la protéine HBx issue d’une région non tumorale (HBx-NT) et la protéine HBx issue d’une région tumorale (HBx-T) d’un même patient. En particulier, nous nous sommes intéressés à la régénération hépatique après hépatectomie partielle et à la carcinogenèse hépatique dans un modèle murin transgénique. Nous avons démontré l’absence d’impact de la forme tronquée de la protéine HBx sur la régénération hépatique. Nous avons démontré que la protéine HBx entière avait la capacité d’activer la sécrétion d’IL-6 dans la phase d’initiation de la régénération hépatique, conduisant à l’hyperactivation de STAT3, l’accumulation de SOCS3 et la diminution de phosphorylation de ERK. Au final, la protéine HBx entière induit un retard de régénération hépatique. Nous avons démontré une cinétique d’apparition de tumeurs plus rapide chez les souris HBx-T que chez les souris HBx-NT après injection d’un carcinogène chimique. Nous avons aussi pu observer que les deux formes HBx-T et HBx-NT sensibilisaient les hépatocytes à l’apoptose, au cours d’un dommage hépatique aigue, et que cette sensibilisation à l’apoptose pouvait en partie rendre compte de l’effet co-carcinogène observé chez les souris HBx-T et HBx-NT. L’ensemble de mes résultats a permis de mieux comprendre les mécanismes par lesquels la protéine HBx participe au développement de CHC. / Hepatitis B virus (HBV) is a worldwide health issue, as it is estimated that 350 millions people are chronically infected. Among the viral proteins, HBx is thought to be involved in hepatocellular carcinoma (HCC) development. In this work, we were interested in biological differences between HBx sequence from non tumoral region (HBx-NT) compared to HBx from tumoral region (HBx-T) from a single patient. In particular, we studied liver regeneration after partial hepatectomy et hepatocarcinogenesis in a transgenic mice model. We demonstrated that HBx-T did not modulate liver regenereation. We also showed that HBx-NT induced IL-6 overexpression during priming phase of liver regeneration, and that IL-6 overexpression was involved in STAT3 hyperactivation, SOCS3 accumulation and inhibition of ERK. Overall, HBx-NT induced IL-6 overexpression was responsible for a delay in liver regeneration. Moreover, we showed that HBx-T induced a faster development of hepatic tumor after DEN initiation, compared to HBx-NT. Both HBx forms were involved in an apoptosis sensibilization during acute liver injury, that could be involved in co-carcinogenic effect of HBx-T and HBx-NT. Overall, my results participate to the comprehension of HBx impact on liver carcinogenesis