Search

The Notch signaling pathway is an evolutionary conserved pathway that plays essential roles during vascular development and in the regulation of normal and pathological angiogenesis in the adulthood. These roles include angioblast specification, arteriovenous differentiation, regulation of blood vessel sprouting and branching, as well as control of vessel maintenance. In mammals there are four Notch receptors (Notch1 – 4), but only Notch1 and Notch4 are expressed in endothelial cells. Endothelial-specific loss of Notch1 is embryonic lethal, demonstrating its pivotal function in the vascular system. Nevertheless, the role of Notch1 signaling in postnatal vascular physiology is not fully understood. Inducible deletion of Notch1 in mice has been shown to cause nodular regenerative hyperplasia (NRH), which is a histopathological entity of the liver also seen in humans. NRH is thought to appear secondary to microcirculatory disturbances, however the exact pathogenesis is not known. NRH is an important cause of non-cirrhotic portal hypertension. The increased portal pressure can lead to complications including splenomegaly, ascites, or variceal bleeding; the latter is associated with high mortality. The aim of this thesis is to elucidate the role of Notch1 signaling in the hepatic microcirculation in normal and pathologic conditions. Further, we want to shed some light on the molecular mechanisms implicated in the development of NRH. In a conditional Notch1 knockout (KO) mouse model we investigated the impact of Notch1 signaling deficiency on the blood vessel homeostasis in the liver. LSEC are normally quiescent. After observing LSEC activation in livers of Notch1 KO mice, resulting in increased cell proliferation, we wanted to explore the ultrastructure of sinusoids in more detail. Scanning electron microscopy analysis of the liver microvasculature revealed phenotypic changes of LSEC, identified by loss of fenestrae. By performing vascular casts, we could discover three-dimensional changes of the hepatic microvasculature. While livers of control mice showed a highly differentiated vascular network, the liver vasculature of Notch1 KO mice was remodeled showing increased branching with larger vessel diameters. In addition, we identified features of intussusceptive angiogenesis in Notch1 KO casts. Time course experiments revealed that vascular changes occur first and that development of portal hypertension and NRH is a secondary phenomenon. To exclude that the observed phenotypic changes are due to loss of Notch1 in cells other than LSEC, a hepatocyte-specific Notch1 KO mouse was generated. This mouse had a completely normal phenotype. Furthermore, different cell populations were isolated from global Notch1 KO mice. Gene expression analysis of the different cell types confirmed that loss of Notch1 mainly affects the endothelium. Vascular dedifferentiation was found to be mediated by Notch, ephrin, and TEK signaling, all of which are known to regulate LSEC differentiation and quiescence. A very crucial finding, supporting our hypothesis that the NRH phenotype is driven by vascular changes, is the spontaneous development of liver angiosarcoma in Notch1 KO mice. Disruption of Notch1 signaling is sufficient enough to induce malignant transformation of endothelial cells in the liver, reflecting the pivotal role of Notch1 in the hepatic microcirculation. In a translational study using liver biopsies from NRH patients we assessed the vascular contribution to the development of NRH. Using morphological and molecular approaches, this part of the thesis addressed two main questions: one from a clinical point of view, the other from a basic science perspective. First, we assessed whether there is an association between the presence of portal hypertension and NRH severity. So far, no one has investigated the relationship between pathologic features and the clinical condition in NRH. Notably, histological assessed nodular transformation correlates well with the presence of portal hypertension. Since most complications occurring in NRH patients are due to increased portal pressure, patients presenting with advanced nodular transformation should be advised to undergo endoscopic screening for varices, since they can cause life-threatening complications. Based on our findings from the animal study we have hypothesized that NRH is caused by a vascular injury of the sinusoids. In our NRH mouse model we identified dysregulation of a number of genes upon Notch1 deletion, which are involved in endothelial differentiation. Therefore, we wanted to explore if the same set of genes is also regulated in human NRH. To our surprise, the same genes were also found to be dysregulated in the liver of NRH patients, irrespective of the underlying cause of disease. Thus we conclude: Despite different etiologic factors associated with NRH, there is evidence that in all cases the hepatic condition can be traced back to an endotheliopathy mediated by the final common pathway of Notch1/Dll4 and EphrinB2/EphB4 signaling. Taken together, our study identifies Notch1 as an important player in LSEC differentiation and quiescence. We provide first insights into the molecular mechanism of human NRH, which are in line with our findings from the NRH mouse model. In addition, we showed an oncosuppressive role of Notch1 in the murine liver endothelium, resulting in vascular neoplasms after loss of Notch1.

Hepatic angiosarcoma (AS) is a rare and highly aggressive tumor of endothelial origin with dismal prognosis. Studies of the molecular biology of AS and treatment options are limited as animal models are rare. We have previously shown that inducible knockout of Notch1 in mice leads to spontaneous formation of hepatic AS. The aims of this study were to: (1) establish and characterize a cell line derived from this murine AS, (2) identify molecular pathways involved in the pathogenesis and potential therapeutic targets, and (3) generate a tumor transplantation model. AS cells retained specific endothelial properties such as tube formation activity, as well as expression of CD31 and Von Willebrand factor. However, electron microscopy analysis revealed signs of dedifferentiation with loss of fenestrae and loss of contact inhibition. Microarray and pathway analysis showed substantial changes in gene expression and revealed activation of the Myc pathway. Exposing the AS cells to sorafenib reduced migration, filopodia dynamics, and cell proliferation but did not induce apoptosis. In addition, sorafenib suppressed ERK phosphorylation and expression of cyclin D2. Injection of AS cells into NOD/SCID mice resulted in formation of undifferentiated tumors, confirming the tumorigenic potential of these cells. In summary, we established and characterized a murine model of spontaneous AS formation and hepatic AS cell lines as a useful in vitro tool. Our data demonstrate antitumor activity of sorafenib in AS cells with potent inhibition of migration, filopodia formation, and cell proliferation, supporting further evaluation of sorafenib as a novel treatment strategy. In addition, AS cell transplantation provides a subcutaneous tumor model useful for in vivo preclinical drug testing.Laboratory Investigation advance online publication, 24 November 2014; doi:10.1038/labinvest.2014.141.