Mechanisms driving notch disorders and therapeutic implications
Author: Iqbal, Afshan
Date: 2024-03-19
Location: Lecture hall Peter Reichard, Biomedicum, Solnavägen 9, Karolinska Institutet, Solna
Time: 13.00
Department: Inst för cell- och molekylärbiologi / Dept of Cell and Molecular Biology
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Thesis (1.149Mb)
Abstract
Biliary tree is an intricate tubular structure lined by cholangiocytes or biliary epithelial cells. The differentiation, development and morphogenesis of bile ducts is controlled by Notch signaling and other pathways. Alagille syndrome (ALGS) is among many diseases associated with aberrant Notch signaling. ALGS is caused due to mutations in JAG1 (97%) and NOTCH2 (2-3%), core components of Notch signaling pathway. ALGS is a multisystemic genetic disease. Patients have short stature with hepatic, vascular, ocular, renal, and vertebral anomalies. Hepatic disease is due to peripheral bile ducts paucity, due to which bile gets accumulated in the liver leading to cholestasis and liver damage. In some patients, the biliary tree is recovered spontaneously by adulthood. The mouse model of ALGS, Jag1Ndr/Ndr mimic human disease in many systems. Similarly, some Jag1Ndr/Ndr mice regenerate bile ducts by adulthood. Our lab has shown previously that regeneration in hilum and periphery of biliary tree occur through different architectural mechanisms, but it remains poorly understood whether the regenerated bile ducts exhibit molecular differences as compared to native bile ducts. Furthermore, our lab has shown that in both humans and mice IGF-1 is downregulated at RNA and protein level, thereby identifying it as a potential therapeutic target.
In Paper I, we have addressed the molecular differences between hilar and peripheral intrahepatic bile ducts using organoids culture and RNA sequencing. Proliferation in Jag1Ndr/Ndr ICOs is far less as compared to Jag1+/+ ICOs as evaluated by organoid size analysis and EdU incorporation assay. Among Jag1Ndr/Ndr ICOs, hICOs were least proliferative which was also confirmed by transcriptional analysis as cell cycle genes were significantly downregulated. Treatment with IGF-1 specifically rescues the survival and proliferation of Jag1Ndr/Ndr pICOs, while hICOs remains unresponsive. Jag1Ndr/Ndr hICOs exhibited less Notch signaling and hepatocyte like nature with enrichment of pathways in IGF uptake and transport proteins, which may explain non-responsive behavior towards IGF-1 treatment. Analysis of portal tract near hilum in vivo revealed ectopic hepatocyte like nuclei which were Hnf4α and IGF-1 positive.
Paper II describes a technique developed by our lab, double resin casting micro computed tomography (μCT) (DUCT) for visualizing and quantifying the 3D architecture of liver vasculature and biliary tree. This method can be used to assess the efficacy of potential therapeutic target for ALGS. The protocol entails two parts. In the first part, two tubular systems, PV and bile ducts are injected with two different radiopaque resins followed by fixation. In second part, the hardened liver is subjected to μCT. The data is then segmented by the custom-written pipeline and analyzed. DUCT injection at P15 and adult Jag1+/+ liver shows that both vascular and biliary systems are well-aligned from hilum towards periphery until adulthood. The biliary tree and vasculature must expand and lengthen further to fill the whole liver lobe to the periphery. DUCT can also be used to study tubular systems in other organs, for example, lung airways.
In Paper III, we determine whether an integrated assessment of multiple liver architecture parameters would correlate better with serum markers of cholestasis than bile duct paucity (BD/PV) alone. In this regard, we suggest an improved immunofluorescence staining method to visualize and quantify portal veins independent of any associated structure. We also developed pipeline for automated quantification of liver architecture. At P30, Jag1Ndr/Ndr displays a wide spectrum of disease as seen in human patients. The Jag1Ndr/Ndr mice can be classified into mild, intermediate, or severely affected categories based on their body size and presence or absence of bile infarcts. Bile ducts/mm2 near hilum and bile acid levels in mild/intermediate Jag1Ndr/Ndr mice positively correlate indicating ongoing regeneration associated with peak bile acid levels, but rescued bilirubin levels. However, the periphery of intermediate and hilar/periphery in severe Jag1Ndr/Ndr mice did not correlate with any of the parameters indicating low biliary regeneration. Manual quantification was compared with the results from automated analysis, which supported these findings, yet it is an ongoing project and needs some modifications to improve the analysis by automated quantification pipeline.
In summary, my thesis shows that hilar and regenerated peripheral bile ducts in adult Jag1Ndr/Ndr mice are distinct. Our findings, both in vitro and in vivo, indicate that hilar bile ducts formed in the absence of Notch signaling exhibit reduced commitment to biliary fate. Additionally, this thesis demonstrates the potential of IGF-1 to rescue bile ducts in ALGS. Our research lays the groundwork for further studies to assess the effectiveness of IGF-1 in vivo. Furthermore, we have introduced an innovative technique for studying tubular systems in three dimensions within the liver, which can also be applicable to other organs. Additionally, we emphasize the significance of the P30 stage in mice as it serves as a representative stage of disease in humans, exhibiting a broad spectrum of liver disease. The excessive number of bile ducts near the hilum in Jag1Ndr/Ndr mice indicates an ongoing ductular reaction.
In Paper I, we have addressed the molecular differences between hilar and peripheral intrahepatic bile ducts using organoids culture and RNA sequencing. Proliferation in Jag1Ndr/Ndr ICOs is far less as compared to Jag1+/+ ICOs as evaluated by organoid size analysis and EdU incorporation assay. Among Jag1Ndr/Ndr ICOs, hICOs were least proliferative which was also confirmed by transcriptional analysis as cell cycle genes were significantly downregulated. Treatment with IGF-1 specifically rescues the survival and proliferation of Jag1Ndr/Ndr pICOs, while hICOs remains unresponsive. Jag1Ndr/Ndr hICOs exhibited less Notch signaling and hepatocyte like nature with enrichment of pathways in IGF uptake and transport proteins, which may explain non-responsive behavior towards IGF-1 treatment. Analysis of portal tract near hilum in vivo revealed ectopic hepatocyte like nuclei which were Hnf4α and IGF-1 positive.
Paper II describes a technique developed by our lab, double resin casting micro computed tomography (μCT) (DUCT) for visualizing and quantifying the 3D architecture of liver vasculature and biliary tree. This method can be used to assess the efficacy of potential therapeutic target for ALGS. The protocol entails two parts. In the first part, two tubular systems, PV and bile ducts are injected with two different radiopaque resins followed by fixation. In second part, the hardened liver is subjected to μCT. The data is then segmented by the custom-written pipeline and analyzed. DUCT injection at P15 and adult Jag1+/+ liver shows that both vascular and biliary systems are well-aligned from hilum towards periphery until adulthood. The biliary tree and vasculature must expand and lengthen further to fill the whole liver lobe to the periphery. DUCT can also be used to study tubular systems in other organs, for example, lung airways.
In Paper III, we determine whether an integrated assessment of multiple liver architecture parameters would correlate better with serum markers of cholestasis than bile duct paucity (BD/PV) alone. In this regard, we suggest an improved immunofluorescence staining method to visualize and quantify portal veins independent of any associated structure. We also developed pipeline for automated quantification of liver architecture. At P30, Jag1Ndr/Ndr displays a wide spectrum of disease as seen in human patients. The Jag1Ndr/Ndr mice can be classified into mild, intermediate, or severely affected categories based on their body size and presence or absence of bile infarcts. Bile ducts/mm2 near hilum and bile acid levels in mild/intermediate Jag1Ndr/Ndr mice positively correlate indicating ongoing regeneration associated with peak bile acid levels, but rescued bilirubin levels. However, the periphery of intermediate and hilar/periphery in severe Jag1Ndr/Ndr mice did not correlate with any of the parameters indicating low biliary regeneration. Manual quantification was compared with the results from automated analysis, which supported these findings, yet it is an ongoing project and needs some modifications to improve the analysis by automated quantification pipeline.
In summary, my thesis shows that hilar and regenerated peripheral bile ducts in adult Jag1Ndr/Ndr mice are distinct. Our findings, both in vitro and in vivo, indicate that hilar bile ducts formed in the absence of Notch signaling exhibit reduced commitment to biliary fate. Additionally, this thesis demonstrates the potential of IGF-1 to rescue bile ducts in ALGS. Our research lays the groundwork for further studies to assess the effectiveness of IGF-1 in vivo. Furthermore, we have introduced an innovative technique for studying tubular systems in three dimensions within the liver, which can also be applicable to other organs. Additionally, we emphasize the significance of the P30 stage in mice as it serves as a representative stage of disease in humans, exhibiting a broad spectrum of liver disease. The excessive number of bile ducts near the hilum in Jag1Ndr/Ndr mice indicates an ongoing ductular reaction.
List of papers:
I. Afshan Iqbal , Noemi Van Hul, Lenka Belicova, Agustin A. Corbat, Simona Hankeova, Emma R. Andersson. Spatially segregated defects and IGF-1-responsiveness of hilar and peripheral biliary organoids from a model of Alagille syndrome. Liver International. 2024, Volume 44(2): pp 541-558.
Fulltext (DOI)
Pubmed
View record in Web of Science®
II. Simona Hankeova, Jakub Salplachta, Noemi Van Hul, Michaela Kavkova, Afshan Iqbal, Tomas Zikmund, Josef Kaiser, Emma R. Andersson. DUCT: Double Resin Casting followed by Micro-Computed Tomography for 3D Liver Analysis. Journal of Visualized Experiments. 2021 (175). Volume 175:e62941.
Fulltext (DOI)
Pubmed
View record in Web of Science®
III. Afshan Iqbal, Noemi Van Hul, Agustin A. Corbat, Lukas Poppe, Linde Sevenants, Petter Ranefall, Emma R. Andersson. Hepatic vascular and biliary density determines liver disease severity in a model of Alagille syndrome. [Manuscript]
I. Afshan Iqbal , Noemi Van Hul, Lenka Belicova, Agustin A. Corbat, Simona Hankeova, Emma R. Andersson. Spatially segregated defects and IGF-1-responsiveness of hilar and peripheral biliary organoids from a model of Alagille syndrome. Liver International. 2024, Volume 44(2): pp 541-558.
Fulltext (DOI)
Pubmed
View record in Web of Science®
II. Simona Hankeova, Jakub Salplachta, Noemi Van Hul, Michaela Kavkova, Afshan Iqbal, Tomas Zikmund, Josef Kaiser, Emma R. Andersson. DUCT: Double Resin Casting followed by Micro-Computed Tomography for 3D Liver Analysis. Journal of Visualized Experiments. 2021 (175). Volume 175:e62941.
Fulltext (DOI)
Pubmed
View record in Web of Science®
III. Afshan Iqbal, Noemi Van Hul, Agustin A. Corbat, Lukas Poppe, Linde Sevenants, Petter Ranefall, Emma R. Andersson. Hepatic vascular and biliary density determines liver disease severity in a model of Alagille syndrome. [Manuscript]
Institution: Karolinska Institutet
Supervisor: Andersson, Emma
Co-supervisor: Van Hul, Noemi; Masek, Jan; Ellis, Ewa; Fischler, Björn; Nemeth, Antal
Issue date: 2024-02-21
Rights:
Publication year: 2024
ISBN: 978-91-8017-267-7
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