Project A02

Mechanisms of vascular Wnt signaling during liver homeostasis and tumorigenesis

Project leader: Hellmut Augustin

Abstract

Endothelial cell (EC) Wnt signaling and EC-derived Wnt ligands have in recent years emerged as autocrine acting regulator of vascular function as well as paracrine (angiocrine) gatekeeper of surrounding parenchymal cells, respectively. The liver has in this context become the prototypic model organ to study mechanisms of angiocrine signaling due to its unique dual vascular supply. At the beginning of the first funding period, EC-derived Wnt ligands had been firmly established as angiocrine regulators of liver metabolic zonation. Based on these findings, we performed spatial transcriptomics, proteomics and phosphoproteomics of liver EC (L-EC) to generate a systems map of zonal vascular signaling. Building on these multiomics analyses in combination with challenge experiments in vivo (liver regeneration and tumorigenesis), we identified Angiopoietin/Tie signaling (through FoxO1 and Stat3) as specific and selective upstream regulator of vascular Wnt ligand expression (notably Wnt2 and Wnt9b). Importantly, Angpt/Tie signaling does not just regulate Wnt ligand production in the liver vasculature, but acts as Wnt code specifier in the vasculature of different organs.

In the second branch of the project, we generated EC mouse mutants of the Wnt secretion factor Evi/Wls as well as the Wnt signaling enhancer Rspo3 (single and double mutants) and studied the consequences of such manipulation on L-EC zonation analytically by scRNAseq and experimentally in tumor challenge experiments. Based on this work, we will in the second funding period study the mechanisms and consequences of vascular Wnt signaling in the control of liver function during homeostatic maintenance and in adaptation to pathologic challenge, most notably liver regeneration, fibrosis and hepatocellular carcinoma. To mechanistically dissect autocrine and angiocrine vascular Wnt signaling, we will perform comparative multiomics analyses of L-EC and hepatocytes harvested from WT and L-EC Wnt mutant mice. The resulting expression-based interactome analysis (RNA & proteome) will be confirmed in silico by alignment with the respective phosphoproteome map and validated experimentally. Corresponding pathology challenge experiments in WT and iECKO mice in vivo (regeneration and fibrosis), ex vivo (organ perfusion) and in vitro (2D and 3D cell culture with freshly isolated L-EC and hepatocytes) will be pursued to assess the role of microenvironmental milieu factors (notably, varying oxygen and shear stress) on vascular Wnt ligand production and consequently angiocrine signaling. Long-term in vivo experiments will reveal the role of vascular Wnt signaling on hepatocyte turnover and organ size. Lastly, to translate these physiology-related experiments towards studying the role of vascular Wnt signaling in disease, we will perform comparative tumor experiments in WT and genetically vascular Wnt signaling challenged mice with a focus on hepatocellular carcinoma primary tumor development as well as lung and liver metastatic dissemination.

Project-related publications

  • Inverso D, Shi J, Lee KH, Jakab M, Ben-Moshe S, Kulkarni SR, Schneider M, Wang G, Komeili M, Vélez PA, Riedel M, Spegg C, Ruppert T, Schaeffer-Reiss C, Helm D, Singh I, Boutros M, Chintharlapalli S, Heikenwalder M, Itzkovitz S, Augustin HG. 2021. A spatial vascular transcriptomic, proteomic, and phosphoproteomic atlas unveils an angiocrine Tie–Wnt signaling axis in the liver. Dev Cell, 56(11):1677-1693.e10. PMID: 34038707
  • Singhal, M., N. Gengenbacher, S. La Porta, S. Gehrs, J. Shi, M. Kamiyama, D.M. Bodenmiller, A. Fischl, B. Schieb, E. Besemfelder, S. Chintharlapalli, H.G. Augustin. 2020. Preclinical validation of a novelmetastasis inhibiting Tie1 function-blocking antibody. EMBO Mol Med, 12: e11163. PMID: 32302470.
  • Abdul Pari, A.A., M. Singhal, C. Hübers, C. Mogler, B. Schieb, A. Gampp, N. Gengenbacher, L.E. Reynolds, D.Terhardt, C. Géraud, J. Utikal, M. Thomas, S. Goerdt, K.M. Hodivala-Dilke, H.G. Augustin#, M. Felcht#. 2020. Tumor cell-derived Angiopoietin-2 promotes metastasis in melanoma. Cancer Res, 80:2586-2598 (#equally contributing last authors). PMID: 32303578.
  • Jakab, M., H.G. Augustin. 2020. Understanding angiodiversity: Insights from single cell biology. Development, 147:dev146621. PMID: 32792338.
  • Singhal, M.#, X. Liu#, D. Inverso, K. Jiang, J. Dai, H. He, S. Bartels, W. Li, A.A. Abdul Pari, N. Gengenbacher, E. Besemfelder, L. Hui, H.G. Augustin§, J. Hu§. 2018. Endothelial cell fitness dictates the source of regenerating liver vasculature. J Exp Med, 215: 2497-2508 (#equally contributing first authors; §equally contributing senior authors). PMID: 30194265.
  • Schlereth, K., D. Weichenhan, T. Bauer, T. Heumann, E. Giannakouri, D. Lipka, S. Jaeger, M. Schlesner, P. Aloy, R. Eils, C. Plass#, and H.G. Augustin#. 2018. The transcriptomic and epigenetic map of vascular quiescence in the continuous lung endothelium. Elife, pii: e34423 (#equally contributing last authors). PMID: 29749927.