| dc.description.abstracteng | Lymphangiogenesis – the development of lymphatic vessels – takes place in response to lymphangiogenic growth factors during embryogenesis, wound healing, inflammation and cancer. It has been shown that WNT signalling plays important roles during embryonic development and cancer, and there are also a number of studies that have shown that WNT signalling controls angiogenesis. However, only very few investigations on WNT signalling on lymphangiogenesis exist, and these have not provided detailed analyses on the mechanisms how WNTs regulate the formation of lymphatics. A previous study of my lab has revealed that Wnt5a, a member of the β-catenin-independent Wnt pathway, is an essential regulator of lymphatic development in the dermis of mice. At embryonic day (ED) 18.5, Wnt5a-null-mice possess non-functional, highly dilated lymphatics, in contrast to functional lymphatics with small lumen observed in Wnt5a+/- and wild-type mice. With whole-mount immunofluorescence staining, I can show that Wnt5a-/- embryos exhibit blood-filled, often cyst-like lymphatics in their dermis, which fail to form proper interconnections. With fluorescence micro-lymphangiography I can show that these lymphatics are non-functional, whereas in Wnt5a+/- and wt-embryos lymphatic drainage is functional already at ED 17.5.
I developed a new ex vivo culture of embryonic dermis, where I could show that Wnts, and especially Wnt5a, are important regulators of lymphangiogenesis. The application of WNT5A protein to the dermis of ED 15.5 Wnt5a-null mice induces flow-independent development of slender, elongated lymphatic networks after two days, in contrast to an immature lymphatic plexus in the controls. Thereby, morphology of individual lymphatic endothelial cells (LECs) changes from round to elongated. Reversely, the application of the PORCN-inhibitor LGK974 to wild type-mouse dermis significantly disrupts lymphatic network maturation and elongation. Using various in vitro lymphangiogenesis assays with three different human LEC lines, I was able to show that WNTs, and prominently WNT5A, are potent regulators of lymphangiogenic mechanisms including sprouting, migration and tube formation. These mechanisms are independent of proliferation, which shows that non-canonical WNT signalling regulates various aspects of the morphogenesis of lymphatics. I also show that activation of the canonical pathway inhibits the effects of the non-canonical pathway. My studies on the intracellular cascade of WNT5A signalling in migrating LECs with scratch assays show that pharmacological inhibition of the RHO-GTPase RAC, or the c-Jun N-terminal kinase JNK significantly reduce migration, which leads to the assumption that WNT5A regulates morphogenesis of lymphatics via RAC and JNK. This is supported by the finding that WNT5A induces transient phosphorylation of JNK.
Finally, global mRNAseq analyses of LECs show that WNTs regulate 116 genes. Among these there are genes that control migration, but also angiocrine growth factors that indicate an active control of LECs for the growth of neighbouring tissues. Taken together, my data clearly show that WNT signalling is an essential regulator of lymphvascular morphogenesis. | de |