Tabrizi, Zarah Beth
(2024)
Investigating the role of ZEB1 in Lymphangiogenesis.
PhD thesis, University of Nottingham.
Abstract
Lymphangiogenesis is the growth of new lymphatic vessels from the existing lymphatic vasculature. Lymphatic endothelial cells (LECs) line all lymphatic vessels and are essential regulators of vessel homeostasis. The switch between LEC quiescence and activation is highly regulated, controlled by multiple signalling pathways and transcription factors. The blood and lymphatic vasculature have shared origins, therefore often governed by similar mechanisms of growth and maintenance. Blood vessel remodelling has been shown to occur via a partial endothelial to mesenchymal transition (EndMT), allowing these cells to migrate from an existing vessel to form a new sprout. Lymphatic vessel remodelling has been relatively understudied, but this same remodelling mechanism is thought to occur. Governing this transition are SLUG and SNAIL, which also regulate epithelial to mesenchymal transition (EMT). Another driver of EMT is the transcription factor ZEB1, which has not been studied at present in EndMT or in the lymphatic system. In the blood vasculature, preliminary work has shown that loss of ZEB1 induces an angiogenic phenotype, therefore we hypothesise that, unlike SLUG and SNAIL, it is loss of ZEB1 which induces a partial EndMT mechanism involved in lymphatic vessel remodelling.
In this thesis, we used a mouse model of inducible endothelial cell knockout (iECKO), and primary human dermal LECs to investigate the role of ZEB1 in the lymphatic vasculature. In vitro, following siRNA knockdown of ZEB1, the transcriptome was pro-lymphangiogenic, with a migratory and proliferative phenotype suggested. At a protein level, SLUG and SNAIL were significantly upregulated, and the cell junctional marker VE-Cadherin was unaffected, suggesting a role of ZEB1 in partial EndMT. Mediators of lymphatic growth and identity, VEGFR3 and PROX1, were also dysregulated. In the ZEB1iECKO mice, no change was detected in lymphatic morphology in the dermis of P5 and adult mice. However, in the skeletal muscle, an increase in density of lymphatic vessels following loss of ZEB1 was discovered. Additionally, these lymphatic vessels in the skeletal muscle of both Control and ZEB1iECKO mice were positive for the immune infiltration marker CD45, which has been suggested as a novel marker of EndMT. This is a significant finding and should be researched further, as this has implications of the fundamental endothelial biology underlying many cell isolation techniques.
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