Identification and characterization of novel regulators of lymphatic vessel development

Abstract

A functional vascular system in the vertebrate is pivotal for normal development and maturation. Failure in the structure and function of such a system will inevitably lead to pathophysiological conditions, and in severe cases to death. The blood and lymphatic system are the two circulatory systems of vertebrates. Lymphatic vessels in particular are important routes for fluid drainage, lipid homeostasis and immune surveillance. Dysfunctional lymphatics will lead to numerous diseases, including lymphedema, metastasis of cancer cells and various inflammatory disorders. Mutations in SRY-box containing gene 18 (SOX18), Vascular Endothelial Growth Factor C (VEGFC) and Vascular Endothelial Growth Factor Receptor 3 (VEGFR3) underlie the hereditary lymphatic disorders hypotrichosis–lymphedema–telangiectasia (HLT), Milroylike lymphedema and Milroy disease, respectively. Genes responsible for hereditary lymphedema are key regulators of lymphatic vascular development in the embryo. To identify novel modulators of lymphangiogenesis, in Chapter 3 of this thesis, a mouse model of HLT (Ragged Opossum) was used and gene expression profiling of aberrant dermal lymphatic vessels was performed. 289 genes were found dysregulated in purified lymphatic endothelial cells isolated from heterozygous Sox18 mutant mice (Sox18RaOp+/-) compared to wild-type. These genes were recognized as having known roles in other processes during development, but had previously not been implicated in lymphatic vessel development. Subsequent expression studies and functional analysis in zebrafish revealed one candidate, ArfGAP with RhoGAP domain, Ankyrin repeat and PH domain 3 (ARAP3), which was down-regulated in HLT mouse lymphatic vessels and necessary for lymphatic vascular development in zebrafish. To further understand how gene expression may alter vessel network formation, an in vivo model system was developed in Chapter 4. The dermal vascular assay allowed examination of changes in vessel morphology over time. Blood vessels remodelled into a dense network of loops at 14.5 days post coitum (dpc) and displayed a completed interconnected blood vascular network by 15.5 dpc. Following this, the first lymphatic vessels anastomosed at the midline of the mouse embryonic skin at 16.5 dpc, displaying a network of larger loops compared to blood vessels. Interestingly, at this time point not all lymphatic vessels appeared to be connected at the midline. These vessels are believed to function as blind-ended lymphatic capillaries.In the final data section, Chapter 5, the function of Arap3 was further examined in mice. A conditional endothelial cell Arap3 knockout mouse was generated and displayed severe lymphatic vascular defects as observed in mouse embryonic skin at 14.5 dpc. Mutant lymphatic vessels appeared to be bulbous in morphology and did not migrate as far to the midline as wild type siblings. Further mechanistic analysis in vitro and in vivo, positioned this known regulator of cell behaviour during migration as a mediator of the cellular response to Vegfc signalling in lymphatic endothelial cells.