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Vascular malformation is a benign overgrowth of blood or lymphatic vessels leading to life-threatening consequences for affected patients. Activating mutations in the TIE2 receptor cause the majority of venous malformations (VMs), while somatic activating mutations in PIK3CA, leading to the overactivation of the PI3K-AKT pathway, cause both VMs and lymphatic malformations (LMs). Although molecular inhibitors targeting the PI3K-AKT-mTOR pathway, such as rapamycin, have shown beneficial effects, they are not curative. This thesis aimed to explore the endothelial cell-autonomous and paracrine mechanisms underlying Pik3ca-driven pathological vascular growth to identify a rationale for improved and curative therapies for vascular malformations.

In Paper I, we reported that one of the most common causative mutations, PIK3CA^H1047R, gives rise to two distinct LM subtypes known as macrocystic and microcystic LM in humans. Using a transgenic mouse model with temporally controlled LEC-specific activation of Pik3ca^H1047R, we found that the growth of microcystic LM is dependent on both the upstream pro-lymphangiogenic VEGF-C-VEGFR3 and the downstream AKT-mTOR signalling. Combination treatment targeting both signalling pathways led to effective inhibition of lesion growth in mice, suggesting a novel therapeutic approach for LM patients. In Paper II, we explored further the endothelial cell-autonomous and paracrine mechanisms underlying microcystic LM growth in mice. Using single-cell RNA sequencing, we identified a new immune-interacting subtype of dermal lymphatic capillary endothelial cells, termed iLECs. We showed that in Pik3ca mutant mice, iLECs produce factors that recruit pro-lymphangiogenic VEGF-C-producing macrophages. Macrophage depletion, inhibition of their recruitment, and anti-inflammatory COX-2 treatment resulted in decreased lymphatic growth, indicating a critical role of paracrine signalling between iLECs and immune cells in the pathogenesis of microcystic LM. In Paper III, we described distinct lymphatic vessel responses to oncogenic PI3K activation in different organs. We observed that while lymphatic vessels in the skin form microcystic LM through vessel sprouting, in certain other organs, they form large cysts reminiscent of macrocystic LM. Finally, we used mice with a BEC-specific activation of Pik3ca^H1047R to compare disease mechanisms in VM to those in LM in Paper II and to focus further on the former in Paper IV.

 Lymphatic vessels are essential for maintaining fluid homeostasis, immune cell trafficking and lipid absorption in the gut. Postnatal expansion of the lymphatic vasculature occurs through sprouting lymphangiogenesis from pre-existing lymphatic networks, which is regulated primarily by vascular endothelial growth factor C (VEGF-C) and its receptors, VEGFR2 and VEGFR3. While the role of VEGFR3 in lymphangiogenesis is well established, the function of VEGFR2 remains less understood. In Paper I, we use high-fidelity conditional genetics for VEGFR2 deletion and adeno-associated viruses (AAVs) overexpressing selective VEGFR2 and VEGFR3 ligands to reveal a critical role of VEGFR2 in lymphatic biology. In Paper II, we extend our studies to the mature lymphatic vasculature, composed of specialized lymphatic capillaries and collecting vessels. Fluid absorption occurs in lymphatic capillaries, which are composed of oak leaf shaped lymphatic endothelial cells (LECs) connected by discontinuous junctions. However, it is unclear how these capillaries maintain endothelial integrity while taking up fluid from the interstitial space. We show that capillary LECs dynamically remodel their shape during homeostasis and in response to increased interstitial fluid in a process driven by cytoskeletal actin remodelling. We further identify isotropic stretch as an upstream regulator of LEC cell shape and use mathematical modelling to show that the oak leaf cell shape provides increased resilience, preventing luminal collapse upon increased pressure on the vessel wall. While the development of blood and lymphatic vasculature is tightly controlled, certain pathologies are associated with aberrant expansion of these vessels. In Paper III and IV, we investigate the mechanisms underlying vascular malformations, which are a spectrum of diseases characterised by focal lesions of malformed blood or lymphatic vessels. The majority of vascular malformations are caused by somatic activating mutations in genes involved in (lymph-)angiogenesis, leading to ectopic growth of endothelial cells. Using genetic mouse models of vascular malformations, Paper III characterized organ-specific responses of LECs driving lymphatic malformations, while Paper IV identified a venous-specific feedback loop that amplifies upstream growth factor signalling, promoting venous malformations. These results illustrate that the same activating mutations can elicit distinct responses in endothelial cells depending on the organs or vessel type involved. In summary, by using various in vivo genetic models coupled with advanced imaging techniques, this thesis work uncovers critical new molecular mechanisms and the underlying cellular dynamics involved in the development, maintenance and pathological expansion of the blood and lymphatic vasculature.