Molecular and mechanical regulators of lymphatic biology

Lymphatic vessels exist in nearly all tissues, yet, despite their omnipresence, there remains a large knowledge gap between the described fundamental roles of lymphatic capillaries and our understanding of their functional biology, adaptive ability, and pathological response. This thesis addressed these shortcomings by utilizing an integrative biomedical engineering approach to examine molecular and mechanical regulators of lymphatic capillaries using in vivo models of lymphatic capillary biology, function, and adaptation. Using a model of skin regeneration in the mouse tail, we demonstrated that slow interstitial flow created by lymphatic drainage was necessary for lymphatic capillary organization. This novel model permitted the identification of spatial, temporal and chemical factors governing lymphangiogenesis. In contrast to the sprouting mechanism of blood angiogenesis, lymphatic endothelial cells (LECs) were demonstrated to organize in a vasculogenesis-like manner, migrating in the direction of interstitial flow and then organizing into functional lymphatic capillaries. Lymphangiogenesis was inhibited by blocking vascular endothelial growth factor (VEGF)-C signaling from day 0, but initiation of receptor blockade once LECs had already migrated did not prevent vessel organization. This uniquely demonstrated the need for a biochemical mediator (VEGF-C) to initiate lymphangiogenesis, but that an important biomechanical force, interstitial flow, was necessary for functional capillary organization. Further insight into the necessity of interstitial flow in LEC biology was found in the response of lymphatic capillary to induced lymphedema, wherein lymphatic drainage is significantly reduced. In a mouse tail model of secondary lymphedema, we demonstrated that the edematous environment – characterized by extracellular matrix breakdown, lipid accumulation, and reduced interstitial flow – resulted in hyperplasia of LECs but concurrent poor function due to the lack of interstitial flow as an organizational guiding cue. Similar dermal matrix adaptations to dysfunctional lymphatic drainage were also noted in two mouse models of congenital lymphedema, the Chy and VEGFR-3-Ig mice, further demonstrating the intimate connection of lymphatic capillary function with tissue maintenance and remodeling. To quantitatively demonstrate the changes in lymphatic capillary uptake and tissue hydraulic conductivity found in these and other transgenic mouse models, we developed a poroelastic model of interstitial transport. Tissue hydraulic conductivity was also calculated in tissues lacking lymphatics using an unsteady-state solution, demonstrating that lymphedema causes a significant increase in tissue conductivity. This model was then utilized to assess the effects of a high fat diet, metabolic disorders, and lymphatic dysfunction on the tissue and on lymphatic capillary function. We discovered that lymphatic capillary uptake function was significantly reduced with dyslipidemia, suggesting a novel interplay between lymphatic function and lipid metabolism. Additionally, we uncovered a new and critical role for lymphangiogenesis and lymphatic transport in reproduction. We demonstrated that lymphangiogenesis is a regular, non-pathological event during folliculogenesis in the ovary. These new lymphatic capillaries are seemingly necessary for hormone transport from the ovary – an essential feedback mechanism during pregnancy. Blockade of lymphangiogenesis resulted in decreased systemic progesterone and estradiol levels and resulted in failed fetal development. In conclusion, this work highlights the critical roles of the lymphatic circulation and demonstrates the interplay between lymphatic biology and the biochemical and biophysical environment in which lymphatic capillaries reside. Interstitial flow and the interstitium modulate lymphatic behavior, and lymphatic function, in turn, controls the tissue microenvironment.