FOXC2 and fluid shear stress stabilize postnatal lymphatic vasculature

Biomechanical forces, such as fluid shear stress, govern multiple aspects of endothelial cell biology. In blood vessels, disturbed flow is associated with vascular diseases, such as atherosclerosis, and promotes endothelial cell proliferation and apoptosis. Here, we identified an important role for disturbed flow in lymphatic vessels, in which it cooperates with the transcription factor FOXC2 to ensure lifelong stability of the lymphatic vasculature. In cultured lymphatic endothelial cells, FOXC2 inactivation conferred abnormal shear stress sensing, promoting junction disassembly and entry into the cell cycle. Loss of FOXC2-dependent quiescence was mediated by the Hippo pathway transcriptional coactivator TAZ and, ultimately, led to cell death. In murine models, inducible deletion of Foxc2 within the lymphatic vasculature led to cell-cell junction defects, regression of valves, and focal vascular lumen collapse, which triggered generalized lymphatic vascular dysfunction and lethality. Together, our work describes a fundamental mechanism by which FOXC2 and oscillatory shear stress maintain lymphatic endothelial cell quiescence through intercellular junction and cytoskeleton stabilization and provides an essential link between biomechanical forces and endothelial cell identity that is necessary for postnatal vessel homeostasis. As FOXC2 is mutated in lymphedema-distichiasis syndrome, our data also underscore the role of impaired mechanotransduction in the pathology of this hereditary human disease.

In vitro lymphatic endothelial morphogenesis

Carolyn Yong Pullin

Cell organization into functional multicellular three-dimensional structures is a long-standing challenge. In particular, engineering of vascularized tissues requires both blood and lymphatic neovascular network formation in a simultaneous and coordinated fashion. While extensive in vitro investigations in blood and lymphangiogenesis have identified a vast array of cellular phenomena and key cellular and extracellular bioactive substances, the dynamic natural cellular environment and its regulatory role in governing cellular response and interactions remain largely unclear. Furthermore, the wealth of existing knowledge describing the molecular regulation of vessel morphogenesis is heavily biased towards the angiogenic growth of blood capillaries, despite the importance of the lymphatic system. This thesis first addresses the differential gene regulation of isolated microvascular lymphatic endothelial cells (LECs) in response to the lymphatic growth factor VEGF-C and to fluid shear stress using gene arrays and QRT-PCR. First, characteristic of a response to a growth factor stimulus, the largest numbers of differentially expressed genes in response to VEGF-C stimulation were transcription factors and cell cycle related. A number of genes known to be important in angiogenesis, tumorigenesis and tumor invasion, and transport of proteins, solutes, and lipids were also affected. Interestingly, a number of genes related to lipid metabolism as well as neurogenesis were also responsive to VEGF-C stimulation. Further analysis of these genes may not only provide insight into the molecular mechanisms underlying lymphangiogenesis and associated pathogenesis, but may also identify other important roles of VEGF-C. The ability of lymphatic endothelium to sense and actively regulate drainage function is poorly understood, and shear stress is likely a key indicator of lymphatic drainage function. Thus, LECs were exposed to 0, 2 and 20 dyn/cm2 shear stress as representative of chronic lymphedema, normal, and acute inflammatory conditions, respectively. Important adaptive responses to both low and high shear stress conditions that were correlated to lymphatic function, including changes in gene expression patterns related to water and solute transport, cell-cell and cell-matrix adhesion, inflammatory cytokines and cell cycle were found. These changes were consistent with increased transport function, both active and passive, in high shear conditions and indicate that during lymphedema, lymphatic endothelium shuts down transport functions. These data demonstrate a functional-adaptive response of lymphatic endothelium to flow conditions, thus indicating that the lymphatic endothelium plays an active role in regulating their drainage function. It has been previously shown in our lab that in vitro blood and lymphatic morphogenesis are differentially enhanced by interstitial flow. Using a three-dimensional tissue culture model, the molecular mechanisms orchestrating endothelial cell morphogenesis, specifically gene regulation of the lymphatic microvascular system was investigated, focusing on genes implicated in both cellular morphogenesis and lymphatic development. Finally, concurrent angiogenesis and lymphangiogenesis were studied in vitro to examine the influences and crosstalk between blood and lymphatic microvascular development. The data suggest that both blood endothelial cell (BEC) and LEC in vitro capillary morphogenesis are enhanced by the presence of BEC-secreted factors, and these effects are, in part, mediated through proliferative and migratory responses of which the latter was induced by VEGFR-2 and VEGFR-3 signaling for BECs and LECs, respectively. When maintained in mixed three-dimensional cultures, both homotypic and heterotypic interactions between LECs and BECs were observed. Furthermore, capillary morphogenesis and interactions were enhanced and distinct when under the additional influence of interstitial flow. In conclusion, the novelties of the work presented in this dissertation include studies of 1) overall LEC gene response to its primary growth factor VEGF-C and biophysical factor, fluid flow and 2) the genetic regulation underlying the morphogenetic processes of LECs under interstitial flow in vitro, by using unique approaches which combine bioengineering principles to address fundamental biological questions and thereby contribute to this novel niche which has so far been neglected. Furthermore, the importance of biophysical processes along with crucial biochemical signal regulation is demonstrated in the concurrent induction of angiogenesis and lymphangiogenesis. Such are the requisites for the understanding of microvascular development and the determination of useful design principles for the in vitro vascularization of engineered tissues.

EPFL2011

Molecular and mechanical regulators of lymphatic biology

Joseph Rutkowski

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.

EPFL2008

Investigation of cell death mechanisms in human lymphatic endothelial cells undergoing photodynamic therapy

Witold Waldemar Kilarski

Background: Photodynamic therapy (PDT) has been shown to induce ablation and functional occlusion of tumor-associated lymphatic vessels. However, direct effects of PDT on lymphatic endothelial cells (LECs) have not been studied so far. The aim of this study was to elucidate molecular mechanisms of cell death induced by PDT in human LECs. Methods: Verteporfin was used as a photosensitizer to investigate PDT-mediated damage of lymphatic vessels in mice using immunofluorescent staining and stereomicroscopy. In vitro dose-response studies were carried-out with crystal violet staining. Immunofluorescence, flow cytometry, immunoblotting and DNA electrophoresis were used to investigate the mechanisms of cell death in human LECs undergoing PDT. Results: PDT induced an increase in the number of propidium iodide positive lymphatic endothelial cells in the mouse dermis. In in vitro studies dose-dependent cytotoxic effects of PDT towards LECs were observed. Typical hallmarks of apoptotic cell death, including Annexin V binding, loss of mitochondrial membrane potential, caspase activation, cleavage of PARP as well as DNA fragmentation were observed in LECs when PDT was used at high irradiation conditions, causing >80% cell death. At lower light fluencies causing