In vitro lymphatic endothelial morphogenesis

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.

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

Biophysical Regulation of Lymphatic Vessel Function

Dimana Miteva

Lymphatic capillaries collect interstitial fluid and dendritic cells from the periphery and deliver them to the lymph nodes for immune surveillance and tolerance maintenance. Upon injury and inflammation, lymphatic drainage can increase rapidly, and thus the lymphatic capillaries experience a broad range of fluid stresses and need to respond rapidly to such changes. Our understanding of their functional biology, adaptive ability and response to pathological changes in the biophysical environment is still very limited. This thesis focuses on how the lymphatic capillary endothelium senses and responds to changes of its biophysical environment with respect to its function, including fluid and cell transport, in the context of physiological and pathophysiological conditions. Using in vitro and in vivo models, we demonstrate that lymphatic endothelium is exquisitely sensitive to transmural flow, modulating both fluid and cell transport functions even in the absence of inflammatory cytokines. Flow increased lymphatic permeability and dendritic cell (DC) transmigration, which were coincident with changes in gene and protein expression of factor known to be involved in these, including chemoattractant chemokines, adhesion molecules and junctional proteins. These data provide the first evidence that lymphatic endothelium can regulate its transport functions in response to flow cues. They also show for the first time that transmural flow can, in the absence of inflammatory cues like TNF-a, drive expression of DC adhesion molecules like ICAM-1 and chemokines like CCL21 by lymphatic endothelium. Based on these findings we suggest that lymphatic flow is an important mediator of lymphatic function, particularly with respect to DC recruitment and trafficking to the lymph node. Furthermore, the response of the lymphatic endothelium to flow may be specific to the type of low, as lymphatic endothelial cells (LECs) might react differently to luminal shear flow, at physiological and pathophysiological levels, compared to transmural flow. To address this we used laminar shear stress chambers to expose only the apical surface of the lymphatic endothelial cells (LECs) to shear stress and demonstrated that lymphatic endothelium is indeed sensitive to luminal flow, fine-tuning its expression of adhesion molecules to modulate DC adhesion according to the shear stress. Specifically, at extremely low shear, adhesion was enhanced, unlike at higher shear, adhesion was downregulated. This is consistent with the notion that DC adhesion to LECs is only needed for entry but not transport in conducting vessels, since only the absorbing capillaries have low shear stresses. The interactions between DCs and LECs were strongly mediated by CCL21 and its receptor CCR7. These findings suggest that luminal shear stress acts as an active mechanosignal in LECs to potentially facilitate the traffic of immune cells inside of the lymphatic vessel to reach the lymph node and trigger an immune response. Additionally, we demonstrated that lymphatic vessels respond differentially to immunogenic and tolerogenic inflammatory stimulus. We used in vitro models to evaluate lymphatic uptake and lymphatic participation in dendritic cell transmigration in the presence of various inflammatory stimuli. We found that lymphatic endothelium exposed to TNF-a or complement activation were more conductive to dendritic cell transmigration, while LPS and TGF-β had opposite effect, affecting DCs to decrease DC transmigration. We further presented some insights in similarities in tumor and dendritic cells intravasation into inflamed lymphatic capillaries. These findings imply that lymphatic activation and participation in immune cells transport is not a universal phenomenon, but is strongly dependent on the nature of the inflammation. In conclusion, this work highlights the critical role of biophysical environment in lymphatic endothelial function, and how exquisitely sensitive the lymphatic capillaries are to their extracellular environment and how they use multiple cues to sense inflammation and danger, modulating their transport functions accordingly to regulate the delivery of antigens and immune cells to the lymph nodes.

EPFL2010

Effects of dynamic environments on extracellular morphogen gradients

Mark Fleury

Tissue morphogenesis and remodeling is often orchestrated by cell-secreted proteins, referred to as morphogens that can trigger cellular responses in addition to modifying the local extracellular matrix (ECM). Of specific interest is the distribution of cell secreted proteins in the cell microenvironment and how this distribution can lead to varied responses. Morphogens often provide directional cues, and as such their specific distribution is critical to the signaling that they induce, in other words cells are sensitive to morphogen gradients and not just absolute amounts. Natural cellular environments in native tissue involve three dimensional ECM and are typically subjected to dynamic conditions such as interstitial flow (IF). Despite the importance of the extracellular environment in signaling, very little is known regarding the effects of mechanical stresses and subtle interstitial flows. This thesis examines the role of IF on morphogen gradients using mass transport models to elucidate mechanisms responsible for several specific biological phenomena including in vitro capillary formation and lymphatic metastasis of tumor cells. In vitro capillary formation had been shown in our lab to be separately enhanced by bound VEGF alone and slow IF alone, with the combination of these two conditions resulting in marked increase in organization. Using our computational model we were able to propose a novel mechanistic model to explain this synergy. Specifically showed how, through the combined effects of IF and matrix-bound growth factors, cells could create their own biased chemokine gradients without recourse to internal amplification methods. This process, which we have termed "autologous chemotaxis," drives directional signaling and migration in the direction of flow. This can occur even at very low Peclet numbers, when diffusion still dominates the overall transport, but convection changes the shape of the gradient, which is what a cell responds to. We then examined the effects of IF on tumor cell migration to explore a novel mechanism of lymphatic metastasis. Tumors often produce high amounts of proteoglycans creating a microenvironment is rich in morphogen binding sites, and tumors are also a source of interstitial flow due to their leaky vasculature. The lymphatic system drains interstitial fluid, therefore IF is always directed toward lymphatic capillaries. The lymphatic system is also a common route of tumor metastasis, which is what lead us to study the role of IF mediated chemotaxis. A computational model of an in vitro tumor invasion assay was constructed that predicted tumor cell migration that was in very good agreement with the in vitro experimental results. This phenomenon was further studied by modeling an in vivo geometry and examining the factors controlling autologous gradient formation in a physiological setting. In conclusion, we have demonstrated the importance of convective mass transport at very low flow velocities in a biological context. While these velocities are low enough that the convective effects would typically be ignored, their subtle effects on pericellular mass transport can be translated into relevant and observable morphogenic responses.

EPFL2007