Experimental Drainage Device to Reduce Lymphoedema in a Rat Model

Objective: Despite recent advances in pharmacological research and microsurgery, lymphoedema remains an incurable disease that deeply affects quality of life. There is an urgent need for innovative approaches to restore continuous lymph flow in affected tissues. To this end, the efficacy of a subcutaneously implanted draining device in reducing lymphoedema volume in a rat hindlimb lymphoedema model was tested. Methods: A rat model of chronic lymphoedema was developed by surgical removal of popliteal and inguinal lymph nodes, followed by irradiation. The model was characterised by monitoring limb volume via tape measure, skin water content via dielectric constant measurement, and lymphatic drainage via lymphofluoroscopy. After lymphoedema establishment in 16 Wistar rats, a device made of fenestrated tubing equipped with a miniaturised pumping system, was implanted subcutaneously in the affected limb to restore continuous recirculation of interstitial fluid. Results: Lymphofluoroscopy imaging showed impaired lymphatic drainage following lymphadenectomy and irradiation. Affected limb volume and skin water content increased significantly compared with the untreated limb, with a median (interquartile range) of 3.85 (0.38) cm(3) versus 3.03 (0.43) cm(3) for volume (n = 16, p = .001) and 26.6 (9.1) versus 16.6 (3.7) cm(3) for skin dielectric constant (n = 16, p = .001). Treatment of lymphoedema with the implanted drainage device showed that 5 weeks post-implant excess volume was significantly reduced by 51 +/- 18% compared with the pre-implant situation (n = 9 sham group, n = 7 pump group). Conclusion: Lymphoedema volume in the rat model was significantly reduced by restoring continuous drainage of excess fluid using a novel subcutaneously implanted device, opening the way to the development of an artificial lymphatic vessel.

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

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

Lymphatic drainage function and its immunological implications: From dendritic cell homing to vaccine design

Jeffrey Alan Hubbell, Sai Reddy, Melody Swartz

The slow interstitial flow that drains fluid from the blood capillaries into the lymphatic capillaries provides transport of macromolecular nutrients to cells in the interstitium. We discuss herein how this flow also provides continuous access to immune cells residing in the lymph nodes of antigens from self or from pathogens residing in the interstitium. We also address mechanisms by which dendritic cells in the periphery sense interstitial flow to home efficiently into the lymphatics after activation, and how lymphatic endothelium can be activated by this flow, including how it can act as a lymphatic morphoregulator. Further, we present concepts on how interstitial flow can be exploited with biomaterial systems to deliver antigen and adjuvant molecules directly into the lymphatics, to target dendritic cells residing in the lymph nodes rather than in the peripheral tissues, using particles that are small enough to be carried along by flow through the network structure of the interstitium. Finally, we present recent work on lymphatic and lymphoid tissue engineering, including how interstitial flow can be used as a design principle. Thus, an understanding of the physiological processes that govern transport in the interstitium guides new understanding of both immune cell interactions with the lymphatics as well as therapeutic interventions exploiting the lymphatics as a target.