RESUMEN
Introduction: Lymphatic vessels (LVs) maintain fluid homeostasis by draining excess interstitial fluid, which is accomplished by two distinct LVs: initial LVs and collecting LVs. The interstitial fluid is first drained into the initial LVs through permeable "button-like" lymphatic endothelial cell (LEC) junctions. Next, the drained fluid ("lymph") transports to lymph nodes through the collecting LVs with less permeable "zipper-like" junctions that minimize loss of lymph. Despite the significance of LEC junctions in lymphatic drainage and transport, it remains unclear how luminal or interstitial flow affects LEC junctions in vascular endothelial growth factors A and C (VEGF-A and VEGF-C) conditions. Moreover, it remains unclear how these flow and growth factor conditions impact lymphatic sprouting. Methods: We developed a 3D human lymphatic vessel-on-chip that can generate four different flow conditions (no flow, luminal flow, interstitial flow, both luminal and interstitial flow) to allow an engineered, rudimentary LV to experience those flows and respond to them in VEGF-A/C. Results: We examined LEC junction discontinuities, lymphatic sprouting, LEC junction thicknesses, and cell contractility-dependent vessel diameters in the four different flow conditions in VEGF-A/C. We discovered that interstitial flow in VEGF-C generates discontinuous LEC junctions that may be similar to the button-like junctions with no lymphatic sprouting. However, interstitial flow or both luminal and interstitial flow stimulated lymphatic sprouting in VEGF-A, maintaining zipper-like LEC junctions. LEC junction thickness and cell contractility-dependent vessel diameters were not changed by those conditions. Conclusions: In this study, we provide an engineered lymphatic vessel platform that can generate four different flow regimes and reveal the roles of interstitial flow and VEGF-A/C for lymphatic sprouting and discontinuous junction formation. Supplementary Information: The online version contains supplementary material available at 10.1007/s12195-023-00780-0.
RESUMEN
OBJECTIVE: Lymphatic vessels (LVs) maintain fluid homeostasis by draining interstitial fluid. A failure in lymphatic drainage triggers lymphatic diseases such as lymphedema. Since lymphatic drainage is regulated by lymphatic barrier function, developing experimental models that assess lymphatic barrier function is critical for better understanding of lymphatic physiology and disease. METHODS: We built a lymphatic vessel-on-chip (LV-on-chip) by fabricating a microfluidic device that includes a hollow microchannel embedded in three-dimensional (3D) hydrogel. Employing luminal flow in the microchannel, human lymphatic endothelial cells (LECs) seeded in the microchannel formed an engineered LV exhibiting 3D conduit structure. RESULTS: Lymphatic endothelial cells formed relatively permeable junctions in 3D collagen 1. However, adding fibronectin to the collagen 1 apparently tightened LEC junctions. We tested lymphatic barrier function by introducing dextran into LV lumens. While LECs in collagen 1 showed permeable barriers, LECs in fibronectin/collagen 1 showed reduced permeability, which was reversed by integrin α5 inhibition. Mechanistically, LECs expressed inactivated integrin α5 in collagen 1. However, integrin α5 is activated in fibronectin and enhances barrier function. Integrin α5 activation itself also tightened LEC junctions in the absence of fibronectin. CONCLUSIONS: Lymphatic vessel-on-chip reveals integrin α5 as a regulator of lymphatic barrier function and provides a platform for studying lymphatic barrier function in various conditions.
