Investigating lymphangiogenesis in vitro and in vivo using engineered human lymphatic vessel networks

Abstract

The lymphatic system is involved in various biological processes, including fluid transport from the interstitium into the venous circulation, lipid absorption, and immune cell trafficking. Despite its critical role in homeostasis, lymphangiogenesis (lymphatic vessel formation) is less widely studied than its counterpart, angiogenesis (blood vessel formation). Although the incorporation of lymphatic vasculature in engineered tissues or organoids would enable more precise mimicry of native tissue, few studies have focused on creating engineered tissues containing lymphatic vessels. Here, we populated thick collagen sheets with human lymphatic endothelial cells, combined with supporting cells and blood endothelial cells, and examined lymphangiogenesis within the resulting constructs. Our model required just a few days to develop a functional lymphatic vessel network, in contrast to other reported models requiring several weeks. Coculture of lymphatic endothelial cells with the appropriate supporting cells and intact PDGFR-β signaling proved essential for the lymphangiogenesis process. Additionally, subjecting the constructs to cyclic stretch enabled the creation of complex muscle tissue aligned with the lymphatic and blood vessel networks, more precisely biomimicking native tissue. Interestingly, the response of developing lymphatic vessels to tensile forces was different from that of blood vessels; while blood vessels oriented perpendicularly to the stretch direction, lymphatic vessels mostly oriented in parallel to the stretch direction. Implantation of the engineered lymphatic constructs into a mouse abdominal wall muscle resulted in anastomosis between host and implant lymphatic vasculatures, demonstrating the engineered construct's potential functionality in vivo. Overall, this model provides a potential platform for investigating lymphangiogenesis and lymphatic disease mechanisms.

Occurrence of Lymphangiogenesis in Peripheral Nerve Autografts Contrasts Schwann Cell-Induced Apoptosis of Lymphatic Endothelial Cells In Vitro

“…To further investigate the reduced lymphatic network density in tri-cultures of LECs, ASCs, and SCs, the addition of another cell type into the positive control hydrogels (LEC + ASC) would be important, to determine whether this effect is related to an increased cell density within the hydrogel. Additionally, Landau et al recently reported that cultivation of LECs with fibroblasts failed to induce lymphatic vessel formation, and they hypothesized that the inhibition of vessel formation was due to the production of dense collagen layers and excessive tension present in these cultures [ 40 ]. Consequently, to screen for the presence of different extracellular matrix (ECM) proteins, and the potential differences thereof between the different cultures, would allow identifying a correlation between ECM secretion and lymphangiogenesis.…”

Engineering the multiscale complexity of vascular networks

“…Increased understanding of the role of lymphatic vessels in development, homeostasis and disease will be important for engineering vascular networks. Emerging work is already looking towards building human lymphatic vessel networks using both technological and self-assembly-based approaches 162 , 163 .…”

Tissue Engineering Approaches to Uncover Therapeutic Targets for Endothelial Dysfunction in Pathological Microenvironments

“…The lymphatic system is responsible for numerous biological processes, including lipid transportat, immune cell trafficking, and maintaining interstitial fluid homeostasis [93][94][95][96]. Recent efforts have been focused on engineering the lymphatic vasculature [97][98][99] due to its potential to rescue various disease phenotypes, such as Alzheimer's, lymphedema, cardiovascular disease, and impaired wound healing [96,98]. Alderfer et al [98] generated tunable hyaluronic acid (HA)-based hydrogels with a wide range of stiffness to study lymphatic cord formation in vitro.…”

“…vasculature [97][98][99] due to its potential to rescue various disease phenotypes, such as Alzheimer's, lymphedema, cardiovascular disease, and impaired wound healing [96,98]. Alderfer et al [98] generated tunable hyaluronic acid (HA)-based hydrogels with a wide range of stiffness to study lymphatic cord formation in vitro.…”