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

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.

The lymphatic and blood vascular systems are two distinct vessel network systems that work in synchrony to maintain tissue homeostasis. Blood vessels transport oxygen and nutrients around the body, while lymphatic vessels collect leaked fluid and macromolecules from the interstitial space and return them to the blood circulation, maintaining interstitial fluid homeostasis (1). Furthermore, the lymphatic system plays a central role in immune responses, inflammation regulation, and lipid absorption (2). While many in vitro models have been created to study angiogenesis, fewer attempts have been made to engineer an in vitro platform to study lymphangiogenesis. Such engineered models are critical for both fundamental research and the development of clinically implantable tissue to treat various diseases involving the lymphatic system. One such disease is lymphedema, a chronic condition that affects 200 million people worldwide (3). Lymphedema is characterized by tissue swelling resulting from a compromised lymphatic system. The condition is mainly caused by complications during cancer treatment but may also develop due to genetic disorders. The condition is progressive and incurable, with a high risk of infection. Implantation of engineered lymphatic tissue can serve as a treatment for such disease (4).Lymph flow is primarily driven by pressures generated by lymphatic contractions of the smooth muscle cells surrounding the vessels (5). Impaired contractility thus reduces lymph flow and may cause lymphedema. Previous computational studies have investigated the correlation between lymphatic vessel contractility and mechanical stimulation, such as mechanical loading, pressure gradients, and shear stress amplitudes (6, 7). Furthermore, studies have investigated lymphatic vessel capacity to distend under mechanical loading conditions. In addition, the microenvironment composition has been shown to play an important role in enabling lymphatic vessel functionality (4).Thus far, several groups have been able to engineer lymphatic tissues. Marino et al. created dermo-epidermal skin grafts with lymphatic and blood vessels embedded in a fibrin-collagen gel (8). Others created a lymphatic vessel network within multilayered fibroblast sheets (9, 10). Another study demonstrated that different hydrogel compositions are required for the optimal growth and development of blood and lymphatic endothelial cells (BECs and LECs, respectively) (11). However, no studies have investigated the influence of the supporting cells, the secreted extracellular matrix (ECM), and the mechanical environment on the forming lymphatic vessels. Since lymphatic pathologies are known to correlate with mechanically impaired lymphatic vessels (4), it is important to create lymphatic models with a biomimetic microenvironment.In this study, lymphatic vessel networks were engineered to investigate fundamental questions concerning lymphangiogenesis, including the influence of different supporting cells on the formation of lymphatic vessels and the role of PDGFR-β, an important receptor associated with support cells recruitment, in vessel formation. In addition, a complex tissue designed to better mimic native tissue was generated and lymphatic and blood vessel development along with muscle formation were monitored. In addition, the impact of the application of cyclic stretch on the organization and alignment of lymphatic-blood-vessel-muscle tissue was assessed. Finally, the penetration and anastomosis of the engineered lymphatic vessels were monitored following their implantation into mice.