Molecular players and signaling pathways involved in mammalian LEC specification
Notch signaling is required for arterial EC (in pink) identity, and COUP-TFII promotes venous EC (in light blue) fate by suppressing Notch signaling. The transcription factor Sox18 cooperates with COUP-TFII to activate Prox1 expression in a subpopulation of venous ECs. These cells become LEC progenitors (in light blue with yellow nuclei). These progenitors later on bud off from the cardinal vein (budding LECs in yellow). Prox1 activity is necessary and sufficient for LEC fate, and it regulates its own expression. Vegfr3 signaling is important for the maintenance of LEC fate specification, and it is directly regulated by Prox1. Notch signaling, miR181, miR31, and miR466 negatively regulate Prox1 in LECs. Biofactors such as blood flow and/or extracellular matrix (ECM) can cause LECs to lose lymphatic characteristics and/or acquire BEC features, resulting in a hybrid EC phenotype. Kaposi sarcoma-associated herpes virus (KSHV) infects both LECs and BECs, and transcriptionally reprograms these cells into hybrid ECs.

Flow is important for lymphatic fate maintenance
A: lymphovenous valves (LVV) form at the junction of the internal jugular vein (IJV) and subclavian veins (SCV). Platelet thrombi block retrograde blood flow into lymphatic vessels (LV). These structures ensure the drainage of lymph into venous circulation. B: usually, extracellular fluid drains into lymphatic vessels (left). When lymphovenous homeostasis is defective, blood enters into lymphatic vessels and disrupts forward lymph flow (right). LECs will lose their lymphatic fate and adopt BEC characteristics, such as acquiring mural coverage (arrow indicates directional lymph flow and retrograde flow). C: flow-rate laminar flow causes calcium influx in LECs, which upregulates Notch E3 ligase heterodimer encoded by Dtx1and Dtx3l, suppressing Notch1 activity. Notch1 is known as a negative regulator of Prox1. Loss of the chromatin-remodeling enzyme CHD4 results in upregulation of uPAR and subsequently elevated plasmin activity to degrade fibrin-rich thrombi that help seal the LVVs. Pdpn and T-synthase, the enzyme that is required for its O-glycosylation, activate platelets via the CLEC-2 receptor. CLEC-2 activates the SYK-Slp76-PLC-γ2 cascade to promote platelet aggregation, a process important for lymphovenous homeostasis. Loss of the platelet thrombi results in blood backflow in lymphatic vessels, leading to reprogramming of LECs into BECs.