Temporal and spatial regulation of epsin abundance and VEGFR3 signaling are required for lymphatic valve formation and function

Abstract

Lymphatic valves prevent the backflow of the lymph fluid and ensure proper lymphatic drainage throughout the body. Local accumulation of lymphatic fluid in tissues, a condition called lymphedema, is common in individuals with malformed lymphatic valves. The vascular endothelial growth factor receptor 3 (VEGFR3) is required for the development of lymphatic vascular system. The abundance of VEGFR3 in collecting lymphatic trunks is high before valve formation and, except at valve regions, decreases after valve formation. We found that in mesenteric lymphatics, the abundance of epsin 1 and 2, which are ubiquitin-binding adaptor proteins involved in endocytosis, was low at early stages of development. After lymphatic valve formation, the initiation of steady shear flow was associated with an increase in the abundance of epsin 1 and 2 in collecting lymphatic trunks, but not in valve regions. Epsin 1 and 2 bound to VEGFR3 and mediated the internalization and degradation of VEGFR3, resulting in termination of VEGFR3 signaling. Mice with lymphatic endothelial cell-specific deficiency of epsin 1 and 2 had dilated lymphatic capillaries, abnormally high VEGFR3 abundance in collecting lymphatics, immature lymphatic valves, and defective lymph drainage. Deletion of a single Vegfr3 allele or pharmacological suppression of VEGFR3 signaling restored normal lymphatic valve development and lymph drainage in epsin-deficient mice. Our findings establish a critical role for epsins in the temporal and spatial regulation of VEGFR3 abundance and signaling in collecting lymphatic trunks during lymphatic valve formation.

Lymphatic Vessel Network Structure and Physiology

“…Loss of Epsins resulted in the maintenance of high Vegfr3 expression in collecting lymphatic vessels, which blocked their maturation and the formation of the valves, which caused defective lymphatic drainage. Interestingly, decreased activity of Vegfr3 in these mice restored normal lymphatic valve formation and improved lymphatic function (617). This study indeed revealed that the posttranscriptional modification of the lymphatic genes affects lymphatic valve development.…”

“…In the case of Milroy disease, caused by mutations of either VEGFR3 or its agonist VEGF-C (131, 328, 384, 516), both impaired fluid absorption in the initial lymphatics was initially proposed as a disease mechanism (684). However, more recent findings that VEGFR3 contributes to lymphatic valve formation and function suggest that valve dysfunction may also be a critical factor (617). In lymphedema distichiasis syndrome, caused by mutation of FOXC2 (317), patients present with aberrant eyelashes at birth, but the onset of lymphedema typically occurs at puberty or later in life, sometimes as late as over 50 years of age (132).…”

Endothelial epsins as regulators and potential therapeutic targets of tumor angiogenesis

“…Besides blood vessels, the lymphatic route provides an important, yet under-appreciated role for facilitating tumor development and progression. The finding that temporal and spatial regulation of epsin abundance, as well as the VEGFR3 signaling required for lymphatic valve formation and function, provides a hint that epsins may also affect tumor-associated lymphangiogenesis [53]. The increasing understanding of additional extrinsic roles of epsins will further contribute together in maximizing tumor inhibition.…”

Selective Targeting of a Novel Epsin–VEGFR2 Interaction Promotes VEGF-Mediated Angiogenesis

“…Selectivity towards ubiquitinated proteins is mediated by two evolutionarily conserved ubiquitin interacting motifs (UIMs) located between the membrane-localizing epsin NH 2 -terminal homology (ENTH) domain and carboxyl-terminal clathrin- and AP-2 binding motifs 9, 16, 17, 19, 23, 25, 26 . Mammals express three epsins: epsins 1 and 2, which are ubiquitously expressed and redundant in function, and epsin 3, which is expressed primarily in the stomach and epidermis 9, 10, 14, 19, 27-30 . Germ line epsins 1 and 2 double knockout (DKO) mice exhibit severe vascular defects resulting in embryonic lethality 10, 27 .…”