VEGFR-3 neutralization inhibits ovarian lymphangiogenesis, follicle maturation, and murine pregnancy.

Lymphatic vessels surround follicles within the ovary, but their roles in folliculogenesis and pregnancy, as well as the necessity of lymphangiogenesis in follicle maturation and health, are undefined. We used systemic delivery of mF4-31C1, a specific antagonist vascular endothelial growth factor receptor 3 (VEGFR-3) antibody to block lymphangiogenesis in mice. VEGFR-3 neutralization for 2 weeks before mating blocked ovarian lymphangiogenesis at all stages of follicle maturation, most notably around corpora lutea, without significantly affecting follicular blood angiogenesis. The numbers of oocytes ovulated, fertilized, and implanted in the uterus were normal in these mice; however, pregnancies were unsuccessful because of retarded fetal growth and miscarriage. Fewer patent secondary follicles were isolated from treated ovaries, and isolated blastocysts exhibited reduced cell densities. Embryos from VEGFR-3-neutralized dams developed normally when transferred to untreated surrogates. Conversely, normal embryos transferred into mF4-31C1-treated dams led to the same fetal deficiencies observed with in situ gestation. Although no significant changes were measured in uterine blood or lymphatic vascular densities, VEGFR-3 neutralization reduced serum and ovarian estradiol concentrations during gestation. VEGFR-3-mediated lymphangiogenesis thus appears to modulate the folliculogenic microenvironment and may be necessary for maintenance of hormone levels during pregnancy; both of these are novel roles for the lymphatic vasculature.

Cooperative and redundant roles of VEGFR-2 and VEGFR-3 signaling in adult lymphangiogenesis.

Activation of vascular endothelial growth factor (VEGF) receptor-3 (VEGFR-3) by VEGF-C initiates lymphangiogenesis by promoting lymphatic proliferation and migration. However, it is unclear whether VEGFR-3 signaling is required beyond these initial stages, namely during the organization of new lymphatic endothelial cells (LECs) into functional capillaries. Furthermore, the role of VEGFR-2, which is also expressed on LECs and binds VEGF-C, is unclear. We addressed these questions by selectively neutralizing VEGFR-3 and/or VEGFR-2 for various time periods in an adult model of lymphangiogenesis in regenerating skin. While blocking either VEGFR-2 or VEGFR-3 with specific antagonist mAbs (DC101 and mF4-31C1, respectively) prior to lymphatic migration prevented lymphangiogenesis, blocking VEGFR-3 subsequent to migration did not affect organization into functional capillaries, and VEGFR-2 blocking had only a small hindrance on organization. These findings were confirmed in vitro using human LECs and anti-human antagonist mAbs (IMC-1121a and hF4-3C5): both VEGFR-2 and -3 signaling were required for migration and proliferation, but tubulogenesis in 3D cultures was unaffected by VEGFR-3 blocking and partially hindered by VEGFR-2 blocking. Furthermore, both in vitro and in vivo, while VEGFR-3 blocking had no effect on LEC organization, coneutralization of VEGFR-2, and VEGFR-3 completely prevented lymphatic organization. Our findings demonstrate that cooperative signaling of VEGFR-2 and -3 is necessary for lymphatic migration and proliferation, but VEGFR-3 is redundant with VEGFR-2 for LEC organization into functional capillaries.