VEGFR-3 ligand-binding and kinase activity are required for lymphangiogenesis but not for angiogenesis

Abstract

Although VEGFR-3 deficiency disrupts blood vascular development during early embryogenesis, the underlying mechanism was not clear. To characterize its function in angiogenesis and lymphangiogenesis, we employed two genetically modified mouse models in this study, targeting the coding region for the ligand-binding domain (Vegfr3 ∆LBD ) or the tyrosine kinase domain with an inactivation point mutation (Vegfr3 TKmut ). We show that lymphatic growth was disrupted in Vegfr3 ∆LBD/∆LBD and Vegfr3 TKmut/TKmut mice, but blood vessels developed normally in both embryo and yolk sac. Interestingly, in Vegfr3 ∆LBD/∆LBD but not Vegfr3 TKmut/TKmut mice, lymph sac was present but there was lack of lymphangiogenic sprouting. We further demonstrate that both the wild-type and mutant forms of VEGFR-3 could form heterodimers with VEGFR-2, and decreased the level of phospho-VEGFR-2 and the downstream phospho-Erk1/2 in endothelial cells when they were treated with VEGF-A. These findings indicate that signaling mediated via VEGFR-3 activation by its cognate ligands (VEGF-C/-D) is not required for angiogenesis, and that VEGFR-3 may play a role in this process by modulating VEGFR-2-mediated signals.

Molecular Regulation of Lymphangiogenesis in Development and Tumor Microenvironment

“…Consistently, similar phenotypes were also observed in an independent mutant mouse model in which the kinase activity of VEGFR-3 was ablated by a missense mutation in the tyrosine kinase catalytic domain. Blood vascular development proceeded normally while lymphangiogenesis was disrupted in Vegfr3 TKmut/TKmut mice (Zhang, Zhou et al 2010). Interestingly, in Vegfr3 ΔLBD/ΔLBD mice, lymph sac formation occurred but without lymphatic vessel sprouting due to the loss of ligand sensing ability by LEC expressing the truncated VEGFR-3 (VEGFR-3 ΔLBD ).…”

“…It is also crucial for pathological lymphangiogenesis, such as in tumor (He et al 2004a;He et al 2005). While VEGFR-3 is initially expressed by all the endothelial cells of primary vascular network in mice during the embryonic development, its expression is primarily in LECs at the later stage of embryogenesis and persists in adult lymphatic vessels (Kaipainen et al 1995;Zhang, Zhou et al 2010). It has been found that TBX-1 activates VEGFR-3 transcription in endothelial cells (ECs) by binding to an enhancer element in the Vegfr3 gene .…”

“…VEGFR-3, also known as Fms-related tyrosine kinase (FLT4) (Pajusola et al 1992), mediates a critical pathway for lymphatic vessel growth and maintenance (Karkkainen et al 2000;Karkkainen et al 2001;Makinen et al 2001;Zhang, Zhou et al 2010). It is also crucial for pathological lymphangiogenesis, such as in tumor (He et al 2004a;He et al 2005).…”

“…Mice null for Vegfr3 died at midgestation resulting from the blood vascular defects (Dumont et al 1998;Hamada et al 2000). To further investigate biological functions of VEGFR-3, we generated a conditional knockout mouse model targeting its ligand binding domain (LBD) (Zhang, Zhou et al 2010). Mice homozygous for the LBD deletion (Vegfr3 ΔLBD/ΔLBD ) showed only disruption of lymphatic vessel growth, but blood vascular development in both embryo and yolk sac was not affected.…”

Pyogenic granuloma, an impaired wound healing process, linked to vascular growth driven by FLT4 and the nitric oxide pathway

“…In this situation, and in contrast to FLT-1, signaling mediated through FLT4 does not require activation by any of its recognized ligands, VEGFB or VEGFC. 30 Finally, FLT4 missense somatic mutations have been found in infantile hemangiomas, 31 further implicating this gene in the pathogenesis of pyogenic granuloma.…”