Dissecting Vascular Endothelial Growth Factor Receptor-3 Functions In Angiogenesis And Lymphangiogenesis

Blood vasculature formation during early embryo development could be generally divided into two steps:vasculogenesis and angiogenesis. Vasculogenesis is the formation of primary vascular plexus from the endothelial precursor cells present in the blood islands, angiogenesis is the remodeling of the primary plexus into a hierarchical mature vascular network. Vascular Endothelial Growth Factor Receptor-3 (VEGFR-3) belongs to receptor tyrosine kinase (RTK) family, which is highly expressed on blood endothelial cells during this period. Genetic inactivation of VEGFR-3 led to abnormal blood vascular growth/remodeling and mice died at midgestation (embryonic day, E9.5-10.5). This reveals that VEGFR-3 is essential for blood vascular development, but the underlying mechanism was not clear. Lymphatic vessel development occurs later than blood vasculature (From E10.5), and initiates by the process of polarized endothelial cells budding from the cardinal vein. These cells express high levels of VEGFR-3 and migrate toward the chemotactic Vascular Endothelial Growth Factor-C (VEGF-C). Both VEGF-C and VEGF-D are the known cognate ligands for VEGFR-3. However, VEGF-C/D double knockout mice only exhibit lymphatic vascular defects, and the blood vasculature is quite normal which does not resemble the VEGFR-3 knockout mice phenotype. This suggests either the existence of unidentified ligands for VEGFR-3 or a ligand independent function of VEGFR-3 during blood vascular development. Furthermore, VEGFR-3 has also been found to be expressed by blood endothelial cells at pathological conditions such as cancer. Antibodies against VEGF-C/VEGFR-3 as an intervention target are under imperative study, in addition to anti-VEGF therapy. Therefore it would be important to find out the underlying mechanism of VEGFR-3 in angiogenesis and elucidate its differential functions in angiogenesis and lymphangiogenesis. This will improve our understandings of VEGFR-3 and facilitate drug discovery targeting this pathway.In this study, we first generated a VEGFR-3 conditional knockout model targeting coding region for the ligand-binding domain (Vegfr3△LBD). We have validated that VEGFR3△LBD lose the ability of binding ligand such as VEGF-C, theoretically also should not bind to any undiscovered VEGF-C/D homologue in vivo if exists. Proper targeting of the LBD region was confirmed in Vegfr3△LBD embryos at both mRNA and protein levels, however these mice only showed lymphatic vasculature defects, evidenced by irregular lymph sacs formation and lacking lymphatic vessels in all the tissues. The residual ability of lymphatic endothelial cells budding from the cardinal vein might be mediated by another VEGF-C receptor Nuropilin-2 or other mediators which requires further investigation. The blood vascular development in embryo and yolk sac was not affected at both E10.5 and E15.5, mice finally died due to unresolved edema. This result suggests that there is unlikely to exist an unknown ligand. In order to further confirm our proposition, we employed another genetic model-Vegfr3TKmut, this mice have an I1053F mutation at the ATP binding motif within the intracellular kinase domain which completely abolishes VEGFR-3 phosphorylation ability. VEGFR-3TKmut also produce a dominant negative effect by forming heterodimers with the wild type(WT) VEGFR-3. Vegfr3+/- mice develop normal lymphatic network, but Vegfr3+/TKmut mice show chylous and abnormal lymphatic vessels. In addition, we have also checked retinal angiogenesis during postnatal and xenograft tumor angiogenesis, and found no obvious difference between Vegfr3+/TKmut and Vegfr3+/+mice. We further investigated homozygous VEGFR-3TKmut mice and as expected Vegfr3TKmut/TKmut mice had normal blood vasculature at both E10.5 and E15.5, but without lymphatic vasculature and lymph sac formation. The mice died perinatally due to severe edema.Above loss of function genetic models provide us evidence that VEGF-C signaling through VEGFR-3 is crucial for lymphatic vessel but not blood vessel growth. Since VEGFR-3 is expressed on blood endothelial cells during early embryogenesis and down-regulated after E10.5, we have generated a conditional VEGFR-3 transgenic mouse model. This mice express Luciferase(Luc) in all the tissues under the CAG promoter, and Luc can be removed to initiate VEGFR-3 expression when Cre recombinase is expressed. After mating with TEKCre (endothelial specific) mice, we detected exogenous VEGFR-3 expression on blood endothelial cells and found the double transgenic mice died around E11.5 with severe anemia and disrupted angiogenesis. This result suggests overexpression of VEGFR-3 also disrupts normal angiogenesis. To answer the question on cellular and molecular levels, we mated conditional Tsa58t(SV40) transgenic mice with our gene targeted and transgenic mice in order to get the immortalized endothelial cells with different VEGFR-3 forms and expression levels. Because primary endothelial cells are difficult to isolate and propagate, we also use viral vectors to overexpress VEGFR-3WT, VEGFR-3△LBD and VEGFR-3TKmut in primary Human Umbilical Vein Endothelial Cells (HUVECs). We found both WT and mutant VEGFR-3 could form heterodimers with VEGFR-2, and decreased the level of phospho-VEGFR-2 as well as the downstream phospho-Erkl/2 in endothelial cells when treated with VEGF-A.In conclusion, through generating loss of function and overexpression genetic models we provide solid evidence showing that VEGFR-3 mediated signaling is crucial for lymphatic growth. However, in blood endothelial cells VEGFR-3 may mainly function via forming heterodimers with VEGFR-2 to modulate its downstream signals.