Characterization of indolinones which preferentially inhibit VEGF-C- and VEGF-D-induced activation of VEGFR-3 rather than VEGFR-2

VEGF-C and VEGF-D are lymphangiogenic factors that bind to and activate VEG FR-3, a fins-like tyrosine kinase receptor whose expression is limited almo st exclusively to lymphatic endothelium in the adult. Processed forms of VE GF-C and VEGF-D can also activate VEGFR-2, a key player in the regulation o f angiogenesis. There is increasing evidence to show that these receptor-li gand interactions play a pivotal role in a number of pathological situation s. Inhibition of receptor activation by VEGF-C and VEGF-D could therefore b e pharmaceutically useful. Furthermore, to understand the different roles o f VEGF-C, VEGF-D, VEGFR-2 and VEGFR-3 in pathological situations it will be necessary to dissect the complex interactions of these ligands and their r eceptors. To facilitate such studies we cloned, sequenced and characterized the expression of rat VEGF-C and VEGF-D. We showed that Cys152 --> Ser mut ants of processed rat VEGF-C can activate VEGFR-3 but not VEGFR-2, while th e corresponding mutation in rat VEGF-D inhibits its ability to activate bot h VEGFR-2 and VEGFR-3. We also synthesized and characterized indolinones th at differentially block VEGF-C- and VEGF-D-induced VEGFR-3 kinase activity compared to that of VEGFR-2. These tools should be useful in analysing the different activities and roles of VEGF-C, VEGF-D and their ligands, and in blocking VEGFR-3-mediated lymphangiogenesis.