Venousization enables angiogenesis in the absence of VEGFR signalling

Abstract The VEGF ligand and its main receptor VEGFR2 are considered to be essential for endothelial differentiation, proliferation, sprouting and survival. Blood vessels cannot form in growing embryos or tissues when VEGF/VEGFR2 signalling is compromised in all cells. The Anti-VEGF blocking antibody is one of the most widely used antibodies in the clinics, blocking angiogenesis in cancer, wound healing or in ischemic diseases. However, vascular resistance to Anti-VEGF has been reported. Here we used iFlpMosaics and iSuRe-HadCre to induce and track genetic mosaics of endothelial cells (ECs) lacking VEGFR2 during the entire embryonic and postnatal development. Surprisingly, Vegfr2 KO ECs adapt, proliferate normally and compete with wild-type cells over time, ultimately forming a substantial portion of the capillary network in most organs. We found that Vegfr2 KO ECs are not able to sprout during the first wave of angiogenesis in most tissues, because it is highly VEGF-dependent and favours the growth and mobilization of wild-type ECs. However, due to their inability to respond to VEGF, over time Vegfr2 KO ECs become dominant in veins, which provide for a long term and continuous source of ECs for subsequent waves of VEGF-independent angiogenesis. Comparative scRNAseq analysis of ECs with acute and long-term loss of VEGFR2, revealed both common and organ-specific molecular mechanisms of adaptation and resistance to VEGF signalling loss. This included significant endothelial venousization and the upregulation of ligands for VEGFR1 and VEGFR3. VEGFR1 only partially compensated for VEGFR2 loss in capillary ECs, whereas VEGFR3 only compensated in arterial ECs. Loss of the three VEGF receptors did not compromise venous growth. This work changes our understanding of the role of VEGF signaling and its receptors in angiogenesis and reveals mechanisms of adaptation and resistance to their loss.