CIRB
Molecular control of vascular development
Anne EICHMANN, DR1 Inserm
Our lab has been interested in the mechanisms allowing blood vessels to develop stereotyped branched networks, as seen in developing chick, mouse and zebrafish embryos (Figure 1). We know that specialized endothelial cells (EC) called tip cells located at the extremities of growing capillary sprouts mediate guided vascular patterning. Tip cells exhibit characteristic features, including extension of filopodia that explore the tip cell environment, lack of a lumen and a slow proliferation rate. Following behind tip cells, other EC termed stalk cells form the capillary lumen and proliferate. Tip cell selection is induced by VEGF signaling through VEGFR2. Work by several groups, including ours, has shown that tip cells also express high levels of the endothelial-specific Notch ligand Delta-like 4 (Dll4). Inactivation of one allele of the Dll4 gene in mice and pharmacological inhibition of Notch signaling using gamma-secretase inhibitors causes excessive formation of tip cells (Figure 2). Dll4 inactivation leads to alterations in VEGF receptor levels, suggesting that Dll4 negatively regulates the sensitivity of ECs to VEGF signaling and so acts as a ‘brake’ on VEGF signaling during capillary sprouting to ensure that only a limited number of cells form tips (1, 2). Isolation of EC from Dll4+/- mice and microarray analysis has allowed us to identify novel tip cell marker genes that we are currently characterizing.
Capillary sprouting also shows morphological similarities to axon guidance. Like endothelial tip cells, axonal growth cones extend filopodia that sense and respond to extracellular guidance cues (Figure 3). By screening for the expression of axon guidance molecules in blood vessel EC, we have identified several key molecules regulating capillary guidance. The Netrin receptor UNC5B is expressed in arterial EC and in tip cells. Loss of function of unc5b in mouse and zebrafish embryos leads to ectopic filopodial extension from endothelial tip cells and excessive branching of blood vessels. Treatment of endothelial tip cells with the UNC5B ligand Netrin-1 leads to filopodial retraction, an effect that is lost in unc5b mutants. UNC5B thus functions as a repulsive guidance receptor controlling morphogenesis of the vascular system (3, 4). The capacity to direct blood vessel growth may have important therapeutic implications for the anti-angiogenic treatment of solid tumors. We are currently studying a novel UNC5B ligand that we have identified.
Our group has also shown the selective expression of the VEGF co-receptors Neuropilin (Nrp) in EC of arteries (Nrp1) and of veins/lymphatics (Nrp2). Homozygous nrp1 mutants show defects in arteriogenesis, while nrp2 mutant embryos exhibit selective defects in the formation of lymphatic vessels (5, 6). Lymphatic vessels are aberrantly positioned and enlarge rather than form branches. These defects are reproduced by antibodies blocking VEGF-C binding to Nrp2 and in Nrp2-Vegfr3 double heterozygous mice, indicating that binding of VEGF to Nrp2 and signal transduction through VEGFR3 mediate lymphatic vessel sprouting (6). We are currently crossing nrp2 mutants to transgenic mice expressing GFP in their lymphatic vessels to analyze live lymphatic vessel development in normal and mutant animals by multiphoton imaging.
Using confocal time-lapse video-microscopy of pericytes expressing red fluorescent protein, we have shown that treatment with thalidomide increases pericyte motility in vitro and pericyte recruitment to blood vessels in vivo in a PDGF-B dependent manner. Treatment of a mouse model of Hereditary Hemorraghic Telangieectasia (HHT) with thalidomide increases pericyte recruitment to damaged vessels and reduced bleeding in HHT patients. These results warrant clinical trials of HHT patients with thalidomide (7).
Selected publications 2004-2010
- Xu Y., Yuan L., Mak J., Pardanaud L., Caunt M., Kasman I., Larrivée B., Suchting S., del Toro R., Medvinsky A., Yang J., Kolodkin A., Thomas J.L., Koch A., Alitalo K., Eichmann* A. & Bagri* A. (2010), Neuropilin-2 mediates VEGF-C induced lymphatic sprouting together with VEGFR3. J Cell Biol, 188:115-30.
*equal contribution.
- Lebrin F., Srun S., Raymond K., Martin S., van den Brink S., Freitas C., Bréant C., Mathivet T., Larrivée B., Thomas J.L., Arthur H.A., Westermann C.J.J., Disch F., Mager J.J., Snijder R.J., Eichmann* A. & Mummery* C. (2010), Thalidomide enhances mural cell recruitment and reduces epistaxis in patients with hereditary hemorrhagic telangieectasia. Nat Med, 16: 420-8.
- Tammela T., Zarkada G., Murtomäki A., Wallgard E., Suchting S., Wirzeius M., Waltari M., Hellstrom M., Schomber T., Peltonen R., Duarte A., Freitas C., Isonemi H., Laakonen P., Christofori G., Yla-Hertuala S., Shibuya M., Pytowski B., Eichmann A., Betsholtz C. & Alitalo K. (2008), Inhibition of VEGFR-3 suppresses angiogenic sprouting and vascular network formation. Nature, 454: 656-660.
- Jones E.A.V., Yuan L., Bréant C., Watts R.J. & Eichmann A. (2008), Separating genetic and hemodynamic defects in neuropilin-1 knockout embryos. Development, 135: 2479-2488.
- Larrivée B., Freitas C., Trombe M., Lv X., Delafarge B., Yuan L., Bouvrée K., Bréant C., Del Toro R., Bréchot N., Germain S., Bono F., Dol F., Claes F., Fischer C., Autiero M., Thomas J.L., Carmeliet P., Tessier-Lavigne M. & Eichmann A. (2007), Activation of the UNC5B receptor by Netrin-1 inhibits sprouting angiogenesis. Genes & Dev, 21: 2433-2447.
- Suchting S., Freitas C., LeNoble F., Benedito R., Bréant C., Duarte A. & Eichmann A. (2007), The Notch ligand Delta-like 4 negatively regulates endothelial tip cell formation and vessel branching. Proc Natl Acad Sci USA, 104: 3225-3230.
- Lu X., LeNoble F., Yuan L., Jiang Q., de Lafarge B., Sugiyama D., Bréant C., Claes F., De Smet F., Thomas J.L., Autiero M., Carmeliet P., Tessier-Lavigne M. & Eichmann A. (2004), The Netrin receptor UNC5B mediates guidance events controlling morphogenesis of the vascular system. Nature, 432: 179-186.