Combinatory action of VEGFR2 and MAP kinase pathways maintains endothelial-cell integrity

Abstract

Blood vessels normally maintain stereotyped lumen diameters and their stable structures are crucial for vascular function. However, very little is known about the molecular mechanisms controlling the maintenance of vessel diameters and the integrity of endothelial cells. We investigated this issue in zebrafish embryos by a chemical genetics approach. Small molecule libraries were screened using live Tg(kdrl:GRCFP)(zn1) transgenic embryos in which endothelial cells are specifically labeled with GFP. By analyzing the effects of compounds on the morphology and function of embryonic blood vessels after lumen formation, PP1, a putative Src kinase inhibitor, was identified as capable of specifically reducing vascular lumen size by interrupting endothelial-cell integrity. The inhibitory effect is not due to Src or general VEGF signaling inhibition because another Src inhibitor and Src morpholino as well as several VEGFR inhibitors failed to produce a similar phenotype. After profiling a panel of 22 representative mammalian kinases and surveying published data, we selected a few possible new candidates. Combinational analysis of these candidate kinase inhibitors established that PP1 induced endothelial collapse by inhibiting both the VEGFR2 and MAP kinase pathways. More importantly, combinatory use of two clinically approved drugs Dasatinib and Sunitinib produced the same phenotype. This is the first study to elucidate the pathways controlling maintenance of endothelial integrity using a chemical genetics approach, indicating that endothelial integrity is controlled by the combined action of the VEGFR2 and MAP kinase pathways. Our results also suggest the possible side effect of the combination of two anticancer drugs on the circulatory system.

Figure 1

PP1 blocked established blood circulation. PP1 was added to embryos at 30 hpf when blood circulation was already established. At 48 hpf the blood circulation of PP1-treated embryos stopped. (A and C) Control embryos. (B and D) PP1-treated embryos. Except blood cells stuck at sinus venous (black arrow), PP1-treated embryos looked normal. (C) Fluorescein isothiocyanate dextran (MW=2 000 000 Da) was observed in circulation, labeling the whole vasculature. (D) Fluorescein isothiocyanate dextran was stuck in heart (white arrow), indicating the lumen of dorsal aorta reduced and did not allow the dye to pass.

Figure 2

PP1 caused dorsal aorta to reduce while SU6656 and Sunitinib did not. All these three small molecules were added to Tg(kdrl:GRCFP)^zn1 embryos at 3 dpf and images were taken at 4 dpf. The trunk region above yolk extension was shown. White arrows point to the dorsal aorta. In PP1-treated embryos the dorsal aorta looked thinner than in control. In SU6656- or Sunitinib-treated embryos, the dorsal aortas remained the same.

Figure 3

Cross section showed PP1 did cause vascular lumens to collapse. The lower row is the magnification of upper row. White arrows point to the lumen of dorsal aorta. In PP1-treated embryos, both the dorsal aorta and cardinal vein collapsed, while the dorsal aorta collapsed more severely than the cardinal vein. The lumen of dorsal aorta disappeared. In other groups, the dorsal aorta and cardinal vein remained intact.

Figure 4

The ultra-structural changes of endothelial cells in aorta revealed by electron microscopy. The images were transverse sections with dorsal to the top. The lumen changes between the control embryos (A) and the PP1-treated embryos (B) at 4 dpf. (C and D) The structural changes of endothelial cells boxed in A and B, respectively. Note that the endothelial cell in D (black arrow head) was short and thick compared with that in C (white arrow head). The stars indicated the cell junctions between endothelial cells. N, notochord; S, somite. Scale bars are 2 μm.

Figure 5

Combinational treatment of kinase inhibitors induces the similar phenotype produced by PP1. All images are lateral view with dorsal to the top and anterior to the left. The combinational treatment of Dasatinib (D) or U0126 (U) with Sunitinib (SU), PTK787 (PTK), or ZM323881 (Z) resulted in the shrinkage of dorsal aorta.