Ischemia is not required for arteriogenesis in zebrafish embryos

Abstract

Objective: The role of ischemia in collateral vessel development (arteriogenesis) is a contentious issue that cannot be addressed using mammalian models. To investigate this, we developed models of arteriogenesis using the zebrafish embryo, which gains sufficient oxygenation via diffusion to prevent ischemia in response to arterial occlusion.

Methods and results: We studied gridlock mutant embryos that suffer a permanently occluded aorta and show that these restore aortic blood flow by collateral vessels. We phenocopied gridlock mutants by laser-induced proximal aortic occlusion in transgenic Fli1:eGFP/GATA1:dsRED embryos. Serial imaging showed these restore aortic blood flow via collateral vessels by recruitment of preexisting endothelium in a manner similar to gridlocks. Collateral aortic blood flow in gridlock mutants was dependent on both nitric oxide and myeloid cells. Confocal microscopy of transgenic gridlock/Fli1:eGFP mutants demonstrated no aberrant angiogenic response to the aortic occlusion. qPCR of HIF1alpha expression confirmed the absence of hypoxia in this model system.

Conclusions: We conclude that NO and myeloid cell-dependent collateral vessel development is an evolutionarily ancient response to arterial occlusion and is able to proceed in the absence of ischemia.

Figure 1

A. The percentage of gridlock mutants with blood flow in the distal aorta from 2-5 days post fertilisation (dpf). B. Angiograms generated by digital motion analysis of one wildtype embryo (upper panel) and two representative gridlock mutants (middle and lower panel) at 5 days post fertilisation. Head is to the right, tail to the left. Collateral vessels are indicated by red arrows.

Figure 2

Confocal microangiography of 3dpf wildtype and gridlock mutant embryos. Figures show dorsal view of proximal trunk, just above yolk sac (position shown on Supplementary Figure 2). Despite the fact that this gridlock embryo had no detectible blood flow in the distal aorta due to the occlusion (double arrow indicates absence of antegrade aortic flow), microspheres are passing via afferent intersegmental vessels into the distal aorta (arrow). A – aorta, CV – cardinal vein, DLAV – dorsal longitudinal anastamotic vessel.

Figure 3

Angiogram generated by digital motion analysis of representative 5dpf wildtype embryo 22 hours after laser-induced occlusion of the proximal aorta (arrow), showing collateral aortic blood flow arising from two communications with the intestinal vasculature (asterisks).

Figure 4

A. The effect of nitric oxide synthase inhibition on recovery of aortic blood flow in gridlock mutant embryos. Groups of 20-30 embryos were incubated in L-NAME, L-Arginine, control, or both at the doses indicated from 1-5 days post fertilisation. B. The timing of nitric oxide dependence of collateral aortic blood flow in gridlock mutant embryos. Data shows mean percentage of embryos with collateral flow ± SEM (3 replicates). Asterisks indicates p<0.05 compared to control.

Figure 5

A. The effect of pu.1 knockdown on macrophage number. Gridlock mutant embryos were injected with morpholino antisense to either control or pu.1 (that prevents myeloid cell differentiation), and stained with neutral red. Figure shows representative micrographs of dorsal and lateral views of representative 2dpf control or pu.1 morphant embryos. Arrows indicate neutral red staining macrophages. B. The effect of pu.1 knockdown on recovery of aortic blood flow in gridlock mutant embryos (n=30-40 per group, 4 replicates). Asterisk indicates p<0.05.