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 HIF1␣ 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. (Arterioscler Thromb Vasc Biol. 2007;27:2135-2141.)Key Words: collateral circulation Ⅲ angiogenesis Ⅲ nitric oxide Ⅲ blood flow Ⅲ zebrafish A fter arterial occlusion, "collateral vessels" can restore some blood flow to the occluded artery. These arise from preexisting endothelial communications by arteriogenesis 1 initiated by shear stress, 2 and which subsequently remodel into mature collateral vessels. This remodeling is dependent on nitric oxide 3-5 and certain leukocyte subtypes, notably the monocyte/macrophage. 6,7 In the 18th century, Hunter observed collateral vessels by injecting dye post mortem into the circulation of a stag, having previously ligated its carotid artery. 8 Current models of arteriogenesis still closely resemble Hunter's, using arterial ligation followed by detection of collateral vessels by some (usually invasive) technique. These models are intuitively relevant but suffer from significant disadvantages, particularly technical difficulty in visualizing arteriogenesis. Most use post mortem perfusion-fixed angiography in a state of maximum vasodilatation, which abolishes physiological regulation of flow and is impossible to perform serially.Arterial ligation in mammals inevitably induces ischemia. This induces angiogenesis, inflammation, and necrosis. The contribution of these to arteriogenesis is unclear. Some studies suggest arteriogenesis proceeds independently of the downstream consequences of ischemia 9 ; others that molecules released in the hypoxic region induce local angiogenesis and upstream arteriogenesis. 10,11 Using current models, it is impossible to separate angiogenesis from arteriogenesis, as both are consequent on arterial occlusion.The zebrafish embryo possesses unique advantages for the study of vas...

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