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...

The response to hemodynamic force is implicated in a number of pathologies including collateral vessel development. However, the transcriptional effect of hemodynamic force is extremely challenging to examine in vivo in mammals without also detecting confounding processes such as hypoxia and ischemia. We therefore serially examined the transcriptional effect of preventing cardiac contraction in zebrafish embryos which can be deprived of circulation without experiencing hypoxia since they obtain sufficient oxygenation by diffusion. Morpholino antisense knock-down of cardiac troponin T2 (tnnt2) prevented cardiac contraction without affecting vascular development. Gene expression in whole embryo RNA from tnnt2 or control morphants at 36, 48, and 60 h postfertilization (hpf) was assessed using Affymetrix GeneChip Zebrafish Genome Arrays (>14,900 transcripts). We identified 308 differentially expressed genes between tnnt2 and control morphants. One such (CXCR4a) was significantly more highly expressed in tnnt2 morphants at 48 and 60 hpf than controls. In situ hybridization localized CXCR4a upregulation to endothelium of both tnnt2 morphants and gridlock mutants (which have an occluded aorta preventing distal blood flow). This upregulation appears to be of functional significance as either CXCR4a knock-down or pharmacologic inhibition impaired the ability of gridlock mutants to recover blood flow via collateral vessels. We conclude absence of hemodynamic force induces endothelial CXCR4a upregulation that promotes recovery of blood flow.

The zebrafish is a well established model of vertebrate development, but has recently emerged as a powerful tool for cardiovascular research and in vivo cardiovascular drug discovery. The zebrafish embryo's low cost, small size and permeability to small molecules coupled with the ability to generate thousands of embryos per week, and improved automation of assays of cardiovascular development and performance allow drug screening for a number of cardiovascular effects. Such studies have already led to discovery of novel cardiovascular drugs with potentially clinically beneficial effects. In this review we summarise the advantages and disadvantages of the zebrafish for drug discovery using some patents, previous literature on zebrafish-based drug screening and assess where the zebrafish will fit into existing drug discovery programmes.

The Deciphering the Mechanisms of Developmental Disorders programme has analysed the morphological and molecular phenotypes of embryonic and perinatal lethal mouse mutant lines in order to investigate the causes of embryonic lethality. Here we show that individual whole-embryo RNA-seq of 73 mouse mutant lines (>1000 transcriptomes) identifies transcriptional events underlying embryonic lethality and associates previously uncharacterised genes with specific pathways and tissues. For example, our data suggest that Hmgxb3 is involved in DNA-damage repair and cell-cycle regulation. Further, we separate embryonic delay signatures from mutant line-specific transcriptional changes by developing a baseline mRNA expression catalogue of wild-type mice during early embryogenesis (4–36 somites). Analysis of transcription outside coding sequence identifies deregulation of repetitive elements in Morc2a mutants and a gene involved in gene-specific splicing. Collectively, this work provides a large scale resource to further our understanding of early embryonic developmental disorders.

We sought to determine a role for platelets in in vivo angiogenesis, quantified by changes in the capillary to fibre ratio (C∶F) of mouse skeletal muscle, utilising two distinct forms of capillary growth to identify differential effects. Capillary sprouting was induced by muscle overload, and longitudinal splitting by chronic hyperaemia. Platelet depletion was achieved by anti-GPIbα antibody treatment. Sprouting induced a significant increase in C∶F (1.42±0.02 vs. contralateral 1.29±0.02, P<0.001) that was abolished by platelet depletion, while the significant C∶F increase caused by splitting (1.40±0.03 vs. control 1.28±0.03, P<0.01) was unaffected. Granulocyte/monocyte depletion showed this response was not immune-regulated. VEGF overexpression failed to rescue angiogenesis following platelet depletion, suggesting the mechanism is not simply reliant on growth factor release. Sprouting occurred normally following antibody-induced GPVI shedding, suggesting platelet activation via collagen is not involved. BrdU pulse-labelling showed no change in the proliferative potential of cells associated with capillaries after platelet depletion. Inhibition of platelet activation by acetylsalicylic acid abolished sprouting, but not splitting angiogenesis, paralleling the response to platelet depletion. We conclude that platelets differentially regulate mechanisms of angiogenesis in vivo, likely via COX signalling. Since endothelial proliferation is not impaired, we propose a link between COX1 and induction of endothelial migration.