Abstract 301: Identifying Novel Regulators of Arteriogenesis Using the Zebrafish
Introduction. We have established a model of arteriogenesis (collateral vessel development) using the gridlock (Hey2) mutant zebrafish. This has a permanently occluded proximal aorta. We have shown that mutants restore distal aortic blood flow by recruiting pre-existing collateral communications with intestinal and dorsal vasculature. As arteriogenesis is driven by altered hemodynamic forces, we identified potential modulators of arteriogenesis by microarray comparison of embryos with and without blood flow. We evaluated the effect of knockdown of two such candidates (WASP and CXCR4a) in our model of arteriogenesis.
Methods; Microarray. We injected wildtype zebrafish embryos with a morpholino against cardiac troponinT2 (CTTN2). This prevents any cardiac contraction though embryos survive without ischemia due to passive oxygen diffusion. The vasculature of CTTN2 morphants thus never experiences blood flow. We extracted total RNA from 120–150 embryos injected with CTTN2 or control morpholino (n=3 per group) at 36, 48, and 60 hours post fertilisation (8, 20, 32 hours after onset of blood flow in control). Expression of over 14500 genes was assessed using Affymetrix chips. Candidate modulators of arteriogenesis were defined as displaying more than two-fold change in expression in response to blood flow. Candidates were knocked down in gridlock mutants by morpholino injection immediately post fertilisation to assess the effect on arteriogenesis.
Results. We identified more than 150 candidate genes differentially expressed in response to blood flow. Many relate to myeloid cell function, known to be essential for arteriogenesis. We evaluated two such candidates; CXCR4a (x2 down regulated in control morphants), and WASP (x4 up-regulated in controls). Morpholino knockdown of CXCR4a in gridlock mutants significantly reduced the percentage of embryos that recovered collateral blood flow to the occluded aorta (24±4% of control p<0.05). WASP morpholino knockdown increased the percentage of embryos which recovered blood flow in the occluded aorta 151±5% of control p<0.01).
Conclusions. Blood flow upregulates WASP, whilst down-regulating CXCR4a. Knockdown of CXCR4a reduces, whilst knockdown of WASP enhances, arteriogenesis in a zebrafish model.