Introduction: There is increasing evidence that the endothelium is able to transduce circulatory force into signals which influence angiogenic behaviour. The Von Hippel-Lindau homozygous mutant zebrafish (vhl-/-) has increased HIF-1α signalling and aberrant angiogenesis. In this study I therefore set out to characterise the angiogenic phenotype of the vhl-/- mutant and examine the effect of manipulating blood flow. Both nitric oxide (NO) and Notch regulated intracellular signalling have been shown to influence endothelial angiogenic behaviour in development and disease, so I studied the role of these on development of the angiogenic phenotype in the vhl-/- mutant.
Hypothesis: Does angiogenesis stimulated by up-regulated hypoxic signalling require blood flow?
Methods: vhl mutant zebrafish were crossed with endothelial reporter transgenics to assess and quantify the angiogenic phenotype. Blood flow was manipulated using genetic and pharmacological manipulation in developing zebrafish embryos. To study the effect of flow on endothelial notch signalling I utilised a novel transgenic line Tg(CSL:venus), which expresses a fluorescent reporter upon activation of a Notch regulated transcription factor. The role of individual notch signalling components was investigated using reverse transcription quantitative polymerase chain reaction (RT-qPCR) for various ligands in developing zebrafish tissue during development in the absence of flow and in control embryos.
Results: The vhl-/- mutant showed early vessel changes including enlargement and sprouting. These angiogenic vessel changes were dependent on the presence of circulatory flow and are lost in mutant embryos treated with troponin t2 morpholino or 2,3-Butanedione monoxime (BDM). Imaging of aortic endothelium showed a significant up regulation of notch signalling in the Tg(CSL:venus) line in the absence of flow, induced by troponin t2 knockdown or BDM treatment. This increase in endothelial notch signalling in the absence of flow was mediated by increased expression of the notch ligand dll4 in RT-qPCR and whole mount in-situ hybridisation experiments.
Conclusion: The aberrant angiogenesis stimulated by increased HIF-1α signalling in the vhl-/- mutant is dependent on blood flow. My data suggests a novel flow dependent regulation of notch signalling in the developing vasculature mediated by the suppression of dll4 by blood flow.
