Alk1 regulation of embryonic angiogenesis

University Of Pittsburgh At Pittsburgh, Pittsburgh PA

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Abstract

DESCRIPTION (provided by applicant): Homozygous inactivation of the endothelial-specific TGFB type I receptor, Alk1, results in embryonic lethality stemming from vascular malformations, and heterozygous inactivation results in a potentially lethal human vascular dysplasia, hereditary hemorrhagic telangiectasia type 2 (HHT2). Clearly, AIk1 plays a necessary role in building and maintaining the vertebrate vasculature; however, the nature of that role is currently unclear. The objective of this proposal is to use the accessible zebrafish embryo, which has a well described, stereotypical vertebrate vasculature, to define the effects of Alkl activation on endothelial cell functional state and transcript expression. Specific Aim 1 comprises experiments designed to test the hypothesis that TGFB signaling through Alk1 is required for angiogenic resolution. Studies in support of this Aim will include time lapse confocal analysis of endothelial sprouting and proliferation in violet beauregarde (vbg) mutants, which harbor a mutation in alk1; and assessment of endothelial migration, proliferation, sprouting, and basement membrane formation in zebrafish embryos overexpressing dominant negative and/or constitutively active forms of alk1, alk5 (the canonical TGFB type I receptor that reportedly competes with Alk1 in determining endothelial state), and tgfB within the endothelium. Specific Aim 2 comprises experiments designed to test the hypothesis that Alk1 is induced by shear stress and plays a role in vascular remodeling. The effect of blood flow on alk1 expression and remodeling of cranial vessels in zebrafish embryos will be determined, and the effect of shear stress on Alk1expression in cultured mammalian cells will be assessed. Further experiments will identify molecular components of the pathway that translates mechanical shear stress into Alk1 induction. Specific Aim 3 comprises experiments designed to identify downstream components of Alk1 signaling. Expression levels of genes reportedly regulated by constitutively active Alk1 in mammalian cell culture will be compared in vbg mutants and wild type siblings, and novel alkl-regulated genes will be sought by comparing the endothelial transcriptome in vbg mutants and wild type siblings. Taken together, the proposed experiments will determine whether Alk1 plays a role in angiogenic activation, resolution, and/or remodeling, and, with the definition of downstream targets, will promote a better understanding of the molecular pathways of normal vessel development and HHT2 pathogenesis.