Introduction and Objectives Pulmonary arterial hypertension (PAH) is a rare but severe condition, often fatal within 3–5 years due to right heart failure. Mutations in the bone morphogenetic protein (BMP) type II receptor (BMPR-II) underlie heritable forms of the disease but the mechanisms leading to vascular disease remain obscure from studies in mice and humans. Here we use zebrafish, which have a well-documented pattern of angiogenesis, as a model organism to address this question.
Methods The transgenic Tg(fli1a:egfp) zebrafish line, where the fli1a promoter drives GFP expression in vascular endothelial and blood cells, was used throughout this work. A variety of methods were used to dissect the role of BMP signalling in vascular development including: (i) BMP receptor inhibitors (dorsomorphin and LDN193189), (ii) antisense morpholino oligonucleotides (morpholinos) and (iii) transgenic zebrafish engineered with heat shock inducible dominant-negative BMP receptors. To identify BMP responsive transcripts from vascular endothelial cells in vivo we developed a system to allow FACS isolation of GFP+ve cells dissociated from Tg(fli1a:egfp) zebrafish following incubation in LDN193189 or DMSO solvent control. The mRNA transcripts in GFP+ve cells were determined using massively parallel (Illumina) sequencing, mapped to the Zv8 zebrafish genome and differences in transcript abundance between LDN193189 and DMSO treated embryos were determined using cufflinks.
Results Inhibition of BMP signalling with LDN193189 in zebrafish embryos after dorso-ventral patterning has occurred blocked venous but not arterial angiogenesis. This phenotype was reproduced in BMPR2 dominant-negative zebrafish and following knockdown of the zebrafish homologues of BMPR2, bmpr2a and bmpr2b with morpholinos. Illumina sequencing identified the BMP responsive genes in vascular endothelial cells and the gene(s) responsible for the venous angiogenic phenotype are being determined by knockdown of these genes with morpholinos.
Conclusion BMP signalling, via BMPR2, is critical for venous but not arterial angiogenesis in zebrafish. BMPR2 mutations are recognised as causative in patients with heritable PAH, where venous involvement is recognised, but also in pulmonary veno-occlusive disease. Our findings provide novel insights onto the potential role of the pulmonary venous system in pulmonary hypertension and implicate abnormal venous angiogenesis as a novel mechanism underlying this disease.
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