EGFL7 Mediates BMP9-Induced Sprouting Angiogenesis of Endothelial Cells Derived from Human Embryonic Stem Cells

摘要

class="head no_bottom_margin" id="sec1title">IntroductionHuman embryonic stem cells (hESCs) are pluripotent and therefore offer opportunities to investigate early human development, model genetic diseases, and provide approaches to regenerative medicine. Thorough understanding of vascular differentiation of hESCs will yield new insights into how human vasculature develops and regenerates and lead to improved methods for producing vascular cells for transplantation. Gene ablation in mice has shown that the transforming growth factor β (TGF-β) family, which also includes bone morphogenetic proteins (BMPs), nodal, and activin, plays a fundamental role in mesodermal development and vascular commitment. Mutations in Endoglin and ALK1 have been linked to a human vascular disorder, hereditary hemorrhagic telangiectasia (HHT1 and HHT2, respectively), often resulting in arteriovenous malformation (AVM) (, ). Also, mutations in the BMP type II receptor gene are responsible for pulmonary arterial hypertension (PAH) ().TGF-β, activin, and nodal all activate SMAD2/3 to promote hESC self-renewal (). BMPs activate SMAD1/5, which upregulate the expression of ID proteins (inhibitors of DNA) that in turn inhibit bHLH transcription factors (). Data on the role of the BMP family in mediating the vascular commitment of hESCs is sparse. Vascular cells have a mesoderm origin. BMP4 induces mesodermal differentiation and patterning and promotes epithelial to mesenchymal transition (EMT) in hESCs in part via SLUG and MSX2 (). It is well known that vascular endothelial growth factor (VEGF) also plays a pivotal role in vascular differentiation. Vascular expansion of hESC-derived endothelial cells (ECs) can be induced by inhibition of the TGF-β/SMAD2/3 pathway (). The BMP9/ALK1/SMAD1/5 pathway has been studied intensively in ECs and in mouse ESC-derived ECs (reviewed in ). The fundamental mechanism by which BMP9/ALK1 signaling can induce or inhibit angiogenesis is, however, still unclear and presumably context dependent. This pathway has so far not been studied in the process of differentiation of hESC toward vascular cells.The BMP effectors SMAD1/5 have a pivotal role in stalk cell competence by regulating EC migration and activating the target genes of the NOTCH intracellular domain (NICD) in stalk cells in mice (). In addition, ALK1 signaling inhibits angiogenesis in mice by cooperating with the NOTCH pathway (). The molecular mechanism driving differential expression of target genes in tip and stalk cells remains unclear although several NICD-dependent genes are also BMP-induced SMAD1/5 targets as shown by chromatin immunoprecipitation sequencing (ChIP-seq) studies in human ECs (). ID proteins are targets of BMP-SMAD signaling that play a crucial role in determining the two EC types during the angiogenic phase (, ).Epidermal growth factor-like domain 7 (EGFL7) is a secreted angiogenic factor that is expressed by and acts on ECs. Expression of EGFL7 is highest in proliferating vasculature and regulates tubulogenesis in zebrafish and vascular patterning and integrity in mice (). Interestingly, microRNA-126, an EC-specific miRNA, resides in intron 7 of the EGFL7 gene and regulates vascular integrity via the VEGF regulators SPRED1 and PIK3R2 (, href="#bib29" rid="bib29" class=" bibr popnode">Wang et al., 2008). Ectopic EGFL7 interacts with the extracellular domain of NOTCH and as a result, functions as an antagonist of NOTCH activation (href="#bib24" rid="bib24" class=" bibr popnode">Schmidt et al., 2009). Here, we used hESCs as a model for human vascular commitment to study the underlying molecular mechanisms of BMP9 in vascular development and disease. We demonstrate that BMP9/ALK1/SMAD1/5-induced endothelial sprouting critically depends on EGFL7. We link the BMP signaling pathway for the first time with EGFL7.