IntroductionVascular endothelial growth factor (VEGF) signaling is a key regulator of vascular development during embryogenesis as well as neovascularization in the adult. [1][2][3] Intensity of VEGF signaling is strictly controlled during vascular development through ligandreceptor interaction. 4,5 Flk1 (also designated as VEGF receptor-2) is tyrosine-phosphorylated much more efficiently than Flt1 (VEGF receptor-1) upon VEGF binding and is thought to be the major receptor in endothelial cells (ECs) for VEGF-induced responses. [6][7][8] Whereas Flk1-null mice die at embryonic day 8.5 (E8.5) to E9.5 with no organized blood vessels, 9 Flt1-null mice die at midgestation with vascular overgrowth and disorganization. 10,11 Flt1 tyrosine kinase-deficient homozygous mice, in which VEGF can bind to the cell-surface domain of Flt1 but cannot conduct kinase signaling, developed normal vessels and survived, 12 indicating that VEGF signal intensity on Flk1 is regulated by absorption of VEGF to the higher affinity receptor, Flt1. VEGF-A heterozygotes die early in gestation due to failure in vascular system formation. 13 On the other hand, 2-to 3-fold overexpression of VEGF-A from its endogenous locus results in aberrant heart development and lethality at E12.5 to E14, 14 indicating that strictly balanced VEGF function is important in normal embryogenesis.Neuropilin-1 (NRP1) is a type 1 membrane protein, which is expressed in particular classes of developing neurons 15,16 and functions as a receptor for the class 3 semaphorins mediating semaphorin-elicited inhibitory axon guidance signals to neurons. 17,18 NRP1 is also expressed in ECs of blood vessels and endocardial cells of the heart. 15,16,19 NRP1, together with Flk1, forms a specific receptor for VEGF 165 , an isoform of VEGF, and the Flk1-VEGF 165 -NRP1 complex potently enhances Flk1 signaling. 20 Coexpression of NRP1 with Flk1 in cultured ECs enhanced VEGF 165 binding to Flk1 and VEGF-elicited mitogenic and chemotactic activities. 20 Overexpression of NRP1 in mouse embryos resulted in an excess production of blood vessels and malformed hearts. 15 NRP1-null mice die midway through gestation at E10.5 to E12.5 and exhibit defects in the heart, vasculature, and nervous system. 16 These findings indicate that NRP1 plays an important role in regulating vascular development, and Flk1/NRP1 system would be important for controlling VEGF signal intensity. However, the regulatory mechanisms of Flk1/NRP1 expression in vascular development are not fully elucidated.In the early embryo and in differentiating embryonic stem (ES) cells, Flk1 expression marks a common progenitor for both blood and endothelium. [21][22][23][24] To elucidate the mechanisms underlying vascular development, we have developed a novel ES cell differentiation system that exhibits early vascular development using Flk1 ϩ cells as common progenitors for vascular cells. 25 ES cell-derived Flk1 ϩ cells can differentiate into both ECs and mural cells (MCs: vascular smooth muscle cells and pericytes) and form mature...

Outcome of TGFβ1 signaling is context dependent and differs between individuals due to germ-line genetic variation. To explore innate genetic variants that determine differential outcome of reduced TGFβ1 signaling, we dissected the modifier locus Tgfbm3, on mouse chromosome 12. On a NIH/OlaHsd genetic background, the Tgfbm3b C57 haplotype suppresses prenatal lethality of Tgfb1 −/− embryos and enhances nuclear accumulation of mothers against decapentaplegic homolog 2 (Smad2) in embryonic cells. Amino acid polymorphisms within a disintegrin and metalloprotease 17 (Adam17) can account, at least in part, for this Tgfbm3b effect. ADAM17 is known to down-regulate Smad2 signaling by shedding the extracellular domain of TGFβRI, and we show that the C57 variant is hypomorphic for down-regulation of Smad2/3-driven transcription. Genetic variation at Tgfbm3 or pharmacological inhibition of ADAM17, modulates postnatal circulating endothelial progenitor cell (CEPC) numbers via effects on TGFβRI activity. Because CEPC numbers correlate with angiogenic potential, this suggests that variant Adam17 is an innate modifier of adult angiogenesis, acting through TGFβR1. To determine whether human ADAM17 is also polymorphic and interacts with TGFβ signaling in human vascular disease, we investigated hereditary hemorrhagic telangiectasia (HHT), which is caused by mutations in TGFβ/bone morphogenetic protein receptor genes, ENG, encoding endoglin (HHT1), or ACVRL1 encoding ALK1 (HHT2), and considered a disease of excessive abnormal angiogenesis. HHT manifests highly variable incidence and severity of clinical features, ranging from small mucocutaneous telangiectases to life-threatening visceral and cerebral arteriovenous malformations (AVMs). We show that ADAM17 SNPs associate with the presence of pulmonary AVM in HHT1 but not HHT2, indicating genetic variation in ADAM17 can potentiate a TGFβ-regulated vascular disease.

Vascular endothelial growth factor receptor 2 (VEGFR2) transmits signals of crucial importance to vasculogenesis, including proliferation, migration, and differentiation of vascular progenitor cells. Embryonic stem cell–derived VEGFR2+ mesodermal cells differentiate into mural lineage in the presence of platelet derived growth factor (PDGF)–BB or serum but into endothelial lineage in response to VEGF-A. We found that inhibition of H-Ras function by a farnesyltransferase inhibitor or a knockdown technique results in selective suppression of VEGF-A–induced endothelial specification. Experiments with ex vivo whole-embryo culture as well as analysis of H-ras −/− mice also supported this conclusion. Furthermore, expression of a constitutively active H-Ras[G12V] in VEGFR2+ progenitor cells resulted in endothelial differentiation through the extracellular signal-related kinase (Erk) pathway. Both VEGF-A and PDGF-BB activated Ras in VEGFR2+ progenitor cells 5 min after treatment. However, VEGF-A, but not PDGF-BB, activated Ras 6–9 h after treatment, preceding the induction of endothelial markers. VEGF-A thus activates temporally distinct Ras–Erk signaling to direct endothelial specification of VEGFR2+ vascular progenitor cells.

Vascular endothelial growth factor receptor 2 (VEGFR2) plays crucial roles in vasculogenesis, a process involving cell proliferation, migration and differentiation. However, the molecular mechanism by which VEGFR2 signaling directs vascular endothelial differentiation of VEGFR2+ mesodermal progenitors is not well understood. In this study, we examined the signal transduction pathway downstream of VEGFR2 for endothelial differentiation using an in vitro differentiation system of mouse embryonic stem-cell-derived VEGFR2+ cells. Using chimeric receptors composed of VEGFR2 and VEGFR3, the third member of the VEGFR family, we found that signaling through tyrosine 1175 (Y1175, corresponding to mouse Y1173) of VEGFR2 is crucial for two processes of endothelial differentiation: endothelial specification of VEGFR2+ progenitors, and subsequent survival of endothelial cells (ECs). Furthermore, we found that phospholipase Cγ1 (PLCγ1), which interacts with VEGFR2 through phosphorylated Y1175, is an inducer of endothelial specification. In contrast to VEGFR2, VEGFR3 does not transmit a signal for endothelial differentiation of VEGFR2+ cells. We found that VEGFR3 does not activate PLCγ1, although VEGFR3 has the ability to support endothelial cell survival. Taken together, these findings indicate that VEGFR2-PLCγ1 signal relay gives rise to the unique function of VEGFR2, thus enabling endothelial differentiation from vascular progenitors.