Objective-To elucidate the downstream mechanisms of vascular endothelial growth factor receptor 2 (VEGFR2), a key receptor in angiogenesis, which has been associated with atherosclerotic plaque growth and instability. Methods and Results-By using a yeast-2-hybrid assay, we identified A Disintegrin And Metalloprotease 10 (ADAM10) as a novel binding partner of VEGFR2. ADAM10 is a metalloprotease with sheddase activity involved in cell migration; however, its exact function in endothelial cells (ECs), angiogenesis, and atherosclerosis is largely unknown. For the first time to our knowledge, we show ADAM10 expression in human atherosclerotic lesions, associated with plaque progression and neovascularization. We demonstrate ADAM10 expression and activity in ECs to be induced by VEGF; also, ADAM10 mediates the ectodomain shedding of VEGFR2. Furthermore, VEGF induces ADAM10-mediated cleavage of vascular endothelium (VE)-cadherin, which could increase vascular permeability and facilitate EC migration. Indeed, VEGF increases vascular permeability in an ADAM10-and ADAM17-dependent way; inhibition of ADAM10 reduces EC migration and chemotaxis. Conclusion-These data provide the first evidence of ADAM10 expression in atherosclerosis and neovascularization. Key Words: angiogenesis Ⅲ atherosclerosis Ⅲ endothelial function Ⅲ growth factors Ⅲ metalloproteinases A ngiogenesis is associated with tumor growth, metastasis, and growth and instability of atherosclerotic plaques. The latter contribute to plaque rupture and its clinical complications. 1 Expression of the major angiogenic factor, vascular endothelial growth factor (VEGF) A, increases during atherogenesis. In endothelial cells (ECs), VEGF exerts its stimulatory effect mostly via VEGF receptor (VEGFR) 2 (kinase insert domain receptor [KDR]/fetal liver kinase-1 ) and is a potent angiogenic mediator of EC migration, proliferation, and survival. 2 VEGF binding induces VEGFR2 dimerization, followed by (auto)phosphorylation on tyrosine residues. Activation of VEGFR2 induces signaling via phospholipase C (PLC)␥, activation of the mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) 1/2 pathway, and EC proliferation. Furthermore, activation of p38 -mitogen-activated protein kinase or phosphatidylinositol 3-kinase (PI3K) leads to EC migration, survival, and increased permeability. 2,3 See accompanying article on page 2087In addition to angiogenesis, VEGF and its receptors are associated with development of atherosclerosis, plaque angiogenesis, and plaque instability. Recently, it was suggested that VEGF may induce a more vulnerable plaque phenotype by promoting leukocyte recruitment. 4 6 The neutralizing VEGF antibody bevacizumab (Avastin) is an effective anticancer therapy that inhibits tumor angiogenesis; however, it is associated with an increase in cardiovascular adverse effects based on thrombosis and plaque instability. 7 Unraveling downstream VEGFR signaling mechanisms involved in angiogenesis and atherosclerosis will identify novel therapeutic...

Mechanical force is a determinant of Notch signalling but the mechanism of force detection and its coupling to Notch are unclear. We propose a role for Piezo1 channels, which are mechanically-activated non-selective cation channels. In cultured microvascular endothelial cells, Piezo1 channel activation by either shear stress or a chemical agonist Yoda1 activated a disintegrin and metalloproteinase domain-containing protein 10 (ADAM10), a Ca2+-regulated transmembrane sheddase that mediates S2 Notch1 cleavage. Consistent with this observation, we found Piezo1-dependent increase in the abundance of Notch1 intracellular domain (NICD) that depended on ADAM10 and the downstream S3 cleavage enzyme, γ-secretase. Conditional endothelial-specific disruption of Piezo1 in adult mice suppressed the expression of multiple Notch1 target genes in hepatic vasculature, suggesting constitutive functional importance in vivo. The data suggest that Piezo1 is a mechanism conferring force sensitivity on ADAM10 and Notch1 with downstream consequences for sustained activation of Notch1 target genes and potentially other processes.

Erythro-myeloid progenitors (EMPs) were recently described to arise from the yolk sac endothelium, just prior to vascular remodeling, and are the source of adult/post-natal tissue resident macrophages. Questions remain, however, concerning whether EMPs differentiate directly from the endothelium or merely pass through. We provide the first evidence in vivo that EMPs can emerge directly from endothelial cells (ECs) and demonstrate a role for these cells in vascular development. We find that EMPs express most EC markers but late EMPs and EMP-derived cells do not take up acetylated low-density lipoprotein (AcLDL), as ECs do. When the endothelium is labelled with AcLDL before EMPs differentiate, EMPs and EMP-derived cells arise that are AcLDL+. If AcLDL is injected after the onset of EMP differentiation, however, the majority of EMP-derived cells are not double labelled. We find that cell division precedes entry of EMPs into circulation, and that blood flow facilitates the transition of EMPs from the endothelium into circulation in a nitric oxide-dependent manner. In gain-of-function studies, we inject the CSF1-Fc ligand in embryos and found that this increases the number of CSF1R+ cells, which localize to the venous plexus and significantly disrupt venous remodeling. This is the first study to definitively establish that EMPs arise from the endothelium in vivo and show a role for early myeloid cells in vascular development.

During angiogenesis, endothelial tip cells start sprouting and express delta-like 4 (DLL4) downstream of vascular endothelial growth factor (VEGF). DLL4 subsequently activates Notch in the adjacent stalk cells suppressing sprouting. VEGF also activates A disintegrin and metalloproteases (ADAMs) that induce Notch ectodomain shedding. Although two major ADAMs, i.e. ADAM10 and ADAM17, have been implicated in Notch-signalling activation, their apparent different roles in angiogenesis have not been fully understood yet. The objective of this study was to determine the roles of ADAM10 and ADAM17 activity in angiogenesis. In mouse retinas, ADAM10 or γ-secretase inhibition induced vascular sprouting and density in vivo, whereas attenuation of both ADAM10 and ADAM17 activity produced the opposite phenotype. Retinal blood vessel analysis in ADAM17 hypomorphic mice confirmed the requirement for ADAM17 activity in angiogenesis. However, ADAM17 inhibition did not phenocopy blood vessel increase by Notch blockage. These observations suggest that ADAM17 regulates other fundamental players during angiogenesis besides Notch, which were not affected by ADAM10. By means of an angiogenesis proteome assay, we found that ADAM17 inhibition induced the expression of a naturally occurring inhibitor of angiogenesis Thrombospondin 1 (TSP1), whereas ADAM10 inhibition did not. Accordingly, ADAM17 overexpression downregulated TSP1 expression, and the TSP1 inhibitor LSKL rescued angiogenesis in the tube formation assay downstream of VEGF in the presence of ADAM17 inhibition. Finally, genetic and pharmacological ADAM17 blockade resulted in increased TSP1 expression in mouse retina. Altogether, our results show that ADAM10 and ADAM17 have opposite effects on sprouting angiogenesis that may be unrelated to Notch signalling and involves differentially expressed anti-angiogenic proteins such as TSP1.

Impaired ALK1 (activin receptor-like kinase-1)/Endoglin/BMP9 (bone morphogenetic protein 9) signaling predisposes to arteriovenous malformations (AVMs). Activation of SMAD1/5 signaling can be enhanced by shear stress. In the genetic disease hereditary hemorrhagic telangiectasia, which is characterized by arteriovenous malformations, the affected receptors are those involved in the activation of mechanosensitive SMAD1/5 signaling. To elucidate how genetic and mechanical signals interact in arteriovenous malformation formation, we sought to identify targets differentially regulated by BMP9 and shear stress. APPROACH AND RESULTS: We identify Cx37 (Connexin37) as a differentially regulated target of ligand-induced and mechanotransduced SMAD1/5 signaling. We show that stimulation of endothelial cells with BMP9 upregulated Cx37, whereas shear stress inhibited this expression. This signaling was SMAD1/5-dependent, and in the absence of SMAD1/5, there was an inversion of the expression pattern. Ablated SMAD1/5 signaling alone caused arteriovenous malformationlike vascular malformations directly connecting the dorsal aorta to the inlet of the heart. In yolk sacs of mouse embryos with an endothelial-specific compound heterozygosity for SMAD1/5, addition of TNFα (tumor necrosis factor-α), which downregulates Cx37, induced development of these direct connections bypassing the yolk sac capillary bed. In wild-type embryos undergoing vascular remodeling, Cx37 was globally expressed by endothelial cells but was absent in regions of enlarging vessels. TNFα and endothelial-specific compound heterozygosity for SMAD1/5 caused ectopic regions lacking Cx37 expression, which correlated to areas of vascular malformations. Mechanistically, loss of Cx37 impairs correct directional migration under flow conditions. CONCLUSIONS: Our data demonstrate that Cx37 expression is differentially regulated by shear stress and SMAD1/5 signaling, and that reduced Cx37 expression is permissive for capillary enlargement into shunts.