Alice Plein

NRP1 acts cell autonomously in endothelium to promote tip cell function during sprouting angiogenesis

Neuropilin (NRP) 1 is a receptor for the vascular endothelial growth factor (VEGF)-A and is essential for normal angiogenesis. Previous in vitro experiments identified NRP1 interactions with VEGF-As main signaling receptor VEGFR2 within endothelial cells, but also between nonendothelial NRP1 and endothelial VEGFR2. Consistent with an endothelial role for NRP1 in angiogenesis, we found that VEGFR2 and NRP1 were coexpressed in endothelial tip and stalk cells in the developing brain. In addition, NRP1 was expressed on two cell types that interact with growing brain vessels-the neural progenitors that secrete VEGF-A to stimulate tip cell activity and the pro-angiogenic macrophages that promote tip cell anastomosis. Selective targeting of Nrp1 in each of these cell types demonstrated that neural progenitor- and macrophage-derived NRP1 were dispensable, whereas endothelial NRP1 was essential for normal brain vessel growth. NRP1 therefore promotes brain angiogenesis cell autonomously in endothelium, independently of heterotypic interactions with nonendothelial cells. Genetic mosaic analyses demonstrated a key role for NRP1 in endothelial tip rather than stalk cells during vessel sprouting. Thus, NRP1-expressing endothelial cells attained the tip cell position when competing with NRP1-negative endothelial cells in chimeric vessel sprouts. Taken together, these findings demonstrate that NRP1 promotes endothelial tip cell function during angiogenesis.

Neuropilin 1 (NRP1) hypomorphism combined with defective VEGF-A binding reveals novel roles for NRP1 in developmental and pathological angiogenesis

Neuropilin 1 (NRP1) is a receptor for class 3 semaphorins and vascular endothelial growth factor (VEGF) A and is essential for cardiovascular development. Biochemical evidence supports a model for NRP1 function in which VEGF binding induces complex formation between NRP1 and VEGFR2 to enhance endothelial VEGF signalling. However, the relevance of VEGF binding to NRP1 for angiogenesis in vivo has not yet been examined. We therefore generated knock-in mice expressing Nrp1 with a mutation of tyrosine (Y) 297 in the VEGF binding pocket of the NRP1 b1 domain, as this residue was previously shown to be important for high affinity VEGF binding and NRP1-VEGFR2 complex formation. Unexpectedly, this targeting strategy also severely reduced NRP1 expression and therefore generated a NRP1 hypomorph. Despite the loss of VEGF binding and attenuated NRP1 expression, homozygous Nrp1Y297A/Y297A mice were born at normal Mendelian ratios, arguing against NRP1 functioning exclusively as a VEGF164 receptor in embryonic angiogenesis. By overcoming the mid-gestation lethality of full Nrp1-null mice, homozygous Nrp1Y297A/Y297A mice revealed essential roles for NRP1 in postnatal angiogenesis and arteriogenesis in the heart and retina, pathological neovascularisation of the retina and angiogenesis-dependent tumour growth.

The embryonic mouse hindbrain as a qualitative and quantitative model for studying the molecular and cellular mechanisms of angiogenesis

The mouse embryo hindbrain is a robust and adaptable model for studying sprouting angiogenesis. It permits the spatiotemporal analysis of organ vascularization in normal mice and in mouse strains with genetic mutations that result in late embryonic or perinatal lethality. Unlike postnatal models such as retinal angiogenesis or Matrigel implants, there is no requirement for the breeding of conditional knockout mice. The unique architecture of the hindbrain vasculature allows whole-mount immunolabeling of blood vessels and high-resolution imaging, as well as easy quantification of angiogenic sprouting, network density and vessel caliber. The hindbrain model also permits the visualization of ligand binding to blood vessels in situ and the analysis of blood vessel growth within a natural multicellular microenvironment in which endothelial cells (ECs) interact with non-ECs to refine the 3D organ architecture. The entire procedure, from embryo isolation to imaging and through to results analysis, takes approximately 4 d.

Neuropilin Regulation of Angiogenesis, Arteriogenesis, and Vascular Permeability

The formation of the cardiovasculature, consisting of both the heart and blood vessels, is a critical step in embryonic development and relies on three processes termed vasculogenesis, angiogenesis, and vascular remodeling. The transmembrane protein NRP1 is an essential modulator of embryonic angiogenesis with additional roles in vessel remodeling and arteriogenesis. NRP1 also enhances arteriogenesis in adults to alleviate pathological tissue ischemia. However, in certain circumstances, vascular NRP1 signaling can be detrimental, as it may promote cancer by enhancing tumor angiogenesis or contribute to tissue edema by increasing vascular permeability. Understanding the mechanisms of NRP1 signaling is, therefore, of profound importance for the design of therapies aiming to control vascular functions. Previous work has shown that vascular NRP1 can variably serve as a receptor for two secreted glycoproteins, the VEGF‐A and SEMA3A, but it also has a poorly understood role as an adhesion receptor. Here, we review current knowledge of NRP1 function during blood vessel growth and homeostasis, with special emphasis on the vascular roles of its multiple ligands and signaling partners.

Neural crest–derived SEMA3C activates endothelial NRP1 for cardiac outflow tract septation

In mammals, the outflow tract (OFT) of the developing heart septates into the base of the pulmonary artery and aorta to guide deoxygenated right ventricular blood into the lungs and oxygenated left ventricular blood into the systemic circulation. Accordingly, defective OFT septation is a life-threatening condition that can occur in both syndromic and nonsyndromic congenital heart disease. Even though studies of genetic mouse models have previously revealed a requirement for VEGF-A, the class 3 semaphorin SEMA3C, and their shared receptor neuropilin 1 (NRP1) in OFT development, the precise mechanism by which these proteins orchestrate OFT septation is not yet understood. Here, we have analyzed a complementary set of ligand-specific and tissue-specific mouse mutants to show that neural crest-derived SEMA3C activates NRP1 in the OFT endothelium. Explant assays combined with gene-expression studies and lineage tracing further demonstrated that this signaling pathway promotes an endothelial-to-mesenchymal transition that supplies cells to the endocardial cushions and repositions cardiac neural crest cells (NCCs) within the OFT, 2 processes that are essential for septal bridge formation. These findings elucidate a mechanism by which NCCs cooperate with endothelial cells in the developing OFT to enable the postnatal separation of the pulmonary and systemic circulation.

Neural crest cells in cardiovascular development.

Cardiac neural crest cells (NCCs) are a transient, migratory cell population exclusive to vertebrate embryos. Ablation, transplantation, and lineage-tracing experiments in chick and mouse have demonstrated their essential role in the remodeling of the initially bilateral and symmetric pharyngeal artery pairs into an aortic arch and for the septation of the cardiac outflow tract into the base of the pulmonary artery and aorta. Accordingly, defective cardiac NCC function is a common cause of congenital birth defects. Here, we review our current understanding of cardiac NCC-mediated vascular remodeling and signaling pathways important for this process. We additionally discuss their contribution to the cardiac valves as well as the still contentious role of cardiac NCCs in the development of the myocardium and conductive system of the heart.

The Mouse Hindbrain: An In Vivo Model to Analyze Developmental Angiogenesis

Angiogenesis, defined as the sprouting of new blood vessels from preexisting ones, is a biological process of great clinical relevance due to its involvement in disease as well as its therapeutic potential for revascularizing ischemic tissues. The embryonic mouse hindbrain provides an excellent model to study the molecular and cellular mechanisms of angiogenesis in vivo due the simple geometry of the hindbrain vasculature and its easy accessibility for fluorescent or histochemical staining, and for image capture and quantitation. This chapter outlines protocols for dissection, staining, and analysis of the mouse embryo hindbrain vasculature.

Regulation and Function of Cardiac Neural Crest Cells

The cardiac neural crest cells (NCCs), also known as circumpharyngeal NCCs, make an essential contribution to cardiovascular development in vertebrates. These cells delaminate from the developing neural tube to migrate into the pharyngeal arches (PAs) of the embryo, where they induce the remodeling of the pharyngeal arch arteries (PAAs) into the great vessels that distribute blood from the heart into the lung and systemic vasculature. A subset of cardiac NCCs continues to migrate into the cardiac outflow tract (OFT) to induce its septation into the arterial and pulmonary trunks that connect to the remodeled PAAs. Owing to their essential role in cardiovascular development, cardiac NCC defects cause congenital heart disease. In this article, we describe current knowledge of the molecular and cellular mechanisms that underlie cardiac NCC function.