The formation of arterial and venous (AV) branches must be exquisitely coordinated to generate proper AV circuitry essential for vascular function. The mechanism of AV coordination particularly that between paired, parallel arteries and veins is poorly understood. Our long-term objective is to elucidate the genetic program of mammalian AV circuitry. In the previous funding period, we reported that luminal sizes of the developing dorsal aorta (DA) and cardinal vein (CV) are synchronized. Notch signaling controls arterial specification and the allocation of both arterial and venous endothelial cells (ECs) into their respective vessels, thereby balancing the sizes of the developing DA and CV. We have also obtained preliminary data in mice suggesting that DA and CV formation is not initiated by pre-determined arterial and venous ECs, as previously thought. Instead, our work suggests a new step-wise model of mammalian parallel AV pair morphogenesis: the primitive unspecified artery assembles prior to the vein;followed by a phase of mixed AV identities in both vessels;finally the mixed ECs are segregated into uniformly-specified vessels with coordinated sizes.
The specific aims of this grant are designed to test this new paradigm and to define the cellular mechanisms mediated by AV signaling in the morphogenesis of parallel AV pairs in mice. Our strategy is to take a cross- disciplinary approach including cutting-edge mouse genetics, cell lineage fate mapping, and imaging technologies. We recently built a custom 2-photon excited fluorescence microscope that is capable of imaging vasculature 1000 5m deep in living mouse tissue, achieving unprecedented resolution of previously inaccessible vascular structures.
Aim 1 Examine Vascular Endothelial Growth Factor (VEGF)-mediated cell differentiation as a mechanism underlying heterogeneous arterial- and venous- fated ECs in the primordial DA (pDA) and CV (pCV).
Aim 2 Examine cell segregation as a mechanism to sort venous-fated ECs in the pDA to the pCV.
Aim 3 Determine the role of Notch signaling in coordinating the development of parallel artery and vein pairs.
Aim 4 Determine the requirement of endothelial Notch1 and Coup-TFII in AV specification of adult parallel artery and vein pairs. Successful completion of this study will conceptually advance our knowledge of the morphogenesis and maintenance of parallel AV pairs, providing evidence regarding the origins of arteries and veins. Basic knowledge of the molecular mechanism of AV specification will inspire novel approaches to study blood vessel regeneration and vein graft engineering in disease settings. The combination of 2-photon high-resolution imaging with cutting-edge cell lineage tracing in living mouse embryos will be a major technological innovation for the field of mammalian vascular development at large.

Public Health Relevance

Our study aims to reveal the role of the Notch signaling pathway in vascular differentiation and maintenance and illuminate potential molecular mechanisms underlying these processes. In the future, this basic understanding of Notch in pre- and post-natal mammalian vascular function will guide investigations into its function in vessel regeneration under pathological conditions, such as heart attack, stroke, and other ischemic diseases. Ultimately, our understanding of the Notch pathway may lead to the identification of novel drug targets and therapeutic interventions for cardiovascular diseases.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Research Project (R01)
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Cardiovascular Differentiation and Development Study Section (CDD)
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Reid, Diane M
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University of California San Francisco
Schools of Medicine
San Francisco
United States
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Hwa, Jennifer J; Beckouche, Nathan; Huang, Lawrence et al. (2017) Abnormal arterial-venous fusions and fate specification in mouse embryos lacking blood flow. Sci Rep 7:11965
Nielsen, Corinne M; Huang, Lawrence; Murphy, Patrick A et al. (2016) Mouse Models of Cerebral Arteriovenous Malformation. Stroke 47:293-300
Cuervo, Henar; Nielsen, Corinne M; Simonetto, Douglas A et al. (2016) Endothelial notch signaling is essential to prevent hepatic vascular malformations in mice. Hepatology 64:1302-1316
Murphy, Patrick A; Kim, Tyson N; Huang, Lawrence et al. (2014) Constitutively active Notch4 receptor elicits brain arteriovenous malformations through enlargement of capillary-like vessels. Proc Natl Acad Sci U S A 111:18007-12
Lin, Yuankai; Jiang, Weiya; Ng, Jennifer et al. (2014) Endothelial ephrin-B2 is essential for arterial vasodilation in mice. Microcirculation 21:578-86
Nielsen, Corinne M; Cuervo, Henar; Ding, Vivianne W et al. (2014) Deletion of Rbpj from postnatal endothelium leads to abnormal arteriovenous shunting in mice. Development 141:3782-92
Lindskog, Henrik; Kim, Yung Hae; Jelin, Eric B et al. (2014) Molecular identification of venous progenitors in the dorsal aorta reveals an aortic origin for the cardinal vein in mammals. Development 141:1120-8
Kim, Tyson N; Goodwill, Patrick W; Chen, Yeni et al. (2012) Line-scanning particle image velocimetry: an optical approach for quantifying a wide range of blood flow speeds in live animals. PLoS One 7:e38590
Murphy, Patrick A; Kim, Tyson N; Lu, Gloria et al. (2012) Notch4 normalization reduces blood vessel size in arteriovenous malformations. Sci Transl Med 4:117ra8
Clever, Jared L; Sakai, Yuki; Wang, Rong A et al. (2010) Inefficient skeletal muscle repair in inhibitor of differentiation knockout mice suggests a crucial role for BMP signaling during adult muscle regeneration. Am J Physiol Cell Physiol 298:C1087-99

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