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Control of angiogenesis by AIBP-mediated cholesterol efflux

Fang, Longhou; Choi, Soo-Ho; Baek, Ji Sun; Liu, Chao; Almazan, Felicidad; Ulrich, Florian; Wiesner, Philipp; Taleb, Adam; Deer, Elena; Pattison, Jennifer; Torres-Vazquez, Jesus; Li, Andrew C; Miller, Yury I
Cholesterol is a structural component of the cell and is indispensable for normal cellular function, although its excess often leads to abnormal proliferation, migration, inflammatory responses and/or cell death. To prevent cholesterol overload, ATP-binding cassette (ABC) transporters mediate cholesterol efflux from the cells to apolipoprotein A-I (apoA-I) and the apoA-I-containing high-density lipoprotein (HDL). Maintaining efficient cholesterol efflux is essential for normal cellular function. However, the role of cholesterol efflux in angiogenesis and the identity of its local regulators are poorly understood. Here we show that apoA-I binding protein (AIBP) accelerates cholesterol efflux from endothelial cells to HDL and thereby regulates angiogenesis. AIBP- and HDL-mediated cholesterol depletion reduces lipid rafts, interferes with VEGFR2 (also known as KDR) dimerization and signalling and inhibits vascular endothelial growth factor-induced angiogenesis in vitro and mouse aortic neovascularization ex vivo. Notably, Aibp, a zebrafish homologue of human AIBP, regulates the membrane lipid order in embryonic zebrafish vasculature and functions as a non-cell-autonomous regulator of angiogenesis. aibp knockdown results in dysregulated sprouting/branching angiogenesis, whereas forced Aibp expression inhibits angiogenesis. Dysregulated angiogenesis is phenocopied in Abca1 (also known as Abca1a) Abcg1-deficient embryos, and cholesterol levels are increased in Aibp-deficient and Abca1 Abcg1-deficient embryos. Our findings demonstrate that secreted AIBP positively regulates cholesterol efflux from endothelial cells and that effective cholesterol efflux is critical for proper angiogenesis.
PMID: 23719382
ISSN: 0028-0836
CID: 563832

Sphingosine-1-Phosphate Receptors S1pr1 and S1pr2 Cooperatively Regulate Embryonic Vascular Development

Mendelson, Karen; Zygmunt, Tomasz; Torres-Vazquez, Jesus; Evans, Todd; Hla, Timothy
Sphingosine-1-phosphate (S1P) binds G-protein coupled S1pr1-5 receptors to regulate a multitude of physiological effects, especially those in the vascular and immune systems. S1P receptors in the vascular system have been characterized primarily in mammals. Here we report that the S1P receptors and metabolic enzymes are conserved in the genome of zebrafish Danio rerio. Bioinformatic analysis identified seven S1P receptor-like sequences in the zebrafish genome, including duplicated orthologs of receptors 3 and 5. Sphingolipidomic analysis detected erythrocyte and plasma S1P as well as high plasma ceramides and sphingosine. Morpholino-mediated knockdown of s1pr1 causes global and pericardial edema, loss of blood circulation, and vascular defects characterized by both reduced vascularization in intersegmental vessels, decreased proliferation of intersegmental and axial vessels, and hypersprouting in the caudal vein plexus. The s1pr2 gene was previously characterized as a regulator of cell migration and heart development but its role in angiogenesis is not known. However, when expression of both s1pr1 and s1pr2 is suppressed, severely reduced vascular development of the intersegmental vessels was observed with doses of the s1pr1 morpholino that alone did not cause any discernible vascular defects, suggesting that s1pr1 and s1pr2 function cooperatively to regulate vascular development in zebrafish. Similarly, the S1P transporter, spns2, also cooperated with s1pr1. We propose that extracellular S1P acts through vascular S1P receptors to regulate vascular development.
PMID: 23229546
ISSN: 0021-9258
CID: 204392

In parallel interconnectivity of the dorsal longitudinal anastomotic vessels requires both VEGF signaling and circulatory flow

Zygmunt, Tomasz; Trzaska, Sean; Edelstein, Laura; Walls, Johnathon; Rajamani, Saathyaki; Gale, Nicholas; Daroles, Laura; Ramirez, Craig; Ulrich, Florian; Torres-Vazquez, Jesus
Blood vessels deliver oxygen, nutrients, hormones and immunity factors throughout the body. To perform these vital functions, vascular cords branch, lumenize and interconnect. Yet, little is known about the cellular, molecular and physiological mechanisms that control how circulatory networks form and interconnect. Specifically, how circulatory networks merge by interconnecting in parallel along their boundaries remains unexplored. To examine this process we studied the formation and functional maturation of the plexus that forms between the Dorsal Longitudinal Anastomotic Vessels (DLAVs) in the zebrafish. We find that the migration and proliferation of endothelial cells within the DLAVs and their Segmental (Se) vessel precursors drives DLAV plexus formation. Remarkably, the presence of Se vessels containing only endothelial cells of the arterial lineage is sufficient for DLAV plexus morphogenesis, suggesting that endothelial cells from the venous lineage make a dispensable or null contribution to this process. The discovery of a circuit that integrates the inputs of circulatory flow and Vascular Endothelial Growth Factor (VEGF) signaling to modulate aortic arch angiogenesis, together with the expression of components of this circuit in the trunk vasculature, prompted us to interrogate the role of these inputs and their relationship during DLAV plexus formation. We find that circulatory flow and VEGF signaling make additive contributions to DLAV plexus morphogenesis, rather than acting as essential inputs with equivalent contributions as they do during aortic arch angiogenesis. Our observations underscore the existence of context-dependent differences in the integration of physiological stimuli and signaling cascades during vascular development.
PMID: 22899709
ISSN: 0021-9533
CID: 175743

CDP-diacylglycerol synthetase-controlled phosphoinositide availability limits VEGFA signaling and vascular morphogenesis

Pan, Weijun; Pham, Van N; Stratman, Amber N; Castranova, Daniel; Kamei, Makoto; Kidd, Kameha R; Lo, Brigid D; Shaw, Kenna M; Torres-Vazquez, Jesus; Mikelis, Constantinos M; Gutkind, J Silvio; Davis, George E; Weinstein, Brant M
Understanding the mechanisms that regulate angiogenesis and translating these into effective therapies are of enormous scientific and clinical interests. In this report, we demonstrate the central role of CDP-diacylglycerol synthetase (CDS) in the regulation of VEGFA signaling and angiogenesis. CDS activity maintains phosphoinositide 4,5 bisphosphate (PIP2) availability through resynthesis of phosphoinositides, whereas VEGFA, mainly through phospholipase Cgamma1, consumes PIP2 for signal transduction. Loss of CDS2, 1 of 2 vertebrate CDS enzymes, results in vascular-specific defects in zebrafish in vivo and failure of VEGFA-induced angiogenesis in endothelial cells in vitro. Absence of CDS2 also results in reduced arterial differentiation and reduced angiogenic signaling. CDS2 deficit-caused phenotypes can be successfully rescued by artificial elevation of PIP2 levels, and excess PIP2 or increased CDS2 activity can promote excess angiogenesis. These results suggest that availability of CDS-controlled resynthesis of phosphoinositides is essential for angiogenesis.
PMID: 22649102
ISSN: 0006-4971
CID: 177779

The novel transmembrane protein Tmem2 is essential for coordination of myocardial and endocardial morphogenesis

Totong, Ronald; Schell, Thomas; Lescroart, Fabienne; Ryckebusch, Lucile; Lin, Yi-Fan; Zygmunt, Tomasz; Herwig, Lukas; Krudewig, Alice; Gershoony, Dafna; Belting, Heinz-Georg; Affolter, Markus; Torres-Vazquez, Jesus; Yelon, Deborah
Coordination between adjacent tissues plays a crucial role during the morphogenesis of developing organs. In the embryonic heart, two tissues - the myocardium and the endocardium - are closely juxtaposed throughout their development. Myocardial and endocardial cells originate in neighboring regions of the lateral mesoderm, migrate medially in a synchronized fashion, collaborate to create concentric layers of the heart tube, and communicate during formation of the atrioventricular canal. Here, we identify a novel transmembrane protein, Tmem2, that has important functions during both myocardial and endocardial morphogenesis. We find that the zebrafish mutation frozen ventricle (frv) causes ectopic atrioventricular canal characteristics in the ventricular myocardium and endocardium, indicating a role of frv in the regional restriction of atrioventricular canal differentiation. Furthermore, in maternal-zygotic frv mutants, both myocardial and endocardial cells fail to move to the midline normally, indicating that frv facilitates cardiac fusion. Positional cloning reveals that the frv locus encodes Tmem2, a predicted type II single-pass transmembrane protein. Homologs of Tmem2 are present in all examined vertebrate genomes, but nothing is known about its molecular or cellular function in any context. By employing transgenes to drive tissue-specific expression of tmem2, we find that Tmem2 can function in the endocardium to repress atrioventricular differentiation within the ventricle. Additionally, Tmem2 can function in the myocardium to promote the medial movement of both myocardial and endocardial cells. Together, our data reveal that Tmem2 is an essential mediator of myocardium-endocardium coordination during cardiac morphogenesis
PMID: 21896630
ISSN: 1477-9129
CID: 137082

Neurovascular development in the embryonic zebrafish hindbrain

Ulrich, Florian; Ma, Leung-Hang; Baker, Robert G; Torres-Vazquez, Jesus
The brain is made of billions of highly metabolically active neurons whose activities provide the seat for cognitive, affective, sensory and motor functions. The cerebral vasculature meets the brain's unusually high demand for oxygen and glucose by providing it with the largest blood supply of any organ. Accordingly, disorders of the cerebral vasculature, such as congenital vascular malformations, stroke and tumors, compromise neuronal function and survival and often have crippling or fatal consequences. Yet, the assembly of the cerebral vasculature is a process that remains poorly understood. Here we exploit the physical and optical accessibility of the zebrafish embryo to characterize cerebral vascular development within the embryonic hindbrain. We find that this process is primarily driven by endothelial cell migration and follows a two-step sequence. First, perineural vessels with stereotypical anatomies are formed along the ventro-lateral surface of the neuroectoderm. Second, angiogenic sprouts derived from a subset of perineural vessels migrate into the hindbrain to form the intraneural vasculature. We find that these angiogenic sprouts reproducibly penetrate into the hindbrain via the rhombomere centers, where differentiated neurons reside, and that specific rhombomeres are invariably vascularized first. While the anatomy of intraneural vessels is variable from animal to animal, some aspects of the connectivity of perineural and intraneural vessels occur reproducibly within particular hindbrain locales. Using a chemical inhibitor of VEGF signaling we determine stage-specific requirements for this pathway in the formation of the hindbrain vasculature. Finally, we show that a subset of hindbrain vessels is aligned and/or in very close proximity to stereotypical neuron clusters and axon tracts. Using endothelium-deficient cloche mutants we show that the endothelium is dispensable for the organization and maintenance of these stereotypical neuron clusters and axon tracts in the early hindbrain. However, the cerebellum's upper rhombic lip and the optic tectum are abnormal in clo. Overall, this study provides a detailed, multi-stage characterization of early zebrafish hindbrain neurovascular development with cellular resolution up to the third day of age. This work thus serves as a useful reference for the neurovascular characterization of mutants, morphants and drug-treated embryos
PMID: 21745463
ISSN: 1095-564x
CID: 137873

Semaphorin-PlexinD1 Signaling Limits Angiogenic Potential via the VEGF Decoy Receptor sFlt1

Zygmunt, Tomasz; Gay, Carl Michael; Blondelle, Jordan; Singh, Manvendra K; Flaherty, Kathleen McCrone; Means, Paula Casey; Herwig, Lukas; Krudewig, Alice; Belting, Heinz-Georg; Affolter, Markus; Epstein, Jonathan A; Torres-Vazquez, Jesus
Sprouting angiogenesis expands the embryonic vasculature enabling survival and homeostasis. Yet how the angiogenic capacity to form sprouts is allocated among endothelial cells (ECs) to guarantee the reproducible anatomy of stereotypical vascular beds remains unclear. Here we show that Sema-PlxnD1 signaling, previously implicated in sprout guidance, represses angiogenic potential to ensure the proper abundance and stereotypical distribution of the trunk's segmental arteries (SeAs). We find that Sema-PlxnD1 signaling exerts this effect by antagonizing the proangiogenic activity of vascular endothelial growth factor (VEGF). Specifically, Sema-PlxnD1 signaling ensures the proper endothelial abundance of soluble flt1 (sflt1), an alternatively spliced form of the VEGF receptor Flt1 encoding a potent secreted decoy. Hence, Sema-PlxnD1 signaling regulates distinct but related aspects of angiogenesis: the spatial allocation of angiogenic capacity within a primary vessel and sprout guidance
PMID: 21802375
ISSN: 1878-1551
CID: 136640

Beyond guidance: A novel role for Sema-PlxnD1 signaling in vascular development [Meeting Abstract]

Zygmunt, Tomasz; Gay, Carl M; Blondelle, Jordan; Flaherty, Kathleen McCrone; Means, Paula Casey; Herwig, Lukas; Krudewig, Alice; Belting, Heinz-Georg; Affolter, Markus; Torres-Vazquez, Jesus
ISSN: 0012-1606
CID: 2611772

Diverse functions for the semaphorin receptor PlexinD1 in development and disease

Gay, Carl M; Zygmunt, Tomasz; Torres-Vazquez, Jesus
Plexins are a family of single-pass transmembrane proteins that serve as cell surface receptors for Semaphorins during the embryonic development of animals. Semaphorin-Plexin signaling is critical for many cellular aspects of organogenesis, including cell migration, proliferation and survival. Until recently, little was known about the function of PlexinD1, the sole member of the vertebrate-specific PlexinD (PlxnD1) subfamily. Here we review novel findings about PlxnD1's roles in the development of the cardiovascular, nervous and immune systems and salivary gland branching morphogenesis and discuss new insights concerning the molecular mechanisms of PlxnD1 activity
PMID: 20880496
ISSN: 1095-564x
CID: 116204

Genetic determinants of hyaloid and retinal vasculature in zebrafish

Alvarez, Yolanda; Cederlund, Maria L; Cottell, David C; Bill, Brent R; Ekker, Stephen C; Torres-Vazquez, Jesus; Weinstein, Brant M; Hyde, David R; Vihtelic, Thomas S; Kennedy, Breandan N
BACKGROUND: The retinal vasculature is a capillary network of blood vessels that nourishes the inner retina of most mammals. Developmental abnormalities or microvascular complications in the retinal vasculature result in severe human eye diseases that lead to blindness. To exploit the advantages of zebrafish for genetic, developmental and pharmacological studies of retinal vasculature, we characterised the intraocular vasculature in zebrafish. RESULTS: We show a detailed morphological and developmental analysis of the retinal blood supply in zebrafish. Similar to the transient hyaloid vasculature in mammalian embryos, vessels are first found attached to the zebrafish lens at 2.5 days post fertilisation. These vessels progressively lose contact with the lens and by 30 days post fertilisation adhere to the inner limiting membrane of the juvenile retina. Ultrastructure analysis shows these vessels to exhibit distinctive hallmarks of mammalian retinal vasculature. For example, smooth muscle actin-expressing pericytes are ensheathed by the basal lamina of the blood vessel, and vesicle vacuolar organelles (VVO), subcellular mediators of vessel-retinal nourishment, are present. Finally, we identify 9 genes with cell membrane, extracellular matrix and unknown identity that are necessary for zebrafish hyaloid and retinal vasculature development. CONCLUSION: Zebrafish have a retinal blood supply with a characteristic developmental and adult morphology. Abnormalities of these intraocular vessels are easily observed, enabling application of genetic and chemical approaches in zebrafish to identify molecular regulators of hyaloid and retinal vasculature in development and disease
PMID: 17937808
ISSN: 1471-213x
CID: 95023