Combination of Reverse and Chemical Genetic Screens Reveals Angiogenesis Inhibitors and Targets

Kalén, Mattias; Wallgard, Elisabet; Asker, Noomi; Nasevicius, Aidas; Athley, Elisabet; Billgren, Erik; Larson, Jon D.; Wadman, Shannon A.; Norseng, Elizabeth; Clark, Karl J.; He, Liqun; Karlsson-Lindahl, Linda; Häger, Ann Katrin; Weber, Holger; Augustin, Hellmut G.; Samuelsson, Tore; Kemmet, Chelsy K.; Utesch, Carly M.; Essner, Jeffrey J.; Hackett, Perry B.; Hellström, Mats

doi:10.1016/j.chembiol.2009.02.010

Cited by 43 publications

(42 citation statements)

References 49 publications

(51 reference statements)

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“…Now, knowing that the expression pattern of Paladin is abundant in the vasculature in late embryogenesis and adulthood, one could speculate that one additional function of Paladin is to regulate insulin signaling in the vasculature. Furthermore, we have previously shown that, in the zebrafish, Paladin is essential for normal vascular development and vascular perfusion (Kalen et al, 2009). Therefore, it is possible that Paladin has a broader role in regulating receptor tyrosine kinase and AKT phosphorylation in the vasculature.…”

Section: Discussionmentioning

confidence: 99%

Paladin (X99384) is expressed in the vasculature and shifts from endothelial to vascular smooth muscle cells during mouse development

Wallgard

Nitzsche

Larsson

et al. 2012

Developmental Dynamics

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Background: Angiogenesis is implicated in many pathological conditions. The role of the proteins involved remains largely unknown, and few vascular‐specific drug targets have been discovered. Previously, in a screen for angiogenesis regulators, we identified Paladin (mouse: X99384, human: KIAA1274), a protein containing predicted S/T/Y phosphatase domains. Results: We present a mouse knockout allele for Paladin with a β‐galactosidase reporter, which in combination with Paladin antibodies demonstrate that Paladin is expressed in the vasculature. During mouse embryogenesis, Paladin is primarily expressed in capillary and venous endothelial cells. In adult mice Paladin is predominantly expressed in arterial pericytes and vascular smooth muscle cells. Paladin also displays vascular‐restricted expression in human brain, astrocytomas, and glioblastomas. Conclusions: Paladin, a novel putative phosphatase, displays a dynamic expression pattern in the vasculature. During embryonic stages it is broadly expressed in endothelial cells, while in the adult it is selectively expressed in arterial smooth muscle cells. Developmental Dynamics 241:770–786, 2012. © 2012 Wiley Periodicals, Inc.

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Section: Discussionmentioning

confidence: 99%

Paladin (X99384) is expressed in the vasculature and shifts from endothelial to vascular smooth muscle cells during mouse development

Wallgard

Nitzsche

Larsson

et al. 2012

Developmental Dynamics

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“…On the contrary, statin use was found to be associated with a reduced risk of prostate (40) and colorectal cancer (41). Statins have recently been found to inhibit HUVEC proliferation (16), spheroid sprouting (19) and intersegmental vessel growth in zebrafish embryos (29). In contrast to angiogenesis, the effect of statins on lymphangiogenesis and lymphangiogenic sprouting had not been investigated yet.…”

Section: Discussionmentioning

confidence: 99%

“…Previously, several studies were performed based on in vitro compound screens with blood vascular endothelial cells, including proliferation assays of human umbilical vein endothelial cells (HUVEC) (16), HUVEC/vascular smooth muscle cell cocultures, human microvascular endothelial cell tube formation assays (17,18), and HUVEC spheroid sprouting assays (19). While the tube formation assay has been considered as a model of vasculogenesis rather than angiogenesis (20), the other assays attempt to measure different aspects of blood vessel formation.…”

Section: Discussionmentioning

confidence: 99%

Phenotype-based high-content chemical library screening identifies statins as inhibitors of in vivo lymphangiogenesis

Schulz

Reisen

Zgraggen

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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Lymphangiogenesis plays an important role in promoting cancer metastasis to sentinel lymph nodes and beyond and also promotes organ transplant rejection. We used human lymphatic endothelial cells to establish a reliable three-dimensional lymphangiogenic sprouting assay with automated image acquisition and analysis for inhibitor screening. This high-content phenotype-based assay quantifies sprouts by automated fluorescence microscopy and newly developed analysis software. We identified signaling pathways involved in lymphangiogenic sprouting by screening the Library of Pharmacologically Active Compounds ðLOPACÞ 1280 collection of pharmacologically relevant compounds. Hit characterization revealed that mitogen-activated protein kinase kinase (MEK) 1/2 inhibitors substantially block lymphangiogenesis in vitro and in vivo. Importantly, the drug class of statins, for the first time, emerged as potent inhibitors of lymphangiogenic sprouting in vitro and of corneal and cutaneous lymphangiogenesis in vivo. This effect was mediated by inhibition of the 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase and subsequently the isoprenylation of Rac1. Supplementation with the enzymatic products of HMG-CoA reductase functionally rescued lymphangiogenic sprouting and the recruitment of Rac1 to the plasma membrane.high-content screening | small compound screening | lymphatic endothelium | chemical genetics

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“…A variety of recent studies have taken advantage of the optical clarity of zebrafish embryos and larvae to carry out live imaging of tubulogenesis in developing vessels within living animals (Ellertsdottir et al, 2010;Herbert and Stainier, 2011;Herwig et al, 2011;Jin et al, 2005Jin et al, , 2007Kalen et al, 2009;Kamei et al, 2006;Lenard et al, 2013;Wang et al, 2010). These studies have provided in vivo evidence substantiating both the cell hollowing and the cord hollowing models for lumenogenesis.…”

Section: Introductionmentioning

confidence: 96%

Single cell analysis of endothelial morphogenesis in vivo

Castranova

Pham

et al. 2015

Development

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Vessel formation has been extensively studied at the tissue level, but the difficulty in imaging the endothelium with cellular resolution has hampered study of the morphogenesis and behavior of endothelial cells (ECs) in vivo. We are using endothelial-specific transgenes and high-resolution imaging to examine single ECs in zebrafish. By generating mosaics with transgenes that simultaneously mark endothelial nuclei and membranes we are able to definitively identify and study the morphology and behavior of individual ECs during vessel sprouting and lumen formation. Using these methods, we show that developing trunk vessels are composed of ECs of varying morphology, and that single-cell analysis can be used to quantitate alterations in morphology and dynamics in ECs that are defective in proper guidance and patterning. Finally, we use singlecell analysis of intersegmental vessels undergoing lumen formation to demonstrate the coexistence of seamless transcellular lumens and single or multicellular enclosed lumens with autocellular or intercellular junctions, suggesting that heterogeneous mechanisms contribute to vascular lumen formation in vivo. The tools that we have developed for single EC analysis should facilitate further rigorous qualitative and quantitative analysis of EC morphology and behavior in vivo.

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Combination of Reverse and Chemical Genetic Screens Reveals Angiogenesis Inhibitors and Targets

Cited by 43 publications

References 49 publications

Paladin (X99384) is expressed in the vasculature and shifts from endothelial to vascular smooth muscle cells during mouse development

Paladin (X99384) is expressed in the vasculature and shifts from endothelial to vascular smooth muscle cells during mouse development

Phenotype-based high-content chemical library screening identifies statins as inhibitors of in vivo lymphangiogenesis

Single cell analysis of endothelial morphogenesis in vivo

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