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A role for planar cell polarity signaling in angiogenesis

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Abstract

The planar cell polarity (PCP) pathway is a highly conserved signaling cascade that coordinates both epithelial and axonal morphogenic movements during development. Angiogenesis also involves the growth and migration of polarized cells, although the mechanisms underlying their intercellular communication are poorly understood. Here, using cell culture assays, we demonstrate that inhibition of PCP signaling disrupts endothelial cell growth, polarity, and migration, all of which can be rescued through downstream activation of this pathway by expression of either Daam-1, Diversin or Inversin. Silencing of either Dvl2 or Prickle suppressed endothelial cell proliferation. Moreover, loss of p53 rescues endothelial cell growth arrest but not the migration inhibition caused by PCP disruption. In addition, we show that the zebrafish Wnt5 mutant (pipetail (ppt)), which has impaired PCP signaling, displays vascular developmental defects. These findings reveal a potential role for PCP signaling in the coordinated assembly of endothelial cells into vascular structures and have important implications for vascular remodeling in development and disease.

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Abbreviations

Dvl:

Dishevelled

PCP:

Planar cell polarity

NHDF:

Normal human dermal fibroblasts

MPE:

Murine pulmonary endothelial

GFP:

Green fluorescent protein

MetAP-2:

Methionine aminopeptidase 2

IS:

Intersegmental

MO:

Morpholino

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Acknowledgments

We would like to thank John Hines for his critical review of the manuscript. This work was supported by grants from the NIH (CA083049 to CMC and CA112369 to DCS). PC is the recipient of the Leukemia and Lymphoma Society Fellowship. The authors have declared no competing interests.

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Authors and Affiliations

  1. Department of Molecular, Cellular and Developmental Biology, Yale University, Kline Biology Tower Room 400, P.O. Box 208103, New Haven, CT, 06520-8103, USA

    Pasquale Cirone, Yi Zhang & Craig M. Crews

  2. Department of Biology, University of Iowa, Iowa City, IA, 52242, USA

    Shengda Lin, Hilary L. Griesbach & Diane C. Slusarski

  3. Department of Chemistry, Yale University, New Haven, CT, 06520-8103, USA

    Craig M. Crews

  4. Department of Pharmacology, Yale University, New Haven, CT, 06520-8103, USA

    Craig M. Crews

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  1. Pasquale Cirone
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Correspondence to Craig M. Crews.

Electronic supplementary material

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10456_2008_9116_MOESM1_ESM.doc

Supplemental Fig S1 Localization of the MOTC in TNP-470-treated Endothelial Cells. In vehicle-treated migrating cells the MOTC was localized at the lamellipodial side (L) of the cell’s nucleus, as opposed to the trailing end (T). After cells are treated with TNP-470, they were predominantly rounded and not actively migrating with the MOTC unperturbed and on a random side of the nucleus. MOTC was identified using γ-tubulin,Cy3-conjugated antibody as per the manufacture’s instructions for immunohistochemistry (ab11319, Abcam) (DOC 1703 kb)

10456_2008_9116_MOESM2_ESM.doc

Supplemental Fig S2 Knockdown of Dvl2 or Prickle Suppresses Endothelial Cell Proliferation. (Upper) Validation of shRNA constructs for Dvl2 (duplex: 5′-CTTTGAGAACATGAGCAA-3′) and prickle (duplex: 5′-AATTTATGCGCCAGCGGAGCT-3′). Mock controls are pSUPER-retro-puro infections. Multiple bands are observed for FLAG-tagged prickle constructs, which are consistently knocked down with the introduction of prickle shRNA. (Lower) Tritiated thymidine incorporation assays were performed on HUVEC cells with shRNA-mediated suppressed Dvl2 or prickle expression. Knockdown of either PCP mediator suppressed endothelial cell proliferation (DOC 265 kb)

10456_2008_9116_MOESM3_ESM.doc

Supplemental Fig S3 Knockdown of Dvl2 on Endothelial Cell Migration and Coordination on Matrigel. A) Silencing of Dvl2 did not suppress migration of endothelial cells although the silencing did rescue from inhibition by TNP-470. (*: p < 0.05, n = 5 random fields of view x4 wells/group and duplicate experiments). HUVECs were visualized by use of Calcein AM (Molecular Probes). (B) Although silencing of Dvl2 could produce noticeable breaks within the cellular extensions observed in Matrigel, the quantification of intact cellular extensions was not statistically meaningful. However, like for the migration assay, the knockdown of Dvl2 did serve to protect endothelial cells from the inhibitory effects of TNP-470. (*: p < 0.05, n =4 per group, performed in duplicate experiments) (DOC 366 kb)

10456_2008_9116_MOESM4_ESM.doc

Supplemental Fig S4 Transplants of Wnt5-MO injected Cells Disrupts Angiogenesis in Zebrafish. Cells injected with Wnt5-MO co-mixed with lineage marker were grafted from donor embryos into the blastodermal margin of host fli-EGFP embryos at sphere stage. Host embryos were examined with fluorescence stereomicroscopy at 24hpf and embryos with transplanted cells (as detected by lineage marker) incorporated into the somite region were fixed and subjected to confocal imaging. Regions of red indicate areas of Wnt5 depletion, green identifies the vasculature (DOC 93 kb)

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Cirone, P., Lin, S., Griesbach, H.L. et al. A role for planar cell polarity signaling in angiogenesis. Angiogenesis 11, 347–360 (2008). https://doi.org/10.1007/s10456-008-9116-2

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