June 2015
Volume 56, Issue 7
Free
ARVO Annual Meeting Abstract  |   June 2015
Endomucin Plays a Role in Developmental Retinal Vascularization and in VEGF-Induced Endothelial Cell Migration, Growth, and Morphogenesis In Vitro
Author Affiliations & Notes
  • Cindy Park-Windhol
    Ophthalmology, Schepens Eye Research Institute/Massachusetts Eye and Ear/Harvard Medical School, Boston, MA
  • Jinling Yang
    Ophthalmology, Schepens Eye Research Institute/Massachusetts Eye and Ear/Harvard Medical School, Boston, MA
  • Vincent Primo
    Ophthalmology, Schepens Eye Research Institute/Massachusetts Eye and Ear/Harvard Medical School, Boston, MA
  • Yin Shan Eric Ng
    Ophthalmology, Schepens Eye Research Institute/Massachusetts Eye and Ear/Harvard Medical School, Boston, MA
  • Magali Saint-Geniez
    Ophthalmology, Schepens Eye Research Institute/Massachusetts Eye and Ear/Harvard Medical School, Boston, MA
  • Patricia A D'Amore
    Ophthalmology, Schepens Eye Research Institute/Massachusetts Eye and Ear/Harvard Medical School, Boston, MA
    Pathology, Harvard Medical School, Boston, MA
  • Footnotes
    Commercial Relationships Cindy Park-Windhol, None; Jinling Yang, None; Vincent Primo, None; Yin Shan Eric Ng, None; Magali Saint-Geniez, None; Patricia D'Amore, AGTC (C), Eleven Biotherapeutics (S)
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science June 2015, Vol.56, 3404. doi:
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    • Get Citation

      Cindy Park-Windhol, Jinling Yang, Vincent Primo, Yin Shan Eric Ng, Magali Saint-Geniez, Patricia A D'Amore; Endomucin Plays a Role in Developmental Retinal Vascularization and in VEGF-Induced Endothelial Cell Migration, Growth, and Morphogenesis In Vitro. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):3404.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: Angiogenesis is complex and proceeds through several stages including migration, proliferation and morphogenesis of endothelial cells (EC). It is central to both normal and pathologic processes. Endomucin-1 (EMCN), a type I O-glycosylated sialic-rich glycoprotein and a component of the endothelial glycocalyx, is specifically expressed by venous and capillary endothelium. Although EC express high levels of EMCN, the role of EMCN in vascular development is unclear. Thus, we examined the potential role of EMCN in angiogenesis.

Methods: C57BL/6J mice were injected intravitreally with siEMCN or scrambled siRNA (siCtrl) at postnatal day four (P4). Two days after injection, retinas were flat-mounted and vascular radial expansion, vessel density, branch point number, and filopodia number were evaluated. In a series of in vitro studies aimed at elucidating mechanism, EMCN was knocked down in human retinal EC (HREC) by transfection with siEMCN. EC migration was assessed in a wound-healing assay, proliferation was determined by cell counting, and tube morphogenesis was examined using the collagen EC tube formation assay. Levels of apoptosis were evaluated by Annexin-V staining and quantified using the Muse analyzer. For all experiments, the siRNA knockdown efficiency was confirmed on mRNA and protein levels.

Results: Knockdown of EMCN mRNA in the retina was significant at 48 and 72 hrs after intravitreal injection compared to siCtrl treatment. Delay in radial expansion of the developing mouse vasculature in siEMCN-injected P6 mice (51±2.2% vs. 69±1.8%, P<0.0001) was observed and was accompanied by reduced vessel density (49±1.9% vs. 66±4.8%, P<0.01), decreased branch point number (43±1.1 mm2 vs. 64±4.0 mm2, P<0.0001), and reduced number of filopodia (18±0.6 mm vs. 28± 2.1 mm, P<0.05) when compared to siCtrl. Knockdown of EMCN in HREC led to a reduction in VEGF-induced migration (22±1.3% vs. 59±2.7%, P<0.0001), proliferation (1.51 x 104±125.0 vs. 2.25 x 104±1500 cell/cm2, P<0.05), and morphogenesis (7201±86 mm vs. 9157±273.0 mm, P<0.005) compared to siCtrl-treated cells, without compromising cell survival. VEGF stimulation of siEMCN transfected ECs showed reduction in phospho-VEGFR2, phospho-ERK1/2 and phospho-Akt levels.

Conclusions: Our study indicates a novel role for EMCN as an important regulator of angiogenesis.

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