Rap1 GTPase Activation and Barrier Enhancement in RPE Inhibits Choroidal Neovascularization In Vivo

Erika S. Wittchen, Eiichi Nishimura, Manabu McCloskey, Haibo Wang, Lawrence Quilliam, Magdalena Chrzanowska-Wodnicka, M. Elizabeth Hartnett

Research output: Contribution to journalArticle

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Abstract

Loss of barrier integrity precedes the development of pathologies such as metastasis, inflammatory disorders, and blood-retinal barrier breakdown present in neovascular age-related macular degeneration. Rap1 GTPase is involved in regulating both endothelial and epithelial cell junctions; the specific role of Rap1A vs. Rap1B isoforms is less clear. Compromise of retinal pigment epithelium barrier function is a contributing factor to the development of AMD. We utilized shRNA of Rap1 isoforms in cultured human retinal pigment epithelial cells, along with knockout mouse models to test the role of Rap1 on promoting RPE barrier properties, with emphasis on the dynamic junctional regulation that is triggered when the adhesion between cells is challenged. In vitro, Rap1A shRNA reduced steady-state barrier integrity, whereas Rap1B shRNA affected dynamic junctional responses. In a laser-induced choroidal neovascularization (CNV) model of macular degeneration, Rap1b-/- mice exhibited larger CNV volumes compared to wild-type or Rap1a-/-. In vivo, intravitreal injection of a cAMP analog (8CPT-2′-O-Me-cAMP) that is a known Rap1 activator significantly reduced laser-induced CNV volume, which correlated with the inhibition of CEC transmigration across 8CPT-2′O-Me-cAMP-treated RPE monolayers in vitro. Rap1 activation by 8CPT-2′-O-Me-cAMP treatment increased recruitment of junctional proteins and F-actin to cell-cell contacts, increasing both the linearity of junctions in vitro and in cells surrounding laser-induced lesions in vivo. We conclude that in vitro, Rap1A may be important for steady state barrier integrity, while Rap1B is involved more in dynamic junctional responses such as resistance to junctional disassembly induced by EGTA and reassembly of cell junctions following disruption. Furthermore, activation of Rap1 in vivo inhibited development of choroidal neovascular lesions in a laser-injury model. Our data suggest that targeting Rap1 isoforms in vivo with 8CPT-2′-O-Me-cAMP may be a viable pharmacological means to strengthen the RPE barrier against the pathological choroidal endothelial cell invasion that occurs in macular degeneration.

Original languageEnglish
Article numbere73070
JournalPLoS One
Volume8
Issue number9
DOIs
StatePublished - Sep 10 2013

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Choroidal Neovascularization
GTP Phosphohydrolases
guanosinetriphosphatase
angiogenesis
lasers
Macular Degeneration
Lasers
Chemical activation
intercellular junctions
Small Interfering RNA
Protein Isoforms
Retinal Pigments
Intercellular Junctions
Endothelial cells
lesions (animal)
endothelial cells
Dynamic response
epithelial cells
Endothelial Cells
Epithelial Cells

ASJC Scopus subject areas

  • Agricultural and Biological Sciences(all)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Medicine(all)

Cite this

Wittchen, E. S., Nishimura, E., McCloskey, M., Wang, H., Quilliam, L., Chrzanowska-Wodnicka, M., & Hartnett, M. E. (2013). Rap1 GTPase Activation and Barrier Enhancement in RPE Inhibits Choroidal Neovascularization In Vivo. PLoS One, 8(9), [e73070]. https://doi.org/10.1371/journal.pone.0073070

Rap1 GTPase Activation and Barrier Enhancement in RPE Inhibits Choroidal Neovascularization In Vivo. / Wittchen, Erika S.; Nishimura, Eiichi; McCloskey, Manabu; Wang, Haibo; Quilliam, Lawrence; Chrzanowska-Wodnicka, Magdalena; Hartnett, M. Elizabeth.

In: PLoS One, Vol. 8, No. 9, e73070, 10.09.2013.

Research output: Contribution to journalArticle

Wittchen, ES, Nishimura, E, McCloskey, M, Wang, H, Quilliam, L, Chrzanowska-Wodnicka, M & Hartnett, ME 2013, 'Rap1 GTPase Activation and Barrier Enhancement in RPE Inhibits Choroidal Neovascularization In Vivo', PLoS One, vol. 8, no. 9, e73070. https://doi.org/10.1371/journal.pone.0073070
Wittchen, Erika S. ; Nishimura, Eiichi ; McCloskey, Manabu ; Wang, Haibo ; Quilliam, Lawrence ; Chrzanowska-Wodnicka, Magdalena ; Hartnett, M. Elizabeth. / Rap1 GTPase Activation and Barrier Enhancement in RPE Inhibits Choroidal Neovascularization In Vivo. In: PLoS One. 2013 ; Vol. 8, No. 9.
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