September 2016
Volume 57, Issue 12
Open Access
ARVO Annual Meeting Abstract  |   September 2016
Characterising the mode of action of Connexin43 mimetic Peptide5 to target retinal ischemia-reperfusion injury
Author Affiliations & Notes
  • Yeri Kim
    Department of Ophthalmology and New Zealand National Eye Centre, University of Auckland, Auckland, New Zealand
  • Jarred M Griffin
    Centre for Brain Research and the Department of Anatomy with Radiology, University of Auckland, Auckland, New Zealand
  • Paul WR Harris
    School of Chemical Sciences, University of Auckland, Auckland, New Zealand
    Maurice Wilkins Centre for Molecular Biodiscovery, University of Auckland, Auckland, New Zealand
  • Sin Hang Crystal Chan
    School of Chemical Sciences, University of Auckland, Auckland, New Zealand
    Maurice Wilkins Centre for Molecular Biodiscovery, University of Auckland, Auckland, New Zealand
  • Louise FB Nicholson
    Centre for Brain Research and the Department of Anatomy with Radiology, University of Auckland, Auckland, New Zealand
  • Simon J O'Carroll
    Centre for Brain Research and the Department of Anatomy with Radiology, University of Auckland, Auckland, New Zealand
  • Margaret Brimble
    School of Chemical Sciences, University of Auckland, Auckland, New Zealand
    Maurice Wilkins Centre for Molecular Biodiscovery, University of Auckland, Auckland, New Zealand
  • Colin Green
    Department of Ophthalmology and New Zealand National Eye Centre, University of Auckland, Auckland, New Zealand
  • Footnotes
    Commercial Relationships   Yeri Kim, None; Jarred Griffin, None; Paul Harris, None; Sin Hang Chan, None; Louise Nicholson, None; Simon O'Carroll, None; Margaret Brimble, None; Colin Green, CoDa Therapeutics, Inc (C), CoDa Therapeutics, Inc (I), CoDa Therapeutics, Inc (P)
  • Footnotes
    Support  University of Auckland Doctoral Scholarship to YK and the CatWalk Spinal Cord Injury Trust to JMG.
Investigative Ophthalmology & Visual Science September 2016, Vol.57, 4592. doi:
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      Yeri Kim, Jarred M Griffin, Paul WR Harris, Sin Hang Crystal Chan, Louise FB Nicholson, Simon J O'Carroll, Margaret Brimble, Colin Green; Characterising the mode of action of Connexin43 mimetic Peptide5 to target retinal ischemia-reperfusion injury. Invest. Ophthalmol. Vis. Sci. 2016;57(12):4592.

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

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Abstract

Purpose : Connexin43 (Cx43) mimetic Peptide5 is a 12-residue sequence (VDCFLSRPTEKT) derived from the second extracellular loop of the human Cx43 protein. We have previously demonstrated in a preclinical model of retinal ischemia-reperfusion injury that Peptide5 prevents endothelial cell loss and protects against vascular permeability. However, the molecular mode of action of Peptide5 remains unknown. We hypothesised that Peptide5 binds to the second extracellular loop (EL) of Cx43 and inhibits Cx43 hemichannel mediated ATP release that contribute to ischemia-reperfusion injury.

Methods : Human cerebral endothelial cells (hCMVEC) were exposed to a hypoxic, acidic, ion-shifted Ringer solution for 2 hours to simulate ischemia, or further exposed to 2 hours of standard ringer solution to simulate ischemia-reperfusion in vitro. ATP released from hemichannels was quantified in a bioluminescence assay. Peptide5 (100µM) was tested under ischemic and reperfusion conditions (n=6). To determine the binding site, ischemic hCMVEC cells were incubated in equimolar concentrations of Peptide5 and synthetic Cx43 EL fragments in competition assays (n=6). To identify binding motifs crucial for Peptide5 efficacy, ischemic hCMVEC cells were incubated in modified and truncated analogues of Peptide5 (100µM)(n=8). One-way ANOVA and Tukey’s statistical test was used.

Results : Peptide5 significantly reduced ATP release when compared to ischemia (73.9 ± 2.1%, p<0.0001) and ischemia-reperfusion (70.3 ± 4.4%, p=0.02) injuries. Equimolar concentrations of Peptide5 and EL1 fragments were not statistically different (p > 0.5), but EL2 fragments significantly inhibited Peptide5 function by 42.2 ± 5.9% (p < 0.0001). Modified Peptide5 with single alanine substitutions significantly increased ATP release by at least 33.3 ± 10.9% when compared to native Peptide5 (p < 0.035). Truncated Peptide5, SRPTEKT (21.5 ± 4.6%, p = 0.0002), VDCFLSRP (21.7 ± 5.9%, p = 0.0084) and VDCFLS (26.3 ± 5.1%, p < 0.0001) also showed reduced efficacy when compared to the full length Peptide5.

Conclusions : Peptide5 inhibits ATP released from open Cx43 hemichannels under ischemic-reperfusion conditions by binding to the EL2 of Cx43. Results from modified analogues suggest that Peptide5 displays tight binding specificity. An understanding of the mode of action of Peptide5 supports its development as a treatment for retinal injury and disease.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.

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