April 2014
Volume 55, Issue 13
Free
ARVO Annual Meeting Abstract  |   April 2014
P2X7 is elevated under high glycemic conditions and alters the wound edge phenotype
Author Affiliations & Notes
  • Vickery E Trinkaus-Randall
    Ophthalmology L904, Boston University School of Med, Boston, MA
    Biochemistry, Boston University School of Medicine, Boston, MA
  • Martin Minns
    Biochemistry, Boston University School of Medicine, Boston, MA
  • Celeste Rich
    Biochemistry, Boston University School of Medicine, Boston, MA
  • Footnotes
    Commercial Relationships Vickery Trinkaus-Randall, None; Martin Minns, None; Celeste Rich, None
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science April 2014, Vol.55, 4708. doi:
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      Vickery E Trinkaus-Randall, Martin Minns, Celeste Rich; P2X7 is elevated under high glycemic conditions and alters the wound edge phenotype. Invest. Ophthalmol. Vis. Sci. 2014;55(13):4708.

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

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Abstract

Purpose: Diabetes may complicate wound repair by slowing the healing process and impairing regeneration. We postulate that defective diabetic healing is in part due to changes in the regulation of signaling pathways including ion and G-protein coupled receptors. P2X, an ionotropic receptor, is activated by ATP that is released in stress conditions. Previously we found that P2X7 mRNA is significantly elevated in the cornea of unwounded diabetic patients. However, the specific relationships between the disease and purinoreceptors have yet to be elucidated. Our goal is to demonstrate the expression of P2X7 and relate it to wound repair.

Methods: Experiments were performed using corneal organ culture and in vitro wound healing studies. Real-time PCR and ddCt analysis, western blot analysis and immunohistochemical analysis using confocal microscopy were performed on tissue and cells from stratified cultures at specific times after injury to determine expression of protein and RNA. Measurements were averaged and standard statistical tests were performed.

Results: Under normal wound organ culture abrasion models, wound repair occurs, P2X7 transcript and protein decrease with injury. However the decrease in P2X7 is not detected in wounded diabetic epithelium. An analysis of the leading edge indicates that P2X7 is present along the plasma membrane under unwounded conditions and becomes cytosolic with injury. When organ cultures were incubated under high glycemic conditions P2X7 mRNA was significantly elevated in the epithelium, localization at the leading edge was modified and wound healing was significantly delayed. In addition when control cultures were treated with oxidized-ATP there was a reduction in wound repair rate. Furthermore, we have shown that P2Y2, a G-protein coupled purinoreceptor, is inversely correlated with levels of P2X7. Moreover, when P2X7 was overexpressed there was a decrease in beta1 integrin mRNA expression.

Conclusions: The elevated P2X7 receptor expression in diabetic tissue and/or glycemic conditions is a predictor for impaired wound repair and wound edge. We predict that the regulation of P2X7 and P2Y2 are required for proper healing in normal corneas and that impaired regulation is involved in defective diabetic healing.

Keywords: 765 wound healing • 446 cell adhesions/cell junctions  
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