Corneal disease and injury remain a major cause of blindness affecting over 10 million people worldwide according to the W.H.O.. Diabetes further complicates 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 and the composition of their underlying matrix. To study this problem we will examine the role of purinoreceptors in wound repair in a diabetic animal model and examine the changes in YAP (yes associated protein), a transcriptional regulator that is activated by changes in composition of the matrix that occur with diabetes. In the past grant period we made the novel observation that P2X7, an ion channel purinoreceptor, is significantly upregulated in the unwounded human diabetic corneal epithelium. A similar elevation in expression was detected in the corneal epithelium of diabetic rodents. We then made the novel observation that both the P2X7 transcript and protein drop rapidly after injury to control corneas, however the decrease in P2X7 levels does not occur in wounded diabetic epithelium. Furthermore, we have shown that P2Y2, a G-protein coupled purinoreceptor, is inversely correlated with levels of P2X7. We predict that this regulation of P2X7 and P2Y2 are required for proper healing in normal corneas and that impaired regulation is involved in defective diabetic healing. The P2X and P2Y purinoreceptor families are two classes of receptors that respond to changes in extracellular ATP due to cellular stress and trigger the phosphorylation of kinases in distinct downstream signaling pathways including paxillin, FAK, Src, and MAPK . Collectively these data indicate that the decrease in P2X7 may be one of the key regulatory events that mediate the early wound response and successful wound repair in the cornea and that this control does not occur in diabetic corneas. While it is known that the composition of the basement membrane zone (BMZ) changes in diabetic tissue, it is not understood how cells sense that change and respond. Previously investigators proposed that the release of soluble factors promote remodeling leading to changes in activation of YAP. Our preliminary data demonstrate that YAP is present in the cytoplasm of unwounded mouse corneal epithelium and moves to the nucleus in cells at the wound margin;however it remains cytoplasmic in epithelial wound margin epithelium from DIO mice. These indicate that the mechanoregulation which links signaling with cell adhesion and cell migration that occurs during wound repair is altered in diabetic epithelia.

Public Health Relevance

Injury to the cornea can be both painful and compromise vision. We are investigating a protein called P2X7 that is present at higher levels in diabetic corneas than control corneas. As wound repair in diabetic eyes is often impaired, our goal is to determine the role of the P2X7 receptor in the repair of injured tissue.

Agency
National Institute of Health (NIH)
Institute
National Eye Institute (NEI)
Type
Research Project (R01)
Project #
2R01EY006000-27A1
Application #
8755285
Study Section
(DPVS)
Program Officer
Mckie, George Ann
Project Start
1986-06-01
Project End
2018-06-30
Budget Start
2014-08-01
Budget End
2015-06-30
Support Year
27
Fiscal Year
2014
Total Cost
Indirect Cost
Name
Boston University
Department
Ophthalmology
Type
Schools of Medicine
DUNS #
City
Boston
State
MA
Country
United States
Zip Code
02118
Minns, Martin S; Teicher, Gregory; Rich, Celeste B et al. (2016) Purinoreceptor P2X7 Regulation of Ca(2+) Mobilization and Cytoskeletal Rearrangement Is Required for Corneal Reepithelialization after Injury. Am J Pathol 186:285-96
Derricks, Kelsey E; Trinkaus-Randall, Vickery; Nugent, Matthew A (2015) Extracellular matrix stiffness modulates VEGF calcium signaling in endothelial cells: individual cell and population analysis. Integr Biol (Camb) 7:1011-25
Lee, Albert; Derricks, Kelsey; Minns, Martin et al. (2014) Hypoxia-induced changes in Ca(2+) mobilization and protein phosphorylation implicated in impaired wound healing. Am J Physiol Cell Physiol 306:C972-85
Stepp, Mary Ann; Zieske, James D; Trinkaus-Randall, Vickery et al. (2014) Wounding the cornea to learn how it heals. Exp Eye Res 121:178-93
Sanderson, Julie; Dartt, Darlene A; Trinkaus-Randall, Vickery et al. (2014) Purines in the eye: recent evidence for the physiological and pathological role of purines in the RPE, retinal neurons, astrocytes, Müller cells, lens, trabecular meshwork, cornea and lacrimal gland. Exp Eye Res 127:270-9
Karamichos, D; Hutcheon, A E K; Rich, C B et al. (2014) In vitro model suggests oxidative stress involved in keratoconus disease. Sci Rep 4:4608
Chi, Cheryl; Trinkaus-Randall, Vickery (2013) New insights in wound response and repair of epithelium. J Cell Physiol 228:925-9
Kehasse, Amanuel; Rich, Celeste B; Lee, Albert et al. (2013) Epithelial wounds induce differential phosphorylation changes in response to purinergic and EGF receptor activation. Am J Pathol 183:1841-52
Oswald, Duane J; Lee, Albert; Trinidad, Monique et al. (2012) Communication between corneal epithelial cells and trigeminal neurons is facilitated by purinergic (P2) and glutamatergic receptors. PLoS One 7:e44574
Mankus, Courtney; Chi, Cheryl; Rich, Celeste et al. (2012) The P2X(7) receptor regulates proteoglycan expression in the corneal stroma. Mol Vis 18:128-38

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