Regulation by P2X₇: Epithelial Migration and Stromal Organization in the Cornea

¹ Biochemistry, Boston University School of Medicine, Boston, Massachusetts, United States
² Department of Anatomy and Reparative Medicine, George Washington University, Washington, District of Columbia, United States
³ Department of Ophthalmology, Boston University School of Medicine, L903, 80 East Concord Street, Boston, Massachusetts, 02118-2394, United States

^* To whom correspondence should be addressed. E-mail: vickery{at}bu.edu.

	Abstract

Purpose: Previously we demonstrated that BzATP, a P2X7 receptor agonist, enhances corneal epithelial cell migration in vitro. Our goal is to characterize the role of the P2X7 receptor in the repair of in vivo corneal epithelial debridement wounds and in the structural organization of the corneal stroma. Methods: Epithelial debridement was performed on P2X7 knock out (P2X7-/-) and wild type (WT) mice and the eyes were harvested after 16 hr. Corneas were stained with Richardson's vital stain and the wound area was recorded. Corneas were fixed and prepared for light microscopic, immunohistochemical, and electron microscopic analysis. Cuprolinic blue staining was performed to analyze proteoglycans (PGs) in the stroma. Real time PCR was performed to examine the expression of stromal collagens. Results: As expected, P2X7 was present in the WT corneal epithelium but was not detected in P2X7-/- mice. Pannexin-1, a protein demonstrated to interact with P2X7, was absent from the wound edge in P2X7-/-. This was associated with a trend toward delayed corneal re-epithleializatiion. The stromal ultrastructure and collagen alignment was altered in P2X7-/-. In addition, collagen fibrils had a reduced diameter with a larger interfibrillar distance. Expression of collagen alpha1(I) and alpha3(v) was reduced. Cuprolinic blue staining revealed 30% fewer sulfated PGs along fibrils in the P2X7-/- stroma. Conclusion: In the absence of the P2X7 receptor, expression of proteins in the corneal epithelium was altered and wound healing was compromised. Loss of the receptor resulted in morphological changes in the stroma including changes in alignment of collagen fibrils, decreased expression of collagen and smaller fibrils with fewer PGs per fibril.

Key Words: corneal stroma, corneal wound healing, extracellular matrix, electron microscopy, purinergic receptors, P2X7

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