Regulation by P2X7: epithelial migration and stromal organization in the cornea.
OBJECTIVE
Previously, the authors demonstrated that BzATP, a P2X(7) receptor agonist, enhanced corneal epithelial migration in vitro. The goal here was to characterize the role of the P2X(7) 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 P2X(7) knockout (P2X(7)(-/-)) and wild-type (WT) mice, and eyes were harvested after 16 hours. Corneas were stained with Richardson 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 stromal proteoglycans (PGs). Real-time PCR was performed to examine the expression of stromal collagens.


RESULTS
P2X(7) was present in the WT corneal epithelium but was not detected in P2X(7)(-/-) mice. Pannexin-1, a protein demonstrated to interact with P2X(7), was absent from the wound edge in P2X(7)(-/-). This was associated with a trend toward delayed corneal reepithelialization. Stromal ultrastructure and collagen alignment were altered in P2X(7)(-/-), and collagen fibrils had smaller diameters with a larger interfibrillar distances. Expression of collagen alpha1(I) and alpha3(v) was reduced. There were 30% fewer sulfated PGs along fibrils in the P2X(7)(-/-) stroma.


CONCLUSIONS
In the absence of the P2X(7) receptor, the expression of proteins in the corneal epithelium was altered and wound healing was compromised. Loss of receptor resulted in morphologic changes in the stroma, including changes in alignment of collagen fibrils, decreased expression of collagen, and smaller fibrils with fewer PGs per fibril.
Mayo
Courtney
C
Departments of Biochemistry, Boston University School of Medicine, Boston, Massachusetts 02118, USA.
Ren
Ruiyi
R
Rich
Celeste
C
Stepp
Mary Ann
MA
Trinkaus-Randall
Vickery
V
D000818:Animals; D002465:Cell Movement / Q000502:physiology*; D003094:Collagen / Q000235:genetics / Q000648:ultrastructure; D024042:Collagen Type I / Q000235:genetics / Q000378:metabolism; D024062:Collagen Type V / Q000235:genetics / Q000378:metabolism; D017630:Connexins / Q000378:metabolism; D003315:Cornea / Q000601:surgery*; D003319:Corneal Stroma / Q000378:metabolism* / Q000648:ultrastructure; D003646:Debridement; D019573:Epithelium, Corneal / Q000502:physiology*; D019084:Fluorescent Antibody Technique, Indirect; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D009419:Nerve Tissue Proteins / Q000378:metabolism; D011509:Proteoglycans / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D018048:Receptors, Purinergic P2 / Q000502:physiology*; D058486:Receptors, Purinergic P2X7; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014945:Wound Healing / Q000502:physiology*
D024042:Collagen Type I; D024062:Collagen Type V; D017630:Connexins; D009419:Nerve Tissue Proteins; C546469:P2rx7 protein, mouse; C503631:Panx1 protein, mouse; D011509:Proteoglycans; D012333:RNA, Messenger; D018048:Receptors, Purinergic P2; D058486:Receptors, Purinergic P2X7; D003094:Collagen