Regulation by P2X7: epithelial migration and stromal organization in the cornea

Abstract

Purpose: 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.

Figures

FIGURE 1

(A) Light micrographs of Masson trichrome-stained corneas 16 hours after injury. Arrow: separation in the anterior stroma at P2X7 −/− wound edge. Images are representative of 12 corneas in each group. (B) Light micrographs of unwounded corneas. Scale bar, 75 µm for each image.

FIGURE 2

Immunohistochemistry of proteins localized to the wound margin. Representative images from paraffin sections were probed with antibodies specific for P2X7, P2Y4, pannexin-1, p-paxillin, and p-FAK, followed by FITC-conjugated IgG. Nuclei were counterstained with nuclear dye (To-Pro 3AM; Invitrogen) and pseudocolored blue. Asterisk: leading edge of wound. S: stroma. Pannexin 1 (inset): 105 µm distal to leading edge shows punctate localization in P2X7 −/−. p-Paxillin (inset): 2 × zoom, 105 µm distal to leading edge, shows localization along epithelial-stromal border in WT. Negative controls represent areas distal to the wound, probed only with secondary antibody and counterstained with nuclear dye. Scale bar, 10 µm. Images are representative of three experiments.

FIGURE 3

Electron micrographs of hemidesmosomes and anterior stroma. Hemidesmosomes and anterior stromal collagen organization were evaluated distal to the wound edge. Asterisk: hemidesmosomal complexes. E, epithelium; BL, basal lamina; S, stroma. Micrographs are representative of at least three animals. Scale bar, 250 nm.

FIGURE 4

Electron micrographs of collagen lamellae from WT and P2X7 −/− mice. (A) Lamellae are more numerous in WT than P2X7 −/− in the middle of the central stroma. Scale bar, 500 nm. (B) Mean lamellae thickness. A minimum of 34 orthogonal and perpendicular lamellae throughout the stroma were measured for each group. Data are presented as ± SEM. *P < 0.001; t-test.

FIGURE 5

Electron micrographs of central stromal collagen from unwounded WT and P2X7 −/− mice. (A) Low magnification of middle stroma depicts swirling of fibrils in P2X7 −/−. (B) Higher magnification of three distinct regions. A, anterior; M, middle; P, posterior. Scale bar: (A) 500 nm; (B) 100 nm.

FIGURE 6

Distribution of collagen fibril diameter in unwounded WT and P2X7 −/− stromas. Fibril diameter was measured and averaged for each nonoverlapping region of the stroma. A, anterior; M, middle; P, posterior. At least 21 fibrils were counted in each region.

FIGURE 7

Interfibrillar distance in unwounded WT and P2X7 −/− stromas. Interfibrillar distance in each nonoverlapping region was measured. (A) Frequency of interfibrillar distance. (B) Mean ± SEM interfibrillar distance of each region is graphed. A, anterior; M, middle; P, posterior. At least 40 measurements were performed for each group. *P < 0.001; t-test.

FIGURE 8

Expression of collagen is decreased in P2X7 −/− unwounded stromas. Negative controls were performed minus the reverse transcriptase. Results are presented as relative expression normalized to 18s rRNA and were calculated using the ΔΔCt method.

FIGURE 9

Electron micrographs of unwounded, central stromas stained with cuprolinic blue. Tissue was stained with cuprolinic blue before it was processed for electron microscopy. (A) Micrographs of middle stroma. Closed arrows: PGs associated with collagen fibrils. Open arrows: elongated filaments running along fibrils. Arrowheads: elongated filaments seen in cross-section. Scale bar, 250 nm. (B) PU/collagen fibril ratio in WT and P2X7 −/− stromas. The length of collagen fibrils was measured, and the number of PG units along that length was counted. PG per micrometer is graphed ± SEM. At last 22 measurements were performed for each group. *P < 0.001; t-test.