Abstract
Purpose: :
Previously we demonstrated that the lack of P2X7 receptor impairs epithelial wound healing and stromal collagen organization. However, the regulatory effects of P2X7 on specific stromal proteins are currently unknown. Here, we investigate potential causes of stromal disorganization in P2X7-/- mice. We hypothesize that deficiency of P2X7 alters the collagen organization by mediating the expression of proteoglycans and lysyl oxidase in addition to collagen itself.
Methods: :
Unwounded corneas from P2X7-/- and C57BL/6J wild type mice (WT) were evaluated for analysis of expression at the mRNA and protein level. For Real Time PCR, RNA was extracted from WT and P2X7-/- corneal stromas, amplified with TaqMan probes, and analyzed using the ΔΔCt method to measure the relative expression of each transcript. Values were given as the mean +/- SEM using Students’ t-test (p < 0.05). Immunohistochemistry was performed and imaged using confocal microscopy. Sulfation of glycosaminoglycans was examined using Cuprolinic Blue and electron microscopy to measure the number of filaments per 100 nm of collagen on corneal samples digested with Keratanase I or Chondroitinase ABC. Data are representative of three independent samples.
Results: :
P2X7-/- mice showed decreased expression in collagen types I and V and small leucine-rich proteoglycans decorin, keratocan, and lumican. In contrast, there was a 2.5-fold increase in lysyl oxidase, but lysyl oxidase-like proteins were minimally changed. In addition there were 3-fold increases in expression of type III collagen, syndecan 1, perlecan, and biglycan, which is structurally similar to decorin. Perlecan, typically present along the basement membrane, was not detected. However, expression was enhanced in the posterior stroma. Similar increases in lysyl oxidase and perlecan were also detected in hypoxic 3-D organ cultures. Overall sulfation was decreased in P2X7-/- stromas, reflecting expression of proteoglycan cores. Digestion with lyases reduced sulfation by a greater percentage in the WT than P2X7-/- samples. The WT corneas demonstrated greater susceptibility to Chondroitinase ABC and Keratanase digestion than the P2X7-/- corneas.
Conclusions: :
These results indicate that the P2X7 receptor plays a crucial role in regulating the composition and architecture of the normal corneal stroma. Loss of the P2X7 receptor alters the expression of matrix proteins in the corneal stroma in a manner similar to that seen in wounded or hypoxic phenotypes, marked by increases in the expression of syndecan 1, perlecan, and lysyl oxidase.
Keywords: cornea: stroma and keratocytes • extracellular matrix • proteoglycans/glycosaminoglycans