Altered KSPG expression by keratocytes following corneal injury.
OBJECTIVE
Keratocytes synthesize keratan-sulfate proteoglycans (KSPG), lumican and keratocan, to develop and maintain proper collagen interfibrillar spacing and fibril diameter characteristics of the transparent cornea. The purposes of this study are to compare the expression patterns of KSPGs and keratin 12 (K12) respectively by corneal keratocytes and epithelial cells after three different types of injuries; partial and total epithelial debridement and alkali burn.


METHODS
Corneas of 8-12 week old C57Bl/6J or FVBN mice were wounded by partial epithelial (2 mm in diameter) and total epithelial debridement, and alkali burn (0.1 M NaOH, 30 s) and were allowed to heal for various periods of time, from 1 to 84 days. The corneas were then subjected to light microscopy, in situ and Northern hybridization and RT-PCR for examining the expression of K12 and KSPG in the corneal epithelium and stroma, respectively. Immunohistochemistry with anti-alpha-smooth muscle actin (alpha-SMA) was used to identify myofibroblasts in the stroma of injured cornea.


RESULTS
In 2-3 days, partial epithelial denuded corneas were resurfaced by corneal epithelium positive for K12, and stromal edema caused by debridement disappeared. Total epithelial debridement wounded corneas were resurfaced by conjunctival epithelial cells in 2 weeks. Stromal edema in the total epithelial debridement corneas began to subside after 6 weeks. Corneal epithelial cells resurfaced alkali burned corneas within 3-5 days. In situ and Northern hybridization showed a decrease in keratocan and lumican expression at 6 weeks and increased at 12 weeks post-injury in all wound types. Alpha-SMA positive myofibroblasts in the cornea were detected via immunostaining at the time point when KSPG expression was lowest, 6 weeks post-injury.


CONCLUSIONS
The results suggest keratocan and lumican are down-regulated during wound healing at 6 weeks and returned to higher levels at 12 weeks post-injury; implicating that the cells repopulating the injured corneal stroma regained the characteristic function of keratocytes independent of the wound types. However, complete epithelial removal results in irreversible loss of K12 expression.
Carlson
Eric C
EC
Department of Ophthalmology, University of Cincinnati, Cincinnati, OH, USA.
Wang
I-Jong
IJ
Liu
Chia-Yang
CY
Brannan
Paul
P
Kao
Candace W C
CW
Kao
Winston W Y
WW
D000818:Animals; D015152:Blotting, Northern; D002057:Burns, Chemical / Q000378:metabolism / Q000473:pathology; D011508:Chondroitin Sulfate Proteoglycans / Q000235:genetics* / Q000378:metabolism; D003315:Cornea / Q000473:pathology; D065306:Corneal Injuries; D003319:Corneal Stroma / Q000378:metabolism* / Q000473:pathology; D015536:Down-Regulation; D019573:Epithelium, Corneal / Q000378:metabolism / Q000473:pathology; D005126:Eye Burns / Q000139:chemically induced; D005131:Eye Injuries / Q000378:metabolism* / Q000473:pathology; D005136:Eye Proteins / Q000235:genetics / Q000378:metabolism; D005347:Fibroblasts / Q000378:metabolism; D007124:Immunoenzyme Techniques; D017403:In Situ Hybridization; D007632:Keratan Sulfate / Q000235:genetics* / Q000378:metabolism; D053551:Keratin-12; D007633:Keratins / Q000235:genetics / Q000378:metabolism; D000071234:Lumican; D051379:Mice; D008810:Mice, Inbred C57BL; D011509:Proteoglycans / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012972:Sodium Hydroxide; D014945:Wound Healing
D011508:Chondroitin Sulfate Proteoglycans; D005136:Eye Proteins; C507495:KRT12 protein, mouse; C099280:Kera protein, mouse; D053551:Keratin-12; C000604307:Lum protein, mouse; D000071234:Lumican; D011509:Proteoglycans; D012333:RNA, Messenger; D012972:Sodium Hydroxide; D007633:Keratins; D007632:Keratan Sulfate