Regulation of 1-cys peroxiredoxin expression in the process of stromal wound healing after photorefractive keratectomy.
OBJECTIVE
To investigate 1-cys peroxiredoxin (1-cysPrx) expression during the corneal wound-healing process after PRK and the effect of growth factors on 1-cysPrx expression in cultured bovine keratocytes (BKs).


METHODS
Rat corneas were excised at 4 hours, 12 hours, 1 day, 3 days, and 7 days after PRK. Expression of 1-cysPrx in the corneas was examined by immunohistochemical, Northern blot, and immunoblot analyses. Keratocytes were isolated from bovine corneas and subcultured to study the effects of TGF-beta1, keratinocyte growth factor (KGF), hepatocyte growth factor (HGF), platelet-derived growth factor (PDGF), and H2O2 on 1-cysPrx expression at different concentrations and time intervals. Generation and proliferation of intracellular reactive oxygen species (ROS) in cultured BKs stimulated by these growth factors were measured by the DCF (2',7'-dichlorofluorescein) assay, the CCK-8 assay, and immunoblot analysis with a polyclonal proliferating cell nuclear antigen (PCNA) antibody, respectively.


RESULTS
Intense staining of 1-cysPrx was observed in the epithelia and the anterior stromas of wounded corneas 4 hours after PRK and had extended to the entire stroma by day 3. By day 7, the expression almost returned to nonsurgical control level in epithelia, although notable expression was still detectable in the stroma. Concomitant augmentation of 1-cysPrx mRNA and protein was seen in the corneas at 12 hours to 7 days. Growth factor treatment in cultured BKs resulted in 1-cysPrx induction in a dose- and time-dependent manner. Growth factor-stimulated cells showed strong DCF fluorescence and increased proliferation during a 24-hour incubation, during which an upregulation of 1-cysPrx occurred.


CONCLUSIONS
These observations provide new information for the regulation of 1-cysPrx expression during the corneal wound-healing process.
Department of Ophthalmology and the Asan Institute for Life Sciences, University of Ulsan College of Medicine, Asan Medical Center, Seoul, Korea.
Hungwon
H
Tchah
Myoung Joon
MJ
Kim
Tae-im
TI
Kim
Hyun-jeung
HJ
Choi
Jae Yong
JY
Kim
Mi Jung
MJ
Kim
Jhang Ho
JH
Pak
D000818:Animals; D015152:Blotting, Northern; D002417:Cattle; D049109:Cell Proliferation; D002478:Cells, Cultured; D003319:Corneal Stroma / Q000166:cytology / Q000293:injuries / Q000378:metabolism*; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism; D019084:Fluorescent Antibody Technique, Indirect; D006133:Growth Substances / Q000494:pharmacology; D015151:Immunoblotting; D054018:Lasers, Excimer; D008297:Male; D010544:Peroxidases / Q000378:metabolism*; D054464:Peroxiredoxins; D018903:Photorefractive Keratectomy; D011183:Postoperative Complications; D051381:Rats; D017207:Rats, Sprague-Dawley; D017382:Reactive Oxygen Species / Q000378:metabolism; D014945:Wound Healing / Q000502:physiology*
D006133:Growth Substances; D017382:Reactive Oxygen Species; D010544:Peroxidases; D054464:Peroxiredoxins