Measurement of mRNAs for TGFss and extracellular matrix proteins in corneas of rats after PRK.
OBJECTIVE
To assess the role of the transforming growth factor (TGF)ss system in formation of corneal haze after excimer laser photorefractive keratectomy (PRK), levels of mRNAs for three TGFss isoforms (TGFss1, TGFss2, and TGFss3), the TGFss type II receptor (TssRII), and extracellular matrix (ECM) genes including fibronectin (FN), collagen I, collagen III, and collagen IV were measured in rat corneas.


METHODS
Corneas were graded for corneal haze at 0, 1.5, 7, 21, 42, and 91 days after PRK. Total RNA was isolated from pooled corneas, and the levels of mRNAs were measured using competition-based quantitative reverse transcription-polymerase chain reaction (RT-PCR).


RESULTS
Severe corneal haze developed by day 42 and persisted to day 91. Levels of TGFss1 mRNA were high in rat corneas before PRK and remained relatively constant. In contrast, levels of TGFss2 and TGFss3 mRNAs were very low in normal corneas, increased 300-fold and 25-fold, respectively, on day 21, and remained elevated on day 91. Levels of mRNA for TssRII increased, with a peak elevation of 50-fold on day 42 after PRK. Levels of mRNAs for ECM proteins also increased. Fibronectin mRNA was nondetectable in normal corneas but rapidly increased to 675 copies/cell on day 7 and remained elevated to day 91. Collagen III mRNA levels peaked on day 21 with a 700-fold increase compared with a very low level of expression in normal cornea, and then decreased on day 91. Expression of collagen I mRNA lagged expression of collagen III mRNA and peaked at day 42 after PRK with a 1200-fold increase over normal cornea. In contrast, mRNA for collagen alpha(1)IV, a major component in basement membranes, remained relatively stable through day 21 and then increased slightly on days 42 and 91.


CONCLUSIONS
The synchronized increase in mRNA synthesis for both the TGFss system and key ECM genes supports the hypothesis that TGFss is a key growth factor promoting stromal haze formation in corneas after PRK and suggests that limiting TGFss system may reduce corneal scarring after excimer laser ablation.
Chen
C
C
Institute of Wound Research, Department of Obstetrics and Gynecology University of Florida, Gainesville, USA.
Michelini-Norris
B
B
Stevens
S
S
Rowsey
J
J
Ren
X
X
Goldstein
M
M
Schultz
G
G
D000818:Animals; D003315:Cornea / Q000378:metabolism* / Q000601:surgery; D015715:Corneal Edema / Q000209:etiology / Q000378:metabolism; D003318:Corneal Opacity / Q000209:etiology / Q000378:metabolism; D017931:DNA Primers / Q000737:chemistry; D016326:Extracellular Matrix Proteins / Q000235:genetics*; D005136:Eye Proteins / Q000235:genetics*; D005784:Gene Amplification; D015870:Gene Expression; D054018:Lasers, Excimer; D008297:Male; D018903:Photorefractive Keratectomy / Q000009:adverse effects*; D020033:Protein Isoforms / Q000235:genetics; D012313:RNA / Q000302:isolation & purification; D012333:RNA, Messenger / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016212:Transforming Growth Factor beta / Q000235:genetics*; D014945:Wound Healing
D017931:DNA Primers; D016326:Extracellular Matrix Proteins; D005136:Eye Proteins; D020033:Protein Isoforms; D012333:RNA, Messenger; D016212:Transforming Growth Factor beta; D012313:RNA