Y El-Shabrawi1, C L Kublin, C Cintron. 1. Schepens Eye Research Institute and the Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts 02114, USA.
Abstract
PURPOSE: Type VI and XII collagens and beta ig, major components of the interfibrillar matrix, may maintain proper spacing among collagen fibrils, necessary for corneal transparency. During normal corneal stroma development and healing, changes in mRNA levels of these proteins were measured to determine whether differences in steady state levels are indicative of the unique structure produced by each corneal tissue. METHODS: A full-thickness excision wound was made in each cornea of six adult rabbits and allowed to heal for 1, 2, or 4 weeks. Scar tissue from two rabbits (four scars) were used from each time period and processed for RNA extraction. Total RNA from 23-day-old fetal rabbit corneas (equivalent to approximately 1 week of stromal development) and 8-day-old neonate corneas (equivalent to approximately 3.5 weeks of stromal development) was also extracted. Relative quantities of alpha1(VI) collagen, alpha1(XII) collagen, beta ig, and beta-actin mRNAs were determined by competitive reverse transcriptase-polymerase chain reaction. Glyceraldehyde-3-phosphate dehydrogenase was used as a housekeeping gene. RESULTS: Increased mRNA levels for alpha1(VI) and alpha1(XII) collagens, beta ig, and beta-actin were seen during the first 2 weeks of healing and were followed by a decrease in 4-week-old scars. Similar increases were seen in fetal corneas with a further increase in the neonate. Differences in the beta ig mRNA levels relative to that of alpha1(XII) collagen in fetal stroma and in comparison with 1-week-old wounds suggest a higher production of beta ig in early healing tissue. CONCLUSIONS: Alterations of mRNA levels during healing and development are consistent with the cellular events and deposition of extracellular matrices in these corneal tissues. Assuming that extracellular matrix protein production is regulated at the transcriptional level, relative changes in beta ig and collagen mRNA levels reflect differences in protein deposition in early fetal and healing tissues. This is consistent with differences in the organization of the interfibrillar matrices of these tissues and their transparency.
PURPOSE: Type VI and XII collagens and beta ig, major components of the interfibrillar matrix, may maintain proper spacing among collagen fibrils, necessary for corneal transparency. During normal corneal stroma development and healing, changes in mRNA levels of these proteins were measured to determine whether differences in steady state levels are indicative of the unique structure produced by each corneal tissue. METHODS: A full-thickness excision wound was made in each cornea of six adult rabbits and allowed to heal for 1, 2, or 4 weeks. Scar tissue from two rabbits (four scars) were used from each time period and processed for RNA extraction. Total RNA from 23-day-old fetal rabbit corneas (equivalent to approximately 1 week of stromal development) and 8-day-old neonate corneas (equivalent to approximately 3.5 weeks of stromal development) was also extracted. Relative quantities of alpha1(VI) collagen, alpha1(XII) collagen, beta ig, and beta-actin mRNAs were determined by competitive reverse transcriptase-polymerase chain reaction. Glyceraldehyde-3-phosphate dehydrogenase was used as a housekeeping gene. RESULTS: Increased mRNA levels for alpha1(VI) and alpha1(XII) collagens, beta ig, and beta-actin were seen during the first 2 weeks of healing and were followed by a decrease in 4-week-old scars. Similar increases were seen in fetal corneas with a further increase in the neonate. Differences in the beta ig mRNA levels relative to that of alpha1(XII) collagen in fetal stroma and in comparison with 1-week-old wounds suggest a higher production of beta ig in early healing tissue. CONCLUSIONS: Alterations of mRNA levels during healing and development are consistent with the cellular events and deposition of extracellular matrices in these corneal tissues. Assuming that extracellular matrix protein production is regulated at the transcriptional level, relative changes in beta ig and collagen mRNA levels reflect differences in protein deposition in early fetal and healing tissues. This is consistent with differences in the organization of the interfibrillar matrices of these tissues and their transparency.