Abstract
We sought to determine if connective tissue growth factor (CTGF) is necessary for the formation of corneal haze after corneal injury. Mice with post-natal, tamoxifen-induced, knockout of CTGF were subjected to excimer laser phototherapeutic keratectomy (PTK) and the corneas were allowed to heal. The extent of scaring was observed in non-induced mice, heterozygotes, and full homozygous knockout mice and quantified by macrophotography. The eyes from these mice were collected after euthanization for re-genotyping to control for possible Cre-mosaicism. Primary corneal fibroblasts from CTGF knockout corneas were established in a gel plug assay. The plug was removed, simulating an injury, and the rate of hole closure and the capacity for these cells to form light reflecting cells in response to CTGF and platelet-derived growth factor B (PDGF-B) were tested and compared to wild-type cells. We found that independent of genotype, each group of mice was still capable of forming light reflecting haze in the cornea after laser ablation (p = 0.40). Results from the gel plug closure rate in primary cell cultures of knockout cells were not statistically different from serum starved wild-type cells, independent of treatment. Compared to the serum starved wild-type cells, stimulation with PDGF-BB significantly increased the KO cell culture’s light reflection (p = 0.03). Most interestingly, both reflective cultures were positive for α-SMA, but the cellular morphology and levels of α-SMA were distinct and not in proportion to the light reflection seen. This new work demonstrates that corneas without CTGF can still form sub-epithelial haze, and that the light reflecting phenotype can be reproduced in culture. These data support the possibilities of growth factor redundancy and that multiple pro-haze pathways exist.
Highlights
Based on observations from initial testing in cell culture systems, connective tissue growth factor (CTGF) was demonstrated to possess pro-fibrotic activities, and possibly to be a necessary factor for transforming growth factor-β’s (TGF-β) well-known pro-fibrotic activities[1,2,3,4,5,6,7,8,9,10,11,12,13]
platelet-derived growth factor (PDGF)-BB has been found in the tears of patients with corneas healing after photorefractive keratectomy (PRK) surgery[19, 20]
Our data did not resolve the source of residual haze in the mouse corneas, but did demonstrate in vitro, a plausible hypothesis centered on the action of PDGF-BB
Summary
Based on observations from initial testing in cell culture systems, connective tissue growth factor (CTGF) was demonstrated to possess pro-fibrotic activities, and possibly to be a necessary factor for transforming growth factor-β’s (TGF-β) well-known pro-fibrotic activities[1,2,3,4,5,6,7,8,9,10,11,12,13]. The activity required the actions of yet other growth factors, resulting in a PLOS ONE | DOI:10.1371/journal.pone.0172304. The activity required the actions of yet other growth factors, resulting in a PLOS ONE | DOI:10.1371/journal.pone.0172304 February 16, 2017
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