Connective tissue growth factor is not necessary for haze formation in excimer laser wounded mouse corneas

<div><p>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.</p></div

Light reflecting cells qualitative differences.

<p>These images were equivalently globally enhanced for better visualization. A) serum starved cells and b) PDGF-BB treated. C&E) the light reflective cells in the main body of the wells. D&F) the remaining gel-plug hole which has yet to be completely closed. G) A detailed image of the 3 distinct portions of the PDGF-BB treated wells which shows very haze-like cell phenotype which surrounds cells without such a phenotype (upper-left), in contrast to the still acellular hole.</p

CTGF KO mouse corneas still form haze.

<p>A) <i>Post hoc</i> genotyping results in the injury corneas reveals good recombination in the homozygous KO corneas, but some degree of Cre-mosaicism in the heterozygous corneas. B) Quantitative analysis via macrophotography reveals increase haze in the absence of CTGF. An ANOVA did not detect any statistical significance among the groups.</p

CTGF KO <i>versus</i> wild-type primary corneal fibroblast cultures.

<p>A) The rate at which primary fibroblasts can cover a circular defect in a monolayer culture varies by cell type. B) Representative images for each of the test conditions at the first (left) and last (right) time point. C) The results of <i>post hoc</i> comparisons among the test conditions.</p

Light reflection and α-SMA levels are not correlated.

<p>The phenotypic nature is grossly similar in that both reflect light and have α-SMA, but the arrangement and level of reflection are drastically different.</p

Representative optical coherence tomography (A and B) and transmission electron microscopy (C and D) images of rabbits receiving AAV5-gfp or AAV5-dcn.

<p>Optical coherence tomography was performed in live rabbits on day 10 after the AAV5-gfp or AAV5-dcn application and transmission electron microscopy was performed in corneas collected 14 days after AAV5-gfp or AAV5-dcn application. The optical scans of no decorin-delivered (A) and decorin-delivered (B) cornea suggest that tissue-selective targeted delivery of decorin gene with AAV5 in the rabbit stroma does not induce changes in corneal structure, thickness or shape. The TEM did not detect significant differences in collagen fibril diameter and arrangement in the naive (C) and decorin-delivered rabbit corneas (D) indicating that localized therapeutic levels of decorin delivery in the cornea does not appear compromise corneal health. dcn = decorin.</p

Representative stereomicroscopy images showing VEGF-induced CNV in no decorin-delivered control (A, C and E) and decorin-delivered (B, D and F) rabbit corneas.

<p>Rabbit eyes were imaged early (5 day, Panels A, B), mid (10 day, Panels C, D), and late (14 day, Panels E, F) stages after VEGF pellet implantation. The 100 µl AAV5 viral titer (5×10¹² vg/ml) expressing no gene/gfp or decorin was topically applied onto the cornea after removing corneal epithelium for single application for 2 minutes. A statistically significant inhibition of neovascularization was observed at three tested early, mid and late stages of the CNV. Scale bar denotes 2 mm. dcn = decorin.</p

Representative images showing CNV in rabbit corneas treated AAV5+/−decorin.

<p>Lectin (A–F) and collagen type IV (G–L) immunostaining was carried out in corneal tissue sections obtained from AAV5-gfp and AAV5-dcn treated rabbit corneas collected 14 days after VEGF implantation. As expected very high lectin (A–C) and collagen type IV (G–I) immunostaining was detected in the corneal sections of AAV5-gfp-treated (no decorin-delivered) corneas due CNV induced by the VEGF implantation. The AAV5-mediated decorin gene therapy significantly reduced (p<0.01) the CNV as evident from the markedly less lectin+ (D–F) and collagen type IV+ (J–L) cells in decorin-delivered rabbit corneal sections. DAPI stained nuclei are shown in blue whereas lectin and collagen type IV+ cells in red. Scale bar denotes 100 µm. dcn = decorin.</p

CD31 Western blot for naive, AAV5+/−decorin-delivered rabbit corneal tissues collected 10 and 14 days after VEGF-implantation.

<p>Panel on left side shows immunoblot and on right side shows quantification of western blotting data collected from corneal tissues of day-10 and day-14. A significant decrease in expression of CD31 on day-10 (62%, p<0.05) and day-14 (66%, p<0.05) was detected in decorin-delivered corneas compared to control corneas suggesting that AAV5-mediated decorin gene therapy is efficient in decreasing CNV. β-actin was used to confirm equal loading of protein in each well and normalization of the data. dcn = decorin.</p

Quantification of mRNA expression of pro- and anti-angiogenic genes by real-time PCR.

<p>Real-time PCR was carried out in naive, control (no decorin-delivered) and decorin-delivered rabbit corneal tissues collected 14 days after VEGF implantation. Decorin gene therapy delivered with AAV5 showed marked decrease in the mRNA expression of VEGF and MCP-1 and less pronounced alteration in angiopoietin and PEDF genes. Our data suggests anti-angiogenic effects of decorin gene therapy are mediated by the down-regulation of pro-angiogeneic (VEGF, MCP1 and angiopoietin) and up-regulation of anti-angiogenic (PEDF) genes. * = p<0.001 compared to naive; ζ = p<0.05 compared to naive; ψ = p<0.05 compared to no decorin (dcn)-delivered.</p