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Daniel J. Gibson, Sriniwas Sriram, Liya Pi, Edward W. Scott, Gregory S. Schultz; CTGF Synthesis and Binding in Wounded and Healing Corneas. Invest. Ophthalmol. Vis. Sci. 2012;53(14):4203.
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In order to improve our understanding of the role of CTGF in scarring, the loci of CTGF synthesis and binding were measured immediately after wounding in rabbit and mouse corneas.
Insight into the location of CTGF mRNA synthesis was obtained using a reporter mouse with enhanced green fluorescent protein (eGFP) under control of CTGF’s promoter (pCTGF-eGFP). Confocal fluorescent micrographs were obtained from DAPI counterstained, unwounded, tissue mounts. The distribution of CTGF / GAPDH production was also measured by gross dissection of unwounded rabbit corneas and subsequent qRT-rtPCR analysis of the grossly-separated cell layers. Changes in CTGF transcription and binding location were measured in wounded pCTGF-eGFP mice which were anesthetized and received a central 1.0 mm, 40 µm deep, excimer laser wound. Cryosections from these mice at day 5 were immunofluorescently stained for CTGF protein using a biotinylated antibody and Tex Red avidin. The sections were counterstained with DAPI and imaged using fluorescent confocal micrography with the green channel representing CTGF promoter activity, the red channel representing CTGF protein, and the blue channel representing nuclei. Initial changes in CTGF / GAPDH mRNA expression were also measured by qRT-rtPCR in excimer wounded (6.0 mm x 125 µm) rabbit corneas grossly dissected 30 - 60 min after wounding.
In the unwounded reporter mouse corneas and in the grossly dissected rabbit corneas the corneal endothelium was the site of highest GAPDH normalized CTGF synthesis in the cornea (18.5 fold vs. epithelium, 11.3 fold vs. stroma). In the wounded corneas, the level of CTGF mRNA was increased in all three layers compared to the unwounded corneas, with the stroma having the greatest increase (70.2 fold increase vs. unwounded), but the endothelium slightly remained the cell layer of highest average expression (1.5 fold vs. stroma). While the endothelium was the site of highest CTGF synthesis, the basal epithelium was the primary location of CTGF protein binding.
Since CTGF is constitutively expressed in the cornea and lens of unwounded eyes, CTGF is not sufficient to generate a fibrotic response. The dominance of the endothelium in CTGF synthesis, and the epithelium in CTGF binding, calls into question our studies to date using fibroblast cultures as a model system. However, since the stroma is the location of greatest increase, using the fibroblasts as a model of the source of CTGF might be justified and may support our therapeutic strategy which has centered on targeting stromal fibroblasts with anti-mRNA technologies. The immediate increase in CTGF expression following wounding, supports the urgency of either immediate, or pretreatment of the cornea with anti-CTGF mRNA therapies.
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