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H. B. Hindman, J. N. Swanton, R. P. Phipps, K. R. Huxlin; Anterior and Posterior Cultured Cat Keratocytes Respond Differently to TGF-B Stimulation. Invest. Ophthalmol. Vis. Sci. 2009;50(13):5697.
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© ARVO (1962-2015); The Authors (2016-present)
To characterize differences between anterior and posterior cat corneal keratocytes in terms of myofibroblast transformation and fibronectin synthesis in response to TGFβ stimulation.
Six clinically clear corneas from 3 cats were obtained. Lamellar dissection was performed manually (2 eyes) or with the Moria microkeratome (4 eyes). Anterior and posterior cells were cultured separately in serum free medium. When the cells reached 80% confluence, they were incubated with various TGFβ concentrations (0, 0.1, 1, and 2 ug/mL). To determine cellular proliferation response, the percent of Ki-67-positive nuclei was determined by calculating the ratio of Ki67-positive cells to total cells (DAPI stain) daily for 13 days. Cell phenotype and fibronectin synthesis were studied using immunostains and Western blot for Thy-1, SMA, and fibronectin after 72 hours of TGFβ incubation. Semi-quantitiative expression of SMA, Thy-1, and fibronectin was assayed using densitometry measurements relative to GAPDH.
TGFβ increased proliferation of both anterior and posterior keratocytes by Ki-67 staining, with higher concentrations of TGFβ inducing greater proliferation. For each concentration of TGFβ, posterior keratocytes reached maximum proliferation at an earlier timepoint (6 days) than did anterior keratocytes (8 days). After 72 hours incubation with TGFβ, increasing TGFβ concentrations induced increasing expression of Thy-1, SMA, and fibronectin in both anterior and posterior keratocytes by immunohistochemistry. However, staining was apparent at a lower concentration of TGFβ (0.1 ug/ml) in anterior keratocytes as compared to posterior keratocytes (1.0 ug/ml). These results were confirmed by Western blot where aterior keratocytes demonstrated significantly greater relative expression of SMA, Thy-1, and fibronectin at 0.1 ug/mL TGFβ.
Posterior keratocytes demonstrated an earlier proliferation response to TGFβ than anterior keratocytes. However, anterior keratocytes became activated and transformed into wound healing myofibroblasts at lower concentrations of TGFβ. These findings suggest that anterior and posterior keratocytes, heretofore assumed to be similar cells, respond very differently to growth factor stimuli.
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