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E. A. Andreae, D. J. Warejcka, S. S. Twining; Thrombin Induces the Expression of Cyr61 in Human Corneal Cells. Invest. Ophthalmol. Vis. Sci. 2009;50(13):5698.
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: Prothrombin is synthesized by corneal cells and is activated to thrombin following corneal wounding. Thrombin has been shown to have proangiogenic properties and to induce synthesis of another proangiogenic molecule, CYR61 (CCN-1) in cells of many organs but has not been studied in corneal cells. The purpose of this study is to determine whether thrombin induces the synthesis and secretion of CYR61 in human corneal cells.
A corneal epithelial cell line (HCEC) was purchased from ATCC for use in these experiments. Corneal stromal cells were isolated from human donor corneas and expanded in culture. Cells were plated on collagen coated wells and cultured in serum free media containing FGF2 for fibroblasts (FB) or TGFβ for myofibroblasts (MYO) for seven days. Cells were treated with -thrombin, DFP-inactivated thrombin or hirudin treated thrombin and supernatant samples were collected at timed intervals. Samples were precipitated with TCA, separated on 14% SDS-PAGE, transferred to nitrocellulose membranes and probed with an antibody to CYR61.
CYR61 was endogenously expressed in cultures of HCECs. Thrombin addition to the cultures increased expression with a peak expression at 0.1 unit/ml thrombin. Peak expression was seen after 7 hours, with decreased levels by 11 hours. Treatment of cells with DFP-inactivated thrombin or hirudin and thrombin had no effect on HCEC CYR61 expression. In contrast to the HCECs, FBs and MYOs do not express CYR61 in control cultures. Upon addition of thrombin, CYR61 expression was induced with highest expression seen at 1 u/ml thrombin. Peak expression was seen at 15 hours after thrombin addition with decreased expression at 24 hours. As with the HCECs, DFP-inactivated thrombin and hirudin plus thrombin were not effective in inducing CYR61 expression.
Proteolytically active thrombin induces CYR61 expression in corneal stromal fibroblasts and myofibroblasts and increases expression of CYR61 in a corneal epithelial cell line. The epithelial cells are more sensitive to thrombin than the wound healing stromal cells. Interference with this process may be important for inhibiting angiogenesis in the cornea.
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