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J. He, H. E. P. Bazan; Up-Regulated Expression of Non-Collagenous Extracellular Matrix Components in Alkali-Induced Retrocorneal Membrane (RCM) and Endothelia-Derived Myofibroblasts. Invest. Ophthalmol. Vis. Sci. 2007;48(13):1480.
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© ARVO (1962-2015); The Authors (2016-present)
A RCM is a fibrous proliferation on the posterior surface of the cornea. Although it has been found in various clinical conditions associated with corneal disorders, its pathogenesis remains unclear. Recently, we have reported in a rabbit model of corneal alkali burn that the injured endothelium was replaced by RCM made up of α SMA-positive cells (Arch Opthalmol, 124: 70, 2006). Here, we examine the expression of ECM components by histochemistry in alkali-induced RCM as well as in endothelia-derived myofibroblasts with the attempt to clarify the cellular and molecular mechanisms of RCM formation.
Corneal alkali burns were made in the right eye of rabbits. Corneas were collected 3 and 4 weeks after injury and prepared for ECM analysis by immunochemistry. For in vitro studies, pieces of Descemet’s membranes with attached endothelial cells were isolated from normal rabbit eyes, endothelial cells were isolated and suspended in DMEM/F12 added with 15% FBS and 50µg/ml of gentamicin, and then seeded in 6- or 24-well plates. When cells grew to 50-60% confluence, they were starved for 24 hrs, and then treated with growth factors including TGF-ß1(10ng/ml), EGF(20ng/ml), bFGF(50ng/ml), PEDF(100ng/ml), and cytokines including IL-1ß (10ng/ml), TNF-α (10ng/ml) and cPAF(100nM), respectively for 48hrs. Cell proliferation and differentiation were evaluated with anti-PCNA and α-SMA antibodies. ECM including keratan sulfate (KS), chondriotin sulfate (CS), fibronectin (FN), laminin (LM), tenascin (TN), and thrombospondin-1(TSP-1), were analyzed by immunochemistry and immunoblotting.
As we already reported, after alkali burn the injured endothelia were replaced by a RCM, consisting of α-SMA-positive cells. Immunofluorescence showed the RCM to be strongly stained for CS, FN, LM, TN and TSP-1, with KS weakly stained. In vitro experiments showed when the endothelia cultured with serum, the cells were transformed into myofibroblasts and constitutively expressed FN, LM, TSP-1, TN, as well as CS and KS. TGF-ß1 or EGF significantly enhanced the expression of α-SMA, FN and TSP-1. EGF also promoted cell proliferation as identified by the increased expression of PCNA. TNF-α up-regulated the expression of FN while it down-regulated the expression of TSP-1. PEDF, bFGF, IL-1ß and cPAF have no significant effect on the ECM expression.
The results suggest that TGF-ß1, EGF and TNF-α may play an important role in the formation of RCM during corneal endothelial wound healing, while IL-1ß and PAF are not involved in the synthesis of non collagen ECM.
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