Corneal Epithelial Cells and Stromal Fibroblasts but Not Stromal Myofibroblasts Efficiently Convert Plasminogen to Angiostatin

Abstract

Abstract: : Purpose: Controlling neovascularization in the cornea is essential to maintain corneal transparency. This is especially important during wound healing when factors that induce angiogenesis are present, such as FGF–2 and extracellular proteases. Proteolysis of the kringle region of plasminogen generates angiostatins, molecules that control angiogenesis in the cornea during wound healing. Epithelial, stromal and endothelial cells of human corneas synthesize plasminogen. Angiostatins are found in extracts of the three layers of the cornea and in serum–free conditioned medium from human corneal organ culture. The purpose of this study was to compare the ability of different types of corneal cells to convert plasminogen to angiostatins. Methods: Primary human corneal epithelial cells were cultured in keratinocyte medium. Primary human corneal stromal cells were cultuered in serum–free media with supplements and were converted to fibroblasts with FGF–2 or to myofibroblasts with TGF–ß. Human plasminogen (33nM) was added to cultured cells and medium was collected at 24, 48 and 72 hrs. Medium proteins were separated under non–reducing conditions and identified on Western blots. The angiostatins were isolated using an ε–aminocaproic acid–Sepharose column. FGF–2 was used to stimulate human umbilical vascular endothelial cell (HUVEC) proliferation. Results: Confluent cultures of epithelial cells converted the plasminogen to angiostatin by 24 hrs while stromal fibroblasts converted the added plasminogen to angiostatin by 48 hrs. Even at 72 hrs, stromal myofibroblasts converted very little plasminogen to angiostatin. The epithelial and stromal fibroblast products were very similar. The initial cleavage removed the N–terminal peptide and secondary cleavages formed products that contained kringles 1–4 plus part of kringle 5 (52 and 49 kDa), kringles 1–4 (45 and 41 kDa) and kringles 1–3 (33 kDa). Over the 72 hr period, the larger polypeptides were further cleaved. Purified angiostatin cleavage products were able to inhibit FGF–2 induced proliferation of HUVECs. Conclusions: These results suggest that the motile cells of the cornea, the epithelial cells and stromal fibroblasts in culture can produce angiostatins from plasminogen, while the less mobile myofibroblasts do not have the proteases to carry out this conversion.

Keywords: cornea: stroma and keratocytes • cornea: epithelium • proteolysis