Search

Purpose: Previous research in our laboratory indicated that prothrombin and other coagulation enzymes required to activate prothrombin to thrombin are synthesized by the cornea and that apoptotic human corneal stromal cells can provide a surface for prothrombin activation through the intrinsic and extrinsic coagulation pathways. The purpose of the work reported here is to study the role of thrombin activity in the regulation of matricellular protein Cyr61 (CCN1) produced by wounded phenotype human corneal stromal fibroblasts and myofibroblasts., Methods: Stromal cells from human donor corneas were converted to defined wounded phenotype fibroblasts and myofibroblasts with fetal bovine serum, followed by basic fibroblast growth factor (bFGF) and transforming growth factor beta-1 (TGFβ-1), respectively, and stimulated with varying concentrations (0-10.0 units (U)/ml) of thrombin from 1-7 h. Cyr61 transcript levels were determined using reverse transcriptase-PCR (RT-PCR) and quantitative PCR (qPCR) while protein forms were analyzed using western blot data. Protease activities were characterized via protease class-specific inhibitors and western blot analysis. Thrombin activity was quantified using the fluorogenic peptide Phe-Pro-Arg-AFC. Protease-activated receptor (PAR) agonist peptides-1 and -4 were used to determine whether cells increased Cyr61 through PAR signaling pathways. The PAR-1 antagonist SCH 79797 was used to block the thrombin cleavage of the receptor. PCR data were analyzed using MxPro software and western blot data were analyzed using Image Lab™ and Image J software. Student t test and one- and two-way ANOVA (with or without ranking, depending on sample distribution), together with Dunnett's test or Tukey comparison tests for post-hoc analysis, were used to determine statistical significance., Results: Full-length Cyr61 is expressed by human corneal stromal fibroblasts and myofibroblasts and is significantly upregulated by active thrombin stimulation at the message (p<0.03) and protein (p<0.03) levels for fibroblasts and myofibroblasts. Inhibition by the allosteric thrombin-specific inhibitor hirudin prevented the thrombin-associated increase in the Cyr61 protein expression, indicating that the proteolytic activity of thrombin is required for the increase of the Cyr61 protein level. PAR-1 agonist stimulation of fibroblasts and myofibroblasts significantly increased cell-associated Cyr61 protein levels (p<0.04), and PAR-1 antagonist SCH 79797 significantly inhibited the thrombin stimulated increase of Cyr61 in fibroblasts but not in myofibroblasts. In the fibroblast and myofibroblast conditioned media, Cyr61 was detected as the full-length 40 kDa protein in the absence of thrombin, and mainly at 24 kDa in the presence of thrombin at ≥0.5 U/ml, using an antibody directed toward the internal linker region between the von Willebrand factor type C and thrombospondin type-1 domains. Although known to undergo alternative splicing, Cyr61 that is synthesized by corneal fibroblasts and myofibroblasts is not alternatively spliced in response to thrombin stimulation nor is Cyr61 directly cleaved by thrombin to generate its 24 kDa form; instead, Cyr61 is proteolytically processed into 24 kDa N- and 16 kDa C-terminal fragments by a thrombin activated leupeptin-sensitive protease present in conditioned media with activity distinct from the proteolytic activity of thrombin., Conclusions: In cultured human corneal stromal fibroblasts and myofibroblasts, thrombin regulates Cyr61 through two mechanisms: 1) thrombin increases the Cyr61 expression at the message and protein levels, and 2) thrombin increases the activation of a leupeptin-sensitive protease that stimulates the cleavage of Cyr61 into N- and C-terminal domain populations in or near the thrombospondin type-1 domain. Generation of Cyr61 peptides during corneal injury stimulation may reveal additional functions of the protein, which modulate corneal wound healing activities or decrease activities of the full-length Cyr61 form., (Copyright © 2020 Molecular Vision.)

Purpose: Maintenance of avascularity of the normal cornea and control of neovascularization during wound healing depend on a balance of angiogenic and antiangiogenic factors. The purpose of this paper is to determine the ability of corneal cells to convert plasminogen to angiostatins and to compare these products with those made by intact corneas., Methods: RT-PCR was performed using plasminogen specific primers and the generated cDNA was sequenced. The proteins in corneal extracts, cornea conditioned medium, and medium from corneal epithelial cells, stromal fibroblasts, and myofibroblasts incubated with plasminogen were separated by SDS-PAGE and electroblotted. Western blots used monoclonal antibodies to kringles 1-3 to detect plasminogen and angiostatins. Angiostatins were isolated and tested for activity in a vascular endothelial cell proliferation inhibition assay., Results: Plasminogen, its mRNA and angiostatins were found in human corneal tissue extracts from the epithelial, stromal, and endothelial layers and from cornea conditioned medium, but not in medium from cultured epithelial cells, stromal fibroblasts, or myofibroblasts. However, cultures of corneal epithelial cells and stromal fibroblasts were able to convert exogenously added plasminogen to angiostatins, whereas cultured myofibroblasts did not. Angiostatins of 38 and 34 kDa were found under all angiostatin generating conditions; however other angiostatins differed in size. Further, the angiostatins isolated from fibroblast culture supernatants inhibited vascular endothelial cell proliferation., Conclusions: Conversion of plasminogen to angiostatin is cell-type dependent. Because corneal cells generate angiostatins, use of human angiostatins may be a means of treating abnormal corneal neovascularization without the risk of side effects.