Investigative ophthalmology & visual science · 1993
Fibrin induction of thrombospondin in corneal endothelial cells in vitro.
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Ramsby M L, Kreutzer D L
Аннотация
PURPOSE: Fibrin deposition in the anterior chamber of the eye occurs in response to injury or inflammation and can permanently damage the corneal endothelium. Fibrin functions as a mediator of inflammation and wound healing by affecting cell morphology and function in a variety of cells, including corneal endothelial cells. We hypothesized that fibrin can directly induce corneal endothelial cells to express injury-related proteins (eg, thrombospondin [TSP]) necessary for corneal repair processes.
METHODS: Bovine corneal endothelial cells (BCECs) were pulse- or continuously labeled with 35S-methionine in the presence or absence of in situ polymerized fibrin (2 mg/ml). BCECs were harvested after 3-48 hr, and 35S-labeled proteins were analyzed by SDS-PAGE, autoradiography, and immunochemical techniques.
RESULTS: Fibrin selectively induced BCECs to express a high molecular weight (MW) protein that was present extracellularly in conditioned medium and fibrin matrix. This induction represented a 3-5 fold increase relative to nonfibrin-treated BCECs, was not accompanied by corresponding changes in 35S-labeled intracellular proteins, and was evident at early (3 hr) or late times (24 hr) post-fibrin treatment. The induced protein had an apparent MW of 180 kD (reduced) and > 420 kD (nonreduced), consistent with the characteristics of TSP. A polyclonal antibody to human TSP recognized the reduced form (180 kD) on Western blots and the native form (> 420 kD) in immunoprecipitation studies.
CONCLUSIONS: Fibrin induces BCECs to express TSP, a matrix protein involved in cell-cell and cell-matrix interactions and implicated in wound healing.
METHODS: Bovine corneal endothelial cells (BCECs) were pulse- or continuously labeled with 35S-methionine in the presence or absence of in situ polymerized fibrin (2 mg/ml). BCECs were harvested after 3-48 hr, and 35S-labeled proteins were analyzed by SDS-PAGE, autoradiography, and immunochemical techniques.
RESULTS: Fibrin selectively induced BCECs to express a high molecular weight (MW) protein that was present extracellularly in conditioned medium and fibrin matrix. This induction represented a 3-5 fold increase relative to nonfibrin-treated BCECs, was not accompanied by corresponding changes in 35S-labeled intracellular proteins, and was evident at early (3 hr) or late times (24 hr) post-fibrin treatment. The induced protein had an apparent MW of 180 kD (reduced) and > 420 kD (nonreduced), consistent with the characteristics of TSP. A polyclonal antibody to human TSP recognized the reduced form (180 kD) on Western blots and the native form (> 420 kD) in immunoprecipitation studies.
CONCLUSIONS: Fibrin induces BCECs to express TSP, a matrix protein involved in cell-cell and cell-matrix interactions and implicated in wound healing.