Fibrin induction of thrombospondin in corneal endothelial cells in vitro.
OBJECTIVE
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.
Ramsby
M L
ML
Department of Pathology, University of Connecticut Health Center, Farmington 06030.
Kreutzer
D L
DL
D000818:Animals; D001345:Autoradiography; D015153:Blotting, Western; D002417:Cattle; D015815:Cell Adhesion Molecules / Q000096:biosynthesis*; D002478:Cells, Cultured; D004591:Electrophoresis, Polyacrylamide Gel; D004728:Endothelium, Corneal / Q000187:drug effects / Q000378:metabolism*; D005136:Eye Proteins / Q000378:metabolism; D005337:Fibrin / Q000494:pharmacology*; D008715:Methionine / Q000378:metabolism; D008970:Molecular Weight; D010980:Platelet Membrane Glycoproteins / Q000096:biosynthesis*; D011233:Precipitin Tests; D013462:Sulfur Radioisotopes; D019699:Thrombospondins
D015815:Cell Adhesion Molecules; D005136:Eye Proteins; D010980:Platelet Membrane Glycoproteins; D013462:Sulfur Radioisotopes; D019699:Thrombospondins; D005337:Fibrin; D008715:Methionine