Keratocyte matrix interactions and thrombospondin 2.
OBJECTIVE
To determine whether human keratocytes synthesize thrombospondins 2 and 3 (TSP-2, TSP-3) in a collagen matrix and the effect of addition of antibodies to TSP-2 and TSP-3 on keratocyte-populated collagen matrix contraction.


METHODS
Keratocyte-populated collagen matrices were evaluated for TSP-2 and TSP-3 mRNA. Sections of matrices were stained by immunohistochemistry for TSP-2 and TSP-3. Keratocyte populated collagen matrices were treated with antibodies specific for TSP-2 and TSP-3 and these preparations were evaluated for contraction and keratocyte morphology.


RESULTS
Keratocyte derived fibroblasts in collagen matrices contained TSP-2 and TSP-3 mRNA, and the cells were immunoreactive for both proteins. Compared to controls, an antibody specific to the N-terminal domain of TSP-2 significantly inhibited matrix contraction for up to 10 days at a concentration of 20 microg/ml antibody. At 2 microg/ml TSP-2 antibody concentration significant inhibition occurred up to 3 days. Removal of the antibody from the media reversed the inhibitory effect. Cultured keratocytes in TSP-2 treated collagen matrices appeared more rounded than keratocytes in control matrices. An antibody specific to TSP-3 had no effect on matrix contraction or keratocyte morphology.


CONCLUSIONS
Keratocyte derived fibroblasts synthesize TSP-2 and TSP-3 when seeded in collagen matrices. Antibody specific to TSP-2 reversibly inhibits matrix contraction. TSP-2 may play a key role in keratocyte/collagen matrix interactions, as may occur during corneal stromal repair.
Armstrong
David J
DJ
Unit of Ophthalmology, Department of Medicine, University of Liverpool, Liverpool, UK. David@Armstrongonline.co.uk
Hiscott
Paul
P
Batterbury
Mark
M
Kaye
Stephen
S
D015815:Cell Adhesion Molecules / Q000096:biosynthesis* / Q000235:genetics; D002450:Cell Communication / Q000502:physiology; D002452:Cell Count; D002478:Cells, Cultured; D024042:Collagen Type I / Q000378:metabolism; D003319:Corneal Stroma / Q000166:cytology*; D005109:Extracellular Matrix / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D007124:Immunoenzyme Techniques; D007074:Immunoglobulin G / Q000494:pharmacology; D012333:RNA, Messenger / Q000096:biosynthesis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019699:Thrombospondins / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D000099262:Thrombospondin 2
D015815:Cell Adhesion Molecules; D024042:Collagen Type I; D007074:Immunoglobulin G; D012333:RNA, Messenger; D019699:Thrombospondins; D000099262:Thrombospondin 2; C101863:thrombospondin 3