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J.R. Hassell, B. Berryhill, B. Kane; Increased Perlecan Production an in vitro Model of Corneal Stromal Fibrosis . Invest. Ophthalmol. Vis. Sci. 2003;44(13):874.
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Purpose: Perlecan is a heparan sulfate containing proteoglycan located on the cell surface/extracellular matrix and is involved in sequestering or delivering growth factors. We evaluated the synthesis of perlecan by keratocytes in the in vitro corneal stromal fibrosis model system. Methods: Collagenase isolated keratocytes from bovine corneas were plated in DMEM containing attachment factors. The media was changed the next day to either DMEM to retain the quiescent phenotype, DMEM containing 10% fetal bovine serum to promote the fibroblast (FB) phenotype or DMEM containing 10% FBS and 5ng/ml TGFß to promote the myofibroblast (MFB) phenotype. Perlecan precursor protein synthesis was measured by 35S-methionine/cysteine pulse labeling on day 3. Cells were lysed, perlecan immunoprecipated with antisera to perlecan and resolved by SDS/PAGE autoradiography. Perlecan proteoglycan synthesis was measured by 35S04 labeling on days 2 to 4. Perlecan was purified from media and cell layer by CsC1 density gradient centrifugation and chromatography on Superose 6. The migration position of perlecan was monitored by immunodotblot and the synthesis of perlecan determined by 35S04 incorporation. Results: Compared to keratocytes, perlecan precursor protein synthesis was 5 fold higher in FBs and 11 fold higher in MFBs. Similarly, 35S04 incorporation into perlecan secreted and retained by the cell layer was 8 fold higher in FBs and 45 fold higher in MFBs. 35S04 incorporation into perlecan secreted into the media was 30 fold higher in FBs and 180 fold higher in MFBs. Conclusions: The results of this studies indicate perlecan synthesis by keratocytes in vitro is substantially upregulated by factors in fetal bovine serum and even further upregulated by TGFß in the presence of fetal bovine serum . These observations suggest perlecan likely plays an important role in keratocyte mediated corneal fibrosis and repair.
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