Insulin-like growth factor-I (IGF-I) and transforming growth factor-beta (TGF-beta) modulate tenascin-C and fibrillin-1 in bullous keratopathy stromal cells in vitro.
OBJECTIVE
Pseudophakic bullous keratopathy (PBK) is a major indication for corneal transplantation. Previous studies showed that PBK corneas had increased levels of insulin-like growth factor-I (IGF-I), bone morphogenetic protein-4 (BMP-4), transforming growth factor-beta (TGF-beta), interleukin-1alpha (IL-1alpha) and IL-8. The PBK corneas also had accumulations of tenascin-C (TN-C), fibrillin-1 (Fib-1), matrix metalloproteinase-2 (MMP-2), inflammatory cells but not myofibroblasts. Our goal is to determine if the growth factors/cytokines that are elevated in PBK corneas alter the expression of extracellular matrix (ECM) and/or degradative enzymes in vitro.


METHODS
Stromal cell cultures from normal and PBK human corneas were established and treated for 6 days with IGF-I, BMP-4, IL-1alpha, IL-8 or TGF-beta1/beta2. Immunostaining, Western blot and dot blot analyses for TN-C, Fib-1, alpha-smooth muscle actin (alpha-SMA, a marker for myofibroblasts) or tissue inhibitor of metalloproteinase-1 (TIMP-1) were performed. RNAs were collected and analyzed with Northern blots for TN-C, Fib-1 and beta(2)-microglobulin. Culture media were analyzed using gelatin zymography with or without ethylenediaminetetraacetic acid (EDTA). Some samples were activated with p-aminophenylmercuric acetate (APMA) and reduction/alkylation, and the degradative activities were measured by the MMP-gelatinase activity assay kit.


RESULTS
The IGF-I and TGF-beta1/TGF-beta2 increased (a) TN-C protein deposition, and (b) Fib-1 protein and RNA levels, but (c) had no significant affect on TIMP-1, matrix metalloproteinase-2 (MMP-2) or gelatinase activities. TGF-beta1/TGF-beta2 induced alpha-SMA protein (myofibroblasts) while IGF-I did not. BMP-4, IL-1alpha and IL-8 had little affect on the cells.


CONCLUSIONS
Based upon our data, the fibrotic markers, TN-C and Fib-1, found in PBK corneas may be accounted for by IGF-I and TGF-beta. These growth factors promote fibrosis and ECM deposition without promoting proteolysis. While the other growth factors/cytokines are elevated in PBK corneas, their role(s) in PBK pathogenesis are not clear. In addition, exogenous IGF-I most closely elicited a response that was most similar to the characteristics of the PBK/ABK corneas, i.e. accumulation of TN-C and Fib-1 proteins in the absence of myofibroblasts.
Kenney
M Cristina
MC
Department of Ophthalmology, College of Medicine, University of California, Irvine, 101 The City Drive, Building 55, Room 220, Orange, CA 92868, USA. mkenney@uci.edu
Zorapapel
Nadia
N
Atilano
Shari
S
Chwa
Marilyn
M
Ljubimov
Alexander
A
Brown
Donald
D
D015152:Blotting, Northern; D015153:Blotting, Western; D002478:Cells, Cultured; D003316:Corneal Diseases / Q000378:metabolism* / Q000473:pathology; D005109:Extracellular Matrix / Q000378:metabolism; D000071838:Fibrillin-1; D000071837:Fibrillins; D006801:Humans; D007334:Insulin-Like Growth Factor I / Q000494:pharmacology*; D020782:Matrix Metalloproteinases / Q000378:metabolism; D008840:Microfilament Proteins / Q000378:metabolism*; D019591:Pseudophakia / Q000378:metabolism / Q000473:pathology; D017154:Stromal Cells / Q000187:drug effects / Q000378:metabolism; D019063:Tenascin / Q000378:metabolism*; D016212:Transforming Growth Factor beta / Q000494:pharmacology*; D054392:Adipokines
D000071838:Fibrillin-1; D000071837:Fibrillins; D007334:Insulin-Like Growth Factor I; D020782:Matrix Metalloproteinases; D008840:Microfilament Proteins; D019063:Tenascin; D016212:Transforming Growth Factor beta; C000605722:FBN1 protein, human; D054392:Adipokines