Insulin-like growth factor-I (IGF-I) and transforming growth factor-β (TGF-β) modulate tenascin-C and fibrillin-1 in bullous keratopathy stromal cells in vitro

Purpose. 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-β (TGF-β), interleukin-1α (IL-1α) 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-1α, IL-8 or TGF-β1/β2. Immunostaining, Western blot and dot blot analyses for TN-C, Fib-1, α-smooth muscle actin (α-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 β 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-β1/TGF-β2 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-β1/TGF-β2 induced α-SMA protein (myofibroblasts) while IGF-I did not. BMP-4, IL-1α 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-β. 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.