S B Lee1, D Q Li, D T Tan, D C Meller, S C Tseng. 1. Bascom Palmer Eye Institute, Department of Ophthalmology, Ocular Surface and Tear Center, University of Miami School of Medicine, Miami, Florida, USA.
Abstract
PURPOSE: When used as an alternative substrate following bare sclera removal of pterygium and other ocular surface diseases, amniotic membrane transplantation can reduce scarring on the reconstructed conjunctival surface. This study was carried out to determine if the amniotic membrane (AM) suppresses the expression of the TGFb signaling system in cultured normal conjunctival (HCF) and pterygial body fibroblasts (PBF). METHODS: HCF and PBF were cultured on AM and plastic wells in serum-containing and serum-free DMEM with or without TGF-beta1. Total RNA was extracted and subjected to Northern hybridization with probes of TGF-beta1, b2 and b3; TGF-beta receptors (TGF- beta R) type I, II and III; a-smooth muscle actin (alpha-SM), b1-integrin, CD44, fibroblast growth factor receptor 1 (FGF-R1/ flg) and platelet-derived growth factor receptor b (PDGFR-beta); and GAPDH as a loading control. MTT assay was used for cell proliferation. RESULTS: Amniotic membrane markedly suppressed the transcript expression of TGF-beta2, b3 and all three types of TGF-beta receptors by both fibroblasts as compared to their cultures on plastic surface. In addition, expression of CD44 transcript was also markedly suppressed while that of b1 integrin, a-SM actin, and FGFR1/flg was mildly suppressed. In contrast, expression of TGF-beta1 and PDGFR-beta remained largely unchanged. The cell proliferation of HCF and PBF grown on AM was also significantly suppressed. CONCLUSIONS: Amniotic membrane matrix uniquely suppresses TGF- beta signaling in both types of fibroblasts. It may also suppress signaling via CD44, b1 integrin and FGFR1/flg. As a result, the phenotype may become less mitogenic, contractile and fibrogenic. These data support in part why amniotic membrane transplantation has an anti-scarring effect for conjunctival surface reconstruction.
PURPOSE: When used as an alternative substrate following bare sclera removal of pterygium and other ocular surface diseases, amniotic membrane transplantation can reduce scarring on the reconstructed conjunctival surface. This study was carried out to determine if the amniotic membrane (AM) suppresses the expression of the TGFb signaling system in cultured normal conjunctival (HCF) and pterygial body fibroblasts (PBF). METHODS: HCF and PBF were cultured on AM and plastic wells in serum-containing and serum-free DMEM with or without TGF-beta1. Total RNA was extracted and subjected to Northern hybridization with probes of TGF-beta1, b2 and b3; TGF-beta receptors (TGF- beta R) type I, II and III; a-smooth muscle actin (alpha-SM), b1-integrin, CD44, fibroblast growth factor receptor 1 (FGF-R1/ flg) and platelet-derived growth factor receptor b (PDGFR-beta); and GAPDH as a loading control. MTT assay was used for cell proliferation. RESULTS: Amniotic membrane markedly suppressed the transcript expression of TGF-beta2, b3 and all three types of TGF-beta receptors by both fibroblasts as compared to their cultures on plastic surface. In addition, expression of CD44 transcript was also markedly suppressed while that of b1 integrin, a-SM actin, and FGFR1/flg was mildly suppressed. In contrast, expression of TGF-beta1 and PDGFR-beta remained largely unchanged. The cell proliferation of HCF and PBF grown on AM was also significantly suppressed. CONCLUSIONS: Amniotic membrane matrix uniquely suppresses TGF- beta signaling in both types of fibroblasts. It may also suppress signaling via CD44, b1 integrin and FGFR1/flg. As a result, the phenotype may become less mitogenic, contractile and fibrogenic. These data support in part why amniotic membrane transplantation has an anti-scarring effect for conjunctival surface reconstruction.
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