Induced expression of insulin-like growth factor-1 by amniotic membrane-conditioned medium in cultured human corneal epithelial cells

PURPOSE. To determine the effect of amniotic membrane-conditioned medium (AMCM), via insulin-like growth factor (IGF)-1 induction, on human corneal epithelial cell (HCEC) proliferation. METHODS. HCECs were cultured from corneal limbal tissue with supplemented hormonal epithelial medium (SHEM). After administration of AMCM, cell proliferation was evaluated with an MTT assay and DNA synthesis with mcthyl-[ 3 H]-thymidine incorporation assay. RT-PCR and Western immunoblot analyses were performed, to determine potential inducible factors that may be associated with AMCM-induced cell proliferation. Neutralizing anti-IGF-1 antibody and small interfering (si)RNA were also used to clarify the role of IGF-1 in AMCM-induced HCEC proliferation. RESULTS. HCEC proliferation increased after AMCM treatment. Of the cytokines known to be associated with HCEC proliferation, only IGF-1 expression was upregulated in response to AMCM in a dose- and time-dependent manner. The IGF-1 induction effect was found on both AMCM from live AM and from cryopreserved AM. HCEC proliferation was also increased by addition of exogenous IGF-1. AMCM-induced HCEC proliferation was inhibited in the presence of neutralizing anti-IGF-1 antibody and IGF-1 siRNA. Finally, Akt phosphorylation was increased in HCECs after AMCM treatment and was inhibited by IGF-1 siRNA. CONCLUSIONS. IGF-1 is induced by AMCM during HCEC proliferation, and this induction may play an important role in the amniotic membrane during HCEC proliferation and migration in several intractable corneal epithelial defects.