Amniotic membrane matrix effects on calcineurin-NFAT-related gene expressions of SHED treated with VEGF for endothelial differentiation

The nuclear factor of activated T-cell (NFAT) signaling pathway is involved in angiogenesis following initiation by vascular endothelial growth factor (VEGF). A number of angiogenic genes have been associated with calcineurin in the NFAT pathway, forming a calcineurin-NFAT pathway. This study aims to investigate the involvement of four angiogenic genes within the calcineurin-NFAT pathway in the endothelial-like differentiation of stem cells from human exfoliated deciduous teeth (SHED) cultured on a human amniotic membrane (HAM) induced by VEGF. SHED were induced with VEGF for 24 h, then cultured on the stromal side of HAM. The cells were then further induced with VEGF until days 1 and 14. To understand the role of calcineurin, its potent inhibitor, cyclosporin A (CsA), was added into the culture. Results from SEM and H&E analyses showed SHED grew on HAM surface. Gene expression study of Cox-2 showed a drastically reduced expression with CsA treatment indicating Cox-2 involvement in the calcineurin-NFAT pathway. Meanwhile, IL-8 was probably controlled by another pathway as it showed no CsA inhibition. In contrast, high expression of ICAM-1 and RCAN1.4 by VEGF and CsA implied that these genes were not controlled by the calcineurin-NFAT-dependent pathway. In conclusion, the results of this study suggest the involvement of Cox-2 in the calcineurin-NFAT-dependent pathway while RCAN1.4 was controlled by NFAT molecule in endothelial-like differentiation of SHED cultured on HAM with VEGF induction.