S100A8 and S100A9 promote endothelial cell activation through the RAGE-mediated mammalian target of rapamycin complex 2 pathway

Abstract

S100 calcium binding protein A8 (S100A8) and A9 (S100A9) belong to the S100 family of calcium-binding proteins and have important roles in inflammation. They increase endothelial cell proliferation, thereby affecting inflammation, angiogenesis and tumorigenesis. However, the mechanism of action of S100A8/9 in endothelial cells needs further study. Therefore, the present study sought to investigate the effects of S100A8/9 on the proliferation and angiogenesis of human umbilical vein endothelial cells (HUVEC s) and their mechanism of action. The viability of HUVEC s was determined through a Cell Counting Kit-8 assay. The effect of S100A8/9 on the proliferation of HUVEC s was detected by flow cytometry. Migration was evaluated by a Transwell migration assay. Apoptosis was evaluated by Annexin V-FITC and PI staining via flow cytometry. Western blot analysis and reverse transcription-quantitative polymerase chain reaction assays were performed to evaluate the activation of the phosphatidylinositol 3-phosphate kinase (PI3K)/Akt/mTOR pathway and mTOR complex 2 (mTORC 2). We previously confirmed that S100A8/9 were consistently overexpressed at 1 and 7 days post-surgery in a rabbit vein graft model, which is the period when apoptosis changes to proliferation in neointimal hyperplasia. In the present study, proliferation, viability and migration were increased after treating HUVEC s with S100A8/9. S100A8/9 stimulated the PI3K/Akt/mTOR pathway and mTORC2, which was significantly suppressed by a receptor for advanced glycation end products (RA GE)-blocking antibody. Furthermore, depleting expression of RA GE or mTORC 2 protein components (rapamycin-insensitive companion of mTOR ) by small interfering RNA was found to reduce the cell viability, migration and angiogenesis of S100A8/9-treated HUVEC s. The development of neointimal hyperplasia is a complex process initiated by damage to endothelial cells. In conclusion, S100A8/9 has an important role in intimal hyperplasia by promoting cell growth and angiogenesis via RA GE signaling and activation of mTORC 2.