Indoxyl Sulfate-Induced Extracellular Vesicles Released from Endothelial Cells Stimulate Vascular Smooth Muscle Cell Proliferation by Inducing Transforming Growth Factor-Beta Production

Vascular access stenosis predominantly occurs as a result of neointimal hyperplasia (NH) formation at the anastomosis. Moreover, in the presence of NH, transforming growth factor-beta (TGF-beta) promotes vascular smooth muscle cell (VSMC) proliferation. Extracellular vesicles (EVs) released by endothelial cells are closely associated with vascular dysfunction. Here, we investigated the effects of EVs on TGF-beta signaling and VSMC proliferation. Specifically, EVs were collected from the culture medium of indoxyl sulfate (IS)-treated human umbilical vein endothelial cells and used (2 x 10(6)) to stimulate human aortic smooth muscle cells (SMCs) (1 x 10(6)). Western blotting was performed to assess the levels of Akt, ERK1/2, p38 MAPK, and Smad3. BrdU proliferation assays, quantitative PCR, and ELISA assays were performed to evaluate SMC proliferation and TGF-beta production. The IS-induced EVs stimulated the proliferation of aortic SMCs in a concentration-dependent manner. The EVs both contained TGF-beta and promoted TGF-beta production by SMCs by phosphorylating Akt, ERK1/2, p38 MAPK, and Smad3, which was significantly inhibited by an anti-TGF-beta antibody. SMC proliferation was suppressed by both an anti-TGF-beta antibody and inhibitors of the downstream factors. These results suggest that EVs are involved in the pathogenesis of vascular access stenosis by modulating TGF-beta signaling in VSMCs under uremic conditions.