The effects of indoxyl sulfate-induced endothelial microparticles on neointimal hyperplasia formation in an ex vivo model

Purpose: Neointimal hyperplasia (NH) is considered to be one of the main causes of vascular access occlusion in patients receiving hemodialysis. Endothelial injury and TGF-β-mediated proliferation of vascular smooth muscle cells (VSMCs) induce NH. Endothelial microparticles (EMPs) are also increased by endothelial injury. We aimed to investigate the effects of EMPs and TGF-β expression on VSMC proliferation and their contributions to NH formation in an ex vivo model. Methods: EMPs were collected from the culture media of human umbilical vein endothelial cells treated with indoxyl sulfate (IS, 250 μg/mL) after ultracentrifugation at 100,000 × g. Porcine internal jugular veins were isolated and treated with EMPs (2 × 10⁶/mL) or left untreated for 12 days and subsequently compared with TGF-β (10 ng/mL)-treated venous tissue. Intima-media thickness and NH area were assessed using a digital program. Masson's trichrome staining and immunohistochemistry (IHC) analysis for α-smooth muscle actin, phosphorylated Akt, ERK1/2, p38 mitogen-activated protein kinase (MAPK), and Smad3 were performed on each vein sample. Results: NH and VSMC proliferation developed to a significantly greater degree in EMP-treated veins compared to controls, with similar patterns seen in TGF-β-stimulated samples. IHC analysis demonstrated that EMPs markedly increased phosphorylation of Akt, ERK1/2, p38 MAPK, and Smad3 in areas of venous NH formation. Conclusion: Our results showed that IS-induced EMPs provoked massive VSMC proliferation and NH formation via activation of the TGF-β signaling pathways. Further investigation is needed to elucidate the precise mechanism of EMP activity on vascular access stenosis in vivo.