An investigation into the role of endothelial-derived microparticles in an in vitro model of vascular health and disease

Endothelial microparticles (EMPs) are complex structures with pleiotropic properties and are emerging as an index of endothelial damage. Increased circulating levels of EMPs have been identified in several inflammatory disorders and are reduced following anti-inflammatory treatment. Since they are cell-to-cell communicators, this study aimed to identify specific effects on both endothelial and vascular smooth muscle cell (VSMC) function. We hypothesise that EMPs have a dual role, depending on the stimuli involved in their release, potentially playing a role in vascular homeostasis, but also in exacerbating vascular damage under disease conditions and this can be executed via activating the endothelium, and also in the cross-talk to the smooth muscle layer, in terms of depositing a calcified matrix. The study is presented in two sections: first, to investigate the effect of EMPs on endothelial cells, by studying EMP release, endothelial cell activation and migration, and second, to investigate whether they modulate osteogenic differentiation of VSMCs in vitro, focusing on the mechanistic pathways involved using a microRNA and proteomic screening. To achieve these aims, three different sets of extracellular vesicles were generated (probably containing EMPs and exosomes): i) uEMPs, which were generated from healthy growing untreated human umbilical vein endothelial cells (HUVECs), ii) sEMPs, from Tumor Necrosis Factor alpha (TNFα)- stimulated HUVECs and iii) AoEMPs, from TNFα-stimulated human aortic endothelial cells. In the first study, HUVECs were treated with either uEMPs or sEMPs for 24 hours to investigate their effects on endothelial cell function, while in the second study, VSMCs were treated with AoEMPs for 3 weeks in osteogenic media to assess their effects on vascular calcification. The study confirmed that EMP content, which depends on their cellular origin and the stimuli involved in their release, defines their properties. Both uEMPs and sEMPs increase vascular cell adhesion protein (VCAM-1) and intercellular adhesion molecule 1 (ICAM-1) and cell migration. However, sEMPs increase EMP release and carry elevated Chemokine (C-C motif) Ligand 20 (CCL20), identified in the proteomic screening and validated by flow cytometry, in comparison to uEMPs, thus contributing to the elevated EMP levels and disease pathogenesis via CCL20 and dysregulated inflammatory pathways. In addition, Alizarin Red S and calcium deposition assays demonstrated that VSMCs treated with AoEMPs in osteogenic media for 3 weeks show enhanced calcification in vitro, using Alizarin Red staining and calcium deposition assays. These findings may be in part, linked with miRNA- 3148/osteoprotegerin signalling pathway, as miRNA-3148 was identified in the microRNA screening and using transfection studies, we identified its relationship with its target osteoprotegerin RNA. This study provides improved understanding of the mechanisms in which EMPs affect endothelial function and VSMC calcification in vitro. Further research will help understanding the ultimate role of EMPs on the vessel wall.