M., da Cruz E Silva O. and microcalcification formation in human being cardiovascular cells and acellular three-dimensional collagen hydrogels. Our findings clarify why microcalcifications are more prone to form in vulnerable regions of plaque, regulating essential cardiovascular pathology, and likely extend to additional EV-associated diseases, including NAN-190 hydrobromide autoimmune and neurodegenerative diseases and malignancy. Intro Substantial molecular understanding of membrane vesicle trafficking within and between cells related to cell growth and maintenance, neurotransmission, and controlled insulin secretion has been accomplished (= 5 donors, with representative images shown). Red arrows show EVs that likely budded from plasma membrane (level bars, 500 nm), blue arrows show multivesicular bodies likely being released (level bars, 500 nm), and black arrows show aggregated EVs in acellular collagen ECM (level bars, 100 nm). (C) Transmission electron microscopy images of aggregated and calcifying EVs (yellow arrows indicate EVs with membrane hydroxyapatite formation) in collagen ECM in human being carotid artery and aortic valve cells (= 5 donors, with representative images shown; level bars, 200 nm). (D) Density-dependent scanning electron microscopy images of aggregated microcalcifications (yellow/orange color) in human being carotid artery and aortic valve cells ECM (green color); level bars, 1 m (= 5 donors with two representative images demonstrated). Osteogenic conditions alter human being cardiovascular EV protein composition To investigate EV protein mechanistic contributions to calcification, we assessed whether human being vascular and valvular EV protein composition changed under calcifying conditions. While additional cell types, including macrophages, contribute to calcification pathology (= 3 pooled donors, with representative images demonstrated); level bars, 100 nm. (C) Proteomics protein volcano plot analysis for EVs derived from human being SMC (= 9 donors) and VIC (= 7 donors) conditioned press. Plots show improved, insignificant, and decreased EV protein abundances, along with pie charts of total recognized protein distribution in OM relative to control NM. SMC EV and VIC EV full proteomics datasets and statistical analysis included in data file S1. (D) Heatmap of shared enriched pathways based on significantly changed proteins in OM NAN-190 hydrobromide from EVs from both SMCs (= 9 donors) Rabbit Polyclonal to CNGA1 and VICs (= 7 donors). SMC EV and VIC EV full pathway datasets and statistical analysis included in data file S1, and full labeled pathway networks included in figs. S2 and S3. PTK2, protein tyrosine kinase 2. Human being SMC EVs and VIC EVs contain tethering proteins Next, we examined whether EVs isolated from human being SMCs and VICs experienced tethering proteins that could generate calcified EV aggregates aggregates such as those that NAN-190 hydrobromide we observed in human being cardiovascular cells. We performed quantitative pathway analysis (= 9 donors) and pink circle including proteins recognized in VIC EV (= 7 donors). Annexin proteins (ANXA1, ANXA2, ANXA5, ANXA6, and ANXA7) recognized in both SMC EV and VIC EV and improved in OM in either or both are indicated in dark blue. Additional proteins recognized and improved in OM in SMC EV (green circle), VIC EV (pink circle), or both SMC EV and VIC EV (overlapping region) are indicated in light blue. Full comparative EV proteomics dataset and statistical analysis included in data file S1. (B) Representative single-EV microarray images acquired NAN-190 hydrobromide using the ExoView R100 platform with SMC EV (= 6 donors) and VIC EV (= 5 donors). Top and middle panels display ANXA1+ EV in NM and OM with ANXA1+ capture, and bottom panels show lack of ANXA1+ EV with IgG bad control capture. (C) Package and whisker plots for single-EV microarray assessment of ANXA1+ EV, tetraspanin+ EV (TSP+; CD9/CD63/CD81), and ANXA1+/TSP+ EV from SMCs (= 6 donors, *< 0.05, analyzed by Wilcoxon matched pairs test) and (D) VICs (= 5 donors, *< 0.05 analyzed by Welchs test) cultured in NM or OM. Data offered as a percentage of the total EV count (ANXA1+, TSP+, and ANXA1+/TSP+ EV combined) for each donor. (E) Single-EV microarray EV count for ANXA1+/CD9+ EV in SMCs (= 6 donors, error bars are means SD) and VICs (= 5 donors, error bars are means SD, *< 0.05 analyzed by Welchs.