Research ArticleCELL BIOLOGY

Annexin A1–dependent tethering promotes extracellular vesicle aggregation revealed with single–extracellular vesicle analysis

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Science Advances  16 Sep 2020:
Vol. 6, no. 38, eabb1244
DOI: 10.1126/sciadv.abb1244
  • Fig. 1 Human cardiovascular tissue SMCs and VICs released EVs that aggregated and calcified in acellular ECM.

    (A) SMCs in calcified human carotid artery and (B) VICs in calcified human aortic valve tissue transmission electron microscopy images (n = 5 donors, with representative images shown). Red arrows indicate EVs that likely budded from plasma membrane (scale bars, 500 nm), blue arrows indicate multivesicular bodies likely being released (scale bars, 500 nm), and black arrows indicate aggregated EVs in acellular collagen ECM (scale 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 carotid artery and aortic valve tissues (n = 5 donors, with representative images shown; scale bars, 200 nm). (D) Density-dependent scanning electron microscopy images of aggregated microcalcifications (yellow/orange color) in human carotid artery and aortic valve tissue ECM (green color); scale bars, 1 μm (n = 5 donors with two representative images shown).

  • Fig. 2 Human cardiovascular EV protein composition was altered under osteogenic conditions.

    (A) Nanoparticle tracking analysis of size and abundance of human SMC EV and VIC EV in conditioned media from cells cultured in NM or OM and used in proteomics analysis; error bars are means ± SD from three donors. (B) Transmission electron microscopy images of EVs isolated from SMC and VIC conditioned media and used in proteomics analysis (n = 3 pooled donors, with representative images shown); scale bars, 100 nm. (C) Proteomics protein volcano plot analysis for EVs derived from human SMC (n = 9 donors) and VIC (n = 7 donors) conditioned media. Plots show increased, insignificant, and decreased EV protein abundances, along with pie charts of total detected 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 from EVs obtained from both SMCs (n = 9 donors) and VICs (n = 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.

  • Fig. 3 Human SMC EV and VIC EV contained tethering proteins.

    (A) Venn diagram of detected EV proteins increased in OM, with the green circle including proteins detected in SMC EV (n = 9 donors) and pink circle including proteins detected in VIC EV (n = 7 donors). Annexin proteins (ANXA1, ANXA2, ANXA5, ANXA6, and ANXA7) detected in both SMC EV and VIC EV and increased in OM in either or both are indicated in dark blue. Additional proteins detected and increased 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 obtained using the ExoView R100 platform with SMC EV (n = 6 donors) and VIC EV (n = 5 donors). Top and middle panels show ANXA1+ EV in NM and OM with ANXA1+ capture, and bottom panels show lack of ANXA1+ EV with IgG negative control capture. (C) Box and whisker plots for single-EV microarray assessment of ANXA1+ EV, tetraspanin+ EV (TSP+; CD9/CD63/CD81), and ANXA1+/TSP+ EV from SMCs (n = 6 donors, *P < 0.05, analyzed by Wilcoxon matched pairs test) and (D) VICs (n = 5 donors, *P < 0.05 analyzed by Welch’s t test) cultured in NM or OM. Data presented 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 (n = 6 donors, error bars are means ± SD) and VICs (n = 5 donors, error bars are means ± SD, *P < 0.05 analyzed by Welch’s t test) cultured in NM and OM.

  • Fig. 4 ANXA1 knockdown attenuated human SMC and VIC calcification.

    (A) ANXA1 immunohistochemistry (red color) in calcified (hematoxylin stain, purple color) human carotid artery and aortic valve tissues. Scale bars, 20 μm; n = 5 donors, with representative images shown and additional images included in fig. S5. (B) Human carotid artery and aortic valve tissue ANXA1 immunofluorescence (red color) near aggregated microcalcifications (OsteoSense 680, white color) in collagen ECM (CNA35-OG488, green color). Scale bars, 20 μm; n = 5 donors, with representative images shown. (C) Calcified human carotid artery and aortic valve tissue transmission electron microscopy (EM) of ANXA1 immunogold labeling (black dots) on EV in close proximity (arrow) in collagen ECM. Scale bars, 100 nm; n = 5 donors, with representative images shown, and additional images included in fig. S5. (D) Human SMC and VIC ANXA1 protein from cells cultured in control NM or OM and incubated with control siRNA (control) or ANXA1 siRNA (ANXA1si); quantification is the sum of both bands. Error bars are means ± SD from three donors. (E) Confocal microscopy images of microcalcifications (red color, OsteoSense 680 staining; blue color, DAPI) in SMCs and VICs cultured in NM or OM with control siRNA or ANXA1si; n = 3 donors, error bars are means ± SD; scale bars, 20 μm. (F) Alizarin red calcification stain and quantification for SMCs cultured in NM or OM with control, ANXA1si, or ANXA1si and recombinant human ANXA1 (rANXA1). Scale bars, 100 μm; error bars are means ± SD from three donors. ****P < 0.0001, **P < 0.01, and *P < 0.05, analyzed by analysis of variance (ANOVA).

  • Fig. 5 ANXA1 tethered vesicles and its inhibition suppressed vesicle aggregation.

    (A) Confocal Z stack image of DiR′ [DiIC18(7) 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindotricarbocyanine iodide]–labeled (purple color) swelling produced vesicles with ANXA1-GFP (green color) binding at vesicle tethered sites (white arrow) (x and y planes, 32.4559 μm, and z, 19.2 μm); n = 3 experiments, with representative image shown. Movie of ANXA1-GFP vesicle binding included as movie S1. (B) ANXA1 time- and concentration-dependent vesicle aggregation with extrusion produced vesicles; error bars are means ± SD from three experiments. (C) pH-dependent vesicle aggregation with extrusion produced vesicles with 1 μg of ANXA1 (+ANXA1) or without ANXA1; data are means ± SD from three experiments. (D) EDTA and (E) N-terminal ANXA1 neutralizing antibody aggregation inhibition of extrusion produced vesicles. Error bars are means ± SD from three experiments. ****P < 0.0001, A < 0.0001 versus pH 5.25, B < 0.0001 versus +ANXA1 pH 5.25, C < 0.05 versus +ANXA1 pH 5.25, D < 0.0001 versus pH 6.75, E < 0.0001 versus +ANXA1 pH 6.75, F < 0.0001 versus pH 8.25, analyzed by ANOVA.

  • Fig. 6 ANXA1 inhibition suppressed EV microcalcification in 3D collagen hydrogels.

    (A) Illustration showing 3D collagen hydrogel EV microcalcification experiment design. (B) Nanoparticle tracking analysis of single-EV quantity in conditioned media used for 3D collagen hydrogel EV experiments (n = 3 donors; analyzed by Welch’s t test). Confocal Z stack images and aggregate size quantification for DiR′-labeled (purple color) OM EV aggregates from SMCs [n = 3 donors with 352 control siRNA (control) and 225 ANXA1 siRNA (ANXA1si) EV aggregate areas quantified] and VICs (n = 3 donors with 65 control and 56 ANXA1si EV aggregate areas quantified). Scale bars, 20 μm; error bars are means ± SD; analyzed by Mann-Whitney test, ****P < 0.0001. (C) EV-generated microcalcifications (OsteoSense 680, red color) in 3D collagen hydrogels (CNA35-OG488, green color) using human SMC EV and VIC EV in conditioned NM or OM with control or ANXA1si; scale bars, 20 μm; n = 3 donors; error bars are means ± SD; ***P < 0.001 and *P < 0.05, analyzed by ANOVA. (D) Super-resolution microscopy images of OM conditioned media SMC EV and VIC EV calcification (OsteoSense 680) aggregates in 3D collagen hydrogels (CNA35-OG488). SMC EV x and y planes, 3200 nm; z, 800 nm; and VIC EV x, 10,800 nm, y, 9600 nm, z, 4800 nm; n = 3 donors, with representative images shown. (E) Working model in which altered calcium signaling may lead to increased calcium-binding proteins, including ANXA1 on EVs that promotes tethering of EVs trapped in ECM, leading to EV aggregation and formation and growth of microcalcifications.

Supplementary Materials

  • Supplementary Materials

    Annexin A1–dependent tethering promotes extracellular vesicle aggregation revealed with single–extracellular vesicle analysis

    Maximillian A. Rogers, Fabrizio Buffolo, Florian Schlotter, Samantha K. Atkins, Lang H. Lee, Arda Halu, Mark C. Blaser, Elena Tsolaki, Hideyuki Higashi, Kristin Luther, George Daaboul, Carlijn V.C. Bouten, Simon C. Body, Sasha A. Singh, Sergio Bertazzo, Peter Libby, Masanori Aikawa, Elena Aikawa

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