Fig 2: Lp(a) altered EVs composition in calcifying human SMCs and VICs. (A) Cartoon depicting the single-EV microarray assay in which microarray chips printed with anti-CD29, anti-tetraspanins, and negative control IgG capture antibodies are used to capture single EVs that are subsequently detected for additional proteins on captured EVs using fluorescently conjugated antibodies. (B) Example images of single-EVs captured on microarray chips and fluorescently detected with CD63, CD81, and CD29 or IgG negative control antibodies; scale bars = 1 µm, n = 5 donors. White boxes indicates area of the higher magnification inset images. Quantification of CD29+/tetraspanin+ EVs and CD29+/tetraspanin- EVs detected using single-EV microarray and conditioned media from SMCs and VICs incubated in control (M199) or calcifying BGP containing media with Lp(a) (n = 5 donors); analyzed by Welch's t-test, *P < 0.05, error bars = STDEV.

Fig 3: EV markers and Lp(a) localized near calcification in human cardiovascular tissues. (A) Apolipoprotein(a) (apo[a]) and OsteoSense680 (calcification) confocal immunofluorescence in human atherosclerotic arteries and human aortic valve tissues (n = 5 donors, with example images shown). (B) CD29, CD63, and OsteoSense680 confocal immunofluorescence in human atherosclerotic arteries and human aortic valve tissues (n = 5 donors, with example images shown). Scale bars = 20 µm. Orange arrows indicate CD29 and CD63 immunofluorescence in acellular regions near microcalcification. (C) Density-dependent scanning electron microscopy of calcification (yellow/orange color) in extracellular matrix (green color) of human atherosclerotic arteries and (D) aortic valves (n = 5 donors, with example images shown). Scale bars = 1 µm.