A tailored single-EV microarray assay that can distinguish multivesicular body-derived exosomes and plasma membrane budded microvesicles

Lipoprotein(a) (Lp[a]) blood levels >50 mg/dL is a major cardiovascular disease risk factor in humans. Lp(a) associates with increased cardiovascular calcification, a critical pathology with no clinically available drug therapies. The mechanisms through which Lp(a) increases cardiovascular calcification risk remain undefined. Researchers from the Harvard Medical School and Brigham and Women’s Hospital hypothesized that Lp(a) promotes the release of calcifying extracellular vesicles (EVs) that contribute to formation of microcalcification in cardiovascular tissues. These researchers show Lp(a) increased calcification in both primary human smooth muscle cells (SMCs) and valvular interstitial cells (VICs), potentially through inflammation-related mechanisms that were suppressed with E06 antibody that neutralizes pro-inflammatory oxidized phospholipids. Incubating human SMCs and VICs with Lp(a) altered the composition of EVs, increasing CD29⁺/tetraspanin⁻ microvesicle release, demonstrated with a tailored single-EV microarray assay that can distinguish multivesicular body-derived exosomes and plasma membrane budded microvesicles at a single-vesicle level. Lp(a) stimulation led to release of SMC and VIC EVs that readily calcified in acellular 3D-collagen hydrogels mimicking formation of ectopic microcalcification occurring in extracellular matrix of human atherosclerotic arteries and stenotic aortic valves. This study mechanistically demonstrates that Lp(a) partially mediates cardiovascular calcification formation via inducing the release of calcifying EVs. Additionally, we provide a customized method to assess calcifying EVs at a single-vesicle level that can be more broadly applied to assist in quantitatively differentiating exosome and microvesicle EV subpopulations.

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.