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Updated: Oct 3, 2025
Analysis of Extracellular Vesicle-Mediated Vascular Calcification Using In Vitro and In Vivo Models
Published on: January 27, 2023
Lipoprotein(a) Induces Vesicular Cardiovascular Calcification Revealed With Single-Extracellular Vesicle Analysis
Maximillian A Rogers1, Samantha K Atkins1, Kang H Zheng1,2
1Center for Interdisciplinary Cardiovascular Sciences, Harvard Medical School, Brigham and Women's Hospital, Boston, MA, United States.
Insights
High Lipoprotein(a) levels increase cardiovascular calcification risk by promoting the release of calcifying extracellular vesicles (EVs). This study reveals a new mechanism linking Lp(a) to cardiovascular disease progression.
Area of Science:
- Cardiovascular Biology
- Extracellular Vesicle Biology
- Lipid Metabolism
Background:
- Elevated Lipoprotein(a) [Lp(a)] is a significant human cardiovascular disease risk factor.
- Lp(a) is linked to increased cardiovascular calcification, a pathology lacking effective drug treatments.
- The precise mechanisms by which Lp(a) drives cardiovascular calcification are not fully understood.
Purpose of the Study:
- To investigate the hypothesis that Lp(a) promotes the release of calcifying extracellular vesicles (EVs) contributing to cardiovascular microcalcification.
- To elucidate the role of Lp(a) in modulating EV composition and calcification potential.
- To develop and apply a novel method for single-EV analysis.
Main Methods:
- Utilized primary human smooth muscle cells (SMCs) and valvular interstitial cells (VICs).
- Stimulated cells with Lp(a) and analyzed EV release and composition using a tailored single-EV microarray assay.
- Assessed EV calcification potential in 3D-collagen hydrogels.
- Investigated the effect of E06 antibody, which neutralizes oxidized phospholipids, on Lp(a)-induced effects.
Main Results:
- Lp(a) stimulation increased calcification in human SMCs and VICs, with effects partially suppressed by the E06 antibody.
- Lp(a) altered EV composition, increasing the release of CD29+/tetraspanin- microvesicles.
- Released SMC and VIC EVs readily calcified in vitro, mimicking ectopic microcalcification in cardiovascular tissues.
- The single-EV microarray assay successfully differentiated exosome and microvesicle subpopulations.
Conclusions:
- Lp(a) partially mediates cardiovascular calcification by inducing the release of calcifying EVs from SMCs and VICs.
- Inflammation-related mechanisms, potentially involving oxidized phospholipids, contribute to Lp(a)-induced EV release and calcification.
- The developed single-EV analysis method provides a quantitative approach to differentiate EV subpopulations and assess their calcification potential.
Abstract:
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. We hypothesized that Lp(a) promotes the release of calcifying extracellular vesicles (EVs) that contribute to formation of microcalcification in cardiovascular tissues. Here, we 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. Our 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.
