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.
Highlights
Ectopic calcification occurs in multiple diseases, as well as in advanced age
To assess whether extracellular vesicles (EVs) are mechanistically involved in Lp(a) induction of cardiovascular calcification we used a model in which primary human cardiovascular cells were incubated with commercially obtained Lp(a) particles (≥ 95% pure after isolation from human blood)
To validate the purity of the Lp(a) particles, we performed proteomics and verified that Lp(a) particles purified from human blood contained both apolipoprotein(a) and apoliprotein B, key protein components of Lp(a), as well as multiple other proteins reported to be on Lp(a) (20) (Supplementary Figure 1A; Supplementary Excel File 1)
Summary
Ectopic calcification occurs in multiple diseases, as well as in advanced age. Despite increasing cardiovascular disease mortality, cardiovascular calcification has no drug therapies available. Cardiovascular calcification occurs as vascular calcification in arteries. Lipoprotein(a) Induces Vesicular Cardiovascular Calcification and as valvular calcification in the heart. Microcalcification in atherosclerotic plaques may lead to plaque rupture (1), which in turn can lead to heart attack and stroke. One of the leading causes of heart valve failure is macrocalcification, notably occurring in aortic valves causing aortic valve stenosis due to calcific aortic valve disease (CAVD). Progression of aortic valve stenosis leads to heart failure and death. A greater understanding of the molecular mechanisms driving cardiovascular calcification may led to development of first-in-class and life-saving therapies to treat these critical pathologies
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