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

Abstract

Extracellular vesicles (EVs) including plasma membrane–derived microvesicles and endosomal-derived exosomes aggregate by unknown mechanisms, forming microcalcifications that promote cardiovascular disease, the leading cause of death worldwide. Here, we show a framework for assessing cell-independent EV mechanisms in disease by suggesting that annexin A1 (ANXA1)–dependent tethering induces EV aggregation and microcalcification. We present single-EV microarray, a method to distinguish microvesicles from exosomes and assess heterogeneity at a single-EV level. Single-EV microarray and proteomics revealed increased ANXA1 primarily on aggregating and calcifying microvesicles. ANXA1 vesicle aggregation was suppressed by calcium chelation, altering pH, or ANXA1 neutralizing antibody. ANXA1 knockdown attenuated EV aggregation and microcalcification formation in human cardiovascular cells and acellular three-dimensional collagen hydrogels. Our findings explain why microcalcifications are more prone to form in vulnerable regions of plaque, regulating critical cardiovascular pathology, and likely extend to other EV-associated diseases, including autoimmune and neurodegenerative diseases and cancer.

Nanoanalytical analysis of bisphosphonate-driven alterations of microcalcifications using a 3D hydrogel system and in vivo mouse model

“…However, we did visualize that EVs had an affinity for collagen fibrils and used these fibrils as scaffolding as the EVs aggregate and mineralize. We recently showed that calcifying EVs contain annexin A1, which functioned as a membrane-binding protein that mediated EV aggregation ( 28 ). Other annexins were also implicated in interactions with collagen in the extracellular matrix and have been hypothesized to mediate calcium influx into EVs ( 11 , 26 , 28 , 53 ).…”

“…Macrophages have also been shown to release procalcifying vesicles ( 20 , 21 ). Thus, just as bone formation is hypothesized to be an active, cell-driven process ( 22 , 23 ), mediated by calcifying matrix vesicles, atheroma-associated calcification may similarly be initiated by the production and aggregation of calcifying EVs ( 11 , 20 , 24 – 28 ).…”

Circulating Extracellular Vesicles As Biomarkers and Drug Delivery Vehicles in Cardiovascular Diseases

“…A recent study, using human cardiovascular cells, demonstrated that Annexin A1 induces EVs aggregation and microcalcification formation that promote CVD. These findings could lead to development of therapeutic strategies in CVD [ 120 ].…”

Effects of Chronic Kidney Disease and Uremic Toxins on Extracellular Vesicle Biology

“…Some studies have highlighted the role of EVs derived from valvular interstitial cells (VICs) in VC [ 121 , 125 , 130 ]. Thus, Cui et al performed a proteomic analysis of EVs from calcified VICs and identified the up-regulation of calcification regulators, such as calcium-binding annexins [ 130 ].…”

“…Furthermore, Bouchareb et al found that mineralization of the aortic valve after mechanical strain was promoted by spheroid microvesicles derived from VICs [ 125 ]. Lastly, Rogers et al reported that annexin A1 tethering of VIC-derived EVs induced microcalcification and EV aggregation [ 121 ].…”