1Uniklinik RWTH Aachen Med. Klinik I - Kardiologie, Angiologie und Internistische Intensivmedizin Aachen, Deutschland
Introduction: Cardiovascular (CV) calcification is a serious medical burden worldwide. It is significantly higher in chronic kidney disease patients and is an independent risk factor for CV morbidity and mortality with no clinically available treatments. Calcifying extracellular vesicles (EVs) are essential for the initiation and progression of arterial calcification. We hypothesize that endocytosis and exocytosis are of critical importance for the genesis and calcification propensity of mineral-loaded EVs.
Methods and Results: Using flow cytometry, we developed a method for detecting the mineral load of EVs. Briefly, all vesicles were stained with cell tracker green, and minerals were visualized using the fluorescence-labeled bisphosphonate osteosense. Mineral-loaded EV were defined as double positive events. Increased mineral content (5-fold, p=0.004, Fig. 1A) was observed in extracellular vesicles (EVs) released from calcifying vascular smooth muscle cells (SMCs) compared to control cells. Additionally, we established a turbidity assay to evaluate the aggregation of EVs based on optical density. Nucleation potential was 3.3-fold increased (p < 0.001) in calcifying EVs compared to control EVs. Next, SMCs were calcified in an osteogenic medium. Targeting Dynamin-1 and -2 (Dynasore hydrate) inhibited caveolin/clathrin-dependent endocytosis, whereas targeting the clathrin terminal domain (Pitstop 2) inhibited only clathrin-dependent endocytosis. Exocytosis was inhibited by targeting Rab27a (Nexinhib20) with no effect on SMC viability by any inhibitor. Dynasore reduced matrix mineralization dose-dependently (-97.7 %, Fig. 1B) and lowered mineral-positive EVs by 18%, while Pitstop 2 had no effect suggesting a role of caveolin-dependent endocytosis in EV-mediated calcification. Indeed, silencing of Cav-1 reduced matrix mineralization, and Cav-1 showed a higher abundance in calcifying EVs compared to control EVs. Both inhibitors had no effect on EV number measured by nanoparticle tracking analysis. Inhibition of exocytosis decreased mineral deposition (-32 %, p=0.017), without altering EV mineral and EV number.
Conclusion: We established two assays to characterize EVs on physicochemical level. Our findings suggest that caveolin-dependent endocytosis is involved in the formation of calcifying EVs and subsequent matrix mineralization. The role of Rab27a-mediated exocytosis of EVs remains unclear.