Caveolin-dependent endocytosis regulates arterial calcification

Cardiovascular Research, vol. 120

Abstract

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): SFB219 Introduction Patients with chronic kidney disease (CKD) have a significantly elevated cardiovascular risk, which is primarily due to premature vascular aging with arterial calcification - a substantial public health burden. Calcifying extracellular vesicles (EVs) are required for the onset and progression of calcification. Endocytosis and exocytosis are important for EV genesis and cargo loading, thus controlling EV release and propensities. Purpose We aim to investigate endocytosis, endosomal ripening, and exocytosis as critical mechanisms of EV-dependent mineralization. Methods and Results First, we characterized EVs from calcifying vascular smooth muscle cells (SMCs) compared to control SMCs. Western blot analysis revealed an increase in tissue nonspecific alkaline phosphatase (TNAP) among calcifying EVs but not in flotillin abundance. When compared to control EVs, calcifying EVs showed higher TNAP activity. Flow cytometry and a turbidity assay were then used to evaluate EV mineral cargo and aggregation potential. Compared to the control, EVs produced from SMCs cultivated under calcifying conditions had 5-fold more mineral-positive EVs (p=0.004) and a 3-fold higher nucleation potential (p<0.001). Next, endocytosis was blocked by targeting Dynamin-1 and –2, which inhibited caveolin- and clathrin-dependent endocytosis (Dynasore hydrate), or targeting clathrin-terminal domain, which inhibited solely clathrin-dependent endocytosis (Pitstop 2). Dynasore reduced matrix mineralization dose-dependently and lowered mineral-positive EVs by 18%, whereas Pitstop 2 had no effect, suggesting a role of caveolin-dependent endocytosis in EV-mediated calcification. Indeed, Cav-1 silencing inhibited matrix mineralization and Cav-1 protein was more abundant in calcifying EVs than in control EVs. Dynasore and Pitstop-2 did not affect the EV number measured by nanoparticle tracking analysis. Inhibition of endosomal ripening by Rab7 inhibition did not affect SMC calcification. Exocytosis was inhibited by Rab27a inhibition, which reduced mineral deposition (-32%, p=0.017) without affecting EV mineral or EV quantity. Conclusion EVs released from calcifying SMCs showed a higher abundance of mineral and TNAP proteins, suggesting a role in mineral transport and deposition. Our data suggest that caveolin-dependent endocytosis is essential in EV-mediated SMC calcification. Mineral cargo in EVs is independent of Rab7 and Rab27a pathways.

Authors 4

1. Anton P.M. Gorgels Aachen

   Universitätsklinikum Aachen · RWTH Aachen University

   Affiliation as printed

   RWTH University Hospital Aachen , Aachen , Germany

2. Nicolas Hense Aachen

   Universitätsklinikum Aachen · RWTH Aachen University

   Affiliation as printed

   RWTH University Hospital Aachen , Aachen , Germany

3. Nikolaus Marx Aachen

   Universitätsklinikum Aachen · RWTH Aachen University

   Affiliation as printed

   RWTH University Hospital Aachen , Aachen , Germany

4. Claudia Goettsch Aachen

   Universitätsklinikum Aachen · RWTH Aachen University

   Affiliation as printed

   RWTH University Hospital Aachen , Aachen , Germany