Cardiac progenitor cell-derived extracellular vesicles promote endothelial cell activation through both associated- and co-isolated proteins
Authors: Roefs M , Qin J , Bauza-Martinez J , Van De Wakker SI , Vader P , Sluijter JPG 
Published: June 2022

Abstract

Funding Acknowledgements
Type of funding sources: Public grant(s) – EU funding. Main funding source(s): ERC Consolidator Grant: EVICARE

Background
Extracellular vesicle (EVs) are small, cell-derived lipid bilayer enclosed particles that play a role in intercellular communication through the delivery of their content which includes nucleic acids and proteins. Cardiac progenitor cell (CPC)-derived EVs have been shown to protect the myocardium against ischemia/reperfusion injury via proangiogenic effects. However, the underlying mechanisms for CPC-EV-mediated angiogenesis remain elusive. Here, we investigated protein-mediated effects of CPC-EVs on the endothelium, and explored EV-dependent and –independent recipient cell activation.

Methods
CPCs were stimulated with calcium ionophore (Ca ion-EVs), previously shown to influence EV release, or vehicle (veh-EVs) for 24 hours and crude EVs were isolated using size exclusion chromatography (SEC). EV concentration and size was assessed using nanoparticle tracking analysis and proteomic composition was profiled using mass spectrometry. Following SEC, iodixanol gradient ultracentrifugation was used to separate EVs from free proteins. CPC-EVs deficient in individual proteins were generated using CRISPR/Cas9 machinery. EV- and protein fractions were functionally characterized based on their potency to activate human microvascular endothelial cells (HMEC-1) and induce wound closure. HMEC-1 activation upon EV-delivery was determined by phosphoproteomics.

Results
HMEC-1 displayed increased wound closure and activation of AKT-mTOR and (Insulin/IGF-) MAPK signaling pathways upon stimulation with veh-EVs but not with Ca ion-EVs, confirmed by phosphoproteomic analysis. MS-proteomic analysis identified multiple proteins strongly enriched in veh-EVs compared with Ca ion-EVs. GO analysis of these candidate proteins revealed their involvement cell migration and –adhesion. This raised the question whether these identified proteins were truly associated to CPC-EVs, or merely co-isolated. Pure EVs isolated using iodixanol gradients lost part of their ability to activate HMEC-1 compared to crude EV preparations. This hints towards a co-stimulatory role of co-isolated proteins in recipient cell activation. When investigating the contribution of individual candidate proteins to CPC-EV functionality, knock-out of NID1 did not affect EV function, while knock-out of PAPP-A resulted in CPC-EVs with reduced functionality. The IGF-receptor inhibitor PPP abrogated CPC-EV-induced HMEC-1 activation, supporting the association of EV-associated PAPP-A with the activation of intracellular IGF-1-MAPK signaling.

Conclusions
A specific set of EV proteins including PAPP-A is identified that may be functionally responsible for the activation of endothelial cells upon exposure to CPC-EVs. It is important to identify if these proteins are EV-associated or represent co-isolated factors that contribute to endothelial cell activation. This may lead to a better mechanistic understanding of CPC-EV-mediated cell activation and translation of EV-mediated therapeutics.