Large Endothelial Extracellular Vesicles as Mediators of Excess Phosphate-induced Vascular Inflammation
Background
Excess phosphate (Pi) – most observed in chronic kidney disease (CKD) – is associated with an increased risk for cardiovascular disease (CVD). Pi and CVD are linked to inflammation. Moreover, Pi leads to the release of endothelial cell (EC)-derived extracellular vesicles (Pi-EV). Here we demonstrate Pi-EVs as distinct markers of inflammation and signal amplifiers of Pi overload in ECs.
Methods
ECs (EA.hy926) were incubated with physiologic (0.9mM) or excess Pi (5mM) for 90min. Large EVs were isolated from the conditioned medium using differential centrifugation (up to 20000g) and characterized according to MISEV guidelines. EVs from physiologic conditions (Ct-EVs) and the EV-depleted supernatants (SUP) served as controls. For functional assays, ECs were incubated with EVs or SUPs for 24 hours. The nuclear translocation of NF-kB and the expression of its target genes were determined by immunofluorescence and real-time PCR, respectively. Monocyte (THP-1) adhesion to the ECs was assessed with or without BAY11-7085, a specific NF-kB inhibitor.
Results
NTA demonstrated a consistent increase in absolute particle count for conditioned medium of ECs stimulated with excess Pi compared to physiologic Pi (+39.5% ± 7% [SEM]; p<0.05). No difference was observed regarding mean particle size (172nm ± 2nm [SEM]). Pi-EVs induced the nuclear translocation of NF-kB (5-fold change; p<0.0001) and the up-regulation of interleukin-8, a leukocyte chemoattractant (13-fold change; p<0.0001), and the intercellular adhesion molecule 1 (ICAM-1; 2.3-fold change; p<0.01), both key inflammatory markers. Accordingly, EC monolayers treated with Pi-EVs -but not with Pi-SUP or Ct-EVs- supported an increased adhesion of monocytes (2-fold change; p<0.0001), an effect abrogated by BAY11-7085.
Conclusion
Pi-EVs mediate an inflammatory phenotype in ECs with an increased adherence of immune cells and the activation of the NF-κB pathway. These findings provide insight into the pathophysiology of phosphate-induced vascular inflammation, demonstrate new functional implications of Pi-EVs, and propose a potential novel target for CVD-treatment in CKD.
Keywords
large extracellular vesicles; excess phosphate; endothelial inflammation; NF-κB signaling; cardiovascular disease
Funding/Acknowledgments
This project was supported by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation—project 493624047)
Auhors
Göran Ramin Boeckel1 (Corresponding Author: goeranramin.boeckel[at]ukmuenster[dot]de), Giovana Seno Di Marco1, Katrin Beul1, Amélie Friederike Menke1, Lino Henkel1, Annalen Bleckmann2,3, Hermann Pavenstädt1, Kerstin Menck2,3, Marcus Brand1