Microvesicles Derived from Indoxyl Sulfate Treated Endothelial Cells Induce Endothelial Progenitor Cells Dysfunction.

Andres Carmona,Julia Carracedo,Teresa Obrero,Fatima Guerrero,Paula Buendia,Pedro Aljama

doi:10.3389/fphys.2017.00666

Abstract

Cardiovascular disease is a major cause of mortality in chronic kidney disease patients. Indoxyl sulfate (IS) is a typical protein-bound uremic toxin that cannot be effectively cleared by conventional dialysis. Increased IS is associated with the progression of chronic kidney disease and development of cardiovascular disease. After endothelial activation by IS, cells release endothelial microvesicles (EMV) that can induce endothelial dysfunction. We developed an in vitro model of endothelial damage mediated by IS to evaluate the functional effect of EMV on the endothelial repair process developed by endothelial progenitor cells (EPCs). EMV derived from IS-treated endothelial cells were isolated by ultracentrifugation and characterized for miRNAs content. The effects of EMV on healthy EPCs in culture were studied. We observed that IS activates endothelial cells and the generated microvesicles (IsEMV) can modulate the classic endothelial roles of progenitor cells as colony forming units and form new vessels in vitro. Moreover, 23 miRNAs were contained in IsEMV including four (miR-181a-5p, miR-4454, miR-150-5p, and hsa-let-7i-5p) that were upregulated in IsEMV compared with control endothelial microvesicles. Other authors have found that miR-181a-5p, miR-4454, and miR-150-5p are involved in promoting inflammation, apoptosis, and cellular senescence. Interestingly, we observed an increase in NFκB and p53, and a decrease in IκBα in EPCs treated with IsEMV. Our data suggest that IS is capable of inducing endothelial vesiculation with different membrane characteristics, miRNAs and other molecules, which makes maintaining of vascular homeostasis of EPCs not fully functional. These specific characteristics of EMV could be used as novel biomarkers for diagnosis and prognosis of vascular disease.

Highlights

Patients with chronic kidney disease (CKD) have a high incidence of cardiovascular disease (CVD)
We evaluated whether indoxyl sulfate (IS) mediates oxidative stress in endothelial cells (EC), analyzing its effect on reactive oxygen species (ROS) production by flow cytometry
After 24 h of treatment, we observed a significant increase in the Mean fluorescence intensity (MFI) of HE in IS-treated human umbilical vein endothelial cells (HUVECs) compared with controls (474.0 ± 1.6 vs. 445.3 ± 2.3; p = 0.004) (Figure 1A)

Summary

Introduction

Patients with chronic kidney disease (CKD) have a high incidence of cardiovascular disease (CVD). To maintain vascular homeostasis, damaged EC are replaced by endothelial progenitors cells (EPCs), which circulate in a low percentage in peripheral blood (Urbich and Dimmeler, 2004) This repair mechanism requires an exquisitely regulated intracellular signaling network that maintains an efficient balance between endothelial damage and the release of EPCs. Previous studies showed, in both animal and human endothelial injury models (Ramirez et al, 2007; Nogueras et al, 2008; Noci et al, 2015), a correlation between plasma levels of EMV and the activity of EPCs. We showed the development of severe vascular disease is associated with an increase in EMV that parallels the decrease in EPCs in patients with CKD (Soriano et al, 2014). The factors involved are not known, and it is necessary to identify if uremic toxins, such as IS, could be involved in endothelial damage, releasing EMV that modulate endothelium repair

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