Microvesicles from indoxyl sulfate-treated endothelial cells induce vascular calcification in vitro

Abstract

Vascular calcification (VC), an unpredictable pathophysiological process and critical event in patients with cardiovascular diseases (CVDs), is the leading cause of morbi-mortality and disability in chronic kidney disease (CKD) patients worldwide. Currently, no diagnostic method is available for identifying patients at risk of VC development; the pathology is detected when the process is irreversible. Extracellular vesicles (EVs) from endothelial cells might promote VC. Therefore, their evaluation and characterization could be useful for designing new diagnostic tools. The aim of the present study is to investigate whether microvesicles (MVs) from endothelial cells damaged by uremic toxin and indoxyl sulfate (IS) could induce calcification in human vascular smooth muscle cells (VMSCs). Besides, we have also analyzed the molecular mechanisms by which these endothelial MVs can promote VC development. Endothelial damage has been evaluated according to the percentage of senescence in endothelial cells, differential microRNAs in endothelial cells, and the amount of MVs released per cell. To identify the role of MVs in VC, VSMCs were treated with MVs from IS-treated endothelial cells. Calcium, inflammatory gene expression, and procalcification mediator levels in VSMCs were determined. IS-treated endothelial cells underwent senescence and exhibited modulated microRNA expression and an increase in the release of MVs. VSMCs exposed to these MVs modulated the expression of pro-inflammatory genes and some mediators involved in calcification progression. MVs produced by IS-treated endothelial cells promoted calcification in VSMCs.

Keywords: Calcification; Endothelial cells; Microvesicles; Uremic toxins; Vascular cells.

Graphical abstract

Fig. 1

Schematic diagram of the experimental design to obtain for endothelial cells (HUVECs) model.

Fig. 2

Senescence in HUVECs. HUVECs develop a senescence phenotype after IS treatment (250 µM; 3 days). The percentage of senescent HUVECs was determined by senescence-associated β-galactosidase staining. (A) Phase-contrast microscopy pictures. (B) The data represent means ± SD and are expressed as a percentage of total cells and fold induction, with regard to control values. Control (n = 6); IS-induced intermediate senescence (n = 4); IS-induced elevated senescence (n = 4); 8 random fields each; magnification, x100. (C) Lamin B1 and Cyclin D1 representative Western blots under different conditions. After probing with GAPDH, it was confirmed that equal amounts of proteins were loaded. (D) The graphs show that densitometric band analysis levels were normalized to GAPDH levels in arbitrary units (AU). In B and D, data represent means ± SD values and are expressed as the fold induction, as compared to control values. Control (n = 6); IS-induced intermediate senescence (n = 4); IS-induced elevated senescence (n = 4). *p < 0.05, **p < 0.01, and ***p < 0.001. Control HUVECs vs. IS-treated HUVECs.

Fig. 3

miRNAs in IS-treated HUVECs. qPCR analysis of miR-126-3p (A), miR-126-5p (B), miR-21-3p (C), miR-21-5p (D), miR-155 (E), and miR-210 (F) was performed in control and IS-treated endothelial cells using the ΔCt method; U6 snRNA was used for normalization in HUVECs. *p < 0.05 and ***p < 0.001 for Control vs. IS-induced intermediate senescence. Control (n = 6); IS-induced intermediate senescence (n = 4); IS-induced elevated senescence (n = 4).

Fig. 4

Characterization and measurement (MVs per cell ratio) of MVs generated by IS treatment in HUVECs. Representative graphs showing the results of flow cytometry analysis of MVs (A-D). Fluorescence-gated beads with sizes between 0.22 µm and 1.35 µm were used for determining gates(A,B). MVs were plotted using an FSC logarithmic (FS log)/SSC logarithmic (SS log) dot-plot histogram and were gated in the window selected above; it was necessary to use bead counts in each experiment, to calculate the concentration of MVs per unit volume of the sample (C). Size-selected MVs are plotted as a function of their double fluorescence for specific Annexin A5-PE binding and CD31-FITC (D). (E) The number of HUVEC-derived MVs was assessed by flow cytometry, and the number of endothelial cells was measured using the Neubauer chamber. The ratio was expressed as the fraction of MVs number/HUVECs number in each experiment (MVs per cell ratio). Control (n = 6); IS-induced intermediate senescence (n = 4); IS-induced elevated senescence (n = 4). **p < 0.01, control HUVECs vs. IS-induced elevated senescence. #p < 0.001, IS-induced intermediate senescence vs. IS-induced elevated senescence.

Fig. 5

Induction of calcification in HASMCs. HASMCs were cultivated with 50,000 MVs/mL from the control and IS-treated HUVECs for a different number of days. (A) Qualitative calcification was performed by staining cells with alizarin red. A representative from five different experiments is shown. (B) Calcium content was determined by spectrophotometry using phenolsulphonphthalein dye after 30 days of treatment. The graphs present the calcium content of the cells, expressed as μg/mg protein. The data represent the means ± SD of 3 independent experiments. **p < 0.05 vs. control w/o MVs; *** p < 0.001 vs. control w/o MVs, and # p < 0.001 vs. IS-induced intermediate senescence MVs at the same time.

Fig. 6

HASMCs calcification markers. (A) qPCR analysis of Runx2 and BMP2 mRNA obtained from MVs-treated HASMCs using the ΔCt method. HPRT1 mRNA was used for normalization. n = 2 per duplicate. **p < 0.05 vs. control MVs; ***p < 0.001 vs. control MVs; #p < 0.05 vs. IS-induced intermediate senescence MVs at the same time. (B) Western blot analysis of SM22α from MVs-treated HASMCs was performed for different days. Equal protein loading was confirmed after probing with α-tubulin. The graph presents data regarding densitometric bands analysis normalized to α-tubulin in terms of arbitrary units (AU). n = 2 per duplicate. ***p < 0.001 vs. 9 days at the same time.

Fig. 7

Effect of MVs on pro-inflammatory genes in HASMCs. qPCR analysis of inflammatory mRNAs in HASMCs treated with control MVs and IS-treated HUVECs using the ΔCt method; HPRT1 mRNA was used for normalization. n = 2 per duplicate. **p < 0.05 vs. control MVs; ***p < 0.001 vs. control MVs; #p < 0.05 vs. MVs obtained simultaneously from IS-induced intermediate senescent cells.

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