RNA-seq analysis of extracellular vesicles from hyperphosphatemia-stimulated endothelial cells provides insight into the mechanism underlying vascular calcification

Abstract

Background Hyperphosphatemia (HP) is associated with vascular calcification (VC) in chronic kidney disease (CKD). However, relationship between HP-induced-endothelial extracellular vesicles (HP-EC-EVs) and VC is unclear, and miR expression in HP-EC-EVs has not been determined. Methods We isolated HP-EC-EVs from endothelial cells with HP and observed that HP-EC-EVs were up-taken by vascular smooth muscle cells (VSMCs). HP-EC-EVs inducing calcium deposition was characterized by Alizarin Red S, colourimetric analysis and ALP activity. To investigate the mechanism of HP-EC-EVs-induced VSMC calcification, RNA-sequencing for HP-EC-EVs was performed. Results We first demonstrated that HP-EC-EVs induced VSMC calcification in vitro. RNA-seq analysis of HP-EC-EVs illustrated that one known miR (hsa-miR-3182) was statistically up-regulated and twelve miRs were significantly down-regulated, which was verified by qRT-PCR. We predicted 58,209 and 74,469 target genes for those down- and up-regulated miRs respectively through miRDB, miRWalk and miRanda databases. GO terms showed that down- and up-regulated targets were mostly enriched in calcium-dependent cell–cell adhesion via plama membrane cell-adhesion molecules (GO:0,016,338, BP) and cell adhesion (GO:0,007,155, BP), plasma membrane (GO:0,005,886, CC), and metal ion binding (GO:0,046,914, MF) and ATP binding (GO:0,005,524, MF) respectively. Top-20 pathways by KEGG analysis included calcium signaling pathway, cAMP signaling pathway, and ABC transporters, which were closely related to VC. Conclusion Our results indicated that those significantly altered miRs, which were packaged in HP-EC-EVs, may play an important role in VC by regulating related pathways. It may provide novel insight into the mechanism of CKD calcification.

Extracellular vesicles in atherosclerosis and vascular calcification: the versatile non-coding RNAs from endothelial cells and vascular smooth muscle cells

“…It is no coincidence that both research studies shared several common miRNAs: 7 miRNAs were up-regulated (miR-92b-5p, miR-204-3p, miR-296-3p, miR-494-3p, miR-667-5p, miR-702-5p, and miR-770-3p) while 6 miRNAs were down-regulated (let-7b-5p, miR-19b-3p, miR-24-3p, miR-30e-5p, miR-99b, and miR-199a-5p). Especially, miR-99b and miR-24-3p were decreased in VC condition in previous studies ( 104 , 159 ). It also suggested that Runx2 was controlled by miR-30e, and the downregulation of miR-30e indicated osteoblast differentiation regulation ( 175 ).…”

“…MiR-29b (miR-29b-3p) and miR-126-5p in EC-EVs also negatively regulated VC ( 102 , 103 ). Additionally, we detected the profile of miRNAs in HP-EVs from HUVECs by RNA-seq, finding that miR-3182 increased while 13 known miRNAs decreased in HP-EVs from HUVECs ( 104 ), indicating the potential function of miRNAs encapsulated in EC-EVs during VC.…”

“…To determine whether the HP condition altered the profile of miRNAs, we ( 104 ) detected the upregulation expression of has-miR-3182, while the downregulation expression of 13 known hsa-miRs (including miR-10a-5p, miR-10b-5p, miR-30a-3p, miR-30a-5p, miR-30c-2-3p, miR-99b-5p, miR-143-3p, miR-193b-5p, miR-365a-5p, miR-486-5p, miR-941, and miR-7706) in the HP-EC-EVs group compared to the PBS-EC-EVs group by miRNA-seq bioinformatics analysis. Target mRNAs for hsa-miR-3182 included acyloxyacyl hydrolase (AOAH), nucleotide-binding oligomerization domain containing 2 (NOD2), and RNA-binding motif protein 47 (RBM47), implying that miR-3182 might be a novel miRNA in an HP-induced VC.…”

Vascular calcification: from the perspective of crosstalk

“…And the combined use of miR-143 and miR-145 mimics was similar to the combined use of miR-221 and miR-222 inhibitors, which could more significantly block the aggravating effects of EV UR on CM-induced calcification [ 172 ]. For Hp-EC-EVs (hyperphosphatemia-induced-endothelial extracellular vesicles) miRNA-seq analysis showed that miRNAs expression were changed: 12 down-regulated miRs (has-miR193b-5p, hsa-miR-941, hsa-miR-99b-5p, hsa-miR-365a-5p, hsa-miR-30c-2-3p, hsa-miR-30a-3p, hsa-miR-30a-5p, hsa-miR-486-5p, hsa-miR-7706, hsa-miR-10a-5p, hsa-miR-10b-5p and including hsa-miR-143-3p as confirmed above) and an up-regulated hsa-miR-3182, suggesting that these miRs may be potential markers for CDK-VC; KEGG analysis of Top-20 signaling pathways showed that calcium signaling pathway, cAMP signaling pathway, and ABC transporters may be closely related to VC [ 261 ]. A recent study also shown that miR-670-3p released from ECs-EVs plays an important role in regulating VC and may be a potential target for arterial calcification in patients with CKD [ 68 ].…”