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

Zhong Peng; Yingjie Duan; Shuzhu Zhong; Juan Chen; Jianlong Li; Zhangxiu He

doi:10.1186/s12882-022-02823-6

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

BMC Nephrol. 2022 May 21;23(1):192. doi: 10.1186/s12882-022-02823-6.

Authors

Zhong Peng^#¹, Yingjie Duan^#², Shuzhu Zhong², Juan Chen³, Jianlong Li^{4

5}, Zhangxiu He⁶

Affiliations

¹ The First Affiliated Hospital, Department of Gastroenterology, Hengyang Medical School, University of South China, Hengyang, Hunan, 421001, China.
² The First Affiliated Hospital, Department of Nephrology, Hengyang Medical School, University of South China, Hengyang, Hunan, 421001, China.
³ School of Food and Biological Engineering, Hefei University of Technology, Hefei, 230002, China.
⁴ Department of Orthopedic Surgery, Nanfang Hospital, Southern Medical University, Guangzhou, 510515, China.
⁵ Department of Pediatrics, Weill Cornell Medicine, New York, NY, 10021, USA.
⁶ The First Affiliated Hospital, Department of Nephrology, Hengyang Medical School, University of South China, Hengyang, Hunan, 421001, China. hezhangxiu20150724@126.com.

^# Contributed equally.

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.

Keywords: Extracellular vesicles (EVs); High-throughput sequencing (HTS); Hyperphosphatemia; Vascular calcification.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Calcium / metabolism
Cells, Cultured
Endothelial Cells / metabolism
Extracellular Vesicles* / metabolism
Humans
Hyperphosphatemia* / genetics
Hyperphosphatemia* / metabolism
MicroRNAs* / genetics
MicroRNAs* / metabolism
Muscle, Smooth, Vascular
Myocytes, Smooth Muscle / metabolism
Renal Insufficiency, Chronic* / genetics
Renal Insufficiency, Chronic* / metabolism
Sequence Analysis, RNA
Vascular Calcification* / genetics
Vascular Calcification* / metabolism

Substances

MicroRNAs
Calcium