MO548: Endothelial Cell Derived Exosomes Promote Vascular Smooth Muscle Cell Calcification and Senescence by Regulating the Β-Catenin Signaling Pathway Through miRNA-29B-3p
Authors: Qin Zheng , Liao Ruoxi , Su Baihai 
Published: May 2022

Abstract

BACKGROUND AND AIMS
Vascular calcification is an important risk factor for high incidence of cardiovascular events and death in patients with chronic kidney disease (CKD). The mechanism of how the calcification signal in early CKD is transmitted to vascular smooth muscle cells (VSMCs) from endothelial cells in the tunica intima to initiate the whole process of vascular calcification remains to be elucidated. The aim of this study was to investigate the effect of miRNA-29B-3p in exosomes derived from vascular endothelial cells under high phosphorus stimulation on VSMC calcification and senescence.

METHOD
Firstly, human umbilical vein endothelial cells (HUVECs) were divided into the high-phosphorus group (HP-HUVEC, 3mmol/L) and the control group (NP-HUVEC) for exosomes isolation by superspeed centrifugation. Transmission electron microscopy, molecular size analysis and Western blot analysis of exosome markers were used to identify exosomes. Secondly, VSMCs were treated with HP-HUVEC-Exo, NP-HUVEC-Exo and blank control. Then, HUVECs were transfection with miR-29b-3p mimic, miR-29b-3p inhibitor and their controls for the extraction of exosomes. VSMCs were further treated with Exo-miR-29b-3p mimic, Exo-miR-29b-3p inhibitor and their controls. Western Blot was used to detect the expressions of calcification-related protein Runx2, senescence-related protein P21 and β-catenin pathway-related proteins (GSK-3β and β-catenin). Alizarin red staining, ALP activity detection and calcium (Ca) content determination were used to detect the calcification of VSMC. The senescence of VSMC was detected by SA-β-gal staining and the expression of miR-29b-3p was detected by qRT-PCR.

RESULTS
The expression of marker proteins CD9 and CD63 was observed by Western Blot, the diameter of particle size analysis was about 172.3 ± 72.5 nm and the structure of typical lipid double-layer membrane was observed, suggesting that exosomes were extracted. Compared with the NP-HUVEC-Exo and blank control group, Western Blot showed that the expression of Runx2, P21 and β-catenin was up-regulated and the expression of GSK-3β was down-regulated in VSMC treated with HP-HUVEC-Exo. VSMC treated with HP-HUVEC-Exo also showed up-regulated miR-29b-3p expression, higher ALP activity and Ca content, suggesting exosomes from high phosphorus-stimulated endothelial cells induced calcification and senescence of VSMCs. Compared with the control group, the expression of Runx2 and P21 was up-regulated and showed more significant calcification and aging characteristics and the expression of GSK-3β was down-regulated and the expression of β-catenin was up-regulated in VSMC treated with Exo-miR-29b-3p mimic. VSMC treated with Exo-miR-29b-3p inhibitor showed the opposite results, indicating that miR-29b-3p could repress GSK-3β and activate β-catenin pathway to promoted VSMC calcification/senescence.

CONCLUSION
Exosomes produced from high phosphorus stimulates endothelial cells could induce the calcification and senescence of VSMC by conveying miR-29b-3p to VSMC and then regulating the β-catenin signaling pathway.