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RAGE/galectin-3 yields intraplaque calcification transformation via sortilin

  • Zhen Sun
  • Zhongqun WangEmail author
  • Lihua Li
  • Jinchuan Yan
  • Chen Shao
  • Zhengyang Bao
  • Lele Jing
  • Qiwen Pang
  • Yue Geng
  • Lili Zhang
Original Article

Abstract

Aims

Macrocalcification and microcalcification present different clinical risks, but the regulatory of their formation was unclear. Therefore, this study explored the underlying mechanisms of macrocalcification and microcalcification in diabetes mellitus.

Methods

Anterior tibial arteries of amputated diabetic feet were collected. According to the calcium content, patients were divided into less-calcification group and more-calcification group. And calcification morphology in plaques was observed. For further study, an in vivo mouse diabetic atherosclerosis model and an in vitro primary mouse aortic smooth muscle cell model were established. After the receptors for AGEs (RAGE) or galectin-3 were silenced, calcified nodule sizes and sortilin expression were determined. Scanning electron microscopy (SEM) was performed to detect the aggregation of matrix vesicles with the inhibition or promotion of sortilin.

Results

Both macro- and microcalcification were found in human anterior tibial artery plaques. Macrocalcification formed after the silencing of RAGE, and microcalcification formed after the silencing of galectin-3. In the process of RAGE- or galcetin-3-induced calcification, sortilin played an important role downstream. SEM showed that sortilin promoted the aggregation of MVs in the early stage of calcification and formed larger calcified nodules.

Conclusion

RAGE downregulated sortilin and then transmitted microcalcification signals, whereas galectin-3 upregulated sortilin, which accelerated the aggregation of MVs in the early stage of calcification and mediated the formation of macrocalcifications, These data illustrate the progression of two calcification types and suggest sortilin as a potential target for early intervention of calcification and as an effective biomarker for the assessment of long-term clinical risk and prognosis.

Keywords

Vascular calcification Matrix vesicle RAGE Galectin-3 Sortilin 

Abbreviations

VSMCs

Vascular smooth muscle cells

CML

Nε-carboxymethyl-lysine

AGEs

Advanced glycation end products

RAGE

Receptor for advanced glycation end products

GWAS

Genome-wide association study

SPF

Specific pathogen free

STZ

Streptozotocin

AAV

Adeno-associated viral

OM

Osteogenic medium

LV

Lentivirus vector

oxLDL

Oxidized low-density lipoprotein

CoIP

Coimmunoprecipitation

SEM

Scanning electron microscopy

NTA

Nanoparticle tracking analysis

MVs

Matrix vesicles

HFD

High-fat diet

RUNX2

Runt-related transcription factor 2

PBS

Phosphate-buffered saline

TNAP

Tissue nonspecific alkaline phosphatase

EVs

Extracellular vesicles

CCK-8

Cell counting kit-8

MOI

Multiplicity of infection

Notes

Author contributions

ZS performed, and analyzed experiments, produced figures, and wrote the manuscript. LL contributed to section preparation and immunofluorescence analysis. JY helped the in vivo and in vitro models establishment. CS contributed to gene silencing of cells and mice. ZB helped with SEM and data analysis. LJ contributed to MVs isolation and NTA. YG helped with clinical data collection and analysis. PQ and LZ provided suggestions for experimental design. ZW designed and supervised experiments and wrote the manuscript. All authors read and approved the final manuscript.

Funding

This work was supported by the foundations as follows: the National Natural Science Foundation of China (Grant Nos. 81770450, 81370408, 81670405), the Foundation of Jiangsu Province (WSN-044, QNRC2016836), the Open Program of Key Laboratory of Nuclear Medicine, Ministry of Health and Jiangsu Key Laboratory of Molecular Nuclear Medicine (KF201504) and Graduate Student Scientific Research Innovation Projects of Jiangsu Province (KYCX17_1801, SJCX18_0754).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Human and animal rights disclosure

Human studies conform to the principles outlined in the Declaration of Helsinki (1964) and was approved by the Ethical Committee of the Affiliated Hospital of Jiangsu University. All animal experiments were approved by the Animal Health and Utilization Committee of the Affiliated Hospital of Jiangsu University, and carried out in accordance with the guidelines from Directive 2010/63/EU and “Principles of laboratory animal care” (NIH publication No. 86-23, revised 1985).

Informed consent

All patients gave consent prior to inclusion.

Supplementary material

592_2018_1273_MOESM1_ESM.docx (4.6 mb)
Supplementary material 1 (DOCX 4718 KB)

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Copyright information

© Springer-Verlag Italia S.r.l., part of Springer Nature 2019

Authors and Affiliations

  • Zhen Sun
    • 1
  • Zhongqun Wang
    • 1
    Email author
  • Lihua Li
    • 2
  • Jinchuan Yan
    • 1
  • Chen Shao
    • 1
  • Zhengyang Bao
    • 1
  • Lele Jing
    • 1
  • Qiwen Pang
    • 1
  • Yue Geng
    • 1
  • Lili Zhang
    • 1
  1. 1.Department of CardiologyAffiliated Hospital of Jiangsu UniversityZhenjiangChina
  2. 2.Department of PathologyAffiliated Hospital of Jiangsu UniversityZhenjiangChina

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