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RAGE/galectin-3 yields intraplaque calcification transformation via sortilin

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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.

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Data availability

All data and materials are available upon request.

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

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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).

Author information

Author notes

  1. Zhen Sun and Zhongqun Wang contributed equally to this work.

Authors and Affiliations

  1. Department of Cardiology, Affiliated Hospital of Jiangsu University, 212001, Zhenjiang, China

    Zhen Sun, Zhongqun Wang, Jinchuan Yan, Chen Shao, Zhengyang Bao, Lele Jing, Qiwen Pang, Yue Geng & Lili Zhang

  2. Department of Pathology, Affiliated Hospital of Jiangsu University, 212001, Zhenjiang, China

    Lihua Li

Authors
  1. Zhen Sun
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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.

Corresponding author

Correspondence to Zhongqun Wang.

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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).

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Sun, Z., Wang, Z., Li, L. et al. RAGE/galectin-3 yields intraplaque calcification transformation via sortilin. Acta Diabetol 56, 457–472 (2019). https://doi.org/10.1007/s00592-018-1273-1

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  • DOI: https://doi.org/10.1007/s00592-018-1273-1

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