RAGE/galectin-3 yields intraplaque calcification transformation via sortilin
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.
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.
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.
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.
KeywordsVascular calcification Matrix vesicle RAGE Galectin-3 Sortilin
Vascular smooth muscle cells
Advanced glycation end products
Receptor for advanced glycation end products
Genome-wide association study
Specific pathogen free
Oxidized low-density lipoprotein
Scanning electron microscopy
Nanoparticle tracking analysis
Runt-related transcription factor 2
Tissue nonspecific alkaline phosphatase
Cell counting kit-8
Multiplicity of infection
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.
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).
All patients gave consent prior to inclusion.