Soluble RAGE attenuates Ang II-induced arterial calcification via inhibiting AT1R-HMGB1-RAGE axis
Graphical abstract
Introduction
Vascular calcification is the deposition of calcium phosphate crystals in the aortic wall and cardiovascular tissue [1], which is mainly caused by aging, atherosclerosis, diabetes, and chronic kidney disease. Vascular calcification is also a strong independent predictor of increased cardiovascular morbidity and mortality [2,3]. The two main types of vascular calcification are intimal and medial. Intimal arterial calcification is associated with atherosclerosis, which is characterized by lipid deposition, inflammation, and cellular necrosis. In contrast, medial arterial calcification usually presents with systemic mineral imbalance caused by chronic renal disease, resulting in the accumulation of calcium and phosphate in the extracellular matrix [4].
It is well known that vascular smooth muscle cells (VSMCs) play an important role in vascular calcification. In the normal state, most VSMCs in blood vessels exhibit a contractile phenotype, which allows them to maintain vascular tone and blood pressure. However, during inflammation-stimulated processes, such as atherosclerotic calcification, VSMCs switch from a contractile to a synthetic phenotype [5,6]. During phenotypic switching, differentiated VSMCs undergo trans-differentiation into osteoblast-like cells, resulting in vascular calcification. This process is accompanied by a decrease in the expression of contractile proteins, such as smooth muscle α-actin (α-SMA) and SM22α, and an increase in the expression of bone-related genes, such as bone morphogenetic protein 2 (BMP2) and osteonectin [7]. Runt-related transcription factor 2 (Runx2) and osterix are well established as key genes for determination of the osteoblast phenotype. Both are known to regulate the expression of osteoblastic marker genes, such as osteocalcin, osteopontin, collagen type 1, bone sialoprotein, and receptor activator of nuclear factor kappa B ligand (RANKL) [8,9]. In addition, alkaline phosphatase (ALP) is an important early marker of osteoblast differentiation [10].
Angiotensin II (Ang II), an important vasoconstrictor of the renin-angiotensin system (RAS), regulates blood pressure, cardiovascular homeostasis, plasma volume, and inflammation, which are highly associated with the development of hypertension, cardiovascular disease, and kidney disease [11]. Ang II is also known to mediate multifunctional responses in VSMCs via the Ang II type 1 receptor (AT1R)-activated intracellular pathways [12]. Previous studies have reported that Ang II plays a critical role in promoting VSMC phenotype switching, which can contribute to the formation of arterial intimal calcification (AIC) over the atherosclerotic plaque [13,14]. One molecule downstream of Ang II, receptor for advanced glycation end-product (RAGE), is an important pattern recognition receptor that has been demonstrated to crosstalk with RAS, particularly AT1R, and is suggested to have important implications in the pathogenesis of atherosclerosis [[15], [16], [17]]. RAGE, a multi-ligand signal transduction receptor, recognizes various endogenous ligands, such as high-molecular-weight group box 1 (HMGB1), advanced glycation end-product (AGE), and S100/calgranulins, which then activate various signaling pathways through oxidative stress and inflammation-mediated factors, such as tumor necrosis factor alpha (TNF-α), interleukin (IL)-1, IL-6, monocyte chemoattractant protein-1 (MCP-1), nuclear factor-kappa B (NF-κB), and activated protein-1 (AP1) [18,19]. Recently, an association of RAGE with vascular calcification was strongly supported by experiments using AGE or S100A12, which are representative ligands for RAGE in vitro and in vivo [20,21]. However, although a few studies have provided circumstantial evidence of possible crosstalk between AT1R-mediated signaling and RAGE-mediated signaling in Ang II-induced VSMC calcification, this link has not been examined, and the underlying mechanism remains unclear.
Therefore, we examined the crosstalk between the RAGE-mediated signaling pathway and the AT1R-mediated signaling pathway in Ang II-induced calcifying VSMCs, and demonstrated the therapeutic efficacy of soluble RAGE (sRAGE), a decoy receptor for RAGE, in an Ang II-induced vascular calcification model.
Section snippets
Cell culture
Human aortic smooth muscle cells (HAoSMCs) were commercially obtained from ATCC (Manassas, VA, USA) and were cultured in smooth muscle cell growth medium (ATCC) supplemented with 5% FBS, 5 ng/mL rh FGF-basic, 5 μg/mL rh insulin, 5 ng/mL EGF, 10 mM l-glutamine, and 50 μg/mL ascorbic acid. Cells at passages 5–7 were used for the experiments.
Statistical analysis
ImageJ (version 10.0, NIH, Bethesda, MD, USA) and GraphPad Prism (version 7.04, Prism Software, San Diego, CA, USA) were used for data analysis and
sRAGE attenuates the Ang II-induced osteogenic differentiation of vascular smooth muscle cells
As RAGE activation has been shown to induce osteogenic differentiation of HAoSMCs, we investigated whether sRAGE, a decoy receptor of RAGE, can attenuate Ang II-induced vascular calcification, using an in vitro HAoSMC model. First, we confirmed that Ang II is a critical inducer of vascular calcification, by testing our in vitro model using alizarin red as a calcification marker. We confirmed that CaCl2 and β-GP did not induce alizarin red-positive staining either alone or in combination.
Discussion
In the present study, we demonstrate, for the first time, the association between AT1R- and RAGE-mediated signaling pathways, as well as the therapeutic efficacy of sRAGE in Ang II-induced VSMC osteogenic phenotype switching and vascular calcification. The results demonstrate a novel mechanism for Ang II induced arterial calcification and present a potential therapeutic target in modulating atherosclerosis associated arterial calcification. Activation of RAGE via AT1R-induced HMGB1 secretion
Financial support
This work was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) grant funded NRF-2018R1A1A1A05078230 (S. Lim), NRF-2020R1A2C1007103 (S. Park), NRF-2019R1A2C1088428 (M. Seo), and by the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI) grant funded by the Ministry of Health & Welfare (HI20C1566, Y. Jang).
CRediT authorship contribution statement
Jisu Jeong: Methodology, Formal analysis, Investigation, Writing – original draft, Visualization. Soyoung Cho: Methodology, Validation. Miran Seo: Validation, Resources, Funding acquisition. Bok-Sim Lee: Investigation. Yangsoo Jang: Validation, Funding acquisition. Soyeon Lim: Conceptualization, Methodology, Validation, Formal analysis, Resources, Writing – original draft, Writing – review & editing, Visualization, Supervision, Funding acquisition. Sungha Park: Conceptualization, Resources,
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
The authors thank Medical Illustration & Design, part of the Medical Research Support Services of Yonsei University College of Medicine, for all artistic support related to this work.
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