Metformin, resveratrol, and exendin-4 inhibit high phosphate-induced vascular calcification via AMPK-RANKL signaling

https://doi.org/10.1016/j.bbrc.2020.07.136Get rights and content

Highlights

  • High inorganic phosphate (Pi) concentrations induce calcification in VSMCs.

  • Metformin, resveratrol, and exendin-4 decreased calcium deposition in the Pi-treated cells.

  • Metformin, resveratrol, and exendin-4 reduced the expression of osteoblast differentiation-associated factors.

  • Metformin, resveratrol, and exendin-4 increase the expression of AMPK.

  • AMPK inhibition reversed the reduction of the expression of Rankl by metformin, resveratrol, and exendin-4.

Abstract

Vascular calcification increases the risk of developing cardiovascular disease, and it is closely associated with metabolic disorders such as diabetes mellitus and non-alcoholic fatty liver disease. We investigated whether the activators of AMP-activated protein kinase (AMPK), metformin, resveratrol, and exendin-4, improved inorganic phosphate (Pi)-induced vascular calcification in rat vascular smooth muscle cells (VSMCs) and whether these effects were via AMPK. Pi increased calcium deposition in a dose-dependent manner, and metformin, resveratrol, and exendin-4 significantly decreased calcium deposition in the Pi-treated VSMCs. Moreover, metformin and exendin-4 increased the expression of a SMC marker gene, α-smooth muscle actin, and Ampk and reduced the receptor activator of nuclear factor kappa-Β ligand (Rankl)/osteoprotegerin ratio. Metformin, resveratrol, and exendin-4 reduced the expression of osteoblast differentiation-associated factors, such as runt-related transcription factor 2, bone morphogenic protein-2, p-small mothers against decapentaplegic 1/5/8, and Rankl. Inhibition of AMPK by siRNA adversely affected the anti-calcification effects of metformin, resveratrol, and exendin-4 and reversed the reduction of the expression of Rankl by metformin and exendin-4 in the Pi-treated VSMCs. These data suggest that metformin, resveratrol, and exendin-4 ameliorate Pi-induced vascular calcification by inhibiting osteoblast differentiation of VSMCs, which is mediated by AMPK.

Introduction

Vascular calcification occurs with aging and is characterized by bone-like mineral deposition on arterial walls [1]. Vascular calcification increases the risk of developing cardiovascular disease (CVD) through the increase in arterial stiffness, and arterial calcification suggests increased atherosclerotic burden in humans [2]. In a multi-ethnic study of atherosclerosis, coronary artery calcification was closely related with future CVD events, and previous studies show the association of vascular calcification with metabolic disorders such as diabetes and non-alcoholic fatty liver disease [[3], [4], [5]].

High levels of inorganic phosphate, calcium, and glucose are factors that induce intracellular calcium deposition, which result in vascular smooth muscle cells (VSMCs) changing into osteoblast-like cells due to alterations in the smooth muscle cell (SMC) phenotype [6,7]. In this process, the expressions of multiple osteogenic pathway-associated markers, including alkaline phosphatase (ALP), bone morphogenic protein 2 (BMP2), osteocalcin, runt-related transcription factor-2 (RUNX2), and receptor activator of nuclear factor κ-B ligand (RANKL), are increased [8]. RANKL belongs to the tumor necrosis factor ligand superfamily, and it plays an important role in bone regeneration and remodeling [9]. In VSMCs, RANKL increases calcification in a dose-dependent manner by binding to its receptor, RANK, whereas osteoprotegerin (OPG), a decoy receptor for RANKL, inhibits RANKL-induced calcification by preventing osteoclast differentiation [10,11]. Recently, several studies demonstrated that an increase in plasma BMP2 [12] and ALP levels and the RANKL/OPG concentration ratio [13,14] is associated with vascular calcification in patients with type 2 diabetes mellitus.

AMP-activated protein kinase (AMPK), which plays a critical role in maintaining energy homeostasis, helps in the defense against diabetes, obesity, and non-alcoholic fatty liver disease (NAFLD) via regulation of glucose and lipid metabolism [15]. AMPK also contributes to the health and survival of cardiomyocytes [16]; AMPK activation by AICAR and metformin promotes bone formation and bone mass, leading to the amelioration of osteoporosis [17,18]. Previous studies suggest AMPK attenuates the osteoblastic differentiation of VSMCs [19].

Metformin, an anti-diabetic agent, ameliorates obesity, diabetes, and hepatic steatosis via increasing fatty acid oxidation and glucose uptake and reducing lipogenesis by AMPK activation [20,21]. Resveratrol, a polyphenol found in grapes, reduces oxidative stress and inflammation, and it helps to ameliorate diabetes or diabetes complications in humans and mice [22,23]. The protective effects of resveratrol in metabolic disorders are mediated by SIRT and AMPK [24], and resveratrol also inhibits vascular calcification by activating SIRT and NRF2 [25]. Exendin-4, a glucagon-like protein-1 receptor agonist (GLP-1RA), is used to treat diabetes, and it regulates appetite, induces weight loss, and increases insulin secretion and glucagon release [26]. GLP-1RAs have cardio-protective effects on patients with type 2 diabetes [27] and regulate lipid and glucose metabolism via AMPK [28].

In the present study, we investigated the protective effects of the AMPK activators metformin, resveratrol, and exendin-4 on Pi-induced vascular calcification and analyzed the functional role and molecular mechanisms of AMPK increased by metformin, resveratrol, and exendin-4 in rat VSMCs.

Section snippets

Cell culture and in vitro calcification

A-10 cells, rat vascular smooth muscle cells (RVSMCs), were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA) and maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (Gibco) and 1% penicillin-streptomycin (Gibco, Grand Island, NY, USA). To construct an in vitro model for vascular calcification, the A-10 cells were treated with inorganic phosphate (Pi) (4 mM; Sigma-Aldrich, St. Louis, MO, USA) for

Metformin, resveratrol, and exendin-4 reduce Pi-induced calcium deposition in RVSMCs

To examine the effects of phosphate on vascular calcification, A-10 cells were exposed to different concentrations of Pi. Calcification increased in the cells treated with Pi (4–8 mM) when compared with the control (Fig. 1A–C), and the expression of Pit-1, a phosphate transporter, was increased by Pi in a dose-dependent manner (Fig. 1E). However, Pi induced apoptosis at concentration of 8 mM (Fig. 1D). Therefore, these results suggest that high Pi concentrations induce calcification in VSMCs

Discussion

In this study, we demonstrated that metformin, resveratrol, and exendin-4, which are AMPK activators, improve vascular calcification in rat VSMCs treated with a high concentration of Pi and AMPK is a major regulator of the defense against vascular calcification as it reduces RANKL expression. Metformin, resveratrol, and exendin-4 inhibited calcium deposition and decreased the Rankl/Opg ratio and expressions of Bmp2, p-Smad1/5/8, and Rankl, which are involved in osteoblastic differentiation, in

Funding sources

This study was supported by the National Research Foundation (NRF), which is funded by the Korean government (NRF-2017R1A2B4011799) (http://www.nrf.re.kr), and was supported by the Medical Research Funds from Kangbuk Samsung Hospital. The funders had no role in the study design, data collection, and analysis, the decision to publish, or preparation of the manuscript.

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.

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    These authors contributed equally to the work reported; both should be considered as the corresponding authors.

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