SORT1 axis regulates vascular smooth muscle cell calcification in vitro and in vivo

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The miR-182/SORT1 axis regulates vascular smooth muscle cell calcification in vitro and in vivo Zhanman Zhang1, Wenhong Jiang1, Han Yang, Qiuning Lin, Xiao Qin

⁎

Department of Vascular Surgery, the First Affiliated Hospital of Guangxi Medical University, Nanning 530021, Guangxi Province, China

A R T I C L E I N F O

A B S T R A C T

Keywords: MiR-182 SORT1 Vascular smooth muscle cell Calcification

Arterial calcification is a common feature of cardiovascular disease. Sortilin is involved in the development of atherosclerosis, but the specific mechanism is unclear. In this study, we established calcification models in vivo and in vitro by using vitamin D3 and β-glycerophosphate, respectively. In vivo, the expression of SORT1 was upregulated and the expression of miR-182 was down-regulated in calcified arterial tissues. Meanwhile there was a negative correlation between SORT1 expression and miR-182 levels. In vitro, downregulating SORT1 expression using shRNA inhibited β-glycerophosphoric induced vascular smooth muscle cells (VSMCs) calcification. Moreover, reduced sortilin levels followed transfection of miR-182 mimics, whereas there was a significant increase in sortilin levels after transfection of miR-182 inhibitors. A luciferase reporter assay confirmed that SORT1 is the direct target of miR-182. Our study suggests that SORT1 plays a vital role in the development of arterial calcification and is regulated by miR-182.

1. Introduction Atherosclerosis, vascular calcification, and osteoporosis are among the most common diseases in the elderly population, associated with significant morbidity and mortality. Various histological [1], animal [2] and clinical [3] studies support the overlap of osteoporosis and vascular calcification, and vascular calcification is now seen as a process of emphasizing the height of bone formation. Thus, better molecular understanding of the common processes underlying these comorbidities may lead to more effective therapeutic strategies. Vascular calcification is an active cell regulatory process in which vascular smooth muscle cells (VSMCs) can lose the expression of marker genes such as α-smooth muscle actin, deposit mineralized bone matrix and gain osteogenic markers [4]. The central role of the dedifferentiation of VSMCs in generation of the osteoblast-like phenotype that promotes vascular calcification has been demonstrated. Recent genome-wide association studies (GWAS) found that possession of the 1p13 site of the SORT1 gene encoding the protein sortilin was associated with myocardial infarction [5], plasma low density lipoprotein cholesterol levels [6], aortic aneurysm [7] and coronary artery calcification [8]. Sortilin is a multi-ligand receptor rich in Golgi compartment which is a member of the Vps10p domain receptor family, characterized by a 10-bladed β-propeller that forms a cavity for binding

a soluble ligand, and a C-terminal cytoplasmic domain comprising an array motif that is responsible for the expression of the recipient subcellular tail [9]. Sortilin plays a variety of cellular roles, including intracellular protein sorting, such as PCSK9 acidic sphingomyelinase and apolipoprotein B100 (apoB100), and as a common receptor in the cell surface receptor conduction complex [10–12]. The complete removal of sortilin can reduce the secretion of very low density lipoprotein (VLDL) from the liver [11] and reduce atherosclerosis in mice [13]. Thus, the regulation of SORT1 expression is associated with the prevention and treatment of vascular calcification. However, few studies have reported the involvement of SORT1 in vascular calcification after transcriptional regulation. MicroRNAs (miRNAs) are small endogenous RNA molecules with a length of 22 nucleotides that anneal to an incomplete complementary sequence in the 3′ untranslated region (3′-UTR) of its target mRNA to mediate post-transcriptional gene expression [14]. It has been found that miRNAs play an important role in vascular biology and disease [15,16]. In particular, some miRNAs are considered to be novel biomarkers or potential drug targets for the prevention and treatment of vascular diseases such as atherosclerosis, thrombosis and restenosis [17]. Goettsch et al. Reported that miR-125b regulates vascular calcification and provides potential for new treatments for atherosclerosis using miRNA-based strategies [18]. However, the expression of miR-

Abbreviations: qRT-PCR, Quantitative real-time polymerase chain reaction; β-GP, β-glycerophosphoric; VSMCs, Vascular smooth muscle cells ⁎ Corresponding author. E-mail address: [email protected] (X. Qin). 1 These authors contribute to this work equally. https://doi.org/10.1016/j.yexcr.2017.11.033 Received 18 September 2017; Received in revised form 23 November 2017; Accepted 25 November 2017 0014-4827/ © 2017 Elsevier Inc. All rights reserved.

Please cite this article as: Zhang, Z., Experimental Cell Research (2017), https://doi.org/10.1016/j.yexcr.2017.11.033

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2.4. Cell culture and calcification determination

mediated SORT1 in arterial calcification requires further characterization. MiR-182 belongs to the miR-183 family consisting of miR-96, miR182 and miR-183. This cluster was initially identified as specifically expressed in the sensory organs [19]. However, recent studies have shown that miR-182 is carcinogenic in different types of human cancers, including cancers of the prostate, breast, bladder, liver, and ovary, as well as glioma, while other target genes such as RASA1 and RECK are inhibited to promote tumorigenesis [20–22]. Furthermore, Li et al. Reported that miR-182 is the target of endothelial cell and cardiomyocyte exchange in the activation of the Akt / mTORC1 pathway and induces hypertrophy of cardiovascular angiogenesis [23]. More importantly, miR-182 is a negative regulator of osteoblast proliferation, differentiation, and skeletogenesis in mesenchymal stem cells [24]. However, the mechanism by which miR-182 induces changes in atherosclerosis remains unclear. To investigate the role of miRNAs that modulate SORT1 resistance, we used rats and VSMCs. In this study, we report the identification of a key miRNA which acts by inhibiting SORT1 regulation of arterial calcification. This reveals the positive effect of miR-182 on the calcification process and demonstrates that SORT1 promotes vascular calcification in vitro and in vivo and identifies it as a potential therapeutic intervention.

VSMCs (A7r5; ATCC CRL-1444) (Aolu Biotech, Shanghai, China) were grown in Dulbecco's modified Eagle's medium (DMEM, Gibco BRL, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS, Gibco), 50 μg/mL streptomycin and 50 U/mL penicillin at 37 °C in 5% CO2. At confluence, the calcification of VSMCs was induced by culture in the presence of calcification medium containing 10 mM βglycerophosphoric (β-GP). for 7, 14 or 21 days, the calcification status was detected by staining with Alizarin red according to the protocol supplied with the Alizarin red staining kit (Sigma-Aldrich, USA). 2.5. Transfection miR-182 mimic, miR-182 inhibitor and the negative control were obtained from RiboBio (Guangzhou, China). miRNA transfection was performed using a CP transfection kit (RiboBio) according to the manufacturer’s instructions. After transfection for 48 h, total RNAs and protein were extracted and prepared for real-time reverse transcription–polymerase chain reaction (RT–qPCR) and western blotting analysis. 2.6. Lentiviral transduction

2. Materials and methods

When the HEK-293T cells were ~90% confluent, the cells were cotransfected with 0.5 μg of pRSV, 0.5 μg of pMDL, 0.5 μg pMD2. G–VSVG and 2.0 μg of a lentiviral construct encoding short hairpin RNA (shRNA; GeneCopoeia, Rockville, MD, USA) to produce lentiviral particles. Particles were harvested after 48 h transfection by 0.22 µm filtration of conditioned medium. Virus-containing filtrate was mixed with complete medium containing 10 μg/mL polybrene in a 1:1 ratio, added to cells for 24 h, and cultured with complete medium for 48 h. Cells were screened with 2 μg/mL puromycin for 3 days.

2.1. Animals and ethical statements All animal studies were performed according to the China National Institutes of Health guidelines for the care and use of laboratory animals with the approval of the Ethics Committee of Guangxi Medical University (Approval Number: 201511018). Specific antigen-free (SPF) male Sprague Dawley (SD) rats weighing approximately 180 g were provided by the experimental animal center of Guangxi Medical University, and housed in an SPF environment with food and water supplied.

2.7. RNA Isolation and RT-qPCR According to the manufacturer’s instructions (Invitrogen, Carlsbad, CA, USA), total RNA containing the miRs was isolated from frozen aorta tissues and cells using TRIzol®. First strand cDNA was reverse transcribed from total RNA using a first strand synthesis kit and 2 µL of the first strand cDNA was used for RT-qPCR (ABI 7500 Fast; Applied Biosystems, Foster City, CA, USA) using a FastStart SYBR Green Kit (Roche, Basel, Switzerland) to detect mRNA and miRNA expression. The specific PCR primers used were: rat SORT1 (forward, 5′-AGGCCTATGAACCATGCTGGAC-3′, reverse, 5′-CACTCTGGCGCTTGTTGGAA-3′); rat RUNX2 (forward, 5′-CATGGC CGGGAATGATGAG-3′, reverse, 5′-TGTGAAGACCGTTATGGTCAAA GTG-3′); rat β-actin (forward, 5′-GGAGATTACTGCCCTGGCTCCTA-3′, reverse, 5′-GACTCATCGTACTCCTGCTTGCTG-3′). The primers of U6 and miR-182 were purchased from GeneCopoeia. Expression of SORT1 and miR-182 were normalized to β-actin and U6, respectively, and the relative expression was calculated by the comparative CT (2-ΔΔCT) method.

2.2. Construction and sampling of the vascular calcification model A rat vascular calcification model was created as previously described with modifications [25]. In brief, rats received 300,000 IU/kg vitamin D3 once a day by subcutaneous injection for 7 consecutive days. For control rats, an equal volume of saline solution was administered by the same route over the same time period. At the end of the week rats were anesthetized and the tissues of the thoracic aorta were fixed in 4% paraformaldehyde for at least 24 h at room temperature. Histological analysis of mineral accumulation and histopathological staining (HE and von Kossa staining) were performed to determine the success of the rat thoracic aorta calcification model. Other aortic structures were immediately frozen in liquid nitrogen until the RNA and protein content were analyzed.

2.3. Von Kossa and HE staining

2.8. Western blot analysis

Paraffin blocks were cut into 8 mm aortic tissue sections and stained according to the protocol provided with a von Kossa staining kit (Jianchengbio, Nanjing, China). In brief, the slides were deparaffinized, hydrated with distilled water, and immersed in 5% silver nitrate solution for 1 h under intense sunlight. The stained sections were then washed three times with deionized water, and 5% thiosulphate solution was added for 10 min to remove un-reacted silver. Other sections were stained with hematoxylin and eosin (HE). Slides were observed using a light microscope, and black-stained areas were identified as calcium phosphate deposits.

For detection of sortilin and runt related transcription factor 2 (RUNX2) protein expression, proteins were isolated from the rat aorta and from cell extracts and western blotting was performed according to the Abcam western blot instructions. Total protein (50 µg) was electrophoresed on 8% polyacrylamide gels and transferred onto PVDF membranes (0.22 µm, Merck-Millipore, Darmstadt, Germany). After blocking with 5% fat-free milk solution, the antibodies used were antisortilin (ab16640; Abcam, Cambridge, MA, USA), anti-RUNX2 (ab37150; Abcam), anti-actin (KC-5A08, Kang Cheng Bio-tech, Shanghai, China).and a corresponding goat anti-rabbit peroxidase2

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linked secondary antibody (1: 10,000; EarthOx, Millbrae, CA, USA). Image-Pro Plus 6.0 software (Media Cybernetics Inc., Rockville, MD, USA) was used to quantitate the band densities.

the vascular calcification model was successfully induced in rats after injection with vitamin D3.

2.9. Luciferase reporter assay

3.2. SORT1 and miR-182 are involved in arterial calcification

Using the public database (TargetScan.www.targetScan.org) to predict miRNAs used to modulate rat SORT1.HEK293T cells were grown to 60% confluence in 24-well plates in high-glucose Dulbecco’s Modified Eagle’s Medium (Gibco, New York, USA) supplemented with 10% fetal bovine serum, 50 µg/mL streptomycin, and 50 U/mL penicillin. For luciferase the reporter assay, HEK293T cells were transfected with wild-type SORT1 3′-UTR or mutant SORT1 3′-UTR and miR-182 mimics or their corresponding negative controls. Cells were collected 48 h after transfection and assayed with the Luciferase Reporter Assay System (Promega Corporation, Madison, Wisconsin, USA). The tests were repeated in triplicate.

Expression of the osteogenic differentiation marker gene Runx2 was significantly upregulated at both the mRNA (Fig. 2B) and the protein level (Fig. 2C). SORT1 and miR-182 are associated with calcitriol-induced vascular calcification in rats. Compared with the control group, the expression of SORT1 mRNA and protein in the vitamin D3 treatment group was up-regulated (P = 0.019 and 0.005, respectively, Fig. 2D). Moreover, miR-182 was significantly decreased (P = 0.47) in calcified rats. We also investigated whether SORT1 expression is associated with miR-182 levels, as this may be associated with post-transcriptional regulation of target gene expression. Correlation analysis showed that SORT1 mRNA and sortilin level was negatively correlated with miR182 expression (r = −0.510 and −0.508, respectively; P = 0.044 and 0.044, respectively), as shown in Fig. 2. These results suggest that miR182 may participate in vascular calcification by mediating SORT1 expression.

2.10. Statistical analyses All experiments were performed with three replicates in duplicate, and the results are expressed as the mean ± SD. Comparisons were made using a two-sample t-test or one-way ANOVA. The relationship between miR-182, SORT1 mRNA and sortilin was determined using linear least squares regression analyses. The results are reported graphically, with a correlation constant and a probability value. All analyses were performed using SPSS statistical software for Windows, version 20.0 (SPSS, IBM Corp, Armonk, NY, USA). A value of P < 0.05 was considered significant.

3.3. β-GP induced SORT1 expression and inhibited the expression of miR182 during calcification of VSMCs in a time-dependent manner Matrix mineralization is a marker of osteoblast differentiation and was evaluated using Alizarin Red S staining, which showed that β-GP increased mineralized nodule formation in VSMCs at day 14 after treatment compared with VSMCs without β-GP treatment (Fig. 3A). We also found upregulation of the osteoblastic marker RUNX2, at both mRNA and protein levels, in VSMCs 14 and 21 days after treatment (Fig. 3C and D, respectively). These results suggest the successful establishment of calcification of VSMCs. RT-qPCR and western blot analysis showed that SORT1 mRNA and sortilin expression were both significantly upregulated in VSMCs after 14 days of exposure to β-GP and then gradually increased over a period of 21 days (Fig. 3E and F, respectively). However, the relative expression of miR-182 was reduced in β-GP-induced calcified VSMCs (Fig. 3G).

3. Results 3.1. Construction of a vascular calcification(VC) rat model As shown in Fig. 1, evaluation of VC by pathological assay (von Kossa staining and HE staining) showed that, in the experimental group, there was mineral deposition, elastic fibers, and extracellular matrix degradation in the aorta media, uneven rupture of the intima, and the muscular rupture calcium spots were dramatically increased in the VC group compared to the control group. The results suggested that

Fig. 1. Rat aortic sections were stained with von Kossa and hematoxylin and eosin (HE). Von Kossa staining of aortic sections in the control group (A) and vitamin D3-treated group (C). HE staining of aortic sections in the control group (B) and vitamin D3treated group (D).

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Fig. 2. Analyses of RUNX2, SORT1 and miR182 expression in the vitamin D3-treated group and control group. (A) Western blot analyses of sortilin and RUNX2 protein levels in rat aortas from the control group (C1, C2, C3) and the vitamin D3-treated group (M1, M2, M3). (B) Scatter diagram showing the expression of SORT1 and miR-182. SORT1 expression was significantly increased and miR-182 was significantly downregulated in the vitamin D3-treated group. The expression of RUNX2 mRNA (C) and protein (D). Correlation analyses to determine the relationships between miR-182 and SORT1 mRNA levels (E) and sortilin (F) in the control and vitamin D3-treated groups (SORT1 mRNA versus miR-182, r = −0.510, P = 0.044; sortilin versus miR-182, r = −0.508, P = 0.044).

3.5. Attenuation of SORT1 expression by miR-182

3.4. Knockdown of SORT1 attenuated RUNX2 to inhibit VSMC calcification

To investigate the effect of miR-182 on sortilin levels, we transfected mouse VSMCs with miR-182 mimics or inhibitors and their negative control, and used bioinformatics and a miRNA target prediction database to determine whether SORT1 was the target gene for miR-182. In VSMCs overexpressing miR-182, sortilin levels were significantly inhibited, whereas in miR-182 inhibited cells, sortilin levels were upregulated (P = 0.038 and 0.047, Fig. 5C). In contrast, transfection of miR-182 oligos had no significant effect on SORT1 mRNA expression (Fig. 5B), indicating that miR-182 attenuated the translation of SORT1 proteins in VSMCs.

To determine SORT1 involvement in vascular calcification, shRNASORT1 and its negative control were transfected into VSMCs by lentiviral transfection. SORT1 shRNA transfection significantly inhibited βGP-induced nodule formation, and analysis of mineralization using Alizarin Red S showed that SORT1 shRNA transfection also significantly inhibited β-GP-induced mineralization (Fig. 2A). Runx2 is a common marker used to identify calcification of VSMCs. qRT-PCR and western blot analysis showed that silencing of SORT1 expression significantly decreased Runx2 mRNA and protein expression in VSMCs treated with or without 10 mM β-GP for 21 days (P = 0.030 and 0.001, respectively, Fig. 4E and F). These results suggested that SORT1 might function as a modulator of VSMC calcification.

3.6. miR-182 directly regulates SORT1 expression Bioinformatics prediction showed that the seven bases of rno-miR182 in the seed region are complementary to the SORT1 3′-UTR base at positions 1497–1503. The luciferase assay was performed to determine 4

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Fig. 3. Calcification of VSMCs induced by treatment with β-glycerophosphate (β-GP) for 7–21 days. Compared with control medium, 10 mM β-GP led to calcium deposition, revealed by Alizarin Red S staining (A). A representative western blot of sortilin and RUNX2 protein expression with or without β-GP treatment (B). Detection of miR-182 expression with U6 as the loading control (C). The expression of RUNX2 (D) and SORT1 (F) mRNA levels. The expression of RUNX2 protein (E) and sortilin (G).

4. Discussion

that miR-182 was used as a mechanism to regulate SORT1 expression directly with SORT1 3′-UTR, Overexpression of miR-182 in wild-type 3′-UTR of the SORT1 group significantly inhibited relative luciferase activity compared to the SORT1 3′-UTR negative control group and the SORT1 3′-UTR mutant group (P < 0.001). These results indicated that miR-182 bind to the 3′-UTR of SORT1 to inhibit translation (Fig. 6).

It is currently believed that the phenotypic diversity of VSMCs depends on natural genetic variation and the effects of the environment and that vascular calcification is an active regulatory process rather than passive calcium deposition and degradation. Although various molecular mechanisms have been reported to be involved in the process of vascular calcification, including phosphate metabolism or abnormal 5

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Fig. 4. SORT1 knockdown suppresses calcification of VSMCs induced by β-GP for 21 days. (A) Representative Alizarin red S staining of VSMCs treated with β-GP. (B) A representative western blot of sortilin and RUNX2 protein levels in VSMCs treated with β-GP and transfected with negative control shRNA (shRNA-NC) or untransfected control (NC) or SORT1 shRNA (shRNA-SORT1). SORT1 shRNA strikingly decreased RUNX2 mRNA (C) and protein (D) expression. SORT1 shRNA significantly decreased SORT1 mRNA (E) and sortilin (F) expression in VSMCs.

calcium levels, apoptosis, osteogenesis of VSMCs, oxidative stress, inflammation and loss of mineralization inhibitors, knowledge of related mechanisms is not comprehensive [26]. In addition, no effective prevention or treatment of vascular calcification has yet been developed. In this study, we found that the expression of SORT1 was up-regulated and the expression of miR-182 was down-regulated compared with the calcineuria-induced vascular calcification rat model, and the two were negatively correlated. Furthermore, there was a negative correlation between miR-182 expression and SORT1 expression. GWAS revealed correlation of SORT1 site variation with cardiovascular calcification [8]. However, it remains unclear how single nucleotide polymorphisms are associated with the level of sortilin in calcified vasculature. In rat VSMCs, overexpression or inhibition of miR-182 resulted in downregulation or upregulation of sortilin, respectively. Overall, the results showed that miR-182 acts as a negative regulator of vascular calcification by repressing SORT1 expression. Previously, the strong association between LDL-C levels and SNP clusters located in chromosome 1 (1p13.3) has attracted wide attention. There are four genes in the region, CELSR2, PSRC1, MYBPHL and SORT1. SORT1 encodes sortilin, which is associated with coronary artery disease, especially atherosclerosis [8]. Therefore, this study may provide biological and molecular explanations for the observed link. Sortilin is closely related to arterial disease. Jones et al. [7] reported that SORT1 is an independent predictor of human abdominal aortic aneurysms. The relationship between SORT1 and abdominal aortic aneurysm is very strong, given the mixed conditions of heart disease,

Fig. 5. Regulation of sortilin levels by miR-182 in VSMCs. VSMCs were transfected with control miR mimics or miR-182 mimics, control miR inhibitors or miR-182 inhibitors. A representative western blot of sortilin in VSMCs (A), and subjected to RT–qPCR analysis of SORT1 mRNA (B), and harvested for the examination of sortilin by western blot analysis (C).

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sortilin binds, sorts and degrades the inducing molecules, including calcification inhibitors, to promote vascular calcification. Recently, emerging evidence suggests that miRNAs play a role in atherosclerotic plaque formation, including inflammation, [36] apoptosis, [37] and angiogenesis [38]. The role of miRNA in medial arterial calcification has not been studied. We focus on the effects of miRNAs on VSMC calcification, which is critical for a variety of cardiovascular lesions. To understand the molecular mechanism that underlies the effects of SORT1 on vascular calcification, we searched for potential targets of SORT1 using the miRNA target prediction algorithms, Targetscan. Interestingly, in the search for a predicted target, we identified miR-182, one member of the miRNA-183 family, which plays a key role in osteogenesis-related proliferation and differentiation. In our experiment, there was considerable evidence to support SORT1 as the most important target for miR-182 in VSMCs. First, miR-182 has been implicated in a variety of cancers including breast cancer [39], hepatocellular cancer [21] and osteosarcoma [40]. It is also reported that miR182 is a negative regulator of osteoblast proliferation, differentiation, and skeletogenesis through targeting FoxO1 [24], a member of the family of forkhead box O (FOXO) proteins, involved in development of atherosclerotic vascular calcification as demonstrated previously; secondly, our finding that miR-182 was downregulated in vascular calcification was consistent with the results of Dong et al. [41], who established that miR-182 effectively suppressed the proliferation and migration of rat VSMCs under both quiescent conditions and PDGF-BB stimulation and prevents VSMCs phenotypic modulation via FGF9/ PDGFRβ signaling. Thirdly, miR-182 binds to SORT1 3′-UTR-binding sites by base pairing in the miR-182 seed region, indicating that it might directly regulate SORT1 expression; In our study, the overexpression of miR-182 significantly inhibited the luciferase activity of the WT-3′-UTR of the SORT1 reporter gene but did not show the luciferase activity of the MUT- SORT1-3′-UTR reporter gene; finally, miR-182 overexpression or inhibition decreased or elevated sortilin levels, but the mRNA expression level was not significant, indicating post-transcriptional modulation. In short, these clues support the theory that miR-182 acts through down-regulating its target SORT1 to inhibit VSMC calcification. However, the possibility that miR-182 may also target other genes cannot be completely ruled out because miRNAs typically target several related genes to regulate specific pathways. This study has some limitations. Vascular calcification is one of the common symptoms of a variety of chronic inflammatory diseases. In view of the fact that the pathogenesis of each disease is unique, different diseases are likely to trigger vascular calcification through different mechanisms. In the present study, vitamin D3 and β-glycerophosphate were used to establish calcification models in vivo and in vitro. Therefore, whether the model is suitable for a variety of diseases, vascular calcification remains to be further defined. Our results show that sortilin is involved in arterial calcification and is negatively correlated with miR-182, but there is currently no reported direct pathway associated with sortilin-mediated arterial calcification. In addition, miR-182 is required for further study of in vivo and in vitro vascular calcification. In a prospective study, we planned to overexpress and knock-down the lentivirus miR-182 in VSMCs and then test whether miR-182 plays a role in the underlying mechanisms of vascular calcification and detects sortilin signaling pathways involved in vascular calcification.

Fig. 6. Target verification between miR-182 and SORT1 in HEK293T cells (A) Schematic of the miR-182 putative target site in SORT1 3′-UTR and alignment of miR-182 with WT and MUT SORT1 3′-UTR showing complementary pairing. (B) HEK293T cells were cotransfected with the luciferase reporter carrying WT- SORT1-3′-UTR or MUT SORT1-3′UTR, psiCHECK™−2, and miR-182 mimic or negative control. Data was expressed as the mean ± SD, and each experiment was repeated three time.

concurrent dyslipidemia and other risk factors [6]. Kjolby et al. described a 60% reduction in plaque area in LDLR-deficient mice after systemic exclusion of sortilin [10], while Ai et al. have shown that nonvascular sortilin expression is inhibited in the high-fat diet model of adipose-dependent obesity [27]. The present study shows that sortilin has the potential to promote calcification in vivo and validated the relevance of a regulatory mechanism in vitro. Our findings demonstrated high sortilin expression in calcification animal models, induced by a toxic dose of vitamin D3. RUNX2 is an essential transcription factor for osteoblastic differentiation, and is associated with high sortilin expression. Our data are in line with the evidence associated with increased sortilin levels in calcified human atheroma and in calcified arteries from patients and experimental animals with chronic renal disease via its recruitment into extracellular vesicles [28]. Aortic calcium accumulation and osteogenic transcription factors and proteins decreased when SORT1 expression and activity were inhibited. Similarly, a previous study revealed that sortilin regulates the inflammatory status of macrophages, attenuating the inflammatory response and promoting atherosclerotic lesion formation [29]. In addition, sortilin associated mineralization pathology is specific to the vasculature, with less arterial calcification in Sortilin-deficient mice, while its bone cell function and skeletal structure remain unchanged [28]. Furthermore, sortilin has been found to bind TGF-β family precursor proteins and promote their trafficking to the lysosome for degradation [30]. TGF-β plays a crucial role in bone matrix production in a high phosphate environment. Phosphate, acting through the stimulation of osteogenesis/cartilage formation and differentiation, matrix vesicle release, apoptosis, inhibitor loss and extracellular matrix degradation, has a direct impact on VSMC calcification [26]. Sortilin is considered to be a key component in the control of neuronal survival of the signal complex [31]. The apoptotic body provides a binding site for minerals involved in the first step of vascular calcification [32]. Luisa et al. Showed that sortilin expression characterizes human atherosclerotic lesions and rat aortic endothelium injury after neointima development and indicates that sortilin represents proNGF-induced SMC apoptosis and is an important regulator of arterial remodeling [33]. Evidence suggests that sortilin helps to select the target protein in the secretory and / or endosomal pathway. Sortilin binds to lipoprotein lipase and mediates endocytosis [34], thereby promoting osteogenesis of mesenchymal stem cells and inhibiting adipogenesis [35]. Thus, it is reasonable to suggest that

5. Conclusion This study revealed a previously unknown function of sortilin in vascular calcification. The expression of SORT1 is closely related to vascular calcification and miR-182 was decreased in a vascular calcification model in rats, which consequently increased the expression of its target SORT1. The mechanism revealed by this study has identified sortilin as a therapeutic target for the prevention or treatment of calcification. 7

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Acknowledgments [18]

This work was supported by The National Natural Science Foundation of China No: 81160047).

[19]

Author contributions [20]

Zhanman Zhang, Wenhong Jiang and Xiao Qin conceived and designed the study. Zhanman Zhang, Wenhong Jiang performed the experiments. Zhanman Zhang analyzed the data and wrote the paper. Zhanman Zhang, Wenhong Jiang, Han Yang, Qiuning Lin, and Xiao Qin reviewed and edited the manuscript. All authors read and approved the manuscript.

[21]

[22]

Conflicts of interest

[23]

The authors declare no conflict of interest. [24]

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