Ets1 axis regulates transdifferentiation and calcification of vascular smooth muscle cells in a high-phosphate environment

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miR-125b/Ets1 axis regulates transdifferentiation and calcification of vascular smooth muscle cells in a high-phosphate environment Ping Wen, Hongdi Cao, Li Fang, Hong Ye, Yang zhou, Lei Jiang, Weifang Su, Hongying Xu, Weichun He, Chunsun Dai, Junwei Yang www.elsevier.com/locate/yexcr

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S0014-4827(14)00045-7 http://dx.doi.org/10.1016/j.yexcr.2014.01.025 YEXCR9542

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Experimental Cell Research

Received date: 28 August 2013 Revised date: 24 December 2013 Accepted date: 22 January 2014 Cite this article as: Ping Wen, Hongdi Cao, Li Fang, Hong Ye, Yang zhou, Lei Jiang, Weifang Su, Hongying Xu, Weichun He, Chunsun Dai, Junwei Yang, miR125b/Ets1 axis regulates transdifferentiation and calcification of vascular smooth muscle cells in a high-phosphate environment, Experimental Cell Research, http://dx.doi.org/10.1016/j.yexcr.2014.01.025 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

miR-125b/Ets1 axis regulates transdifferentiation and calcification of vascular smooth muscle cells in a high-phosphate environment

Ping Wen, Hongdi Cao, Li Fang, Hong Ye, Yang zhou, Lei Jiang, Weifang Su, Hongying Xu, Weichun He, Chunsun Dai, Junwei Yang*

Center for Kidney Disease, 2nd Affiliated Hospital, Nanjing Medical University, 262 Zhongshan North Road, Nanjing, Jiangsu 210003, China

Running title: miR125b/Ets1 axis in VSMCs

* To whom correspondence should be addressed: Junwei Yang, M.D/Ph.D, Center for Kidney Disease, 2nd Affiliated Hospital, Nanjing Medical University, 262 Zhongshan North Road, Nanjing, Jiangsu 210003, China Phone (Office): 8625-83345110, Email: [email protected].

Key words: miR-125b, VSMCs, calcification Subject codes: none. Abbreviations: VSMCs——vascular smooth muscle cells; β-GP——β-glycerophosphoric acid. Word count: 3009 (except material and methods). Number of figures: 5; Number of tables: 0.

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Abstract Objectives: Vascular calcification is highly prevalent in patients with chronic kidney disease (CKD) and contributes to increased risk of cardiovascular disease and mortality. Accumulated evidences suggested that vascular smooth muscle cells (VSMCs) to osteoblast-like cells transdifferentiation (VOT) plays a crucial role in promoting vascular calcification. MicroRNAs (miRNAs) are a novel class of small RNAs that negatively regulate gene expression via repression of the target mRNAs. In the present work, we sought to determine the role of miRNAs in VSMCs phenotypic transition and calcification induced by β-glycerophosphoric acid. Approach and results: Primary cultured rat aortic VSMCs were treated with β-glycerophosphoric acid for different periods of time. In VSMCs, after β-glycerophosphoric acid treatment, the expressions of cbfα1, osteocalcin and osteopontin were significantly increased and SM-22α expression was decreased. ALP activity was induced by β-glycerophosphoric acid in a time or dose dependent manner. Calcium deposition was detected in VSMCs incubated with calcification media; Then, miR-125b expression was detected by real-time RT PCR. miR-125b expression was significantly decreased in VSMCs after incubated with β-glycerophosphoric acid. Overexpression of miR-125b could inhibit β-glycerophosphoric acid-induced osteogenic markers expression and calcification of VSMCs whereas knockdown of miR-125b promoted the phenotypic transition of VSMCs and calcification. Moreover, miR-125b targeted Ets1 and regulated its protein expression in VSMCs. Downregulating Ets1 expression by its siRNA inhibited β-glycerophosphoric acid-induced the VSMCs phenotypic transition and calcification. Conclusion: Our study suggests that down-regulation of miR-125b after β-glycerophosphoric acid treatment facilitates VSMCs transdifferentiation and calcification through targeting Ets1. Abbreviations: VSMCs——vascular smooth muscle cells; β-GP——β-glycerophosphoric acid.

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Introduction Vascular calcification is highly prevalent in patients with chronic kidney disease (CKD) and contributes to increased risk of cardiovascular disease and mortality[1,2]. Medial artery calcification is more prevalent in CKD and ESRD (end stage renal disease) patients, and this phenotype is also known as Monckeberg’s medial sclerosis[3,4]. Vascular smooth muscle cells (VSMCs) are the predominant cells in the tunica media of arteries. They play a critical role in regulating the blood vessel tone, which in turn influences blood pressure. VSMCs are of mesenchymal origin and under stress could differentiate to different mesenchymal-derived cell types such as osteoblasts, chondrocytes, and adipocytes, leading to calcification, altered matrix production, and lipid accumulation in the vessel wall[4,5]. In response to high phosphate stimulus, VSMCs increase expression of osteogenic genes such as cbfα1, osteopontin, and osteocalcin, meanwhile decrease expression of VSMCs differentiation marker genes including smooth muscle α-actin (α-SMA), SM-22α and calponin. Therefore, instead of a passive process involving spontaneous calcium phosphate precipitation in necrotic tissue, calcification is an active regulated process of osteoblastic differentiation of VSMCs. However, the specific pathways governing this active process is still unknown. MicroRNAs are a novel class of small RNAs that negatively regulate gene expression via repression of the target mRNAs[6,7]. Numerous miRNAs have been identified or predicted; however, their cellular targets, biologic roles and disease relevance are still under investigation. In VSMCs, key miRNAs such as miR-21, miR-143, miR-145, and miR-221 have recently been shown to play important roles in phenotypic changes, migration, proliferation, and neointimal thickening[8-11]. Besides, miR-125b has been described as a marker in vascular calcification[12], and has been demonstrated as inhibitors of osteoblastic differentiation[13]. Another study demonstrated a novel upstream role for miR-125b in the epigenetic regulation of inflammatory genes in VSMC of db/db mice[14]. In the present study, we sought to determine whether microRNAs (miRNAs) play a role during the process of VSMCs phenotypic transition and calcification induced by β-glycerophosphoric acid (β-GP). β-GP as a donor of organic phosphate has been confirmed to induce VSMCs transdifferentiate into osteoblasts via several mechanisms [5,15,16]. Ets1, as the first member of ets family, is a evolutionarily related, DNA-binding transcriptional factor, and is involved in regulating a wide variety of biological processes, including cellular growth, migration and differentication[17,18]. During the process of bone formation, Ets1 is expressed in proliferating preosteoblastic cells and mainly promotes osteoblasts proliferation, differentiation and mineralization. Previous studies have shown that Ets1 plays critical role in mediating vascular inflammation and remodeling[19,20]. However, the role of Ets1 on VSMCs phenotypic transition has not been elucidated in details. Yan Zhang etal reported that Ets1 was a novel direct target of miR-125b and explored the role of miR-125b/Ets1 axis in breast cancer[21]. However, the contribution of miR-125b/Ets1 axis to vascular calcification has not been explored. Evidences from our studies suggest that β-GP induces osteoblastic transition of VSMCs, but down-regulates miR-125b expression in VSMCs. Ectopic expression of miR-125b inhibits VSMCs transdifferentiation and calcification induced by high phosphorus. Manipulating the expression of miR-125b changes the expression of Ets1, a reported target, in vitro. Our studies identify miR-125b as a pivotal mediator for regulating VSMCs calcification. 3

Material and Methods Cell culture and Treatment Rat aortic SMCs were cultured as described previously[22,23]. Cell cultures were maintained in DMEM/F12 containing 20% fetal bovine serum (FBS), 100U/ml penicillin, 100mg/ml streptomycin and neomycin. Cells were grown to confluence and used from passages 3-8. For high phosphorus treatment, cells were incubated with 10mmol/L β-glycerophosphoric acid (Sigma-Aldrich), after different time of treatment, cells were harvested for real-time RT-PCR or Western blotting. MiR-125b mimics, inhibitors and Ets1 siRNA was transient transfected using Lipofectamine 2000 according to the instructions by the manufacturer (Invitrogen). Calcification assay Rat aortic SMCs calcification was induced as previously described[24]. Briefly, cells (6-well format) were incubated for 6-10 days with high-glucose DMEM, supplemented with 15% FBS, 10mM β-glycerophosphoric acid, 50mg/ml ascorbic acid, 10-7 mol/L insulin, 10 mmol/L sodium pyruvate and 100U/ml penicillin, 100mg/ml streptomycin and neomycin (calcification media). Von kossa and Alizarin Red S staining For von kossa staining, radial arteries were embedded in paraffin blocks. The blocks were then sectioned and stained with 5% silver nitrate solution in a clear glass coplin jar placed under ultraviolet light for 1 hour. Remove un-reacted silver with 5% sodium thiosulfate for 5 min. Counterstaining was performed in nuclear fast red for 2 min. For Alizarin Red S staining, cells were incubated with 10% neutral buffered formalin for 10 min, then incubated with a 2% aqueous Alizarin red solution (pH 4.2) for 10-15 min. Quantification of calcium deposition Cells grown on 10mm dishes were washed twice with phosphate buffered saline and decalcified with 0.6 mol/l HCl for 24 h. Calcium content of the supernatants was determined by the QuantiChrome Calcium Assay Kit (Bioassay Systems, DICA-500). After decalcification, cells were solubilized with a solution of 0.1 mol/l NaOH and 0.1% sodium dodecyl sulfate, and protein content of the samples were measured with the BCA protein assay kit (Thermo Scientific, Rockford, IL). Calcium content of the cells was normalized to protein content and expressed as μg/mg protein. Real-time RT-PCR Total RNA was used for real-time RT-PCR. Primer sequences for cbfα1, collagen I, osteopontin and Ets1 were as follows: cbfα1-F, 5’- TGTGTGCCTCCAACCTGTGT-3’; cbfα1-R, 5’CTTTCCCCCTCAATTTGTGTCA-3’; collagen I-F, 5’CTGTTCTGTTCCTTGTGTAACTGTGTT-3’; collagen I-R, 5’- GCCCCGGTGACACATCAA-3’; osteopontin-F, 5’GAGGAGGCAGAGCACAGCAT-3’; osteopontin-R, 5’TTGGCTGAGAAGGCTGCAA-3’; Ets1-F, 5’-GGGTGGGAGGAAAACAGTTT-3’; Ets1-R, 5’-CCTTCCATTAAAGTCCATCTTTGTT-3’, GAPDH-F, 5’CAGCAAGGATACTGAGAGCAAGAG-3’; GAPDH-R, 5’GGATGGAATTGTGAGGGAGATG-3’. Assays to quantify the mature miRNAs were conducted as previously decribed[25]. All the primers were acquired from Qiagen. 4

Western blotting Western blotting was performed as described previously[25]. Antibodies used were as follows: cbfα1 (Abcam), osteocalcin (Santa cruz), osteopontin (Santa cruz), SM-22α (Abcam), Ets1 (Santa cruz), GAPDH (Santa cruz). Immunofluorescence staining Indirect immunofluorescence staining was performed using an established protocol[26]. Briefly, cells cultured on coverslips were fixed with cold methanol: acetone (1:1) for 10 minutes at -20°C and blocked with 2% bovine serum albumin in PBS for 30 minutes. Cells were then incubated with the specific primary antibodies against Ets1. Sections were washed in PBS extensively before incubated for 1 hour with affinity-purified secondary antibodies (Santa cruz) at a dilution of 1:100 in PBS containing 1% BSA. As a negative control, the primary antibody was substituted with nonimmune IgG. Random samples were selected and double stained with DAPI (4’,6’-diamidino-2- phenylindole, HCL) to visualize the nuclei. Slides were viewed and photographed with an Eclipse 80i epifluorescence microscope equipped with a digital camera (Nikon). Alkaline phosphatase (ALP) activity assay ALP activity assay was performed as previously described[27]. Rat aortic SMCs were cultured in 96 well plate for 24 hours and then treated with different dose of β-glycerophosphoric acid for 72 hours. After treatment, cells were washed with PBS for three times and lysed with 50 μL ALP lysis buffer (150 mM NaCl, 3mM NaHCO3, 0.2% Triton X-100) for 30 min at 37°C. Pre-chilled p-nitrophenyl phosphate substrate (Sigma-Aldrich) 100 μL/well was added. After incubating at 37°C for 5-10 minutes, absorbance was measured at 405 nm. Boyden chamber for motility assay Cell motility and migration were evaluated using a Boyden chamber motogenicity assay with tissue culture-treated Transwell filters (Costar). Rat aortic smooth muscle cells (1 × 104) were seeded onto the filters (8-μm pore size, 0.33-cm2 growth area) in the top compartment of the chamber. After 24 hours of incubation with or without β-GP plus Ets1 siRNA in the lower compartment at 37°C, filters were fixed with 3% paraformaldehyde in PBS and stained with 0.1% Coomassie blue in 10% methanol and 10% actic acid, and the upper surface of the filters was carefully wiped with a cotton-tipped applicator. Cells that passed the Transwell filter pores toward the lower surface of the filters were counted in 5 nonoverlapping ×10 fields and photographed with a Nikon microscope. The experiments were performed in triplicate.

Statistical analysis Data collected were expressed as mean±SEM. Western blot and immunofluorescence staining were performed at least three times independently. Western blot analysis was completed by scanning and analyzing the intensity of hybridization signals using the NIH Image program. Statistical analyses of data were performed using Sigma Stat software (Jandel Scientific Software). Comparison between groups was made using one-way ANOVA, 5

followed by the t test. P<0.05 was considered significant. Results High phosphorus induces VSMCs transdifferentiation to osteoblast-like cells. The initial aim of the present studies was to determine whether our cultured rat aortic SMCs had an ability to convert into osteogenic cells under high phosphate concentration, as described previously by other laboratories[4,5,15,28-31]. We first examined the effect of phosphorus on osteogenic gene expression by incubating rat aortic SMCs with β-glycerophosphoric acid (10.0mmol/L) for 72 hours. As shown in Figure 1a, β-glycerophosphoric acid induced cbfα1, collagen I and osteopontin expression examined by real-time RT-PCR. Incubation of VSMCs with β-glycerophosphoric acid for 6 days increased osteocalcin and osteopontin protein expression while decreased SM-22α expression (Figure 1b). ALP activity was also detected by spectrophotometry and it was found that β-glycerophosphoric acid increased ALP activity in a dose or time dependent manner (Figure 1c). In addition, high phosphorus induced calcium deposition in VSMCs revealed by Alizarin Red S staining (Figure 1d). These results indicate that incubation with high phosphorus concentration decreased expression of VSMCs differentiation marker gene, whereas it increased expression of osteogenic genes and thereby induced calcification in our cultured VSMCs. MiR-125b inhibits VSMCs transdifferentiation and calcification induced by high phosphorus. To determine if miR-125b is associated with VSMCs transdifferentiation, total RNA was extracted for real-time RT-PCR analysis. The results showed that expression of miR-125b was significantly decreased in VSMCs after incubated with β-glycerophosphoric acid (Figure 2a). To investigate miR-125b’s effect on VSMCs transdifferentiation, miR-125b mimics or inhibitors and their controls were transfected to rat aortic SMCs respectively. Real-time RT-PCR results demonstrated significantly increased miR-125b level in VSMCs transfected with miR-125b mimics and decreased miR-125b level with miR-125b inhibitors (Figure 2b). Overexpression of miR-125b significantly inhibited osteocalcin and osteopontin protein expression induced by β-GP while knockdown of miR-125b expression was sufficient to promote their expression in VSMCs (Figure 2c). Ectopic expression of miR-125b restored SM-22α protein expression inhibited by β-GP whereas lack of miR-125b resulted in suppression of SM-22α in VSMCs (Figure 2d). Afterwards, the effect of miR-125b on ALP activity in VSMCs was explored. As shown in Figure 2e, abundant miR-125b attenuated ALP overactivity induced by β-GP but absence of miR-125b was adequate to induce ALP activity in VSMCs. Overexpression of miR-125b inhibited calcium deposition stimulated by calcification media whereas knockdown of miR-125b expression aggravated calcium deposition in VSMCs (Figure 2f). The calcium contents were measured by a quantitative method that showed similar trend with the staining result (Figure 2g). Taken together, our results suggest that miR-125b could play a key role in regulating phenotypic switching of VSMCs into osteogenic cells under high phosphorus conditions. Ets1,

a

target

gene

of

miR-125b,

is 6

induced

in

VSMCs

treated

with

β-glycerophosphoric acid. Since Target Scan indicates that V-ets erythroblastosis virus E26 oncogene homolog 1 (Ets1) is a target gene of miR-125b and it was confirmed by luciferase report in human invasive breast cancer[21]. Therefore, the expression of Ets1 was detected in VSMCs treated with β-glycerophosphoric acid. After rat aortic SMCs were treated with β-glycerophosphoric acid, both the mRNA and protein levels were increased in a dose or time dependent manner (Figure 3a, b, c, d). Immunofluorescence staining also revealed that Ets1 expression was remarkably induced and predominantly localized in the cytosol of VSMCs treated with either β-glycerophosphoric acid or inorganic phosphorus (Figure 3e). To investigate the effect of miR-125b on expression of Ets1, miR-125b mimics or inhibitors and their controls were transfected in VSMCs, respectively. As shown in Figure 3f, mRNA expression of Ets1 was not regulated by miR-125b while overexpression of miR-125b essentially abolished β-GP-induced Ets1 protein expression. Conversely, knockdown of miR-125b markedly induced Ets1 protein expression, which mimicking the effect of β-GP on up-regulating Ets1 protein expression in VSMCs (Figure 3g). Accordingly, Ets1 was induced in VSMCs under a high-phosphate condition, which was directly regulated by downregulation of miR-125b. Knockdown of Ets1 blocks VSMCs phenotypic switching into osteogenic cells induced by high phosphorus. Previous studies showed that Ets1 negatively regulated transcription of multiple smooth muscle cell differentiation marker genes[32]. We examined whether Ets1 acted to repress VSMCs differentiation marker gene by transfected Ets1 siRNA. Immunoblotting showed that the protein level of Ets1 was significantly down-regulated in cells transfected with Ets1 siRNA (Figure 4a). In addition, knockdown of Ets1 restored SM-22α protein level inhibited by β-GP (Figure 4a). Of interest, ALP overactivity and calcium deposition induced by β-GP were suppressed by down-regulation of Ets1 in VSMCs (Figure 4b, c and d). In the process of transdifferentiation, the synthetic phenotype is concomitant with accelerated migration of VSMCs[33,34], which is consistent with our result. Furthermore, the migration of VSMCs induced by β-glycerophosphoric acid was inhibited by knockdown of Ets1 (Figure 4e). These results indicated that Ets1 could play a crucial role in the pathogenesis of VSMCs transdifferentiaion and calcification. Expression of miR-125b is decreased in radial arteries of uremia patients with vascular calcification. Radial arteries tissues were obtained from hemodialysis patients in the artery-venous fistula surgery. Von kossa staining was used to assess the calcification of radial arteries. Among the 10 arteries samples, calcium deposition was observed in 5 arteries (Figure 5a). Total RNA was extracted from these arteries tissue and real-time RT-PCR was used to quantify miR-125b levels in the arteries. As presented in Figure 5b, miR-125b levels were significantly decreased in arteries with calcification compared with those without calcification. These results confirmed that miR-125b is down-regulated in VSMCs during calcification process both in vivo and in vitro.

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Discussion Accumulated evidences suggested that VSMCs to osteoblast-like cells transdifferentiation (VOT) plays a crucial role in promoting vascular calcification[4,5,15]. Many recent studies have shown that elevated serum phosphorus level is one of the causative agents of vascular calcification[33,35]. However, the precise mechanism involved in phosphorus-induced VOT remained undefinite. In our study, we presented persuasive evidence suggesting that through repressing Ets1 expression, miR-125b acts as an important regulator implicated in the phenotypic switching and calcification of VSMCs induced by high phosphorus. Unlike either skeletal or cardiac muscle cells that are terminally differentiated, VSMCs retain more extensive plasticity to undergo modulation of their phenotype in response to changes in local environmental cues[36]. Here we confirmed that incubation of VSMCs with β-glycerophosphoric acid, a donor of organic phosphorus, promoted osteogenic transition and calcification of VSMCs. Previous studies have implied that apoptosis[37,38], reactive oxygen species[39], and migration[34] are important mechanisms related to vascular calcification. MiRNAs have been verified involving in several physiological and pathophysiological processes associated with vascular calcification. Recently, emerging evidences suggest that miRNAs play a role in atherosclerotic plaque formation[40] and medium calcification[41,42]. MiR-125b has been implicated in a variety of cancers like breast cancer[21,43], hepatocellular cancer[44,45] and inflammation[46]. It is also reported that miR-125b was involved in osteoblastic differentiation[13] and regulated the transdifferentiation of VSMCs to osteoblast-like cells[12]. Consistent with these previous studies, our study demonstrated that miR-125b was downregulated in calcified arteries of human. To our knowledge, this is the first report which is describing the expression pattern of miR-125b in human arteries. Moreover, here we first demonstrated that overexpression of miR-125b attenuated VSMCs phenotypic transition and calcification induced by high phosphorus. In contrast, inhibition of miR-125b promoted osteogenic transdifferentiation and calcification of VSMCs in vitro, which suggests that knockdown of miR-125b could be an important facilitator of high phosphorus in regulating transdifferentiation and calcification of VSMCs. MiRNAs regulates gene expression through binding to the 3’-UTR sites of specific mRNA targets. Target scan predicts that Ets1 is a target gene of miR-125b and this has been demonstrated in breast cancer by luciferase reporter assay[21]. Ets1 is the founding member of the ets family and was originally discovered as a part of the avian E26 retrovirus genome[47]. Ets1 plays an important role in essential biological processes such as growth, transformation, apoptosis, differentiation, and organogenesis. During bone and cartilage specific development, Ets1 acts as either a regulator involving in proliferation or an effector of a signal transduction pathway, which affects the osteoblast function[48,49]. In addition, several Ets family members are expressed in cells of vascular origin, including endothelial cells and vascular smooth muscle cells, where they regulate the expression of a number of vascular-specific genes. Our results displayed that miR-125b regulated Ets1 protein expression but not mRNA expression, suggesting that miR-125b plays a role of post-transcriptional modulation for Ets1 gene. Knockdown of Ets1 significantly inhibited the ALP activity and calcium deposition in VSMCs treated with high phosphorus. Gary K. etal reported that PDGF-BB and Ets1 induced potent coordinate repression of multiple SMC 8

differentiation marker genes in cultured VSMCs[32], that was confirmed by our results. Accordingly, we presume the possible mechanism involved is that overexpression of miR-125b , which in turn inhibition of Ets1, could restore the differentiation marker gene SM-22α and inhibition of migration induced by high phosphorus. In summary, we provide novel evidence that miR-125b is a pivotal mediator which contributes to high phosphorus-induced phenotypic switching of VSMCs to osteoblast-like cells and high phosphorus-induced calcification of VSMCs in vitro and in human arteries, hereby providing a novel target for therapeutic intervention of a variety of cardiovascular disease related to vascular calcification. Acknowledgments and sources of funding This work was supported by scientific research fund of Jiangsu provincial science and technology department to Yang J (BL2013037). Natural Science Foundation of Jiangsu Province of China (BK2012870) to W. He.

Disclosures None. Figures and figure legends Figure 1 β-glycerophosphoric acid induced VSMCs osteogenic transdifferentiation and calcification. mRNA levels of cbfα1, collagen I and osteopontin were increased in VSMCs treated with 10.0 mmol/L β-GP for 24 hours and 72 hours (a); After incubated with β-GP for 6 days, expressions of osteocalcin and osteopontin were increased and protein level of SM-22α was decreased significantly (b); ALP activities measured by OD405 augmented significantly in a time or dose dependent manner, * P<0.05 (c); Alizarin Red S staining showed obvious calcium deposition in VSMCs incubated with calcification media (B) compared with those with control media (A) (d).

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Figure 2 The protective effect of miR-125b against VSMCs phenotypic transition and calcification. The expression of miR-125b was decreased significantly in VSMCs treated with 10.0 mmol/L β-GP for different periods of time (a); overexpression of miR-125b strikingly increased miR-125b levels in VSMCs compared with NC (negative control) while knockdown miR-125b significantly down-regulated miR-125b expression, * P<0.05 (b); The upregulations of osteoblast markers osteocalcin and osteopontin induced by β-GP were inhibited by miR-125b mimic whereas these two proteins can be induced by miR-125b inhibitor alone. The semi-quantitative analysis showed significant differences between groups, * P<0.05 (c); The expression of VSMCs differentiation marker SM-22α was restored by miR-125b mimic while inhibited by miR-125b inhibitor. The semi-quantitative analysis showed significant differences between groups, * P<0.05 (d); ALP activity was increased after treated with β-GP, overexpression of miR-125b significantly reduced ALP activity while knockdown miR-125b raised ALP activity, * P<0.05 (e); Compared with control media (A), calcification media lead to calcium depositing presented by Alizarin Red S staining (B), VSMCs transfected with miR-125b mimic incubated in calcification media displayed less calcium deposition compared with cells transfected with negative control (C). In contrast, cells transfected with miR-125b inhibitor showed aggressive calcium deposition (D). Calcium content was measured and shown as means±standard deviation of three independent experiments; * P<0.05 (g).

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Figure 3 The expression of Ets1 in VSMCs induced by β-GP and the regulation of miR-125b on Ets1 expression. After incubated with 10 mM β-GP for different periods of time, mRNA expression of Ets1 was increased significantly at 12 hours and 72 hours, * P<0.05 (a); When SMCs were cultured with different doses of β-GP for 6 days, mRNA expression of Ets1 was increased in a dose dependent manner, * P<0.05 (b); The protein expression of Ets1 was also 11

induced by β-GP in a similar manner with mRNA expression (c, d); Immunofluorescence staining showed that Ets1 expression was raised in cells treated with either β-GP or inorganic phosphorus (e); mRNA expression of Ets1 induced by β-GP was not regulated by miR-125b (f) while protein expression was inhibited by miR-125b mimic and miR-125b inhibitor could increase Ets1 protein expression in VSMCs (g); The semi-quantification of Ets1 protein expression treated with miR-125b mimic and inhibitor, * P<0.05 (h).

Figure 4 Ets1 knockdown blocks VSMCs phenotypic switching into osteogenic cells induced by high phosphorus. Ets1 siRNA significantly decreased Ets1 expression in VSMCs and restored SM-22α expression that was inhibited by β-GP (a); ALP activities were increased after treated with β-GP for 72 hours and Ets1 siRNA decreased the ALP activities strikingly, * P<0.05 (b); Ets1 siRNA attenuated calcium deposition (c). Compared with control media (A), 12

cells incubated in calcification media for 6 days (B) showed obviously calcium deposition, however, cells transfected with Ets1 siRNA displayed slightly calcium deposition (C). Calcium content measured by a quantitative method showed similar trend with the staining results, * P<0.05 (d). VSMCs were seeded on the Transwell filters of a Boyden chamber in different conditions for 24 hours. The cells or cell extensions that passed through the pores of filters were counted after staining. Graphic presentation of the numbers of cells or cell extensions migrated through the pores of the filters showed the migration induced by β-GP was inhibited by Ets1 siRNA, * P<0.05 (e).

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Figure 5 Expression of miR-125b in radial arteries of uremia patients. Von kossa staining was applied to detect calcium deposition in radial arteries of uremia patients (a). Ten artery tissues were stained and figure A presented negative result while figure B presented positive result. Real time quantitative RT-PCR showed that miR-125b expression was significantly decreased in arteries with calcification compared with those without calcification, * P<0.05 (b).

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Highlights z z z z z

β-glycerophosphoric acid can induce rat aortic SMCs transdifferentiated to osteoblasts. microRNA 125b regulated VSMCs phenotypic transition and calcification. Ets1 was upregulated during the VSMCs phenotypic transition and regulated by miR-125b. Knockdown Ets1 in VSMCs inhibited VSMCs phenotypic transition and calcification induced by β-glycerophosphoric acid. The expressions of miR-125b were down-regulated in human calcified radial arteries.

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