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MicroRNA-223 inhibits tissue factor expression in vascular endothelial cells
Atherosclerosis 237 (2014) 514e520
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Atherosclerosis journal homepage: www.elsevier.com/locate/atherosclerosis
MicroRNA-223 inhibits tissue factor expression in vascular endothelial cells Sufang Li a, Hong Chen a, *, Jingyi Ren a, Qiang Geng a, Junxian Song a, Chongyou Lee a, Chengfu Cao a, Jing Zhang a, Ning Xu b a b
Department of Cardiology, Peking University People's Hospital, No 11. Xizhimen South Street, Xicheng District, Beijing 100044, China Department of Medicine, Karolinska Institutet, Stockholm, Sweden
a r t i c l e i n f o
a b s t r a c t
Article history: Received 20 May 2014 Received in revised form 9 September 2014 Accepted 30 September 2014 Available online 18 October 2014
Objective: Atherosclerosis is a chronic inflammatory process, in which vascular endothelial cells (ECs) become dysfunctional owing to the effects of chemical substances, such as inflammatory factor and growth factors. Tissue factor (TF) expression is induced by the above chemical substances in activated ECs. TF initiates thrombosis on disrupted atherosclerotic plaques which plays an essential role during the onset of acute coronary syndromes (ACS). Increasing evidences suggest the important role of microRNAs as epigenetic regulators of atherosclerotic disease. The aim of our study is to identify if microRNA-223 (miR-223) targets TF in ECs. Methods and results: Bioinformatic analysis showed that TF is a target candidate of miR-223. Western blotting analysis revealed that tumor necrosis factor a (TNF-a) increased TF expression in aorta of C57BL/6J mice and cultured ECs (EA.hy926 cells and HUVEC) after 4 h treatment. In TNF-a treated ECs, TF mRNA was also increased measured by real-time PCR. Real-time PCR results showed that miR-223 levels were downregulated in TNF-a-treated aorta of C57BL/6J mice and cultured ECs. Transfection of ECs with miR-223 mimic or miR-223 inhibitor modified TF expression both in mRNA and protein levels. Luciferase assays confirmed that miR-223 suppressed TF expression by binding to the sequence of TF 30 -untranslated regions (30 UTR). TF procoagulant activity was inhibited by overexpressing miR-223 with or without TNF-a stimulation. Conclusions: MiR-223-mediated suppression of TF expression provides a novel molecular mechanism for the regulation of coagulation cascade, and suggests a clue against thrombogenesis during the process of atherosclerotic plaque rupture. © 2014 Elsevier Ireland Ltd. All rights reserved.
Keywords: Tissue factor microRNA-223 TNF-a Atherosclerosis Thrombogenesis
1. Introduction Atherosclerosis is a chronic inflammatory disease of the arterial wall [1]. Risk factors of atherosclerosis, such as hyperlipidemia and hypertension, provoke vascular cells to produce cytokines. Inflammatory cytokines (e.g. TNF-a, interleukin-1, interleukin-6) could further aggravate vascular cells dysfunction and atherogenesis. Endothelial cells (ECs) play a key role in the initiation of atherosclerosis and ultimately thrombosis [2]. Vascular endothelium stand as the first barrier to inflammation and thrombosis. Once endothelium structure or function is impaired, it will act as the earliest facilitator of vascular inflammation and thrombosis. Tissue factor (TF), formerly known as thromboplastin, is the initiator of extrinsic coagulation pathway [3,4]. Under a variety of stimulus, TF expression and activity can be significantly induced in vascular smooth muscle cells, endothelial cells and monocytes. TF * Corresponding author. E-mail address: [email protected] (H. Chen). http://dx.doi.org/10.1016/j.atherosclerosis.2014.09.033 0021-9150/© 2014 Elsevier Ireland Ltd. All rights reserved.
has been shown to play important role in the pathogenesis of atherosclerosis and thrombosis [5]. Once atherosclerotic plaque ruptures, TF in ruptured plaque will be released to the blood flow [5,6]. As soon as TF contacts with circulating activated factor VII (VIIa), coagulation cascade is rapidly initiated at the injured site of blood vessel [7]. There are some transcriptional factors which regulate TF expression in transcriptional level [8e10]. TF promoter region contains the binding sites of nuclear factor kB (NF-kB), activator protein 1 (AP-1) and specificity protein 1 (SP-1), which could mediate TNF-a or lipopolysaccharide (LPS)-induced TF expression in ECs. However, the post-transcriptional regulation mechanism(s) of TF expression is largely unknown. microRNAs (miRNAs) are single-stranded, non-coding, small RNAs which regulate gene expression in post-transcriptional level by binding preferentially to the 30 UTR of their target gene, destabilizing mRNAs and/or inhibiting translation [11,12]. miRNAs are involved in almost all phases of atherogenesis e from lesion initiation, through progression, to ultimately clinical complications of this
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disease [13]. Thrombosis is a critical pathological process of acute coronary syndromes (ACS) which is one of the serious complications of atherosclerosis [5]. A recent study showed that miR-223 may be involved in the occurrence of atherosclerosis during the course of chronic kidney disease (CKD) in experimental mice [14]. Another study reported that miR-223 promoted HUVEC apoptosis by targeting the insulin-like growth factor 1 receptor [15]. These results suggest that there may be close relationship between miR-223 and cardiovascular diseases. In the present study, we observed that miR223 was downregulated, whereas TF was upregulated induced by TNF-a in C57BL/6J mice and cultured ECs. We further identified miR223 as a novel regulator of TF expression in ECs. miR-223 could at least partially block the TNF-a-induced procoagulant activity of TF, which suggested a promising potential treatment for inhibiting thrombogenesis in patients with atherosclerosis. 2. Materials and methods 2.1. Animal experiments The animal protocol was approved by the Medical Ethics Committee of Peking University People's Hospital. Male C57BL/6J mice
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at 8 weeks old were purchased from Charles River Laboratories in China and were housed in pathogen-free barrier facilities. Recombinant mouse TNF-a (PeproTech, USA) was i.p. injected (4 mg/ mouse) for 4 h. Aorta arches and descending aorta were snapfrozen separately for total RNA and proteins extraction. 2.2. Cell culture and transfection Human umbilical vein endothelial cells (HUVEC) and EA.hy926 cells (fusion cell line derived from HUVEC and lung carcinoma cells) were obtained from Shanghai Institutes for Biological Sciences (CAS, China). HUVEC were cultured in endothelial cell medium (ECM) (Sciencell, USA) supplemented with 5% fetal bovine serum (FBS) (Sciencell, USA), 1% endothelial cell growth supplement (ECGS) (Sciencell, USA), penicillin (100 units/mL) and streptomycin (100 mg/mL). For all experiments, HUVEC were used between passages 4 to 6. EA.hy926 cells were cultured in DMEM supplemented with 10% FBS, penicillin (100 units/mL), and streptomycin (100 mg/ mL). All cells were cultured in a 5% CO2, 37 C water-saturated atmosphere. Lipofectamine 2000 (Invitrogen, USA) at a final concentration of 3 mg/mL was used for transfection following manufacturer's recommendation. Negative control (NC) mimic,
Fig. 1. The putative binding sites of miR-223 to TF mRNA and the levels of TF and miR-223 in aorta of C57BL/6J mice treated with TNF-a for 4 h. (A) Schematic representation of the 30 untranslated region (30 UTR) of TF mRNA (gray bar) with the predicted target sites for miR-223 (red lines). Nucleotide resolution of the predicted target sites: the seed sequence (green letters); the target sequence (red letters); the evolutionarily conserved regions (blue boxes). (B) The protein levels of TF in descending aorta of the mice. TF protein expression was examined by western blotting and normalized with respect to GAPDH protein. *p < 0.05. (C) The expression of miR-223 in aorta arches of the mice was detected by real-time PCR and normalized with respect to U6 snRNA. *p < 0.05. The data shown were all as mean ± SEM. Vehicle group: n ¼ 4, TNF-a group: n ¼ 4. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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Fig. 2. The levels of TF and miR-223 in cultured EA.hy926 cells and HUVEC treated with TNF-a for 4 h (A and D) TF mRNA levels in EA.hy926 cells (A) and HUVEC (D) were measured by real-time PCR and normalized with respect to GAPDH mRNA. n ¼ 3. ***p < 0.001. (B and E) TF protein levels in EA.hy926 cells (B) and HUVEC (E) were examined by western blotting and normalized with respect to GAPDH protein. n ¼ 3. *p < 0.05. (C and F) miR-223 levels in EA.hy926 cells (C) and HUVEC (F) were tested by real-time PCR and normalized with respect to RNU6B. *p < 0.05. n ¼ 3. The data shown were all as mean ± SEM.
miR-223 mimic, NC inhibitor and miR-223 inhibitor (complementary antagonist) (ABI, USA) were transfected at a final concentration of 30 pmol/mL. Cells were treated with recombinant human TNF-a (PeproTech, USA) at a final concentration of 10 ng/mL. 2.3. Real-time PCR Total RNA was extracted from vascular tissue and cells using miRNeasy Mini Kit (Qiagen, USA). Real-time PCR reactions were performed on an Applied Biosystems system (ViiA7). Values are expressed as 2△△CT. Amplification conditions and primers were shown in the Supplementary data. 2.4. Western blotting Transfected EA.hy926 cells and HUVEC were lysed and centrifuged at 12,000 g for 10 min at 4 C. The supernatants were collected and protein concentration was determined with bicinchoninic acid (BCA) protein assay kit (Pierce Biotechnology Inc, USA). 20e30 mg proteins were separated by 10% SDSpolyacrylamide gel and electrophoretically transferred to PVDF membranes (Millipore, USA). The membranes were probed against TF (Abcam, USA) and reprobed against GAPDH (Santa Cruz Biotechnology, USA) as a loading control. 2.5. Plasmid construct and luciferase reporter assay Firefly luciferase reporter plasmid containing TF 30 UTR (TF-luciWT) and mutated TF 30 UTR (TF-luci-MUT) were constructed
(Details were described in the Supplementary data). EA.hy926 cells and HUVEC plated in 24-well plate were cotransfected with NC or miR-223 mimic (final concentration: 60 pmol/mL) and firefly luciferase reporter plasmid (final concentration: 300 ng/mL) along with renilla luciferase control plasmid (final concentration: 10 ng/ mL; Promega, USA) using lipofectamine 2000 (final concentration: 4 mg/mL). After 24 h, luciferase activity was measured according to the manufacturer's instructions (Dual Luciferase Assay System; Promega, USA). Each measured firefly luciferase activity was normalized by the Renilla luciferase activity in the same well. 2.6. Assay of TF procoagulant activity TF procoagulant activity was analyzed using Cell Tissue Factor Assay Kit (Genmed Scientifics Inc, USA) following manufacturers instructions. Briefly, EA.hy926 cells were lysed and 50 mg proteins of each sample were used for measuring TF activity. Samples were incubated with prothrombin complex (containing Factor II, VII, IX, X) and CaCl2. Reaction was terminated by the addition of EDTA buffer. Lastly, a chromogenic substrate (Spectrozyme factor Xa) was added and absorbance at 405 nm was measured. 2.7. Statistical analysis Quantitative data are presented as mean ± SEM. Two groups were compared using Student's t-test. Between-group comparison of means was performed by one-way ANOVA, followed by the Tukey multiple-comparisons test. Prism 5.0 was used for all statistical analyses. Statistical significance was defined as p < 0.05.
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Fig. 3. The effect of modulated miR-223 on TF expression in cultured EA.hy926 cells and HUVEC. Transfection of EA.hy926 cells and HUVEC with miR-223 mimic (miR-223-m) for 24 h (A-B, E-F) or miR-223 inhibitor (miR-223-i) for 36 h (C-D, G-H) modified TF expression. (A, C, E and G) TF mRNA levels were measured by real-time PCR and normalized with respect to GAPDH. n ¼ 3. **p < 0.01 vs NC mimic (NC-m) or NC inhibitor (NC-i) group. (B, D, F and H) TF protein levels were measured by western blotting with GAPDH as a loading control. Densitometry was performed and normalized with respect to GAPDH expression level. n ¼ 3. *p < 0.05 vs NC-i group, **p < 0.01 vs NC-m or NC-i group, ***p < 0.01 vs NC-m group. The normalized data were expressed as changes relative to the data of cells transfected with NC-m or NC-i and shown as mean ± SEM.
3. Results
3.3. MiR-223 inhibits TF expression in ECs
3.1. MiR-223 expression is reduced in aorta of C57BL/6J mice treated with TNF-a
To prove that the inhibitory effect of miR-223 on TF expression, we performed gain- and loss-of-function experiments by transfecting miR-223 mimics or miR-223 inhibitors in EA.hy926 cells and HUVEC, respectively. miR-223 levels were significantly increased after transfecting miR-223 mimics for 24 h (**p < 0.01, ***p < 0.001, Supplementary Fig. 1) and decreased after transfecting miR-223 inhibitors for 36 h (*p < 0.05, Supplementary Fig. 2). Overexpression of miR-223 inhibited TF mRNA expression by 33% in EA.hy926 cells (**p < 0.01, Fig. 3A) and 43% in HUVEC (**p < 0.01, Fig. 3E), respectively, whereas miR-223 inhibitors increased TF expression by 183% in EA.hy926 cells (**p < 0.01, Fig. 3C) and 324% in HUVEC (**p < 0.01, Fig. 3G), respectively. In agreement with these results, the protein levels of TF were reduced by 34% in EA.hy926 cells (**p < 0.01, Fig. 3B) and 28% in HUVEC (***p < 0.001, Fig. 3F) by overexpressing miR-223 compared with negative control (NC mimics) (Fig. 1D). Nevertheless, the protein levels of TF were increased by 31% in EA.hy926 cells (**p < 0.01, Fig. 3E) and 77% in HUVEC (*p < 0.05, Fig. 3H) by inhibiting miR-223 level compared with negative control (NC inhibitors).
For identifying the potential miRNA targeting TF gene, we performed in silico analysis using two different miRNA target prediction algorithms, TargetScan (http://www.targetscan.org/) and miRanda (http://miracle.igib.res.in/miracle/). Results showed a putative miR-223 binding sites within the 30 UTR of TF mRNA (Fig. 1A). To determine whether TF expression might be regulated by miR223, C57BL/6J mice were i.p. injected with TNF-a (TNF-a group, n ¼ 4) or saline (Vehicle group, n ¼ 4) to induce TF upregulation, and miR-223 levels were simultaneously tested by real-time PCR. We observed that TNF-a increased TF protein levels (protein from descending aorta) for approximately two fold, compared with controls (*p < 0.05, Fig. 1B). Conversely, miR-223 levels (RNA from aorta arches) were reduced by 30% (*p < 0.05, Fig. 1C). 3.2. MiR-223 expression is reduced in ECs treated with TNF-a To further confirm the correlation between miR-223 and TF in vascular endothelium, we stimulated cultured EA.hy926 cells and HUVEC with TNF-a for 4 h, respectively. We found that during the upregulation of TF both in mRNA (***p < 0.001, Fig. 2A and D) and protein levels (*p < 0.05, Fig. 2B and E), miR-223 levels were decreased by 21% in EA.hy926 cells (*p < 0.05, Fig. 2C) and 22% in HUVEC (*p < 0.05, Fig. 2F).
3.4. TF is a direct target of miR-223 To elucidate whether TF is a direct target of miR-223, we cloned the putative miR-223 binding site on TF 30 UTR or its mutational sequences into luciferase reporter plasmid and co-transfected it with miR-223 mimics into EA.hy926 cells and HUVEC, respectively (Fig. 4A). Results showed that overexpression of miR-223 decreased
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Fig. 4. Luciferase reporter assays of miR-223 binding sites on human TF 30 UTR. (A) The scheme of constructing luciferase reporter plasmid. TF-luci-WT plasmid contained human TF 30 UTR sequences and green letters were the seed sequence, TF-luci-MUT plasmid contained human TF 30 UTR sequences with mutated miR-223-binding site (gray boxes). (B and C) EA.hy926 cells (B) and HUVEC (C) were transfected with either wild-type 30 UTR TF (TF-luci-WT) or Mutant 30 UTR TF (TF-luci-MUT), along with miR-223 mimic (miR-223-m). Luciferase activities were normalized to renilla activities. The normalized data were expressed as changes relative to the data of cells co-transfected with NC mimic (NC-m) and TFluci-WT plasmid. Results shown were as mean ± SEM. n ¼ 4. **p < 0.01, ***p < 0.001, #p < 0.05, ###p < 0.001. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
the expression of luciferase reporter gene to approximately 65% in EA.hy926 cells (***p < 0.001, Fig. 4B) and 53% in HUVEC (**p < 0.01, Fig. 4C), which contained the wild-type binding site of miR-223. However, when the putative binding site of miR-223 was mutated, miR-223 mediated inhibition of luciferase gene expression was absolutely rescued in the ECs (#p < 0.05, ###p < 0.001, Fig. 4B and C). These data demonstrated that TF was a direct target of miR-223.
3.5. MiR-223 suppresses procoagulant activity in TNF-a-stimulated ECs To evaluate the effect of miR-223 on procoagulant activity of TF, we firstly transfected miR-223 mimic or NC mimic for 24 h in EA.hy926 cells using lipofectamine 2000. Results showed that miR223 decreased the procoagulant activity of TF by 25% in comparison with NC group (**p < 0.01, Fig. 5). We further stimulated cultured EA.hy926 cells with TNF-a for 4 h after miR-223 overexpressed for 24 h and found that miR-223 could partially blocked TNF-ainduced increase of TF pro-coagulant activity by 21% (#p < 0.05, Fig. 5).
4. Discussion In ACS, the plasma concentrations of TNF-a are increased at the site of coronary artery occlusion, which to such an extent induce TF expression in vascular cells [16]. As soon as plaque ruptures, high levels of TF in activated vascular ECs, VSMCs and macrophage is released to circulation which triggers the process of ACS by prompting thrombosis [5,17]. Therefore, exploring the regulation mechanism(s) of TF expression is very meaningful for constraining atherothrombosis. miRNAs have recently emerged as a novel class of gene regulators that posttranscriptionally regulate expression of multiple target genes. Increasing evidences suggest an important role of miRNAs in regulating atherosclerotic disease [18]. Enhancing disease-induced downregulated vascular miRNAs or inhibition of upregulated might be a promising strategy to cure atherosclerotic disease [19]. Herein, we reported that miRNA-223 may serve as a potent regulator of thrombosis followed with atherosclerotic plaque rupture by targeting TF, an initiator of exogenous coagulation cascade. Increased circulating TF antigen and activity were detected in patients with unstable angina or myocardial infarction compared with those with stable angina [20], which suggested a
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hypercoagulable state of patients with ACS [21]. Decreased TF expression may delay the process of atherosclerosis and consequently thrombosis [17]. More recently, some studies revealed that miRNAs were involved in hypercoagulable diseases by regulating haemostatic proteins levels [22e24]. Teruel R et al. reported that miR-19b and miR-20a could inhibit TF expression in THP1 cells, which may explain why systemic lupus erythematosus and antiphospholipid syndrome patients with decreased miR-19b and miR20a were in the hypercoagulatable state [22]. In fact, the miRNAs targeting TF are far from being discovered. Identifying more miRNAs involved in TF expression is very useful for understanding the mechanism of atherothrombosis and improving treatment. For finding new miRNAs targeting TF, we firstly performed bioinformatic prediction using two miRNA target prediction algorithms, TargetScan and miRanda. The results showed that the 30 UTR of TF mRNA contains target sites of miR-223. miR-223 binding site across several species including human, mouse, rat and dog suggested an evolutionarily conserved importance for miR-223. Recently, Shi L et al. reported that miR-223 could resist angiogenesis by targeting b1 integrin and preventing growth factor signaling in ECs [25] and Pan Y et al. found that platelet-secreted miR-223 promoted endothelial cell apoptosis induced by advanced glycation end products (AGEs) [15]. These reports imply a variety of biological role of miR-223 in the occurrence and development of athrosclerosis. To clarify the correlation of miR-223 and TF, we measured miR-223 levels in aorta from TNF-a-treated C57BL/6J mice and found that, during the upregulation of TF, miR-223 levels were significantly downregulated, which was not in line with the results reported by Taïbi F et al. [14]. In their study, miR-223 levels were increased in the aorta from ApoE/ mice with sham operations or CKD compared to wild type C57BL/6J mice with sham operations. We analyzed the reason leading to the opposite phenomenon that is mainly duo to the different time course of pathological stimulus.
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In our study, we chose to observe miR-223 levels in mice treated with TNF-a for 4 h when TF was obviously upregulated. However, in Fatiha Taïbi's study, miR-223 levels were increased in ApoE/ mice, at least, after two weeks of exposure to CKD compared to wild type controls. More importantly, instability and rupture of atherosclerotic vulnerable plaques are the pathologic basis of ACS [26], and we previously reported that the circulating miRNAs expression profiles in patients with unstable angina were different from patients with stable angina [27]. However, ApoE/ mice are not optimal models to reproduce plaque instability observed in humans [28]. Hence, we treated C57BL/6J mice with TNF-a to mimic the acute inflommation state during the rupture of plaques [16]. ECs activation and dysfunction have been linked to a variety of vascular inflammatory disease states [2] and miR-223 was reported to be correlated with inflammation [29]. So, we further observed miR-223 levels in cultured ECs incubated with TNF-a. We found that miR-223 expression decreased in ECs, whereas TF protein increased at the moment. The above in vivo and in vitro assays, combined with bioinformatics analysis, suggested that decreased miR-223 may be correlated with TF upgregulation. Next, we examined the effect of miR-223 on TF expression in ECs by using gain- and loss-of-function experiments. Overexpressed miR-223 induced TF downregulation both in mRNA and protein level. In contrast, TF levels were upregulated due to decreased miR223. To further illustrate the regulation mechanism of TF expression by miR-223, we performed luciferase reporter assay and the results indicated that miR-223 could inhibit TF expression by directly interacting with the “seed region” of TF 30 UTR. Lastly, we tested the effect of miR-223 on TF procoagulate activity. Results showed that miR-223 could not only inhibit TF procoagulate activity, but also partly block TNF-a-induced increase of TF procoagulate activity in ECs. Our results may partially explain that miR223 was inversely associated with myocardial infarction risk reported by Zampetaki et al. [30]. Inflammation could increase TF expression and activity in vascular cells. TF upregulation may reversely enhance inflammation by prompting the deposition and formation of proinlammatory fragments of fibrin in vascular wall, and by generating coagulation proteases, such as FVIIa, FXa and thrombin, which activate protease-dependent receptors [31]. Furthermore, TF is involved in cell proliferation and angiogenesis by activating MAPK pathway [32,33]. Therefore, other than antagonizing atherothrombosis, miR223 may mediate multiple biology effects in the progress of atherosclerosis through regulating TF expression. In conclusion, we discovered a novel mechanism for the regulation of TF expression by miR-223. Our study suggests an inhibitory role for miR-223 in thrombotic complications of atherosclerosis. Therapeutic interventions by elevating miR-223 in patients with atherosclerotic disease may reduce atherothrombotic events via suppressing TF expression in ECs. Sources of funding This work was supported by National Science and Technology “Creation of Major New Dugs” (No. 2012ZX09303019), Beijing Science and Technology Project (No. D141100003014002), National Natural Science Foundation of China (501100001809) (No. 81270274, No. 81270276), Beijing Natural Science Foundation (No. 7132225, No. 7122198).
Fig. 5. The effect of overexpressed miR-223 on TF procoagulant activity of EA.hy926 cells with or without TNF-a administration for 4 h. FXa generation was quantified at 405 nm after addition of FXa substrate. The procoagulant activity of TF was expressed as changes relative to the activity of cells transfected with NC mimic. Results shown were as mean ± SEM. n ¼ 4. *p < 0.05 vs NC mimic group, **p < 0.01 vs NC mimic group, #p < 0.05 vs NC mimic þ TNF-a group.
Author contributions Hong Chen and Jingyi Ren designed research and critically revised the manuscript. Sufang Li designed research, performed research, analyzed data and wrote the manuscript. Ning Xu and
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Junxian Song critically revised the manuscript. Qiang Geng Chengfu Cao, Chongyou Lee, Jing Zhang performed research. Disclosure of conflict of interests The authors state that they have no conflict of interest. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.atherosclerosis.2014.09.033. References [1] Libby P. Inflammation in atherosclerosis. Arterioscler. Thromb. Vasc. Biol. 2012;32:2045e51. [2] Hopkins PN. Molecular biology of atherosclerosis. Physiol. Rev. 2013;93: 1317e542. [3] Eilertsen KE, Østerud B. Tissue factor: (patho)physiology and cellular biology. Blood Coagul. Fibrinolysis 2004;15:521e38. [4] Mandal SK, Pendurthi UR, Rao LV. Cellular localization and trafficking of tissue factor. Blood 2006;107:4746e53. [5] Libby P. Mechanisms of acute coronary syndromes and their implications for therapy. N. Engl. J. Med. 2013;368:2004e13. [6] Furie B, Furie BC. Mechanisms of thrombus formation. N. Engl. J. Med. 2008;359:938e49. [7] Bierhaus A, Zhang Y, Deng Y, et al. Mechanism of the tumor necrosis factor alpha-mediated induction of endothelial tissue factor. J. Biol. Chem. 1995;270: 26419e32. [8] Moll T, Czyz M, Holzmüller H, et al. Regulation of the tissue factor promoter in endothelial cells. Binding of NF kappa B-, AP-1-, and Sp1-like transcription factors. J. Biol. Chem. 1995;270:3849e57. [9] Sakamoto T, Ishibashi T, Sakamoto N, et al. Endogenous NO blockade enhances tissue factor expression via increased Ca2þ influx through MCP-1 in endothelial cells by monocyte adhesion. Arterioscler. Thromb. Vasc. Biol. 2005;25: 2005e11. [10] Li YD, Ye BQ, Zheng SX, et al. NF-kappaB transcription factor p50 critically regulates tissue factor in deep vein thrombosis. J. Biol. Chem. 2009;284: 4473e83. [11] Bartel DP. MicroRNAs: target recognition and regulatory functions. Cell 2009;136:215e33. [12] Pillai RS, Bhattacharyya SN, Filipowicz W. Repression of protein synthesis by miRNAs: how many mechanisms? Trends Cell. Biol. 2007;17:118e26. [13] Santovito D, Mezzetti A, Cipollone F. MicroRNAs and atherosclerosis: new actors for an old movie. Nutr. Metab. Cardiovasc Dis. 2012;22:937e43. [14] Taïbi F, Metzinger-Le Meuth V, M'Baya-Moutoula E, et al. Possible involvement of microRNAs in vascular damage in experimental chronic kidney disease. Biochim. Biophys. Acta 2014;1842:88e98. [15] Pan Y, Liang H, Liu H, et al. Platelet-secreted microRNA-223 promotes endothelial cell apoptosis induced by advanced glycation end products via targeting the insulin-like growth factor 1 receptor. J. Immunol. 2014;192: 437e46.
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Atherosclerosis journal homepage: www.elsevier.com/locate/atherosclerosis
MicroRNA-223 inhibits tissue factor expression in vascular endothelial cells Sufang Li a, Hong Chen a, *, Jingyi Ren a, Qiang Geng a, Junxian Song a, Chongyou Lee a, Chengfu Cao a, Jing Zhang a, Ning Xu b a b
Department of Cardiology, Peking University People's Hospital, No 11. Xizhimen South Street, Xicheng District, Beijing 100044, China Department of Medicine, Karolinska Institutet, Stockholm, Sweden
a r t i c l e i n f o
a b s t r a c t
Article history: Received 20 May 2014 Received in revised form 9 September 2014 Accepted 30 September 2014 Available online 18 October 2014
Objective: Atherosclerosis is a chronic inflammatory process, in which vascular endothelial cells (ECs) become dysfunctional owing to the effects of chemical substances, such as inflammatory factor and growth factors. Tissue factor (TF) expression is induced by the above chemical substances in activated ECs. TF initiates thrombosis on disrupted atherosclerotic plaques which plays an essential role during the onset of acute coronary syndromes (ACS). Increasing evidences suggest the important role of microRNAs as epigenetic regulators of atherosclerotic disease. The aim of our study is to identify if microRNA-223 (miR-223) targets TF in ECs. Methods and results: Bioinformatic analysis showed that TF is a target candidate of miR-223. Western blotting analysis revealed that tumor necrosis factor a (TNF-a) increased TF expression in aorta of C57BL/6J mice and cultured ECs (EA.hy926 cells and HUVEC) after 4 h treatment. In TNF-a treated ECs, TF mRNA was also increased measured by real-time PCR. Real-time PCR results showed that miR-223 levels were downregulated in TNF-a-treated aorta of C57BL/6J mice and cultured ECs. Transfection of ECs with miR-223 mimic or miR-223 inhibitor modified TF expression both in mRNA and protein levels. Luciferase assays confirmed that miR-223 suppressed TF expression by binding to the sequence of TF 30 -untranslated regions (30 UTR). TF procoagulant activity was inhibited by overexpressing miR-223 with or without TNF-a stimulation. Conclusions: MiR-223-mediated suppression of TF expression provides a novel molecular mechanism for the regulation of coagulation cascade, and suggests a clue against thrombogenesis during the process of atherosclerotic plaque rupture. © 2014 Elsevier Ireland Ltd. All rights reserved.
Keywords: Tissue factor microRNA-223 TNF-a Atherosclerosis Thrombogenesis
1. Introduction Atherosclerosis is a chronic inflammatory disease of the arterial wall [1]. Risk factors of atherosclerosis, such as hyperlipidemia and hypertension, provoke vascular cells to produce cytokines. Inflammatory cytokines (e.g. TNF-a, interleukin-1, interleukin-6) could further aggravate vascular cells dysfunction and atherogenesis. Endothelial cells (ECs) play a key role in the initiation of atherosclerosis and ultimately thrombosis [2]. Vascular endothelium stand as the first barrier to inflammation and thrombosis. Once endothelium structure or function is impaired, it will act as the earliest facilitator of vascular inflammation and thrombosis. Tissue factor (TF), formerly known as thromboplastin, is the initiator of extrinsic coagulation pathway [3,4]. Under a variety of stimulus, TF expression and activity can be significantly induced in vascular smooth muscle cells, endothelial cells and monocytes. TF * Corresponding author. E-mail address: [email protected] (H. Chen). http://dx.doi.org/10.1016/j.atherosclerosis.2014.09.033 0021-9150/© 2014 Elsevier Ireland Ltd. All rights reserved.
has been shown to play important role in the pathogenesis of atherosclerosis and thrombosis [5]. Once atherosclerotic plaque ruptures, TF in ruptured plaque will be released to the blood flow [5,6]. As soon as TF contacts with circulating activated factor VII (VIIa), coagulation cascade is rapidly initiated at the injured site of blood vessel [7]. There are some transcriptional factors which regulate TF expression in transcriptional level [8e10]. TF promoter region contains the binding sites of nuclear factor kB (NF-kB), activator protein 1 (AP-1) and specificity protein 1 (SP-1), which could mediate TNF-a or lipopolysaccharide (LPS)-induced TF expression in ECs. However, the post-transcriptional regulation mechanism(s) of TF expression is largely unknown. microRNAs (miRNAs) are single-stranded, non-coding, small RNAs which regulate gene expression in post-transcriptional level by binding preferentially to the 30 UTR of their target gene, destabilizing mRNAs and/or inhibiting translation [11,12]. miRNAs are involved in almost all phases of atherogenesis e from lesion initiation, through progression, to ultimately clinical complications of this
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disease [13]. Thrombosis is a critical pathological process of acute coronary syndromes (ACS) which is one of the serious complications of atherosclerosis [5]. A recent study showed that miR-223 may be involved in the occurrence of atherosclerosis during the course of chronic kidney disease (CKD) in experimental mice [14]. Another study reported that miR-223 promoted HUVEC apoptosis by targeting the insulin-like growth factor 1 receptor [15]. These results suggest that there may be close relationship between miR-223 and cardiovascular diseases. In the present study, we observed that miR223 was downregulated, whereas TF was upregulated induced by TNF-a in C57BL/6J mice and cultured ECs. We further identified miR223 as a novel regulator of TF expression in ECs. miR-223 could at least partially block the TNF-a-induced procoagulant activity of TF, which suggested a promising potential treatment for inhibiting thrombogenesis in patients with atherosclerosis. 2. Materials and methods 2.1. Animal experiments The animal protocol was approved by the Medical Ethics Committee of Peking University People's Hospital. Male C57BL/6J mice
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at 8 weeks old were purchased from Charles River Laboratories in China and were housed in pathogen-free barrier facilities. Recombinant mouse TNF-a (PeproTech, USA) was i.p. injected (4 mg/ mouse) for 4 h. Aorta arches and descending aorta were snapfrozen separately for total RNA and proteins extraction. 2.2. Cell culture and transfection Human umbilical vein endothelial cells (HUVEC) and EA.hy926 cells (fusion cell line derived from HUVEC and lung carcinoma cells) were obtained from Shanghai Institutes for Biological Sciences (CAS, China). HUVEC were cultured in endothelial cell medium (ECM) (Sciencell, USA) supplemented with 5% fetal bovine serum (FBS) (Sciencell, USA), 1% endothelial cell growth supplement (ECGS) (Sciencell, USA), penicillin (100 units/mL) and streptomycin (100 mg/mL). For all experiments, HUVEC were used between passages 4 to 6. EA.hy926 cells were cultured in DMEM supplemented with 10% FBS, penicillin (100 units/mL), and streptomycin (100 mg/ mL). All cells were cultured in a 5% CO2, 37 C water-saturated atmosphere. Lipofectamine 2000 (Invitrogen, USA) at a final concentration of 3 mg/mL was used for transfection following manufacturer's recommendation. Negative control (NC) mimic,
Fig. 1. The putative binding sites of miR-223 to TF mRNA and the levels of TF and miR-223 in aorta of C57BL/6J mice treated with TNF-a for 4 h. (A) Schematic representation of the 30 untranslated region (30 UTR) of TF mRNA (gray bar) with the predicted target sites for miR-223 (red lines). Nucleotide resolution of the predicted target sites: the seed sequence (green letters); the target sequence (red letters); the evolutionarily conserved regions (blue boxes). (B) The protein levels of TF in descending aorta of the mice. TF protein expression was examined by western blotting and normalized with respect to GAPDH protein. *p < 0.05. (C) The expression of miR-223 in aorta arches of the mice was detected by real-time PCR and normalized with respect to U6 snRNA. *p < 0.05. The data shown were all as mean ± SEM. Vehicle group: n ¼ 4, TNF-a group: n ¼ 4. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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Fig. 2. The levels of TF and miR-223 in cultured EA.hy926 cells and HUVEC treated with TNF-a for 4 h (A and D) TF mRNA levels in EA.hy926 cells (A) and HUVEC (D) were measured by real-time PCR and normalized with respect to GAPDH mRNA. n ¼ 3. ***p < 0.001. (B and E) TF protein levels in EA.hy926 cells (B) and HUVEC (E) were examined by western blotting and normalized with respect to GAPDH protein. n ¼ 3. *p < 0.05. (C and F) miR-223 levels in EA.hy926 cells (C) and HUVEC (F) were tested by real-time PCR and normalized with respect to RNU6B. *p < 0.05. n ¼ 3. The data shown were all as mean ± SEM.
miR-223 mimic, NC inhibitor and miR-223 inhibitor (complementary antagonist) (ABI, USA) were transfected at a final concentration of 30 pmol/mL. Cells were treated with recombinant human TNF-a (PeproTech, USA) at a final concentration of 10 ng/mL. 2.3. Real-time PCR Total RNA was extracted from vascular tissue and cells using miRNeasy Mini Kit (Qiagen, USA). Real-time PCR reactions were performed on an Applied Biosystems system (ViiA7). Values are expressed as 2△△CT. Amplification conditions and primers were shown in the Supplementary data. 2.4. Western blotting Transfected EA.hy926 cells and HUVEC were lysed and centrifuged at 12,000 g for 10 min at 4 C. The supernatants were collected and protein concentration was determined with bicinchoninic acid (BCA) protein assay kit (Pierce Biotechnology Inc, USA). 20e30 mg proteins were separated by 10% SDSpolyacrylamide gel and electrophoretically transferred to PVDF membranes (Millipore, USA). The membranes were probed against TF (Abcam, USA) and reprobed against GAPDH (Santa Cruz Biotechnology, USA) as a loading control. 2.5. Plasmid construct and luciferase reporter assay Firefly luciferase reporter plasmid containing TF 30 UTR (TF-luciWT) and mutated TF 30 UTR (TF-luci-MUT) were constructed
(Details were described in the Supplementary data). EA.hy926 cells and HUVEC plated in 24-well plate were cotransfected with NC or miR-223 mimic (final concentration: 60 pmol/mL) and firefly luciferase reporter plasmid (final concentration: 300 ng/mL) along with renilla luciferase control plasmid (final concentration: 10 ng/ mL; Promega, USA) using lipofectamine 2000 (final concentration: 4 mg/mL). After 24 h, luciferase activity was measured according to the manufacturer's instructions (Dual Luciferase Assay System; Promega, USA). Each measured firefly luciferase activity was normalized by the Renilla luciferase activity in the same well. 2.6. Assay of TF procoagulant activity TF procoagulant activity was analyzed using Cell Tissue Factor Assay Kit (Genmed Scientifics Inc, USA) following manufacturers instructions. Briefly, EA.hy926 cells were lysed and 50 mg proteins of each sample were used for measuring TF activity. Samples were incubated with prothrombin complex (containing Factor II, VII, IX, X) and CaCl2. Reaction was terminated by the addition of EDTA buffer. Lastly, a chromogenic substrate (Spectrozyme factor Xa) was added and absorbance at 405 nm was measured. 2.7. Statistical analysis Quantitative data are presented as mean ± SEM. Two groups were compared using Student's t-test. Between-group comparison of means was performed by one-way ANOVA, followed by the Tukey multiple-comparisons test. Prism 5.0 was used for all statistical analyses. Statistical significance was defined as p < 0.05.
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Fig. 3. The effect of modulated miR-223 on TF expression in cultured EA.hy926 cells and HUVEC. Transfection of EA.hy926 cells and HUVEC with miR-223 mimic (miR-223-m) for 24 h (A-B, E-F) or miR-223 inhibitor (miR-223-i) for 36 h (C-D, G-H) modified TF expression. (A, C, E and G) TF mRNA levels were measured by real-time PCR and normalized with respect to GAPDH. n ¼ 3. **p < 0.01 vs NC mimic (NC-m) or NC inhibitor (NC-i) group. (B, D, F and H) TF protein levels were measured by western blotting with GAPDH as a loading control. Densitometry was performed and normalized with respect to GAPDH expression level. n ¼ 3. *p < 0.05 vs NC-i group, **p < 0.01 vs NC-m or NC-i group, ***p < 0.01 vs NC-m group. The normalized data were expressed as changes relative to the data of cells transfected with NC-m or NC-i and shown as mean ± SEM.
3. Results
3.3. MiR-223 inhibits TF expression in ECs
3.1. MiR-223 expression is reduced in aorta of C57BL/6J mice treated with TNF-a
To prove that the inhibitory effect of miR-223 on TF expression, we performed gain- and loss-of-function experiments by transfecting miR-223 mimics or miR-223 inhibitors in EA.hy926 cells and HUVEC, respectively. miR-223 levels were significantly increased after transfecting miR-223 mimics for 24 h (**p < 0.01, ***p < 0.001, Supplementary Fig. 1) and decreased after transfecting miR-223 inhibitors for 36 h (*p < 0.05, Supplementary Fig. 2). Overexpression of miR-223 inhibited TF mRNA expression by 33% in EA.hy926 cells (**p < 0.01, Fig. 3A) and 43% in HUVEC (**p < 0.01, Fig. 3E), respectively, whereas miR-223 inhibitors increased TF expression by 183% in EA.hy926 cells (**p < 0.01, Fig. 3C) and 324% in HUVEC (**p < 0.01, Fig. 3G), respectively. In agreement with these results, the protein levels of TF were reduced by 34% in EA.hy926 cells (**p < 0.01, Fig. 3B) and 28% in HUVEC (***p < 0.001, Fig. 3F) by overexpressing miR-223 compared with negative control (NC mimics) (Fig. 1D). Nevertheless, the protein levels of TF were increased by 31% in EA.hy926 cells (**p < 0.01, Fig. 3E) and 77% in HUVEC (*p < 0.05, Fig. 3H) by inhibiting miR-223 level compared with negative control (NC inhibitors).
For identifying the potential miRNA targeting TF gene, we performed in silico analysis using two different miRNA target prediction algorithms, TargetScan (http://www.targetscan.org/) and miRanda (http://miracle.igib.res.in/miracle/). Results showed a putative miR-223 binding sites within the 30 UTR of TF mRNA (Fig. 1A). To determine whether TF expression might be regulated by miR223, C57BL/6J mice were i.p. injected with TNF-a (TNF-a group, n ¼ 4) or saline (Vehicle group, n ¼ 4) to induce TF upregulation, and miR-223 levels were simultaneously tested by real-time PCR. We observed that TNF-a increased TF protein levels (protein from descending aorta) for approximately two fold, compared with controls (*p < 0.05, Fig. 1B). Conversely, miR-223 levels (RNA from aorta arches) were reduced by 30% (*p < 0.05, Fig. 1C). 3.2. MiR-223 expression is reduced in ECs treated with TNF-a To further confirm the correlation between miR-223 and TF in vascular endothelium, we stimulated cultured EA.hy926 cells and HUVEC with TNF-a for 4 h, respectively. We found that during the upregulation of TF both in mRNA (***p < 0.001, Fig. 2A and D) and protein levels (*p < 0.05, Fig. 2B and E), miR-223 levels were decreased by 21% in EA.hy926 cells (*p < 0.05, Fig. 2C) and 22% in HUVEC (*p < 0.05, Fig. 2F).
3.4. TF is a direct target of miR-223 To elucidate whether TF is a direct target of miR-223, we cloned the putative miR-223 binding site on TF 30 UTR or its mutational sequences into luciferase reporter plasmid and co-transfected it with miR-223 mimics into EA.hy926 cells and HUVEC, respectively (Fig. 4A). Results showed that overexpression of miR-223 decreased
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Fig. 4. Luciferase reporter assays of miR-223 binding sites on human TF 30 UTR. (A) The scheme of constructing luciferase reporter plasmid. TF-luci-WT plasmid contained human TF 30 UTR sequences and green letters were the seed sequence, TF-luci-MUT plasmid contained human TF 30 UTR sequences with mutated miR-223-binding site (gray boxes). (B and C) EA.hy926 cells (B) and HUVEC (C) were transfected with either wild-type 30 UTR TF (TF-luci-WT) or Mutant 30 UTR TF (TF-luci-MUT), along with miR-223 mimic (miR-223-m). Luciferase activities were normalized to renilla activities. The normalized data were expressed as changes relative to the data of cells co-transfected with NC mimic (NC-m) and TFluci-WT plasmid. Results shown were as mean ± SEM. n ¼ 4. **p < 0.01, ***p < 0.001, #p < 0.05, ###p < 0.001. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
the expression of luciferase reporter gene to approximately 65% in EA.hy926 cells (***p < 0.001, Fig. 4B) and 53% in HUVEC (**p < 0.01, Fig. 4C), which contained the wild-type binding site of miR-223. However, when the putative binding site of miR-223 was mutated, miR-223 mediated inhibition of luciferase gene expression was absolutely rescued in the ECs (#p < 0.05, ###p < 0.001, Fig. 4B and C). These data demonstrated that TF was a direct target of miR-223.
3.5. MiR-223 suppresses procoagulant activity in TNF-a-stimulated ECs To evaluate the effect of miR-223 on procoagulant activity of TF, we firstly transfected miR-223 mimic or NC mimic for 24 h in EA.hy926 cells using lipofectamine 2000. Results showed that miR223 decreased the procoagulant activity of TF by 25% in comparison with NC group (**p < 0.01, Fig. 5). We further stimulated cultured EA.hy926 cells with TNF-a for 4 h after miR-223 overexpressed for 24 h and found that miR-223 could partially blocked TNF-ainduced increase of TF pro-coagulant activity by 21% (#p < 0.05, Fig. 5).
4. Discussion In ACS, the plasma concentrations of TNF-a are increased at the site of coronary artery occlusion, which to such an extent induce TF expression in vascular cells [16]. As soon as plaque ruptures, high levels of TF in activated vascular ECs, VSMCs and macrophage is released to circulation which triggers the process of ACS by prompting thrombosis [5,17]. Therefore, exploring the regulation mechanism(s) of TF expression is very meaningful for constraining atherothrombosis. miRNAs have recently emerged as a novel class of gene regulators that posttranscriptionally regulate expression of multiple target genes. Increasing evidences suggest an important role of miRNAs in regulating atherosclerotic disease [18]. Enhancing disease-induced downregulated vascular miRNAs or inhibition of upregulated might be a promising strategy to cure atherosclerotic disease [19]. Herein, we reported that miRNA-223 may serve as a potent regulator of thrombosis followed with atherosclerotic plaque rupture by targeting TF, an initiator of exogenous coagulation cascade. Increased circulating TF antigen and activity were detected in patients with unstable angina or myocardial infarction compared with those with stable angina [20], which suggested a
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hypercoagulable state of patients with ACS [21]. Decreased TF expression may delay the process of atherosclerosis and consequently thrombosis [17]. More recently, some studies revealed that miRNAs were involved in hypercoagulable diseases by regulating haemostatic proteins levels [22e24]. Teruel R et al. reported that miR-19b and miR-20a could inhibit TF expression in THP1 cells, which may explain why systemic lupus erythematosus and antiphospholipid syndrome patients with decreased miR-19b and miR20a were in the hypercoagulatable state [22]. In fact, the miRNAs targeting TF are far from being discovered. Identifying more miRNAs involved in TF expression is very useful for understanding the mechanism of atherothrombosis and improving treatment. For finding new miRNAs targeting TF, we firstly performed bioinformatic prediction using two miRNA target prediction algorithms, TargetScan and miRanda. The results showed that the 30 UTR of TF mRNA contains target sites of miR-223. miR-223 binding site across several species including human, mouse, rat and dog suggested an evolutionarily conserved importance for miR-223. Recently, Shi L et al. reported that miR-223 could resist angiogenesis by targeting b1 integrin and preventing growth factor signaling in ECs [25] and Pan Y et al. found that platelet-secreted miR-223 promoted endothelial cell apoptosis induced by advanced glycation end products (AGEs) [15]. These reports imply a variety of biological role of miR-223 in the occurrence and development of athrosclerosis. To clarify the correlation of miR-223 and TF, we measured miR-223 levels in aorta from TNF-a-treated C57BL/6J mice and found that, during the upregulation of TF, miR-223 levels were significantly downregulated, which was not in line with the results reported by Taïbi F et al. [14]. In their study, miR-223 levels were increased in the aorta from ApoE/ mice with sham operations or CKD compared to wild type C57BL/6J mice with sham operations. We analyzed the reason leading to the opposite phenomenon that is mainly duo to the different time course of pathological stimulus.
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In our study, we chose to observe miR-223 levels in mice treated with TNF-a for 4 h when TF was obviously upregulated. However, in Fatiha Taïbi's study, miR-223 levels were increased in ApoE/ mice, at least, after two weeks of exposure to CKD compared to wild type controls. More importantly, instability and rupture of atherosclerotic vulnerable plaques are the pathologic basis of ACS [26], and we previously reported that the circulating miRNAs expression profiles in patients with unstable angina were different from patients with stable angina [27]. However, ApoE/ mice are not optimal models to reproduce plaque instability observed in humans [28]. Hence, we treated C57BL/6J mice with TNF-a to mimic the acute inflommation state during the rupture of plaques [16]. ECs activation and dysfunction have been linked to a variety of vascular inflammatory disease states [2] and miR-223 was reported to be correlated with inflammation [29]. So, we further observed miR-223 levels in cultured ECs incubated with TNF-a. We found that miR-223 expression decreased in ECs, whereas TF protein increased at the moment. The above in vivo and in vitro assays, combined with bioinformatics analysis, suggested that decreased miR-223 may be correlated with TF upgregulation. Next, we examined the effect of miR-223 on TF expression in ECs by using gain- and loss-of-function experiments. Overexpressed miR-223 induced TF downregulation both in mRNA and protein level. In contrast, TF levels were upregulated due to decreased miR223. To further illustrate the regulation mechanism of TF expression by miR-223, we performed luciferase reporter assay and the results indicated that miR-223 could inhibit TF expression by directly interacting with the “seed region” of TF 30 UTR. Lastly, we tested the effect of miR-223 on TF procoagulate activity. Results showed that miR-223 could not only inhibit TF procoagulate activity, but also partly block TNF-a-induced increase of TF procoagulate activity in ECs. Our results may partially explain that miR223 was inversely associated with myocardial infarction risk reported by Zampetaki et al. [30]. Inflammation could increase TF expression and activity in vascular cells. TF upregulation may reversely enhance inflammation by prompting the deposition and formation of proinlammatory fragments of fibrin in vascular wall, and by generating coagulation proteases, such as FVIIa, FXa and thrombin, which activate protease-dependent receptors [31]. Furthermore, TF is involved in cell proliferation and angiogenesis by activating MAPK pathway [32,33]. Therefore, other than antagonizing atherothrombosis, miR223 may mediate multiple biology effects in the progress of atherosclerosis through regulating TF expression. In conclusion, we discovered a novel mechanism for the regulation of TF expression by miR-223. Our study suggests an inhibitory role for miR-223 in thrombotic complications of atherosclerosis. Therapeutic interventions by elevating miR-223 in patients with atherosclerotic disease may reduce atherothrombotic events via suppressing TF expression in ECs. Sources of funding This work was supported by National Science and Technology “Creation of Major New Dugs” (No. 2012ZX09303019), Beijing Science and Technology Project (No. D141100003014002), National Natural Science Foundation of China (501100001809) (No. 81270274, No. 81270276), Beijing Natural Science Foundation (No. 7132225, No. 7122198).
Fig. 5. The effect of overexpressed miR-223 on TF procoagulant activity of EA.hy926 cells with or without TNF-a administration for 4 h. FXa generation was quantified at 405 nm after addition of FXa substrate. The procoagulant activity of TF was expressed as changes relative to the activity of cells transfected with NC mimic. Results shown were as mean ± SEM. n ¼ 4. *p < 0.05 vs NC mimic group, **p < 0.01 vs NC mimic group, #p < 0.05 vs NC mimic þ TNF-a group.
Author contributions Hong Chen and Jingyi Ren designed research and critically revised the manuscript. Sufang Li designed research, performed research, analyzed data and wrote the manuscript. Ning Xu and
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Junxian Song critically revised the manuscript. Qiang Geng Chengfu Cao, Chongyou Lee, Jing Zhang performed research. Disclosure of conflict of interests The authors state that they have no conflict of interest. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.atherosclerosis.2014.09.033. References [1] Libby P. Inflammation in atherosclerosis. Arterioscler. Thromb. Vasc. Biol. 2012;32:2045e51. [2] Hopkins PN. Molecular biology of atherosclerosis. Physiol. Rev. 2013;93: 1317e542. [3] Eilertsen KE, Østerud B. Tissue factor: (patho)physiology and cellular biology. Blood Coagul. Fibrinolysis 2004;15:521e38. [4] Mandal SK, Pendurthi UR, Rao LV. Cellular localization and trafficking of tissue factor. Blood 2006;107:4746e53. [5] Libby P. Mechanisms of acute coronary syndromes and their implications for therapy. N. Engl. J. Med. 2013;368:2004e13. [6] Furie B, Furie BC. Mechanisms of thrombus formation. N. Engl. J. Med. 2008;359:938e49. [7] Bierhaus A, Zhang Y, Deng Y, et al. Mechanism of the tumor necrosis factor alpha-mediated induction of endothelial tissue factor. J. Biol. Chem. 1995;270: 26419e32. [8] Moll T, Czyz M, Holzmüller H, et al. Regulation of the tissue factor promoter in endothelial cells. Binding of NF kappa B-, AP-1-, and Sp1-like transcription factors. J. Biol. Chem. 1995;270:3849e57. [9] Sakamoto T, Ishibashi T, Sakamoto N, et al. Endogenous NO blockade enhances tissue factor expression via increased Ca2þ influx through MCP-1 in endothelial cells by monocyte adhesion. Arterioscler. Thromb. Vasc. Biol. 2005;25: 2005e11. [10] Li YD, Ye BQ, Zheng SX, et al. NF-kappaB transcription factor p50 critically regulates tissue factor in deep vein thrombosis. J. Biol. Chem. 2009;284: 4473e83. [11] Bartel DP. MicroRNAs: target recognition and regulatory functions. Cell 2009;136:215e33. [12] Pillai RS, Bhattacharyya SN, Filipowicz W. Repression of protein synthesis by miRNAs: how many mechanisms? Trends Cell. Biol. 2007;17:118e26. [13] Santovito D, Mezzetti A, Cipollone F. MicroRNAs and atherosclerosis: new actors for an old movie. Nutr. Metab. Cardiovasc Dis. 2012;22:937e43. [14] Taïbi F, Metzinger-Le Meuth V, M'Baya-Moutoula E, et al. Possible involvement of microRNAs in vascular damage in experimental chronic kidney disease. Biochim. Biophys. Acta 2014;1842:88e98. [15] Pan Y, Liang H, Liu H, et al. Platelet-secreted microRNA-223 promotes endothelial cell apoptosis induced by advanced glycation end products via targeting the insulin-like growth factor 1 receptor. J. Immunol. 2014;192: 437e46.
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