- Email: [email protected]
145 Gene Cluster Downregulation during Aortic Dissection Formation
Accepted Manuscript P38 MAPK signaling pathway mediates AngiotensinⅡ induced miR143/145 gene cluster down-regulation during aortic dissection formation Bowen Li, Zhiwei Wang, Zhipeng Hu, Min Zhang, Zongli Ren, Zhen Zhou, Jizhen Huang, Xiaoping Hu PII:
S0890-5096(17)30119-X
DOI:
10.1016/j.avsg.2016.09.016
Reference:
AVSG 3128
To appear in:
Annals of Vascular Surgery
Received Date: 15 June 2016 Revised Date:
18 August 2016
Accepted Date: 9 September 2016
Please cite this article as: Li B, Wang Z, Hu Z, Zhang M, Ren Z, Zhou Z, Huang J, Hu X, P38 MAPK signaling pathway mediates AngiotensinⅡ induced miR143/145 gene cluster down-regulation during aortic dissection formation, Annals of Vascular Surgery (2017), doi: 10.1016/j.avsg.2016.09.016. 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 proof before it is published in its final 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.
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P38 MAPK signaling pathway mediates Angiotensin
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down-regulation during aortic dissection formation
induced miR143/145 gene cluster
3 Bowen Li1, Zhiwei Wang1,Zhipeng Hu,Min Zhang,Zongli Ren,Zhen Zhou,Jizhen Huang,Xiaoping Hu
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Department of Cardiovascular Surgery, Renmin Hospital of Wuhan University, Wuhan 430060, Hubei
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Province, PR.China
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Corresponding author : Zhiwei Wang
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Department of Cardiovascular Surgery, Renmin Hospital of Wuhan University,238 Jiefang Road,
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Wuhan 430060, Hubei Province, PR.China.
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E-mail address: [email protected]
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Conflicts of interest:The authors declare that they have no conflict of interest.
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Funding:This study was supprted by grants from the National Natural Science Foundation of China
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(no.81570428).
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Keywords: p38 MAPK signaling pathway;angiotension
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muscle cell
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;miR143/145 gene cluster;vascular smooth
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Abstract :
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Objectives:
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We endeavored to prove that angiotensin
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and differentiation of vascular smooth muscle cells (VSMCs) during the formation of aortic
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dissection. We also studied the contribution the p38 MAPK signaling pathway has towards this
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process.
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Methods:
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Ascending aortic tissues were harvested from the patients with aortic dissection (AD) and organ
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donors.Tissues were immunostained with labeled antibodies targeting p38 MAPK,phospho-p38
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MAPK,alpha-smooth muscle actin (α-SMA), and osteopontin (OPN). Next, we treated mouse
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aortic VSMCs with different regimens of Ang
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expression levels of micro-RNA143/145 (miR143/145) and VSMCs phenotype marker proteins
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(α-SMA and OPN) by quantitative PCR and/or Western blotting. SB203580 was used to inhibit
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the p38 MAPK signaling pathway. Finally, the VSMC phenotype was validated by
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immunofluorescence microscopy.
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Results:
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Expression of phospho-p38 MAPK was significantly greater in the aortic dissection tissue.Ang
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induced the phenotypic swithcing of VSMCs along with the down-regulation of a miR143/145
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gene cluster. Expression of OPN and phospho-p38 was significantly increased in VSMCs treated
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with 0.1µM Ang
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down-regulated by Ang
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pre-treatment with an SB203580 inhibitor, the expression of miR143, miR145, and VSMC
) regulates both the expression of miR143/145
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(Ang
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(duration and dosages) in vitro and determined
for 12 hours. Furthermore, expression of miR143 and miR145 was treatment. When the p38 MAPK signaling pathway was blocked by
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ACCEPTED MANUSCRIPT phenotypic markers was not affected by Ang . Immunohistochemical staining of aortic tissues
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donated by AD patients and healthy donors showed that the expression of α-SMA decreased in
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pathological tissue, while the OPN increased and the arrangment of the smooth muscle cells of the
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media was dysregulated, Which we verified in vitro.
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Conclusion:
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Ang
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VSMCs via the p38 MAPK signaling pathway. This may play an important role in the
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pathogenesis of aortic dissection.
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could regulate the expression of miR143/145 gene cluster and the phenotypic switching of
1. Introduction
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Aortic dissection (AD) occurs when a tear in the tunica intima of the aorta enables blood to breach the
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tunica media , rendering the vascular stratum vulnerable to separation and degeneration [1]. Despite
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advances in treatments, AD remains a medical emergency wherein rapid intervention is required to
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avoid mortality.
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Many have hypothesized that smooth muscle cells (SMCs) play a pivotal role in the development of
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arterial diseases owing to their function to remodel the vascular wall. Other studies meanwhile have
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demonstrated that aortic media SMCs transition from a contractile to synthetic phenotype during aortic
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dissection/aneurysm in a process known as ‘phenotypic switching’[2][3], Currently, however, the exact
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mechanism(s) underlying AD remains uncertain.
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Experiments conducted in vitro and in vivo have shown that the Angiotensin II (Ang II) growth factor
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induces cellular proliferation, differentiation, and apoptosis in addition to promoting hypertrophy of
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ACCEPTED MANUSCRIPT vascular smooth muscle cells (VSMCs) [4]. Acute stimulation with Ang II regulates salt/water
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homeostasis and vasoconstriction, modulating blood pressure, while chronic stimulation promotes
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hyperplasia and hypertrophy of VSMCs [5][6]. Ang II infusion increases aortic atherosclerosis and
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aneurysm formation, independent of blood pressure [7]. More recently, Ang II was reported to activate
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signaling cascades that activate MAPKs, most notably the extracellular signal-regulated kinase
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(ERK1/2), JNK, and p38 MAPK that are implicated in VSMC differentiation, proliferation, migration,
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and fibrosis [8],[9]. Furthermore, we have previously reported that the concentration of Ang II is
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significantly elevated in the serum of patients with AD [10]. Herein, we endeavor to clarify if the Ang
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II contributes to the phenotypic switching of VSMCs during the development of aortic dissection via
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MAPK signaling.
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Several studies have proven that p38 MAPK activity is critical for normal immune and inflammatory
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responses. There are four p38 MAP kinases in mammals: α, β, γ, and δ. Among all p38 MAPK
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isoforms, p38α is the best characterized and is expressed in most cell types. Although p38α-knockout
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murine models have been generated, they tend to die at mid-gestation [11-13], but tissue-specific
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knockouts have implicated p38α in the proliferation and survival of cardiomyocytes [14][15].
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Moreover, the p38 MAPK pathway has been implicated in the conversion of myoblasts to
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differentiated myotubes during myogenic progression [16-19]. Nevertheless, targets of the p38 MAPK
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pathway vary, both in the cytoplasm and in the nucleus, thereby suggesting that multiple cellular
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functions are under their control. One recent study reported evidence for p38 MAPK signaling
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controlling the expression of a specific set of muscle-specific genes, acting not only at the
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transcriptional level but also on mRNA turnover. In our preliminary experiment, we found that
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phospho-p38 MAPK was increased in tissues suffering AD, leading us to speculate that the p38 MAPK
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There is strong evidence for the post-transcriptional regulation of gene expression by microRNAs
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(miRNAs); a set ~22 nucleotide-long, non-coding RNAs that repress protein expression of target
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mRNAs by mRNA degradation or translational repression [20]. Many miRNAs are expressed in a
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tissue-specific manner and play pivotal roles in the control of proliferation and differentiation of
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different cell types [21-24]. The miR143/145 gene cluster includes miR143 and miR145, which lie
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within a highly conserved, 1.7-kb region on the mouse chromosome 18 [25], is restricted to adult
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SMCs lineages during their development. This family of miRNAs may promote the differentiation of
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VSMCs in part by increasing Myocd protein and functioning in a feed-forward reinforcement of its
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own expression by the SRF-myocd complex. Most notably, Boettger and colleagues have reported that
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the recently discovered miR143/145 gene cluster promotes the acquisition of a contractile phenotype of
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murine VSMCs [26].
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Thus, we make such assumptions that the Ang II regulates miR143/145 expression via the p38 MAPK
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pathway, consequently inducing the differentiation of VSMCs and thereby contributing to the
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formation of ADs.
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2. Materials and methods
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2.1 Immunohistochemistry
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Ten pathological aortic tissues were obtained from patients who underwent ascending aorta
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replacement, whilst normal were from donors who had no aortic diseases (n=10). The baseline
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ACCEPTED MANUSCRIPT characteristics of these AD patients and organ donors are presented in Table1. Tissue samples were
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fixed with 4% paraformaldehyde/0.1 M PBS (pH 7.4) for 24h. Next, fixed sections were dehydrated,
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embedded in paraffin, sectioned, dewaxed, and hydrated with 3% hydrogen peroxide to remove
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endogenous peroxidase. Prepares slides were incubated with ~50µl (1:100 dilution) rabbit anti-human
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primary antibody overnight at 25 . Then, stained slides were incubated with ~50µl goat anti-rabbit
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IgG antibody for 1h at 37
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hematoxylin and dehydrated with gradient alcohol and xylene. Immunohistochemical analysis was
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conducted as previously described [27].
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and developed with diaminobenzidine.Finally,slides were stained with
2.2 VSMC culture and isolation
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Vascular smooth muscle cells (VSMCs) were retrieved from the aorta of young, C57BL/6 mice (8
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weeks old,20-25g). Briefly, the thoracic aorta was separated under sterile conditions and washed with
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phosphate-bufered saline (PBS). Next, the adventitia was removed from the aorta and digested in a
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5-mL solution containing 0.125 mg/ml elastase,0.25 mg/ml soybean trypsin inhibor,10 mg/ml
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collagense
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cells were then filtered through a sterile 100-µm nylon mesh,centrifuged at 1,000 rpm for 10 min, and
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washed twice in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal calf serum.
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Finally, prepared VSMCs were cultured in DMEM containing 10% fetal bovine serum (FBS).VSMCs
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were first identified by staining tissues with an immunofluorescent monoclonal antibody targeting the
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α-SMA maker (Clone: ab5694; Abcam, England). Isolated VSMCs were then incubated in DMEM
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containing 10% FBS, 1% mycillin, 2mM glutamine, 50µg/ml gentamycin, and 50µL/mL
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. Digested
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amphotericin-B at 37
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experiments.
in a 5% CO2 atmosphere. Seven cell passages were used throughout most
134 2.3 Ang
treatments
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Cells were seeded in a 50mL culture flask and cultured to 70% confluence. Following serum starvation
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overnight, cells were traeted with one of 1µM, 0.1µM or 0.01µM Ang
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respectively.To study the impact of p38 MAPK (α and β) inhibition, cells were pre-treated with 10µM
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SB203580 (Selleck) 30 min prior to Ang
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for 6h, 12h or 24h,
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Cells were rinsed twice with PBS, and protein was extracted with ice-cold lysisbuffer containing a 1%
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protease inhibitor mixture (Sigma, USA) as previously described [28]. Protein concetration was
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determined with a BCA Protein Assay Kit (Takara, Japan) compatible with a detergent-protein assay
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(Perkin Elmer). Equal amounts of protein were resolved by SDS-PAGE, transferred onto
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polyvinylidene fluoride (PVDF) membranes, blocked with 5% non-fat milk for 1h, and incubated with
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primary antibody against p38 MAPK (Clone: D13E1; Cell Signaling Technology, USA), phospho-p38
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MAPK (Clone: 28B10; Cell Signaling Technology, USA), α-SMA (Clone: ab5694; Abcam, England),
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and OPN (Clone: ab91655; Abcam, England). Overnight at 4 . Membranes were then incubate with a
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secondary antibody (Licor, USA) for 1h and blot signals were revealed by addition of an enhanced
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chemiluminescence reagent (Odyssey).
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2.5 RNA extraction and RT-PCR
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and use as a template from which cDNA was reverse transcribed using a First Strand cDNA Synthesis
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Kit (Takara, Japan). miR levels were subsequently determined by quantitative, SYBR Green-based
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real-time PCR (ABI 7500). The U6 gene was used as the control housekeeping gene. Data was
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normalized as per the 2-△△Ct method for previously described [29]. The primers and their sequences are
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listed in the Supplementary Table.2.
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VSMCs were dispersed under sterile glass coverslips and grown to 70% confluence. After 12h of
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serum starvation, cells were stimulated with 0.1µM AngⅡfor 12h. Immunofluorescent labeling of
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α-SMA and OPN was conducted as described previously [28]. Briefly, cells were washed twice with
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PBS (PH 7.4), fixed with freshly prepared 4% paraformaldehyde for 20min, rinsed thrice with
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PBS-Tween 20, and permeabilized with 0.1% Triton X-100 for 10 min. Slides were then stained with
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either a 1:100 dilution of rabbit anti-mouse α-SMA or a 1:150 dilution of anti-OPN IgG. Nuclear
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profiles were revealed with a brief incubation in DAPI (Molecular Probes, USA) prior to microscopic
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obserbvation. Fluorescence signals were visualized with an upright Olympus BX51 fluorescence
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microscope and imaged for direct importation into Adobe Photoshop. All images were processed in a
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standardized manner to faithfully capture the real-time images of each sample.
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2.7 Statistical analyses
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Data were expressed as mean ± SD. Paired and/or unpaired Student’s t-tests were used to evaluate the
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statistical significance of differences between the means of 2 groups, while analysis of variance
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(ANOVA) was performed to determine significance across multiple groups. A P-value <0.05 was
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considered statistically significant.All graphs were fitted with Sigma Plot Version 10.0 software.
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3.1 The p38 MAPK pathway is active during AD
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This was determined by perfoming a immunohistochemistry. There was a significant increase in the
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expression of phospho-p38 MAPK, whilst that of p38 MAPK decreased slightly (P < 0.05; Fig.1),
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thereby indicating that the p38 MAPK pathway is active during AD.
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3.2 The miR143/145 gene cluster is downregulated during the phenotypic switching of VSMCs in
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AD
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For the purpose of verifying the expression of the miR143/145 gene cluster and phenotypic switching
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of VSMCs during AD, we immunostained VSMC undergoing AD for the α-SMA and OPN contractile
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and synthetic phenotypic markers, respectively. Our data showed that the expression of α-SMA was
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decreased in the media of the pathological aortic tissue, whilst that of
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significantly compared the normal group. Furthermore, the arrangment of the smooth muscle cells was
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disordered in all of the pathological tissues (P < 0.05; Fig.2). We also performed the q-PCR to
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determine the expression of miR143 and miR145 in AD samples, This data showeds that the miR143
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and miR145 were significantly downregulated in the pathological specimens ( P < 0.05; Fig.3).
OPN was increased
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3.3 Ang
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vitro
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In order to demonstrate that Ang
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phenotypic switching,we treated mouse aortic VSMCs in vitro, with either 0, 1µM, 0.1µM, 0.01µM
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Ang
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p38 MAPK, phospho-p38 MAPK, α-SMA, and OPN. Whilst the expression of p38 MAPK decreased
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significantly (P < 0.05) post-treament with 0.1µM Ang
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significantly increased (P < 0.05; Fig.4). There was a tendency of α-SMA to be upregulated
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post-treatment with Ang
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significant. To the contrary, expression of OPN markedly increased post-treatment with 0.1µM Ang
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for 12h (P < 0.05; Fig.5). Immunofluorescent staining control group highlighted that the signal of OPN
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was notably weaker to that of α-SMA. This fluorescent signal was greater post-treatment with
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Ang .When VSMCs were pre-treated with a p38 MAPK inhibitor before Ang , the expression of
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α-SMA decreased, while that of OPN significantly increased, whilst the expression of OPN was not
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afftected by Ang
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could activate the p38 MAPK pathway and trigger downstream
for 0h, 6h, 12h or 24h, respectively. Thereafter, we probed Western blots with antibodies against
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activated the p38 MAPK pathway and induced the phenotypic switching of VSMCs in
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3.4 The p38 MAPK pathway mediates the Ang
-induced down-regulation of miR143 and miR
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145 during the phenotypic switching of VSMCs
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To verify the impact of Ang
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q-PCR to determine these expression profiles in VSMCs treated with 0.1µM Ang
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showed that the expression of miR143 and miR145 was markedly decreased compared to the control
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group (P < 0.05; Fig.7). We then pre-treated VSMCs with a specific inhibitor of the p38 MAPK
stimulation on the expression of the miR143/145 gene cluster,we used
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for 12h. Our data
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pathway immediately prior to addition of 10µM Ang
. Our Western blotting data showed that p38
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MAPK was all but inhibited (P < 0.001). Furthermore, the p38 MAPK inhibitor blocked the activity of
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Ang
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Western-blotting revealed that the expression of α-SMA was decreased (P < 0.05), while that of OPN
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increased (P < 0.05; Fig.9). q-PCR showed that the expression of miR143 miR145 were decreased
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when compared with the control group (P < 0.05) .
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225 4. Discussion
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The present study has discovered a possible mechanism underlying AD. Ang , an important member
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of the renin-angiotensin system and heavily implicated in the pathogenesis of AD, can induce the
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phenotypic switching of VSMCs via the downregulation of miR143/145 gene cluster. This down
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regulation was mediated by the p38 MAPK signaling pathway.
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Cells respond to changes in their physical and chemical environment by altering many cellular
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programs, including cell survival, proliferative, differentiation, metabolism, interactions with other
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cells, and numerous homeostatic loops. VSMCs switch their phenotype - from contractile to synthetic -
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in respond to the local environment cues, in a process known as phenotypic modulation or switching.
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We have demonstrated that the expression of OPN was much greater, and α-SMA lower, in the media
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of specimens taken from tissues undergoing AD than their non-pathological controls, thereby alluding
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that a phenotypic switch had taken place. Our finding is consistent with that reported by Lesauskaite
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and co-workers [2], of whom also observed the phenotypic switching of VSMCs in AD/aneurysm
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tissues. We then treated VSMCs in vitro with Ang
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that this intervention tended to elevate expression of α-SMA, albeit non-significantly. Meanwhile, we
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, and subsequently observed by Western-blotting
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ACCEPTED MANUSCRIPT also observed that OPN was significantly elevated post-treatment, thereby suggesting that high dosage
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of Ang II promotes the contract of vascular and induces the phenotypic switching of VSMCs from
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contractile to synthetic.
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Ang II induces the differentiation of VSMCs in numerous cell culture models. However, there are
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almost no studies that have examined the genes underlying the differentiation of VSMCs induced Ang
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-treated VSMCs, and nothing is known about its modulation by micoRNAs. We thus sought to characterize the phenotypic response of VSMCs to Ang
stimulation. The p38 MAPK pathway is one
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of the stress-activated protein knases and is strongly activated in vivo by environmental stresses and
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inflammatory cytokines [30]. It has been reported to function as a molecular switch to activate the
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muscle stem cells [31-33], but these studies were mostly restricted to the VSMCs of
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artery[34]. Here, we found that the expression of phospho-p38 MAPK was increased in tissues
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undergoing AD, suggesting that the p38 MAPK pathway was activated too. In addition, stimulating by
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Ang
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in vitro, meanwhile the expression of OPN increased as well. And since the expression of OPN tended
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to still increase despite pretreatment with an inhibitor of p38 MAPK before Ang
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involvement of the p38 MAPK pathway in the Ang
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MicroRNAs regulate the fate of cardiovascular cell, albeit inefficiently [22,35-39]. Kimberly and
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colleagues demonstrated that the miR143/145 gene cluster regulate the fate of VSMCs in regards to
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their proliferation, differentiation, migration, and so on [25]. Here, we have provided evidence that Ang
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stimulation downregulates transcription of the miR143/145 gene cluster. We also observed that the
coronary
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, this data favors the
-induced phenotypic switching of aortic VSMCs.
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enhanced the expression of phospho-p38 MAPK of mouse aortic VSMCs increased significantly
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expression of miR143/145 gene cluster reduced in tissues undergoing AD. Our in vitro experiments
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showed it affects the expression of phenotypic markers of VSMCs. Stimulation of VSMCs by Ang
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significantly downregulated the expression of miR143 and miR145 in combination with their
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phenotype switching from contractile to synthetic, thereby suggesting that the miR143/145 gene cluster
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is a downstream factor of Ang
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blocking the p38 MAPK pathway with an inhibitor suppressed this modulation of miR143 and miR145
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by Ang
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pathway. All of these findings have many implications for the p38 MAPK pathway, most notably that it
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may play a key role on the miR143/145 gene cluster regulated phenotypic modulation of aortic VSMCs
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induced by Ang
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The dysfunction of VSMCs leads to a varitey of artery diseaseas of humans, including atherosclerosis,
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AD/aneurysm, cancer, and Alzheimer’s disease [40-43]. AD is an acute and catastrophic illness, and a
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serious threat to human health. Normal aorta contains an elastic layer (mainly composed of smooth
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muscle cells), and a layer of elastic fiber that links the continuous frame structure. This the
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arrangement, however, along with the morphology of the fiber layer was irregular in AD samples.
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During the formation of AD and the characteristic degeneration of the aortic media, the elastic fiber
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breakage loss, phenotypic modulation of SMCs, loss of SMCs and proteoglycans material gathered
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characterized. The pathogenesis of AD is likely to be multifaceted, though, with other researchers
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reporting that inflammation genes are switched on, extracellular matrix protein degrades, and
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transformation and transcription are upregulated, while the genes encoding the extracellular matrix, cell
281
adhesion proteins and cytoskeleton proteins are downregulated, thereby indicating that inflammatory
282
response promotes the development of AD as well [44]. Ang
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AD/aneurysm wherein it can contribute to this disease through inflammatory and non-inflammatory
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approaches (Fig.10). In the present study, we aim to demonstrate the mechanism that underlying the
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has long been thought as a risk factor of
ACCEPTED MANUSCRIPT phenotypic switching of VSMCs during the development of AD. For this, we used Ang
as the
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stimulus of in our in vitro experiments, subsequently linking the p38 MAPK pathway to the
287
miR143/145 gene cluster during the phenotypic switching of mouse aortic VSMCs. Our preliminary
288
experiments proved first that the miR143/145 gene cluster is a positive factor in the differentiation of
289
VSMCs and secondly that the Ang
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the p38 MAPK pathway.
291
We acknowledge our investigations are limited by the nature of the study’s representative design.
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Accordingly, it is impossible using our data to determine if the activation of p38 MAPK signaling
293
occurred when the downregulation of miR143/145 gene cluster occurred in the pathogenesis of AD-
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whether before or after its onset. Experimentation of a p38 MAPK-knockdown animal model will be
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critical in verifying our findings.
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-induced downregulation of miR143 and miR145 is regulated by
5. Conclusions
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We have shown that Ang II affects the expression of the miR143/145 gene cluster in mouse aortic
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VSMCs and promotes the phenotypic switching of VSMCs. Our data supports the involvement of the
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p38 MAPK signaling pathway amongst this. These outcomes may offer another mechanism of Ang
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II-induced VSMCs phenotypic modulation during the formation of AD. Our study suggests that the
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miR143 /145 gene cluster may be an attractive target for strategies aiming to prevent the formation of
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AD.
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6. Acknowledgements
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The authors would like to thank all the patients and donors and the Ethics Committee of Renmin
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Hospital of WuHan University, China for supportting this study.
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42.Pauletto P, Sarzani R, Rappelli A, Chiavegato A,Pessina AC,Sartore S .Differentiation and growth of
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vascular smooth muscle cells in experimental hypertension. Am J Hypertens.1994; 7(7 Pt 1):661–674.
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43.Xu Q. Stem cells and transplant arteriosclerosis. Circ Res.2008; 102(9):1011–1024.
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differentiation. Nat. Genet.2006;
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44.Müller BT, Modlich O, Prisack HB, Bojar H,Schipke JD,Goecke T,et al. Gene expression profiles in
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the acutely dissected human aorta. Eur J Vasc Endovasc Surg.2002; 24(4): 356-364.
418 Figure legends:
420
Fig.1 There is no expression of pp38 MAPK in normal aorta (A),the expression of pp38MAPK significant increased in aortic dissection
421
tissue (B; *P<0.05); the expression of p38MAPK decreased in aortic dissection tissue (D) compared with the normal tissue (C; *P<0.05).
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Fig.2 The expression of OPN, the marker of synthetic phenotype of VSMCs,increased significantly in the pathological tissue (D)
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when compared to the normal tissue (C; *P<0.05); the expression of α-SMA is abundantly in the aorta (A ),while decreased significantly
425
in the aortic dissection tissue (B; *P<0.05); the arrangment of smooth muscle cells were disorded in the pathological tissue.
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Fig.3 The miR143 and miR145 were down-regulated (*P<0.05)in the aortic dissection tissues when compared to the donor’s aorta.
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Fig.4 After stimulate by Ang at different concentrations for 6h,12h,24h,respectivly,the expression of p38 MAPK decreased,the 0.1M for
430
12h has the most significant decrease (*P<0.05) (A).Meanwhile,the expression of phospho-p38 MAPK was increased (*P<0.05) with
431
Ang
432
vitro.
interven,the most significant increase showing at the 0.1M for 12h (B),indicating that Ang
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activate the p38 MAPK pathway in
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Fig.5 The Ang induced the phenotypic marker of VSMCs transfored.When stimulate by Ang
435
each concentration and time,but there is no significant difference of each group .The Ang
436
siginificantly (*P<0.05) in vitro,and the most difference showing at 0.1M for 12h,indicating that the phenotypic swithcing of mouse
437
aortic smooth muscle cells.
20
,the expression of α-SMA increased at
induced the expression of OPN increased
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Fig.6 Immunofluorescence showed the brightness of the α-SMA in the control group is greater than the OPN obviously;while in the 0.1M
440
Ang
441
weaker than the control group,and the expression of OPN increased;in the inhibitor with 0.1M Ang
442
brightness of OPN compared with the inhibitor alone group.
group,there is more brightness of OPN compared to the control group;after added the inhibitor,the brightness of α-SMA become
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group,there is no difference of
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Fig.7 After stimulate by Ang for 12h,the expression of miR143 and miR145 was significantly decreased (*P<0.05),the miR145 has the
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more obvious decrease.
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Fig.8 After using the inhibitor,SB203580,the expression of p38 MAPK pathway was blocked (*P<0.001),and there is no phospho-p38
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MAPK expression (A).When the p38 MAPK pathway was blocked ,the expression of OPN increased significantly (***P<0.05),while the
449
α-SMA decreased (**P<0.05),and the expression of phenotypic marker of VSMCs didn’t affected by the stimulation of Ang
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(B).
Fig.9 When added the inhibitor,the expression of miR143 and miR145 decreased significantly (P<0.05),and the miR145 is lower than
452
the miR143,then stimulated by the Ang ,the expression of miRs have no difference with the inhibitor alone group.
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contribute to aortic diseases through inflammatory and non-inflammatory approaches.
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21
ACCEPTED MANUSCRIPT Aortic dissection group
Organ donors group
50.14±6.48
49.71±7.28
Age(year)
10/0 58.62±11.08
Type of aortic disease(n)
10
0
Acute aortic dissection
10
0
Marfan Syndrome
0
0
Thoracic aortic aneurysm
0
0
Comorbidities (n) Hypertensive disease
10
Diabetes mellitus
0
Heart disease
0
COPD
0
Renal insufficiency
0
10
COPD= chronic obstructive pulmonary disease
0 0 0 0 0
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Weight(kg)
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Male/female(n)
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Table.1 Characteristics of the aortic dissection patients and organ donors
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Primer sequences
miR143
RT-primer
5’-CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGCCAGAGAT-3’
Forward-primer 5’-ACACTCCAGCTGGGGGTGCAGTGCTGCAT-3’
miR145
Reverse-primer
5’-TGGTGTCGTGGAGTCG-3’
RT-primer
5’-CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGAGGGATTC-3’
U6
Reverse-primer
5’-TGGTGTCGTGGAGTCG-3’
RT-primer
5’-AACGCTTCACGAATTTGCGT-3’
Forward-primer 5’-CTCGCTTCGGCAGCACA-3’ Reverse-primer
5’-AACGCTTCACGAATTTGCGT-3’
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Table.1 The primers used for different target miRNAs.
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Forward-primer 5’-ACACTCCAGCTGGGGTCCAGTTTTCCCAGGA-3’
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S0890-5096(17)30119-X
DOI:
10.1016/j.avsg.2016.09.016
Reference:
AVSG 3128
To appear in:
Annals of Vascular Surgery
Received Date: 15 June 2016 Revised Date:
18 August 2016
Accepted Date: 9 September 2016
Please cite this article as: Li B, Wang Z, Hu Z, Zhang M, Ren Z, Zhou Z, Huang J, Hu X, P38 MAPK signaling pathway mediates AngiotensinⅡ induced miR143/145 gene cluster down-regulation during aortic dissection formation, Annals of Vascular Surgery (2017), doi: 10.1016/j.avsg.2016.09.016. 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 proof before it is published in its final 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.
ACCEPTED MANUSCRIPT 1
P38 MAPK signaling pathway mediates Angiotensin
2
down-regulation during aortic dissection formation
induced miR143/145 gene cluster
3 Bowen Li1, Zhiwei Wang1,Zhipeng Hu,Min Zhang,Zongli Ren,Zhen Zhou,Jizhen Huang,Xiaoping Hu
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Department of Cardiovascular Surgery, Renmin Hospital of Wuhan University, Wuhan 430060, Hubei
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Province, PR.China
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Corresponding author : Zhiwei Wang
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Department of Cardiovascular Surgery, Renmin Hospital of Wuhan University,238 Jiefang Road,
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Wuhan 430060, Hubei Province, PR.China.
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E-mail address: [email protected]
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Conflicts of interest:The authors declare that they have no conflict of interest.
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Funding:This study was supprted by grants from the National Natural Science Foundation of China
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(no.81570428).
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Keywords: p38 MAPK signaling pathway;angiotension
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muscle cell
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;miR143/145 gene cluster;vascular smooth
ACCEPTED MANUSCRIPT 23
Abstract :
24
Objectives:
25
We endeavored to prove that angiotensin
26
and differentiation of vascular smooth muscle cells (VSMCs) during the formation of aortic
27
dissection. We also studied the contribution the p38 MAPK signaling pathway has towards this
28
process.
29
Methods:
30
Ascending aortic tissues were harvested from the patients with aortic dissection (AD) and organ
31
donors.Tissues were immunostained with labeled antibodies targeting p38 MAPK,phospho-p38
32
MAPK,alpha-smooth muscle actin (α-SMA), and osteopontin (OPN). Next, we treated mouse
33
aortic VSMCs with different regimens of Ang
34
expression levels of micro-RNA143/145 (miR143/145) and VSMCs phenotype marker proteins
35
(α-SMA and OPN) by quantitative PCR and/or Western blotting. SB203580 was used to inhibit
36
the p38 MAPK signaling pathway. Finally, the VSMC phenotype was validated by
37
immunofluorescence microscopy.
38
Results:
39
Expression of phospho-p38 MAPK was significantly greater in the aortic dissection tissue.Ang
40
induced the phenotypic swithcing of VSMCs along with the down-regulation of a miR143/145
41
gene cluster. Expression of OPN and phospho-p38 was significantly increased in VSMCs treated
42
with 0.1µM Ang
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down-regulated by Ang
44
pre-treatment with an SB203580 inhibitor, the expression of miR143, miR145, and VSMC
) regulates both the expression of miR143/145
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(Ang
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(duration and dosages) in vitro and determined
for 12 hours. Furthermore, expression of miR143 and miR145 was treatment. When the p38 MAPK signaling pathway was blocked by
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ACCEPTED MANUSCRIPT phenotypic markers was not affected by Ang . Immunohistochemical staining of aortic tissues
46
donated by AD patients and healthy donors showed that the expression of α-SMA decreased in
47
pathological tissue, while the OPN increased and the arrangment of the smooth muscle cells of the
48
media was dysregulated, Which we verified in vitro.
49
Conclusion:
50
Ang
51
VSMCs via the p38 MAPK signaling pathway. This may play an important role in the
52
pathogenesis of aortic dissection.
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could regulate the expression of miR143/145 gene cluster and the phenotypic switching of
1. Introduction
55
Aortic dissection (AD) occurs when a tear in the tunica intima of the aorta enables blood to breach the
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tunica media , rendering the vascular stratum vulnerable to separation and degeneration [1]. Despite
58
advances in treatments, AD remains a medical emergency wherein rapid intervention is required to
59
avoid mortality.
60
Many have hypothesized that smooth muscle cells (SMCs) play a pivotal role in the development of
61
arterial diseases owing to their function to remodel the vascular wall. Other studies meanwhile have
62
demonstrated that aortic media SMCs transition from a contractile to synthetic phenotype during aortic
63
dissection/aneurysm in a process known as ‘phenotypic switching’[2][3], Currently, however, the exact
64
mechanism(s) underlying AD remains uncertain.
65
Experiments conducted in vitro and in vivo have shown that the Angiotensin II (Ang II) growth factor
66
induces cellular proliferation, differentiation, and apoptosis in addition to promoting hypertrophy of
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ACCEPTED MANUSCRIPT vascular smooth muscle cells (VSMCs) [4]. Acute stimulation with Ang II regulates salt/water
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homeostasis and vasoconstriction, modulating blood pressure, while chronic stimulation promotes
69
hyperplasia and hypertrophy of VSMCs [5][6]. Ang II infusion increases aortic atherosclerosis and
70
aneurysm formation, independent of blood pressure [7]. More recently, Ang II was reported to activate
71
signaling cascades that activate MAPKs, most notably the extracellular signal-regulated kinase
72
(ERK1/2), JNK, and p38 MAPK that are implicated in VSMC differentiation, proliferation, migration,
73
and fibrosis [8],[9]. Furthermore, we have previously reported that the concentration of Ang II is
74
significantly elevated in the serum of patients with AD [10]. Herein, we endeavor to clarify if the Ang
75
II contributes to the phenotypic switching of VSMCs during the development of aortic dissection via
76
MAPK signaling.
77
Several studies have proven that p38 MAPK activity is critical for normal immune and inflammatory
78
responses. There are four p38 MAP kinases in mammals: α, β, γ, and δ. Among all p38 MAPK
79
isoforms, p38α is the best characterized and is expressed in most cell types. Although p38α-knockout
80
murine models have been generated, they tend to die at mid-gestation [11-13], but tissue-specific
81
knockouts have implicated p38α in the proliferation and survival of cardiomyocytes [14][15].
82
Moreover, the p38 MAPK pathway has been implicated in the conversion of myoblasts to
83
differentiated myotubes during myogenic progression [16-19]. Nevertheless, targets of the p38 MAPK
84
pathway vary, both in the cytoplasm and in the nucleus, thereby suggesting that multiple cellular
85
functions are under their control. One recent study reported evidence for p38 MAPK signaling
86
controlling the expression of a specific set of muscle-specific genes, acting not only at the
87
transcriptional level but also on mRNA turnover. In our preliminary experiment, we found that
88
phospho-p38 MAPK was increased in tissues suffering AD, leading us to speculate that the p38 MAPK
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ACCEPTED MANUSCRIPT pathway may 1) regulate genes pertinent to VSMCs and 2) contribute to the formation of ADs.
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There is strong evidence for the post-transcriptional regulation of gene expression by microRNAs
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(miRNAs); a set ~22 nucleotide-long, non-coding RNAs that repress protein expression of target
92
mRNAs by mRNA degradation or translational repression [20]. Many miRNAs are expressed in a
93
tissue-specific manner and play pivotal roles in the control of proliferation and differentiation of
94
different cell types [21-24]. The miR143/145 gene cluster includes miR143 and miR145, which lie
95
within a highly conserved, 1.7-kb region on the mouse chromosome 18 [25], is restricted to adult
96
SMCs lineages during their development. This family of miRNAs may promote the differentiation of
97
VSMCs in part by increasing Myocd protein and functioning in a feed-forward reinforcement of its
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own expression by the SRF-myocd complex. Most notably, Boettger and colleagues have reported that
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the recently discovered miR143/145 gene cluster promotes the acquisition of a contractile phenotype of
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murine VSMCs [26].
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Thus, we make such assumptions that the Ang II regulates miR143/145 expression via the p38 MAPK
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pathway, consequently inducing the differentiation of VSMCs and thereby contributing to the
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formation of ADs.
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2. Materials and methods
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2.1 Immunohistochemistry
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Ten pathological aortic tissues were obtained from patients who underwent ascending aorta
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replacement, whilst normal were from donors who had no aortic diseases (n=10). The baseline
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ACCEPTED MANUSCRIPT characteristics of these AD patients and organ donors are presented in Table1. Tissue samples were
112
fixed with 4% paraformaldehyde/0.1 M PBS (pH 7.4) for 24h. Next, fixed sections were dehydrated,
113
embedded in paraffin, sectioned, dewaxed, and hydrated with 3% hydrogen peroxide to remove
114
endogenous peroxidase. Prepares slides were incubated with ~50µl (1:100 dilution) rabbit anti-human
115
primary antibody overnight at 25 . Then, stained slides were incubated with ~50µl goat anti-rabbit
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IgG antibody for 1h at 37
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hematoxylin and dehydrated with gradient alcohol and xylene. Immunohistochemical analysis was
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conducted as previously described [27].
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2.2 VSMC culture and isolation
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Vascular smooth muscle cells (VSMCs) were retrieved from the aorta of young, C57BL/6 mice (8
122
weeks old,20-25g). Briefly, the thoracic aorta was separated under sterile conditions and washed with
123
phosphate-bufered saline (PBS). Next, the adventitia was removed from the aorta and digested in a
124
5-mL solution containing 0.125 mg/ml elastase,0.25 mg/ml soybean trypsin inhibor,10 mg/ml
125
collagense
126
cells were then filtered through a sterile 100-µm nylon mesh,centrifuged at 1,000 rpm for 10 min, and
127
washed twice in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal calf serum.
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Finally, prepared VSMCs were cultured in DMEM containing 10% fetal bovine serum (FBS).VSMCs
129
were first identified by staining tissues with an immunofluorescent monoclonal antibody targeting the
130
α-SMA maker (Clone: ab5694; Abcam, England). Isolated VSMCs were then incubated in DMEM
131
containing 10% FBS, 1% mycillin, 2mM glutamine, 50µg/ml gentamycin, and 50µL/mL
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. Digested
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amphotericin-B at 37
133
experiments.
in a 5% CO2 atmosphere. Seven cell passages were used throughout most
134 2.3 Ang
treatments
136
Cells were seeded in a 50mL culture flask and cultured to 70% confluence. Following serum starvation
137
overnight, cells were traeted with one of 1µM, 0.1µM or 0.01µM Ang
138
respectively.To study the impact of p38 MAPK (α and β) inhibition, cells were pre-treated with 10µM
139
SB203580 (Selleck) 30 min prior to Ang
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for 6h, 12h or 24h,
140 2.4 Western blotting
142
Cells were rinsed twice with PBS, and protein was extracted with ice-cold lysisbuffer containing a 1%
143
protease inhibitor mixture (Sigma, USA) as previously described [28]. Protein concetration was
144
determined with a BCA Protein Assay Kit (Takara, Japan) compatible with a detergent-protein assay
145
(Perkin Elmer). Equal amounts of protein were resolved by SDS-PAGE, transferred onto
146
polyvinylidene fluoride (PVDF) membranes, blocked with 5% non-fat milk for 1h, and incubated with
147
primary antibody against p38 MAPK (Clone: D13E1; Cell Signaling Technology, USA), phospho-p38
148
MAPK (Clone: 28B10; Cell Signaling Technology, USA), α-SMA (Clone: ab5694; Abcam, England),
149
and OPN (Clone: ab91655; Abcam, England). Overnight at 4 . Membranes were then incubate with a
150
secondary antibody (Licor, USA) for 1h and blot signals were revealed by addition of an enhanced
151
chemiluminescence reagent (Odyssey).
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2.5 RNA extraction and RT-PCR
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and use as a template from which cDNA was reverse transcribed using a First Strand cDNA Synthesis
156
Kit (Takara, Japan). miR levels were subsequently determined by quantitative, SYBR Green-based
157
real-time PCR (ABI 7500). The U6 gene was used as the control housekeeping gene. Data was
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normalized as per the 2-△△Ct method for previously described [29]. The primers and their sequences are
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listed in the Supplementary Table.2.
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160 2.6 Immunofluorescence microscopy
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VSMCs were dispersed under sterile glass coverslips and grown to 70% confluence. After 12h of
163
serum starvation, cells were stimulated with 0.1µM AngⅡfor 12h. Immunofluorescent labeling of
164
α-SMA and OPN was conducted as described previously [28]. Briefly, cells were washed twice with
165
PBS (PH 7.4), fixed with freshly prepared 4% paraformaldehyde for 20min, rinsed thrice with
166
PBS-Tween 20, and permeabilized with 0.1% Triton X-100 for 10 min. Slides were then stained with
167
either a 1:100 dilution of rabbit anti-mouse α-SMA or a 1:150 dilution of anti-OPN IgG. Nuclear
168
profiles were revealed with a brief incubation in DAPI (Molecular Probes, USA) prior to microscopic
169
obserbvation. Fluorescence signals were visualized with an upright Olympus BX51 fluorescence
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microscope and imaged for direct importation into Adobe Photoshop. All images were processed in a
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standardized manner to faithfully capture the real-time images of each sample.
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2.7 Statistical analyses
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Data were expressed as mean ± SD. Paired and/or unpaired Student’s t-tests were used to evaluate the
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statistical significance of differences between the means of 2 groups, while analysis of variance
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(ANOVA) was performed to determine significance across multiple groups. A P-value <0.05 was
177
considered statistically significant.All graphs were fitted with Sigma Plot Version 10.0 software.
178 3. Results
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3.1 The p38 MAPK pathway is active during AD
182
This was determined by perfoming a immunohistochemistry. There was a significant increase in the
183
expression of phospho-p38 MAPK, whilst that of p38 MAPK decreased slightly (P < 0.05; Fig.1),
184
thereby indicating that the p38 MAPK pathway is active during AD.
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3.2 The miR143/145 gene cluster is downregulated during the phenotypic switching of VSMCs in
187
AD
188
For the purpose of verifying the expression of the miR143/145 gene cluster and phenotypic switching
189
of VSMCs during AD, we immunostained VSMC undergoing AD for the α-SMA and OPN contractile
190
and synthetic phenotypic markers, respectively. Our data showed that the expression of α-SMA was
191
decreased in the media of the pathological aortic tissue, whilst that of
192
significantly compared the normal group. Furthermore, the arrangment of the smooth muscle cells was
193
disordered in all of the pathological tissues (P < 0.05; Fig.2). We also performed the q-PCR to
194
determine the expression of miR143 and miR145 in AD samples, This data showeds that the miR143
195
and miR145 were significantly downregulated in the pathological specimens ( P < 0.05; Fig.3).
OPN was increased
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3.3 Ang
198
vitro
199
In order to demonstrate that Ang
200
phenotypic switching,we treated mouse aortic VSMCs in vitro, with either 0, 1µM, 0.1µM, 0.01µM
201
Ang
202
p38 MAPK, phospho-p38 MAPK, α-SMA, and OPN. Whilst the expression of p38 MAPK decreased
203
significantly (P < 0.05) post-treament with 0.1µM Ang
204
significantly increased (P < 0.05; Fig.4). There was a tendency of α-SMA to be upregulated
205
post-treatment with Ang
206
significant. To the contrary, expression of OPN markedly increased post-treatment with 0.1µM Ang
207
for 12h (P < 0.05; Fig.5). Immunofluorescent staining control group highlighted that the signal of OPN
208
was notably weaker to that of α-SMA. This fluorescent signal was greater post-treatment with
209
Ang .When VSMCs were pre-treated with a p38 MAPK inhibitor before Ang , the expression of
210
α-SMA decreased, while that of OPN significantly increased, whilst the expression of OPN was not
211
afftected by Ang
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could activate the p38 MAPK pathway and trigger downstream
for 0h, 6h, 12h or 24h, respectively. Thereafter, we probed Western blots with antibodies against
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regardless of dosage and duration, though this was not statistically
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(Fig.6).
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activated the p38 MAPK pathway and induced the phenotypic switching of VSMCs in
213
3.4 The p38 MAPK pathway mediates the Ang
-induced down-regulation of miR143 and miR
214
145 during the phenotypic switching of VSMCs
215
To verify the impact of Ang
216
q-PCR to determine these expression profiles in VSMCs treated with 0.1µM Ang
217
showed that the expression of miR143 and miR145 was markedly decreased compared to the control
218
group (P < 0.05; Fig.7). We then pre-treated VSMCs with a specific inhibitor of the p38 MAPK
stimulation on the expression of the miR143/145 gene cluster,we used
10
for 12h. Our data
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pathway immediately prior to addition of 10µM Ang
. Our Western blotting data showed that p38
220
MAPK was all but inhibited (P < 0.001). Furthermore, the p38 MAPK inhibitor blocked the activity of
221
Ang
222
Western-blotting revealed that the expression of α-SMA was decreased (P < 0.05), while that of OPN
223
increased (P < 0.05; Fig.9). q-PCR showed that the expression of miR143 miR145 were decreased
224
when compared with the control group (P < 0.05) .
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, wherein the expression of miR143 and miR145 was not notably affected (Fig.8).
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225 4. Discussion
227
The present study has discovered a possible mechanism underlying AD. Ang , an important member
228
of the renin-angiotensin system and heavily implicated in the pathogenesis of AD, can induce the
229
phenotypic switching of VSMCs via the downregulation of miR143/145 gene cluster. This down
230
regulation was mediated by the p38 MAPK signaling pathway.
231
Cells respond to changes in their physical and chemical environment by altering many cellular
232
programs, including cell survival, proliferative, differentiation, metabolism, interactions with other
233
cells, and numerous homeostatic loops. VSMCs switch their phenotype - from contractile to synthetic -
234
in respond to the local environment cues, in a process known as phenotypic modulation or switching.
235
We have demonstrated that the expression of OPN was much greater, and α-SMA lower, in the media
236
of specimens taken from tissues undergoing AD than their non-pathological controls, thereby alluding
237
that a phenotypic switch had taken place. Our finding is consistent with that reported by Lesauskaite
238
and co-workers [2], of whom also observed the phenotypic switching of VSMCs in AD/aneurysm
239
tissues. We then treated VSMCs in vitro with Ang
240
that this intervention tended to elevate expression of α-SMA, albeit non-significantly. Meanwhile, we
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, and subsequently observed by Western-blotting
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ACCEPTED MANUSCRIPT also observed that OPN was significantly elevated post-treatment, thereby suggesting that high dosage
242
of Ang II promotes the contract of vascular and induces the phenotypic switching of VSMCs from
243
contractile to synthetic.
244
Ang II induces the differentiation of VSMCs in numerous cell culture models. However, there are
245
almost no studies that have examined the genes underlying the differentiation of VSMCs induced Ang
246
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-treated VSMCs, and nothing is known about its modulation by micoRNAs. We thus sought to characterize the phenotypic response of VSMCs to Ang
stimulation. The p38 MAPK pathway is one
248
of the stress-activated protein knases and is strongly activated in vivo by environmental stresses and
249
inflammatory cytokines [30]. It has been reported to function as a molecular switch to activate the
250
muscle stem cells [31-33], but these studies were mostly restricted to the VSMCs of
251
artery[34]. Here, we found that the expression of phospho-p38 MAPK was increased in tissues
252
undergoing AD, suggesting that the p38 MAPK pathway was activated too. In addition, stimulating by
253
Ang
254
in vitro, meanwhile the expression of OPN increased as well. And since the expression of OPN tended
255
to still increase despite pretreatment with an inhibitor of p38 MAPK before Ang
256
involvement of the p38 MAPK pathway in the Ang
257
MicroRNAs regulate the fate of cardiovascular cell, albeit inefficiently [22,35-39]. Kimberly and
258
colleagues demonstrated that the miR143/145 gene cluster regulate the fate of VSMCs in regards to
259
their proliferation, differentiation, migration, and so on [25]. Here, we have provided evidence that Ang
260
stimulation downregulates transcription of the miR143/145 gene cluster. We also observed that the
coronary
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, this data favors the
-induced phenotypic switching of aortic VSMCs.
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enhanced the expression of phospho-p38 MAPK of mouse aortic VSMCs increased significantly
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expression of miR143/145 gene cluster reduced in tissues undergoing AD. Our in vitro experiments
262
showed it affects the expression of phenotypic markers of VSMCs. Stimulation of VSMCs by Ang
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significantly downregulated the expression of miR143 and miR145 in combination with their
264
phenotype switching from contractile to synthetic, thereby suggesting that the miR143/145 gene cluster
265
is a downstream factor of Ang
266
blocking the p38 MAPK pathway with an inhibitor suppressed this modulation of miR143 and miR145
267
by Ang
268
pathway. All of these findings have many implications for the p38 MAPK pathway, most notably that it
269
may play a key role on the miR143/145 gene cluster regulated phenotypic modulation of aortic VSMCs
270
induced by Ang
271
The dysfunction of VSMCs leads to a varitey of artery diseaseas of humans, including atherosclerosis,
272
AD/aneurysm, cancer, and Alzheimer’s disease [40-43]. AD is an acute and catastrophic illness, and a
273
serious threat to human health. Normal aorta contains an elastic layer (mainly composed of smooth
274
muscle cells), and a layer of elastic fiber that links the continuous frame structure. This the
275
arrangement, however, along with the morphology of the fiber layer was irregular in AD samples.
276
During the formation of AD and the characteristic degeneration of the aortic media, the elastic fiber
277
breakage loss, phenotypic modulation of SMCs, loss of SMCs and proteoglycans material gathered
278
characterized. The pathogenesis of AD is likely to be multifaceted, though, with other researchers
279
reporting that inflammation genes are switched on, extracellular matrix protein degrades, and
280
transformation and transcription are upregulated, while the genes encoding the extracellular matrix, cell
281
adhesion proteins and cytoskeleton proteins are downregulated, thereby indicating that inflammatory
282
response promotes the development of AD as well [44]. Ang
283
AD/aneurysm wherein it can contribute to this disease through inflammatory and non-inflammatory
284
approaches (Fig.10). In the present study, we aim to demonstrate the mechanism that underlying the
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during the process of phenotypic modulation of VSMCs. Furthermore,
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,making it clear that the miR143/145 gene cluster is regulated by the activation of p38 MAPK
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has long been thought as a risk factor of
ACCEPTED MANUSCRIPT phenotypic switching of VSMCs during the development of AD. For this, we used Ang
as the
286
stimulus of in our in vitro experiments, subsequently linking the p38 MAPK pathway to the
287
miR143/145 gene cluster during the phenotypic switching of mouse aortic VSMCs. Our preliminary
288
experiments proved first that the miR143/145 gene cluster is a positive factor in the differentiation of
289
VSMCs and secondly that the Ang
290
the p38 MAPK pathway.
291
We acknowledge our investigations are limited by the nature of the study’s representative design.
292
Accordingly, it is impossible using our data to determine if the activation of p38 MAPK signaling
293
occurred when the downregulation of miR143/145 gene cluster occurred in the pathogenesis of AD-
294
whether before or after its onset. Experimentation of a p38 MAPK-knockdown animal model will be
295
critical in verifying our findings.
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-induced downregulation of miR143 and miR145 is regulated by
5. Conclusions
298
We have shown that Ang II affects the expression of the miR143/145 gene cluster in mouse aortic
299
VSMCs and promotes the phenotypic switching of VSMCs. Our data supports the involvement of the
300
p38 MAPK signaling pathway amongst this. These outcomes may offer another mechanism of Ang
301
II-induced VSMCs phenotypic modulation during the formation of AD. Our study suggests that the
302
miR143 /145 gene cluster may be an attractive target for strategies aiming to prevent the formation of
303
AD.
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6. Acknowledgements
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The authors would like to thank all the patients and donors and the Ethics Committee of Renmin
307
Hospital of WuHan University, China for supportting this study.
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44.Müller BT, Modlich O, Prisack HB, Bojar H,Schipke JD,Goecke T,et al. Gene expression profiles in
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418 Figure legends:
420
Fig.1 There is no expression of pp38 MAPK in normal aorta (A),the expression of pp38MAPK significant increased in aortic dissection
421
tissue (B; *P<0.05); the expression of p38MAPK decreased in aortic dissection tissue (D) compared with the normal tissue (C; *P<0.05).
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Fig.2 The expression of OPN, the marker of synthetic phenotype of VSMCs,increased significantly in the pathological tissue (D)
424
when compared to the normal tissue (C; *P<0.05); the expression of α-SMA is abundantly in the aorta (A ),while decreased significantly
425
in the aortic dissection tissue (B; *P<0.05); the arrangment of smooth muscle cells were disorded in the pathological tissue.
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Fig.3 The miR143 and miR145 were down-regulated (*P<0.05)in the aortic dissection tissues when compared to the donor’s aorta.
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Fig.4 After stimulate by Ang at different concentrations for 6h,12h,24h,respectivly,the expression of p38 MAPK decreased,the 0.1M for
430
12h has the most significant decrease (*P<0.05) (A).Meanwhile,the expression of phospho-p38 MAPK was increased (*P<0.05) with
431
Ang
432
vitro.
interven,the most significant increase showing at the 0.1M for 12h (B),indicating that Ang
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activate the p38 MAPK pathway in
434
Fig.5 The Ang induced the phenotypic marker of VSMCs transfored.When stimulate by Ang
435
each concentration and time,but there is no significant difference of each group .The Ang
436
siginificantly (*P<0.05) in vitro,and the most difference showing at 0.1M for 12h,indicating that the phenotypic swithcing of mouse
437
aortic smooth muscle cells.
20
,the expression of α-SMA increased at
induced the expression of OPN increased
ACCEPTED MANUSCRIPT 438 439
Fig.6 Immunofluorescence showed the brightness of the α-SMA in the control group is greater than the OPN obviously;while in the 0.1M
440
Ang
441
weaker than the control group,and the expression of OPN increased;in the inhibitor with 0.1M Ang
442
brightness of OPN compared with the inhibitor alone group.
group,there is more brightness of OPN compared to the control group;after added the inhibitor,the brightness of α-SMA become
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group,there is no difference of
443
Fig.7 After stimulate by Ang for 12h,the expression of miR143 and miR145 was significantly decreased (*P<0.05),the miR145 has the
445
more obvious decrease.
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446
Fig.8 After using the inhibitor,SB203580,the expression of p38 MAPK pathway was blocked (*P<0.001),and there is no phospho-p38
448
MAPK expression (A).When the p38 MAPK pathway was blocked ,the expression of OPN increased significantly (***P<0.05),while the
449
α-SMA decreased (**P<0.05),and the expression of phenotypic marker of VSMCs didn’t affected by the stimulation of Ang
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450
(B).
Fig.9 When added the inhibitor,the expression of miR143 and miR145 decreased significantly (P<0.05),and the miR145 is lower than
452
the miR143,then stimulated by the Ang ,the expression of miRs have no difference with the inhibitor alone group.
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contribute to aortic diseases through inflammatory and non-inflammatory approaches.
457
21
ACCEPTED MANUSCRIPT Aortic dissection group
Organ donors group
50.14±6.48
49.71±7.28
Age(year)
10/0 58.62±11.08
Type of aortic disease(n)
10
0
Acute aortic dissection
10
0
Marfan Syndrome
0
0
Thoracic aortic aneurysm
0
0
Comorbidities (n) Hypertensive disease
10
Diabetes mellitus
0
Heart disease
0
COPD
0
Renal insufficiency
0
10
COPD= chronic obstructive pulmonary disease
0 0 0 0 0
SC
Weight(kg)
RI PT
7/3 60.39±9.15
Male/female(n)
AC C
EP
TE D
M AN U
Table.1 Characteristics of the aortic dissection patients and organ donors
0
ACCEPTED MANUSCRIPT Gene
Primer sequences
miR143
RT-primer
5’-CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGCCAGAGAT-3’
Forward-primer 5’-ACACTCCAGCTGGGGGTGCAGTGCTGCAT-3’
miR145
Reverse-primer
5’-TGGTGTCGTGGAGTCG-3’
RT-primer
5’-CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGAGGGATTC-3’
U6
Reverse-primer
5’-TGGTGTCGTGGAGTCG-3’
RT-primer
5’-AACGCTTCACGAATTTGCGT-3’
Forward-primer 5’-CTCGCTTCGGCAGCACA-3’ Reverse-primer
5’-AACGCTTCACGAATTTGCGT-3’
AC C
EP
TE D
M AN U
SC
Table.1 The primers used for different target miRNAs.
RI PT
Forward-primer 5’-ACACTCCAGCTGGGGTCCAGTTTTCCCAGGA-3’
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT