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SHP2 Inhibitor PHPS1 Protects Against Atherosclerosis by Inhibiting Smooth Muscle Cell Proliferation Jia Chen1, Zhiyong Cao2 and Jingshu Guan1*

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SHP2 Inhibitor PHPS1 Protects Against Atherosclerosis by Inhibiting Smooth Muscle Cell Proliferation Jia Chen1, Zhiyong Cao2 and Jingshu Guan1* Chen et al. BMC Cardiovascular Disorders (2018) 18:72 https://doi.org/10.1186/s12872-018-0816-2 RESEARCH ARTICLE Open Access SHP2 inhibitor PHPS1 protects against atherosclerosis by inhibiting smooth muscle cell proliferation Jia Chen1, Zhiyong Cao2 and Jingshu Guan1* Abstract Background: Smooth muscle cells play an important role in the development of atherosclerosis. SHP2 is known to regulate the proliferation and migration of smooth muscle cells. The purpose of this study was to determine whether the SHP2 inhibitor PHPS1 has a pro-atherosclerotic or an atheroprotective effect in vivo and in vitro. Methods: After exposure to a high-cholesterol diet for 4 weeks, LDL receptor-deficient (Ldlr−/−)micewere exposed to the SHP2 inhibitor PHPS1 or vehicle. Body weight, serum glucose and lipid levels were determined. The size and composition of atherosclerotic plaques were measured by en face analysis, Movat staining and immunohistochemistry. The phosphorylation of SHP2 and related signaling molecules was analyzed by Western blot. Mechanistic analyses were performed in oxLDL-stimulated cultured vascular smooth muscle cells (VSMCs) with or without 10 mM PHPS1 pretreatment. Protein phosphorylation levels were detected by Western blot, and VSMC proliferation was assessed by BrdU staining. Results: PHPS1 decreased the number of atherosclerotic plaques without significantly affecting body weight, serum glucose levels or lipid metabolism. Plaque composition analysis showed a significant decrease in the number of VSMCs in atherosclerotic lesions of Ldlr−/− mice treated with PHPS1. Stimulation with oxLDL induced a dose-dependent increase in the number of VSMCs and in SHP2 and ERK phosphorylation levels, and these effects were blocked by PHPS1. Conclusion: The SHP2 inhibitor PHPS1 exerts a protective effect against atherosclerosis by reducing VSMC proliferation via SHP2/ERK pathway activation. Keywords: SHP2,PHPS1,Atherosclerosis,Smoothmusclecells,Proliferation,ERK Background and intracellular signaling pathways [5]. Smooth muscle Cardiovascular disease is a threat to human health and cells (SMCs) play an important role in the development of seriously impacts quality of life [1]. Atherosclerosis (AS) is AS [6, 7]. a major pathological basis of cardiovascular and cerebro- Protein tyrosine phosphorylation and dephosphoryla- vascular diseases [2]. Although the mechanism underlying tion play significant roles in numerous intracellular sig- AS is complex, the basic pathology of AS is fibrous prolif- naling pathways and are influenced by several opposing eration caused by the presence of progressive lipid de- kinases, such as protein tyrosine kinases (PTKs) and protein posits, accumulation of inflammatory cells and deposition tyrosine phosphatases (PTPs) [8, 9]. PTKs catalyze tyrosine of extracellular matrix [3, 4]. AS is not a separate patho- phosphorylation, whereas PTPs catalyze dephosphorylation logical process; rather, it is regulated by a variety of cells [10]. Src homology 2 domain-containing protein tyrosine phosphatase 2 (SHP2), also known as protein tyrosine phos- * Correspondence: [email protected] phatase N 11 (PTPN11), is an important PTP that acts 1Department of Cardiology, Shanghai Baoshan Hospital of Integrated Traditional Chinese and Western Medicine, Friendship Road 181, Baoshan downstream of various growth factors, cytokines and tyro- District, Shanghai, China sine kinases. It plays an integral role in multiple cellular Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Chen et al. BMC Cardiovascular Disorders (2018) 18:72 Page 2 of 9 events that regulate various functions, including migration, containing 2 mM EDTA and centrifuged (13,000 g) for differentiation, survival and metabolism [11–13]. Schramm 15 min at 4 °C. Plasma aliquots were separated and found that pharmacological reduction of SHP2/FAK/ stored at − 80 °C. The serum glucose level was deter- Akt/mTOR signaling at all levels of the signaling cas- mined using the glucose oxidase method (Beckman). cade effectively prevents cardiomyocyte hypertrophy Levels of triglycerides (TG), total cholesterol (TC), low- [14]. In recent years, SHP2 inhibitors have become density lipoprotein cholesterol (LDL-C) and high-density available for research applications. A SHP2 inhibitor lipoprotein cholesterol (HDL-C) were determined by phenylhydrazono pyrazolone sulfonate 1 (PHPS1) is a high-performance liquid chromatography (HPLC). potent and cell permeable inhibitor that is specific for SHP2 over the closely related tyrosine phosphatases AS quantification SHP1 and PTP1B [15]. In neutrophils, inflammatory Quantification of AS in the heart and aorta was monocytes and pDCs, the level of SHP2 expression is performed by staining. Briefly, the heart and aorta were muchlowerthanSHP1[16]. PTP1B is an enzyme perfused with phosphate-buffered saline (PBS) from the that negatively regulates insulin signaling and is likely left ventricle and dissected from the aortic arch to the involved in the pathways leading to insulin resistance iliac bifurcation. The adventitia was carefully cleaned [17]. PHPS1 remarkably suppresses E2-induced gene from the surrounding material. The aorta was sliced into transcription, rapid DNA synthesis and late effects on sections, fixed in 4% paraformaldehyde for 48 h, stained cell growth. The finding introduces a new mechanism with Oil Red O, incubated for 30 min at 37 °C, and for SHP2 oncogenic action and sheds new light on washed with PBS several times; theareaoftheatheroscler- extranuclear ER-initiated actions in breast cancer [18]. otic lesion was compared to the total area of the aorta using SHP2 regulates the acute pulmonary inflammation in- a dissection microscope and Adobe Photoshop software. duced by cigarette smoke through the ERK1/2 path- The heart was embedded in optimal cutting temperature way. PHPS1 significantly inhibits ERK1/2 activation (OCT) compound and frozen at − 80 °C. Serial sections and attenuates the inflammatory response induced in (8 μm) were taken from the aortic sinus and valve region, mouse lungs [19]. and sections in which all three valve leaflets were visible The purpose of this study was to determine whether were used for Movat staining. the SHP2 inhibitor PHPS1 has a pro-atherosclerotic or an atheroprotective effect in vivo and in vitro by evaluat- Western blot analysis ing the effect of phosphorylation or dephosphorylation Protein samples (30 μg) from cultured cells or aortic on the development of AS in LDL receptor-deficient specimens were electrophoresed on a 12% polyacryl- − − (Ldlr / ) mice. amide gel by SDS-PAGE and blotted onto a nitrocellu- lose membrane. After being blocked with 5% BSA for Methods 2 h at room temperature, the membrane was incubated Animal preparation overnight at 4 °C with primary antibodies (PRS3901, − − Ldlr / (005061) mice were purchased from Jackson M5670, SAB4500491, and SAB4502398; 1:1,000 dilution; Laboratory. All animal protocols were approved by the Sigma-Aldrich), washed with TBST 3 times, incubated Ethics Committee for Animal Experimentation at the with anti-rabbit secondary antibody (1:5,000 dilution) for Second Military Medical University. Five mice were 1 h at room temperature and then washed with TBST 3 housed in a cage on a 12-h light/dark cycle with free ac- times. cess to water and food. The animals received a high-fat diet containing 1.25% cholesterol for 4 weeks. The SHP2 Cell culture inhibitor PHPS1 was purchased from Sigma-Aldrich. Mouse aorta vascular smooth muscle cells (VSMCs) PHPS1 was dissolved in saline with 0.5% DMSO. Thirty were purchased from ATCC. The cells were grown in 6- mice were randomly divided into three groups: an AS well culture plates in RPMI 1640 supplemented with group, an AS+Vehicle group and an AS+PHPS1 group. 10% FBS, penicillin and streptomycin at 37 °C in a hu- Mice in the AS+PHPS1 group received an intraperito- midified atmosphere of 5% CO2. The medium was chan- neal (i.p.) injection of 3 mg/kg PHPS1 every day during ged daily, and the cells were passaged after treatment the last week on the high-fat diet, and those in the AS with 0.05% trypsin-0.02% EDTA solution. Cells at pas- +Vehicle group received an equal volume of saline with sage 5-8 were used in the subsequent experiments. Cells 0.5% DMSO on the same days. were confirmed as SMCs by their typical “hill-and-val- ley” morphological features and by the expression of Measurement of serum glucose and lipids smooth muscle α-actin by immunofluorescence. Cells The mice were euthanized with an overdose of pento- were made quiescent by a 48-h incubation in RPMI barbital. Blood samples were collected in EP tubes 1640 medium containing 0.1% FBS. Chen et al. BMC Cardiovascular Disorders (2018) 18:72 Page 3 of 9 Cytotoxicity assay the last 3 h of the 24-h stimulation period. BrdU incorpor- Cells were seeded in 96-well culture plates at a concen- ation into the cells was quantified using the BrdU cell pro- tration of 5 × 105 cells/ml 24 h prior to experiments. liferation assay kit (Roche Diagnostics, Indianapolis, IN, The cultured cells were stimulated with 10 μM PHPS1 USA). The cells were counted under a microscope using a or an equal volume of DMSO for 30 min and then with hemocytometer as previously described [20]. BrdU-labeled 100 μg/ml oxLDL for 48 h. Then, cells were washed 3 and unlabeled SMCs were counted in each section. The times with PBS. To evaluate the cytotoxicity of PHPS1 proliferation index was calculated by dividing the number and DMSO, the number of cells was determined using of BrdU-labeled cells by the number of unlabeled cells.
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