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Hypoxia decrease expression of cartilage oligomeric matrix protein to promote phenotype switching of pulmonary arterial smooth muscle cells
Accepted Manuscript Title: Hypoxia Decrease Expression of Cartilage Oligomeric Matrix Protein to Promote Phenotype Switching of Pulmonary Arterial Smooth Muscle Cells Author: Hang Yu Qingbo Jia Xiaoqian Feng Hongxia Chen Liang Wang Xiuqin Ni Wei Kong PII: DOI: Reference:
S1357-2725(17)30194-2 http://dx.doi.org/doi:10.1016/j.biocel.2017.08.007 BC 5196
To appear in:
The International Journal of Biochemistry & Cell Biology
Received date: Revised date: Accepted date:
13-2-2017 30-7-2017 10-8-2017
Please cite this article as: Yu, H., Jia, Q., Feng, X., Chen, H., Wang, L., Ni, X., and Kong, W.,Hypoxia Decrease Expression of Cartilage Oligomeric Matrix Protein to Promote Phenotype Switching of Pulmonary Arterial Smooth Muscle Cells, International Journal of Biochemistry and Cell Biology (2017), http://dx.doi.org/10.1016/j.biocel.2017.08.007 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.
Hypoxia Decrease Expression of Cartilage Oligomeric Matrix Protein to Promote Phenotype Switching g of Pulmonary Arterial Smooth Muscle Cells
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Hang Yua, Qingbo Jiab, Xiaoqian Fengc, Hongxia Chend, Liang Wangc, Xiuqin Nic*, Wei Konge* Department of Physiology, Harbin Medical University-Daqing, Daqing, Heilongjiang Province, China
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Department of Chest Surgery, the Fifth Affiliated Hospital of Harbin Medical University, Daqing,
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a
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Heilongjiang Province, China
Department of Anatomy, Harbin Medical University-Daqing, Daqing, Heilongjiang Province, China
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Department of Physiology and Pathophysiology, Harbin Medical University-Daqing, Daqing,
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c
Heilongjiang Province, China
of Physiology and Pathophysiology, Basic Medical College of Peking University, Beijing,
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e Department
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China
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Authors' E-mail addresses: Hang Yu: [email protected]
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Qingbo Jia: [email protected]
Xiaoqian Feng: [email protected]
Hongxia Chen: [email protected] Liang Wang: [email protected]
Xiuqin Ni: [email protected]
Wei Kong: [email protected] Co-corresponding Author: Wei Kong and Xiuqin Ni Pro. Wei Kong (the first corresponding author) Department of Physiology and Pathophysiology, Basic Medical College of Peking University 1 1
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Hypoxia Decrease Expression of Cartilage Oligomeric Matrix Protein to Promote Phenotype Switching of Pulmonary Arterial Smooth Muscle Cells
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Hang Yua, Qingbo Jiab, Xiaoqian Fengc, Hongxia Chend, Liang Wangc, Xiuqin Nic*, Wei Konge* Department of Physiology, Harbin Medical University-Daqing, Daqing, Heilongjiang Province, China
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Department of Chest Surgery, the Fifth Affiliated Hospital of Harbin Medical University, Daqing,
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a
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Heilongjiang Province, China
Department of Anatomy, Harbin Medical University-Daqing, Daqing, Heilongjiang Province, China
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Department of Physiology and Pathophysiology, Harbin Medical University-Daqing, Daqing,
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c
Heilongjiang Province, China
of Physiology and Pathophysiology, Basic Medical College of Peking University, Beijing,
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e Department
China
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Authors' E-mail addresses: Hang Yu: [email protected]
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Qingbo Jia: [email protected]
Xiaoqian Feng: [email protected]
Hongxia Chen: [email protected] Liang Wang: [email protected]
Xiuqin Ni: [email protected]
Wei Kong: [email protected] Co-corresponding Author: Wei Kong and Xiuqin Ni Pro. Wei Kong (the first corresponding author) Department of Physiology and Pathophysiology, Basic Medical College of Peking University 2
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38 Xueyuan Road, Haidian District, Beijing 100191 Beijing, 3HRSOHಬV5HSXEOLFRI&KLQD
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Tel: +86 10 82805594 Fax: +86 10 82805594
(the second corresponding author)
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Pro. Xiuqin Ni
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E-mail: [email protected]
Department of Anatomy, Harbin Medical University-Daqing, Daqing
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39 Xinyang Road, Gaoxin District, Daqing 163311 Heilongjiang Province, China
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Tel: +86 459 8153068
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E-mail: [email protected]
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Fax: +86 459 8153366
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Running title:: COMP and PASMC Phenotype
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Abstract Extracellular matrix proteins play important roles in the development of pulmonary hypertension(PH).
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However, the role of Cartilage oligomeric matrix protein (COMP) in the development of hypoxia-induced PH is largely unknown. We tested the hypothesis that COMP deficiency induced by
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hypoxia leads to the phenotype switching of pulmonary arterial smooth muscle cells (PASMCs). The
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expression of COMP decreased in a chronic hypoxia rat PH model (P˘0.05) and in PASMCs under hypoxia (3%O2) (P˘0.05). The expressions of differentiated marker proteins reduced in the pulmonary
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arteries from 5 month old COMP-/- mice and in PASMCs under hypoxia or with the siRNA of COMP treatment under normoxia, but increased in PASMCs with adenovirus-increased COMP under hypoxia.
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The absorbance of cell counting kit-8 at 450 nm and the expressions of proliferating cell nuclear antigen (PCNA) and osteopontin increased in PASMCs with the siRNA of COMP under normoxia (P㸺
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0.05). PCNA and osteopontin decreased in PASMCs with adenovirus-increased COMP under hypoxia (P㸺0.05). Additionally, the expression of bone morphogenetic protein receptor 2 (BMPR2) was
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reduced in COMP-/- mice (P㸺0.01). Both mRNA and protein levels of bone morphogenetic protein 2 (BMP2) were lower in PASMCs with the siRNA of COMP (P㸺0.05). The protein level of BMP2 could be reversed by adenovirus-increased COMP under hypoxia (P㸺0.05). These data suggest that COMP could normally have a protective role against PASMC phenotype switching and maintain BMP2/BMPR2 signaling, and these protective actions could be lost as a result of hypoxia promoting a depletion of COMP.
Keywords Cartilage Oligomeric Matrix Protein; Pulmonary Hypertension; Phenotype Switching; Pulmonary Arterial Smooth Muscle Cells; Bone Morphogenetic Protein Receptor 2 4
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Introduction
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Pulmonary arterial hypertension (PAH) is a rare, progressive, and fatal disease that is characterized by increased pulmonary artery pressure in the absence of common causes of pulmonary hypertension
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(PH), such as chronic heart, lung, or thromboembolic disease. A major hallmark of PAH is irreversible
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pulmonary arterial remodeling, manifested by excessive cellular proliferation, apoptotic resistance and abnormal contractile-to-synthetic vascular smooth muscle cell (VSMC) phenotype switching(Mandegar
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et al.,2004; Stenmark et al.,2006; Rabinovitch,2007). Although diverse environmental signals including sustained hypoxia and alterations in extracellular matrix are known to regulate SMC phenotype
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dedifferentiation are not well understood.
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(Lann«r et al.,2005; Schultz et al.,2006; Zhou et al.,2009), the mechanisms that control SMC
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Vascular smooth muscle cells (VSMCs), unlike cardiac or skeletal muscle cells, have a unique property
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of plasticity and can undergo reversible changes in phenotype(Owens,1995). Normally, they are mainly restricted to the media of adult blood vessels, express a repertoire of contractile proteins such DVVPRRWKPXVFOH 60 P\RVLQKHDY\FKDLQ60˞-DFWLQ60˞DQGFDOSRQLQDQGKDYHDORZUDWHRI replication. However, on various environmental cues, VSMCs can undergo a transition from a quiescent, contractile/differentiated phenotype to a synthetic/dedifferentiated phenotype, with a high rate of migration/proliferation and a concomitant reduction in expression of VSMC marker proteins(Owens,1995; Hao et al.,2003). Phenotype switching of VSMCs plays an essential role in the development of cardiovascular diseases such as atherosclerosis, postangioplastic restenosis and hypertension. Studies have demonstrated the contribution to cell dedifferentiated of growth factors, mitogenic cytokines, reactive oxygen species, stretch or injury(Owens et al.,2004; Rzucidlo et 5
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al.,2007). VSMC functional contractility depends on extracellular mechanical properties(Steucke et al.,2015) and
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extracellular matrix (ECM) presentation modulates vascular smooth muscle cell mechanotransduction (Sazonova et al.,2015). In vitro, non-confluent VSMCs cultured on stiff substrates express contractile
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phenotype markers at higher levels than on soft substrates, though this difference declines in confluent
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VSMCs(Sazonova et al.,2011). Phenotypic changes in VSMCs induced by vessel injury results in proliferation and deposition of ECM (Collins et al.,2012). One example of the proteins is
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thrombospondins (TSPs), a family of multifunctional matricellular glycoproteins. TSPs do not contribute to the normal ECM structural architecture, they do function in cellular signaling by interacting with other
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ECM proteins(Stenina-Adognravi,2014). The TSP family consists of 5 member proteins. TSP-5 also known as cartilage oligomeric matrix protein (COMP) is a secreted glycoprotein. Previous studies have
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identified COMP in normal rat aortas(Oldberg et al.,1992), human VSMCs and the atherosclerotic plaques of ApoE-/-mice (Riessen et al.,2001), implicating an importance to vascular pathology and a
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potential target for therapy. COMP has recently been identified as a normal component of the human artery wall and secreted by VSMCs. COMP affects cellular attachment, proliferation, and influences chondrogenesis (Kipnes et al.,2003; Chen et al.,2005; Xu et al.,2007). Extracellular modulators have been a particular focus of investigation, including proteins that affect receptor stability, ligand function, or ligand availability. Transforming growth factor ˟-like ligands, the bone morphogenetic proteins (BMPs), is the direct modulation of ligand activity by extracellular factors (De Robertis EM and Sasai,1996). Mice that are heterozygous null for BMP2 (BMP2 (+/-)) develop more severe hypoxia PH than their wild-type littermates, but not BMP4 (LacZ/+) mutant mice. The protective effects of BMP2 are mediated by increasing eNOS expression and activity in the hypoxic pulmonary vasculature
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(Anderson et al.,2010). BMPR2 acts as one of the receptors of BMP2 and is involved in PAH. We have documented evidence suggesting that COMP is essential for maintaining the differentiated
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phenotype of systemic VSMCs; inhibits vascular smooth muscle calcification by interacting with BMP2; prevents vascular aging and vascular smooth muscle cells senescence (Wang et al.,2010; Du Y et
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al.,2011; Wang et al.,2016).
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However, there are many known differences between the systemic system and pulmonary system. It is still not clear whether COMP is expressed in pulmonary artery smooth muscle cells (PASMCs) and
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also involved in the phenotype switching of PASMCs. Here, we show that COMP was also expressed in the media of pulmonary arteries. The deficiency of COMP switched the PASMC phenotype and
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contributed to pulmonary artery remodeling. Additionally, the absence of COMP in pulmonary arteries led to the downregulation of BMP2 and BMPR2. The results of this study appear to identify properties
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of a potentially novel role for ECM proteins on pulmonary hypertension development that have potential for therapeutic targeting.
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Materials and Methods
Material and Reagents
Smooth muscle cell growth medium was purchased from Cell Applications, Inc. (San Diego, CA, USA). Dulbecco's Modified Eagle Medium (DMEM), used as serum-free media (SFM), trypsin and trypsin neutralizing solution were purchased from Lonza (Walkersville, MD, USA). Cells were made quiescent by incubation in SFM for 48 h. Antibodies were purchased from companies as following: COMP (Abcam Inc. Catalog: ab74524 60˞-actin (Santa Cruz. Catalog: sc-53142 60˞ (Abcam Inc. Catalog: ab14106), calponin (Santa Cruz. Catalog: sc-16604-R), OPN (Santa Cruz. Catalog: sc-10591), PCNA (Santa Cruz. Catalog: sc-9857), BMP2 (Santa Cruz. Catalog: sc-6895), BMPR2 7
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(Santa Cruz. Catalog: sc-5682), ˟-actin (Santa Cruz. Catalog: sc-81178) and GAPDH (Santa Cruz. Catalog: sc-47724). All other reagents were from common commercial sources.
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Animals
Male or female Sprague-Dawley rats were housed in the Animal Resource Center of the Harbin
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Medical University. Rats (230-250g) were exposed to chronic hypoxia (10% O2) in a ventilated
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chamber. The hypoxic environment was a mixture of air and nitrogen. The control rats were raised in another chamber where the FiO2 was 21% (normoxia). After 21 days, animals were anesthetized with
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4% halothane after heparin was administered intraperitoneally. The systemic arterial systolic blood
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pressure was measured via caudal artery and the mean pulmonary arterial systolic blood pressure was measured from right heart catheterization (Rats were anesthetized with isoflurane and a micro-tip
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pressure transducer was inserted into the right jugular vein and advanced to the right ventricle. Right
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ventricular pressure was continuously monitored for 10 min and data were analyzed). Then animals
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were weighed and the chest was opened for the removal of the heart and lungs en bloc. The hearts and lungs were placed in an iced modified Krebs solution (mmol/L: KCl 4.7㸪MgSO4 0.57㸪KH2PO4 1.16, CaCl2 2.5, NaHCO3 24, glucose 10 and NaCl 118, PH 7.4) and transferred to the laboratory for microdissection of selected tissues. COMP-/- (KO) mice with a C57BL/6J background were kindly provided by Professor Oldberg Ake (Department of Cell and Molecular Biology, University of Lund, Sweden). KO mice (5 months of age) and COMP+/+ littermates as wild type (WT) were used for experiments. Animals were genotyped using polymerase chain reaction via respective primers. All animal studies followed the guidelines of the Animal Care and Use Committee of Harbin Medical University.
Image analysis of small pulmonary arteries 8
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Six small pulmonary arteries each animal (diameter between 30~4˩P, near hilus of the lung) were randomly measured with 2D image analysis software to get the data of external diameter and wall
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thickness. The times of external diameter compared with wall thickness were calculated.
Cell culture
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Briefly, the pulmonary arteries (PAs) of rats were cut into small pieces after cleaned of their connective
containing collagenase II (10 U/ml DWr&IRU1 hours.
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tissue and removed the endothelium by rubbing the lumen. Then PAs were dispersed by solution The dispersed vascular smooth muscle cells
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were then centrifuged for 10 min to get cell pellet. The cells were resuspended in DMEM with 20%
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serum and 1% penicillin/streptomycin and plated in T25 culture flask to culture. Cell viability (usually greater than 98%) was determined by Trypan Blue exclusion㸬
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Recombinant adenovirus construction and infection
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The adenovirus for full-length COMP (Ad-COMP; NM_016685.2) was constructed and amplified
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according to the manufacturer's protocol (BD Biosciences). An adenovirus that carried b-galactosidase (Ad-LacZ) was used as a negative control. Cells cultured at approximately 80% confluence were infected with recombinant adenovirus (50 multiplicity of infection) for 48 h.
Transfection of small interfering RNA
SiRNA was purchased from GenePharma Co., Ltd (Shanghai). The sequences that corresponded to the siRNA of rat COMP (siRNA-COMP) were seQVHಬ- AGAAACUUGAGCUGUGUUGAUGCC-ಬ and anti-VHQVHಬ- GGCUAUCAAGACAGCUCAAGUUUCU-ಬ$VFUDPEOHVWHDOWK51$LGXSOH[ served as a negative control. In vitro siRNA transfection (50 nM) of rat PASMCs was performed using RNAi MAX (Invitrogen, CA, USA). The transfection procedures followed the manufacturers' 9
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instructions.
RNA isolation and cDNA reverse transcription
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PASMCs were made quiescent by incubation in serum free media (SFM) for 24h. Quiescent cells were exposed to SFM, Ad-COMP, siRNA-COMP for 24h. Media was decanted and cells frozen to -ഒ
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RNAs were extracted using Trizol from pulmonary arteries or PASMCs. Quality of the RNA samples
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was assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA). CDNA was
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generated using Prime6FULSWറ57UHDJHQW.LW 7$.$5$%,2&DWDORJ 55$
Real-time quantitative reverse transcriptase-polymerase chain reaction
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Real-WLPHTXDQWLWDWLYHSRO\PHUDVHFKDLQUHDFWHGZLWK6<%5p3UHPL[([7DT70 7$.$5$%,2
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Catalog: RR420A). Cycle and threshold were REWDLQHGDFFRUGLQJWRWKHPDQXIDFWXUHUಬVLQVWUXFWLRQV
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Changes in expression given are the average of three biological replicates. (The primers are listed in
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the supplemental table 1 on line.)
Agarose Electrophoresis
PCR products were separated by electrophoresis on 3% agarose/TBE gels. Gels were photographed using the Chemi Imager 5500 gel image analysis instrument (AlPha InnCh). The integrated density value (IDV) of PCR product bands was measured by Chemi Imager 5500 V2.03 software (AlPha InnCh), to assess the expressions of long non-coding RNAs (lncRNAs). (The primers are listed in the supplemental table 1 on line.)
Western Blot Assessment
:HVHSDUDWHG˩g protein extracted from PAs (without adventitia and intima) or PASMCs by a 10% or
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15% SDS-PAGE gel and transferred it to a PVDF membrane. The membrane was probed with specific SULPDU\DQWLERGLHVIRU&20360˞-DFWLQ60˞FDOSRQLQ2313&1$%03DQG%035
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respectively, and appropriate secondary antibodies were conjugated with horseradish peroxidase. The VSHFLILFEDQGVZHUHYLVXDOL]HGZLWKDQHQKDQFHGFKHPLOXPLQHVFHQWUHDFWLRQDQGQRUPDOL]HGWR˟-actin
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or GAPDH.
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Immunohistochemistry Assay
Fresh tissues were fixed in 4% Paraformaldehyde solution for 24h at 4r& and then were dehydrated in
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30% sucrose solutions for 48h at 4r&. Sections were cut on cryostat at 7 ˩PRIWKLFNQHVVDW-r&7KH
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sections were mounted onto coated slides and were prepared for histology and immunohistochemistry. The sections were stained with hematoxylin and eosin to confirm the presence of microvessles. The
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serial sections to those above were used for immunohistochemistry. They were rehydrated in PBS
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before immunostaining and endogenous peroxidase activity was quenched with 3% H 2O2 in PBS.
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Sections were blocked with 5% donkey VHUXPDQGLQFXEDWHGRYHUQLJKWDWr&ZLWKUDEELWDQWL-COMP antibody (Abcam Inc. Catalog: ab11056). Sections were incubated with biotinylated donkey anti-rabbit secondary antibody, followed by horse radish peroxidase-streptavidin (ZSGB-BIO, China). Samples of 10-20 arterioles, less than100 mm in size, were assessed. The slides were photographed and the integrated optical density of yellow or brown staining was measured using a computer-assisted image analyzing system (Motic Images Advanced 3.2).
Cell Proliferation Assay
Incubate PASMCs (3000 cells/well) in a 96-ZHOOSODWH DWr&&22). Add 10 ˩ORIcell counting kit-8 (CCK-8) solution to each well of the plate. Incubate the plate for 2 hours in the incubator. And then,
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the absorbance was measured at 450 nm using an ELISA reader (Anthros, Durham, NC, USA). All analyses were performed in at least three independent replicate cultures. The optical density value
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(ODV) of treated to non-treated control cells was calculated and presented in terms of relative cell viability.
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Statistical analysis
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Statistical analysis was performed with GraphPad Prism 5 software. All values were expressed as mean s SEM. Statistical analyses between WZRJURXSVZHUHSHUIRUPHGZLWKSDLUHG6WXGHQWಬV t-test,
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and a one-way ANOVA with Newman Keuls correction was used for comparison between multiple
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groups. A value of P<0.05 was used to establish statistical significance.
Results
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COMP Protein Level Is Decreased in Chronic hypoxia-induced PH Rat Arteries and PASMCs
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To investigate the role of COMP in PH, we first compared the expression of COMP in protein level
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between chronic hypoxia-induced PH rats and normoxia-raised rats by immunohistochemistry (Figure 1A) and western blot (Figure 1B & 1C). The data showed that COMP was mainly expressed in the media of pulmonary arteries and degraded in chronic hypoxia-induced PH rats. Furthermore, we tested the expression of COMP in PASMCs under hypoxia (supplement Figure 1). The data demonstrated that COMP was apparently decreased after hypoxia 48h compared with the expression under normoxia. In addition, hematoxylin and eosin (HE) staining was applied to compare the vascular wall thickness between the COMP knockout (COMP-/-) and wide type mice in pulmonary small arteries (Figure 1D & 1E). As shown in HE staining, the wall of pulmonary small arteries in COMP-/- mice was thicker than those in WT mice.
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These results implied that COMP was expressed in pulmonary arteries and played a role in PH. COMP deficiency was correlation with pulmonary artery remodeling. Combined with our recent observation
the phenotype switching of PASMCs.
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COMP Is Required for Maintenance of the PASMC Differentiated Phenotype
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that COMP is necessary to maintain the contractile phenotype of VSMCs, COMP may also regulate
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After observing an association of COMP expression with PH, we next investigated the potential effect of COMP on PASMC phenotype transition by small interfering RNA (siRNA) knockdown. COMP
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mRNA level was inhibited by 70% with the siRNA of rat COMP (siRNA-COMP) knockdown (Figure 2A, 2B and 2C). The mRNA levels of SM ˞-actin, calponin, and SM22˞were downregulated in parallel
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(Figure 2A). Similarly, the protein levels of SM ˞-actin, calponin, and SM22˞were markedly reduced by siRNA-COMP treatment as compared with the scramble siRNA treatment (Figure 2B & 2C). These
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data indicate a causal role of COMP in altering the mRNA and protein level of differentiated marker genes. Thus, COMP is necessary for differentiated marker expression. Additionally, the pulmonary
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arteries of COMP-/- mice and WT mice were separated to analyze the expressions of phenotype markers. In the absence of COMP, the protein levels of SM ˞-actin and calponin were significantly downregulated. These data further support COMP as being required for maintenance of the PASMC differentiated phenotype.
COMP Overexpression Retains the Characteristics of the Differentiated Phenotype of PASMCs under hypoxia For PASMC dedifferentiated with COMP knockdown, we next asked whether adenoviral overexpression of COMP could retain the PASMC differentiated state challenged by hypoxia, which leads to PASMC phenotype switching. Western blot analysis revealed COMP protein level was higher
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in PASMCs with the adenovirus for full-length COMP (Ad-COMP) than with the adenovirus that carried b-galactosidase (Ad-lacZ) infection (Figure 3C & 3D). Interestingly, Ad-COMP significantly
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circumvented hypoxia-induced suppression of differentiated marker at protein level in PASMCs (Figure 3A, 3B, 3C and 3D). Thus, COMP overexpression could restore hypoxia-induced PASMC
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dedifferentiated and render differentiated in cells. It suggests that the protective effect of COMP
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maintains the contractile/differentiated phenotype of PASMCs.
COMP Inhibits PASMCs Excessive Proliferation
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With siRNA-COMP knockdown under normoxia, the optical density value (ODV) of CCK8 at 450 nm increased in PASMCs (Figure A). However, Ad-COMP significantly decreased the ODV under hypoxia
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(Figure 4B). We also detected the expressions of proliferation proteins (osteopontin and PCNA). As shown in Figure 4C & 4D, the expression of PCNA and osteopontin (OPN) in protein level increased
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with siRNA-COMP treatment. And we got the similar data from COMP-/- mice and WT mice (Figure 4E & 4F). It suggests that COMP negatively regulates the PASMC proliferation.
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Absence of COMP Reduces the Expressions of BMP2 and BMPR2 To investigate the mechanisms of COMP in PAH, the BMPR2 was measured, which has been defined to be involved in PAH. It is interesting that BMPR2 was downregulated at protein level in COMP-/- mice compared with that in WT mice (Figure 5A & 5B). In our previous paper, we have reported that COMP directly bound to BMP2 through the C-terminal domain in rat systemic VSMCs. So we also tested the expression of BMP2 in PASMCs. Our data showed that both mRNA (supplement figure 2) and protein levels of BMP2 (Figure 5C & 5D) were lower in PASMCs with siRNA-COMP treatment than those with the scramble siRNA transfection. The BMP2 protein level could be reversed by Ad-COMP under hypoxia (Figure 5E & 5F, supplement figure 3).
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There Is No Difference in Pulmonary Hemodynamics between COMP-/- Mice and Wild Type Mice We measured several parameters of pulmonary hemodynamics in COMP-/- mice and wild type mice (5
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months of age) to observe the effect of COMP on pulmonary function in vivo. The data showed that there were no obvious differences between KO mice and WT mice in body weight, right ventricle
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weight / body weight (RV/BW), right ventricle weight / left ventricle plus ventricular septum weight (RV /
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LV+S), mean pulmonary arterial systolic blood pressure (mPAP), mean systemic systolic blood pressure (SBP) of caudal artery (Figure 6). But there was increasing trend of mPAP in KO mice.
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(Figure 6D) Discussion
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VSMCs are highly specialized cells that regulate vascular tone and participate in vessel remodeling in physiological and pathological conditions. Although many reports have described key factors involved
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in the regulation of the SMC phenotype switching (Alexander and Owens,2012), the detailed molecular mechanisms driving this process are not yet fully understood. In this study, we show that COMP, a
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macromolecular ECM protein, was necessary for maintaining the PASMC differentiated phenotype. Moreover, overexpression of COMP could rescue the hypoxia-induced cell phenotype switching to a dedifferentiated state in vitro. These protective effects may be partly due to the suppression of BMP2/BMPR2 signaling pathway. The causal relationship between them needs further study. Our results reveal intrinsic ECM-associated mechanisms regulating the plasticity of the PASMC phenotype. VSMC in mature animals is a highly specialized cell whose principal function is contraction and regulation of blood vessel diameter, blood pressure, and blood flow distribution. Although there are likely many alternative phenotypic states of SMCs, in general SMC phenotypic switching is characterized by markedly reduced expressions of SMC-selective differentiated marker genes and
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increased SMC proliferation, migration, and synthesis of ECM components required for vascular repair (Alexander and Owens,2012). SMCs within adult animals retain remarkable plasticity and can undergo
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profound and reversible changes in phenotype, a process referred to as phenotypic switching, in response to vascular injury (Owens,1995). The extensive plasticity exhibited by the fully mature SMC
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is an inherent property of the cell that likely evolved in higher organisms because it conferred a survival
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advantage or disease. However, an unfortunate consequence of this plasticity is that it predisposes the SMC to environmental cues/signals that can induce adverse phenotype switching and contribute to
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development and/or progression of vascular disease (Bijli et al.,2016). In the present study, the novel ECM factor COMP was found to modulate the PASMC phenotype switching. In the absence of COMP,
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the differentiated/contractile SMC phenotype marker proteins were down-regulated and proliferation proteins such as PCAN and OPN were up-regulated both in vitro and in vivo. In addition, our data
PASMCs.
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supported COMP as being required to maintain the characteristics of the differentiated phenotype of
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Our data showed that COMP was mainly expressed in the media of the pulmonary artery wall in rats. Hypoxia stimulates pulmonary hypertension, in part by increasing the proliferation of human pulmonary artery smooth muscle cells (Newman et al.,2004). We therefore tested the expression of COMP in PH model rats and PASMCs under hypoxia (24h, 48h, and 72h) and found the decreased COMP level both in hypoxia-induced PH model rats (as compared with control rats) and in PASMCs under hypoxia (48h and 72h) (as compared with a control under normoxia). The data suggested COMP was involved in PH, but its biological relevance to phenotype-related vascular remodeling needed further to be illuminated. The following data found that the differentiated markers were down-regulated in PASMCs after COMP gene knockdown with siRNA. Moreover, PASMCs under hypoxia also reduced the
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expressions of differentiated marker and this could be reversed by adenoviral over-expression of COMP for 24 hours before hypoxia (3%O2). The cell proliferation experiment was done to clarify the
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relationship between COMP and proliferation in PASMCs. The data showed that PASMC proliferation was enhanced after COMP gene knockdown with siRNA or in knockout mice. The increased
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proliferation of PASMCs under hypoxia could be reversed by adenoviral-increased COMP for 24 hours
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before hypoxia (3%O2). This further demonstrates the inhibitory effect of COMP on PASMC phenotype switching. The uniqueness of our study lies in providing a systemic and dynamic evaluation of the
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alteration in the level of the ECM protein COMP during PASMC phenotype switching. Also, our results emphasize the importance of the integrity of the ECM in phenotype-related vascular remodeling such
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as in PH. In our data of COMP-/- mice and WT mice, there were no obvious differences in several pulmonary function parameters, which may be due to the mice age. But there was increasing trend of
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mPAP in COMP-/- mice. In conclusion, COMP was normally expressed in pulmonary arteries and necessary for PASMCs to maintain their differentiated phenotype.
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Although ECM constituents such as COMP are important biological cues regulating the phenotype plasticity of PASMCs, little is known about the mechanisms. BMPR2 plays an important role in PH. The BMPR2 mutation is thought to increase susceptibility to idiopathic PAH in the context of environmental or other genetic factors (Alastalo et al.,2011). It is important to note that even PAH patients without a BMPR2 mutation have reduced expression of this receptor (Atkinson et al.,2002; Du Y et al.,2011). Interestingly, our study on COMP-/- mice and littermate mice showed that BMPR2 was reduced in pulmonary arteries in the absence of COMP. BMPR2 can act as one receptors of BMP2. BMP2 plays a protective role in the hypoxic pulmonary vasculature by increasing eNOS expression and activity (Anderson et al.,2010). In our previous study, we reported that COMP physically interacts with BMP2
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through the C-terminal domain of COMP, prevents BMP2 receptor binding, suppresses Smad1/5/8 phosphorylation, and inhibits the expression of the downstream transcriptional factors Runx-2 and
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Msx-2 (Du Y et al.,2011). Thus, we tested the expression of BMP2 in PASMCs. The data showed that the expression of BMP2 reduced after COMP gene knockdown with siRNA and adenoviral
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over-expression of COMP for 24 hours before hypoxia (3%O2) could reverse the decrease of BMP2
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under hypoxia. This suggests that the deficiency of COMP suppressed BMP2/BMPR2 signaling. The mechanism of COMP on PASMCs is different from that on systemic VSMCs. Further studies are
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needed to define the causal relationship between BMP2/BMPR2 signaling and phenotype switching in PASMCs and elucidate the mechanisms of hypoxia on the decrease of COMP.
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The present study identified a critical role of COMP on PASMC retaining a differentiated phenotype and maintaining BMP2/BMPR2 signaling. The findings reveal a self-protective mechanism of PASMCs
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in response to various environmental cues such as hypoxia and identify properties of a potentially novel role for extracellular matrix proteins on pulmonary hypertension development that have potential
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for therapeutic targeting. Acknowledgments
We appreciated Dr. Ake Oldberg from Lund University for kindly providing COMP-/- mice for our vivo experiments, Dr. Daling Zhu from Harbin medical university-Daqing for generously providing guidance on experiments.
This research was supported by funding from National Natural Science Foundation of China (31400989); Natural Science Foundation of Heilongjiang Province (C2015069); Natural Science Foundation of Heilongjiang Province (LBH-Q15087) Disclosures
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All authors have no conflicts of interest.
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Figure legends: Figure 1 COMP decreases in pulmonary small arteries from chronic hypoxia-induced PH rats. (n=5
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each group) (A) Representative immunohistochemistry staining and (B) western blot of COMP protein in pulmonary arteries from PH model and control rats. (C) Quantitative analysis of full-length COMP in
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pulmonary arteries from PH model and control rats. Relative COMP protein level was normalized to
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WKDWRI˟-actin. *P㸺0.05 vs control. (D) Representative hematoxylin and eosin staining and (E) quantitative analysis of diameter/wall thickness of pulmonary arteries in COMP-/- (KO) and wild type
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(WT) mice (n=5 each group). *P㸺0.05 vs WT mice. (CTL: control, PH: pulmonary hypertension model)
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Figure 2 COMP is required for PASMCs maintaining a differentiated phenotype. (A) Specific knockdown of COMP by siRNA but not scramble siRNA. *P㸺0.05 vs scramble siRNA. Quantification
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of differentiated marker genes in mRNA levels during siRNA-COMP treatment. *P㸺0.05 vs scramble siRNA. (B) Representative western blot and (C) quantitative analysis of COMP, differentiated marker
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protein expression after COMP gene knockdown with siRNA. Results are from 3 independent experiments performed in duplicate. *P㸺0.05, **P㸺0.01 vs scramble siRNA. (D) Representative western blot and (E) quantitative analysis of differentiated marker protein expression in COMP-/- (KO) and wild type (WT) mice. (n=5 each group) *P㸺0.05 vs WT mice.
Figure 3 COMP over-expression retains the characteristics of the differentiated phenotype in PASMCs under hypoxia. (A) Representative western blot and (B) quantitative analysis of PASMC differentiated markers normalized to GAPDH (SM22 ˞) or ˟-actin (Calponin). PASMCs were under hypoxia (3%O2) for 24, 48, and 72 hours. *P˘0.05, **P˘0.01 vs normoxia. (C) Representative western blot and (D)
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quantitative analysis of COMP DQGGLIIHUHQWLDWHGPDUNHUVQRUPDOL]HGWRWKDWRI˟-actin. PASMCs were infected with Ad-lacZ or Ad-COMP for 24 hours before hypoxia (3%O2) for additional 48 hours. Results
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are from 3 independent experiments performed in duplicate. *P㸺0.05, **P㸺0.01 vs Ad-lacZ. (N:
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normaxia, H: hypoxia, Ad: adenovirus)
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Figure 4 COMP inhibits the excessive proliferation of PASMCs. (A) Quantitative analysis of the optical density value (ODV) of CCK8 at 450 nm in PASMCs after COMP gene knockdown with siRNA under
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normoxia . *P㸺0.05 vs scramble siRNA. (B) Quantitative analysis of ODV, PASMCs infected with Ad-lacZ or Ad-COMP for 24 hours before hypoxia (3%O2) for additional 48 hours. **P㸺0.01 vs
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Ad-lacZ. (C) Representative western blot and (D) quantitative analysis of the proliferation protein expressions after COMP gene knockdown with siRNA. Results are from 3 independent experiments
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performed in duplicate. **P㸺0.01 vs scramble siRNA. (E) Representative western blot and (F) quantitative analysis of the proliferation protein expressions in COMP-/- (KO) and wild type (WT) mice.
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(n=5 each group) *P㸺0.05 vs WT mice.
(Ad: adenovirus)
Figure 5 Absence of COMP reduces the expressions of BMP2 and BMPR2. (A) Representative western blot and (B) quantitative analysis of BMPR2 in the pulmonary arteries from COMP-/- (KO) and wild type (WT) mice. (n=5 each group) **P㸺0.01 vs WT mice. (C) Representative western blot and (D) quantitative analysis of BMP2 after COMP gene knockdown with siRNA. *P㸺0.05 (E) Representative western blot and (F) quantitative analysis of BMP2 normalized to that of GAPDH, PASMCs were infected with Ad-lacZ or Ad-COMP for 24 hours before hypoxia (3%O2) for additional 48 hours. Results are from 3 independent experiments performed in duplicate. *P㸺0.05 vs Ad-lacZ. (Ad: adenovirus)
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Figure 6 Pulmonary hemodynamics in COMP-/- mice and wild type mice (5 months of age, n=8 each
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group). (A) Body weight (g/g); (B) the weight of right ventricle / body weight (mg/g); (C) the weight ratio of right ventricle and left ventricle plus ventricle septum (mg/mg); (D) the mean pulmonary arterial
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systolic blood pressure; (E) the mean systemic arterial systolic blood pressure. (RV: right ventricle;
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V+S: left ventricle plus ventricle septum; MPAP: the mean pulmonary arterial systolic blood pressure;
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SBP: the mean systemic arterial systolic blood pressure; KO: COMP-/- mice; WT: wild type mice)
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S1357-2725(17)30194-2 http://dx.doi.org/doi:10.1016/j.biocel.2017.08.007 BC 5196
To appear in:
The International Journal of Biochemistry & Cell Biology
Received date: Revised date: Accepted date:
13-2-2017 30-7-2017 10-8-2017
Please cite this article as: Yu, H., Jia, Q., Feng, X., Chen, H., Wang, L., Ni, X., and Kong, W.,Hypoxia Decrease Expression of Cartilage Oligomeric Matrix Protein to Promote Phenotype Switching of Pulmonary Arterial Smooth Muscle Cells, International Journal of Biochemistry and Cell Biology (2017), http://dx.doi.org/10.1016/j.biocel.2017.08.007 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.
Hypoxia Decrease Expression of Cartilage Oligomeric Matrix Protein to Promote Phenotype Switching g of Pulmonary Arterial Smooth Muscle Cells
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Hang Yua, Qingbo Jiab, Xiaoqian Fengc, Hongxia Chend, Liang Wangc, Xiuqin Nic*, Wei Konge* Department of Physiology, Harbin Medical University-Daqing, Daqing, Heilongjiang Province, China
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Department of Chest Surgery, the Fifth Affiliated Hospital of Harbin Medical University, Daqing,
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a
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Heilongjiang Province, China
Department of Anatomy, Harbin Medical University-Daqing, Daqing, Heilongjiang Province, China
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Department of Physiology and Pathophysiology, Harbin Medical University-Daqing, Daqing,
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Heilongjiang Province, China
of Physiology and Pathophysiology, Basic Medical College of Peking University, Beijing,
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e Department
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China
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Authors' E-mail addresses: Hang Yu: [email protected]
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Qingbo Jia: [email protected]
Xiaoqian Feng: [email protected]
Hongxia Chen: [email protected] Liang Wang: [email protected]
Xiuqin Ni: [email protected]
Wei Kong: [email protected] Co-corresponding Author: Wei Kong and Xiuqin Ni Pro. Wei Kong (the first corresponding author) Department of Physiology and Pathophysiology, Basic Medical College of Peking University 1 1
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Hypoxia Decrease Expression of Cartilage Oligomeric Matrix Protein to Promote Phenotype Switching of Pulmonary Arterial Smooth Muscle Cells
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Hang Yua, Qingbo Jiab, Xiaoqian Fengc, Hongxia Chend, Liang Wangc, Xiuqin Nic*, Wei Konge* Department of Physiology, Harbin Medical University-Daqing, Daqing, Heilongjiang Province, China
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Department of Chest Surgery, the Fifth Affiliated Hospital of Harbin Medical University, Daqing,
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a
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Heilongjiang Province, China
Department of Anatomy, Harbin Medical University-Daqing, Daqing, Heilongjiang Province, China
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Department of Physiology and Pathophysiology, Harbin Medical University-Daqing, Daqing,
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c
Heilongjiang Province, China
of Physiology and Pathophysiology, Basic Medical College of Peking University, Beijing,
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e Department
China
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Authors' E-mail addresses: Hang Yu: [email protected]
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Qingbo Jia: [email protected]
Xiaoqian Feng: [email protected]
Hongxia Chen: [email protected] Liang Wang: [email protected]
Xiuqin Ni: [email protected]
Wei Kong: [email protected] Co-corresponding Author: Wei Kong and Xiuqin Ni Pro. Wei Kong (the first corresponding author) Department of Physiology and Pathophysiology, Basic Medical College of Peking University 2
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38 Xueyuan Road, Haidian District, Beijing 100191 Beijing, 3HRSOHಬV5HSXEOLFRI&KLQD
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Tel: +86 10 82805594 Fax: +86 10 82805594
(the second corresponding author)
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Pro. Xiuqin Ni
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E-mail: [email protected]
Department of Anatomy, Harbin Medical University-Daqing, Daqing
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39 Xinyang Road, Gaoxin District, Daqing 163311 Heilongjiang Province, China
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Tel: +86 459 8153068
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E-mail: [email protected]
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Fax: +86 459 8153366
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Running title:: COMP and PASMC Phenotype
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Abstract Extracellular matrix proteins play important roles in the development of pulmonary hypertension(PH).
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However, the role of Cartilage oligomeric matrix protein (COMP) in the development of hypoxia-induced PH is largely unknown. We tested the hypothesis that COMP deficiency induced by
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hypoxia leads to the phenotype switching of pulmonary arterial smooth muscle cells (PASMCs). The
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expression of COMP decreased in a chronic hypoxia rat PH model (P˘0.05) and in PASMCs under hypoxia (3%O2) (P˘0.05). The expressions of differentiated marker proteins reduced in the pulmonary
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arteries from 5 month old COMP-/- mice and in PASMCs under hypoxia or with the siRNA of COMP treatment under normoxia, but increased in PASMCs with adenovirus-increased COMP under hypoxia.
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The absorbance of cell counting kit-8 at 450 nm and the expressions of proliferating cell nuclear antigen (PCNA) and osteopontin increased in PASMCs with the siRNA of COMP under normoxia (P㸺
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0.05). PCNA and osteopontin decreased in PASMCs with adenovirus-increased COMP under hypoxia (P㸺0.05). Additionally, the expression of bone morphogenetic protein receptor 2 (BMPR2) was
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reduced in COMP-/- mice (P㸺0.01). Both mRNA and protein levels of bone morphogenetic protein 2 (BMP2) were lower in PASMCs with the siRNA of COMP (P㸺0.05). The protein level of BMP2 could be reversed by adenovirus-increased COMP under hypoxia (P㸺0.05). These data suggest that COMP could normally have a protective role against PASMC phenotype switching and maintain BMP2/BMPR2 signaling, and these protective actions could be lost as a result of hypoxia promoting a depletion of COMP.
Keywords Cartilage Oligomeric Matrix Protein; Pulmonary Hypertension; Phenotype Switching; Pulmonary Arterial Smooth Muscle Cells; Bone Morphogenetic Protein Receptor 2 4
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Introduction
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Pulmonary arterial hypertension (PAH) is a rare, progressive, and fatal disease that is characterized by increased pulmonary artery pressure in the absence of common causes of pulmonary hypertension
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(PH), such as chronic heart, lung, or thromboembolic disease. A major hallmark of PAH is irreversible
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pulmonary arterial remodeling, manifested by excessive cellular proliferation, apoptotic resistance and abnormal contractile-to-synthetic vascular smooth muscle cell (VSMC) phenotype switching(Mandegar
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et al.,2004; Stenmark et al.,2006; Rabinovitch,2007). Although diverse environmental signals including sustained hypoxia and alterations in extracellular matrix are known to regulate SMC phenotype
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dedifferentiation are not well understood.
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(Lann«r et al.,2005; Schultz et al.,2006; Zhou et al.,2009), the mechanisms that control SMC
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Vascular smooth muscle cells (VSMCs), unlike cardiac or skeletal muscle cells, have a unique property
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of plasticity and can undergo reversible changes in phenotype(Owens,1995). Normally, they are mainly restricted to the media of adult blood vessels, express a repertoire of contractile proteins such DVVPRRWKPXVFOH 60 P\RVLQKHDY\FKDLQ60˞-DFWLQ60˞DQGFDOSRQLQDQGKDYHDORZUDWHRI replication. However, on various environmental cues, VSMCs can undergo a transition from a quiescent, contractile/differentiated phenotype to a synthetic/dedifferentiated phenotype, with a high rate of migration/proliferation and a concomitant reduction in expression of VSMC marker proteins(Owens,1995; Hao et al.,2003). Phenotype switching of VSMCs plays an essential role in the development of cardiovascular diseases such as atherosclerosis, postangioplastic restenosis and hypertension. Studies have demonstrated the contribution to cell dedifferentiated of growth factors, mitogenic cytokines, reactive oxygen species, stretch or injury(Owens et al.,2004; Rzucidlo et 5
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al.,2007). VSMC functional contractility depends on extracellular mechanical properties(Steucke et al.,2015) and
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extracellular matrix (ECM) presentation modulates vascular smooth muscle cell mechanotransduction (Sazonova et al.,2015). In vitro, non-confluent VSMCs cultured on stiff substrates express contractile
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phenotype markers at higher levels than on soft substrates, though this difference declines in confluent
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VSMCs(Sazonova et al.,2011). Phenotypic changes in VSMCs induced by vessel injury results in proliferation and deposition of ECM (Collins et al.,2012). One example of the proteins is
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thrombospondins (TSPs), a family of multifunctional matricellular glycoproteins. TSPs do not contribute to the normal ECM structural architecture, they do function in cellular signaling by interacting with other
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ECM proteins(Stenina-Adognravi,2014). The TSP family consists of 5 member proteins. TSP-5 also known as cartilage oligomeric matrix protein (COMP) is a secreted glycoprotein. Previous studies have
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identified COMP in normal rat aortas(Oldberg et al.,1992), human VSMCs and the atherosclerotic plaques of ApoE-/-mice (Riessen et al.,2001), implicating an importance to vascular pathology and a
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potential target for therapy. COMP has recently been identified as a normal component of the human artery wall and secreted by VSMCs. COMP affects cellular attachment, proliferation, and influences chondrogenesis (Kipnes et al.,2003; Chen et al.,2005; Xu et al.,2007). Extracellular modulators have been a particular focus of investigation, including proteins that affect receptor stability, ligand function, or ligand availability. Transforming growth factor ˟-like ligands, the bone morphogenetic proteins (BMPs), is the direct modulation of ligand activity by extracellular factors (De Robertis EM and Sasai,1996). Mice that are heterozygous null for BMP2 (BMP2 (+/-)) develop more severe hypoxia PH than their wild-type littermates, but not BMP4 (LacZ/+) mutant mice. The protective effects of BMP2 are mediated by increasing eNOS expression and activity in the hypoxic pulmonary vasculature
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(Anderson et al.,2010). BMPR2 acts as one of the receptors of BMP2 and is involved in PAH. We have documented evidence suggesting that COMP is essential for maintaining the differentiated
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phenotype of systemic VSMCs; inhibits vascular smooth muscle calcification by interacting with BMP2; prevents vascular aging and vascular smooth muscle cells senescence (Wang et al.,2010; Du Y et
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al.,2011; Wang et al.,2016).
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However, there are many known differences between the systemic system and pulmonary system. It is still not clear whether COMP is expressed in pulmonary artery smooth muscle cells (PASMCs) and
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also involved in the phenotype switching of PASMCs. Here, we show that COMP was also expressed in the media of pulmonary arteries. The deficiency of COMP switched the PASMC phenotype and
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contributed to pulmonary artery remodeling. Additionally, the absence of COMP in pulmonary arteries led to the downregulation of BMP2 and BMPR2. The results of this study appear to identify properties
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of a potentially novel role for ECM proteins on pulmonary hypertension development that have potential for therapeutic targeting.
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Materials and Methods
Material and Reagents
Smooth muscle cell growth medium was purchased from Cell Applications, Inc. (San Diego, CA, USA). Dulbecco's Modified Eagle Medium (DMEM), used as serum-free media (SFM), trypsin and trypsin neutralizing solution were purchased from Lonza (Walkersville, MD, USA). Cells were made quiescent by incubation in SFM for 48 h. Antibodies were purchased from companies as following: COMP (Abcam Inc. Catalog: ab74524 60˞-actin (Santa Cruz. Catalog: sc-53142 60˞ (Abcam Inc. Catalog: ab14106), calponin (Santa Cruz. Catalog: sc-16604-R), OPN (Santa Cruz. Catalog: sc-10591), PCNA (Santa Cruz. Catalog: sc-9857), BMP2 (Santa Cruz. Catalog: sc-6895), BMPR2 7
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(Santa Cruz. Catalog: sc-5682), ˟-actin (Santa Cruz. Catalog: sc-81178) and GAPDH (Santa Cruz. Catalog: sc-47724). All other reagents were from common commercial sources.
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Animals
Male or female Sprague-Dawley rats were housed in the Animal Resource Center of the Harbin
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Medical University. Rats (230-250g) were exposed to chronic hypoxia (10% O2) in a ventilated
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chamber. The hypoxic environment was a mixture of air and nitrogen. The control rats were raised in another chamber where the FiO2 was 21% (normoxia). After 21 days, animals were anesthetized with
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4% halothane after heparin was administered intraperitoneally. The systemic arterial systolic blood
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pressure was measured via caudal artery and the mean pulmonary arterial systolic blood pressure was measured from right heart catheterization (Rats were anesthetized with isoflurane and a micro-tip
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pressure transducer was inserted into the right jugular vein and advanced to the right ventricle. Right
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ventricular pressure was continuously monitored for 10 min and data were analyzed). Then animals
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were weighed and the chest was opened for the removal of the heart and lungs en bloc. The hearts and lungs were placed in an iced modified Krebs solution (mmol/L: KCl 4.7㸪MgSO4 0.57㸪KH2PO4 1.16, CaCl2 2.5, NaHCO3 24, glucose 10 and NaCl 118, PH 7.4) and transferred to the laboratory for microdissection of selected tissues. COMP-/- (KO) mice with a C57BL/6J background were kindly provided by Professor Oldberg Ake (Department of Cell and Molecular Biology, University of Lund, Sweden). KO mice (5 months of age) and COMP+/+ littermates as wild type (WT) were used for experiments. Animals were genotyped using polymerase chain reaction via respective primers. All animal studies followed the guidelines of the Animal Care and Use Committee of Harbin Medical University.
Image analysis of small pulmonary arteries 8
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Six small pulmonary arteries each animal (diameter between 30~4˩P, near hilus of the lung) were randomly measured with 2D image analysis software to get the data of external diameter and wall
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thickness. The times of external diameter compared with wall thickness were calculated.
Cell culture
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Briefly, the pulmonary arteries (PAs) of rats were cut into small pieces after cleaned of their connective
containing collagenase II (10 U/ml DWr&IRU1 hours.
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tissue and removed the endothelium by rubbing the lumen. Then PAs were dispersed by solution The dispersed vascular smooth muscle cells
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were then centrifuged for 10 min to get cell pellet. The cells were resuspended in DMEM with 20%
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serum and 1% penicillin/streptomycin and plated in T25 culture flask to culture. Cell viability (usually greater than 98%) was determined by Trypan Blue exclusion㸬
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Recombinant adenovirus construction and infection
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The adenovirus for full-length COMP (Ad-COMP; NM_016685.2) was constructed and amplified
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according to the manufacturer's protocol (BD Biosciences). An adenovirus that carried b-galactosidase (Ad-LacZ) was used as a negative control. Cells cultured at approximately 80% confluence were infected with recombinant adenovirus (50 multiplicity of infection) for 48 h.
Transfection of small interfering RNA
SiRNA was purchased from GenePharma Co., Ltd (Shanghai). The sequences that corresponded to the siRNA of rat COMP (siRNA-COMP) were seQVHಬ- AGAAACUUGAGCUGUGUUGAUGCC-ಬ and anti-VHQVHಬ- GGCUAUCAAGACAGCUCAAGUUUCU-ಬ$VFUDPEOHVWHDOWK51$LGXSOH[ served as a negative control. In vitro siRNA transfection (50 nM) of rat PASMCs was performed using RNAi MAX (Invitrogen, CA, USA). The transfection procedures followed the manufacturers' 9
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instructions.
RNA isolation and cDNA reverse transcription
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PASMCs were made quiescent by incubation in serum free media (SFM) for 24h. Quiescent cells were exposed to SFM, Ad-COMP, siRNA-COMP for 24h. Media was decanted and cells frozen to -ഒ
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RNAs were extracted using Trizol from pulmonary arteries or PASMCs. Quality of the RNA samples
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was assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA). CDNA was
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generated using Prime6FULSWറ57UHDJHQW.LW 7$.$5$%,2&DWDORJ 55$
Real-time quantitative reverse transcriptase-polymerase chain reaction
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Real-WLPHTXDQWLWDWLYHSRO\PHUDVHFKDLQUHDFWHGZLWK6<%5p3UHPL[([7DT70 7$.$5$%,2
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Catalog: RR420A). Cycle and threshold were REWDLQHGDFFRUGLQJWRWKHPDQXIDFWXUHUಬVLQVWUXFWLRQV
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Changes in expression given are the average of three biological replicates. (The primers are listed in
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the supplemental table 1 on line.)
Agarose Electrophoresis
PCR products were separated by electrophoresis on 3% agarose/TBE gels. Gels were photographed using the Chemi Imager 5500 gel image analysis instrument (AlPha InnCh). The integrated density value (IDV) of PCR product bands was measured by Chemi Imager 5500 V2.03 software (AlPha InnCh), to assess the expressions of long non-coding RNAs (lncRNAs). (The primers are listed in the supplemental table 1 on line.)
Western Blot Assessment
:HVHSDUDWHG˩g protein extracted from PAs (without adventitia and intima) or PASMCs by a 10% or
10
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15% SDS-PAGE gel and transferred it to a PVDF membrane. The membrane was probed with specific SULPDU\DQWLERGLHVIRU&20360˞-DFWLQ60˞FDOSRQLQ2313&1$%03DQG%035
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respectively, and appropriate secondary antibodies were conjugated with horseradish peroxidase. The VSHFLILFEDQGVZHUHYLVXDOL]HGZLWKDQHQKDQFHGFKHPLOXPLQHVFHQWUHDFWLRQDQGQRUPDOL]HGWR˟-actin
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or GAPDH.
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Immunohistochemistry Assay
Fresh tissues were fixed in 4% Paraformaldehyde solution for 24h at 4r& and then were dehydrated in
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30% sucrose solutions for 48h at 4r&. Sections were cut on cryostat at 7 ˩PRIWKLFNQHVVDW-r&7KH
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sections were mounted onto coated slides and were prepared for histology and immunohistochemistry. The sections were stained with hematoxylin and eosin to confirm the presence of microvessles. The
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serial sections to those above were used for immunohistochemistry. They were rehydrated in PBS
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before immunostaining and endogenous peroxidase activity was quenched with 3% H 2O2 in PBS.
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Sections were blocked with 5% donkey VHUXPDQGLQFXEDWHGRYHUQLJKWDWr&ZLWKUDEELWDQWL-COMP antibody (Abcam Inc. Catalog: ab11056). Sections were incubated with biotinylated donkey anti-rabbit secondary antibody, followed by horse radish peroxidase-streptavidin (ZSGB-BIO, China). Samples of 10-20 arterioles, less than100 mm in size, were assessed. The slides were photographed and the integrated optical density of yellow or brown staining was measured using a computer-assisted image analyzing system (Motic Images Advanced 3.2).
Cell Proliferation Assay
Incubate PASMCs (3000 cells/well) in a 96-ZHOOSODWH DWr&&22). Add 10 ˩ORIcell counting kit-8 (CCK-8) solution to each well of the plate. Incubate the plate for 2 hours in the incubator. And then,
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the absorbance was measured at 450 nm using an ELISA reader (Anthros, Durham, NC, USA). All analyses were performed in at least three independent replicate cultures. The optical density value
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(ODV) of treated to non-treated control cells was calculated and presented in terms of relative cell viability.
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Statistical analysis
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Statistical analysis was performed with GraphPad Prism 5 software. All values were expressed as mean s SEM. Statistical analyses between WZRJURXSVZHUHSHUIRUPHGZLWKSDLUHG6WXGHQWಬV t-test,
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and a one-way ANOVA with Newman Keuls correction was used for comparison between multiple
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groups. A value of P<0.05 was used to establish statistical significance.
Results
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COMP Protein Level Is Decreased in Chronic hypoxia-induced PH Rat Arteries and PASMCs
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To investigate the role of COMP in PH, we first compared the expression of COMP in protein level
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between chronic hypoxia-induced PH rats and normoxia-raised rats by immunohistochemistry (Figure 1A) and western blot (Figure 1B & 1C). The data showed that COMP was mainly expressed in the media of pulmonary arteries and degraded in chronic hypoxia-induced PH rats. Furthermore, we tested the expression of COMP in PASMCs under hypoxia (supplement Figure 1). The data demonstrated that COMP was apparently decreased after hypoxia 48h compared with the expression under normoxia. In addition, hematoxylin and eosin (HE) staining was applied to compare the vascular wall thickness between the COMP knockout (COMP-/-) and wide type mice in pulmonary small arteries (Figure 1D & 1E). As shown in HE staining, the wall of pulmonary small arteries in COMP-/- mice was thicker than those in WT mice.
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These results implied that COMP was expressed in pulmonary arteries and played a role in PH. COMP deficiency was correlation with pulmonary artery remodeling. Combined with our recent observation
the phenotype switching of PASMCs.
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COMP Is Required for Maintenance of the PASMC Differentiated Phenotype
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that COMP is necessary to maintain the contractile phenotype of VSMCs, COMP may also regulate
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After observing an association of COMP expression with PH, we next investigated the potential effect of COMP on PASMC phenotype transition by small interfering RNA (siRNA) knockdown. COMP
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mRNA level was inhibited by 70% with the siRNA of rat COMP (siRNA-COMP) knockdown (Figure 2A, 2B and 2C). The mRNA levels of SM ˞-actin, calponin, and SM22˞were downregulated in parallel
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(Figure 2A). Similarly, the protein levels of SM ˞-actin, calponin, and SM22˞were markedly reduced by siRNA-COMP treatment as compared with the scramble siRNA treatment (Figure 2B & 2C). These
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data indicate a causal role of COMP in altering the mRNA and protein level of differentiated marker genes. Thus, COMP is necessary for differentiated marker expression. Additionally, the pulmonary
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arteries of COMP-/- mice and WT mice were separated to analyze the expressions of phenotype markers. In the absence of COMP, the protein levels of SM ˞-actin and calponin were significantly downregulated. These data further support COMP as being required for maintenance of the PASMC differentiated phenotype.
COMP Overexpression Retains the Characteristics of the Differentiated Phenotype of PASMCs under hypoxia For PASMC dedifferentiated with COMP knockdown, we next asked whether adenoviral overexpression of COMP could retain the PASMC differentiated state challenged by hypoxia, which leads to PASMC phenotype switching. Western blot analysis revealed COMP protein level was higher
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in PASMCs with the adenovirus for full-length COMP (Ad-COMP) than with the adenovirus that carried b-galactosidase (Ad-lacZ) infection (Figure 3C & 3D). Interestingly, Ad-COMP significantly
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circumvented hypoxia-induced suppression of differentiated marker at protein level in PASMCs (Figure 3A, 3B, 3C and 3D). Thus, COMP overexpression could restore hypoxia-induced PASMC
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dedifferentiated and render differentiated in cells. It suggests that the protective effect of COMP
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maintains the contractile/differentiated phenotype of PASMCs.
COMP Inhibits PASMCs Excessive Proliferation
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With siRNA-COMP knockdown under normoxia, the optical density value (ODV) of CCK8 at 450 nm increased in PASMCs (Figure A). However, Ad-COMP significantly decreased the ODV under hypoxia
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(Figure 4B). We also detected the expressions of proliferation proteins (osteopontin and PCNA). As shown in Figure 4C & 4D, the expression of PCNA and osteopontin (OPN) in protein level increased
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with siRNA-COMP treatment. And we got the similar data from COMP-/- mice and WT mice (Figure 4E & 4F). It suggests that COMP negatively regulates the PASMC proliferation.
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Absence of COMP Reduces the Expressions of BMP2 and BMPR2 To investigate the mechanisms of COMP in PAH, the BMPR2 was measured, which has been defined to be involved in PAH. It is interesting that BMPR2 was downregulated at protein level in COMP-/- mice compared with that in WT mice (Figure 5A & 5B). In our previous paper, we have reported that COMP directly bound to BMP2 through the C-terminal domain in rat systemic VSMCs. So we also tested the expression of BMP2 in PASMCs. Our data showed that both mRNA (supplement figure 2) and protein levels of BMP2 (Figure 5C & 5D) were lower in PASMCs with siRNA-COMP treatment than those with the scramble siRNA transfection. The BMP2 protein level could be reversed by Ad-COMP under hypoxia (Figure 5E & 5F, supplement figure 3).
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There Is No Difference in Pulmonary Hemodynamics between COMP-/- Mice and Wild Type Mice We measured several parameters of pulmonary hemodynamics in COMP-/- mice and wild type mice (5
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months of age) to observe the effect of COMP on pulmonary function in vivo. The data showed that there were no obvious differences between KO mice and WT mice in body weight, right ventricle
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weight / body weight (RV/BW), right ventricle weight / left ventricle plus ventricular septum weight (RV /
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LV+S), mean pulmonary arterial systolic blood pressure (mPAP), mean systemic systolic blood pressure (SBP) of caudal artery (Figure 6). But there was increasing trend of mPAP in KO mice.
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(Figure 6D) Discussion
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VSMCs are highly specialized cells that regulate vascular tone and participate in vessel remodeling in physiological and pathological conditions. Although many reports have described key factors involved
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in the regulation of the SMC phenotype switching (Alexander and Owens,2012), the detailed molecular mechanisms driving this process are not yet fully understood. In this study, we show that COMP, a
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macromolecular ECM protein, was necessary for maintaining the PASMC differentiated phenotype. Moreover, overexpression of COMP could rescue the hypoxia-induced cell phenotype switching to a dedifferentiated state in vitro. These protective effects may be partly due to the suppression of BMP2/BMPR2 signaling pathway. The causal relationship between them needs further study. Our results reveal intrinsic ECM-associated mechanisms regulating the plasticity of the PASMC phenotype. VSMC in mature animals is a highly specialized cell whose principal function is contraction and regulation of blood vessel diameter, blood pressure, and blood flow distribution. Although there are likely many alternative phenotypic states of SMCs, in general SMC phenotypic switching is characterized by markedly reduced expressions of SMC-selective differentiated marker genes and
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increased SMC proliferation, migration, and synthesis of ECM components required for vascular repair (Alexander and Owens,2012). SMCs within adult animals retain remarkable plasticity and can undergo
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profound and reversible changes in phenotype, a process referred to as phenotypic switching, in response to vascular injury (Owens,1995). The extensive plasticity exhibited by the fully mature SMC
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is an inherent property of the cell that likely evolved in higher organisms because it conferred a survival
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advantage or disease. However, an unfortunate consequence of this plasticity is that it predisposes the SMC to environmental cues/signals that can induce adverse phenotype switching and contribute to
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development and/or progression of vascular disease (Bijli et al.,2016). In the present study, the novel ECM factor COMP was found to modulate the PASMC phenotype switching. In the absence of COMP,
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the differentiated/contractile SMC phenotype marker proteins were down-regulated and proliferation proteins such as PCAN and OPN were up-regulated both in vitro and in vivo. In addition, our data
PASMCs.
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supported COMP as being required to maintain the characteristics of the differentiated phenotype of
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Our data showed that COMP was mainly expressed in the media of the pulmonary artery wall in rats. Hypoxia stimulates pulmonary hypertension, in part by increasing the proliferation of human pulmonary artery smooth muscle cells (Newman et al.,2004). We therefore tested the expression of COMP in PH model rats and PASMCs under hypoxia (24h, 48h, and 72h) and found the decreased COMP level both in hypoxia-induced PH model rats (as compared with control rats) and in PASMCs under hypoxia (48h and 72h) (as compared with a control under normoxia). The data suggested COMP was involved in PH, but its biological relevance to phenotype-related vascular remodeling needed further to be illuminated. The following data found that the differentiated markers were down-regulated in PASMCs after COMP gene knockdown with siRNA. Moreover, PASMCs under hypoxia also reduced the
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expressions of differentiated marker and this could be reversed by adenoviral over-expression of COMP for 24 hours before hypoxia (3%O2). The cell proliferation experiment was done to clarify the
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relationship between COMP and proliferation in PASMCs. The data showed that PASMC proliferation was enhanced after COMP gene knockdown with siRNA or in knockout mice. The increased
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proliferation of PASMCs under hypoxia could be reversed by adenoviral-increased COMP for 24 hours
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before hypoxia (3%O2). This further demonstrates the inhibitory effect of COMP on PASMC phenotype switching. The uniqueness of our study lies in providing a systemic and dynamic evaluation of the
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alteration in the level of the ECM protein COMP during PASMC phenotype switching. Also, our results emphasize the importance of the integrity of the ECM in phenotype-related vascular remodeling such
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as in PH. In our data of COMP-/- mice and WT mice, there were no obvious differences in several pulmonary function parameters, which may be due to the mice age. But there was increasing trend of
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mPAP in COMP-/- mice. In conclusion, COMP was normally expressed in pulmonary arteries and necessary for PASMCs to maintain their differentiated phenotype.
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Although ECM constituents such as COMP are important biological cues regulating the phenotype plasticity of PASMCs, little is known about the mechanisms. BMPR2 plays an important role in PH. The BMPR2 mutation is thought to increase susceptibility to idiopathic PAH in the context of environmental or other genetic factors (Alastalo et al.,2011). It is important to note that even PAH patients without a BMPR2 mutation have reduced expression of this receptor (Atkinson et al.,2002; Du Y et al.,2011). Interestingly, our study on COMP-/- mice and littermate mice showed that BMPR2 was reduced in pulmonary arteries in the absence of COMP. BMPR2 can act as one receptors of BMP2. BMP2 plays a protective role in the hypoxic pulmonary vasculature by increasing eNOS expression and activity (Anderson et al.,2010). In our previous study, we reported that COMP physically interacts with BMP2
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through the C-terminal domain of COMP, prevents BMP2 receptor binding, suppresses Smad1/5/8 phosphorylation, and inhibits the expression of the downstream transcriptional factors Runx-2 and
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Msx-2 (Du Y et al.,2011). Thus, we tested the expression of BMP2 in PASMCs. The data showed that the expression of BMP2 reduced after COMP gene knockdown with siRNA and adenoviral
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over-expression of COMP for 24 hours before hypoxia (3%O2) could reverse the decrease of BMP2
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under hypoxia. This suggests that the deficiency of COMP suppressed BMP2/BMPR2 signaling. The mechanism of COMP on PASMCs is different from that on systemic VSMCs. Further studies are
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needed to define the causal relationship between BMP2/BMPR2 signaling and phenotype switching in PASMCs and elucidate the mechanisms of hypoxia on the decrease of COMP.
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The present study identified a critical role of COMP on PASMC retaining a differentiated phenotype and maintaining BMP2/BMPR2 signaling. The findings reveal a self-protective mechanism of PASMCs
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in response to various environmental cues such as hypoxia and identify properties of a potentially novel role for extracellular matrix proteins on pulmonary hypertension development that have potential
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for therapeutic targeting. Acknowledgments
We appreciated Dr. Ake Oldberg from Lund University for kindly providing COMP-/- mice for our vivo experiments, Dr. Daling Zhu from Harbin medical university-Daqing for generously providing guidance on experiments.
This research was supported by funding from National Natural Science Foundation of China (31400989); Natural Science Foundation of Heilongjiang Province (C2015069); Natural Science Foundation of Heilongjiang Province (LBH-Q15087) Disclosures
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All authors have no conflicts of interest.
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Figure legends: Figure 1 COMP decreases in pulmonary small arteries from chronic hypoxia-induced PH rats. (n=5
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each group) (A) Representative immunohistochemistry staining and (B) western blot of COMP protein in pulmonary arteries from PH model and control rats. (C) Quantitative analysis of full-length COMP in
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pulmonary arteries from PH model and control rats. Relative COMP protein level was normalized to
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WKDWRI˟-actin. *P㸺0.05 vs control. (D) Representative hematoxylin and eosin staining and (E) quantitative analysis of diameter/wall thickness of pulmonary arteries in COMP-/- (KO) and wild type
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(WT) mice (n=5 each group). *P㸺0.05 vs WT mice. (CTL: control, PH: pulmonary hypertension model)
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Figure 2 COMP is required for PASMCs maintaining a differentiated phenotype. (A) Specific knockdown of COMP by siRNA but not scramble siRNA. *P㸺0.05 vs scramble siRNA. Quantification
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of differentiated marker genes in mRNA levels during siRNA-COMP treatment. *P㸺0.05 vs scramble siRNA. (B) Representative western blot and (C) quantitative analysis of COMP, differentiated marker
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protein expression after COMP gene knockdown with siRNA. Results are from 3 independent experiments performed in duplicate. *P㸺0.05, **P㸺0.01 vs scramble siRNA. (D) Representative western blot and (E) quantitative analysis of differentiated marker protein expression in COMP-/- (KO) and wild type (WT) mice. (n=5 each group) *P㸺0.05 vs WT mice.
Figure 3 COMP over-expression retains the characteristics of the differentiated phenotype in PASMCs under hypoxia. (A) Representative western blot and (B) quantitative analysis of PASMC differentiated markers normalized to GAPDH (SM22 ˞) or ˟-actin (Calponin). PASMCs were under hypoxia (3%O2) for 24, 48, and 72 hours. *P˘0.05, **P˘0.01 vs normoxia. (C) Representative western blot and (D)
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quantitative analysis of COMP DQGGLIIHUHQWLDWHGPDUNHUVQRUPDOL]HGWRWKDWRI˟-actin. PASMCs were infected with Ad-lacZ or Ad-COMP for 24 hours before hypoxia (3%O2) for additional 48 hours. Results
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are from 3 independent experiments performed in duplicate. *P㸺0.05, **P㸺0.01 vs Ad-lacZ. (N:
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normaxia, H: hypoxia, Ad: adenovirus)
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Figure 4 COMP inhibits the excessive proliferation of PASMCs. (A) Quantitative analysis of the optical density value (ODV) of CCK8 at 450 nm in PASMCs after COMP gene knockdown with siRNA under
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normoxia . *P㸺0.05 vs scramble siRNA. (B) Quantitative analysis of ODV, PASMCs infected with Ad-lacZ or Ad-COMP for 24 hours before hypoxia (3%O2) for additional 48 hours. **P㸺0.01 vs
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Ad-lacZ. (C) Representative western blot and (D) quantitative analysis of the proliferation protein expressions after COMP gene knockdown with siRNA. Results are from 3 independent experiments
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performed in duplicate. **P㸺0.01 vs scramble siRNA. (E) Representative western blot and (F) quantitative analysis of the proliferation protein expressions in COMP-/- (KO) and wild type (WT) mice.
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(n=5 each group) *P㸺0.05 vs WT mice.
(Ad: adenovirus)
Figure 5 Absence of COMP reduces the expressions of BMP2 and BMPR2. (A) Representative western blot and (B) quantitative analysis of BMPR2 in the pulmonary arteries from COMP-/- (KO) and wild type (WT) mice. (n=5 each group) **P㸺0.01 vs WT mice. (C) Representative western blot and (D) quantitative analysis of BMP2 after COMP gene knockdown with siRNA. *P㸺0.05 (E) Representative western blot and (F) quantitative analysis of BMP2 normalized to that of GAPDH, PASMCs were infected with Ad-lacZ or Ad-COMP for 24 hours before hypoxia (3%O2) for additional 48 hours. Results are from 3 independent experiments performed in duplicate. *P㸺0.05 vs Ad-lacZ. (Ad: adenovirus)
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Figure 6 Pulmonary hemodynamics in COMP-/- mice and wild type mice (5 months of age, n=8 each
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group). (A) Body weight (g/g); (B) the weight of right ventricle / body weight (mg/g); (C) the weight ratio of right ventricle and left ventricle plus ventricle septum (mg/mg); (D) the mean pulmonary arterial
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systolic blood pressure; (E) the mean systemic arterial systolic blood pressure. (RV: right ventricle;
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V+S: left ventricle plus ventricle septum; MPAP: the mean pulmonary arterial systolic blood pressure;
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SBP: the mean systemic arterial systolic blood pressure; KO: COMP-/- mice; WT: wild type mice)
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