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Available online at www.sciencedirect.com

journal homepage: www.elsevier.com/locate/yexcr

Research Article

The sGC activator inhibits the proliferation and migration, promotes the apoptosis of human pulmonary arterial smooth muscle cells via the up regulation of plasminogen activator inhibitor-2$ Shuai Zhanga,b, Lihui Zouc,d, Ting Yanga,b, Yuanhua Yanga,b, Zhenguo Zhaia,b, Fei Xiaoc,d, Chen Wanga,b,d,n a Beijing Institute of Respiratory Medicine, Beijing Chao-yang Hospital, Capital Medical University, 8 Gongti South Rd, Beijing, PR China b Beijing Key Laboratory of Respiratory and Pulmonary Circulation Disorders, 8 Gongti South Rd, Beijing, PR China c Institute of Geriatrics, Beijing Hospital, 1 Dahua Rd, Beijing, PR China d National Clinical Research Center for Respiratory Diseases, 1 Dahua Rd, Beijing, PR China

article information

abstract

Article Chronology:

Background: Different types of pulmonary hypertension (PH) share the same process of

Received 6 November 2014

pulmonary vascular remodeling, the molecular mechanism of which is not entirely clarified by

Received in revised form

far. The abnormal biological behaviors of pulmonary arterial smooth muscle cells (PASMCs) play

23 January 2015

an important role in this process.

Accepted 10 February 2015

Objectives: We investigated the regulation of plasminogen activator inhibitor-2 (PAI-2) by the sGC

Available online 19 February 2015

activator, and explored the effect of PAI-2 on PASMCs proliferation, apoptosis and migration.

Keywords:

Methods: After the transfection with PAI-2 overexpression vector and specific siRNAs or treatment

Soluble guanylate cyclase

with BAY 41-2272 (an activator of sGC), the mRNA and protein levels of PAI-2 in cultured human

Pulmonary arterial smooth muscle

PASMCs were detected, and the proliferation, apoptosis and migration of PASMCs were investigated.

cells

Results: BAY 41-2272 up regulated the endogenous PAI-2 in PASMCs, on the mRNA and protein level.

Plasminogen activator inhibitor-2

In PAI-2 overexpression group, the proliferation and migration of PASMCs were inhibited significantly,

Proliferation

and the apoptosis of PASMCs was increased. In contrast, PAI-2 knockdown with siRNA increased

Apoptosis

PASMCs proliferation and migration, inhibited the apoptosis.

Migration

Conclusions: PAI-2 overexpression inhibits the proliferation and migration and promotes the apoptosis of human PASMCs. Therefore, sGC activator might alleviate or reverse vascular remodeling in PH through the up-regulation of PAI-2. & 2015 Elsevier Inc. All rights reserved.

☆ Funding/Support: This work was supported by National High Technology Research and Development Program [Grant 2012AA02A511], National Department Public Benefit Research Foundation by Ministry of Health PR China [Grant 201302008] and National Key Technology Research and Development Program [Grant 2013BAI09B00]. n Corresponding author at: Department of Respiratory Medicine, Capital Medical University; National Clinical Research Center for Respiratory Diseases; China-Japan Friedship Hospital, No. 2, Yinghuayuan East Street, Chaoyang District, Beijing 100029, PR China. Fax: þ86 010 64289840. E-mail address: [email protected] (C. Wang).

http://dx.doi.org/10.1016/j.yexcr.2015.02.006 0014-4827/& 2015 Elsevier Inc. All rights reserved.

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Introduction Pulmonary arterial hypertension (PAH) and chronic thromboembolic pulmonary hypertension (CTEPH), two different types of pulmonary hypertension (PH), share the same pathological process of pulmonary vascular remodeling [1]. Vascular remodeling is characterized by medial hypertrophy, intimal proliferative changes, adventitial thickening, complex lesions and thrombotic lesions [2]. The enhanced proliferation and reduced apoptosis of pulmonary arterial smooth muscle cells (PASMCs) in the medial layer of vessel wall lead to vascular remodeling, which results in vessel stiffness, occlusion, and increased vascular resistance. However, its underlying mechanisms remain to be fully elucidated. The dysregulation of nitric oxide (NO) production, soluble guanylate cyclase (sGC) activity and cyclic guanosine monophosphate (cGMP) degradation in PH demonstrate the crucial role of the NO/sGC/cGMP pathway in the development of PH [3]. The sGC activators have been shown to be beneficial in the treatment of CTEPH [4] and PAH [5] in clinical trials. However, the downstream genes of the NO/sGC/cGMP pathway involved in the pathogenesis of PH are rarely described. It is generally assumed that the plasminogen activator (PA) system plays an important role in tissue remodeling, cell migration and angiogenesis, wound healing [6,7]. Plasminogen activator inhibitor-2 (PAI-2), also known as SerpinB2, is a member of a large family of serine protease inhibitors (serpins), which are recognized to be key regulators of a range of biological processes such as complement activation, fibrinolysis, cellular differentiation, tumor suppression, apoptosis, and cell motility [8–10]. Forty years ago, PAI-2 was isolated from human placenta and first described as an inhibitor of urokinase-type plasminogen activator (uPA) [11]. Subsequent investigations revealed that PAI-2 could also inhibit tissue plasminogen activator (tPA) [12]. With the finding that PAI-1 had a faster and stronger inhibition on PA [13] and was present in normal plasma, whereas the highest expression levels of PAI-2 were found intracellularly in vitro [14], a new focus into the intracellular PAI-2 function was invoked. In recent decades, it has been reported that PAI-2 has effect on the proliferation [15], apoptosis [16–18], differentiation [18], migration [19], adhesion [20], tumor cells invasion [21] and other biological behaviors [22,23] of various cells. Our previous study found that PAI-2 was one of the candidate downstream genes in NO/sGC/cGMP pathway. In this report, we hypothesized that PAI-2 could function as a regulator of PASMCs proliferation, apoptosis and migration, and thereby might contribute to the pathologic pulmonary vascular remodeling in PAH. In order to confirm our hypothesis, we used cultured human PASMCs to identify the effect of sGC activators on PAI-2 expression, and the effect of PAI-2 overexpression and knockdown on the cell proliferation, apoptosis and migration.

Materials and methods Cell culture PASMCs purchased from ScienCell Research Laboratories (Carlsbad, CA, USA) are isolated from human pulmonary arteries, and characterized by an immune-fluorescent method with antibodies to α-smooth muscle actin and desmin. PASMCs were cultured in

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smooth muscle cell medium (SMCM) (ScienCell Research Laboratories, Carlsbad, CA, USA) supplemented with 2% fetal bovine serum and 1% smooth muscle cell growth supplement, enriched at 37 1C in a 5% CO2 atmosphere. Passages three to seven were used for experiments.

Recombinant plasmids construction and cell transfection To explore the effect of PAI-2 on PASMCs proliferation, apoptosis and migration, PAI-2 overexpression vector and specific small interference RNAs (siRNAs) were constructed (shown in Table 1). The fulllength cDNA sequence of the human PAI-2 gene and green fluorescent protein (GFP) was cloned. PAI-2–GFP was amplified in the recombinant PCR, digested with restriction enzymes, and cloned between NheI and BamHI sites of pcDNA 3.1(-) (Invitrogen) and named pcDNA–PAI-2–GFP. The synthetic PAI-2 gene was confirmed by DNA sequencing. Recombinant plasmid pcDNA–GFP was constructed as a control. PAI-2 specific siRNAs, negative control (NC) siRNA, FAM-labeled NC siRNA and glyceraldehyde–phosphate dehydrogenase (GAPDH) positive control (PC) siRNA were designed. Different siRNAs targeting PAI-2 were tested and the one with the most efficient knock-down was used to transiently transfect PASMCs. All siRNAs were purchased from GenePharma (Shanghai, China). PASMCs of approximately 80–90% confluency were transfected using Lipofectamine 3000 Reagent (Invitrogen Life Technologies, Carlsberg, CA, USA) following the manufacturer's instructions. In each well of 96-well plate, 0.1 μg DNA and 0.3 μl Lipofectamine 3000 (Invitrogen) were added to 10 μl SMCM (Invitrogen). Cells were harvested 24 h later. PASMCs were treated with sGC activators, BAY 41-2272 (5 μmol/L, 10 μmol/L, 20 μmol/L and 40 μmol/L) or BAY 582667 (5 μmol/L, 10 μmol/L, 20 μmol/L and 40 μmol/L). BAY 41-2272 and BAY 58-2667 were dissolved in DMSO, stored at 100 μmol/L at 201C and used at the concentration needed for 24 h, and then cells were harvested at the indicated time points. PASMCs were used to perform the PCR and western blot 24 h after the transfection or treatment with sGC activators.

RNA extraction, reverse transcription and real-time PCR Total RNA was isolated from PASMCs, using TRIzol (Invitrogen Life Technologies, Carlsberg, CA, USA) according to the manufacturer's instructions (Invitrogen). The Moloney Murine Leukemia Virus reverse transcription kit (Invitrogen Life Technologies, Carlsberg, CA, USA) was used in the reverse transcription of RNA. Real-time PCR was performed to quantify the interested gene PAI-2, the internal control gene β-actin and GAPDH by an iQ5 Multicolor Real-time PCR Detection System (Bio-Rad Laboratories, Hercules, CA, USA). The primers for PAI-2, β-actin and GAPDH were purchased from Invitrogen Life Technologies (Carlsberg, CA, USA). Specific primers used for sequence detection were: β-actin Table 1 – Small inference RNAs used in the transfection. siRNA

Sense

PAI-2 siRNA 1# PAI-2 siRNA 2# PAI-2 siRNA 3# GAPDH positive control siRNA Negative control siRNA

50 -GCUUCCGGGAAGAAUAUAUTT-30 50 -GGUCAAGACUCAAACCAAATT-30 50 -GCACACCUGUACAGAUGAUTT-30 50 -GUAUGACAACAGCCUCAAGTT-30 50 -UUCUCCGAACGUGUCACGUTT-30

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50 -TGGCACCACACCTTCTACAA-30 (forward), 50 -CCAGAGGCGTACAGGGATAG-30 (reverse); PAI-2 50 -TCCATTCATCCTTCCGCTCT-30 (forward), 50 -AAGTCTACTGCCTGGGGTTC-30 (reverse); GAPDH 50 -ACCACAGTCCATGCCATCAC-30 (forward), 50 -TCCACCACCCTGTTGCTGTA-30 (reverse). The PCR conditions were as follows: 95 1C for 5 min, followed by 40 cycles of 5 s at 95 1C and 30 s at 60 1C. Experiments were performed in triplicate and were repeated at least three times. A comparative CT method was used to relatively quantify the expression of these genes. The change fold of PAI-2 after transfection or treatment was analyzed.

Protein isolation and western blot The protein was isolated using the protein lysis buffer (including 1% NP-40, 1 μg/ml aprotinin, 1 μg/ml leupeptin, and 100 μg/ml PMSF). Its concentration was estimated with a Bio-Rad DC Protein Assay (Bio-Rad, Hercules, CA, USA). Protein extracts (50 μg) from PASMCs 24 h after transfection or treatment were subjected to the sodium dodecyl sulfate–polyacrylamide gel electrophoresis and then transferred to polyvinylidene difluoride membranes by electroblotting (Millipore Corp., Boston, MA, USA). Following blocking with 5% nonfat milk for 30 min, the membranes were incubated overnight with primary antibodies at 4 1C, including 2 μg/ml anti-PAI-2 antibody (abcam, Cambridge, MA, USA) and 1:1000 GAPDH antibody (cwbiotech, Beijing, China). The membranes were washed three times with Tris-buffered saline containing Tween 20 and then incubated with 1:5000 peroxidase-conjugated anti-IgG antibody (zsbio, Beijing, China) at 4 1C for 2 h. After washing with Tris-buffered saline containing Tween 20 for three times, the membranes were exposed to enhanced chemiluminescent reagents (Millipore Corp., Boston, MA, USA) for the detection of protein bands. GAPDH was used as an internal control.

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Migration assay The 8-μm pore size polycarbonate membrane of Transwell Permeable Support (24 mm; Corning, Inc., Corning, Cambridge, MA, USA) was used to examine the PASMCs migration. Control and transfected PASMCs were isolated and added to the upper well of a Transwell at 2  105 cells/well. Fibronectin (ProSpec-Tany TechnoGene Ltd., Ness-Ziona, Israel) was added to the lower chamber at a concentration of 5 μg/ml and the cells were allowed to migrate for 24 h. In experiments with the sGC activator BAY 412272, the PASMCs were pre-incubated with BAY 41-2272 in the upper well of a Transwell for 30 min before the addition of fibronectin into the lower chamber. The migration was quantified by staining the cells with 0.5% crystal violet following microscopic cell counting on eight random fields (200  ) in each well. Experiments were performed in triplicate and were repeated at least three times.

Data analysis All data obtained in triplicate independent experiments were evaluated using GraphPad Prism 5.01 for Windows (GraphPad Software, Inc., La Jolla, CA, USA). All data fitted normal distribution are presented as mean7standard deviation. Statistical comparisons were performed with the Student t test for analyzing two-group data, whereas one-way ANOVA was used to test differences among multiple independent groups with Po0.05 regarded as significant.

Results PAI-2 is significantly up regulated in gene expression profile

Proliferation assay We used the Cell Counting Kit-8 (DOJINDO Molecular Technologies, Inc., Kumamoto, Japan) to detect the proliferation of cells. PASMCs in logarithmic phase were seeded into 96-well plates at the density of 3  103 per well. 24 h after transfection, 10 μl CCK-8 reagent was added to each well. After culture for 4 h, the optical density (OD) was measured at 450 nm with an ultramark microplate reader (Bio-Rad, Hercules, CA, USA). Experiments were performed in triplicate and were repeated at least three times.

Apoptosis assay Apoptotic cells were quantified by Annexin V/propidium iodide double staining 24 h after the transfection or treatment with sGC activators. The double-staining procedure was performed as follows. The cells were collected and washed twice in the cold phosphate buffer solution. Cell pellets were resuspended in 200 μl 1  binding buffer and resuspended cells were gently vortexed and stained with 5 μl Annexin V-fluorescein isothiocyanate (Jiamei Biotech Co., Ltd., Beijing, China) in the dark at room temperature, and then with 5 μl propidium iodide (Jiamei Biotech Co., Ltd., Beijing, China) for 15 min in the dark at room temperature. The early and late apoptosis of PASMCs was detected by BD Accuri C6 flow cytometer (BD Accuri, Ann Arbor, Michigan, USA).

In the gene expression profile, PAI-2 was one of the differentially expressed genes, which were obviously up regulated in cultured human PASMCs after the treatment with 8-Br-cGMP (100 μm for 8 h). The agarose gel electrophoresis result of Real-time PCR and PCR is shown in Fig. 1.

The regulation of PAI-2 by sGC activators in the PASMCs PASMCs were treated with different concentrations of BAY 412272 and BAY 58-2667 (sGC activator) (5 μmol/L, 10 μmol/L, 20 μmol/L and 40 μmol/L). The results of Real-time PCR are shown in Fig. 2A. The mRNA level of PAI-2 was increased after the treatment with BAY 41-2272 in a dose-dependent fashion. The increase of PAI-2 level in the BAY 58-2667 group was not as significant as that in the BAY 41-2272 group. Based on the results of Real-time PCR, we chose 20 μmol/L BAY 41-2272 to treat the cells, extracted the protein and performed Western blotting. PAI-2 also increased on the protein level after the treatment with BAY 41-2272 (Fig. 2B).

The overexpression and knockdown of PAI-2 in the PASMCs Fig. 3A and B shows the amplification chart and melting curve of Real-time PCR after the transfection of PAI-2 specific siRNAs. The

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Ct values of triplicate independent Real-time PCRs are provided in Supplementary tables 1–3. PAI-2 was significantly decreased on the mRNA level in PASMCs transfected with PAI-2 siRNAs (Fig. 3C). Western blotting shows that the exogenous PAI-2 (72 kD) was overexpressed in the PASMCs transfected with pcDNA3.1 ( )-PAI2–GFP, and knocked down in the PASMCs transfected with PAI-2 specific siRNA (Fig. 3D). HEK-293 cells and K 562 cells were also transfected to confirm the overexpression and knockdown of PAI2 (Data not shown).

The overexpression of PAI-2 inhibits PASMCs proliferation in vitro The results of cell proliferation assay are shown in Fig. 4. In PAI-2 overexpression groups (pcDNA–PAI-2–GFP group and BAY 41-2272 group), the proliferation of PASMCs was inhibited significantly. In the PAI-2 knockdown group, PASMCs proliferation was promoted, as compared with the PASMCs transfected with NC siRNA.

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The overexpression of PAI-2 promotes PASMCs apoptosis in vitro Fig. 5 shows the results of cell apoptosis detection. Both the early and late apoptosis of PASMCs was increased by PAI-2 overexpression. In the PAI-2 knockdown group, PASMCs apoptosis was decreased compared with the PASMCs transfected with NC siRNA. The apoptosis of PASMCs was promoted by BAY 41-2272, and the promotion effect enhanced as the concentration of BAY 41-2272 increased (Fig. 5E and F).

The overexpression of PAI-2 inhibits PASMCs migration in vitro In the next step, we investigated the effect of PAI-2 on PASMCs migration, a function that also contributes to the pathologic vascular remodeling. We observed the migration of PASMCs after cells staining by crystal violet (Fig. 6A–F). After cell counting, the cell number of each group was compared (Fig. 6G). The migration of PASMCs was inhibited after the transfection of PAI-2 overexpression vector, while promoted after the knockdown of PAI-2. BAY 41-2272 20 μmol/L treatment for 24 h also induced a significant decrease in the migration of PASMCs, compared with the control medium.

Discussion

Fig. 1 – The up-regulation of endogenous PAI-2 in the PASMCs after the treatment with 8-Br-cGMP. Total RNA was extracted from the PASMCs treated with a cGMP analog, 8-Br-cGMP (100 μmol/L for 8 h). Real-time PCR and PCR were performed to amplify β-actin and PAI-2. Real-time PCR (A) and PCR (B) products were subjected to the agarose gel electrophoresis. The expression levels of PAI-2 were normalized to the β-actin levels.

In the present study, we demonstrate that the expression level of PAI-2 in PASMCs was significantly up regulated after the induction of the sGC activator or cGMP analog. The sGC activator, BAY 412272, inhibits the migration of PASMCs stimulated with fibronectin and promotes the apoptosis of PASMCs. The proliferation of PASMCs is also inhibited by BAY 41-2272 in a concentrationdependent manner. Our study demonstrates for the first time that the sGC activator regulates PAI-2 and therefore is responsible for the vascular remodeling. To our knowledge, there are 4 analogs of cGMP. Rp-8-Br-cGMPS is a cGMP analog which blocks cGMP dependent protein kinase. Sp-8-BrcGMPS is an unspecific activator of cAMP-dependent protein kinase.

Fig. 2 – The regulation of PAI-2 by sGC activators in the PASMCs. The PASMCs were treated with sGC activators, BAY 41-2272 (5 μmol/L, 10 μmol/L, 20 μmol/L and 40 μmol/L) or BAY 58-2667 (5 μmol/L, 10 μmol/L, 20 μmol/L and 40 μmol/L). 24 h after the treatment with sGC activators, mRNA were isolated from the PASMCs, and subjected to Real-time PCR (A). Based on the result of Real-time PCR, the protein of the PASMCs treated with BAY 41-2272 20 μmol/L was isolated and subjected to Western blotting. The GAPDH levels were measured as a loading control (B). **Po0.01 vs. the control group. ***Po0.001 vs. the control group.

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Fig. 3 – The over-expression and knock-down of PAI-2 in the PASMCs. The amplification chart of β-actin, PAI-2 and GAPDH after transfection of PAI-2 specific siRNA (A). Melting curve of real-time PCR (B). The PASMCs were transiently transfected with PAI-2 siRNA 1#, PAI-2 siRNA 2# and PAI-2 siRNA 3#. The expression levels of PAI-2 were normalized to the β-actin levels after Real-time PCR. The data were obtained from three individual experiments and were expressed as mean7SD (C). PAI-2 protein levels in pcDNA3.1(-)–PAI-2–GFP and PAI-2 specific siRNA cells were analyzed using immunoblotting assay. The GAPDH levels were measured as loading control (D). ***Po0.001 vs. the negative control siRNA group.

8-pCPT–cGMPNa is a selective activator of cGMP-dependent protein kinase. 8-Br-cGMP is a selective activator of cGMP-dependent protein kinase, a cell-permeable cGMP analog with greater resistance to hydrolysis by phosphodiesterases than cGMP. After comparing the function and characters of these four analogs, we chose 8-Br-cGMP to treat PASMCs in the gene expression profile analysis. In previous studies, the expression of sGC in PASMCs has been proved [24–26]. The activation of sGC and up-regulation of cGMP by BAY 41-2272 and BAY 58-2667 have also been proved in previous studies [27]. Thus, we did not design relevant experiments in the present study. A distinct difference in cellular localization of PAI-2 has been noted: 47 kD PAI-2

being intracellular and 60kD PAI-2 being secreted [13]. Based on our previous finding and the literature review, PAI-2 mainly exists inside the PASMCs, while the concentration of its secreted form is quite low. Therefore, in this study, we focused on the function of intracellular PAI-2 and did not collect the medium to detect extracellular PAI-2.

The role of PASMCs in pulmonary vascular remodeling Vascular remodeling is the main pathological change of PAH and can be seen in CTEPH as well. It involves structural and functional changes to the normal structure of the arterial walls that lead to

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Fig. 4 – The overexpression of PAI-2 inhibits PASMCs proliferation. The PASMCs in logarithmic phase were seeded into 96-well plates and transfected with the recombinant plasmids, siRNAs or treated with BAY 41-2272 (20 μmol/L). 24 h after transfection, CCK-8 reagent was added to each well. After culture for 4 h, the optical density (OD) was measured at 450 nm with an ultramark microplate reader, representing the proliferation of the PASMCs. Experiments were performed in triplicate and were repeated at least three times. The data were expressed as mean7SD. **Po0.01 vs. the control group or the negative control siRNA group. ***Po0.001 vs. the control group or the negative control siRNA group.

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present study, we found the anti-proliferative and pro-apoptosis effect of PAI-2 on PASMCs, through which PAI-2 could prevent the progression of medial hypertrophy and the plexiform lesion. Previous studies have demonstrated that the expression of PAI-2 in THP-1 monocyte-like cells decreased the cell proliferation [20]. This anti-proliferative effect of PAI-2 was attenuated by treating the PAI-2-expressing THP-1 cells with recombinant urokinase (u-PA), suggesting that PAI-2 was disruptive of a u-PA/u-PA receptor signaling pathway initiated on the cell surface [20]. In this study, we found that the pro-apoptotic effect on PASMCs by BAY 41-2272 is statistically significant; however, it seems not very strong. We believe that the function of PAI-2 in the regulation of pulmonary vascular remodeling is mainly realized through its antiproliferation effect on PASMCs. Moreover, if BAY 41-2272 had strong effect on PASMCs apoptosis, the normal morphology and structure of pulmonary artery might be damaged by it. The effect of PAI-2 on apoptosis is still under debate. Earlier publications provided in vitro evidence that PAI-2 inhibits the TNFinduced apoptosis in HT-1080 fibrosarcoma cells [16] and HeLa cells [34]. However, some studies provided contradictory data [35]. PAI-2 in endothelial cells induced with inflammatory stimuli can inhibit the proteasome and thus tilt the balance favoring pro-apoptotic signaling [18]. One argument in the interpretation of the effect of PAI-2 on apoptosis is the level of enforced expression of PAI-2 in cells. In most of these in vitro studies, PAI-2 was overexpressed in cells that either did not produce PAI-2 at all or were expressed to levels that well exceeded endogenous expression levels. Under these conditions, whether the in vivo role of PAI-2 is genuinely reflected can be reasonably debated.

The anti-migratory effect of PAI-2 on PASMCs increased muscularization of the muscular pulmonary arteries, muscularization of the peripheral nonmuscular arteries, formation of plexiform lesions and neointima [28]. PASMCs make up the medial layer of distal pulmonary arteries. The precise role of PASMCs in the initial cause of PH is controversial; however, the hypertrophy, proliferation, migration, and resistance to apoptosis of PASMCs contribute to the development of pulmonary vascular remodeling in PH. It will lead to increased thickness of the smooth muscle component of the vessel wall and abnormal muscularization of the normally nonmuscularized, diatal pulmonary arteries. Therefore, we focus on the biological behaviors of PASMCs and try to find another therapeutic effect of sGC activators in PH, in addition to the relaxation of vascular smooth muscle.

The anti-proliferative and pro-apoptotic effect of PAI-2 on PASMCs A balance between PASMCs proliferation and death is necessary for normal development and function of tissues. The imbalance leads to disease states such as PH. The uncontrolled proliferation and resistance to apoptosis of PASMCs, which leads to the increase of pulmonary vascular resistance, is a major contributor to pulmonary vascular remodeling [29,30]. In animal experiments, the promotion of apoptosis or coupling with the inhibition of the proliferation of PASMCs prevents the progression of medial hypertrophy [31,32]. A histological hallmark of PAH, the plexiform lesion, is caused by monoclonal proliferation of endothelial cells, migration and proliferation of PASMCs, and accumulation of circulating cells [33]. In the

The migration of vascular smooth muscle cells (VSMCs) occurs during development, vascular injury and remodeling. It is possible that the migration of VSMCs plays a role in neointima formation, muscularization of distal, normally nonmuscular arteries, and formation of plexiform lesions [28]. Previous studies indicated that proliferating cells arising in the media or adventitia of the arteries migrate to the subendothelial space to contribute to the neointima [36,37]. Whether VSMCs migrate in vivo, and in which direction, is probably controlled by the balance of pro-migratory and anti-migratory influences. Many pro-migratory factors, including cytokines [38], peptide growth factors [39,40], and extracellular matrix (ECM) components [41] have been identified. The anti-migratory factors include heparin in ECM [42] and tissue inhibitors of metalloproteinases [43]. In idiopathic PAH, plasminogen activator inhibitor-1 (PAI-1) negatively regulates PASMCs proliferation and increases PASMCs migration [44]. The effect of PASMCs phenotype regulated by PAI-2 has not been reported before. Although the evidence for the antimigratory effect of PAI-2 on PASMCs is rare, uPA and uPA receptor (uPAR) have been reported to be important components of a proteolytic system that regulates the matrix remodeling during the cell migration, wound healing, developmental tissue remodeling, tumor invasion and other processes [45–47]. The effect of PAI-2 on the migration of cells, via the inhibition of uPA, is mainly studied in tumor so far [48,49]. The uPA system is involved at multiple steps in cancer progression. In particular, uPA has been implicated in the remodeling of ECM, enhancing both cell proliferation and migration and modulating cell adhesion [50]. The anti-migratory effects of

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Fig. 5 – The overexpression of PAI-2 promotes PASMCs apoptosis. Apoptotic cells were quantified by Annexin V/propidium iodide double staining 24 h after the transfection or treatment with sGC activators. The early and late apoptosis of PASMCs was detected by the flow cytometer. The cells were as follows: the left lower quadrant, viable; and the right lower quadrant, early apoptotic; the right upper quadrant, late apoptotic or necrotic; the left upper quadrant, mechanically damaged (A and B). Apoptotic cells were calculated by qualitative flow cytometry. Experiments were performed in triplicate and were repeated at least three times. The data were expressed as mean7SD (C–F). * Po0.05 vs. the control group or the negative control siRNA group. ** Po0.01 vs. the control group or the negative control siRNA group.

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Fig. 6 – The PAI-2 overexpression inhibits PASMCs migration. The PASMCs migrated through the polycarbonate membrane, stimulated with fibronectin, were stained by 0.5% crystal violet and observed under a microscope (A–F, 200  ). After microscopic cell counting on eight random fields (200  ) in each well, the mean number of migrated cells was compared (F). Experiments were performed in triplicate and were repeated at least three times. The data were expressed as mean7SD. **Po0.01 vs. the control group or the negative control siRNA group. ***Po0.001 vs. the control group or the negative control siRNA group. PAI-2 on PASMCs we found in this study may be closely linked to the regulation of cytoskeleton dynamics and ECM proteolysis via the inhibition of uPA. In animal models, the periadventitial application to the injured carotid artery of recombinant uPA stimulated the neointima formation as well as the proliferation and migration of smooth muscle cells [47]. The mechanism involved need to be investigated in further studies.

Based on the characteristic of vascular remodeling, the agents that could regulate the proliferation, apoptosis or migration of PASMCs are potential targets for therapy in patients with PH. The inhalation of combined PDE3/4 inhibitor, tolafentrine, reverses PH induced by monocrotaline in rats. Tolafentrine blocked the enhanced PASMC migratory response when assessed in vitro [51]. Sildenafil exerts an anti-proliferative effect on human PASMCs that is mediated by an

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interaction between the cGMPPKG and the cAMP–PKA activated pathways, leading to inhibition of PDGF-mediated activation of the ERK [52]. The finding of our study might provide a new insight in the development of biomarkers and target agents in the vascular remodeling of PH.

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[3]

[4]

Limitations There are several limitations in the present study. First, only one method was used in the functional analysis of PASMCs. However, the methods we chose are relatively classic. Moreover, all data were obtained in at least triplicate independent experiments. Secondly, it would be more supportive to test for the effect of exogenous NO donors and compare findings with sGC activators. Based on the findings of Stasch [27], although BAY 58-2667 does not activate sGC as strongly as does NO, concentrations as low as 1 nM activate sGC sufficiently to yield biologically important increases in cGMP. In the gene expression profile analysis, the effect of sGC activators and 8Br-cGMP is almost the same. In addition, we mainly focus on the downstream of NO/sGC/cGMP pathway and its effect on PASMC phenotype. Therefore, we only used sGC activators to analyze the change of PASMC phenotype. Moreover, to strength our finding, the mechanism that cGMP induces PAI-2 transcription and the loss of function of sGC on PAI-2 expression, activity and PASMC phenotype are investigated in another study by our team, including the knockdown of sGC in the cell experiment and the knock-out of sGC in the animal experiments.

[5]

[6]

[7]

[8]

[9]

[10]

Conclusions In conclusion, we demonstrate for the first time that PAI-2 inhibits the proliferation and migration and promotes the apoptosis of cultured human PASMCs. Moreover, sGC activators could alleviate or reverse the vascular remodeling in PH through the upregulation of PAI-2 in PASMCs. Thus, the study might provide a novel insight into the molecular mechanisms of pulmonary vascular remodeling in PAH and CTEPH. The results of our study suggest that PAI-2 is a potent regulator of PASMCs and the disturbance in its expression levels may lead to the development of pulmonary vascular remodeling. These observations indicate that PAI-2 may be a promising target for therapeutic intervention through the NO/sGC/cGMP signaling pathway in PH.

[11]

[12]

[13]

[14]

[15]

Appendix A.

Supporting information [16]

Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.yexcr.2015.02.006.

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