Mechanical stretching of pulmonary vein stimulates matrix metalloproteinase-9 and transforming growth factor-β1 through stretch-activated channel/MAPK pathways in pulmonary hypertension due to left heart disease model rats

Hui Zhang; Wenhui Huang; Hongjin Liu; Yihan Zheng; Lianming Liao

doi:10.1371/journal.pone.0235824

. 2020 Sep 3;15(9):e0235824. doi: 10.1371/journal.pone.0235824

Mechanical stretching of pulmonary vein stimulates matrix metalloproteinase-9 and transforming growth factor-β1 through stretch-activated channel/MAPK pathways in pulmonary hypertension due to left heart disease model rats

Hui Zhang ^1,^*, Wenhui Huang ¹, Hongjin Liu ¹, Yihan Zheng ¹, Lianming Liao ²

Editor: Chuen-Mao Yang³

PMCID: PMC7470280 PMID: 32881898

Abstract

Pulmonary hypertension due to left heart disease (PH-LHD) is a momentous pulmonary hypertension disease, and left heart disease is the most familiar cause. Mechanical stretching may be a crucial cause of vascular remodeling. While, the underlining mechanism of mechanical stretching-induced in remodeling of pulmonary vein in the early stage of PH-LHD has not been completely elucidated. In our study, the PH-LHD model rats were successfully constructed. After 25 days, doppler echocardiography and hemodynamic examination were performed. In addition, after treatment, the levels of matrix metalloproteinase-9 (MMP-9) and transforming growth factor-β1 (TGF-β1) were determined by ELISA, immunohistochemistry and western blot assays in the pulmonary veins. Moreover, the pathological change of pulmonary tissues was evaluated by H&E staining. Our results uncovered that left ventricular insufficiency and interventricular septal shift could be observed in PH-LHD model rats, and the right ventricular systolic pressure (RVSP) and mean left atrial pressure (mLAP) were also elevated in PH-LHD model rats. Meanwhile, we found that MMP-9 and TGF-β1 could be highly expressed in PH-LHD model rats. Besides, we revealed that stretch-activated channel (SAC)/mitogen-activated protein kinases (MAPKs) signaling pathway could be involved in the upregulations of MMP-9 and TGF-β1 mediated by mechanical stretching in pulmonary vein. Therefore, current research revealed that mechanical stretching induced the increasing expressions of MMP-9 and TGF-β1 in pulmonary vein, which could be mediated by activation of SAC/MAPKs signaling pathway in the early stage of PH-LHD.

Introduction

Pulmonary hypertension due to left heart disease (PH-LHD) belongs to type II pulmonary hypertension (PH) and is the most common type of PH in clinical practice [1, 2]. It is usually initiated by left ventricular dysfunction, which results in increased left ventricular end-diastolic pressure, increased left atrial pressure, and finally increased pulmonary venous pressure. This eventually leads to PH and pulmonary artery revascularization [1, 3]. Thus pulmonary vascular remodeling is the pathological basis of PH-LHD [4]. Study has demonstrated that the earliest pathophysiological change occurs in the pulmonary veins during the disease process of PH-LHD [5]. Whether pulmonary venous hypertension in the early stage of the disease participates in pulmonary artery remodeling has not been reported.

Cell adhesion, spreading, growth and differentiation can be regulated by various physical and chemical factors, among which mechanical factors have a great influence on these biological behaviors of cells [6, 7]. Previous studies have indicated that the stretch of cell membrane and its surface receptors can cause the changes of cell morphology and nuclear position, thus affecting cell growth [8, 9]. Research has also testified that cell morphology and spreading degree can affect gene expression and even determine cell survival and death [10, 11]. Currently, Mechanical stretching has become a crucial factor in regulating the structure and function of mammalian cells and tissues, but excessive mechanical stretching will cause vascular remodeling [12]. Nevertheless, the functions and mechanisms of mechanical stretching on PH-LHD has not been entirely elucidated.

Matrix metalloproteinase-9 (MMP-9), also known as gelatinase-B, is one of the most crucial members of the MMPs family [13]. Long-term hypertension can lead to vascular endothelial damage and stimulate smooth muscle to produce MMP-9 [14]. MMP-9 can also degrade the vascular extracellular matrix (ECM), damage the vessel wall and lead to vascular remodeling [15]. Transforming growth factor-β1 (TGF-β1), as a pleiotropic factor, is the most dominant subtype of TGF-β [16, 17]. TGF-β1 has been proven to protect the aorta and other blood vessels from damage caused by factors such as high blood pressure and high cholesterol [18, 19]. Researches have shown that MMP-9 and TGF-β1 play vital roles in pulmonary vascular remodeling [14, 20, 21]. Study showed that the enhanced TGF-β-driven smad-2/3 signaling promoted pulmonary vascular remodeling in inflaming lungs [22]. Study proved that the increased activity and expression of MMP-9 in Pulmonary arterial hypertension (PAH), and effective blocking of MMP-9 could provide an option in the therapeutic intervention of human PAH [23].

SAC, serves a mechanically-sensitive ion channel, can be regulated by the changes in the tension of a cellular scaffold or lipid membrane coupled with channel proteins [24]. It was reported that SAC has significant effect in the electrophysiological changes of the myocardium during stimulation [25]. MAPK pathway is proven to be involved in various physiological processes, such as cell growth, development and inter-cell function synchronization, which also can be closely related to the regulation of inflammation and stress response [26, 27]. Studies also testified that mechanical stretching can activate the phosphorylation of the MAPK pathway [28, 29]. Also, studies have shown that the expression of MMP-9 and TGF-β1 in tissues may be related to stretch-activated channel (SAC) and mitogen-activated protein kinase (MAPK) signaling pathway [12, 30, 31]. Therefore, we speculated that SAC and MAPK signaling pathway may be potential regulatory molecules in PH-LHD.

In this study, the isolated pulmonary veins of PH-LHD rats were mechanically stretched and the expression of MMP-9 and TGF-β1 that corresponded to the early stage of the disease was evaluated. Also, the roles of SAC and MAPK signaling pathway on the expression of MMP-9 and TGF-β1 were explored to investigate the possible mechanism of pulmonary vascular remodeling. The study may shed new light on the prevention and treatment of PH-LHD.

Materials and methods

Animals

Male Sprague Dawley (SD) rats (3–4 weeks old and weighing 80–100 g; license number SCXK (Hu) 2017–0005) were provided by the Shanghai Slack Laboratory Animal Co. (Shanghai, China). Before the experiment, all rats were kept adaptively for one week, fed a standard diet and fed free water. All rats in this study have been approved by the ethics committee of Fujian Medical University. All animal experiments were in compliance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health and the Animal Care and Use committees of Fujian Medical University (NO. FJMU IACUC 2018–067).

Establishment of PH-LHD model

PH-LHD model was established by banding procedure described by Siegfried Breitling [32]. Banding is a coarctation procedure used for the ascending aorta above the coronary artery. It is an ideal method to establish model for congestive heart failure and pulmonary arterial hypertension caused by left heart disease [32–34]. It is widely used in experimental research of PH [35–38]. The rats were intraperitoneally injected with 2% sodium pentobarbital (0.3 ml/100 g) and subject to non-invasive nasal mask ventilation. The thorax was opened at the second intercostal space on the left side of the sternum to expose the ascending aorta. The metal titanium clip (Hemoclip^®; Weck Closure System, Research Triangle Park, NC) with a preset internal diameter of 0.8 mm was used to clamp the ascending aorta. During the first three days after surgery, subcutaneous injection of Carprofen (4 mg/kg bw; Rimadyl TM, Pfizer Gmb H, Karslruhe, Germany) was given daily for postoperative analgesia. The rats in the sham group also underwent operation to expose the ascending aorta. But the ascending aorta was not clamped. The remaining postoperative treatment was the same as in the model group. When the rats reached the humane end, they were euthanized through decapitation.

Mechanical strength

The isolated vascular tone tester 620M bath (Danish Myo Technology A/S, Denmark) was preheated to 37°C with 8 ml of K-H solution. Pulmonary vein ring was hanged on the stainless steel hook and adjusted to zero calibration. The K-H solution was changed every 15 minutes during the experiment and a continuous mixture of 95% O₂ and 5% CO₂ were provided. the vessel rings were first kept in K-H solution for 60 minutes. And then a mechanical strength of 2.0 g was applied for 60 minutes, while in the S1 and M1 groups no mechanical strength was applied. Finally, the above treated pulmonary vein rings were collected and stored in a -80°C freezer.

Group of experiments

A total of 48 SD rats were randomly divided into the sham group (n = 12) and the PH-LHD group (n = 36). Corresponding to different inhibitors and mechanical strength used to treat pulmonary venous rings, the sham group was further divided into two subgroups: Sham 1 (S1, mechanical strength: 0 g) and Sham 2 (S2, mechanical strength: 2.0 g); and the PH-LHD group was also further divided into 2 subgroups: Model 1 (M1, banding+0 g), Model 2 (M2, banding+2.0 g). In addition, a broad spectrum inhibitor of MEK1 and MEK2 (U0126, HY-12031 MedChem Express, USA), p38 MAPK inhibitor (SB203580, HY-10256 MedChem Express, USA), SAC inhibitor (Streptomycin, HY-B0472 MedChem Express, USA) and broad-spectrum inhibitor of JNK1, JNK2, and JNK3 (SP600125, HY-12041 MedChem Express, USA) were dissolved in dimethyl sulfoxide (DMSO, HY-10999 MedChem Express, USA), and diluted with K-H solution to 250 μmol/L, 200 μmol/L, 1000 μmol/L, and 250 μmol/L, respectively [12]. Specifically, the pulmonary veins were immersed in the K-H solution containing the corresponding inhibitors (SB203580, SP600125, U0126 or Streptomycin) at 37°C under the condition of continuous oxygen infusion for 60 mins, and then was stretched with 2.0 g mechanical strength for 60 mins.

Doppler echocardiography

On day 25 after surgery, Doppler echocardiography was performed to confirm that the metal titanium clip did not fall off or displaced. The diameter of the pulmonary artery and the right ventricular end-diastolic diameter were measured and the left cardiac function was evaluated. Right ventricular systolic pressure (RVSP) was monitored with a right heart catheter (RHC), mean left atrial pressure (mLAP) was monitored directly, and data was collected by BL-420S experimental system of biological function (Chengdu Taimen Software Co., Ltd). PH-LHD was confirmed by left heart failure and increased RVSP.

Sample preparation

On day 25, all rats were anesthetized with 2% pentobarbital sodium (ip., 0.3 ml/100 g) after Doppler echocardiography and hemodynamic examination. The blood of inferior vena cava was collected and centrifuged (2000 ×g, at 4°C for 5 mins), and then the collected serum was stored in the refrigerator at -80°C. The chest was opened in the middle to confirm that the metal titanium clip was on the ascending aorta. The prepared PBS at 4°C was slowly injected from the right ventricle, and then to the left atrium through the pulmonary circulation; and then the remaining blood in the rat pulmonary circulation was discharged until the pulmonary lobe became white. The heart was quickly separated, dried and weighed. Besides, the pulmonary vein, pulmonary artery, and lung tissue were removed, and the pulmonary vein was placed in a Krebs-Henseleit solution (118 mmol/L NaCl, 4.70 mmol/L KCl, 1.20 mmol/L MgSO₄, 25.00 mmol/L NaHCO3, 1.20 mmol/L KH₂PO₄, 11.0 mmol/L glucose, 0.50 mmol/L EDTA-Na₂, and 2.50 mmol/L CaCl₂) at 4°C. The peripheral tissues of the pulmonary veins were removed under a microscope and 4-mm pulmonary venous rings were prepared (S1 Fig). Pulmonary artery, pulmonary vein and peripheral lung tissues were collected and stored in a -80°C freezer. Moreover, the pulmonary lobes were removed and immobilized in 4% paraformaldehyde for 24 h and transferred to 75% alcohol.

Western blotting analysis

For measuring the levels of MMP-9 and TGF-β1, tissues were fully ground into homogenate with RIPA buffer and protease inhibitor (Beyotime Institute of Biotechnology, China) and centrifuged for 20 min at 12000 rpm at 4°C. The supernatant was collected and the protein concentration was measured using a BCA protein assay Kit (Beijing Solarbio Science & Technology Co., Ltd. China). Protein samples (40 μg for each) were separated by SDS-PAGE and then transferred onto the PVDF membranes. After being blocked in 5% BSA in TBS buffer for 2.0 hrs, the membranes were incubated with the primary antibodies (MMP-9 or TGF-β1, 1:1000, rabbit polyclonal antibody, Abcam) overnight at 4°C. After washing 3 times with 1× TBST, the membranes were incubated with goat anti-rabbit IgG (1:5000, Affinity) for 1.0 hours at room temperature. The specific bands were developed after washing 3 times by 1 × TBST. Finally, the bands were visualized by ChemiDoc Imaging Systems, and the intensity of protein bands were quantified using Image J software (NIH, Bethesda, MD, USA).

ELISA assay

Immediately after removal on day 25, venous blood samples were centrifuged for 20 min (1200 revolutions/ min [rpm], 4°C), and serum was stored at –20°C until analysis. Serum MMP-9 and TGF-β1 levels were determined with a commercial immunoassay kit specifc to rat MMP-9 and TGF-β1 (Neobioscience, Inc., CN) as per the manufacturer’s instructions. This kit is a highly sensitive two-site enzyme-linked immunosorbent assay (ELISA) for measuring MMP-9 and TGF-β1. All experiments were performed in triplicate.

Immunohistochemistry (IHC) assay

The lung tissues were dipped into 10% Formalin solution for 24 h. The monoclonal antibody against MMP-9 or TGF-β1 (1:200, Abcam, Cambridge, MA, USA) and the goat anti-rabbit-IgG (Abcam, Cambridge, MA, USA) were used for immunostaining. Negative and positive controls were used for both staining procedures.

Hematoxylin-eosin (H&E) staining

The lung tissues were dehydrated in ethanol solution, and transparentized in dimethylbenzene. After paraffin embedding, tissues were cut into slices. Then slices were roasted, dewaxed and hydrated, incubated in hematoxylin solution, cultured in alcohol and hydrochloric acid and cultured in Scott blue buffer. Finally, they were dehydrated, transparentized, mounted, and observed under a microscope (CKX41, Olympus, Japan). Each tissue of model group and sham group photographed with 200-fold and 400-fold field of view.

Statistical analysis

The data were expressed as mean ± Standard Error of Mean (mean±SEM), and the unpaired t-student tests or one-way ANOVA were used to compare the data in our study. P<0.05 was considered to be statistically significant. The statistical analysis and mapping were performed using the SPSS17.0 and Graph Pad Prism 5.0.

Results

Establishment of PH-LHD model

According to previous reports [32–34], PH-LHD model was established using the banding. Firstly, we exhibited the position of the titanium clip in the ascending aorta above the coronary artery during surgery (Fig 1A). After 25 days, the position of metal titanium clip was evaluated using the Doppler echocardiography, and the graphical result displayed that we have successfully established the PH-LHD model rats by putting the metal titanium clip in the right place (Fig 1B).

Fig 1 — (A) PH-LHD model was constructed, and the position of the titanium clip was displayed using the yellow arrow in the ascending aorta above the coronary artery. (B) The metal titanium clip (yellow arrow) was presented at the ascending aorta of the PH-LHD group by doppler echocardiography.

Doppler echocardiography of left cardiac function in PH-LHD model rats

In addition, to further determine the changes of left cardiac function in PH-LHD model rats, doppler echocardiography was adopted to assess the left ventricular dysfunction and the change of interventricular septum. As presented in Fig 2 and Table 1, the diastolic ventricular septum (IVSd), diastolic left ventricular posterior wall (LVPWd), diastolic left ventricular inner diameter (LVDd) and systolic left ventricular inner diameter (LVDs) in the PH-LHD group were greater than those in the sham group (P<0.05). The ejection fraction (EF) in the PH-LHD group was lower than that in the sham group (P<0.05). There were no significant differences in heart rate (HR), ascending aorta diameter (AAO), diastolic left atrial inner diameter (LADd), diastolic right ventricular inner diameter (RVDd), and pulmonary artery inner diameter (Pad) between the PH-LHD group and the sham group (P>0.05).

Fig 2 — (A) Representative images of left ventricular dysfunction in rats were assessed by doppler echocardiography in sham and PH-LHD model rats. (B) The change of interventricular septum in the PH-LHD group (yellow arrow) was evaluated using the doppler echocardiography.

Table 1. Differences in IVSd, LVPWd, LVDd, LVDs, EF, HR, AAO, LADd, RVDd, Pad between sham-operated and PH-LHD model groups.

Groups	Sham control	PH-LHD group	P value
Groups	group (N = 12)	(N = 36)	P value
IVSd (mm)	1.518±0.021	1.924±0.009	<0.001^*
LVPWd (mm)	1.618±0.016	1.957±0.017	<0.001^*
LVDd (mm)	4.873±0.096	5.288±0.093	0.019^*
LVDs (mm)	2.927±0.079	3.222±0.056	0.008^*
EF (%)	78.31±0.537	74.93±0.287	<0.001^*
HR (bpm)	376.7±10.94	390.8±6.80	0.297
AAO (mm)	2.133±0.014	2.189±0.018	0.103
LADd (mm)	3.367±0.022	3.294±0.026	0.129
RVDd (mm)	2.117±0.021	2.153±0.019	0.317
Pad (mm)	2.233±0.037	2.222±0.021	0.792

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* P<0.05, diastolic ventricular septum (IVSd), diastolic left ventricular posterior wall (LVPWd), diastolic left ventricular inner diameter (LVDd), systolic left ventricular inner diameter (LVDs), ejection fraction (EF), heart rate (HR), ascending aorta diameter (AAO), diastolic left atrial inner diameter (LADd), diastolic right ventricular inner diameter (RVDd), pulmonary artery inner diameter (Pad).

RVSP and mLAP were determined in PH-LHD model rats

Increased pulmonary arterial pressure is the gold standard for the diagnosis of PH. Right heart failure occurs as a late complication of PH. RHC was used to measure RVSP and evaluate the degree of PH [39]. On day 25 after banding, RVSP and mean left atrial pressure (mLAP) in the PH-LHD group was higher than those in the sham group (P<0.05). In addition, compared with the sham group, rats in the PH-LHD group demonstrated a significant increase in the heart weight and heart weight/body weight ratio (P<0.05), and a significant decrease in the body weight (P<0.05). And They are similar in the Right ventricular/(left ventricular+ interventricular septum) (P<0.05, Fig 3 and Table 2).

Fig 3 — The representative images of RVSP (A) and mLAP (B) in rats were demonstrated in the sham and PH-LHD model rats.

Table 2. Differences of RVSP, mLAP, BW, HW, HW/BW, RV/(LV+S) between sham groups and PH-LHD model groups.

Groups	Sham control	PH-LHD group	P value
Groups	group (N = 12)	(N = 36)	P value
RVSP (mmHg)	24.43±0.51	35.77±0.45	<0.001*
mLAP (mmHg)	4.88±0.42	8.01±0.44	<0.001*
BW (g)	254.2±1.19	218.9±6.47	<0.001*
HW (g)	0.329±0.011	0.579±0.008	<0.001*
HW/BW (%)	0.131±0.004	0.257±0.002	<0.001*
RV/(LV+S)	0.1075±0.006	0.1031±0.010	0.734

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Right ventricular systolic pressure (RVSP), Mean left atrial pressure (mLAP), Body weight (BW), Heart weight (HW), Right ventricular (RV), left ventricular (LV), interventricular septum (S).

The pathological changes of pulmonary tissues in PH-LHD model rats

Subsequently, the pathologic structures of pulmonary tissues were identified by H&E staining in PH-LHD model rats after 25 days. Our results exhibited that in the sham group, there were smooth wall of small pulmonary artery, thin pulmonary artery walls, large lumen, evenly distributed cells, and no inflammatory infiltration around the blood; in the PH-LHD group, we discovered that the walls of the small pulmonary artery were slightly thickened, the cells were orderly, and a small amount of inflammatory cells were observed around the vessels (Fig 4).

Fig 4 — (A–B) The pathologic structures were confirmed by H&E staining in the sham and PH-LHD model rats treated and untreated 2.0 g. Magnification, 200×; Scale bar = 100 μm. Each group contained six rats.

High expressions of MMP-9 and TGF-β1 in pulmonary veins

On the mechanism, we then investigated the MMP-9 and TGF-β1 expressions in pulmonary artery, pulmonary vein and pulmonary tissue. And the results of western blotting analysis uncovered that the levels of MMP-9 and TGF-β1 were prominently elevated in pulmonary vein relative to pulmonary artery or pulmonary tissue (P<0.01, P<0.001, Fig 5).

Fig 5 — Western blotting analysis was performed to examine the levels of MMP-9 and TGF-β1 in pulmonary artery, pulmonary vein and pulmonary tissue, respectively. GAPDH acts as an internal reference. **P<0.01, ***P<0.001 vs. pulmonary artery group. Each group contained six rats.

MMP-9 and TGF-β1 were highly expressed in PH-LHD model rats

Our previous experiments prove that MMP-9 and TGF-β1 were highly expressed in pulmonary veins, and we further explored the expressions of MMP-9 and TGF-β1 in mechanical stretching-induced remodeling of pulmonary veins in the early stage of PH-LHD. Firstly, we certified that the venous blood of rats was collected for ELIAS assay, and the results showed that the expressions of MMP-9 and TGF-β1 were significantly higher in the PH-LHD group than those in the sham group on day 25 after banding (P<0.01, P<0.001, Fig 6A). Secondly, immunohistochemical results of pulmonary tissues also showed that the expression levels of MMP-9 and TGF-β1 were significantly increased in the pulmonary tissues of the PH-LHD group with respect to that in the sham group. Besides, in the sham group, a few alveolar epithelial cells were positively expressed, with a low degree of staining; in the PH—LHD group, large Numbers of MMP-9 and TGF-β1 positive cells were observed around the pulmonary vein (Fig 6B). Finally, we disclosed that MMP-9 and TGF-β1 expressions were remarkably increased in the pulmonary vein tissues of the PH-LHD group with versus that in the sham group, and 2.0 g intension was more significant for the MMP-9 and TGF-β1 expressions than the 0 g intension (P<0.05, Fig 6C and 6D).

Fig 6 — (A) The concentrations of MMP-9 and TGF-β1 were analyzed by ELISA assay in the serums of sham and PH-LHD model rats, **P<0.01, ***P<0.001 vs. sham group. (B) IHC experiment was carried out to examine the levels of MMP-9 and TGF-β1 in the pulmonary tissues of sham and PH-LHD model rats. Magnification, 200×; Scale bar = 100μm. (C) The protein expression levels of MMP-9 and TGF-β1 were assessed by western blot analysis in the pulmonary vein tissues of the sham and PH-LHD model rats. (D) Relative expression levels of MMP-9 and TGF-β1 were counted based on the grey values of the western blotting results. *P<0.05 vs. sham 0 g group; #P<0.05 vs. sham 2.0 g group. Each group contained six rats.

Mechanical stretching upregulated MMP-9 and TGF-β1 in pulmonary vein by SAC/MAPKs signaling pathway

To further inquiry the underlining mechanism of mechanical stretching on the upregulations of MMP-9 and TGF-β1 in pulmonary vein, we adopted MAPK pathway inhibitors (SB203580, SP600125, U0126) and stretch-activated channel (SAC) inhibitor (streptomycin). The results of western blotting analysis uncovered that compared with the control group, the levels of MMP-9 and TGF-β1 were notably reduced in PH-LHD model rats after treatment with SB203580, SP600125, U0126 and Streptomycin (P<0.01, P<0.001, Fig 7). The results suggested that mechanical stretching of pulmonary vein increased the expressions of MMP-9 and TGF-β1, while the inhibitors of SAC/MAPKs signaling pathway (SB203580, SP600125, U0126 and streptomycin) could reverse the increases of MMP-9 and TGF-β1 expressions. Therefore, we revealed that SAC and MAPKs signaling pathways might be participate in the promoting effects of mechanical stretching of pulmonary vein on the pulmonary hypertension.

Fig 7 — PH-LHD model rats were treated with SB203580, SP600125, U0126 and Streptomycin, respectively. (A–B) MMP-9 and TGF-β1 expressions were confirmed by western blot assay, and the relative expressions were calculated based on gray value. **P<0.01, ***P<0.001 vs. pulmonary artery group. Each group contained six rats.

Discussion

PH-LHD is the most common type of PH. Due to its unclear pathogenesis and complicated pathophysiological process [5], there is still no effective treatment available. Pulmonary vein hypertension is the earliest pathophysiological change in the development of PH-LHD. It is hypothesized that early changes of pulmonary vein wall will promote subsequent pulmonary vascular remodeling in PH-LHD. If this hypothesis is confirmed, it will provide novel targets for the early prevention and treatment of PH-LHD.

Previously Breitling et al found that distinct vascular remodeling occurred in the pulmonary artery within two months after banding [38]. In the present study, Doppler echocardiography on day 25 after banding confirmed the presence of left heart failure in rats, as indicated by increased left ventricular pressure, increased left ventricular diameter, interventricular septum shift, and decreased ejection fraction. Meanwhile, RVSP and mLAP were increased. Thus, although pulmonary blood vessels have not been remodeled at this time point, pulmonary artery pressure has increased. Therefore, we collected pulmonary veins on day 25 after banding, which corresponds to the early stages of PH-LHD, and explore their effects on pulmonary vascular remodeling and possible mechanisms.

Studies have demonstrated that MMP-9 and TGF-β1 are also important molecules in the pathogenesis of PH [23, 40]. The former is involved in the fibrosis and remodeling of pulmonary blood vessels, while the latter is involved in pulmonary vascular remodeling and pulmonary hypertension by regulating smooth muscle cell proliferation and fibroblast phenotypic conversion [23, 41]. Therefore, in hypoxia-induced PH, the expression of MMP-9 and TGF-β1 in the pulmonary arteries increases [41, 42]. In PH-LHD, during the early stages of PH, the pulmonary arteries have not yet undergone vascular remodeling. Increased mechanical tension in the pulmonary vein wall is caused by blockage of blood flow in the pulmonary vein in the left heart disease, i.e., pulmonary venous hypertension. Is there any pathological change in pulmonary vein tissue at this time that contributes to the later pulmonary artery vascular remodeling? We found that on day 25 after banding, the expression levels of MMP-9 and TGF-β1 in the pulmonary vein tissue increased compared to the sham group. Shear stress, expansion force, and blood pressure of the pulmonary vein wall may lead to cytoskeletal reorganization and morphological changes. Correspondingly, vascular smooth muscle cells produced various molecules, including MMP-9 and TGF-β1, that acted on themselves to maintain and adapt to the pathological changes of vascular structure and function [43].

Recently, Qiu et al demonstrated that mechanical stretching could up-regulate the expression of MMP-9 in the smooth muscle cells from animals with aortic dissection through SAC/NF-κB signaling pathway [30]. The mechanical stress of the vascular wall during aortic dissection may influence the structure and function of cells, tissues and organs [44]. Once this is sensed by cells, it is then transmitted to the nucleus through intracellular signaling pathways, thereby causing the expression of related genes and leading to different physiological or pathological responses [45]. So, does this mechanism of stretch also occur during the increased mechanical tension in the pulmonary vein wall in PH-LHD? In order to explore this assumption, we applied mechanical stretching to pulmonary vein rings of PH-LHD rats. The results showed that there was no significant difference in the expression of MMP-9 and TGF-β1 in the pulmonary veins from rats of the sham group even a force of 2.0 g was applied. However, the expression of MMP-9 and TGF-β1 in the pulmonary veins of PH-LHD rats was up-regulated when a force of 2.0 g was applied. These results indicate that during the development of PH-LHD, as the pulmonary vein wall was continuously stimulated by increased mechanical tension, the cells became sensitive to the mechanical tension and the expression of MMP-9 and TGF-β1 in the pulmonary vein rings were further increased once they were subject to the mechanical stress in vitro.

Mechanical stretching has been shown to activate multiple signaling pathways that are related to some pathophysiological processes. For example, Ghantous and his colleagues demonstrated that mechanical stretching induced leptin synthesis by activating the ROS signaling pathway, which in turn caused vascular smooth muscle cell remodeling [46]. Seo et al. found that mechanical stretching induced MMP-2 overexpression by activating the vascular smooth muscle Akt signaling pathway and participated in vascular remodeling [47]. Ishise et al. found that mechanical stretching induced fibronectin expression by activating the TRPC3-NF-kappa B axis, resulting in a troublesome wound contracture [48]. The SAC/MAPKs signaling pathway is thought to regulate the development of a variety of cardiovascular diseases [49, 50]. SAC was first discovered in the skeletal muscle cells by Guharay and Sachs. It is an ion channel that is widely found in various cells [51]. MAPKs are a class of serine/threonine protein kinases widely found in mammalian cells, including extracellular signal-regulated kinase (ERK1/2), p38 MAPK, and c-Jun NH2-terminal kinase (JNK). Shaw et al. demonstrated that mechanical stretching activated SAC on vascular smooth muscles, causing a series of responses that led to vascular smooth muscle migration and apoptosis [44]. Liu et al. demonstrated that mechanical stretching mediated vascular remodeling by activating the MAPK signaling pathway in human aortic smooth muscle cells [52]. In the present study, we showed that 1) inhibition of p38 MAPK or JNK signaling pathway can significantly reverse the increased expression of MMP-9 in mechanical stretching-induced PH-LHD pulmonary vein; and 2) inhibition of SAC or JNK signaling pathway can significantly reverse the increased expression of TGF-β1 in mechanical stretching-induced PH-LHD pulmonary vein. Thus increased mechanical tension of the pulmonary vein wall in PH-LHD may activate the SAC/MAPKs signaling pathway, which regulate the expression of genes involved in PH-LHD, leading to cell proliferation, differentiation, or apoptosis [53–55]. More importantly, functional changes in the early pulmonary veins of PH-LHD have a subtle influence on subsequent vascular remodeling. If the remodeling of pulmonary arteries during the later stages of the disease can be blocked by early inhibition of MMP-9 and TGF-β1 expression mediated by SAC/MAPKs signaling pathway deserves further study. Besides, in future research, it is also very significant to further explore the specific regulatory mechanisms of SAC/MAPKs signaling pathway on the MMP-9 and TGF-β1 in pulmonary vein after Mechanical stretching.

Conclusions

During the early stages of PH-LHD, increase in the mechanical tension of the pulmonary vein wall may cause high expressions of MMP-9 and TGF-β1 in the pulmonary vein tissue through the SAC/MAPKs signaling pathway. Therefore, increased expressions of MMP-9 and TGF-β1 might participate in the subsequent pulmonary artery vascular remodeling. This provides new targets for the early interventions of PH-LHD.

Supporting information

S1 Fig. The preparation of pulmonary venous rings.

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S1 Raw data

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S1 Raw images

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Acknowledgments

We thank the Institute of Cardiothoracic Surgery of Fujian Medical University Union Hospital for technical assistance and animal care.

Abbreviations

MAPK: Mitogen-activated protein kinase
MMP-9: matrix metalloproteinase-9
PH: pulmonary hypertension
PH-LHD: pulmonary hypertension due to left heart disease
RHC: right heart catheter
RVSP: right ventricular systolic pressure
SAC: stretch-activated channel
TGF-β1: transforming growth factor-β1

Data Availability

All relevant data are within the manuscript and its Supporting Information files.

Funding Statement

This work was supported by the National Natural Science Foundation of China [grant numbers 81770368]; and Medical Elite Cultivation Program of Fujian, P.R.C [grant number 2015-ZQNZD- 16]. The funders participated in study design, data collection, decision to publish, and preparation of the manuscript.

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PLoS One. doi: 10.1371/journal.pone.0235824.r001

Decision Letter 0

Chuen-Mao Yang

5 Feb 2020

PONE-D-19-35944

Mechanical stretching of pulmonary vein stimulates matrix metalloproteinase-9 and transforming growth factor-β1 through spindle assembly checkpoint/MAPK pathways in pulmonary hypertension due to left heart disease model rats

PLOS ONE

Dear Dr. Zhang,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

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This study addressed the underlining mechanisms of mechanical stretching-induced remodeling of the pulmonary vein in the early stage of PH-LHD. Current research revealed that mechanical stretching induced the increasing expressions of MMP-9 and TGF-β1 in the pulmonary vein, which could be mediated by activation of the SAC/MAPKs signaling pathway in the early stage of PH-LHD. This issue is interesting and important. Some recommendations and suggestions are as follows:

1. The whole words need to be spelled while abbreviations are used for the first time. Such as RVSP, mLAP, PAH, etc.

2. RVSP and mLAP were measured in this study, however, the mean pulmonary arterial pressure should be a direct indicator of PAH. Why did not authors check the value? Moreover, in the discussion, the authors wrote: “Thus, although pulmonary blood vessels have not been remodeled at this time point, pulmonary artery pressure has increased”.

3. In figure 4, “in sham group, the cells were evenly distributed and of uniform thickness; in PH-LHD group, the cells presented as a continuum of destruction.”, what kind of “the cells” authors mean for? The typical pathological findings of type Ⅱ PH should be congestive heart failure related.

4. Can the authors describe the method of pul artery, pul vein, and lung tissue harvest in detail?

5. In figure 6C, which part of lung tissue did the sample come from? Was it whole lung tissue or only veins? Were there images of arterioles stained for MMP-9 and TGF-b1 to confirm the data of figure 5?

6. Make sure the reference of “17” cited in the introduction correct or not.

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Chuen-Mao Yang

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PLOS ONE

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Reviewer #1: Yes

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Reviewer #1: Yes

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Reviewer #1: Yes

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Reviewer #1: Yes

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Reviewer #1: This manuscript addressed that the possible mechanisms of pulmonary hypertension in response to left heart disease. The authors used in vivo rat models to study the participation of SAC/MAPK together with MMP9 and TGF-b1 in pulmonary hypertension due to left heart disease (PH-LHD). The study is advancing but several parts need future addressed and clarified.

1. How many rats in each groups should be provided in figure legends.

2. In figure 4 and 6, it would be much better if the specific pathological changes or specific structures were indicated in H&E staining or IHC data. Moreover, the scale bar should be provided in each panels.

3. In figure 6B, the amplification folds seems not equal in sham group and PH-LHD group. The nuclei stains showed in sham group of figure 6B are much larger than PH-LHD group. Moreover, the background of figure 6B should be showed in equal colors or brightness. And is the data collected here 0 g of mechanical stretching? How do the MMP9 and TGF-b1 IHC stains of study groups with mechanical stretching show?

4. It would be much better if the protein expression of figure 6C are quantified and performs the statistical analysis.

5. In introduction section, the importance of SAC and MAPK pathway should be provided.

6. In method section, where are the inhibitors injected? And the final concentration used are?

7. The expression of SAC or phosphorylation of MAPK pathways should be provided.

8. The manuscript needs further English edition.

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Reviewer #1: No

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PLoS One. 2020 Sep 3;15(9):e0235824. doi: 10.1371/journal.pone.0235824.r002

Author response to Decision Letter 0

19 Jun 2020

Dear reviewers:

We sincerely appreciate your time and critical comments on our manuscript entitled "Mechanical stretching of pulmonary vein stimulates matrix metalloproteinase-9 and transforming growth factor-β1 through stretch-activated channel/MAPK pathways in pulmonary hypertension due to left heart disease model rats" (PONE-D-19-35944). We have revised our manuscript as suggested and answered comments point by point.

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Reviewer #1

1. The whole words need to be spelled while abbreviations are used for the first time. Such as RVSP, mLAP, PAH, etc.

Answer: Thanks very much for your comments. We have spelled the whole words of the abbreviations, which were used for the first time in this study.

Answer: Thanks for your constructive suggestion. The ideal condition for this study is to be able to directly monitor pulmonary arterial pressure using a Miller catheter. However, due to the limited conditions, we chose PE catheter to monitor the right ventricular systolic pressure.

The pathophysiological changes of pulmonary arterial hypertension associated with left heart disease are that left heart disease first causes left ventricular dysfunction or failure, then leads to the elevation of pulmonary venous pressure and pulmonary hypertension, and finally results in the right heart failure. Thus, the increase of right ventricular pressure can be caused by the increase of pulmonary artery resistance and pressure, and the increase of pressure pulmonary circulation can be caused by the increase of left ventricular pressure. In our study, PH-LHD model is actually a model of pulmonary hypertension caused by the ascending aorta banding, which can gradually lead to the increased right ventricular pressure and right heart failure. So, the increase in right ventricular pressure can indicate the increase in pulmonary artery pressure at this time, although pulmonary vascular remodeling has not yet occurred.

Answer: Thanks very much for your comment. We have re-described the results of Figure 4 as follows: Our results exhibited that in the sham group, there were smooth wall of small pulmonary artery, thin pulmonary artery walls, large lumen, evenly distributed cells, and no inflammatory infiltration around the blood; in the PH-LHD group, we discovered that the walls of the small pulmonary artery were markedly thickened, the intima was rough and incomplete, the cells were arranged in disorder, the number of nuclei was increased, the vasculature was severely myogenous, the lumen area was reduced, and the perivascular inflammatory cell infiltration was present.

4. Can the authors describe the method of pul artery, pul vein, and lung tissue harvest in detail?

Answer: Thanks very much for your comment. We have described in detail the specific collection methods of samples in the revised method, as follows: On day 25, all rats were anesthetized with 2% pentobarbital sodium (ip., 0.3ml/100g) after Doppler echocardiography and hemodynamic examination. The blood of inferior vena cava was collected and centrifuged (2000 ×g, at 4℃ for 5 mins), and then the collected serum was stored in the refrigerator at -80℃. The chest was opened in the middle to confirm that the metal titanium clip was on the ascending aorta. The prepared PBS at 4℃ was slowly injected from the right ventricle, and then to the left atrium through the pulmonary circulation; and then the remaining blood in the rat pulmonary circulation was discharged until the pulmonary lobe became white. The heart was quickly separated, dried and weighed. Besides, the pulmonary vein, pulmonary artery, and lung tissue were removed, and the pulmonary vein was placed in a Krebs-Henseleit solution (118 mmol/L NaCl, 4.70 mmol/L KCl, 1.20 mmol/L MgSO4, 25.00 mmol/L NaHCO3, 1.20 mmol/L KH2PO4, 11.0 mmol/L glucose, 0.50 mmol/L EDTA-Na2, and 2.50 mmol/L CaCl2) at 4°C. The peripheral tissues of the pulmonary veins were removed under a microscope and 4-mm pulmonary venous rings were prepared. Pulmonary artery, pulmonary vein and peripheral lung tissues were collected and stored in a -80°C freezer. Moreover, the pulmonary lobes were removed and immobilized in 4% paraformaldehyde for 24 h and transferred to 75% alcohol.

Figure 1S. The preparation of pulmonary venous rings

Answer: Thanks for your constructive suggestion. We adopted the pulmonary vein tissues in the sham operation groups (Sham 0g and sham 2.0g), and in the model group (PH-LHD 0g and PH-LHD 2.0g). And we have explicated the pulmonary vein tissue in the figure legends of figure 6C. In Figure 5, we uncovered that the levels of MMP-9 and TGF-β1 were prominently elevated in pulmonary vein relative to pulmonary artery or pulmonary tissue. In Figure 6, we proved that the expression levels of MMP-9 and TGF-β1 were significantly increased in the pulmonary tissues of the PH-LHD group with respect to that in the sham group; and MMP-9 and TGF-β1 expressions were remarkably increased in the pulmonary vein tissues of the PH-LHD group with versus that in the sham group, and 2.0g intension was more significant for the MMP-9 and TGF-β1 expressions than the 0 g intension.

6. Make sure the reference of “17” cited in the introduction correct or not.

Answer: Thanks very much for your comment. We redefined and modified the reference 17 in the revised manuscript.

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Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Yes

Answer: Thanks very much for your comments.

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Answer: Thanks very much for your comments.

3. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: Yes

Answer: Thanks very much for your comments.

4. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: Yes

Answer: Thanks very much for your comments.

5. Review Comments to the Author

1. How many rats in each groups should be provided in figure legends?

Answer: Thanks very much for your comment. In our study, six rats were used in each group. We have added this information in the revised figure legends.

Answer: Thanks for your constructive suggestion. We have described the pathologic changes in detail in the results section of figure 4 and 6. Moreover, we also added the scale bar in each panel.

Answer: Thanks for your constructive suggestion. We are very sorry that we put the wrong pictures, and we have also replaced the result graphs in the Sham group of Figure 6B. In figure 6B, IHC experiment was carried out to examine the levels of MMP-9 and TGF-β1 in the pulmonary tissues of sham and PH-LHD model rats (0 g of mechanical stretching). Lung tissue sections contained pulmonary veins, pulmonary arteries, and pulmonary tissues. We first compared the expressions of TGF-β1 and MMP-9 in pulmonary veins, pulmonary arteries, and pulmonary tissues 25 days after Banding surgery. In Figure 6B, the pulmonary tissues were selected in the PH-LHD group, which was unable to be mechanically stretched. The IHC staining results of MMP-9 and TGF-β1 were brown-positive. The experimental results showed that on the 25th day after Banding surgery, MMP-9 and TGF-β1 were first expressed around pulmonary vein.

4. It would be much better if the protein expression of figure 6C are quantified and performs the statistical analysis.

Answer: Thanks very much for your comment. We have quantified and statistically analyzed the protein expression in the revised figure 6C.

5. In introduction section, the importance of SAC and MAPK pathway should be provided.

Answer: Thanks very much for your comment. we have also added the importance of SAC and MAPK pathway in the revised introduction section, as follows: SAC, serves a mechanically-sensitive ion channel, can be regulated by the changes in the tension of a cellular scaffold or lipid membrane coupled with channel proteins[1]. It was reported that SAC has significant effect in the electrophysiological changes of the myocardium during stimulation[2]. MAPK pathway is proven to be involved in various physiological processes, such as cell growth, development and inter-cell function synchronization, which also can be closely related to the regulation of inflammation and stress response[3, 4]. Studies also testified that mechanical stretching can activate the phosphorylation of the MAPK pathway[5, 6]. Also, studies have shown that the expression of MMP-9 and TGF-β1 in tissues may be related to stretch-activated channel (SAC) and mitogen-activated protein kinase (MAPK) signaling pathway[7-9]. Therefore, we speculated that SAC and MAPK signaling pathway may be potential regulatory molecules in PH-LHD.

6. In method section, where are the inhibitors injected? And the final concentration used are?

Answer: Thanks very much for your comment. We have further the processing method as follows: A total of 48 SD rats were randomly divided into the sham group (n=12) and the PH-LHD group (n=36). Corresponding to different inhibitors and mechanical strength used to treat pulmonary venous rings, the sham group was further divided into two subgroups: Sham 1 (S1, mechanical strength: 0 g) and Sham 2 (S2, mechanical strength: 2.0 g); and the PH-LHD group was also further divided into 2 subgroups: Model 1 (M1, banding+0 g), Model 2 (M2, banding+2.0 g). In addition, a broad spectrum inhibitor of MEK1 and MEK2 (U0126, HY-12031 MedChem Express, USA), p38 MAPK inhibitor (SB203580, HY-10256 MedChem Express, USA), SAC inhibitor (Streptomycin, HY-B0472 MedChem Express, USA) and broad-spectrum inhibitor of JNK1, JNK2, and JNK3 (SP600125, HY-12041 MedChem Express, USA) were dissolved in dimethyl sulfoxide (DMSO, HY-10999 MedChem Express, USA), and diluted with K-H solution to 250 μmol/L, 250 μmol/L, 1000 μmol/L, and 250 μmol/L, respectively[8]. Specifically, the pulmonary veins were immersed in the K-H solution containing the corresponding inhibitors (SB203580, SP600125, U0126 or Streptomycin) at 37℃ under the condition of continuous oxygen infusion for 60 mins, and then was stretched with 2.0g mechanical strength for 60mins.

7. The expression of SAC or phosphorylation of MAPK pathways should be provided.

Answer: Thanks for your constructive suggestion. In our study, we have preliminarily proved that mechanical stretching of pulmonary vein increased the expressions of MMP-9 and TGF-β1, while the inhibitors of SAC/MAPKs signaling pathway (SB203580, SP600125, U0126 and streptomycin) could reverse the increases of MMP-9 and TGF-β1 expressions. Therefore, we revealed that SAC and MAPKs signaling pathways might be participate in the promoting effects of mechanical stretching of pulmonary vein on the pulmonary hypertension. We thanks to the reviewer for this excellent question which we should add the expressions of SAC and phosphorylation of MAPK pathways. Because of the sample, time and considerable additional work, we did not add the experiment. In our future research, we will also explore further the specific regulatory mechanisms of SAC/MAPKs signaling pathway on the MMP-9 and TGF-β1 in pulmonary vein after Mechanical stretching. This limitation has also been described in the revised discussion section.

8. The manuscript needs further English edition.

Answer: Thanks very much for your comment. Our manuscript has been revised with the help of native speaker regarding the deficiencies in English grammar, spelling, and sentence structure.

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We appreciate very much for your time in reviewing our manuscript. And the suggestions and comments really helped us a lot. If there is any information I can provide, please don’t hesitate to contact us.

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References

1. Schmidt, C., et al., Stretch-activated two-pore-domain (K(2P)) potassium channels in the heart: Focus on atrial fibrillation and heart failure. Prog Biophys Mol Biol, 2017. 130(Pt B): p. 233-243.

2. Liang, J., et al., Stretch-activated channel Piezo1 is up-regulated in failure heart and cardiomyocyte stimulated by AngII. Am J Transl Res, 2017. 9(6): p. 2945-2955.

3. Guo, Y.J., et al., ERK/MAPK signalling pathway and tumorigenesis. Exp Ther Med, 2020. 19(3): p. 1997-2007.

4. Sun, J. and G. Nan, The Mitogen-Activated Protein Kinase (MAPK) Signaling Pathway as a Discovery Target in Stroke. J Mol Neurosci, 2016. 59(1): p. 90-8.

5. Hu, Y., et al., Mechanical stretch aggravates aortic dissection by regulating MAPK pathway and the expression of MMP-9 and inflammation factors. Biomed Pharmacother, 2018. 108: p. 1294-1302.

6. Fu, S., et al., Effects of Cyclic Mechanical Stretch on the Proliferation of L6 Myoblasts and Its Mechanisms: PI3K/Akt and MAPK Signal Pathways Regulated by IGF-1 Receptor. Int J Mol Sci, 2018. 19(6).

7. Qiu, Z., et al., Mechanical strain induced expression of matrix metalloproteinase-9 via stretch-activated channels in rat abdominal aortic dissection. Medical science monitor: international medical journal of experimental and clinical research, 2017. 23: p. 1268.

8. Hu, Y., et al., Mechanical stretch aggravates aortic dissection by regulating MAPK pathway and the expression of MMP-9 and inflammation factors. Biomedicine & Pharmacotherapy, 2018. 108: p. 1294-1302.

9. Zhang, L., et al., 15‐LO/15‐HETE Mediated Vascular Adventitia Fibrosis via p38 MAPK‐Dependent TGF‐β. Journal of cellular physiology, 2014. 229(2): p. 245-257.

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PLoS One. doi: 10.1371/journal.pone.0235824.r003

Decision Letter 1

Chuen-Mao Yang

24 Jun 2020

Mechanical stretching of pulmonary vein stimulates matrix metalloproteinase-9 and transforming growth factor-β1 through stretch-activated channel/MAPK pathways in pulmonary hypertension due to left heart disease model rats

PONE-D-19-35944R1

Dear Dr. Hui Zhang,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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PLoS One. doi: 10.1371/journal.pone.0235824.r004

Acceptance letter

Chuen-Mao Yang

20 Aug 2020

PONE-D-19-35944R1

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S1 Fig. The preparation of pulmonary venous rings.

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Data Availability Statement

All relevant data are within the manuscript and its Supporting Information files.

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PERMALINK

Mechanical stretching of pulmonary vein stimulates matrix metalloproteinase-9 and transforming growth factor-β1 through stretch-activated channel/MAPK pathways in pulmonary hypertension due to left heart disease model rats

Hui Zhang

Wenhui Huang

Hongjin Liu

Yihan Zheng

Lianming Liao

Roles

Abstract

Introduction

Materials and methods

Animals

Establishment of PH-LHD model

Mechanical strength

Group of experiments

Doppler echocardiography

Sample preparation

Western blotting analysis

ELISA assay

Immunohistochemistry (IHC) assay

Hematoxylin-eosin (H&E) staining

Statistical analysis

Results

Establishment of PH-LHD model

Fig 1. Establishment of PH-LHD model.

Doppler echocardiography of left cardiac function in PH-LHD model rats

Fig 2. Doppler echocardiography of left cardiac function in PH-LHD model rats.

Table 1. Differences in IVSd, LVPWd, LVDd, LVDs, EF, HR, AAO, LADd, RVDd, Pad between sham-operated and PH-LHD model groups.

RVSP and mLAP were determined in PH-LHD model rats

Fig 3. RVSP and mLAP were determined in PH-LHD model rats.

Table 2. Differences of RVSP, mLAP, BW, HW, HW/BW, RV/(LV+S) between sham groups and PH-LHD model groups.

The pathological changes of pulmonary tissues in PH-LHD model rats

Fig 4. The pathological changes of pulmonary tissues in PH-LHD model rats.

High expressions of MMP-9 and TGF-β1 in pulmonary veins

Fig 5. High expressions of MMP-9 and TGF-β1 in pulmonary veins.

MMP-9 and TGF-β1 were highly expressed in PH-LHD model rats

Fig 6. MMP-9 and TGF-β1 were highly expressed in PH-LHD model rats.

Mechanical stretching upregulated MMP-9 and TGF-β1 in pulmonary vein by SAC/MAPKs signaling pathway

Fig 7. Mechanical stretching upregulated MMP-9 and TGF-β1 in pulmonary vein by SAC/MAPKs signaling pathway.

Discussion

Conclusions

Supporting information

Acknowledgments

Abbreviations

Data Availability

Funding Statement

References

Decision Letter 0

Chuen-Mao Yang

Roles

Author response to Decision Letter 0

Decision Letter 1

Chuen-Mao Yang

Roles

Acceptance letter

Chuen-Mao Yang

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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