# S-Nitroso-L-Cysteine Ameliorated Pulmonary Hypertension in the MCT-Induced Rats through Anti-ROS and Anti-Inflammatory Pathways

Authors: Moran Wang; Pengcheng Luo; Wei Shi; Junyi Guo; Shengqi Huo; Dan Yan; Lulu Peng; Cuntai Zhang; Jiagao Lv; Li Lin; Sheng Li
Journal: Oxidative Medicine and Cellular Longevity
 (2021)

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## Abstract

Pulmonary hypertension (PH) is a progressive and life-threatening chronic disease in which increased pulmonary artery pressure (PAP) and pulmonary vasculature remodeling are prevalent. Inhaled nitric oxide (NO) has been used in newborns to decrease PAP in the clinic; however, the effects of NO endogenous derivatives, S-nitrosothiols (SNO), on PH are still unknown. We have reported that S-nitroso-L-cysteine (CSNO), one of the endogenous derivatives of NO, inhibited RhoA activity through oxidative nitrosation of its C16/20 residues, which may be beneficial for both vasodilation and remodeling. In this study, we presented data to show that inhaled CSNO attenuated PAP in the monocrotaline- (MCT-) induced PH rats and, moreover, improved right ventricular (RV) hypertrophy and fibrosis induced by RV overloaded pressure. In addition, aerosolized CSNO significantly inhibited the hyperactivation of signal transducers and activators of transduction 3 (STAT3) and extracellular regulated protein kinases (ERK) pathways in the lung of MCT-induced rats. CSNO also regulated the expression of smooth muscle contractile protein and improved aberrant endoplasmic reticulum (ER) stress and mitophagy in lung tissues following MCT induction. On the other hand, CSNO inhibited reactive oxygen species (ROS) production in vitro, which is induced by angiotensin II (AngII) as well as interleukin 6 (IL-6). In addition, CSNO inhibited excessive ER stress and mitophagy induced by AngII and IL-6 in vitro; finally, STAT3 and ERK phosphorylation was inhibited by CSNO in a concentration-dependent manner. Taken together, CSNO led to pulmonary artery relaxation and regulated pulmonary circulation remodeling through anti-ROS and anti-inflammatory pathways and may be used as a therapeutic option for PH treatment.


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## Body

## 1. Introduction

Pulmonary hypertension (PH) is a life-threatening cardiopulmonary disease characterized by pulmonary artery vascular contraction and remodeling, possibly due to pulmonary vascular endothelial cell dysfunction, smooth muscle cell proliferation, and perivascular inflammation [1, 2]. Pulmonary artery vascular remodeling results in a continuous increase in pulmonary vascular resistance, eventually leading to right ventricular failure and even death. So far, the strategy which can improve PH vascular remodeling and survival is limited and needs to be explored. Therefore, it is urgent to hunt for novel treatments for PH.The nitric oxide (NO) signaling pathway is widely studied in PH. An expanding body of knowledge has related deficient NO signaling to PH pathogenesis. Endothelial cell injury is regarded as the initiating trigger of pulmonary hypertension, while continuous contraction, proliferation, and migration of smooth muscle cells mainly contribute to pulmonary hypertension progression. In addition, endothelial dysfunction may lead to a decreased output of NO and its endogenous derivatives, S-nitrosothiols (SNO). SNO not only leads to vasodilation as NO does but also is able to modify protein-free thiol groups by S-nitrosylation, which may further regulate cell bioactivities and functions, including reactive oxygen species (ROS) production, proliferation, and inflammation. Previously, we reported that CSNO can transport NO equivalents into intact vascular cells and regulate constriction of vascular smooth muscle cells by S-nitrosylation [3, 4], suggesting a role of SNO in the pathogenesis of pulmonary hypertension and its treatment. So far, NO nebulizer therapy is only implemented in the pulmonary hypertension crisis in neonates. Whether aerosolized CSNO can effectively improve the development and progression of PH still remains uncertain.Pulmonary vascular remodeling is a characteristic of PH development. Perivascular inflammation is now recognized to contribute greatly to PH development in patients and experimental animals. Abundant evidence implicates that IL-6 plays an important role in pulmonary vascular remodeling. IL-6 recognizes IL-6R and gp130, further leads to phosphorylation modification of STAT3, and eventually regulates downstream genes. The overactivated STAT3 pathway contributes to PH development; moreover, binding of p-STAT3 to the eNOS promoter impairs eNOS activity, leading to decreased eNOS protein levels and NO production [5]. It was reported that the activation of the ERK signaling pathway contributes to arterial wall remodeling in rats with hypertension. In addition, p-STAT3 and p-ERK both contribute to the smooth muscle cell (SMC) phenotype switch, which was associated with the progression of experimental occlusive pulmonary vascular disease [6].In recent decades, evidence indicating endoplasmic reticulum stress in pulmonary arterial hypertension has been accumulated, but targeted clinical treatment is still lacking [7]. Endoplasmic reticulum (ER) stress participates in many known PAH-triggering and PAH-facilitating processes. For example, both inflammation and hypoxia are closely associated with ER stress. Moreover, the loss-of-function mutations in BMPRII lead to ER stress as well [8]. It was reported that the inhibition of ER stress signaling prevented and reversed vascular remodeling in PH [7]. The unfolded protein response (UPR) triggered by ER stress serves as an adaptive mechanism to protect the cell from stress and restore ER homeostasis in the initial stages [9]; however, excessive ER stress would trigger UPR termination, cell death, and mitochondrial collapse, involving disturbance of mitochondria-associated endoplasmic reticulum membrane (MAM) and resultant mitochondrial dysfunction [10]. ER structural remodeling leads to decreased influx of Ca2+ from the ER to mitochondria and resultant decreasing mitochondrial Ca2+ (Ca2+m), contributing to mitochondrial suppression, as many Ca2+-dependent mitochondrial enzymes are inhibited.Emerging evidence from clinical and basic medical research has suggested mitophagy serves as a double-edged sword in pulmonary diseases. As a highly conserved process mediated under various cellular stress conditions, mitophagy leads to beneficial or detrimental consequences in different diseases. Mitophagy plays an essential role in cell survival through the maintenance of energy homeostasis, while excessive mitophagy promotes cell death [11]. It was reported that cigarette smoke would induce mitophagy through stabilization of the mitophagy regulator PINK1 and increasing the expression of BNIP3L in chronic obstructive pulmonary disease (COPD) development, which leads to mitochondrial dysfunction and cell injury. Moreover, it was reported that Parkin translocates to mitochondria upon dissipation of the mitochondrial membrane potential (ΔΨm) by the uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP) or in response to ROS. The precise role of mitophagy in PH is still uncertain and needs further study.Previously, we reported that CSNO regulated constriction of vascular smooth muscle cells by oxidative nitrosation of RhoA, and CSNO caused Keap1 thiol modification, which activated the antioxidant response element (ARE), leading to transcriptional upregulation of cytoprotective and antioxidant genes. In this study, we investigated the effect of CSNO on pulmonary vessel constriction and remodeling and explored the underlying mechanisms through which CSNO attenuated mPAP, alternated pulmonary vascular remodeling, and improved RV hypertrophy and PH development.


## 2. Materials and Methods

### 2.1. Ethics Statement

All studies using Sprague-Dawley rats were approved by the institutional animal care and use committee of Tongji Medical College, Huazhong University of Science and Technology, and performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.


### 2.2. Animals and Treatment

Male Sprague-Dawley rats (age: 8-10 weeks; weight: 220-250 g) were allowed to acclimate to the experimental environment for 1 week. Then, they received 1 intraperitoneal injection of MCT at 60 mg/kg to produce the pulmonary hypertension model. According to previous studies, stable pulmonary hypertension was established in rats after two weeks after the injection of MCT.Rats were randomly divided into four groups (n=6 per group) as described below. (1) In the negative control (NC) group, a single dose of solvent but without MCT was given intraperitoneally on day 1 of the experiment. (2) In the diseased nontreatment (NT) group, a single dose of MCT (60 mg/kg) was given intraperitoneally on day 1 of the experiment. (3) In the diseased early treatment (ET) group, a single dose of MCT (60 mg/kg) was given intraperitoneally on day 1, and aerosol inhalation of CSNO was administrated from day 1 to day 28 (88 ppm for 20 minutes per day). (4) In the diseased late treatment (LT) group, a single dose of MCT (60 mg/kg) was given intraperitoneally on day 1, and aerosol inhalation of CSNO was administrated from day 14 to day 28 (88 ppm for 20 minutes per day).Sodium nitrite was obtained from Sigma-Aldrich, and L-cysteine was obtained from Tokyo Chemical Industry. CSNO was synthesized as previously described. Monocrotaline, obtaining from MCE, was dissolved in solvent (ethanol and 0.9% NaCl at 1 : 4).


### 2.3. Hemodynamic Measurements

Pulmonary hemodynamic studies were conducted on day 28; all animals were anesthetized by isoflurane inhalation (1.5-2%) and then euthanized by cervical dislocation. A PE-50 tube, with specific angles, was carefully inserted into the right jugular vein, through the right ventricle, eventually to the pulmonary artery to continuously monitor the hemodynamic parameters. Cardiac output (CO) was measured using the thermodilution method with the CO pod (ML313C, ADInstruments) and the PowerLab/4SP data acquisition system [12]. Pulmonary vascular resistance is calculated by the ratio of mPAP and CO: PVR=mPAP/CO.


### 2.4. HE and EVG Staining

Animals were sacrificed after hemodynamic measurements on day 28. Middle and lower portions of the left lungs were embedded in paraffin, followed by staining with both hematoxylin and eosin (HE) staining and Elastic van Gieson (EVG) staining in a standard manner. Medial wall thickness was determined in distal pulmonary arteries with diameters between 50 and 150μm. Image capture was performed using the EVOS FL Auto Imaging System (Life Technologies, Thermo Fisher Scientific).Two indexes reflecting the vessel thickness were calculated:theratioofvascularwallthicknessWT%=100%×outerdiameterofthepulmonaryarterioles−innerdiameterofthepulmonaryarterioles/outerdiameterofthepulmonaryarterioles; theratioofvascularwallareaWA%=100%×transectionareaofthewallsofpulmonaryarterioles/cross−sectionalareaofpulmonaryarterioles.


### 2.5. Masson’s Trichrome Staining

Paraffin-embedded lung tissues and frozen right ventricle sections were stained with Masson’s trichrome staining in a standard manner.Two indexes reflecting the collagen deposition level were calculated:tissuecollagenvolumefractionCVF%=collagenarea/entirearea×100%; perivascularcollagenareaversusvascularlumenareaPVCA/VA%=collagenareasurroundingthevessel/vascularlumenarea×100%.


### 2.6. Immunohistochemistry Staining

Monoclonal antibodies againstα-smooth muscle actin (α-SMA) (Denmark, 1 : 200 dilution) were used to perform immunohistochemical studies in paraffin-embedded lung tissues. The categorization of pulmonary arteries is based on the degree of muscularization. The vessels were categorized as fully muscularized, partially muscularized, or nonmuscularized vessels, according to previous studies. Immunohistochemistry photomicrographs were captured with EVOS FL Auto Imaging System (Life Technologies, Thermo Fisher Scientific).


### 2.7. Measurement of RVHI

The hearts without residuary blood were quickly separated, and each chamber was weighed to determine the grade of the right ventricular hypertrophy index (RVHI). The index of RVH was expressed as the weight ratio of RV to LV plus the septum (RV/LV+S).


### 2.8. Western Blot Analysis

Lung tissue homogenates and collected cultured cells were lysed in the RIPA lysis buffer. Equal amounts of proteins were loaded for gel electrophoresis using 10% Bis-Tris SDS-PAGE, transferred to a PVDF membrane, and probed with antibodies. Prestained Protein Ladder (Thermo Fisher Scientific), p-JAK2 (Tyr1007/1008, CST, #3776, 1 : 1000 dilution), p-STAT3 (Tyrosine 705, CST, #9145, 1 : 1000 dilution), p-MEK1/2 (Ser217/221, CST, #9154, 1 : 1000 dilution), p-ERK1/2 (Thr202/Tyr204, CST, #4370, 1 : 1000 dilution), SMMHC (Proteintech, #21404-1-AP, 1 : 1000 dilution), SM22α (Proteintech, #55135-1-AP, 1 : 1000 dilution), MMP2 (Santa Cruz Biotechnology, sc-10736, 1 : 1000 dilution), MMP9 (Abcam, ab38898, 1 : 1000 dilution), iNOS (Abcam, ab49999, 1 : 1000 dilution), Bip (CST, #3183, 1 : 1000 dilution), CHOP (CST, #2895, 1 : 1000 dilution), p-EIF2α (Ser51, CST, #3398P, 1 : 1000 dilution), p-PERK(Thr982, Affinity, DF7576, 1 : 1000 dilution), PINK1 (Abcam, ab23707, 1 : 1000 dilution), Parkin (CST, #4211, 1 : 1000 dilution), BNIP3 (Abcam, ab10433, 1 : 1000 dilution), BNIP3L (CST, #12396, 1 : 1000 dilution), FUNDC1 (CST, #49240, 1 : 1000 dilution), LC3B (CST, #3868, 1 : 1000 dilution), P62 (CST, #23214, 1 : 1000 dilution), β-actin (Santa Cruz Biotechnology, sc-47778, 1 : 2000 dilution), and GAPDH (CST, #2118 L, 1 : 2000 dilution) antibodies and HRP-conjugated secondary antibodies were used.


### 2.9. Quantitative PCR

Quantitative PCR studies were performed as we recently delineated. Primer sequences are listed as follows: Rat Fibronectin Forward 5′-AGGCACAAGGTCCGAGAAGAGG-3′, Reverse 5′-CATGAGTCATCCGTAGGCTGGTTC-3′; Rat COL1A1 Forward 5′-TGTTGGTCCTGCTGGCAAGAATG-3′, Reverse 5′-GTCACCTTGTTCGCCTGTCTCAC-3′; Rat COL3A1 Forward 5′-GACACGCTGGTGCTCAAGGAC-3′, Reverse 5′-GTTCGCCTGAAGGACCTCGTTG-3′; Rat iNOS Forward 5′-GTGTTCCACCAGGAGATGTTG-3′, Reverse 5′-GAAGGCGTAGCTGAACAAGG-3′; Rat NOX4 Forward 5′-TGCATGGTGGTGGTATTGTTCCTC-3′, Reverse 5′-AGCAGCAGCAGCATGTAGAAGAC-3′; Rat IL-1β Forward 5′-TCCATGAGCTTTGTACAAGG-3′, Reverse 5′-GGTGCTGATGTACCAGTTGG-3′; Rat IL-6 Forward 5′-TGTTCTCAGGGAGATCTTGG-3′, Reverse 5′-TCCAGGTAGAAACGGAACTC-3′; Rat TNFα Forward 5′-GATCGGTCCCAACAAGGAGG-3′, Reverse 5′-TTTGCTACGACGTGGGCTAC-3′; and Rat GAPDH Forward 5′-GACATGCCGCCTGGAGAAAC-3′, Reverse 5′-AGCCCAGGATGCCCTTTAGT-3′.


### 2.10. Cell Culture

The A7R5 cell line was purchased from the American Type Culture Collection and cultured in DMEM (Dulbecco’s modified Eagle’s medium) containing 10% FBS (fetal bovine serum, HyClone) and 1% penicillin/streptomycin in a humidified 37°C incubator with 95% air and 5% CO2. N-Acetyl-cysteine (NAC) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Dihydroethidium (DHE) was purchased from MCE.


### 2.11. Wound Healing Assay

The wound healing assay was performed as previously described. A7R5 cells were seeded in 6-well plates (1.5×105/well) and were grown to confluence. A 10 μL pipette tip was used to scratch the monolayer cells, followed by PBS rinsing to remove floating cells.


### 2.12. Statistical Analysis

Data was represented asmean±SEM performed in quadruplicate. Statistical analysis was performed with SPSS software (version 21.0). One-way ANOVA and two-way ANOVA followed by Bonferroni’s post hoc test were used to test for significant differences among test groups. Statistical significance was defined as P<0.05. Quantitative assessments were performed by ImageJ and Image-Pro Plus (6.0).


## 2.1. Ethics Statement

All studies using Sprague-Dawley rats were approved by the institutional animal care and use committee of Tongji Medical College, Huazhong University of Science and Technology, and performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.


## 2.2. Animals and Treatment

Male Sprague-Dawley rats (age: 8-10 weeks; weight: 220-250 g) were allowed to acclimate to the experimental environment for 1 week. Then, they received 1 intraperitoneal injection of MCT at 60 mg/kg to produce the pulmonary hypertension model. According to previous studies, stable pulmonary hypertension was established in rats after two weeks after the injection of MCT.Rats were randomly divided into four groups (n=6 per group) as described below. (1) In the negative control (NC) group, a single dose of solvent but without MCT was given intraperitoneally on day 1 of the experiment. (2) In the diseased nontreatment (NT) group, a single dose of MCT (60 mg/kg) was given intraperitoneally on day 1 of the experiment. (3) In the diseased early treatment (ET) group, a single dose of MCT (60 mg/kg) was given intraperitoneally on day 1, and aerosol inhalation of CSNO was administrated from day 1 to day 28 (88 ppm for 20 minutes per day). (4) In the diseased late treatment (LT) group, a single dose of MCT (60 mg/kg) was given intraperitoneally on day 1, and aerosol inhalation of CSNO was administrated from day 14 to day 28 (88 ppm for 20 minutes per day).Sodium nitrite was obtained from Sigma-Aldrich, and L-cysteine was obtained from Tokyo Chemical Industry. CSNO was synthesized as previously described. Monocrotaline, obtaining from MCE, was dissolved in solvent (ethanol and 0.9% NaCl at 1 : 4).


## 2.3. Hemodynamic Measurements

Pulmonary hemodynamic studies were conducted on day 28; all animals were anesthetized by isoflurane inhalation (1.5-2%) and then euthanized by cervical dislocation. A PE-50 tube, with specific angles, was carefully inserted into the right jugular vein, through the right ventricle, eventually to the pulmonary artery to continuously monitor the hemodynamic parameters. Cardiac output (CO) was measured using the thermodilution method with the CO pod (ML313C, ADInstruments) and the PowerLab/4SP data acquisition system [12]. Pulmonary vascular resistance is calculated by the ratio of mPAP and CO: PVR=mPAP/CO.


## 2.4. HE and EVG Staining

Animals were sacrificed after hemodynamic measurements on day 28. Middle and lower portions of the left lungs were embedded in paraffin, followed by staining with both hematoxylin and eosin (HE) staining and Elastic van Gieson (EVG) staining in a standard manner. Medial wall thickness was determined in distal pulmonary arteries with diameters between 50 and 150μm. Image capture was performed using the EVOS FL Auto Imaging System (Life Technologies, Thermo Fisher Scientific).Two indexes reflecting the vessel thickness were calculated:theratioofvascularwallthicknessWT%=100%×outerdiameterofthepulmonaryarterioles−innerdiameterofthepulmonaryarterioles/outerdiameterofthepulmonaryarterioles; theratioofvascularwallareaWA%=100%×transectionareaofthewallsofpulmonaryarterioles/cross−sectionalareaofpulmonaryarterioles.


## 2.5. Masson’s Trichrome Staining

Paraffin-embedded lung tissues and frozen right ventricle sections were stained with Masson’s trichrome staining in a standard manner.Two indexes reflecting the collagen deposition level were calculated:tissuecollagenvolumefractionCVF%=collagenarea/entirearea×100%; perivascularcollagenareaversusvascularlumenareaPVCA/VA%=collagenareasurroundingthevessel/vascularlumenarea×100%.


## 2.6. Immunohistochemistry Staining

Monoclonal antibodies againstα-smooth muscle actin (α-SMA) (Denmark, 1 : 200 dilution) were used to perform immunohistochemical studies in paraffin-embedded lung tissues. The categorization of pulmonary arteries is based on the degree of muscularization. The vessels were categorized as fully muscularized, partially muscularized, or nonmuscularized vessels, according to previous studies. Immunohistochemistry photomicrographs were captured with EVOS FL Auto Imaging System (Life Technologies, Thermo Fisher Scientific).


## 2.7. Measurement of RVHI

The hearts without residuary blood were quickly separated, and each chamber was weighed to determine the grade of the right ventricular hypertrophy index (RVHI). The index of RVH was expressed as the weight ratio of RV to LV plus the septum (RV/LV+S).


## 2.8. Western Blot Analysis

Lung tissue homogenates and collected cultured cells were lysed in the RIPA lysis buffer. Equal amounts of proteins were loaded for gel electrophoresis using 10% Bis-Tris SDS-PAGE, transferred to a PVDF membrane, and probed with antibodies. Prestained Protein Ladder (Thermo Fisher Scientific), p-JAK2 (Tyr1007/1008, CST, #3776, 1 : 1000 dilution), p-STAT3 (Tyrosine 705, CST, #9145, 1 : 1000 dilution), p-MEK1/2 (Ser217/221, CST, #9154, 1 : 1000 dilution), p-ERK1/2 (Thr202/Tyr204, CST, #4370, 1 : 1000 dilution), SMMHC (Proteintech, #21404-1-AP, 1 : 1000 dilution), SM22α (Proteintech, #55135-1-AP, 1 : 1000 dilution), MMP2 (Santa Cruz Biotechnology, sc-10736, 1 : 1000 dilution), MMP9 (Abcam, ab38898, 1 : 1000 dilution), iNOS (Abcam, ab49999, 1 : 1000 dilution), Bip (CST, #3183, 1 : 1000 dilution), CHOP (CST, #2895, 1 : 1000 dilution), p-EIF2α (Ser51, CST, #3398P, 1 : 1000 dilution), p-PERK(Thr982, Affinity, DF7576, 1 : 1000 dilution), PINK1 (Abcam, ab23707, 1 : 1000 dilution), Parkin (CST, #4211, 1 : 1000 dilution), BNIP3 (Abcam, ab10433, 1 : 1000 dilution), BNIP3L (CST, #12396, 1 : 1000 dilution), FUNDC1 (CST, #49240, 1 : 1000 dilution), LC3B (CST, #3868, 1 : 1000 dilution), P62 (CST, #23214, 1 : 1000 dilution), β-actin (Santa Cruz Biotechnology, sc-47778, 1 : 2000 dilution), and GAPDH (CST, #2118 L, 1 : 2000 dilution) antibodies and HRP-conjugated secondary antibodies were used.


## 2.9. Quantitative PCR

Quantitative PCR studies were performed as we recently delineated. Primer sequences are listed as follows: Rat Fibronectin Forward 5′-AGGCACAAGGTCCGAGAAGAGG-3′, Reverse 5′-CATGAGTCATCCGTAGGCTGGTTC-3′; Rat COL1A1 Forward 5′-TGTTGGTCCTGCTGGCAAGAATG-3′, Reverse 5′-GTCACCTTGTTCGCCTGTCTCAC-3′; Rat COL3A1 Forward 5′-GACACGCTGGTGCTCAAGGAC-3′, Reverse 5′-GTTCGCCTGAAGGACCTCGTTG-3′; Rat iNOS Forward 5′-GTGTTCCACCAGGAGATGTTG-3′, Reverse 5′-GAAGGCGTAGCTGAACAAGG-3′; Rat NOX4 Forward 5′-TGCATGGTGGTGGTATTGTTCCTC-3′, Reverse 5′-AGCAGCAGCAGCATGTAGAAGAC-3′; Rat IL-1β Forward 5′-TCCATGAGCTTTGTACAAGG-3′, Reverse 5′-GGTGCTGATGTACCAGTTGG-3′; Rat IL-6 Forward 5′-TGTTCTCAGGGAGATCTTGG-3′, Reverse 5′-TCCAGGTAGAAACGGAACTC-3′; Rat TNFα Forward 5′-GATCGGTCCCAACAAGGAGG-3′, Reverse 5′-TTTGCTACGACGTGGGCTAC-3′; and Rat GAPDH Forward 5′-GACATGCCGCCTGGAGAAAC-3′, Reverse 5′-AGCCCAGGATGCCCTTTAGT-3′.


## 2.10. Cell Culture

The A7R5 cell line was purchased from the American Type Culture Collection and cultured in DMEM (Dulbecco’s modified Eagle’s medium) containing 10% FBS (fetal bovine serum, HyClone) and 1% penicillin/streptomycin in a humidified 37°C incubator with 95% air and 5% CO2. N-Acetyl-cysteine (NAC) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Dihydroethidium (DHE) was purchased from MCE.


## 2.11. Wound Healing Assay

The wound healing assay was performed as previously described. A7R5 cells were seeded in 6-well plates (1.5×105/well) and were grown to confluence. A 10 μL pipette tip was used to scratch the monolayer cells, followed by PBS rinsing to remove floating cells.


## 2.12. Statistical Analysis

Data was represented asmean±SEM performed in quadruplicate. Statistical analysis was performed with SPSS software (version 21.0). One-way ANOVA and two-way ANOVA followed by Bonferroni’s post hoc test were used to test for significant differences among test groups. Statistical significance was defined as P<0.05. Quantitative assessments were performed by ImageJ and Image-Pro Plus (6.0).


## 3. Results

### 3.1. CSNO Attenuated Pulmonary Hemodynamic Changes and Right Ventricular Hypertrophy Induced by MCT In Vivo

To assess the effect of CSNO on PH development, a catheter with specific angles was inserted into the pulmonary artery through the right ventricle to directly measure pulmonary artery pressure (PAP). As shown in Figure1(a), MCT administration significantly elevated mean pulmonary artery pressure (mPAP), indicating that the PH model was established successfully as expected. When diseased animals were treated with CSNO, the mPAP decreased significantly compared to the nontreatment group, with the trend being more effective in the early treatment group (Figures 1(a) and 1(b)). To further confirm the effect of CSNO on hemodynamic changes in pulmonary arteries and avoid potential bias resulting from a different baseline, we calculated pulmonary vascular resistance (PVR) in different groups (Figure 1(c)). In agreement with mPAP changes, the PVR index was elevated in the diseased nontreatment group and, importantly, decreased in both CSNO treatment groups significantly, with the trend being more effective in the early treatment group (Figure 1(c)).Figure 1
Effect of CSNO on pulmonary hemodynamic parameters in MCT-induced PH animals. Representative images of pulmonary artery pressure (PAP) measurement interface from rats on day 28 by the PowerLab/4SP data acquisition system (a). Comparison of mean pulmonary artery pressure (mPAP) (b), pulmonary vascular resistance (PVR) (c), RVHI (d), and the ratio of RV/BW (e) in each group. Data are presented asmean±SEM. n=6 rats. ∗P<0.05 vs. the NC group, #P<0.05 vs. the NT group.
(a)(b)(c)(d)(e)RVHI, the index of RV hypertrophy, was also increased in the NT group than in the NC group. When diseased animals were treated with CSNO, RVHI was significantly decreased in both the ET and LT groups (Figure1(d)), indicating that CSNO treatment improved right ventricular hypertrophy. In addition, MCT-induced PH animals exhibited a significantly increased RV/BW ratio; again, CSNO treatment significantly reduced this ratio, with the trend being more effective in the early treatment group (Figure 1(e)). These data indicate that CSNO treatment can effectively improve hemodynamic alterations in the process of PH in MCT-induced rats.


### 3.2. CSNO Ameliorated Vascular Remodeling and Regulated Smooth Muscle Contractile Protein Expression and MMP Expression

Pulmonary vascular remodeling, characterized by proliferation and migration of vascular smooth muscle cells, leading to continuous progress and deterioration of PH, is the key process in the pathophysiology development of PH. As shown in Figures2(a) and 2(b), pulmonary artery wall thickness, the main manifestation of vascular remodeling, was significantly increased in the NT group, but when treated with CSNO, the remodeling in these rats was markedly ameliorated. CSNO treatment obtained therapeutic effects in both the ratio of vascular wall thickness (WT%) (Figure 2(c)) and ratio of vascular wall area (WA%) (Figure 2(d)) indexes, which are both critical indexes to evaluate pulmonary vascular remodeling. The above effects were further confirmed by hematoxylin and eosin (HE) staining (Figure 2(a)) and Elastic van Gieson (EVG) staining (Figure 2(b)). In addition, the fracture of the pulmonary artery vascular wall external elastic layer was also improved in rats following CSNO treatment (Figure 2(b)).Figure 2
Effect of CSNO on pulmonary vascular remodeling in MCT-induced PH animals. (a) Hematoxylin and eosin (HE) staining for rat pulmonary arterioles. Scale bar: 20μm. (b) Elastic van Gieson (EVG) staining of the elastic fiber of rat pulmonary arterioles. Scale bar: 20 μm. Comparison of (c) vascular wall thickness and (d) vascular wall area in each group as indicated. (e) Lung tissue SMMHC, SM22α, MMP2, and MMP9 expression in each group as indicated. (f–i) Schematic representation of the quantitative expression of indicated proteins. Data are presented as mean±SEM. n=6 rats. ∗P<0.05 vs. the NC group, #P<0.05 vs. the NT group.To further investigate the role of smooth muscle in vascular remodeling, we explored smooth muscle contractile protein expression by Western blot. As shown in Figure2(e), CSNO treatment significantly increased the expression of SMMHC and SM22α, which are both contractile markers for the smooth muscle phenotype switch. In addition, CSNO treatment significantly inhibited PH animals’ lung tissue MMP2 and MMP9 expression, where levels are closely related to the ability of vascular smooth muscle cells to migrate (Figures 2(e)–2(i)). These data further confirm the effect of CSNO on pulmonary vascular remodeling in PH animals.


### 3.3. CSNO Ameliorated Pulmonary Vascular Muscularization and Collagen Deposition

Lung vascular muscularization and pulmonary and right ventricular collagen deposition are all the key features of pulmonary vascular remodeling. Compared to the NC group, MCT-induced animals showed an increased pulmonary muscularization degree, vascular partial muscularization and full muscularization distribution (Figures3(a) and 3(d)). Notably, both the early and late CSNO interventions significantly improved the muscularization degree, which may be due to the increased percentage of nonmuscularization and decreased percentage of full muscularization (Figure 3(d)). CSNO treatment also inhibited collagen volume fraction (CVF) (Figure 3(e)) and the ratio of perivascular collagen area versus vascular lumen area (PVCA/VA) in the pulmonary perivascular area (Figures 3(b) and 3(f)) and, in addition, CVF in the right ventricle (Figures 3(c) and 3(g)).Figure 3
Effect of CSNO on muscularization and collagen deposition. (a) Immunohistochemical staining ofα-SMA in lung tissues. Scale bar: 20 μm. (b) Masson’s trichrome staining of lung tissues. (c) Masson’s trichrome staining of RV tissues. (d) Categorization of pulmonary arteries based on the degree of muscularization, as fully muscularized, partially muscularized, or nonmuscularized vessels. (e, f) Indexes to evaluate collagen deposition in the lung tissues and pulmonary perivascular area. (g) Index to evaluate collagen deposition in RV. (h–j) A comparison of mRNA levels of COL1A1, COL3A1, and Fibronectin by RT-qPCR analysis in lung tissues. Magnification=400x. Bar=20μm. Data are displayed as mean±SEM. n=6 rats. ∗P<0.05 vs. the NC group, #P<0.05 vs. the NT group.To further explore the inhibition of CSNO on collagen deposition in lung tissues, we determined the mRNA levels of COL1A1, COL3A1, and Fibronectin in each group. As shown in Figures3(h)–3(j), both the early and late CSNO treatments attenuated the elevated mRNA levels of COL1A1, COL3A1, and Fibronectin in lung tissues of rats with PH. The amelioration of CSNO on the pulmonary vascular muscularization, collagen deposition, and right ventricular collagen deposition may contribute to the improvement of CSNO on PH development.


### 3.4. CSNO Treatment Ameliorated Oxidative Stress and Inflammatory Pathways in Lung Tissues

Oxidative stress plays an important role in PH development and pathophysiological processes in PH. We first investigated the transcription levels of NOX4 and iNOS in lung tissues by RT-PCR and found that they were both elevated in MCT-induced PH groups (Figures4(g) and 4(h)). Notably, CSNO intervention decreased the transcription of these proteins in both the early treatment group and the late treatment group (Figures 4(g) and 4(h)). Next, we explored the protein expression of iNOS in lung tissues and found that, in agreement with the mRNA change, CSNO treatment decreased the expression of iNOS when compared with the nontreatment group (Figures 4(a) and 4(b)). Further, we investigated the levels of IL-1β, IL-6, and TNFα in lung tissues to evaluate local inflammation. As shown in Figures 4(i)–4(k), IL-1β, IL-6, and TNFα were all elevated in the MCT-induced PH groups and significantly decreased in both the ET and LT groups, indicating that CSNO treatment ameliorated lung tissue inflammation in PH.Figure 4
CSNO inhibited ROS and cytokine production and mediated STAT3 and ERK signaling pathways. (a–f) iNOS, p-JAK2, p-STAT3 (Y705), p-MEK1/2, p-ERK1/2, and GAPDH protein expressions were determined by immunoblotting. (g–k) NOX4, iNOS, IL-1β, IL-6, and TNFα mRNA levels were determined by RT-qPCR. Data are presented as mean±SEM. n=6 rats. ∗P<0.05 vs. the NC group, #P<0.05 vs. the NT group.
(a)(b)(c)(d)(e)(f)(g)(h)(i)(j)(k)To elucidate the underlying mechanisms of the phenomena observed above, we explored the alternations of JAK2/STAT3 and MEK/ERK pathways in the lung tissues of these animals. CSNO inhibited the phosphorylation of JAK2 and STAT3 in lung tissues induced by MCT induction (Figures4(a), 4(c), and 4(d)). In addition, p-MEK1/2 and p-ERK1/2 in lung tissues were remarkably enhanced in the MCT-induced groups and suppressed in both the ET and LT groups (Figures 4(a), 4(e), and 4(f)), suggesting CSNO may achieve its effects through multiple signaling pathways.


### 3.5. CSNO Treatment Improved Aberrant ER Stress and Mitophagy in Lung Tissues of MCT-Induced PH Rats

ER stress has been reported as an important cellular response in PH development. Figure5(a) shows that the expression of Bip, p-PERK, p-EIF2α, and CHOP remarkably elevated in MCT-induced PH rat lung tissues. Notably, their expression was significantly decreased in rats with CSNO inhalation, which indicated that CSNO improved ER stress occurring in lung tissues of MCT-induced PH rats (Figures 5(a)–5(e)).Figure 5
CSNO attenuated ER stress and mitophagy in MCT-induced rats. (a) Representative images of Bip, p-PERK, p-EIF2α, and CHOP expression in MCT-induced rat lung tissues. (b–e) Quantification of corresponding images. (f) Representative images of PINK1, Parkin, BNIP3, BNIP3L, FUNDC1, and LC3B expression in MCT-induced rat lung tissues. (g–l) Quantification of corresponding images. Data were presented as mean±SEM. n=6 rats. ∗P<0.05 vs. the NC group, #P<0.05 vs. the NT group.
(a)(b)(c)(d)(e)(f)(g)(h)(i)(j)(k)(l)ER stress and mitophagy are closely related and interact with each other. We further investigated the changes of mitophagy in PH. As shown in Figure5(f), mitophagy-related proteins including PINK1, Parkin, BNIP3, BNIP3L, FUNDC1, and LC3B were elevated in the NT group. In the lung tissues of PH rats treated with CSNO inhalation, their expression was decreased, suggesting CSNO improved mitophagy in MCT-induced rat lung tissues (Figures 5(f)–5(l)).


### 3.6. CSNO Inhibited Aberrant ROS Production, Wound Healing, ER Stress, and Mitophagy In Vitro

To confirm the beneficial effects of CSNO observed in vivo, we further investigated its effects in vitro using cultured smooth muscle cells. We first evaluated ROS production induced by AngII and IL-6 in A7R5 cells. As shown in Figures6(a)–6(d), CSNO treatment significantly inhibited ROS production in these cells. Next, we investigated the effect of CSNO on A7R5 cell migration by the wound healing assay. As shown in Figures 6(e) and 6(f), CSNO suppressed A7R5 cell migration in a dose-dependent manner. Furthermore, we determined the STAT3 and ERK phosphorylation and found that CSNO intervention significantly decreased the elevated phosphorylation of ERK1/2 and STAT3 induced by either AngII or IL-6 (Figures 6(g)–6(j)).Figure 6
CSNO suppressed ROS generation, cell migration, and phosphorylation of STAT3 and ERK in vitro. (a, c) A7R5 cells were pretreated with CSNO (50μmol/L) or NAC (5 mmol/L) for 1 hour, then stimulated with 0.5 μmol/L AngII or 25 ng/mL IL-6 for an additional 24 hours. Cytosolic ROS production was detected using the DHE fluorescence probe. (b, d) Quantification of DHE fluorescence in A7R5 cells. (e) A7R5 cells were treated with different concentrations of CSNO as indicated, and cell migration was determined by the wound healing assay. (f) Quantitative analysis of corresponding images. (g) A7R5 cells were pretreated with CSNO (50 μmol/L) for 1 hour and incubated with 0.5 μmol/L AngII for an additional 5 minutes; then, ERK1/2 phosphorylation was determined by immunoblotting. (h) Quantification of corresponding images. (i) A7R5 cells were pretreated with CSNO (50 μmol/L) for 1 hour and incubated with 25 ng/mL IL-6 for an additional 30 minutes; then, STAT3 phosphorylation was determined by immunoblotting. (j) Quantification of corresponding images. These data are calculated from three independent experiments and are displayed as mean±SEM. ∗P<0.05 vs. controls, #P<0.05 vs. AngII or IL-6 treatment.
(a)(b)(c)(d)(e)(f)(g)(h)(i)(j)Finally, we explored the effects of CSNO on ER stress and mitophagy in A7R5 cells. As shown in Figure7, AngII and IL-6 were effective stimuli leading to ER stress and mitophagy in A7R5 cells; however, when cells were treated with CSNO, the excessively expressed ER stress-related proteins and mitophagy-related proteins were significantly inhibited (Figures 7(a)–7(l)). Further, the transcription levels of Bip, CHOP, and XBP1 in A7R5 cells were elevated following AngII and IL-6 induction; when cells were treated with CSNO, the transcription of these proteins was inhibited (Figures 7(m)–7(r)).Figure 7
CSNO inhibited mitophagy and ER stress in vitro. (a) A7R5 cells were pretreated with CSNO (50μmol/L) for 1 hour and incubated with 0.5 μmol/L AngII for an additional 24 hours. Protein expression was determined by immunoblotting using specific antibodies as indicated. Representative images of PINK1, Bip, CHOP, LC3B, and P62 expression. (c–g) Quantification of corresponding images. (b) A7R5 cells were pretreated with CSNO (50 μmol/L) for 1 hour and incubated with 25 ng/mL IL-6 for an additional 24 hours. Protein expression was determined by immunoblotting using specific antibodies as indicated. (h–l) Quantification of corresponding images. (m–r) Bip, CHOP, and XBP1 mRNA levels were determined by RT-qPCR. These data are calculated from three independent experiments and are displayed as mean±SEM. ∗P<0.05 vs. controls, #P<0.05 vs. AngII or IL-6 treatment.
(a)(b)(c)(d)(e)(f)(g)(h)(i)(j)(k)(l)(m)(n)(o)(p)(q)(r)


## 3.1. CSNO Attenuated Pulmonary Hemodynamic Changes and Right Ventricular Hypertrophy Induced by MCT In Vivo

To assess the effect of CSNO on PH development, a catheter with specific angles was inserted into the pulmonary artery through the right ventricle to directly measure pulmonary artery pressure (PAP). As shown in Figure1(a), MCT administration significantly elevated mean pulmonary artery pressure (mPAP), indicating that the PH model was established successfully as expected. When diseased animals were treated with CSNO, the mPAP decreased significantly compared to the nontreatment group, with the trend being more effective in the early treatment group (Figures 1(a) and 1(b)). To further confirm the effect of CSNO on hemodynamic changes in pulmonary arteries and avoid potential bias resulting from a different baseline, we calculated pulmonary vascular resistance (PVR) in different groups (Figure 1(c)). In agreement with mPAP changes, the PVR index was elevated in the diseased nontreatment group and, importantly, decreased in both CSNO treatment groups significantly, with the trend being more effective in the early treatment group (Figure 1(c)).Figure 1
Effect of CSNO on pulmonary hemodynamic parameters in MCT-induced PH animals. Representative images of pulmonary artery pressure (PAP) measurement interface from rats on day 28 by the PowerLab/4SP data acquisition system (a). Comparison of mean pulmonary artery pressure (mPAP) (b), pulmonary vascular resistance (PVR) (c), RVHI (d), and the ratio of RV/BW (e) in each group. Data are presented asmean±SEM. n=6 rats. ∗P<0.05 vs. the NC group, #P<0.05 vs. the NT group.
(a)(b)(c)(d)(e)RVHI, the index of RV hypertrophy, was also increased in the NT group than in the NC group. When diseased animals were treated with CSNO, RVHI was significantly decreased in both the ET and LT groups (Figure1(d)), indicating that CSNO treatment improved right ventricular hypertrophy. In addition, MCT-induced PH animals exhibited a significantly increased RV/BW ratio; again, CSNO treatment significantly reduced this ratio, with the trend being more effective in the early treatment group (Figure 1(e)). These data indicate that CSNO treatment can effectively improve hemodynamic alterations in the process of PH in MCT-induced rats.


## 3.2. CSNO Ameliorated Vascular Remodeling and Regulated Smooth Muscle Contractile Protein Expression and MMP Expression

Pulmonary vascular remodeling, characterized by proliferation and migration of vascular smooth muscle cells, leading to continuous progress and deterioration of PH, is the key process in the pathophysiology development of PH. As shown in Figures2(a) and 2(b), pulmonary artery wall thickness, the main manifestation of vascular remodeling, was significantly increased in the NT group, but when treated with CSNO, the remodeling in these rats was markedly ameliorated. CSNO treatment obtained therapeutic effects in both the ratio of vascular wall thickness (WT%) (Figure 2(c)) and ratio of vascular wall area (WA%) (Figure 2(d)) indexes, which are both critical indexes to evaluate pulmonary vascular remodeling. The above effects were further confirmed by hematoxylin and eosin (HE) staining (Figure 2(a)) and Elastic van Gieson (EVG) staining (Figure 2(b)). In addition, the fracture of the pulmonary artery vascular wall external elastic layer was also improved in rats following CSNO treatment (Figure 2(b)).Figure 2
Effect of CSNO on pulmonary vascular remodeling in MCT-induced PH animals. (a) Hematoxylin and eosin (HE) staining for rat pulmonary arterioles. Scale bar: 20μm. (b) Elastic van Gieson (EVG) staining of the elastic fiber of rat pulmonary arterioles. Scale bar: 20 μm. Comparison of (c) vascular wall thickness and (d) vascular wall area in each group as indicated. (e) Lung tissue SMMHC, SM22α, MMP2, and MMP9 expression in each group as indicated. (f–i) Schematic representation of the quantitative expression of indicated proteins. Data are presented as mean±SEM. n=6 rats. ∗P<0.05 vs. the NC group, #P<0.05 vs. the NT group.To further investigate the role of smooth muscle in vascular remodeling, we explored smooth muscle contractile protein expression by Western blot. As shown in Figure2(e), CSNO treatment significantly increased the expression of SMMHC and SM22α, which are both contractile markers for the smooth muscle phenotype switch. In addition, CSNO treatment significantly inhibited PH animals’ lung tissue MMP2 and MMP9 expression, where levels are closely related to the ability of vascular smooth muscle cells to migrate (Figures 2(e)–2(i)). These data further confirm the effect of CSNO on pulmonary vascular remodeling in PH animals.


## 3.3. CSNO Ameliorated Pulmonary Vascular Muscularization and Collagen Deposition

Lung vascular muscularization and pulmonary and right ventricular collagen deposition are all the key features of pulmonary vascular remodeling. Compared to the NC group, MCT-induced animals showed an increased pulmonary muscularization degree, vascular partial muscularization and full muscularization distribution (Figures3(a) and 3(d)). Notably, both the early and late CSNO interventions significantly improved the muscularization degree, which may be due to the increased percentage of nonmuscularization and decreased percentage of full muscularization (Figure 3(d)). CSNO treatment also inhibited collagen volume fraction (CVF) (Figure 3(e)) and the ratio of perivascular collagen area versus vascular lumen area (PVCA/VA) in the pulmonary perivascular area (Figures 3(b) and 3(f)) and, in addition, CVF in the right ventricle (Figures 3(c) and 3(g)).Figure 3
Effect of CSNO on muscularization and collagen deposition. (a) Immunohistochemical staining ofα-SMA in lung tissues. Scale bar: 20 μm. (b) Masson’s trichrome staining of lung tissues. (c) Masson’s trichrome staining of RV tissues. (d) Categorization of pulmonary arteries based on the degree of muscularization, as fully muscularized, partially muscularized, or nonmuscularized vessels. (e, f) Indexes to evaluate collagen deposition in the lung tissues and pulmonary perivascular area. (g) Index to evaluate collagen deposition in RV. (h–j) A comparison of mRNA levels of COL1A1, COL3A1, and Fibronectin by RT-qPCR analysis in lung tissues. Magnification=400x. Bar=20μm. Data are displayed as mean±SEM. n=6 rats. ∗P<0.05 vs. the NC group, #P<0.05 vs. the NT group.To further explore the inhibition of CSNO on collagen deposition in lung tissues, we determined the mRNA levels of COL1A1, COL3A1, and Fibronectin in each group. As shown in Figures3(h)–3(j), both the early and late CSNO treatments attenuated the elevated mRNA levels of COL1A1, COL3A1, and Fibronectin in lung tissues of rats with PH. The amelioration of CSNO on the pulmonary vascular muscularization, collagen deposition, and right ventricular collagen deposition may contribute to the improvement of CSNO on PH development.


## 3.4. CSNO Treatment Ameliorated Oxidative Stress and Inflammatory Pathways in Lung Tissues

Oxidative stress plays an important role in PH development and pathophysiological processes in PH. We first investigated the transcription levels of NOX4 and iNOS in lung tissues by RT-PCR and found that they were both elevated in MCT-induced PH groups (Figures4(g) and 4(h)). Notably, CSNO intervention decreased the transcription of these proteins in both the early treatment group and the late treatment group (Figures 4(g) and 4(h)). Next, we explored the protein expression of iNOS in lung tissues and found that, in agreement with the mRNA change, CSNO treatment decreased the expression of iNOS when compared with the nontreatment group (Figures 4(a) and 4(b)). Further, we investigated the levels of IL-1β, IL-6, and TNFα in lung tissues to evaluate local inflammation. As shown in Figures 4(i)–4(k), IL-1β, IL-6, and TNFα were all elevated in the MCT-induced PH groups and significantly decreased in both the ET and LT groups, indicating that CSNO treatment ameliorated lung tissue inflammation in PH.Figure 4
CSNO inhibited ROS and cytokine production and mediated STAT3 and ERK signaling pathways. (a–f) iNOS, p-JAK2, p-STAT3 (Y705), p-MEK1/2, p-ERK1/2, and GAPDH protein expressions were determined by immunoblotting. (g–k) NOX4, iNOS, IL-1β, IL-6, and TNFα mRNA levels were determined by RT-qPCR. Data are presented as mean±SEM. n=6 rats. ∗P<0.05 vs. the NC group, #P<0.05 vs. the NT group.
(a)(b)(c)(d)(e)(f)(g)(h)(i)(j)(k)To elucidate the underlying mechanisms of the phenomena observed above, we explored the alternations of JAK2/STAT3 and MEK/ERK pathways in the lung tissues of these animals. CSNO inhibited the phosphorylation of JAK2 and STAT3 in lung tissues induced by MCT induction (Figures4(a), 4(c), and 4(d)). In addition, p-MEK1/2 and p-ERK1/2 in lung tissues were remarkably enhanced in the MCT-induced groups and suppressed in both the ET and LT groups (Figures 4(a), 4(e), and 4(f)), suggesting CSNO may achieve its effects through multiple signaling pathways.


## 3.5. CSNO Treatment Improved Aberrant ER Stress and Mitophagy in Lung Tissues of MCT-Induced PH Rats

ER stress has been reported as an important cellular response in PH development. Figure5(a) shows that the expression of Bip, p-PERK, p-EIF2α, and CHOP remarkably elevated in MCT-induced PH rat lung tissues. Notably, their expression was significantly decreased in rats with CSNO inhalation, which indicated that CSNO improved ER stress occurring in lung tissues of MCT-induced PH rats (Figures 5(a)–5(e)).Figure 5
CSNO attenuated ER stress and mitophagy in MCT-induced rats. (a) Representative images of Bip, p-PERK, p-EIF2α, and CHOP expression in MCT-induced rat lung tissues. (b–e) Quantification of corresponding images. (f) Representative images of PINK1, Parkin, BNIP3, BNIP3L, FUNDC1, and LC3B expression in MCT-induced rat lung tissues. (g–l) Quantification of corresponding images. Data were presented as mean±SEM. n=6 rats. ∗P<0.05 vs. the NC group, #P<0.05 vs. the NT group.
(a)(b)(c)(d)(e)(f)(g)(h)(i)(j)(k)(l)ER stress and mitophagy are closely related and interact with each other. We further investigated the changes of mitophagy in PH. As shown in Figure5(f), mitophagy-related proteins including PINK1, Parkin, BNIP3, BNIP3L, FUNDC1, and LC3B were elevated in the NT group. In the lung tissues of PH rats treated with CSNO inhalation, their expression was decreased, suggesting CSNO improved mitophagy in MCT-induced rat lung tissues (Figures 5(f)–5(l)).


## 3.6. CSNO Inhibited Aberrant ROS Production, Wound Healing, ER Stress, and Mitophagy In Vitro

To confirm the beneficial effects of CSNO observed in vivo, we further investigated its effects in vitro using cultured smooth muscle cells. We first evaluated ROS production induced by AngII and IL-6 in A7R5 cells. As shown in Figures6(a)–6(d), CSNO treatment significantly inhibited ROS production in these cells. Next, we investigated the effect of CSNO on A7R5 cell migration by the wound healing assay. As shown in Figures 6(e) and 6(f), CSNO suppressed A7R5 cell migration in a dose-dependent manner. Furthermore, we determined the STAT3 and ERK phosphorylation and found that CSNO intervention significantly decreased the elevated phosphorylation of ERK1/2 and STAT3 induced by either AngII or IL-6 (Figures 6(g)–6(j)).Figure 6
CSNO suppressed ROS generation, cell migration, and phosphorylation of STAT3 and ERK in vitro. (a, c) A7R5 cells were pretreated with CSNO (50μmol/L) or NAC (5 mmol/L) for 1 hour, then stimulated with 0.5 μmol/L AngII or 25 ng/mL IL-6 for an additional 24 hours. Cytosolic ROS production was detected using the DHE fluorescence probe. (b, d) Quantification of DHE fluorescence in A7R5 cells. (e) A7R5 cells were treated with different concentrations of CSNO as indicated, and cell migration was determined by the wound healing assay. (f) Quantitative analysis of corresponding images. (g) A7R5 cells were pretreated with CSNO (50 μmol/L) for 1 hour and incubated with 0.5 μmol/L AngII for an additional 5 minutes; then, ERK1/2 phosphorylation was determined by immunoblotting. (h) Quantification of corresponding images. (i) A7R5 cells were pretreated with CSNO (50 μmol/L) for 1 hour and incubated with 25 ng/mL IL-6 for an additional 30 minutes; then, STAT3 phosphorylation was determined by immunoblotting. (j) Quantification of corresponding images. These data are calculated from three independent experiments and are displayed as mean±SEM. ∗P<0.05 vs. controls, #P<0.05 vs. AngII or IL-6 treatment.
(a)(b)(c)(d)(e)(f)(g)(h)(i)(j)Finally, we explored the effects of CSNO on ER stress and mitophagy in A7R5 cells. As shown in Figure7, AngII and IL-6 were effective stimuli leading to ER stress and mitophagy in A7R5 cells; however, when cells were treated with CSNO, the excessively expressed ER stress-related proteins and mitophagy-related proteins were significantly inhibited (Figures 7(a)–7(l)). Further, the transcription levels of Bip, CHOP, and XBP1 in A7R5 cells were elevated following AngII and IL-6 induction; when cells were treated with CSNO, the transcription of these proteins was inhibited (Figures 7(m)–7(r)).Figure 7
CSNO inhibited mitophagy and ER stress in vitro. (a) A7R5 cells were pretreated with CSNO (50μmol/L) for 1 hour and incubated with 0.5 μmol/L AngII for an additional 24 hours. Protein expression was determined by immunoblotting using specific antibodies as indicated. Representative images of PINK1, Bip, CHOP, LC3B, and P62 expression. (c–g) Quantification of corresponding images. (b) A7R5 cells were pretreated with CSNO (50 μmol/L) for 1 hour and incubated with 25 ng/mL IL-6 for an additional 24 hours. Protein expression was determined by immunoblotting using specific antibodies as indicated. (h–l) Quantification of corresponding images. (m–r) Bip, CHOP, and XBP1 mRNA levels were determined by RT-qPCR. These data are calculated from three independent experiments and are displayed as mean±SEM. ∗P<0.05 vs. controls, #P<0.05 vs. AngII or IL-6 treatment.
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## 4. Discussion

Pulmonary hypertension, known as a “malignant tumor of the cardiovascular system” [13], is a chronic disorder accompanied by poor prognosis. So far, the strategies are mainly aimed at relieving symptoms and improving prognosis, neglecting characteristic lesions that are closely associated with the overall survival [14]. Therefore, new therapies are needed for PH based on its pathogenesis.NO has been suggested to exert vasodilation and antiproliferation effects. It has been reported that inhaled NO can selectively dilate pulmonary vessels in both patients with PH and animal models of PH [15]. However, the oxygen-rich environment of the airway and lung may predispose it toward toxicity by forming reactive nitrogen/oxygen species; thus, so far, NO is mainly utilized in pulmonary hypertension crises in neonates. SNO including CSNO are endogenous NO derivatives that are relatively resistant to toxic reactions with oxygen. In addition, besides leading to vasodilation through the cGMP-dependent pathway as NO does, CSNO also regulates protein function by S-nitrosylation of thiols, an effect through protein posttranslational modification which NO gas lacks, so it may mimic the whole function of the endogenous nitric oxide species and restore the endothelial dysfunction which has been reported in PH. Previously, we have reported that CSNO inhibited RhoA activity through oxidative nitrosylation of C16/20 residues, which may be beneficial for both vasodilation and remodeling [4]. We also observed CSNO could induce upregulation of cytoprotective and antioxidant genes by thiol modification of Keap1 [16]. Whether CSNO could regulate aberrant redox and ameliorate PH progression is unknown.Inflammation can be exacerbated by endothelial dysfunction and NO deficiency. Notably, endothelial dysfunction in PH leads to the decrease of SNO production, while the expression and activity of GSNOR (S-nitrosoglutathione reductase), which can degrade SNO as an enzyme, increased [17], suggesting that SNO insufficiency may be involved in the progression of PH. In this study, we found that CSNO exogenous supplement improved pulmonary hemodynamics and vascular remodeling in the PH animals and further demonstrated the importance of SNO in maintaining vasculature resistant balance in PH pathogenesis.Torok et al. [18] reported that L-Leu, a LAT inhibitor that blocks CSNO transmembrane movement, inhibited pulmonary vasodilation in L-cysteine-exposed lungs following NO inhalation. Moreover, Jankov et al. [19] reported that NaNO2, a CSNO precursor, reversed chronic hypoxic PH partly via upregulated SNO-LTA4H. Another SNO precursor such as O-nitrosoethanol has been explored to maintain the lung function in PH [20]. We previously reported that CSNO inhibited RhoA activity through SNO-RhoA modification in vitro, which suggested CSNO may contribute to reversing vascular remodeling. In this study, we presented direct data to show that CSNO improved pulmonary vasoconstriction and remodeling in PH. To our knowledge, our study is the first one showing that inhaled CSNO could alleviate the development of PH.In decades, greater attention has been focused on inflammation in patients and experimental animals with PH. Inflammation precedes vascular remodeling and elevated pulmonary artery pressures in experimental PH [21, 11]. An expanding body of knowledge has related inflammatory factors to worse clinical outcomes, quality of life-related symptoms, and death in PH patients. IL-6 was reported to trigger smooth muscle cell proliferation [22] and vascular remodeling [23]. Moreover, IL-6 overexpression induced PH in rodents [22]. Consistent with other studies, our data showed that IL-6 expression was significantly elevated in lung tissues of PH rats, accompanied by increased IL-1β and TNFα. And importantly, the expressions of these inflammatory proteins were significantly decreased following CSNO treatment.Abundant studies have reported STAT3 and ERK abnormal activation in PH, and inhibition of their activities contributed to the reversal of PH [24, 25]. Consistent with previous studies, our data showed excessive phosphorylation of STAT3 and ERK in lung tissues of MCT-induced rats, and we further found that overactivation can be reversed by CSNO intervention. Our results suggested that CSNO improved PH development by regulating STAT3 and ERK activity in the lungs. In addition, CSNO showed inhibition of hyperactivated STAT3 and ERK in A7R5 cells, which is consistent with the data from our study in vivo. Interestingly, both STAT3 [26] and ERK [27] could be modified by S-nitrosylation, leading to the reduction of phosphorylation on Tyr705 and Thr202/Tyr204 modification, respectively. Moreover, STAT3 and ERK S-nitrosylation modification was accompanied by cell growth inhibition [28, 26], suggesting that CSNO may regulate STAT3 and ERK1/2 S-nitrosylation modification to improve PH.The inhibition of ER stress improving PH progression suggests its contribution to PH development. Consistent with previous studies, excessive ER stress was observed in lung tissues following MCT injection. In this study, we found that ER stress-related proteins significantly increased in lung tissues in MCT-induced PH animals. It is reported that the PERK pathway plays an important role in the pathogenesis of chronic hypoxia-induced PH [29]. Inflammation is regarded as a trigger factor of ER stress. Interestingly, ER stress and inflammatory markers were largely mimicked by chronic stimulation of PDGF, which is a stimulus used to induce the smooth muscle phenotype switch.Moderate ER stress is an adaptive strategy for the accumulation of UPR in the ER. Excessive ER stress injures mitochondrial function and contributes to pulmonary vascular remodeling in PH [7]. Moreover, severe ER stress leads to mitochondrial dysfunction by abnormal Ca+ influx from ER to mitochondrial and aberrant ROS regulation. Mitochondrial dysfunction has been theorized as a crucial player in PH development, which leads to the increasing production of ROS [30].The increasing evidence suggested mitochondrial dysfunction contributes to the development and progression of pulmonary hypertension. Reactive oxygen species generated by mitochondria was increased in the vascular smooth muscle cells isolated from the pulmonary vessels of rats with PH [31]. Hsu et al. [32] reported that transplantation of viable mitochondria improved right ventricular performance and pulmonary artery remodeling in rats with established PH. Interestingly, it was reported that S-nitrosylation of PINK1 led to activity inhibition and further reduced Parkin translocation to mitochondria [33]. In our study, we also observed that CSNO inhibited the excessive elevation of mitophagy-related proteins including PINK1, Parkin, BNIP3, BNIP3L, FUNDC1, and LC3B in the lung tissues of PH rats. These data suggested a role of SNO in regulating mitochondrial function.Accumulating evidence indicated that mitophagy, including PINK1/Parkin- and BNIP3L-dependent mitophagy, was involved in the pathogenesis of COPD and other cigarette smoke-associated pulmonary diseases [34, 35]. PINK1 and Parkin were increased in the lungs of smokers, patients with COPD, and chronic cigarette smoke-exposed mouse lungs [36]. PINK1/Parkin-mediated mitophagy was reported to participate in proliferation and atherosclerotic lesion deterioration [37]. In the present study, data showed that the expression of mitophagy-related proteins was increased following MCT induction, and CSNO application partly reversed their excessively increased expression in vivo. Moreover, amelioration on AngII-induced proliferation of vascular smooth muscle cells was accompanied by downregulation of the expression of PINK1, Parkin, and LC3B. AngII could induce the contractile-synthetic phenotypic switch and proliferation of VSMC in vitro [38]. Previous studies have documented that the possible mechanisms for AngII-mediated autophagy may be associated with the activation of NADPH oxidase, further inducing ROS generation [39]. The mRNA transcription of NOX4 and iNOS decreased in lung tissues when rats were treated with inhaled CSNO, indicating that CSNO treatment improved oxidative stress and may further contribute to the amelioration of PH development. We also presented data on ROS production detected by the DHE probe, which was induced by AngII and IL-6 in vitro. Notably, CSNO treatment alleviated ROS production in A7R5 cells, in accordance with our data from the study in vivo. Moreover, our data showed increased expression of PINK1 and Parkin in A7R5 cells induced by AngII and IL-6, indicating that aberrant ROS relates to excessive mitophagy. Furthermore, we presented data that CSNO treatment improved excessive ROS production and aberrant mitophagy in vivo and in vitro. The precise mechanisms of the interaction of oxidative stress and mitophagy need further investigation.Some limitations of this article are as follows. First, we only chose one of the PH animal models, MCT-induced PH in rats, to test whether CSNO could improve PH. So far, no model has been able to fully simulate PH in humans. Different PH models mimic distinct characteristics occurring in human PH. Among these models, the MCT-induced model simulates relatively high PAP and severe PH development to a great extent. Second, in this study, we did not compare the difference of improvements in CSNO, sodium nitrite, and L-cysteine treatment in MCT-induced rats, respectively. Sodium nitrite and L-cysteine are the precursors of CSNO, and they were reported to improve PH by S-nitrosylation of the target protein. However, CSNO is a functional SNO and may directly modify related proteins by S-nitrosylation, which might be more effective than NaNO2/L-cysteine.


## 5. Conclusions

In conclusion, we presented data showing that CSNO attenuated pulmonary artery blood pressure and pulmonary vascular wall remodeling as well as RV hypertrophy; we also showed that the beneficial effects of CSNO on PH may occur through inhibition of oxidative stress, inflammation, and mitophagy.



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*Source: 6621232-2021-01-28.xml*