Forsythoside B Mitigates Monocrotaline-Induced Pulmonary Arterial Hypertension via Blocking the NF-κB Signaling Pathway to Attenuate Vascular Remodeling.

Purpose: Pulmonary arterial hypertension (PAH) is a devastating disease with little effective treatment. The proliferation of pulmonary artery smooth muscle cells (PASMCs) induced by the nuclear factor-κB (NF-κB) signaling activation plays a pivotal role in the pathogenesis of PAH. Forsythoside B (FTS•B) possesses inhibitory effect on NF-κB signaling pathway. The present study aims to explore the effects and mechanisms of FTS•B in PAH. Methods: Sprague-Dawley rats received monocrotaline (MCT) intraperitoneal injection to establish PAH model, and FTS•B was co-treated after MCT injection. Right ventricular hypertrophy and pulmonary artery pressure were measured by echocardiography and right heart catheterization, respectively. Histological alterations were detected by H&E staining and immunohistochemistry. FTS•B's role in PASMC proliferation and migration were evaluated by CCK-8 and wound healing assay. To investigate the underlying mechanisms, Western blotting, immunofluorescence staining and ELISA were conducted. The NF-κB activator PMA was used to investigate the role of NF-κB in FTS•B's protective effects against PAH. Results: FTS•B markedly alleviated MCT-induced vascular remodeling and pulmonary artery pressure, and improved right ventricular hypertrophy and survival. FTS•B also reversed PDGF-BB-induced PASMC proliferation and migration, decreased PCNA and CyclinD1 expression in vitro. The elevated levels of IL-1ß and IL-6 caused by MCT were decreased by FTS•B. Mechanistically, MCT-triggered phosphorylation of p65, IκBα, IKKα and IKKß was blunted by FTS•B. FTS•B also reversed MCT-induced nuclear translocation of p65. However, all these protective effects were blocked by PMA-mediated NF-κB activation. Conclusion: FTS•B effectively attenuates PAH by suppressing the NF-κB signaling pathway to attenuate vascular remodeling. FTS•B might be a promising drug candidate with clinical translational potential for the treatment of PAH.

Hydroxycitric Acid Tripotassium Hydrate Attenuates Monocrotaline and Hypoxia-Induced Pulmonary Hypertension in Rats.

This study investigated the effects of hydroxycitric acid tripotassium hydrate on right ventricular function, myocardial and pulmonary vascular remodeling in rats with pulmonary hypertension, and possible mechanisms. METHODS: Pulmonary hypertension was induced in male Sprague-Dawley rats by a single subcutaneous injection of monocrotaline or hypoxic chamber. In vivo, inflammatory cytokine (including TNF-α, IL-1ß, IL-6, and TGF-ß, the level of SOD) expression, superoxide dismutase and hydrogen peroxide levels, and p-IκBα and p65 expressions were detected. In vitro, pulmonary artery smooth muscle cell proliferation and migration, ROS production, and hypoxia-inducible factor-1 expression were also studied. RESULTS: Hydroxycitric acid tripotassium hydrate decreased right ventricular systolic pressure and reduced right ventricular fibrosis and pulmonary vascular remodeling in rats with two kinds of pulmonary hypertension. Moreover, the expression of both inflammatory and oxidative stress factors was effectively reduced, and the p65 signaling pathway was found to be inhibited in this study. Additionally, hydroxycitric acid tripotassium hydrate inhibited human pulmonary artery smooth cell proliferation and migration in vitro. CONCLUSIONS: This study shows that hydroxycitric acid tripotassium hydrate can alleviate pulmonary hypertension caused by hypoxia and monocycloline in rats, improve remodeling of the right ventricle and pulmonary artery, and inhibit pulmonary artery smooth muscle cell proliferation and migration. The protective effects may be achieved by regulating inflammation and oxidative stress through the p65 signaling pathway.

Ntsr1 contributes to pulmonary hypertension by enhancing endoplasmic reticulum stress via JAK2-STAT3-Thbs1 signaling.

Pulmonary hypertension (PH) is a severe clinical syndrome with pulmonary vascular remodeling and poor long-term prognosis. Neurotensin receptor 1 (Ntsr1), serve as one of the G protein-coupled receptors (GPCRs), implicates in various biological processes, but the potential effects of Ntsr1 in PH development are unclear. The Sugen/Hypoxia (SuHx) or monocrotaline (MCT) induced rat PH model was used in our study and the PH rats showed aggravated pulmonary artery remodeling and increased right ventricular systolic pressure (RVSP). Our results revealed that Ntsr1 induced endoplasmic reticulum (ER) stress response via ATF6 activation contributed to the development of PH. Moreover, RNA-sequencing (RNA-seq) and phosphoproteomics were performed and the Ntsr1-JAK2-STAT3-thrombospondin 1 (Thbs1)-ATF6 signaling was distinguished as the key pathway. In vitro, pulmonary artery smooth muscle cells (PASMCs) under hypoxia condition showed enhanced proliferation and migration properties, which could be inhibited by Ntsr1 knockdown, JAK2 inhibitor (Fedratinib) treatment, STAT3 inhibitior (Stattic) treatment, Thbs1 knockdown or ATF6 knockdown. In addition, adeno-associated virus 1 (AAV1) were used to knockdown the expression of Ntsr1, Thbs1 or ATF6 in rats and reversed the phenotype of PH. In summary, our results reveal that Ntsr1-JAK2-STAT3-Thbs1 pathway can induce enhanced ER stress via ATF6 activation and increased PASMC proliferation and migration capacities, which can be mechanism of the pulmonary artery remodeling and PH. Targeting Ntsr1 might be a novel therapeutic strategy to ameliorate PH.

SCUBE1 promotes pulmonary artery smooth muscle cell proliferation and migration in acute pulmonary embolism by modulating BMP7.

Objectives: After an episode of acute pulmonary embolism (APE), activated platelets have the ability to release various bioactive factors that can stimulate both proliferation and migration of pulmonary artery smooth muscle cells (PASMCs). SCUBE1 has been previously reported to engage in platelet-platelet interactions, potentially contributing to the activation of platelets in early onset thrombi. The purpose of this study was to examine the alterations in SCUBE1 expression in PASMCs after APE, as well as understand the mechanism behind these changes. Methods: The platelet-rich plasma samples of both APE patients and healthy individuals were collected. A hyperproliferative model of PASMCs was established by using platelet-derived growth factor (PDGF) as a stimulator and various assays were used to investigate how SCUBE1-mediated BMP7 can regulate PDGF-induced PASMC proliferation and migration. Results: Elevated level of SCUBE1 were observed in platelet-rich plasma from patients with APE and in PASMCs induced by PDGF. SCUBE1 interference ameliorated PDGF-driven cell proliferation and migration, and also downregulated PCNA expression. Additionally, mechanistic studies demonstrated that SCUBE1 could directly bind to bone morphogenetic protein 7 (BMP7) and enhance BMP7 expression, which completely abolished the impact of SCUBE1 silencing on proliferation and migration ability of PASMCs after PDGF treatment. Conclusion: In the PDGF-induced proliferation of PASMCs, the expression of SCUBE1 and BMP7 was upregulated. Silencing of SCUBE1 impeded PDGF-induced proliferation and migration of PASMCs by restraining BMP7.

Platelet-derived growth factor-stimulated pulmonary artery smooth muscle cells regulate pulmonary artery endothelial cell dysfunction through extracellular vesicle miR-409-5p.

Platelet-derived growth factor (PDGF)-induced changes in vascular smooth muscle cells (VSMCs) stimulate vascular remodeling, resulting in vascular diseases such as pulmonary arterial hypertension. VSMCs communicate with endothelial cells through extracellular vesicles (EVs) carrying cargos, including microRNAs. To understand the molecular mechanisms through which PDGF-stimulated pulmonary artery smooth muscle cells (PASMCs) interact with pulmonary artery endothelial cells (PAECs) under pathological conditions, we investigated the crosstalk between PASMCs and PAECs via extracellular vesicle miR-409-5p under PDGF stimulation. miR-409-5p expression was upregulated in PASMCs upon PDGF signaling, and it was released into EVs. The elevated expression of miR-409-5p was transported to PAECs and led to their impaired function, including reduced NO release, which consequentially resulted in enhanced PASMC proliferation. We propose that the positive regulatory loop of PASMC-extracellular vesicle miR-409-5p-PAEC is a potential mechanism underlying the proliferation of PASMCs under PDGF stimulation. Therefore, miR-409-5p may be a novel therapeutic target for the treatment of vascular diseases, including pulmonary arterial hypertension.

Exosomes derived from mesenchymal stromal cells exert a therapeutic effect on hypoxia-induced pulmonary hypertension by modulating the YAP1/SPP1 signaling pathway.

OBJECTIVE: Hypoxic pulmonary hypertension (HPH) is a progressive and life-threatening disease characterized by perivascular inflammation, pulmonary vascular remodeling, and occlusion. Mesenchymal stromal cell-derived exosomes (MSC-exo) have emerged as potential therapeutic agents due to their role in cell communication and the transportation of bioactive molecules. In this study, we aimed to investigate the therapeutic effects of MSC-exo against HPH and elucidate the underlying molecular mechanism. METHODS: Exosomes were isolated from conditioned media of human bone mesenchymal stromal cells using ultracentrifugation and characterized through western blotting, transmission electron microscopy (TEM), and nanoparticle tracking analysis (NTA). An HPH animal model was established in male SD rats, and MSC-exo or phosphate-buffered saline (PBS) were administered via the tail vein for three weeks. Subsequently, right ventricular systolic pressure (RVSP), right ventricular hypertrophy index (RVHI), and pulmonary vascular remodeling were evaluated. Lung tissues from HPH rats and normal rats underwent high-throughput sequencing and transcriptomic analysis. Gene Ontology (GO) analysis was employed to identify upregulated differentially expressed genes. Additionally, rat pulmonary artery smooth muscle cells (PASMC) exposed to platelet-derived growth factor-BB (PDGF-BB) were used to simulate HPH-related pathological behavior. In vitro cellular models were established to examine the molecular mechanism of MSC-exo in HPH. RESULTS: MSC-exo administration protected rats from hypoxia-induced increases in RVSP, RVHI, and pulmonary vascular remodeling. Additionally, MSC-exo alleviated PDGF-BB-induced proliferation and migration of PASMC. Transcriptomic analysis revealed 267 upregulated genes in lung tissues of HPH rats compared to control rats. Gene Ontology analysis indicated significant differences in pathways associated with Yes Associated Protein 1 (YAP1), a key regulator of cell proliferation and organ size. RT-qPCR and western blot analysis confirmed significantly increased expression of YAP1 in HPH lung tissues and PASMC, which was inhibited by MSC-exo treatment. Furthermore, analysis of datasets demonstrated that Secreted Phosphoprotein 1 (SPP1), also known as Osteopontin (OPN), is a downstream binding protein of YAP1 and can be upregulated by PDGF-BB. MSC-exo treatment reduced the expression of both YAP1 and SPP1. Lentivirus-mediated knockdown of YAP1 inhibited PDGF-BB-induced PASMC proliferation, migration, and SPP1 protein levels. CONCLUSION: Our findings demonstrate that MSC-exo exert a therapeutic effect against hypoxia-induced pulmonary hypertension by modulating the YAP1/SPP1 signaling pathway. The inhibition of YAP1 and downstream SPP1 expression by MSC-exo may contribute to the attenuation of pulmonary vascular remodeling and PASMC proliferation and migration. These results suggest that MSC-exo could serve as a potential therapeutic strategy for the treatment of HPH. Further investigations are warranted to explore the clinical applicability of MSC-exo-based therapies in HPH patients.

Inhibition of YTHDF1 prevents hypoxia-induced pulmonary artery smooth muscle cell proliferation by regulating Foxm1 translation in an m6A-dependent manner.

Pulmonary arterial hypertension (PAH) is a chronic disease characterized by pulmonary vascular remodeling. It refers to the abnormal proliferation of pulmonary artery smooth muscle cells (PASMCs), and hypoxia is an important risk factor for this progression. The present study aims to investigate the role of YTHDF1 in the regulation of hypoxic PASMC proliferation and the underlying mechanism. Human PASMCs were transfected with si-YTHDF1/2/3 followed by treatment of hypoxia, and the PASMC proliferation and Foxm1 expression were detected. Through RNA pull-down, RNA immunoprecipitation, and protein synthesis assay, the mechanism of YTHDF1 regulating Foxm1 was explored. Next, Foxm1 was inhibited by thiostrepton, and cell proliferation was detected. In vivo, mice received a tail vein injection of adenovirus containing si-YTHDF1 and were exposed to hypoxia treatment. Pulmonary vascular changes, right ventricular systolic pressure (RVSP), and genes involving proliferation were analyzed. YTHDF1 silencing reduced more hypoxic PASMC proliferation and Foxm1 protein level than YTHDF2/3 silencing. Mechanical results showed that YTHDF1 interacted with Foxm1 mRNA and up-regulated Foxm1 protein level by enhancing the translation efficiency in an m6A-dependent manner. Furthermore, YTHDF1 facilitated hypoxic PASMC proliferation and proliferation marker expressions through up-regulation of Foxm1 in an m6A-dependent manner. In vivo, the YTHDF1 silencing alleviated pulmonary vascular changes and fibrosis, reduced RVSP, inhibited the interaction of YTHDF1 and Foxm1, and reduced proliferation marker levels, as compared to the PAH group. In conclusion, YTHDF1 silencing inhibits hypoxic PASMC proliferation by regulating Foxm1 translation in an m6A-dependent manner.

Sphingosine Kinase 1 Deficiency in Smooth Muscle Cells Protects against Hypoxia-Mediated Pulmonary Hypertension via YAP1 Signaling.

Sphingosine kinase 1 (SPHK1) and the sphingosine-1-phosphate (S1P) signaling pathway have been shown to play a role in pulmonary arterial hypertension (PAH). S1P is an important stimulus for pulmonary artery smooth muscle cell (PASMC) proliferation and pulmonary vascular remodeling. We aimed to examine the specific roles of SPHK1 in PASMCs during pulmonary hypertension (PH) progression. We generated smooth muscle cell-specific, Sphk1-deficient (Sphk1f/f TaglnCre+) mice and isolated Sphk1-deficient PASMCs from SPHK1 knockout mice. We demonstrated that Sphk1f/f TaglnCre+ mice are protected from hypoxia or hypoxia/Sugen-mediated PH, and pulmonary vascular remodeling and that Sphk1-deficient PASMCs are less proliferative compared with ones isolated from wild-type (WT) siblings. S1P or hypoxia activated yes-associated protein 1 (YAP1) signaling by enhancing its translocation to the nucleus, which was dependent on SPHK1 enzymatic activity. Further, verteporfin, a pharmacologic YAP1 inhibitor, attenuated the S1P-mediated proliferation of hPASMCs, hypoxia-mediated PH, and pulmonary vascular remodeling in mice and hypoxia/Sugen-mediated severe PH in rats. Smooth muscle cell-specific SPHK1 plays an essential role in PH via YAP1 signaling, and YAP1 inhibition may have therapeutic potential in treating PH.

MicroRNA miR-627-5p restrains pulmonary artery smooth muscle cell dysfunction by targeting MAP 2 K4 and PI3K/AKT signaling.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is characterized by pulmonary vascular remodeling, which can be caused by abnormal proliferation and migration of pulmonary artery smooth muscle cells (PASMCs). Several microRNAs were demonstrated to regulate the PASMC dysfunction. Our study intends to evaluate whether miR-627-5p affects cigarette smoke extract (CSE)-induced aberrant biological behaviors of PASMCs. METHODS: PASMCs was treated with CSE to create the in vitro cellular model of COPD. The viability and LDH release of PASMCs was detected by CCK-8 assay and LDH release assay. MiR-627-5p and MAP 2 K4 expression in CSE (2%)-treated PASMCs was detected by qRT-PCR. PASMC proliferation was observed under a microscope, and PASMC migration was assessed by Transwell migration assays. The binding of miR-627-5p on MAP 2 K4 was verified by dual-luciferase reporter assay. Protein levels of MAP2K4 and the PI3K/AKT signaling markers were examined by western blotting. RESULTS: The viability of PASMCs treated with 2% CSE reached a peak. CSE dose-dependently downregulated miR-627-5p expression in PASMCs. MiR-627-5p overexpression attenuated the CSE-induced abnormal proliferation and migration of PASMCs. However, MAP2K4 overexpression antagonized the effects of miR-627-5p on PASMC dysfunction. Importantly, miR-627-5p inhibited CSE-stimulated activation of the PI3K/AKT pathway via downregulating MAP2K4. CONCLUSION: MiR-627-5p improves CSE-induced abnormal proliferation and migration of PASMCs by inhibiting MAP2K4 expression and the PI3K/AKT pathway.

Inhibition of KIR2.1 decreases pulmonary artery smooth muscle cell proliferation and migration.

The investigation of effective therapeutic drugs for pulmonary hypertension (PH) is critical. KIR2.1 plays crucial roles in regulating cell proliferation and migration, and vascular remodeling. However, researchers have not yet clearly determined whether KIR2.1 participates in the proliferation and migration of pulmonary artery smooth muscle cells (PASMCs) and its role in pulmonary vascular remodeling (PVR) also remains elusive. The present study aimed to examine whether KIR2.1 alters PASMC proliferation and migration, and participates in PVR, as well as to explore its mechanisms of action. For the in vivo experiment, a PH model was established by intraperitoneally injecting Sprague­Dawley rats monocrotaline (MCT). Hematoxylin and eosin staining revealed evidence of PVR in the rats with PH. Immunofluorescence staining and western blot analysis revealed increased levels of the KIR2.1, osteopontin (OPN) and proliferating cell nuclear antigen (PCNA) proteins in pulmonary blood vessels and lung tissues following exposure to MCT, and the TGF­ß1/SMAD2/3 signaling pathway was activated. For the in vitro experiments, the KIR2.1 inhibitor, ML133, or the TGF­ß1/SMAD2/3 signaling pathway blocker, SB431542, were used to pre­treat human PASMCs (HPASMCs) for 24 h, and the cells were then treated with platelet­derived growth factor (PDGF)­BB for 24 h. Scratch and Transwell assays revealed that PDGF­BB promoted cell proliferation and migration. Immunofluorescence staining and western blot analysis demonstrated that PDGF­BB upregulated OPN and PCNA expression, and activated the TGF­ß1/SMAD2/3 signaling pathway. ML133 reversed the proliferation and migration induced by PDGF­BB, inhibited the expression of OPN and PCNA, inhibited the TGF­ß1/SMAD2/3 signaling pathway, and reduced the proliferation and migration of HPASMCs. SB431542 pre­treatment also reduced cell proliferation and migration; however, it did not affect KIR2.1 expression. On the whole, the results of the present study demonstrate that KIR2.1 regulates the TGF­ß1/SMAD2/3 signaling pathway and the expression of OPN and PCNA proteins, thereby regulating the proliferation and migration of PASMCs and participating in PVR.

The substitution of SERCA2 redox cysteine 674 promotes pulmonary vascular remodeling by activating IRE1α/XBP1s pathway.

Pulmonary hypertension (PH) is a life-threatening disease characterized by pulmonary vascular remodeling, in which hyperproliferation of pulmonary artery smooth muscle cells (PASMCs) plays an important role. The cysteine 674 (C674) in the sarcoplasmic/endoplasmic reticulum Ca2+ ATPase 2 (SERCA2) is the critical redox regulatory cysteine to regulate SERCA2 activity. Heterozygous SERCA2 C674S knock-in mice (SKI), where one copy of C674 was substituted by serine to represent partial C674 oxidative inactivation, developed significant pulmonary vascular remodeling resembling human PH, and their right ventricular systolic pressure modestly increased with age. In PASMCs, substitution of C674 activated inositol requiring enzyme 1 alpha (IRE1α) and spliced X-box binding protein 1 (XBP1s) pathway, accelerated cell cycle and cell proliferation, which reversed by IRE1α/XBP1s pathway inhibitor 4µ8C. In addition, suppressing the IRE1α/XBP1s pathway prevented pulmonary vascular remodeling caused by substitution of C674. Similar to SERCA2a, SERCA2b is also important to restrict the proliferation of PASMCs. Our study articulates the causal effect of C674 oxidative inactivation on the development of pulmonary vascular remodeling and PH, emphasizing the importance of C674 in restricting PASMC proliferation to maintain pulmonary vascular homeostasis. Moreover, the IRE1α/XBP1s pathway and SERCA2 might be potential targets for PH therapy.

Interplay of Low-Density Lipoprotein Receptors, LRPs, and Lipoproteins in Pulmonary Hypertension.

The low-density lipoprotein receptor (LDLR) gene family includes LDLR, very LDLR, and LDL receptor-related proteins (LRPs) such as LRP1, LRP1b (aka LRP-DIT), LRP2 (aka megalin), LRP4, and LRP5/6, and LRP8 (aka ApoER2). LDLR family members constitute a class of closely related multifunctional, transmembrane receptors, with diverse functions, from embryonic development to cancer, lipid metabolism, and cardiovascular homeostasis. While LDLR family members have been studied extensively in the systemic circulation in the context of atherosclerosis, their roles in pulmonary arterial hypertension (PAH) are understudied and largely unknown. Endothelial dysfunction, tissue infiltration of monocytes, and proliferation of pulmonary artery smooth muscle cells are hallmarks of PAH, leading to vascular remodeling, obliteration, increased pulmonary vascular resistance, heart failure, and death. LDLR family members are entangled with the aforementioned detrimental processes by controlling many pathways that are dysregulated in PAH; these include lipid metabolism and oxidation, but also platelet-derived growth factor, transforming growth factor ß1, Wnt, apolipoprotein E, bone morpohogenetic proteins, and peroxisome proliferator-activated receptor gamma. In this paper, we discuss the current knowledge on LDLR family members in PAH. We also review mechanisms and drugs discovered in biological contexts and diseases other than PAH that are likely very relevant in the hypertensive pulmonary vasculature and the future care of patients with PAH or other chronic, progressive, debilitating cardiovascular diseases.

Human pulmonary artery smooth muscle cell dysfunction is regulated by miR-509-5p in hypoxic environment.

Reportedly, dysfunction of human pulmonary arterial smooth muscle cells (PASMCs) is associated with the pathogenesis of pulmonary arterial hypertension (PAH). Herein, the role of miR-509-5p in hypoxia-induced PASMCs and the underlying mechanism were explored. PASMCs were cultured under both normoxia and hypoxia conditions. Quantitative real-time polymerase-chain reaction (qPCR) was employed for quantifying the expressions of miR-509-5p and DNMT1 mRNA in the serum of PAH patients and PASMCs. MiR-509-5p mimics and inhibitors were then, respectively, transfected into PAMSCs, and CCK-8 and Transwell assays were utilized to detect PASMCs' proliferation and migration. Flow cytometry was executed for evaluating PASMCs' apoptosis. Interrelation between miR-509-5p and DNMT1 was determined utilizing bioinformatics analysis and dual-luciferase reporter assay. Western blot assay was used to detect the expression of DNMT1 or SOD2. MiR-509-5p in serum samples of patients with PAH as well as hypoxia-induced PASMCs was significantly down-regulated, whereas DNMT1 was markedly up-regulated. MiR-509-5p mimics reduces the proliferation and migration of PASMCs, but promotes the apoptosis; conversely, miR-509-5p inhibitors exerted opposite effects. DNMT1 was identified as a target gene of miR-509-5p, and overexpression of DNMT1 reversed the biological functions of miR-509-5p in regulating the phenotypes of PAMSCs. MiR-509-5p up-regulated the expression of SOD2 by down-regulating DNMT1. MiR-509-5p regulates the proliferation, migration and apoptosis of PASMCs, and restoration of miR-509-5p may be a promising strategy to treat PAH.

RAS protein activator like 2 promotes the proliferation and migration of pulmonary artery smooth muscle cell through AKT/mammalian target of Rapamycin complex 1 pathway in pulmonary hypertension.

RAS protein activator like 2 (Rasal2) exerts pro-proliferative effect in several types of cells. However, whether Rasal2 is involved in the regulation of pulmonary artery smooth muscle cell (PASMC) remains unclear. In the current study, we explored the role of Rasal2 in proliferation and migration of PASMC during the development of pulmonary arterial hypertension (PAH). We found that the protein level of Rasal2 was increased in both pulmonary arteries of chronic hypoxia-induced pulmonary hypertension (CH-PH) mice and hypoxia-challenged PASMC. Overexpression of Rasal2 caused enhanced proliferation and migration of PASMC after hypoxia exposure. Mechanistically, we found elevated phosphorylation of AKT and two downstream effectors of mammalian target of Rapamycin complex 1 (mTORC1), S6 and 4E-Binding Protein 1 (4EBP1) after Rasal2 overexpression in hypoxia-challenged PASMC. Inactivation of mTORC1 abolished Rasal2-mediated enhancement of proliferation and migration of PASMC. Furthermore, we also demonstrated that AKT might act downstream of Rasal2 to enhance the activity of mTORC1. Once AKT was inactivated by MK-2206 application, overexpression of Rasal2 failed to further increase the phosphorylation level of S6 and 4EBP1. Finally, inhibition of AKT also blocked Rasal2-induced proliferation and migration in hypoxia-challenged PASMC. In conclusion, Rasal2 promotes the proliferation and migration of PASMC during the development of PAH via AKT/mTORC1 pathway.

Pathogenic Mechanisms of Pulmonary Arterial Hypertension: Homeostasis Imbalance of Endothelium-Derived Relaxing and Contracting Factors.

Pulmonary arterial hypertension (PAH) is a progressive and fatal disease. Sustained pulmonary vasoconstriction and concentric pulmonary vascular remodeling contribute to the elevated pulmonary vascular resistance and pulmonary artery pressure in PAH. Endothelial cells regulate vascular tension by producing endothelium-derived relaxing factors (EDRFs) and endothelium-derived contracting factors (EDCFs). Homeostasis of EDRF and EDCF production has been identified as a marker of the endothelium integrity. Impaired synthesis or release of EDRFs induces persistent vascular contraction and pulmonary artery remodeling, which subsequently leads to the development and progression of PAH. In this review, the authors summarize how EDRFs and EDCFs affect pulmonary vascular homeostasis, with special attention to the recently published novel mechanisms related to endothelial dysfunction in PAH and drugs associated with EDRFs and EDCFs.

Isoflurane Inhibits Pyroptosis of Human Pulmonary Artery Smooth Muscle Cells Through the Nrf2/HO⁃1/ROS Pathway / 中国生物化学与分子生物学报

Previous studies have demonstrated that isoflurane inhale anesthesia can effectively attenuate the ischemia-reperfusion-induced pulmonary hypertension (PAH), indicating a protective effect of isoflurane on pulmonary circulation. Pulmonary artery smooth muscle cells (PASMCs) play an important role in pulmonary vascular remodeling and PAH. The abnormality of PASMC structure and function may greatly contribute to the development of PAH. This study aims to explore the effects of isoflurane on hypoxia-induced PASMC pyroptosis and the underlying mechanisms, and to find potential therapeutic target for the treatment of PAH. PASMCs were cultured at 37 ℃, 5%CO

Research progress on therapeutic strategies for improving pulmonary vascular remodeling in pulmonary hypertension / 中国药理学通报

Pulmonary hypertension(PH)is a chronic,progressive,high-mortality disease characterized by a continuous increase in pulmonary vascular pressure. All types of PH have the same characteristics,i.e.,the excessive proliferation,anti-apoptosis and inflammation of pulmonary artery endothelial cells and smooth muscle cells,which leads to progressive thickening of pulmonary small vessels,resulting in pulmonary vascular remodeling and increased pulmonary vascular resistance,ultimately leading to right ventricular hypertrophy,heart failure,and death. The drugs used to treat PH mainly include L-type calcium channel blockers,phosphodiesterase 5 inhibitors,guanosine cyclase activators,endothelin receptor antagonists,and synthetic prostacyclin and its analogues. These drugs reduce pulmonary artery pressure by relaxing pulmonary blood vessels but do not cure the patient,and their prognosis remains poor. Therefore,the development of drugs that can effectively improve or even reverse pulmonary vascular remodeling is the key to treating PH. In recent years,studies on pulmonary vascular remodeling mainly included(1)the synthesis of new small-molecule compounds;(2)the transformation of mature drugs,such as the use of drug combinations and dosage form transformation,etc.;(3)the pharmacodynamic evaluation of traditional Chinese medicines and derived compounds based on the theory of "lung distension";(4)research into monomers of traditional Chinese medicine; and(5)research into new targets.

The substitution of SERCA2 redox cysteine 674 promotes pulmonary vascular remodeling by activating IRE1α/XBP1s pathway

Pulmonary hypertension (PH) is a life-threatening disease characterized by pulmonary vascular remodeling, in which hyperproliferation of pulmonary artery smooth muscle cells (PASMCs) plays an important role. The cysteine 674 (C674) in the sarcoplasmic/endoplasmic reticulum Ca2+ ATPase 2 (SERCA2) is the critical redox regulatory cysteine to regulate SERCA2 activity. Heterozygous SERCA2 C674S knock-in mice (SKI), where one copy of C674 was substituted by serine to represent partial C674 oxidative inactivation, developed significant pulmonary vascular remodeling resembling human PH, and their right ventricular systolic pressure modestly increased with age. In PASMCs, substitution of C674 activated inositol requiring enzyme 1 alpha (IRE1α) and spliced X-box binding protein 1 (XBP1s) pathway, accelerated cell cycle and cell proliferation, which reversed by IRE1α/XBP1s pathway inhibitor 4μ8C. In addition, suppressing the IRE1α/XBP1s pathway prevented pulmonary vascular remodeling caused by substitution of C674. Similar to SERCA2a, SERCA2b is also important to restrict the proliferation of PASMCs. Our study articulates the causal effect of C674 oxidative inactivation on the development of pulmonary vascular remodeling and PH, emphasizing the importance of C674 in restricting PASMC proliferation to maintain pulmonary vascular homeostasis. Moreover, the IRE1α/XBP1s pathway and SERCA2 might be potential targets for PH therapy.

Upregulation of IRF9 Contributes to Pulmonary Artery Smooth Muscle Cell Proliferation During Pulmonary Arterial Hypertension.

Abnormal proliferation of pulmonary artery smooth muscle cells (PASMCs) is a critical pathological feature in the pathogenesis of pulmonary arterial hypertension (PAH), but the regulatory mechanisms remain largely unknown. Herein, we demonstrated that interferon regulatory factor 9 (IRF9) accelerated PASMCs proliferation by regulating Prohibitin 1 (PHB1) expression and the AKT-GSK3ß signaling pathway. Compared with control groups, the rats treated with chronic hypoxia (CH), monocrotaline (MCT) or sugen5416 combined with chronic hypoxia (SuHx), and mice challenged with CH had significantly thickened pulmonary arterioles and hyperproliferative PASMCs. More importantly, the protein level of IRF9 was found to be elevated in the thickened medial wall of the pulmonary arterioles in all of these PAH models. Notably, overexpression of IRF9 significantly promoted the proliferation of rat and human PASMCs, as evidenced by increased cell counts, EdU-positive cells and upregulated biomarkers of cell proliferation. In contrast, knockdown of IRF9 suppressed the proliferation of rat and human PASMCs. Mechanistically, IRF9 directly restrained PHB1 expression and interacted with AKT to inhibit the phosphorylation of AKT at thr308 site, which finally led to mitochondrial dysfunction and PASMC proliferation. Unsurprisingly, MK2206, a specific inhibitor of AKT, partially reversed the PASMC proliferation inhibited by IRF9 knockdown. Thus, our results suggested that elevation of IRF9 facilitates PASMC proliferation by regulating PHB1 expression and AKT signaling pathway to affect mitochondrial function during the development of PAH, which indicated that targeting IRF9 may serve as a novel strategy to delay the pathological progression of PAH.

Small-molecule inhibitor LF3 restrains the development of pulmonary hypertension through the Wnt/ß-catenin pathway.

Pulmonary hypertension (PH) associated with congenital heart disease is a progressive hemodynamic disease that can lead to increased pulmonary vascular resistance, vascular remodeling, and even right heart failure and death. LF3 is a novel inhibitor of the reporter gene activity of ß-catenin/TCF4 interaction in the Wnt/ß-catenin signal pathway. However, whether this action of LF3 can prevent PH development remains unclear. In this study, we investigated the therapeutic effect of LF3 in rat primary pulmonary artery smooth muscle cells (PASMCs) of the PH model. We found that LF3 inhibited the decrease in pulmonary artery acceleration time and ejection time by ultra-high-resolution ultrasound imaging and blocked the increase of pulmonary artery systolic pressure by using the BL420 biological function experimental system and right ventricular hypertrophy index by the electronic scales. Simultaneously, it prevented the increase of α-smooth muscle actin and fibronectin and the decrease of elastin in pulmonary arteries of rats in the PH group, as revealed by an immunohistochemical analysis. Moreover, cell proliferation and migration assays showed that LF3 significantly reduced the proliferation and migration of PASMCs. Western blotting and quantitative real-time polymerase chain reaction analyses revealed that LF3 suppressed the expression of proliferating cell nuclear antigens and Bcl-2 and increased the expression of Bax but did not alter the expressions of ß-catenin and TCF4. Taken together, LF3 can reduce the migration and proliferation of PASMCs and induce their apoptosis to prevent the development of PH. It would be worthwhile to explore the potential use of LF3 in the treatment of PH.