SOX9 promotes hypoxic pulmonary hypertension through stabilization of DPP4 in pulmonary artery smooth muscle cells.

Pulmonary hypertension (PH) is a progressive cardiopulmonary disorder characterized by pulmonary vascular remodeling (PVR), primarily due to the excessive proliferation of pulmonary artery smooth muscle cells (PASMCs). This study aimed to investigate the role and molecular mechanism of SOX9 in hypoxic PH in rats. The findings revealed that SOX9 was upregulated in the pulmonary arteries and PASMCs of hypoxia-exposed rats. SOX9 knockdown inhibited hypoxia-induced proliferation and migration of PASMCs, reduced PVR, and subsequently alleviated hypoxia-induced PH in rats, suggesting that SOX9 plays a critical role in PH. Further investigation demonstrated that SOX9 interacted with DPP4, preventing its ubiquitin degradation in hypoxia-exposed PASMCs. DPP4 knockdown inhibited hypoxia-induced PASMC proliferation and migration, and administration of the DPP4 inhibitor sitagliptin (5 mg/kg) significantly reduced PVR and alleviated hypoxia-induced PH in rats, indicating that SOX9 contributes to PH by stabilizing DPP4. The results also showed that hypoxia induced YAP1 expression and dephosphorylation, leading to YAP1 nuclear localization. YAP1 knockdown promoted the degradation of HIF-1α in hypoxia-exposed PASMCs and inhibited hypoxia-induced proliferation and migration of PASMCs. Additionally, HIF-1α, as a transcription factor, promoted SOX9 expression by binding to the SOX9 promoter in hypoxia-exposed PASMCs. In conclusion, hypoxia promotes the proliferation and migration of PASMCs through the regulation of the YAP1/HIF-1α/SOX9/DPP4 signaling pathway, leading to PH in rats. These findings suggest that SOX9 may serve as a potential prognostic marker and therapeutic target for PH.

Pulmonary artery smooth muscle cell pyroptosis promotes the proliferation of PASMCs by paracrine IL­1ß and IL­18 in monocrotaline­induced pulmonary arterial hypertensive rats.

Pulmonary arterial hypertension (PAH) is a common vascular disease, and pulmonary vascular remodeling is a pivotal pathophysiological mechanism of PAH. Major pathological changes of pulmonary arterial remodeling, including proliferation, hypertrophy and enhanced secretory activity, can occur in pulmonary artery smooth muscle cells (PASMCs). Multiple active factors and cytokines play important roles in PAH. However, the regulatory mechanisms of the active factors and cytokines in PAH remain unclear. The present study aimed to reveal the crucial role of PASMC pyroptosis in PAH and to elucidate the intrinsic mechanisms. To establish the PAH rat models, Sprague-Dawley rats were injected intraperitoneally with monocrotaline (MCT) at a dose of 60 mg/kg. The expression of proteins and interleukins were detected by western blotting and ELISA assay. The results indicated that the pyroptosis of PASMCs is significantly increased in MCT-induced PAH rats. Notably, pyroptotic PASMCs can secret IL-1ß and IL-18 to promote the proliferation of PASMCs. On this basis, inhibiting the secretion of IL-1ß and IL-18 can markedly inhibit PASMC proliferation. Collectively, the findings of the present study indicate a critical role for PASMC pyroptosis in MCT-induced PAH rats, prompting a new preventive and therapeutic strategy for PAH.

The protective mechanism of sevoflurane in pulmonary arterial hypertension via downregulation of TRAF6.

Pulmonary arterial hypertension (PAH) is an obstructive vasculopathy that, if not promptly treated, culminates in right heart failure. Therefore, pre-clinical studies are needed to support and optimize therapeutic approaches of PAH. Here, we explore a prospective function of sevoflurane in experimental PAH through regulating TRAF6. Monocrotaline (MCT)-induced PAH rats were subjected to sevoflurane inhalation and intratracheal instillation of lentivirus overexpressing TRAF6. Platelet-derived growth factor (PDGF)-treated pulmonary artery smooth muscle cells (PASMCs) were exposed to sevoflurane and genetically manipulated for TRAF6 overexpression. It was found that MCT and PDGF challenge upregulated the levels of TRAF6 in rat lung tissues and PASMCs, but sevoflurane treatment led to reduced TRAF6 expression. Sevoflurane inhalation in MCT-induced rats resulted in alleviative pulmonary vascular remodeling, mitigated right ventricular dysfunction and hypertrophy, improved mitochondrial function and dynamics, and inactivation of NF-κB pathway. In vitro studies confirmed that exposure to sevoflurane repressed PDGF-induced proliferation, migration, and phenotype switching of PASMCs, and suppressed mitochondrial dysfunction and NF-κB activation in PDGF-stimulated PASMCs. The beneficial impact of sevoflurane on pathological changes of lung and cell phenotype of PASMCs were reversed by overexpression of TRAF6. In summary, our study suggested the protective properties of sevoflurane in targeting PAH by downregulating TRAF6 expression, providing a novel avenue for the management of PAH.

Study on the Regulatory Mechanism of the PDK1-Mediated TGF-ß/Smad Signaling Pathway in Hypoxia-Induced Yak Lungs.

The aim of this study was to investigate the effects of hypoxia-induced phenotype, glucose metabolism, ROS levels, and the PDK1-mediated regulation of TGF-ß/Smad signaling in yellow cattles, yaks, and those overexpressing PDK1 PASMCs using growth curves, flow cytometry, scratch experiments, glucose and lactic acid assays, RT-qPCR, and Western blotting. The results showed that hypoxia significantly promoted proliferation, migration, antiapoptosis, ROS levels, glucose consumption, and lactate production in yellow cattle PASMCs (p < 0.05), and the cells were dedifferentiated from the contractile phenotype; conversely, hypoxia had no significant effect on yak PASMCs (p > 0.05). PDK1 overexpression significantly promoted proliferation, antiapoptosis, glucose consumption, and lactate production in yak PASMCs under normoxia and hypoxia (p < 0.05), decreased their migration levels under hypoxia (p < 0.05), and dedifferentiated the contractile phenotype of the cells. Overexpression of PDK1 in yak PASMCs is detrimental to their adaptation to hypoxic environments. Yak PASMCs adapted to the effects of hypoxia on lung tissue by downregulating the expression of genes related to the PDK1 and TGF-ß/Smad signaling pathways. Taken together, the regulation of PDK1-mediated TGF-ß/Smad signaling may be involved in the process of yaks' adaptation to the hypoxic environment of the plateau, reflecting the good adaptive ability of yaks. The present study provides basic information to further elucidate the mechanism of PDK1-mediated TGF-ß/Smad signaling induced by hypoxia in the lungs of yaks, as well as target genes for the treatment of plateau diseases in humans and animals.

Cell Death in Pulmonary Arterial Hypertension.

Pulmonary arterial hypertension (PAH) is a severe pulmonary vascular disease characterized by increased pulmonary vascular resistance because of vascular remodeling and vasoconstriction. Subsequently, PAH leads to right ventricular hypertrophy and heart failure. Cell death mechanisms play a significant role in development and tissue homeostasis, and regulate the balance between cell proliferation and differentiation. Several basic and clinical studies have demonstrated that multiple mechanisms of cell death, including pyroptosis, apoptosis, autophagy, ferroptosis, anoikis, parthanatos, and senescence, are closely linked with the pathogenesis of PAH. This review summarizes different cell death mechanisms involved in the death of pulmonary artery smooth muscle cells (PASMCs) and pulmonary artery endothelial cells (PAECs), the primary target cells in PAH. This review summarizes the role of these cell death mechanisms, associated signaling pathways, unique effector molecules, and various pro-survival or reprogramming mechanisms. The aim of this review is to summarize the currently known molecular mechanisms underlying PAH. Further investigations of the cell death mechanisms may unravel new avenues for the prevention and treatment of PAH.

Effect of the TGF-ß/BMP Signaling Pathway on the Proliferation of Yak Pulmonary Artery Smooth Muscle Cells under Hypoxic Conditions.

To survive in low-oxygen environments, yaks effectively avoid hypoxia-induced pulmonary arterial hypertension through vascular remodeling. The TGF-ß/BMP signaling pathway plays a key role in maintaining the homeostasis of pulmonary artery smooth muscle cells (PASMCs). However, little is known about the molecular regulatory mechanisms by which the TGF-ß/BMP signaling pathway contributes to the proliferation of yak PASMCs. In this study, yak PASMCs were cultured in vitro, and a hypoxia model was constructed to investigate the effect of TGFß/BMP signaling on yak PASMC proliferation. Hypoxia treatment increased the proliferation of yak PASMCs significantly. As the duration of hypoxia increased, the expression levels of TGF-ß1 and the phosphorylation levels of Smad2/3 were upregulated significantly. The BMP signaling pathway was transiently activated by hypoxia, with increases in BMPR2 expression and Smad1/5 phosphorylation, and these changes were gradually reversed with prolonged hypoxia exposure. In addition, exogenous TGF-ß1 activated the TGF-ß signaling pathway, increased the phosphorylation levels of the downstream proteins Smad2 and Smad3, and increased the proliferation and migration rates of yak PASMCs significantly. Finally, treatment with noggin (an inhibitor of BMP signaling) significantly reduced BMPR2 protein expression levels and Smad1/5 phosphorylation levels and increased yak PASMC proliferation and migration rates. In summary, these results revealed that under hypoxic conditions, the dynamic regulation of the TGF-ß/BMP signaling pathway promotes the proliferation of yak PASMCs.

Roles of LncRNAs in the Pathogenesis of Pulmonary Hypertension.

Pulmonary hypertension (PH) is a persistently progressive, incurable, multifactorial associated fatal pulmonary vascular disease characterized by pulmonary vascular remodeling. Long noncoding RNAs (lncRNAs) are involved in regulating pathological processes such as pulmonary vasoconstriction, thickening, remodeling, and inflammatory cell infiltration in PH by acting on different cell types. Because of their differential expression in PH patients, as demonstrated by the observation that some lncRNAs are significantly upregulated while others are significantly downregulated in PH patients, lncRNAs are potentially useful biomarkers for assessing disease progression and diagnosis or prognosis in PH patients. This article provides an overview of the different mechanisms by which lncRNAs are involved in the pathogenesis of PH.

Luteolin ameliorates hypoxic pulmonary vascular remodeling in rat via upregulating KV1.5 of pulmonary artery smooth muscle cells.

BACKGROUND: Hypoxic pulmonary vascular remodeling (HPVR) is a key pathological feature of hypoxic pulmonary hypertension (HPH). Oxygen-sensitive potassium (K+) channels in pulmonary artery smooth muscle cells (PASMCs) play a crucial role in HPVR. Luteolin (Lut) is a plant-derived flavonoid compound with variety of pharmacological actions. Our previous study found Lut alleviated HPVR in HPH rat. PURPOSE: To elucidate the mechanism by which Lut mitigated HPVR, focusing on oxygen-sensitive voltage-dependent potassium channel 1.5 (Kv1.5). METHODS: HPH rat model was established using hypobaric chamber to simulate 5000 m altitude. Isolated perfused/ventilated rat lung, isolated pulmonary arteriole ring was utilized to investigate the impact of Lut on K+ channels activity. Kv1.5 level in lung tissue and pulmonary arteriole of HPH rat was assessed. CyclinD1, CDK4, PCNA, Bax, Bcl-2, cleaved caspase-3 levels in lung tissue of HPH rat were tested. The effect of Lut on Kv1.5, cytoplasmic free calcium concentration ([Ca2+]cyt), CyclinD1, CDK4, PCNA, Bax/Bcl-2 was examined in PASMCs under hypoxia, with DPO-1 as a Kv1.5 specific inhibitor. The binding affinity between Lut and Kv1.5 in PASMCs was detected by drug affinity responsive target stability (DARTS). The overexpression of KCNA5 gene (encoding Kv1.5) in HEK293T cells was utilized to confirm the interaction between Lut and Kv1.5. Furthermore, the impact of Lut on mitochondrial structure, SOD, GSH, GSH-Px, MDA and HIF-1α levels were evaluated in lung tissue of HPH rat and PASMCs under hypoxia. RESULTS: Lut dilated pulmonary artery by directly activating Kv and Ca2+-activated K+ channels (KCa) in smooth muscle. Kv1.5 level in lung tissue and pulmonary arteriole of HPH rat was upregulated by Lut. Lut downregulated CyclinD1, CDK4, PCNA while upregulating Bax/Bcl-2/caspase-3 axis in lung tissue of HPH rat. Lut decreased [Ca2+]cyt, reduced CDK4, CyclinD1, PCNA, increased Bax/Bcl-2 ratio, in PASMCs under hypoxia, by upregulating Kv1.5. The binding affinity and the interaction between Lut and Kv1.5 was verified in PASMCs and in HEK293T cells. Lut also decreased [Ca2+]cyt and inhibited proliferation via targeting Kv1.5 of HEK293T cells under hypoxia. Furthermore, Lut protected mitochondrial structure, increased SOD, GSH, GSH-Px, decreased MDA, in lung tissue of HPH rat. Lut downregulated HIF-1α level in both lung tissue of HPH rat and PASMCs under hypoxia. CONCLUSION: Lut alleviated HPVR by promoting vasodilation of pulmonary artery, reducing cellular proliferation, and inducing apoptosis through upregulating of Kv1.5 in PASMCs.

miR-221-3p is upregulated in acute pulmonary embolism complicated with pulmonary hypertension and promotes pulmonary arterial smooth muscle cells proliferation and migration by inhibiting PTEN.

Pulmonary arterial smooth muscle cells (PASMCs) functions are associated with the pathogenesis of pulmonary hypertension (PH) which is a life-threatening complication of acute pulmonary embolism (APE). This study sought to explore the expression pattern of microRNA (miR)-221-3p in APE-PH patients and its role in PASMCs proliferation and migration. The clinical data and venous blood of APE-PH patients were collected. The expression levels of miR-221-3p and phosphatase and tensin homolog (PTEN) in serum were determined, followed by receiver operator characteristic curve analysis of miR-221-3p diagnostic efficacy. PASMCs were transfected with miR-221-3p mimics and PTEN-overexpressed vector, followed by assessment of cell viability, proliferation, and migration through cell counting kit-8, 5-ethynyl-2'-deoxyuridine, Transwell, and wound healing assays. The binding between miR-221-3p and PTEN 3'UTR region was testified by the dual-luciferase assay. miR-221 was upregulated in the serum of APE-PH patients and presented with good diagnostic efficacy with 1.155 cutoff value, 66.25% sensitivity, and 67.50% specificity. miR-221 was negatively correlated with PTEN in APE-PH patients. miR-221 overexpression facilitated PASMCs proliferation and migration in vitro. miR-221-3p bound to PTEN 3'UTR region to decrease PTEN protein levels. PTEN overexpression abolished the promotive role of miR-221-3p in PASMCs. Overall, miR-221-3p targeted PTEN to facilitate PASMC proliferation and migration.

Metallothionein 3 Potentiates Pulmonary Artery Smooth Muscle Cell Proliferation by Promoting Zinc-MTF1-ATG5 Axis-mediated Autophagosome Formation.

Abnormal proliferation of pulmonary artery smooth muscle cells (PASMCs) is one of the critical pathological mechanisms of pulmonary hypertension (PH), and therefore is gradually being adopted as an important direction for the treatment of PH. Metallothioneins (MTs) have been reported to be associated with PH, but the underlying mechanisms are not fully understood. Here, we demonstrated that the expression level of metallothionein 3 (MT3) was significantly increased in pulmonary arterioles from PH patients and chronic hypoxia-induced rat and mouse PH models, as well as in hypoxia-treated human PASMCs. Knockdown of MT3 significantly inhibited the proliferation of human PASMCs by arresting the cell cycle in the G1 phase, while overexpression of MT3 had the opposite effect. Mechanistically, we found that MT3 increased the intracellular zinc (Zn2+) concentration to enhance the transcriptional activity of metal-regulated transcription factor 1 (MTF1), which promoted the expression of autophagy-related gene 5 (ATG5), facilitating autophagosome formation. More importantly, MT3-induced autophagy and proliferation of human PASMCs were largely prevented by knockdown of MTF1 and ATG5. Therefore, in this study, we identified MT3-Zinc-MTF1-ATG5 as a novel pathway that affects PASMC proliferation by regulating autophagosome formation, suggesting that MT3 may be a novel target for the treatment of PH.

Fluorinated perhexiline derivative attenuates vascular proliferation in pulmonary arterial hypertension smooth muscle cells.

Increased proliferation and reduced apoptosis of pulmonary artery smooth muscle cells (PASMCs) is recognised as a universal hallmark of pulmonary arterial hypertension (PAH), in part related to the association with reduced pyruvate dehydrogenase (PDH) activity, resulting in decreased oxidative phosphorylation of glucose and increased aerobic glycolysis (Warburg effect). Perhexiline is a well-recognised carnitine palmitoyltransferase-1 (CPT1) inhibitor used in cardiac diseases, which reciprocally increases PDH activity, but is associated with variable pharmacokinetics related to polymorphic variation of the cytochrome P450-2D6 (CYP2D6) enzyme, resulting in the risk of neuro and hepatotoxicity in 'slow metabolisers' unless blood levels are monitored and dose adjusted. We have previously reported that a novel perhexiline fluorinated derivative (FPER-1) has the same therapeutic profile as perhexiline but is not metabolised by CYP2D6, resulting in more predictable pharmacokinetics than the parent drug. We sought to investigate the effects of perhexiline and FPER-1 on PDH flux in PASMCs from patients with PAH. We first confirmed that PAH PASMCs exhibited increased cell proliferation, enhanced phosphorylation of AKTSer473, ERK 1/2Thr202/Tyr204 and PDH-E1αSer293, indicating a Warburg effect when compared to healthy PASMCs. Pre-treatment with perhexiline or FPER-1 significantly attenuated PAH PASMC proliferation in a concentration-dependent manner and suppressed the activation of the AKTSer473 but had no effect on the ERK pathway. Perhexiline and FPER-1 markedly activated PDH (seen as dephosphorylation of PDH-E1αSer293), reduced glycolysis, and upregulated mitochondrial respiration in these PAH PASMCs as detected by Seahorse analysis. However, both perhexiline and FPER-1 did not induce apoptosis as measured by caspase 3/7 activity. We show for the first time that both perhexiline and FPER-1 may represent therapeutic agents for reducing cell proliferation in human PAH PASMCs, by reversing Warburg physiology.

Mesenchymal stem cell-derived exosomes ameliorate hypoxic pulmonary hypertension by inhibiting the Hsp90aa1/ERK/pERK pathway.

Hypoxic pulmonary hypertension (HPH) is a serious and life-threatening chronic cardiopulmonary disease characterized by progressive elevation of pulmonary artery pressure and pulmonary vascular remodeling. Mesenchymal stem cell- derived exosomes (MSC-Exos) can relieve HPH by reversing pulmonary vascular remodeling. The HPH model was established in healthy male Sprague-Dawley (SD) rats aged 6 to 8 weeks. The rats were placed in a room with oxygen concentration of (10 ± 1) % for 8 hours a day over 28 days, were then injected intravenously with MSC-Exos (100 ug protein/kg) or equal-volume phosphate buffer saline (PBS) once a day over 1 week. Right ventricular systolic pressure (RVSP), right ventricular hypertrophy index (RVHI) and pulmonary vascular remodeling were observed after anesthesia. In addition, platelet-derived growth factor BB (PDGF-BB) was used to stimulate rat pulmonary artery smooth muscle cells (PASMCs) to construct HPH pathological cell models. The results showed that MSC-Exos could not only reduce the elevation of RVSP, right ventricular hypertrophy and the degree of pulmonary vascular remodeling in HPH rats, but also reduce the proliferation, migration and apoptosis resistance of PASMCs. Finally, GSE53408 and GSE113439 datasets were analyzed and showed that the expression of Hsp90aa1 and pERK/ERK were significantly increased in HPH, also could be inhibited by MSC-Exos. Meanwhile, inhibition of Hsp90aa1 also reduced PASMCs migration and pERK/ERK protein level. In conclusion, MSC-Exos alleviated HPH by suppressing PASMCs proliferation, migration and apoptosis resistance through inhibiting the Hsp90aa1/ERK/pERK pathway.

CXCR7 promotes pulmonary vascular remodeling via targeting p38/MMP2 pathway in pulmonary arterial hypertension.

Background: A hallmark feature of pulmonary arterial hypertension (PAH) is the excessive proliferation of pulmonary artery smooth muscle cells (PASMCs) in the pulmonary arteries. The exact role of C-X-C motif chemokine ligand 12 (CXCL12)/chemokine receptor type 7 (CXCR7) in the PASMCs remains unknown. This study was conducted to investigate CXCR7's role in p38/MMP2 pathway and its effect on PASMCs. Methods: In this study, we examined the expression profile of CXCL12/CXCR7 in both hypoxic rats and PASMCs. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was used to measure the level of proliferation in PASMCs. Enzyme-linked immunosorbent assay (ELISA) and western blotting assays were applied to investigate the protein expression of the related molecules. Results: We found that a high level of CXCR7 was correlated with remodeled pulmonary arterioles in hypoxic rats. Moreover, CXCR7 protein levels were significantly increased by the induction of CXCL12, indicating that the CXCL12-CXCR7 axis participates in PAH. During hypoxia-PAH, CXCR7 inhibition reduces right ventricular systolic pressure (RVSP), the Fulton index, and pulmonary arteriosclerosis remodeling. Further study indicated inhibition CXCR7 reduced PASMCs by downregulating MMP2, via p38 MAPK pathway. It was additionally found that CXCL12/CXCR7 stimulated the phosphorylation of the p38 MAPK pathway, which was a contributing factor to the decrease in MMP2 expression following preconditioning with SB203580, which inhibited p38 MAPK. Conclusions: In summary, these findings suggest that CXCL12/CXCR7 plays a critical role in PAH, the therapy of which can be developed further by targeting its potential targets.

Effect of IL-17 on pulmonary artery smooth muscle cells and connective tissue disease-associated pulmonary arterial hypertension.

OBJECTIVE: To explore the role of interleukin (IL)-17 in connective tissue disease-associated pulmonary arterial hypertension (CTD-PAH) and to investigate its possible mechanism on pulmonary artery smooth muscle cells (PASMCs). METHODS: Enzyme-linked immunosorbent assay (ELISA) were used to compare levels of serum IL-17 in patients with CTD-PAH and healthy controls (HCs). After treatment for 3 months, the serum IL-17 levels were tested in CTD-PAH. ELISA and immunohistochemistry were used to compare levels of serum IL-17 and numbers of pulmonary artery IL-17+ cells, respectively, in a rat model of monocrotaline-induced PAH and untreated rats. Proliferation, migration, and inflammatory factors expression of PASMCs were assessed after stimulation with different concentrations of IL-17 for various time periods. Proteins in the mitogen-activated protein kinase (MAPK) pathway were examined by western blot. RESULTS: Levels of IL-17 were upregulated in patients with CTD-PAH compared to HCs. After 3 months of treatment, serum IL-17 levels were downregulated with pulmonary artery pressure amelioration. Moreover, serum IL-17 levels and numbers of IL-17+ cells infiltrating lung arterioles were increased in PAH model rats. IL-17 could dose- and time-dependently promote proliferation and migration of PASMCs as well as time-dependently induce IL-6 and intercellular cell adhesion molecule-1 (ICAM-1) expression. The levels of MKK6 increased after IL-17 treatment. Inhibition of MAPK decreased proliferation of PASMCs. CONCLUSION: Levels of IL-17 may increase in CTD-PAH, and IL-17 promotes proliferation, migration, and secretion of IL-6 and ICAM in PASMCs, respectively, which likely involves the p-38 MAPK pathway.

Neutrophil extracellular traps promote proliferation of pulmonary smooth muscle cells mediated by CCDC25 in pulmonary arterial hypertension.

BACKGROUND: Previous studies have indicated that neutrophil extracellular traps (NETs) play a pivotal role in pathogenesis of pulmonary arterial hypertension (PAH). However, the specific mechanism underlying the impact of NETs on pulmonary artery smooth muscle cells (PASMCs) has not been determined. The objective of this study was to elucidate underlying mechanisms through which NETs contribute to progression of PAH. METHODS: Bioinformatics analysis was employed in this study to screen for potential molecules and mechanisms associated with occurrence and development of PAH. These findings were subsequently validated in human samples, coiled-coil domain containing 25 (CCDC25) knockdown PASMCs, as well as monocrotaline-induced PAH rat model. RESULTS: NETs promoted proliferation of PASMCs, thereby facilitating pathogenesis of PAH. This phenomenon was mediated by the activation of transmembrane receptor CCDC25 on PASMCs, which subsequently activated ILK/ß-parvin/RAC1 pathway. Consequently, cytoskeletal remodeling and phenotypic transformation occur in PASMCs. Furthermore, the level of NETs could serve as an indicator of PAH severity and as potential therapeutic target for alleviating PAH. CONCLUSION: This study elucidated the involvement of NETs in pathogenesis of PAH through their influence on the function of PASMCs, thereby highlighting their potential as promising targets for the evaluation and treatment of PAH.

The Xanthine Derivative KMUP-1 Inhibits Hypoxia-Induced TRPC1 Expression and Store-Operated Ca2+ Entry in Pulmonary Arterial Smooth Muscle Cells.

Exposure to hypoxia results in the development of pulmonary arterial hypertension (PAH). An increase in the intracellular Ca2+ concentration ([Ca2+]i) in pulmonary artery smooth muscle cells (PASMCs) is a major trigger for pulmonary vasoconstriction and proliferation. This study investigated the mechanism by which KMUP-1, a xanthine derivative with phosphodiesterase inhibitory activity, inhibits hypoxia-induced canonical transient receptor potential channel 1 (TRPC1) protein overexpression and regulates [Ca2+]i through store-operated calcium channels (SOCs). Ex vivo PASMCs were cultured from Sprague-Dawley rats in a modular incubator chamber under 1% O2/5% CO2 for 24 h to elucidate TRPC1 overexpression and observe the Ca2+ release and entry. KMUP-1 (1 µM) inhibited hypoxia-induced TRPC family protein encoded for SOC overexpression, particularly TRPC1. KMUP-1 inhibition of TRPC1 protein was restored by the protein kinase G (PKG) inhibitor KT5823 (1 µM) and the protein kinase A (PKA) inhibitor KT5720 (1 µM). KMUP-1 attenuated protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA, 1 µM)-upregulated TRPC1. We suggest that the effects of KMUP-1 on TRPC1 might involve activating the cyclic guanosine monophosphate (cGMP)/PKG and cyclic adenosine monophosphate (cAMP)/PKA pathways and inhibiting the PKC pathway. We also used Fura 2-acetoxymethyl ester (Fura 2-AM, 5 µM) to measure the stored calcium release from the sarcoplasmic reticulum (SR) and calcium entry through SOCs in hypoxic PASMCs under treatment with thapsigargin (1 µM) and nifedipine (5 µM). In hypoxic conditions, store-operated calcium entry (SOCE) activity was enhanced in PASMCs, and KMUP-1 diminished this activity. In conclusion, KMUP-1 inhibited the expression of TRPC1 protein and the activity of SOC-mediated Ca2+ entry upon SR Ca2+ depletion in hypoxic PASMCs.

Pirfenidone and nintedanib attenuates pulmonary artery endothelial and smooth muscle cells transformations induced by IL-11.

Idiopathic pulmonary fibrosis (IPF) associated to pulmonary hypertension (PH) portends a poor prognosis, characterized by lung parenchyma fibrosis and pulmonary artery remodeling. Serum and parenchyma levels of Interleukin 11 (IL-11) are elevated in IPF-PH patients and contributes to pulmonary artery remodeling and PH. However, the effect of current approved therapies against IPF in pulmonary artery remodeling induced by IL-11 is unknown. The aim of this study is to analyze the effects of nintedanib and pirfenidone on pulmonary artery endothelial and smooth muscle cell remodeling induced by IL-11 in vitro. Our results show that nintedanib (NTD) and pirfenidone (PFD) ameliorates endothelial to mesenchymal transition (EnMT), pulmonary artery smooth muscle cell to myofibroblast-like transformation and pulmonary remodeling in precision lung cut slices. This study provided also evidence of the inhibitory effect of PFD and NTD on IL-11-induced endothelial and muscle cells proliferation and senescence. The inhibitory effect of these drugs on monocyte arrest and angiogenesis was also studied. Finally, we observed that IL-11 induced canonical signal transducer and activator of transcription 3 (STAT3) and non-canonical mitogen-activated protein kinase 1/2 (ERK1/2) phosphorylation, but, PFD and NTD only inhibited ERK1/2 phosphorylation. Therefore, this study provided evidence of the inhibitory effect of NTD and PFD on markers of pulmonary artery remodeling induced by IL-11.

Non-canonical IKB kinases regulate YAP/TAZ and pathological vascular remodeling behaviors in pulmonary artery smooth muscle cells.

Pulmonary arterial hypertension (PAH) causes pulmonary vascular remodeling, increasing pulmonary vascular resistance (PVR) and leading to right heart failure and death. Matrix stiffening early in the disease promotes remodeling in pulmonary artery smooth muscle cells (PASMCs), contributing to PAH pathogenesis. Our research identified YAP and TAZ as key drivers of the mechanobiological feedback loop in PASMCs, suggesting targeting them could mitigate remodeling. However, YAP/TAZ are ubiquitously expressed and carry out diverse functions, necessitating a cell-specific approach. Our previous work demonstrated that targeting non-canonical IKB kinase TBK1 reduced YAP/TAZ activation in human lung fibroblasts. Here, we investigate non-canonical IKB kinases TBK1 and IKKε in pulmonary hypertension (PH) and their potential to modulate PASMC pathogenic remodeling by regulating YAP/TAZ. We show that TBK1 and IKKε are activated in PASMCs in a rat PH model. Inflammatory cytokines, elevated in PAH, activate these kinases in human PASMCs. Inhibiting TBK1/IKKε expression/activity significantly reduces PAH-associated PASMC remodeling, with longer-lasting effects on YAP/TAZ than treprostinil, an approved PAH therapy. These results show that non-canonical IKB kinases regulate YAP/TAZ in PASMCs and may offer a novel approach for reducing vascular remodeling in PAH.

RNA m6A methylation and regulatory proteins in pulmonary arterial hypertension.

m6A (N6­methyladenosine) is the most common and abundant apparent modification in mRNA of eukaryotes. The modification of m6A is regulated dynamically and reversibly by methyltransferase (writer), demethylase (eraser), and binding protein (reader). It plays a significant role in various processes of mRNA metabolism, including regulation of transcription, maturation, translation, degradation, and stability. Pulmonary arterial hypertension (PAH) is a malignant cardiopulmonary vascular disease characterized by abnormal proliferation of pulmonary artery smooth muscle cells. Despite the existence of several effective and targeted therapies, there is currently no cure for PAH and the prognosis remains poor. Recent studies have highlighted the crucial role of m6A modification in cardiovascular diseases. Investigating the role of RNA m6A methylation in PAH could provide valuable insights for drug development. This review aims to explore the mechanism and function of m6A in the pathogenesis of PAH and discuss the potential targeting of RNA m6A methylation modification as a treatment for PAH.

Transcriptome-Wide N6-Methyladenosine Alternations in Pulmonary Arteries of Monocrotaline-Induced Pulmonary Arterial Hypertension in Rats and Novel Therapeutic Targets.

N6-methyladenosine (m6A) is a post-transcriptional epigenetic change with transcriptional stability and functionality regulated by specific m6A-modifying enzymes. However, the significance of genes modified by m6A and enzymes specific to m6A regulation in the context of pulmonary arterial hypertension (PAH) remains largely unexplored. MeRIP-seq and RNA-seq were applied to explore variances in m6A and RNA expression within the pulmonary artery tissues of control and monocrotaline-induced PAH rats. Functional enrichments were analyzed using the Gene Ontology and Kyoto Encyclopedia of Genes and Genomes. To screen candidate m6A-related genes, the STRING and Metascape databases were used to construct a protein-protein interaction network followed by a real-time PCR validation of their expression. The expression level of an m6A regulator was further investigated using immunohistochemical staining, immunofluorescence, and Western blot techniques. Additionally, proliferation assays were conducted on primary rat pulmonary artery smooth muscle cells (PASMCs). We identified forty-two differentially expressed genes that exhibited either hypermethylated or hypomethylated m6A. These genes are predominantly related to the extracellular matrix structure, MAPK, and PI3K/AKT pathways. A candidate gene, centromere protein F (CENPF), was detected with increased expression in the PAH group. Additionally, we first identified an m6A reader, leucine rich pentatricopeptide repeat containing (LRPPRC), which was downregulated in the PAH rat model. The in vitro downregulation of Lrpprc mediated by siRNA resulted in the enhanced proliferation and elevated expression of Cenpf mRNA in primary rat PASMCs. Our study revealed a modified transcriptome-wide m6A landscape and associated regulatory mechanisms in the pulmonary arteries of PAH rats, potentially offering a novel target for therapeutic strategies in the future.