4-hydroxysesamin protects rat with right ventricular failure due to pulmonary hypertension by inhibiting JNK/p38 MAPK signaling.

The specific mechanism of 4-hydroxysesamin (4-HS), a modification of Sesamin, on right ventricular failure due to pulmonary hypertension (PH) is ominous. By creating a rat model of PH in vivo and a model of pulmonary artery smooth muscle cell (PASMC) hypoxia and inflammation in vitro, the current work aimed to investigate in depth the molecular mechanism of the protective effect of 4-HS. In an in vitro model of hypoxia PASMC, changes in cell proliferation and inflammatory factors were detected after treatment with 4-HS, followed by changes in the JNK/p38 MAPK signaling pathway as detected by Western blot signaling pathway. The findings demonstrated that 4-HS was able to minimize PASMC cell death, block the JNK/p38 MAPK signaling pathway, and resist the promoting effect of hypoxia on PASMC cell proliferation. Following that, we found that 4-HS could both mitigate the right ventricular damage brought on by MCT and had a protective impact on rats Monocrotaline (MCT)-induced PH in in vivo investigations. The key finding of this study is that 4-HS may protect against PH by inhibiting the JNK/p38 MAPK signaling pathway.

N6-methyladenosine modification of KLF2 may contribute to endothelial-to-mesenchymal transition in pulmonary hypertension.

BACKGROUND: Pulmonary hypertension (PH) is a progressive disease characterized by pulmonary vascular remodeling. Increasing evidence indicates that endothelial-to-mesenchymal transition (EndMT) in pulmonary artery endothelial cells (PAECs) is a pivotal trigger initiating this remodeling. However, the regulatory mechanisms underlying EndMT in PH are still not fully understood. METHODS: Cytokine-induced hPAECs were assessed using RNA methylation quantification, qRT-PCR, and western blotting to determine the involvement of N6-methyladenosine (m6A) methylation in EndMT. Lentivirus-mediated silencing, overexpression, tube formation, and wound healing assays were utilized to investigate the function of METTL3 in EndMT. Endothelial-specific gene knockout, hemodynamic measurement, and immunostaining were performed to explore the roles of METTL3 in pulmonary vascular remodeling and PH. RNA-seq, RNA Immunoprecipitation-based qPCR, mRNA stability assay, m6A mutation, and dual-luciferase assays were employed to elucidate the mechanisms of RNA methylation in EndMT. RESULTS: The global levels of m6A and METTL3 expression were found to decrease in TNF-α- and TGF-ß1-induced EndMT in human PAECs (hPAECs). METTL3 inhibition led to reduced endothelial markers (CD31 and VE-cadherin) and increased mesenchymal markers (SM22 and N-cadherin) as well as EndMT-related transcription factors (Snail, Zeb1, Zeb2, and Slug). The endothelial-specific knockout of Mettl3 promoted EndMT and exacerbated pulmonary vascular remodeling and hypoxia-induced PH (HPH) in mice. Mechanistically, METTL3-mediated m6A modification of kruppel-like factor 2 (KLF2) plays a crucial role in the EndMT process. KLF2 overexpression increased CD31 and VE-cadherin levels while decreasing SM22, N-cadherin, and EndMT-related transcription factors, thereby mitigating EndMT in PH. Mutations in the m6A site of KLF2 mRNA compromise KLF2 expression, subsequently diminishing its protective effect against EndMT. Furthermore, KLF2 modulates SM22 expression through direct binding to its promoter. CONCLUSIONS: Our findings unveil a novel METTL3/KLF2 pathway critical for protecting hPAECs against EndMT, highlighting a promising avenue for therapeutic investigation in PH.

A correlated light and electron microscopy approach to study the subcellular localization of phosphorylated vimentin in human lung tissue.

Correlative microscopy is an important approach for bridging the resolution gap between fluorescence light and electron microscopy. Here, we describe a fast and simple method for correlative immunofluorescence and immunogold labeling on the same section to elucidate the localization of phosphorylated vimentin (P-Vim), a robust feature of pulmonary vascular remodeling in cells of human lung small arteries. The lung is a complex, soft and difficult tissue to prepare for transmission electron microscopy (TEM). Detailing the molecular composition of small pulmonary arteries (<500µm) would be of great significance for research and diagnostics. Using the classical methods of immunochemistry (either hydrophilic resin or thin cryosections), is difficult to locate small arteries for analysis by TEM. To address this problem and to observe the same structures by both light and electron microscopy, correlative microscopy is a reliable approach. Immunofluorescence enables us to know the distribution of P-Vim in cells but does not provide ultrastructural detail on its localization. Labeled structures selected by fluorescence microscope can be identified and further analyzed by TEM at high resolution. With our method, the morphology of the arteries is well preserved, enabling the localization of P-Vim inside pulmonary endothelial cells. By applying this approach, fluorescent signals can be directly correlated to the corresponding subcellular structures in areas of interest.

GPS2 ameliorates cigarette smoking-induced pulmonary vascular remodeling by modulating the ras-Raf-ERK axis.

BACKGROUND: Mitogen-activated protein kinase (MAPK)signaling-mediated smoking-associated pulmonary vascular remodeling (PVR) plays an important role in the pathogenesis of group 3 pulmonary hypertension (PH). And G protein pathway suppressor 2 (GPS2) could suppress G-protein signaling such as Ras and MAPK, but its role in cigarette smoking -induced PVR (CS-PVR) is unclear. METHODS: An in vivo model of smoke-exposed rats was constructed to assess the role of GPS2 in smoking-induced PH and PVR. In vitro, the effects of GPS2 overexpression and silencing on the function of human pulmonary arterial smooth cells (HPASMCs) and the underlying mechanisms were explored. RESULTS: GPS2 expression was downregulated in rat pulmonary arteries (PAs) and HPASMCs after CS exposure. More importantly, CS-exposed rats with GPS2 overexpression had lower right ventricular systolic pressure (RVSP), right ventricular hypertrophy index (RVHI), and wall thickness (WT%) than those without. And enhanced proliferation and migration of HPASMCs induced by cigarette smoking extract (CSE) can be evidently inhibited by overexpressed GPS2. Besides, GPS2siRNA significantly enhanced the proliferation, and migration of HPASMCs as well as activated Ras and Raf/ERK signaling, while these effects were inhibited by zoledronic acid (ZOL). In addition, GPS2 promoter methylation level in rat PAs and HPASMCs was increased after CS exposure, and 5-aza-2-deoxycytidine (5-aza) inhibited CSE-induced GPS2 hypermethylation and downregulation in vitro. CONCLUSIONS: GPS2 overexpression could improve the CS-PVR, suggesting that GPS2 might serve as a novel therapeutic target for PH-COPD in the future.

CircST6GAL1 knockdown alleviates pulmonary arterial hypertension by regulating miR-509-5p/multiple C2 and transmembrane domain containing 2 axis.

BACKGROUND: Hypertension is a main contributing factor of cardiovascular diseases; deregulated circular RNAs are involved in the pathogenesis of pulmonary arterial hypertension (PAH). Herein, we evaluated the function and mechanism of circST6GAL1 in PAH process. METHODS: Human pulmonary artery smooth muscle cells (HPASMCs) were cultured in hypoxic environment for functional analysis. The cell counting kit-8, 5-ethynyl-2'-deoxyuridine, wound healing, and flow cytometry assays were used to investigate cell proliferation, migration, and apoptosis. qRT-PCR and Western blotting analyses were used for level measurement of genes and proteins. The binding between miR-509-5p and circST6GAL1 or multiple C2 and transmembrane domain containing 2 (MCTP2) was analyzed by dual-luciferase reporter, RNA immunoprecipitation, and pull-down assays. The monocrotaline (MCT)-induced PAH mouse models were established for in vivo assay. RESULTS: CircST6GAL1 was highly expressed in PAH patients and hypoxia-induced HPASMCs. Functionally, circST6GAL1 deficiency reversed hypoxia-induced proliferation and migration, as well as apoptosis arrest in HPASMCs. Mechanistically, circST6GAL1 directly targeted miR-509-5p, and MCTP2 was a target of miR-509-5p. Rescue assays showed that the regulatory effects of circST6GAL1 deficiency on hypoxia-induced HPASMCs were abolished. Moreover, forced expression of miR-509-5p suppressed HPASMC proliferation and migration and induced cell apoptosis under hypoxia stimulation, while these effects were abolished by MCTP2 overexpression. Moreover, circST6GAL1 silencing improved MCT-induced pulmonary vascular remodeling and PAH. CONCLUSION: CircST6GAL1 deficiency reversed hypoxia-induced proliferation and migration, as well as apoptosis arrest in HPASMCs, and alleviated pulmonary vascular remodeling in MCT-induced PAH mouse models through the miR-509-5p/MCTP2 axis, indicating a potential therapeutic target for PAH.

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.

OPN silencing reduces hypoxic pulmonary hypertension via PI3K-AKT-induced protective autophagy.

Hypoxic pulmonary hypertension (HPH) is a pulmonary vascular disease primarily characterized by progressive pulmonary vascular remodeling in a hypoxic environment, posing a significant clinical challenge. Leveraging data from the Gene Expression Omnibus (GEO) and human autophagy-specific databases, osteopontin (OPN) emerged as a differentially expressed gene, upregulated in cardiovascular diseases such as pulmonary arterial hypertension (PAH). Despite this association, the precise mechanism by which OPN regulates autophagy in HPH remains unclear, prompting the focus of this study. Through biosignature analysis, we observed significant alterations in the PI3K-AKT signaling pathway in PAH-associated autophagy. Subsequently, we utilized an animal model of OPNfl/fl-TAGLN-Cre mice and PASMCs with OPN shRNA to validate these findings. Our results revealed right ventricular hypertrophy and elevated mean pulmonary arterial pressure (mPAP) in hypoxic pulmonary hypertension model mice. Notably, these effects were attenuated in conditionally deleted OPN-knockout mice or OPN-silenced hypoxic PASMCs. Furthermore, hypoxic PASMCs with OPN shRNA exhibited increased autophagy compared to those in hypoxia alone. Consistent findings from in vivo and in vitro experiments indicated that OPN inhibition during hypoxia reduced PI3K expression while increasing LC3B and Beclin1 expression. Similarly, PASMCs exposed to hypoxia and PI3K inhibitors had higher expression levels of LC3B and Beclin1 and suppressed AKT expression. Based on these findings, our study suggests that OPNfl/fl-TAGLN-Cre effectively alleviates HPH, potentially through OPN-mediated inhibition of autophagy, thereby promoting PASMCs proliferation via the PI3K-AKT signaling pathway. Consequently, OPN emerges as a novel therapeutic target for HPH.

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.

Endothelial HIFα/PDGF-B to smooth muscle Beclin1 signaling sustains pathological muscularization in pulmonary hypertension.

Mechanisms underlying maintenance of pathological vascular hypermuscularization are poorly delineated. Herein, we investigated retention of smooth muscle cells (SMCs) coating normally unmuscularized distal pulmonary arterioles in pulmonary hypertension (PH) mediated by chronic hypoxia with or without Sugen 5416, and reversal of this pathology. With hypoxia in mice or culture, lung endothelial cells (ECs) upregulated hypoxia-inducible factor 1α (HIF1-α) and HIF2-α, which induce platelet-derived growth factor B (PDGF-B), and these factors were reduced to normoxic levels with re-normoxia. Re-normoxia reversed hypoxia-induced pulmonary vascular remodeling, but with EC HIFα overexpression during re-normoxia, pathological changes persisted. Conversely, after establishment of distal muscularization and PH, EC-specific deletion of Hif1a, Hif2a, or Pdgfb induced reversal. In human idiopathic pulmonary artery hypertension, HIF1-α, HIF2-α, PDGF-B, and autophagy-mediating gene products, including Beclin1, were upregulated in pulmonary artery SMCs and/or lung lysates. Furthermore, in mice, hypoxia-induced EC-derived PDGF-B upregulated Beclin1 in distal arteriole SMCs, and after distal muscularization was established, re-normoxia, EC Pdgfb deletion, or treatment with STI571 (which inhibits PDGF receptors) downregulated SMC Beclin1 and other autophagy products. Finally, SMC-specific Becn1 deletion induced apoptosis, reversing distal muscularization and PH mediated by hypoxia with or without Sugen 5416. Thus, chronic hypoxia induction of the HIFα/PDGF-B axis in ECs is required for non-cell-autonomous Beclin1-mediated survival of pathological distal arteriole SMCs.

Canagliflozin inhibits PASMCs proliferation via regulating SGLT1/AMPK signaling and attenuates artery remodeling in MCT-induced pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) was a devastating disease characterized by artery remodeling, ultimately resulting in right heart failure. The aim of this study was to investigate the effects of canagliflozin (CANA), a sodium-glucose cotransporter 2 inhibitor (SGLT2i) with mild SGLT1 inhibitory effects, on rats with PAH, as well as its direct impact on pulmonary arterial smooth muscle cells (PASMCs). PAH rats were induced by injection of monocrotaline (MCT) (40 mg/kg), followed by four weeks of treatment with CANA (30 mg/kg/day) or saline alone. Pulmonary artery and right ventricular (RV) remodeling and dysfunction in PAH were alleviated with CANA, as assessed by echocardiography. Hemodynamic parameters and structural of pulmonary arteriole, including vascular wall thickness and wall area, were reduced by CANA. RV hypertrophy index, cardiomyocyte hypertrophy, and fibrosis were decreased with CANA treatment. PASMCs proliferation was inhibited by CANA under stimulation by platelet-derived growth factor (PDGF)-BB or hypoxia. Activation of AMP kinase (AMPK) was induced by CANA treatment in cultured PASMCs in a time- and concentration-dependent manner. These effects of CANA were attenuated when treatment with compound C, an AMPK inhibitor. Abundant expression of SGLT1 was observed in PASMCs and pulmonary arteries, while SGLT2 expression was undetectable. SGLT1 increased in response to PDGF-BB or hypoxia stimulation, while PASMCs proliferation was inhibited and beneficial effects of CANA were counteracted by knockdown of SGLT1. Our research demonstrated for the first time that CANA inhibited the proliferation of PASMCs by regulating SGLT1/AMPK signaling and thus exerted an anti-proliferative effect on MCT-induced PAH.

TRPC4 aggravates hypoxic pulmonary hypertension by promoting pulmonary endothelial cell apoptosis.

Pulmonary hypertension (PH) is a devastating disease that lacks effective treatment options and is characterized by severe pulmonary vascular remodeling. Pulmonary arterial endothelial cell (PAEC) dysfunction drives the initiation and pathogenesis of pulmonary arterial hypertension. Canonical transient receptor potential (TRPC) channels, a family of Ca2+-permeable channels, play an important role in various diseases. However, the effect and mechanism of TRPCs on PH development have not been fully elucidated. Among the TRPC family members, TRPC4 expression was markedly upregulated in PAECs from hypoxia combined with SU5416 (HySu)-induced PH mice and monocrotaline (MCT)-treated PH rats, as well as in hypoxia-exposed PAECs, suggesting that TRPC4 in PAECs may participate in the occurrence and development of PH. In this study, we aimed to investigate whether TRPC4 in PAECs has an aggravating effect on PH and elucidate the molecular mechanisms. We observed that hypoxia treatment promoted PAEC apoptosis through a caspase-12/endoplasmic reticulum stress (ERS)-dependent pathway. Knockdown of TRPC4 attenuated hypoxia-induced apoptosis and caspase-3/caspase-12 activity in PAECs. Accordingly, adeno-associated virus (AAV) serotype 6-mediated pulmonary endothelial TRPC4 silencing (AAV6-Tie-shRNA-TRPC4) or TRPC4 antagonist suppressed PH progression as evidenced by reduced right ventricular systolic pressure (RVSP), pulmonary vascular remodeling, PAEC apoptosis and reactive oxygen species (ROS) production. Mechanistically, unbiased RNA sequencing (RNA-seq) suggested that TRPC4 deficiency suppressed the expression of the proapoptotic protein sushi domain containing 2 (Susd2) in hypoxia-exposed mouse PAECs. Moreover, TRPC4 activated hypoxia-induced PAEC apoptosis by promoting Susd2 expression. Therefore, inhibiting TRPC4 ameliorated PAEC apoptosis and hypoxic PH in animals by repressing Susd2 signaling, which may serve as a therapeutic target for the management of PH.

Corosolic acid attenuates platelet-derived growth factor signaling in macrophages and smooth muscle cells of pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is a progressive and life-threatening disease that is characterized by vascular remodeling of the pulmonary artery. Pulmonary vascular remodeling is primarily caused by the excessive proliferation and migration of pulmonary arterial smooth muscle cells (PASMCs), which are facilitated by perivascular inflammatory cells including macrophages. Corosolic acid (CRA) is a natural pentacyclic triterpenoid that exerts anti-inflammatory effects. In the present study, the effects of CRA on the viability of macrophages were examined using monocrotaline (MCT)-induced PAH rats and human monocyte-derived macrophages. Although we previously reported that CRA inhibited signal transducer and activator of transcription 3 (STAT3) signaling and ameliorated pulmonary vascular remodeling in PAH, the inhibitory mechanism remains unclear. Therefore, the underlying mechanisms were investigated using PASMCs from idiopathic PAH (IPAH) patients. In MCT-PAH rats, CRA inhibited the accumulation of macrophages around remodeled pulmonary arteries. CRA reduced the viability of human monocyte-derived macrophages. In IPAH-PASMCs, CRA attenuated cell proliferation and migration facilitated by platelet-derived growth factor (PDGF)-BB released from macrophages and PASMCs. CRA also downregulated the expression of PDGF receptor ß and its signaling pathways, STAT3 and nuclear factor-κB (NF-κB). In addition, CRA attenuated the phosphorylation of PDGF receptor ß and STAT3 following the PDGF-BB simulation. The expression and phosphorylation levels of PDGF receptor ß after the PDGF-BB stimulation were reduced by the small interfering RNA knockdown of NF-κB, but not STAT3, in IPAH-PASMCs. In conclusion, CRA attenuated the PDGF-PDGF receptor ß-STAT3 and PDGF-PDGF receptor ß-NF-κB signaling axis in macrophages and PASMCs, and thus, ameliorated pulmonary vascular remodeling in PAH.

NGF increases Connexin-43 expression and function in pulmonary arterial smooth muscle cells to induce pulmonary artery hyperreactivity.

AIMS: Pulmonary hypertension (PH) is characterised by an increase in pulmonary arterial pressure, ultimately leading to right ventricular failure and death. We have previously shown that nerve growth factor (NGF) plays a critical role in PH. Our objectives here were to determine whether NGF controls Connexin-43 (Cx43) expression and function in the pulmonary arterial smooth muscle, and whether this mechanism contributes to NGF-induced pulmonary artery hyperreactivity. METHODS AND RESULTS: NGF activates its TrkA receptor to increase Cx43 expression, phosphorylation, and localization at the plasma membrane in human pulmonary arterial smooth muscle cells, thus leading to enhanced activity of Cx43-dependent GAP junctions as shown by Lucifer Yellow dye assay transfer and fluorescence recovery after photobleaching -FRAP- experiments. Using both in vitro pharmacological and in vivo SiRNA approaches, we demonstrate that NGF-dependent increase in Cx43 expression and activity in the rat pulmonary circulation causes pulmonary artery hyperreactivity. We also show that, in a rat model of PH induced by chronic hypoxia, in vivo blockade of NGF or of its TrkA receptor significantly reduces Cx43 increased pulmonary arterial expression induced by chronic hypoxia and displays preventive effects on pulmonary arterial pressure increase and right heart hypertrophy. CONCLUSIONS: Modulation of Cx43 by NGF in pulmonary arterial smooth muscle cells contributes to NGF-induced alterations of pulmonary artery reactivity. Since NGF and its TrkA receptor play a role in vivo in Cx43 increased expression in PH induced by chronic hypoxia, these NGF/Cx43-dependent mechanisms may therefore play a significant role in human PH pathophysiology.

Functions and novel regulatory mechanisms of key glycolytic enzymes in pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is a progressive vascular disease characterized by remodeling of the pulmonary vasculature and elevated pulmonary arterial pressure, ultimately leading to right heart failure and death. Despite its clinical significance, the precise molecular mechanisms driving PAH pathogenesis warrant confirmation. Compelling evidence indicates that during the development of PAH, pulmonary vascular cells exhibit a preference for energy generation through aerobic glycolysis, known as the "Warburg effect", even in well-oxygenated conditions. This metabolic shift results in imbalanced metabolism, increased proliferation, and severe pulmonary vascular remodeling. Exploring the Warburg effect and its interplay with glycolytic enzymes in the context of PAH has yielded current insights into emerging drug candidates targeting enzymes and intermediates involved in glucose metabolism. This sheds light on both opportunities and challenges in the realm of antiglycolytic therapy for PAH.

Effect of free and nanoemulsified ß-caryophyllene on monocrotaline-induced pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is characterized by increased pulmonary vascular resistance (PVR), right ventricular (RV) failure and premature death. Compounds with vasodilatory characteristics, such as ß-caryophyllene, could be promising therapeutics for PAH. This study aimed to determine the effects of free and nanoemulsified ß-caryophyllene in lung oxidative stress and heart function in PAH rats. Male Wistar rats (170 g, n = 6/group) were divided into four groups: control (CO), monocrotaline (MCT), monocrotaline + ß-caryophyllene (MCT-Bcar) and monocrotaline + nanoemulsion with ß-caryophyllene (MCT-Nano). PAH was induced by MCT (60 mg/kg i.p.), and 7 days later, treatment with ß-caryophyllene, either free or in a nanoemulsion (by gavage, 176 mg/kg/day) or vehicle was given for 14 days. Echocardiographic and hemodynamic measurements were performed, and after, the RV was collected for morphometry and the lungs for evaluation of oxidative stress, antioxidant enzymes, total sulfhydryl compounds, nitric oxide synthase (NOS) activity and endothelin-1 receptor expression. RV hypertrophy, increased PVR and RV systolic and diastolic pressures (RVSP and RVEDP, respectively) and increased mean pulmonary arterial pressure (mPAP) were observed in the MCT group. Treatment with both free and nanoemulsified ß-caryophyllene reduced RV hypertrophy, mPAP, RVSP and lipid peroxidation. The reduction in RVSP was more pronounced in the MCT-Nano group. Moreover, RVEDP decreased only in the MCT-Nano group. These treatments also increased superoxide dismutase, catalase and NOS activities and decreased endothelin-1 receptors expression. Both ß-caryophyllene formulations improved mPAP, PVR and oxidative stress parameters. However, ß-caryophyllene in a nanoemulsion was more effective in attenuating the effects of PAH.

Effect of RNA interference with HIF-1α on the growth of pulmonary artery endothelial cells in broiler chickens.

Pulmonary artery remodeling is a characteristic feature of broiler ascites syndrome (BAS). Pulmonary artery endothelial cells (PAECs) regulated by HIF-1α play a critical role in pulmonary artery remodeling, but the underlying mechanisms of HIF-1α in BAS remain unclear. In this experiment, primary PAECs were cultured in vitro and were identified by coagulation factor VIII. After hypoxia and RNA interference, the mRNA and protein expression levels of HIF-1α and VEGF were determined by qPCR and Western blotting. The transcriptome profiles of PAECs were obtained by RNA sequencing. Our results showed that the positive rate of PAECs was more than 90%, hypoxia-induced promoted the proliferation and apoptosis of PAECs, and RNA interference significantly downregulated the expression of HIF-1α, inhibited the proliferation of PAECs, and promoted the apoptosis of PAECs. In addition, transcriptome sequencing analysis indicated that HIF-1α may regulate broiler ascites syndrome by mediating COL4A, vitronectin, vWF, ITGα8, and MKP-5 in the ECM, CAMs and MAPK pathways in PAECs. These studies lay the foundation for further exploration of the mechanisms of pulmonary artery remodeling, and HIF-1α may be a potentially effective gene for the prevention and treatment of BAS.

Spermidine attenuates monocrotaline-induced pulmonary arterial hypertension in rats by inhibiting purine metabolism and polyamine synthesis-associated vascular remodeling.

Ensuring the homeostatic integrity of pulmonary artery endothelial cells (PAECs) is essential for combatting pulmonary arterial hypertension (PAH), as it equips the cells to withstand microenvironmental challenges. Spermidine (SPD), a potent facilitator of autophagy, has been identified as a significant contributor to PAECs function and survival. Despite SPD's observed benefits, a comprehensive understanding of its protective mechanisms has remained elusive. Through an integrated approach combining metabolomics and molecular biology, this study uncovers the molecular pathways employed by SPD in mitigating PAH induced by monocrotaline (MCT) in a Sprague-Dawley rat model. The study demonstrates that SPD administration (5 mg/kg/day) significantly corrects right ventricular impairment and pathological changes in pulmonary tissues following MCT exposure (60 mg/kg). Metabolomic profiling identified a purine metabolism disorder in MCT-treated rats, which SPD effectively normalized, conferring a protective effect against PAH progression. Subsequent in vitro analysis showed that SPD (0.8 mM) reduces oxidative stress and apoptosis in PAECs challenged with Dehydromonocrotaline (MCTP, 50 µM), likely by downregulating purine nucleoside phosphorylase (PNP) and modulating polyamine biosynthesis through alterations in S-adenosylmethionine decarboxylase (AMD1) expression and the subsequent production of decarboxylated S-adenosylmethionine (dcSAM). These findings advocate SPD's dual inhibitory effect on PNP and AMD1 as a novel strategy to conserve cellular ATP and alleviate oxidative injuries, thus providing a foundation for SPD's potential therapeutic application in PAH treatment.

FAK mediates hypoxia-induced pulmonary artery smooth muscle cell proliferation by modulating mitochondrial transcription termination factor 1/cyclin D1.

This study aimed to investigate the mechanism of FAK-dependent hypoxia-induced proliferation on human pulmonary artery smooth muscle cells (HPASMCs). Primary HPASMCs were isolated and cultured in vitro under normal and hypoxia conditions to assess cell proliferation with cell counting kit-8. FAK and mitochondrial transcription termination factor 1 (mTERF1) were silenced with siRNA, mRNA, and protein levels of FAK, mTERF1, and cyclin D1 were determined. HPASMC proliferation increased under hypoxia compared to normal conditions. Knocking down FAK or mTERF1 with siRNA led to decreased cell proliferation under both normal and hypoxia conditions. FAK knockdown led to the reduction of both mTERF1 and cyclin D1 expressions under the hypoxia conditions, whereas mTERF1 knockdown led to the downregulation of cyclin D1 expression but not FAK expression under the same condition. However, under normal conditions, knocking down either FAK or mTERF1 had no impact on cyclin D1 expression. These results suggested that FAK may regulate the mTERF1/cyclin D1 signaling pathway to modulate cell proliferation in hypoxia.

Blockade of ZFX Alleviates Hypoxia-Induced Pulmonary Vascular Remodeling by Regulating the YAP Signaling.

High expression of the zinc finger X-chromosomal protein (ZFX) correlates with proliferation, aggressiveness, and development in many types of cancers. In the current report, we investigated the efficacy of ZFX in mouse pulmonary artery smooth muscle cells (PASMCs) proliferation during pulmonary arterial hypertension (PAH). PASMCs were cultured in hypoxic conditions. Real-time PCR and western blotting were conducted to detect the expression of ZFX. Cell proliferation, apoptosis, migration, and invasion were, respectively, measured by CCK-8, flow cytometry, wound scratchy, and transwell assays. Glycolytic ability was validated by the extracellular acidification rate and oxygen consumption rate. Transcriptome sequencing technology was used to explore the genes affected by ZFX knockdown. Luciferase and chromatin immunoprecipitation assays were utilized to verify the possible binding site of ZFX and YAP1. Mice were subjected to hypoxia for 21 days to induce PAH. The right ventricular systolic pressure (RVSP) was measured and ratio of RV/LV + S was calculated. The results show that ZFX was increased in hypoxia-induced PASMCs and mice. ZFX knockdown inhibited the proliferation, migration, and invasion of PASMC. Using RNA sequencing, we identify glycolysis and YAP as a key signaling of ZFX. ZFX knockdown inhibited Glycolytic ability. ZFX strengthened the transcription activity of YAP1, thereby regulating the YAP signaling. YAP1 overexpression reversed the effect of ZFX knockdown on hypoxia-treated PASMCs. In conclusion, ZFX knockdown protected mice from hypoxia-induced PAH injury. ZFX knockdown dramatically reduced RVSP and RV/(LV + S) in hypoxia-treated mice.