Hypoxia-induced Pulmonary Artery Smooth Muscle Research Articles

Methyltransferase-Like 3-Mediated N6-Methyladenosine RNA Methylation Regulates Hypoxia-Induced Pulmonary Arterial Smooth Muscle Cell Pyroptosis by Targeting PTEN.

Pulmonary hypertension is a rare, progressive disorder that can lead to right ventricular hypertrophy, right heart failure, and even sudden death. N6-methyladenosine modification and the main methyltransferase that mediates it, methyltransferase-like (METTL) 3, exert important effects on many biological and pathophysiological processes. However, the role of METTL3 in pyroptosis remains unclear. Here, we characterized the role of METTL3 and the underlying cellular and molecular mechanisms of pyroptosis, which is involved in pulmonary hypertension. METTL3 was downregulated in a pulmonary hypertension mouse model and in hypoxia-exposed pulmonary artery smooth muscle cell. The small interfering RNA-induced silencing of METTL3 decreased the m6A methylation levels and promoted pulmonary artery smooth muscle cell pyroptosis, mimicking the effects of hypoxia. In contrast, overexpression of METTL3 suppressed hypoxia-induced pulmonary artery smooth muscle cell pyroptosis. Mechanistically, we identified the phosphate and tension homology deleted on chromosome 10 (PTEN) gene as a target of METTL3-mediated m6A modification, and methylated phosphate and tension homology deleted on chromosome 10 mRNA was subsequently recognized by the m6A "reader" protein insulin-like growth factor 2 mRNA-binding protein 2, which directly bound to the m6A site on phosphate and tension homology deleted on chromosome 10 mRNA and enhanced its stability. These results identify a new signaling pathway, the METTL3/phosphate and tension homology deleted on chromosome 10/insulin-like growth factor 2 mRNA-binding protein 2 axis, that participates in the regulation of hypoxia-induced pyroptosis.

Levosimendan mediates the BMP/Smad axis through upregulation of circUSP34-targeted miR-1298 to alleviate pulmonary hypertension

BackgroundPulmonary hypertension (PH) is a long-term disease that impacts approximately 1% of the world’s population. Currently, levosimendan (Lev) is proposed for PH treatment. However, the mechanism of Lev in the treatment of PH is unknown.MethodsWe used hypoxia-induced pulmonary artery smooth muscle cells (PASMCs) to establish a PH cell model. A number of cell biology methods were performed to assay alterations in cell proliferation, migration and apoptosis after Lev treatment. qRT-PCR and WB were performed to test the levels of circUSP34 and miR-1298, and BMP/Smad protein respectively. In addition, the regulatory relationship between circUSP34 or BMPR2 with miR-1298 was verified through the use of double luciferase as well as RIP assay. In addition, we explored the regulatory effect of Lev on the circUSP34/miR-1298/BMP/Smad axis using a rat PH model.ResultsOur results demonstrate that Lev inhibited PASMCs cell proliferation, migration and promoted apoptosis exposed to hypoxia. In hypoxia-treated PASMCs, circUSP34 expression got downregulated while miR-1298 upregulated, whereas the addition with Lev resulted in upregulation of circUSP34 expression and downregulation of miR-1298 expression, indicating that circUSP34 can target and regulate miR-1298. In addition, miR-1298 targets and regulates the expression of BMPR2. In a rat PH model induced by hypoxia combined with SU5416, Lev upregulated circUSP34 targeting miR-1298-mediated BMP/Smad axis to alleviate the PH phenotype.ConclusionWe have shown that Lev can be used as a therapeutic drug for PH patients, which works through the circUSP34/miR-1298/BMP/Smad axis to alleviate PH symptoms.

Urolithin A Protects against Hypoxia-Induced Pulmonary Hypertension by Inhibiting Pulmonary Arterial Smooth Muscle Cell Pyroptosis via AMPK/NF-κB/NLRP3 Signaling.

Recent studies confirmed that pyroptosis is involved in the progression of pulmonary hypertension (PH), which could promote pulmonary artery remodeling. Urolithin A (UA), an intestinal flora metabolite of ellagitannins (ETs) and ellagic acid (EA), has been proven to possess inhibitory effects on pyroptosis under various pathological conditions. However, its role on PH remained undetermined. To investigate the potential of UA in mitigating PH, mice were exposed to hypoxia (10% oxygen, 4 weeks) to induce PH, with or without UA treatment. Moreover, in vitro experiments were carried out to further uncover the underlying mechanisms. The in vivo treatment of UA suppressed the progression of PH via alleviating pulmonary remodeling. Pyroptosis-related genes were markedly upregulated in mice models of PH and reversed after the administration of UA. In accordance with that, UA treatment significantly inhibited hypoxia-induced pulmonary arterial smooth muscle cell (PASMC) pyroptosis via the AMPK/NF-κB/NLRP3 pathway. Our results revealed that UA treatment effectively mitigated PH progression through inhibiting PASMC pyroptosis, which represents an innovative therapeutic approach for PH.

CircLMBR1 inhibits phenotypic transformation of hypoxia-induced pulmonary artery smooth muscle via the splicing factor PUF60

Phenotypic transformation of pulmonary artery smooth muscle cells (PASMCs) contributes to vascular remodeling in hypoxic pulmonary hypertension (PH). Recent studies have suggested that circular RNAs (circRNAs) may play important roles in the vascular remodeling of hypoxia-induced PH. However, whether circRNAs cause pulmonary vascular remodeling by regulating the phenotypic transformation in PH has not been investigated. Microarray and RT-qPCR analysis identified that circLMBR1, a novel circRNA, decreased in mouse lung tissues of the hypoxia-SU5416 PH model, as well as in human PASMCs and mouse PASMCs exposed to hypoxia. Overexpression of circLMBR1 in the Semaxinib (SU5416) mouse model ameliorated hypoxia-induced PH and vascular remodeling in the lungs. Notably, circLMBR1 was mainly distributed in the nucleus and bound to the splicing factor PUF60. CircLMBR1 suppressed the phenotypic transformation of human PASMCs and vascular remodeling by inhibiting PUF60 expression. Furthermore, we identified U2AF65 as the downstream regulatory factor of PUF60. U2AF65 directly interacted with the pre-mRNA of the contractile phenotype marker smooth muscle protein 22-α (SM22α) and inhibited its splicing. Meanwhile, hypoxia exposure increased the formation of the PUF60–U2AF65 complex, thereby inhibiting SM22α production and inducing the transition of human PASMCs from a contractile phenotype to a synthetic phenotype. Overall, our results verified the important role of circLMBR1 in the pathological process of PH. We also proposed a new circLMBR1/PUF60–U2AF65/pre-SM22α pathway that could regulate the phenotypic transformation and proliferation of human PASMCs. This study may provide new perspectives for the diagnosis and treatment of PH.

Effect of 2-phenylethyl-beta-glucopyranoside isolated from Huaizhong No. 1 Rehmannia glutinosa on hypoxic pulmonary hypertension by regulating PI3K/Akt/m TOR/HIF-1α pathway

The study investigated the protective effect and mechanism of 2-phenylethyl-beta-glucopyranoside(Phe) from Huaizhong No.1 Rehmannia glutinosa on hypoxic pulmonary hypertension(PH), aiming to provide a theoretical basis for clinical treatment of PAH. Male C57BL/6N mice were randomly divided into normal group, model group, positive drug(bosentan, 100 mg·kg~(-1)) group, and low-and high-dose Phe groups(20 and 40 mg·kg~(-1)). Except for the normal group, all other groups were continuously subjected to model induction in a 10% hypoxic environment for 5 weeks, with oral administration for 14 days starting from the 3rd week. The cardiopulmonary function, right ventricular pressure, cough and asthma index, lung injury, cell apoptosis, oxidative stress-related indicators, immune cells, and phosphatidylinositol 3-kinase(PI3K)/protein kinase B(Akt)/mammalian target of rapamycin(mTOR)/hypoxic inducible factor 1α(HIF-1α) pathway-related proteins or mRNA levels were examined. Furthermore, hypoxia-induced pulmonary arterial smooth muscle cell(PASMC) were used to further explore the mechanism of Phe intervention in PH combined with PI3K ago-nist(740Y-P). The results showed that Phe significantly improved the cardiopulmonary function of mice with PH, decreased right ventricular pressure, cough and asthma index, and lung injury, reduced cell apoptosis, oxidative stress-related indicators, and nuclear levels of phosphorylated Akt(p-Akt) and phosphorylated mTOR(p-mTOR), inhibited the expression levels of HIF-1α and PI3K mRNA and proteins, and maintained the immune cell homeostasis in mice. Further mechanistic studies revealed that Phe significantly reduced the viability and migration ability of hypoxia-induced PASMC, decreased the expression of HIF-1α and PI3K proteins and nuc-lear levels of p-Akt and p-mTOR, and this effect was blocked by 740Y-P. Therefore, it is inferred that Phe may exert anti-PH effects by alleviating the imbalance of oxidative stress and apoptosis in lung tissues and regulating immune levels, and its mechanism may be related to the regulation of the PI3K/Akt/mTOR/HIF-1α pathway. This study is expected to provide drug references and research ideas for the treatment of PH.

Exosomes enriched by miR-429-3p derived from ITGB1 modified Telocytes alleviates hypoxia-induced pulmonary arterial hypertension through regulating Rac1 expression

BackgroundRecent studies have emphasized the critical role of Telocytes (TCs)-derived exosomes in organ tissue injury and repair. Our previous research showed a significant increase in ITGB1 within TCs. Pulmonary Arterial Hypertension (PAH) is marked by a loss of microvessel regeneration and progressive vascular remodeling. This study aims to investigate whether exosomes derived from ITGB1-modified TCs (ITGB1-Exo) could mitigate PAH.MethodsWe analyzed differentially expressed microRNAs (DEmiRs) in TCs using Affymetrix Genechip miRNA 4.0 arrays. Exosomes isolated from TC culture supernatants were verified through transmission electron microscopy and Nanoparticle Tracking Analysis. The impact of miR-429-3p-enriched exosomes (Exo-ITGB1) on hypoxia-induced pulmonary arterial smooth muscle cells (PASMCs) was evaluated using CCK-8, transwell assay, and inflammatory factor analysis. A four-week hypoxia-induced mouse model of PAH was constructed, and H&E staining, along with Immunofluorescence staining, were employed to assess PAH progression.ResultsForty-five miRNAs exhibited significant differential expression in TCs following ITGB1 knockdown. Mus-miR-429-3p, significantly upregulated in ITGB1-overexpressing TCs and in ITGB1-modified TC-derived exosomes, was selected for further investigation. Exo-ITGB1 notably inhibited the migration, proliferation, and inflammation of PASMCs by targeting Rac1. Overexpressing Rac1 partly counteracted Exo-ITGB1’s effects. In vivo administration of Exo-ITGB1 effectively reduced pulmonary vascular remodeling and inflammation.ConclusionsOur findings reveal that ITGB1-modified TC-derived exosomes exert anti-inflammatory effects and reverse vascular remodeling through the miR-429-3p/Rac1 axis. This provides potential therapeutic strategies for PAH treatment.Graphical 1. Identification of Differentially Expressed microRNAs (DEmiRs) in ITGB1 overexpressed TCs.2. Effects of Exo-ITGB1 or miR-429-3p on Hypoxia-Induced PASMCs in vitro.3. Exo-ITGB1 inhibits the hyper-proliferation and migration of PASMCs through regulating miR-429-3p/Rac1 axis in vitro.4. The therapeutic potential of Exo-ITGB1 in hypoxia-induced PAH model in vivo.

Super enhancer-associated circRNA-circLrch3 regulates hypoxia-induced pulmonary arterial smooth muscle cells pyroptosis by formation of R-loop with host gene

BackgroundPulmonary hypertension (PH) is a complex vascular disorder, characterized by pulmonary vessel remodeling and perivascular inflammation. Pulmonary arterial smooth muscle cells (PASMCs) pyroptosis is a novel pathological mechanism implicated of pulmonary vessel remodeling. However, the involvement of circRNAs in the process of pyroptosis and the underlying regulatory mechanisms remain inadequately understood. MethodsWestern blotting, PI staining and LDH release were used to explore the role of circLrch3 in PASMCs pyroptosis. Moreover, S9.6 dot blot and DRIP-PCR were used to assess the formation of R-loop between circLrch3 and its host gene Lrch3. Chip-qPCR were used to evaluate the mechanism of super enhancer-associated circLrh3, which is transcriptionally activated by the transcription factor Tbx2. ResultsCircLrch3 was markedly upregulated in hypoxic PASMCs. CircLrch3 knockdown inhibited hypoxia induced PASMCs pyroptosis in vivo and in vitro. Mechanistically, circLrch3 can form R-loop with host gene to upregulate the protein and mRNA expression of Lrch3. Furthermore, super enhancer interacted with the Tbx2 at the Lrch3 promoter locus, mediating the augmented transcription of circLrch3. ConclusionOur findings clarify the role of a super enhancer-associated circLrch3 in the formation of R-loop with the host gene Lrch3 to modulate pyroptosis in PASMCs, ultimately promoting the development of PH.

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 invitro 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.

SOCS5, targeted by miR-155-5p, plays a negative regulatory role in pulmonary hypertension through inhibiting JAK2/STAT3 signaling pathway

Pulmonary hypertension (PH) is a chronic pulmonary vascular disease and causes massive deaths. Here, we intended to investigate the function and mechanism of SOCS5 in PH. We engineered a hypoxia-induced PH model in mice. HE staining were implemented to evaluate pathological alterations in the lung tissues. The potential mechanism of SOCS5 in regulating hypoxia-induced pulmonary artery smooth muscle cell (PASMC) function was explored in vitro. RT-qPCR and western blot revealed that the level of SOCS5 was decreased both in PH mice and hypoxia-induced HPASMCs. Functional assays were performed for confirming the role of SOCS5 in modulating the cell phenotype and JAK2/STAT3 pathway in HPASMCs. Results revealed that overexpression of SOCS5 suppressed proliferation, migration and contraction of HPASMCs and negatively regulated the JAK2/STAT3 signaling pathway in HPASMCs under hypoxia in vitro, while knockdown of SOCS5 accelerated it. As evidenced by mechanism studies, SOCS5 was targeted and regulated by miR-155-5p, hence affecting on HPASMC proliferation, migration and contraction. These outcomes indicated that the decreased level of SOCS5 in hypoxia-induced HPASMCs promoted the cell proliferation, cell migration, and cell contraction through activating JAK2/STAT3 signaling pathway. Moreover, SOCS5 was targeted by miR-155-5p. All in all, our work hinted that miR-155-5p/SOCS5/JAK2/STAT3 axis played a crucial part in PH.

Matrine alleviates hypoxia-induced inflammation and pulmonary vascular remodelling via RPS5/NF-κB signalling pathway.

Hypoxia-induced vasoconstriction and vascular remodelling are the main pathological features of hypoxic pulmonary arterial hypertension (HPAH), and inflammation is participated in the occurrence of pulmonary vascular remodelling (PVR). Matrine is an alkaloid with the effects of anti-inflammation, antifibrosis and antitumour. But, few studies have explored the role of matrine in regulating PVR, and the related mechanisms are still unknown. In this study, we found that hypoxia-induced pulmonary artery smooth muscle cells(PASMCs) proliferation and inhibited its apoptosis, reduced the expression of ribosomal protein s5 and activated the nuclear factor kappa-B (NF-κB) signalling. Matrine, sildenafil and NF-κB inhibitor Bay 11-7082 could reverse these changes and impel the cell cycle in phase S retardation, and reduced the expression of p50, p65, proliferating cell nuclear antigen (PCNA), Bcl-2. In addition, matrine could lower right ventricular systolic pressure and mean pulmonary artery pressureof rats,α-smooth muscle actinand PCNA expression in pulmonary artery media, the levels of tumor necrosis factor-α and interleuki-1β, thus improved hypoxia-induced PVR. This study indicated that matrine could alleviate inflammation and improve PVR through reversing the imbalance of proliferation and apoptosis of PASMCs, thus it had a therapeutic effect on HPAH.

Inhibitory Effect of PPARδ Agonist GW501516 on Proliferation of Hypoxia-induced Pulmonary Arterial Smooth Muscle Cells by Regulating the mTOR Pathway.

This study aimed to investigate the effects of the peroxisome proliferator-activated receptor δ (PPARδ) agonist GW501516 on the proliferation of pulmonary artery smooth muscle cells (PASMCs) induced by hypoxia, in order to search for new drugs for the treatment and prevention of pulmonary vascular remodeling. PASMCs were incubated with different concentrations of GW501516 (10, 30, 100 nmol/L) under the hypoxic condition. The proliferation was determined by a CCK-8 assay. The cell cycle progression was analyzed by flow cytometry. The expression of PPARδ, S phase kinase-associated protein 2 (Skp2), and cell cycle-dependent kinase inhibitor p27 was detected by Western blotting. Then PASMCs were treated with 100 nmol/ L GW501516, 100 nmol/L mammalian target of rapamycin (mTOR) inhibitor rapamycin and/or 2 µmol/L mTOR activator MHY1485 to explore the molecular mechanisms by which GW501516 reduces the proliferation of PASMCs. The presented data demonstrated that hypoxia reduced the expression of PPARδ in an oxygen concentration- and time-dependent manner, and GW501516 decreased the proliferation of PASMCs induced by hypoxia by blocking the progression through the G0/G1 to S phase of the cell cycle. In accordance with these findings, GW501516 downregulated Skp2 and upregulated p27 in hypoxia-exposed PASMCs. Further experiments showed that rapamycin had similar effects as GW501516 in inhibiting cell proliferation, arresting the cell cycle, regulating the expression of Skp2 and p27, and inactivating mTOR in hypoxia-exposed PASMCs. Moreover, MHY1485 reversed all the beneficial effects of GW501516 on hypoxia-stimulated PASMCs. GW501516 inhibited the proliferation of PASMCs induced by hypoxia through blocking the mTOR/Skp2/p27 signaling pathway.

The HIF-1α/miR-26a-5p/PFKFB3/ULK1/2 axis regulates vascular remodeling in hypoxia-induced pulmonary hypertension by modulation of autophagy.

Pulmonary arterial hypertension (PAH) is a progressive and life-threatening disease characterized by pulmonary vascular remodeling, which may cause right heart failure and even death. Accumulated evidence confirmed that microRNA-26 family play critical roles in cardiovascular disease; however, their function in PAH remains largely unknown. Here, we investigated the expression of miR-26 family in plasma from PAH patients using quantitative RT-PCR, and identified miR-26a-5p as the most downregulated member, which was also decreased in hypoxia-induced pulmonary arterial smooth muscle cell (PASMC) autophagy models and lung tissues of PAH patients. Furthermore, chromatin immunoprecipitation (ChIP) analysis and luciferase reporter assays revealed that hypoxia-inducible factor 1α (HIF-1α) specifically interacted with the promoter of miR-26a-5p and inhibited its expression in PASMCs. Tandem mRFP-GFP-LC3B fluorescence microscopy demonstrated that miR-26a-5p inhibited hypoxia-induced PAMSC autophagy, characterized by reduced formation of autophagosomes and autolysosomes. In addition, results showed that miR-26a-5p overexpression potently inhibited PASMC proliferation and migration, as determined by cell counting kit-8, EdU staining, wound-healing, and transwell assays. Mechanistically, PFKFB3, ULK1, and ULK2 were direct targets of miR-26a-5p, as determined by dual-luciferase reporter gene assays and western blots. Meanwhile, PFKFB3 could further enhance the phosphorylation level of ULK1 and promote autophagy in PASMCs. Moreover, intratracheal administration of adeno-miR-26a-5p markedly alleviated right ventricular hypertrophy and pulmonary vascular remodeling in hypoxia-induced PAH rat models in vivo. Taken together, the HIF-1α/miR-26a-5p/PFKFB3/ULK1/2 axis plays critical roles in the regulation of hypoxia-induced PASMC autophagy and proliferation. MiR-26a-5p may represent as an attractive biomarker for the diagnosis and treatment of PAH.

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.

Circ-calm4 regulates hypoxia-induced pulmonary artery smooth muscle autophagy by binding Purb

Pulmonary hypertension (PH) is a serious and fatal disease characterized by pulmonary vasoconstriction and pulmonary vascular remodeling. The excessive autophagy of pulmonary artery smooth muscle cells (PASMCs) is one of the important factors of pulmonary vascular remodeling. A number of studies have shown that circular RNA (circRNA) can participate in the onset of PH. Our previous studies have shown that circRNA calmodulin 4 (circ-calm4) is involved in the progression of hypoxic PH. However, the role of circ-calm4 on regulation of hypoxic PH autophagy has not been reported. In this study, we demonstrated for the first time that hypoxia-mediated upregulated circ-calm4 expression has a key regulatory effect on autophagy in hypoxia-induced PASMCs and hypoxic PH mouse models. Knockdown of circ-calm4 both in vivo and in vitro can inhibit the autophagy in PASMCs induced by hypoxia. We also performed bioinformatics predictions and conducted experiments to verify that circ-calm4 bound to the purine-rich binding protein (Purb) to promote its expression in the nucleus, thereby initiating the transcription of autophagy-related protein Beclin1. Interestingly, we found that Beclin1 transcription initiated by Purb was accompanied by a modification of Beclin1 super-enhancer to improve transcription activity and efficiency. Overall, our results confirm that the circ-calm4/Purb/Beclin1 signal axis is involved in the occurrence of hypoxia-induced PASMCs autophagy, and the novel regulatory mechanisms and signals transduction pathways in PASMC autophagy induced by hypoxia.

SIRT6 inhibits hypoxia-induced pulmonary arterial smooth muscle cells proliferation via HIF-1α/PDK4 signaling

SIRT6 is an NAD+-dependent protein that plays a vital role in regulating the cell proliferation, differentiation and apoptosis. Abnormal proliferation of pulmonary artery smooth muscle cells (PASMCs) in peripheral vascular is one of the major pathological findings of pulmonary vascular remodeling in pulmonary arterial hypertension (PAH). However, whether SIRT6 is involved in hypoxia-induced proliferation of PASMCs and its possible mechanisms remain unknown. In the present study, we found that the expression of SIRT6 was decreased in both hypoxia-induced PAH rats model and HPASMCs. Hypoxia promoted the proliferation of HPASMCs in a time-dependent manner, inhibited the activity of caspase-3 and the production of PDH, increased the activity of LDH, ROS level, mitochondrial membrane potential(MMP) and the expression of HIF-1α and PDK4, which induced glycolysis. SIRT6 over-expression could inhibit the proliferation of HPASMCs and increase the apoptosis rate, impelled the retardation of cell cycle in phase G1. Meanwhile, SIRT6 over-expression reduced LDH activity, the levels of ROS and MMP, which simultaneously increased the production of PDH, the expression of HIF-1α, PDK4, Cyclin D1 and PCNA in hypoxia-induced HPASMCs. Moreover, SIRT6 over-expression inhibited the transcriptional activation of HIF-1α/PDK4 signaling. In addition, SIRT6 knockdown with SIRT6 siRNA exhibited the same effect as hypoxia. Together, our results indicated that SIRT6 was participant in regulating hypoxia-induced imbalance of proliferation and apoptosis of HPASMCs, which was associated with the activation of HIF-1α/PDK4 signaling pathway. Targeting at SIRT6 gene and regulating the downstream metabolism signaling pathway may be a novel strategy for the treatment of hypoxia-induced PAH.

MiR-155-5p regulates hypoxia-induced pulmonary artery smooth muscle cell function by targeting PYGL

ABSTRACT Pulmonary arterial hypertension (PAH) is a cardiovascular disease that has high incidence and causes massive deaths. miR-155-5p/PYGL pathway was revealed to play a crucial role in PAH by weighted gene co-expression network analysis (WGCNA). The potential mechanism of miR-155-5p in regulating hypoxia-induced pulmonary artery smooth muscle cell (PASMC) function was analyzed through in vitro experiments. Hypoxia treatment stimulated the proliferation of PASMCs and increased the expression of vascular endothelial growth factor (VEGF) and hypoxia-inducible factor-1α (HIF-1α). At the same time, revealed by qRT-PCR and western blot, the level of miR-155-5p was raised, and the level of PYGL was decreased in hypoxia-induced PASMCs. Through CCK-8 assay, transwell assay and flow cytometry, it was revealed that miR-155-5p inhibitor remarkably inhibited the cell proliferation and migration and decreased the proportion of hypoxia-stimulated PASMCs in S and G2/M phases. Dual-luciferase reporter system was subsequently applied to validate the straight regulation of miR-155-5p on PYGL based on the analysis of online database. Furthermore, siPYGL was revealed to reverse the influence of miR-155-5p inhibitor on hypoxia-induced PASMCs. These outcomes indicate that the increased level of miR-155-5p in hypoxia-stimulated PASMCs could enhance the cell proliferation, cell migration, and cell cycle progression by targeting PYGL directly. This study may supply novel treatment strategies for PAH. Abbreviations: PH, pulmonary hypertension; PAH, pulmonary arterial hypertension; WGCNA, weighted gene co-expression network analysis; PASMCs, pulmonary artery smooth muscle cells; VEGF, vascular endothelial growth factor; HIF-1α, hypoxia-inducible factor-1α; SMCs, smooth muscle cells; DEGs, differentially expressed genes; GEO, Gene Expression Omnibus; GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; FBS, fetal bovine serum; OD, optical density; BCA, bicinchoninic acid; PVDF, polyvinylidene fluoride; PBS, phosphate-buffered saline; BP, biological process; MF, molecular function; CC, cell component.

The MFF-SIRT1/3 axis, regulated by miR-340-5p, restores mitochondrial homeostasis of hypoxia-induced pulmonary artery smooth muscle cells

AbstractMitochondrial dynamics and quality control play a central role in the maintenance of the proliferation–apoptosis balance, which is closely related to the progression of pulmonary arterial hypertension (PAH). However, the exact mechanism of this balance remains unknown. Pulmonary artery smooth muscle cells (PASMCs) were cultured in hypoxia condition for constructing a PAH model in vitro. The expression of genes and proteins were determined by qRT-PCR and western bolt assays. Cell proliferation–apoptosis balance were tested by MTT, EdU and TUNEL assays. The mitochondrial functions were assessed by flow cytometry, JC-1, Mito tracker red staining, and corresponding kits. Besides, the molecular interaction was validated by dual-luciferase reporter assay. MFF was overexpressed in hypoxia-treated PAMSCs. Knockdown of MFF significantly repressed the excessive proliferation but enhanced cell apoptosis in hypoxia-treated PAMSCs. Moreover, MFF silencing improved mitochondrial function of hypoxia-treated PAMSCs by increasing ATP production and decreasing ROS release and mitochondrial fission. Mechanistically, MFF was a directly target of miR-340-5p, and could negatively regulate SIRT1/3 expression. Subsequently, functional rescue assays showed that the biological effects of MFF in hypoxia-treated PAMSCs were negatively regulated by miR-340-5p and depended on the regulation on SIRT1/3 pathway. These results provided evidences that miR-340-5p regulated MFF-SIRT1/3 axis to improve mitochondrial homeostasis and proliferation–apoptosis imbalance of hypoxia-treated PAMSCs, which provided a theoretical basis for the prevention and treatment of PAH.The imbalance between the proliferation and apoptosis of pulmonary arterial smooth muscle cells (PASMCs) is an important pathological process in pulmonary arterial hypertension (PAH). Mitochondrial dynamics and quality control play important roles in maintaining the cell proliferation–apoptosis balance. This paper reveals that miR-340-5p upregulates SIRT1/3 by targeting MFF, therefore improving mitochondrial dysfunction to maintain the proliferation–apoptosis balance of hypoxia-treated PAMSCs.

Decrease in LINC00963 attenuates the progression of pulmonary arterial hypertension via microRNA-328-3p/profilin 1 axis.

BackgroundPulmonary arterial hypertension (PAH) is a severe cardiopulmonary disease characterized by vascular hyperplasia and remodeling. Long noncoding RNA LINC00963 can regulate cell proliferation and metastasis in nonsmall cell lung cancer. However, the function of LINC00963 on PAH progression is rarely reported.MethodsQuantitative real‐time PCR was used to determine the expression levels of LINC00963, microRNA (miRNA)‐328‐3p, and profilin 1 (PFN1), as well as vascular endothelial growth factor (VEGF), fibroblast growth factor 2 (FGF‐2), and hypoxia‐inducible factor (HIF)‐α. The protein level of PFN1 was measured by western blotting. The viability and migration of hypoxia‐induced pulmonary arterial smooth muscle cells (PASMCs) were assessed by 3‐(4, 5‐dimethyl‐2‐thiazolyl)‐2, 5‐diphenyl‐2‐h‐tetrazolium bromide, and transwell assays, respectively. The target relationships between miR‐328‐3p and LINC00963/PFN1 were confirmed by dual‐luciferase reporter assay. A PAH mouse model was conducted to explore the effects of hypoxia on cardiopulmonary functions.ResultsIn hypoxia‐induced PASMCs and PAH mouse model, high expression levels of LINC00963 and PFN1, and low expression of miR‐328‐3p, were determined. The viability, migration of hypoxia‐induced PASMCs, the expression of VEGF, FGF‐2, and HIF‐α were significantly repressed by transfection of si‐LINC00963 or miR‐328‐3p mimics. The inhibitory effects of LINC00963 silencing on cell viability, migration, and the levels of VEGF, FGF‐2, and HIF‐α were partly eliminated by miR‐328‐3p inhibitor or increasing the expression of PFN1. Hypoxia treatment increased the levels of RVSP, mPAP, and RV/(LV+S), as well as the thickness of pulmonary artery wall.ConclusionsSilencing of LINC00963 ameliorates PAH via modulating miR‐328‐3p/PFN1.

Tanreqing Injection Regulates Cell Function of Hypoxia-Induced Human Pulmonary Artery Smooth Muscle Cells (HPASMCs) through TRPC1/CX3CL1 Signaling Pathway.

Hypoxia-induced pulmonary arterial hypertension (HPAH) is due to hypoxia caused by vascular endothelial cell remolding and damage. Previous studies have suggested that CX3CL1 plays an important role in HPAH which is affected by oxidative stress. Ca2+ channel activation correlated with increasing NF-κB levels induced by ROS. Tanreqing injection (TRQ) is a traditional Chinese medicine (TCM) for acute upper respiratory tract infection and acute pneumonia. In the present study, we explored the effect of TRQ on human pulmonary artery smooth muscle cells (HPASMCs) undergoing hypoxia and feasible molecular mechanisms involved in. Cell proliferation was assayed using CCK8 kits. Immunofluorescence and western blotting along with ELISA assay were performed to investigate the effect of TRQ on hypoxia-induced ROS, Ca2+, hydroxyl free radicals, and the expression of Ca2+ channel protein TRPC1, CX3CR1, HIF-1α, NF-κBp65, and p-NF-κBp65 in HPASMCs. Human CX3CL1 and the inhibitor of TRPC1 as SKF96365 were used for further investigation. TRQ inhibited hypoxia-induced increasing cell adhesion, ROS, Ca2+, hydroxyl free radicals, CX3CR1, HIF-1α, NF-κBp65 activation, and even on TRPC1 expression in HPASMC which tended to be attenuated even reversed by CX3CL1. Our results suggested that TRQ might help to attenuate remodeling of HPASMC through inhibiting the ROS and TRPC1/CX3CL1 signaling pathway.

Ubiquitinated AIF is a major mediator of hypoxia-induced mitochondrial dysfunction and pulmonary artery smooth muscle cell proliferation

BackgroundExcessive proliferation of pulmonary artery smooth muscle cells (PASMCs) is the main cause of hypoxic pulmonary hypertension (PH), and mitochondrial homeostasis plays a crucial role. However, the specific molecular regulatory mechanism of mitochondrial function in PASMCs remains unclear.MethodsIn this study, using the CCK8 assay, EdU incorporation, flow cytometry, Western blotting, co-IP, mass spectrometry, electron microscopy, immunofluorescence, Seahorse extracellular flux analysis and echocardiography, we investigated the specific involvement of apoptosis-inducing factor (AIF), a mitochondrial oxidoreductase in regulating mitochondrial energy metabolism and mitophagy in PASMCs.ResultsIn vitro, AIF deficiency in hypoxia leads to impaired oxidative phosphorylation and increased glycolysis and ROS release because of the loss of mitochondrial complex I activity. AIF was also downregulated and ubiquitinated under hypoxia leading to the abnormal occurrence of mitophagy and autophagy through its interaction with ubiquitin protein UBA52. In vivo, treatment with the adeno-associated virus vector to overexpress AIF protected pulmonary vascular remodeling from dysfunctional and abnormal proliferation.ConclusionsTaken together, our results identify AIF as a potential therapeutic target for PH and reveal a novel posttranscriptional regulatory mechanism in hypoxia-induced mitochondrial dysfunction.