Background/Aims: Increasing evidence has demonstrated a significant role of long non-coding RNAs (lncRNAs) in diverse biological processes, and many of which are likely to have functional roles in vascular remodeling. However, their functions in pulmonary arterial hypertension (PAH) remain largely unknown. Pulmonary vascular remodeling is an important pathological feature of PAH, leading to increased vascular resistance and reduced compliance. Pulmonary artery smooth muscle cells (PASMCs) dysfunction is involved in vascular remodeling. Long noncoding RNAs are potential regulators of PASMCs function. Herein, we determined whether long noncoding RNA–maternally expressed gene 3 (MEG3) was involved in PAH-related vascular remodeling. Methods: The arterial wall thickness was examined by hematoxylin and eosin (H&E) staining in distal pulmonary arteries (PAs) isolated from lungs of healthy volunteers and PAH patients. The expression level of MEG3 was analyzed by qPCR. The effects of MEG3 on human PASMCs were assessed by cell counting Kit-8 assay, BrdU incorporation assay, flow cytometry, scratch-wound assay, immunofluorescence, and western blotting in human PASMCs. Results: We revealed that the expression of MEG3 was significantly downregulated in lung and PAs of patients with PAH. MEG3 knockdown affected PASMCs proliferation and migration in vitro. Moreover, inhibition of MEG3 regulated the cell cycle progression and made more smooth muscle cells from the G₀/G₁ phase to the G₂/M+S phase and the process could stimulate the expression of PCNA, Cyclin A and Cyclin E. In addition, we found that the p53 pathway was involved in MEG3–induced smooth muscle cell proliferation. Conclusions: This study identified MEG3 as a critical regulator in PAH and demonstrated the potential of gene therapy and drug development for treating PAH.

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Long noncoding RNAs (lncRNAs) have been discovered to be playing important role in various biological processes. However, the contribution of lncRNAs to pulmonary artery hypertension (PAH) remains largely unknown. Pulmonary vascular remodeling is an important pathological feature of PAH, leading to increased vascular resistance and reduced compliance. Here, we investigated the biological role of lncRNAs in PAH. Differences in the lncRNAs and mRNAs between hypoxia PAH rats and normoxia rats were screened using microarray analysis. The results showed that 36 lncRNAs and 519 mRNAs were upregulated in the pulmonary arteries (PAs) of hypoxia PAH rats, whereas 111 lncRNAs and 246 mRNAs were downregulated. Expressions of the screened lncRNAs, including TCONS_00034812, were validated by real-time PCR. We revealed that the expression of TCONS_00034812 was significantly downregulated in PAs of PAH rats and hypoxia pulmonary artery smooth muscle cells (PASMCs). TCONS_00034812 knockdown promoted proliferation and inhibited apoptosis of PASMCs in vitro. Moreover, TCONS_00034812 regulated PASMCs function in vitro. We found that TCONS_00034812 increased the expression of transcription factors Stox1. TCONS_00034812 and Stox1 knockdown mediated PASMCs function through MAPK signaling. Our findings imply lncRNA as a critical regulator in PAH and demonstrate the potential of gene therapy and drug development for treating PAH. The present study reveals a novel mechano responsive lncRNA-TCONS_00034812, which modulates PASMCs proliferation and apoptosis, and participates in vascular remodeling during PAH.

Previous studies have shown that metformin (MET) prevents experimental pulmonary arterial hypertension (PAH) and that activation of autophagy is involved in the development of pulmonary vascular remodeling. However, the mechanism of how MET inhibits autophagy and reverses pulmonary vascular remodeling is still unclear. The objective of the present study was to investigate the role of autophagy in MET-induced hypoxia PAH protection and the underlying mechanisms. To examine the effects of MET on hypoxia, we treated rats with MET (100 mg/kg/day) after 3 weeks of hypoxia. Hemodynamic changes, weight of the right ventricle/left ventricle plus septum (RV/LV+S) ratio, and lung morphological features were examined after 3 weeks. In addition, alpha smooth muscle actin (α-SMA), p62, and PCNA were assessed by immunofluorescence and immunohistochemistry staining. BECN-1, LC3B, p62, and activation of adenosine monophosphate-activated protein kinase (AMPK) were analyzed by Western blotting. Cell proliferation was detected using the Cell Counting Kit-8 (CCK-8) and the 5-ethynyl-2′-deoxyuridine staining kit assay. Hypoxia induced increases in right ventricular systolic pressure and the RV/LV+S ratio, which were attenuated by MET treatment. MET also inhibited hypoxia-induced pulmonary vascular remodeling, collagen deposition, proliferation of pulmonary arterial smooth muscle cells, elevation of BECN-1 and the LC3B-II/-I ratio, and downregulation of p62. Further studies found that this process was mediated by inhibition of autophagy and activation of the AMPK signaling pathway.

Atherosclerosis (AS) is a systemic disease associated with lipid metabolic disorders and abnormal proliferation of smooth muscle cells. Baicalin is a flavonoid compound isolated from the dry roots of Scutellaria baicalensis Georgi and exerts anti-proliferative effects in various types of cells. However, the effect of baicalin on AS remains unclear. In the present study, serum samples were collected from patients with AS and an in vitro model of AS was established using oxidized low-density lipoprotein (ox-LDL)-treated human aorta vascular smooth muscle cells (HA-VSMCs). The siRNA transfection and overexpression efficiency of endogenous maternally expressed gene 3 (MEG3) and the expression level of MEG3 were analyzed by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The effects of alterations in expression levels of MEG3 were assessed by MTT assay, bromodeoxyuridine incorporation assay, 5-ethynyl-2′-deoxyuridine staining, wound healing assay, immunofluorescence and western blotting in HA-VSMCs. qPCR indicated that the expression of MEG3 was reduced in serum samples from patients with AS and ox-LDL-treated HA-VSMCs, compared with serum samples from healthy patients and untreated HA-VSMCs, respectively. Further experiments indicated that ox-LDL-induced decrease of MEG3 expression was reversed by treatment with baicalin in a concentration-dependent manner. Following treatment with ox-LDL, decreased expression of MEG3 promoted proliferation and migration, and suppressed apoptosis in HA-VSMCs. Furthermore, treatment with baicalin reversed these effects on proliferation and apoptosis in ox-LDL-treated HA-VSMCs. The current study indicated that downregulated expression of MEG3 increased cell cycle-associated protein expression. However, treatment with baicalin inhibited the expression of cell-cycle associated proteins in HA-VSMCs with MEG3 knockdown. In addition, baicalin activated the p53 signaling pathway and promoted the expression and transport of p53 from the cytoplasm to nucleus following MEG3 knockdown in ox-LDL-treated HA-VSMCs. Baicalin inhibited proliferation and promoted apoptosis by regulating the expression of MEG3/p53, indicating that baicalin may serve a role in AS by activating the MEG3/p53 signaling pathway. The present study suggested a potential mechanism underlying the protective role of baicalin in the in vitro model of AS, and these results may be used to develop novel therapeutic approaches for the affected patients.

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Methylophiopogonanone A (MOA) is a naturally occurring homoisoflavonoid from the Chinese herb Ophiopogon japonicus, which has been demonstrated to attenuate myocardial apoptosis. However, the metabolism of MOA remains unknown. The goal of the present work was to investigate the in vitro metabolism of MOA using liver microsomes and hepatocytes. Methods:The metabolites were generated by incubating MOA with rat, monkey and human liver microsomes or hepatocytes. The resulting samples were analyzed by using a quadrupole-orbitrap high-resolution mass spectrometer. The metabolites were identified through the measurements of the exact mass, elemental composition and product ions.Results: A total of 15 metabolites were detected and identified. Among these metabolites, M7 (demethylenation) was the most abundant metabolite in liver microsomes, while M6 (hydroxylation) was the predominant metabolite in hepatocytes, and glucuronidation metabolites (M9 and M10) were also the main metabolites in hepatocytes. The metabolic pathways of MOA included hydroxylation, demethylenation, glucuronidation, methylation, sulfation and glutathione conjugation.Conclusions: This study for the first time provides valuable data on the metabolites of MOA, which will be of great importance for a better understanding of its disposition and to predict human pharmacokinetics.