NDUFA4L2 in smooth muscle promotes vascular remodeling in hypoxic pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is characterized by a progressive increase in pulmonary vascular resistance and obliterative pulmonary vascular remodelling (PVR). The imbalance between the proliferation and apoptosis of pulmonary artery smooth muscle cells (PASMCs) is an important cause of PVR leading to PAH. Mitochondria play a key role in the production of hypoxia-induced pulmonary hypertension (HPH). However, there are still many issues worth studying in depth. In this study, we demonstrated that NADH dehydrogenase (ubiquinone) 1 alpha subcomplex 4 like 2 (NDUFA4L2) was a proliferation factor and increased in vivo and in vitro through various molecular biology experiments. HIF-1α was an upstream target of NDUFA4L2. The plasma levels of 4-hydroxynonene (4-HNE) were increased both in PAH patients and hypoxic PAH model rats. Knockdown of NDUFA4L2 decreased the levels of malondialdehyde (MDA) and 4-HNE in human PASMCs in hypoxia. Elevated MDA and 4-HNE levels might be associated with excessive ROS generation and increased expression of 5-lipoxygenase (5-LO) in hypoxia, but this effect was blocked by siNDUFA4L2. Further research found that p38-5-LO was a downstream signalling pathway of PASMCs proliferation induced by NDUFA4L2. Up-regulated NDUFA4L2 plays a critical role in the development of HPH, which mediates ROS production and proliferation of PASMCs, suggesting NDUFA4L2 as a potential new therapeutic target for PAH.

Metformin Prevents Progression of Experimental Pulmonary Hypertension via Inhibition of Autophagy and Activation of Adenosine Monophosphate-Activated Protein Kinase.

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.

LncRNA-TCONS_00034812 in cell proliferation and apoptosis of pulmonary artery smooth muscle cells and its mechanism.

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.

The lncRNA PAHRF functions as a competing endogenous RNA to regulate MST1 expression by sponging miR-23a-3p in pulmonary arterial hypertension.

BACKGROUND: Emerging evidence has shown that long non-coding RNA (lncRNA) plays important roles in the development of pulmonary arterial hypertension (PAH). However, some new lncRNAs in patients with PAH are still lacking research. Herein, we examined the expression and role of lncRNA (pulmonary arterial hypertension related factor, PAHRF) in PAH. METHODS: LncRNA PAHRF expression and localization were analyzed by realtime PCR and fluorescence in situ hybridization. Proliferation and apoptosis were detected by MTT, CCK-8, EDU staining, JC-1 assay, flow cytometry and western blotting. Luciferase activity assay was used to identify PAHRF/ miR-23a-3p/serine/threonine kinase 4 (STK4/MST1) interaction. RESULTS: LncRNA PAHRF was down-regulated in both the PAs of PAH patients and hypoxic human pulmonary artery smooth muscle cells (PASMCs). The overexpression of PAHRF inhibited the proliferation and promoted the apoptosis of PASMCs. Similarly, we also found PAHRF overexpression decreased the proliferation under hypoxia condition. Knockdown of PAHRF exerted the opposite effects. Luciferase activity assay proved molecular binding between PAHRF and hsa-miR-23a-3p. Moreover, MST1 was confirmed to be the putative target gene and regulated by PAHRF/miR-23a-3p. In addition, we explored the molecular mechanism regulating the expression of miR-23a-3p, and found that lncRNA PAHRF acted as an endogenous sponge for miR-23a-3p, and silencing lncRNA PAHRF could up-regulate the expression of miR-23a-3p. On the contrary, PAHRF-overexpressing plasmid inhibited the expression of miR-23a-3p in hypoxia. CONCLUSIONS: Our present study reveals a novel PAH regulating model that is composed of PAHRF, miR-23a-3p, and MST1. The aim of this study is probably going to provide a new explanation and give a further understanding of the occurrence of vascular remodeling in PAH from the perspective competing endogenous RNA hypothesis.

Baicalin inhibits proliferation and promotes apoptosis of vascular smooth muscle cells by regulating the MEG3/p53 pathway following treatment with ox­LDL.

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 anti­proliferative 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 low­density lipoprotein (ox­LDL)­treated human aorta vascular smooth muscle cells (HA­VSMCs). The siRNA transfection and overexpression efficiency of endogenous maternally expressed gene 3 (MEG3) and the expression level of MEG3 were analyzed by reverse transcription­quantitative polymerase chain reaction (RT­qPCR). The effects of alterations in expression levels of MEG3 were assessed by MTT assay, bromodeoxyuridine incorporation assay, 5­ethynyl­2'­deoxyuridine staining, wound healing assay, immunofluorescence and western blotting in HA­VSMCs. qPCR indicated that the expression of MEG3 was reduced in serum samples from patients with AS and ox­LDL­treated HA­VSMCs, compared with serum samples from healthy patients and untreated HA­VSMCs, respectively. Further experiments indicated that ox­LDL­induced decrease of MEG3 expression was reversed by treatment with baicalin in a concentration­dependent manner. Following treatment with ox­LDL, decreased expression of MEG3 promoted proliferation and migration, and suppressed apoptosis in HA­VSMCs. Furthermore, treatment with baicalin reversed these effects on proliferation and apoptosis in ox­LDL­treated HA­VSMCs. The current study indicated that downregulated expression of MEG3 increased cell cycle­associated protein expression. However, treatment with baicalin inhibited the expression of cell­cycle associated proteins in HA­VSMCs 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 ox­LDL­treated HA­VSMCs. 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.

Transforming growth factor-beta1 upregulation triggers pulmonary artery smooth muscle cell proliferation and apoptosis imbalance in rats with hypoxic pulmonary hypertension via the PTEN/AKT pathways.

Transforming growth factor-beta1 (TGFß1) and Phosphatase and Tensin homolog deleted on chromosome ten (PTEN) are involved in the regulation of proliferation, differentiation, migration and apoptosis of various cell types. In previous studies, we have shown that TGFß1 and PTEN play an important role in the progression of pulmonary vascular remodeling induced by pulmonary artery smooth muscle cells (PASMCs). However, the mechanisms involved in the activation of PASMCs between TGFß1 and PTEN pathways remain unknown. We found that pulmonary vascular walls in hypoxic pulmonary arterial hypertension (PAH) rats were thicker than the vessels from normal rats in vivo. Substantially higher levels of TGFß1 and significant loss of PTEN expression were observed in the lungs of PAH rats when compared with normoxia. Meanwhile, AKT, a downstream proliferative signaling protein of the PTEN antagonist PI3K, was markedly activated in the lungs of PAH rats. In vitro studies using PASMCs showed that TGFß1 increased cell proliferation in PTEN-dependent manner. Moreover, we found that TGFß1 enhanced cell survival, up-regulated the expression of Bcl-2 and procaspase-3, decreased the number of TUNEL-positive cells and caspase-3 expression in PASMCs under serum-deprived (SD) condition via PI3K/AKT pathway. The results further establish that TGFß1 promoted PAH by decreasing PTEN expression and increasing PI3K/AKT activation in the lung. In conclusion, TGFß1 mediated PTEN inactivation and resistance to apoptosis seems to be key mediators of lung vascular remodeling associated with PAH. These findings further clarify molecular mechanisms that support targeting PTEN/AKT signaling pathway to attenuate pathogenic derangements in PAH.

Detection of mRNAs of GA733 genes by RT-PCR in exfoliated cells of pleural and peritoneal effusions and its clinical values.

OBJECTIVE: To evaluate the diagnostic values of the detection of mRNAs of GA733 gene family in exfoliated cells of pleural and peritoneal effusions. METHODS: Sixty specimens of pleural and peritoneal fluids from 60 patients were collected. GA733-1 and GA733-2 mRNA in the exfoliated cells were detected by qualitative RT-PCR, and their diagnostic values were assessed. Patients Sixty patients with pleural or peritoneal effusions, from May 2003 and August 2004, aged 23-85 (average 56.5 years). RESULTS: GA733-1 and GA733-2 mRNA were positive in 5 (13.9%) and 27 (75.0%) of 36 malignant specimens, and in 1 and 7 of 11 cause-unknown specimens, respectively, but both of them were negative in all 13 benign specimens, and the difference of GA733-2 mRNA positive rates among the three groups was significant (P<0.005), but that of GA733-1 mRNA was not (P>0.05). GA733-1 and GA733-2 mRNA were positive in 4 (15.4%) and 22 (84.6%) of 26 cytological positive specimens, and in 1 and 6 of 6 suspicious specimens, and in 1 (3.5%) and 6 (21.4%) of 28 negative specimens, respectively and the difference of GA733-2 mRNA positive rates among the three groups was significant (P<0.005), but that of GA733-1 mRNA was not (P>0.05). Sensitivity, specificity and accuracy of detection for GA733-2 mRNA for diagnosis of malignant effusions were 75.0%, 100% and 81.6%, respectively. CONCLUSIONS: The detection of GA733-2 mRNA by qualitative RT-PCR is sensitive and highly specific for the diagnosis of malignant pleural and peritoneal effusions, while the diagnostic value of GA733-1 mRNA needs to be further investigated.