Apoptosis Of Pulmonary Cells Research Articles

Baicalein exerts a protective role in pneumonia caused by Streptococcus pneumoniae.

Streptococcus pneumoniae is the most common pathogenic bacterium of pneumonia and can cause death. Here, we examined the impact of Baicalein on mouse mortality caused by Streptococcus pneumoniae infection. Baicalein protected mice from Streptococcus pneumoniae-induced deaths. Baicalein reduced alveolocapillary membrane permeability, lung infiltration of inflammatory cells, inflammatory cytokine production and pulmonary cell apoptosis. These evidences show that Baicalein is useful in the therapy of a severe form of pneumonia that can cause deaths.

3-Bromopyruvate Attenuates Experimental Pulmonary Hypertension via Inhibition of Glycolysis.

The shift of metabolism from mitochondrial oxidative phosphorylation to glycolysis and mitochondria binding partner of hexokinase are features common to cancer. These have been seen in pulmonary hypertension (PH) as well. An inhibitor of hexokinase 2 (HK 2), the small molecule 3-bromopyruvate (3-BrPA) is an incredibly powerful and swift-acting anticancer agent. However, whether it could be of potential benefit to PH has still been unknown. Sprague-Dawley rats with monocrotaline (MCT)-induced PH were administered 2 oral doses of 3-BrPA (15 and 30 mg/kg/day, respectively) for 14 days. Hemodynamic parameters were obtained by right heart catheterization. Histopathology, immunohistochemistry, transmission electron microscopy, flow cytometry, and assessments of relative protein expressions were conducted. Compared with MCT treatment, 3-BrPA decreased mean pulmonary arterial pressure and pulmonary vascular resistance, and increased cardiac output. 3-BrPA significantly suppressed proliferation in addition to enhancing apoptosis of pulmonary artery smooth muscle cells, attenuating small pulmonary artery remodeling and right ventricular hypertrophy. Treatment with 3-BrPA markedly reduced the mitochondrial membrane potential and restored mitochondrial structure. Furthermore, 3-BrPA significantly inhibited HK 2 expression but not HK 1. The expression of both pyruvate dehydrogenase kinase and lactate dehydrogenase was decreased whereas that of pyruvate dehydrogenase and cytosolic cytochrome c was upregulated with 3-BrPA administration. This study demonstrates the reversal of PH by 3-BrPA is related to alteration in glycolysis and improved mitochondria function, indicating the "metabolic targeting" as a rational therapeutic strategy for PH.

A Potential Role for Exosomal Translationally Controlled Tumor Protein Export in Vascular Remodeling in Pulmonary Arterial Hypertension.

Pulmonary arterial hypertension (PAH) is characterized by increased proliferation and resistance to apoptosis of pulmonary vascular cells. Increased expression of translationally controlled tumor protein (TCTP), a prosurvival and antiapoptotic mediator, has recently been demonstrated in patients with heritable PAH; however, its role in the pathobiology of PAH remains unclear. Silencing of TCTP in blood outgrowth endothelial cells (BOECs) isolated from control subjects led to significant changes in morphology, cytoskeletal organization, increased apoptosis, and decreased directionality during migration. Because TCTP is also localized in extracellular vesicles, we isolated BOEC-derived extracellular vesicles (exosomes and microparticles) by sequential ultracentrifugation. BOECs isolated from patients harboring BMPR2 mutations released more exosomes than those derived from control subjects in proapoptotic conditions. Furthermore, TCTP expression was significantly higher in exosomes than in microparticles, indicating that TCTP is mainly exported via exosomes. Coculture assays demonstrated that exosomes transferred TCTP from ECs to pulmonary artery smooth muscle cells, suggesting a role for endothelial-derived TCTP in conferring proliferation and apoptotic resistance. In an experimental model of PAH, rats treated with monocrotaline demonstrated increased concentrations of TCTP in the lung and plasma. Consistent with this finding, we observed increased circulating TCTP levels in patients with idiopathic PAH compared with control subjects. Therefore, our data suggest an important role for TCTP in regulating the critical vascular cell phenotypes that have been implicated in the pathobiology of PAH. In addition, this research implicates TCTP as a potential biomarker for the onset and development of PAH.

MiR‑760 mediates hypoxia-induced proliferation and apoptosis of human pulmonary artery smooth muscle cells via targeting TLR4.

MicroRNAs (miRNAs) have a key role in the pathogenesis of pulmonary arterial hypertension (PAH), a disease characterized by enhanced proliferation and reduced apoptosis of pulmonary artery smooth muscle cells. In the present study, miR-760 was demonstrated to be downregulated in PAH lung tissues compared with normal lung tissues, an effect that may be associated with the development of PAH. Hypoxia is an important stimulus for human pulmonary artery smooth muscle cell (hPASMC) proliferation and the occurrence of PAH. Therefore, the effect of miR-760 in hypoxia-treated and normal hPASMCs was investigated. Expression of exogenous miR-760 decreased cell proliferation in hypoxia-induced hPASMCs, and promoted cell apoptosis with an increase in the BCL2 associated X/BCL2 ratio and the expression levels of Caspase-3 and Caspase-9. In addition, overexpression of miR-760 suppressed the migration of hPASMCs under hypoxic conditions. Furthermore, miR-760 was demonstrated to mediate its anti-proliferation effect via the regulation of toll-like receptor 4 (TLR4), a direct target of miR-760. The results revealed that knockdown of TLR4 restrained the hypoxia-induced hPASMC proliferation and induced cell apoptosis. The present study uncovered a novel regulatory pathway involving miR-760 and suggested that miR-760 may be explored as a potential therapy for PAH in the future.

Possible mechanisms mediating apoptosis of bronchial epithelial cells in chronic obstructive pulmonary disease – A next-generation sequencing approach

PurposeChronic obstructive pulmonary disease (COPD) is a chronic inflammatory airway disease characterized by persistent airflow limitation. Apoptosis of pulmonary structural cells contributes to pulmonary destruction and dysfunction. This study aimed to explore the possible mechanisms underlying decreased cell proliferation and increased apoptosis of bronchial epithelial cells of COPD. Materials and methodsThe expression profiles of mRNAs and microRNAs in bronchial epithelial cells from a COPD patient and a normal subject were identified using next-generation sequencing (NGS) and analyzed using bioinformatic tools. ResultsWe identified 233 significantly upregulated and 204 significantly downregulated genes in COPD bronchial epithelial cells. The PI3K-Akt pathway was one of the most important dysregulated pathways in bronchial epithelial cells. We further identified that 3 genes involved in the PI3K-Akt signaling pathway, including IL6, F2R, and FGFR3, might be associated with inhibition of cell proliferation in bronchial epithelial cells, while 5 genes involved in the PI3K-Akt signaling pathway, including TLR4, IL6, F2R, FGFR3, and FGFR1, might be associated with apoptosis of bronchial epithelial cells. FGFR1 was also a predicted target for some up-regulated miRNAs in COPD bronchial epithelial cells, including hsa-miR-195-5p, hsa-miR-424-5p, and hsa-miR-6724-5p. ConclusionOur findings suggest PI3K-Akt signaling pathway plays an important role in COPD. We observed altered expression of apoptosis and cell proliferation-related genes that might contribute to the pathogenesis of COPD.

Shikonin‑mediated inhibition of nestin affects hypoxia‑induced proliferation of pulmonary artery smooth muscle cells.

The imbalance between the proliferation and apoptosis of pulmonary artery smooth muscle cells (PASMCs) is of importance in pulmonary vascular remodeling. Shikonin, a naphthoquinone compound extracted from the Chinese medicinal herb Lithospermumerythrorhizon, inhibits the proliferation of rat smooth muscle cells (SMCs). The present study was designed to investigate the effects of shikonin on the proliferation of rat PASMCs and the possible mechanisms involved. Rat PASMCs were cultured under the following five treatment conditions: Normal control; hypoxia for 24h; hypoxia + 1µM shikonin for 24h; hypoxia + 2µM shikonin for 24h; and hypoxia + 4µM shikonin for 24h. The viability of PASMCs was measured using the Cell Counting Kit‑8 assay, the mRNA expression of nestin (NES) in each group was measured by reverse transcription‑polymerase chain reaction and the protein expression of NES was measured by western blotting. The proliferation of hypoxic PASMCs transfected with NES‑specific small interfering (si)RNA decreased compared with the non‑transfected group. These results indicated that hypoxia induced the proliferation of PASMCs through the enhancement of NES expression. The treatment of hypoxic PASMCs with shikonin resulted in a significant downregulation of NES expression and the inhibition of PASMC proliferation.

Mitochondrial HSP90 Accumulation Promotes Vascular Remodeling in Pulmonary Arterial Hypertension.

Pulmonary arterial hypertension (PAH) is a vascular remodeling disease with a poor prognosis and limited therapeutic options. Although the mechanisms contributing to vascular remodeling in PAH are still unclear, several features, including hyperproliferation and resistance to apoptosis of pulmonary artery smooth muscle cells (PASMCs), have led to the emergence of the cancer-like concept. The molecular chaperone HSP90 (heat shock protein 90) is directly associated with malignant growth and proliferation under stress conditions. In addition to being highly expressed in the cytosol, HSP90 exists in a subcellular pool compartmentalized in the mitochondria (mtHSP90) of tumor cells, but not in normal cells, where it promotes cell survival. We hypothesized that mtHSP90 in PAH-PASMCs represents a protective mechanism against stress, promoting their proliferation and resistance to apoptosis. Expression and localization of HSP90 were analyzed by Western blot, immunofluorescence, and immunogold electron microscopy. In vitro, effects of mtHSP90 inhibition on mitochondrial DNA integrity, bioenergetics, cell proliferation and resistance to apoptosis were assessed. In vivo, the therapeutic potential of Gamitrinib, a mitochondria-targeted HSP90 inhibitor, was tested in fawn-hooded and monocrotaline rats. We demonstrated that, in response to stress, HSP90 preferentially accumulates in PAH-PASMC mitochondria (dual immunostaining, immunoblot, and immunogold electron microscopy) to ensure cell survival by preserving mitochondrial DNA integrity and bioenergetic functions. Whereas cytosolic HSP90 inhibition displays a lack of absolute specificity for PAH-PASMCs, Gamitrinib decreased mitochondrial DNA content and repair capacity and bioenergetic functions, thus repressing PAH-PASMC proliferation (Ki67 labeling) and resistance to apoptosis (Annexin V assay) without affecting control cells. In vivo, Gamitrinib improves PAH in two experimental rat models (monocrotaline and fawn-hooded rat). Our data show for the first time that accumulation of mtHSP90 is a feature of PAH-PASMCs and a key regulator of mitochondrial homeostasis contributing to vascular remodeling in PAH.

Effects of PKCα/c-fos, Bax/Bcl-2 expression on the proliferation and apoptosis of rat pulmonary arterial smooth muscle cells in hypoxia

To explore the effects of PKCα/c-fos, Bax/Bcl-2 on the proliferation and apoptosis of rat pulmonary arterial smooth musclecells(PASMCs) in hypoxia. The PASMCs of rats had been isolated and cultured, and then were cultured under normoxia (5%CO2、21%O2、74%N2)and hypoxia(5%CO2,2% O2,93%N2)condition for 24 h and 48 h, respectively. The proliferation of PASMCs was tested by methylthiazolyltetrazolium (MTT). The changes of PASMCs apoptosis were detected by Annexin V-FITC/PI staining combined flow cytometry. RT-PCR and Western blot analysis were performed to detect the gene and protein levels of PKCα/c-fos, Bax/Bcl-2, respectively. All experiments were repeated three times with at least triplicate samples. The proliferation of PASMCs in hypoxia group were stronger than that of normoxia group after 24 h and 48 h (P < 0.01), while the apoptosis rate did not change significantly. Meanwhile, the higher levels of PKCα, c-fos, Bcl-2 mRNA and proteins after hypoxia for 24 h and 48 h were detected by RT-PCR and Western blot analysis, while the ex-pression levels of Bax had no significant difference under normoxic and hypoxic conditions. Elevated proliferation and expression of PKCα, c-fos, Bcl-2 was observed in rat PASMCs in hypoxia, and while the apoptosis rate had no significant change.

Regulatory role of autophagy in development of pulmonary artery hypertension

Pulmonary arterial hypertension (PAH) is a multi-etiological chronic disease characterized by a progressive elevation in pulmonary resistance and vascular remodeling. Its pathogenesis is complicated. Recently, emerging researches suggest that autophagy, as a self-protection mechanism maintaining the intracellular environment homeostasis in eukaryotes, participate in the occurrence and development of various types of PAH. Autophagy can regulate the survival, apoptosis of pulmonary vascular wall cells and secretion of vasoactive substances and inflammatory cytokines, thus influencing pulmonary vascular homeostasis. Some drugs based on regulating autophagy activity can effectively improve the prognosis of PAH. In this article, the regulatory role of autophagy on the development of pulmonary hypertension is reviewed to provide insight into PAH and its treatment.

Topotecan alleviates ventilator-induced lung injury via NF-κB pathway inhibition

ObjectiveWe investigated the effect of topotecan on injury and inflammation in a model of ventilator-inducedlunginjury (VILI). MethodsAcute lung injury (ALI) was induced in mice by high-tidal volume ventilation, and the mice were then treated with topotecan or PBS. Lung tissue and bronchoalveolar lavage fluid were collected to assess pulmonary vascular leaks, inflammation, and cell apoptosis. ResultsCompared to PBS treatment, topotecan significantly decreased the ALI score, myeloperoxidase (MPO) content, total protein concentration, and presence of inflammatory cells and inflammatory cytokines in bronchoalveolar lavage fluid. Topotecan also reduced caspase-3 activation and type Ⅱ alveolar epithelial cell apoptosis. Moreover, topotecan inhibited NF-κB expression and activation in the VILI model. ConclusionTopotecan alleviates acute lung injury in the model of VILI through the inhibition of the NF-κB pathway.

Rutaecarpine Suppresses Proliferation and Promotes Apoptosis of Human Pulmonary Artery Smooth Muscle Cells in Hypoxia Possibly Through HIF-1α-Dependent Pathways.

The aim of this study is to investigate the potential roles of Rutaecarpine (Rut) in hypoxia-induced human pulmonary artery smooth muscle cells (HPASMCs) model. HPASMCs were cultured with or without hypoxia followed by Rut administration. Cytotoxicity and cell proliferation were assessed by CCK-8 and Cell counting method. Flow cytometry was used for the measurement of cell apoptosis rates. The mRNA expression of hypoxia-induced factor (HIF)-1α and protein levels of HIF-1α, p53, p21, erythropoietin, and vascular endothelial growth factor were determined by quantitative real-time polymerase chain reaction and Western blot, respectively. Rut inhibited the proliferation of HPASMCs with IC50 value of 43.5 μmol·L. Hypoxia significantly increased proliferation and decreased apoptosis in HPASMCs, whereas Rut rescued this phenomenon at the appropriate concentration. Meanwhile, Rut effectively decreased the protein and mRNA expressions of HIF-1α. Knockdown of HIF-1α expression by small interfering RNA (siRNA) significantly enhanced the proapoptotic effect rather than antiproliferation effect of Rut in HPASMCs. Moreover, Rut simultaneously reduced proliferating cell nuclear antigen protein expression, whereas increased p53 and p21 protein levels. However, no significant difference was observed in the protein levels of vascular endothelial growth factor and erythropoietin. Our results demonstrated that Rut exerted protective effects on HPASMCs against hypoxia partly through the HIF-1α-dependent signaling pathway.

A novel role of endocan in alleviating LPS-induced acute lung injury

AimsEndotoxin induced acute lung injury (ALI) is a critical complication of some clinical illnesses. Endothelial cell dysfunction and excessive pro-inflammation cytokine release are pivotal to the injury of alveolar-capillary membrane which is the typical characteristic of endotoxic lung injury. As a potential marker of endothelial cells, endocan plays an important role in many endothelial-dependent pathophysiological diseases. We speculated that endocan have anti-inflammatory property in ALI. Here, we investigated the role of endocan in LPS-induced ALI. Materials and methodsMice were randomly divided into 4 groups. LPS were used to construct ALI mice model by aerosolization for 20 min. Endocan was intraperitoneal injected at 30 min before LPS exposure. Levels of TNF-α, IFN-γ, IL-1β, IL-6 and MPO activities were detected by indicated ELISA. Cell apoptotic rate was determined by Annexin V/PI kit, ROS level and MPTP were detected by DCFH-DA and JC-1 kit, respectively. Seahorse XF96 was applied to evaluate the alteration of OCR and ECAR. Western blot and qRT-PCR were used to detect indicated molecules. Key findingsEndocan effectively decreased TNF-α, IFN-γ, IL-1β, and IL-6 levels as well as relieved pulmonary epithelium cell apoptosis caused by LPS exposure. Endocan significantly reversed LPS induced UPRmt and promoted cell metabolism reprogramming which were crucial for the protective characteristic of endocan in ALI mice model. SignificanceThe above findings suggested endocan could significantly suppress inflammatory response in ALI model through attenuating UPRmt associated apoptosis and switch cellular bioenergetics, indicating endocan could be considered as a promising compound against LPS induced ALI.

Aquaporin 1 regulates pulmonary arterial smooth muscle cell apoptosis

Pulmonary arterial hypertension (PAH) is a devastating disease, characterized by functional and structural vascular changes that lead to right heart failure and death. PAH pathogenesis involves remodeling of distal small diameter pulmonary arteries, including the development of vaso‐occlusive lesions featuring proliferation, migration and reduced apoptosis of pulmonary arterial smooth muscle cells (PASMCs). Patients often present with late disease, where targeting apoptosis to reverse remodeling would be of benefit; unfortunately, no such therapy currently exists. Apoptosis is a complex process, often involving disruption of the mitochondrial membrane and release of cytochrome c to activate the downstream effector, caspase 3, the final executioner of apoptosis. Formerly, we showed that the water channel protein, aquaporin 1 (AQP1), is a key molecule mediating PASMC proliferation and migration by increasing β‐catenin levels. Others have shown in different cell types that activating β‐catenin prevent apoptosis and improved cell survival. Therefore, in this study, we explored whether AQP1 also regulates PASMC survival and/or susceptibility to apoptosis. Primary cultured rat distal PASMCs were isolated from the SU5416‐hypoxia (SuHx) rat model of PAH or control rats. PASMCs from SuHx rats exhibited lack of apoptosis, measured by: 1) visualizing nuclear morphology via Hoechst 33342 staining; 2) cleaved caspase 3 expressions; and 3) caspase 3 activity, both at baseline and in response to the apoptotic stimuli staurosporine (ST) or H2O2. AQP1 mRNA and protein expression were significantly elevated in PASMCs from SuHx rats. Not surprisingly, β‐catenin levels were also elevated in these cells. Compared to non‐targeting siRNA, depleting AQP1 induced apoptosis at baseline in both control and SuHx rat PASMCs. Furthermore, reduction of AQP1 sensitized SuHx PASMCs to apoptotic stimuli. Our results indicate that AQP1 is a critical regulator of apoptosis in PASMCs, controlling caspase 3 activation, potentially through a pathway involving β‐catenin. Moreover, these findings suggest the possibility that targeting AQP1 might provide an attractive therapeutic option to both stop the progression of, and even reverse, vascular remodeling in PAH.Support or Funding InformationHL126514This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

MiR-125a-5p ameliorates monocrotaline-induced pulmonary arterial hypertension by targeting the TGF-\u03b21 and IL-6/STAT3 signaling pathways

Pulmonary vascular remodeling due to excessive proliferation and resistance to apoptosis of pulmonary artery smooth muscle cells (PASMCs) is the hallmark feature of pulmonary arterial hypertension (PAH). Recent evidence suggests that miR-125a-5p plays a role in a rat model of monocrotaline-induced PAH (MCT-PAH); however, the underlying mechanism is currently unknown. Here, we examined the expression profile of miR-125a-5p in MCT-PAH rats and investigated the putative therapeutic effect of miR-125a-5p using the miR-125a-5p agomir. In addition, the miR-125a-5p agomir or antagomir was transfected into rat PASMCs, and proliferation and apoptosis were measured. Activity of the miR-125a-5p target STAT3 was measured using a luciferase reporter assay, and the expression of downstream molecules was measured using RT–qPCR and/or western blot analysis. Importantly, inducing miR-125a-5p expression in vivo slowed the progression of MCT-PAH by reducing systolic pulmonary arterial pressure, the Fulton index, and pulmonary vascular remodeling. Moreover, overexpressing miR-125a-5p inhibited the proliferation and promoted the apoptosis of PASMCs. In addition, stimulating PASMCs with TGF-β1 or IL-6 upregulated miR-125a-5p expression, whereas overexpressing miR-125a-5p reduced TGF-β1 and IL-6 production, as well as the expression of their downstream targets STAT3 and Smad2/3; in contrast, downregulating miR-125a-5p increased TGF-β1 and IL-6 production. Finally, a dual-luciferase reporter assay revealed that miR-125a-5p targets the 3′-UTR of STAT3, suppressing the downstream molecules PCNA, Bcl-2, and Survivin. Taken together, these findings suggest that miR-125a-5p ameliorates MCT-PAH in rats, has a negative feedback regulation with TGF-β1 and IL-6, and regulates the proliferation and apoptosis of PASMCs by directly targeting STAT3.

Deregulation of microRNA‑31a‑5p is involved in the development of primary hypertension by suppressing apoptosis of pulmonary artery smooth muscle cells via targeting TP53.

The present study aimed to identify the association between microRNA (miRNA/miR)-31a-5p and the development of hypertension, and its potential molecular mechanism. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot analyses were performed to validate the candidate miRNA and genes involved in hypertension, following which an online miRNA database search, luciferase assay, and RT-qPCR and western blot analyses were performed to confirm the interaction between miR-31a-5p and TP53. A MTT assay and flow cytometric analysis were utilized to determine the effect of miR-31a-5p on cell growth and apoptosis. The results revealed that miR-31a-5p and TP53 were the candidate miRNA and gene regulating hypertension, and that TP53 was the virtual target gene of miR-31a-5p with a binding site located in the TP53 3′ untranslated region (3′UTR). It was confirmed by luciferase activity that miR-31a-5p markedly reduced the luciferase activity of the Luc-wild-type-TP53-3′UTR, whereas the mutated putative miR-31a-5p binding located on the TP53-3′UTR was found to eliminate such an inhibitory effect. miR-31a-5p had no effect on specificity protein 1, E2F transcription factor 2 or forkhead box P3 luciferase activity. Smooth muscle cells collected from spontaneously hypertensive rats treated with gold nano-particles containing anti-rno-miR-31a-5p exhibited a lower growth rate and a higher apoptotic rate. The results of the RT-qPCR and western blot analyses showed that miR-31a-5p negatively regulated the expression of TP53, and transfection with the hsa-miR-31a-5p mimic significantly promoted cell growth and inhibited cell apoptosis, whereas transfection with the anti-hsa-miR-31a-5p mimic significantly suppressed cell growth and induced cell apoptosis. Taken together, these findings indicated that miR-31a-5p is involved in hypertension via the accelerated proliferation of arterial smooth muscle cells and inhibition of apoptosis through targeting TP53.

Upregulation of Klotho potentially inhibits pulmonary vascular remodeling by blocking the activation of the Wnt signaling pathway in rats with PM2.5-induced pulmonary arterial hypertension.

We evaluated the effects of Klotho on pulmonary vascular remodeling and cell proliferation and apoptosis in rat models with PM2.5-induced pulmonary arterial hypertension (PAH) via the Wnt signaling pathway. After establishing rat models of PM2.5-induced PAH, these Sprague-Dawley male rats were randomized into control and model groups. Cells extracted from the model rats were sub-categorized into different groups. Activation of Wnt/β-catenin signaling transcription factor was detected by a TOPFlash/FOPFlash assay. A serial of experiment was conducted to identify the mechanism of Klotho on PHA via the Wnt signaling pathway. VEGF levels and PaCO2 content were higher in the model group, while PaO2, NO2- /NO3- content and Klotho level was lower compared to the control group. In comparison to the control group, the model group had decreased Klotho and Bax levels, and elevated Wnt-1, β-catenin, bcl-2, survivin, and PCNA expression, VEGF, IL-6, TNF-α, TNF-β1, and bFGF levels, as well as the percentage of pulmonary artery ring contraction. The Klotho vector, DKK-1 and DKK-1 + Klotho vector groups exhibited reduced cell proliferation, luciferase activity, and the expression of Wnt-1, β-catenin, bcl-2, survivin, and PCNA, as well as shortened S phase compared with the blank and NC groups. Compared with the Klotho vector and DKK-1 groups, the DKK-1 + Klotho vector groups had reduced cell proliferation, luciferase activity, and the expression of Wnt-1, β-catenin, bcl-2, survivin, and PCNA, as well as a shortened S phase. Conclusively, Klotho inhibits pulmonary vascular remodeling by inactivation of Wnt 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.

Inhibition of miR-361-5p suppressed pulmonary artery smooth muscle cell survival and migration by targeting ABCA1 and inhibiting the JAK2/STAT3 pathway

MicroRNAs play a crucial role in the progression of pulmonary arterial hypertension (PAH). The aim of this study was to investigate the effect of miR-361-5p on the proliferation, migration and apoptosis of pulmonary artery smooth muscle cells (PASMCs) that under the treatment of hypoxia and explore the underlying mechanisms. The results proved that hypoxia noticeably up-regulated the expression of miR-361-5p in PASMCs in comparison to the normoxia-treated cells, while TNF-α and IL-6 stimulation had no obvious effects on miR-361-5p level. Hypoxia induced miR-361-5p elevation in a HIF-1α-dependent manner. Inhibition of miR-361-5p dramatically inhibited hypoxia-induced cell proliferation and migration. miR-361-5p inhibition also rescued hypoxia exposure caused suppression of PASMCs apoptosis. In addition, the results showed that ABCA1 was a direct target of miR-361-5p and was down-regulated in hypoxia-induced PASMCs. Hypoxia and TNF-α or IL-6 stimulation significantly inhibited ABCA1 expression. In addition, overexpression of ABCA1 enhanced the effect of miR-361-5p on hPASMCs. Furthermore, the inhibition of miR-361-5p significantly down-regulated the expression level of p-JAK2 and p-STAT3. In conclusion, it may suggest that the suppression of miR-361-5p suppressed PASMC survival and migration by targeting ABCA1 and inhibiting the JAK2/STAT3 pathway.

Effect of miR-29b on the Proliferation and Apoptosis of Pulmonary Artery Smooth Muscle Cells by Targeting Mcl-1 and CCND2.

The proliferation and apoptosis of pulmonary artery smooth muscle cells (PASMCs) are considered to be key steps in the progression of pulmonary arterial hypertension (PAH). MicroRNAs (e.g., miR-29b) have been identified in various diseases to be critical modulators of cell growth and apoptosis by targeting Mcl-1 and CCND2. However, the role of miR-29b in PAH remains unknown. So we try to investigate the effect of miR-29b on Mcl-1 and CCND2 protein in PASMCs, analyze the effect of miR-29b on the proliferation of PASMCs, and explore the significance of miR-29b in the proliferation, apoptosis, and gene therapy of PAH. It was observed that gene chip analysis showed miR-29b expression in pulmonary artery tissue. The expression of miR-29b was significantly reduced in PAH model mice. MiR-29b inhibited the proliferation of PASMCs and promoted the apoptosis of PASMCs. Mechanically, miR-29b could inhibit the expression of Mcl-1 and CCND2 protein and silenced Mcl-1 and CCND2 could abolish the change of proliferation and apoptosis of PASMCs. These results demonstrate that miR-29b suppressed cellular proliferation and promoted apoptosis of PASMCs, possibly through the inhibition of Mcl-1 and CCND2. Therefore, miR-29b may serve as a useful therapeutic tool to treat PAH.

Calcineurin/NFAT Signaling Modulates Pulmonary Artery Smooth Muscle Cell Proliferation, Migration and Apoptosis in Monocrotaline-Induced Pulmonary Arterial Hypertension Rats

Background/Aims: Pulmonary arterial hypertension (PAH) is a severe and debilitating disease characterized by remodeling of the pulmonary vessels, which is driven by excessive proliferation and migration and apoptosis resistance in pulmonary artery smooth muscle cells (PASMCs). The calcineurin (CaN)/nuclear factor of activated T-cells (NFAT) signaling pathway is the most important downstream signaling pathway of store-operated Ca²⁺ entry (SOCE), which is increased in PAH. CaN/NFAT has been reported to contribute to abnormal proliferation in chronic hypoxia (CH)-induced PAH. However, the effect of CaN/NFAT signaling on PASMC proliferation, migration and apoptosis in monocrotaline (MCT)-induced PAH remains unclear. Methods: PAH rats were established by a single intraperitoneal injection of MCT for 21 days. PASMCs were isolated and cultured in normal and MCT-induced PAH Sprague-Dawley rat. PASMCs were treated with CsA targeting CaN and siRNA targeting NFATc2-4 gene respectively by liposome. We investigated the expression of calcineurin/NFAT signaling by immunofluorescence, qRT-PCR and Western blotting methods. Cell proliferation was monitored using MTS reagent or by assessing proliferating cell nuclear antigen (PCNA) expression. Cell apoptosis was evaluated with an Annexin V - FITC/propidium iodide (PI) apoptosis kit by flow cytometry. PASMC migration was assessed with a Transwell chamber. Results: MCT successfully induced PAH and pulmonary vascular remodeling in rats. CaN phosphatase activity and nuclear translocation of NFATc2-4 were increased in PASMCs derived from MCT-treated rats. In addition, CaNBβ/NFATc2-4 expression was amplified at the mRNA and protein levels. PASMC proliferation and migration were markedly inhibited in a dosedependent manner by cyclosporin A (CsA). Furthermore, siRNA targeting NFATc2 and NFATc4 attenuated the excessive proliferation and migration and apoptosis resistance in PASMCs derived from both CON and MCT-treated rats, while NFATc3 knockdown specifically affected MCT-PASMCs. Conclusion: Our results demonstrate that CaN/NFAT signaling is activated and involved in the modulation of PASMC proliferation, migration and apoptosis in MCT-induced PAH.