DNA-PKcs participated in hypoxic pulmonary hypertension.

BACKGROUND: Hypoxic pulmonary hypertension (HPH) is a common complication of chronic lung disease, which severely affects the survival and prognosis of patients. Several recent reports have shown that DNA damage and repair plays a crucial role in pathogenesis of pulmonary arterial hypertension. DNA-dependent protein kinase catalytic subunit (DNA-PKcs) as a part of DNA-PK is a molecular sensor for DNA damage that enhances DSB repair. This study aimed to demonstrate the expression and potential mechanism of DNA-PKcs on the pathogenesis of HPH. METHODS: Levels of DNA-PKcs and other proteins in explants of human and rats pulmonary artery from lung tissues and pulmonary artery smooth muscle cells (PASMC) were measured by immunohistochemistry and western blot analysis. The mRNA expression levels of DNA-PKcs and NOR1 in PASMCs were quantified with qRT-PCR. Meanwhile, the interaction among proteins were detected by Co-immunoprecipitation (Co-IP) assays. Cell proliferation and apoptosis was assessed by cell counting kit-8 assay(CCK-8), EdU incorporation and flow cytometry. Rat models of HPH were constructed to verify the role of DNA-PKcs in pulmonary vascular remodeling in vivo. RESULTS: DNA-PKcs protein levels were both significantly up-regulated in explants of pulmonary artery from HPH models and lung tissues of patients with hypoxemia. In human PASMCs, hypoxia up-regulated DNA-PKcs in a time-dependent manner. Downregulation of DNA-PKcs by targeted siRNA or small-molecule inhibitor NU7026 both induced cell proliferation inhibition and cell cycle arrest. DNA-PKcs affected proliferation by regulating NOR1 protein synthesis followed by the expression of cyclin D1. Co-immunoprecipitation of NOR1 with DNA-PKcs was severely increased in hypoxia. Meanwhile, hypoxia promoted G2 + S phase, whereas the down-regulation of DNA-PKcs and NOR1 attenuated the effects of hypoxia. In vivo, inhibition of DNA-PKcs reverses hypoxic pulmonary vascular remodeling and prevented HPH. CONCLUSIONS: Our study indicated the potential mechanism of DNA-PKcs in the development of HPH. It might provide insights into new therapeutic targets for pulmonary vascular remodeling and pulmonary hypertension.

Resveratrol prevented experimental pulmonary vascular remodeling via miR-638 regulating NR4A3/cyclin D1 pathway.

OBJECTIVE: Resveratrol has shown benefit for pulmonary hypertension improvement. Our previous reports showed NR4A3/cyclin D1 pathway promoted pulmonary arterial smooth muscle cells (PASMCs) proliferation. This study tried to explore the mechanism underlying this process, focusing on the role of resveratrol in regulation of miRNA and NR4A3. METHODS: Rats were injected with monocrotaline (MCT) to establish pulmonary hypertension (PH) models. Resveratrol was used to prevent pulmonary vascular remodeling. Primary rat PASMCs were cultured in vitro and stimulated by platelet-derived growth factor (PDGF) with or without resveratrol. Cells proliferation and expression of miR-638 as well as NR4A3 were evaluated. RESULTS: MCT resulted in significant pulmonary vascular remodeling and down-regulation of miR-638, which could be suppressed by resveratrol. Moreover, PDGF-induced PASMC proliferation and miR-638 down-regulation were both significantly prevented by resveratrol treatment in vitro. MiR-638 mimics markedly inhibited PASMC proliferation and percentage of PCNA-positive cells in vitro. But anti-miR-638 could markedly promote cells proliferation and percentage of PCNA-positive cells. The luciferase reporter assay showed that NR4A3 was a direct target of miR-638. The loss-of-function and gain-of-function experiments indicated that NR4A3 promoted proliferation via cyclin D1 pathway. CONCLUSION: Our data indicated that resveratrol prevented MCT-induced pulmonary vascular remodeling via miR-638 regulating NR4A3/cyclin D1 pathway.

Neuron-derived orphan receptor 1 promoted human pulmonary artery smooth muscle cells proliferation.

AIMS: As a transcription factor of the nuclear receptor superfamily, neuron-derived orphan receptor 1 (NOR1) is induced rapidly in response to various extracellular stimuli. But, it is still unclear its role in pulmonary artery smooth muscle cells proliferation. MATERIALS AND METHODS: Human PASMCs were cultured in vitro and stimulated by serum. The special antisense oligodeoxynucleotides (AS-ODNs) were used to knockdown human NOR1 gene expression. Real-time PCR and Western-blot were used to evaluate the gene expression and protein levels. RESULTS: Fetal bovine serum (FBS) induced human PASMCs proliferation in a dose dependent manner. Furthermore, FBS promoted NOR1 gene expression in a dose dependent manner and a time dependent manner. 10% FBS induced a maximal NOR1 mRNA levels at 2 h. FBS also induced a significant higher NOR1 protein levels as compared with control. The NOR1 over-expressed plasmid significantly promoted DNA synthesis and cells proliferation. Moreover, the special AS-ODNs against human NOR1 not only prevented NOR1 expression but also inhibited DNA synthesis and cells proliferation significantly. The NOR1 over-expression plasmid could up-regulate cyclin D1 expression markedly, but the AS-ODNs inhibited cyclin D1 expression significantly. CONCLUSION: So, we concluded that NOR1 could promote human PASMCs proliferation. Cyclin D1 might be involved in this process.

Circular RNA Expression of Peripheral Blood Mononuclear Cells Associated with Risk of Acute Exacerbation in Smoking Chronic Obstructive Pulmonary Disease.

Purpose: Circular RNAs (circRNAs) are newly identified endogenous non-coding RNAs that function as crucial gene modulators in the development of several diseases. By assessing the expression levels of circRNAs in peripheral blood mononuclear cells (PBMCs) from patients with chronic obstructive pulmonary disease (COPD), this study attempted to find new biomarkers for COPD screening. Patients and Methods: We confirmed altered circRNA expression in PBMCs of COPD (n=41) vs controls (n=29). Further analysis focused on the highest and lowest circRNA expression levels. The T-test is used to assess the statistical variances in circRNAs among COPD patients in the smoking and non-smoking cohorts. Additionally, among smokers, the Spearman correlation test assesses the association between circRNAs and clinical indicators. Results: Two circRNAs, hsa_circ_0042590 and hsa_circ_0049875, that were highly upregulated and downregulated in PBMCs from COPD patients were identified and verified. Smokers with COPD had lower hsa_circ_0042590 and higher hsa_circ_0049875, in comparison to non-smokers. There was a significant correlation (r=0.52, P<0.01) between the number of acute exacerbations (AEs) that smokers with COPD experienced in the previous year and the following year (r=0.67, P<0.001). Moreover, hsa_circ_0049875 was connected to the quantity of AEs in the year prior (r=0.68, P<0.0001) as well as the year after (r=0.72, P<0.0001). AUC: 0.79, 95% CI: 0.1210-0.3209, P<0.0001) for hsa_circ_0049875 showed a strong diagnostic value for COPD, according to ROC curve analysis. Hsa_circ_0042590 showed a close second with an AUC of 0.83 and 95% CI: -0.1972--0.0739 (P <0.0001). Conclusion: This research identified a strong correlation between smoking and hsa_circ_0049875 and hsa_circ_0042590 in COPD PBMCs. The number of AEs in the preceding and succeeding years was substantially linked with the existence of hsa_circ_0042590 and hsa_circ_0049875 in COPD patients who smoke. Additionally, according to our research, hsa_circ_0049875 and hsa_circ_0042590 may be valuable biomarkers for COPD diagnosis.

Suppression of cyclin D1 by plasmid-based short hairpin RNA ameliorated experimental pulmonary vascular remodeling.

Our previous study has demonstrated that a plasmid-based short hairpin RNA (shRNA) against cyclin D1 could attenuate the pulmonary artery smooth muscle cell (PASMC) proliferation and pulmonary vascular remodeling in smoking rats. In this report, we examined the efficiency of this shRNA plasmid in monocrotaline-induced pulmonary vascular remodeling. A single injection of monocrotaline induced pulmonary vascular remodeling and cyclin D1 over-expression in pulmonary vascular smooth muscle. The shRNA successfully suppressed the up-regulation of cyclin D1 in pulmonary vessels of monocrotaline-treated rats. Moreover, this shRNA decreased the percentage of muscularized vessels and the wall thickness of pulmonary vessels. So, we concluded that plasmid-based shRNA against cyclin D1 ameliorated pulmonary vascular remodeling in monocrotaline-treated rats. Cyclin D1 might be a potential target for the therapy of pulmonary vascular remodeling and pulmonary hypertension.

Interleukin (IL)-4 induces production of cytokine-induced neutrophil chemoattractants (CINCs) and intercellular adhesion molecule (ICAM)-1 in lungs of asthmatic rats.

The present study aimed to examine the effect of interleukin (IL)-4 on neutrophil chemotaxis in airway inflammation in asthmatic rats and the possible mechanism. Male Wistar rats were intranasally instilled with recombinant rat (rr) IL-4 (rrIL-4) at different doses [2, 4 or 8 µg/animal, dissolved in 200 µL normal saline (NS)] or rrIL-4 at 4 µg/animal (dissolved in 200 µL NS). NS (200 µL) and LPS (6 mg/kg/animal, dissolved in 200 µL NS) were intranasally given respectively in the negative and positive control groups. Moreover, the asthmatic lung inflammation was induced in rats which were then intranasally treated with rrIL-4 (4 µg/animal) or LPS (6 mg/kg/animal). The normal rats treated with different doses of rrIL-4 and those asthmatic rats were sacrificed 6 h later. And animals instilled with rrIL-4 at 4 µg were sacrificed 6, 12 or 24 h later. The bronchoalveolar lavage fluid (BALF) and lungs were harvested for detection of leukocyte counts by Wright-Giemsa staining and lung histopathology by haematoxylin-eosin (HE) staining. The levels of cytokine-induced neutrophil chemoattractant (CINC)-1 and intercellular adhesion molecule (ICAM)-1 in BALF were determined by ELISA. Real-time PCR was used to measure the mRNA expression of CINCs (CINC-1, CINC-2α, CINC-2ß, CINC-3) and ICAM-1 in lung tissues. The results showed that the intranasal instillation of IL-4 did not induce a recruitment of neutrophils in BALF in rats. However, IL-4 could increase the CINC-1 level in BALF in a dose-dependent manner at 6 h. But the mRNA expression levels of CINC-1, CINC-2α, CINC-2ß, CINC-3 were not significantly increased in lungs of IL-4-treated rats relative to NS negative control group. Moreover, IL-4 was found to augment the mRNA expression of ICAM-1 in lungs and the ICAM-1 level in BALF at 6 h. However, the increase in CINC-1 and ICAM-1 levels in BALF of IL-4-treated asthmatic rats was not significantly different from that in untreated asthmatic rats. These findings indicate that IL-4 does not directly recruit neutrophils in the rat lungs, but it may contribute to airway neutrophilia through up-regulation of CINC-1 and ICAM-1.

The role of tracheal wall injury in the development of benign airway stenosis in rabbits.

To investigate the role of tracheal wall injury in the development of benign airway stenosis in rabbits. Prospective study. We injured the tracheal walls of 28 New Zealand white rabbits using four different methods. Experimental group: Group A (n = 7, mild injury of tracheal mucosa by ordinary brush under bronchoscopy); Group B (n = 7, severe injury of tracheal mucosa by nylon brush under tracheotomy); Group C (n = 7, tracheal cartilage was injured by vascular clamp after tracheotomy); Group D (n = 7, the tracheal cartilage was injured with vascular forceps and the tracheal mucosa was injured with a nylon brush after tracheotomy). Bronchoscopy was performed on each experimental rabbit at 1, 2, 3 and 4 weeks after operation. High-resolution computed tomography (HRCT) and endobronchial optical coherence tomography (EB-OCT) were performed at 4 weeks, and the rabbits were sacrificed after the examination. Their gross and histological findings were comparatively determined whether the experimental rabbit stenosis was established. No airway stenosis was observed in group A. In group B, 28.57% of experimental rabbits developed tracheal stenosis (granulation tissue proliferation was observed in rabbits No. 2 and No. 6 at 1, 2 and 3 weeks after operation, and the tracheal scar contracture was observed in No.6 rabbit at 4 weeks after operation). Fourteen rabbits in group C and group D had tracheal stenosis caused by granulation tissue proliferation at 1, 2 and 3 weeks after operation. At the fourth week after operation, 71.43% of experimental rabbits had tracheal stenosis due to granulation tissue hyperplasia, 7.14% of experimental rabbits had tracheal stenosis due to scar contracture and granulation hyperplasia, and 21.43% of experimental rabbits had tracheal stenosis due to scar contracture. EB-OCT scan showed that the cartilage layer with low signal reflection band was discontinuous. The injury of cartilage is the key factor of benign airway stenosis. Acute injury of airway mucosa alone is unlikely to cause airway stenosis, but combined with cartilage injury may aggravate airway stenosis. EB-OCT can clearly identify the airway layers of rabbits, which is helpful to evaluate the damage of tracheal cartilage and mucosa. The diagnostic potential of this technique makes EB-OCT a promising approach for the study and monitoring of airway diseases.

CCN2 promotes cigarette smoke-induced proliferation of rat pulmonary artery smooth muscle cells through upregulating cyclin D1 expression.

Cigarette smoke has been demonstrated to induce pulmonary vascular remodeling, which is characterized by medial thickening of the pulmonary arteries mainly resulting from the abnormal proliferation of pulmonary artery smooth muscle cells (PASMCs). However, the molecular mechanism underlying this process is still unclear. In the present study, we investigated whether CCN2 regulated rat PASMCs (rPASMCs) proliferation induced by cigarette smoke extract (CSE) and nicotine by upregulating cyclin D1 in vitro. CCN2 siRNA or cyclin D1 siRNA were transfected to rPASMCs which were then exposed to CSE and nicotine. Both mRNA and protein expressions of CCN2 were significantly increased in rPASMCs treated with 2% CSE or 1 µM nicotine, which markedly promoted the proliferation of rPASMCs. CCN2 siRNA inhibited the proliferation of rPASMCs induced by CSE or nicotine. Furthermore, CCN2 siRNA markedly suppressed the mRNA and protein expressions of cyclin D1 in rPASMCs and led to cell cycle arrest in G0/G1 phase resulting in reduced rPASMCs proliferation. These findings suggest that CCN2 contributes to the CSE and nicotine-induced proliferation of rPASMCs at least in part by upregulating cyclin D1 expression.

Comparison of operation time, efficacy and safety between through-the-scope stent and over-the-while stent in malignant central airway obstruction: a multi-center randomized control trial.

Background: Self-expandable metallic (SEM) airway stents are an important approach to treating malignant central airway obstruction (CAO). Standard over-the-while (OTW) stent needs the guidance of a guide-wire. It should be implanted under flouroscopy or the guidance of bronchoscope visualization. In this study, we evaluated the operation time and safety between OTW stent and a novel through-the-scope (TTS) SEM airway stent. Methods: In this multi-center, randomized, parallel-group superiority study, malignant CAO patients were enrolled randomly assigned (2:1) to the TTS stent implantation group (TTS group) or the standard OTW stent group (OTW group) in six sites across China. The entire process of all surgical procedures was recorded by video. Primary endpoint was the operation time of the airway stent implantation and secondary endpoint was the success rate of the stent implantation as well as its efficacy and safety. Results: From May 15, 2017, to December 30, 2018, 148 patients were enrolled from the six sites. We analyzed 134 patients (including 91 patients from the TTS group and 43 patients from the OTW group) according to the per-protocol set. There were no significant differences in the ages, genders, underlying diseases, and stenosis sites between the two groups. The operation time in the TTS group was significantly shorter than that in the OTW group (104±68 vs. 252±111 seconds, P<0.001). Compared to the OTW group, the efficacy of stent implantation (97.80% vs. 90.70%, P=0.093) and rate of first-time successful stent implantation (78.02% vs. 74.42%, P=0.668) were higher in the TTS group, but did not reach statistically significance. The rates of granulation (28.57% vs. 41.86%, P=0.128) and restenosis (15.38% vs. 30.23%, P=0.064) in the TTS group were slightly lower as compared with the OTW group without achieving statistical significance. Conclusions: The TTS stent implantation procedure time was significantly shorter than that of the OTW airway stent with similar efficacy and complications, which might reduce the risk and flexibility of stent implantation. Trial Registration: Chinese Clinical Trial Registry ChiCTR-IOR-17011431.