Fractional exhaled nitric oxide in idiopathic pulmonary arterial hypertension and mixed connective tissue disease complicating pulmonary hypertension.

BACKGROUND: Fractional exhaled nitric oxide (FeNO) has been extensively studied in various causes of pulmonary hypertension (PH), but its utility as a noninvasive marker remains highly debated. The objective of our study was to assess FeNO levels in patients with idiopathic pulmonary arterial hypertension (IPAH) and mixed connective tissue disease complicating pulmonary hypertension (MCTD-PH), and to correlate them with respiratory functional data, disease severity, and cardiopulmonary function. METHODS: We collected data from 54 patients diagnosed with IPAH and 78 patients diagnosed with MCTD-PH at the Shanghai Pulmonary Hospital Affiliated to Tongji University. Our data collection included measurements of brain natriuretic peptide (pro-BNP), cardiopulmonary exercise test (CPET), pulmonary function test (PFT), impulse oscillometry (IOS), and FeNO levels. Additionally, we assessed World Health Organization functional class (WHO-FC) of each patient. RESULTS: (1) The fractional exhaled concentration of nitric oxide was notably higher in patients with IPAH compared to those with MCTD-PH. Furthermore, within the IPAH group, FeNO levels were found to be lower in cases of severe IPAH compared to mild IPAH (P = 0.024); (2) In severe pulmonary hypertension as per the WHO-FC classification, FeNO levels in IPAH exhibited negative correlations with FEV1/FVC (Forced Expiratory Velocity at one second /Forced Vital Capacity), MEF50% (Maximum Expiratory Flow at 50%), MEF25%, and MMEF75/25% (Maximum Mid-expiratory Flow between 75% and 25%), while in severe MCTD-PH, FeNO levels were negatively correlated with R20% (Resistance at 20 Hz); (3) ROC (Receiving operator characteristic curve) analysis indicated that the optimal cutoff value of FeNO for diagnosing severe IPAH was 23ppb; (4) While FeNO levels tend to be negatively correlated with peakPETO2(peak end-tidal partial pressure for oxygen) in severe IPAH, in mild IPAH they had a positive correlation to peakO2/Heart rate (HR). An interesting find was observed in cases of severe MCTD-PH, where FeNO levels were negatively correlated with HR and respiratory exchange ratio (RER), while positively correlated with O2/HR throughout the cardiopulmonary exercise test. CONCLUSION: FeNO levels serve as a non-invasive measure of IPAH severity. Although FeNO levels may not assess the severity of MCTD-PH, their significant makes them a valuable tool when assessing severe MCTD-PH.

Up-regulated oxidized USP2a can increase Mdm2-p60-p53 to promote cell apoptosis.

Mdm2 promotes the ubiquitination and degradation of p53, while Mdm2-p60 can bind to p53 and reduce the Mdm2-induced p53 ubiquitination to improve its stability. USP2a can deubiquitinate and stabilize Mdm2, whether USP2a can regulate Mdm2-p60 needs to be further confirmed and elucidated. We found that oxidative stress can up-regulate USP2a at the post-transcriptional level and induce USP2a to be oxidized by forming inter-subunit disulfide bonds. The oxidized USP2a is closely related with cell apoptosis. In apoptotic cells, oxidized USP2a has enhanced protein stability and further stabilizes Mdm2-p60 through deubiquitination, and the USP2a-Mdm2-p60-p53 axis plays a role in cell apoptosis. Altogether USP2a is oxygen sensitive, oxidized USP2a exerts apoptotic effects through the Mdm2-p60-p53 axis, which provides an experimental basis for regulating p53 apoptotic signaling by targeting USP2a.

Long-term variability of impulse oscillometry and spirometry in stable COPD and asthma.

BACKGROUND: While optimizing spirometry is a challenge for lung function labs, long-term variability if any between IOS (impulse oscillometry) parameters and spirometry is not clearly known in stable COPD (chronic obstructive pulmonary disease) and chronic asthma. The forced oscillation technique is increasingly employed in routine lung function testing. Our aim in this study was to determine the variability in oscillometric parameters between clinic visits over weeks or months in two patient groups during a period of clinical stability. Moreover, the research assessed relationships between IOS parameters long-term variability and COPD severity. METHODS: We used data from 73 patients with stable COPD and 119 patients with stable asthma at the Shanghai Pulmonary Hospital Affiliated to Tongji University. Patients were included if they had three or more clinic visits where spirometry and IOS were performed during a clinically stable period. Data recorded from the first three visits were used. The standard deviation (SDbv), the coefficient of variation (COV), intraclass correlation coefficient (ICC) and the coefficient of repeatability (COR) were calculated, Wilcoxon Mann-Whitney test was used for data that did not conform to normality of distributions, Kruskal Wallis test was used to compare with multiple groups, post hoc comparison was analyzed by Bonferroni, Spearman correlation coefficients for non-parametric data, the multiple regression analyses to determine the relationship between long-term variability and airflow obstruction. RESULTS: (1) The repeatability of IOS resistance parameters with ICC values > 0.8 was high in COPD and asthma. ICC values of IOS resistance parameters were higher than IOS reactance parameters; (2) the repeatability of spirometry parameters with ICC values < 0.8 was lower than IOS resistance parameters in different GOLD (the Global Initiative for Chronic Obstructive Lung Disease) stages, the higher the stage the worse the repeatability; (3) the severity of airflow obstruction was correlated with long-term variability of R5 (R at 5 Hz) (P < 0.05) in GOLD4, not with long-term variability of R20 (R at 20 Hz) (P > 0.05) and R5-R20 (P > 0.05). CONCLUSION: IOS resistance parameters have good long-term repeatability in asthma and COPD. Additionally, repeatability of spirometry parameters is lower than IOS resistance parameters in different GOLD stages.

Variation of PetCO2 during incremental exercise and severity of IPAH and CTEPH.

BACKGROUND AND OBJECTIVE: End-tidal PCO2 (PetCO2) patterns during exercise testing as well as ventilatory equivalents for CO2 have been reported for different pulmonary vascular diseases but seldomly for the significant differences in exercise response depending on the etiology of pulmonary hypertension. We aimed to compare PetCO2 change pattern in IPAH and CTEPH with varying severity during incremental cardiopulmonary exercise testing (CPET). METHODS: 164 IPAH patients and 135 CTEPH patients referred to Shanghai Pulmonary Hospital between 2012 and 2019 were retrospectively recruited into the study. All patients performed CPET and also underwent right-heart catheterization (RHC). Forty-four healthy subjects also performed CPET and were included as controls. RESULTS: PetCO2 was significantly lower in IPAH and CTEPH patients as compared to normal subjects. Moreover, the PetCO2 did not rise, in fact fell from rest to anaerobic threshold (AT), then further decreased until peak in both IPAH and CTEPH. PetCO2 value at rest, unloaded, AT and peak were proportionately reduced as the World Health Organization functional class (WHO-Fc) increased in both IPAH and CTEPH patients. The PETCO2 in IPAH patients had significant differences during all phases of exercise between WHO-Fc I-II and III-IV subgroup. CTEPH also demonstrated significant difference except for PetCO2 at peak. PetCO2 values were significantly higher in IPAH during all phases of exercise as compared to CTEPH patients (all P < 0.001). PeakVO2%pred correlated significantly with PetCO2 at rest (r = 0.477, P < 0.001), AT (r = 0.609, P < 0.001) and peak exercise (r = 0.576, P < 0.001) in IPAH. N-terminal natriuretic peptide type-B (NT-proBNP) also correlated markedly with PetCO2, with a correlation coefficient of - 0.326 to - 0.427 (all P < 0.001). Additionally, PetCO2 at rest, at AT and at peak correlated positively with peakVO2%pred and showed an inverse correlation with NT-proBNP in CTEPH patients (all P < 0.05). CONCLUSIONS: PetCO2 during exercise in IPAH and CTEPH patients was significantly different from normal subjects. Moreover, PetCO2 values were significantly higher in IPAH during all phases of exercise as compared to CTEPH patients (all P < 0.001). PetCO2 was progressively more abnormal with increasing disease severity according to peakVO2%pred and WHO-Fc.

Cardiopulmonary exercise testing and pulmonary function testing for predicting the severity of CTEPH.

BACKGROUND: Cardiopulmonary exercise testing (CPET) and pulmonary function testing (PFT) are noninvasive methods to evaluate the respiratory and circulatory systems. This research aims to evaluate and monitor chronic thromboembolic pulmonary hypertension (CTEPH) noninvasively and effectively by these two methods. Moreover, the research assesses the predictive value of CPET and PFT parameters for severe CTEPH. METHODS: We used data from 86 patients with CTEPH (55 for test set, and 31 for validation set) at the Shanghai Pulmonary Hospital Affiliated to Tongji University. The clinical, PFT and CPET data of CTEPH patients of different severity classified according to pulmonary artery pressure (PAP) (mm Hg) were collected and compared. Logistic regression analysis was performed to appraise the predictive value of each PFT and CPET parameter for severe CTEPH. The performance of CPET parameters for predicting severe CTEPH was determined by receiver operating characteristic (ROC) curves and calibration curves. RESULTS: Data showed that minute ventilation at anaerobic threshold (VE @ AT) (L/min) and oxygen uptake at peak (VO2 @ peak) (mL/kg/min) were independent predictors for severe CTEPH classified according to PAP (mm Hg). Additionally, the efficacy of VE @ AT (L/min) and VO2 @ peak (mL/kg/min) in identifying severe CTEPH was found to be moderate with the area under ROC curve (AUC) of 0.769 and 0.740, respectively. Furthermore, the combination of VE @ AT (L/min) and VO2 @ peak (mL/kg/min) had a moderate utility value in identifying severe CTEPH with the AUC of 0.843. CONCLUSION: Our research suggests that CPET and PFT can noninvasively and effectively evaluate, monitor and predict the severity of CTEPH.

Oridonin induces Mdm2-p60 to promote p53-mediated apoptosis and cell cycle arrest in neuroblastoma.

Oridonin could induce NB (neuroblastoma) cells growth inhibition by inducing apoptosis and cell cycle arrest, and the molecular mechanisms behind the effects deserve to be further explored. Here, oridonin was confirmed to cause the reactivation of p53 (cellular tumor antigen p53) to promote the expression of a series of apoptosis- and cell cycle arrest-related proteins for the biological effects. During the process, oridonin relied on the caspase activation to cleave p53-induced Mdm2 (E3 ubiquitin-protein ligase Mdm2) to generate Mdm2-p60. The generation of Mdm2-p60 stabilized p53, and resulted in p53 accumulation for p53 continuous activation. In our research, it was also found that the reactivation of p53 induced by oridonin was closely related with the generation of ROS (reactive oxygen species). Taken together, these findings explain that oridonin exerts its anticancer activity partially by targeting the Mdm2-p53 axis in NB cells, which lay an experimental base for future research of exploring the effects and molecular mechanisms of oridonin.

Regulatory Molecules and Corresponding Processes of BCR-ABL Protein Degradation.

The BCR-ABL fusion protein with strong tyrosine kinase activity is one of the molecular biological bases of leukemia. Imatinib (Gleevec), a specific targeted drug for the treatment of chronic myeloid leukemia (CML), was developed for inhibiting the kinase activity of the BCR-ABL fusion protein. Despite the positive clinical efficacy of imatinib, the proportion of imatinib resistance has gradually increased. The main reason for the resistance is a decrease in sensitivity to imatinib caused by mutation or amplification of the BCR-ABL gene. In response to this phenomenon, the new generation of tyrosine kinase inhibitors (TKIs) targeting the BCR-ABL fusion protein was developed to solve the problem. However this strategy only selectively inhibits the tyrosine kinase activity of the BCR-ABL protein without eliminating the BCR-ABL protein, it does not fundamentally cure the BCR-ABL-positive leukemia patients. With the accumulation of the knowledge of cellular molecular biology, it has become possible to specifically eliminate certain proteins by cellular proteases in a specific way. Therefore, the therapeutic strategy to induce the degradation of the BCR-ABL fusion protein is superior to the strategy of inhibiting its activity. The protein degradation strategy is also a solution to the TKI resistance caused by different BCR-ABL gene point mutations. In order to provide possible exploration directions and clues for eliminating the BCR-ABL fusion protein in tumor cells, we summarize the significant molecules involved in the degradation pathway of the BCR-ABL protein, as well as the reported potent compounds that can target the BCR-ABL protein for degradation.

The Molecular Mechanisms of Regulation on USP2's Alternative Splicing and the Significance of Its Products.

Ubiquitin-specific protease 2 (USP2) has a regulatory function in cell growth or death and is involved in the pathogenesis of various diseases. USP2 gene can generate 7 splicing variants through alternative splicing, and 5 variants respectively as USP2-201, USP2-202, USP2-204, USP2-205, USP2-206 can encode proteins. The influence of circadian rhythm, nutrition and androgen on specific signaling molecules or cytokines can regulate the alternative splicing of USP2. Specifically, PKC activator, IL-1ß, TNF-α, PDGF-BB, TGF-ß1 are all regulatory factors for USP2's alternative splicing. USP2-201 plays a crucial role in cell cycle progression, and is also of great significance in EGFR recycling. USP2-202 can activate apoptosis signaling pathway to participate in cell apoptosis, and USP2-204 can induce cell anti-virus reaction to decrease. In general, we collect and summarize the factors involved in the alternative splicing of USP2 in this review to further understand the mechanism behind the USP2's alternative splicing.

GSK3ß-dependent cyclin D1 and cyclin E1 degradation is indispensable for NVP-BEZ235 induced G0/G1 arrest in neuroblastoma cells.

Cyclin D1 and cyclin E1, as vital regulatory factors of G1-S phase cell cycle progression, are frequently constitutive expressed and associated with pathogenesis and tumorigenesis in most human cancers and they have been regarded as promising targets for cancer therapy. In this study, we established NVP-BEZ235, a potent dual kinase inhibitor, could induce neuroblastoma cells proliferation inhibition without apoptosis activation. Moreover, we showed NVP-BEZ235 could induce neuroblastoma cells arrested at G0/G1 phase accompanied with significant reduction of the cyclin D1 and E1 proteins in a dose dependent manner at nanomole concentration. Additionally we found that GSK3ß was dephosphorylated and activated by NVP-BEZ235 and then triggered cyclin D1 and cyclin E1 degradation through ubiquitination proteasome pathway, based on the evidences that NVP-BEZ235 induced downregulation of cyclin D1 and cyclin E1 were obviously recovered by proteasome inhibitor and the blockade of GSK3ß contributed to remarkable rescue of cyclin D1 and cyclin E1. Analogous results about its anti-proliferation effects and molecular mechanism were observed on neuroblastoma xenograft mouse model in vivo. Therefore, these results indicate that NVP-BEZ235-induced cyclin D1 and cyclin E1 degradation, which happened through activating GSK3ß, and GSK3ß-dependent down-regulation of cyclin D1 and cyclin E1 should be available for anticancer therapeutics.

MiR-17-92 cluster promotes hepatocarcinogenesis.

MiR-17-92 cluster is an oncogenic miRNA cluster that is implicated in several cancers, although its role in hepatocarcinogenesis has not been clearly defined. In this study, we show that the miR-17-92 cluster is highly expressed in human hepatocellular carcinoma (HCC) tissues compared to the non-tumorous liver tissues by RT-PCR and in situ hybridization analyses. Increased miR-17-92 cluster expression in HCC tissues was further confirmed by analysis of the RNA-sequencing data of 319 patients available from the Cancer Genome Atlas (TCGA) Data Portal (https://tcga-data.nci.nih.gov/tcga/). To create an animal model that resembles enhanced miR-17-92 in the liver, we developed liver-specific miR-17-92 transgenic mice and the animals were treated with the hepatic carcinogen, diethylnitrosamine (DEN). We observed that the liver-specific miR-17-92 transgenic mice showed significantly increased hepatocellular cancer development compared to the matched wild-type control mice. Forced overexpression of the miR-17-92 cluster in cultured human hepatocellular cancer cells enhanced tumor cell proliferation, colony formation and invasiveness in vitro, whereas inhibition of the miR-17-92 cluster reduced tumor cell growth. By analyzing the miRNA and mRNA sequencing data from the 312 hepatocellular cancer patients available from the TCGA database, we observed that the expression levels of the miR-17-92 cluster members and host gene in the tumor tissues are negatively correlated with several target genes, including CREBL2, PRRG1, NTN4. Our findings demonstrate an important role of the miR-17-92 cluster in hepatocarcinogenesis and suggest the possibility of targeting this pivotal miRNA cluster for potential therapy.

miR-17-92 cluster promotes cholangiocarcinoma growth: evidence for PTEN as downstream target and IL-6/Stat3 as upstream activator.

miR-17-92 is an oncogenic miRNA cluster implicated in the development of several cancers; however, it remains unknown whether the miR-17-92 cluster is able to regulate cholangiocarcinogenesis. This study was designed to investigate the biological functions and molecular mechanisms of the miR-17-92 cluster in cholangiocarcinoma. In situ hybridization and quantitative RT-PCR analysis showed that the miR-17-92 cluster is highly expressed in human cholangiocarcinoma cells compared with the nonneoplastic biliary epithelial cells. Forced overexpression of the miR-17-92 cluster or its members, miR-92a and miR-19a, in cultured human cholangiocarcinoma cells enhanced tumor cell proliferation, colony formation, and invasiveness, in vitro. Overexpression of the miR-17-92 cluster or miR-92a also enhanced cholangiocarcinoma growth in vivo in hairless outbred mice with severe combined immunodeficiency (SHO-Prkdc(scid)Hr(hr)). The tumor-suppressor, phosphatase and tensin homolog deleted on chromosome 10 (PTEN), was identified as a bona fide target of both miR-92a and miR-19a in cholangiocarcinoma cells via sequence prediction, 3' untranslated region luciferase activity assay, and Western blot analysis. Accordingly, overexpression of the PTEN open reading frame protein (devoid of 3' untranslated region) prevented miR-92a- or miR-19a-induced cholangiocarcinoma cell growth. Microarray analysis revealed additional targets of the miR-17-92 cluster in human cholangiocarcinoma cells, including APAF-1 and PRDM2. Moreover, we observed that the expression of the miR-17-92 cluster is regulated by IL-6/Stat3, a key oncogenic signaling pathway pivotal in cholangiocarcinogenesis. Taken together, our findings disclose a novel IL-6/Stat3-miR-17-92 cluster-PTEN signaling axis that is crucial for cholangiocarcinogenesis and tumor progression.

miR-101 inhibits cholangiocarcinoma angiogenesis through targeting vascular endothelial growth factor (VEGF).

Recent evidence has suggested an important role of miRNAs in liver biology and diseases, although the implication of miRNAs in cholangiocarcinoma remains to be defined further. This study was designed to examine the biological function and molecular mechanism of miR-101 in cholangiocarcinogenesis and tumor progression. In situ hybridization and quantitative RT-PCR were performed to determine the expression of miR-101 in human cholangiocarcinoma tissues and cell lines. Compared with noncancerous biliary epithelial cells, the expression of miR-101 is decreased in 43.5% of human cholangiocarcinoma specimens and in all three cholangiocarcinoma cell lines used in this study. Forced overexpression of miR-101 significantly inhibited cholangiocarcinoma growth in severe combined immunodeficiency mice. miR-101-overexpressed xenograft tumor tissues showed decreased capillary densities and decreased levels of vascular endothelial growth factor (VEGF) and cyclooxygenase-2 (COX-2). The VEGF and COX-2 mRNAs were identified as the bona fide targets of miR-101 in cholangiocarcinoma cells by both computational analysis and experimental assays. miR-101 inhibits cholangiocarcinoma angiogenesis by direct targeting of VEGF mRNA 3'untranslated region and by repression of VEGF gene transcription through inhibition of COX-2. This study established a novel tumor-suppressor role of miR-101 in cholangiocarcinoma and it suggests the possibility of targeting miR-101 and related signaling pathways for future therapy.

Natural killer cells are involved in acute lung immune injury caused by respiratory syncytial virus infection.

It is known that respiratory syncytial virus (RSV) is the main cause of bronchiolitis and pneumonia in young children. RSV infection often leads to severe acute lung immunopathology, but the underlying immune mechanisms are not yet fully elucidated. Here, we found that RSV infection induced severe acute lung immune injury and promoted the accumulation and activation of lung natural killer (NK) cells at the early stage of infection in BALB/c mice. Activated lung NK cells highly expressed activating receptors NKG2D and CD27 and became functional NK cells by producing a large amount of gamma interferon (IFN-γ), which was responsible for acute lung immune injury. NK cell depletion significantly attenuated lung immune injury and reduced infiltration of total inflammatory cells and production of IFN-γ in bronchoalveolar lavage fluid (BALF). These data show that NK cells are involved in exacerbating the lung immune injury at the early stage of RSV infection via IFN-γ secretion.

MicroRNA miR-155 inhibits bone morphogenetic protein (BMP) signaling and BMP-mediated Epstein-Barr virus reactivation.

MicroRNA miR-155 is expressed at elevated levels in human cancers including cancers of the lung, breast, colon, and a subset of lymphoid malignancies. In B cells, miR-155 is induced by the oncogenic latency gene expression program of the human herpesvirus Epstein-Barr virus (EBV). Two other oncogenic herpesviruses, Kaposi's sarcoma-associated herpesvirus and Marek's disease virus, encode functional homologues of miR-155, suggesting a role for this microRNA in the biology and pathogenesis of these viruses. Bone morphogenetic protein (BMP) signaling is involved in an array of cellular processes, including differentiation, growth inhibition, and senescence, through context-dependent interactions with multiple signaling pathways. Alteration of this pathway contributes to a number of disease states including cancer. Here, we show that miR-155 targets the 3' untranslated region of multiple components of the BMP signaling cascade, including SMAD1, SMAD5, HIVEP2, CEBPB, RUNX2, and MYO10. Targeting of these mediators results in the inhibition of BMP2-, BMP6-, and BMP7-induced ID3 expression as well as BMP-mediated EBV reactivation in the EBV-positive B-cell line, Mutu I. Further, miR-155 inhibits SMAD1 and SMAD5 expression in the lung epithelial cell line A549, it inhibits BMP-mediated induction of the cyclin-dependent kinase inhibitor p21, and it reverses BMP-mediated cell growth inhibition. These results suggest a role for miR-155 in controlling BMP-mediated cellular processes, in regulating BMP-induced EBV reactivation, and in the inhibition of antitumor effects of BMP signaling in normal and virus-infected cells.