E3 ubiquitin ligase MDM2 acts through p53 to control respiratory progenitor cell number and lung size.

The respiratory lineage initiates from the specification of NKX2-1+ progenitor cells that ultimately give rise to a vast gas-exchange surface area. How the size of the progenitor pool is determined and whether this directly impacts final lung size remains poorly understood. Here, we show that epithelium-specific inactivation of Mdm2, which encodes an E3 ubiquitin ligase, led to lethality at birth with a striking reduction of lung size to a single vestigial lobe. Intriguingly, this lobe was patterned and contained all the appropriate epithelial cell types. The reduction of size can be traced to the progenitor stage, when p53, a principal MDM2 protein degradation target, was transiently upregulated. This was followed by a brief increase of apoptosis. Inactivation of the p53 gene in the Mdm2 mutant background effectively reversed the lung size phenotype, allowing survival at birth. Together, these findings demonstrate that p53 protein turnover by MDM2 is essential for the survival of respiratory progenitors. Unlike in the liver, in which genetic reduction of progenitors triggered compensation, in the lung, respiratory progenitor number is a key determinant factor for final lung size.

Alveolar Epithelial Type 2 Cell Dysfunction in Idiopathic Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a progressive and irreversible pulmonary interstitial disease that seriously affects the patient's quality of life and lifespan. The pathogenesis of IPF has not been clarified, and its treatment is limited to pirfenidone and nintedanib, which only delays the decline of lung function. Alveolar epithelial type 2 (AT2) cells are indispensable in the regeneration and lung surfactant secretion of alveolar epithelial cells. Studies have shown that AT2 cell dysfunction initiates the occurrence and progression of IPF. This review expounds on the AT2 cell dysfunction in IPF, involving senescence, apoptosis, endoplasmic reticulum stress, mitochondrial damage, metabolic reprogramming, and the transitional state of AT2 cells. This article also briefly summarizes potential treatments targeting AT2 cell dysfunction.

Ceramide induces MMP-9 expression through JAK2/STAT3 pathway in airway epithelium.

BACKGROUND: Ceramide, a bioactive lipid, plays an essential role in the development of several pulmonary inflammatory diseases. Matrix metallopeptidase 9 (MMP-9) regulates the synthesis and degradation of extracellular matrix, and is associated with airway remodeling and tissue injury. This study was conducted to investigate the effects and underlying mechanisms of ceramide on MMP-9 expression in airway epithelium. METHODS: BEAS-2B cells, normal human bronchial epithelium cell lines, were pretreated with AG490, a selective janus tyrosine kinase 2 (JAK2) inhibitor, or Stattic, a selective signal transducer and activator of transcription 3 (STAT3) inhibitor. The cells were then stimulated with C6-ceramide. The levels of MMP-9 were determined by ELISA and real-time quantitative PCR (RT-qPCR). JAK2, phosphorylated JAK2 (p-JAK2), STAT3, and phosphorylated STAT3 (p-STAT3) expression was examined by Western blotting. BALB/c mice were pretreated with AG490 or Stattic before intratracheally instillated with C6-ceramide. Pathological changes in lung tissues were examined by Hematoxylin and Eosin staining, Periodic-acid Schiff staining, and Masson's trichrome staining. MMP-9, JAK2, p-JAK2, STAT3, and p-STAT3 expression in the lung tissues was examined by Western blotting. RESULTS: The expression of MMP-9, p-JAK2 and p-STAT3 in BEAS-2B cells was significantly increased after the treatment of C6-ceramide. Furthermore, the increased expression of MMP-9 induced by C6-ceramide was inhibited by AG490 and Stattic. Similar results were obtained in the lung tissues of C6-ceramide-exposed mice which were treated with AG490 or Stattic. CONCLUSIONS: Ceramide could up-regulate MMP-9 expression through the activation of the JAK2/STAT3 pathway in airway epithelium. Targeted modulation of the ceramide signaling pathway may offer a potential therapeutic approach for inhibiting MMP-9 expression. This study points to a potentially novel approach to alleviating airway remodeling in inflammatory airway diseases.

ß-Arrestin2 Inhibits Expression of Inflammatory Cytokines in BEAS-2B Lung Epithelial Cells Treated with Cigarette Smoke Condensate via Inhibition of Autophagy.

BACKGROUND/AIMS: ß-arrestin2 has been shown to have a role in human inflammatory disease. However, the role of ß-arrestin2 in cigarette smoke-induced inflammation in the lung remains unknown. The aims of this study were to investigate the effects of ß-arrestin2 on cigarette smoke condensate (CSC)-induced expression of inflammatory cytokines in the BEAS-2B human bronchial epithelial cell line in vitro, and the mechanisms involved. METHODS: The MTT assay determined cell viability of cultured BEAS-2B cells. Autophagy was assessed by western blot, adenoviral mRFP-GFP-LC3 transfection, and immunofluorescence. The effects of ß-arrestin2 shRNA knockdown were studied by western blot and real-time reverse transcription-polymerase chain reaction (RT-PCR). Western blot evaluated the AMPK/mTOR signaling pathway. Levels of inflammatory cytokines, interleukin (IL)-6, IL-8, and MCP-1 were measured in cell culture supernatants by enzyme-linked immunosorbent assay (ELISA). RESULTS: CSC suppressed expression of ß-arrestin2 in BEAS-2B cells, activated the AMPK/mTOR signaling pathway, increased cell autophagy and the expression of IL-6, IL-8, and MCP-1,pretreatment with the ß-arrestin2 biased ligands, propranolol, and ICI118551 reversed these changes. Inhibition of autophagy reduced the expression of inflammatory cytokines following CSC. CONCLUSION: In the human bronchial epithelial cell line, BEAS-2B, ß-arrestin2 reduced the expression of CSC-induced inflammatory cytokines by inhibiting autophagy, most likely via the AMPK/mTOR signaling pathway.

14,15-Epoxyeicosatrienoic acid suppresses cigarette smoke condensate-induced inflammation in lung epithelial cells by inhibiting autophagy.

Epoxyeicosatrienoic acids (EETs) are metabolic products of free arachidonic acid, which are produced through cytochrome P-450 (CYP) epoxygenases. EETs have anti-inflammatory, antiapoptotic, and antioxidative activities. However, the effect of EETs on cigarette smoke-induced lung inflammation is not clear. Autophagy is believed to be involved in the pathogenesis of chronic obstructive pulmonary disease. In addition, nuclear erythroid-related factor 2 (Nrf2), a transcription factor that regulates many antioxidant genes, is thought to regulate antioxidant defenses in several lung diseases. In addition, interaction between EETs, autophagy, and Nrf2 has been reported. The aim of this study was to explore the effect of 14,15-EET on cigarette smoke condensate (CSC)-induced inflammation in a human bronchial epithelial cell line (Beas-2B), and to determine whether the underlying mechanisms involved in the regulation of Nrf2 through inhibition of autophagy. Autophagy and expression of autophagy signaling pathway proteins (LC3B, p62, PI3K, Akt, p-Akt, and p-mTOR) and anti-inflammatory proteins (Nrf2 and HO-1) were assessed via Western blot analysis. Autophagosomes and autolysosomes were detected by adenoviral mRFP-GFP-LC3 transfection. Inflammatory factors (IL-6, IL-8, and MCP-1) were detected by ELISA. Lentiviral vectors carrying p62 short hairpin RNA were used to interfere with p62 expression to evaluate the effect of p62 on Nrf2 expression. Nrf2 expression was determined through immunocytochemistry. 14,15-EET treatment resulted in a significant reduction in IL-6, IL-8, and MCP-1 secretion, and increased accumulation of Nrf2 and expression of HO-1. In addition, 14,15-EET inhibited CSC-induced autophagy in Beas-2B cells. The mechanism of the anti-inflammatory effect of 14,15-EET involved inhibition of autophagy and an increase in p62 levels, followed by translocation of Nrf2 into the nucleus, which then upregulated expression of the antioxidant enzyme HO-1. 14,15-EET protects against CSC-induced lung inflammation by promoting accumulation of Nrf2 via inhibition of autophagy.

14,15-epoxyeicosatrienoic Acid suppresses cigarette smoke extract-induced apoptosis in lung epithelial cells by inhibiting endoplasmic reticulum stress.

BACKGROUND/AIMS: Epoxyeicosatrienoic acids (EETs), a type of lipid mediators produced by cytochrome P450 epoxygenases, exert anti-inflammatory, angiogenic, anti-oxidative and anti-apoptotic effects. However, the role of EETs in cigarette smoke-induced lung injury and the underlying mechanisms are not fully known. The aim of this study was to explore the effects of CYP2J2-EETs on cigarette smoke extracts (CSE)-induced apoptosis in human bronchial epithelial cell line (Beas-2B) and the possible mechanisms involved. METHODS: Cytochrome P450 epoxygenase 2J2 (CYP2J2) and its metabolites EETs were assessed by western blotting or LC-MS-MS. Cell viability and apoptosis were determined by MTT assay and AnnexinV-PI staining. Reactive oxygen species (ROS) were assessed by measuring H2DCFDA. Caspase-3, HO-1, MAPK and endoplasmic reticulum (ER) stress-related markers GRP78, p-elF2a, and CHOP were evaluated by western blotting. RESULTS: CSE suppressed expression of both CYP2J2 and EET by Beas-2B cells. CSE also induced apoptosis, the generation of ROS and the ER stress in Beas-2B cells. These changes were abolished by pretreatment with exogenous 14,15-EET while pretreatment with 14,15-EEZE, a selective EET antagonist, abolished the protective effects of 14,15-EET. In addition, EETs increased the expression of antioxidant enzyme HO-1. Furthermore, 14,15-EET reduced CSE-induced activation of p38 and JNK. CONCLUSION: The data suggest that CYP2J2-derived EETs protect against CSE-induced lung injury possibly through attenuating ER stress.

Aging of alveolar type 2 cells induced by Lonp1 deficiency exacerbates pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a progressive and chronic disease that significantly impacts patient quality of life, and its incidence is on the rise. The pathogenesis of IPF remains poorly understood. Alveolar type 2 (AT2) cells are crucial in the onset and progression of IPF, yet the specific mechanisms involved are not well defined. Lon protease 1 (LONP1), known for its critical roles in various diseases, has an unclear function in IPF. Our research investigated the impact of Lonp1 gene deletion on AT2 cell functionality and its subsequent effect on IPF development. We generated a bleomycin-induced pulmonary fibrosis mouse model with a targeted Lonp1 knockout in AT2 cells and assessed the consequences on AT2 cell function and fibrosis progression. Additionally, we constructed the MLE12 cells with stable Lonp1 knockdown and utilized transcriptome sequencing to identify pathways altered by the Lonp1 knockdown. Our results indicated that mice with AT2 cell-specific Lonp1 knockout exhibited more severe fibrosis compared to controls. These mice exhibited a reduction in AT2 and AT1 cell populations, along with an increase in p53- and p21-positive AT2 cells. Lonp1 knockdown in MLE12 cells led to the upregulation of aging-associated pathways, with fibroblast growth factor 2 (Fgf2) gene emerging as a central gene interconnecting these pathways. Therefore, loss of Lonp1 appears to promote AT2 cell aging and exacerbate bleomycin-induced pulmonary fibrosis. Fgf2 emerges as a pivotal downstream gene associated with cellular senescence. This study uncovers the role of the Lonp1 gene in pulmonary fibrosis, presenting a novel target for investigating the pathological mechanisms and potential therapeutic approaches for IPF.

Predictive biomarkers of disease progression in idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a chronic interstitial disease that cannot be cured, and treatment options for IPF are very limited. Early diagnosis, close monitoring of disease progression, and timely treatment are therefore the best options for patients due to the irreversibility of IPF. Effective markers help doctors judge the development and prognosis of disease. Recent research on traditional biomarkers (KL-6, SP-D, MMP-7, TIMPs, CCL18) has provided novel ideas for predicting disease progression and prognosis. Some emerging biomarkers (HE4, GDF15, PRDX4, inflammatory cells, G-CSF) also provide more possibilities for disease prediction. In addition to markers in serum and bronchoalveolar lavage fluid (BALF), some improvements related to the GAP model and chest HRCT also show good predictive ability for disease prognosis.

A novel gene signature based on the hub genes of COVID-19 predicts the prognosis of idiopathic pulmonary fibrosis.

Background: Increasing evidence has demonstrated that there was a strong correlation between COVID-19 and idiopathic pulmonary fibrosis (IPF). However, the studies are limited, and the real biological mechanisms behind the IPF progression were still uncleared. Methods: GSE70866 and GSE 157103 datasets were downloaded. The weight gene co-expression network analysis (WGCNA) algorithms were conducted to identify the most correlated gene module with COVID-19. Then the genes were extracted to construct a risk signature in IPF patients by performing Univariate and Lasso Cox Regression analysis. Univariate and Multivariate Cox Regression analyses were used to identify the independent value for predicting the prognosis of IPF patients. What's more, the Kyoto Encyclopedia of Genes and Genomes (KEGG), Gene Ontology (GO), and gene set enrichment analysis (GSEA) were conducted to unveil the potential biological pathways. CIBERSORT algorithms were performed to calculate the correlation between the risk score and immune cells infiltrating levels. Results: Two hundred thirty three differentially expressed genes were calculated as the hub genes in COVID-19. Fourteen of these genes were identified as the prognostic differentially expressed genes in IPF. Three (MET, UCHL1, and IGF1) of the fourteen genes were chosen to construct the risk signature. The risk signature can greatly predict the prognosis of high-risk and low-risk groups based on the calculated risk score. The functional pathway enrichment analysis and immune infiltrating analysis showed that the risk signature may regulate the immune-related pathways and immune cells. Conclusion: We identified prognostic differentially expressed hub genes related to COVID-19 in IPF. A risk signature was constructed based on those genes and showed great value for predicting the prognosis in IPF patients. What's more, three genes in the risk signature may be clinically valuable as potential targets for treating IPF patients and IPF patients with COVID-19.

Loss of Thy-1 may reduce lung regeneration after pneumonectomy in mice.

BACKGROUND: Lung regeneration plays an important role in lung repair after injury. It is reliant upon proliferation of multiple cell types in the lung, including endothelium, epithelium, and fibroblasts, as well as remodeling of the extracellular matrix. METHODS: Lung regeneration following injury progresses via an initial inflammatory response during which macrophages clear the tissue of cellular debris. This process continues through cellular proliferation when existing cells and progenitors act to repopulate cells lost during injury, followed by tissue maturation in which newly formed cells achieve a differentiated phenotype. RESULTS: Signaling pathways critical for lung regeneration include FGF, EGF, WNT, and NOTCH. In addition, HDACs, miRNAs, ELASTIN, and MMP14 have been shown to regulate lung regeneration. Partial pneumonectomy (PNX) has been used as a therapeutic and investigational tool for several decades. Following PNX the remaining lung increases in size to compensate for loss of volume and respiratory capacity. CONCLUSIONS: Much has been learned about the triggers and mechanisms regulating pulmonary regeneration. However, the role of thymocyte differentiation antigen-1 (Thy-1) in post-PNX lung growth remains incompletely characterized. Thy-1 is a phosphatidylinositol glycoprotein with a relative molecular weight of 25000~37000 Da, which is expressed in almost all types of fibroblasts and regulates many biological functions. It not only supports the structure of fibroblasts, but also can balance cell proliferation, migration and regulate the synthesis of immune inflammatory mediators.

Intercept of minute ventilation versus carbon dioxide output relationship as an index of ventilatory inefficiency in chronic obstructive pulmonary disease.

BACKGROUND: Ventilatory inefficiency contributes to exercise intolerance in chronic obstructive pulmonary disease (COPD). The intercept of the minute ventilation (V˙ E) vs. carbon dioxide output (V˙ CO2) plot is a key ventilatory inefficiency parameter. However, its relationships with lung hyperinflation (LH) and airflow limitation are not known. This study aimed to evaluate correlations between the V˙ E/V˙ CO2 intercept and LH and airflow limitation to determine its physiological interpretation as an index of functional impairment in COPD. METHODS: We conducted a retrospective analysis of data from 53 COPD patients and 14 healthy controls who performed incremental cardiopulmonary exercise tests (CPETs) and resting pulmonary function assessment. Ventilatory inefficiency was represented by parameters reflecting the V˙ E/V˙ CO2 nadir and slope (linear region) and the intercept of V˙ E/V˙ CO2 plot. Their correlations with measures of LH and airflow limitation were evaluated. RESULTS: Compared to control, the slope (30.58±3.62, P<0.001) and intercept (4.85±1.11 L/min, P<0.05) were higher in COPDstages1-2, leading to a higher nadir (31.47±4.47, P<0.01). Despite an even higher intercept in COPDstages3-4 (7.16±1.41, P<0.001), the slope diminished with disease progression (from 30.58±3.62 in COPDstages1-2 to 26.84±4.96 in COPDstages3-4, P<0.01). There was no difference in nadir among COPD groups and higher intercepts across all stages. The intercept was correlated with peak V˙ E/maximal voluntary ventilation (MVV) (r=0.489, P<0.001) and peak V˙ O2/Watt (r=0.354, P=0.003). The intercept was positively correlated with residual volume (RV) % predicted (r=0.571, P<0.001), RV/total lung capacity (TLC) (r=0.588, P<0.001), peak tidal volume (VT)/FEV1 (r=0.482, P<0.001) and negatively correlated with rest inspiratory capacity (IC)/TLC (r=-0.574, P<0.001), peak VT/TLC (r=-0.585, P<0.001), airflow limitation forced expiratory volume in 1 s (FEV1) % predicted (r=-0.606, P<0.001), and FEV1/forced vital capacity (FVC) (r=-0.629, P<0.001). CONCLUSIONS: V˙ E/V˙ CO2 intercept was consistently correlated with worsening static and dynamic LH, pulmonary gas exchange, and airflow limitation in COPD. The V˙ E/V˙ CO2 intercept emerged as a useful index of ventilatory inefficiency in COPD patients.

RS4651 suppresses lung fibroblast activation via the TGF-ß1/SMAD signalling pathway.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a progressive disease resulting in respiratory failure with no efficient treatment options. We investigated the protective effect of RS4651 on pulmonary fibrosis in mice and the mechanism. METHODS: Intratracheal injection of bleomycin (BLM) was used to induce pulmonary fibrosis in mice. RS4561 was administered intraperitoneally at different doses. Histopathological changes were observed. The level of alpha-smooth muscle actin (α-SMA) were also tested. In vitro, the proliferation and migratory effects of RS4651 treatment on MRC-5 cells pre-treated with transforming growth factor (TGF-ß1) were examined. RNA-sequencing was used to detect differentially expressed target genes. Then, the expression of α-SMA, pSMAD2 and SMAD7 were analysed during RS4651 treatment of MRC-5 cells with or without silencing by SMAD7 siRNA. RESULTS: Histopathological staining results showed decreased collagen deposition in RS4651 administered mice. Additionally, a lower level of α-SMA was also observed compared to the BLM group. The results of in vitro studies confirmed that RS4651 can inhibit the proliferation and migration, as well as α-SMA and pSMAD2 expression in MRC-5 cells treated with TGF-ß1. RNA-sequencing data identified the target gene SMAD7. We found that RS4651 could upregulate SMAD7 expression and inhibit the proliferation and migration of MRC-5 cells via SMAD7, and RS4651 inhibition of α-SMA and pSMAD2 expression was blocked in SMAD7-siRNA MRC-5 cells. In vivo studies further confirmed that RS4651 could upregulate SMAD7 expression in BLM-induced lung fibrosis in mice. CONCLUSIONS: Our data suggest that RS4651 alleviates BLM-induced pulmonary fibrosis in mice by inhibiting the TGF-ß1/SMAD signalling pathway.

Soluble Thy-1 reverses lung fibrosis via its integrin-binding motif.

Loss of Thy-1 expression in fibroblasts correlates with lung fibrogenesis; however, the clinical relevance of therapeutic targeting of myofibroblasts via Thy-1-associated pathways remains to be explored. Using single (self-resolving) or repetitive (nonresolving) intratracheal administration of bleomycin in type 1 collagen-GFP reporter mice, we report that Thy-1 surface expression, but not mRNA, is reversibly diminished in activated fibroblasts and myofibroblasts in self-resolving fibrosis. However, Thy-1 mRNA expression is silenced in lung with nonresolving fibrosis following repetitive bleomycin administration, associated with persistent activation of αv integrin. Thy1-null mice showed progressive αv integrin activation and myofibroblast accumulation after a single dose of bleomycin. In vitro, targeting of αv integrin by soluble Thy-1-Fc (sThy-1), but not RLE-mutated Thy-1 or IgG, reversed TGF-ß1-induced myofibroblast differentiation in a dose-dependent manner, suggesting that Thy-1's integrin-binding RGD motif is required for the reversibility of myofibroblast differentiation. In vivo, treatment of established fibrosis induced either by single-dose bleomycin in WT mice or by induction of active TGF-ß1 by doxycycline in Cc10-rtTA-tTS-Tgfb1 mice with sThy-1 (1000 ng/kg, i.v.) promoted resolution of fibrosis. Collectively, these findings demonstrate that sThy-1 therapeutically inhibits the αv integrin-driven feedback loop that amplifies and sustains fibrosis.

Correlation between autophagy levels in peripheral blood mononuclear cells and clinical parameters in patients with chronic obstructive pulmonary disease.

Autophagy serves a role in the pathogenesis of chronic inflammatory diseases. The aim of the present study was to compare the autophagy levels in the peripheral blood mononuclear cells (PBMCs) of patients with chronic obstructive pulmonary disease (COPD) and healthy individuals and to assess the association between autophagy and the clinical parameters of COPD. Samples of peripheral blood from 20 patients with stable COPD and 20 healthy controls were collected. PBMCs were harvested using Ficoll density gradient centrifugation. Levels of the autophagy­associated proteins ubiquitin­binding protein p62 (p62), microtubule­associated proteins 1A/1B light chain 3A (LC3I/II) and beclin­1 in PBMCs were detected by western blotting. Enzyme­linked immunosorbent assay kits were used to detect the serum concentrations of interleukin (IL)­6, IL­8 and tumor necrosis factor (TNF)­α. Associations between the levels of autophagy and forced expiratory volume in 1 sec % predicted (FEV1%) and pro­inflammatory factors were assessed. Western blotting demonstrated that the protein expression of p62 was decreased, but LC3II/I and beclin­1 levels increased in patients with COPD compared with healthy controls. Serum levels of IL­6, IL­8 and TNF­α were increased in patients with COPD. The extent of PBMC autophagy was negatively correlated with FEV1% predicted, but positively correlated with levels of pro­inflammatory cytokines. The levels of autophagy in PBMCs in patients with COPD were increased and were negatively correlated with FEV1% predicted and positively correlated with circulating levels of pro­inflammatory cytokines. Autophagy may serve a role as a biomarker of the severity of COPD or as a therapeutic target for treatment of COPD.

αvß3 Integrin drives fibroblast contraction and strain stiffening of soft provisional matrix during progressive fibrosis.

Fibrosis is characterized by persistent deposition of extracellular matrix (ECM) by fibroblasts. Fibroblast mechanosensing of a stiffened ECM is hypothesized to drive the fibrotic program; however, the spatial distribution of ECM mechanics and their derangements in progressive fibrosis are poorly characterized. Importantly, fibrosis presents with significant histopathological heterogeneity at the microscale. Here, we report that fibroblastic foci (FF), the regions of active fibrogenesis in idiopathic pulmonary fibrosis (IPF), are surprisingly of similar modulus as normal lung parenchyma and are nonlinearly elastic. In vitro, provisional ECMs with mechanical properties similar to those of FF activate both normal and IPF patient-derived fibroblasts, whereas type I collagen ECMs with similar mechanical properties do not. This is mediated, in part, by αvß3 integrin engagement and is augmented by loss of expression of Thy-1, which regulates αvß3 integrin avidity for ECM. Thy-1 loss potentiates cell contractility-driven strain stiffening of provisional ECM in vitro and causes elevated αvß3 integrin activation, increased fibrosis, and greater mortality following fibrotic lung injury in vivo. These data suggest a central role for αvß3 integrin and provisional ECM in overriding mechanical cues that normally impose quiescent phenotypes, driving progressive fibrosis through physical stiffening of the fibrotic niche.

Reliability and Physiological Interpretation of Pulmonary Gas Exchange by "Circulatory Equivalents" in Chronic Heart Failure.

BACKGROUND: Peak ratios of pulmonary gas-exchange to ventilation during exercise (V˙O2/V˙E and V˙CO2/V˙E, termed "circulatory equivalents") are sensitive to heart failure (HF) severity, likely reflecting low and/or poorly distributed pulmonary perfusion. We tested whether peak V˙O2/V˙E and V˙CO2/V˙E would: (1) distinguish HF patients from controls; (2) be independent of incremental exercise protocol; and (3) correlate with lactate threshold (LT) and ventilatory compensation point (VCP), respectively. METHODS AND RESULTS: Twenty-four HF patients (61±11 years) with reduced ejection fraction (31±8%) and 11 controls (63±7 years) performed ramp-incremental cycle ergometry. Eighteen HF patients also performed slow (5±1 W/min), medium (9±4 W/min), and fast (19±6 W/min) ramps. Peak V˙O2/V˙E and V˙CO2/V˙E from X-Y plot, and LT and VCP from 9-panel plot, were determined by 2 independent, blinded, assessors. Peak V˙O2/V˙E (31.2±4.4 versus 41.8±4.8 mL/L; P<0.0001) and V˙CO2/V˙E (29.3±3.0 versus 36.9±4.0 mL/L; P<0.0001) were lower in HF than controls. Within individuals, there was no difference across 3 ramp rates in peak V˙O2/V˙E (P=0.62) or V˙CO2/V˙E (P=0.97). Coefficient of variation (CV) in peak V˙O2/V˙E was lower than for LT (5.1±2.1% versus 8.2±3.7%; P=0.014), and coefficient of variation in peak V˙CO2/V˙E was lower than for VCP (3.3±1.8% versus 8.7±4.2%; P<0.001). In all participants, peak V˙O2/V˙E was correlated with, but occurred earlier than, LT (r2=0.94; mean bias, -0.11 L/min), and peak V˙CO2/V˙E was correlated with, but occurred earlier than, VCP (r2=0.98; mean bias -0.08 L/min). CONCLUSIONS: Peak circulatory equivalents during exercise are strongly associated with (but not identical to) LT and VCP. Peak circulatory equivalents are reliable, objective, effort-independent indices of gas-exchange abnormality in HF.

Cigarette Smoke-Induced Pulmonary Inflammation and Autophagy Are Attenuated in Ephx2-Deficient Mice.

Cigarette smoke (CS) increases the risk of chronic obstructive pulmonary disease (COPD) by causing inflammation, emphysema, and reduced lung function. Additionally, CS can induce autophagy which contributes to COPD. Arachidonic acid-derived epoxyeicosatrienoic acids (EETs) have promising anti-inflammatory properties that may protect the heart and liver by regulating autophagy. For this reason, the effect of decreased soluble epoxide hydrolase (sEH, Ephx2)-mediated EET hydrolysis on inflammation, emphysema, lung function, and autophagy was here studied in CS-induced COPD in vivo. Adult male wild-type (WT) C57BL/6J and Ephx2-/- mice were exposed to air or CS for 12 weeks, and lung inflammatory responses, air space enlargement (emphysema), lung function, and autophagy were assessed. Lungs of Ephx2-/- mice had a less pronounced inflammatory response and less autophagy with mild distal airspace enlargement accompanied by restored lung function and steady weight gain. These findings support the idea that Ephx2 may hold promise as a therapeutic target for COPD induced by CS, and it may be protective property by inhibiting autophagy.

Thy-1 dependent uptake of mesenchymal stem cell-derived extracellular vesicles blocks myofibroblastic differentiation.

Bone marrow-derived mesenchymal stem cells (MSC) have been promoted for multiple therapeutic applications. Many beneficial effects of MSCs are paracrine, dependent on extracellular vesicles (EVs). Although MSC-derived EVs (mEVs) are beneficial for acute lung injury and pulmonary fibrosis, mechanisms of mEV uptake by lung fibroblasts and their effects on myofibroblastic differentiation have not been established. We demonstrate that mEVs, but not fibroblast EVs (fEVs), suppress TGFß1-induced myofibroblastic differentiation of normal and idiopathic pulmonary fibrosis (IPF) lung fibroblasts. MEVs display increased time- and dose-dependent cellular uptake compared to fEVs. Removal or blocking of Thy-1, or blocking Thy-1-beta integrin interactions, decreased mEV uptake and prevented suppression of myofibroblastic differentiation. MicroRNAs (miRs) 199a/b-3p, 21-5p, 630, 22-3p, 196a-5p, 199b-5p, 34a-5p and 148a-3p are selectively packaged in mEVs. In silico analyses indicated that IPF lung fibroblasts have increased expression of genes that are targets of mEV-enriched miRs. MiR-630 mimics blocked TGFß1 induction of CDH2 in normal and IPF fibroblasts, and antagomiR-630 abrogated the effect of mEV on CDH2 expression. These data suggest that the interaction of Thy-1 with beta integrins mediates mEV uptake by lung fibroblasts, which blocks myofibroblastic differentiation, and that mEVs are enriched for miRs that target profibrotic genes up-regulated in IPF fibroblasts.

Decreased Histone Deacetylase 2 (HDAC2) in Peripheral Blood Monocytes (PBMCs) of COPD Patients.

BACKGROUND: Histone deacetylase 2 (HDAC2) is a class I histone deacetylase family member that plays a critical role in suppressing inflammatory gene expression in the airways, lung parenchyma, and alveolar macrophages in patients with chronic obstructive pulmonary disease (COPD). However, the expression of HDAC2 in peripheral blood monocytes (PBMCs), nuclear factor kappa B (NF-κB) p65, and serum inflammatory cytokine levels in COPD patients, smokers, and non-smokers remains unclear. METHODS: PBMCs were obtained from COPD patients, healthy smokers, and healthy nonsmokers. The HDAC2 and NF-κB p65 expression were quantified by Western Blot. HDAC activity was assessed by an HDAC fluorometric immunoprecipitation activity assay kit. Serum tumor necrosis factor-alpha (TNF-α) and interleukin-8 (IL-8) levels were measured by ELISA. RESULTS: HDAC2 expression and HDAC activity were decreased in PBMCs in COPD patients compared with smokers and non-smokers. Increased NF-κB p65 expression, serum TNF-α and IL-8 levels were observed in COPD patients compared with nonsmokers. The FEV1%pred was positively correlated with HDAC2 expression and HDAC activity in COPD patients. Smokers had decreased HDAC activity, increased NF-κB p65 expression and serum TNF-α compared with nonsmokers. CONCLUSIONS: HDAC2 expression was decreased in PBMCs of COPD patients and was correlated with disease severity. The reduction of HDAC2 expression not only directly enhances the expression of inflammatory genes, but may account for the activation of NF-κB mediated inflammation. Decreased HDAC2 may serve as a potential biomarker of COPD and predict the decline of lung function.

[Cardiopulmonary exercise testing as predictors for lung resection in patients with lung cancer].

Cardiopulmonary exercise testing (CPET) has become an important clinical tool to evaluate exercise capacity and predict outcome for patients with lung cancer being considered for lung resection. CPET can help in stratifying the surgical risk and identify those high-risk patients with lung resection. Preoperative screening using CPET allow the selection of appropriate therapeutic approach to decrease surgical complications and mortality.