Search

The detection rate of the solitary pulmonary nodule (SPN) is increasing with the popularization of CT scanning. Malignancy risk stratification for SPN is a major clinical difficulty.There have been several guidelines for SPN assessment. Inconsistency of these guidelines makes the clinical application difficult and confusing.In this Rapid Recommendation, solid and subsolid SPNs are recommended to be evaluated respectively. Six factors, namely the combination of age with sex, smoking history, history of malignancy, family history of malignancy, and nodule size, are recommended for malignancy risk stratification for both kinds of SPNs; the border of nodules (spiculation and lobulation) is recommended for evaluating solid SPNs and the density of nodules (pure or mixed ground-glass nodule) is recommended for subsolid nodules. Among them, smoking history and radiologic features (nodule diameter, border, and density) are of relatively higher importance. A scoring system was proposed to assist malignancy risk stratification of SPNs, with a total score ranging from six points to 15 points (if solid) or 17 points (if subsolid). For each SPN, regardless of solid or subsolid in nature, a total score of ≤ 7 points suggested a low risk of being malignant, while 7 to 9 points suggested medium risk, and ≥ 9 points suggested high risk.This rapid recommendation was developed using the MAGIC (Making GRADE the Irresistible Choice) methodological framework. First, a clinical subcommittee identified the topic of recommendation and requested evidence. Then, an independent evidence synthesis subcommittee performed a comprehensive literature review and evaluated the evidence. Finally, based on findings from the systematic review and use of real-world data, the clinical subcommittee formulated recommendations, including the scoring system, through a consensus procedure.A total of 13857 patients with SPNs were included in the meta-analysis and the association between 12 candidate factors and the risk of SPNs being malignant was studied. Eventually, seven factors were recommended for SPNs evaluation, and a scoring system was proposed.The parameters included are objective. Therefore, this recommendation is feasible in clinical practice. However, there are several uncertainties, such as a lack of further verification. It might be misclassified by the scoring system. Clinicians could choose the most suitable scheme according to the recommendation, along with their own experience in specific situations.

Omentin-1 is an adipokine expressed by the adipose tissue and is reduced in obesity. This study was designed to calculate the protective efficiency and mechanism of omentin-1 against inflammation of the adipose tissue in obese mice. A transgenic mouse model with omentin-1 protein overexpression was established by crossing omentin-1 transgenic mice with Fabp4-Cre mice. Obesity was induced in the mice by feeding them a high-fat diet for 10 weeks. Fabp4-Cre-mediated overexpression of omentin-1 significantly increased serum omentin-1 level, serum anti-inflammatory factor levels, and expression of M2-specific mRNAs; inhibited body weight and adipose tissue weight gain; improved glucose tolerance, insulin tolerance, and insulin sensitivity; decreased serum levels of insulin and proinflammatory factors, adipocyte size, and expression of M1-specific mRNAs; suppressed macrophage infiltration; downregulated expression of proinflammatory factors; upregulated expression of anti-inflammatory factors; and inhibited thioredoxin-interacting protein (TXNIP)/NOD-like receptor 3 (NLRP3) signaling in the adipose tissue of obese mice. An NLRP3 inhibitor (20 mg/kg MCC950) exhibited the same effects as overexpression of omentin-1. Pretreatment with omentin-1 inhibited lipopolysaccharide-induced inflammation via TXNIP/NLRP3 signaling in RAW 264.7 macrophages. These findings suggest that omentin-1 suppresses adipose tissue inflammation in obese mice, at least partly, via inhibiting the TXNIP/NLRP3 signaling pathway.

Objectives Chemoresistance induced by cisplatin has become the major impediment to lung cancer chemotherapy. This study explored the potential chemoresistant genes and underlying mechanisms of chemoresistance in NSCLC. Materials and methods Gene expression profile was integrated with DNA methylation profile to screen the candidate chemoresistant genes. Bioinformatic analysis and immunohistochemistry were used to analyse the association of a candidate gene with the characteristics of NSCLC patients. Recombinant lentivirus vectors were utilized to overexpress or silence candidate gene. Microarrays and immunoblotting were applied to explore the downstream targets of candidate gene. Xenograft models were established to validate the findings in vitro. Results An increased ZNF300 expression was detected in three chemoresistant cell lines of NSCLC, and the higher expression of ZNF300 was associated with poor OS of NSCLC patients. Cells with upregulated ZNF300 presented chemoresistance and enhanced aggressive growth compared to cells with downregulated ZNF300. ZNF300 inhibited MAPK/ERK pathways and activated CDK1 through inhibiting WEE1 and MYT1 and modulating MYC/AURKA/BORA/PLK1 axis. ICA and ATRA improved the anti‐tumour effect of cisplatin on chemoresistant cells by inducing differentiation. Conclusions ZNF300 promotes chemoresistance and aggressive behaviour of NSCLC through regulation of proliferation and differentiation by downregulating MAPK/ERK pathways and regulation of slow‐cycling phenotype via activating CDK1 by inhibiting WEE1/MYT1 and modulating MYC/AURKA/BORA/PLK1 axis. Cisplatin, combined with ATRA and ICA, might be beneficial in chemoresistant cases of NSCLC., ZNF300 mediates chemoresistance of NSCLC. ZNF300 promotes aggressive growth of tumour cells correlating to poor prognosis of patients with NSCLC. ZNF300 inhibits MAPK/ERK signalling pathway to suppress proliferation and differentiation of chemoresistant cells. ZNF300 activates CDK1 by inhibiting WEE1 and MYT1 and modulating MYC/AURKA/BORA/PLK1 axis to regulate cell cycle of chemoresistant cells. ICA and ATRA improves anti‐tumour effect of cisplatin on chemoresistant cells by inducing differentiation.

Background: Chemo-resistance induced by cisplatin (DDP) has become the major impediment to lung cancer chemotherapy. The study aimed to unearth an alternative route for reversing chemo-resistance and suppressing the malignant progression of tumors. Methods: Gene expression profile was integrated with DNA methylation profile of A549/DDP cells of human adenocarcinoma. Immunohistochemistry were used to analyze the association of gene with characteristics of NSCLC patients. Cell viability, apoptosis, cell cycle, migration, engulfment, and senescence were assessed between the different groups of cells. Genes related with differentiation and cell cycle, screened by microarrays, were examined by immunoblotting. Xenograft model was utilized to validate the results in vitro. Findings: ZNF300 was association with poor overall survival of NSCLC patients. Multidrug-resistant cells exhibited a more malignant behavior related to chemo-sensitive cells. Most of genes related with differentiation, cyclin-dependent kinase inhibitor, and MAPK/ERK pathways were downregulated, while most of genes related with cell cycle and cancer stem cell were upregulated in multidrug-resistant cells compared to chemo-sensitive cells. ICA improved the antitumor effect of cisplatin on multidrug-resistant cells. Interpretation: ZNF300 promoted the malignant behavior of multidrug-resistant cells through inhibiting differentiation via supressing MAPK/ERK pathways and enhancing cancer stem cell-like properties via upregulating Nanog and OCT4. Additionally, CDK1 activated by ZNF300 via inhibiting WEE1 and MYT1 and modulating AURKA/BORA/PLK1/CDC25 cascade promoted cells to enter cell cycle and escape the apoptosis caused by chemotherapeutic drug. ICA improved the antitumor effect of cisplatin on multidrug resistant cells by inducing differentiation. Funding Statement: This work was supported by the National Natural Scientific Fund of China (№:81372499), the Cultivating Project for Young Scholar at Hubei University of Medicine ( № :2018QDJZR01 and 2018QDJZR11), and the Open Project of Hubei Key Laboratory of Wudang Local Chinese Medicine Research (Hubei University of Medicine) (№:WDCM2018002). Declaration of Interests: The authors declare no conflict of interest. Ethics Approval Statement: The informed consents were obtained from all the patients undertaking surgery and participating in this study. All animal experiments were authorized by the Experimental Animal Ethics Committee of Xinqiao Hospital of the Army Medical University [SYXK(YU)2012-0011] and Hubei University of Medicine [SYXK(E)2016-0031].

Background: So far, the platinum-based chemotherapy (e.g. cisplatin-etoposide doublet) is still the backbone for SCLC management due to its high respond rate both in LS-SCLC and ES-SCLC. However, cisplatin treatment often results in the development of chemo-resistance, leading to therapeutic failure and becoming the main obstacle to improve the therapeutic efficacy. Currently, little has been known about the genome-wide abnormal methylation of SCLC induced by cisplatin, which might provide prospective layouts to discover the potential genes and the signal pathways related with chemo-resistance of SCLC. Results: A total of 58,401 sites was identified to be differentially methylated (|Δβ| ≧ 0.20) in H446/DDP cells compared with that of H446 cells, of which 25,991 genes were found to be hypomethylated and 32,410 genes were shown to be hypermethylated. KEGG enrichment displayed that the differentially hypomethylated genes were mainly gathered in MAPK signaling pathway, ECM-receptor interaction, and Focal adhesion, while the differentially hypermethylated genes were clustered in Neuroactive ligand-receptor interaction, Type I diabetes mellitus, Focal adhesion, Allograft rejection, ECM-receptor interaction, CAMs, Graft-versus-host disease, Intestinal immune network for IgA production, ARVC, and Viral myocarditis (KEGG enrichment, qvalue < 0.05). Among the 152 genes which were selected as the MDR-related candidate genes for qRT-PCR to testify whether the abnormal methylation regulated the expression of related genes at the mRNA level, 69 hypomethylated genes were revealed to be significantly increased and the other 54 hypermethylated genes evidently decreased in H446/DDP cells compared with that of H446 cells. Moreover, the upregulated genes with the hypomethylated sites were found to be mainly clustered in Pathways in cancer, MAPK signaling pathway, Cytokine-cytokine receptor interaction, and Cell adhesion molecules (CAMs), while the downregulated genes with the hypermethylated sites were mainly clustered in MAPK signaling pathway, Pathways in cancer, Melanoma, Osteoclast differentiation, and Prostate cancer. Conclusions: Cisplatin could induce a large-scale abnormal methylation in the whole genome of SCLC. Pathways in cancer, MAPK signaling pathway, Cytokine-cytokine receptor interaction, and Cell adhesion molecules (CAMs) were most likely to be affected by cisplatin via. methylation to contribute to the development of chemo-resistance and other malignant biological behavior of SCLC cells.

Autophagy plays a key role in pulmonary vascular remodeling via regulation of apoptosis and hyperproliferation of pulmonary arterial endothelial cells, which are the subject of increased attention. Autophagy-related 101 (ATG101) is an essential gene for the initiation of autophagy. Although the structure of ATG101 has been well-characterized, its exact biological function in autophagy is still unknown. In this study, an ATG101 single-stranded antisense RNA-loaded DNA triangular nanoparticle (ssATG101-TNP) is constructed to knock down the ATG101 gene expression. ssATG101-TNP can be effectively transfected into human pulmonary arterial endothelial cells (HPAECs) in time- and dose-dependent manners. Knockdown of ATG101 promotes cell apoptosis as well as inhibits cell autophagy and proliferation with hypoxic stimulation. Additionally, the hedgehog/Gli signal pathway is involved in ATG101-mediated macroautophagy and HPAEC proliferation. This study found that ATG101, an important member of the autophagy gene family, can regulate cell macroautophagy, apoptosis, and growth in HPAECs. ssATG101-TNP is demonstrated to be a nontoxic, highly efficient, gene-delivery vehicle for HPAECs. These findings also suggest that ATG101 might be a potential therapeutic target in diseases involving endothelial injury.

Fibromodulin (FMOD), an ECM small leucine-rich proteoglycan (SLRP), was reported to promote angiogenesis not only during wound healing, but also in optical and cutaneous angiogenesis-dependent diseases. However, whether it plays important roles in tumor angiogenesis remains unclear. To explore the role of FMOD in tumor angiogenesis of human small cell lung cancer (SCLC), initially the study analyzed the relationship of FMOD expression in cancer tissues of SCLC with clinical characteristics. The analysis revealed that the positive FMOD expression was significantly associated with extensive stage of SCLC and higher vascular density. In mouse models, xenograft tumors developed with FMOD-silenced H446 cells (H446-shFMOD) exhibited slowed growth rate, decreased microvessel density, and reduced blood perfusion related to that of controls (H446-shCON). Additionally, compared with that of controls, the decreased secretion of FMOD in conditioned medium (CM) from H446-shFMOD inhibited proliferation, migration, and invasion of human umbilical vessel endothelial cells (HUVECs). Moreover, the decreased secretion of FMOD downregulated the expression of VEGF, TGF-β1, FGF-2, and PDGF-B in HUVECs. The findings strongly suggested that the autocrine FMOD of cancer cells may promote tumor angiogenesis of SCLC by upregulating the expression of angiogenic factors that act in concert to facilitate the angiogenic phenotype of endothelial cells as a proangiogenic factor. Therefore, silencing FMOD may be a potentially clinical therapy for repressing tumor angiogenesis.

// Li Yang 1, * , Qiao Zhang 2, * , Li Bai 2 , Ting-Yuan Li 1 , Chuang He 1 , Qian-Li Ma 2 , Liang-Shan Li 1 , Xue-Quan Huang 1 , Gui-Sheng Qian 2 1 Interventional Radiology Department, the First Hospital of the Third Military Medical University, Chongqing 400038, China 2 Institute of Respiratory Diseases, the Second Hospital of the Third Military Medical University, Chongqing 400038, China * These authors are contributed equally to these work Correspondence to: Gui-Sheng Qian, email: qiangs1220@163.com Keywords: solitary, pulmonary nodule, malignancy, risk factor, model Received: October 15, 2016 Accepted: January 16, 2017 Published: March 21, 2017 ABSTRACT There are no large samples or exact prediction models for assessing the cancer risk factors of solitary pulmonary nodules (SPNs) in the Chinese population. We retrospectively analyzed the clinical and imaging data of patients with SPNs who underwent computer tomography guided needle biopsy in our hospital from Jan 1st of 2011 to March 30th of 2016. These patients were divided into a development data set and a validation data set. These groups included 1078 and 344 patients, respectively. A prediction model was developed from the development data set and was validated with the validation data set using logistic regression. The predictors of cancer in our model included female gender, age, pack-years of smoking, a previous history of malignancy, nodule size, lobulated and spiculated edges, lobulation alone and spiculation alone. The Area Under the Curves, sensitivity and specificity of our model in the development and validation data sets were significantly higher than those of the Mayo model and VA model ( p 8 mm in size. SPNs in female patients, as well as SPNs featuring a combination of lobulated and spiculated edges or lobulated edges alone, should be evaluated carefully due to the probability that they are malignant.

High-altitude deacclimatization syndrome (HADAS) is involved in hypoxia-reoxygenation injury and inflammatory response, induced a series of symptoms, and has emerged as a severe public health issue. Here, we investigated the mechanism as well as potential means to prevent HADAS using Shenqi pollen capsules (SPCs) in subjects with HADAS in a multicenter, double-blinded, randomized, placebo-controlled study. All subjects were at the same high altitude (3650 m) for 4-8 months before returning to lower altitudes. Subjects (n=288) in 20 clusters were diagnosed with mild or moderate HADAS on the third day of the study. We randomly allocated 20 clusters of subjects (1 : 1) to receive SPCs or a placebo for 7 weeks, and they were then followed up to the 14th week. The primary endpoints were subjects’ HADAS scores recorded during the 14 weeks of follow-up. Compared with the placebo, SPC treatment significantly decreased the subjects’ HADAS scores and reduced the incidence of symptom persistence. SPC therapy also reduced the serum levels of CK, CK-MB, LDH, IL-17A, TNF-α, and miR-155 and elevated IL-10 and miR-21 levels. We thus demonstrate that SPCs effectively ameliorated HADAS symptoms in these subjects via suppression of the hypoxia-reoxygenation injury and inflammatory response.

Objective The aim of this study was to investigate whether autophagy is enhanced in alveolar epithelial cells as well as its role in alveolar barrier function of in lipopolysaccharide (LPS)-induced ALI mice. Materials and methods Autophagy inhibitors, including 3-methyladenine (3-MA) and chloroquine (CLQ), and LPS were intraperitoneally administered to mice. Histological evaluation and confocal microscopy, Western blot, transmission electron microscopy, and ELISA were performed for analysis. First, the mouse model of ALI was established. Then, autophagy level changes in the mouse lung as well as the effects of autophagy inhibition on indirect ALI and alveolar epithelial barrier function induced by LPS were assessed. Finally, pro-inflammatory factors in BALF from ALI mice after autophagy inhibition by 3-MA or CLQ administration were detected. Results The experimental animal model of LPS-induced ALI had the expected features. In addition, autophagy in alveolar epithelial cells in ALI mice was enhanced. Furthermore, autophagy in alveolar epithelial cells promoted alveolar epithelial barrier dysfunction in LPS-induced ALI. Finally, autophagy inhibition resulted in reduced LPS-induced lung tissue inflammation. Conclusion These findings suggest that autophagy inhibition protects from alveolar barrier dysfunction in LPS-induced ALI mice by targeting alveolar epithelial cells.

The mechanisms leading to diabetic neuropathy are complex. As an active component in several traditional Chinese medicines, berberine has a beneficial effect in the treatment of diabetes with hyperlipidemia. This study evaluated the protective effects of berberine on diabetic neuropathy induced by streptozotocin and a high-carbohydrate/high-fat diet in rats. Diabetic neuropathy was induced in rats by intraperitoneal injection of 35 mg/kg streptozotocin and a high-carbohydrate/high-fat diet. Two weeks after diabetes induction, rats were treated with berberine (100 mg/kg) and rosiglitazone (4 mg/kg) for 24 weeks. Rats were studied using evoked potentials, the Morris water maze, transmission electron microscopy, real-time PCR, and Western blotting. Blood glucose, glycated hemoglobin, lipid profile, body weight, evoked potentials, and memory were altered in diabetic rats, as was the hippocampal expression of neuritin mRNA, p38 mitogen-activated protein kinase mRNA, c-Jun N-terminal kinase (JNK) mRNA, extracellular signal-regulated kinase mRNA and the phospho-proteins of p38, JNK, and extracellular signal-regulated kinase. In diabetic rats, berberine decreased body weight and the blood levels of glucose, glycated hemoglobin, triglyceride, and total cholesterol, improved memory and affected evoked potential by decreasing latency. Berberine decreased the mRNA expression of neuritin, p38, and JNK and the protein expression of neuritin, p-p38, and p-JNK. Slight micropathological changes were observed in the hippocampus of berberine-treated diabetic rats. These findings suggest that berberine has a beneficial effect against diabetic neuropathy by improving micropathology and increasing neuritin expression via the mitogen-activated protein kinase signaling pathway.

Excessive inflammatory response induced by lipopolysaccharide (LPS) plays a critical role in the development of acute lung injury (ALI). Paralemmin-3 (PALM3) is a novel protein that can modulate LPS-stimulated inflammatory responses in alveolar epithelial A549 cells. However, it remains unclear whether it is involved in the progression of ALI in vivo. Therefore, we studied the role of PALM3 in the pathogenesis of ALI induced by LPS. ALI was induced by LPS peritoneal injection in C57BL/6J mice. Lentivirus-mediated small interfering RNA (siRNA) targeting the mouse PALM3 gene and a negative control siRNA were intranasally administered to the mice. We found that the expression of PALM3 was up-regulated in the lung tissues obtained from the mouse model of LPS-induced ALI. The LPS-evoked inflammatory response (neutrophils and the concentrations of proinflammatory cytokines [IL-6, IL-1β, TNF-α, MIP-2] in the bronchoalveolar lavage fluid [BALF]), histologic lung injury (lung injury score), permeability of the alveolar capillary barrier (lung wet/dry weight ratio and BALF protein concentration) and mortality rates were attenuated in the PALM3 siRNA-treated mice. These results indicate that PALM3 contributes to the development of ALI in mice challenged with LPS. Inhibiting PALM3 through the intranasal application of specific siRNA protected against LPS-induced ALI.

Silent mating type information regulation 2 homolog-1 (SIRT1) is involved in a wide range of cellular processes because of its role as a deacetylated histone and its association with a variety of transcription factors. SIRT1 has essential roles in autophagy, including in the formation of autophagic vacuoles and the assembly of autophagy-related gene (ATG) protein complexes. The present study focused on the role of SIRT1 in autophagy in lipopolysaccharide (LPS)-induced mouse type II alveolar epithelial cells (AECII). We designed experiments using SIRT1-overexpressing mice and wild-type mice, and AECII were isolated from these two types of mouse for in vitro LPS injury trials. Our results suggest that levels of the autophagy proteins, Beclin1 and LC3B, as well as those of the inflammatory factors, IL-6 and TNF-α, were increased in LPS-induced mouse AECII, and that SIRT1 protected against damage in mice with acute respiratory distress syndrome and in mouse AECII in vitro following LPS treatment. Subsequently, we screened multiple inflammatory, apoptotic, and unclassified genes (including Atg7), which interacted with SIRT1 in LPS-injured mouse AECII, as assessed by mRNA microarray analysis. These results demonstrate that LPS can reduce the levels of SIRT1 and ATG7 in vivo and in vitro and indicate that SIRT1 is involved in autophagy through regulation of ATG7 in AECII in response to LPS.

Type 2 diabetes increases the risk of developing cognitive dysfunction in the elderly in the form of short-term memory and executive function impairment. Genetic and diet-induced models of type 2 diabetes further support this link, displaying deficits in working memory, learning, and memory performance. The risk factors for diabetic cognitive dysfunction include vascular disease, hypoglycaemia, hyperlipidaemia, adiposity, insulin resistance, lifestyle factors, and genetic factors. Using neuronal imaging technologies, diabetic patients with cognitive dysfunction show atrophy of the whole brain, particularly the grey matter, hippocampus and amygdala; increased volume of the ventricular and white matter; brain infarcts; impaired network integrity; abnormal microstructure; and reduced cerebral blood flow and amplitude of low-frequency fluctuations. The pathogenesis of type 2 diabetes with cognitive dysfunction involves hyperglycaemia, macrovascular and microvascular diseases, insulin resistance, inflammation, apoptosis, and disorders of neurotransmitters. Large clinical trials may offer further proof of biomarkers and risk factors for diabetic cognitive dysfunction. Advanced neuronal imaging technologies and novel disease animal models will assist in elucidating the precise pathogenesis and to provide better therapeutic interventions and treatment.

Microvascular barrier dysfunction is the central pathophysiological feature of acute lung injury (ALI). RAB26 is a newly identified small GTPase involved in the regulation of endothelial cell (EC) permeability. However, the mechanism behind this protection has not been clearly elucidated. Here we found that RAB26 promoted the integrity of adherens junctions (AJs) in a macroautophagy/autophagy-dependent manner in ALI. RAB26 is frequently downregulated in mouse lungs after LPS treatment. Mice lacking Rab26 exhibited phosphorylated SRC expression and increased CDH5/VE-cadherin phosphorylation, leading to AJ destruction. rab26-null mice showed further aggravation of the effects of endotoxin insult on lung vascular permeability and water content. Depletion of RAB26 resulted in upregulation of phosphorylated SRC, enhancement of CDH5 phosphorylation, and aggravation of CDH5 internalization, thereby weakening AJ integrity and endothelial barrier function in human pulmonary microvascular endothelial cells (HPMECs). RAB26 overexpression caused active interaction between SRC and the autophagy marker LC3-II and promoted degradation of phosphorylated SRC. Furthermore, RAB26 was involved in a direct and activation-dependent manner in autophagy induction through interaction with ATG16L1 in its GTP-bound form. These findings demonstrate that RAB26 exerts a protective effect on endothelial cell (EC) permeability, which is in part dependent on autophagic targeting of active SRC, and the resultant CDH5 dephosphorylation maintains AJ stabilization. Thus, RAB26-mediated autophagic targeting of phosphorylated SRC can maintain barrier integrity when flux through the RAB26-SRC pathway is protected. These findings suggest that activation of RAB26-SRC signaling provides a new therapeutic opportunity to prevent vascular leakage in ALI. Abbreviations: AJs: adherens junctions; ALI: acute lung injury; ARDS: acute respiratory distress syndrome; ATG5: autophagy related 5; ATG12: autophagy related 12; ATG 16L1: autophagy related 16 like; 1 BALF: bronchoalveolar lavage fluidCQ: chloroquine; Ctrl: control; EC: endothelial cell; GFP: green fluorescent protein; HA-tagged; RAB26WT: HA-tagged wild-type; RAB26 HA-tagged; RAB26QL: HA-tagged; RAB26Q123LHA-tagged; RAB26NI: HA-tagged; RAB26N177IHPMECs: human pulmonary microvascular endothelial cells; H&E: hematoxylin & eosin; IgG: immunoglobulin; GIF: immunofluorescence; IP: immunoprecipitationi;. p.: intraperitoneal; LPS: lipopolysaccharide; PBS: phosphate-buffered salinesi; RNA: small interfering;RNASQSTM1/p62, sequestosome; 1TBS: Tris-buffered saline; VEGF: vascular endothelial growth factor; WB: western blot; WT: wild-type

Diabetes impairs the bone marrow (BM) architecture and function as well as the mobilization of immature cells into the bloodstream and number of potential regenerative cells. Circadian regulation of bone immature cell migration is regulated by β-adrenergic receptors, which are expressed on haematopoietic stem cells, mesenchymal stem cells, and osteoblasts in the BM. Diabetes is associated with a substantially lower number of sympathetic nerve terminal endings in the BM; thus, diabetic neuropathy plays a critical role in BM dysfunction. Treatment with mesenchymal stem cells, BM mononuclear cells, haematopoietic stem cells, and stromal cells ameliorates the dysfunction of diabetic neuropathy, which occurs, in part, through secreted neurotrophic factors, growth factors, adipokines, and polarizing macrophage M2 cells and inhibiting inflammation. Inflammation may be a therapeutic target for BM stem cells to improve diabetic neuropathy. Given that angiogenic and neurotrophic effects are two major barriers to effective diabetic neuropathy therapy, targeting BM stem cells may provide a novel approach to develop these types of treatments.

Hepatopulmonary syndrome (HPS) is characterized by an arterial oxygenation defect induced by intrapulmonary vasodilation (IPVD) that increases morbidity and mortality. In our previous study, it was determined that both the proliferation and the myogenic differentiation of pulmonary microvascular endothelial cells (PMVECs) play a key role in the development of IPVD. However, the molecular mechanism underlying the relationship between IPVD and the myogenic differentiation of PMVECs remains unknown. Additionally, it has been shown that bone morphogenic protein-2 (BMP2), via the control of protein expression, may regulate cell differentiation including cardiomyocyte differentiation, neuronal differentiation and odontoblastic differentiation. In this study, we observed that common bile duct ligation (CBDL)-rat serum induced the upregulation of the expression of several myogenic proteins (SM-α-actin, calponin, SM-MHC) and enhanced the expression levels of BMP2 mRNA and protein in PMVECs. We also observed that both the expression levels of Smad1/5 and the activation of phosphorylated Smad1/5 were significantly elevated in PMVECs following exposure to CBDL-rat serum, which was accompanied by the down-regulation of Smurf1. The blockage of the BMP2/Smad signaling pathway with Noggin inhibited the myogenic differentiation of PMVECs, a process that was associated with relatively low expression levels of both SM-α-actin and calponin in the setting of CBDL-rat serum exposure, although SM-MHC expression was not affected. These findings suggested that the BMP2/Smad signaling pathway is involved in the myogenic differentiation of the PMVECs. In conclusion, our data highlight the pivotal role of BMP2 in the CBDL-rat serum-induced myogenic differentiation of PMVECs via the activation of both Smad1 and Smad5 and the down-regulation of Smurf1, which may represent a potential therapy for HPS-induced pulmonary vascular remodeling.

Hepatopulmonary syndrome (HPS) is characterized by a triad of severe liver disease, intrapulmonary vascular dilation and hypoxaemia. Pulmonary vascular remodelling (PVR) is a key feature of HPS pathology. Our previous studies have established the role of the pulmonary artery smooth muscle cell (PASMC) phenotypic modulation and proliferation in HPS-associated PVR. Myocardin, a robust transcriptional coactivator of serum response factor, plays a critical role in the vascular smooth muscle cell phenotypic switch. However, the mechanism regulating myocardin upstream signalling remains unclear. In this study, treatment of rat PASMCs with serum drawn from common bile duct ligation rats, which model symptoms of HPS, resulted in a significant increase in miR-9 expression correlated with a decrease in expression of myocardin and the phenotypic markers SM-α-actin and smooth muscle-specific myosin heavy chain (SM-MHC). Furthermore, miRNA functional analysis and luciferase reporter assay demonstrated that miR-9 effectively regulated myocardin expression by directly binding to its 3′-untranslated region. Both the knockdown of miR-9 and overexpression of myocardin effectively attenuated the HPS rat serum-induced phenotype switch and proliferation of PASMCs. Taken together, the findings of our present study demonstrate that miR-9 is required in HPS rat serum-induced phenotypic modulation and proliferation of PASMCs for targeting of myocardin and that miR-9 may serve as a potential therapeutic target in HPS.

Background: Alveolar epithelial cell death plays a critical role in the pathogenesis of lipopolysaccharide (LPS)-induced acute lung injury. Increased autophagy has a dual effect on cell survival. However, it is not known whether autophagy promotes death or survival in human alveolar epithelial cells exposed to LPS. Methods: Genetic and pharmacological approaches were used to evaluate the effect of autophagy on A549 cell viability upon LPS exposure. The endoplasmic reticulum (ER) stress and unfolded protein response (UPR) pathways were examined with immunoblotting studies to further explore underlying mechanisms. Results: Treatment with LPS (50 µg/ml) led to autophagy activation and decreased cell viability in A549 cells. Blocking autophagy via short interfering RNA or inhibitor significantly decreased, whereas rapamycin increased, the LPS-induced effect on viability. ER stress was activated in LPS-stimulated A549 cells, and ER stress inhibitor reduced LPS-induced autophagy. LPS activated only the PERK pathway and had rarely effect on the ATF6 and IRE1 branches of the UPR in A549 cells. Moreover, the knockdown of PERK and ATF4 attenuated LPS-induced autophagy and promoted cell survival. Conclusion: In human alveolar epithelial A549 cells, LPS induces autophagic cell death that depends on the activation of the PERK branch of the UPR upon ER stress.

The small GTPase Rab5 has been well defined to control the vesicle-mediated plasma membrane protein transport to the endosomal compartment. However, its function in the internalization of vascular endothelial (VE)-cadherin, an important component of adherens junctions, and as a result regulating the endothelial cell polarity and barrier function remain unknown. Here, we demonstrated that lipopolysaccharide (LPS) simulation markedly enhanced the activation and expression of Rab5 in human pulmonary microvascular endothelial cells (HPMECs), which is accompanied by VE-cadherin internalization. In parallel, LPS challenge also induced abnormal cell polarity and dysfunction of the endothelial barrier in HPMECs. LPS stimulation promoted the translocation of VE-cadherin from the plasma membrane to intracellular compartments, and intracellularly expressed VE-cadherin was extensively colocalized with Rab5. Small interfering RNA (siRNA)-mediated depletion of Rab5a expression attenuated the disruption of LPS-induced internalization of VE-cadherin and the disorder of cell polarity. Furthermore, knockdown of Rab5 inhibited the vascular endothelial hyperpermeability and protected endothelial barrier function from LPS injury, both in vitro and in vivo. These results suggest that Rab5 is a critical mediator of LPS-induced endothelial barrier dysfunction, which is likely mediated through regulating VE-cadherin internalization. These findings provide evidence, implicating that Rab5a is a potential therapeutic target for preventing endothelial barrier disruption and vascular inflammation.

Lung inflammation and alveolar epithelial cell death are critical events in the development and progression of acute lung injury (ALI). Although angiopoietin-like protein 4 (ANGPTL4) participates in inflammation, whether it plays important roles in ALI and alveolar epithelial cell inflammatory injury remains unclear. We therefore investigated the role of angptl4 in lipopolysaccharide (LPS)-induced ALI and the associated mechanisms. Lentivirus-mediated short interfering RNA targeted to the mouse angptl4 gene (AngsiRNA) and a negative control lentivirus (NCsiRNA) were intranasally administered to mice. Lung inflammatory injury and the underlying mechanisms for regulation of angptl4 on the LPS-induced ALI were subsequently determined. We reported that angptl4 levels were increased both in human alveolar epithelial A549 cells and lung tissues obtained from a mouse model of LPS-induced ALI. Angptl4 expression was induced by LPS in alveolar epithelial cells, whereas LPS-induced lung inflammation (neutrophils infiltration in the lung tissues, tumor necrosis factor α, interleukin 6), lung permeability (lung wet/dry weight ratio and bronchoalveolar lavage fluid (BALF) protein concentration), tissue damage (caspase3 activation), and mortality rates were attenuated in AngsiRNA-treated mice. The inflammatory reaction (tumor necrosis factor α, interleukin 6) and apoptosis rates were reduced in AngsiRNA(h)-treated A549 cells. Moreover, angptl4 promoted NF-kBp65 expression and suppressed SIRT1 expression both in mouse lungs and A549 cells. Additionally, SIRT1 antagonist nicotinamide (NAM) attenuated the inhibitory effects of AngsiRNA both on LPS-induced NF-kBp65 expression and IL6 expression. These findings suggest that silencing angptl4 protects against LPS-induced ALI via regulating SIRT1/NF-kB signaling pathway. J. Cell. Physiol. 230: 2390–2402, 2015. © 2015 Wiley Periodicals, Inc.

Contactin-1 (CNTN-1), a glycosyl phosphatidylinositol anchor neural cell adhesion molecule (ACAM), is thought to function not only in nervous system development but also in the invasion and metastasis of several tumours. To investigate whether CNTN-1 is involved in multidrug resistance (MDR) in lung adenocarcinoma, CNTN-1 expression was compared between MDR human lung adenocarcinoma A549/cisplatin (A549/DDP) cells and its progenitor A549 cells. The comparison showed that CNTN-1 expression in A549/DDP cells was significantly higher than in A549 cells both at the mRNA level and the protein level. In order to confirm the physiological function of the abnormal expression, lentivirus-mediated short hairpin RNA (shRNA) was used to silence CNTN-1. Cell cytotoxicity assay and cell apoptosis assay revealed that silencing CNTN-1 both in A549 cells and in A549/DDP cells not only rendered cells more sensitive to cisplatin than the negative control, but also increased the cisplatin-induced apoptosis. Metastasis and invasion assays demonstrated that CNTN-1 knockdown reduced metastasis and invasion but did not affect A549 or A549/DDP cell proliferation. To investigate whether the abnormal expression of CNTN-1 is associated with characteristics of patients with non-small cell lung cancer (NSCLC), immunohistochemistry was used to detect CNTN-1 expression in 143 tissue samples from NSCLC patients and the results showed that the degree of CNTN-1 expression positively correlated with lymphatic invasion in patients with lung adenocarcinoma who received adjuvant cisplatin- or carboplatin-based treatment after surgery. Thus, we concluded that CNTN-1 is closely related with MDR of lung adenocarcinoma. Additionally, CNTN-1 is a novel marker to predict chemotherapeutic efficacy of patients with lung adenocarcinoma, especially with regard to cisplatin- or carboplatin-based regimens.

Hepatopulmonary syndrome (HPS) is characterized by arterial oxygenation defects induced by intrapulmonary vascular dilation (IPVD). Pulmonary vascular remodeling (PVR) is an important pathological feature of IPVD; however, the details regarding the underlying mechanisms of this process remain undefined. Recent studies have determined that the abnormal migration of pulmonary arterial smooth muscle cells (PASMCs) plays a role in the pathogenesis of the PVR associated with HPS. Additionally, aquaporin 1 (AQP1) not only functions as a water channel molecule but also promotes cell migration by facilitating water transport in the lamellipodia of migrating cells. Common bile duct ligation (CBDL) rat is a well-accepted HPS model; we determined that the immunoperoxidase labeling of AQP1 was enhanced in the media of the pulmonary vessels in CBDL rats. HPS rat serum mediated the overexpression of AQP1 in PASMCs, and also upregulated PASMC migration. Small interfering RNAs (siRNAs) that targeted rat AQP1 caused significant downregulation of AQP1, which resulted in decreased PASMC migration. Furthermore, the inhibition of the p38-MAPK pathway abolished AQP1-dependent PASMC migration. In conclusion, this study demonstrated that AQP1 enhanced PASMC migration via the p38-MAPK pathway in rat with HPS and may represent a potential therapeutic strategy in the setting of pulmonary vascular remodeling associated with HPS.

Background: As an active component from traditional Chinese medicine, trigonelline has a protective effect on diabetes. This study evaluated the protective effects of trigonelline on diabetic mice during pregnancy. Methods: Diabetes was induced in female mice by intraperitoneal injection for continuous 5-day of 40 mg/kg/day streptozotocin. Female mice were divided into 4 groups after they were allowed to mate with normal male mice: nondiabetic, nondiabetic treated with trigonelline (70 mg/kg) for 18 days, diabetic, and diabetic treated with trigonelline (70 mg/kg). Results: Diabetic pregnant mice had significantly higher levels of blood glucose, serum total cholesterol, triglyceride, insulin, and leptin but lower serum omentin-1 level and insulin sensitivity index than the nondiabetic ones. Trigonelline improved the hyperglycemia, dyslipidemia, insulin resistance, and adipocytokine of diabetic pregnant mice. Diabetic pregnant mice had significantly reduced fetus numbers, fetal weight, and fetal/placental ratio, which were reversed by trigonelline. Trigonelline prevented the increase in proinflammatory cytokines and reduced interleukin-10 level in placenta of diabetic pregnant mice. Trigonelline increased β-cell replication and the decreased β-cell mass, and decreased the β-cell apoptosis of diabetic pregnant mice. Conclusion: These findings suggest that trigonelline protects diabetic pregnancy partly by suppressing inflammation, regulating the secretion of adipocytokines, increasing β-cell mass, replication, and decreasing β-cell apoptosis.

N -methyl- d -aspartate (NMDA) receptor subunits GluN1 and GluN2B in hippocampal neurons play key roles in anxiety. Our previous studies show that rhynchophylline, an active component of the Uncaria species, down-regulates GluN2B expression in the hippocampal CA1 area of amphetamine-induced rat. The effects of rhynchophylline on expressions of GluN1 and GluN2B in primary hippocampal neurons in neonatal rats in vitro were investigated. Neonatal hippocampal neurons were cultured with neurobasal-A medium. After incubation for 6 h or 48 h with rhynchophylline (non-competitive NMDAR antagonist) and MK-801 (non-competitive NMDAR antagonist with anxiolytic effect, as the control drug) from day 6, neuron toxicity, mRNA and protein expressions of GluN1 and GluN2B were analyzed. GluN1 is mainly distributed on neuronal axons and dendritic trunks, cytoplasm and cell membrane near axons and dendrites. GluN2B is mainly distributed on the membrane, dendrites, and axon membranes. GluN1 and GluN2B are codistributed on dendritic trunks and dendritic spines. After 48 h incubation, a lower concentration of rhynchophylline (lower than 400 μmol/L) and MK-801 (lower than 200 μmol/L) have no toxicity on neonatal hippocampal neurons. Rhynchophylline up-regulated GluN1 mRNA expression at 6 h and mRNA and protein expressions at 48 h, but down-regulated GluN2B mRNA and protein expressions at 48 h. However, GluN1 and GluN2B mRNA expressions were down-regulated at 6 h, and mRNA and protein expressions were both up-regulated by MK-801 at 48 h. These findings show that rhynchophylline reciprocally regulates GluN1 and GluN2B expressions in hippocampal neurons, indicating a potential anxiolytic property for rhynchophylline.

High altitude illness can be life-threatening if left untreated. Acute mountain sickness and high altitude pulmonary hypertension are two syndromes of high altitude illness. Recent clinical studies showed the beneficial effects of phosphodiesterase type 5 (PDE-5) inhibitors on the treatment of pulmonary hypertension. In this report, we performed a meta-analysis to evaluate the clinical efficacy of PDE-5 inhibitors on high altitude hypoxia and its complications.Randomized controlled trials evaluating the efficacy of PDE-5 inhibitor in the setting of high altitude were identified by searching Cochrane Central Register of Controlled Trials (September 2013), PubMed (from 1990 to September 2013), and EMBASE (from 1990 to September 2013). Extracted outcomes from selected studies for meta-analysis included arterial oxygen saturation, pulmonary artery systolic pressure, heart rate, and Lake Louise Consensus AMS symptom score. Weighted mean differences with 95% confidence intervals were presented for the continuous outcomes.Five clinical trials that met the selection criteria were identified for the meta-analysis. All of these studies used sildenafil as the PDE-5 inhibitor. A total of 60 subjects received sildenafil, and 72 subjects were given placebo. In accordance with previous report, short-term treatment with sildenafil (1-2 days) significantly reduced pulmonary artery systolic pressure at rest (MD -4.53; 95% CI -6.72, -2.34; p0.0001). However, treatment with sildenafil (1-2 days) did not improve oxygen saturation after exposure to high altitude (MD 0.07; 95% CI -1.26, 1.41; p=0.91). Moreover, no significant difference was observed in heart rate between sildenafil and placebo-treated group (MD 6.95; 95% CI -3.53, 17.43; p=0.19). AMS score did not improve after treatment at different time points.Short-term treatment with sildenafil can attenuate the altitude-induced high pulmonary systolic arterial pressure, but has no significant beneficial effects on arterial oxygen saturation, heart rate, and acute mountain sickness.

Background: Hepatopulmonary syndrome (HPS) is a serious complication of advanced liver disease that is characterised by intrapulmonary vascular dilatation (IPVD) and arterial hypoxemia. Pulmonary vascular remodelling (PVR) is an important pathological feature of HPS, but the potential mechanisms underlying PVR remain undefined. Recent findings have established the essential role of changes in Annexin A2 (ANXA2) in controlling the phenotypic modulation of pulmonary artery smooth muscle cells (PASMCs) in PVR associated with HPS. However, the mechanism by which upstream signalling regulates ANXA2 is unclear. Methods: In the present study, computational analysis was used to predict which miRNA might target the 3´-untranslated region (3´-UTR) of the ANXA2 mRNA. Real-time PCR and western blotting were performed to study the level of correlation between ANXA2 and the differentiation marker with the predicted miRNAs in PASMCs stimulated with serum from normal rats or those with HPS. Functional analysis of the miRNA and a luciferase reporter assay were performed to demonstrate that the predicted miRNA suppressed ANXA2 expression by directly targeting the predicted 3´-UTR site of the ANXA2 mRNA. Results: Computational analysis predicted that miR-206 would target the 3´-UTR of ANXA2 mRNA. In HPS rat serum-stimulated PASMCs, the expression of miR-206 displayed an inverse correlation with ANXA2, while a positive correlation was observed with the phenotypic marker smooth muscle α-actin (SM α-actin). The miRNA functional analysis and luciferase reporter assay demonstrated that miR-206 effectively downregulated the expression of ANXA2 by binding to the 3´-UTR of the ANXA2 mRNA. Consistently, miR-206 effectively inhibited the HPS rat serum-induced phenotypic modulation and proliferation, while these effects were reversed in ANXA2-overexpressing PASMCs. Conclusion: This study demonstrates that miR-206 inhibits the HPS rat serum-induced phenotypic modulation and proliferation in PASMCs by down-regulating ANXA2 gene expression.

The small GTPase Rab26 is involved in multiple processes, such as vesicle-mediated secretion and autophagy. However, the mechanisms and functions of Rab26 in the human pulmonary microvascular endothelial cells (HPMVECs) are not clear. In this study, we thoroughly investigated the role and novel mechanism of Rab26 in permeability and apoptosis of HPMVECs using a self-assembled Rab26 siRNA loaded DNA Y-motif nanoparticle (siRab26-DYM) and Rab26 adenovirus. We found that siRab26-DYM could be efficiently transfected into HPMVECs in a time- and dose-dependent manner. Importantly, the siRab26-DYM nanovector markedly aggravated the LPS-induced apoptosis and hyper-permeability of HPMVECs by promoting the nuclear translocation of Foxo1, and subsequent activation of Toll-like receptor 4 (TLR4) signal pathway. Overexpression of Rab26 by Rab26 adenoviruses partially inactivated LPS-induced TLR4 signaling pathway, suppressed the cell apoptosis and attenuated the hyperpermeability of HPMVECs. These results suggest that the permeability and apoptosis of HPMVECs can be modulated by manipulating Rab26 derived TLR4 signaling pathway, and that Rab26 can be potential therapeutic target for the treatment of vascular diseases related to endothelial barrier functions.

// Chaowen Jiang 1, * , Shilong Yu 1, * , Pin Qian 1, * , Ruiling Guo 2 , Ruijie Zhang 3 , Zhi Ao 1 , Qi Li 1 , Guoming Wu 1 , Yan Chen 1 , Jin Li 1 , Changzheng Wang 1 , Wei Yao 1 , Jiancheng Xu 1 , Guisheng Qian 1 , Fuyun Ji 1 1 Institute of Human Respiratory Disease, Xinqiao Hospital, The Third Military Medical University, Chongqing 400037, China 2 Department of Respiratory Diseases, 324 th Hospital of People’s Liberation Army (No.324 Hospital of PLA), Chongqing 400020, China 3 Department of Respiratory Medicine, The Third Xiangya Hospital, Central South University, Changsha, Hunan 410013, China * These authors have contributed equally to this work Correspondence to: Fuyun Ji, email: JiF@email.chop.edu , jifuyun@263.net Guisheng Qian, email: qiangs1220@163.com Keywords: lung cancer, risk, TOX3/LOC643714, polymorphisms, breast cancer Received: February 02, 2016 Accepted: July 09, 2016 Published: July 28, 2016 ABSTRACT It has been well established that besides environmental factors, genetic factors are also associated with lung cancer risk. However, to date, the prior identified genetic variants and loci only explain a small fraction of the familial risk of lung cancer. Hence it is vital to investigate the remaining missing heritability to understand the development and process of lung cancer. In the study, to test our hypothesis that the previously identified breast cancer risk-associated genetic polymorphisms at the TOX3/LOC643714 locus might contribute to lung cancer risk, 16 SNPs at the TOX3/LOC643714 locus were evaluated in a Han Chinese population based on a case-control study. Pearson’s chi-square test or Fisher’s exact test revealed that rs9933638, rs12443621, and rs3104746 were significantly associated with lung cancer risk ( P < 0.001, P < 0.001, and P = 0.005, respectively). Logistic regression analyses displayed that lung cancer risk of individuals with rs9933638(GG+GA) were 1.89 times higher than that of rs9933638AA carriers (OR = 1.893, 95% CI = 1.308-2.741, P = 0.001). Similar findings were manifested for rs12443621 (OR = 1.824, 95% CI = 1.272-2.616, P = 0.001, rs12443621(GG+GA) carriers vs. rs12443621AA carriers) and rs3104746 (OR = 1.665, 95% CI = 1.243-2.230, P = 0.001, rs3104746TT carriers vs. rs3104746(TA+AA) carriers). The study discovered for the first time that three SNPs (rs9933638, rs12443621, and rs3104746) at the TOX3/LOC643714 locus contributed to lung cancer risk, providing new evidences that lung cancer and breast cancer are linked at the molecular and genetic level to a certain extent.

Background This review provides a critical overview of problem-based learning (PBL) practices in Chinese pharmacy education. PBL has yet to be widely applied in pharmaceutical education in China. The results of those studies that have been conducted are published in Chinese and thus may not be easily accessible to international researchers. Therefore, this meta-analysis was carried out to review the effectiveness of PBL. Methods Databases were searched for studies in accordance with the inclusion criteria. Two reviewers independently performed the study identification and data extraction. A meta-analysis was conducted using Revman 5.3 software. Results Sixteen randomized controlled trials were included. The meta-analysis revealed that PBL had a positive association with higher theoretical scores (SMD = 1.17, 95 % CI [0.77, 11.57], P

PRISMA 2009 Checklist. (DOC 65Â kb)

The interplay of a complex genetic basis with the environmental factors of chronic obstructive pulmonary disease (COPD) may account for the differences in individual susceptibility to COPD. Mitochondrial DNA (mtDNA) contributes to an individual's ability to resist oxidation, an important determinant that affects COPD susceptibility. To investigate whether mtDNA haplogroups play important roles in COPD susceptibility, the frequencies of mtDNA haplogroups and an 822-bp mtDNA deletion in 671 COPD patients and 724 control individuals from southwestern China were compared. Multivariate logistic regression analysis revealed that, whereas mtDNA haplogroups A and M7 might be associated with an increased risk for COPD (OR=1.996, 95% CI=1.149-2.831, p=0.006, and OR=1.754, 95% CI=1.931-2.552, p=0.021, respectively), haplogroups F, D, and M9 might be associated with a decreased risk for COPD in this population (OR=0.554, 95% CI=0.390-0.787, p=0.001; OR=0.758, 95% CI=0.407-0.965, p=0.002; and OR=0.186, 95% CI=0.039-0.881, p=0.034, respectively). Additionally, the increased frequency of the 822-bp mtDNA deletion in male cigarette-smoking subjects among COPD patients and controls of haplogroup D indicated that haplogroup D might increase an individual's susceptibility to DNA damage from external reactive oxygen species derived from heavy cigarette smoking. We conclude that haplogroups A and M7 might be risk factors for COPD, whereas haplogroups D, F, and M9 might decrease the COPD risk in this Han Chinese population.

The distinction between mild pathogenic mtDNA mutations and population polymorphisms can be ambiguous because both are homoplasmic, alter conserved functions, and correlate with disease. One possible explanation for this ambiguity is that the same variant may have different consequences in different contexts. The NADH dehydrogenase subunit 1 (ND1) nucleotide 3394 T > C (Y30H) variant is such a case. This variant has been associated with Leber hereditary optic neuropathy and it reduces complex I activity and cellular respiration between 7% and 28% on the Asian B4c and F1 haplogroup backgrounds. However, complex I activity between B4c and F1 mtDNAs, which harbor the common 3394T allele, can also differ by 30%. In Asia, the 3394C variant is most commonly associated with the M9 haplogroup, which is rare at low elevations but increases in frequency with elevation to an average of 25% of the Tibetan mtDNAs (odds ratio = 23.7). In high-altitude Tibetan and Indian populations, the 3394C variant occurs on five different macrohaplogroup M haplogroup backgrounds and is enriched on the M9 background in Tibet and the C4a4 background on the Indian Deccan Plateau (odds ratio = 21.9). When present on the M9 background, the 3394C variant is associated with a complex I activity that is equal to or higher than that of the 3394T variant on the B4c and F1 backgrounds. Hence, the 3394C variant can either be deleterious or beneficial depending on its haplogroup and environmental context. Thus, this mtDNA variant fulfills the criteria for a common variant that predisposes to a “complex” disease.

Our previous work has demonstrated that the cellular phenotype changes of human pulmonary artery smooth muscle cells (PASMCs) play an important role during pulmonary vascular remodelling. However, little is known about the role of PASMCs phenotype modulation in the course of hypoxia-induced migration and its behind molecular mechanisms. In this study, we have shown that cGMP-dependent protein kinase (PKG) Iα transfection significantly attenuated the hypoxia-induced down-regulation of the expressions of SM-α-actin, MHC and calponin. Hypoxia-induced PASMC migration was also suppressed by PKGIα overexpression. Furthermore, this overexpression attenuated ANX A1 upregulation under hypoxic conditions. All those effects were reversed by a PKG inhibitor KT5823. Our data indicate that manipulating upstream entity e.g., PKGIa, may have a potential therapeutic value to prevent hypoxia–associated pulmonary arterial remodeling for pulmonary hypertension development.

Recent evidence suggests that TGF-β1 has a dual role in regulating cell response to Fas/Fas ligand (FasL)-induced apoptosis. TGF-β1 may play a positive or negative role on cell sensitivity to apoptosis via Fas/FasL system, depending on cell types and their specific environment. TGF-β1 and the Fas/FasL system are also involved in pathological processes of acute lung injury (ALI) and interstitial lung diseases including early lung injury and subsequent tissue repair. However, it is not well understood how TGF-β1 regulates Fas/FasL mediated apoptotic signaling in lung epithelium. In this study, we found that TGF-β1 could affect the sensitivity of lung epithelial A549 cells to Fas/FasL mediated apoptosis in a time-dependent manner. Apoptosis of A549 cells could be enhanced significantly by co-treatment with TGF-β1 and FasL, or pretreatment with TGF-β1 followed by FasL exposure, as evidenced by markedly increased caspase-8 and JNK activities. However, prolonged exposure to TGF-β1 could result in an obvious inhibition of the Fas/FasL-induced apoptosis, accompanied by down-regulation of Fas and up-regulation of c-Flip. Our results also showed that the effect of TGF-β1 on cell sensitivity to Fas-mediated apoptosis was independent of Akt pathway activation. These findings suggest that timely interplay of TGF-β1 and the Fas/FasL system could determine the final outcomes of cell survival/death signaling, for example, switching cell death signaling to survival signaling during early injury and later repair process of lung epithelium.

The objectives of this study were to detect the immunogenicity of a lipopolysaccharide (LPS) binding protein (LBP) mimic epitope peptide vaccine and evaluate its effect on controlling excessive and uncontrolled inflammatory reactions in mice with acute lung injury. The vaccine was prepared by mixing self-made LBP mimic epitope multiple antigen peptide (MAP) and Freund adjuvants in a proper proportion. Healthy mice were inoculated with the vaccine and the dynamic changes of anti-MAP antibody were measured using ELISA. Anti-MAP antibody was prepared from the immune serum of the mice based on the standard antibody preparation program. Western blot assay was used to identify LBP specificity of anti-MAP antibody. Anti-MAP antibody bioactivity was analyzed using in vitro binding activity test. Following the vaccine inoculation, the mice were injected with LPS to induce acute lung injury. Anti-MAP antibody prepared was also used to immune the mice with LPS-induced acute lung injury. TNF-α and IL-1β contents in serum and lung tissue homogenate were measured using double antibody sandwiched ELISA at different time-points after LPS challenge. At 8h time-point, total white blood cell counts, polymorphonuclear leucocyte count and protein content in bronchoalveolar lavage fluid were measured and pulmonary morphological changes were evaluated. The antibody titer was gradually rising, reaching to its peak at 8th week and lasting to the tenth week. The antibody possessed strong immunogenicity, high specificity and favorable biologic activity. Whole range inoculation of LBP mimic epitope peptide vaccine or anti-MAP antibody intervention partly eliminates LPS-mediated acute lung injury. In conclusion, LBP mimic epitope peptide vaccine successfully induced highly specific antibody with high bioactivity in mice. LBP mimic epitope peptide vaccine and anti-MAP antibody inhibited excessive and uncontrolled inflammatory reactions from LPS-mediated acute lung injury in mice.

Energy metabolism is the foundation of survival for all organisms, and mitochondria are the most important energy-supplying organelles in eukaryotic cells. However, the mitochondrial and energy/metabolism-related properties of cancer stem cells (CSCs), the stem cell-like subpopulation in tumor masses, remain unknown. In our study, we compared the masses of mitochondria and mitochondrial DNA (mtDNA), the mitochondrial membrane potential (Δψm), oxygen/glucose consumption, and the concentration of reactive oxygen species (ROS) and ATP between lung CSCs (LCSCs) and non-LCSCs. In addition, the change in features during differentiation was examined. Some mitochondrial and energy metabolism-related properties, such as perinuclear mitochondrial distribution, a lower quantity of mtDNA, higher Δψm, lower oxygen/glucose consumption, and lower intracellular concentrations of ROS and ATP, can be used as indicators of LCSCs.

Previous studies have shown that single immunoglobulin IL-1 receptor-related molecule (SIGIRR) is a negative regulator of Toll-Interleukin-1 receptor signaling. Nevertheless, the molecular mechanism of the negatively regulatory effect of SIGIRR remains unknown. Using a yeast two-hybrid screen, we identified paralemmin-3 (PALM3) as a novel binding protein of SIGIRR. This interaction of SIGIRR with PALM3 was confirmed by coimmunoprecipitation in mammalian cells. In addition, the PALM3 mRNA expression was upregulated by lipopolysaccharide (LPS)-stimulation in a human alveolar epithelial cell line (A549 cells). Furthermore, silencing PALM3 by RNA interference inhibited the release of inflammatory cytokines in A549 cells after LPS-stimulation. These results suggest that PALM3 may function as an adaptor in the LPS- Toll-like receptor 4 signaling and the interaction of SIGIRR with PALM3 may partly account for the mechanism of the negatively regulatory effect of SIGIRR.

Hypoxia down-regulates the expression of the growth arrest-specific homeobox (Gax) in pulmonary arterial smooth muscle cells (PASMCs), resulting in increased cell proliferation and decreased apoptosis, but the mechanism for this response remains unclear. The present study investigated the role of Gax in the development of hypoxia-induced pulmonary hypertension (PH). We found that hypoxia suppressed the expression of endogenous Gax in rats, but not in those pretreated intratracheally with a Gax construct (Ad-Gax). Hypoxic rats pretreated with Ad-Gax were resistant to hypoxia-induced PH, right ventricular hypertrophy, increased wall thickness, and the muscularization of pulmonary arterioles. Hypoxia-induced PASMC proliferation and suppression of Gax expression were blocked by the Mitogen-activated protein kinase (MEK) inhibitor U0126. The PASMCs with Ad-Gax transfection exhibited hyperexpression of the Bcl-2-associated X protein (Bax) and hypoexpression of B-cell lymphoma 2 (Bcl-2), leading to cell apoptosis. Thus, our data indicate that the enhanced expression of Gax inhibits hypoxia-induced PASMC proliferation, probably via the extracellular-signal-regulated kinase (ERK) 1/2 pathway, and induces the apoptosis of hypoxic PASMCs via the Bcl-2/Bax pathway. Gax may be a potential new therapeutic target for pulmonary hypertension.

Recent findings identify the role of proliferation of pulmonary artery smooth muscle cells (PASMCs) in pulmonary vascular remodeling. Phosphoinositide 3 kinase (PI3K) and serine/threonine kinase (Akt) proteins are expressed in vascular smooth muscle cells. In addition, phosphatase and tensin homolog deleted on chromosome 10 (PTEN) has been identified as a negative regulator of cytokine signaling that inhibits the PI3K-Akt pathway. However, little is known about the role of PTEN/Akt signaling in hypoxia-associated vascular remodeling. In this study, we found that hypoxia-induced the expression of Akt1 mRNA and phosphorylated protein by at least twofold in rat PASMCs. Phospho-PTEN significantly decreased in the nuclei of PASMCs after hypoxic stimulation. After forcing over-expression of PTEN by adenovirus-mediated PTEN (Ad-PTEN) transfection, the expression of phospho-Akt1 was significantly suppressed in PASMCs at all time-points measured. Additionally, we showed here that hypoxia increased proliferation of PASMCs by nearly twofold and over-expression of PTEN significantly inhibited hypoxia-induced PASMCs proliferation. These findings suggest that phospho-PTEN loss in the nuclei of PASMCs under hypoxic conditions may be the major cause of aberrant activation of Akt1 and may, therefore, play an important role in hypoxia-associated pulmonary arterial remodeling. Finally, the fact that transfection with Ad-PTEN inhibits the phosphorylation of Akt1 in PASMCs suggests a potential therapeutic effect on hypoxia-associated pulmonary arterial remodeling.

It is known that Rab1 regulates the expression and function of beta-adrenoceptors (beta-ARs) in many cells. However, the effect of these changes in rat pulmonary microvascular endothelial cells (RPMVECs) is not known. In the present study, we investigated the role of Rab1, a Ras-like GTPase that coordinates protein transport from the endoplasmic reticulum (ER) to the Golgi body and regulates the cell-surface targeting and function of endogenous beta-ARs in RPMVECs in the presence of lipopolysaccharide (LPS). We found that lentivirus-driven expression of wild-type Rab1 (Rab1WT) in RPMVECs strongly enhanced the amount of beta-ARs on the cell surface, whereas the dominant-negative mutant Rab1N124I significantly attenuated beta-ARs expression on the cell surface. In addition, LPS stimulation significantly reduced beta-ARs expression on the cell surface in RPMVECs; however, this effect was reversed by over-expression of wild-type Rab1WT. Fluorescent microscopy analysis demonstrated that expression of Rab1N124I and Rab1 small interfering RNA (siRNA) significantly induced the accumulation of green fluorescent protein (GFP)-tagged beta(2)-AR in the ER. Consistent with their effects on beta-ARs export, Rab1WT and Rab1N124I differentially modified the beta-AR-mediated activation of extracellular signal-regulated kinase1/2 (ERK1/2). Importantly, over-expression of Rab1WT markedly reduced LPS-induced hyper-permeability of RPMVECs by increasing the expression of beta(2)-AR on the cell surface. These data reveal that beta-ARs function in RPMVECs could be modulated by manipulating beta-ARs traffic from the ER to the Golgi body. We propose the ER-to-Golgi transport as a regulatory site for control of permeability of RPMVECs.

Human airway epithelial cells (HAEC) may contribute to acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) through toll-like receptors (TLRs)-mediated molecular mechanisms. TLRs exist on the surface of HAEC where binding to their cognate ligands initiates airway inflammation. Single immunoglobulin interleukin-1 receptor-related protein (SIGIRR) is a member of the toll-interleukin-1 receptor (TIR) family that can negatively modulate the immune response. We carried out studies to characterize SIGIRR modulation of TLR-mediated immune response in HAEC and to define its mechanisms of action. Following treatment with various concentrations of LPS, flagellin and CpG DNA, the levels of cognate TLRs 4, 5, and 9 were measured in the supernatants of HAEC over-expressing the SIGIRR molecule. Moreover, the interaction of the TLR adaptor myeloid differentiation factor 88 (MyD88) with SIGIRR in response to LPS-, flagellin- and CpG DNA-stimulation was examined by co-immunoprecipitation. The findings from this study revealed that overexpression of SIGIRR in HAEC stimulated by LPS, flagellin or CpG DNA resulted in attenuated production of the inflammatory mediators IL-6 and TNF-α. This attenuation was not the result of decreased expression of TLR4, 5 or 9, but rather a sequestration of MyD88 to the TLRs. In conclusion, SIGIRR can inhibit TLR4, 5, and 9-mediated immune responses in HAEC and may be a valuable therapeutic target for the prevention of ALI/ARDS.

This study aimed to investigate the mechanism by which the human lung cancer drug resistance-related gene BC006151 regulates chemosensitivity by down-regulating BC006151 expression via antisense gene transfer in H446/(C)DDP cells. A retroviral vector containing the antisense BC006151 sequence was constructed and transfected into H446/(C)DDP cells. Transfection of the empty vector served as a negative control. The two groups of transfected cells were treated with various chemotherapeutic agents, after which morphological changes in cell ultrastructure were compared by transmission electron microscopy, cell proliferation and chemosensitivity to particular chemotherapeutic agents were compared by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method, the effects of chemotherapy on cell cycle and apoptosis were compared by flow cytometry, and Bcl-2 was evaluated by immunohistochemistry and Western blot analysis. Results showed that apoptotic body-like structures were identified by transmission electron microscopy in the antisense gene-transfected cells. MTT founded that these cells exhibited a significantly lower level of proliferation than the control cells (p0.01), together with a markedly increased sensitivity to various chemotherapeutic agents (p0.01). Flow cytometry analysis revealed that a G1 phase arrest accounted for the reduction in proliferation in the antisense gene-transfected cells; increased apoptosis was also detected (p0.01). Both immunohistochemistry and western blot analysis confirmed that Bcl-2 expression was significantly down-regulated in the antisense gene-transfected cells compared to controls. In a word, down-regulation of BC006151 can significantly inhibit proliferation and increase apoptosis of H446/(C)DDP cells after chemotherapy, and this gene may play an important role in the development of multidrug resistance in lung cancer.