Core planar cell polarity genes VANGL1 and VANGL2 in predisposition to congenital vertebral malformations.

Congenital scoliosis (CS), affecting approximately 0.5 to 1 in 1,000 live births, is commonly caused by congenital vertebral malformations (CVMs) arising from aberrant somitogenesis or somite differentiation. While Wnt/ß-catenin signaling has been implicated in somite development, the function of Wnt/planar cell polarity (Wnt/PCP) signaling in this process remains unclear. Here, we investigated the role of Vangl1 and Vangl2 in vertebral development and found that their deletion causes vertebral anomalies resembling human CVMs. Analysis of exome sequencing data from multiethnic CS patients revealed a number of rare and deleterious variants in VANGL1 and VANGL2, many of which exhibited loss-of-function and dominant-negative effects. Zebrafish models confirmed the pathogenicity of these variants. Furthermore, we found that Vangl1 knock-in (p.R258H) mice exhibited vertebral malformations in a Vangl gene dose- and environment-dependent manner. Our findings highlight critical roles for PCP signaling in vertebral development and predisposition to CVMs in CS patients, providing insights into the molecular mechanisms underlying this disorder.

Effects of sequential vs single pneumococcal vaccination on cardiovascular diseases among older adults: a population-based cohort study.

BACKGROUND: Recommendations around the use of 23-valent pneumococcal polysaccharide vaccine (PPSV23) and 13-valent pneumococcal conjugate vaccine (PCV13) seldom focus on potential benefits of vaccine on comorbidities. We aimed to investigate whether sequential vaccination with PCV13 and PPSV23 among older adults would provide protection against cardiovascular diseases (CVD) compared with using a single pneumococcal vaccine. METHODS: We conducted a Hong Kong-wide retrospective cohort study between 2012 and 2020. Adults aged ≥65 years were identified as receiving either a single or sequential dual vaccination and followed up until the earliest CVD occurrence, death or study end. To minimize confounding, we matched each person receiving a single vaccination to a person receiving sequential vaccination according to their propensity scores. We estimated the hazard ratio (HR) of CVD risk using Cox regression and applied structural equation modelling to test whether the effect of sequential dual vaccination on CVD was mediated via the reduction in pneumonia. RESULTS: After matching, 69 390 people remained in each group and the median (interquartile range) follow-up time was 1.89 (1.55) years. Compared with those receiving a single vaccine, those receiving sequential dual vaccination had a lower risk of CVD [HR (95% CI): 0.75 (0.71, 0.80), P < 0.001]. Post-hoc mediation analysis showed strong evidence that the decreased CVD risk was mediated by the reduction in all-cause pneumonia. CONCLUSIONS: Sequential dual pneumococcal vaccination was associated with lower risk of CVD compared with single-dose PCV13 or PPSV23 in older adults. Such additional CVD benefits should be considered when making decisions about pneumococcal vaccination.

Novel treatments against airway inflammation in COPD based on drug repurposing.

Chronic obstructive pulmonary disease (COPD) is a major cause of death and reduces quality of life that contributes to a health problem worldwide. Chronic airway inflammation is a hallmark of COPD, which occurs in response to exposure of inhaled irritants like cigarette smoke. Despite accessible to the most up-to-date medications, none of the treatments is currently available to decrease the disease progression. Therefore, it is believed that drugs which can reduce airway inflammation will provide effective disease modifying therapy for COPD. There are many broad-range anti-inflammatory drugs including those that inhibit cell signaling pathways like inhibitors of p38 mitogen-activated protein kinase (MAPK), nuclear factor-κB (NF-κB), and phosphoinositide-3-kinase (PI3K), are now in phase III development for COPD. In this chapter, we review recent basic research data in the laboratory that may indicate novel therapeutic pathways arisen from currently used drugs such as selective monoamine oxidase (MAO)-B inhibitors and drugs targeting peripheral benzodiazepine receptors [also known as translocator protein (TSPO)] to reduce airway inflammation. Considering the impact of chronic airway inflammation on the lives of COPD patients, the potential pharmacological candidates for new anti-inflammatory targets should be further investigated. In addition, it is crucial to consider the phenotypes/molecular endotypes of COPD patients together with specific outcome measures to target novel therapies. This review will enhance our knowledge on how cigarette smoke affects MAO-B activity and TSPO activation/inactivation with specific ligands through regulation of mitochondrial function, and will help to identify new potential treatment for COPD in future.

Mitochondrial Transfer Regulates Bioenergetics in Healthy and Chronic Obstructive Pulmonary Disease Airway Smooth Muscle.

Mitochondrial dysfunction has been reported in chronic obstructive pulmonary disease (COPD). Transfer of mitochondria from mesenchymal stem cells to airway smooth muscle cells (ASMCs) can attenuate oxidative stress-induced mitochondrial damage. It is not known whether mitochondrial transfer can occur between structural cells in the lungs or what role this may have in modulating bioenergetics and cellular function in healthy and COPD airways. Here, we show that ASMCs from both healthy ex-smokers and subjects with COPD can exchange mitochondria, a process that happens, at least partly, via extracellular vesicles. Exposure to cigarette smoke induces mitochondrial dysfunction and leads to an increase in the donation of mitochondria by ASMCs, suggesting that the latter may be a stress response mechanism. Healthy ex-smoker ASMCs that receive mitochondria show increases in mitochondrial biogenesis and respiration and a reduction in cell proliferation, irrespective of whether the mitochondria are transferred from healthy ex-smoker or COPD ASMCs. Our data indicate that mitochondrial transfer between structural cells is a homeostatic mechanism for the regulation of bioenergetics and cellular function within the airways and may represent an endogenous mechanism for reversing the functional consequences of mitochondrial dysfunction in diseases such as COPD.

Obstructive sleep apnea, intermittent hypoxia and non-alcoholic fatty liver disease.

With the current epidemic of obesity worldwide, the prevalence of various obesity-related diseases is constantly increasing. Obesity remains the strongest phenotypic risk factor in both obstructive sleep apnea (OSA) and non-alcoholic fatty liver disease (NAFLD). In OSA, intermittent hypoxia-reoxygenation and sleep fragmentation, as a result of recurrent episodes of upper airway obstruction during sleep, may give rise to a plethora of metabolic derangements downstream. Intermittent hypoxia (IH) is postulated to be an important mechanistic trigger for potential systemic impact on organs or tissues in OSA, and has served as a useful experimental model for seeking evidence for downstream effects of OSA. This narrative review focuses on the clinical association between OSA and NAFLD, and the role of IH in the progression of NAFLD in lean and diet-induced obese animal models. Understanding the roles of obesity and IH on NAFLD would advance our limited knowledge on the potential health consequences of OSA, a disease which is afflicting more and more people globally, and also in devising effective therapeutic strategies for this progressively common liver condition.

ORMDL3 regulates cigarette smoke-induced endoplasmic reticulum stress in airway smooth muscle cells.

BACKGROUND: Orosomucoid 1-like protein 3 (ORMDL3), a transmembrane protein localized in the endoplasmic reticulum (ER), has been genetically associated with chronic obstructive pulmonary disease (COPD), in addition to childhood-onset asthma. However, the functional role of ORMDL3 in the pathogenesis of COPD is still unknown. OBJECTIVE: Because cigarette smoke is the major risk factor for COPD, we aimed to investigate the role of ORMDL3 in cigarette smoke-induced human airway smooth muscle cell (HASMC) injury. METHODS: The mRNA and protein expression of ORMDL3 was examined in HASMCs from nonsmokers and smokers without or with COPD. Knockdown of ORMDL3 in primary healthy HASMCs was performed using small interfering RNA before exposure to cigarette smoke medium (CSM) for 24 hours. Inflammatory, proliferative/apoptotic, ER stress, and mitochondrial markers were evaluated. RESULTS: Elevation of ORMDL3 mRNA and protein expression was observed in HASMCs of smokers without or with COPD. CSM caused significant upregulation of ORMDL3 expression in healthy nonsmokers. ORMDL3 knockdown regulated CSM-induced inflammation, cell proliferation, and apoptosis. Silencing ORMDL3 led to reduction of CSM-induced ER stress via inhibition of unfolded protein response pathways such as activating transcription factor 6 and protein kinase RNA-like ER kinase. ORMDL3 was also involved in CSM-induced mitochondrial dysfunction via the mitochondrial fission process. CONCLUSIONS: We report the induction of ORMDL3 in HASMCs after cigarette smoke exposure. ORMDL3 may mediate cigarette smoke-induced activation of unfolded protein response pathways during airway smooth muscle cell injury.

Study of Mesenchymal Stem Cell-Mediated Mitochondrial Transfer in In Vitro Models of Oxidant-Mediated Airway Epithelial and Smooth Muscle Cell Injury.

Mesenchymal stem cells (MSCs) have emerged as an attractive candidate for cell-based therapy. In the past decade, many animal and pilot clinical studies have demonstrated that MSCs are therapeutically beneficial for the treatment of obstructive lung diseases such as asthma and chronic obstructive pulmonary disease (COPD). However, due to the scarcity of adult human MSCs, human-induced pluripotent stem cells mesenchymal stem cells (iPSCs) are now increasingly used as a source of MSCs. iPSCs are derived by reprogramming somatic cells from a wide variety of tissues such as skin biopsies and then differentiating them into iPSC-MSCs. One of the mechanisms through which MSCs exert their protective effects is mitochondrial transfer. Specifically, transfer of mitochondria from iPSC-MSCs to lung cells was shown to protect lung cells against oxidative stress-induced mitochondrial dysfunction and apoptosis and to reduce lung injury and inflammation in in vivo models of lung disease. In this chapter, we detail our methods to visualize and quantify iPSC-MSC-mediated mitochondrial transfer and to study its effects on oxidant-induced airway epithelial and smooth muscle cell models of acute airway cell injury.

Cigarette smoke induces apoptosis via 18 kDa translocator protein in human bronchial epithelial cells.

AIMS: The 18 kDa translocator protein (TSPO) - also known as peripheral benzodiazepine receptor, is found to be expressed in lung epithelium and pneumocytes, which is closely associated with the mitochondrial permeability transition pore (mPTP) and apoptosis. Cigarette smoking, a key risk factor for the development of chronic obstructive pulmonary disease (COPD), is known to induce apoptosis. We aimed to investigate TSPO subcellular localization and to examine whether cigarette smoke medium (CSM) induce apoptosis via TSPO in airway epithelial cells. MAIN METHODS: TSPO subcellular localization and expression were evaluated using immunofluorescent staining and Western blot analysis respectively. TSPO ligands either PK 11195 (a specific antagonist) or AC-5216 (a specific agonist) were pre-incubated in human bronchial epithelial cells before treating with 2% CSM for measurements of apoptotic cells, mitochondrial membrane potential (ΔΨm), cytoplasmic/mitochondrial reactive oxygen species (ROS) and inflammatory marker interleukin (IL)-8 respectively. KEY FINDINGS: TSPO was localized around the nucleus and overlapped with mitochondria in BEAS-2B cells. CSM caused an increase in apoptotic cells along with elevation of TSPO protein expression. Pretreatment of PK 11195 suppressed while AC-5216 potentiated CSM-induced apoptosis, collapse of ΔΨm, elevation of cytoplasmic/mitochondrial ROS levels and IL-8 release. In support, knockdown of TSPO caused a significant suppression of CSM-induced IL-8 release in BEAS-2B cells. SIGNIFICANCE: The findings suggest that TSPO may play a crucial role in the regulation of cigarette smoke-induced mitochondrial dysfunction via mPTP. Therefore, the development of specific TSPO antagonists like PK11195 may be beneficial to combat smoking-related diseases, such as COPD.

Inhaled Therapies for Asthma and Chronic Obstructive Pulmonary Disease.

Asthma and chronic obstructive pulmonary disease (COPD) are obstructive lung diseases which are characterized by chronic inflammation and an increase in mucus production, and are highly prevalent conditions. Despite recent advances and multiple available therapies, there remains a significant unmet medical need. Over the past 40 years, the introduction of new classes of safe and effective therapy is insufficient. In spite of the high burden of asthma and COPD among patients, there are fewer new approved therapies in comparison to cardiovascular, metabolic and neurological diseases due to few drug candidates and a higher failure rate in the development of respiratory medicine. Lung diseases are amongst the leading causes of death globally with asthma being one of the most prevalent respiratory diseases, which affects people of all ages but, despite effective therapies available, many patients are poorly controlled and have a low quality of life. COPD is currently ranked as the fourth cause of death worldwide and predicted to become the third leading cause of death in 2030. The development of more effective treatments is urgently needed in order to reduce the high mortality rate and the enormous suffering from asthma and COPD. Various inhalation devices with different classes of medications are the foundation as therapies in both asthma and COPD. This article gives a comprehensive review of the promising inhaled therapies in the treatment of asthma and COPD. However, the lack of disease control in asthma and COPD patients may be due to numerous reasons. The association between non-adherence to guidelines on the part of the health care provider and poor inhalation technique and/or non-adherence to the prescribed treatment plan by the patients is common. It is therefore essential to discuss the different delivery systems and the methods used in asthma and COPD patients.

Protective effect of selegiline on cigarette smoke-induced oxidative stress and inflammation in rat lungs in vivo.

BACKGROUND: Cigarette smoke (CS)-induced build-up of oxidative stress is the leading cause of chronic obstructive pulmonary disease (COPD). Monoamine oxidases (MAOs) are novel sources of reactive oxygen species (ROS) due to the production of hydrogen peroxide (H2O2). However, it remains unclear whether MAO signaling is involved in CS-induced oxidative stress in vivo. This study aimed at investigating the impact of selegiline, a selective MAO-B inhibitor, on CS-induced lung oxidative stress and inflammation in vivo and its underlying mechanism. METHODS: Sprague Dawley rats were randomly divided into four groups: saline plus sham air (Saline/air), saline plus cigarette smoke (Saline/CS), selegiline plus sham air (Slg/air) and selegiline plus cigarette smoke (Slg/CS). Rats from Saline/air and Saline/CS groups were intraperitoneally injected with saline (2 mL/kg body weight) while rats from Slg/air and Slg/CS groups were injected with selegiline (2 mg/kg body weight) about 30 min prior to exposure daily. The Saline/air and Slg/air groups were exposed to atmospheric air while the Saline/CS and Slg/CS groups were exposed to mainstream CS generated from the whole body inExpose smoking system (SCIREQ, Canada) for twice daily (each for 1 hour with 20 cigarettes). After 7 days, rats were sacrificed to collect bronchoalveolar lavage (BAL) and lung tissues for the measurement of oxidative/anti-oxidative and inflammatory/anti-inflammatory makers respectively. RESULTS: CS caused significant elevation of MAO-B activity, reduction of total antioxidant capacity (T-AOC) and rGSH/GSSG ratio, and enhancement of superoxide dismutase (SOD) activity in rat lung. Selegiline significantly only reversed CS-induced elevation of MAO-B activity and reduction of rGSH/GSSG ratio. The CS-induced elevation of heme oxygenase-1 (HO-1) and NAD(P)H quinone dehydrogenase 1 (NQO1) expression via nuclear factor erythroid 2-related factor 2 (Nrf2) was also reversed by selegiline. Despite of CS-induced increase in total cell counts, especially the number of macrophages, selegiline had no effect. Selegiline attenuated CS-induced elevation of pro-inflammatory mediators (CINC-1, MCP-1 and IL-6) and restored CS-induced reduction of anti-inflammatory mediator IL-10 in BAL, which was driven through MAPK and NF-κB. CONCLUSIONS: Inhibition of MAO-B may provide a promising therapeutic strategy for CS-mediated oxidative stress and inflammation in acute CS-exposed rat lungs.

The acute effects of cigarette smoke exposure on muscle fiber type dynamics in rats.

Reduced exercise capacity is common in people with chronic obstructive pulmonary diseases (COPD) and chronic smokers and is suggested to be related to skeletal muscle dysfunction. Previous studies using human muscle biopsies have shown fiber-type shifting in chronic smokers particularly those with COPD. These results, however, are confounded with aging effects because people with COPD tend to be older. In the present study, we implemented an acute 7-day cigarette smoke-exposed model using Sprague-Dawley rats to evaluate early effects of cigarette smoking on soleus muscles. Rats (n = 5 per group) were randomly assigned to either a sham air (SA) or cigarette smoking (CS) groups of three different concentrations of total particulate matters (TPM) (CSTPM2.5, CSTPM5, CSTPM10). Significantly lower percentages of type I and higher type IIa fiber were detected in the soleus muscle in CS groups when compared with SA group. Of these, only CSTMP10 group exhibited significantly lower citrate synthase activity and higher muscle tumor necrosis factor-α level than that of SA group. Tumor necrosis factor-α level was correlated with the percentage of type I and IIa fibers. However, no significant between-group differences were found in fiber cross-sectional area, physical activities, or lung function assessments. In conclusion, acute smoking may directly trigger the onset of glycolytic fiber type shift in skeletal muscle independent of aging.

Circulating adipocyte fatty acid-binding protein is reduced by continuous positive airway pressure treatment for obstructive sleep apnea-a randomized controlled study.

PURPOSE: The circulating level of adipocyte fatty acid-binding protein (AFABP), a biomarker with prognostic and therapeutic importance in metabolic disorders, has been shown to be elevated in obstructive sleep apnea (OSA). This randomized controlled study aimed to investigate the effect of continuous positive airway pressure (CPAP) treatment for OSA on AFABP levels. METHODS: Consecutive subjects attending sleep study were invited if they were confirmed to have severe OSA and were free of metabolic diseases. Participants were randomized (1:1) into CPAP or observation group for 4 weeks. Demographics, anthropometric data, and circulating biomarkers were checked at baseline and after the 4-week study period. RESULTS: Ninety subjects were randomized. The mean age was 46 ± 9 years old; 82% were male. Their mean body mass index (BMI) was 29 ± 5 kg/m2. By intention-to-treat approach, the CPAP group showed significant reductions in Epworth sleepiness scale and morning systolic blood pressure (- 7.2 mmHg, - 12.7 to - 1.7 mmHg, p = 0.011), but no significant difference in AFABP, adiponectin, C-reactive protein (CRP), and 8-isoprostane levels. In the per-protocol analysis, when only those who were compliant to CPAP were included, a significant reduction in AFABP (- 7.32 ng/ml, - 13.58, - 1.06, p = 0.023) were found in the CPAP-treated group compared with the control group, along with improvements in clinical parameters. Changes in AFABP were independently associated with both systolic blood pressure (ß = 0.289, p = 0.028) and diastolic blood pressure (ß = 0.217, p = 0.030). CONCLUSION: CPAP therapy used regularly over 4 weeks for severe OSA lowered circulating AFABP level, suggesting a potential beneficial effect of OSA treatment on alleviating metabolic risks. TRIAL REGISTRATION: The research protocol was registered at the National Institutes of Health clinical trials registry (NCT01173432).

Dysregulation of cardiac lipid parameters in high-fat high-cholesterol diet-induced rat model.

BACKGROUND: Lipid dysregulation is a classical risk factor for cardiovascular disease (CVD), yet scanty evidence existed regarding cardiac lipid metabolism that is directly related to heart damage. Recently, the relationship between dyslipidemia and pro-inflammatory insults has led to further understanding on the CVD-predisposing effects of dyslipidemia. The aims of the present study were to investigate whether high-fat high-cholesterol (HFHC) diet-induced hyperlipidemia would cause heart damage and to study the potential role of local cardiac lipid dysregulation in the occurrence of cellular injury. METHODS: Male Sprague-Dawley rats were divided into normal chow or HFHC diet groups, and sacrificed after 2 or 4 weeks, respectively. Lipid peroxidation marker level was measured. Lipid parameters in the rat hearts were detected. Cardiac damage was evaluated. RESULTS: HFHC diet increased serum levels of cholesterol and free fatty acids (FFAs) and led to systemic oxidative stress and pro-inflammatory status. Cardiac lipid dysregulation, which was characterized by elevated levels of cholesterol and adipocyte (A)- and heart (H)-fatty acid binding proteins (FABPs), occurred after HFHC diet for 4 weeks. Cardiac damage was further evident with elevated circulating H-FABP levels, increased cardiac interstitial fibrosis and the loss of troponin I. CONCLUSION: Our data demonstrated that HFHC diet led to systemic and cardiac lipid dysregulation, accompanied by systemic oxidative and pro-inflammatory stresses, and these may finally cooperate to cause a series of pathological changes of the heart tissue. Our findings suggest that maintenance of lipid regulation may be essential in the prevention of heart damage.

Untreated Obstructive Sleep Apnea Is Associated With Myocardial Injury Independent of Blood Pressure Control in Hypertension.

STUDY OBJECTIVES: Obstructive sleep apnea (OSA) and hypertension are independent risk factors of cardiovascular morbidities. This study aims to investigate the relationship between OSA, blood pressure (BP) control, and myocardial injury in patients with difficult-to-control hypertension. METHODS: Patients with hypertension who required three or more medications were prospectively recruited at a tertiary referral center. In-laboratory polysomnography, followed by blood tests for fasting glucose, glycated hemoglobin, lipids, high-sensitivity troponin I (hsTnI), B-type natriuretic peptide (BNP), C-reactive protein, and advanced oxidation protein products were performed. After polysomnography, 24-hour ambulatory BP monitoring was arranged. RESULTS: A total of 98 participants were analyzed, with mean age 51 ± 9 years and body mass index 30 ± 5 kg/m2. Previously undiagnosed severe OSA (apneahypopnea index [AHI] ≥ 30 events/h) was present in 51 patients (52%). hsTnI was negatively correlated with nocturnal dip in systolic BP (r = -.205, P = .048). After controlling for confounders, including BP control, AHI and oxygen desaturation index (ODI) were positively correlated with hsTnI (r = .282, P = .009 and r = .279, P = .010, respectively) and C-reactive protein (r = .302, P = .005 and r = .285, P = .008, respectively), but not with BNP or advanced oxidation protein products. Age, ODI, and loss of nocturnal systolic BP dip were significant determinants of hsTnI level (ß = .225, P = .022; ß = .293, P = .003; and ß = -.215, P = .029; R2 = .151). Age, female sex, 24-hour mean diastolic BP, and metabolic syndrome, but not indices of apnea severity, were predictors of BNP level. CONCLUSIONS: Unrecognized severe OSA was common in patients with difficult-to-control hypertension, and OSA severity was associated with myocardial injury, independent of BP control with medications. CLINICAL TRIAL REGISTRATION: Registry: ClinicalTrials.gov, Title: A Cross-sectional Study of the Occurrence and Effect of Obstructive Sleep Apnea in Subjects With Resistant Hypertension, Identifier: NCT00843583, URL: https://clinicaltrials.gov/ct2/show/NCT00843583.

Low but not high frequency of intermittent hypoxia suppresses endothelium-dependent, oxidative stress-mediated contractions in carotid arteries of obese mice.

Obstructive sleep apnea is characterized by intermittent hypoxia (IH) during sleep and predisposes to endothelial dysfunction. Obesity is a major risk factor for the occurrence of sleep apnea. The present study compared the functional impact of low- (IH10; 10 hypoxic events/h) and high-frequency (IH60; 60 hypoxic events/h) IH for 4 wk on endothelial function in male C57BL/6 mice with or without high-fat (HF) diet-induced obesity. Mean arterial blood pressure (tail cuff method) was increased in obese mice after IH60 exposure, i.e., HF + IH60 group. The serum levels of the oxidative stress marker malondialdehyde were augmented in lean IH60 and HF groups, with a further increase in HF + IH60 but a reduction in HF + IH10 mice compared with the HF group. Vascular responsiveness was assessed as changes in isometric tension in isolated arteries. Relaxations to the endothelium-dependent vasodilator acetylcholine were impaired in HF + IH60 aortae. Endothelium-dependent contractions (EDC; response to acetylcholine in the presence of the nitric oxide synthase inhibitor l-NAME) in carotid arteries were augmented in the HF group, but this HF-induced augmentation was suppressed by low-frequency IH exposure. The addition of apocynin (antioxidant) reduced EDC in HF and HF + IH60 groups but not in HF + IH10 group. In conclusion, these findings suggest that exposure of obese mice to mild IH exerts preconditioning-like suppression of endothelium-dependent and oxidative stress-mediated contractions. When IH severity increases, this suppression diminishes and endothelial dysfunction accelerates. NEW & NOTEWORTHY The present study demonstrates, for the first time, that low-frequency intermittent hypoxia may exert a preconditioning-like suppression of oxidative stress-induced endothelium-dependent contractions in mice with diet-induced obesity. This relative suppression was diminished as intermittent hypoxia became more severe, and a deleterious effect on endothelial function emerged.

Low-Frequency Intermittent Hypoxia Promotes Subcutaneous Adipogenic Differentiation.

Obstructive sleep apnea (OSA), characterized by intermittent hypoxia (IH), is associated with obesity and metabolic disorders. The mass and function of adipose tissue are largely dependent on adipogenesis. The impact of low-frequency IH on adipogenesis is unknown. Sprague-Dawley rats were subjected to IH (4 min for 10% O2 and 2 min for 21% O2) or intermittent normoxia (IN) for 6 weeks. The degree of adipogenic differentiation was evaluated by adipogenic transcriptional factors, adipocyte-specific proteins, and oily droplet production in both subcutaneous adipose tissue (SAT) and visceral adipose tissue (VAT). Upregulation of proadipogenic markers (CEBPα, PPARγ, and FABP4) and downregulation of antiadipogenic markers CHOP in line with smaller size of adipocytes were found in IH-exposed SAT. In vitro experiments using human preadipocytes (HPAs) of subcutaneous lineage during differentiation phase, subjected to IH (1% O2 for 10 min and 21% O2 for 5 min; 5% CO2) or IN treatment, were done to investigate the insulin-like growth factor 1 receptor (IGF-1R)/Akt pathway in adipogenesis. IH promoted the accumulation of oily droplets and adipogenesis-associated markers. IGF-1R kinase inhibitor NVP-AEW541 attenuated the proadipogenic role in IH-exposed HPAs. In summary, relatively low frequency of IH may enhance adipogenesis preferentially in SAT.

Mesenchymal stem cells alleviate oxidative stress-induced mitochondrial dysfunction in the airways.

BACKGROUND: Oxidative stress-induced mitochondrial dysfunction can contribute to inflammation and remodeling in patients with chronic obstructive pulmonary disease (COPD). Mesenchymal stem cells protect against lung damage in animal models of COPD. It is unknown whether these effects occur through attenuating mitochondrial dysfunction in airway cells. OBJECTIVE: We sought to examine the effect of induced pluripotent stem cell-derived mesenchymal stem cells (iPSC-MSCs) on oxidative stress-induce mitochondrial dysfunction in human airway smooth muscle cells (ASMCs) in vitro and in mouse lungs in vivo. METHODS: ASMCs were cocultured with iPSC-MSCs in the presence of cigarette smoke medium (CSM), and mitochondrial reactive oxygen species (ROS) levels, mitochondrial membrane potential (ΔΨm), and apoptosis were measured. Conditioned medium from iPSC-MSCs and transwell cocultures were used to detect any paracrine effects. The effect of systemic injection of iPSC-MSCs on airway inflammation and hyperresponsiveness in ozone-exposed mice was also investigated. RESULTS: Coculture of iPSC-MSCs with ASMCs attenuated CSM-induced mitochondrial ROS, apoptosis, and ΔΨm loss in ASMCs. iPSC-MSC-conditioned medium or transwell cocultures with iPSC-MSCs reduced CSM-induced mitochondrial ROS but not ΔΨm or apoptosis in ASMCs. Mitochondrial transfer from iPSC-MSCs to ASMCs was observed after direct coculture and was enhanced by CSM. iPSC-MSCs attenuated ozone-induced mitochondrial dysfunction, airway hyperresponsiveness, and inflammation in mouse lungs. CONCLUSION: iPSC-MSCs offered protection against oxidative stress-induced mitochondrial dysfunction in human ASMCs and in mouse lungs while reducing airway inflammation and hyperresponsiveness. These effects are, at least in part, dependent on cell-cell contact, which allows for mitochondrial transfer, and paracrine regulation. Therefore iPSC-MSCs show promise as a therapy for oxidative stress-dependent lung diseases, such as COPD.

From Pulmonary Surfactant, Synthetic KL4 Peptide as Effective siRNA Delivery Vector for Pulmonary Delivery.

Pulmonary delivery of small interfering RNA (siRNA) has huge potential for the treatment of a wide range of respiratory diseases. The ability of naked siRNA to transfect cells in the lungs without a delivery vector has prompted the investigation of whether an endogenous component is at least partially responsible for the cellular uptake of siRNA, and whether a safe and efficient delivery system could be developed from this component to further improve the transfection efficiency. Surfactant protein B (SP-B), a positively charged protein molecule found in lung surfactant, is one of the possible candidates. While the role of SP-B in siRNA transfection remains to be determined, the SP-B mimic, synthetic KL4 peptide, was investigated in this study as a potential siRNA carrier. KL4 is a 21-residue cationic peptide that was able to bind to siRNA to form nanosized complexes. It mediated siRNA transfection effectively in vitro on human lung epithelial cells, A549 cells, and BEAS-2B cells, which was comparable to Lipofectamine 2000. When commercial pulmonary surfactant (Infasurf) was added in the transfection medium, the gene silencing effect of siRNA in cells transfected with Lipofectamine 2000 was completely abolished, whereas those transfected with KL4 remained unaffected. At 4 °C, KL4 failed to deliver siRNA into the cells, indicating that an energy-dependent process was involved in the uptake of the complexes. Chlorpromazine (inhibitor of chathrin-mediated endocytosis), but not nystatin (inhibitor of caveolae-mediated endocytosis), inhibited the uptake of KL4/siRNA complexes, suggesting that they entered cells through clathrin-mediated endocytosis. There was no sign of cytotoxicity or immune response caused by KL4 and KL4/siRNA complexes. Overall, this study demonstrated that synthetic KL4 peptide is a promising candidate for siRNA carrier for pulmonary delivery and could be a potential platform for delivering other types of nucleic acid therapeutics.

(-)-Epigallocatechin-3-gallate Reduces Cigarette Smoke-Induced Airway Neutrophilic Inflammation and Mucin Hypersecretion in Rats.

Background: Cigarette smoking is the leading cause of chronic obstructive pulmonary disease. (-)-Epigallocatechin-3-gallate (EGCG), the major catechins in Chinese green tea, has been studied for its anti-oxidative and anti-inflammatory properties in cell and animal models. In this study, we aimed to analyze the effects of EGCG on cigarette smoke (CS)-induced airway inflammation and mucus secretion in the CS-exposed rat model. Methods: Male Sprague-Dawley rats were randomly divided into either sham air (SA) or CS exposure. EGCG (50 mg/kg b.wt.) was given by oral gavage every other day in both SA and CS-exposed animals. Oxidative stress and inflammatory markers were determined in serum and/or bronchoalveolar lavage fluid by biochemical assays or ELISA. Lung morphological changes were examined by Periodic Acid-Schiff, Masson's Trichrome staining and immunohistochemical analysis. Western blot analysis was performed to explore the effects of EGCG on epidermal growth factor receptor (EGFR)-mediated signaling pathway. Results: (-)-Epigallocatechin-3-gallate treatment attenuated CS-induced oxidative stress, lung cytokine-induced neutrophil chemoattractant-1 release and neutrophil recruitment. CS exposure caused an increase in the number of goblet cells in line with MUC5AC upregulation, and increased lung collagen deposition, which were alleviated in the presence of EGCG. In addition, CS-induced phosphorylation of EGFR in rat lung was abrogated by EGCG treatment. Conclusion: (-)-Epigallocatechin-3-gallate treatment ameliorated CS-induced oxidative stress and neutrophilic inflammation, as well as airway mucus production and collagen deposition in rats. The present findings suggest that EGCG has a therapeutic effect on chronic airway inflammation and abnormal airway mucus production probably via inhibition of EGFR signaling pathway.