Dysfunction of Endothelial Progenitor Cells from Smokers and Chronic Obstructive Pulmonary Disease Patients Due to Increased DNA Damage and Senescence

Cardiovascular disease (CVD) is a major cause of death in smokers, particularly in those with chronic obstructive pulmonary disease (COPD). Circulating endothelial progenitor cells (EPC) are required for endothelial homeostasis, and their dysfunction contributes to CVD. To investigate EPC dysfunction in smokers, we isolated and expanded blood outgrowth endothelial cells (BOEC) from peripheral blood samples from healthy nonsmokers, healthy smokers, and COPD patients. BOEC from smokers and COPD patients showed increased DNA double-strand breaks and senescence compared to nonsmokers. Senescence negatively correlated with the expression and activity of sirtuin-1 (SIRT1), a protein deacetylase that protects against DNA damage and cellular senescence. Inhibition of DNA damage response by silencing of ataxia telangiectasia mutated (ATM) kinase resulted in upregulation of SIRT1 expression and decreased senescence. Treatment of BOEC from COPD patients with the SIRT1 activator resveratrol or an ATM inhibitor (KU-55933) also rescued the senescent phenotype. Using an in vivo mouse model of angiogenesis, we demonstrated that senescent BOEC from COPD patients are dysfunctional, displaying impaired angiogenic ability and increased apoptosis compared to cells from healthy nonsmokers. Therefore, this study identifies epigenetic regulation of DNA damage and senescence as pathogenetic mechanisms linked to endothelial progenitors' dysfunction in smokers and COPD patients. These defects may contribute to vascular disease and cardiovascular events in smokers and could therefore constitute therapeutic targets for intervention. 

Development of a Novel Assay for the Detection of Active Neutrophil Elastase in Patients with Chronic Obstructive Pulmonary Disease

Neutrophil elastase (NE), a biomarker of infection and inflammation, correlates with the severity of several respiratory diseases including chronic obstructive pulmonary disease (COPD). However, it’s detection and quantification in biological samples is confounded by a lack of reliable and robust methodologies. Standard assays using chromogenic or fluorogenic substrates are not specific when added to complex clinical samples containing multiple proteolytic and hydrolytic enzymes which have the ability to hydrolyse the substrate, thereby resulting in an over-estimation of the target protease. Furthermore, ELISA systems measure total protease levels which can be a mixture of latent, active and protease-inhibitor complexes. Therefore, we have developed a novel immunoassay (ProteaseTag™ Active NE Immunoassay) which is selective and specific for the capture of active NE in sputum and Bronchoalveolar Lavage (BAL) in patients with COPD. The objective of this study was to clinically validate ProteaseTag™ Active NE Ultra Immunoassay for the detection of NE in sputum from COPD patients. 20 matched sputum sol samples were collected from 10 COPD patients (M=6, F=4; 73 ± 6 years) during stable and exacerbation phases. Samples were assayed for NE activity utilising both ProteaseTag™ Active NE Ultra Immunoassay and a fluorogenic substrate-based kinetic activity assay. Both assays detected elevated levels of NE in the majority of patients (n=7) during an exacerbation (mean=217.2 μg/ml ±296.6) compared to their stable phase (mean=92.37 μg/ml ±259.8). However, statistical analysis did not show this difference to be significant (p=0.07, ProteaseTag™ Active NE Ultra Immunoassay; p=0.06 kinetic assay), most likely due to the low study number. A highly significant correlation was found between the 2 assay types (p≤0.0001, r=0.996). NE as a primary efficacy endpoint in clinical trials or as a marker of inflammation within the clinic has been hampered by the lack of a robust and simple to use assay. ProteaseTag™ Active NE Immunoassay specifically measures only active NE in clinical samples, is quick and easy to use (< 3 hours) and has no dependency on a kinetic readout. ProteaseTag™ technology is currently being transferred to a lateral flow device for use at Point of Care.

Community dynamics and the lower airway microbiota in stable chronic obstructive pulmonary disease, smokers and healthy non-smokers

RATIONALE: The role bacteria play in the progression of COPD has increasingly been highlighted in recent years. However, the microbial community complexity in the lower airways of patients with COPD is poorly characterised.

OBJECTIVES: To compare the lower airway microbiota in patients with COPD, smokers and non-smokers.

METHODS: Bronchial wash samples from adults with COPD (n=18), smokers with no airways disease (n=8) and healthy individuals (n=11) were analysed by extended-culture and culture-independent Illumina MiSeq sequencing. We determined aerobic and anaerobic microbiota load and evaluated differences in bacteria associated with the three cohorts. Culture-independent analysis was used to determine differences in microbiota between comparison groups including taxonomic richness, diversity, relative abundance, 'core' microbiota and co-occurrence.

MEASUREMENT AND MAIN RESULTS: Extended-culture showed no difference in total load of aerobic and anaerobic bacteria between the three cohorts. Culture-independent analysis revealed that the prevalence of members of Pseudomonas spp. was greater in the lower airways of patients with COPD; however, the majority of the sequence reads for this taxa were attributed to three patients. Furthermore, members of Bacteroidetes, such as Prevotella spp., were observed to be greater in the 'healthy' comparison groups. Community diversity (α and β) was significantly less in COPD compared with healthy groups. Co-occurrence of bacterial taxa and the observation of a putative 'core' community within the lower airways were also observed.

CONCLUSIONS: Microbial community composition in the lower airways of patients with COPD is significantly different to that found in smokers and non-smokers, indicating that a component of the disease is associated with changes in microbiological status.

Quality of life and Chronic Obstructive Pulmonary Disease

The Chronic Obstructive Pulmonary Disease (COPD) has a progressive and irreversible character and it’s associated to the triad of dyspnea, exercise limitation and the evident deterioration of quality of life. In the United States the prevalence of COPD in adult population is approximately of 6% in men, and 1 to 3% in women and it’s the fourth cause of mortality by no transmissible chronic diseases. In 1993, the National Health Interview Surgery considered that 12 millions of Americans suffer from chronic bronchitis and 2 million had emphysema. These two affections are responsible for more than 13% of the hospitalizations. As this affection progresses, patients experience a diminution in quality of life related to health (CVRS), their capacity to work get worse and their participation in physical and social activities reduces. Nevertheless, it has been confirmed that the isolated evaluation of COPD seriousness, defined by the reduction of the Forced Expiratory Volume in the First Second (FEV1), does not provide enough information to know the health state perceived by the patients. The fact that the CVRS is the result of the interaction of multiple physical, psychological and social factors, unique for each individual, can explain this finding. This paper is a general and updated approach to the integral handling of patients with COPD, and it discusses the concept of quality of life, related to health improvement.

Muscle atrophy and hypertrophy signaling in patients with chronic obstructive pulmonary disease

Rationale: The molecular mechanisms of muscle atrophy in chronic obstructive pulmonary disease (COPD) are poorly understood. In wasted animals, muscle mass is regulated by several AKT-related signaling pathways.
Objectives: To measure the protein expression of AKT, forkhead box class O (FoxO)-1 and -3, atrogin-1, the phosphophrylated form of AKT, p70^S6K glycogen synthase kinase-3ß (GSK-3ß), eukaryotic translation initiation factor 4E binding protein-1 (4E-BP1), and the mRNA expression of atrogin-1, muscle ring finger (MuRF) protein 1, and FoxO-1 and -3 in the quadriceps of 12 patients with COPD with muscle atrophy and 10 healthy control subjects. Five patients with COPD with preserved muscle mass were subsequently recruited and were compared with six patients with low muscle mass.
Methods: Protein contents and mRNA expression were measured by Western blot and quantitative polymerase chain reaction, respectively.
Measurements and Main Results: The levels of atrogin-1 and MuRF1 mRNA, and of phosphorylated AKT and 4E-BP1 and FoxO-1 proteins, were increased in patients with COPD with muscle atrophy compared with healthy control subjects, whereas atrogin-1, p70^S6K, GSK-3ß, and FoxO-3 protein levels were similar. Patients with COPD with muscle atrophy showed an increased expression of p70^S6K, GSK-3ß, and 4E-BP1 compared with patients with COPD with preserved muscle mass.
Conclusions: An increase in atrogin-1 and MuRF1 mRNA and FoxO-1 protein content was observed in the quadriceps of patients with COPD. The transcriptional regulation of atrogin-1 and MuRF1 may occur via FoxO-1, but independently of AKT. The overexpression of the muscle hypertrophic signaling pathways found in patients with COPD with muscle atrophy could represent an attempt to restore muscle mass.

Factors affecting the offer of pulmonary rehabilitation to patients with chronic obstructive pulmonary disease by primary care professionals : a qualitative study

Aim: To explore health professionals’ experiences of barriers and facilitators to referring patients for pulmonary rehabilitation in a primary care setting.

Background: Pulmonary rehabilitation involves a multidisciplinary teamwork approach to improving
the quality of life for people with chronic obstructive pulmonary disease. This study aimed to find out about health care professionals’ experiences when referring patients. Reports suggest that a health care professional’s attitude towards a treatment affects the willingness of patients to accept advice.

Methods: Five focus group interviews were undertaken with 21 health professionals from North Midlands, UK. Data were analysed using a thematic analysis drawing on the techniques of grounded theory.

Findings: Chronic disease management has been delegated to Practice Nurses in many cases leaving some nurses feeling unsupported and some General Practitioners feeling deskilled. Problems with communication, a lack of adequate and timely local service provision, a difficult referral process, time pressures and lack of information were barriers to health care professionals making an offer of pulmonary rehabilitation. An explanatory model is proposed to describe how addressing barriers to referral may improve health care professionals views about pulmonary rehabilitation and therefore may mean that they present it in a more positive manner.

Early predictors of hospital admission in emergency department patients with chronic obstructive pulmonary disease

Background: Streamlining emergency department (ED) care of patients with chronic obstructive pulmonary disease (COPD) may be an important strategy in managing the increasing burden of this disease.

Study objectives: The aim of this study was to identify factors predictive of hospital admission in ED patients with COPD, specifically factors that can be used early in the ED episode of care.

Methods: Using retrospective regression analysis, case data from 321 randomly selected medical records from five Australian EDs were analysed. Patient characteristics, triage and ED system features, physiological status, and ED treatment during the first four hours of ED care were compared between discharged and admitted patients.

Results: Factors available on ED arrival associated with increased likelihood of admission were: age (OR = 1.04, p = 0.008) respiratory symptoms affecting activities of daily living (OR = 1.8, p = 0.043) and signs of respiratory dysfunction (OR = 2.5, p = 0.005). Factors available from the first four hours of ED care associated with increased likelihood of admission were: age (OR = 1.04, p = 0.021), oxygen use at four hours (OR = 3.5, p = 0.002) and IV antibiotic administration (OR = 2.6, p = 0.026). There were conflicting findings regarding the association between ambulance transport and admission.

Descriptive analysis of emergency department oxygen use in acute exacerbation of chronic obstructive pulmonary disease

Background: Inconsistencies in oxygen therapy recommendations in acute exacerbation of chronic obstructive pulmonary disease (COPD) may result in variability in emergency department (ED) oxygen management of patients with COPD. The aim of this study was to describe oxygen management in the first 4 h of ED care for patients with exacerbation of COPD.
Methods: A retrospective medical record audit was conducted at four public and one private ED in Melbourne, Australia. Participants were 273 adult ED patients with COPD presenting with a primary complaint of shortness of breath from July 2006 to July 2007. Outcome measures were physiological data, including oxygen saturation (SpO2), oxygen delivery devices and flow rates on ED arrival, 1 and 4 h.
Results: Oxygen was used in 82.0% of patients. Patients who required oxygen had higher incidence of ambulance transport (P < 0.001), triage category 2 (P = 0.006), home oxygen use (P < 0.001), and increased work of breathing on ED arrival (P < 0.001), and higher median respiratory rate (P < 0.001) and heart rate (P = 0.001). SpO2 > 90% occurred in the majority of patients (87.5%; 96.4%; 95.6%); however, a considerable number of patients with SpO2 < 90% were not given oxygen (61.8%; 30%; 45.5%).
Conclusions: A number of patients with documented hypoxaemia were not given oxygen and there may be variables other than oxygen saturation that may influence oxygen use. Future research should focus on increasing the evidence-based supporting
oxygen use and better understanding of clinicians’ oxygen decision-making in patients with COPD.

Enhanced cytotoxic function of natural killer and natural killer T-like cells associated with decreased CD94 (Kp43) in the chronic obstructive pulmonary disease airway

Background and objective: Natural killer (NK) and natural killer T (NKT)-like cells represent a small but important proportion of effector lymphocytes that we have previously shown to be major sources of pro-inflammatory cytokines and granzymes. We hypothesized that these cells would be increased in the airway in chronic obstructive pulmonary disease (COPD), accompanied by reduced expression of the inhibitory receptor CD94 (Kp43) and increased expression of cytotoxic mediators granzyme B and perforin.
Methods: We measured NK and NKT-like cells and their expression of CD94 in the blood of COPD patients (n = 71; 30 current and 41 ex-smokers), smokers (16) and healthy controls (25), and bronchoalveolar lavage fluid (BALF) from a cohort of subjects (19 controls, 12 smokers, 33 COPD). Activation was assessed by measuring CD69 in blood and the cytotoxic potential of NK cells by measuring granzymes A and B, and using a cytotoxicity assay in blood and BALF.
Results: In blood in COPD, there were no significant changes in the proportion of NK or NKT-like cells or expression of granzyme A or NK cytotoxic potential versus controls. There was, however, increased expression of granzyme B and decreased expression of CD94 by both cell types versus controls. The proportion of NK and NKT-like cells were increased in BALF in COPD, associated with increased NK cytotoxicity, increased expression of granzyme B and decreased expression of the inhibitory receptor CD94 by both cell types.
Conclusions: Treatment strategies that target NK and NKT-like cells, their cytotoxicity and production of inflammatory mediators in the airway may improve COPD morbidity.