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Persistence of circulating endothelial microparticles in COPD despite smoking cessation

Strulovici-Barel Y, Staudt MR, Krause A, Gordon C, Tilley AE, et al. Thorax 2016 ; 0 :1-8. Doi :10.1136/thoraxjnl-2015-208274

Background: Chronic obstructive pulmonary disease (COPD) is commonly thought of as a disease of the airways and the pulmonary parenchyma. Increasing attention has been given to the pulmonary vasculature, particularly endothelial cell apoptosis, as a major player in the pathogenesis of COPD. Prior work has demonstrated that COPD patients have an increase in levels of circulating endothelial microparticles (EMPs) compared to non-smokers. The current study sought to investigate whether EMP levels changed in COPD patients who quit smoking.

Methods: This was a 1-year prospective observational study of 28 non-smokers, 61 smokers without COPD, and 49 smokers with COPD (all GOLD stage I or II). Pulmonary function testing and CT scans to quantify emphysema were obtained at baseline in all study subjects. EMPs were quantified at baseline, 3, 6, and 12 months. This was accomplished by differential centrifugation and flow cytometry; EMPs were defined as microparticles <1.5 µm in size, expressing either CD31+ or CD62E+ but not CD42b. Furthermore, EMPs were analyzed by the presence of ACE+ (for pulmonary capillary origin) and also classified by their CD62E/CD31 ratio, with a low ratio reflecting “apoptotic EMPs.”

Results: Over the course of 1-year follow up, 17 healthy smokers and 18 COPD smokers successfully quit smoking by using varenicline, as evidenced by urinary cotinine levels. Baseline EMP levels were significantly higher in healthy smokers compared to non-smokers. Interestingly, COPD smokers also had elevated levels of EMPs, but these values were lower than those of healthy smokers. There were no correlations between EMP levels and any pulmonary function or demographic parameter. Levels of EMPs derived mainly from the pulmonary capillaries (ACE+) were highest in COPD smokers, and the amount of apoptotic EMPs was equally high in the COPD smokers and healthy smokers.

At the 1-year time point, healthy smokers who quit had significantly lower levels of EMPs compared to healthy smokers who continued to smoke, with levels no different than those of never-smokers. Additionally, the percentage of EMPs derived from apoptotic endothelial cells was lower in healthy smokers who quit versus those who continued smoking. In contrast to healthy smokers, COPD smokers who quit smoking had persistently elevated EMP levels, no different than those who continued to smoke.

Conclusions: This study confirms the concept that smoking is injurious to the pulmonary capillary bed, and shows for the first time that endothelial damage (as measured by EMP levels) may be reversible in smokers without COPD. However, despite smoking cessation, COPD patients continue to have elevated levels of EMPs, suggesting that endothelial cell apoptosis persists unabated even in the absence of continued smoke exposure.

Commentary: There is both human and animal model evidence to support a role for lung endothelial apoptosis in the development of emphysema. The physiologic measure of loss of pulmonary microvessels (DLCO) is difficult to interpret due to confounding factors that alter the measurement, such as carboxyhemoglobin levels in smokers. Radiologic measures of rarefaction of the pulmonary circulation, such as Hounsfield units calculated from HRCT, may lack sensitivity. Thus, there is a need for a clinically useful and convenient measure of lung endothelial damage that could be used to better characterize emphysema phenotype and also to assess the effects of therapies. The previously published observation by the authors of this study raised the suggestion that circulating microparticles could be used as a measure of loss of pulmonary microvessels in smokers. They reported that circulating endothelial cell-derived microparticles bearing apoptosis markers are increased among smokers and that the levels were inversely correlated with DLCO. The current results indicate that smoking cessation results in return of circulating endothelial derived microparticles to healthy control levels, but that this is not seen in quitters with already established COPD. This important observation suggests that circulating endothelial microparticles might be a sensitive measure of ongoing lung vascular injury in COPD.

There are some methodological concerns that need to be considered. Both the previous study and the current paper used a low ratio of CD45-CD62+/CD45-CD31+ as a measure of microparticles derived from apoptotic lung endothelial cells (as opposed to endothelial cells active by cytokines). This assertion is based on a previously published paper by other authors (Jimenez. Thrombosis Res. 109:175, 2003) who studied activated and apoptotic cultured renal microvascular endothelial cells. The validity of this measure would be strengthened by further validation of the low ratio of CD45-CD62+/CD45-CD31+ markers in circulating microparticles as a measure of endothelial apoptosis using other markers of apoptosis.

The authors use positive expression of anti-human angiotensin converting enzyme as a measure of lung capillary origin of circulating microparticles, with 75% of circulating CD45-CD31+ microparticles displaying anti-human ACE (CD143) positivity. It is worth noting that studies of human tissues obtained at the time of autopsy revealed ACE immunoreactivity only in EC, with more immunoreactivity in capillaries than in conduit vessels (Metzger. Microcirculation Res. 81:206, 2011).  Most organs displayed ACE immunoreactivity in about 20% of capillary EC with the exception of kidney (no EC ACE detected) and lung (100% of capillary EC). Thus, although ACE (CD143) immunoreactivity is more likely to be derived from lung vascular endothelium, it is not specific to the lung endothelium. Indeed, it is possible that the circulating endothelial microparticles found in continuing smokers with and without COPD might also be markers for ongoing injury to non-pulmonary vascular beds in addition to the pulmonary circulation.

Nevertheless, despite these methodologic concerns, the paper by Strulovici-Barel and colleagues raises the intriguing possibility that measurement of circulating endothelial microparticles may be a clinically useful tool to assess the effects of therapy on cigarette smoke-induced vascular injury.

Article summary by: Matthew Lammi MD, MSCR; Assistant Professor of Medicine, Louisiana State University Health Sciences Center, Comprehensive Pulmonary Hypertension Center-University Medical Center

Expert commentary by: Dr. Sharon Rounds MD, Professor of Medicine, Professor of Pathology and Laboratory Medicine at Alpert Medical School of Brown University