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Previous studies have suggested a role for an increased apoptosis of the endothelial cells in the pulmonary capillaries of the alveolar septa in the pathogenesis of human pulmonary emphysema.1 In animal models, circulating endothelial stem cells, characterised by the concomitant expression of CD34+, CD133 and vascular endothelial growth factor receptor 2 (VEGF-R2), may contribute to the repair of lung damage.2 However, it is unknown if a decrease in the blood of these stem cells contributes to the pathogenesis of pulmonary emphysema in humans. The aim of our study was to investigate by flow cytometry the number of total (CD34+) and endothelial stem (triple positive for CD34+/CD133/VEGF-R2) cells in the peripheral venous blood of current and former smokers of similar age, with or without pulmonary emphysema.
All the recruited subjects were free from concomitant diseases or drugs able to interfere with the number of circulating stem cells. Venous blood samples were obtained from 37 subjects (mean (SD) age 66.8 (1.4) years, 25M/12F, mean (SD) 33.11 (3.2) pack-years, 12 current and 25 ex-smokers). All former smokers had stopped smoking for more than 1 year. Twenty-two subjects (59.5%) had chronic obstructive pulmonary disease (COPD) according to the criteria of the Global Initiative for Chronic Obstructive Lung Disease guidelines3 (mean post-bronchodilator forced expiratory volume in 1 s/forced vital capacity (FEV1/FVC) ratio 56.8 (2.7)%) whereas 39.5% (n = 15) had normal lung function (FEV1/FVC ratio 77.1 (1.4)%).
The presence and the severity of pulmonary emphysema was determined using high-resolution CT (HRCT) scans of the chest with density mask and the National Emphysema Treatment Trial Research Group score (0–4).4 The mean (SD) HRCT score was 1.7 (0.4). Blood sampling and flow cytometry were performed as previously described.5 Briefly, quantification of peripheral blood CD34+ cells was performed with double labelling with FITC-anti-CD45 and phycoerythrin-anti-CD34 monoclonal antibodies (Becton Dickinson, Milan, Italy) on a FACSCalibur flow cytometer (Becton Dickinson) according to standardised procedures. Enumeration of endothelial stem cells was performed as CD34+ cells co-expressing CD133 and VEGF-2. It was performed on immunomagnetically purified peripheral blood CD34+ cells (Miltenyi Biotech, Bologna, Italy) by triple labelling with peridinin chlorophyll protein-conjugated anti-CD34, phycoerythrin-conjugated anti-CD133 (Miltenyi Biotech) and unconjugated anti-VEGFR-2 (Santa Cruz Biotechnology, Milan, Italy), followed by FITC-conjugated swine anti-rabbit (Dako, Milan, Italy) as secondary reagent.
We found a significant correlation between the absolute number of circulating CD34+ cells and the absolute number of circulating endothelial stem cells (r2 = 0.593, p<0.0001; see figure 1 in online supplement). There was also a significant correlation between the percentage of circulating endothelial stem cells and the number of pack-years smoked (r2 = 0.42, p<0.05; see figure 2 in online supplement). No significant correlation was found between total and endothelial stem cell numbers and HRCT score of pulmonary emphysema (figure 1), lung function data or smoking status (current vs ex-smokers). These data indicate that the number of circulating endothelial stem cells is not related to the presence and/or severity of the pulmonary emphysema or the presence or absence of COPD.
Competing interests None.
Ethics approval The study was approved by the local ethics committee of the University Hospital of Ferrara and written informed consent was obtained from each participant in accordance with the principles outlined in the Declaration of Helsinki.
Provenance and Peer review Not commissioned; externally peer reviewed.