Circulating Progenitor Cells Are Reduced in Patients with Severe Lung Disease - 《stemcells》干细胞学杂志干细胞之家



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作者：Gian Paolo Fadinia, Marco Schiavonb, Marcella Cantinic, Ilenia Baessod, Monica Faccod, Marta Miorind, Mauro Tassinatod, Saula Vigili de Kreutzenberga, Angelo Avogaroa, Carlo Agostinid作者单位：aDepartment of Clinical and Experimental Medicine, Division of Metabolic Diseases,bDepartment of Cardiac, Thoracic and Vascular Sciences, Thoracic Surgery Branch,dDepartment of Clinical and Experimental Medicine, Clinical Immunology and Hematology, University of Padova School of Medicine, Padova, It + y5 h, J2 U* M% L, f$ p

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      【摘要】1 U0 a( R( d0 C8 A8 b  c6 H  H5 h
   Patients with chronic severe lung disease are prone to develop pulmonary vascular remodeling, possibly through pulmonary endothelial dysfunction. Circulating endothelial progenitor cells (EPCs) are involved in maintenance of endothelial homeostasis. The aim of this study was to assess whether obstructive and restrictive lung diseases are associated with modification of EPC number in peripheral blood. The study was cross-sectional and involved patients with obstructive (n = 15) and restrictive (n = 15) lung disease on oxygen therapy and 15 control subjects. Circulating EPCs were defined by the surface expression of CD34, CD133, and kinase-insert domain receptor. Results from spirometric tests, blood gas analyses, and blood cell counts have been related to EPC numbers. Patients with chronic hypoxia and severe lung disease showed lower levels of all progenitors than do control subjects. A consensual further reduction of EPC was found in restrictive patients in comparison with obstructive patients. Among restrictive patients, EPC reduction was related to reduced lung volumes and impaired alveolo-arterial diffusion, whereas progenitor cell levels were directly related to erythrocyte number. Considering obstructive patients, significant correlations were found between progenitor cell levels and bronchial obstruction and between progenitor cell levels and arterial oxygen tension. These findings demonstrate a reduction of EPCs in patients with chronic lung disease and long-lasting hypoxia. This alteration was more evident in restrictive patients and correlated to disease severity. Depletion of circulating EPCs may be involved in altered endothelial homeostasis of pulmonary circulation in these disorders. ; T& N7 y! I. R2 C- @6 o
      【关键词】 Endothelial progenitor cells Stem cells Endothelial dysfunction Pulmonary disease
   INTRODUCTION  `% v8 f* V; v  A2 ]: p

In most diffuse obstructive and restrictive lung diseases, progressive pulmonary vascular remodeling and neovascularization may be observed .
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Endothelial progenitor cells (EPCs) have been found to take part in human neovascularization and maintenance of vascular integrity .

In this study we tested the hypothesis that altered pulmonary vascular homeostasis may be attributed to a decrease in the number of circulating EPCs in patients with chronic hypoxemia due to severe lung disease.

Our data cannot confirm the hypothesis that chronic hypoxia stimulates EPCs as does tissue ischemia. On the contrary, patients with both obstructive and restrictive pulmonary disease had lower levels of EPCs in peripheral blood than did control subjects. Such an alteration may contribute to pulmonary endothelial dysfunction, lung disease progression, and increased cardiovascular risk in these patients.

MATERIALS AND METHODS

Patients

The study involved a total of 30 patients with chronic hypoxia due to lung disease and 15 healthy control subjects. All patients were recruited from the Social Service of Pneumology of the Unit¨¤ Locale Socio-Sanitaria 17 Veneto Region, whereas control subjects were recruited from the local community. Ethics committee approval and informed consent from all subjects were obtained. The criterion for patient inclusion was the presence of chronic lung disease on oxygen therapy for at least 6 months. Chronic obstructive pulmonary disease (COPD) (n = 15) and restrictive lung diseases (RLDs) (n = 15) were included. International guidelines have been used to validate the diagnosis. Functional diagnosis of COPD was considered in the presence of a history of resting or exertional dyspnea and reduced forced expiratory volume in 1 second to forced vital capacity (FEV1/FVC) ratio with an irreversible increased airway resistance on a standard spyrovolumetric test. According to the American Thoracic Society statement , diagnosis of chronic bronchitis was made on the basis of a history of persistent cough with sputum production for at least 3 months a year for at least 2 consecutive years. Diagnosis of pulmonary emphysema was considered when functional diagnosis of COPD was not associated with clinical features of chronic bronchitis; diagnosis was clinically supported by the evidence of prolonged exhalation, hyperinflation, and decreased breath sounds and was confirmed in the presence of an increased residual volume/thin-layer chromatography ratio on a standard spyrovolumetric test; other features suggestive of pulmonary emphysema were increased dyafanic transparence, flat-shaped diaphragms, and increased anterior-posterior over lateral chest diameter on a standard posterior-anterior chest radiograph.9 B# p; E9 p; V9 D8 v
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Diagnosis of RLD  was considered in the presence of consensual reduction in lung volumes (total lung volume, forced vital capacity, and residual volume) and an altered carbon monoxide diffusion test. For interstitial lung disease, reticular opacities with volume loss were considered a suggestive radiographic feature, and the findings on the standard x-ray film were always confirmed by a high-resolution computed tomography performed after the administration of contrast material in accordance with established guidelines and interpreted by a radiologist experienced in the evaluation of diffuse lung diseases: diffused reticular alterations, ground glass opacities, and honeycombing aspect were considered suggestive patterns.
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Control subjects were free from clinical and instrumental evidence of pulmonary disease and were, on the average, matched with patients by age and sex.( g: Z7 J; x# J( k
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Exclusion criteria were subject¡¯s refusal, acute illnesses, already diagnosed or highly presumed neoplasm, age over 80, recent surgery or vascular intervention, risk factors for or established cardiovascular disease, dialysis, acute infections, autoimmune diseases, and organ transplant receipt. Cardiovascular disease and risk factors were ruled out by minimal criteria, such as history evaluation and a basal clinical examination, except when cardiac abnormalities were considered secondary to pulmonary disease.
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Patients underwent standard evaluation by means of spiroergometric test, carbon monoxide diffusion test, arterial and venous blood gas analyses, and electrocardiogram. When available, results from echocardiograms were also recorded. Blood samples were drawn for determinations of white and red blood cell counts, hematocrit, and hemoglobin. Pharmacological data and smoking habit were also recorded.

Quantification of Peripheral Blood Progenitor Cells

After overnight fast, patients were admitted at the outpatient clinic. Oxygen therapy was discontinued for 1 hour before blood collection. Venous blood samples were obtained from a forearm vein and processed after 1¨C2 hours. Peripheral blood progenitor cells were analyzed for the expression of cell surface antigens with direct three-color analysis using fluorescein isothiocyanate (FITC)-conjugated, phycoerythrin (PE)-conjugated and allophycocyanin (APC)-conjugated monoclonal antibodies (mAbs) by flow cytometry analysis (FACSCalibur; Becton, Dickinson and Company, Franklin Lakes, NJ, http://www.bd.com), as previously reported . Briefly, before staining with specific monoclonal antibodies, cells were treated with fetal calf serum for 10 minutes, and then the samples were washed with buffer containing phosphate-buffered saline and 0.5% bovine albumin. Then, 150 µl of peripheral blood was incubated with 10 µl of FITC-conjugated anti-human CD34 mAb (Becton, Dickinson and Company), with 5 µl of APC-conjugated anti-human CD133 mAb (Miltenyi Biotec, Bergisch Gladbach, Germany, http://www.miltenyibiotec.com), and 10 µl of PE-conjugated anti-human KDR mAb (R&D Systems Inc., Minneapolis, http://www.rndsystems.com), followed by incubation at 4¡ãC for 30 minutes. Unlabeled cells or anti-isotype antibody served as a control. The frequency of peripheral blood cells positive for the above reagents was determined by a two-dimensional side scatter-fluorescence dot plot analysis of the samples, after appropriate gating (Fig. 1A¨C1C). After morphological gating to exclude granulocyte and cell debris, we gated CD34  peripheral blood cells and then examined the resulting population for dual and triple expression of KDR and CD133 (Fig. 1A¨C1G). Circulating progenitor cells were defined as CD34  or CD133  or CD34 CD133  cells, whereas endothelial progenitor cells were defined as CD34 KDR or CD133 KDR  and CD133 CD34 KDR  cells. For fluorescence-activated cell sorting analysis, 500,000 cells were acquired and scored using a FACSCalibur analyzer (Becton, Dickinson and Company). Data were processed using the Macintosh CELLQuest software program (Becton, Dickinson and Company). The instrument setup was optimized daily by analyzing the expression of peripheral blood lymphocytes labeled with anti-CD4 FITC/CD8 PE/CD3 PECy5/CD45 APC four-color combination. The same trained operator, who was blind to the clinical status of the patients, performed all the tests throughout the study.. r0 g! |1 S' q0 t1 f

Figure 1. Representative cytograms for the determination of CD34 , CD133  CD133 KDR , CD34 KDR , and CD133 Annexin V  cells obtained from a healthy control. After morphological gating to exclude granulocyte and cell debris (A), we gated CD34  (C) or CD133  (E) peripheral blood cells according to their respective isotype controls (B and D, respectively). Then, we examined the resulting population for expression of KDR (F and G). (H): Annexin V binding to CD133  cells. The resulting cell counts were as follows: 687 CD34  cells per 106 total events; 252 CD133  cells per 106 events; 14 CD133 KDR  cells per 106 events; 54 CD34 KDR  cells per 106 events; 22 CD133 Annexin V  cells per 106 events, representing 10.3% of total CD133  cells. Abbreviations: APC, allophycocyanin; FITC, fluorescein isothiocyanate; FSC-H, forward scatter; PE, phycoerythrin; SSC-H, side scatter.; C$ ^% j# U, G% `, T* u5 H# F4 p
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Detection of Progenitor Cells Apoptosis

Apoptosis of progenitor cells was detected by flow cytometry through the analysis of Annexin V binding to externalized phosphatidylserine on apoptotic cells using a commercially available Annexin V Apoptosis Detection kit (BD Pharmingen, San Diego, http://www.bdbiosciences.com/pharmingen) in accordance with the manufacturer¡¯s instructions, as previously reported . Briefly, cells were labeled with Annexin V-FITC, anti-human CD34 PE-Cy5, or anti-human CD133 APC (Miltenyi Biotech) and anti-human KDR PE mAbs (R&D Systems); resuspended in binding buffer; and then analyzed using the FACSCalibur Analyzer. Cell debris was excluded from analysis by appropriate forward light scatter threshold setting. At least 5 x 105 cells were analyzed in each condition. Four quadrants of the cytograms were set using negative controls. CD34 , CD133 , CD34 KDR , and CD133 KDR  cells were analyzed for Annexin V binding. Proportions of cells in each quadrant were expressed as the percentage of the total population (Fig. 1H).
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Statistical Analyses  r9 Y& A# Q9 `6 s# U- I
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Data are expressed as mean ¡À SEM. All results from flow cytometry analyses are reported as number of cells per 1,000,000 cytometric events. Comparisons between two or more groups were performed by unpaired Student¡¯s t test and analysis of variance, respectively. The 2 test was used for dichotomous variables. Statistical correlations between clinical data and cell counts were examined by univariate analysis using the linear regression. Statistical significance was accepted if the null hypothesis could be rejected at p  .05.

RESULTS

Patient Characteristics
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Patient characteristics are reported in Table 1. Significant differences between obstructive and restrictive patients reflected the diverse pathobiology of the two conditions. COPD patients were heavier smokers than RLD patients. Patients with RLD had the following diagnoses: six patients (40%) had a diffuse interstitial lung disease (five with idiopathic pulmonary fibrosis and one with sarcoidosis); two patients (13.3%) had fibrothorax due to tuberculosis; and seven patients (46.7%) had mechanical ventilatory impairment due to severe obesity (four patients), amyotrophic lateral sclerosis (one patient), or severe kyphoscoliosis (two patients). Patients with COPD had chronic bronchitis (eight patients) or pulmonary emphysema (seven patients).! M4 T% Y6 @; f% ^/ {) o! ]7 X8 \  X
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Table 1. Patient characteristics
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Levels of Circulating Progenitor Cells in Patients with COPD or RLD

In the whole study sample, range variations for progenitor cells were as follows: CD34  (42¨C2668 per 106 cells), CD133  (24¨C1737 per 106 cells), CD34 CD133  (19¨C1353 per 106 cells), CD34 KDR  (9¨C337 per 106 cells), CD34 CD133  (1¨C245 per 106 cells), and CD34 CD133 KDR  (0¨C121 per 106 cells).- b0 o* Q" ~8 C& C% d
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In general terms, patients with chronic systemic hypoxia were characterized by a marked, consensual, and significant reduction of all progenitor cell subtypes in peripheral blood compared with healthy control subjects (Fig. 2A). CD133 CD34 KDR  cells, which better correspond to the definition of EPCs, showed the largest difference (6.75 ¡À 1.24 vs. 19.38 ¡À 5.26;  = 65%; p = .01). Patients with respiratory failure due to an RLD showed the lowest number of all circulating progenitor cells. Moreover, RLD was characterized by a consistent reduction of all cell subtypes with respect to COPD, although statistical significance was reached only for CD34 , CD133 , and CD34 CD133  cells. To determine whether an increased cell death was involved in the decrease of circulating progenitors, Annexin V binding assay was performed. The apoptotic rate of CD133 KDR  cells was significantly higher in patients (49.18% ¡À 6.61%) versus controls (31.87% ¡À 9.09%), whereas apoptosis of CD34 KDR  progenitor cells was selectively increased in RLD patients (34.64% ¡À 13.4%) versus control subjects (18.0% ¡À 6.85%) and versus COPD patients (18.61% ¡À 3.96%) (Fig. 2B).

Figure 2. Reduction of circulating progenitor cells in patients with severe lung disease. (A): Comparisons of the levels of all progenitor cell subtypes in control subjects, in all patients with hypoxemia due to pulmonary disease, in patients with RLD, and in patients with COPD. Analysis of variance (ANOVA) p & U- p9 L  @$ L) W2 ?! A; P

Correlations Between Progenitor Cell Counts and Disease Severity

In the subgroup of patients with COPD, CD133 CD34 KDR  cells were correlated with arterial oxygen tension (r = ¨C.55; p = .03) and with the flux of oxygen therapy (r = .73; p = .002), whereas CD133 KDR  cells correlated to FEV1/FVC ratio (r = ¨C.52; p = .04). (Fig. 3). No differences were present between pulmonary emphysema and chronic bronchitis.5 C: C  L3 S( u# ~, z8 G

Figure 3. Significant linear correlations between clinical/instrumental data and progenitor cell counts in patients with chronic obstructive pulmonary disease. Abbreviation: pO2, arterial oxygen tension.$ D) v7 `- _0 o- q3 C2 Y
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In the subgroup of patients with RLD, CD133 CD34 KDR  cells were correlated with total lung capacity (r = .64; p = .008), residual volume (r = .64; p = .01), and carbon monoxide diffusion (r = .68; p = .005), red blood cell number (r = .70; p = .003), blood hemoglobin concentration (r = .90; p 8 B6 N' P$ [2 @( @8 T
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Figure 4. Significant linear correlations between laboratory/instrumental data and progenitor cell counts in patients with restrictive lung disease. Abbreviation: DLco, carbone monoxide diffusion.( w. R7 ?% s7 `7 O9 m' B# H

Effects of Smoking on Progenitor Cell Levels
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COPD patients with current smoking habits displayed a tendency to a further consistent reduction of all progenitors compared with nonsmokers, which was statistically significant for CD133 CD34 KDR  cells (4.67 ¡À 2.46 vs. 10.33 ¡À 1.78;  = 55%; p = .049), whereas the difference in CD133 KDR  cells was nearly significant (p = .07), and CD34 KDR  cells were not affected (Fig. 5A). The percentage of CD133 KDR  cells positive for Annexin V binding was higher in smokers than in nonsmokers (70.7% ¡À 5.8% vs. 30.5% ¡À 2.3%; p = .005) (Fig. 5B).1 A$ i( J) w8 m( f% t' p

Figure 5. Among patients with chronic obstructive pulmonary disease (COPD), progenitor cells from smokers displayed a consensual trend towards lower levels in comparison with nonsmokers, which was statistically significant for CD34 CD133 KDR  cells (A). Annexin V binding assay revealed that smoking is associated with increased apoptosis of CD133 KDR  compared with nonsmoking in COPD patients (B). Smoking patients with restrictive lung disease (RLD) showed a tendency to increased levels of circulating progenitor cells compared with nonsmokers, which was significant for CD133 , CD34 KDR , and CD133 KDR  cells (C). Consistently, apoptosis of progenitor cells was not increased in RLD smokers, as shown by Annexin V binding assay (D). *, p
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Among RLD patients, smokers tended to have higher levels of circulating progenitors than nonsmokers, which was statistically significant for CD133  (241.2 ¡À 61.5 vs. 131 ¡À 18.3; p = .03), CD34 KDR  (70.8 ¡À 15.1 vs. 35.5 ¡À 9; p = .03), and CD133 KDR  cells (24.4 ¡À 8.8 vs. 8 ¡À 1.5; p = .02) (Fig. 5C). These differences were not associated with increased apoptosis of any progenitor cell subtype (Fig. 5D).' ^5 W" ^* r$ x) i* t/ X; K5 C
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When comparison of groups was repeated excluding all smokers, patients continued to have significantly lower levels of all progenitor subtypes than controls, but the difference in CD34 CD133 KDR  cells between COPD patients and healthy subjects was not statistically significant.
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DISCUSSION

In this work, we demonstrate that circulating EPCs are reduced in patients with hypoxia due to severe lung disease. An emerging concept is that pulmonary endothelial dysfunction represents the earliest pathological alteration of the pulmonary vasculature .
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EPCs provide a pool of circulating cells that restore the damaged or dysfunctional endothelium . Therefore, the contribution of mature endothelial cells to CD34 KDR  cell count can be considered negligible.8 ]5 V9 _0 d+ N% l

We found that, irrespective of the disease pattern (obstructive or restrictive), circulating EPC levels were lower in hypoxic patients than in control subjects. This result may reflect the increased cardiovascular risk in these patients . On the contrary, when patients with COPD were considered, correlations of EPCs with indicators of bronchial obstruction and arterial oxygen tension indicated that more severe patterns were associated with higher numbers of progenitor cells, suggesting an intriguing compensatory effect of hypoxia on EPC mobilization. Consistently, intensity of oxygen therapy, reflecting severity of hypoxia, was directly correlated with progenitor cell count. This different behavior of progenitor cells in relation to disease severity explains in part why patients with RLD displayed a more profound EPC reduction than did COPD patients.' N1 t5 E# _+ {5 _! ?

Consistent with the major role played by smoke in the pathogenesis of COPD . Moreover, even if disease severity did not differ in smokers versus nonsmokers, patients with persistent smoking habits, despite a need for oxygen therapy, may display a milder disease pattern and a slower progression. Nonetheless, as apoptosis of progenitor cells was not affected by smoking in RLD patients, a different susceptibility to the effects of smoking on progenitor cells, as well as on airway remodeling, may characterize COPD versus RLD.
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Besides reduced bone marrow mobilization and shortened peripheral survival, we cannot exclude that also a small difference in age between patients and controls also contributed to the difference in progenitor cell counts, as an age-dependent EPC exhaustion has previously been reported .7 K( [- c9 A# ~

A number of data suggest the contribution of extrapulmonary progenitor cells to the development of vascular lesions characterizing PH . Therefore, it is clear how an exhausted EPC pool, as that which we demonstrate in patients with severe chronic lung disease, may contribute to disease progression and worsening.
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Alternatively, it is possible that EPC reduction in peripheral blood reflects localization of progenitor cells in the lungs. Studies performed on lung biopsy and bronchoalveolar lavage fluid of patients with obstructive and restrictive disease demonstrated either increased or decreased levels of one of the most potent stimuli for EPC mobilization and recruitment, vascular endothelial growth factor (VEGF) . To clarify this point, we are evaluating whether there is a relationship between the EPC and VEGF levels in the bronchoalveolar lavage of patients with different lung diseases.
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CONCLUSION
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Our study demonstrates that circulating EPCs are reduced in end-stage chronic lung disease, especially in patients with RLD, possibly suggesting defective mobilization and shortened peripheral survival of progenitor cells. Compensatory attempts to increase EPCs may rely on erythropoietin in RLD and on hypoxia in COPD. We also report a differential impact of smoking on EPC levels and apoptotic rate, depending upon the disease pattern.
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Animal models of PH have shown that EPC administration prevents anatomical lesions and improves animal survival . To better understand and take advantage of the therapeutic potential of EPC administration in humans with diffuse lung disease and hypoxia is a major challenge for researchers involved in the clinical management of these disorders.
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DISCLOSURES

The authors indicate no potential conflicts of interest.7 w& |) x# d" n1 Y5 Z6 K: p
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