Stem Cells http://www.peprotech.com/
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

First published online April 6, 2006
Stem Cells Vol. 24 No. 7 July 2006, pp. 1806 -1813
doi:10.1634/stemcells.2005-0440; www.StemCells.com
© 2006 AlphaMed Press

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
2005-0440v1
24/7/1806    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Reprints/Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Fadini, G. P.
Right arrow Articles by Agostini, C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Fadini, G. P.
Right arrow Articles by Agostini, C.

TRANSLATIONAL AND CLINICAL RESEARCH

Circulating Progenitor Cells Are Reduced in Patients with Severe Lung Disease

Gian Paolo Fadinia, Marco Schiavonb, Marcella Cantinic, Ilenia Baessod, Monica Faccod, Marta Miorind, Mauro Tassinatod, Saula Vigili de Kreutzenberga, Angelo Avogaroa, Carlo Agostinid

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, Italy;
cUnità Locale Socio-Sanitaria 17 Veneto Region (Italy), Social Pneumology Service, Italy

Key Words. Endothelial progenitor cells • Stem cells • Endothelial dysfunction • Pulmonary disease

Correspondence: Gian Paolo Fadini, M.D.,Dipartimento di Medicina Clinica e Sperimentale, Divisione di Malattie del Metabolismo, Università degli Studi di Padova, Via Giustiniani 2, 35128 Padova, Italy. Telephone: 0039-333-5682517 Fax: 0039-049-8754179 email: gianpaolofadini{at}hotmail.com

Received September 8, 2005; accepted for publication March 27, 2006.
First published online in STEM CELLS EXPRESS   April 6, 2006.


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
 Disclosures
 References
 
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.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Conclusion
 Disclosures
 References
 
In most diffuse obstructive and restrictive lung diseases, progressive pulmonary vascular remodeling and neovascularization may be observed [1–4]. The functional consequence is secondary pulmonary hypertension (PH), which may contribute to premature mortality and added morbidity [5, 6]. A variety of factors have been involved in the development of secondary PH, in both obstructive and restrictive lung diseases, including persistent pulmonary vasoconstriction due to hypoxia [7–9]. In particular, it is thought that hypoxia-induced endothelial dysfunction sets the stage for vascular remodeling processes [10–13], which are due, in part, to an imbalance in the production and release of endothelium-derived vasoactive molecules [14–16].

Endothelial progenitor cells (EPCs) have been found to take part in human neovascularization and maintenance of vascular integrity [17–19]. They are considered a subset of stem cells capable of differentiating into mature endothelial cells, and they can be defined on the basis of the expression of surface markers reflecting immaturity and endothelial commitment [20, 21]. Many events and mediators, including tissue ischemia and cytokines, are able to mobilize EPCs from the bone marrow [22], but it is not known whether chronic systemic hypoxia plays a role in the mobilization of EPCs. Recently, EPC reduction has been demonstrated to be related to endothelial dysfunction in the systemic circulation [23], and interestingly, elevated markers of endothelial dysfunction have been demonstrated in patients with PH [11, 24].

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
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Conclusion
 Disclosures
 References
 
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 Unità 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 [25], 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.

Diagnosis of RLD [26] 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.

Control subjects were free from clinical and instrumental evidence of pulmonary disease and were, on the average, matched with patients by age and sex.

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.

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 1–2 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 [27, 28]. 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°C 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–1C). 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–1G). 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.


Figure 1
View larger version (53K):
[in this window]
[in a new window]
  		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.

 
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 [29]. 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).

Statistical Analyses
Data are expressed as mean ± 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 {chi}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
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Conclusion
 Disclosures
 References
 
Patient Characteristics
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).


View this table:
[in this window]
[in a new window]
  		Table 1. Patient characteristics

 
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+ (42–2668 per 106 cells), CD133+ (24–1737 per 106 cells), CD34+CD133+ (19–1353 per 106 cells), CD34+KDR+ (9–337 per 106 cells), CD34+CD133+ (1–245 per 106 cells), and CD34+CD133+KDR+ (0–121 per 106 cells).

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 ± 1.24 vs. 19.38 ± 5.26; {Delta} = 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% ± 6.61%) versus controls (31.87% ± 9.09%), whereas apoptosis of CD34+KDR+ progenitor cells was selectively increased in RLD patients (34.64% ± 13.4%) versus control subjects (18.0% ± 6.85%) and versus COPD patients (18.61% ± 3.96%) (Fig. 2B).


Figure 2
View larger version (26K):
[in this window]
[in a new window]
  		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 < .05 for all cell types. (B): Percentages of progenitor cells positive for Annexin V binding in the same four groups. ANOVA p < .05 for CD34+KDR+ and CD133+KDR+ cells. *, p < .05 for t test comparing patients with controls. {ddagger}, p < .05 for t test comparing COPD with RLD patients. Abbreviations: COPD, chronic obstructive pulmonary disease; CTRL, control subjects; RLD, restrictive lung disease.

 
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 = –.55; p = .03) and with the flux of oxygen therapy (r = .73; p = .002), whereas CD133+KDR+ cells correlated to FEV1/FVC ratio (r = –.52; p = .04). (Fig. 3). No differences were present between pulmonary emphysema and chronic bronchitis.


Figure 3
View larger version (14K):
[in this window]
[in a new window]
  		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.

 
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 < .001), and hematocrit value (r = .91; p < .001) (Fig. 4), whereas no significant correlation was present with white blood cell count (not shown). No differences were present between parenchymal and mechanical RLD.


Figure 4
View larger version (19K):
[in this window]
[in a new window]
  		Figure 4. Significant linear correlations between laboratory/instrumental data and progenitor cell counts in patients with restrictive lung disease. Abbreviation: DLco, carbone monoxide diffusion.

 
Effects of Smoking on Progenitor Cell Levels
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 ± 2.46 vs. 10.33 ± 1.78; {Delta} = 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% ± 5.8% vs. 30.5% ± 2.3%; p = .005) (Fig. 5B).


Figure 5
View larger version (18K):
[in this window]
[in a new window]
  		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 < .05 for t test.

 
Among RLD patients, smokers tended to have higher levels of circulating progenitors than nonsmokers, which was statistically significant for CD133+ (241.2 ± 61.5 vs. 131 ± 18.3; p = .03), CD34+KDR+ (70.8 ± 15.1 vs. 35.5 ± 9; p = .03), and CD133+KDR+ cells (24.4 ± 8.8 vs. 8 ± 1.5; p = .02) (Fig. 5C). These differences were not associated with increased apoptosis of any progenitor cell subtype (Fig. 5D).

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.


   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Conclusion
 Disclosures
 References
 
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 [10, 11], as peripheral endothelial dysfunction is for the atherosclerotic process in the systemic vasculature [30]. The term endothelial dysfunction refers to a subclinical condition in which the endothelium loses its normal homeostatic role in maintaining vessel tone, adhesion, and reactivity, transforming into a vasoconstricting and prothrombotic state. Dysfunction of endothelial cells, which can be induced in experimental models of hypoxia [31], has been demonstrated in patients with chronic obstructive pulmonary disease or primary PH [11, 24].

EPCs provide a pool of circulating cells that restore the damaged or dysfunctional endothelium [19, 20]. Currently, there is no consensus on the definition of EPCs and on methods for their identification. Flow cytometry has been extensively used for this purpose and validated in several studies [27, 28]. Although acquisition of 500,000 events is considered adequate for EPC enumeration by flow cytometry [32], larger analyses may be required given the low number of positive cells in peripheral blood. We chose the triple detection of CD133, CD34, and KDR as the most selective and restrictive criteria for EPC enumeration in peripheral blood. Adding further markers would lead to a critical reduction in the number of positive cells, approaching the sensitivity threshold of the instrument. On the other hand, our data suggest that KDR is the least widely expressed surface antigen of the three and also that the CD34+KDR+ cells and CD133+KDR+ cells may be considered endothelial progenitors. CD34 and KDR may also identify mature endothelial cells, but it has been reported that cells positive for CD146, the specific marker for those cells, are extremely rare in peripheral blood (1–10 cells per ml), and a very small fraction (1%–2%) express CD34 [33]. Therefore, the contribution of mature endothelial cells to CD34+KDR+ cell count can be considered negligible.

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 [34]. All the subtypes of progenitor cells that were evaluated showed a similar trend of reduction, and a consensual further decrease was seen in patients with RLD. It is noteworthy that these patients have more severe alterations in the pulmonary vessels and a greater tendency to develop PH [2]. Even though the patient groups were somewhat heterogeneous, the subdivision of patients into RLD and COPD revealed relationships between progenitor cells and pulmonary function that were consistent with the differential pulmonary pathophysiology of these disorders. Among patients with RLD, EPC decrease was related to disease severity, defined as reduction in pulmonary volumes and impairment in alveolo-arterial diffusion. A similar correlation with carbon monoxide diffusion has been reported previously by Del Papa et al. in patients with systemic sclerosis [35]. In our study, EPC count was also strongly correlated to red blood cell number, hemoglobin level, and hematocrit value. Unfortunately, we were not able to determine erythropoietin levels in our patients, but our data suggest that a compensatory increase in erythropoietin may influence EPC mobilization [36, 37]. 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.

Consistent with the major role played by smoke in the pathogenesis of COPD [38], we have shown that smokers had lower levels of circulating triple-positive EPCs than did nonsmokers among patients with COPD. Indeed, EPC reduction in the COPD group may be attributed mainly to smoking patients, because when smokers were excluded from the analyses, difference in CD34+CD133+KDR+ cells was no longer significant. Apoptosis of progenitor cells seemed to be increased in smokers, since CD133+KDR+Annexin V+ cell numbers were higher than in nonsmokers. The effect of smoke on EPC reduction has been already reported, being related to a smoke-induced endothelial dysfunction [27]. Demonstrating that apoptosis of EPCs is increased in smokers, we provide a plausible mechanistic explanation for the decreased EPCs levels in COPD. Surprisingly, smoking was associated with increased progenitor cells in RLD patients. This unexpected result is consistent with previous reports indicating that smoking may have favorable and protective effects on certain interstitial RLDs [39]. 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.

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 [40].

A number of data suggest the contribution of extrapulmonary progenitor cells to the development of vascular lesions characterizing PH [41–47]. One working hypothesis is that hypoxia stimulates EPC mobilization from the bone marrow and localization at the damaged pulmonary vascular endothelium, as tissue ischemia does. From a functional point of view, there are in vitro data indicating that EPCs cultured under low oxygen tension display increased efficiency in proliferation and differentiation [48]. Our results are in contrast with this in vitro finding. To explain this apparent paradox, we may postulate that low levels of circulating EPCs reflect a reduced production or mobilization from bone marrow. It should be noted that the majority of in vitro studies have been performed on endothelial cells isolated from normal subjects, whereas we have shown that the apoptotic rate of EPCs is increased in patients with diffuse lung disease, likely as a reflection of disease chronicity. Otherwise, it is possible that the continuous hypoxic stimulation of the bone marrow led to an exhaustion of the precursor pool. Studies are in progress in our laboratory to evaluate this hypothesis. Recent data suggest that EPCs are mobilized during lung injury and that a poor progenitor cell mobilization is associated with persistent fibrotic changes after pneumonia [49, 50]. Moreover, myelosuppression was found to increase susceptibility to bleomycin (Blenoxane; Bristol-Myers Squibb; Princeton, NJ)-mediated lung injury in mice, which was instead prevented by administration of stem cells [51]. 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.

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) [52, 53]. Recently, an increased number of vascular progenitors have been found in pulmonary arteries of patients with COPD [54]. 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.


   CONCLUSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
Disclosures
 References
 
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.

Animal models of PH have shown that EPC administration prevents anatomical lesions and improves animal survival [55–57]. 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.


   DISCLOSURES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
 Disclosures
References
 
The authors indicate no potential conflicts of interest.


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
 Disclosures
 References
 

  1. Hopkins N, McLoughlin P. The structural basis of pulmonary hypertension in chronic lung disease: Remodelling, rarefaction or angiogenesis? J Anat 2002;201:335–348.[CrossRef][Medline]

  2. Dorfmuller P, Humbert M, Capron F et al. Pathology and aspects of pathogenesis in pulmonary arterial hypertension. Sarcoidosis Vasc Diffuse Lung Dis 2003;20:9–19.[Medline]

  3. Keane MP. Angiogenesis and pulmonary fibrosis: Feast or famine? Am J Respir Crit Care Med 2004;170:207–209.[Free Full Text]

  4. Renzoni EA. Neovascularization in idiopathic pulmonary fibrosis: Too much or too little? Am J Respir Crit Care Med 2004;169:1179–1180.[Free Full Text]

  5. Chaouat A, Bugnet AS, Kadaoui N et al. Severe pulmonary hypertension and chronic obstructive pulmonary disease. Am J Respir Crit Care Med 2005;172:189–194.[Abstract/Free Full Text]

  6. Barbera JA, Peinado VI, Santos S. Pulmonary hypertension in chronic obstructive pulmonary disease. Eur Respir J 2003;21:892–905.[Abstract/Free Full Text]

  7. Howell K, Preston RJ, McLoughlin P. Chronic hypoxia causes angiogenesis in addition to remodelling in the adult rat pulmonary circulation. J Physiol 2003;547:133–145.[Abstract/Free Full Text]

  8. Molthen RC, Karau KL, Dawson CA. Quantitative models of the rat pulmonary arterial tree morphometry applied to hypoxia-induced arterial remodeling. J Appl Physiol 2004;97:2372–2384.[Abstract/Free Full Text]

  9. Meyrick B, Reid L. The effect of continued hypoxia on rat pulmonary arterial circulation. An ultrastructural study. Lab Invest 1978;38:188–200.[Medline]

  10. Budhiraja R, Tuder RM, Hassoun PM. Endothelial dysfunction in pulmonary hypertension. Circulation 2004;109:159–165.[Free Full Text]

  11. Cella G, Bellotto F, Tona F et al. Plasma markers of endothelial dysfunction in pulmonary hypertension. Chest 2001;120:1226–1230.[CrossRef][Medline]

  12. Taraseviciene-Stewart L, Kasahara Y, Alger L et al. Inhibition of the VEGF receptor 2 combined with chronic hypoxia causes cell death-dependent pulmonary endothelial cell proliferation and severe pulmonary hypertension. FASEB J 2001;15:427–438.[Abstract/Free Full Text]

  13. Murata T, Hori M, Sakamoto K et al. Dexamethasone blocks hypoxia-induced endothelial dysfunction in organ-cultured pulmonary arteries. Am J Respir Crit Care Med 2004;170:647–655.[Abstract/Free Full Text]

  14. Humbert M, Morrell NW, Archer SL et al. Cellular and molecular pathobiology of pulmonary arterial hypertension. J Am Coll Cardiol 2004;43:13S–24S.[Abstract/Free Full Text]

  15. Farber HW, Loscalzo J. Pulmonary arterial hypertension. N Engl J Med 2004;351:1655–1665.[Free Full Text]

  16. Malmstrom RE, Tornberg DC, Settergren G et al. Endogenous nitric oxide release by vasoactive drugs monitored in exhaled air. Am J Respir Crit Care Med 2003;168:114–120.[Abstract/Free Full Text]

  17. Asahara T, Murohara T, Sullivan A et al. Isolation of putative progenitor endothelial cells for angiogenesis. Science 1997;275:964–967.[Abstract/Free Full Text]

  18. Crosby JR, Kaminski WE, Schatteman G et al. Endothelial cells of hematopoietic origin make a significant contribution to adult blood vessel formation. Circ Res 2000;87:728–730.[Abstract/Free Full Text]

  19. Cho HJ, Kim HS, Lee MM et al. Mobilized endothelial progenitor cells by granulocyte-macrophage colony stimulating factor accelerate reendothelization and reduce vascular inflammation after intravascular radiation. Circulation 2003;108:2918–2925.[Abstract/Free Full Text]

  20. Urbich C, Dimmeler S. Endothelial progenitor cells: Characterization and role in vascular biology. Circ Res 2004;95:343–353.[Abstract/Free Full Text]

  21. Peichev M, Naiyer AJ, Pereira D et al. Expression of VEGFR-2 and AC133 by circulating human CD34(+) cells identify a population of functional endothelial precursors. Blood 2000;95:952–958.[Abstract/Free Full Text]

  22. Takahashi T, Kalka C, Masuda H et al. Ischemia- and cytokine-induced mobilization of bone marrow-derived endothelial progenitor cells for neovascularization. Nat Med 1999;5:434–438.[CrossRef][Medline]

  23. Hill JM, Zalos G, Halcox JP et al. Circulating endothelial progenitor cells, vascular function, and cardiovascular risk. N Engl J Med 2003;348:593–600.[Abstract/Free Full Text]

  24. Cella G, Sbarai A, Mazzaro G et al. Plasma markers of endothelial dysfunction in chronic obstructive pulmonary disease. Clin Appl Thromb Hemost 2001;7:205–208.[Abstract/Free Full Text]

  25. Ferguson GT. Recommendations for the management of COPD. Chest 2000;117:23S–8S.[Medline]

  26. American Thoracic Society; European Respiratory Society. American Thoracic Society/European Respiratory Society International multidisciplinary consensus classification of the idiopathic interstitial pneumonias. Am J Respir Crit Care Med 2002;165:277–304.[Free Full Text]

  27. Kondo T, Hayashi M, Takeshida K et al. Smoking cessation rapidly increases circulating progenitor cells in peripheral blood in chronic smokers. Arterioscler Thromb Vasc Biol 2004;24:1–6.[Free Full Text]

  28. Lambiase PD, Edwards RJ, Anthopoulos P et al. Circulating humoral factors and endothelial progenitor cells in patients with differing coronary collateral support. Circulation 2004;109:2993–2999.[Abstract/Free Full Text]

  29. Kravtsov VD, Daniel TO, Koury MJ. Comparative analysis of different methodological approaches to the in vitro study of drug-induced apoptosis. Am J Pathol 1999;155:1327–1339.[Abstract/Free Full Text]

  30. Landmesser U, Hornig B, Drexler H. Endothelial function: A critical determinant in atherosclerosis? Circulation 2004;109:1127–1133.[Abstract/Free Full Text]

  31. Peinado VI, Santos S, Ramirez J et al. Response to hypoxia of pulmonary arteries in chronic obstructive pulmonary disease: An in vitro study. Eur Respir J 2002;20:332–338.[Abstract/Free Full Text]

  32. Khan SS, Solomon MA, McCoy JP. Detection of circulating endothelial cells and endothelial progenitor cells by flow cytometry. Cytometry B Clin Cytom 2005;64:1–8.[Medline]

  33. Lee KW, Lip GYH, Tayebjee M et al. Circulating endothelial cells, von Willebrand factor, interleukin-6, and prognosis in patients with acute coronary syndromes. Blood 2005;105:526–532.[Abstract/Free Full Text]

  34. Hunninghake DB. Cardiovascular disease in chronic obstructive pulmonary disease. Proc Am Thorac Soc 2005;2: 44–49.

  35. Del Papa N, Colombo G, Fracchiolla N et al. Circulating endothelial cells as a marker of ongoing vascular disease in systemic sclerosis. Arthritis Rheum 2004;50:1296–1304.[CrossRef][Medline]

  36. Bahlmann FH, De Groot K, Spandau JM et al. Erythropoietin regulates endothelial progenitor cells. Blood 2004;103:921–926.[Abstract/Free Full Text]

  37. Heeschen C, Aicher A, Lehmann R et al. Erythropoietin is a potent physiologic stimulus for endothelial progenitor cell mobilization. Blood 2003;102:1340–1346.[Abstract/Free Full Text]

  38. Willemse BW, Ten Hacken NT, Rutgers B et al. Association of current smoking with airway inflammation in chronic obstructive pulmonary disease and asymptomatic smokers. Respir Res 2005;6:38.[Medline]

  39. Selman M. The spectrum of smoking-related interstitial lung disorders. Chest 2003;124:1184–1187.

  40. Heiss C, Keymel S, Niesler U et al. Impaired progenitor cell activity in age-related endothelial dysfunction. J Am Coll Cardiol 2005;45:1441–1448.[Abstract/Free Full Text]

  41. Epperly MW, Guo H, Gretton JE et al. Bone marrow origin of myofibroblasts in irradiation pulmonary fibrosis. Am J Respir Cell Mol Biol 2003;29:213–224.[Abstract/Free Full Text]

  42. Suratt BT, Cool CD, Serls AE et al. Human pulmonary chimerism after hematopoietic stem cell transplantation. Am J Respir Crit Care Med 2003;168:318–322.[Abstract/Free Full Text]

  43. Abe S, Boyer C, Liu X et al. Cells derived from the circulation contribute to the repair of lung injury. Am J Respir Crit Care Med 2004;170:1158–1163.[Abstract/Free Full Text]

  44. Ebina M, Shimizukawa M, Shibata N et al. Heterogeneous increase in CD34-positive alveolar capillaries in idiopathic pulmonary fibrosis. Am J Respir Crit Care Med 2004;169:1203–1208.[Abstract/Free Full Text]

  45. Neuringer IP, Randell SH. Stem cells and repair of lung injuries. Respir Res 2004;20:5–6.[CrossRef]

  46. Hashimoto N, Jin H, Liu T et al. Bone marrow-derived progenitor cells in pulmonary fibrosis. J Clin Invest 2004;113:243–252.[CrossRef][Medline]

  47. Phillips RJ, Burdick MD, Hong K et al. Circulating fibrocytes traffic to the lungs in response to CXCL12 and mediate fibrosis. J Clin Invest 2004;114:438–446.[CrossRef][Medline]

  48. Akita T, Murohara T, Ikeda H et al. Hypoxic preconditioning augments efficacy of human endothelial progenitor cells for therapeutic neovascularization. Lab Invest 2003;83:65–73.[Medline]

  49. Yamada M, Kubo H, Ishizawa K et al. Increased circulating endothelial progenitor cells in patients with bacterial pneumonia: Evidence than bone marrow derived cells contribute to lung repair. Thorax 2005;60:410–413.[Abstract/Free Full Text]

  50. Burnham EL, Taylor WR, Quyyumi AA et al. Increased circulating endothelial progenitor cells are associated with survival in acute lung injury. Am J Respir Crit Care Med 2005;172:854–860.[Abstract/Free Full Text]

  51. Rojas M, Xu J, Woods CR et al. Bone marrow-derived mesenchymal stem cells in repair of the injured lung. Am J Respir Cell Mol Biol 2005;33:145–152.[Abstract/Free Full Text]

  52. Santos S, Peinado VI, Ramirez J et al. Enhanced expression of vascular endothelial growth factor in pulmonary arteries of smokers and patients with moderate chronic obstructive pulmonary disease. Am J Respir Crit Care Med 2003;167:1250–1256.[Abstract/Free Full Text]

  53. Koyama S, Sato E, Haniuda M et al. Decreased level of vascular endothelial growth factor in bronchoalveolar lavage fluid of normal smokers and patients with pulmonary fibrosis. Am J Respir Crit Care Med 2002;166:382–385.[Abstract/Free Full Text]

  54. Peinado V, Ramirez J, Roca J et al. Identification of vascular progenitor cells in pulmonary arteries of patients with chronic obstructive pulmonary disease. Am J Respir Cell Mol Biol 2006;34:257–263.[Abstract/Free Full Text]

  55. Nagaya N, Kangawa K, Kanda M et al. Hybrid cell-gene therapy for pulmonary hypertension based on phagocytosing action of endothelial progenitor cells. Circulation 2003;108:889–895.[Abstract/Free Full Text]

  56. Takahashi M, Nakamura T, Toba T et al. Transplantation of endothelial progenitor cells into the lung to alleviate pulmonary hypertension in dogs. Tissue Eng 2004;10:771–779.[CrossRef][Medline]

  57. Zhao YD, Courtman DW, Deng Y et al. Rescue of monocrotaline-induced pulmonary arterial hypertension using bone marrow-derived endothelial-like progenitor cells: Efficacy of combined cell and eNOS gene therapy in established disease. Circ Res 2005;96:442–450.[Abstract/Free Full Text]




This article has been cited by other articles:


Home page
 Proc Am Thorac SocHome page
D. J. Weiss, J. K. Kolls, L. A. Ortiz, A. Panoskaltsis-Mortari, and D. J. Prockop
Stem Cells and Cell Therapies in Lung Biology and Lung Diseases
Proceedings of the ATS, July 15, 2008; 5(5): 637 - 667.
[Full Text] [PDF]

Home page
 Ann Rheum DisHome page
G Coppolino, S Campo, D Bolignano, A Sturiale, M S Giacobbe, S Loddo, and M Buemi
Effect of immunoglobulin treatment on endothelial progenitor cells in systemic lupus erythematosus
Ann Rheum Dis, July 1, 2008; 67(7): 1047 - 1048.
[Full Text] [PDF]

Home page
 HeartHome page
W Wojakowski, M Kucia, M Kazmierski, M Z Ratajczak, and M Tendera
Circulating progenitor cells in stable coronary heart disease and acute coronary syndromes: relevant reparatory mechanism?
Heart, January 1, 2008; 94(1): 27 - 33.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Respir. Crit. Care Med.Home page
G. P. Fadini, M. Schiavon, F. Rea, A. Avogaro, and C. Agostini
Depletion of Endothelial Progenitor Cells May Link Pulmonary Fibrosis and Pulmonary Hypertension
Am. J. Respir. Crit. Care Med., October 1, 2007; 176(7): 724 - 725.
[Full Text] [PDF]

Home page
 Am. J. Respir. Crit. Care Med.Home page
B. Thebaud and S. H. Abman
Bronchopulmonary Dysplasia: Where Have All the Vessels Gone? Roles of Angiogenic Growth Factors in Chronic Lung Disease
Am. J. Respir. Crit. Care Med., May 15, 2007; 175(10): 978 - 985.
[Abstract] [Full Text] [PDF]

Home page
 Diabetes CareHome page
G. P. Fadini, S. Sartore, C. Agostini, and A. Avogaro
Significance of Endothelial Progenitor Cells in Subjects With Diabetes
Diabetes Care, May 1, 2007; 30(5): 1305 - 1313.
[Full Text] [PDF]

Home page
 Am. J. Physiol. Lung Cell. Mol. Physiol.Home page
V. Balasubramaniam, C. F. Mervis, A. M. Maxey, N. E. Markham, and S. H. Abman
Hyperoxia reduces bone marrow, circulating, and lung endothelial progenitor cells in the developing lung: implications for the pathogenesis of bronchopulmonary dysplasia
Am J Physiol Lung Cell Mol Physiol, May 1, 2007; 292(5): L1073 - L1084.
[Abstract] [Full Text] [PDF]

Home page
 J Am Coll CardiolHome page
A. Avogaro and G. P. Fadini
The Janus Face of Nicotinic Angiogenesis
J. Am. Coll. Cardiol., December 19, 2006; 48(12): 2561 - 2563.
[Full Text] [PDF]

Home page
 Proc Am Thorac SocHome page
S. H. Randell
Airway Epithelial Stem Cells and the Pathophysiology of Chronic Obstructive Pulmonary Disease
Proceedings of the ATS, November 1, 2006; 3(8): 718 - 725.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Respir. Cell Mol. Bio.Home page
G. P. Fadini, A. Avogaro, and C. Agostini
Pathophysiology of circulating progenitor cells in pulmonary disease and parallels with cardiovascular disease.
Am. J. Respir. Cell Mol. Biol., September 1, 2006; 35(3): 403 - 404.
[Full Text] [PDF]

Home page
 Arterioscler. Thromb. Vasc. Bio.Home page
G. P. Fadini, S. Sartore, M. Albiero, I. Baesso, E. Murphy, M. Menegolo, F. Grego, S. Vigili de Kreutzenberg, A. Tiengo, C. Agostini, et al.
Number and Function of Endothelial Progenitor Cells as a Marker of Severity for Diabetic Vasculopathy
Arterioscler. Thromb. Vasc. Biol., September 1, 2006; 26(9): 2140 - 2146.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
2005-0440v1
24/7/1806    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Reprints/Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Fadini, G. P.
Right arrow Articles by Agostini, C.
Right arrow Search for Related Content
PubMed