Published ahead of print on October 20, 2005, doi:10.1165/rcmb.2005-0255OC
© 2006 American Thoracic Society DOI: 10.1165/rcmb.2005-0255OC Identification of Vascular Progenitor Cells in Pulmonary Arteries of Patients with Chronic Obstructive Pulmonary DiseaseDepartments of Pulmonary Medicine and Pathology, Hospital Clinic, Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Universitat de Barcelona, Barcelona, Spain Correspondence and requests for reprints should be addressed to Dr. Joan A. Barberà, Servei de Pneumologia, Hospital Clínic, Villarroel, 170. 08036 Barcelona, Spain. E-mail: jbarbera{at}clinic.ub.es
Progenitor cells of bone marrow origin migrate to injured vessels, where they may contribute to endothelial maintenance and vessel remodeling through vascular endothelial growth factor (VEGF)-related signals. To what extent progenitor cells may play a role in vascular changes occurring in patients with chronic obstructive pulmonary disease (COPD) remains undetermined. In this study we sought to identify vascular progenitor cells in pulmonary arteries of patients with COPD and to investigate whether the presence of these cells could be related to changes in endothelial function or the expression of VEGF. Pulmonary arteries of nine patients with COPD and six control subjects were studied. Scanning electron microscopy demonstrated areas of denuded endothelium in the arteries of patients with COPD. Vascular progenitor cells were identified by immunohistochemistry and immunogold using antibodies against AC133, CD34, and CD45. AC133+ cells were localized in the endothelial surface, close to denuded areas. The number of AC133+ and CD45+ cells in pulmonary arteries was greater in patients with COPD than in control subjects. The number of AC133+ cells correlated with the response of pulmonary artery rings to hypoxic stimulus. AC133+ and CD45+ cells were also identified in the intimal layer. The wall thickness correlated with the number of progenitor cells in the intima and with VEGF and VEGF receptor-2 mRNA expression. We conclude that patients with COPD show an increased number of bone marrow–derived progenitor cells in pulmonary arteries. These cells seem to contribute to ongoing endothelial repair, but they might also be involved in the pathogenesis of pulmonary vascular remodeling.
Key Words: cigarette smoking • endothelium • pulmonary hypertension • vascular remodeling
Chronic obstructive pulmonary disease (COPD) is frequently associated with abnormalities in the pulmonary circulation. These abnormalities lead to pulmonary hypertension and right ventricular failure, complications that entail greater mortality risk and worse clinical evolution (1). Pulmonary hypertension in COPD is produced by alterations in pulmonary vessel structure (intimal hyperplasia of muscular arteries and muscularization of small arterioles). Intimal hyperplasia is produced by the proliferation of poorly differentiated smooth muscle cells (SMCs) and the deposition of collagen and elastic fibers (2). To what extent SMCs proliferating in the intima exert any physiologic role remains undetermined, although intimal hyperplasia is usually associated with endothelial dysfunction (3). Because endothelium plays a key role in regulating cell growth in the vessel wall, it has been hypothesized that endothelial dysfunction might be an initiating event that promotes vessel remodeling and pulmonary hypertension in COPD (1). The pathobiology of pulmonary vascular remodeling in COPD is not fully understood. A potential source of SMCs proliferating in the intima could be the adjacent medial layer. According to this hypothesis, upon endothelial injury, SMCs might dedifferentiate, migrate to the intima, proliferate, and synthesize extracellular matrix. However, few studies have documented SMCs migrating across the internal elastic lamina from the media into the intima (4, 5). Furthermore, partial or complete muscularization develops in small arterioles that in normal conditions lack of muscular cells. Asahara and colleagues (6) demonstrated that circulating bone marrow–derived progenitor cells have the capability to differentiate in vitro into endothelial cells. Upon vascular injury, bone marrow cells give rise to vascular progenitor cells (VPCs) that have the capacity to mobilize to sites of vascular lesion and differentiate into endothelial cells, facilitating vascular repair (7). However, bone marrow–derived VPCs may also contribute to vascular lesion formation by inducing SMC proliferation and neointimal formation at sites of vascular injury (8–11). Accordingly, VPCs might exert opposite effects on injured vessels (i.e., repair and impairment). Cigarette smoking is a well established risk factor for vascular impairment in systemic and pulmonary vessels. The products of cigarette smoke may also damage the pulmonary endothelium and initiate the sequence of events evolving to pulmonary hypertension in COPD (12). We hypothesized that in pulmonary vessels of patients with COPD that are damaged by cigarette smoke products, VPCs might contribute to endothelial repair but also to vessel remodeling. The potential contribution of VPCs in the pathobiology of pulmonary vascular abnormalities associated with COPD has not been evaluated. The present study was addressed to identify and characterize VPCs in pulmonary arteries of patients with COPD and to investigate whether the presence of these cells is related to endothelial function or the expression of angiogenic factors. The present study demonstrates the presence of VPCs in the endothelial surface and the intimal space of pulmonary arteries of patients with COPD. The number of progenitor cells was associated with the response to hypoxic stimulus but also with the enlargement of the arterial wall. These findings suggest that VPCs might be involved in the mechanisms of pulmonary vessel repair and remodeling in COPD. Preliminary results of the study have been previously reported in the form of abstracts (13, 14).
Subjects Pulmonary arteries dissected from lung specimens of 15 patients (12 men and 3 women) who underwent lung resection for localized carcinoma were studied. All patients underwent pulmonary function testing (forced spirometry, body plethysmography, carbon monoxide diffusing capacity, and arterial blood gas analysis) the days before surgery. The study was approved by the Committee on Human Research of our institution. None of the patients had clinical evidence of pulmonary hypertension. Patients were divided into two groups: (1) a control group (n = 6; four smokers and two nonsmokers) with normal pulmonary function and (2) a COPD group (n = 9; all smokers) with mild to moderate airflow obstruction (Table 1).
Tissue Preparation After lung resection, a pulmonary artery segment  3 cm long with an external diameter of 2 mm, corresponding to the fourth or fifth division, was identified and carefully dissected. Segments were cleaned of surrounding tissue under microscope and cut into several rings 3 mm long. Four rings were fixed in 4% paraformaldehyde buffered in PBS at pH = 7.4, cryo-embedded in ornithine carbamyl transferase (OCT), and frozen at –80°C for morphologic and immunohistochemical analysis. Four serial rings were selected for pharmacologic studies in the organ bath. The remaining rings were preserved in RNA later solution (Ambion Inc., Austin, TX) and frozen at –20°C for gene expression analysis. In 11 cases, additional rings were processed for endothelial surface harvesting. Briefly, pulmonary artery segments were longitudinally cut, extended on a rubber surface, secured with histologic needles, and fixed in PBS-4% formaldehyde overnight. After fixation, the artery was extended on a slide treated with poly-L-lysine with the luminal side in contact with the glass. After dehydration at room temperature, the tissue was slowly picked up by one corner, and the media layer was carefully detached from the endothelium, which remained firmly adhered to the slide. The integrity of endothelial surface was checked by immunofluorescence using antibodies against vimentin and von Willebrand factor.
Assessment of Vascular Reactivity
Morphometric Analyses
Immunohistochemistry
Immunofluorescence
Scanning Electron Microscopy Studies Immunogold studies were performed in seven patients. After fixation, pulmonary artery segments were incubated overnight with a primary antibody against AC133 or CD34, another vascular progenitor cell marker (17). Primary antibody was revealed using a secondary antibody labeled with 40-nm gold particles (British Biocell International, Stanstead, UK). Then vessels were dehydrated, dried by the critical point method, and carbon coated (model 12E6; Edwards, Crawley, UK). Observations were made in a scanning electron microscope (model JSM-850; Jeol) at acceleration voltages of 13–15 kV.
RT-PCR
Data Analysis
Patients with COPD were heavy smokers (mean cumulative cigarette consumption, 49 ± 20 pack-years) and showed mild to moderate airflow obstruction and arterial hypoxemia (Table 1). Pulmonary artery rings from the COPD group exhibited a moderate degree of endothelial dysfunction, as shown by lower relaxation to histamine as compared with controls (Table 2), although differences did not reach statistical significance (P = 0.1). The response to hypoxic stimulus did not differ between groups. Morphometric analysis of pulmonary arteries revealed thicker intimas in patients with COPD (Table 2).
Endothelial Lesions in Pulmonary Arteries The endothelial surface of pulmonary arteries was examined by scanning electronic microscopy. In nonsmokers of the control group, the endothelial surface tended to be smooth (Figure 1A), occasionally with small cytoplasmic structures (similar to microvilli) protruding toward the lumen. By contrast, in patients with COPD, detachments between endothelial cells were frequently observed (Figures 1B and 1C), occasionally with circulating cells adhered to them (Figure 1D) and large areas of denudation (Figure 1E). These changes were also seen in smokers with normal lung function. In pulmonary arteries with macroscopically thick walls, the endothelial surface was irregular, with numerous endothelial cells detached from subendothelial tissue (Figure 1F).
Identification of Vascular Progenitor Cells Immunogold studies revealed the presence of the vascular progenitor cell marker AC133 (19) on cells adhered to the endothelial surface, frequently close to denuded areas (Figures 2A and 2B). These cells were small ( 7 µm) compared with other cells adhered to endothelium and showed large microvillous projections. Cells with similar morphology showed immunoreactivity to CD34 antigen (Figures 2C and 2D).
Immunofluorescence studies of the endothelium also revealed the presence of small mononuclear cells (
The presence of AC133 and CD45+ cells in the arterial wall was evaluated by immunofluorescence in transverse sections of pulmonary arteries. Cells expressing the AC133 antigen were identified in the endothelium and in the intima. In some instances, they were also identified in the media (Figure 4B).
Number of AC133+ and CD45+ Cells in Pulmonary Artery Wall The number of cells showing positive immunoreactivity to AC133 and CD45 antibodies adhered to the endothelium or in the intima was assessed in serial transverse sections of pulmonary arteries from all patients (Figures 4C and 4D). The number of AC133+ cells attached to the endothelium or infiltrating the intima was greater in patients with COPD than in control subjects (Table 2 and Figure 4E). The number of AC133+ cells in the endothelium or in the intima was inversely related to FEV1 (r = –0.63 and r = –0.70, respectively; P < 0.01) and directly related to the wall thickness (r = 0.58 and r = 0.56, respectively; P < 0.05) (Figure 5). Furthermore, the number of AC133+ cells in the endothelium and in the intima correlated with the magnitude of the response to hypoxia (r = 0.61 and r = 0.76, respectively; P < 0.05 and P < 0.01, respectively) (Figure 5). Similar correlations were observed with the number of CD45+ cells, confirming the results of AC133 (data not shown).
VEGF and VEGFR2 mRNA Expression in Pulmonary Arteries We examined semiquantitatively in homogenates of pulmonary artery segments the mRNA expression of VEGF and its receptor VEGFR2 as potential molecules implicated in vascular remodeling and in the differentiation of the cellular components of pulmonary arteries. There were no differences in the expression of VEGF and VEGFR2 mRNA between control subjects and patients with COPD (Table 2). Nevertheless, VEGF and VEGFR2 mRNA expression were related to wall thickness (Figure 6), and VEGFR2 mRNA expression was inversely related to the endothelium-dependent vascular relaxation (r = –0.70, P < 0.01). Moreover, there was an inverse correlation between the density of AC133+ cell infiltrating the intima and VEGF mRNA expression (Figure 6).
In the present study, we sought to identify VPCs in pulmonary arteries of patients with COPD because they might contribute to endothelial repair and vessel remodeling. We initially screened for endothelial lesions because they could be potential sites for circulating vascular progenitor cell homing. Evaluation of the endothelial surface revealed areas of denuded endothelium in pulmonary arteries of patients with COPD (Figure 1). We identified cells showing positive immunoreaction to the progenitor cell marker AC133 antigen (16, 19) adhered to the endothelial surface in the vicinity of denuded areas (Figure 2). The number of AC133+ cells adhered to the endothelial surface of pulmonary arteries was greater in patients with COPD than in control subjects and correlated with the severity of airflow obstruction (Figures 4 and 6). The AC133 antigen is expressed by poorly differentiated circulating bone marrow–derived cells that have the potential to differentiate into mature endothelial cells (16, 19). This cellular marker, in combination with CD34, has been frequently used to identify progenitor endothelial cells in peripheral blood (6, 16, 17, 20). Our study demonstrates an increased number of AC133+ cells in pulmonary arteries of patients with COPD. Positive cells were not confined to the endothelial surface; they were also identified in the vessel wall. The number of AC133+ cells identified in the endothelium showed an inverse relationship with FEV1 (Figure 5), indicating potential for tissue repair by bone marrow–derived cells in patients with COPD, at least in those with moderate airflow obstruction. To our knowledge, this is the first study demonstrating the presence of vascular progenitor cells in human pulmonary arteries, specifically in patients with COPD. We cannot extrapolate our findings to patients with end-stage disease in whom the potential for tissue repair might be eventually impaired. The increased number of AC133+ cells adhered to the endothelium is consistent with the notion that VPCs could contribute to endothelial repair in patients with COPD who show endothelial dysfunction in pulmonary arteries at different degrees of disease severity (3, 21). There is evidence that endothelial damage in systemic arteries induces the mobilization of VPCs and their homing in injured vessels (7, 22, 23). VPCs seem to be essential in the process of endothelial function repair in mature blood vessels (24). In chronic vascular diseases where continuous injury can stimulate VPCs recruitment at sites of lesion, an insufficient number of circulating vascular progenitor cells could compromise vessel repair (24). In our study, a higher number of VPCs in the endothelium was associated with a greater response to hypoxic stimulus in vitro (Figure 5), likely suggesting that in COPD dysfunctional reactivity of pulmonary arteries might be preserved by homing of VPCs in injured vessels. In contrast, we did not find a significant relationship between the number of AC133+ cells and endothelium-dependent vascular relaxation. A greater number of VPCs in pulmonary arteries could imply greater repair capacity, but it might also be associated with more extensive endothelial damage. Overall, these findings suggest that VPCs contribute to pulmonary endothelium maintenance in COPD. AC133+ and CD45+ cells were not confined to the endothelial surface; they were also identified within the intimal layer (Figure 4). To what extent VPCs can give rise to SMCs proliferating in the intima is controversial (25). Yet, the finding that pulmonary artery wall enlargement was associated with a greater number of VPCs (Figure 5) may suggest that these cells could play a role in pulmonary vessel remodeling in COPD. Smooth muscle cells might differentiate from several sources, such as mesenchymal cells, endothelial cells, bone marrow precursors, or macrophages. In vitro, common vascular progenitor cells may give rise to endothelial cells upon exposure to VEGF and to SMCs when treated with platelet-derived growth factor-BB (26). Accordingly, an imbalance between growth factors, facilitated by the disruption of endothelial function and/or oxidative stress generated by cigarette smoke products, could determine the fate of a common vascular progenitor cell in pulmonary vessels. Increased concentrations of growth factors may accelerate locally the process of cell differentiation, thereby diminishing the number of progenitor cells infiltrating the vessel wall. Indeed, we found an inverse correlation between VEGF expression and the density of AC133+ cells in the intima but not in the endothelium, likely suggesting a potential role of VEGF in promoting cell differentiation (Figure 6). In agreement with previous observations (18), the expression of VEGF mRNA and its receptor, VEGFR2, correlated with the pulmonary artery wall thickness (Figure 6), suggesting a potential role of this growth factor in pulmonary vascular remodeling. We postulated that, in patients with COPD, cigarette smoke products may be at the origin of pulmonary vascular impairment through a direct injury of endothelial cells or the release of inflammatory mediators (12, 27). Repeated injury by high concentrations of cigarette smoke products in pulmonary vessels could represent a strong stimulus to give rise of molecular signals, such as VEGF (28), a molecule capable of mobilizing, recruiting, and differentiating bone marrow VPCs in pulmonary vessels (22). In summary, the present study shows the presence of VPCs adhered to the endothelial surface and within the intimal layer in pulmonary arteries of patients with COPD. The presence of progenitor cells in the vicinity of areas of endothelium denudation and their relation with the response to hypoxic stimulus suggest that these cells may contribute to an ongoing process of endothelium repair. We also observed an increased number of VPCs in the intima of pulmonary arteries, which was associated with the enlargement of the vessel wall. The latter finding suggests that VPCs could also be involved in the pathogenesis of intimal hyperplasia, presumably through VEGF-related signals.
The authors thank Anna Martínez, Blanca Reyes, and Belén González for their expert technical assistance. The authors also thank the Servei Científic Tècnic from the Universitat de Barcelona, especially Ramón Fontarnau and Nuria Cortadellas, for their assistance in scanning electronic microscopy studies.
This work was supported by grants FIS 03/0549, SEPAR-2002, FUCAP-2003, MTV 04-316, and ISCIII-RTIC 03/11. This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org Originally Published in Press as DOI: 10.1165/rcmb.2005-0255OC on October 20, 2005 Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript. Received in original form July 11, 2005 Accepted in final form October 10, 2005
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Abstract
Introduction
-actin band to account for variations in gel loading. 
