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cells (coexpressing CD146,. CD31, and CD105) is enriched for ECFC activity (24). Among all current putative EPCs, ECFCs appear to function as a circulat-.
Translational Review Endothelial Progenitor Cells in Regeneration after Acute Lung Injury Do They Play a Role? ¨ nshoff1, Angelika Bierhaus2, and Grietje C. Beck3 Neysan Rafat1, Burkhard To 1 Department of Pediatrics I, University Children’s Hospital Heidelberg, Heidelberg, Germany; 2Department of Medicine I and Department of Clinical Chemistry, University of Heidelberg, Heidelberg, Germany; and 3Department of Anesthesiology, Dr. Horst Schmidt Kliniken, Wiesbaden, Germany

Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are common disorders in patients requiring critical care. The clinical management of these disorders is difficult and unrewarding, and thus they are among the most common causes of death in intensive care units. The activation and damage of pulmonary endothelium comprise the hallmark of ALI/ARDS. Therefore, the recruitment of circulating endothelial progenitor cells (EPCs) to these lesions may exert a beneficial effect on the clinical course of ALI/ARDS. Consequently, cell-based therapies using stem cells to regenerate lung tissue have emerged as potential novel treatment strategies. Although initial studies suggested implantations of exogenously administered bone marrow– derived progenitor cells into damaged vessel walls, recent evidence indicates that this is rather a rare occurrence with uncertain physiologic significance. In the past few years, different populations of progenitor cells were identified, with different functional capacities. This review (1) highlights the different populations of EPCs identified or administered in different models of ALI/ARDS, (2) reports on whether beneficial effects of EPCs could be demonstrated, and (3) puts the conflicting results of different studies into perspective. Keywords: endothelial progenitor cells; acute lung injury; neovascularization; endothelial regeneration

Acute lung injury (ALI) and endstage acute respiratory distress syndrome (ARDS) commonly develop in patients with sepsis, multiple trauma, burn injury, and aspiration, and are among the most common causes of death in intensive care units. Despite years of well-conducted clinical trials, no specific medical therapies yet exist (1, 2). During the acute exudative phase (i.e., the first 24–48 h) of ALI/ARDS, histologic changes in lung tissues are characterized by an infiltration of inflammatory cells and the destruction of pulmonary endothelium (3, 4). Injury to the alveolar– capillary barrier leads to increased pulmonary vascular permeability, pulmonary edema, and hypoxemia. At this stage, endothelial cells (ECs) can be detached from the vasculature, and thus appear in the circulation (3, 5, 6). The inadequate formation of focal adhesion contacts (7), the proteolysis of the endothelial basal membrane (8, 9), the apoptosis of ECs (10), and the production of antiangiogenic factors (11) are among the (Received in original form April 20, 2011 and in final form October 11, 2012) N.R. was supported by a research scholarship from the Post Doc Program of the University of Heidelberg. A.B. was supported by European Union FP7–proposal number 223326 (CASCADE). Correspondence and requests for reprints should be addressed to Neysan Rafat, M.D., Ph.D., Department of Pediatrics I, University Children’s Hospital Heidelberg, Im Neuenheimer Feld 430, 69120 Heidelberg, Germany. E-mail: Neysan.Rafat@med. uni-heidelberg.de Am J Respir Cell Mol Biol Vol 48, Iss. 4, pp 399–405, Apr 2013 Copyright ª 2013 by the American Thoracic Society Originally Published in Press as DOI: 10.1165/rcmb.2011-0132TR on October 25, 2012 Internet address: www.atsjournals.org

various causes for the release of ECs into the circulation. Simultaneous with these pathologic processes, reconstitution of the endothelial layer is initiated. This reconstitution generally involves two biologic paradigms, referred to as angiogenesis and vasculogenesis. Angiogenesis involves the formation of new blood vessels via the migration and proliferation of the endothelia of preexisting vessels. The capacity of mature ECs to proliferate, however, depends on the presence of endothelial colony–forming cells (ECFCs) that give rise to endothelial progeny (12–14). Because heterogeneity may be evident in the level of proliferative potential, depending, for instance, on the vascular bed, adequate vascular repair may require additional support. In this context, adult vasculogenesis, that is, the de novo formation of blood vessels from endothelial progenitor cells (EPCs), has been demonstrated to play a role. Great numbers of clinical and animal studies have been performed, either to quantify concentrations of EPCs and their association with clinical outcomes, or to investigate the potential of mobilizing or administering EPCs in the context of lung regeneration for different lung disorders. The present overview will attempt to examine the different populations of EPCs identified or administered in different models of ALI/ARDS, and to describe whether beneficial effects could be demonstrated.

ENDOTHELIAL PROGENITOR CELLS The pioneering work of Asahara and colleagues in 1997 (15) described for the first time the presence of circulating blood cells with the ability to promote vascular repair and regeneration. Because the identified cells displayed a variety of seemingly endothelialspecific cell-surface antigens, they were referred to as EPCs. In the next decade, numerous studies performed in a variety of animal models appeared to support the hypothesis that bone marrow– derived cells may be recruited and incorporated into sites of active neovascularization during tissue ischemia, vascular trauma, tumor growth, inflammation, and other conditions. In parallel, a multitude of studies in human subjects identified EPCs as a biomarker for clinical disorders such as cardiovascular disease (16), cerebrovascular disease (17, 18), sepsis (19), and numerous types of cancer (20, 21) because the concentrations of circulating EPCs correlated with the severity or risk of adverse outcomes in each particular disease. Although bone marrow transplantation experiments have shown unequivocally that bone marrow–derived cells are recruited to sites of active neovascularization and can differentiate into vascular cells in situ, the frequency of this phenomenon and the identification of the cell types involved remain a matter of debate (22). Different Populations of Endothelial Progenitor Cells

A major limiting factor in this field has been the lack of a specific marker to identify circulating EPCs. In the first description of EPCs, CD34 and vascular endothelial growth factor–2 receptor

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(KDR) expression in human peripheral blood mononuclear cells was adopted for the EPC phenotype (15). Other groups suggested the phenotype CD341 AC1331 KDR1 to distinguish EPCs from mature endothelial cells (23). This cell-surface phenotype has gained wide use as a means to measure circulating EPCs in human subjects. In the past several years, it has become evident that populations of CD34 1 AC133 1 KDR 1 cells do not form capillary-like structures with lumens in vitro nor in human blood vessels in vivo upon implantation in a collagen/fibronectin scaffold (24–26). Although these cells may be recruited to denuded vessels in ischemic sites, they do not directly become persistent vascular endothelial cells or display de novo in vivo vasculogenic potential. Rather, they display potent paracrine properties regulating new vessel formation via angiogenesis (25, 27) (Figure 1). These cells are referred to as “proangiogenic hematopoietic cells.” The nomenclature has not yet been universally accepted, but we will make use of the terms proposed by Yoder (28), Timmermans and colleagues (29), and Richardson and Yoder (30). Other populations of EPCs were identified using colony-forming assays. In the original study (15), plated human CD341 peripheral blood cells formed cellular clusters in vitro. These clusters binding acetylated low-density lipoprotein (acLDL) were presented as evidence of CD341 peripheral blood cells differentiating into spindle-shaped endothelial cells. Others have modified this assay by adding a preplating period, and replating the nonadherent fraction for 7 days on fibronectin-coated dishes (16, 31). The emerging cell clusters were referred to as EPC colony–forming units (CFUs) or CFU–Hill. Ingram and colleagues (14) identified yet another type of cell colony emerging from plated peripheral blood mononuclear cells in 14–21 days when adult blood samples were plated, or in 6 days when umbilical cord blood samples were used. This

cell colony emerges as tightly adherent with a typical cobblestone appearance, and is referred to as endothelial colony–forming cells (ECFCs), late outgrowth cells, or blood outgrowth endothelial cells (30). Not only do these cells possess vessel-forming ability, but the vessels also connect to the host immunodeficient murine vessels and become part of the systemic circulation of the host animal (12, 32) (Figure 1). This functional capacity makes this cell colony a true EPC by its actual definition. Although distinguishing the cellsurface phenotype of the ECFC progeny from vascular endothelial cells is difficult, recent approaches have succeeded to some extent by enriching this population first, and by depleting monocytes, erythrocytes, dead cells, and CD451 blood cells (25). Therefore, the population of CD34hiCD452 cells (coexpressing CD146, CD31, and CD105) is enriched for ECFC activity (24). Among all current putative EPCs, ECFCs appear to function as a circulating precursor with in vivo human vessel–forming ability, and exhibit the most features of a human postnatal vasculogenic cell. Origin of Endothelial Progenitor Cells

Endothelial progenitor cells are thought to originate primarily from bone marrow, and to circulate in the peripheral blood (33, 34). However, evidence has also been presented that vessel wall–derived endothelial cell populations contain a complete hierarchy of endothelial progenitor cells, enabling them to proliferate rapidly (13). Recently, the presence of resident endothelial progenitor cells was reported in the pulmonary microvascular endothelium. Yoder (35) has presented an elaborate review of resident progenitor cells in the pulmonary circulation. These resident progenitor cells have been identified as endothelial cells with high proliferative potential (ECFCs). Controversy persists

Figure 1. Multistep homing process in subsets of endothelial progenitor cells (EPCs). The recruitment and incorporation of proangiogenic hematopoietic cells (PHCs) from bone marrow into ischemic or injured tissue require a coordinated multistep process, including mobilization, chemotaxis, adhesion to the endothelium, transendothelial migration, invasion, and in situ differentiation. PHCs release growth factors that stimulate the angiogenic acitivity of resting mature endothelial cells (ECs). Different factors that are thought to regulate the distinct steps are indicated. Endothelial colony–forming cells (ECFCs) have proliferative potential, and divide and migrate to repair the injured area. They are resident in the vascular bed, but can also be found as circulating cells and have been isolated from cord blood. Both cell types seem to be involved in the repair of endothelial denudation injury. Whereas PHCs are first recruited to the site of injury to facilitate repair, ECFCs have vascular-forming ability, and integrate into denuded vasculatures. CXCR-4, C-X-C chemokine receptor–4; HGF, hepatocyte growth factor; IGF-1, insulin-like growth factor-1; MMP-9, matrix metalloproteinase–9; SDF-1, stromal cell–derived factor-1; VEGF, vascular endothelial growth factor.

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in regard to whether ECFCs are progenitor cells. Because a similar clonal hierarchical proliferative potential was found when comparing human adult aorta and cord blood artery and vein endothelial cells with circulating ECFCs derived from cord blood and adult peripheral blood (13), it is plausible to specify the vascular endothelium as the origin of circulating ECFCs. Future studies need to clarify the relationship between resident and circulating ECFCs, and to confirm whether they can be defined as progenitor cells.

ENDOTHELIAL PROGENITOR CELLS IN ACUTE LUNG INJURY Understanding how circulating and resident progenitor cells accumulate and differentiate into the parenchymal cells of organs has become an exciting and intriguing field of research. Initially, it was speculated that bone marrow–derived cells could structurally engraft as mature, differentiated airway and alveolar epithelial cells or as vascular endothelial cells. As such, a vast number of studies have evaluated the concentrations of bone marrow–derived progenitor cells in several lung disorders, correlating these concentrations with the clinical outcomes for that particular disease. Other studies used techniques that evaluated histologic demonstrations of tagged donor-derived bone marrow cells in recipient lungs or the intratracheal administration of tagged donor bone marrow cells. Contrary to the initial hypothesis, it has become evident that the engraftment of exogenously administered or endogenously mobilized bone marrow cells as airway, alveolar epithelial, or endothelial cells is generally a rare occurrence of uncertain physiologic significance (22, 36–41). Here, we discuss some of these previous studies that investigated the role of bone marrow–derived or resident progenitor cells in ALI/ARDS, and we put their conflicting results into perspective. In Vitro Studies

Various in vitro studies have been performed to investigate the role of bone marrow–derived or resident EPCs in lung regeneration. Zhao and colleagues demonstrated that murine bone marrow–derived progenitor cells (BMPCs) positive for the antigen markers CD133, stem-cell antigen–1 (Sca-1), and CD34, when added to either LPS-induced (42) or thrombin-induced (43) endothelial monolayers, prevented the increase in pulmonary endothelial permeability and formation of edema. Their results suggest that BMPC transplantation or mobilization can induce endothelial barrier protection under the proinflammatory conditions that may be present during acute lung injury. Alvarez and colleagues discovered the presence of resident microvascular endothelial progenitor cells (RMEPCs) within the population of pulmonary microvascular endothelial cells (PMVECs),

which were highly proliferative and capable of reconstituting the entire proliferative hierarchy of PMVECs (44). These RMEPCs seem to form an enriched source for homeostasis and repair in the lung microcirculation. Similar results were found by Schniedermann and colleagues in murine lung microvascular endothelial cells (45). The resident EPCs in their study possessed the capacity to integrate into various types of vessels, including blood and lymph vessels (45). The same group recently studied the secretome of RMEPCs isolated from the lungs of adult mice by nanoflow liquid chromatographic mass spectrometry, and provided a platform for the in-depth analysis of EPCs (46). Clinical Studies

Many descriptive studies have been performed to quantify circulating EPCs in the peripheral blood of patients with ALI/ARDS (see Table 1 for definitions of the EPC populations used in the original studies and for our proposed terms). Burnham and colleagues showed that EPC CFU numbers were significantly higher in patients with ALI compared with healthy control subjects, but these numbers did not differ between patients with ALI and intubated control subjects (47). However, in the patients with ALI, improved survival was correlated with a higher CFU count, suggesting that an increased number of circulating EPCs in ALI is associated with improved survival (47). In a recent study (48), the same group showed increased CFU counts in patients with ALI compared with patients with severe sepsis, which may have resulted from higher lung injury scores in this patient population. We have demonstrated significantly higher levels of circulating CD341CD1331KDR1 EPCs in septic patients (of whom more than half exhibited pulmonary involvement), compared with nonseptic Intensive Care Unit patients and healthy control subjects (19). Furthermore, sepsis survivors exhibited significantly greater numbers of EPCs than did nonsurvivors, suggesting that sepsisinduced vascular damage stimulates the release of EPCs, and that circulating EPCs may predict clinical outcomes in critically ill patients (19). In a subsequent study, we confirmed these findings, and identified an association between the up-regulation of vascular cell adhesion molecule (VCAM)–1 and C-X-C chemokine receptor– 4 by CD341CD1331 cells and survival (manuscript submitted for publication). Similar results were found in neonates with respiratory distress syndrome (RDS), who exhibited elevated EPC concentrations (defined as CD341 cells) compared with preterm control patients (49). Furthermore, RDS survivors exhibited significantly higher concentrations of EPCs than did nonsurvivors, and low EPC concentrations in RDS were correlated with prolonged duration of ventilation, suggesting that EPCs may be involved in the regeneration of neonatal lung injury (49). Table 1 presents an overview of these clinical studies, including definitions

TABLE 1. DESCRIPTIVE CLINICAL STUDIES OF PUTATIVE ENDOTHELIAL PROGENITOR CELLS IN PATIENTS WITH ALI/ARDS AND SEPSIS Authors (Ref. no.) Burnham and colleagues (47) Burnham and colleagues (48) Rafat and colleagues (19) Rafat and colleagues (submitted for publication) Qi and colleagues (49)

Disease ALI ALI/sepsis Sepsis Sepsis

Neonatal RDS

Methods Used to Isolate Colony-forming unit assay Colony-forming unit assay Polychromatic flow cytometry Polychromatic flow cytometry Monochromatic flow cytometry

Marker to Define

Term

acLDL

EPC CFUs

acLDL

EPC CFUs

CD34, CD133, KDR CD34, CD133, CXCR-4, VCAM

EPCs

CD34, CD45low

CD341 cells

PHCs

Clinical Outcome

Proposed Term

High numbers of CFUs associated with improved survival High numbers of CFUs demonstrated a trend toward improved survival Increased EPC numbers were associated with improved survival Increased EPC numbers were associated with improved survival

Proangiogenic hematopoietic Proangiogenic hematopoietic Proangiogenic hematopoietic Proangiogenic hematopoietic

Increased numbers of CD341 cells were associated with improved survival

Proangiogenic hematopoietic cells

cells cells cells cells

Definition of abbreviations: acLDL, acetylated low-density lipoprotein; ALI, acute lung injury; ARDS, acute respiratory distress syndrome; CFUs, colony-forming units; CXCR-4, C-X-C chemokine receptor–4; EPCs, endothelial progenitor cells; PHCs, proangiogenic hematopoietic cells; RDS, respiratory distress syndrome; VCAM, vascular cell adhesion molecule.

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of EPCs, methods of isolation, our proposed terms, and clinical outcomes. Whereas all these clinical studies we have described only quantified EPCs and their correlations with disease outcomes, Suratt and colleagues provided the first evidence in support of the capacity of progenitor cells to replenish lung cells (50). In a retrospective study, lung biopsies were examined after male-to-female hematopoietic stem-cell transplantations in three subjects (50). Their approach involved the immunohistochemical staining of cells for cytokeratin to identify epithelial cells, the immunohistochemical staining of platelet endothelial cell adhesion molecules to identify endothelial cells, and fluorescent in situ hybridization analysis to identify male cells (50). The results demonstrated that 2.5–8.0% of lung epithelial cells and 37.5–42.3% of lung endothelial cells were male, indicating that the lung tissue contained cells derived from the transplanted stem cells, forming a chimera (50). Although the small sample size of that study limited its definitive conclusions, the results offer evidence that pulmonary lung cells (especially pulmonary endothelium) can be derived from BMPCs. Animal Studies

Based on the detection of increased populations of EPCs in different lung disorders and their association with clinical outcomes,

a number of animal studies have been performed to evaluate the effect of BMPC/EPC mobilization or administration in lung regeneration (Table 2). Many of these studies used an endotoxininduced model of ALI. Yamada and colleagues showed that LPS in the lung airways in mice induces a rapid mobilization of Sca-11KDR1 BMPCs into the circulation, which then accumulate within the inflammatory site and mediate regeneration and to some extent differentiate to become endothelial and epithelial cells (51). When BMPCs were suppressed by sublethal irradiation before intrapulmonary LPS, the tissue structure was disrupted and presented emphysema-like changes, whereas the reconstitution of the bone marrow prevented these changes (51). Therefore, Yamada and colleagues suggested that BMPCs are important, and indeed are required for lung repair after LPS-induced lung injury (51). In two studies, Lam and colleagues demonstrated that the transplantation of autologous acLDL-binding cultured EPCs can restore pulmonary endothelial function, preserve the integrity of the alveolocapillary barrier, and suppress the lung inflammatory response in a rabbit model of ALI, leading to improved gas exchange (52, 53). We have also demonstrated in two studies a beneficial effect of CD1331 BMPCs in an LPS-induced isolated ALI model in rats (unpublished results, and Ref. 54). BMPC transplantation not only improved oxygenation, but also led to decreased concentrations of the proinflammatory cytokines IL-1 and IL-6 and a down-

TABLE 2. PRECLINICAL THERAPEUTIC STUDIES OF BONE MARROW–DERIVED PROGENITOR CELLS IN ANIMAL MODELS OF ALI/ARDS Authors (Ref. no.)

Animal Model

Methods Used to Isolate

Yamada and colleagues (52)

LPS-induced ALI in mice

Lam and colleagues (53)

Oleic acid-induced Plating of PBMCs on ALI in rabbits fibronectin-coated dishes, and harvest of adherent cells on Day 7 LPS-induced Plating of PBMCs on ALI in rabbits fibronectin-coated dishes and at Day 7 harvested adherent cells

Lam and colleagues (54)

Rafat and LPS-induced colleagues (submitted ALI in rats for publication) Rafat and LPS-induced colleagues (54) ALI in rats

Kahler and colleagues (55)

Mao and colleagues (56)

Wary and colleagues (57)

Cell sorting of PBMC/GFP 1 BM donors

Immunomagnetic bead separation of CD1331 bone marrow cells Immunomagnetic bead separation of CD1331 bone marrow cells

Marker to Define

Term

Sca-1, KDR

BMPCs

acLDL, KDR

Early EPCs

acLDL, KDR

Early EPCs

CD133

PHCs

CD133

EPCs

Clinical Outcome Accumulation within inflammatory site, differentiation to become ECs, inhibited development of emphysematous lesions in the lungs Preserved endothelial function, maintained integrity of pulmonary alveolar–capillary barrier Preserved integrity of alveolocapillary barrier, suppressed lung inflammatory response, improved pulmonary gas exchange Down-regulation of adhesion molecules in pulmonary alveolar tissue Reduced mortality and proinflammatory cytokine expression, improved pulmonary gas exchange Incorporation into injured vasculature and destruction of lung tissue

Proposed Term ECFCs

PHCs

PHCs

PHCs

PHCs

acLDL, CD31, vWF, BM-derived ECFCs Left-sided lung 48-hour preplating period before CD146, CD133, EPCs transplantation– replating nonadherent BM cells KDR, CD106 induced ALI on fibronectin-coated dishes, colony formation 7–10 days later PHCs acLDL, KDR, vWF Early EPCs Reduced pulmonary edema, LPS-induced Plating of BM cells on inflammation, hemorrhage, ALI in rats fibronectin-coated dishes; and formation of hyaline at 96 hours, membranes, increased survival nonadherent cells were removed; on Day 7, adherent cells were harvested ECFCs Late-outgrowth Reduced lung vasculature LPS-induced Plating of BM cells on gelatin-coated CD34, KDR, VE-cadherin EPCs injury and extravascular ALI in rats dishes; replating adherent water content, increased cells on Day 7 to new gelatin-coated survival dishes; passage up to passage 3

Definition of abbreviations: acLDL, acetylated low-density lipoprotein; ALI, acute lung injury; ARDS, acute respiratory distress syndrome; BM, bone marrow; BMPCs, bone marrow–derived progenitor cells; CFUs, colony-forming units; ECFCs, endothelial colony–forming cells; EPCs, endothelial progenitor cells; GFP, green fluorescent protein; KDR, kinase insert domain receptor; LPS, lipopolysaccharide; PBMCs, peripheral blood mononuclear cells; PHCs, proangiogenic hematopoietic cells; Sca-1, stem-cell antigen–1; VCAM, vascular cell adhesion molecule; VE-cadherin, vascular endothelia cadherin; vWF, von Willebrand factor.

Translational Review

regulation of the adhesion molecules VCAM and intercellular adhesion molecule (ICAM) in pulmonary alveolar tissue. Similar results were found in other endotoxin-induced models of ALI in rats (55, 56) and mice (57). Table 2 presents an overview of these animal studies, including definitions of EPCs, methods of isolation, our proposed terms, and clinical outcomes. Conflicting Results

Despite the positive reports we have described, some studies failed to show a contribution of BMPCs to pulmonary vascular growth and maintenance. In an animal model of bone marrow transplantation, using three different transgenic murine strains (flk-11/lacZ, tie-2/lacZ, and eGFP) as bone marrow donors, Voswinckel and colleagues investigated whether pulmonary endothelial and perivascular cells are derived from circulating progenitors (22). In their model of postpneumonectomy lung growth, BMPCs did not contribute significantly to the generation of endothelial cells, pericytes, vascular smooth muscle cells, or fibroblasts (22). At very low frequencies, however, single enhanced green fluorescent protein– positive endothelial cells could be detected in pulmonary arteries (22). The same group also looked into a murine model of hindlimb ischemia, and could not detect any incorporation of circulating BMPCs in the endothelium or tunica media of growing vessels (38). However, they found a significant perivascular accumulation of GFP-positive cells around growing collateral arteries (38). Many other studies could not show a direct effect of BMPCs on vascular growth or repair (39–41, 58). Several reasons could account for the disparity between these different findings. First, the different studies did not necessarily refer to the same biologic phenomenon (here, lung regeneration). Organs can differ in the way they renew themselves. Moreover, different organ lesions can lead to differences in bone-marrow and stem-cell recruitment (59), suggesting that these cells may behave differently depending not only on the organ, but also on the conditions investigated. Along this line, the isolation and subsequent culture of either progenitor or bone marrow– derived cells under special conditions may likely exert an influence on the properties of these cells in regard to their capacity to incorporate into target tissue. Furthermore, different methodologies were applied. In the animal studies, most investigators used an approach wherein BMPC injections were administered therapeutically for experimental tissue damage. Other investigators studied the contributions of bone marrow– derived cells to a process of regulated regenerative growth in models of bone marrow transplantation, without interfering with endogenous circulating progenitor cell concentrations. In addition, different approaches were applied in clinical settings to detect circulating progenitor cells. Some investigators used cell-culturing methods, whereas others applied flow cytometry to detect these cells. The use of different methods for the detection of circulating progenitor cells makes comparisons of data difficult, because no unique markers define these cells. Both techniques have their advantages and disadvantages. With cell-culturing methods, the initial population in a cell culture is heterogeneous, and thus the determination of the precursor cell that gives rise to EPCs becomes difficult. However, this technique may offer the option of measuring progenitor cell functionality. The use of FACS allows for the identification of a more homogenous progenitor cell population (60), but functionality cannot be determined. In a recent study that sought to determine the reproducibility of and correlations between progenitor cell alternative assay methodologies, Povsic and colleagues (61) reported that progenitor cell enumeration based on flow cytometry (i.e., FACS) was more precise than with culture assays. However, the field would benefit from more precise

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terminology to define the different populations of EPCs, and from better assay standardization (61).

FUTURE PERSPECTIVES The critical question put forward in all studies described here involves the role that circulating and resident progenitor cells play in the resolution of injured pulmonary endothelium and increased pulmonary vascular permeability in the course of ALI/ARDS. Is the administration of EPC populations an option for future therapy regimes? The studies we have mentioned demonstrate that ALI/ARDS increases the number of circulating progenitor cells, which are important in effective lung repair. Furthermore, the administration of BMPCs decreases both the systemic and local inflammatory responses induced by endotoxin, and is associated with improved clinical outcomes. Therefore, the differential release of progenitor cells from the bone marrow offers particular therapeutic potential. Strategies to manipulate the microenvironment to enhance lung repair, particularly in patients who are immunocompromised and have inadequate bone marrow responses, would provide obvious therapeutic benefits. The particular bone marrow–derived progenitor cell populations that are critical in repairing lung injuries remain to be determined. This could lead to important approaches in cell-based treatments where effective bone marrow–derived progenitor cells are infused in patients with ALI/ARDS and other lung diseases. Author disclosures are available with the text of this article at www.atsjournals.org. Acknowledgments: The authors thank Krista Rafat for her editorial assistance during preparation of the manuscript.

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