American Journal of Respiratory and Critical Care Medicine

Rationale: Donor mesenchymal stromal/stem cell (MSC) expansion and fibrotic differentiation is associated with development of bronchiolitis obliterans syndrome (BOS) in human lung allografts. However, the regulators of fibrotic differentiation of these resident mesenchymal cells are not well understood.

Objectives: This study examines the role of endogenous and exogenous prostaglandin (PG)E2 as a modulator of fibrotic differentiation of human lung allograft-derived MSCs.

Methods: Effect of PGE2 on proliferation, collagen secretion, and α-smooth muscle actin (α-SMA) expression was assessed in lung-resident MSCs (LR-MSCs) derived from patients with and without BOS. The response pathway involved was elucidated by use of specific agonists and antagonists.

Measurement and Main Results: PGE2 treatment of LR-MSCs derived from normal lung allografts significantly inhibited their proliferation, collagen secretion, and α-SMA expression. On the basis of pharmacologic and small-interfering RNA approaches, a PGE2/E prostanoid (EP)2/adenylate cyclase pathway was implicated in these suppressive effects. Stimulation of endogenous PGE2 secretion by IL-1β was associated with amelioration of their myofibroblast differentiation in vitro, whereas its inhibition by indomethacin augmented α-SMA expression. LR-MSCs from patients with BOS secreted significantly less PGE2 than non-BOS LR-MSCs. Furthermore, BOS LR-MSCs were found to be defective in their ability to induce cyclooxygenase-2, and therefore unable to up-regulate PGE2 synthesis in response to IL-1β. BOS LR-MSCs also demonstrated resistance to the inhibitory actions of PGE2 in association with a reduction in the EP2/EP1 ratio.

Conclusions: These data identify the PGE2 axis as an important autocrine–paracrine brake on fibrotic differentiation of LR-MSCs, a failure of which is associated with BOS.

Scientific Knowledge on the Subject

Fibrotic obliteration of small airways by aberrant mesenchymal cell proliferation and differentiation leads to chronic allograft failure after lung transplantation. However, factors regulating mesenchymal cell phenotype in human lung allografts are poorly understood.

What This Study Adds to the Field

These data identify prostaglandin E2 as an inhibitory modulator of fibrotic differentiation of mesenchymal progenitors residing in human lung allografts and establish loss of this autocrine brake in chronic allograft rejection in human lung transplants.

The major cause of long-term mortality in lung transplant recipients, bronchiolitis obliterans (BO), is a graft response marked by fibrosis and obliteration of the terminal airways (1). The resulting obstructive ventilatory defect is termed “bronchiolitis obliterans syndrome” (BOS). BO shares many common features with other fibroproliferative diseases including development of a profibrotic milieu and accumulation of myofibroblasts, a differentiated mesenchymal cell with an increased collagen secretory ability and α-smooth muscle actin (α-SMA) expression. However, the pathogenesis of this fibrotic remodeling and the regulators of fibroproliferative pathways in BOS are not well understood.

Adult mesenchymal progenitor cells have the potential to regulate the local microenvironment by their paracrine actions or transdifferentiation to other mesenchymal lineages (2). We have recently demonstrated the presence of multipotent donor-derived mesenchymal stem/stromal cells (MSC) in human lung allografts (3). Similar to other adult MSCs (4), allograft-derived lung-resident MSCs demonstrate an ability to inhibit T-cell proliferation in vitro (5). However, MSCs can also participate in fibrotic responses (6, 7) and several recent findings suggest an important pathogenic role for lung-resident MSCs (LR-MSCs) in fibroproliferative responses marking allograft remodeling. First, an increase in the number of LR-MSCs precedes BOS onset (8). Second, LR-MSCs can differentiate to myofibroblasts and LR-MSCs isolated from patients with BOS demonstrate an altered profibrotic phenotype (9). Third, like LR-MSCs, myofibroblasts in BO lesions demonstrate a local lung rather than recipient origin (9). However, the mechanism of altered LR-MSC phenotype in BO and the mediators driving the ultimate fate of these cells remains to be determined.

Prostaglandins (PGs) are lipid mediators formed by the sequential actions of cyclooxgenase (COX) and specific PG synthases on arachidonic acid. MSCs constitutively produce PGE2 and PGE2 production by MSCs can be potentiated by induction of the COX-2 enzyme in response to various mediators (4, 10). PGE2 is a well-established inhibitor of T-cell proliferation (11, 12) and recent studies have demonstrated that PGE2 secreted by MSCs is important in mediating their T-cell suppressive ability (4, 5, 13, 14). It is also pertinent to note that PGE2 is a well-characterized down-regulator of fibrogenesis. The relevant in vitro actions of PGs include inhibition of fibroblast migration (15), proliferation (16, 17), collagen synthesis (18), and myofibroblast differentiation (19). In animal models, PGE2 has been demonstrated to protect against inflammation-induced fibrotic remodeling of the airway and parenchyma (20, 21). However, the modulation of LR-MSC fibroproliferative responses by PGE2 and the role of this lipid mediator in the pathogenesis of BOS have not been evaluated.

Here we study LR-MSCs derived directly from lung allografts with and without BOS and demonstrate that PGE2 is an important autocrine–paracrine brake on fibrotic differentiation of LR-MSCs. Importantly, dysregulation of PGE2 synthesis and signaling is shown to be associated with BOS. Some of the results of these studies have been previously reported in the form of an abstract (22).

Isolation and Culture of LR-MSCs

The MSCs were derived from the bronchoalveolar lavage (BAL) fluid of lung transplant recipients by plastic adherence and subsequently characterized by cell surface analysis and differentiation under a protocol approved by the University of Michigan Institutional Review Board, as previously described (3, 5, 8, 23). LR-MSCs obtained from individual BAL samples were treated as separate cell lines and cells were studied at passages two through six. Patients were diagnosed with BOS according to the International Society of Heart and Lung Transplantation guidelines (24) on the basis of a persistent decrease in the FEV1 by 20% or more of the peak predicted value after transplantation in the absence of confounding variables, as previously described (25, 26). Patients with pathologic findings of acute rejection based on transbronchial biopsy or evidence of infection on microbiologic cultures at the time of BAL were excluded from the study. No significant difference was noted in time post-transplant (P = 0.20 between BOS and non-BOS subjects included in the study).

Reagents and Conditions

For proliferation assays and protein analysis, the following modulation concentrations were used: 1-μM PGE2; 1-μM butaprost (E prostanoid [EP]2 receptor agonist); 4-μM EP1 receptor agonist (ONO-004); 1-μM EP3 receptor agonist (ONO-248); 1-μM EP4 receptor agonist (ONO-329); 50-μM adenylate cyclase agonist (forskolin); 50-μM nonselective phosphodiesterase inhibitor (3-isobutyl-1-methylxanthine [IBMX]); and 100-μM adenylyl cyclase inhibitor (SQ22536). PGE2 and butaprost were purchased from Cayman Chemical (Ann Arbor, MI). SQ22536 was purchased from Biomol/Enzo Life Sciences (Farmingdale, NY). ONO-004, ONO-AE3–248, and ONO-AE1–329 were provided as a generous gift by Ono Pharmaceuticals (Osaka, Japan). The rest of the reagents were obtained from Sigma (St. Louis, MO). Myofibroblast differentiation was induced by transforming growth factor (TGF)-β at 2 ng/ml and IL-13 at 10 nm/ml, both from R&D Systems (Minneapolis, MN)

Western Blotting

Protein lysates were collected and Western blot analysis for α-SMA and collagen I performed using 1:1,000 dilution of monoclonal α-SMA antibody (Dako, Carpieria, CA) or 1:500 dilution of rabbit polyclonal to Collagen l (Cedarlane, Ontario, Canada) as previously described (27, 28).

Proliferation Assays

The 1 × 104 LR-MSCs were plated per well in 96-well flat-bottom culture-treated plates, serum starved overnight, and then subjected to various treatment conditions. Twenty-four hours post-treatment, [3H]thymidine (1 μCi per well) was added to wells and allowed to incubate for 20 hours before harvesting. [3H]thymidine incorporation was expressed as the mean of six replicates in counts per minute.

Determination of PGE2 Synthesis

LR-MSCs were cultured in the absence or presence of IL-1β (10 ng/ml) for 24 hours and PGE2 quantified in culture supernatants using a highly sensitive and specific enzyme immunoassay kit from Cayman Chemicals.

Real-Time Polymerase Chain Reaction Analysis

Total LR-MSC mRNA was extracted using an RNeasy Kit (Qiagen, Valencia, CA). Primers and probes for each prostanoid receptor were designed and then purchased from Applied Biosystems (Foster City, CA), and sequences are as follows: EP1 forward: 5′-TGG GCC AGC TTG TCG G-3′, EP1 reverse: 5′-CTG CAG GGA GGT AGA GC-3′, EP1 probe: 5′-6FAM-TCA TGG TGG TGT CGT GCA TCT GCT GGA-TAMRA-3′, EP2 forward: 5′-GGT GCT CGC CTG CAA CTT C-3′, EP2 reverse: 5′-TCC GCA GCG GCT TCT C-3′, EP2 probe: 5′-6FAM-TCC GCA TGC ACC GCC GAA TAMRA-3′, EP3 forward: 5′-CCA CCT CTA CTG TGA TTG ATC C-3′, EP3 reverse: 5′-CTC AGC CCT TCC GTT GGT T-3′, EP3 probe: 5′-6FAM-TGG ATT TGT CCT TTC CCG CCA TGT C-TAMRA-3′, EP4 forward: 5′-CAT GTA CGC GGG CTT CAG-3′, EP4 reverse: 5′-GCG AGG TGC GGC GCA TGA AC-3′, EP4 probe: 5′-6FAM-TCG CCA CCG TCC TCT GCA ACG T-TAMRA-3′. All polymerase chain reactions (PCR) were performed on an Applied Biosystems ABI Prism 7000 Thermocycler (Applied Biosystems).

Small-interfering RNA

Small-interfering RNA (siRNA) targeting EP2 receptor and nontargeting scrambled siRNA were purchased from Applied Biosystems. LR-MSCs (60% confluence) were transiently transfected with siRNA using Oligofectamine and OPTI-MEM medium (Invitrogen, Carlsbad, CA). Cells were incubated with the transfecting media for 24 hours. Transfecting media were replaced with serum-free media for 48 hours before cells were treated or collected for experiments. EP2 receptor mRNA expression levels were tested to confirm silencing efficiency. Real-time PCR demonstrated a 56.5 ± 18.22% reduction in EP2 receptor mRNA expression level after silencing.

Statistical Analysis

Data are presented as mean values ± SEM. Statistical significance was analyzed using GraphPad Prism 3 software (La Jolla, CA). Significance was assessed with a Student t test for comparisons of two groups, or with analysis or variance and a post hoc Neuman-Keuls test for three or more groups. P less than 0.05 was considered significant.

LR-MSC Proliferation and Myofibroblast Differentiation Are Inhibited by Exogenous PGE2

We have previously demonstrated that human lung allograft-derived MSCs can differentiate to myofibroblasts in vitro in response to profibrotic mediators TGF-β and IL-13 (9). To determine if PGE2 can modulate the profibrotic activation of human lung allograft-derived MSCs, the effect of exogenous PGE2 on proliferation and myofibroblast differentiation of LR-MSCs isolated from normal lung allografts was investigated.

LR-MSCs isolated from lung allografts without evidence of acute rejection, infection, or BOS (n = 10) treated overnight with PGE2 demonstrated an approximately 70% inhibition of proliferation as measured by [3H]thymidine incorporation (Figure 1A). To examine if PGE2 could also inhibit the transition of LR-MSCs to myofibroblasts, LR-MSCs were incubated with TGF-β1 ± PGE2 for 24 hours, and their α-SMA and collagen I protein expression measured by Western blot analysis. Significantly lower collagen I and α-SMA protein expression was also noted in TGF-β1–stimulated LR-MSCs in the presence of PGE2 (Figures 1B and 1C). PGE2 also led to a decrease in myofibroblast differentiation induced by IL-13 (Figure 1D). All experiments shown were performed using a 1-μM dose of PGE2. This dose of PGE2 was chosen based on previous literature (27, 29) and preliminary experiments demonstrated a dose-dependent effect of PGE2 in the range of 100 nM to 10 μM.

Inhibition of Profibrotic Properties of LR-MSCs by PGE2 Is Mediated by EP2 Receptor Ligation and cAMP Signaling

The cellular actions of PGE2 are transduced on its ligation of one of four 7-transmembrane G-protein–coupled EP receptors, designated EP1–4 (30). Examination of the relative expression of EP receptors by real-time PCR demonstrated a relative abundance of the Gαs-coupled receptor EP2, suggesting that the inhibitory effect of PGE2 was likely mediated by this receptor (Figure 2A). To investigate this further, the effect of selective EP receptor agonists on LR-MSC proliferation and differentiation was assessed. As shown in Figure 2B, the EP2 receptor-selective agonist butaprost was able to suppress LR-MSC proliferation in a manner similar to PGE2, whereas agonists selective for EP1, 3, and 4 could not (EP4 data are not shown because of the little to no EP4 receptor expression on LR-MSCs). Butaprost also uniquely demonstrated inhibition of TGF-β–induced α-SMA expression in a similar manner to that of PGE2 (Figure 2C).

To further establish that EP2 is responsible for PGE2-induced inhibition of LR-MSC myofibroblast differentiation, we used gene-specific knockdown by siRNA. Transfection of the siRNA targeting EP2 blocked the ability of PGE2 to inhibit TGF-β–induced α-SMA expression (Figure 2D), thus verifying that the inhibitory effects of PGE2 on LR-MSCs are mediated by EP2.

The EP2 receptor couples to Gs, which stimulates adenylate cyclase, resulting in increased cAMP. To validate that cAMP signaling is capable of mediating antifibrotic actions, LR-MSCs were treated with the direct adenylate cyclase activator forskolin in the presence of the phosphodiesterase inhibitor IBMX (to prevent cAMP degradation). Forskolin suppressed both proliferation (Figure 2 E) and α-SMA expression (Figure 2 F) of LR-MSCs in a similar manner to PGE2. To further pinpoint cAMP as the second messenger eliciting PGE2 antiproliferative effects, LR-MSCs were treated with PGE2 in the presence of SQ22536, an adenylate cyclase inhibitor. Inhibition of adenylate cyclase with SQ22536 was able to abrogate PGE2 effects on LR-MSC proliferation (Figure 2E). These data suggest that EP2 receptor cAMP-mediated signaling is the likely mechanism by which PGE2 elicits antifibrotic effects in LR-MSCs.

Endogenous PGE2 Production and Signaling by EP2 Receptor Limits Profibrotic Differentiation of LR-MSCs

We have previously demonstrated that LR-MSCs secrete PGE2 (5). Interestingly, an increase in baseline α-SMA was seen in LR-MSCs transfected with EP2 siRNA (Figure 2D), suggesting that endogenous PGE2 generated by LR-MSC stimulation acts in an autocrine manner to limit myofibroblast differentiation. Blocking endogenous PGE2 production by treatment with indomethacin, a nonselective COX inhibitor, was also found to increase basal α-SMA protein expression (Figure 3A), reiterating that endogenous PGE2 production is important in maintaining the undifferentiated state of these cells. We have previously demonstrated that PGE2 secretory ability of LR-MSCs is up-regulated in response to IL-1β by induction of COX-2 (5). A significant decrease in TGF-β–induced α-SMA expression was noted in the presence of IL-1β (Figure 3B). This effect of IL-1β was reversed in the presence of the indomethacin demonstrating that up-regulation of endogenous PGE2 generation in LR-MSCs acts as a brake on myofibroblast differentiation induced by profibrotic signals.

LR-MSCs from Patients with BOS Demonstrate Altered PGE2 Secretory Function

LR-MSCs from patients with BOS demonstrate a profibrotic phenotype marked by increased collagen secretory ability and α-SMA expression (9). Because PGE2 secretory function of cells can also be altered in diseases characterized by tissue remodeling (20, 31), we measured PGE2 synthetic capacity of BOS versus non-BOS LR-MSCs. Cells from patients with and without BOS were cultured for 18 hours in the absence or presence of IL-1β, and the conditioned medium was analyzed for PGE2. Baseline levels of PGE2 secretion by BOS LR-MSCs were found to be almost fivefold less than those of normal LR-MSCs (Figure 4A). Furthermore, BOS cells elaborated 22-fold less PGE2 than did non-BOS cells under stimulated conditions (Figure 4A). This failure of LR-MSCs from patients with BOS to up-regulate PGE2 in response to IL-1β suggested failure of induction of COX-2. When COX-2 protein expression was studied, LR-MSCs derived from patients with BOS indeed demonstrated a dramatically blunted induction of COX-2 in response to IL-1β (Figure 4B).

LR-MSCs from Patients with BOS Demonstrate Impaired PGE2 Responsiveness

To investigate if PGE2 modulation of LR-MSC is altered in BOS, the effect of PGE2 on TGF-β–induced α-SMA expression was compared between BOS and normal LR-MSCs. In LR-MSCs derived from normal lung allografts, PGE2 inhibited TGF-β–induced α-SMA expression in 8 out of the 10 lines tested with a mean decrease to 52.95 ± 10.64% of the TGF-β–induced levels. By contrast, no significant decrease in α-SMA expression in TGF-β–stimulated BOS LR-MSCs was noted in the presence of PGE2 (mean = 95.25 ± 7.95% of the TGF-β–induced levels) (Figure 5A). As demonstrated in the dot plot, variability in the response to PGE2 was noted among LR-MSCs derived from individual patients with BOS. LR-MSCs from three patients with BOS demonstrated greater that 30% inhibition of TGF-β–induced α-SMA expression by PGE2. However, in six BOS cell lines, PGE2 actually augmented α-SMA expression.

To understand the mechanism of this altered responsiveness to PGE2, relative EP receptor expression was examined by real-time PCR in the same non-BOS and BOS cell lines. As a group, the BOS cell lines demonstrated a relatively lower EP2/EP1 ratio than did the non-BOS cell lines (P = 0.001) (Figure 5B), and a significant correlation was noted between the EP2/EP1 ratio and PGE2-induced α-SMA expression in the entire cohort (P = 0.0057; R2 = 0.34) (Figure 5C). This alteration in ratio primarily reflected a relative increase in expression of EP1 receptors in BOS MSCs compared with non-BOS MSCs (P = 0.008).

Graft resident donor-derived mesenchymal cells play a key role in fibrogenesis in the transplanted lung (8, 9). In this study we sought to examine the role of PGE2 as an inhibitory modulator of fibrotic phenotype of human lung allograft-derived mesenchymal stem cells. We demonstrate that LR-MSCs derived from normal lung allografts demonstrate abundant expression of the Gαs-coupled receptor EP2, and acting by this cAMP-dependent pathway, exogenous PGE2 inhibits LR-MSC proliferation, collagen synthesis, and myofibroblast differentiation. Stimulation of endogenous PGE2 secretion by IL-1β was associated with amelioration of their myofibroblast differentiation. By contrast, inhibition of endogenous PGE2 synthesis by indomethacin or prevention of its action by EP2 receptor silencing was associated with augmented fibrotic differentiation. Furthermore, impairment in both PGE2 synthesis and responsiveness was found in LR-MSCs isolated from patients with BOS, suggesting an in vivo phenotypic alteration favoring fibrogenesis. Our findings suggest that PGE2, a secretory product of MSCs, acts as an autocrine brake on their fibrotic differentiation under normal circumstances and disruption of this homeostatic axis is associated with BOS. The identification of a dysregulated PG secretory and response pathway in LR-MSCs in association with a BOS provides the first demonstration of a potential role of PGE2 in the pathogenesis of this disease.

Locally resident mesenchymal cells are important cellular participants in tissue repair responses. Lung-specific mesenchymal progenitors can serve important beneficial functions after allogeneic lung transplantation as promoters of epithelial growth (23) and inhibitors of T-cell responses (5). Indeed, by quantitating MSCs in the BAL of human lung allografts, we have demonstrated a significant increase in LR-MSC numbers in the first 3 months after lung transplantation, supporting a role for these cells in repair responses (8). However, an increase in LR-MSCs numbers later after transplant is associated with development of BOS (8), suggesting that a switch in LR-MSC phenotype or local milieu likely perpetuates fibrotic differentiation of these cells. The demonstration of an autocrine mechanism controlling LR-MSC fibrotic differentiation and its dysregulation in BOS, as suggested by the present work, offers a mechanistic explanation for this dual role of these cells in an allograft milieu.

Both exogenous and endogenous PGE2 were shown to inhibit LR-MSC proliferation, collagen secretion, and myofibroblast differentiation in vitro. This suggests that PGE2 secreted by LR-MSCs or from other cellular sources, such as the epithelium, can prevent their expansion and profibrotic differentiation, thus keeping the LR-MSCs recruited to the alveolar spaces in a nondifferentiated state. The microenvironment of a lung allograft milieu has been studied by BAL. The development of BOS, similar to other fibrotic diseases, is associated with a switch from a proinflammatory type 1 immune response to a type 2 immune response (32). Proinflammatory cytokines, such as IL-1β, tumor necrosis factor-α, and IFN-γ, predominate in ischemia reperfusion and acute rejection (3337), whereas profibrotic mediators, such as IL-13 and TGF-β, are linked to development of BO (28, 3841). IL-1β, tumor necrosis factor-α, and IFN-γ are also significant inducers of COX-2 and PGE2 secretion (42). Thus, it can be postulated that LR-MSCs recruited to a proinflammatory milieu are prevented from undergoing fibrotic differentiation because of this inhibitory effect of PGE2 on their proliferation, synthetic function, and α-SMA expression. Indeed, we report herein that IL-1β ameliorates the stimulatory effect of TGF-β on α-SMA expression of LR-MSCs by a COX-dependent mechanism. However, IL-13, a type 2 cytokine that enhances collagen secretion and α-SMA expression in LR-MSCs (9), is a known inhibitor of COX-2 induction and PGE2 synthesis (43, 44). Thus, the profile of soluble mediators in the allograft environment could differentially modulate LR-MSC fibrotic differentiation by their contrasting effects on PGE2 synthetic function.

LR-MSCs isolated from patients with BOS demonstrated a defect in their capacity to synthesize PGE2. Diminished PGE2 secretory ability was noted under basal and stimulated conditions; BOS LR-MSCs failed to demonstrate increases in COX-2 protein expression in response to agonist IL-1β. These findings are similar to those previously described in fibroblasts isolated from lung biopsies of patients with idiopathic pulmonary fibrosis (31). Because PGE2 production by normal lung allograft-derived LR-MSCs has been demonstrated to be important in T-cell inhibition, decreased PGE2 secretion in LR-MSCs from patients with BOS can potentially “switch” the function of these cells from immunoregulatory to profibrotic. PGE2 has also been demonstrated to promote apoptosis by modulating multiple survival pathways in normal human lung fibroblasts (45). Furthermore, inhibition of COX-2 in such cells was shown to significantly increase resistance to FasL-induced apoptosis (46). Whether LR-MSCs demonstrate resistance to apoptosis and whether PGE2 has a role in inducing apoptosis of LR-MSCs recruited to alveolar airspaces remains to be investigated.

The four EP receptors transducing the cellular actions of PGE2 are traditionally associated with specific Gα subunits that activate varying signaling pathways (30): EP1 couples with Gαq, which results in increases in intracellular calcium and release of diacylglycerol (47); EP2 and EP4 couple with Gαs, which stimulates adenyl cyclase, leading to increases in cAMP; and EP3 couples through Gαi, which inhibits adenyl cyclase, leading to decreases in cAMP (48). The integrated response to PGE2 often represents the net effect of additive or opposing signal transduction events, hence the effects of PGE2 can be pleiotropic (49). Furthermore, if expression of EP receptors is altered in disease states, then PGE2 might have opposing effects in normal and disease conditions. Indeed, although PGE2 is a bronchodilator in healthy subjects with no asthma, it results in significant bronchoconstriction in some people with asthma (50). Similarly, contradictory effects of PGE2 on cells isolated from diseased and nondiseased tissues have been described (51). EP2 was found to be the predominant receptor in LR-MSCs derived from normal lung allografts and the antifibrotic effects of PGE2 on these cells were shown to be mediated by EP2-linked cAMP signaling, in keeping with previously published data in this field (19). However, exogenous PGE2 failed to demonstrate significant suppression of myofibroblast differentiation in LR-MSCs isolated from patients with BOS. This resistance to the antifibrotic actions of exogenous PGE2 in BOS is similar to what has been previously reported in tissue fibroblasts from patients with idiopathic pulmonary fibrosis (29) and seems to arise from a reversal of the ratio of inhibitory EP2 to stimulatory EP1 receptors. DNA hypermethylation was recently demonstrated to be responsible for diminished EP2 expression leading to PGE2 resistance in pulmonary fibrosis (52). The mechanisms underlying dysregulated PGE2 production and EP receptor expression in BOS needs further investigation. However, this documentation of defective PGE2 production and response pathways in BOS LR-MSCs provides the first demonstration for a role for this mediator in the pathogenesis of fibroproliferation in human lung allografts.

In summary, by first determining the presence of an antifibrotic mechanism in LR-MSCs isolated from normal lung allografts and then demonstrating its dysregulation in BOS LR-MSCs, we establish a role for altered PGE2 synthetic and response pathways in the pathogenesis of BOS.

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Correspondence and requests for reprints should be addressed to Vibha N. Lama, M.D., M.S., Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine, University of Michigan Health System, 1500 East Medical Center Drive, 3916 Taubman Center, Ann Arbor, MI 48109-0360. E-mail:

Supported by NIH grants RO1 HL094622 (V.N.L.) and RO1 094311 (M.P.-G.); and the American Thoracic Society Research Award, the Scleroderma Research Foundation Award, and the Brian and Mary Campbell and Elizabeth Campbell Carr research gift fund (V.N.L.).

Author Contributions: Conception and design, V.N.L., M.P.-G., N.M.W., and L.N.B.; acquisition of data and analysis and interpretation, V.N.L., M.P.-G., N.M.W., L.N.B., A.W., and S.W.; and drafting of the manuscript, V.N.L., M.P.-G., N.M.W., L.N.B., A.W., and S.W.

Originally Published in Press as DOI: 10.1164/rccm.201105-0834OC on September 22, 2011

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