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Respiratory Physiology & Neurobiology 187 (2013) 190– 198

Contents lists available at SciVerse ScienceDirect

Respiratory Physiology & Neurobiology

jou rn al h omepa g e: www.elsev ier .com/ locate / resphys io l

one marrow-derived mononuclear cells vs. mesenchymal stromal cells inxperimental allergic asthma

oraia C. Abreua, Mariana A. Antunesa, Júlia C. de Castroa, Milena V. de Oliveiraa, Elga Bandeiraa,ebora S. Ornellasa, Bruno L. Diazb, Marcelo M. Moralesc, Débora G. Xistoa,c, Patricia R.M. Roccoa,∗

Laboratory of Pulmonary Investigation, Carlos Chagas Filho Institute of Biophysics, Federal University of Rio de Janeiro, Rio de Janeiro, RJ, BrazilIntermediate Laboratory of Inflammation, Carlos Chagas Filho Institute of Biophysics, Federal University of Rio de Janeiro, Rio de Janeiro, RJ, BrazilLaboratory of Cellular and Molecular Physiology, Carlos Chagas Filho Institute of Biophysics, Federal University of Rio de Janeiro, Rio de Janeiro, RJ, Brazil

a r t i c l e i n f o

rticle history:ccepted 25 March 2013

eywords:sthmallergic lung diseaseirway epithelium

a b s t r a c t

We compared the effects of bone marrow-derived mononuclear cells (BMMCs) and mesenchymal stro-mal cells (MSCs) on airway inflammation and remodeling and lung mechanics in experimental allergicasthma. C57BL/6 mice were sensitized and challenged with ovalbumin (OVA group). A control groupreceived saline using the same protocol. Twenty-four hours after the last challenge, groups were furtherrandomized into subgroups to receive saline, BMMCs (2 × 106) or MSCs (1 × 105) intratracheally. BMMCand MSC administration decreased cell infiltration, bronchoconstriction index, alveolar collapse, collagen

ulmonary eosinophiliafiber content in the alveolar septa, and interleukin (IL)-4, IL-13, transforming growth factor (TGF)-� andvascular endothelial growth factor (VEGF) levels compared to OVA-SAL. Lung function, alveolar collapse,collagen fiber deposition in alveolar septa, and levels of TGF-� and VEGF improved more after BMMCthan MSC therapy. In conclusion, intratracheal BMMC and MSC administration effectively modulatedinflammation and fibrogenesis in an experimental model of asthma, but BMMCs was associated withgreater benefit in terms of reducing levels of fibrogenesis-related growth factors.

. Introduction

Asthma is a chronic inflammatory disease of the airwaysMurphy and O’Byrne, 2010) associated with structural changesuch as subepithelial fibrosis, mucous metaplasia, wall thickening,mooth muscle cell hypertrophy and hyperplasia, myofibroblastyperplasia, vascular proliferation, and extracellular matrix abnor-alities (Al-Muhsen et al., 2011). These changes accelerate decline

n lung function despite treatment with inhaled corticosteroids.herefore, new strategies that can hasten the repair process andttenuate airway inflammation and remodeling are warranted.

Several recent studies have investigated the impact of bonearrow-derived mononuclear cells (BMMCs) (Abreu et al.,

011) or mesenchymal stromal cells (MSCs) (Firinci et al., 2011;oodwin et al., 2011; Ou-Yang et al., 2011; Kapoor et al., 2012)

n experimental allergic asthma. Each has specific advantages.MMCs can be used in autologous transplantation, on the sameay of harvesting, and avoid common complications such as

∗ Corresponding author at: Laboratory of Pulmonary Investigation, Carlos Chagasilho Biophysics Institute, Federal University of Rio de Janeiro, Centro de Ciênciasa Saúde, Avenida Carlos Chagas Filho, s/n, Bloco G-014, Ilha do Fundão 21941-902,io de Janeiro, RJ, Brazil. Tel.: +55 21 2562 6530; fax: +55 21 2280 8193.

E-mail addresses: prmrocco@biof.ufrj.br, prmrocco@gmail.com (P.R.M. Rocco).

569-9048/$ – see front matter © 2013 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.resp.2013.03.014

© 2013 Elsevier B.V. All rights reserved.

graft-versus-host disease, whereas MSCs exhibit multilineagedifferentiation potential and immune-privileged features andenable allogenic use (Mathieu et al., 2009; Lu et al., 2011). So far,however, no study has evaluated the effects of cell type in celltherapy of experimental asthma. Furthermore, most cell therapieshave been studied at the onset of the remodeling process; thereare no data on the effects of cell therapy once the remodelingprocess of asthma is already established. Within this context, thepresent study sought to investigate and compare the therapeuticeffects of BMMCs or MSCs on lung mechanics and histology,collagen fiber content in the airway and alveolar septa, and levelsof cytokines and growth factors in lung tissue in a murine modelof experimental allergic asthma.

2. Materials and methods

This study was approved by the Ethics Committee of the HealthSciences Center, Federal University of Rio de Janeiro.

2.1. Extraction and characterization of BMMCs and MSCs

BMMCs and MSCs were obtained from male C57BL/6 mice(weight 20–25 g, n = 5 per group) and administered on the dayof collection or after 3 passages, respectively. Bone marrow cells

S.C. Abreu et al. / Respiratory Physiology & Neurobiology 187 (2013) 190– 198 191

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ig. 1. Study design. (A) Schematic flow chart and (B) timeline of the study. Experihallenged with ovalbumin; SAL, mice treated with saline; BMMC, mice treated wihe last challenge (1 × 105). All data were analyzed on day 54.

ere aspirated from the femur and tibia by flushing the bonearrow cavity with Dulbecco’s modified Eagle’s medium (DMEM)

Life Technologies, Grand Island, NY, USA). After a homogeneousell suspension was achieved, cells were centrifuged (400 × gor 10 min), plated in DMEM containing 20% fetal bovine serumMSCs) or re-suspended in DMEM (BMMCs) and added to Ficoll-ypaque (Histopaque 1083, Sigma Chemical Co., St. Louis, MO,SA), and again centrifuged and supplemented with phosphate-uffered saline (PBS). Cell characterization was performed byow cytometry using antibodies against CD45 (leukocytes), CD34hematopoietic precursors), CD3, CD8, and CD4 (T lymphocytes),D19 (B lymphocytes), CD14 (monocytes), and CD11b, CD29 andD45 (non-hematopoietic precursors) (BD Biosciences, USA). Thebsence of CD34 and CD45 and the presence of CD14, CD29,nd Sca-1 were used to identify MSCs. Furthermore, MSCs weredentified by the capacity to differentiate into osteoblasts andhondroblasts.

.2. Animal preparation and experimental protocol

Thirty-six female C57BL/6 mice (weight, 20–25 g) were ran-omly assigned to two groups. In the OVA group, mice were

mmunized using an adjuvant-free protocol by intraperitonealnjection of sterile ovalbumin (OVA, 10 �g OVA in 100 �l saline)n 7 alternate days. Forty days after the start of sensitization, 20 �gf OVA in 20 �l of saline were instilled intratracheally. This proce-ure was performed 3 times at 3-day intervals (Xisto et al., 2005).he control group (C) received saline using the same protocol. The Cnd OVA groups were further randomized to receive saline solution0.9% NaCl, 50 �l, SAL), BMMCs (2 × 106 in 50 �l) or MSCs (1 × 105

n 50 �l) intratracheally, 24 h after the last challenge (Fig. 1). For

he administration of saline, BMMCs or MSCs, mice were anes-hetized with sevoflurane, the trachea of each mouse was dissected,nd cells were slowly injected. Furthermore, small aliquots weresed for immunophenotypic flow cytometry characterization of

l groups: C, mice sensitized and challenged with saline; OVA, mice sensitized andMC 24 h after the last challenge (2 × 106); MSC, mice treated with MSCs 24 h after

the injected cell populations and to evaluate the ability of MSCsto differentiate into osteoblasts and chondroblasts (Fig. 2).

2.3. Mechanical parameters

One week after cell therapy, the animals were sedated(diazepam 1 mg i.p.), anesthetized (thiopental sodium 20 mg/kgi.p.), tracheotomized, paralyzed (vecuronium bromide,0.005 mg/kg i.v.), and ventilated with a constant flow ventilator(Samay VR15; Universidad de la Republica, Montevideo, Uruguay)set to the following parameters: frequency 100 breaths/min, tidalvolume (VT) 0.2 mL, and fraction of inspired oxygen (FiO2) 0.21.The anterior chest wall was surgically removed and a positiveend-expiratory pressure of 2 cm H2O applied. Airflow and trachealpressure (Ptr) were measured. Lung mechanics were analyzedby the end-inflation occlusion method. In an open chest prepa-ration, Ptr reflects transpulmonary pressure (PL). Briefly, afterend-inspiratory occlusion, there is a rapid initial decline in PL(�P1,L) from the preocclusion value down to an inflection point(Pi), followed by a slow pressure decay (�P2,L), until a plateau isreached. This plateau corresponds to the elastic recoil pressureof the lung (Pel). �P1,L selectively reflects the pressure used toovercome airway resistance. �P2,L reproduces the pressure spentby stress relaxation, or the viscoelastic properties of the lung, aswell as a small contribution of pendelluft. Static lung elastance(Est,L) was determined by dividing Pel by VT. Lung mechanics mea-surements were obtained 10 times in each animal. All data wereanalyzed using ANADAT software (RHT-InfoData, Inc., Montreal,Quebec, Canada). All experiments lasted less than 15 min.

2.4. Lung histology

Laparotomy was performed immediately after determinationof lung mechanics. Heparin (1000 IU) was injected into the venacava. The trachea was clamped at end-expiration, and the abdom-inal aorta and vena cava were sectioned, producing massive

192 S.C. Abreu et al. / Respiratory Physiology & Neurobiology 187 (2013) 190– 198

F Cs. (A)C fferen

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ig. 2. Flow cytometry analysis and potential of differentiation of the injected MSD14−/CD34−. (D) Cells CD29+ and Sca1+. (E) Osteoblasts and (F) chondroblasts di

emorrhage and terminal bleeding for euthanasia. The right lung

as then removed, fixed in 3% buffered formalin and embed-ed in paraffin; 4-�m-thick slices were cut and stained withematoxylin–eosin.

ig. 3. Lung mechanics. (A) Lung static elastance (Est,L); (B) resistive pressure (�P1,L)hallenged with saline; OVA, mice sensitized and challenged with ovalbumin; SAL, mice gSC, mice treated with MSC 24 h after the last challenge (1 × 105). Box plots show media

-SAL (p < 0.05). **Significantly different from OVA-SAL (p < 0.05). #Significantly different

Cells CD45+ and CD45−. (B) Cells CD45+ and CD14+/CD34+. (C) Cells CD45− andtiation.

Lung histology analysis was performed with an integrating

eyepiece with a coherent system consisting of a grid with 100points and 50 lines (known length) coupled to a conventionallight microscope (Olympus BX51, Olympus Latin America-Inc.,

; (C) viscoelastic (�P2,L) pressures. Experimental groups: C, mice sensitized andiven saline; BMMC, mice treated with BMMC 24 h after the last challenge (2 × 106);ns and interquartile ranges for 6 mice in each group. *Significantly different from

from OVA-BMMC (p < 0.05).

S.C. Abreu et al. / Respiratory Physiology & Neurobiology 187 (2013) 190– 198 193

Table 1Morphometric parameters.

Groups Normal area (%) Alveolar collapse (%) MN (%) PMN (%) Contraction index

CSAL 99 (91–100) 1 (0–1) 32.3 (29.8–37.9) 0.20 (0–0.99) 2.15 (1.9–2.8)BMMC 99 (91–100) 1 (0–1) 28 (24.3–36) 3.48 (2.04–4.01)* 2.44 (1.5–2.5)MSC 99 (91–100) 1 (0–1) 27.7 (23.6–32) 1.03(0.84–2.5)* 2.26 (1.2–2.4)

OVASAL 47 (37–58)* 53 (42–63)* 52.5 (47.3–56.1)* 6.94 (6.15–8.89)* 3.06 (2.8–3.9)*

BMMC 91 (74–95)*,** 9 (8–26)*,** 29.4 (26.4–31) 3.26 (3.03–6.07)*,** 2.08 (1.4–2.9)**

MSC 75 (72–75)*,** ,# 25 (5–27)*,** ,# 29.4 (26.4–32) 3.93 (2.85–4.42)*,** 2.13 (1.8–2.5)**

Values expressed as median (interquartile range) of 6 animals per group. All values were computed in 10 random, non-coincident fields per mouse. Fractional area of normaland collapsed alveoli, mononuclear (MN) and polymorphonuclear (PMN) cells, and contraction index. Experimental groups: C, mice sensitized and challenged with saline;OVA, mice sensitized and challenged with ovalbumin; SAL, mice given saline; BMMC, mice treated with BMMC 24 h after the last challenge (2 × 106); MSC, mice treated withMSC 24 h after the last challenge (1 × 105).

* Significantly different from C-SAL (p < 0.05).** Significantly different from OVA-SAL (p < 0.05).# Significantly different from OVA-BMMC (p < 0.05).

Fig. 4. Representative photomicrographs of lung parenchyma (upper panels) and airways (lower panels), hematoxylin–eosin stain. Collapsed areas in the lung parenchymaand a reduction in central airway diameter, as well as increased cell infiltration, are visible in the OVA-SAL animals. Experimental groups: C, mice sensitized and challengedwith saline; OVA, mice sensitized and challenged with ovalbumin; SAL, mice given saline; BMMC, mice treated with BMMC 24 h after the last challenge (2 × 106); MSC, micetreated with MSC 24 h after the last challenge (1 × 105). Original magnification: 200× and 400× (upper and lower panels, respectively). Bars = 100 �m.

194 S.C. Abreu et al. / Respiratory Physiology & Neurobiology 187 (2013) 190– 198

Fig. 5. Collagen fiber content in the lung parenchyma and airways. Experimental groups: C, mice sensitized and challenged with saline; OVA, mice sensitized and challengedw r the( up. *S(

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ith ovalbumin; SAL, mice given saline; BMMC, mice treated with BMMC 24 h afte1 × 105). Box plots show medians and interquartile ranges for 6 mice in each grop < 0.05). #Significantly different from OVA-BMMC (p < 0.05).

razil). The volume fraction of collapsed and normal pulmonaryreas, the magnitude of bronchoconstriction (contraction index),nd the number of mononuclear and polymorphonuclear cellsn pulmonary tissue were determined by the point-countingechnique across 10 random, non-coincident microscopic fieldsWeibel, 1990; Hsia et al., 2010).

Collagen was quantified in the airways and alveolar septa by theicrosirius polarization method, using Image-Pro Plus 6.0 softwareXisto et al., 2005; Antunes et al., 2009, 2010).

.5. Enzyme-linked immunosorbent assay (ELISA)

Levels of interleukin (IL)-4, IL-13, transforming growth factorTGF)-� and vascular endothelial growth factor (VEGF) in lung tis-ue were evaluated by ELISA using matched antibody pairs fromrepoTech and R&D Systems (Minneapolis, MN, USA), according toanufacturer instructions. Results are expressed as pg/mL.

.6. Statistical analysis

One-way ANOVA on ranks followed by Dunn’s test was used foromparison of between-group differences. Data were expressed asedians and interquartile ranges. All tests were performed using

he SigmaStat 3.1 software package (Jandel Corporation, San Rafael,A, USA), and statistical significance was established as p < 0.05.

. Results

The following subpopulations were identified in theool of injected BMMCs: total lymphocytes (lower SSC,D45+/CD11+/CD29−/CD34− = 19.6%), T lymphocytes (lower SSC/D45+/CD3+/CD34− = 5.4%), T helper lymphocytes (CD3+/CD4+/D8− = 1.98%), cytotoxic T lymphocytes (CD3+/CD4−/CD8+

5.06%), monocytes (CD45+/CD29+/CD11b+ low/CD34−/CD3− 7.24%), hematopoietic progenitors (CD34+/CD45+ = 2.65%), andossible MSCs (CD45−/CD34−/CD11b− = 3.8%). Similarly, MSCsere characterized as CD45−/CD14−/CD34−/CD29+/Sca1+ andere capable of differentiation into osteoblasts and chondroblasts

Fig. 2). The number of MSCs administered was similar to thatresent in the pool of BMMCs.

According to lung function analysis, the OVA-SAL groups exhib-

ted higher Est,L (57%), �P1,L (76%), and �P2,L (53%) as compared

ith the C-SAL group. Both cell therapies were effective for reduc-ion of �P1,L and �P2,L. However, these decrements were moreronounced after BMMC therapy than MSC therapy. Furthermore,

last challenge (2 × 106); MSC, mice treated with MSC 24 h after the last challengeignificantly different from C-SAL (p < 0.05). **Significantly different from OVA-SAL

only BMMC therapy was associated with a significant decrease inEst,L (Fig. 3).

Lung morphometric examination demonstrated a significantincrease in fractional area of alveolar collapse, contraction index,number of mononuclear and polymorphonuclear cells, and colla-gen fiber content in the airways and alveolar septa in the OVA-SALgroup compared to the C-SAL group (Table 1 and Figs. 4 and 5). Bothcell therapies minimized the fractional area of alveolar collapseand polymorphonuclear cell infiltration in lung tissue (Table 1 andFig. 4), and completely reversed changes in the contraction index(Table 1) and airway wall thickness (Fig. 4). Furthermore, both ther-apies decreased the amount of collagen fiber, specifically in thealveolar septa. BMMC therapy led to a more significant reductionin alveolar collapse and collagen fiber deposition in alveolar septaas compared with MSC therapy (Table 1 and Figs. 5 and 6). No sig-nificant difference was observed in the amount of collagen fiber inthe airways after both therapies (Figs. 5 and 6).

Levels of IL-4, IL-13, TGF-� and VEGF in lung tissue werehigher in the OVA-SAL group than in the C-SAL group. BMMC andMSC administration yielded similar reductions in IL-4 and IL-13,whereas TGF-� and VEGF levels presented a greater reduction afterBMMC therapy than after MSC therapy (Fig. 7).

4. Discussion

In the murine model of allergic asthma used herein, BMMC andMSC administration yielded similar beneficial effects on airwayinflammation, as shown by significant reductions in the numberof mononuclear and polymorphonuclear cells and in the levels ofpro-inflammatory cytokines (IL-4 and IL-13) in lung tissue. How-ever, BMMC administration led to greater improvement in lungmechanics and a greater reduction in fractional area of alveolar col-lapse, collagen fiber content in the alveolar septa, and growth factorlevels (TGF-� and VEGF) as compared with MSCs. Our findings sug-gest that both cell types play an important role in the inflammatoryprocess in experimental allergic asthma, but suggest that BMMCsare more effective than MSCs at reducing the remodeling process.

Several studies have investigated the effects of BMMC (Abreuet al., 2011) and MSC (Goodwin et al., 2011; Ou-Yang et al., 2011;Kapoor et al., 2012) administration in experimental asthma. Wehave previously demonstrated that pre-treatment with BMMCscurtails airway inflammation and remodeling and induces lung

repair, thus improving lung mechanics (Abreu et al., 2011). Therationale supporting BMMC therapy relies on the knowledge thatthe functional effects of these cells result from a balance betweendifferent cell types, with involvement of all cells with the potential

S.C. Abreu et al. / Respiratory Physiology & Neurobiology 187 (2013) 190– 198 195

Fig. 6. Representative photomicrographs of lung parenchyma (upper panels) and airways (lower panels), Picrosirius stain. Marked deposition of collagen fibers is visiblein the OVA-SAL groups. Experimental groups: C, mice sensitized and challenged with saline; OVA, mice sensitized and challenged with ovalbumin; SAL, mice given saline;BMMC, mice treated with BMMC 24 h after the last challenge (2 × 106); MSC, mice treated with MSC 24 h after the last challenge (1 × 105). Original magnification: 200× and4

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00× (upper and lower panels, respectively). Bars = 100 �m.

o yield beneficial effects (Mathieu et al., 2009; Araujo et al., 2010;u et al., 2011; Cruz et al., 2012). This hypothesis is supported by therosstalk between multiple cell types that occurs during embryonicevelopment (Rafii and Lyden, 2003). Additionally, BMMCs can bedministered easily and safely, on the day of harvesting, at lowerosts, and without risk of cell rejection (graft-versus-host disease).SCs also lead to beneficial effects in experimental asthma when

dministered during sensitization or before challenge (Firinci et al.,011; Goodwin et al., 2011; Lee et al., 2011). MSCs exhibit multilin-age differentiation potential (Jiang et al., 2002), support adequateissue repair, have immune-privileged features and can be used inllogeneic therapy.

No previous study has compared the effects of BMMCs andSCs in experimental asthma, particularly once the remodeling

rocess is already established. For this purpose, we employed C57BL/6 mouse model of allergic asthma (Abreu et al., 2011),hich features eosinophilia and Th2 pro-inflammatory cytokines

Yu et al., 2006; Allen et al., 2012). Even though early therapy with

MMCs modulates lung inflammation and remodeling regardlessf the route of administration (Abreu et al., 2012), in the presenttudy, both cell types were instilled intratracheally, since a moreirect administration route will ensure delivery of a higher number

of cells to the airway and alveoli (Bonios et al., 2011). To ascertainwhether the effects of BMMC therapy resulted from the balancebetween cell types rather than strictly from the presence of MSCsin the bone marrow pool, approximately the same number of MSCsfound in the bone marrow mononuclear fraction according to flowcytometry was administered to the OVA-MSC group (4% MSCs in2 × 106 BMMCs, approximately 1 × 105 MSCs).

Asthma is an inflammatory disease classically associated withincreased expression of T helper 2 (Th2) cytokines, mainly IL-4 and IL-13. Among other functions, these cytokines induce Th2differentiation bias, fibroblast proliferation, extracellular matrixdeposition, airway hyperresponsiveness, epithelial cell apopto-sis, mucus production, and eosinophil recruitment (Hamid andTulic, 2009). Therefore, they play important roles not only in theinflammatory process, but also in airway remodeling, and are thusconsidered important therapeutic targets (Borowski et al., 2008;Bellini et al., 2011). In this context, both BMMC and MSC cell thera-pies were found to reduce IL-4 and IL-13 levels, possibly as a result

of the decrease in eosinophil infiltration and collagen fiber contentin alveolar septa. Interestingly, these cells were unable to reduceairway fibrosis, which may be explained by the onset of the col-lagen deposition process before initiation of cell therapy, unlike

196 S.C. Abreu et al. / Respiratory Physiology & Neurobiology 187 (2013) 190– 198

Fig. 7. Levels of IL-4, IL-13, TGF-�, and VEGF in lung tissue. Experimental groups: C, mice sensitized and challenged with saline; OVA, mice sensitized and challenged withovalbumin; SAL, mice given saline; BMMC, mice treated with BMMC 24 h after the last challenge (2 × 106); MSC, mice treated with MSC 24 h after the last challenge (1 × 105).B cantl#

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ox plots show medians and interquartile ranges for 6 mice in each group. *SignifiSignificantly different from OVA-BMMC (p < 0.05).

revious studies in which cells were administrated as pretreatmentnd, therefore, before the ultrastructural changes characteristic ofhe remodeling process had occurred (Abreu et al., 2011; Goodwint al., 2011). Further studies are recommended to assess whetherong-term treatment and the administration of repeated doses ofither cell type could further reduce collagen fiber content in theirways.

Both BMMC and MSC administration were effective in mini-izing lung remodeling in the present model of allergic asthma.owever, BMMCs promoted a more marked reduction of TGF-�nd VEGF levels than MSCs. TGF-� is a profibrotic agent, pro-uced by epithelium, fibroblasts and inflammatory cells (mainlyosinophils) (Minshall et al., 1997; Lee et al., 2001). It is capablef inducing epithelial detachment, epithelial–mesenchymal transi-ion, subepithelial fibrosis, and airway smooth muscle hyperplasiand migration, and plays an important role in airway remodel-ng (Halwani et al., 2011). The reduction in TGF-� observed in theresent study was consistent with a previous report (Abreu et al.,011), while another study associated the beneficial effects of MSCherapy with stimulation of TGF-� expression (Nemeth et al., 2010).GF-� also contributes to the increased vascularity of asthmaticirways through induction of VEGF, a key angiogenic moleculeWillems-Widyastuti et al., 2011) that plays a prominent role inhe remodeling process in experimental asthma (Lee et al., 2006).EGF levels also declined after BMMC and MSC therapy, in closeorrelation with the changes observed in TGF-� levels. Therefore,t seems that BMMC and, less efficiently, MSC administration mod-late steps in the airway remodeling pathway involving IL-4, IL-13,osinophils, TGF-�, and VEGF.

Although the pathologic characteristics of asthma are wellnown, the association between these features and physiologicalmpairment is less clear. Furthermore, it is noteworthy that not onlyentral airways, but also distal airways and lung parenchyma, are

y different from C-SAL (p < 0.05). **Significantly different from OVA-SAL (p < 0.05).

involved in the functional changes of asthma (Xisto et al., 2005).In the experimental model of allergic asthma used herein, weobserved histological changes such as increased alveolar collapseand contraction index, which were due to alterations in airwaywall thickness and collagen fiber deposition. These morphologicalchanges led to increased lung static elastance and viscoelastic andresistive pressures respectively. Both cell therapies decreased resis-tive pressure, probably due to an increase in the internal diameter ofthe central airways and a reduction in collagen fiber content in thedistal airways. BMMC therapy led to a more pronounced reductionin viscoelastic pressure and static elastance than MSC administra-tion, a finding that may be associated with explain less alveolarcollapse and reduction in collagen deposition in the alveolar septain the OVA-BMMC group. These results corroborate the findings ofa previous study that evaluated the role of BMMC therapy usingthe same experimental protocol (Abreu et al., 2011). Therefore,the fact that the reduction in these histological changes was morepronounced with BMMC therapy may be associated with greaterimprovement in lung mechanics.

The clinical implication of these findings is associated withthe advantages of using BMMCs over MSCs, namely the fact thatBMMCs may be used in autologous transplantation (thus avoidingpotential cell rejection) and on the same day of harvesting.

This study has some limitations. First, saline was administratedrather than fibroblasts, since fibroblasts have been shown to yieldno beneficial effects (Xu et al., 2007). Furthermore, it is speculatedthat MSCs constitute a unique cell type, distinct from fibroblasts(Martinez et al., 2007). Second, other cytokines and growth fac-tors in addition to those analyzed in this study may be involved

in the airway remodeling process. Third, even though the num-ber of animals in each group was relatively small (n = 6), threesets of experiments were conducted to assess reproducibility andreliability. Finally, BMMCs are a heterogeneous mix that includes

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ematopoietic cells, a variety of inflammatory cell types, and amall number of cells with phenotypic characteristics of MSCs. Pre-linical models have demonstrated that the hematopoietic fractionould differentiate into lineages that could regenerate damagedissue (Lakshmipathy and Verfaillie, 2005), whereas MSCs havemmunomodulatory properties and release trophic factors, accel-rating the repair process and regenerating viable tissue, therebymproving lung function (Ou-Yang et al., 2011). The present study

as unable to evaluate which combination of cells observed in theMMC pool yielded better effects. Further studies are warranted tovaluate additional mechanisms of action of BMMCs and MSCs, asell as the potential role of the combination of different cell types

ound in BMMCs.In conclusion, in the murine model of allergic asthma used

erein, both BMMC and MSC administration were effective ineducing airway inflammation and remodeling and improving lungunction. However, the improvement in lung mechanics and his-ology was more evident after BMMC administration, suggestinghat the interaction between the multiple cell types present inhe bone marrow mononuclear fraction plays an important role inhese processes. These observations have several implications forhe framework of future clinical studies, due to the aforementioneddvantages of BMMCs over MSCs.

cknowledgments

The authors would like to express their gratitude to Mr. Andreenedito da Silva for animal care, Dr. Bruno Paredes for his helpith flow cytometry analysis, Mrs. Ana Lucia Neves da Silva for herelp with microscopy, and Mrs. Moira Elizabeth Schöttler and Ms.laudia Buchweitz for their assistance in editing the manuscript.

Financial support: This study was supported by the Centres ofxcellence Program (PRONEX-FAPERJ), Brazilian Council for Scien-ific and Technological Development (CNPq), Rio de Janeiro Stateesearch Foundation (FAPERJ), Coordination for the Improvementf Higher Education Personnel (CAPES), INCT-INOFAR, Coordi-ation Theme 1 (Health) of the European Community’s FP7TARKINAID).

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