Effects of different mesenchymal stromal cell sources and delivery routes in experimental emphysema

Emphysema, a key feature of chronic obstructive pulmonary disease (COPD), is characterized by the enlargement of air spaces accompanied by destruction of parenchymal structure and impaired pulmonary regeneration [1]. Currently, COPD is the fourth leading cause of death worldwide, and so far there has been no effective therapy for patients with emphysema [2]. One potential therapeutic approach for emphysema has focused on inducing lung repair and regeneration and/or decreasing chronic inflammation by administering mesenchymal stem (stromal) cells (MSCs) of bone marrow or adipose origin [3]. A number of preclinical studies have shown that MSCs attenuate lung inflammation and apoptosis in experimental emphysema [4]-[7]. Furthermore, a recent clinical study showed that the intravenous (IV) administration of non HLA-matched allogeneic bone marrow MSCs in emphysema patients is safe; however, no functional improvement was reported, although a decrease in an inflammatory mediator, C-reactive protein, was observed in treated patients [8]. Nevertheless, depending on the site of origin, MSCs may have different phenotypes, including differences in immunogenicity, anti-inflammatory and regenerative activity, and expansibility in culture [9],[10], which may lead to differing results depending on MSC source. Therefore, further comparative experimental studies are required to better assess the efficacy of different sources of MSCs for use in emphysema.

A further critical aspect for cell transplantation is the selection of the optimal administration route. While IV injection of MSCs is generally utilized in preclinical studies of experimental emphysema, due to ease of administration and subsequent wide biodistribution [4],[6],[11],[12], intratracheal (IT) administration of MSCs also attenuates lung damage [13],[14]. Thus, no definite conclusion has been reached regarding the optimal administration route of MSCs in experimental emphysema.

The aims of the present study were to: (a) comparatively assess the extent to which different sources (bone marrow, adipose, or lung tissue) of MSCs are able to decrease inflammation and promote alveolar epithelium and endothelium repair, thereby improving lung function in elastase-induced emphysema in mice, (b) investigate whether IV versus IT administration of MSCs influences their effectiveness on lung inflammation and remodeling, and (c) evaluate the effects of IV versus IT delivery of the aforementioned different sources of MSCs on elastase (emphysema)-induced changes in cardiac function.

This study was approved by the Ethics Committee of the Health Sciences Centre, Federal University of Rio de Janeiro. All animals received humane care in compliance with the "Principles of Laboratory Animal Care" formulated by the National Society for Medical Research and the U.S. National Research Council "Guide for the Care and Use of Laboratory Animals".

Intravenous administration of lung-derived MSCs led to immediate death of all mice, which may be associated with the larger size of the L-MSCs (see below) or with cellular clumping resulting in pulmonary embolism. Thus, this group was not included in further analysis. Conversely, survival rate in all other groups was 100%. As no significant differences in any endpoint measures were observed between any of the C groups (Table 1), henceforth a single C group, which consists of the average of all C groups, was reported.

To our knowledge, this was the first study to compare the potential therapeutic effects of three different sources of MSCs, delivered through two different administration routes, on lung inflammation and remodeling and on cardiovascular function in experimental emphysema induced by repeated doses of elastase. In contrast to the classical single-dose protocols of elastase-induced emphysema [26], which induce only emphysema-like lesions without systemic [27] or cardiovascular impairment, the present model, developed in our laboratory [11], results in lung histological and ultrastructural changes and cardiac impairment that resemble human emphysema. Using this model, all studied MSC groups (with the exception of the IV L-MSC group), regardless of administration route, exhibited decreased Lm, neutrophil infiltration, and cell apoptosis; increased elastic fiber content; reduced alveolar-capillary membrane and type II epithelial and endothelial cell ultrastructural damage; and decreased KC and TGF-β expression in lung tissue. Therefore, MSC administration can modulate the inflammatory and remodeling processes of emphysema; however, specific beneficial effects can differ depending on MSC source and administration route.

While all MSCs share similar general properties, cells from different sources can exhibit significant differences in anti-inflammatory or regenerative potency depending on the particular injury being addressed [28]. Recent studies have compared the characteristics of adult MSCs from different sources [9],[10],[17],[29], and have demonstrated distinct effects in different experimental models, even when cells have similar proliferation and differentiation capacities [30],[31]. The relevant mechanisms whereby different MSCs populations have distinct actions in the same disease model remain unclear. In our study, the beneficial effects of MSCs varied according to source.

BM-MSCs are well-characterized and currently the most widely used [16]; however, they require an invasive harvesting process and have limited availability. Like BM-MSCs [4],[7],[13], AD-MSCs have also demonstrated promising effects on the maintenance of vascular integrity by secreting anti-apoptotic and pro-angiogenic factors [32], and reduce inflammation in experimental emphysema [6],[33]. In adults, these cells are easy to obtain in large quantities by liposuction, which makes them good candidates for therapeutic use and facilitates autologous transplantation [34]. More recently, highly proliferative and clonogenic MSC populations have also been isolated from explants [35] and allografts [36] of adult lung tissue. L-MSCs are immunoprivileged, do not express MHC II or the co-stimulatory molecules CD80 or CD86 [37], and can inhibit T cell-based allorecognition [36], facilitating the success of allogeneic transplants. Additionally, L-MSCs express several basement membrane proteins and growth factors which seem to amplify their retention in the injured tissue [12],[14], making them promising candidates for cell-based therapy in lung diseases. However, there is limited information regarding the effects of L-MSCs in experimental emphysema [12],[14],[35]. In contrast to the present study, Hoffman et al. (13) observed no death after IV delivery of L-MSCs in mice. The reasons for this discrepancy are unclear; one potential explanation is the route (jugular versus tail vein) chosen for cell administration. Since no significant differences between saline and lung fibroblasts were observed in our pilot studies, nor in previous reports [38],[39], saline was administered as control instead of mouse fibroblasts in the present investigation.

Reports have demonstrated that a direct pathway of delivery - e.g., IT for lung diseases [24] and intra-myocardial for acute ischemia-reperfusion [40] - may result in greater retention of MSCs in the target tissues. However, in an experimental model of ventilator-induced lung injury, MSCs enhanced recovery and repair regardless of administration route (IT vs. IV) [38]. Recent evidence demonstrated that neither IV nor IT administration of BM-MSC is able to revert lung histology in a single dose protocol of elastase-induced emphysema [41], while in the present study, IT administration of BM-MSCs led to a greater reduction in alveolar hyperinflation than IV delivery. This dissociation between the beneficial effects observed in the present study with IT administration versus those obtained with IV administration may be associated with great loss of alveolar membrane surface area in emphysema, resulting in reduced endothelial cell adhesion molecules [42] and, thus, decreased MSC adhesion.

In our emphysema model, the increase in Lm was associated with neutrophil infiltration of lung tissue and M1 macrophage polarization. Conflicting evidence on the effects of smoking in downregulating M1 macrophages has been published [43]-[46]. Macrophages can be activated by various extracellular signals to polarize toward either the M1 (inflammatory and antimicrobial) or the M2 (wound repair and inflammation resolution) phenotype. The enhanced M1 polarization observed in lung tissue in our experiment is in line with some reports, which evinced an increase in pro-inflammatory macrophages and a reduction of chemokine ligand 18 (CCL18), a chemokine expressed by alternatively activated macrophages, in the bronchoalveolar lavage fluid of smokers compared to nonsmokers [43],[44]. In the present study, M1 activation was similarly inhibited by IV delivery of BM-MSC and AD-MSC. However, only BM-MSC therapy stimulated the M2 phenotype, and more effectively when given IV than when administered IT. The differences in M1 and M2 phenotype observed according to the MSC source and route of administration may be explained by the existence of an "environmental-niche memory" in BM-MSCs and an "epithelial" commitment of AD-MSCs, as described in a previous report [29].

In the present study, BM-, AD-, and L-MSCs seemed to differentially modulate production of some chemokines and growth factors associated with the pathophysiology of emphysema. Increasing evidence demonstrates that the pathogenic changes mediated by MSCs are highly sensitive to the microenvironment to which these cells are exposed. For example, MSC-conditioned media may be a rich source of TGF-β secretion and lead to an increase in collagen gene expression [47]. Conversely, in an experimental model of bleomycin-induced fibrosis, BM-MSCs reduced lung tissue TGF-β levels and soluble collagen in lung extracts [48]. In emphysema, increased TGF-β secretion by epithelial cells [49] is associated with progressive small-airway fibrosis. In our study, a similar reduction of TGF-β levels was observed in all MSC-treated groups, regardless of the delivery route; however, it was not accompanied by equal decrease in deposition of collagen fibers in the small airways. Previous reports demonstrated the ability of MSCs to stimulate VEGF production in vitro[13],[33] and in vivo [11],[13] in experimental models of emphysema. We observed that IV administration of BM-MSC and AD-MSC increased VEGF levels in lung tissue, which was not observed with the IT route. Based on previous studies of cardiac revascularization [50],[51], we hypothesize that the systemic injection of BM-MSCs and AD-MSCs results in direct contact with the remaining endothelial cells of the pulmonary vasculature, stimulating them to synthesize VEGF. Intense neutrophilia in the sputum of COPD patients correlates positively with disease severity and high production of IL-8 [52]. This chemokine is released by alveolar macrophages when stimulated by pollutant particles, and is responsible for massive neutrophil recruitment to the lungs. We observed that all MSC therapies similarly reduced KC and neutrophilia in lung tissue.

Only IV administration of BM-MSC and AD-MSC reverted the reduction in the PAT/PET ratio, which may be associated with the inhibition of pulmonary microvasculature muscularization and stimulation of VEGF-induced angiogenesis [4]. Nevertheless, these changes did not result in modifications in right ventricular area, probably due to the timing of analysis and the small number of cells that reach the heart.

Despite the major lung morphology changes induced by our model of emphysema, no significant changes in Est, L were observed. This is in agreement with other studies using different experimental models of emphysema, which showed dissociation between the degree of tissue loss and pulmonary dysfunction [53]-[55].

Several limitations of this study should be considered: (1) the absence of MSC tracking after IT or IV administration, limiting our knowledge regarding the delivery dynamics of each cell lineage; (2) the experimental period of 5 weeks, which may not be enough to understand the late effects of MSC therapy; and (3) only a few specific cytokines and growth factors were evaluated; a wider range of mediators should be analyzed to provide a more complete understanding of the mechanisms associated with each cell type. Additionally, more extensive analysis of the range of soluble mediators released by each MSC type may provide further information on the different effects noted in this model.

In conclusion, all three MSC sources tested (BM-MSC, AD-MSC and L-MSC), regardless of the administration route (with the exception of the IV L-MSC group), attenuated lung damage in this mouse model of elastase-induced experimental emphysema. Nevertheless, MSCs from different sources exhibited distinct effects on the different aspects of lung and cardiovascular injury, through mechanisms that remain unclear. Further research comparing the effects of different MSC sources and routes of administration is required.

MAA participated in the design of the study, carried out the experiments, performed data analyses and drafted the manuscript; SCA and FFF contributed to the study design, carried out the experiments, performed data analyses, and wrote the manuscript; ACT and MLP, carried out the histological analyses and contributed to the manuscript; EB and PCO provided expert assistance during experiments, analyzed flow cytometry data, and helped draft the manuscript; BLD and CMT contributed to the study design, supervised ELISA and histological analysis; IPRGF and NRR performed echocardiographic analysis and helped draft the manuscript; VLC performed the electron microscopy analysis and helped draft the manuscript; DGX participated in the design of the study, carried out the experiments and drafted the manuscript; DJW, MMM, and PRMR contributed to study design, supervised experimental work and statistical analysis, and wrote the manuscript. All authors read and approved the final manuscript.