The major findings of this study are the following. 1) Allogeneic human MSC restored epithelial protein permeability across primary cultures of human alveolar epithelial type II cell monolayer after cytomix-induced injury. 2) Secretion of the paracrine-soluble factor Ang1 by human MSC mediated a major fraction of this protective effect on human ATII permeability after the inflammatory insult. 3) The beneficial effect of Ang1 secretion on human ATII permeability was mediated by a direct effect on cytoskeletal re-organization of both actin and claudin 18, in part through suppression of NFκB activity. To our knowledge, this is the first study to demonstrate the potential therapeutic use of allogeneic human MSC and/or recombinant Ang1 on lung epithelial permeability to protein in ATII cells after an inflammatory insult.
Alveolar barrier integrity is critical for the prevention of permeability pulmonary edema and is maintained by both lung endothelium and epithelium. The alveolar epithelium, consisting of both ATI and ATII cells, forms the tighter membrane of the two. ATII cells exert other critical functions in the lung as well such as surfactant protein synthesis and alveolar fluid clearance through sodium and chloride transport. Disruption of this epithelial barrier occurs due to mechanical and inflammatory stress and leads to the influx of protein-rich fluid and inflammatory cells. This is manifested by hypoxemia, atelectasis, and pulmonary edema, which are the main characteristics of patients with ALI/ARDS. We have reported that human ALI pulmonary edema fluid itself increased protein permeability in our in vitro
model of primary cultures of human alveolar epithelial type II cells (34
). In line with this prior study, we demonstrated that ATII cells exposed to a mixture of the major biologically active cytokines found in ALI pulmonary edema fluid, IL-1β, TNFα, and IFNγ (referred to as cytomix), resulted in a significant increase in protein permeability by 500% over base line (). The most significant finding of the current study was that the simultaneous addition of allogeneic human MSC to the bottom chamber of the Transwell plate restored epithelial protein permeability after cytomix exposure ().
Several studies have demonstrated that stem cell-based therapies may offer an effective intervention to lung diseases (16
). Initially, most early studies postulated that the therapeutic effect was from the capacity of MSC to localize to injured lung tissues and differentiate into specific cell types as the principal mechanism of tissue repair. Different biological mechanisms were postulated to mediate this lung engraftment (6
); 1) trafficking of MSC to a local niche of progenitor cells in the lung, 2) trans-differentiation into specified lung cells, or 3) fusion of MSC with differentiated resident cells. Using bleomycin as a model of lung injury, Ortiz et al.
) found engraftment of MSC in the lung localized to areas of injury. Liebler et al.
) also demonstrated a low level of retention of human bone marrow-derived cells in a murine xenograft transplantation model after bleomycin-induced lung injury. Stromal derived factor-1 levels in the injured tissues seemed responsible for homing and engraftment of CXCR4+ cells (38
). Rojas et al.
) found a high retention and differentiation rate (up to 29%) of intravenously administered MSC in bleomycin-injured murine lungs. In this study the authors could not rule out potential cell fusion between donor and resident cells and hypothesized that additional chemotaxis of endogenous bone marrow cells contributed to the therapeutic effect. However, in most subsequent studies of MSC used in lung injury (19
), engraftment rates approached <5%, suggesting that the magnitude of MSC on repair appeared out of proportion to the number of donor-derived mesenchymal cells found in the lungs.
Therefore, the mechanistic focus underlying the therapeutic benefit of MSC shifted largely to the immunomodulatory properties of secreted paracrine factors such as IL-10 (26
) and IL-1ra (24
). We reported that MSC administration could shift the balance from a pro-inflammatory to an anti-inflammatory cytokine profile in a mouse model of LPS-induced lung injury (26
). In that study, intratracheally administration of MSC resulted in lower levels of TNFα and MIP-2 in bronchoalveolar lavage fluid and plasma, which was associated with an up-regulation of the anti-inflammatory cytokines IL-10, IL-13, and IL-1ra. In another study, Xu et al.
) showed a reduction of the systemic inflammatory response when MSC were injected intravenously in a mouse model of LPS sepsis. The cells localized initially to the lung but did not engraft, and the effect was associated with the reduction in pro-inflammatory cytokines produced by the injured lung tissue, suggesting a paracrine effect. Moreover, it has been subsequently shown that MSC produced and secreted a broad variety of cytokines, chemokines, and growth factors (41
). From these data, we hypothesized that the beneficial effects of MSC treatment might be related to the secretion of paracrine-soluble factors.
In search of such a potential soluble factor, we found that human allogeneic MSC secreted a significant amount of Ang1 that increased by 270% with an inflammatory stimulus (). More importantly, using siRNA techniques, Ang1 secretion by MSC was responsible for the therapeutic effect of MSC on epithelial permeability to protein across primary cultures of polarized human alveolar type II cells injured by cytomix (). Interestingly, exposure of MSC to cytomix in the presence of human ATII cells increased the secretion of Ang1 further than with MSC alone, suggesting a potential cross-talk between human ATII cells and MSC (). The biological importance of Ang1 was further supported by using rhAng1 as a positive control, confirming its anti-permeability effect in alveolar epithelial type II cells ().
Angiopoietin 1–4 are a family of growth factors that function as ligands for the Tie2 receptor. Tie2 is a member of a distinct family of receptor tyrosine kinases and is involved in angiogenesis including destabilization of existing vessels, endothelial cell migration, tube formation, and the subsequent stabilization of newly formed tubes by mesenchymal cells (42
). Tie2 or Ang1 deficiency results in embryonic lethality, the absence of vessel remodeling, and decreased endothelial cell survival (35
). Ang1 (44
) and Ang2 (45
) are the most studied ligands of Tie2. Binding of the agonist Ang1 to Tie2 mediates rapid receptor autophosphorylation. In contrast, binding of Ang2 to Tie2 has an antagonistic effect. Tie2 protein is also present in quiescent endothelial cells in a range of adult tissues and is constitutively activated to maintain a resting state and cell survival (46
). It has been demonstrated that Ang1 is an important stabilizing, anti-inflammatory, and anti-permeability factor targeting the mature vascular endothelium in addition to its angiogenic properties (29
). In our transwell model, in accordance with studies of endothelial cells, we found high levels of Tie2 phosphorylation in alveolar epithelial type II cells at base line. In several models of systemic inflammation (49
), Ang-2 is responsible for Tie2 dephosphorylation resulting in increased vascular permeability. Among ATII cells, we found that cytomix reduced the activated Tie2 protein levels directly, whereas secretion of Ang1 by MSC restored the phosphorylated form of the receptor ().
The potential role of Ang1 in acute lung injury has recently been addressed by other investigators as well. Karmpaliotis et al.
) observed that endogenous Ang1 expression was decreased in response to endotoxin in a murine model of ALI. McCarter et al.
) demonstrated that skin fibroblasts transfected with plasmid vectors overexpressing human Ang1 improved morphological, biochemical, and molecular indices of lung injury and inflammation in a rat model of ALI. In Tie2 heterozygous-deficient mice (Tie2+/−
) or binary transgenic mice in which doxycycline-conditional Ang1 overexpression was targeted to endothelium using the Tie1 promoter, the same authors found that overexpression of Ang1 blunted endothelial adhesion molecule expression, increased HO-1 and endothelial nitric-oxide synthase expression, and decreased ET-1 expression, all of which likely contributed to reduced airspace inflammation, intra-alveolar septal thickening, and early mortality. In similar experiments, Mei et al.
) and Xu et al.
) transfected mouse MSC with human Ang1 to use as a cellular vector in the treatment of murine models of LPS induced lung injury. Mei et al.
) showed that treatment with MSC alone or MSC transfected with Ang1, both, reduced airspace inflammation, but MSC transfected with Ang1 had a more profound effect. In their study, Xu et al.
) reported an improvement in several lung inflammatory markers (alveolar permeability, neutrophil activation, inflammatory cytokines) with MSC transfected with Ang1 after lung injury. In contrast with our findings, these investigators could not detect Ang1 production by mouse MSC alone, possibly due to the species specificity of the ELISA used. Interestingly, the levels of Ang1 produced by transfected MSC were comparable with our levels of Ang1 secretion by human MSC in the Transwell model. In this project, our finding that human alveolar epithelial type II cells contain the Tie2 receptor may have implications in interpreting previous in vivo
studies of lung protein permeability in mice. In the future, studies will need to be performed using transgenic mouse studies using Tie2-cre for endothelial-specific gene deletion experiments to differentiate the contribution of the epithelium and endothelium to lung permeability.
For the first time, in addition to its an anti-permeability effect on endothelium, we have found that the effect of MSC and Ang1 on epithelial permeability to protein in cultured monolayers of ATII cells after cytomix-induced injury depended in part on cytoskeletal reorganization. Epithelial barrier properties are maintained by intercellular junctional complexes composed of tight junctions, adherens junction, and desmosomes. Tight junctions form a physical barrier and limit the diffusion of proteins and lipids (51
). Interaction with several scaffolding proteins ultimately links the tight junction to the actin cytoskeleton, and its integrity is reflected in limited paracellular tracer flux of large molecules such as labeled albumin. Interaction of actin and myosin, the main components of the anchored cytoskeleton, regulates the tension throughout the cells. In the resting state, they form a dense ring encircling the cell. Cellular activation such as by inflammatory insult (cytomix) promotes actin disorganization or stress fiber formation and increases the centripetal tension. This augmented physical stress on the tight junctions might explain the increase in paracellular permeability often seen (52
). This mechanism involves activation of myosin light chain kinase, resulting in phosphorylation of MLC-2 and their interaction with actin fibers. In our experiments, immunofluorescence staining showed this redistribution of actin fibers and its interaction with phospho-MLC-2 after cytomix exposure, which was restored by MSC or rhAng1 treatment (). Two members of the small GTPases, RhoA and Rac1/2/3, have important opposing effects in this process. Activation of RhoA induces stress fiber formation and was activated immediately by cytomix administration (). Activation of Rac1/2/3 counterbalances this and was increased by MSC treatment (36
) (), suggesting one mechanism of MSC for improvement in lung epithelial protein permeability. However, the simultaneous addition of a ROCK inhibitor, Y-27632, preventing RhoA activation, had no beneficial effect on overall epithelial protein permeability after cytomix exposure in our Transwell model (A
). In contrast, the simultaneous addition of a myosin light chain kinase inhibitor, ML-7, fully restored epithelial protein permeability to a normal level after cytomix exposure, suggesting that the effect of MSC may be distal to RhoA activation and/or directly involve the formation of actin stress fibers (B
Based on the lack of effect of a RhoA inhibitor on epithelial protein permeability, we studied the effect of cytomix on tight junction proteins. By immunofluorescence and Western blot analyses, we were unable to detect an effect of cytomix on total protein levels or the distribution of occludin, claudin 3, 4, and 5, and JAM-A or its associated proteins, ZO-1, E-cadherin, or β catenin ( and ). In contrast to the studies by Wray et al.
) in a model of ventilator induced acute lung injury, we were unable to detect an increase in claudin 4 expression in primary cultures of human alveolar type II cells injured with cytomix. Consequently, we were unable to establish the role of claudin 4 in response to an increase in paracellular epithelial permeability. However, in our model cytomix decreased total protein levels of claudin 18 and caused a re-organization away from the periphery to a disorganized state (). The simultaneous addition of MSC or BMS-345541, a NFκB inhibitor, partially restored total protein levels and the distribution of claudin 18 after cytomix exposure (), suggesting an important role of this tight junction protein in barrier permeability. The addition of rhAng1 instead of MSC also had a similar effect on the redistribution of claudin-18 to the periphery or cell junction (), again demonstrating the important role of Ang1 secretion. This conclusion was supported by a recent manuscript by Koval et al.
) who demonstrated that increased claudin-18 localization to the plasma membrane was associated with higher barrier resistance in primary cultures of rat alveolar epithelial type II cells. To determine how MSC restored claudin 18 levels, we measured the effect of simultaneous addition of recombinant Ang1 among primary cultures of human type II cells injured by cytomix on NFκB activation. The addition of rhAng1 partially reduced NFκB activation among human type II cells after cytomix exposure at 3 h, suggesting how MSC may restore lung epithelial permeability. More importantly, the addition of BMS-34551 or a NFκB p50 inhibitory peptide fully restored epithelial protein permeability to control levels among ATII cells after cytomix exposure (, C
). In the future, the individual contributions of “actin stress formation” and/or claudin 18 re-organization on epithelial protein permeability will need to be clarified further.
There are some limitations to the current study. The underlying hypothesis of these studies rested on the contribution of MSC to alveolar epithelial barrier integrity paracrine mechanisms or cell contact independent effects. However, as reported by Németh et al.
), the role of cell-contact may influence the immunomodulatory effect of MSC, which may influence lung epithelial permeability by changing the inflammatory milieu of an injured alveolus. In the future we will need to know the role, if any, of both the immunomodulatory effects and cell contact effect of MSC on epithelial permeability; the Transwell model cannot be used to answer this question due to the lack of other cells types such as macrophages and neutrophils. In addition, the therapeutic efficacy of MSC on epithelial permeability may in part result from the effects of secreted growth factors on alveolar type II cell apoptosis or growth. However, the role of cell death is probably not relevant in this model because we found that cytomix did not cause an increase in human alveolar type II cell apoptosis or necrosis. Finally, our study does not address the role of alveolar epithelial type I cells.
In conclusion, angiopoietin-1 secretion by human MSC can restore epithelial permeability to a normal level in cultured human type II cells. These effects are at least partially regulated at the level of cytoskeletal reorganization. Although it has been well known that Ang1 can restore the barrier properties of endothelial cells, the capacity of Ang1 to restore protein permeability in lung epithelial cells is novel and may provide insight into the mechanisms by which MSC have therapeutic effects in experimental studies of acute lung injury.