Paracrine factors from mesenchymal stem cells: a proposed therapeutic tool for acute lung injury and acute respiratory distress syndrome

Abstract

Despite extensive researches in acute lung injury (ALI) and acute respiratory distress syndrome (ARDS), current pharmacological therapies and respiratory support are still the main methods to treat patients with ALI and ARDS and the effects remain limited. Hence, innovative therapies are needed to decrease the morbidity and mortality. Because of the proven therapeutic effects in other fields, mesenchymal stem cells (MSCs) might be considered as a promising alternative to treat ALI and ARDS. Numerous documents demonstrate that MSCs can exert multiple functions, such as engraftment, differentiation and immunoregulation, but now the key researches are concentrated on paracrine factors secreted by MSCs that can mediate endothelial and epithelial permeability, increase alveolar fluid clearance and other potential mechanisms. This review aimed to review the current researches in terms of the effects of MSCs on ALI and ARDS and to analyse these paracrine factors, as well as to predict the potential directions and challenges of the application in this field.

Properties of mesenchymal stem cells

Mesenchymal stem cells (MSCs) were found originally in bone marrow 1, 2. Currently, in addition to from bone marrow, MSCs could also be isolated from placenta, umbilical cord 3, amniotic fluid, and so on 4. MSCs are adherent cells with a fibroblast‐like morphology 5 and are generally evaluated by three main biological criteria: plastic‐adherent cultured cells, self‐duplication and differentiation into different cells, such as osteoblasts, adipocytes, cartilage cells, nerve cells and myocardial cells. MSCs could positively express cell surface markers including CD73, CD90 and CD105 and negatively express CD45, CD34, CD14 or CD11b surface molecules 6, 7. According to the mesenchymal and tissue Stem Cell Committee of the International Society for Cellular Therapy, the unique surface marker still has not been found in numerous markers.

MSCs have been reported to possess more multifunctions than before both in vivo and in vitro, so they can be potentially used as a promising cell‐based therapeutic candidate in different fields. So far, the premedical experiments and clinical usage are based on the following primary properties: homing to inflammatory sites following injury, differentiating into various cell types in conditioned setting, secreting multiple soluble factors that are capable of repairing injured cells and attenuating injury and performing immunomodulatory as lacking immunogenicity 7.

To date, there have been more than 350 clinical trials of human MSCs registered in ClinicalTrials.gov. MSCs have been used in various fields, such as haematopoietic stem cell transplantation, graft‐versus‐host disease 8, 9, 10 as well as tissue injury including damages of bone, cartilage, joint and myocardium 11, 12. In recent years, MSCs have also been considered to be a potentially powerful new approach for the management of lung diseases including acute lung injury (ALI) and acute respiratory distress syndrome (ARDS).

Characteristics of ALI and ARDS

Aetiology and pathological process

In hospital, ALI is one common complication caused by various reasons, such as trauma, infection, sepsis, extracorporeal circulation and extensive burns excluding cardiogenic factors 13. ALI and ARDS are considered two continuous stages according to pathological process. In comparison to ALI characterised by acute and progressive hypoxaemia, ARDS is characterised by severe hypoxaemia and progressive dyspnoea. However, hypoxaemia is difficult to be reversed. The patients with ALI always evolve into ARDS or even more severe condition, if not treated timely.

Alveolar‐capillary membrane consisting of pulmonary alveolar epithelium and vascular endothelium is always damaged by various inflammatory factors including interleukin‐1β (IL‐1β), tumour necrosis factor‐α (TNF‐α) and interferon‐γ (IFN‐γ). On account of inflammatory insult, pulmonary oedema appears and diffusion dysfunction of carbon dioxide and oxygen will ensue as the promoted permeability of alveolar‐capillary membrane 14. Pulmonary compliance will always be lowered owing to the reduction of alveolar surfactant secreted by type II alveolar epithelial cell (AEC‐II) 15; therefore, pulmonary atelectasis and respiratory failure occur easily.

Treatment and outcome

As the effective treatment for ALI and ARDS is limited, mortality and morbidity remain high. So far, the effectively therapeutic approaches for ALI and ARDS include respiratory support and pharmacological treatments. However, the broad documented application of pharmacological therapies including inhaled surfactant, nitric oxide, prostacyclins, glucocorticoids, ketoconzaole, antioxidants, β‐agonists and pentoxifylline has not been evaluated effectively in reducing mortality for ALI so far. To date, the most significant advances in supporting ALI patient have been associated with improved ventilator management 16. Prognosis varies depending upon numerous causes involved. If the primary inflammation is not controlled in time, prognosis of ALI and ARDS is generally poor. If bone marrow transplantation was complicated by ARDS, mortality is always 100%. In this situation, innovative therapies are therefore needed 17. To date, cell‐based therapy and gene therapy are attractive approaches 18, 19. Many experiments are being in research to find more effective treatment 20 and now MSC‐mediated therapy is considered as a more promising method owing to the potential risks in gene therapy.

Current mechanisms

In preclinical researches, several ALI animal models are always built for researches, such as intratracheal instillation of bleomycin in mice, injecting endotoxin intraperitoneally in rat and administering lipopolysaccharide (LPS) through intrapulmonary in mice 21, 22. Although MSCs have been evidenced to play an important role via various functions in treating ALI, the most effective mechanism is unclear. Hence, the potential mechanisms reported currently are discussed.

Engraftment and differentiation

Researchers administered MSCs from a green fluorescent protein (GFP)‐donor to injured mice lung model induced by bleomycin and found MSCs appear in injured lung by immunofluorescence photographic technique. MSCs found in the lung showed phenotypic characteristics of AECs including type I AEC (AEC‐I), AEC‐II, fibroblasts and endothelial cells. MSCs have been proved to obtain more multiple plasticities in inflammatory situation 23, 24. Bone marrow‐derived cells were proved to engraft into alveolar space and exhibit specific characteristics to differentiate into lung epithelial cells. Additionally, some aspirated bone marrow cells cultured in vitro could express markers including T1 and aquaporin‐5 of AEC‐I 24. Yamada et al. delivered LPS to build ALI model in mice and bone marrow‐suppressed animal model by sublethal irradiation. Disruption of lung tissue structure and emphysema‐like change appeared. They treated the mouse model with bone marrow‐derived cells from GFP‐donor and found abundant GFP‐positive cells in the injured lungs later. Some cells expressed cytokeratin as one marker of epithelial cells, whereas others expressed CD34. This report suggested that marrow‐derived progenitor cells are important in repairing lung injured by LPS 25.

It appears that bone marrow‐derived cells could engraft to injured lung and express specific biomarker of AEC. However, engraftment rate of MSCs to injured alveoli proved still lower 26. It suggests that direct engraftment and differentiation of MSCs in the lung are less likely the exact therapeutic mechanisms.

Regulation of the inflammatory response

MSCs are reported to attenuate the damaged lung because of its anti‐inflammatory properties. For instance, Xu et al. infused LPS intraperitoneally into mice. MSCs were injected intravenously 1 hour later and a significant reduction in neutrophil counts in histological sections appeared 6 hours later. Differences between the experimental groups were larger 24 hours after endotoxaemia and the next time point. LPS caused an acute systemic inflammatory response, which is reflected as increased serum concentrations of the proinflammatory mediators, such as IFN‐α, IL‐1β and macrophage inflammatory protein‐1. Administration of bone marrow‐derived MSCs moderated the increase in each of these proinflammatory mediators in serum. This suggested that allogeneic MSCs might play an important role in repairing inflammatory damage injured by LPS 27.

MSCs therapy could reduce alveolar exudate and the levels of proinflammatory cytokines in plasma. MSCs increased the levels of several anti‐inflammatory cytokines including interleukin‐10 (IL‐10) reported to inhibit the rolling, adhesion and transepithelial migration of neutrophils. In mice model of sepsis through caecal ligation and puncture, bone marrow‐derived MSCs are reported to secrete prostaglandin E2, which reprogrammed alveolar macrophages to secrete IL‐10 28.

In a bleomycin‐induced model, the investigators administered MSCs to mice and found MSCs blocked the production of IL‐1 receptor antagonist. Therefore, MSCs might provide a novel way in treating lung injury 29.

Potential mechanisms

In research of MSCs, more researchers concentrated on the effect on the integrity of alveolar‐capillary membrane, permeability of alveolar‐capillary membrane and the alveolar fluid clearance (AFC) of alveolar epithelium.

For some solutes moving through alveolar epithelial and capillary endothelium, radii of the pores were estimated to be 0·6–1·0 and 4–5·8 nm, respectively. Resistance for liquid through capillary to alveolus mainly originates from the alveolar epithelium and capillary endothelium. In addition, more resistance for albumin moving through the alveolocapillary barrier mainly originates from alveolar epithelium other than capillary wall 30, 31.

Maintain integrity of alveolar‐capillary membrane

Some paracrine soluble factors of MSCs have been reported to improve inflammation. Cell‐based angiopoietin‐1 (Ang‐1) gene transfer improved morphological, biochemical and molecular changes in lung injury and inflammation. These findings were confirmed in a rat model in which Ang‐1 reduced the activation of endothelial cell associated with a marked improvement in airspace inflammation. These results support a critical role of the pulmonary vascular response to lung injury 26, 32.

The alveolar epitheliums normally form a tighter barrier than the capillary endothelium, and its loss of integrity is of great significance in ALI. Some investigators sought to study the effect of MSCs on the alveolar epithelium exposed to the inflammatory cytokines. Protein permeability across the epithelial layer was increased by approximately 500% in this setting. When alveolar epitheliums were co‐cultured with allogeneic MSCs, protein permeability of monolayer was reduced to control level by Ang‐1 secreted by MSCs. MSCs no longer prevented the increased epithelial permeability after blocked by small interfering RNA (siRNA) 33, 34.

Improving AFC

AFC is the resolution to clear pulmonary oedema by alveolar epitheliums consisting of sodium channels, aquaporin and sodium–potassium adenosine triphosphatase (Na‐K‐ATPase). AFC is always damaged in inflammatory situation. The level of AFC impairment has significant prognostic value in determining morbidity and mortality 35. In the alveolar environment, AFC is mainly determined by amiloride‐sensitive, insensitive sodium channels and the activity of Na‐K‐ATPase 36. Both AEC‐II and AEC‐I have been evidenced to exert effect on AFC according to the research in pulmonary oedema. Although AEC‐II played an important role in this report, now more researchers focus on AEC‐I in research of clearing pulmonary oedema. Therefore, AFC of alveolar epitheliums plays an important role in clearing pulmonary oedema. The documented paracrine factors secreted by MSCs in process of ALI are listed in Figure 1.

Figure 1.

Injured alveolus with an influx of protein‐rich oedema fluid is shown. MSCs can be delivered via the circulation and appear in alveolus. The therapeutic property of MSCs mainly relies on both paracrine mechanism in both AEC‐I and AEC‐II. Lung oedema is cleared by active Na⁺ transport from the apical to the basolateral surface mostly via apical ENaC and basolaterally located Na‐K‐ATPase with water following isosmotically the Na⁺ gradient. AEC, alveolar epithelial cell; AQP, aquaporin; ENaC, epithelial sodium channel; Na‐K‐ATPase, sodium–potassium adenosine triphosphatase; MSCs, mesenchymal stem cells.

AEC‐II is cuboidal cells with a diameter of less than 10 µm; nevertheless, AEC‐I is squamous cells with a diameter ranging from 50 to 100 µm. AEC‐I covers more than 90% of the alveolar surface. The numbers of AEC‐I and AEC‐II cell are similar in populating the alveolar area 36, 37, 38.

AEC‐II was responsible for vectorial transport of sodium from the apical to the basolateral surface 39, 40. Transepithelial sodium transport at the apical surface of alveolar epithelium is mediated mainly by amiloride‐sensitive epithelial sodium channels (ENaCs). Subsequently, sodium is pumped actively from the basolateral surface into the lung interstitium by Na‐K‐ATPase. The active transport of sodium through alveolar epithelium provides a major driving force for removing fluid from the alveolar space. ENaC comprised three subunits including α‐, β‐ and γ‐ENaC widely distributed in epithelia of the lung 41, 42, 43.

AEC‐I has the highest permeability to water possibly contributed by aquaporin in mammalian research. This strongly supports the role of AEC‐I in vectorial ion transport 38, 44, 45. Na‐K‐ATPase comprised α‐ and β‐subunits which are expressed in both AEC‐II and AEC‐I and in sodium pumps located on the basolateral surface. AEC‐I pumps sodium out of alveolar epithelium in exchange for potassium influx by consuming ATP to maintain sodium and potassium gradients across the plasma membrane. The basolateral membrane site of Na‐K‐ATPase is critical for alveolar fluid reabsorption, where the sodium transport is followed by outflux of water in an isosmolar manner 46, 47.

It has been reported that paracrine growth factors secreted by MSCs containing epidermal growth factor (EGF) 48, 49, transforming growth factor (TGF) 50, keratinocyte growth factor (KGF) 51, 52 and fibroblast growth factor‐10 could influence sodium uptake and alveolus fluid transport in clearing pulmonary oedema through different mechanisms. Bone marrow‐derived MSCs have been known to produce various growth factors, specifically KGF. KGF was evidenced to reduce lung injury and pulmonary oedema in animal experiments 53. Through intrabronchial instillation of human MSCs 1 hour following endotoxin‐induced lung injury, AFC was restored partly by its secretion of KGF 34.

Paracrine soluble factors

Previous literatures have evidenced paracrine factors that can regulate AFC, reduce permeability of alveolar‐capillary membrane, decrease inflammation and enhance tissue repair. Below, we summarise the soluble factors and their mechanisms. Factors, target cell and its specific function are listed in Table 1.

Table 1.

Important molecules secreted by MSCs and their functions

Bioactive molecules	Target cell	Functions
Angiopoietin‐1	Type II pneumocyte	Restore alveolar fluid clearance
	Type II pneumocyte	Restore alveolar permeability
KGF	Type II pneumocyte	Restore alveolar fluid clearance, epithelial permeability, upregulating α‐ENaC gene expression and Na‐K‐ATPase activity
	Capillary endothelium	Restore endothelial permeability
TSG‐6	Neutrophil	Anti‐inflammation
IL1RN	Macrophage	Block release of TNF‐α
LL‐37	Bacteria	Direct antimicrobial activity
IL‐10	Macrophage	Anti‐inflammation
	Neutrophil	Anti‐inflammation
HGF	Capillary endothelium	Stabilise the integrity of endothelial cells
Prostaglandin E2	Macrophage	Increase the production of IL‐10

MSCs, mesenchymal stem cells; KGF, keratinocyte growth factor; ENaC, epithelial sodium channel; Na‐K‐ATPase, sodium–potassium adenosine triphosphatase; TSG‐6, tumour necrosis factor‐α‐induced protein 6; IL1RN, interleukin‐1 receptor antagonist; TNF‐α, tumour necrosis factor‐α; IL‐10, interleukin‐10; HGF, hepatocyte growth factor.

Ang‐1

Human allogeneic MSCs had been proved that they could restore epithelial permeability of protein through primary culture of human AEC‐II following an inflammatory insult. In this experiment, type II alveolar epitheliums were grown on a transwell plate with an inserted interface and injured by inflammatory factors containing IL‐1β, TNF‐α and IFN‐γ. Co‐culture system of human MSCs and AEC‐II restored permeability of epitheliums to control level for protein. This study revealed that Ang‐1 secretion was responsible for this beneficial effect in part by preventing the formation of actin stress fibre and claudin‐18 disorganisation through suppressing nuclear factor kappa‐light‐chain‐enhancer of activated B cells (NFκB) activity by siRNA. This provided a novel evidence for paracrine function of MSCs in an inflammatory environment 33.

KGF

KGF is the seventh member of the fibroblast growth factor. It has been reported that pulmonary endothelial and epithelial permeability to protein and pulmonary oedema fluid were increased in the middle lobe of right lung exposed to intra‐alveolar endotoxin. The normal capacity of the AEC‐II to remove alveolar fluid was damaged. Permeability of lung microvascular endothelial cell and lung epithelium returned to the normal level. Meanwhile, the rate of AFC was increased to a normal level by delivering intrabronchially allogeneic MSCs 1 hour after instillation of endotoxin, but the control group with human fibroblasts showed no effect. The researchers found that MSCs produced a quantity of KGF and tested the effect of inhibiting release of KGF by siRNA method. Approximately 80% of the effect could be attributed to KGF as it appeared to restore lung endothelial and epithelial permeability and improve the alveolar epitheliums to remove alveolar oedema fluid. Although the function of conditioned medium could be attributed primarily to the release of KGF, further study will be needed to determine whether other factors could work in lung injury 54, 55.

In another animal model of pulmonary oedema, KGF increased alveolar fluid transport partly by upregulating ENaC gene expression and improving Na‐K‐ATPase activity 21, 56. Recombinant human KGF could restore the therapeutic effect of the conditioned medium alone on AFC in the lung injured by endotoxin 57.

Interleukin‐1 receptor antagonist (IL1RN)

Ortiz et al. reported that MSCs inhibited inflammation in mice induced by bleomycin and prevented pulmonary fibrosis in mice. IL1RN was considered as a potential mediator in this process. They found that conditioned medium of MSCs could block proliferation of an IL‐1α‐dependent T‐cell line and inhibit production of TNF‐α in vitro. MSCs protect lung tissue from bleomycin‐induced injury by blocking TNF‐α and IL‐1 in the lung. IL1RN‐expressing human MSC subpopulations may be considered as a novel cellular vector for treating chronic inflammatory diseases for human in future application 32.

LL‐37

Anna Krasnodembskaya found that human bone marrow‐derived MSCs could inhibit bacterial growth. Their antimicrobial effect is mediated partly by an antimicrobial peptide LL‐37 secreted by MSCs. Through stimulation of bacteria, LL‐37 was upregulated. It could improve bacterial clearance in the mice model of Escherichia coli pneumonia after administering MSCs intrabronchially 58.

Marked inhibition of bacterial growth by MSCs as well as by conditioned medium of MSCs was found in comparison with normal human lung fibroblasts. Next, they found that hCAP‐18/LL‐37 in conditioned medium was responsible for the antimicrobial activity. Experimental data showed that the expression of LL‐37 in MSCs increased in both mRNA and protein levels after bacterial insult. Using an in vitro mouse model of E. coli pneumonia, intratracheal delivery of MSCs reduced bacterial growth in the lung homogenates as well as in the bronchoalveolar lavage fluid, based on number of colony‐forming units. Meanwhile, administrating MSCs with the neutralising antibody to LL‐37 resulted in a decrease in clearing bacteria 58.

IL‐10

The beneficial effect of MSCs on mortality following sepsis was eliminated by macrophage depletion or pretreatment with antibodies to IL‐10 or its receptor. IL‐10 secreted mainly by monocytes has also been reported to inhibit the rolling, adhesion and transepithelial migration of neutrophils 22, 59. IL‐10 plays an important role in these experiments.

TNF‐α‐induced protein 6 (TNFAIP6/TSG‐6)

Danchuk et al. treated ALI mice model with MSCs and found that the expression of proinflammatory cytokines, neutrophil counts and total protein in BAL decreased. Pulmonary oedema was also reduced by the administration of MSCs in mice. Anti‐inflammatory effects were attenuated by knocking down TNFAIP6/TSG‐6. In addition, recombinant human TSG‐6 reduced LPS‐induced inflammatory reaction in the lung. This research showed that the beneficial effects of rodent MSCs on ALI were activated at least in part by the secretion of TSG‐6 60.

Challenge and further direction

Some hospitalised patients with ALI or ARDS caused by trauma, infection or extensive burns were eventually dead in the early stage owing to severe pneumonedema. Hence, an optimal approach should be sought urgently to save these patients in severe condition. To date, the effects of pharmacological therapies remain limited. Although lung‐protective ventilation strategies have substantially reduced mortality of ALI patients, there is still a need for new strategies that can further decrease mortality. MSCs have been proven a promising strategy. Although the mechanism is still unclear, MSCs have taken the beneficial effect on ALI or ARDS. According to current data, direct engraftment and differentiation of MSCs in the lung are less likely the exact therapeutic mechanisms. MSCs might play an important role in mediating inflammatory reaction but paracrine factors were considered as a novel application. However, it is still a long way from satisfactory clinical application. Some issues involved with the application of MSCs including critical paracrine factors, AFC and resistance of alveolar‐capillary membrane should be further considered in next research.

According to paracrine factors of MSCs, proteomics research of conditioned medium should be required to analyse the specific function of these critical factors in treating ALI and ARDS, as well as the involved signalling pathways in healing process. Subsequently, overexpression of these critical growth factors in MSCs may be a more effective treatment to repair the injured tissues 61. Furthermore, regulation of these growth factor secretion and delivery by MSCs stably are also of importance 62.

There also remain some challenging problems to be solved in the process of treatment. Epithelial cells including AEC‐I and AEC‐II have been documented to play a vital role in clearing clear pulmonary oedema fluid. Although both of them consist of sodium channels, aquaporin and Na‐K‐ATPase, AEC‐I and AEC‐II exert function by different mechanisms. Therefore, considerable studies should be done to determine the functional units as well as the quantities of sodium channels, aquaporin and Na‐K‐ATPase exist in AEC‐I or AEC‐II, which could be helpful to make a evaluation of injury degree. Meanwhile, valuable biomarkers of sodium channels, aquaporin and Na‐K‐ATPase in plasma should highlight the therapeutic targets for MSC administration.

Because permeability of alveolar‐capillary membrane changes when inflammatory factors contact endothelial cells, studies on the resistance of pulmonary epithelial cells might be more significant. Moreover, cellular junctions are also of importance in resisting production of pulmonary oedema. Thus, an understanding of interaction between MSCs and epithelial structures and functions as well as cellular junction within inflammatory microenvironment will provide critical information in revealing the precise mechanisms of MSC‐mediated therapeutic effects and designing more practical protocols for clinical use of these cells.

In summary, owing to numerous complex problems that will be encountered, there is a long way to go before MSCs can be used as a regular clinical therapy for ALI and ARDS. However, available experimental and clinical significance is encouraging. Besides functions in terms of engraftment, differentiation and immunoregulation, researches in paracrine factors secreted by MSCs, which can mediate endothelial, epithelial permeability, increase AFC and other potential mechanisms, are more attractive. In addition to controlling the primary disease, respiratory support and administering medications, MSCs might be a proper therapeutic tool for ALI and ARDS treatments.

Li J, Huang S, Wu Y, Gu C, Gao D, Feng C, Wu X, Fu X. Paracrine factors from mesenchymal stem cells: a proposed therapeutic tool for acute lung injury and acute respiratory distress syndrome.