In summary, MSC MVs increased alveolar fluid clearance and reduced lung protein permeability, and pretreatment with Poly (I:C) enhanced the antimicrobial activity of MVs in an ex vivo perfused human lung with severe bacteria pneumonia.

Our previous study demonstrated that mesenchymal stem cell (MSC) microvesicles (MV) reduced lung inflammation, protein permeability, and pulmonary edema in endotoxin‐induced acute lung injury in mice. However, the underlying mechanisms for restoring lung protein permeability were not fully understood. In this current study, we hypothesized that MSC MV would restore protein permeability across injured human lung microvascular endothelial cells (HLMVEC) in part through the transfer of angiopoietin‐1 (Ang1) mRNA to the injured endothelium. A transwell coculture system was used to study the effect of MSC MV on protein permeability across HLMVECs injured by cytomix, a mixture of IL‐1β, TNF‐α, and IFN‐γ (50 ng/ml). Our result showed that cytomix significantly increased permeability to FITC‐dextran (70 kDa) across HLMVECs over 24 hours. Administration of MSC MVs restored this permeability in a dose dependent manner, which was associated with an increase in Ang1 mRNA and protein secretion in the injured endothelium. This beneficial effect was diminished when MSC MV was pretreated with an anti‐CD44 antibody, suggesting that internalization of MV into the HLMVEC was required for the therapeutic effect. Fluorescent microscopy showed that MSC MV largely prevented the reorganization of cytoskeleton protein F‐actin into “actin stress fiber” and restored the location of the tight junction protein ZO‐1 and adherens junction protein VE‐cadherin in injured HLMVECs. Ang1 siRNA pretreatment of MSC MV prior to administration to injured HLMVECs eliminated the therapeutic effect of MV. In summary, MSC MVs restored protein permeability across HLMVEC in part by increasing Ang1 secretion by injured HLMVEC. Stem Cells Translational Medicine 2018;7:615–624

Bone marrow-derived mesenchymal stem cells (MSCs), which have beneficial effects in acute lung injury (ALI), can serve as a vehicle for gene therapy. Angiotensin-converting enzyme 2 (ACE2), a counterregulatory enzyme of ACE that degrades angiotensin (Ang) II into Ang 1-7, has a protective role against ALI. Because ACE2 expression is severely reduced in the injured lung, a therapy targeted to improve ACE2 expression in lung might attenuate ALI. We hypothesized that MSCs overexpressing ACE2 would have further benefits in lipopolysaccharide (LPS)-induced ALI mice, when compared with MSCs alone. MSCs were transduced with ACE2 gene (MSC-ACE2) by a lentiviral vector and then infused into wild-type (WT) and ACE2 knockout (ACE2 −/y ) mice following an LPS-induced intratracheal lung injury. The results demonstrated that the lung injury of ALI mice was alleviated at 24 and 72 h after MSC-ACE2 transplantation. MSC-ACE2 improved the lung histopathology and had additional anti-inflammatory effects when compared with MSCs alone in both WT and ACE2 −/y ALI mice. MSC-ACE2 administration also reduced pulmonary vascular permeability, improved endothelial barrier integrity, and normalized lung eNOS expression relative to the MSC group. The beneficial effects of MSC-ACE2 could be attributed to its recruitment into the injured lung and enhanced local expression of ACE2 protein without changing the serum ACE2 levels after MSC-ACE2 transplantation. The biological activity of the increased ACE2 protein decreased the Ang II amount and increased the Ang 1-7 level in the lung when compared with the ALI and MSC-only groups, thereby inhibiting the detrimental effects of accumulating Ang II. Therefore, compared to MSCs alone, the administration of MSCs overexpressing ACE2 resulted in a further improvement in the inflammatory response and pulmonary endothelial function of LPS-induced ALI mice. These additional benefits could be due to the degradation of Ang II that accompanies the targeted overexpression of ACE2 in the lung.

BackgroundAcute respiratory distress syndrome (ARDS) is a life-threatening condition in critically ill patients. Recently, we have found that mesenchymal stem cells (MSC) improved the permeability of human lung microvascular endothelial cells by secreting hepatocyte growth factor (HGF) in vitro. However, the properties and functions of MSC may change under complex circumstances in vivo. Here, we sought to determine the role of the HGF-expressing character of MSC in the therapeutic effects of MSC on ARDS in vivo.MethodsMSC with HGF gene knockdown (MSC-ShHGF) were constructed using lentiviral transduction. The HGF mRNA and protein levels in MSC-ShHGF were detected using quantitative real-time polymerase chain reaction and Western blotting analysis, respectively. HGF levels in the MSC culture medium were measured by enzyme-linked immunosorbent assay (ELISA). Rats with ARDS induced by lipopolysaccharide received MSC infusion via the tail vein. After 1, 6, and 24 h, rats were sacrificed. MSC retention in the lung was assessed by immunohistochemical assay. The lung wet weight to body weight ratio (LWW/BW) and Evans blue dye extravasation were obtained to reflect lung permeability. The VE-cadherin was detected with inmmunofluorescence, and the lung endothelial cell apoptosis was assessed by TUNEL assay. The severity of lung injury was evaluated using histopathology. The cytokines and HGF levels in the lung were measured by ELISA.ResultsMSC-ShHGF with markedly lower HGF expression were successfully constructed. Treatment with MSC or MSC carrying green fluorescent protein (MSC-GFP) maintained HGF expression at relatively high levels in the lung at 24 h. MSC or MSC-GFP decreased the LWW/BW and the Evans Blue Dye extravasation, protected adherens junction VE-cadherin, and reduced the lung endothelial cell apoptosis. Furthermore, MSC or MSC-GFP reduced the inflammation and alleviated lung injury based on histopathology. However, HGF gene knockdown significantly decreased the HGF levels without any changes in the MSC retention in the lung, and diminished the protective effects of MSC on the injured lung, indicating the therapeutic effects of MSC on ARDS were partly associated with the HGF-expressing character of MSC.ConclusionsMSC restores lung permeability and lung injury in part by maintaining HGF levels in the lung and the HGF-expressing character is required for MSC to protect the injured lung.Electronic supplementary materialThe online version of this article (doi:10.1186/s13287-016-0320-5) contains supplementary material, which is available to authorized users.

IntroductionMesenchymal stem cells (MSCs) have potential for re-epithelization and recovery in acute respiratory distress syndrome (ARDS). In a previous in vitro study, the results showed that the canonical Wnt/β-catenin pathway promoted the differentiation of MSCs into type II alveolar epithelial cells, conferred resistance to oxidative stress, and promoted their migration, suggesting that the Wnt/β-catenin pathway might be one of the key mechanisms underling the therapeutic effect of mouse MSCs in ARDS.MethodsMouse MSCs stable transfected with β-catenin or green fluorescent protein control were transplanted intratracheally into the ARDS mice induced by lipopolysaccharide. Lung tissue injury and repair assessment were examined using haematoxylin and eosin staining, lung injury scoring, Masson’s trichrome staining and fibrosis scoring. Homing and differentiation of mouse MSCs were assayed by labelling and tracing MSCs using NIR815 dye, immunofluorescent staining, and Western immunoblot analysis. The inflammation and permeability were evaluated by detecting the cytokine and protein measurements in bronchoalveolar lavage fluid using enzyme-linked immunosorbent assay.ResultsIn this study, β-catenin-overexpressing MSC engraftment led to more significant effects than the GFP controls, including the retention of the MSCs in the lung, differentiation into type II alveolar epithelial cells, improvement in alveolar epithelial permeability, and the pathologic impairment of the lung tissue.ConclusionThese results suggest that the activation of canonical Wnt/β-catenin pathway by mouse MSCs by overexpressing β-catenin could further improve the protection of mouse MSCs against epithelial impair and the therapeutic effects of mouse MSCs in ARDS mice.