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Lats2-Underexpressing Bone Marrow-Derived Mesenchymal Stem Cells Ameliorate LPS-Induced Acute Lung Injury in Mice.

ABSTRACT: The pathophysiology of the acute lung injury (ALI) is characterized by the damage of alveolar epithelial cells, which can be repaired by exogenous bone marrow-derived mesenchymal stem cells (BMSCs). However, the migration and differentiation abilities of BMSCs are not sufficient for the purpose, and a new approach that could strengthen the repair effects of BMSCs in ALI still needs to be clarified. We have previously proved that in vitro large tumor suppressor kinase 2- (Lats2-) underexpressing BMSCs may enhance their tissue repair effects in ALI; thus, in the present study, we tried to explore whether Lats2-underexpressing BMSCs could rescue lipopolysaccharide- (LPS-) induced ALI in vivo. BMSCs from C57BL/6 mice transfected with Lats2-interfering lentivirus vector or lentivirus blank controls were transplanted intratracheally into LPS-induced ALI mice. The retention and differentiation of BMSCs in the lung were evaluated by in vivo imaging, immunofluorescence staining, and Western blotting. The lung edema and permeability were assessed by lung wet weight/body weight ratio (LWW/BW) and measurements of proteins in bronchoalveolar lavage fluid (BALF) using ELISA. Acute lung inflammation was measured by the cytokines in the lung homogenate and BALF using RT-qPCR and ELISA, respectively. Lung injury was evaluated by HE staining and lung injury scoring. Pulmonary fibrosis was evaluated by Picrosirius red staining, immunohistochemistry for ?-SMA and TGF-?1, and hydroxyproline assay and RT-qPCR for Col1?1 and Col3?1. Lats2-mediated inhibition of the Hippo pathway increased the retention of BMSCs and their differentiation toward type II alveolar epithelial cells in the lung. Furthermore, Lats2-underexpressing BMSCs improved lung edema, permeability of the lung epithelium, and lung inflammation. Finally, Lats2-underexpressing BMSCs alleviated lung injury and early pulmonary fibrosis. Our studies suggest that underexpression of Lats2 could further enhance the repair effects of BMSCs against epithelial impair and the therapeutic potential of BMSCs in ALI mice.

SUBMITTER: Dong L

PROVIDER: S-EPMC6854183 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Lats2-Underexpressing Bone Marrow-Derived Mesenchymal Stem Cells Ameliorate LPS-Induced Acute Lung Injury in Mice.

Dong Liang L Li Lang L

Mediators of inflammation 20191021

The pathophysiology of the acute lung injury (ALI) is characterized by the damage of alveolar epithelial cells, which can be repaired by exogenous bone marrow-derived mesenchymal stem cells (BMSCs). However, the migration and differentiation abilities of BMSCs are not sufficient for the purpose, and a new approach that could strengthen the repair effects of BMSCs in ALI still needs to be clarified. We have previously proved that <i>in vitro</i> large tumor suppressor kinase 2- (Lats2-) underexpr ...[more]

PMID: 31772503

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Project description:Chronic hyperglycemia causes a progressive decrease of ?-cell function and mass in type 2 diabetic patients. Growing evidence suggests that augment of autophagy may be an effective approach to protect ? cells against various extra-/intracellular stimuli. In this study, we thus investigated whether bone marrow-derived mesenchymal stem cells (BM-MSCs) could ameliorate chronic high glucose (HG)-induced ?-cell injury through modulation of autophagy. Prolonged exposure to HG decreased cell viability, increased cell apoptosis and impaired basal insulin secretion and glucose-stimulated insulin secretion of INS-1 cells, but BM-MSC treatment significantly alleviated these glucotoxic alternations. In addition, western blotting displayed upregulated expression of Beclin1 and LC3-II in INS-1 cells co-cultured with BM-MSCs. Results from immunofluorescence staining and transmission electronic microscope analysis also revealed that BM-MSCs promoted autophagosomes and autolysosomes formation in HG-treated INS-1 cells. However, it should be noted that inhibition of autophagy significantly diminished the protective effects of BM-MSCs on HG-treated INS-1 cells, suggesting that the improvement of ?-cell function and survival induced by BM-MSCs was mediated through autophagy. Furthermore, our results showed that BM-MSCs improved mitochondrial function and reduced reactive oxygen species production in HG-treated INS-1 cells, largely owing to autophagic clearance of impaired mitochondria. In vivo study was performed in rats with type 2 diabetes (T2D). BM-MSC infusion not only ameliorated hyperglycemia, but also promoted restoration of pancreatic ? cells in T2D rats. Meanwhile, BM-MSC infusion upregulated LAMP2 expression and enhanced formation of autophagosomes and autolysosomes, combined with reduced ?-cell apoptosis and increased number of insulin granules. These findings together indicated that BM-MSCs could protect ? cells against chronic HG-induced injury through modulation of autophagy in vitro and in vivo. This study unveiled novel evidence of BM-MSCs as an ideal strategy to enhance autophagy for treatment of T2D mellitus.

Project description:BackgroundFamilial hemophagocytic lymphohistiocytosis 2 (FHL2) is the most common familial type of hemophagocytic lymphohistiocytosis with immune dysregulation. FHL2 patients have mutations in the perforin gene which cause overactivation and proliferation of cytotoxic T lymphocytes and natural killer cells. Perforin is the key component of the cytolytic granule response function of cytotoxic T lymphocytes and natural killer cells. Perforin dysfunction causes a cytotoxic immune deficiency with a clinical outcome of uncontrolled and continuous immune stimulation response. This excessive stimulation leads to continuous systemic inflammation and, ultimately, multiorgan failure. Radical therapy is hematopoietic stem cell transplantation which is limited by the availability of a donor. Exacerbations of inflammatory attacks require a palliative immunosuppressive regimen. There is a need for an alternative or adjuvant therapy to maintain these patients when immunosuppression is ineffective or a donor is not available. Beneficial actions of mesenchymal stem cells (MSCs) have been shown in autoimmune diseases in clinical trials and are attributed to their immune-modulatory properties. This study aimed to assess the immune-modulatory effect of MSCs in an in-vitro model of FHL2.MethodsWe generated a targeted mutation in the perforin gene of NK92 cells to create an in-vitro FLH2 model using Crispr/Cas technology. A coculture setup was employed to assess the immunomodulatory efficacy of MSCs.ResultsEngineered NK92 clones did not show PRF1 mRNA expression and failed to secrete perforin upon phorbol myristate acetate-ionomycin stimulation, providing evidence for a valid FHL2 model. Coculture media of the engineered cells were investigated for the abundance of several cytokines. Coculture with MSCs revealed a reduction in major proinflammatory cytokines and an induction in anti-inflammatory and immunomodulatory cytokines compared to the parental NK92 cells.ConclusionsThis study shows the ameliorating effect of MSCs as an adjuvant immune modulator toward the therapy of FHL2 patients. MSCs are supportive therapy candidates for FHL2 patients under circumstances where prolonged immunosuppression is required to gain time before allogeneic hematopoietic stem cell transplantation.

Project description:BackgroundHypoxia preconditioning has been proven to be an effective method to enhance the therapeutic action of mesenchymal stem cells (MSCs). However, the beneficial effects of hypoxic MSCs in ischemia/reperfusion (I/R) lung injury have yet to be investigated. In this study, we hypothesized that the administration of hypoxic MSCs would have a positive therapeutic impact on I/R lung injury at molecular, cellular, and functional levels.MethodsI/R lung injury was induced in isolated and perfused rat lungs. Hypoxic MSCs were administered in perfusate at a low (2.5×105 cells) and high (1×106 cells) dose. Rats ventilated with a low tidal volume of 6 ml/kg served as controls. Hemodynamics, lung injury indices, inflammatory responses and activation of apoptotic pathways were determined.ResultsI/R induced permeability pulmonary edema with capillary leakage and increased levels of reactive oxygen species (ROS), pro-inflammatory cytokines, adhesion molecules, cytosolic cytochrome C, and activated MAPK, NF-κB, and apoptotic pathways. The administration of a low dose of hypoxic MSCs effectively attenuated I/R pathologic lung injury score by inhibiting inflammatory responses associated with the generation of ROS and anti-apoptosis effect, however this effect was not observed with a high dose of hypoxic MSCs. Mechanistically, a low dose of hypoxic MSCs down-regulated P38 MAPK and NF-κB signaling but upregulated glutathione, prostaglandin E2, IL-10, mitochondrial cytochrome C and Bcl-2. MSCs infused at a low dose migrated into interstitial and alveolar spaces and bronchial trees, while MSCs infused at a high dose aggregated in the microcirculation and induced pulmonary embolism.ConclusionsHypoxic MSCs can quickly migrate into extravascular lung tissue and adhere to other inflammatory or structure cells and attenuate I/R lung injury through anti-oxidant, anti-inflammatory and anti-apoptotic mechanisms. However, the dose of MSCs needs to be optimized to prevent pulmonary embolism and thrombosis.

Dataset Information

Lats2-Underexpressing Bone Marrow-Derived Mesenchymal Stem Cells Ameliorate LPS-Induced Acute Lung Injury in Mice.

Publications

Lats2-Underexpressing Bone Marrow-Derived Mesenchymal Stem Cells Ameliorate LPS-Induced Acute Lung Injury in Mice.

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