Lung Resident Mesenchymal Stem Cells Research Articles

In Vivo and In Vitro Pro-Fibrotic Response of Lung-Resident Mesenchymal Stem Cells from Patients with Idiopathic Pulmonary Fibrosis.

Lung-resident mesenchymal stem cells (LR-MSC) are thought to participate in idiopathic pulmonary fibrosis (IPF) by differentiating into myofibroblasts. On the other hand, LR-MSC in IPF patients present senescence-related features. It is unclear how they respond to a profibrotic environment. Here, we investigated the profibrotic response of LR-MSC isolated from IPF and control (CON) patients. LR-MSC were inoculated in mice 48 h after bleomycin (BLM) instillation to analyze their contribution to lung damage. In vitro, LR-MSC were exposed to TGFβ. Mice inoculated with IPF LR-MSC exhibited worse maintenance of their body weight. The instillation of either IPF or CON LR-MSC sustained BLM-induced histological lung damage, bronchoalveolar lavage fluid cell count, and the expression of the myofibroblast marker, extracellular matrix (ECM) proteins, and proinflammatory cytokines in the lungs. In vitro, IPF LR-MSC displayed higher basal protein levels of aSMA and fibronectin than CON LR-MSC. However, the TGFβ response in the expression of TGFβ, aSMA, and ECM genes was attenuated in IPF LR-MSC. In conclusion, IPF LR-MSC have acquired myofibroblastic features, but their capacity to further respond to profibrotic stimuli seems to be attenuated. In an advanced stage of the disease, LR-MSC may participate in disease progression owing to their limited ability to repair epithelial damage.

METTL3-mediated m6A RNA methylation induces the differentiation of lung resident mesenchymal stem cells into myofibroblasts via the miR-21/PTEN pathway

BackgroundThe accumulation of myofibroblasts is the key pathological feature of pulmonary fibrosis (PF). Aberrant differentiation of lung-resident mesenchymal stem cells (LR-MSCs) has been identified as a critical source of myofibroblasts, but the molecular mechanisms underlying this process remain largely unknown. In recent years, N6-methyladenosine (m6A) RNA modification has been implicated in fibrosis development across diverse organs; however, its specific role in promoting the differentiation of LR-MSCs into myofibroblasts in PF is not well defined.MethodsIn this study, we examined the levels of m6A RNA methylation and the expression of its regulatory enzymes in both TGF-β1-treated LR-MSCs and fibrotic mouse lung tissues. The downstream target genes of m6A and their related pathways were identified according to a literature review, bioinformatic analysis and experimental verification. We also assessed the expression levels of myofibroblast markers in treated LR-MSCs and confirmed the involvement of the above-described pathway in the aberrant differentiation direction of LR-MSCs under TGF-β1 stimulation by overexpressing or knocking down key genes within the pathway.ResultsOur results revealed that METTL3-mediated m6A RNA methylation was significantly upregulated in both TGF-β1-treated LR-MSCs and fibrotic mouse lung tissues. This process directly led to the aberrant differentiation of LR-MSCs into myofibroblasts by targeting the miR-21/PTEN pathway. Moreover, inhibition of METTL3 or miR-21 and overexpression of PTEN could rescue this abnormal differentiation.ConclusionOur study demonstrated that m6A RNA methylation induced aberrant LR-MSC differentiation into myofibroblasts via the METTL3/miR-21/PTEN signaling pathway. We indicated a novel mechanism to promote PF progression. Targeting METTL3-mediated m6A RNA methylation and its downstream targets may present innovative therapeutic approaches for the prevention and treatment of PF.

Mitochondrial Dysfunction in Lung Resident Mesenchymal Stem Cells from Idiopathic Pulmonary Fibrosis Patients.

Idiopathic pulmonary fibrosis (IPF) is characterized by an aberrant repair response with uncontrolled turnover of extracellular matrix involving mesenchymal cell phenotypes, where lung resident mesenchymal stem cells (LRMSC) have been supposed to have an important role. However, the contribution of LRMSC in lung fibrosis is not fully understood, and the role of LRMSC in IPF remains to be elucidated. Here, we performed transcriptomic and functional analyses on LRMSC isolated from IPF and control patients (CON). Both over-representation and gene set enrichment analyses indicated that oxidative phosphorylation is the major dysregulated pathway in IPF LRMSC. The most relevant differences in biological processes included complement activation, mesenchyme development, and aerobic electron transport chain. Compared to CON LRMSC, IPF cells displayed impaired mitochondrial respiration, lower expression of genes involved in mitochondrial dynamics, and dysmorphic mitochondria. These changes were linked to an impaired autophagic response and a lower mRNA expression of pro-apoptotic genes. In addition, IPF TGFβ-exposed LRMSC presented different expression profiles of mitochondrial-related genes compared to CON TGFβ-treated cells, suggesting that TGFβ reinforces mitochondrial dysfunction. In conclusion, these results suggest that mitochondrial dysfunction is a major event in LRMSC and that their occurrence might limit LRMSC function, thereby contributing to IPF development.

SENP1 regulates the transformation of lung resident mesenchymal stem cells and is associated with idiopathic pulmonary fibrosis progression

BackgroundLung resident mesenchymal stem cells (LR-MSCs) play an important role in idiopathic pulmonary fibrosis (IPF) by transforming into myofibroblasts, thereby losing their repair ability. Evidence suggests that key proteins of multiple signaling pathways are involved in myofibroblast differentiation of LR-MSCs, such as β-Catenin and GLI family zinc finger 1 (GLI1). These proteins are regulated by SUMO (small ubiquitin-like modifier) modification, which is a post-translational modification that promotes protein degradation, while Sumo specific protein 1 (SENP1)-mediated deSUMOylation produces the opposite biological effects. Therefore, we speculated that SENP1 might be a potential target for treating pulmonary fibrosis by preventing the myofibroblast differentiation of LR-MSCs.MethodsLR-MSCs were isolated from mice by using immunomagnetic beads. The extracted LR-MSCs were identified by flow cytometric analysis and multilineage differentiation assays. Lentivirus packaged shRNA silenced the expression of SENP1 in vitro and vivo. The silencing efficacy of SENP1 was verified by real-time quantitative PCR. The effect of down-regulated SENP1 on the myofibroblast differentiation of LR-MSCs was assessed by Immunofluorescence and Western blot. Immunoprecipitation was used to clarify that SENP1 was a key target for regulating the activity of multiple signaling pathways in the direction of LR-MSCs differentiation. LR-MSCs resident in the lung was analyzed with in vivo imaging system. HE and Masson staining was used to evaluate the therapeutic effect of LR-MSCs with SENP1 down-regulation on the lung of BLM mice.ResultsIn this study, we found that the myofibroblast differentiation of LR-MSCs in IPF lung tissue was accompanied by enhanced SENP1-mediated deSUMOylation. The expression of SENP1 increased in LR-MSCs transition of bleomycin (BLM)-induced lung fibrosis. Interfering with expression of SENP1 inhibited the transformation of LR-MSCs into myofibroblasts in vitro and in vivo and restored their therapeutic effect in BLM lung fibrosis. In addition, activation of the WNT/β-Catenin and Hedgehog/GLI signaling pathways depends on SENP1-mediated deSUMOylation.ConclusionsSENP1 might be a potential target to restore the repair function of LR-MSCs and treat pulmonary fibrosis.8mnU6TCL-hGPayjm_r7Fe6Video

Wnt8b regulates myofibroblast differentiation of lung-resident mesenchymal stem cells via the activation of Wnt/β-catenin signaling in pulmonary fibrogenesis

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, and fatal lung disease that is characterized by enhanced changes in stem cell differentiation and fibroblast proliferation. Lung resident mesenchymal stem cells (LR-MSCs) are important regulators of pathophysiological processes including tissue repair and inflammation, and evidence suggests that this cell population also plays an essential role in fibrosis. Our previous study demonstrated that Wnt/β-catenin signaling is aberrantly activated in the lungs of bleomycin-treated mice and induces myofibroblast differentiation of LR-MSCs. However, the underlying correlation between LR-MSCs and the Wnt/β-catenin signaling remains poorly understood. We found that Wnt8b was highly expressed by LR-MSCs undergoing myofibroblast differentiation. In vitro, Wnt8b promoted LR-MSCs differentiate into myofibroblasts via activating Wnt/β-catenin signaling. Moreover, siRNA-mediated inhibition of Wnt8b prevented Transforming growth factor (TGF)-β1-induced myofibroblast differentiation of LR-MSCs in vitro and ameliorated pulmonary fibrotic lesions. Our study identified Wnt proteins and Wnt/β-catenin signaling in pulmonary fibrosis in vitro and in vivo, and highlighted Wnt8b as a potential therapeutic target in pulmonary fibrosis. Moreover, these finding might provide a new perspective in the development of treatment strategies for IPF.

Lung-Resident Mesenchymal Stem Cell Fates within Lung Cancer

Simple SummaryLung cancer remains the leading cause of cancer-related deaths worldwide. Herein, the heterogeneous tumor stroma decisively impacts on tumor progression, therapy resistance, and, thus, poor clinical outcome. Among the numerous non-epithelial cells constructing the complex environment of lung carcinomas, mesenchymal stem cells (MSC) gained attraction being stromal precursor cells that could be recruited and ‘educated’ by lung cancer cells to adopt a tumor-associated MSC phenotype, serve as source for activated fibroblasts and presumably for vascular mural cells finally reinforcing tumor progression. Lung-resident MSCs should be considered as ‘local MSCs in stand by’ ready to be arranged within the cancer stroma.Lung-resident mesenchymal stem cells (LR-MSCs) are non-hematopoietic multipotent stromal cells that predominately reside adventitial within lung blood vessels. Based on their self-renewal and differentiation properties, LR-MSCs turned out to be important regulators of normal lung homeostasis. LR-MSCs exert beneficial effects mainly by local secretion of various growth factors and cytokines that in turn foster pulmonary regeneration including suppression of inflammation. At the same time, MSCs derived from various tissues of origins represent the first choice of cells for cell-based therapeutic applications in clinical medicine. Particularly for various acute as well as chronic lung diseases, the therapeutic applications of exogenous MSCs were shown to mediate beneficial effects, hereby improving lung function and survival. In contrast, endogenous MSCs of normal lungs seem not to be sufficient for lung tissue protection or repair following a pathological trigger; LR-MSCs could even contribute to initiation and/or progression of lung diseases, particularly lung cancer because of their inherent tropism to migrate towards primary tumors and metastatic sites. However, the role of endogenous LR-MSCs to be multipotent tumor-associated (stromal) precursors remains to be unraveled. Here, we summarize the recent knowledge how ‘cancer-educated’ LR-MSCs impact on lung cancer with a focus on mesenchymal stem cell fates.

Pulmonary mesenchymal stem cells are engaged in distinct steps of host response to respiratory syncytial virus infection.

Lung-resident (LR) mesenchymal stem and stromal cells (MSCs) are key elements of the alveolar niche and fundamental regulators of homeostasis and regeneration. We interrogated their function during virus-induced lung injury using the highly prevalent respiratory syncytial virus (RSV) which causes severe outcomes in infants. We applied complementary approaches with primary pediatric LR-MSCs and a state-of-the-art model of human RSV infection in lamb. Remarkably, RSV-infection of pediatric LR-MSCs led to a robust activation, characterized by a strong antiviral and pro-inflammatory phenotype combined with mediators related to T cell function. In line with this, following in vivo infection, RSV invades and activates LR-MSCs, resulting in the expansion of the pulmonary MSC pool. Moreover, the global transcriptional response of LR-MSCs appears to follow RSV disease, switching from an early antiviral signature to repair mechanisms including differentiation, tissue remodeling, and angiogenesis. These findings demonstrate the involvement of LR-MSCs during virus-mediated acute lung injury and may have therapeutic implications.

Targeted Dual Small Interfering Ribonucleic Acid Delivery via Non-Viral Polymeric Vectors for Pulmonary Fibrosis Therapy.

Inhibiting the myofibroblast differentiation of lung-resident mesenchymal stem cells (LR-MSCs) is a promising yet challenging approach for pulmonary fibrosis (PF) therapy. Here, micelles formed by a graft copolymer of multiple PEGs modified branched polyethylenimine are used for delivering runt-related transcription factor-1 (RUNX1) small interfering RNA (siRNA) (siRUNX1) to the lung, aiming to inhibit the myofibroblast differentiation of LR-MSCs. LR-MSC targeting is achieved by functionalizing the micelle surface with an anti-stem-cell antigen-1 antibody fragment (Fab'). Consequently, therapeutic benefits are obtained by successful suppression of myofibroblast differentiation of LR-MSCs in bleomycin-induced PF model mice treated with siRUNX1-loaded micelles. Furthermore, an excellent synergistic effect of PF therapy is achieved for this micelle system loaded siRUNX1 and glioma-associated oncogene homolog-1 (Gli1) small interfering RNA (siGli1), a traditional anti-PF siRNA of glioma-associated oncogene homolog-1. Hence, this work not only provides RUNX1 as a novel PF therapeutic target, but also as a promising dual siRNA-loaded nanocarrier system for the therapy of PF.

NLRP3 inflammasome activation in alveolar epithelial cells promotes myofibroblast differentiation of lung-resident mesenchymal stem cells during pulmonary fibrogenesis

Idiopathic pulmonary fibrosis (IPF) is a lethal and agnogenic interstitial lung disease, which has limited therapeutic options. Recently, the NOD-, LRR- and pyrin domain-containing 3 (NLRP3) inflammasome has been demonstrated as an important contributor to various fibrotic diseases following its persistent activation. However, the role of NLRP3 inflammasome in pulmonary fibrogenesis still needs to be further clarified. Here, we found that the activation of the NLRP3 inflammasome was raised in fibrotic lungs. In addition, the NLRP3 inflammasome was found to be activated in alveolar epithelial cells (AECs) in the lung tissue of both IPF patients and pulmonary fibrosis mouse models. Further research revealed that epithelial cells, following activation of the NLRP3 inflammasome, could induce the myofibroblast differentiation of lung-resident mesenchymal stem cells (LR-MSCs). In addition, inhibiting the activation of the NLRP3 inflammasome in epithelial cells promoted the expression of dickkopf-1 (DKK1), a secreted Wnt antagonist. DKK1 was capable of suppressing the profibrogenic differentiation of LR-MSCs and bleomycin-induced pulmonary fibrosis. In conclusion, this study not only provides a further in-depth understanding of the pathogenesis of pulmonary fibrosis, but also reveals a potential therapeutic strategy for disorders associated with pulmonary fibrosis.

The vascular nature of lung-resident mesenchymal stem cells.

Human lungs bear their own reservoir of endogenous mesenchymal stem cells (MSCs). Although described as located perivascular, the cellular identity of primary lung MSCs remains elusive. Here we investigated the vascular nature of lung‐resident MSCs (LR‐MSCs) using healthy human lung tissue. LR‐MSCs predominately reside within the vascular stem cell niche, the so‐called vasculogenic zone of adult lung arteries. Primary LR‐MSCs isolated from normal human lung tissue showed typical MSC characteristics in vitro and were phenotypically and functionally indistinguishable from MSCs derived from the vascular wall of adult human blood vessels (VW‐MSCs). Moreover, LR‐MSCs expressed the VW‐MSC‐specific HOX code a characteristic to discriminate VW‐MSCs from phenotypical similar cells. Thus, LR‐MSC should be considered as VW‐MSCs. Immunofluorescent analyses of non‐small lung cancer (NSCLC) specimen further confirmed the vascular adventitia as stem cell niche for LR‐MSCs, and revealed their mobilization and activation in NSCLC progression. These findings have implications for understanding the role of MSC in normal lung physiology and pulmonary diseases, as well as for the rational design of additional therapeutic approaches.

Lung-resident mesenchymal stem cells regulated the inflammatory responses in innate and adaptive immune cells through HVEM-BTLA pathway during ARDS

Acute respiratory distress syndrome (ARDS) is an organ failure syndrome caused by overactivation of the immune system. Mesenchymal stem cells (MSCs) have been found to be effective in ARDS therapy due to their excellent immunomodulatory abilities; however, people are concerned about the safety of infusing exogenous cells. We found that rat lung-resident mesenchymal stem cells (LRMSCs) (Sca-1+CD45−CD31−) played important roles in regulating inflammation in the lungs during the pathogenesis of ARDS. LRMSCs could regulate the production of cytokines (TNF-α, MCP-1, and IL-10) by both innate and adaptive immune cells following LPS stimulation in vivo or in vitro. We also found that Herpes Virus Entry Mediator (HVEM) expression in LRMSCs enhanced the immunomodulatory ability of LRMSCs, and expression of the HVEM ligand B and T Lymphocyte Attenuator (BTLA) in innate and adaptive immune cells was required. The clarification of this immunoregulatory mechanism may provide evidence for ARDS therapy mediated by mobilizing endogenous MSCs in the future.

The Shh/Gli signaling cascade regulates myofibroblastic activation of lung-resident mesenchymal stem cells via the modulation of Wnt10a expression during pulmonary fibrogenesis

Lung-resident mesenchymal stem cells (LR-MSCs) are important regulators of lung repair and regeneration, and evidence suggests that this cell population also plays a vital role in fibrosis. Crosstalk between sonic hedgehog (Shh) signaling and wingless/integrated (Wnt) has been demonstrated in idiopathic pulmonary fibrosis (IPF). However, the underlying correlation between LR-MSCs and the Shh-Wnt signaling cascade remains poorly understood. Here, we identified Wnt10a as a key factor in pulmonary fibrosis. Using a bleomycin mouse model, we found that highly expressed Wnt10a was secreted by LR-MSCs undergoing myofibroblastic differentiation. LR-MSCs with myofibroblast characteristics isolated from fibrotic lungs exhibited increased Shh pathway activity, suggesting their role as Shh targets. In vitro, LR-MSCs responded to stimulation by recombinant Shh, acquiring a myofibroblast phenotype. We further demonstrated that the Shh/glioblastoma (Gli) system machinery regulated LR-MSC-to-myofibroblast transition and pulmonary fibrosis via manipulation of Wnt/β-catenin signaling. Accordingly, inhibition of the Shh-Wnt signaling cascade prevented LR-MSC transformation into myofibroblasts and ameliorated pulmonary fibrotic lesions. Moreover, induction of Wnt10a expression and activation of Shh/Gli signaling were confirmed in human pulmonary fibrosis. In summary, this study linking the Shh-Wnt signaling cascade with LR-MSC fibrogenic activity furthered the current understanding of pulmonary fibrosis pathogenesis and might provide a new perspective in the development of treatment strategies for IPF.

Expression analysis of microRNAs and mRNAs in myofibroblast differentiation of lung resident mesenchymal stem cells

Idiopathic pulmonary fibrosis (IPF) is a serious lung disease that involved the myofibroblast differentiation of lung resident mesenchymal stem cells (LR-MSCs). However, the specific molecular mechanisms of myofibroblast differentiation of LR-MSCs still remain a mystery. In this study, a comprehensive analysis of miRNAs and mRNAs changes in LR-MSCs treated with TGF-β1 was performed. Through computational approaches, the pivotal roles of differentially expressed miRNAs that were associated with tight junction, pathways in cancer, focal adhesion, and cytokine-cytokine receptor interaction were shown. Kruppel-like factor 4 (Klf4) and inhibitor of growth family, member 5 (Ing5) may be the targets for the therapy of pulmonary fibrosis by inhibiting myofibroblast differentiation of LR-MSCs and EMT. Collectively, a molecular paradigm for understanding myofibroblast differentiation of LR-MSCs in IPF was provided by the integrated miRNA/mRNA analyses.

Smoking Impairs the Immunomodulatory Capacity of Lung-Resident Mesenchymal Stem Cells in Chronic Obstructive Pulmonary Disease.

Tobacco smoking is the main environmental risk factor for chronic obstructive pulmonary disease (COPD), but not all smokers develop the disease. A population of lung-resident mesenchymal stem cells (LR-MSCs) exist in healthy lungs, but how tobacco smoking affects them and their role in COPD have not been assessed yet. Using a sphere-based culture technique, we isolated LR-MSCs from lung tissue obtained from nonsmokers and current and former smokers with and without COPD (n = 53). The cells were characterized by flow cytometry and Affymetrix arrays. Their immunomodulatory capacity was assessed in vitro using cocultures with T cells and after preincubation with 2.5% and 5% cigarette smoke extract. We were able to isolate LR-MSCs expressing similar phenotypic markers in all of the study groups. LR-MSCs from current smokers with COPD expressed different levels of CX3CL1 and CCL5 cytokines, and were unable to modulate CD8+ T-cell proliferation. Preincubation of LR-MSCs with cigarette smoke extract reduced their immunomodulatory capacity. In conclusion, 1) LR-MSCs can be isolated in similar amounts from never-smokers and smokers with and without COPD; 2) their immunomodulatory capacity is impaired in current smokers with COPD, but not in those with normal lung function; and 3) this is reversible after smoking cessation and is reproducible in vitro.

MiR-124 regulates transforming growth factor-β1 induced differentiation of lung resident mesenchymal stem cells to myofibroblast by repressing Wnt/β-catenin signaling

Lung resident mesenchymal stem cells (LR-MSCs) contribute to the progression of idiopathic pulmonary fibrosis (IPF). We aimed to investigate the molecular mechanism underlying LR-MSCs regulation upon transforming growth factor (TGF)-β1 stimulation. We induced fibrogenic differentiation of LR-MSCs isolated from mice by TGF-β1. Several stem cell markers were detected by flow cytometric analysis. Protein expression level was tested by Western blotting and mRNA level was detected by quantitative real-time polymerase chain reaction (qRT-PCR). Cell viability, proliferation and apoptosis were measured. TGF-β1 promoted fibrogenic differentiation of LR-MSCs and upregulated β-catenin and p-glycogen synthase kinase-3β, suggesting the activation of Wnt signaling. MicroRNA (MiR)-124-3p was significantly upregulated in TGF-β1 treated LR-MSCs compared to untreated cells. Intriguingly, silence of miR-124 reversed the TGF-β1-induced changes in cell viability and proliferation, and also led to a decrease of cell apoptosis. Additionally, in miR-124 silenced cells, α-smooth muscle actin, collagen I and fibronectin were downregulated compared to control cells. We ultimately identified a new target of miR-124, AXIN1, which was repressed by miR-124. In conclusion, miR-124 regulates AXIN1 to activate Wnt signaling and therefore plays a crucial role in the TGF-β1-induced fibrogenic differentiation.

M2 macrophages promote myofibroblast differentiation of LR-MSCs and are associated with pulmonary fibrogenesis

BackgroundIdiopathic pulmonary fibrosis (IPF) is a devastating disease characterized by the histopathological pattern of usual interstitial pneumonia and is associated with a high mortality rate. Recently, lung resident mesenchymal stem cells (LR-MSCs) have been identified as an important contributor to myofibroblast activation in pulmonary fibrosis. Macrophages are also believed to play a critical role in pulmonary fibrosis. However, the underlying connections between LR-MSCs and macrophages in the pathogenesis of pulmonary fibrosis are still elusive.MethodsIn this study, we investigated the interaction between LR-MSCs and macrophages using a bleomycin-induced mouse pulmonary fibrosis model and a coculture system.ResultsHere, we show that blocking pulmonary macrophage infiltration attenuated bleomycin-induced pulmonary fibrosis. In addition, as determined by flow cytometry, we discovered that the recruited macrophages in fibrotic lungs of bleomycin-treated mice were mainly M2 macrophages. In particular, we found that M2, rather than M1 macrophages, promoted myofibroblast differentiation of LR-MSCs. Moreover, we demonstrated that suppression of the Wnt/β-catenin signaling pathway could attenuate myofibroblast differentiation of LR-MSCs induced by M2 macrophages and bleomycin-induced pulmonary fibrosis. Tissue samples from IPF patients confirmed the infiltration of M2 macrophages and activation of Wnt/β-catenin signaling pathway.ConclusionIn summary, this study furthered our understanding of the pulmonary fibrosis pathogenesis and highlighted M2 macrophages as a critical target for treating pulmonary fibrosis.

Treatment of Hyperoxia-Induced Lung Injury with Lung Mesenchymal Stem Cells in Mice.

Objective Bronchopulmonary dysplasia (BPD) is a common chronic lung disease in preterm neonates and has no effective treatment. This study aimed to investigate the therapeutic effects of neonatal mouse lung resident mesenchymal stem cells (L-MSCs) on the hyperoxia-induced lung injury. Methods L-MSCs were separated and identified according to the MSC criterions. Hyperoxia-Induced Lung Injury (HILI) of neonatal KM mice was induced with hyperoxia (FiO2 = 60%) and investigated with pathological methods. Neonatal KM mice were divided into 3 groups (hyperoxia + L-MSC group, hyperoxia + PBS group, and air control group). Mice in the hyperoxia + L-MSC group were treated with L-MSCs at 3, 7, and 14 days after birth. After hyperoxia exposure for 21 days, the lung pathology, Radial Alveolar Count (RAC), CD31 expression, and vascular endothelial growth factor (VEGF) expression were investigated. Results After hyperoxia exposure, the body weight, RAC, CD31 expression, and VEGF expression in the hyperoxia + L-MSC group were significantly better than those in the hyperoxia + PBS group but inferior to those in the air control group significantly. These indicate L-MSCs are partially protective on the lung injury of mice with hyperoxia-induced BPD. Conclusion L-MSCs are helpful for the prevention and treatment of BPD, and endogenous L-MSCs may play a role in the postinjury repair of the lung.

Inhibition of Wnt/\u03b2-catenin signaling suppresses myofibroblast differentiation of lung resident mesenchymal stem cells and pulmonary fibrosis

An emerging paradigm proposes a crucial role for lung resident mesenchymal stem cells (LR-MSCs) via a fibroblastic transdifferentiation event in the pathogenesis of idiopathic pulmonary fibrosis (IPF). Aberrant activation of Wnt/β-catenin signaling occurs in virtually all fibrotic lung diseases and is relevant to the differentiation of mesenchymal stem cells (MSCs). In vitro, by measuring the protein levels of several key components involved in Wnt/β-catenin signaling, we confirmed that this signaling pathway was activated in the myofibroblast differentiation of LR-MSCs. Targeted inhibition of Wnt/β-catenin signaling by a small molecule, ICG-001, dose-dependently impeded the proliferation and transforming growth factor-β1 (TGF-β1)-mediated fibrogenic actions of LR-MSCs. In vivo, ICG-001 exerted its lung protective effects after bleomycin treatment through blocking mesenchymal-myofibroblast transition, repressing matrix gene expression, and reducing cell apoptosis. Moreover, delayed administration of ICG-001 attenuated bleomycin-induced lung fibrosis, which may present a promising therapeutic strategy for intervention of IPF. Interestingly, these antifibrotic actions of ICG-001 are operated by a mechanism independent of any disruption of Smad activation. In conclusion, our study demonstrated that Wnt/β-catenin signaling may be an essential mechanism underlying the regulation of myofibroblast differentiation of LR-MSCs and their further participation in the development of pulmonary fibrosis.

Lung-Resident Mesenchymal Stem Cells Promote Repair of LPS-Induced Acute Lung Injury via Regulating the Balance of Regulatory T cells and Th17 cells.

Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are associated with high morbidity and mortality. Mesenchymal stem cells (MSCs) have been shown to improve ALI, and the imbalance of regulatory T cells (Tregs) and Th17 cells is associated with mortality in ALI/ARDS patients. However, whether administration of lung-resident MSC (LRMSC) improves lung injury and regulates the balance of Tregs and Th17 cells remains unknown. An ALI animal model was induced by LPS, and PBS or LRMSC were administered via tail vein after 4h. LRMSC were subsequently detected in the lungs by a live imaging system (Berthold LB983, Germany). Lung morphology; lung wet-to-dry weight ratio; and total protein concentration, inflammatory cells, and cytokines in bronchoalveolar lavage fluid (BALF) and plasma were determined. The percentage of Tregs in lung and spleen, and of Th17 cells in lung and blood, were also evaluated. The results showed that LRMSC not only attenuated histopathological damage but also mediated the downregulation of lung wet-to-dry weight ratio and the reduction of total protein concentration and inflammatory cells in BALF. LRMSC also decreased inflammatory cytokines in both BALF and plasma and increased KGF-2 and surfactant protein C (SPC) expression in the lung. Flow cytometry revealed the upregulation of Tregs and the downregulation of Th17 cells, and the increase in the ratio of Tregs and Th17 cells. The live imaging system showed that LRMSC migrated to and were retained in the injured area. In conclusion, the results indicated that administration of LRMSC attenuates LPS-induced ALI via upregulating the balance of Tregs and Th17 cells.

TNF-α-induced NF-κB activation promotes myofibroblast differentiation of LR-MSCs and exacerbates bleomycin-induced pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, and irreversible lung disease of unknown cause. It has been reported that both lung resident mesenchymal stem cells (LR-MSCs) and tumor necrosis factor-α (TNF-α) play important roles in the development of pulmonary fibrosis. However, the underlying connections between LR-MSCs and TNF-α in the pathogenesis of pulmonary fibrosis are still elusive. In this study, we found that the pro-inflammatory cytokine TNF-α and the transcription factor nuclear factor kappa B (NF-κB) p65 subunit were both upregulated in bleomycin-induced fibrotic lung tissue. In addition, we discovered that TNF-α promotes myofibroblast differentiation of LR-MSCs through activating NF-κB signaling. Interestingly, we also found that TNF-α promotes the expression of β-catenin. Moreover, we demonstrated that suppression of the NF-κB signaling could attenuate myofibroblast differentiation of LR-MSCs and bleomycin-induced pulmonary fibrosis which were accompanied with decreased expression of β-catenin. Our data implicates that inhibition of the NF-κB signaling pathway may provide a therapeutic strategy for pulmonary fibrosis, a disease that warrants more effective treatment approaches.