Metabolic and Epigenetic Regulation of SMAD7 by STC1 Ameliorates Lung Fibrosis.

As shown in our previous studies, the intratracheal-administration of STC1 (stanniocalcin-1) ameliorates pulmonary fibrosis by reducing oxidative and endoplasmic reticulum stress through the uncoupling of respiration in a bleomycin-treated mouse model. However, the overall effect of STC1 on metabolism was not examined. Therefore, we first conducted a comprehensive metabolomics analysis to screen the overall metabolic changes induced by STC1 in an alveolar epithelial cell line using capillary electrophoresis time-of-flight mass spectrometry. The results were subsequently validated in multiple alveolar epithelial and fibroblast cell lines by performing precise analyses of each substance. STC1 stimulated glycolysis, acetyl-CoA synthesis, and the methionine and cysteine-glutathione pathways, which are closely related to the uncoupling of respiration, modulation of epigenetics, and reduction in oxidative stress. These results are consistent with our previous study. Subsequently, we focused on the inhibitory factor SMAD7, which exerts an antifibrotic effect and is susceptible to epigenetic regulation. STC1 upregulates SMAD7 in an uncoupling protein 2-dependent manner, induces demethylation of the SMAD7 promoter region and acetylation of the SMAD7 protein in human alveolar epithelial and fibroblast cell lines and a bleomycin-treated mouse model, and subsequently attenuates fibrosis. The antifibrotic effects of STC1 may partially depend on the regulation of SMAD7. In the evaluation using lung tissue from patients with idiopathic pulmonary fibrosis, SMAD7 expression and acetylation were high in the alveolar structure-preserving region and low in the fibrotic region. The intratracheal administration of STC1 may prevent the development of pulmonary fibrosis by regulating the metabolism-mediated epigenetic modification of SMAD7 in patients.

Effect of CTLA4-Ig on Obliterative Bronchiolitis in a Mouse Intrapulmonary Tracheal Transplantation Model.

OBJECTIVES: One of the serious problems after lung transplantation is chronic lung allograft dysfunction (CLAD). Most CLAD patients pathologically characterized by obliterative bronchiolitis (OB). Cytotoxic T-lymphocyte-associated antigen 4 (CTLA4)-Ig is a combination protein of the Fc fragment of human IgG1 linked to the extracellular domain of CTLA4. The aim of the study was to examine the effect of CTLA4-Ig therapy on OB using a mouse intrapulmonary tracheal transplantation (IPTT) model. METHODS: IPTT was performed between BALB/c (donor) and C57BL/6 (recipient) mice. Abatacept, which is a commercially available form of CTLA4-Ig, was intraperitoneally injected in recipient mice immediately after surgery, on days 7, 14, and 21. The mice in the control group received human IgG. RESULTS: We performed semi-quantitative analysis of graft luminal obliteration at post-transplant day 28. We calculated the obliteration ratio of the lumen of the transplanted trachea in each case. The obliteration ratio was significantly lower in the CTLA4-Ig group than that in the control group (91.2 ± 2.1% vs. 47.8 ± 7.9%, p = 0.0008). Immunofluorescent staining revealed significantly decreased lymphoid neogenesis in the lung. CONCLUSIONS: CTLA4-Ig therapy attenuated tracheal obliteration with fibrous tissue in the mouse IPTT model. The attenuation of fibrous obliteration was correlated with the inhibition of lymphoid neogenesis.

Targeting stanniocalcin-1-expressing tumor cells elicits efficient antitumor effects in a mouse model of human lung cancer.

Lung cancer is the most common cause of cancer-related death in developed countries; therefore, the generation of effective targeted therapeutic regimens is essential. Recently, gene therapy approaches toward malignant cells have emerged as attractive molecular therapeutics. Previous studies have indicated that stanniocalcin-1 (STC-1), a hormone involved in calcium and phosphate homeostasis, positively regulates proliferation, apoptosis resistance, and glucose metabolism in lung cancer cell lines. In this study, we investigated if targeting STC-1 in tumor cells could be a promising strategy for lung cancer gene therapy. We confirmed that STC-1 levels in peripheral blood were higher in lung cancer patients than in healthy donors and that STC-1 expression was observed in five out of eight lung cancer cell lines. A vector expressing a suicide gene, uracil phosphoribosyltransferase (UPRT), under the control of the STC-1 promoter, was constructed (pPSTC-1 -UPRT) and transfected into three STC-1-positive cell lines, PC-9, A549, and H1299. When stably transfected, we observed significant cell growth inhibition using 5-fluorouracil (5-FU) treatment. Furthermore, growth of the STC-1-negative lung cancer cell line, LK-2 was significantly arrested when combined with STC-1-positive cells transfected with pPSTC-1 -UPRT. We believe that conferring cytotoxicity in STC-1-positive lung cancer cells using a suicide gene may be a useful therapeutic strategy for lung cancer.

Muse Cells and Ischemia-Reperfusion Lung Injury.

Ischemia-reperfusion injury (IRI) is one of the main causes of primary graft dysfunction that accounts for 25% of mortality after lung transplantation. Disruption of blood supply and subsequent reperfusion result in organ damage with activating innate and adaptive immune response, leading to inflammatory insults. The IRI after lung transplantation is primarily manifested by permeability pulmonary edema on the basis of pulmonary vascular endothelial cell injury as seen in acute respiratory distress syndrome (ARDS). Stem cells have potent anti-inflammatory and immunomodulatory properties through local paracrine mechanisms. The application of mesenchymal stem cells (MSCs) for ARDS as well as IRI in various organs, therefore, has been interested and extensively investigated in animal models with promising results. Furthermore, two recent clinical randomized, placebo-controlled pilot studies demonstrated that treatment of ARDS with MSCs appears to be safe and feasible.Muse cells are stress-tolerant and non-tumorigenic endogenous pluripotent-like stem cells. They comprise small proportions of cultured fibroblasts and MSCs and can be isolated from these populations. Muse cells are known to migrate to the damaged tissue after local or systemic administration, spontaneously differentiate into the tissue-compatible cells, and also secrete factors related to immunomodulation and tissue repair. We have recently shown the effect of Muse cells on ameliorating lung IRI in a rat model. With 2 h of warm ischemia and subsequent reperfusion on the left lung, the lung showed severe pulmonary edema. Administration of Muse cell through the left pulmonary artery immediately after reperfusion more significantly improved lung oxygenation capacity, compliance, and histological damage on days 1 and 3 after reperfusion compared with MSCs, and this was associated with higher expression levels of proteins related with anti-inflammation and tissue repair in the lung. Encouraging results of this study advocate further investigation of the ability of Muse cells to prevent and treat IRI after lung transplantation.

Bone marrow mesenchymal stromal cells protect allograft lung transplants from acute rejection via the PD-L1/IL-17A axis.

PURPOSE: Using a rat model of allograft lung transplantation, we investigated the effectiveness of mesenchymal stromal cells (MSCs) as prophylactic and therapeutic agents against the acute rejection of lung grafts. METHODS: Lung grafts were harvested from donor rats and transplanted orthotopically into major histocompatibility complex-mismatched rats. MSCs were administered to the recipients once (on day 0) or twice (on days 0 and 3) after transplantation. The grade of acute rejection was evaluated both macroscopically and microscopically 6 days after transplantation. To elucidate the related mechanism, mRNA levels of inflammatory cytokines and immunomodulatory receptors in the transplanted grafts were measured using quantitative RT-PCR. RESULTS: The lung graft tissue from the rats that received MSCs post-surgically was protected from acute rejection significantly better than that from the untreated controls. Notably, the rats administered MSCs twice after surgery exhibited the least signs of rejection, with a markedly upregulated mRNA level of PD-L1 and a downregulated mRNA level of IL-17A. CONCLUSION: This study assessed MSC protection of lung allografts from acute rejection by modulating T cell activity via enforced expression of PD-L1 in transplants and downregulation of IL-17A.

Animal ethics and welfare education in wet-lab training can foster residents' ethical values toward life.

Live animals are used in surgical skills training in wet lab, which has undeniable effectiveness for the development of future surgeons. However, where such training is provided, animal welfare is a major consideration. Increasingly, institutions that offer wet-lab training are incorporating animal ethics and welfare-related content into their training courses, but the effectiveness of such animal ethics education has yet to be evaluated quantitatively. We investigated whether the animal ethics content of a training course affected trainees by measuring increase in ethical awareness using visual analog scale questionnaires before and after training. Our results demonstrated a significant and positive increase in awareness of animal ethics (significance level of 5%; 0.0380≤P≤0.0016).

[Surgical Training Utilizing Swine Under General Anesthesia and Animal Ethics].

A cross-sectional study is conducted with primary residents attended wet labs at Tohoku University Hospital Advanced Medical Training Center in order to investigate the efficacy of the training, especially focused on the animal ethics. The 41 participants answered questionnaires in regard to non-technical skills, technical skills and ethics before and after the practice. To identify differences in each ethical question between 2 time points, Wilcoxon signed ranks test was used because the data was not normally distributed. As the result of it, all animal ethical questions showed significant differences(0.0016≤p≤0.0380, α=0.05 level of significance). Beside them, only 1 out of 5 general ethical questions showed it (p=0.0137). This outcome verified that the lecture of animal ethics and the observation of animal care in this center fixed in the training curriculum clearly induced participants' psychological movements.

An Lysophosphatidic Acid Receptors 1 and 3 Axis Governs Cellular Senescence of Mesenchymal Stromal Cells and Promotes Growth and Vascularization of Multiple Myeloma.

Mesenchymal stromal cells (MSCs) are multipotent progenitor cells and there is much interest in how MSCs contribute to the regulation of the tumor microenvironment. Whether MSCs exert a supportive or suppressive effect on tumor progression is still controversial, but is likely dependent on a variety of factors that are tumor-type dependent. Multiple myeloma (MM) is characterized by growth of malignant plasma cells in the bone marrow. It has been shown that the progression of MM is governed by MSCs, which act as a stroma of the myeloma cells. Although stroma is created via mutual communication between myeloma cells and MSCs, the mechanism is poorly understood. Here we explored the role of lysophosphatidic acid (LPA) signaling in cellular events where MSCs were converted into either MM-supportive or MM-suppressive stroma. We found that myeloma cells stimulate MSCs to produce autotaxin, an indispensable enzyme for the biosynthesis of LPA, and LPA receptor 1 (LPA1) and 3 (LPA3) transduce opposite signals to MSCs to determine the fate of MSCs. LPA3-silenced MSCs (siLPA3-MSCs) exhibited cellular senescence-related phenotypes in vitro, and significantly promoted progression of MM and tumor-related angiogenesis in vivo. In contrast, siLPA1-MSCs showed resistance to cellular senescence in vitro, and efficiently delayed progression of MM and tumor-related angiogenesis in vivo. Consistently, anti-MM effects obtained by LPA1-silencing in MSCs were completely reproduced by systemic administration of Ki6425, an LPA1 antagonist. Collectively, our results indicate that LPA signaling determines the fate of MSCs and has potential as a therapeutic target in MM. Stem Cells 2017;35:739-753.

Mesenchymal stem cells attenuate ischemia-reperfusion injury after prolonged cold ischemia in a mouse model of lung transplantation: a preliminary study.

PURPOSE: Mesenchymal stem cells (MSCs) suppress inflammation and immune responses. We conducted this study to find out if MSCs attenuate ischemia-reperfusion injury in a mouse model of lung transplantation. METHODS: C57BL/6J mouse lungs perfused with low-potassium dextran glucose solution were preserved at 4 °C for 18 h. Human MSCs were slowly injected into the left pulmonary artery of the lung grafts, and orthotopic left lung transplantation was then performed. The lung isografts were reperfused for 6 h, and bronchoalveolar lavage fluid (BALF) from the left lung graft was collected. We measured the protein concentration, cell count, and proinflammatory cytokine concentrations in the BALF. RESULTS: The protein concentration and cell count in the BALF were significantly lower in the MSC-administered grafts than in the PBS-administered controls. Concentrations of proinflammatory cytokines, including IL-1ß, IL-17A, and TNF-α, in BALF tended to be lower in the MSC-administered grafts than in the controls, but the difference was not significant. CONCLUSION: The pre-transplant administration of MSCs via the pulmonary artery of the lung graft attenuated ischemia-reperfusion injury after prolonged cold ischemia in this mouse model of lung transplantation.

OX40 ligand expressed in glioblastoma modulates adaptive immunity depending on the microenvironment: a clue for successful immunotherapy.

BACKGROUND: Glioblastoma is the most malignant human brain tumor and has a dismal prognosis; however, some patients show long-term survival. The interaction between the costimulatory molecule OX40 and its ligand OX40L generates key signals for T-cell activation. The augmentation of this interaction enhances antitumor immunity. In this present study, we explored whether OX40 signaling is responsible for antitumor adaptive immunity against glioblastoma and also established therapeutic antiglioma vaccination therapy. METHODS: Tumor specimens were obtained from patients with primary glioblastoma (n = 110) and grade III glioma (n = 34). Quantitative polymerase chain reaction (PCR), flow cytometry, and immunohistochemistry were used to analyze OX40L expression in human glioblastoma specimens. Functional consequences of OX40 signaling were studied using glioblastoma cell lines, mouse models of glioma, and T cells isolated from human subjects and mice. Cytokine production assay with mouse regulatory T cells was conducted under hypoxic conditions (1.5% O2). RESULTS: OX40L mRNA was expressed in glioblastoma specimens and higher levels were associated with prolonged progression-free survival of patients with glioblastoma, who had undergone gross total resection. In this regard, OX40L protein was expressed in A172 human glioblastoma cells and its expression was induced under hypoxia, which mimics the microenvironment of glioblastoma. Notably, human CD4 T cells were activated when cocultured in anti-CD3-coated plates with A172 cells expressing OX40L, as judged by the increased production of interferon-γ. To confirm the survival advantage of OX40L expression, we then used mouse glioma models. Mice bearing glioma cells forced to express OX40L did not die during the observed period after intracranial transplantation, whereas all mice bearing glioma cells lacking OX40L died. Such a survival benefit of OX40L was not detected in nude mice with an impaired immune system. Moreover, compared with systemic intraperitoneal injection, the subcutaneous injection of the OX40 agonist antibody together with glioma cell lysates elicited stronger antitumor immunity and prolonged the survival of mice bearing glioma or glioma-initiating cell-like cells. Finally, OX40 triggering activated regulatory T cells cultured under hypoxia led to the induction of the immunosuppressive cytokine IL10. CONCLUSION: Glioblastoma directs immunostimulation or immunosuppression through OX40 signaling, depending on its microenvironment.

Mesenchymal stem cells correct inappropriate epithelial-mesenchyme relation in pulmonary fibrosis using stanniocalcin-1.

Current hypotheses suggest that aberrant wound healing has a critical role in the pathogenesis of idiopathic pulmonary fibrosis (IPF). In these hypotheses, continuous TGF-ß1 secretion by alveolar epithelial cells (AECs) in abnormal wound healing has a critical role in promoting fibroblast differentiation into myofibroblasts. Mesenchymal stem cells (MSCs) home to the injury site and reduce fibrosis by secreting multifunctional antifibrotic humoral factors in IPF. In this study, we show that MSCs can correct the inadequate-communication between epithelial and mesenchymal cells through STC1 (Stanniocalcin-1) secretion in a bleomycin-induced IPF model. Inhalation of recombinant STC1 shows the same effects as the injection of MSCs. Using STC1 plasmid, it was possible to enhance the ability of MSCs to ameliorate the fibrosis. MSCs secrete large amounts of STC1 in response to TGF-ß1 in comparison to AECs and fibroblasts. The antifibrotic effects of STC1 include reducing oxidative stress, endoplasmic reticulum (ER) stress, and TGF-ß1 production in AECs. The STC1 effects can be controlled by blocking uncoupling protein 2 (UCP2) and the secretion is affected by the PI3/AKT/mTORC1 inhibitors. Our findings suggest that STC1 tends to correct the inappropriate epithelial-mesenchymal relationships and that STC1 plasmid transfected to MSCs or STC1 inhalation could become promising treatments for IPF.

GATA2 regulates differentiation of bone marrow-derived mesenchymal stem cells.

The bone marrow microenvironment comprises multiple cell niches derived from bone marrow mesenchymal stem cells. However, the molecular mechanism of bone marrow mesenchymal stem cell differentiation is poorly understood. The transcription factor GATA2 is indispensable for hematopoietic stem cell function as well as other hematopoietic lineages, suggesting that it may maintain bone marrow mesenchymal stem cells in an immature state and also contribute to their differentiation. To explore this possibility, we established bone marrow mesenchymal stem cells from GATA2 conditional knockout mice. Differentiation of GATA2-deficient bone marrow mesenchymal stem cells into adipocytes induced accelerated oil-drop formation. Further, GATA2 loss- and gain-of-function analyses based on human bone marrow mesenchymal stem cells confirmed that decreased and increased GATA2 expression accelerated and suppressed bone marrow mesenchymal stem cell differentiation to adipocytes, respectively. Microarray analysis of GATA2 knockdowned human bone marrow mesenchymal stem cells revealed that 90 and 189 genes were upregulated or downregulated by a factor of 2, respectively. Moreover, gene ontology analysis revealed significant enrichment of genes involved in cell cycle regulation, and the number of G1/G0 cells increased after GATA2 knockdown. Concomitantly, cell proliferation was decreased by GATA2 knockdown. When GATA2 knockdowned bone marrow mesenchymal stem cells as well as adipocytes were cocultured with CD34-positive cells, hematopoietic stem cell frequency and colony formation decreased. We confirmed the existence of pathological signals that decrease and increase hematopoietic cell and adipocyte numbers, respectively, characteristic of aplastic anemia, and that suppress GATA2 expression in hematopoietic stem cells and bone marrow mesenchymal stem cells.

Human marrow stromal cells downsize the stem cell fraction of lung cancers by fibroblast growth factor 10.

The functional interplay between cancer cells and marrow stromal cells (MSCs) has attracted a great deal of interest due to the MSC tropism for tumors but remains to be fully elucidated. In this study, we investigated human MSC-secreted paracrine factors that appear to have critical functions in cancer stem cell subpopulations. We show that MSC-conditioned medium reduced the cancer stem cell-enriched subpopulation, which was detected as a side population and quiescent (G0) cell cycle fraction in human lung cancer cells by virtue of fibroblast growth factor 10 (FGF10). This reduction of the stem cell-enriched fraction was also observed in lung cancer cells supplemented with recombinant human FGF10 protein. Moreover, supplementary FGF10 attenuated the expression of stemness genes encoding transcription factors, such as OCT3/4 and SOX2, and crippled the self-renewal capacity of lung cancer cells, as evidenced by the impaired formation of floating spheres in the suspension culture. We finally confirmed the therapeutic potential of the FGF10 treatment, which rendered lung cancer cells prone to a chemotherapeutic agent, probably due to the reduced cancer stem cell subpopulation. Collectively, these results add further clarification to the molecular mechanisms underlying MSC-mediated cancer cell kinetics, facilitating the development of future therapies.

Flagellin/TLR5 signaling potentiates airway serous secretion from swine tracheal submucosal glands.

Airway serous secretion is essential for the maintenance of mucociliary transport in airway mucosa, which is responsible for the upregulation of mucosal immunity. Although there are many articles concerning the importance of Toll-like receptors (TLRs) in airway immune systems, the direct relationship between TLRs and airway serous secretion has not been well investigated. Here, we focused on whether TLR5 ligand flagellin, which is one of the components of Pseudomonas aeruginosa, is involved in the upregulation of airway serous secretion. Freshly isolated swine tracheal submucosal gland cells were prepared, and the standard patch-clamp technique was applied for measurements of the whole cell ionic responses of these cells. Flagellin showed potentiating effects on these oscillatory currents induced by physiologically relevant low doses of acetylcholine (ACh) in a dose-dependent manner. These potentiating effects were TLR5 dependent but TLR4 independent. Both nitric oxide (NO) synthase inhibitors and cGMP-dependent protein kinase (cGK) inhibitors abolished these flagellin-induced potentiating effects. Furthermore, TLR5 was abundantly expressed on tracheal submucosal glands. Flagellin/TLR5 signaling further accelerated the intracellular NO synthesis induced by ACh. These findings suggest that TLR5 takes part in the airway mucosal defense systems as a unique endogenous potentiator of airway serous secretions and that NO/cGMP/cGK signaling is involved in this rapid potentiation by TLR5 signaling.

Targeting lysophosphatidic acid signaling retards culture-associated senescence of human marrow stromal cells.

Marrow stromal cells (MSCs) isolated from mesenchymal tissues can propagate in vitro to some extent and differentiate into various tissue lineages to be used for cell-based therapies. Cellular senescence, which occurs readily in continual MSC culture, leads to loss of these characteristic properties, representing one of the major limitations to achieving the potential of MSCs. In this study, we investigated the effect of lysophosphatidic acid (LPA), a ubiquitous metabolite in membrane phospholipid synthesis, on the senescence program of human MSCs. We show that MSCs preferentially express the LPA receptor subtype 1, and an abrogation of the receptor engagement with the antagonistic compound Ki16425 attenuates senescence induction in continually propagated human MSCs. This anti-aging effect of Ki16425 results in extended rounds of cellular proliferation, increased clonogenic potential, and retained plasticity for osteogenic and adipogenic differentiation. Expressions of p16(Ink4a), Rb, p53, and p21(Cip1), which have been associated with cellular senescence, were all reduced in human MSCs by the pharmacological inhibition of LPA signaling. Disruption of this signaling pathway was accompanied by morphological changes such as cell thinning and elongation as well as actin filament deformation through decreased phosphorylation of focal adhesion kinase. Prevention of LPA receptor engagement also promoted ubiquitination-mediated c-Myc elimination in MSCs, and consequently the entry into a quiescent state, G(0) phase, of the cell cycle. Collectively, these results highlight the potential of pharmacological intervention against LPA signaling for blunting senescence-associated loss of function characteristic of human MSCs.

Mesenchymal stromal cells protect cancer cells from ROS-induced apoptosis and enhance the Warburg effect by secreting STC1.

Previous studies have demonstrated that mesenchymal stromal cells (MSCs) enhance cell survival through upregulation and secretion of stanniocalcin-1 (STC1). This study shows that MSC-derived STC1 promotes survival of lung cancer cells by uncoupling oxidative phosphorylation, reducing intracellular reactive oxygen species (ROS), and shifting metabolism towards a more glycolytic metabolic profile. MSC-derived STC1 upregulated uncoupling protein 2 (UCP2) in injured A549 cells in an STC1-dependent manner. Knockdown of UCP2 reduced the ability of MSCs and recombinant STC1 (rSTC1) to reduce cell death in the A549 population. rSTC1-treated A549 cells displayed decreased levels of ROS, mitochondrial membrane potential (MMP), and increased lactate production, all of which were dependent on the upregulation of UCP2. Our data suggest that MSCs can promote cell survival by regulating mitochondrial respiration via STC1.

Toll-like receptor 4 potentiates Ca2+-dependent secretion of electrolytes from swine tracheal glands.

Airway surface fluids are mainly secreted from submucosal glands, and play important roles in the defense of airways via the up-regulation of mucociliary transport, resulting in an exclusion of many microbes or foreign substances. Although there are many articles concerning the importance of Toll-like receptors (TLRs) in airway immune systems, whether TLRs directly cooperate with tracheal submucosal glands to increase secretion remains unknown. We investigated the effects of ligands of the three TLR subtypes (TLR2, TLR3, and TLR4) on the physiologic secretion of electrolytes by using a patch-clamp technique. Among these TLRs, only the TLR4 ligand, LPS, showed potentiating effects on acetylcholine (ACh)-induced ionic currents in a dose-dependent manner. These potentiating effects were completely abolished by pretreatment with a specific TLR4 antagonist or the anti-TLR4 antibody. LPS per se exerted no appreciable effect on baseline currents. Next, we demonstrated the abundant expression of TLR4 in submucosal gland acinar cells by using immunofluorescent staining and RT-PCR. Furthermore, we revealed that both nitric oxide synthase inhibitors and cyclic guanosine monophosphate (cGMP)-dependent protein kinase (cGK) inhibitors abolished the LPS-induced potentiating effects completely. Analyses of fluorescence intensities, using an intracellular nitric oxide (NO) indicator, demonstrated that LPS could further increase the ACh-induced synthesis of NO. These findings suggest that TLR4 takes part in airway mucosal defense systems as a unique exogenous potentiator of electrolyte-water secretion from submucosal gland acinar cells, and that NO/cGMP/cGK signaling is involved in this rapid TLR4 signaling pathway.

Mesenchymal stromal cells promote tumor growth through the enhancement of neovascularization.

Mesenchymal stromal cells (MSCs), also called mesenchymal stem cells, migrate and function as stromal cells in tumor tissues. The effects of MSCs on tumor growth are controversial. In this study, we showed that MSCs increase proliferation of tumor cells in vitro and promote tumor growth in vivo. We also further analyzed the mechanisms that underlie these effects. For use in in vitro and in vivo experiments, we established a bone marrow-derived mesenchymal stromal cell line from cells isolated in C57BL/6 mice. Effects of murine MSCs on tumor cell proliferation in vitro were analyzed in a coculture model with B16-LacZ cells. Both coculture with MSCs and treatment with MSC-conditioned media led to enhanced growth of B16-LacZ cells, although the magnitude of growth stimulation in cocultured cells was greater than that of cells treated with conditioned media. Co-injection of B16-LacZ cells and MSCs into syngeneic mice led to increased tumor size compared with injection of B16-LacZ cells alone. Identical experiments using Lewis lung carcinoma (LLC) cells instead of B16-LacZ cells yielded similar results. Consistent with a role for neovascularization in MSC-mediated tumor growth, tumor vessel area was greater in tumors resulting from co-injection of B16-LacZ cells or LLCs with MSCs than in tumors induced by injection of cancer cells alone. Co-injected MSCs directly supported the tumor vasculature by localizing close to vascular walls and by expressing an endothelial marker. Furthermore, secretion of leukemia inhibitory factor, macrophage colony-stimulating factor, macrophage inflammatory protein-2 and vascular endothelial growth factor was increased in cocultures of MSCs and B16-LacZ cells compared with B16-LacZ cells alone. Together, these results indicate that MSCs promote tumor growth both in vitro and in vivo and suggest that tumor promotion in vivo may be attributable in part to enhanced angiogenesis.

Paracrine factors of multipotent stromal cells ameliorate lung injury in an elastase-induced emphysema model.

Multipotent stromal cells (MSCs) ameliorate several types of lung injury. The differentiation of MSCs into specific cells at the injury site has been considered as the important process in the MSC effect. However, although MSCs reduce destruction in an elastase-induced lung emphysema model, MSC differentiation is relatively rare, suggesting that MSC differentiation into specific cells does not adequately explain the recuperation observed. Humoral factors secreted by MSCs may also play an important role in ameliorating emphysema. To confirm this hypothesis, emphysema was induced in the lungs of C57BL/6 mice by intratracheal elastase injection 14 days before intratracheal MSC or phosphate-buffered saline (PBS) administration. Thereafter, lungs were collected at several time points and evaluated. Our results showed that MSCs reduced the destruction in elastase-induced emphysema. Furthermore, double immunofluorescence staining revealed infrequent MSC engraftment and differentiation into epithelial cells. Real-time PCR showed increased levels of hepatocyte growth factor (HGF) and epidermal growth factor (EGF). Real-time PCR and western blotting showed enhanced production of secretory leukocyte protease inhibitor (SLPI) in the lung. In-vitro coculture studies confirmed the in vivo observations. Our findings suggest that paracrine factors derived from MSCs is the main mechanism for the protection of lung tissues from elastase injury.

Serial OX40 engagement on CD4+ T cells and natural killer T cells causes allergic airway inflammation.

RATIONALE: OX40-OX40 ligand (OX40L) interactions have been proposed to support induction of allergic airway inflammation, which may be attributable to OX40 signaling in CD4(+) helper T cells for adaptive immune responses. However, a possible involvement of natural killer T (NKT) cells in the pathogenesis suggests that the underlying mechanisms are not yet fully elucidated. OBJECTIVES: We aimed to characterize the OX40-modulated cellular contribution to allergic airway inflammation in a mouse model of house dust mite (HDM) allergen exposure. METHODS: Mice were sensitized to HDM and, 3 weeks later, challenged with HDM on three consecutive days through the airways. Two days after the last exposure, bronchoalveolar lavage fluids and blood samples and lung tissues were evaluated for the airway inflammation. MEASUREMENTS AND MAIN RESULTS: The development of HDM-induced eosinophilic airway inflammation was dependent on OX40 of both CD4(+) T cells and NKT cells; OX40 engagement on CD4(+) T cells in the sensitization led to pulmonary OX40L augmentation after the allergen challenge, which stimulated pulmonary NKT cells through OX40 to provide the pathogenic cytokine milieu. This was ablated by OX40L blockade by inhalation of the neutralizing antibody during the challenge, suggesting the therapeutic potential of targeting pulmonary OX40-OX40L interactions. Moreover, OX40 expression in CD4(+) T cells, but not in NKT cells, was reciprocally regulated by the helper T cell type 1-skewing transcription factor Runx3. CONCLUSIONS: OX40 on not only CD4(+) T cells but also NKT cells is involved in allergic airway inflammation. Notably, pulmonary blockade of OX40 ligation on NKT cells has therapeutic implications.