Deborah E. Sullivan

Human multipotent stromal cells attenuate lipopolysaccharide-induced acute lung injury in mice via secretion of tumor necrosis factor-α-induced protein 6

IntroductionMultipotent stromal cells (MSCs) are currently in clinical trials for a number of inflammatory diseases. Recent studies have demonstrated the ability of MSCs to attenuate inflammation in rodent models of acute lung injury (ALI) suggesting that MSCs may also be beneficial in treating ALI.MethodsTo better understand how human MSCs (hMSCs) may act in ALI, the lungs of immunocompetent mice were exposed to lipopolysaccharide (LPS) and four hours later bone marrow derived hMSCs were delivered by oropharyngeal aspiration (OA). The effect of hMSCs on lung injury was assessed by measuring the lung wet/dry weight ratio and total protein in bronchoalveolar lavage (BAL) fluid 24 or 48 h after LPS. BAL fluid was also analyzed for the presence of inflammatory cells and cytokine expression by multiplex immunoassay. Microarray analysis of total RNA isolated from treated and untreated lungs was performed to elucidate the mechanism(s) involved in hMSC modulation of lung inflammation.ResultsAdministration of hMSCs significantly reduced the expression of pro-inflammatory cytokines, neutrophil counts and total protein in bronchoalveolar lavage. There was a concomitant reduction in pulmonary edema. The anti-inflammatory effects of hMSCs were not dependent on localization to the lung, as intraperitoneal administration of hMSCs also attenuated LPS-induced inflammation in the lung. Microarray analysis revealed significant induction of tumor necrosis factor (TNF)-α-induced protein 6 (TNFAIP6/ TSG-6) expression by hMSCs 12 h after OA delivery to LPS-exposed lungs. Knockdown of TSG-6 expression in hMSCs by RNA interference abrogated most of their anti-inflammatory effects. In addition, intra-pulmonary delivery of recombinant human TSG-6 reduced LPS-induced inflammation in the lung.ConclusionsThese results show that hMSCs recapitulate the observed beneficial effects of rodent MSCs in animal models of ALI and suggest that the anti-inflammatory properties of hMSCs in the lung are explained, at least in part, by activation of hMSCs to secrete TSG-6.

Multipotent Stromal Cells Are Activated to Reduce Apoptosis in Part by Upregulation and Secretion of Stanniocalcin‐1

Multipotent stromal cells (MSCs) have been shown to reduce apoptosis in injured cells by secretion of paracrine factors, but these factors were not fully defined. We observed that coculture of MSCs with previously UV‐irradiated fibroblasts reduced apoptosis of the irradiated cells, but fresh MSC conditioned medium was unable reproduce the effect. Comparative microarray analysis of MSCs grown in the presence or absence of UV‐irradiated fibroblasts demonstrated that the MSCs were activated by the apoptotic cells to increase synthesis and secretion of stanniocalcin‐1 (STC‐1), a peptide hormone that modulates mineral metabolism and has pleiotrophic effects that have not been fully characterized. We showed that STC‐1 was required but not sufficient for reduction of apoptosis of UV‐irradiated fibroblasts. In contrast, we demonstrated that MSC‐derived STC‐1 was both required and sufficient for reduction of apoptosis of lung cancer epithelial cells made apoptotic by incubation at low pH in hypoxia. Our data demonstrate that STC‐1 mediates the antiapoptotic effects of MSCs in two distinct models of apoptosis in vitro. STEM CELLS 2009;27:670–681

Bone marrow progenitor cells contribute to repair and remodeling of the lung and heart in a rat model of progressive pulmonary hypertension

Infusion of bone marrow stem or progenitor cells may provide powerful therapies for injured tissues such as the lung and heart. We examined the potential of bone marrow‐derived (BMD) progenitor cells to contribute to repair and remodeling of lung and heart in a rat monocrotaline (MCT) model of pulmonary hypertension. Bone marrow from green fluorescent protein (GFP)‐transgenic male rats was transplanted into GFP‐negative female rats. The chi‐meric animals were injected with MCT to produce pulmonary hypertension. Significant numbers of male GFP‐positive BMD cells engrafted in the lungs of MCT‐treated rats. Microarray analyses and double‐im‐munohistochemistry demonstrated that many of the cells were interstitial fibroblasts or myofibroblasts, some of the cells were hematopoietic cells, and some were pulmonary epithelial cells (Clara cells), vascular endothelial cells, and smooth muscle cells. A few BMD cells fused with pulmonary cells from the host, but the frequency was low. In the hypertrophied hearts of MCT‐treated rats, we found a significant increase in the relative numbers of BMD cells in the right ventricle wall as compared with the left ventricle. Some of the BMD cells in the right ventricle were vascular cells and cardiomyocytes. We report BMD cardiomyocytes with a normal chromosome number, fusion of BMD cells with host cardiomyocytes, and, in some cases, nuclear fusion. Spees, J. L., Whitney, M. J., Sullivan, D. E., Lasky, J. A., Laboy, M., Ylostalo, J., Prockop, D. J. Bone marrow progenitor cells contribute to repair and remodeling of the lung and heart in a rat model of progressive pulmonary hypertension. FASEB J. 22, 1226–1236 (2008)

TNF‐α induces TGF‐β1 expression in lung fibroblasts at the transcriptional level via AP‐1 activation

Tumour necrosis factor‐α (TNF‐α) and transforming growth factor‐β1 (TGF‐β1) are peptides with multiple biological activities that influence neoplastic, immunologic and fibroproliferative diseases. There are clear interrelationships and overlap between the actions of TNF‐α and TGF‐β1 in lung fibrosis; therefore, we postulated that TNF‐α may play a significant role in regulating TGF‐β1 expression in lungs. We recently reported that TNF‐α activates the extracellular regulated kinase (ERK)‐specific pathway in fibroblasts resulting in stabilization of TGF‐β1 mRNA and increased expression of TGF‐β1. In the current study, we further investigated the molecular mechanisms involved in TNF‐α regulation of TGF‐β1 expression. Nuclear run‐on assays showed that treatment of Swiss 3T3 fibroblasts with TNF‐α increased transcription of the TGF‐β1 gene in an ERK independent manner. Pre‐treatment with the activator protein‐1 (AP‐1) inhibitor curcumin attenuated TNF‐α induced transcription of the TGF‐β1 gene. TNF‐α induced increased levels of c‐Jun and C‐Fos in the nucleus accompanied by phosphorylation of c‐Jun. In electrophoretic mobility shift assays, AP‐1 binding to an AP‐1 binding site found within the TGF‐β1 promoter was increased in nuclear extracts from Swiss 3T3 fibroblasts treated with TNF‐α. Together, these results suggest that TNF‐α induces expression and DNA binding of AP‐1 resulting in increased transcription of the TGF‐β1 gene. It is essential to know which transcription pathways are activated because of the wide distribution of TNF‐α and TGF‐β1, the general lack of effective treatments for fibroproliferative disease and the possibility that targeting the correct transcription factors could be palliative.

New concepts on the immune modulation mediated by mesenchymal stem cells

Mesenchymal stem cells (MSCs) are the nonhematopoietic multipotent progenitor cells found in various adult tissues. They are characterized by their ease of isolation and their rapid growth in vitro while maintaining their differentiation potential, allowing for extensive expansion in culture that yields large quantities suitable for therapeutic use. This article reviews the immunomodulatory activities associated with MSCs. Numerous studies have demonstrated that MSCs are potently immunosuppressive in vitro and in vivo. However, this article presents a new paradigm in MSC biology, in which MSCs, at least in vitro, can undergo polarization into either a pro-inflammatory or an immunosuppressive phenotype.

Differences in Gastric Carcinoma Microenvironment Stratify According to EBV Infection Intensity: Implications for Possible Immune Adjuvant Therapy

Epstein-Barr virus (EBV) is associated with roughly 10% of gastric carcinomas worldwide (EBVaGC). Although previous investigations provide a strong link between EBV and gastric carcinomas, these studies were performed using selected EBV gene probes. Using a cohort of gastric carcinoma RNA-seq data sets from The Cancer Genome Atlas (TCGA), we performed a quantitative and global assessment of EBV gene expression in gastric carcinomas and assessed EBV associated cellular pathway alterations. EBV transcripts were detected in 17% of samples but these samples varied significantly in EBV coverage depth. In four samples with the highest EBV coverage (hiEBVaGC – high EBV associated gastric carcinoma), transcripts from the BamHI A region comprised the majority of EBV reads. Expression of LMP2, and to a lesser extent, LMP1 were also observed as was evidence of abortive lytic replication. Analysis of cellular gene expression indicated significant immune cell infiltration and a predominant IFNG response in samples expressing high levels of EBV transcripts relative to samples expressing low or no EBV transcripts. Despite the apparent immune cell infiltration, high levels of the cytotoxic T-cell (CTL) and natural killer (NK) cell inhibitor, IDO1, was observed in the hiEBVaGCs samples suggesting an active tolerance inducing pathway in this subgroup. These results were confirmed in a separate cohort of 21 Vietnamese gastric carcinoma samples using qRT-PCR and on tissue samples using in situ hybridization and immunohistochemistry. Lastly, a panel of tumor suppressors and candidate oncogenes were expressed at lower levels in hiEBVaGC versus EBV-low and EBV-negative gastric cancers suggesting the direct regulation of tumor pathways by EBV.

Epidermal growth factor-stimulated extravillous cytotrophoblast motility is mediated by the activation of PI3-K, Akt and both p38 and p42/44 mitogen-activated protein kinases

BACKGROUND Trophoblast invasion is a temporally and spatially regulated scheme of events that can dictate pregnancy outcome. Evidence suggests that the potent mitogen epidermal growth factor (EGF) regulates cytotrophoblast (CTB) differentiation and invasion during early pregnancy. METHODS AND RESULTS In the present study, the first trimester extravillous CTB cell line SGHPL-4 was used to investigate the signalling pathways involved in the motile component of EGF-mediated CTB migration/invasion. EGF induced the phosphorylation of the phosphatidylinositol 3-kinase (PI3-K)-dependent proteins, Akt and GSK-3beta as well as both p42/44 MAPK and p38 mitogen-activated protein kinases (MAPK). EGF-stimulated motility was significantly reduced following the inhibition of PI3-K (P < 0.001), Akt (P < 0.01) and both p42/44 MAPK (P < 0.001) and p38 MAPKs (P < 0.001) but not the inhibition of GSK-3beta. Further analysis indicated that the p38 MAPK inhibitor SB 203580 inhibited EGF-stimulated phosphorylation of Akt on serine 473, which may be responsible for the effect SB 203580 has on CTB motility. Although Akt activation leads to GSK-3beta phosphorylation and the subsequent expression of beta-catenin, activation of this pathway by 1-azakenpaullone was insufficient to stimulate the motile phenotype. CONCLUSION We demonstrate a role for PI3-K, p42/44 MAPK and p38 MAPK in the stimulation of CTB cell motility by EGF, however activation of beta-catenin alone was insufficient to stimulate cell motility.

Human Cytomegalovirus Infection Dysregulates the Canonical Wnt/β-catenin Signaling Pathway

Human Cytomegalovirus (HCMV) is a ubiquitous herpesvirus that currently infects a large percentage of the world population. Although usually asymptomatic in healthy individuals, HCMV infection during pregnancy may cause spontaneous abortions, premature delivery, or permanent neurological disabilities in infants infected in utero. During infection, the virus exerts control over a multitude of host signaling pathways. Wnt/β-catenin signaling, an essential pathway involved in cell cycle control, differentiation, embryonic development, placentation and metastasis, is frequently dysregulated by viruses. How HCMV infection affects this critical pathway is not currently known. In this study, we demonstrate that HCMV dysregulates Wnt/β-catenin signaling in dermal fibroblasts and human placental extravillous trophoblasts. Infection inhibits Wnt-induced transcriptional activity of β-catenin and expression of β-catenin target genes in these cells. HCMV infection leads to β-catenin protein accumulation in a discrete juxtanuclear region. Levels of β-catenin in membrane-associated and cytosolic pools, as well as nuclear β-catenin, are reduced after infection; while transcription of the β-catenin gene is unchanged, suggesting enhanced degradation. Given the critical role of Wnt/β-catenin signaling in cellular processes, these findings represent a novel and important mechanism whereby HCMV disrupts normal cellular function.

The latent form of TGFbeta(1) is induced by TNFalpha through an ERK specific pathway and is activated by asbestos-derived reactive oxygen species in vitro and in vivo.

Tumor necrosis factor-α (TNFα) and transforming growth factor-β1 (TGFβ1) are potent peptide growth factors that are likely to play important roles in the development of interstitial pulmonary fibrosis (IPF). Previously we showed that TNFα and TGFβ1 are up-regulated in macrophages, epithelial and mesenchymal cells early after exposure to chrysotile asbestos, particularly at sites of fiber deposition in vivo. We also showed that TNFα receptor knockout mice are resistant to asbestos-induced fibrosis. Importantly, vectors that over-express TNFα cause inflammation and fibrogenesis along with increased TGFβ1 production in C57Bl/6 mice. Recently we reported that TNFα activates the extracellular regulated kinase pathway in fibroblasts leading to a 200–400% increase in TGFβ1 mRNA and protein. The mechanism of TNFα induction of TGFβ1 expression appears to be complex, involving both transcriptional and post-transcriptional mechanisms. In asbestos-exposed animals, this TGFβ1 is produced on alveolar surfaces in a latent form (controlled by binding of a latent associated peptide [LAP]) that must be activated for the TGFβ1 to bind to its receptors and induce its multiple biological effects. Thus, we recently reported that, in vitro, reactive oxygen species (ROS) derived from chrysotile and crocidolite asbestos activate TGFβ1 by oxidation of the LAP. Now, in preliminary findings, we have shown that over-expression of latent TGFβ1 prior to asbestos exposure of fibrogenic-resistant TNFα receptor knockout mice produces asbestos lesions with the same severity as seen in normal C57/Bl6 mice. This finding plus the demonstration of increased amounts of TGFβ1, increased Smad activation and amelioration of the developing disease by treating the mice with an anti-oxidant all support the concept that, in vivo, latent TGFβ1 is activated by asbestos-generated oxygen radicals and consequently mediates at least a component of the consequent fibrogenesis. Taken together, these findings support the postulate that TNFα controls fibrogenesis by regulating TGFβ1 expression and that one mechanism through which ROS induce lung fibrosis is by activating latent TGFβ 1.

Transforming Growth Factor-β1 Induces Heparan Sulfate 6-O-Endosulfatase 1 Expression in Vitro and in Vivo

Transforming growth factor (TGF)-β1 plays an important role in the development of pulmonary fibrosis. In this study we examined the relationship between TGF-β1 stimulation and the expression of heparan sulfate (HS) 6-O-endosulfatase 1 (Sulf1) in cultured normal human lung fibroblasts (NHLFs) and in murine lungs in vivo. By removing 6-O-sulfates from specific HS intrachain sites on the cell surface, Sulf1 has been shown to modulate the activities of many HS binding growth factors and morphogens including fibroblast growth factor (FGF)-2. Real time reverse transcription-PCR analysis revealed that TGF-β1 increased Sulf1 expression in NHLFs in a dose- and time-dependent manner which was accompanied by a decrease in 6-O-sulfated disaccharides as revealed by high performance liquid chromatography analysis. Decreased ERK activation after FGF-2 stimulation was observed in TGF-β1-treated NHLFs compared with control cells without changes in HS-dependent FGF-2 binding or FGF-2·FR1c complex formation. To study the function of Sulf1, negative control or Sulf1-specific small interference RNA (siRNA)-transfected NHLFs were stimulated with TGF-β1. Enhanced Smad2/3 phosphorylation and elevated total Smad2 protein level were observed in Sulf1 siRNA-transfected cells and were accompanied by enhanced expression of α-smooth muscle actin and fibronectin. In addition, Sulf1 siRNA transfection enhanced the anti-proliferative effect of TGF-β1. Finally Sulf1 expression was up-regulated in the lungs of mice treated with adenovirus encoding active TGF-β1. Taken together, our data indicate that Sulf1 is a TGF-β1-responsive gene both in vitro and in vivo and may function as a negative regulator of TGF-β1-induced fibrogenesis.