Mesenchymal stem cells require MARCKS protein for directed chemotaxis in vitro.

Mesenchymal stem cells (MSCs) reside within tissues such as bone marrow, cord blood, and dental pulp and can differentiate into other mesenchymal cell types. Differentiated MSCs, called circulating fibrocytes, have been demonstrated in human lungs and migrate to injured lung tissue in experimental models. It is likely that MSCs migrate from the bone marrow to sites of injury by following increasing chemokine concentrations. In the present study, we show that primary mouse bone marrow mesenchymal stem cells (BM-MSCs) exhibit directed chemotaxis through transwell inserts toward increasing concentrations of the chemokines complement component 5a, stromal cell-derived factor-1alpha, and monocyte chemotactic protein-1. Prior research has indicated that myristoylated alanine-rich C kinase substrate (MARCKS) protein is critically important for motility in macrophages, neutrophils, and fibroblasts, and here we investigated a possible role for MARCKS in BM-MSC directed chemotaxis. The presence of MARCKS in these cells as well as in human cord blood MSC was verified by Western blotting, and MARCKS was rapidly phosphorylated in these cells after exposure to chemokines. A synthetic peptide that inhibits MARCKS function attenuated, in a concentration-dependent manner, directed chemotaxis of BM-MSCs, while a missense control peptide had no effect. Our results illustrate, for the first time, that MARCKS protein plays an integral role in BM-MSC-directed chemotaxis in vitro.

TNF-alpha induces TGF-beta1 expression in lung fibroblasts at the transcriptional level via AP-1 activation.

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

Mesenchymal stem cells produce Wnt isoforms and TGF-beta1 that mediate proliferation and procollagen expression by lung fibroblasts.

Studies have been carried out previously to determine whether mesenchymal stem cells (MSC) influence the progression of pulmonary fibrosis. Here, we asked whether MSC (derived from mouse bone marrow and human umbilical cord blood) produce factors that mediate lung fibroblast (LF) growth and matrix production. MSC-conditioned media (CM) were found by ELISA to contain significant amounts of PDGF-AA and transforming growth factor-beta1 (TGF-beta1). Proliferation was increased in a concentration-dependent manner in LF cell lines and primary cells cultured in MSC-CM, but neither anti-PDGF antibodies nor PDGF receptor-specific antibodies affected proliferation, nor did a number of other antibodies to well-known mitogenic factors. However, proliferation was significantly inhibited by the Wnt signaling antagonist, secreted frizzled related protein-1 (sFRP-1). In addition, anti-Wnt1 and anti-Wnt2 antibodies attenuated MSC-CM-induced proliferation, and increased expression of Wnt7b was identified. As would be expected in cells activated by Wnt, nuclear beta-catenin was increased. The amount of TGF-beta1 in MSC-CM and its biological activity were revealed by activation at acidic pH. The stem cells synthesized and released TGF-beta1 that increased alpha1-procollagen gene expression by LF target cells. Addition of anti-TGF-beta to the MSC-CM blocked upregulation of collagen gene expression. These data demonstrate that MSC from mice and humans produce Wnt proteins and TGF-beta1 that respectively stimulate LF proliferation and matrix production, two hallmarks of fibroproliferative lung disease. It will be essential to determine whether these factors can play a role in attempts to use MSC for therapeutic approaches.

Laser capture microdissection reveals dose-response of gene expression in situ consequent to asbestos exposure.

The genes that mediate fibroproliferative lung disease remain to be defined. Prior studies from our laboratory showed by in situ hybridization and immunohistochemistry that the genes coding for tumour necrosis factor alpha, transforming growth factor beta, the platelet-derived growth factor A and B isoforms, and alpha-1 pro-collagen are expressed in fibroproliferative lesions that develop quickly after asbestos inhalation. These five genes, along with matrix metalloproteinase 9, a collagenase found to be increased in several lung diseases, are known to control matrix production and cell proliferation in humans and animals. Here we show by laser capture microdissection that (i) The six genes are expressed at significantly higher levels in the asbestos-exposed mice when comparing the same anatomic regions 'captured' in unexposed mice. (ii) The bronchiolar-alveolar duct (BAD) junctions, where the greatest number of fibres initially deposit, were always significantly higher than the other anatomic regions for each gene. The first alveolar duct bifurcation (ADB) generally was higher than the second ADB, the ADBs were always significantly higher than the airway walls and pleura, and the airway walls and pleura were generally higher than the unexposed tissues. (iii) Animals exposed for 3 days always exhibited significantly higher levels of gene expression at the BAD junctions and ADBs than animals exposed for 2 days. To our knowledge, this is the first demonstration of a dose-response to a toxic particle in situ, and this response appears to be dependent on the number of fibres that deposits at the individual anatomic site.

Phospho-Akt overexpression in non-small cell lung cancer confers significant stage-independent survival disadvantage.

PURPOSE: Akt is a signal transduction protein that plays a central role in inhibiting apoptosis in a variety of cell types including human cancer cells. In cell lines derived from human non-small cell lung cancers (NSCLCs), Akt has been shown to confer chemoresistance by inhibition of apoptosis in response to different chemotherapeutic agents including platinum-based agents, which are often the first-line therapy for NSCLCs. Only 20% to 30% of patients with NSCLC treated with chemotherapy have clinical evidence of response. The purpose of this study is to determine whether or not overexpression of activated Akt [i.e., phosphorylated Akt (pAkt)] is correlated with survival. EXPERIMENTAL DESIGN: We studied tumors from 61 patients with NSCLC in three tissue microarrays. All patients were followed for a period of 10 years or until death. The arrays were studied immunohistochemically with antibodies against pAkt, p53, and Ki-67. RESULTS: There was a statistically significant difference in survival between the 14 patients with strong pAkt staining and the 47 patients with weak to absent pAkt staining both by log-rank (P = 0.0416) and Breslow analysis (P = 0.0446). Difference in survival time with respect to pAkt status was also statistically significant even after accounting for stage at diagnosis (P = 0.004). Neither p53 nor Ki-67 was a statistically significant prognostic factor. CONCLUSIONS: Overexpression of pAkt is an independent prognostic factor. Additional studies of human NSCLCs are warranted to drive the development of targeted tumor-specific antineoplastic therapies.

Cigarette smoke represses the innate immune response to asbestos.

Both cigarette smoke (CS) and asbestos cause lung inflammation and lung cancer, and at high asbestos exposure levels, populations exposed to both of these carcinogens display a synergistic increase in the development of lung cancer. The mechanisms through which these two toxic agents interact to promote lung tumorigenesis are poorly understood. Here, we begin to dissect the inflammatory signals induced by asbestos in combination with CS using a rodent inhalation model and in vitro cell culture. Wild-type C57BL/6 mice were exposed to room air as a control, CS, and/or asbestos (4 days per week to CS and 1 day per week to asbestos for 5 weeks). Bronchoalveolar lavage (BAL) fluid was collected following exposure and analyzed for inflammatory mediators. Asbestos-exposed mice displayed an increased innate immune response consistent with NLRP3 inflammasome activation. Compared to mice exposed only to asbestos, animals coexposed to CS + asbestos displayed attenuated levels of innate immune mediators and altered inflammatory cell recruitment. Histopathological changes in CS + asbestos-exposed mice correlated with attenuated fibroproliferative lesion development relative to their counterparts exposed only to asbestos. In vitro experiments using a human monocyte cell line (THP-1 cells) supported the in vivo results in that coexposure to cigarette smoke extract repressed NLRP3 inflammasome markers in cells treated with asbestos. These observations indicate that CS represses central components of the innate immune response to inhaled asbestos.

Chronic treatment in vivo with ß-adrenoceptor agonists induces dysfunction of airway ß(2) -adrenoceptors and exacerbates lung inflammation in mice.

BACKGROUND AND PURPOSE: Inhalation of a ß-adrenoceptor agonist (ß-agonist) is first-line asthma therapy, used for both prophylaxis against, and acute relief of, bronchoconstriction. However, repeated clinical use of ß-agonists leads to impaired bronchoprotection and, in some cases, adverse patient outcomes. Mechanisms underlying this ß(2) -adrenoceptor dysfunction are not well understood, due largely to the lack of a comprehensive animal model and the uncertainty as to whether or not bronchorelaxation in mice is mediated by ß(2) -adrenoceptors. Thus, we aimed to develop a mouse model that demonstrated functional ß-agonist-induced ß(2) -adrenoceptor desensitization in the context of allergic inflammatory airway disease. EXPERIMENTAL APPROACH: We combined chronic allergen exposure with repeated ß-agonist inhalation in allergen-treated BALB/C mice and examined the contribution of ß(2) -adrenoceptors to albuterol-induced bronchoprotection using FVB/NJ mice with genetic deletion of ß(2) -adrenoceptors (KO). Associated inflammatory changes - cytokines (ELISA), cells in bronchoalevolar lavage and airway remodelling (histology) and ß(2) -adrenoceptor density (radioligand binding) - were also measured. KEY RESULTS ß(2) -Adrenoceptors mediated albuterol-induced bronchoprotection in mice. Chronic treatment with albuterol induced loss of bronchoprotection, associated with exacerbation of the inflammatory components of the asthma phenotype. CONCLUSIONS AND IMPLICATIONS: This animal model reproduced salient features of human asthma and linked loss of bronchoprotection with airway pathobiology. Accordingly, the model offers an advanced tool for understanding the mechanisms of the effects of chronic ß- agonist treatment on ß-adrenoceptor function in asthma. Such information may guide the clinical use of ß-agonists and provide insight into development of novel ß-adrenoceptor ligands for the treatment of asthma.

Mesenchymal stem cells modulate lung injury.

Mesenchymal stem cells (MSCs) have been shown to differentiate into a variety of mesenchymal cell types, including fibroblasts, myofibroblasts, osteoblasts, chondroblasts, adipocytes, and myoblasts, as well as epithelial cells. It has been shown that these cells can be recovered from bone marrow as well as umbilical cord blood, and they can be propagated, stored, and administered to animals and patients in clinical trials. It is clear that the cells engraft in the lung, and several laboratories have demonstrated an ameliorating effect in models of acute injury caused by LPS and in chronic lung injury induced by bleomycin and asbestos. However, it is not at all clear under what conditions these cells must be applied to provide an advantage and when using these cells might cause exacerbation of the lung injury. This brief review focuses on the biology of MSCs in vitro, how the cells have been used in some animal models, and the potential for their use in therapeutic strategies for diseases as diverse as lung cancer and interstitial fibrosis.

Characterization of side population cells in human malignant mesothelioma cell lines.

Side population (SP) assay composed of Hoechst 33342 staining and subsequent flow cytometric analysis has been widely utilized for characterizing putative cancer stem cells (CSCs) in various human malignancies. The present study was designed to evaluate the SP assay as a research tool for mesothelial CSCs. A distinct fraction of SP cells was identified in various human malignant mesothelioma (HMM) cell lines, ranging from 0.05 to 1.32%. The sorted mesothelial SP cells exhibited enhanced proliferation potentials and higher expression of stem-cell genes, compared to non-SP (NSP) cells. Cisplatin treatment increased percentage of SP cells in the HMM cell lines. However, tumorigenic potential of SP cells in immunodeficient mice was similar to that of the NSP cells. These data indicated that SP assay may not be appropriate for enriching putative CSCs in HMM cell lines, and thus warrants the development of a novel tool for mesothelial CSC study.

Inhaled carbon nanotubes reach the subpleural tissue in mice.

Carbon nanotubes are shaped like fibres and can stimulate inflammation at the surface of the peritoneum when injected into the abdominal cavity of mice, raising concerns that inhaled nanotubes may cause pleural fibrosis and/or mesothelioma. Here, we show that multiwalled carbon nanotubes reach the subpleura in mice after a single inhalation exposure of 30 mg m(-3) for 6 h. Nanotubes were embedded in the subpleural wall and within subpleural macrophages. Mononuclear cell aggregates on the pleural surface increased in number and size after 1 day and nanotube-containing macrophages were observed within these foci. Subpleural fibrosis unique to this form of nanotubes increased after 2 and 6 weeks following inhalation. None of these effects was seen in mice that inhaled carbon black nanoparticles or a lower dose of nanotubes (1 mg m(-3)). This work suggests that minimizing inhalation of nanotubes during handling is prudent until further long-term assessments are conducted.

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-alpha (TNFalpha) and transforming growth factor-beta(1) (TGFbeta(1)) are potent peptide growth factors that are likely to play important roles in the development of interstitial pulmonary fibrosis (IPF). Previously we showed that TNFalpha and TGFbeta(1) 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 TNFalpha receptor knockout mice are resistant to asbestos-induced fibrosis. Importantly, vectors that over-express TNFalpha cause inflammation and fibrogenesis along with increased TGFbeta(1) production in C57Bl/6 mice. Recently we reported that TNFalpha activates the extracellular regulated kinase pathway in fibroblasts leading to a 200-400% increase in TGFbeta(1) mRNA and protein. The mechanism of TNFalpha induction of TGFbeta(1) expression appears to be complex, involving both transcriptional and post-transcriptional mechanisms. In asbestos-exposed animals, this TGFbeta(1) is produced on alveolar surfaces in a latent form (controlled by binding of a latent associated peptide [LAP]) that must be activated for the TGFbeta(1) 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 TGFbeta(1) by oxidation of the LAP. Now, in preliminary findings, we have shown that over-expression of latent TGFbeta(1) prior to asbestos exposure of fibrogenic-resistant TNFalpha 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 TGFbeta(1), 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 TGFbeta(1) 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 TNFalpha controls fibrogenesis by regulating TGFbeta(1) expression and that one mechanism through which ROS induce lung fibrosis is by activating latent TGFbeta(1).

Knowns and unknowns of the alveolus.

Our current alveolar paradigm includes three highly specialized cell populations. Alveolar type I cells are flat, elongated cells that presumably enable gas exchange. Alveolar type II cells are small, cuboidal cells with metabolic, secretory, progenitor, and immunologic functions. Alveolar fibroblasts secrete extracellular matrix proteins that support alveolar structure. These cells work together to facilitate respiration. Many years of high-quality research have defined our understanding of alveolar biology. However, there is much to be determined about the factors controlling cellular phenotypes and crosstalk. Moreover, specific questions remain regarding origin, repopulation, and previously unrecognized functions of each cell. This article summarizes the current data for each cell type and highlights areas that would benefit from further investigation.

Resident cellular components of the human lung: current knowledge and goals for research on cell phenotyping and function.

The purpose of the workshop was to identify still obscure or novel cellular components of the lung, to determine cell function in lung development and in health that impacts on disease, and to decide promising avenues for future research to extract and phenotype these cells. Since robust technologies are now available to identify, sort, purify, culture, and phenotype cells, progress is now within sight to unravel the origins and functional capabilities of lung cells in developmental stages and in disease. The Workshop's agenda was to first discuss the lung's embryologic development, including progenitor and stem cells, and then assess the functional and structural cells in three main compartments of the lung: (1) airway cells in bronchial and bronchiolar epithelium and bronchial glands (basal, secretory, ciliated, Clara, and neuroendocrine cells); (2) alveolar unit cells (Type 1 cells, Type 2 cells, and fibroblasts in the interstitium); and (3) pulmonary vascular cells (endothelial cells from different vascular structures, smooth muscle cells, and adventitial fibroblasts). The main recommendations were to: (1) characterize with better cell markers, both surface and nonsurface, the various cells within the lung, including progenitor cells and stem cells; (2) obtain more knowledge about gene expression in specific cell types in health and disease, which will provide insights into biological and pathologic processes; (3) develop more methodologies for cell culture, isolation, sorting, co-culture, and immortalization; and (4) promote tissue banks to facilitate the procurement of tissue from normal and from diseased lung for analysis at all levels.

Engraftment of bone marrow progenitor cells in a rat model of asbestos-induced pulmonary fibrosis.

RATIONALE: Bone marrow-derived cells have been shown to engraft during lung fibrosis. However, it is not known if similar cells engraft consequent to inhalation of asbestos fibers that cause pulmonary fibrosis, or if the cells proliferate and differentiate at sites of injury. OBJECTIVES: We examined whether bone marrow-derived cells participate in the pulmonary fibrosis that is produced by exposure to chrysotile asbestos fibers. METHODS: Adult female rats were lethally irradiated and rescued by bone marrow transplant from male transgenic rats ubiquitously expressing green fluorescent protein (GFP). Three weeks later, the rats were exposed to an asbestos aerosol for 5 hours on three consecutive days. Controls were bone marrow-transplanted but not exposed to asbestos. MEASUREMENTS AND MAIN RESULTS: One day and 2.5 weeks after exposure, significant numbers of GFP-labeled male cells had preferentially migrated to the bronchiolar-alveolar duct bifurcations, the specific anatomic site at which asbestos produces the initial fibrogenic lesions. GFP-positive cells were present at the lesions as monocytes and macrophages, fibroblasts, and myofibroblasts or smooth muscle cells. Staining with antibodies to PCNA demonstrated that some of the engrafted cells were proliferating in the lesions and along the bronchioles. Negative results for TUNEL at the lesions confirmed that both PCNA-positive endogenous pulmonary cells and bone marrow-derived cells were proliferating rather than undergoing apoptosis, necrosis, or DNA repair. CONCLUSIONS: Bone marrow-derived cells migrated into developing fibrogenic lesions, differentiated into multiple cell types, and persisted for at least 2.5 weeks after the animals were exposed to aerosolized chrysotile asbestos fibers.

Tumor necrosis factor-alpha induces transforming growth factor-beta1 expression in lung fibroblasts through the extracellular signal-regulated kinase pathway.

Increased expression of transforming growth factor (TGF)-beta(1) and tumor necrosis factor (TNF)-alpha are thought to play important roles in the development of pulmonary fibrosis. We recently reported that TNF-alpha upregulates TGF-beta(1) expression in primary mouse lung fibroblasts (MLFs), a key cell population in fibrogenesis. In the present study, we have investigated signal transduction pathways involved in TNF-alpha upregulation of TGF-beta(1) in both primary MLFs and the Swiss 3T3 fibroblast cell line. Treatment of fibroblasts with TNF-alpha resulted in a significant increase in TGF-beta(1) protein as measured by ELISA. The increase in protein was preceded by a 200-400% increase in TGF-beta(1) mRNA detected by quantitative, real-time, reverse transcriptase-polymerase chain reaction. Western blot analysis showed that TNF-alpha activated the extracellular signal-regulated kinase (ERK), and inhibitors of the ERK-specific mitogen-activated protein kinase pathway (PD98059 or U0126) blocked TNF-alpha induction of TGF-beta(1) mRNA and protein. mRNA stability experiments showed that TNF-alpha increased the half-life of TGF-beta(1) mRNA to more than 24 h compared with approximately 15 h in unstimulated cells. Expression of constitutively active MEK1 that selectively phosphorylates ERK was sufficient for TGF-beta(1) mRNA stabilization in Swiss 3T3 fibroblasts. These results indicate that TNF-alpha activates the ERK-specific mitogen-activated protein kinase pathway leading to increased TGF-beta(1) production in fibroblasts, primarily via a post-transcriptional mechanism that involves stabilization of the TGF-beta(1) transcript.