OBJECTIVE

Abnormal cytokine expression accompanies myelofibrosis and might be a therapeutic target for Janus-associated kinase (JAK) inhibitor drugs. This study describes the spectrum of plasma cytokine abnormalities in primary myelofibrosis (PMF) and examines their phenotypic correlates and prognostic significance.

METHODS

Patients included in this study were required to have archived plasma, bone marrow biopsy, and cytogenetic information available at the time of first referral to the Mayo Clinic. Multiplex biometric sandwich immunoassay was used to measure plasma levels of 30 cytokines.

RESULTS

In total, 127 PMF patients were studied; comparison with normal controls (n = 35) revealed significantly increased interleukin-1β (IL-1β), IL-1RA, IL-2R, IL-6, IL-8, IL-10, IL-12, IL-13, IL-15, tumor necrosis factor α (TNF-α), granulocyte colony-stimulating factor (G-CSF), interferon alfa (IFN-α), macrophage inflammatory protein 1α (MIP-1α), MIP-1β, hepatocyte growth factor (HGF), IFN-γ-inducible protein 10 (IP-10), monokine induced by IFN-γ (MIG), monocyte chemotactic protein 1 (MCP-1), and vascular endothelial growth factor (VEGF) levels and decreased IFN-γ levels. In treatment-naive patients (n = 90), increased levels of IL-8 (P < .001), IL-2R (P < .001), IL-12 (P < .001), IL-15 (P = .001), and IP-10 (P = .003) were independently predictive of inferior survival. A similar multivariable analysis that included all 127 study patients confirmed the prognostic value of these five cytokines, and IL-8, IL-2R, IL-12, and IL-15 remained significant when risk stratification, according to the recently revised Dynamic International Prognostic Scoring System (DIPSS plus), was added to the multivariable model. Leukemia-free survival was predicted by IL-8, which was also the only cytokine associated with ≥ 1% circulating blasts. Other cytokine-phenotype associations included increased IL-8 and constitutional symptoms; IL-2R, IL-12, and transfusion need; IL-2R, IL-8, and leukocytosis; IP-10 and thrombocytopenia; HGF, MIG, IL-1RA, and marked splenomegaly; and IL-1RA, IL-2R, IP-10, MIP-1β, and JAK2V617F. A two-cytokine (IL-8/IL-2R) -based risk categorization delineated prognostically different groups within specific DIPSS plus risk categories.

CONCLUSIONS

This study signifies the presence of specific cytokine-phenotype associations in PMF and a prognostically relevant plasma cytokine signature that might prove useful as a laboratory tool for predicting and monitoring treatment response.

Understanding the mechanisms by which adult stem cells produce growth factors may represent an important way to optimize their beneficial paracrine and autocrine effects. Components of the wound milieu may stimulate growth factor production to promote stem cell-mediated repair. We hypothesized that tumor necrosis factor-alpha (TNF-alpha), endotoxin (LPS), or hypoxia may activate human mesenchymal stem cells (MSCs) to increase release of vascular endothelial growth factor (VEGF), fibroblast growth factor 2 (FGF2), insulin-like growth factor 1 (IGF-1), or hepatocyte growth factor (HGF) and that nuclear factor-kappa B (NF kappa B), c-Jun NH2-terminal kinase (JNK), and extracellular signal-regulated kinase (ERK) mediates growth factor production from human MSCs. To study this, human MSCs were harvested, passaged, divided into four groups (100,000 cells, triplicates) and treated as follows: 1) with vehicle; 2) with stimulant alone [24 h LPS (200 ng/ml), 24 h TNF-alpha (50 ng/ml), or 24 h hypoxia (1% O2)]; 3) with inhibitor alone [NF kappa B (PDTC, 1 mM), JNK (TI-JIP, 10 microM), or ERK (ERK Inhibitor II, 25 microM)]; and 4) with stimulant and the various inhibitors. After 24 h incubation, MSC activation was determined by measuring supernatants for VEGF, FGF2, IGF-1, or HGF (ELISA). TNF-alpha, LPS, and hypoxia significantly increased human MSC VEGF, FGF2, HGF, and IGF-1 production versus controls. Stem cells exposed to injury demonstrated increased activation of NF kappa B, ERK, and JNK. VEGF, FGF2, and HGF expression was significantly reduced by NF kappa B inhibition (50% decrease) but not ERK or JNK inhibition. Moreover, ERK, JNK, and NF kappa B inhibitor alone did not activate MSC VEGF expression over controls. Various stressors activate human MSCs to increase VEGF, FGF2, HGF, and IGF-1 expression, which depends on an NFkB mechanism.

The molecular signals controlling liver regeneration are becoming rapidly defined. Control of growth in regenerating liver has advanced from elusive serum factors and nutrient effects to identification of entirely new growth factors with apparent liver specificity as well as establishment of meaningful gene expression patterns for growth factors already known. Based on studies with hepatocyte cultures and gene expression in regenerating liver, the substances EGF, TGF alpha, HBGF-1 (aFGF), and two new substances (HPTA/HGF and Hepatopoietin B) have been defined as complete mitogens for hepatocytes and implicated in control of liver growth. The amino acid sequence of HPTA/HGF recently became clear and revealed interesting structural homologies in a molecule that might become the largest known growth factor. The plasticity of growth responses seen in liver may be controlled by these factors as well as by comitogenic substances such as norepinephrine which, although nonmitogenic per se, can initiate growth in hepatocytes exposed to the above mitogenic growth factors or mitogenic inhibitors such as TGF beta. The role of the latter in cessation of DNA synthesis in liver regeneration will be discussed, presenting the positive and negative evidence that constitutes the TGF beta paradox of liver regeneration.

Recent observations indicate that several stem cells can differentiate into hepatocytes; thus, cell-based therapy is a potential alternative to liver transplantation. The goal of the present study was to examine the in vitro hepatic differentiation potential of adipose tissue-derived mesenchymal stem cells (AT-MSCs). We used AT-MSCs from different age patients and found that, after incubation with specific growth factors (hepatocyte growth factor [HGF], fibroblast growth factor [FGF1], FGF4) the CD105(+) fraction of AT-MSCs exhibited high hepatic differentiation ability in an adherent monoculture condition. CD105(+) AT-MSC-derived hepatocyte-like cells revealed several liver-specific markers and functions, such as albumin production, low-density lipoprotein uptake, and ammonia detoxification. More importantly, CD105(+) AT-MSC-derived hepatocyte-like cells, after transplantation into mice incorporated into the parenchyma of the liver.

CONCLUSIONS

Adipose tissue is a source of multipotent stem cells that can be easily isolated, selected, and induced into mature, transplantable hepatocytes. The fact that they are easy to procure ex vivo in large numbers makes them an attractive tool for clinical studies in the context of establishing an alternative therapy for liver dysfunction.

Intratumoral hypoxia is an independent indicator of poor patient outcome and increasing evidence supports a role for hypoxia in the development of metastatic disease. Studies suggest that the acquisition of the metastatic phenotype is not simply the result of dysregulated signal transduction pathways, but instead is achieved through a stepwise selection process driven by hypoxia. Hypoxia facilitates disruption of tissue integrity through repression of E-cadherin expression, with concomitant gain of N-cadherin expression which allows cells to escape anoikis. Through upregulation of urokinase-type plasminogen activator receptor (uPAR) expression, hypoxia enhances proteolytic activity at the invasive front and alters the interactions between integrins and components of the extracellular matrix, thereby enabling cellular invasion through the basement membrane and the underlying stroma. Cell motility is increased through hypoxia-induced hepatocyte growth factor (HGF)-MET receptor signaling, resulting in cell migration towards the blood or lymphatic microcirculation. Hypoxia-induced vascular endothelial growth factor (VEGF) activity also plays a critical role in the dynamic tumor-stromal interactions required for the subsequent stages of metastasis. VEGF promotes angiogenesis and lymphangiogenesis in the primary tumor, providing the necessary routes for dissemination. VEGF-induced changes in vascular integrity and permeability promote both intravasation and extravasation, while VEGF-induced angiogenesis in the secondary tissue is essential for cell proliferation and establishment of metastatic lesions. Through regulation of these critical molecular targets, hypoxia promotes each step of the metastatic cascade and selects tumor cell populations that are able to escape the unfavorable microenvironment of the primary tumor.

The liver lobule is formed by parenchymal cells, i.e., hepatocytes and nonparenchymal cells. In contrast to hepatocytes that occupy almost 80% of the total liver volume and perform the majority of numerous liver functions, nonparenchymal liver cells, which contribute only 6.5% to the liver volume, but 40% to the total number of liver cells, are localized in the sinusoidal compartment of the tissue. The walls of hepatic sinusoid are lined by three different cell types: sinusoidal endothelial cells (SEC), Kupffer cells (KC), and hepatic stellate cells (HSC, formerly known as fat-storing cells, Ito cells, lipocytes, perisinusoidal cells, or vitamin A-rich cells). Additionally, intrahepatic lymphocytes (IHL), including pit cells, i.e., liver-specific natural killer cells, are often present in the sinusoidal lumen. It has been increasingly recognized that both under normal and pathological conditions, many hepatocyte functions are regulated by substances released from neighboring nonparenchymal cells. Liver sinusoidal endothelial cells constitute the lining or wall of the hepatic sinusoid. They perform important filtration function due to the presence of small fenestrations that allow free diffusion of many substances, but not of particles of the size of chylomicrons, between the blood and the hepatocyte surface. SEC show huge endocytic capacity for many ligands including glycoproteins, components of the extracellular matrix (ECM; such as hyaluronate, collagen fragments, fibronectin, or chondroitin sulphate proteoglycan), immune complexes, transferrin and ceruloplasmin. SEC may function as antigen-presenting cells (APC) in the context of both MHC-I and MHC-II restriction with the resulting development of antigen-specific T-cell tolerance. They are also active in the secretion of cytokines, eicosanoids (i.e., prostanoids and leukotrienes), endothelin-1, nitric oxide, and some ECM components. Kupffer cells are intrasinusoidally located tissue macrophages with a pronounced endocytic and phagocytic capacity. They are in constant contact with gut-derived particulate materials and soluble bacterial products so that a subthreshold level of their activation in the normal liver may be anticipated. Hepatic macrophages secrete potent mediators of the inflammatory response (reactive oxygen species, eicosanoids, nitric oxide, carbon monoxide, TNF-alpha, and other cytokines), and thus control the early phase of liver inflammation, playing an important part in innate immune defense. High exposure of Kupffer cells to bacterial products, especially endotoxin (lipopolysaccharide, LPS), can lead to the intensive production of inflammatory mediators, and ultimately to liver injury. Besides typical macrophage activities, Kupffer cells play an important role in the clearance of senescent and damaged erythrocytes. Liver macrophages modulate immune responses via antigen presentation, suppression of T-cell activation by antigen-presenting sinusoidal endothelial cells via paracrine actions of IL-10, prostanoids, and TNF-alpha, and participation in the development of oral tolerance to bacterial superantigens. Moreover, during liver injury and inflammation, Kupffer cells secrete enzymes and cytokines that may damage hepatocytes, and are active in the remodeling of extracellular matrix. Hepatic stellate cells are present in the perisinusoidal space. They are characterized by abundance of intracytoplasmic fat droplets and the presence of well-branched cytoplasmic processes, which embrace endothelial cells and provide focally a double lining for sinusoid. In the normal liver HSC store vitamin A, control turnover of extracellular matrix, and regulate the contractility of sinusoids. Acute damage to hepatocytes activates transformation of quiescent stellate cells into myofibroblast-like cells that play a key role in the development of inflammatory fibrotic response. Pit cells represent a liver-associated population of large granular lymphocytes, i.e., natural killer (NK) cells. They spontaneously kill a variety of tumor cells in an MHC-unrestricted way, and this antitumor activity may be enhanced by the secretion of interferon-gamma. Besides pit cells, the adult liver contains other subpopulations of lymphocytes such as gamma delta T cells, and both "conventional" and "unconventional" alpha beta T cells, the latter containing liver-specific NK T cells. The development of methods for the isolation and culture of main liver cell types allowed to demonstrate that both nonparenchymal and parenchymal cells secrete tens of mediators that exert multiple paracrine and autocrine actions. Co-culture experiments and analyses of the effects of conditioned media on cultures of another liver cell type have enabled the identification of many substances released from non-parenchymal liver cells that evidently regulate some important functions of neighboring hepatocytes and non-hepatocytes. To the key mediators involved in the intercellular communication in the liver belong prostanoids, nitric oxide, endothelin-1, TNF-alpha, interleukins, and chemokines, many growth factors (TGF-beta, PDGF, IGF-I, HGF), and reactive oxygen species (ROS). Paradoxically, the cooperation of liver cells is better understood under some pathological conditions (i.e., in experimental models of liver injury) than in normal liver due to the possibility of comparing cellular phenotype under in vivo and in vitro conditions with the functions of the injured organ. The regulation of vitamin A metabolism provides an example of the physiological role for cellular cross-talk in the normal liver. The majority (up to 80%) of the total body vitamin A is stored in the liver as long-chain fatty acid esters of retinal, serving as the main source of retinoids that are utilized by all tissues throughout the body. Hepatocytes are directly involved in the uptake from blood of chylomicron remnants, and the synthesis of retinol-binding protein that transfers retinol to other tissues. However, more than 80% of the liver retinoids are stored in lipid droplets of hepatic stellate cells. HSC are capable of both uptake and release of retinol depending on the body's retinol status. The activity of some major enzymes of vitamin A metabolism have been found to be many times higher per protein basis in stellate cells than in hepatocytes. Despite progress in the understanding of the roles played by these two cell types in hepatic retinoid metabolism, the way in which retinoids move between the parenchymal cells, stellate cells, and blood plasma has not been fully elucidated. Sinusoidal blood flow is, to a great extent, regulated by hepatic stellate cells that can contract due to the presence of smooth muscle alpha-actin. The main vasoactive substances that affect constriction or relaxation of HSC derive both from distant sources and from neighboring hepatocytes (carbon monoxide, leukotrienes), endothelial cells (endothelin, nitric oxide, prostaglandins), Kupffer cells (prostaglandins, NO), and stellate cells themselves (endothelin, NO). The cellular cross-talk reflected by the fine-tuned modulation of sinusoidal contraction becomes disturbed under pathological conditions, such as endotoxemia or liver fibrosis, through the excess synthesis of vasoregulatory compounds and the involvement of additional mediators acting in a paracrine way. The liver is an important source of some growth factors and growth factor-binding proteins. Although hepatocytes synthesize the bulk of insulin-like growth factor I (IGF-I), also other types of nonparenchymal liver cells may produce this peptide. Cell-specific expression of distinct IGF-binding proteins observed in the rat and human liver provides the potential for specific regulation of hepatic IGF-I synthesis not only by growth hormone, insulin, and IGF-I, but also by cytokines released from activated Kupffer (IL-1, TNF-alpha, TGF-beta) or stellate cells (TGF-alpha, TGF-beta). Hepatic stellate cells may affect turnover of hepatocytes through the synthesis of potent positive as well as negative signals such as, respectively, hepatocyte-growth-factor or TGF-beta. Although hepatocytes seem not to produce TGF-beta, a pleiotropic cytokine synthesized and secreted in the latent form by Kupffer and stellate cells, they may contribute to its actions in the liver by the intracellular activation of latent TGF-beta, and secretion of the biologically active isoform. Many mediators that reach the liver during inflammatory processes, such as endotoxins, immune-complexes, anaphylatoxins, and PAF, increase glucose output in the perfused liver, but fail to do so in isolated hepatocytes, acting indirectly via prostaglandins released from Kupffer cells. In the liver, prostaglandins synthesized from arachidonic acid mainly in Kupffer cells in a response to various inflammatory stimuli, modulate hepatic glucose metabolism by increasing glycogenolysis in adjacent hepatocytes. The release of glucose from glycogen supports the increased demand for energetic fuel by the inflammatory cells such as leukocytes, and additionally enables enhanced glucose turnover in sinusoidal endothelial cells and Kupffer cells which is necessary for effective defense of these cells against invading microorganisms and oxidative stress in the liver. Leukotrienes, another oxidation product of arachidonic acid, have vasoconstrictive, cholestatic, and metabolic effects in the liver. A transcellular synthesis of cysteinyl leukotrienes (LTC4, LTD4, and LTE4) functions in the liver: LTA4, an important intermediate, is synthesized in Kupffer cells, taken up by hepatocytes, converted into the potent LTC4, and then released into extracellular space, acting in a paracrine way on Kupffer and sinusoidal endothelial cells. Thus, hepatocytes are target cells for the action of eicosanoids and the site of their transformation and degradation, but can not directly oxidate arachidonic acid to eicosanoids. (ABSTRACT TRUNCATED)

Scatter factor (SF), a secretory protein of fibroblasts, dissociates and increases the motility of epithelial cells and may be involved in cell migration processes during embryogenesis and tumor progression. Hepatocyte growth factor (HGF), a protein isolated from serum of patients with liver failure, is a potent mitogen for hepatocytes and is thought to play a role in liver regeneration. Here we present structural and functional evidence that human SF and human HGF (and also the human lung fibroblast-derived mitogen) are identical proteins encoded by a single gene, since (i) no major difference could be found by protein sequencing, by cDNA analysis, and by immunological comparison and (ii) SF in fact acts as a hepatocyte growth factor--i.e., stimulates DNA synthesis of activity--i.e., dissociates and induces invasiveness of various epithelial cells. The human SF/HGF gene was localized to chromosome bands 7q11.2-21. These results have important consequences for further studies on the involvement of SF/HGF as a modulator of cellular growth and motility in embryonal, malignant, and regenerative processes.

On binding to the cell surface receptor tyrosine kinase (TK) known as c-Met, hepatocyte growth factor (HGF) stimulates mitogenesis, motogenesis, and morphogenesis in a wide range of cellular targets including, epithelial and endothelial cells, hematopoietic cells, neurons, melanocytes, and hepatocytes. These pleiotropic actions are fundamentally important during development, homeostasis, and tissue regeneration. HGF signaling also contributes to oncogenesis and tumor progression in several human cancers and promotes aggressive cellular invasiveness that is strongly linked to tumor metastasis. Our present understanding of c-Met oncogenic signaling supports at least three avenues of pathway selective anticancer drug development: antagonism of ligand/receptor interaction, inhibition of TK catalytic activity, and blockade of intracellular receptor/effector interactions. Potent and selective preclinical drug candidates have been developed using all three strategies, and human clinical trials in two of the three areas are now under way.

Claudins are transmembrane proteins that seal tight junctions, and are critical for maintaining cell-to-cell adhesion in epithelial cell sheets. However, their role in cancer progression remains largely unexplored. Here, we report that Claudin-7 (CLDN-7) expression is lower in invasive ductal carcinomas (IDC) of the breast than in normal breast epithelium, as determined by both RT-PCR (9/10) and Western analysis (6/8). Immunohistochemical (IHC) analysis of ductal carcinoma in situ (DCIS) and IDC showed that the loss of CLDN-7 expression correlated with histological grade in both DCIS (P<0.001, n=38) and IDC (P=0.014, n=31), occurring predominantly in high-grade (Nuclear and Elston grade 3) lesions. Tissue array analysis of 355 IDC cases further confirmed the inverse correlation between CLDN-7 expression and histological grade (P=0.03). This pattern of expression is consistent with the biological function of CLDN-7, as greater discohesion is typically observed in high-grade lesions. In line with this observation, by IHC analysis, CLDN-7 expression was lost in the vast majority (13/17) of cases of lobular carcinoma in situ, which is defined by cellular discohesion. In fact, inducing disassociation of MCF-7 and T47D cells in culture by treating with HGF/scatter factor resulted in a loss of CLDN-7 expression within 24 h. Silencing of CLDN-7 expression correlated with promoter hypermethylation as determined by methylation-specific PCR (MSP) and nucleotide sequencing in breast cancer cell lines (3/3), but not in IDCs (0/5). In summary, these studies provide insight into the potential role of CLDN-7 in the progression and ability of breast cancer cells to disseminate.

A unique property of many adult stem cells is their ability to exist in a non-cycling, quiescent state. Although quiescence serves an essential role in preserving stem cell function until the stem cell is needed in tissue homeostasis or repair, defects in quiescence can lead to an impairment in tissue function. The extent to which stem cells can regulate quiescence is unknown. Here we show that the stem cell quiescent state is composed of two distinct functional phases, G0 and an 'alert' phase we term G(Alert). Stem cells actively and reversibly transition between these phases in response to injury-induced systemic signals. Using genetic mouse models specific to muscle stem cells (or satellite cells), we show that mTORC1 activity is necessary and sufficient for the transition of satellite cells from G0 into G(Alert) and that signalling through the HGF receptor cMet is also necessary. We also identify G0-to-G(Alert) transitions in several populations of quiescent stem cells. Quiescent stem cells that transition into G(Alert) possess enhanced tissue regenerative function. We propose that the transition of quiescent stem cells into G(Alert) functions as an 'alerting' mechanism, an adaptive response that positions stem cells to respond rapidly under conditions of injury and stress, priming them for cell cycle entry.

OBJECTIVE

The delivery of autologous progenitor cells into ischemic tissue of patients is emerging as a novel therapeutic option. Here, we report the potential impact of cultured adipose tissue-derived cells (ADSC) on angiogenic cell therapy.

RESULTS

ADSC were isolated from C57Bl/6 mouse inguinal adipose tissue and showed high expression of ScaI and CD44, but not c-kit, Lin, CD34, CD45, CD11b, and CD31, compatible with that of mesenchymal stem cells from bone marrow. In coculture conditions with ADSC and human aortic endothelial cells (ECs) under treatment with growth factors, ADSC significantly increased EC viability, migration and tube formation mainly through secretion of vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF). At 4 weeks after transplantation of ADSC into the ischemic mouse hindlimb, the angiogenic scores were improved in the ADSC-treated group, which were evaluated with blood flow by laser Doppler imaging (LDI) and capillary density by immunostaining with anti-CD31 antibody. However, injected ADSC did not correspond to CD31, von Willebrand factor, and alpha-smooth muscle actin-positive cells in ischemic tissue.

CONCLUSIONS

These adipose tissue-derived cells demonstrated potential as angiogenic cell therapy for ischemic disease, which appears to be mainly achieved by their ability to secrete angiogenic growth factors.

alpha6beta4 integrin and the Met receptor for HGF have been shown independently to promote invasive growth. We demonstrate here that Met selectively associates with alpha6beta4. In carcinoma cells expressing Met alone, HGF does not exert significant biological effects. Ectopic expression of alpha6beta4 restores HGF-regulated processes. Following Met activation, alpha6beta4 is tyrosine phosphorylated and combines with Shc and PI3K, generating an additional signaling platform that potentiates HGF-triggered activation of Ras- and PI3K-dependent pathways. In the presence of an alpha6beta4 mutant defective for Shc recruitment, Met cannot sustain HGF-mediated responses. Surprisingly, a truncated beta4 unable to bind laminins retains the activity of wild-type alpha6beta4. Such findings invoke an unexpected role for alpha6beta4 in cancer invasion as a functional amplifier of biochemical outputs rather than a mechanical adhesive device.

The scattering of Madin-Darby canine kidney cells in vitro mimics key aspects of epithelial-mesenchymal transitions during development, carcinoma cell invasion, and metastasis. Scattering is induced by hepatocyte growth factor (HGF) and is thought to involve disruption of cadherin-dependent cell-cell junctions. Scattering is enhanced on collagen and fibronectin, as compared with laminin1, suggesting possible cross talk between integrins and cell-cell junctions. We show that HGF does not trigger any detectable decrease in E-cadherin function, but increases integrin-mediated adhesion. Time-lapse imaging suggests that tension on cell-cell junctions may disrupt cell-cell adhesion. Varying the density and type of extracellular matrix proteins shows that scattering correlates with stronger integrin adhesion and increased phosphorylation of the myosin regulatory light chain. To directly test the role of integrin-dependent traction forces, substrate compliance was varied. Rigid substrates that produce high traction forces promoted scattering, in comparison to more compliant substrates. We conclude that integrin-dependent actomyosin traction force mediates the disruption of cell-cell adhesion during epithelial cell scattering.

Trophoblast research over the past decades has underlined the striking similarities between the proliferative, migratory and invasive properties of placental cells and those of cancer cells. This review recapitulates the numerous key molecules, proto-oncogenes, growth factors, receptors, enzymes, hormones, peptides and tumour-associated antigens (TAAs) expressed by both trophoblastic and cancer cells in an attempt to evaluate the genes and proteins forming molecular circuits and regulating the similar behaviours of these cells. Among the autocrine and paracrine loops that might be involved in the strong proliferative capacity of trophoblastic and cancer cells, epidermal growth factor (EGF)/EGF receptor (EGFR), hepatocyte growth factor (HGF)/HGF receptor (HGFR) (Met) and vascular endothelial growth factor (VEGF)/VEGF receptor (VEGFR) loops may play a predominant role. Similar mechanisms of migration and invasion displayed by trophoblastic and malignant cells comprise alterations in the adhesion molecule phenotype, including the increased expression of alpha1beta1 and alphavbeta3 integrin receptors, whereas another critical molecular event is the down-regulation of the cell adhesion molecule E-cadherin. Among proteases that may play an active role in the invasive capacities of these cells, accumulating evidence suggests that matrix metalloproteinase-9 (MMP-9) expression/activation is a prerequisite. Finally, an overview of molecular circuitries shared by trophoblast and cancer cells reveals that the activation of the phosphatidylinositol 3'-kinase (PI3K)/AKT axis has recently emerged as a central feature of signalling pathways used by these cells to achieve their proliferative, migratory and invasive processes.

Hepatocyte growth factor (HGF) induces a three-phase response leading to the formation of branched tubular structures in epithelial cells. The HGF receptor tyrosine kinase works through a Src homology (SH2) docking site that can activate several signalling pathways. The first phase of the response (scattering), which results from cytoskeletal reorganization, loss of intercellular junctions and cell migration, is dependent on phosphatidylinositol-3-OH kinase and Rac activation. The second phase (growth) requires stimulation of the Ras-MAP kinase cascade. Here we show that the third phase (tubulogenesis) is dependent on the STAT pathway. HGF stimulates recruitment of Stat-3 to the receptor, tyrosine phosphorylation, nuclear translocation and binding to the specific promoter element SIE. Electroporation of a tyrosine-phosphorylated peptide, which interferes with both the association of STAT to the receptor and STAT dimerization, inhibits tubule formation in vitro without affecting either HGF-induced 'scattering' or growth. The same result is obtained using a specific 'decoy' oligonucleotide that prevents STAT from binding to DNA and affecting the expression of genes involved in cell-cycle regulation (c-fos and waf-1). Activation of signal transducers that directly control transcription is therefore required for morphogenesis.

Murine mesenchymal stem cells (MSC) have the ability to inhibit allogeneic immune responses. Two different mechanisms, either cell contact-dependent or independent, have been proposed to account for this immunosuppression. The focus of this study was to elucidate the involvement of soluble suppressive factors secreted by murine MSC in an inflammatory setting, and their role in MSC immunomodulation. In a non-inflammatory environment, bone marrow derived murine MSC constitutively expressed low levels of COX-2, PGE-2, TGF-beta1 and HGF, but not IL-10, PD-1, PD-L1 or PD-L2. These MSC were able to significantly reduce alloantigen driven proliferation in mixed lymphocyte reactions as well as mitogen driven proliferation. The pro-inflammatory cytokines IFN-gamma and TNF-alpha did not ablate MSC mediated immunosuppression. MSC expression of PGE-2, IDO and PD-L1 was differentially regulated by these cytokines. COX-2 and PGE-2 expression by MSC were upregulated by both IFN-gamma and TNF-alpha, and using a biochemical inhibitor this was shown to have an essential, non-redundant role in modulating alloantigen-driven proliferation. However, the surface expression of PD-L1 was induced by IFN-gamma but not TNF-alpha and similarly functional IDO expression was only induced by IFN-gamma stimulation. Blocking studies using neutralising antibodies and biochemical antagonists revealed that while PD-L1 induction was not essential, IDO expression was a prerequisite for IFN-gamma mediated MSC immunomodulation. These data demonstrate that murine MSC expression of immunomodulatory factors dramatically changes in a pro-inflammatory environment and that IFN-gamma in particular has an important role in regulating MSC immunomodulatory factor expression.

The maintenance of a healthy corneal epithelium under both normal and wound healing conditions is achieved by a population of stem cells (SC) located in the basal epithelium at the corneoscleral limbus. In the light of the development of strategies for reconstruction of the ocular surface in patients with limbal stem cell deficiency, a major challenge in corneal SC biology remains the ability to identify stem cells in situ and in vitro. Until recently, the identification of limbal stem cells mainly has been based on general properties of stem cells, e.g. lack of differentiation, prolonged label-retaining, indefinite capacity of proliferation exemplified by the clonogenic assay as well as their special role in corneal wound healing. During the last years, a number of molecular markers for the limbal SC compartment has been proposed, however, their role in distinguishing limbal SC from their early progeny is still under debate. Data reported from the literature combined with our own recent observations suggest, that the basal epithelial cells of the human limbus contain ABCG2, K19, vimentin, KGF-R, metallothionein, and integrin alpha9, but do not stain for K3/K12, Cx43, involucrin, P-cadherin, integrins alpha2, alpha6, and beta4, and nestin, when compared to the basal cells of the corneal epithelium. A relatively higher expression level in basal limbal cells was observed for p63, alpha-enolase, K5/14, and HGF-R, whereas there were no significant differences in staining intensity for beta-catenin, integrins alphav, beta1, beta2, and beta5, CD71, EGF-R, TGF-beta-RI, TGF-beta-RII, and TrkA between limbal and corneal basal epithelial cells. Therefore, a combination of differentiation-associated markers (e.g. K3/K12, Cx43, or involucrin) and putative SC-associated markers (e.g. ABCG2, K19, vimentin, or integrin alpha9) may provide a suitable tool for identification of human limbal SC. While most putative SC markers label the majority of limbal basal cells and, therefore, may not distinguish SC from progenitor cells, only ABCG2 was strictly confined to small clusters of basal cells in the limbal epithelium. At present, ABCG2 therefore appears to be the most useful cell surface marker for the identification and isolation of corneal epithelial SC. Moreover, the characteristics of the specific microenvironment of corneal SC, as provided by growth factor activity and basement membrane heterogeneity in the limbal area, could serve as additional tools for their selective enrichment and in vitro expansion for the purpose of ocular surface reconstruction.

Embryonic stem cells (ES cells), bone marrow-derived mesenchymal stem cells, umbilical cord blood-derived mesenchymal stem cells, and hepatic stem cells in liver have been known as a useful source that can induce to differentiate into hepatocytes. In this study, we examined whether human adipose tissue-derived stromal cells (hADSC) can differentiate into hepatic lineage in vitro. hADSC, that were induced to differentiate into hepatocyte-like cells by the treatment of HGF and OSM, had morphology similar to hepatocytes. Addition of DMSO enhanced differentiation into hepatocytes. RT-PCR and immunocytochemical analysis showed that hADSC express albumin and alpha-fetoprotein during differentiation. Differentiated hADSC showed LDL uptake and production of urea. Additionally, transplanted hADSC to CCl4-injured SCID mouse model were able to be differentiated into hepatocytes and they expressed albumin in vivo. Mesenchymal stem cells isolated from human adipose tissue are immunocompatible and are easily isolated. Therefore, hADSC may become an alternative source to hepatocyte regeneration or liver cell transplantation.

Myogenesis in the embryo and the adult mammal consists of a highly organized and regulated sequence of cellular processes to form or repair muscle tissue that include cell proliferation, migration, and differentiation. Data from cell culture and in vivo experiments implicate both FGFs and HGF as critical regulators of these processes. Both factors require heparan sulfate glycosaminoglycans for signaling from their respective receptors. Since syndecans, a family of cell-surface transmembrane heparan sulfate proteoglycans (HSPGs) are implicated in FGF signaling and skeletal muscle differentiation, we examined the expression of syndecans 1-4 in embryonic, fetal, postnatal, and adult muscle tissue, as well as on primary adult muscle fiber cultures. We show that syndecan-1, -3, and -4 are expressed in developing skeletal muscle tissue and that syndecan-3 and -4 expression is highly restricted in adult skeletal muscle to cells retaining myogenic capacity. These two HSPGs appear to be expressed exclusively and universally on quiescent adult satellite cells in adult skeletal muscle tissue, suggesting a role for HSPGs in satellite cell maintenance or activation. Once activated, all satellite cells maintain expression of syndecan-3 and syndecan-4 for at least 96 h, also implicating these HSPGs in muscle regeneration. Inhibition of HSPG sulfation by treatment of intact myofibers with chlorate results in delayed proliferation and altered MyoD expression, demonstrating that heparan sulfate is required for proper progression of the early satellite cell myogenic program. These data suggest that, in addition to providing potentially useful new markers for satellite cells, syndecan-3 and syndecan-4 may play important regulatory roles in satellite cell maintenance, activation, proliferation, and differentiation during skeletal muscle regeneration.

During liver regeneration quiescent hepatocytes undergo one or two rounds of replication and then return to a nonproliferative state. Growth factors regulate this process by providing both stimulatory and inhibitory signals for cell proliferation. EGF, TGF alpha, and HGF stimulate DNA synthesis in hepatocytes in vivo and in culture but the sensitivity of cultured hepatocytes to the mitogenic effects of these factors is much higher than that of quiescent hepatocytes in intact livers. We have proposed that after partial hepatectomy, hepatocytes enter a state of replicative competence ("priming") before they can fully respond to growth factors. The priming step is an initiating event in liver regeneration that involves the activation and DNA binding of NF-kappa B and other transcription factors, which could be induced by TNF or other cytokines. EGF, TGF alpha, and HGF have major effects on liver growth. TGF alpha expression correlates with hepatocyte DNA synthesis during liver development and growth and the constitutive expression of the factor confers proliferative activity to adult hepatocytes in vivo and in culture. The data indicate that the activity of stimulatory and inhibitory growth factors such as TGF beta 1 and activin is low in normal livers but that the expression of both types of factors increase during liver regeneration.

In order to metastasize, cancer cells need to acquire a motile phenotype. Previously, development of this phenotype was thought to rely on the acquisition of selected, random mutations and thus would occur late in cancer progression. However, recent studies show that cancer cells disseminate early, implying the existence of a different, faster route to the metastatic motile phenotype. Using a spontaneous murine model of melanoma, we show that a subset of bone marrow-derived immune cells (myeloid-derived suppressor cells or MDSC) preferentially infiltrates the primary tumor and actively promotes cancer cell dissemination by inducing epithelial-mesenchymal transition (EMT). CXCL5 is the main chemokine attracting MDSC to the primary tumor. In vitro assay using purified MDSC showed that TGF-β, EGF, and HGF signaling pathways are all used by MDSC to induce EMT in cancer cells. These findings explain how cancer cells acquire a motile phenotype so early and provide a mechanistic explanation for the long recognized link between inflammation and cancer progression.

Hepatocyte growth factor/scatter factor (HGF) exerts several functions in physiological and pathological processes, among them the induction of epithelial cell scattering and motility. Its pivotal role in angiogenesis, tumor progression, and metastasis is evident; however, the underlying molecular mechanisms are still poorly understood. Here, we demonstrate that HGF induces scattering of epithelial cells by upregulating the expression of Snail, a transcriptional repressor involved in epithelial-mesenchymal transition (EMT). Snail is required for HGF-induced cell scattering, since shRNA-mediated ablation of Snail expression prevents this process. HGF-induced upregulation of Snail transcription involves activation of the mitogen-activated protein kinase (MAPK) pathway and requires the activity of early growth response factor-1 (Egr-1). Upon induction by Egr-1, Snail represses the expression of E-cadherin and claudin-3 genes. It also binds to the Egr-1 promoter and represses Egr-1 transcription, thereby establishing a negative regulatory feedback loop. These findings indicate that Snail upregulation by HGF is mediated via the MAPK/Egr-1 signaling pathway and that both Snail and Egr-1 play a critical role in HGF-induced cell scattering, migration, and invasion.

BACKGROUND

The optimum treatment for high-risk soft-tissue sarcoma (STS) in adults is unclear. Regional hyperthermia concentrates the action of chemotherapy within the heated tumour region. Phase 2 studies have shown that chemotherapy with regional hyperthermia improves local control compared with chemotherapy alone. We designed a parallel-group randomised controlled trial to assess the safety and efficacy of regional hyperthermia with chemotherapy.

METHODS

Patients were recruited to the trial between July 21, 1997, and November 30, 2006, at nine centres in Europe and North America. Patients with localised high-risk STS >> or = 5 cm, Fédération Nationale des Centres de Lutte Contre le Cancer [FNCLCC] grade 2 or 3, deep to the fascia) were randomly assigned to receive either neo-adjuvant chemotherapy consisting of etoposide, ifosfamide, and doxorubicin (EIA) alone, or combined with regional hyperthermia (EIA plus regional hyperthermia) in addition to local therapy. Local progression-free survival (LPFS) was the primary endpoint. Efficacy analyses were done by intention to treat. This trial is registered with ClinicalTrials.gov, number NCT 00003052.

RESULTS

341 patients were enrolled, with 169 randomly assigned to EIA plus regional hyperthermia and 172 to EIA alone. All patients were included in the analysis of the primary endpoint, and 332 patients who received at least one cycle of chemotherapy were included in the safety analysis. After a median follow-up of 34 months (IQR 20-67), 132 patients had local progression (56 EIA plus regional hyperthermia vs 76 EIA). Patients were more likely to experience local progression or death in the EIA-alone group compared with the EIA plus regional hyperthermia group (relative hazard [RH] 0.58, 95% CI 0.41-0.83; p=0.003), with an absolute difference in LPFS at 2 years of 15% (95% CI 6-26; 76% EIA plus regional hyperthermia vs 61% EIA). For disease-free survival the relative hazard was 0.70 (95% CI 0.54-0.92, p=0.011) for EIA plus regional hyperthermia compared with EIA alone. The treatment response rate in the group that received regional hyperthermia was 28.8%, compared with 12.7% in the group who received chemotherapy alone (p=0.002). In a pre-specified per-protocol analysis of patients who completed EIA plus regional hyperthermia induction therapy compared with those who completed EIA alone, overall survival was better in the combined therapy group (HR 0.66, 95% CI 0.45-0.98, p=0.038). Leucopenia (grade 3 or 4) was more frequent in the EIA plus regional hyperthermia group compared with the EIA-alone group (128 of 165 vs 106 of 167, p=0.005). Hyperthermia-related adverse events were pain, bolus pressure, and skin burn, which were mild to moderate in 66 (40.5%), 43 (26.4%), and 29 patients (17.8%), and severe in seven (4.3%), eight (4.9%), and one patient (0.6%), respectively. Two deaths were attributable to treatment in the combined treatment group, and one death was attributable to treatment in the EIA-alone group.

CONCLUSIONS

To our knowledge, this is the first randomised phase 3 trial to show that regional hyperthermia increases the benefit of chemotherapy. Adding regional hyperthermia to chemotherapy is a new effective treatment strategy for patients with high-risk STS, including STS with an abdominal or retroperitoneal location.

BACKGROUND

Deutsche Krebshilfe, Helmholtz Association (HGF), European Organisation of Research and Treatment of Cancer (EORTC), European Society for Hyperthermic Oncology (ESHO), and US National Institute of Health (NIH).

Mature adult parenchymal hepatocytes, typically of restricted capacity to proliferate in culture, can now enter into clonal growth under the influence of hepatocyte growth factor (scatter factor) (HGF/SF), epidermal growth factor (EGF), and transforming growth factor alpha (TGFalpha) in the presence of a new chemically defined medium (HGM). The expanding populations of hepatocytes lose expression of hepatocyte specific genes (albumin, cytochrome P450 IIB1), acquire expression of markers expressed by bile duct epithelium (cytokeratin 19), produce TGFalpha and acidic FGF and assume a very simplified morphologic phenotype by electron microscopy. A major change associated with this transition is the decrease in ratio between transcription factors C/EBPalpha and C/EBPbeta, as well as the emergence in the proliferating hepatocytes of transcription factors AP1, NFkappaB. The liver associated transcription factors HNFI, HNF3, and HNF4 are preserved throughout this process. After population expansion and clonal growth, the proliferating hepatocytes can return to mature hepatocyte phenotype in the presence of EHS gel (Matrigel). This includes complete restoration of electron microscopic structure and albumin expression. The hepatocyte cultures however can instead be induced to form acinar/ductular structures akin to bile ductules (in the presence of HGF/SF and type I collagen). These transformations affect the entire population of the hepatocytes and occur even when DNA synthesis is inhibited. Similar acinar/ductular structures are seen in embryonic liver when HGF/SF and its receptor are expressed at high levels. These findings strongly support the hypothesis that mature hepatocytes can function as or be a source of bipotential facultative hepatic stem cells (hepatoblasts). These studies also provide evidence for the growth factor and matrix signals that govern these complex phenotypic transitions of facultative stem cells which are crucial for recovery from acute and chronic liver injury.