Hypoxia stimulates pancreatic stellate cells to induce fibrosis and angiogenesis in pancreatic cancer

Pancreatic cancer is characterized by excessive desmoplastic reaction and by a hypoxic microenvironment within the solid tumor mass. Chronic pancreatitis is also characterized by fibrosis and hypoxia. Fibroblasts in the area of fibrosis in these pathological settings are now recognized as activated pancreatic stellate cells (PSCs). Recent studies have suggested that a hypoxic environment concomitantly exists not only in pancreatic cancer cells but also in surrounding PSCs. This study aimed to clarify whether hypoxia affected the cell functions in PSCs. Human PSCs were isolated and cultured under normoxia (21% O(2)) or hypoxia (1% O(2)). We examined the effects of hypoxia and conditioned media of hypoxia-treated PSCs on cell functions in PSCs and in human umbilical vein endothelial cells. Hypoxia induced migration, type I collagen expression, and vascular endothelial growth factor (VEGF) production in PSCs. Conditioned media of hypoxia-treated PSCs induced migration of PSCs, which was inhibited by anti-VEGF antibody but not by antibody against hepatocyte growth factor. Conditioned media of hypoxia-treated PSCs induced endothelial cell proliferation, migration, and angiogenesis in vitro and in vivo. PSCs expressed several angiogenesis-regulating molecules including VEGF receptors, angiopoietin-1, and Tie-2. In conclusion, hypoxia induced profibrogenic and proangiogenic responses in PSCs. In addition to their established profibrogenic roles, PSCs might play proangiogenic roles during the development of pancreatic fibrosis, where they are subjected to hypoxia.     

Exosomes derived from pancreatic cancer cells induce activation and profibrogenic activities in pancreatic stellate cells

Pancreatic cancer cells (PCCs) interact with pancreatic stellate cells (PSCs), which play a pivotal role in pancreatic fibrogenesis, to develop the cancer-conditioned tumor microenvironment. Exosomes are membrane-enclosed nanovesicles, and have been increasingly recognized as important mediators of cell-to-cell communications. The aim of this study was to clarify the effects of PCC-derived exosomes on cell functions in PSCs. Exosomes were isolated from the conditioned medium of Panc-1 and SUIT-2 PCCs. Human primary PSCs were treated with PCC-derived exosomes. PCC-derived exosomes stimulated the proliferation, migration, activation of ERK and Akt, the mRNA expression of α-smooth muscle actin (ACTA2) and fibrosis-related genes, and procollagen type I C-peptide production in PSCs. Ingenuity pathway analysis of the microarray data identified transforming growth factor β1 and tumor necrosis factor as top upstream regulators. PCCs increased the expression of miR-1246 and miR-1290, abundantly contained in PCC-derived exosomes, in PSCs. Overexpression of miR-1290 induced the expression of ACTA2 and fibrosis-related genes in PSCs. In conclusion, PCC-derived exosomes stimulate activation and profibrogenic activities in PSCs. Exosome-mediated interactions between PSCs and PCCs might play a role in the development of the tumor microenvironment.     

Lipopolysaccharide induces the differentiation of hepatic progenitor cells into myofibroblasts via activation of the Hedgehog signaling pathway

Normally, hepatic progenitor cells (HPCs) are activated and differentiate into hepatocytes or bile ductular cells to repair liver damage during liver injury. However, it remains controversial whether the abnormal differentiation of HPCs occurs under abnormal conditions. Lipopolysaccharide (LPS), a component of the microenvironment, promotes liver fibrosis. In the present study, HPCs promoted liver fibrosis in rats following carbon tetrachloride (CCl₄) treatment. Meanwhile, the LPS level in the portal vein was elevated and played a primary role in the fate of HPCs. In vitro, LPS inhibited the hepatobiliary differentiation of HPCs. Concurrently, HPCs co-cultured with LPS for 2 weeks showed a tendency to differentiate into myofibroblasts (MFs). Thus, we conclude that LPS promotes the aberrant differentiation of HPCs into MFs as a third type of descendant. This study provides insight into a novel differentiation fate of HPCs in their microenvironment, and could thus lead to the development of HPCs for treatment methods in liver fibrosis.     

Involvement of host stroma cells and tissue fibrosis in pancreatic tumor development in transgenic mice

Introduction

Stroma cells and extracellular matrix (ECM) components provide the pivotal microenvironment for tumor development. The study aimed to evaluate the importance of the pancreatic stroma for tumor development.

Methods

Pancreatic tumor cells were implanted subcutaneously into green fluorescent protein transgenic mice, and stroma cells invading the tumors were identified through immunohistochemistry. Inhibition of tumor invasion by stroma cells was achieved with halofuginone, an inhibitor of TGFβ/Smad3 signaling, alone or in combination with chemotherapy. The origin of tumor ECM was evaluated with species-specific collagen I antibodies and in situ hybridization of collagen α1(I) gene. Pancreatic fibrosis was induced by cerulean injection and tumors by spleen injection of pancreatic tumor cells.

Results

Inhibition of stroma cell infiltration and reduction of tumor ECM levels by halofuginone inhibited development of tumors derived from mouse and human pancreatic cancer cells. Halofuginone reduced the number only of stroma myofibroblasts expressing both contractile and collagen biosynthesis markers. Both stroma myofibroblasts and tumor cells generated ECM that contributes to tumor growth. Combination of treatments that inhibit stroma cell infiltration, cause apoptosis of myofibroblasts and inhibit Smad3 phosphorylation, with chemotherapy that increases tumor-cell apoptosis without affecting Smad3 phosphorylation was more efficacious than either treatment alone. More tumors developed in fibrotic than in normal pancreas, and prevention of tissue fibrosis greatly reduced tumor development.

Conclusions

The utmost importance of tissue fibrosis and of stroma cells for tumor development presents potential new therapy targets, suggesting combination therapy against stroma and neoplastic cells as a treatment of choice.     

Rapamycin ameliorates kidney fibrosis by inhibiting the activation of mTOR signaling in interstitial macrophages and myofibroblasts

Interstitial fibrosis is an inevitable outcome of all kinds of progressive chronic kidney disease (CKD). Emerging data indicate that rapamycin can ameliorate kidney fibrosis by reducing the interstitial infiltrates and accumulation of extra cellular matrix (ECM). However, the cellular mechanism that regulates those changes has not been well understood yet. In this study, we revealed the persistent activation of mammalian target of rapamycin (mTOR) signaling in the interstitial macrophages and myofibroblasts, but rarely in injured proximal epithelial cells, CD4+ T cells, neutrophils, or endothelial cells, during the development of kidney fibrosis. Administration of rapamycin to unilateral ureteral obstruction (UUO) mice significantly suppressed the immunoreactivity of mTOR signaling, which decreased the inflammatory responses and ECM accumulation in the obstructed kidneys. Isolated macrophages from rapamycin-treated obstructed kidneys presented less inflammatory activity than vehicle groups. In vitro study confirmed that rapamycin significantly inhibited the fibrogenic activation of cultured fibroblasts (NIH3T3 cells), which was induced by the stimulation of TGF-β(1). Further experiment revealed that rapamycin did not directly inhibit the fibrogenesis of HK2 cells with aristolochic acid treatment. Our findings clarified that rapamycin can ameliorate kidney fibrosis by blocking the mTOR signaling in interstitial macrophages and myofibroblasts.     

Laser-induced gene expression in specific cells of transgenic zebrafish

Over the past few years, a number of studies have described the generation of transgenic lines of zebrafish in which expression of reporters was driven by a variety of promoters. These lines opened up the real possibility that transgenics could be used to complement the genetic analysis of zebrafish development. Transgenic lines in which the expression of genes can be regulated both in space and time would be especially useful. Therefore, we have cloned the zebrafish promoter for the inducible hsp70 gene and made stable transgenic lines of zebrafish that express the reporter green fluorescent protein gene under the control of a hsp70 promoter. At normal temperatures, green fluorescent protein is not detectable in transgenic embryos with the exception of the lens, but is robustly expressed throughout the embryo following an increase in ambient temperature. Furthermore, we have taken advantage of the accessibility and optical clarity of the embryos to express green fluorescent protein in individual cells by focussing a sublethal laser microbeam onto them. The targeted cells appear to develop normally: cells migrate normally, neurons project axons that follow normal pathways, and progenitor cells divide and give rise to normal progeny cells. By generating other transgenic lines in which the hsp70 promoter regulates genes of interest, it should be possible to examine the in vivo activity of the gene products by laser-inducing specific cells to express them in zebrafish embryos. As a first test, we laser-induced single muscle cells to make zebrafish Sema3A1, a semaphorin that is repulsive for specific growth cones, in a hsp70-sema3A1 transgenic line of zebrafish and found that extension by the motor axons was retarded by the induced muscle.     

Specific activation of the human HSP70 promoter by copper sulfate in mosaic transgenic zebrafish

Heat shock proteins (HSPs) play a central role in cell protection and repair upon stresses, such as that caused by heat and heavy metals. Copper sulfate inducibility of a pHhsp70 construct expressing the enhanced green fluorescent protein (EGFP) gene under the control of the exogenous human hsp70 promoter was tested in transfected CHSE 214 cells and transgenic zebrafish (Danio rerio). We developed a transient expression system, using mosaically transgenic zebrafish, which allows rapid analysis of transgenic expression. Transfected CHSE 214 cells which had been exposed to 250 nM and 2.5 microM copper sulfate for up to 24h showed increased EGFP expression in a dose-dependent manner. The 1.5 microM copper sulfate caused stronger EGFP fluorescence than the 1.0 microM copper sulfate in transgenic zebrafish. Most of the expression was spotty and was detected in the gills, dorsal and ventral retina, myotubes of the trunk, and skin epithelium. Transgenic zebrafish exposed to copper sulfate exhibited gross dysmorphogenesis, edema and trunk abnormalities, such as spinal lordosis, in vertebral development 5 days after fertilization. This transgenic zebrafish system was sensitive enough to detect copper sulfate at doses below the median lethal concentration (the LC50 was calculated to be 1.2 microM (95% confidence interval of 0.6-1.9 microM)). These results indicate that zebrafish could be useful transgenic biosensor systems for the detection of xenobiotic toxicants in the environment.     

Lysophosphatidic acid accelerates lung fibrosis by inducing differentiation of mesenchymal stem cells into myofibroblasts

Lung fibrosis is characterized by vascular leakage and myofibroblast recruitment, and both phenomena are mediated by lysophosphatidic acid (LPA) via its type‐1 receptor (LPA1). Following lung damage, the accumulated myofibroblasts activate and secrete excessive extracellular matrix (ECM), and form fibrotic foci. Studies have shown that bone marrow‐derived cells are an important source of myofibroblasts in the fibrotic organ. However, the type of cells in the bone marrow contributing predominantly to the myofibroblasts and the involvement of LPA‐LPA1 signalling in this is yet unclear. Using a bleomycin‐induced mouse lung‐fibrosis model with an enhanced green fluorescent protein (EGFP) transgenic mouse bone marrow replacement, we first demonstrated that bone marrow derived‐mesenchymal stem cells (BMSCs) migrated markedly to the bleomycin‐injured lung. The migrated BMSC contributed significantly to α‐smooth muscle actin (α‐SMA)‐positive myofibroblasts. By transplantation of GFP‐labelled human BMSC (hBMSC) or EGFP transgenic mouse BMSC (mBMSC), we further showed that BMSC might be involved in lung fibrosis in severe combined immune deficiency (SCID)/Beige mice induced by bleomycin. In addition, using quantitative‐RT‐PCR, western blot, Sircol collagen assay and migration assay, we determined the underlying mechanism was LPA‐induced BMSC differentiation into myofibroblast and the secretion of ECM via LPA1. By employing a novel LPA1 antagonist, Antalpa1, we then showed that Antalpa1 could attenuate lung fibrosis by inhibiting both BMSC differentiation into myofibroblast and the secretion of ECM. Collectively, the above findings not only further validate LPA1 as a drug target in the treatment of pulmonary fibrosis but also elucidate a novel pathway in which BMSCs contribute to the pathologic process.     

L-cysteine administration attenuates pancreatic fibrosis induced by TNBS in rats by inhibiting the activation of pancreatic stellate cell

Background and Aims

Recent studies have shown that activated pancreatic stellate cells (PSCs) play a major role in pancreatic fibrogenesis. We aimed to study the effect of L-cysteine administration on fibrosis in chronic pancreatitis (CP) induced by trinitrobenzene sulfonic acid (TNBS) in rats and on the function of cultured PSCs.

Methods

CP was induced by TNBS infusion into rat pancreatic ducts. L-cysteine was administrated for the duration of the experiment. Histological analysis and the contents of hydroxyproline were used to evaluate pancreatic damage and fibrosis. Immunohistochemical analysis of α-SMA in the pancreas was performed to detect the activation of PSCs in vivo. The collagen deposition related proteins and cytokines were determined by western blot analysis. DNA synthesis of cultured PSCs was evaluated by BrdU incorporation. We also evaluated the effect of L-cysteine on the cell cycle and cell activation by flow cytometry and immunocytochemistry. The expression of PDGFRβ, TGFβRII, collagen 1α1 and α-SMA of PSCs treated with different concentrations of L-cysteine was determined by western blot. Parameters of oxidant stress were evaluated in vitro and in vivo. Nrf2, NQO1, HO-1, IL-1β expression were evaluated in pancreas tissues by qRT-PCR.

Results

The inhibition of pancreatic fibrosis by L-cysteine was confirmed by histological observation and hydroxyproline assay. α-SMA, TIMP1, IL-1β and TGF-β1 production decreased compared with the untreated group along with an increase in MMP2 production. L-cysteine suppressed the proliferation and extracellular matrix production of PSCs through down-regulating of PDGFRβ and TGFβRII. Concentrations of MDA+4-HNE were decreased by L-cysteine administration along with an increase in GSH levels both in tissues and cells. In addition, L-cysteine increased the mRNA expression of Nrf2, NQO1 and HO-1 and reduced the expression of IL-1β in L-cysteine treated group when compared with control group.

Conclusion

L-cysteine treatment attenuated pancreatic fibrosis in chronic pancreatitis in rats.     

Acinar cells and the development of pancreatic fibrosis

Pancreatic fibrosis is caused by excessive deposition of extracellular matrixes of collagen and fibronectin in the pancreatic tissue as a result of repeated injury often seen in patients with chronic pancreatic diseases. The most common causative conditions include inborn errors of metabolism, chemical toxicity and autoimmune disorders. Its pathophysiology is highly complex, including acinar cell injury, acinar stress response, duct dysfunction, pancreatic stellate cell activation, and persistent inflammatory response. However, the specific mechanism remains to be fully clarified. Although the current therapeutic strategies targeting pancreatic stellate cells show good efficacy in cell culture and animal models, they are not satisfactory in the clinic. Without effective intervention, pancreatic fibrosis can promote the transformation from pancreatitis to pancreatic cancer, one of the most lethal malignancies. In the normal pancreas, the acinar component accounts for 82% of the exocrine tissue. Abnormal acinar cells may activate pancreatic stellate cells directly as cellular source of fibrosis or indirectly via releasing various substances and initiate pancreatic fibrosis. A comprehensive understanding of the role of acinar cells in pancreatic fibrosis is critical for designing effective intervention strategies. In this review, we focus on the role of and mechanisms underlying pancreatic acinar injury in pancreatic fibrosis and their potential clinical significance.     

Repression of Hedgehog signal transduction in T-lineage cells increases TCR-induced activation and proliferation

Hedgehog proteins signal for differentiation, survival and proliferation of the earliest thymocyte progenitors, but their functions at later stages of thymocyte development and in peripheral T-cell function are controversial. Here we show that repression of Hedgehog (Hh) pathway activation in T-lineage cells, by expression of a transgenic repressor form of Gli2 (Gli2δC2), increased T-cell differentiation and activation in response to TCR signalling. Expression of the Gli2δC2 transgene increased differentiation from CD4+CD8+ to single positive thymocyte, and increased peripheral T cell populations. Gli2δC2 T-cells were hyper-responsive to activation by ligation of CD3 and CD28: they expressed cell surface activation markers CD69 and CD25 more quickly, and proliferated more than wild-type T-cells. These data show that Hedgehog pathway activation in thymocytes and T-cells negatively regulates TCR-dependent differentiation and proliferation. Thus, as negative regulators of TCR-dependent events, Hh proteins provide an environmental influence on T-cell fate.     

Hedgehog signal activation coordinates proliferation and differentiation of fetal liver progenitor cells

Hedgehog (Hh) signaling plays crucial roles in development and homeostasis of various organs. In the adult liver, it regulates proliferation and/or viability of several types of cells, particularly under injured conditions, and is also implicated in stem/progenitor cell maintenance. However, the role of this signaling pathway during the normal developmental process of the liver remains elusive. Although Sonic hedgehog (Shh) is expressed in the ventral foregut endoderm from which the liver derives, the expression disappears at the onset of the liver bud formation, and its possible recurrence at the later stages has not been investigated. Here we analyzed the activation and functional relevance of Hh signaling during the mouse fetal liver development. At E11.5, Shh and an activation marker gene for Hh signaling, Gli1, were expressed in Dlk⁺ hepatoblasts, the fetal liver progenitor cells, and the expression was rapidly decreased thereafter as the development proceeded. In the culture of Dlk⁺ hepatoblasts isolated from the E11.5 liver, activation of Hh signaling stimulated their proliferation and this effect was cancelled by a chemical Hh signaling inhibitor, cyclopamine. In contrast, hepatocyte differentiation of Dlk⁺ hepatoblasts in vitro as manifested by the marker gene expression and acquisition of ammonia clearance activity was significantly inhibited by forced activation of Hh signaling. Taken together, these results demonstrate the temporally restricted manner of Hh signal activation and its role in promoting the hepatoblast proliferation, and further suggest that the pathway needs to be shut off for the subsequent hepatic differentiation of hepatoblasts to proceed normally.     

Identification,paracrine generation,and possible function of human breast carcinoma myofibroblasts in culture

Summary Myofibroblasts from human breast carcinomas were identified and experimentally generated in culture, and a possible function was examined. The frequency ofα-smooth muscle actin immunoreactive cells was evaluated as a measure of myofibroblast differentiation in primary culture. Few or noα-smooth muscle actin-positive stromal cells (6.1 ± 8.4%) were identified in primary cultures from normal breast tissue (n=9). In contrast, high frequencies (68.8 ± 15.1%) were observed in primary cultures from carcinomas (n=19). The frequencies of myofibroblasts in primary cultures were almost identical to those obtained in the corresponding cryostat sections (69.1 vs. 68.8%). A possible precursor cell to the myofibroblast was looked for among typical fibroblasts and vascular smooth muscle cells. Purified blood vessels containing both fibroblasts and vascular smooth muscle cells were embedded in collagen gel and incubated with medium conditioned by breast epithelial cells. Fibroblasts rather than smooth muscle cells were recruited from the blood vessels. In medium conditioned by carcinoma cell lines or in co-cultures of carcinoma cell lines and purified fibroblasts,α-smooth muscle actin and the typical myofibroblast phenotype were induced in otherwiseα-smooth muscle actin-negative fibroblasts. The effect of myofibroblasts on cellular movement—essential to neoplastic cells—was analyzed. Spontaneous motility of tumor cells (MCF-7) was entirely suppressed in a collagen gel assay. Under these conditions tumor cell motility was selectively mediated by direct cell-to-cell interaction between tumor cells and myofibroblasts. Under chemically defined conditions, interaction was dependent on the presence of plasminogen. Anti-plasminogen, soybean trypsin inhibitor, and anti-fibronectin partly neutralized the effect of plasminogen. It is concluded that elements of myofibroblast differentiation and function may be studied in culture.     

Liposomes fuse with sperm cells and induce activation by delivery of impermeant agents

Sperm cell activation is a critical step in fertilization. To directly investigate the cell signaling events leading to sperm activation it is necessary to deliver membrane impermeant agents into the cytoplasm. In this study, the use of liposomes as possible agent-loading vectors was examined using (1) the octadecylrhodamine B (R18) and NBD phosphatidylethanolamine (NBD DHPE)/rhodamine phosphatidylethanolamine (rhod DHPE) fusion assays in bulk samples, (2) membrane transfer of fluorescence from liposome membranes labeled with R18 and rhodamine-tagged phosphatidylethanolamine (TRITC DHPE), and (3) lumenal transfer of impermeant calcium ions from liposomes to sperm cells, a process that stimulated sperm cell activation. Intermediate-sized unilamellar liposomes (98.17+/-15.34 nm) were prepared by the detergent-removal technique using sodium cholate as the detergent and a phosphatidylcholine/phosphatidylethanolamine/cholesterol (2:1:1 mole ratio) lipid composition. In the R18 fusion assays, self-quenching increased logarithmically with increasing concentrations of R18 in the liposome membranes; addition of unlabeled sperm to R18-labeled liposomes lead to a rapid release of self-quenching. In the NBD DHPE/rhod DHPE resonance energy transfer (RET) fusion assay, RET was rapidly reduced under similar conditions. In addition, individual sperm became fluorescent when TRITC DHPE-labeled liposomes were incubated with unlabeled sperm cells. Incubation of sperm cells with empty liposomes did not significantly affect sperm cell activation and did not alter cell morphology. However, incubation with Ca (10 mM)-loaded liposomes resulted in a time-dependent increase in sperm cell activation (7.5-fold over controls after 15 min). We conclude that liposomes can be used for direct loading of membrane-impermeant agents into sea squirt sperm cell cytoplasm, and that delivery occurs via fusion and content intermixing.