Reprogramming of Activated Pancreatic Stellate Cells via Mechanical Modulation of Transmembrane Force-sensitive N-cadherin Receptor.

Cancer has been the leading cause of death due mainly to tumor metastasis. The tumor microenvironment plays a key role in tumor metastasis. As the main stromal cells in tumor microenvironment originated from activated fibroblast, cancer-associated fibroblasts (CAFs) play a major role in promoting tumor metastasis. A promising therapeutic avenue is reprogramming of CAFs into tumor-restraining quiescence state. In this study, we observed that CAF-like active pancreatic stellate cells (PSCs) interact with each other via N-cadherin, a force-sensitive transmembrane receptor. Since N-cadherin ligation mediated mechanotransduction has been reported to restrict integrin mediated signalling, we thus hypothesized that the reprogramming of activated PSCs by mechanical modulation of N-cadherin ligation might be possible. To test this hypothesis, we grafted N-cadherin ligand (HAVDI peptide) onto soft polyethylene glycol hydrogel substrate prior to cell adhesion to mimic cell-cell interaction via N-cadherin ligation. We found that the activated PSCs could be reprogrammed to their original quiescent state when transferred onto the substrate with immobilized HAVDI peptide. These results reveal a key role of mechanosensing by intercellular transmembrane receptor in reprogramming of activated PSCs, and provide a potential way for designing novel therapeutic strategies for cancer treatment.

Yap-Myc signaling induces pancreatic stellate cell activation through regulating glutaminolysis.

The accumulation of activated myofibroblastic pancreatic stellate cells (MF-PSCs) induces pancreatic cancer desmoplasia. These MF-PSCs are derived from quiescent pancreatic stellate cells (Q-PSCs). MF-PSCs in pancreatic cancer tend to glycolysis. However, increased glycolysis alone could not be sufficient for the increased metabolic demands of MF-PSCs. Yap and Myc signaling activation is involved in pancreatic cancer metabolism. Since elucidating the metabolic processes of MF-PSCs may be a promising strategy to suppress pancreatic cancer desmoplasia, we explored whether glutaminolysis meets the bioenergetic and biosynthetic demands of Q-PSCs converted into MF-PSCs and whether this is mediated by Yap signaling to Myc. In this study, we found that during the transdifferentiation of Q-PSCs into MF-PSCs, glutaminolysis regulatory genes were upregulated, and suppression of glutaminolysis inhibited transdifferentiation. Disrupting glutaminolysis in MF-PSCs inhibited cell growth, mitochondrial respiration, and fibrogenesis, while treatment of MF-PSCs with DKG (a glutaminolysis metabolite) reversed these activities. The expression of glutaminase (GLS1), a rate-limiting enzyme in glutaminolysis, was upregulated by Yap overexpression. Yap upregulates Myc to regulate the expression of GLS1 in MF-PSCs. Yap and Myc inhibitors disrupted glutaminolysis and inhibited myofibroblastic activities in PSCs. Thus, Yap-Myc signaling controls glutaminolysis to activate PSCs and might be a therapeutic target for pancreatic cancer desmoplasia.

Pancreatic stellate cells exhibit adaptation to oxidative stress evoked by hypoxia.

BACKGROUND INFORMATION: Pancreatic stellate cells play a key role in the fibrosis that develops in diseases such as pancreatic cancer. In the growing tumour, a hypoxia condition develops under which cancer cells are able to proliferate. The growth of fibrotic tissue contributes to hypoxia. In this study, the effect of hypoxia (1% O2 ) on pancreatic stellate cells physiology was investigated. Changes in intracellular free-Ca2+ concentration, mitochondrial free-Ca2+ concentration and mitochondrial membrane potential were studied by fluorescence techniques. The status of enzymes responsible for the cellular oxidative state was analyzed by quantitative reverse transcription-polymerase chain reaction, high-performance liquid chromatography, spectrophotometric and fluorimetric methods and by Western blotting analysis. Cell viability and proliferation were studied by crystal violet test, 5-bromo-2-deoxyuridine cell proliferation test and Western blotting analysis. Finally, cell migration was studied employing the wound healing assay. RESULTS: Hypoxia induced an increase in intracellular and mitochondrial free-Ca2+ concentration, whereas mitochondrial membrane potential was decreased. An increase in mitochondrial reactive oxygen species production was observed. Additionally, an increase in the oxidation of proteins and lipids was detected. Moreover, cellular total antioxidant capacity was decreased. Increases in the expression of superoxide dismutase 1 and 2 were observed and superoxide dismutase activity was augmented. Hypoxia evoked a decrease in the oxidized/reduced glutathione ratio. An increase in the phosphorylation of nuclear factor erythroid 2-related factor and in expression of the antioxidant enzymes catalytic subunit of glutamate-cysteine ligase, catalase, NAD(P)H-quinone oxidoreductase 1 and heme oxygenase-1 were detected. The expression of cyclin A was decreased, whereas expression of cyclin D and the content of 5-bromo-2-deoxyuridine were increased. This was accompanied by an increase in cell viability. The phosphorylation state of c-Jun NH2 -terminal kinase was increased, whereas that of p44/42 and p38 was decreased. Finally, cells subjected to hypoxia maintained migration ability. CONCLUSIONS AND SIGNIFICANCE: Hypoxia creates pro-oxidant conditions in pancreatic stellate cells to which cells adapt and leads to increased viability and proliferation.

Melatonin modulates proliferation of pancreatic stellate cells through caspase-3 activation and changes in cyclin A and D expression.

In this study, the effects of melatonin (1 µM-1 mM) on pancreatic stellate cells (PSC) have been examined. Cell viability and proliferation, caspase-3 activation, and the expression of cyclin A and cyclin D were analyzed. Our results show that melatonin decreased PSC viability in a time- and concentration-dependent manner. This effect was not inhibited by treatment of cells with MT1, MT2, calmodulin, or ROR-alpha inhibitors prior to melatonin addition. Activation of caspase-3 in response to melatonin was detected. The expression of cyclin A and cyclin D was decreased in cells treated with melatonin. Finally, changes in BrdU incorporation into the newly synthesized DNA of proliferating cells were also observed in the presence of melatonin. We conclude that melatonin, at pharmacological concentrations, modulates proliferation of PSC through activation of apoptosis and involving crucial regulators of the cell cycle. These actions might not require specific melatonin receptors. Our observations suggest that melatonin, at high doses, could potentially exert anti-fibrotic effects and, thus, could be taken into consideration as supportive treatment in the therapy of pancreatic diseases.

Co-targeting of CXCR4 and hedgehog pathways disrupts tumor-stromal crosstalk and improves chemotherapeutic efficacy in pancreatic cancer.

Pancreatic cancer (PC) remains a therapeutic challenge because of its intrinsic and extrinsic chemoresistance mechanisms. Here, we report that C-X-C motif chemokine receptor 4 (CXCR4) and hedgehog pathways cooperate in PC chemoresistance via bidirectional tumor-stromal crosstalk. We show that when PC cells are co-cultured with pancreatic stellate cells (PSCs) they are significantly more resistant to gemcitabine toxicity than those grown in monoculture. We also demonstrate that this co-culture-induced chemoresistance is abrogated by inhibition of the CXCR4 and hedgehog pathways. Similarly, the co-culture-induced altered expression of genes in PC cells associated with gemcitabine metabolism, antioxidant defense, and cancer stemness is also reversed upon CXCR4 and hedgehog inhibition. We have confirmed the functional impact of these genetic alterations by measuring gemcitabine metabolites, reactive oxygen species production, and sphere formation in vehicle- or gemcitabine-treated monocultures and co-cultured PC cells. Treatment of orthotopic pancreatic tumor-bearing mice with gemcitabine alone or in combination with a CXCR4 antagonist (AMD3100) or hedgehog inhibitor (GDC-0449) displays reduced tumor growth. Notably, we show that the triple combination treatment is the most effective, resulting in nearly complete suppression of tumor growth. Immunohistochemical analysis of Ki67 and cleaved caspase-3 confirm these findings from in vivo imaging and tumor measurements. Our findings provide preclinical and mechanistic evidence that a combination of gemcitabine treatment with targeted inhibition of both the CXCR4 and hedgehog pathways improves outcomes in a PC mouse model.

Pancreatic stellate cell-potentiated insulin secretion from Min6 cells is independent of interleukin 6-mediated pathway.

Pancreatic stellate cells (PSCs) secrete various factors, which can influence the ß-cell function. The identification of stellate cell infiltration into the islets in pancreatic diseases suggests possible existence of cross-talk between these cells. To elucidate the influence of PSCs on ß-cell function, mouse PSCs were cocultured with Min6 cells using the Transwell inserts. Glucose-stimulated insulin secretion from Min6 cells in response to PSCs was quantified by enzyme-linked immunosorbent assay and insulin gene expression was measured by quantitative polymerase chain reaction. Upon cytometric identification of IL6 in PSC culture supernatants, Min6 cells were cultured with IL6 to assess its influence on the insulin secretion and gene expression. PLC-IP3 pathway inhibitors were added in the cocultures, to determine the influence of PSC-secreted IL6 on Glucose-stimulated insulin secretion from Min6 cells. Increased insulin secretion with a concomitant decrease in total insulin content was noticed in PSC-cocultured Min6 cells. Although increased GSIS was noted from IL6-treated Min6 cells, no change in the total insulin content was noted. Coculture of Min6 cells with PSCs or their exposure to IL6 did not alter either the expression of ß-cell-specific genes or that of miRNA-375. PSC-cocultured Min6 cells, in the presence of PLC-IP3 pathway inhibitors (U73122, Neomycin, and Xestospongin C), did not revoke the observed increase in GSIS. In conclusion, the obtained results indicate that augmented insulin secretion from Min6 cells in response to PSC secretions is independent of IL6-mediated PLC-IP3 pathway.

A modified in vitro tool for isolation and characterization of rat quiescent islet stellate cells.

BACKGROUND: Islet stellate cells (ISCs) play a critical role in islet fibrosis, contributing to the progression of pancreatic diseases. Previous studies have focused on fibrosis-associated activated ISCs obtained by standard islet explant techniques. However, in vitro models of quiescent ISCs (qISCs) are lacking. This study aims to develop a method to isolate qISCs and analyze their phenotype during activation. METHODS: Immunofluorescence staining was applied to localize ISCs in normal human, rat, and mouse islets. qISCs were isolated from rat islets using density gradient centrifugation (DGC) method. qRT-PCR, immunoblotting, proliferation, and migration assays were employed for their characterization. RESULTS: Desmin-positive ISCs were detected in normal human, rat, and mouse islets. Freshly isolated qISCs, obtained by density gradient centrifugation, displayed a polygonal appearance with refringent cytoplasmic lipid droplets and expressed transcriptional markers indicating a low activation/quiescent state. With increasing culture time, the marker expression pattern changed, reflecting ISC activation. qISCs contained more lipid droplets and exhibited lower proliferation and migration abilities compared to spindle-shaped ISCs obtained by traditional explant techniques. CONCLUSIONS: This study describes a new method for efficient isolation of qISCs from rat islets, representing a useful in vitro tool to study the biology of ISCs in more physiological conditions.

Identification of key pathways and genes changes in pancreatic cancer cells (BXPC-3) after cross-talk with primary pancreatic stellate cells using bioinformatics analysis.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most malignant tumors with poor prognosis, and the interaction between activated pancreatic stellate cells (PSCs) and PDAC cells plays an important role in the development of PDAC. The aim of this study was to identify gene changes in BXPC-3 after cross-talk with PSCs and reveal their potential mechanisms. The gene expression profiling analysis of BXPC-3 was completed after co-culture with primary PSCs for 48 h. The gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes pathway (KEGG) enrichment analyses were performed, and the differentially expressed genes (DEGs) were identified by Agilent GeneSpring GX software. In total, 3657 DEGs were identified in BXPC-3, including 1881 up-regulated genes and 1776 downregulated genes. GO analysis results showed that upregulated DEGs were significantly enriched in biological processes (BP), including peptide metabolic process, response to stress and electron transport chain; the downregulated DEGs were significantly enriched in biological processes, including signaling, multicellular organism development and anatomical structure development. KEGG pathway analysis revealed that 19 pathways were upregulated and 32 pathways were downregulated, and that upregulated DEGs were enriched in protein export and glutathione metabolism, while the downregulated DEGs were enriched in axon guidance and focal adhesion. The top 10 upregulated genes and the top 10 downregulated genes were identified. By constructing PPI network, we selected out 10 key genes (TP53, SRC, IL6, JUN, ISG15, CAD, STAT1, OAS3, OAS1, VIM) and significant pathways. The associated survival analysis was performed and the SRC, IL-6, ISG15, STAT1, OAS3, OAS1 and VIM were proved to be related to worse overall survival time of PDAC patients. In conclusion, the present study indicated that the identified DEGs promote our understanding of the molecular mechanisms underlying the interaction between pancreatic cancer cells and PSCs and might be used as molecular targets in the future to study the role of tumor microenvironment in the progression of PDAC.

Cellular context-dependent interaction between cancer and stellate cells in hetero-type multicellular spheroids of pancreatic tumor.

Heterotypic cell-cell interaction between cancer cells and pancreatic stellate cells (PSCs) within tumor microenvironment is considered as a key mechanism for epithelial-mesenchymal transition (EMT) that triggers disease progression and chemoresistance in pancreatic ductal adenocarcinoma (PDAC). Hence, PSCs should be incorporated into in vitro co-culture models to maximize clinical relevance of data obtained using these models. In this study, we developed hetero-type spheroids of pancreatic cancer cells (ductal carcinoma cells PANC-1 and primacy sarcomatoid adenocarcinoma 36473 cells) and PSCs. Effect of PSC co-culture on the formation and growth of multicellular spheroids was cell-line dependent in that growth stimulation effect appeared in PANC-1/PSC spheroids, but not in 36473/PSC spheroids. Spatial distribution of PSCs within spheroids was also cell-line dependent. It was either confined to the center region (PANC-1) or evenly distributed (36473). Changes in expression levels of E-cadherin and vimentin revealed EMT induction in PANC-1/PSC hetero-type spheroids, but not in 36473/PSC spheroids. Gemcitabine sensitivity was increased partially by PSC co-culture. However, PSCs showed relative resistance to gemcitabine compared to PANC-1 cells in PANC-1/PSC spheroids. Overall, our hetero-type spheroid model can be used to study cancer-stroma interaction and their mechanism and evaluate anticancer drug activity. We demonstrated that stromal effect by PSC co-culture might be cellular context dependent with regard to growth stimulation and EMT induction. Hence, anti-stromal therapy should take these differences into consideration.

Melatonin induces reactive oxygen species generation and changes in glutathione levels and reduces viability in human pancreatic stellate cells.

In this study, the effects of pharmacological concentrations of melatonin (1 µM-1 mM) on human pancreatic stellate cells (HPSCs) have been examined. Cell type-specific markers and expression of melatonin receptors were analyzed by western blot analysis. Changes in intracellular free Ca2+ concentration were followed by fluorimetric analysis of fura-2-loaded cells. Reduced glutathione (GSH) and oxidized glutathione (GSSG) levels were determined by fluorescence techniques. Production of reactive oxygen species (ROS) was monitored following 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate acetyl ester and MitoSOX™ Red-derived fluorescence. Cell viability was studied using the AlamarBlue® test. Cultured cells expressed markers typical of stellate cells. However, cell membrane receptors for melatonin could not be detected. Thapsigargin, bradykinin, or melatonin induced changes in intracellular free Ca2+ concentration. In the presence of the indole, a decrease in the GSH/GSSG ratio was observed that depended on the concentration of melatonin used. Furthermore, the indole evoked a concentration-dependent increase in ROS production in the mitochondria and in the cytosol. Finally, melatonin decreased HPSC viability in a time and concentration-dependent manner. We conclude that melatonin, at pharmacological concentrations, induces changes in the oxidative state of HPSC. This might regulate cellular viability and could not involve specific plasma membrane receptors.

Saikosaponin d ameliorates pancreatic fibrosis by inhibiting autophagy of pancreatic stellate cells via PI3K/Akt/mTOR pathway.

Chronic pancreatitis is characterized by pancreatic fibrosis, associated with excessive activation of pancreatic stellate cells (PSCs) and increased expression of transforming growth factor-ß1 (TGF-ß1). Recently, our studies have shown that autophagy inhibitor could inhibit PSCs activation and reduce collagen secretion. Saikosaponin d (SSd), the major active component of bupleurum falcatum (a medicinal plant), has anti-fibrosis effects in liver. However, it is unclear whether SSd has a role in pancreatic fibrosis. This study aimed to investigate the effect of SSd on the autophagy and activation of PSCs in vivo and in vitro. In vivo, a rat chronic pancreatitis model was induced by intravenous injection of dibutyltin dichloride. SSd was administered at a dose of 2.0 mg/kg body weight per day by gavage. After 4 weeks, the pancreas was collected for histological and molecular analysis. In vitro, PSCs were isolated and cultured for treatment with different dosages of SSd. The results showed that SSd inhibited PSCs autophagy and activation while also reducing extracellular matrix (ECM) formation and pancreatic damage. SSd inhibited autophagy through activating the PI3K/Akt/mTOR pathway. SSd also promoted degradation of ECM with an increasing ratio of MMPs/TIMPs and suppressed the TGF-ß1/Smads pathway. From these results, we concluded that SSd prevents pancreatic fibrosis by reducing autophagy of PSCs through PI3K/Akt/mTOR pathway, which has crosstalk with the TGF-ß1/Smads pathway.

IL-6 Signaling Blockade during CD40-Mediated Immune Activation Favors Antitumor Factors by Reducing TGF-ß, Collagen Type I, and PD-L1/PD-1.

IL-6 plays a role in cancer pathogenesis via its connection to proteins involved in the formation of desmoplastic stroma and to immunosuppression by driving differentiation of myeloid suppressor cells together with TGF-ß. Inhibition of IL-6 signaling in the tumor microenvironment may, thus, limit desmoplasia and myeloid suppressor cell differentiation. CD40 signaling can further revert myeloid cell differentiation toward antitumor active phenotypes. Hence, the simultaneous use of IL-6 blockade with CD40 stimuli may tilt the tumor microenvironment to promote antitumor immune responses. In this paper, we evaluated the mechanisms of LOAd713, an oncolytic adenovirus designed to block IL-6R signaling and to provide myeloid cell activation via a trimerized membrane-bound isoleucine zipper (TMZ) CD40L. LOAd713-infected pancreatic cancer cells were killed by oncolysis, whereas infection of stellate cells reduced factors involved in stroma formation, including TGF-ß-1 and collagen type I. Virus infection prevented IL-6/GM-CSF-mediated differentiation of myeloid suppressors, but not CD163 macrophages, whereas infection of dendritic cells led to upregulation of maturation markers, including CD83, CD86, IL-12p70, and IFN-γ. Further, IL-6R blockade prevented upregulation of programed death ligand 1 (PD-L1) and PD-1 on the stimulated dendritic cells. These results suggest that LOAd713 can kill infected tumor cells and has the capacity to affect the tumor microenvironment by stimulating stellate cells and myeloid suppressors with TMZ-CD40L and IL-6R blockade. Gene transfer of murine TMZ-CD40L prolonged survival in an animal model. LOAd713 may be an interesting therapeutic option for cancers connected to IL-6 signaling, such as pancreatic cancer.

FTY720 inhibits the activation of pancreatic stellate cells by promoting apoptosis and suppressing autophagy via the AMPK/mTOR pathway.

AIMS: Pancreatic stellate cells (PSCs) play a critical role in the development of pancreatic fibrosis. Any agents that can affect PSC activation could become potential candidates for treating pancreatic fibrosis. FTY720 can attenuate chronic pancreatic fibrosis by suppressing T-cell infiltration, but its effect on PSCs remains unknown. This study was conducted to investigate the effects of FTY720 on PSC activation in cultured rat PSCs. MAIN METHODS: The viability of PSCs after FTY720 treatment was detected by MTT. Cell proliferation and migration analysis was performed using the iCELLigence System and a Transwell assay. Cell apoptosis was assessed by flow cytometry, western blot and an activity assay. The mitochondrial membrane potential (MMP) was assessed by JC-1 staining. The expression of α-SMA, collagen I, fibronectin, Beclin-1, Atg5, P62 and LC3B were analysed by immunofluorescence, quantitative real-time PCR and western blot. Rapamycin and phenformin hydrochloride were used to determine whether FTY720 inhibits PSC autophagy by the AMPK/mTOR pathway. KEY FINDINGS: FTY720 supressed PSC viability, proliferation and migration. FTY720 inhibited PSC activation, induced PSC apoptosis and supressed PSC autophagy. We also confirmed that FTY720 inhibited PSC autophagy via the AMPK/mTOR pathway. SIGNIFICANCE: Our results indicated that FTY720 inhibited PSC activation by promoting cell apoptosis and inhibiting PSC autophagy by suppressing AMPK and activating the mTOR pathway. These findings may explain the therapeutic mechanisms of FTY720 in treating pancreatic fibrosis and further suggest that targeting autophagy and the related signalling pathways may provide new strategies for the treatment of pancreatic fibrosis.

Effects of the tumor suppressor PTEN on biological behaviors of activated pancreatic stellate cells in pancreatic fibrosis.

Pancreatic stellate cells (PSCs), when activated, are characterized by proliferation and collagen synthesis, and contribute to extracellular matrix deposition in pancreatic fibrosis. Concomitantly, fibrosis is linked with the loss of PTEN (phosphatase and tensin homolog) protein in several organs. This study investigated the association between PTEN protein levels and the activated or apoptotic status of PSCs in a rat model of chronic pancreatitis. In addition, the activation status and biological behaviors of culture-activated PSCs were analyzed after lentiviral transfection with wildtype or mutant (G129E) PTEN for upregulation, or PTEN short hairpin RNA for downregulation, of PTEN. In vivo, PTEN levels gradually decreased during pancreatic fibrosis, which positively correlated with apoptosis of activated PSCs, but negatively with PSC activation. In vitro, activated PSCs with wildtype PTEN showed less proliferation, migration, and collagen synthesis compared with control PSCs, and greater numbers were apoptotic; activated PSCs with mutant PTEN showed similar, but weaker, effects. Furthermore, AKT and FAK/ERK signaling was involved in this process. In summary, activated PSCs during pancreatic fibrosis in vivo have lower levels of PTEN. In vitro, PTEN appears to prevent PSCs from further activation and promotes apoptosis through regulation of the AKT and FAK/ERK pathways.

RB1CC1-enhanced autophagy facilitates PSCs activation and pancreatic fibrogenesis in chronic pancreatitis.

Chronic pancreatitis (CP) is described as a progressive fibro-inflammatory disorder of the exocrine disease, which eventually leads to damage of the gland. Excessive activation of pancreatic stellate cells (PSCs) is a critical participant in the initiation of CP. Autophagy is involved in multiple degeneration and inflammation in acute pancreatitis and CP. In our study, we report that retinoblastoma coiled coil protein 1 (RB1CC1) expression and the autophagic level are elevated in activated PSCs. RB1CC1 is positively correlated with pancreatic fibrogenesis in tissues and plasma of CP patients. Knockdown of RB1CC1 restrains alpha smooth muscle actin (α-SMA) and collagen expressions, and autophagy in activated PSCs in vitro. Furthermore, we show that RB1CC1 induces PSC activation via binding to ULK1 promoter and the direct interaction with ULK1 protein. These suppress ULK1 expression and its kinase activity. In mice, knockdown of RB1CC1 blocks autophagy and then inhibits the pancreatic duct ligation-induced pancreatic fibrosis. Consequently, our study highlights that RB1CC1-mediated autophagy is a key event for the activation of PSCs. Inhibition of RB1CC1 alleviates autophagy, which plays a critical role in anti-fibrotic activation in PSCs and CP progression. RB1CC1 could be a novel strategy for the treatment of pancreatic fibrosis.

Inhibition of Class I Histone Deacetylases Abrogates Tumor Growth Factor ß Expression and Development of Fibrosis during Chronic Pancreatitis.

Pancreatic fibrosis is the hallmark of chronic pancreatitis, a highly debilitating disease for which there is currently no cure. The key event at the basis of pancreatic fibrosis is the deposition of extracellular matrix proteins by activated pancreatic stellate cells (PSCs). Transforming growth factor ß (TGFß) is a potent profibrotic factor in the pancreas as it promotes the activation of PSC; thus, pharmacologic interventions that effectively reduce TGFß expression harbor considerable therapeutic potential in the treatment of chronic pancreatitis. In this study, we investigated whether TGFß expression is reduced by pharmacologic inhibition of the epigenetic modifiers histone deacetylases (HDACs). To address this aim, chronic pancreatitis was induced in C57BL/6 mice with serial injections of cerulein, and the selective class 1 HDAC inhibitor MS-275 was administered in vivo in a preventive and therapeutic manner. Both MS-275 regimens potently reduced deposition of extracellular matrix and development of fibrosis in the pancreas after 4 weeks of chronic pancreatitis. Reduced pancreatic fibrosis was concomitant with lower expression of pancreatic TGFß and consequent reduced PSC activation. In search of the cell types targeted by the inhibitor, we found that MS-275 treatment abrogated the expression of TGFß in acinar cells stimulated by cerulein treatment. Our study demonstrates that MS-275 is an effective antifibrotic agent in the context of experimental chronic pancreatitis and thus may constitute a valid therapeutic intervention for this severe disease.

A Transcriptional Sequencing Analysis of Islet Stellate Cell and Pancreatic Stellate Cell.

BACKGROUND: Our previous studies have shown that islet stellate cell (ISC), similar to pancreatic stellate cell (PSC) in phenotype and biological characters, may be responsible for the islet fibrosis in type 2 diabetes. To further identify the differences between PSC and ISC and for better understanding of the physiological function of ISC, we employed genome-wide transcriptional analysis on the PSCs and ISCs of Wistar rats. METHOD: PSCs and ISCs from each rat were primarily cultured at the same condition. Genome-wide transcriptional sequence of stellate cells was generated. The identified differentially expressed genes were validated using RT-PCR. RESULTS: 32 significant differentially expressed genes between PSCs and ISCs were identified. Moreover, collagen type 11a1 (COL11A1), was found to be expressed 2.91-fold higher in ISCs compared with PSCs, indicating that COL11A1 might be a potential key gene modulating the differences between PSC and ISC. CONCLUSIONS: Our study identified and validated the differences between PSC and ISC in genome-wide transcriptional scale, confirming the assumption that ISC and PSC are similar other than identical. Moreover, our data might be instrumental for further investigation of ISC and islet fibrosis, and some differential expressed genes may provide an insight into new therapeutic targets for type 2 diabetes.

Indometacin inhibits the proliferation and activation of human pancreatic stellate cells through the downregulation of COX-2.

Increasing evidence indicates that pancreatic stellate cells (PSCs) are responsible for the stromal reaction in pancreatic ductal adenocarcinoma (PDAC). The interaction between activated PSCs and PDAC cells and the resultant stromal reaction facilitate cancer progression. Previous findings suggested that cyclooxygenase­2 (COX­2) may have a profound role in regulating the proliferation and activation of PSCs in response to pancreatic cancer. Indometacin, a well­known anti­inflammatory drug and a non­selective inhibitor of COX­2, has been shown to exert anticancer effects in various types of cancer, including PDAC. However, whether indometacin affects PSC activation remains unclear. Using RT­qPCR and western blot analysis, we determined that COX­2 expression was elevated in tandem with the activation of PSCs. Treatment with indometacin suppressed the viability and the migration ability of PSCs in a dose­dependent manner. In addition, the immunoblotting and immunofluorescence results showed that α­SMA expression was markedly decreased by indometacin. A further study indicated that COX­2 expression was decreased in PSCs after indometacin intervention. In conclusion, these data indicate that indometacin serves as an effective drug against PSC activation via the targeting of COX-2.

Basement membrane destruction by pancreatic stellate cells leads to local invasion in pancreatic ductal adenocarcinoma.

Stroma invasion is an important step in pancreatic cancer progression. However, how pancreatic ductal adenocarcinoma (PDAC) with ductal structure invades the surrounding stroma has not been clear. Here, we elucidated the mechanism of stromal invasion of PDAC, using organoids. From resected PDAC specimens, we established human PDAC organoids, which developed ductal and basement membrane (BM) structures. When the organoids were co-cultured with pancreatic stellate cells (PSCs) in a collagen matrix, organoids lost their BM and ductal structures, and invaded collagen matrix more frequently than did mono-cultured organoids. Interestingly, direct contact by PSCs to PDAC organoids was observed before BM destruction. Matrix metalloproteinase (MMP) 2 or membrane type-1 MMP (MT1MMP) knockdown in PSCs significantly attenuated BM destruction by PSCs, and retained the ductal structures in organoids. Our results imply that direct contact by PSCs induces BM destruction and stromal invasion of PDAC via MMP2 which binds to MT1MMP on PSCs.

Calcium signalling in the acinar environment of the exocrine pancreas: physiology and pathophysiology.

KEY POINTS: Ca2+ signalling in different cell types in exocrine pancreatic lobules was monitored simultaneously and signalling responses to various stimuli were directly compared. Ca2+ signals evoked by K+ -induced depolarization were recorded from pancreatic nerve cells. Nerve cell stimulation evoked Ca2+ signals in acinar but not in stellate cells. Stellate cells are not electrically excitable as they, like acinar cells, did not generate Ca2+ signals in response to membrane depolarization. The responsiveness of the stellate cells to bradykinin was markedly reduced in experimental alcohol-related acute pancreatitis, but they became sensitive to stimulation with trypsin. Our results provide fresh evidence for an important role of stellate cells in acute pancreatitis. They seem to be a critical element in a vicious circle promoting necrotic acinar cell death. Initial trypsin release from a few dying acinar cells generates Ca2+ signals in the stellate cells, which then in turn damage more acinar cells causing further trypsin liberation. ABSTRACT: Physiological Ca2+ signals in pancreatic acinar cells control fluid and enzyme secretion, whereas excessive Ca2+ signals induced by pathological agents induce destructive processes leading to acute pancreatitis. Ca2+ signals in the peri-acinar stellate cells may also play a role in the development of acute pancreatitis. In this study, we explored Ca2+ signalling in the different cell types in the acinar environment of the pancreatic tissue. We have, for the first time, recorded depolarization-evoked Ca2+ signals in pancreatic nerves and shown that whereas acinar cells receive a functional cholinergic innervation, there is no evidence for functional innervation of the stellate cells. The stellate, like the acinar, cells are not electrically excitable as they do not generate Ca2+ signals in response to membrane depolarization. The principal agent evoking Ca2+ signals in the stellate cells is bradykinin, but in experimental alcohol-related acute pancreatitis, these cells become much less responsive to bradykinin and then acquire sensitivity to trypsin. Our new findings have implications for our understanding of the development of acute pancreatitis and we propose a scheme in which Ca2+ signals in stellate cells provide an amplification loop promoting acinar cell death. Initial release of the proteases kallikrein and trypsin from dying acinar cells can, via bradykinin generation and protease-activated receptors, induce Ca2+ signals in stellate cells which can then, possibly via nitric oxide generation, damage more acinar cells and thereby cause additional release of proteases, generating a vicious circle.