Transcription profiling of human hepatic and pancreatic stellate cells obtained from tissues of chronic inflammation, and primary or metastatic cancers
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ABSTRACT:
ORGANISM(S): Homo sapiens
SUBMITTER: Erkan M
PROVIDER: S-ECPF-TABM-625 | biostudies-other | 2010
REPOSITORIES: biostudies-other
Similar Datasets
Project description:Differences in the expression profile of hepatic and pancreatic stellate cells are investigated. Aim is to identify organ and disease specific transcriptome signatures of stellate cells, comparing hepatic and pancreatic stellate cells obtained from tissues of chronic inflammation, and primary or metastatic cancers of the pancreas. Tissues of chronic pancreatitis (n=6), pancreatic ductal adenocarcinoma (n=5), liver cirrhosis (n=5) and liver metastasis of pancreatic ductal adenocarcinoma (n=6) were collected and stellate cells were isolated by the outgrowth method. Using cDNA microarrays, differentially expressed genes are identified.
2010-01-15 | E-TABM-625 | biostudies-arrayexpress
Project description:BACKGROUND: Tissue fibrosis is an integral component of chronic inflammatory (liver and pancreas) diseases and pancreatic cancer. Activated pancreatic- (PSC) and hepatic- (HSC) stellate cells play a key role in fibrogenesis. To identify organ- and disease-specific stellate cell transcriptional fingerprints, we employed genome-wide transcriptional analysis of primary human PSC and HSC isolated from patients with chronic inflammation or cancer. METHODS: Stellate cells were isolated from patients with pancreatic ductal adenocarcinoma (n = 5), chronic pancreatitis (n = 6), liver cirrhosis (n = 5) and liver metastasis of pancreatic ductal adenocarcinoma (n = 6). Genome-wide transcriptional profiles of stellate cells were generated using our 51K human cDNA microarray platform. The identified organ- and disease specific genes were validated by quantitative RT-PCR, immunoblot, ELISA, immunocytochemistry and immunohistochemistry. RESULTS: Expression profiling identified 160 organ- and 89 disease- specific stellate cell transcripts. Collagen type 11a1 (COL11A1) was discovered as a novel PSC specific marker with up to 65-fold higher expression levels in PSC compared to HSC (p < 0.0001). Likewise, the expression of the cytokine CCL2 and the cell adhesion molecule VCAM1 were confined to HSC. PBX1 expression levels tend to be increased in inflammatory- vs. tumor- stellate cells. Intriguingly, tyrosine kinase JAK2 and a member of cell contact-mediated communication CELSR3 were found to be selectively up-regulated in tumor stellate cells. CONCLUSIONS: We identified and validated HSC and PSC specific markers. Moreover, novel target genes of tumor- and inflammation associated stellate cells were discovered. Our data may be instrumental in developing new tailored organ- or disease-specific targeted therapies and stellate cell biomarkers.
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Project description:The functions of the liver and the pancreas differ; however, chronic inflammation in both organs is associated with fibrosis. Evidence suggests that fibrosis in both organs is partially regulated by organ-specific stellate cells. We explore the proteome of human hepatic stellate cells (hHSC) and human pancreatic stellate cells (hPaSC) using mass spectrometry (MS)-based quantitative proteomics to investigate pathophysiologic mechanisms. Proteins were isolated from whole cell lysates of immortalized hHSC and hPaSC. These proteins were tryptically digested, labeled with tandem mass tags (TMT), fractionated by OFFGEL, and subjected to MS. Proteins significantly different in abundance (P<0.05) were classified via gene ontology (GO) analysis. We identified 1223 proteins and among them, 1222 proteins were quantifiable. Statistical analysis determined that 177 proteins were of higher abundance in hHSC, while 157 were of higher abundance in hPaSC. GO classification revealed that proteins of relatively higher abundance in hHSC were associated with protein production, while those of relatively higher abundance in hPaSC were involved in cell structure. Future studies using the methodologies established herein, but with further upstream fractionation and/or use of enhanced MS instrumentation will allow greater proteome coverage, achieving a comprehensive proteomic analysis of hHSC and hPaSC.
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Project description:Chronic liver inflammation is a complex pathological process under different stress conditions, and the roles of stellate cells and macrophages in chronic liver inflammation have been widely reported. Moderate liver inflammation can protect the liver from damage and facilitate the recovery of liver injury. However, an inflammatory response that is too intense can result in massive death of hepatocytes, which leads to irreversible damage to the liver parenchyma. Epigenetic regulation plays a key part in liver inflammation. This study reviews the regulation of epigenetics on stellate cells and macrophages to explore the new mechanisms of epigenetics on liver inflammation and provide new ideas for the treatment of liver disease.
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Project description:Chemokines are the inflammatory mediators that modulate liver fibrosis, a common feature of chronic inflammatory liver diseases. CX3CL1/fractalkine is a membrane-associated chemokine that requires step processing for chemotactic activity and has been recently implicated in liver disease. Here, we investigated the potential shedding activities involved in the release of the soluble chemotactic peptides from CX3CL1 in the injured liver. We showed an increased expression of the sheddases ADAM10 and ADAM17 in patients with chronic liver diseases that was associated with the severity of liver fibrosis. We demonstrated that hepatic stellate cells (HSC) were an important source of ADAM10 and ADAM17 and that treatment with the inflammatory cytokine inter-feron-gamma induced the expression of CX3CL1 and release of soluble peptides. This release was inhibited by the metalloproteinase inhibitor batimastat; however, ADAM10/ADAM17 inhibitor GW280264X only partially affected shedding activity. By using selective tissue metalloprotease inhibitors and overexpression analyses, we showed that CX3CL1 was mainly processed by matrix metalloproteinase (MMP)-2, a metalloprotease highly expressed by HSC. We further demonstrated that the CX3CL1 soluble peptides released from stimulated HSC induced the activation of the CX3CR1-dependent signalling pathway and promoted chemoattraction of monocytes in vitro. We conclude that ADAM10, ADAM17 and MMP-2 synthesized by activated HSC mediate CX3CL1 shedding and release of chemotactic peptides, thereby facilitating recruitment of inflammatory cells and paracrine stimulation of HSC in chronic liver diseases.
| S-EPMC3828864 | biostudies-other
Project description:Liver fibrosis (LF) mortality rate is approximately 2 million per year. Irrespective of the etiology of LF, a key element in its development is the transition of hepatic stellate cells (HSCs) from a quiescent phenotype to a myofibroblast-like cell with the production of fibrotic proteins. It is necessary to define optimal isolation and culturing conditions for good HSCs yield and proper phenotype preservation for studying the activation of HSCs in vitro. In the present study, the optimal conditions of HSC isolation and culture were examined to maintain the HSC's undifferentiated phenotype. HSCs were isolated from Balb/c mice liver using Nycodenz, 8, 9.6, and 11%. The efficiency of the isolation procedure was evaluated by cell counting and purity determination by flow cytometry. Quiescent HSCs were cultured in test media supplemented with different combinations of fetal bovine serum (FBS), glutamine (GLN), vitamin A (vitA), insulin, and glucose. The cells were assessed at days 3 and 7 of culture by evaluating the morphology, proliferation using cell counting kit-8, lipid storage using Oil Red O (ORO) staining, expression of a-smooth muscle actin, collagen I, and lecithin-retinol acyltransferase by qRT-PCR and immunocytochemistry (ICC). The results showed that Nycodenz, at 9.6%, yielded the best purity and quantity of HSCs. Maintenance of HSC undifferentiated phenotype was achieved optimizing culturing conditions (serum-free Dulbecco's Modified Eagle's Medium (DMEM) supplemented with glucose (100 mg/dl), GLN (0.5 mM), vitA (100 μM), and insulin (50 ng/ml)) with a certain degree of proliferation allowing their perpetuation in culture. In conclusion, we have defined optimal conditions for HSCs isolation and culture.
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Project description:The activation of pancreatic stellate cells (PSCs) plays a critical role in the progression of pancreatic fibrosis. Nuclear factor-kappa B (NF-κB) is associated with chronic pancreatitis (CP). Previous evidence indicated that NF-κB in acinar cells played a double-edged role upon pancreatic injury, whereas NF-κB in inflammatory cells promoted the progression of CP. However, the effects of NF-κB in PSCs have not been studied. In the present study, using two CP models and RNAi strategy of p65 in cultured PSCs, we found that the macrophage infiltration and MCP-1 expression were increased, and the NF-κBp65 protein level was elevated. NF-κBp65 was co-expressed with PSCs. In vitro, TGF-β1 induced overexpression of the TGF-β receptor 1, phosphorylated TGF-β1-activated kinase 1 (p-TAK1) and NF-κB in the PSCs. Moreover, the concentration of MCP-1 in the supernatant of activated PSCs was elevated. The migration of BMDMs was promoted by the supernatant of activated PSCs. Further knockdown of NF-κBp65 in PSCs resulted in a decline of BMDM migration, accompanied by a lower production of MCP-1. These findings indicate that TGF-β1 can induce the activation of NF-κB pathway in PSCs by regulating p-TAK1, and the NF-κB pathway in PSCs may be a target of chronic inflammation and fibrosis.
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Project description:Activated hepatic stellate cells (aHSC) are the main source of extra cellular matrix in liver fibrosis. Activation is classically divided in two phases: initiation and perpetuation. Currently, HSC-based therapeutic candidates largely focus on targeting the aHSCs in the perpetuation phase. However, the importance of HSC initiation during chronic liver disease (CLD) remains unclear. Here, we identified transcriptional programs of initiating and activated HSCs by RNA sequencing, using in vitro and in vivo mouse models of fibrosis. Importantly, we show that both programs are active in HSCs during murine and human CLD. In human cirrhotic livers, scar associated mesenchymal cells employ both transcriptional programs at the single cell level. Our results indicate that the transcriptional programs that drive the initiation of HSCs are still active in humans suffering from CLD. We conclude that molecules involved in the initiation of HSC activation, or in the maintenance of aHSCs can be considered equally important in the search for druggable targets of chronic liver disease.
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Project description:With their location in the perisinusoidal space of Disse, hepatic stellate cells (HSCs) communicate with all of the liver cell types both by physical association (cell body as well as cytosolic processes penetrating into sinusoids through the endothelial fenestrations) and by producing several cytokines and chemokines. Bacterial lipopolysaccharide (LPS), circulating levels of which are elevated in liver diseases and transplantation, stimulates HSCs to produce increased amounts of cytokines and chemokines. Although recent research provides strong evidence for the role of HSCs in hepatic inflammation and immune regulation, the number of HSC-elaborated inflammatory and immune regulatory molecules may be much greater then known at the present time. Here we report time-dependent changes in the gene expression profile of inflammatory and immune-regulatory molecules in LPS-stimulated rat HSCs, and their validation by biochemical analyses. LPS strongly up-regulated LPS-response elements (TLR2 and TLR7) but did not affect TLR4 and down-regulated TLR9. LPS also up-regulated genes in the MAPK, NFκB, STAT, SOCS, IRAK and interferon signaling pathways, numerous CC and CXC chemokines and IL17F. Interestingly, LPS modulated genes related to TGFβ and HSC activation in a manner that would limit their activation and fibrogenic activity. The data indicate that LPS-stimulated HSCs become a major cell type in regulating hepatic inflammatory and immunological responses by altering expression of numerous relevant genes, and thus play a prominent role in hepatic pathophysiology including liver diseases and transplantation.
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