Glycolysis in hepatic stellate cells coordinates fibrogenic extracellular vesicle release spatially to amplify liver fibrosis
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Orbitrap Exploris 480
ORGANISM(S): Homo Sapiens (human) Bos Taurus (bovine)
TISSUE(S): Hepatic Stellate Cell
SUBMITTER: 
Enis Kostallari
LAB HEAD: Enis Kostallari
PROVIDER: PXD050337 | Pride | 2024-05-16
REPOSITORIES: Pride
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Project description:Liver fibrosis is characterized by the activation of hepatic stellate cells (HSCs) and the release of fibrogenic nano-sized extracellular vesicles (EVs). Activated HSCs increase their glucose metabolism via glycolysis to satisfy high energy demands. Nevertheless, the mechanism of how glycolysis in HSCs coordinates fibrosis amplification in the fibrogenic zones in the liver is elusive and is the scope of this study. Single cell RNA sequencing (scRNAseq) and bulk RNAseq demonstrated that several glycolysis enzymes, including hexokinase 2 (HK2), were upregulated in activated HSCs. HSC-selective glycolysis-deficient mice (HK2ΔHSC) showed abrogated CCl4-mediated fibrosis as compared to littermate controls (HK2fl/fl). Spatial transcriptomics revealed an upregulation of several EV-related pathways in the fibrotic pericentral zone during liver fibrosis in control HK2fl/fl mice. However, glycolysis-deficient HK2ΔHSC mice showed downregulation of these EV-related pathways in the pericentral zone. Consistently, induction of glycolysis in HSCs in vitro, either by glucose or platelet-derived growth factor B (PDGF), upregulated the expression of several extracellular vesicle (EV)-related genes, including RAB31. Glycolysis in HSCs epigenetically enhanced RAB31 expression through histone-3 lysine-9 acetylation (H3K9ac) on the promoter region, leading to increased EV release. Functionally, glycolysis-dependent EVs were enriched with fibrogenic molecules and increased the expression of fibrotic markers in recipient HSCs. Finally, EVs derived from glycolysis-deficient HK2ΔHSC mice abrogated liver fibrosis amplification as compared to EVs derived from littermate control HK2fl/fl mice. In summary, glycolysis in HSCs amplifies liver fibrosis by promoting fibrogenic EV release in the pericentral fibrotic regions in the liver.
2024-03-20 | GSE259363 | GEO
Project description:Activation and migration of hepatic stellate cells (HSCs) followed by matrix deposition are characteristics of liver fibrosis. Several studies have shown the importance of hepatocyte and endothelial cell-derived extracellular vesicles (EVs) in liver pathobiology. However, less is known about the role of HSC-derived EVs in liver diseases. In this study, we investigated the molecules released through HSC-derived EVs and whether these can promote fibrosis.
2018-10-18 | PXD007258 | Pride
Project description:Uncovering the complex cellular mechanisms underlying hepatic fibrogenesis could expedite the development of effective treatments and noninvasive diagnosis for liver fibrosis. The biochemical complexity of extracellular vesicles (EVs) and their role in intercellular communication make them an attractive tool to look for biomarkers as potential alternative to liver biopsies. We developed a solid set of methods to isolate and characterize EVs from differently treated human hepatic stellate cell (HSC) line LX-2, and we investigated their biological effect onto naïve LX-2, proving that EVs do play an active role in fibrogenesis. We mined our proteomic data for EV-associated proteins whose expression correlated with HSC treatment, choosing the matricellular protein SPARC as proof-of-concept for the feasibility of fluorescence nanoparticle-tracking analysis to determine an EV-based HSCs’ fibrogenic phenotype. We thus used EVs to directly evaluate the efficacy of treatment with S80, a polyenylphosphatidylcholines-rich lipid, finding that S80 reduces the relative presence of SPARC-positive EVs. Here we correlated the cellular response to lipid-based antifibrotic treatment to the relative presence of a candidate protein marker associated with the released EVs. Along with providing insights into polyenylphosphatidylcholines treatments, our findings pave the way for precise and less invasive diagnostic analyses of hepatic fibrogenesis.
2023-03-11 | PXD037453 | Pride
Project description:BACKGROUND & AIMS: Nonalcoholic steatohepatitis (NASH) is a chronic liver disease characterized by hepatic lipid accumulation, inflammation, and progressive fibrosis. Acetyl-CoA carboxylase (ACC) catalyzes the rate-limiting step of de novo lipogenesis and regulates fatty-acid beta-oxidation in hepatocytes. ACC inhibition reduces hepatic fat content and markers of liver injury in NASH patients; however, the effect of ACC inhibition on liver fibrosis has not been reported. METHODS: A direct role for ACC in fibrosis was evaluated by measuring de novo lipogenesis, procollagen production, gene expression, glycolysis, and mitochondrial respiration in hepatic stellate cells (HSCs) in the absence or presence of small-molecule inhibitors of ACC. ACC inhibitors were evaluated in rodent models of liver fibrosis induced by diet or the hepatotoxin, DEN. Fibrosis and hepatic steatosis were evaluated by histological and biochemical assessments. RESULTS: In TGF-beta-stimulated HSCs, ACC inhibition reduced activation and collagen production independent of mitochondrial beta-oxidation by blocking de novo lipogenesis. ACC inhibition prevented a metabolic switch necessary for induction of glycolysis and oxidative phosphorylation during HSC activation. Consistent with this direct anti-fibrotic mechanism in HSCs, ACC inhibition reduced liver fibrosis in a rat CDHFD model and in response to chronic DEN-induced liver injury that lacked hepatic lipid accumulation. CONCLUSIONS: In addition to reducing lipid accumulation in hepatocytes, ACC inhibition also directly impairs the pro-fibrogenic activity of HSCs. Small molecule inhibitors of ACC may reduce liver fibrosis by both reducing lipotoxicity in hepatocytes and directly reducing HSC activation, providing a mechanistic rationale for the treatment of patients with advanced liver fibrosis due to NASH.
2020-04-24 | GSE148849 | GEO
Project description:Osteolineage cells represent one of the critical bone marrow niche components that support maintenance of hematopoietic stem and progenitor cells (HSPCs). Recent studies demonstrate that extracellular vesicles (EVs) regulate stem cell development via horizontal transfer of bioactive cargo, including microRNAs (miRNAs). Here, we characterize the miRNA profile of EVs secreted by human osteoblasts and study their biological effect of on human umbilical cord blood-derived CD34+ HSPCs by sequencing, gene expression and biochemical analyses. Using next-generation sequencing we show that osteoblast-derived EVs contain highly abundant miRNAs specifically enriched in EVs, including critical regulators of hematopoietic proliferation (e.g., miR-29a). EV treatment of CD34+ HSPCs alters the expression of candidate miRNA targets, such as HBP1, BCL2 and PTEN. Furthermore, EVs enhance proliferation of CD34+ cells and their immature subsets in growth factor-driven ex vivo expansion cultures. Importantly, EV-expanded cells retain their differentiation capacity in vitro and show successful engraftment in NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice in vivo. These discoveries reveal a novel osteoblast-derived EV-mediated mechanism for regulation of HSPC proliferation and warrant consideration of EV-miRNAs for the development of expansion strategies to treat hematological disorders.
2016-10-05 | E-MTAB-4560 | biostudies-arrayexpress
Project description:Mice were intravenously injected with extracellular vesicles (EVs) isolated from B16V wt (n=3) or BAG6KO (n=3) cells or with PBS (n=3) as a control for 4 weeks on a weekly basis. Since B-16V melanoma cells colonise the lung upon intravenous injection and referring to current literature, we hypothesise that melanoma EVs alter the (immune-) microenvironment of the lung to prepare a pre-metastatic niche. Based on current literature and own previous experiments identifying BAG6 as an immunoregulatory protein that is also involved in the biogenesis of EVs, we are particularly interested in differences between the immune signature upon wt EV treatment compared to BAG6KO EV treatment.
2019-11-29 | E-MTAB-7119 | biostudies-arrayexpress
Project description:Development of liver fibrosis is associated with activation of quiescent Hepatic Stellate Cells (qHSCs) into Collagen Type I-producing myofibroblasts (aHSCs). Cessation of liver injury often results in fibrosis resolution and inactivation of aHSCs/myofibroblasts into a quiescent-like state (iHSCs). To identify the molecular drivers of these HSC phenotypes, we investigated the global gene expression and epigenetic changes in H3K4me2 and H3K27ac marks of primary murine and human HSCs. Motif enrichment identified ETS1/2, GATA4/6, and IRF1/2 transcription factors (TFs) as putative regulators of the HSC lineage identity in murine and human HSCs. shRNA-knockdown of these TFs resulted in marked upregulation of fibrogenic and inflammatory genes and a loss of HSC-specific phenotype in targeted murine HSCs. In support, in vivo HSC-specific ablation of GATA4, GATA6, and ETS1, and ETS1 target genes NF1 (neurofibromin 1) and PPARγ exacerbated development of carbon tetrachloride (CCl4)-induced liver fibrosis in LratΔGATA6, LratETS1+/-, LratΔNF1, and LratΔPPARγ mice (vs wt littermates), but only LratΔGATA6, and LratΔPPARγ mice exhibited a defect in fibrosis resolution due to the lack of HSC inactivation. Therapeutic administration of a PPARγ agonist accelerated regression of liver fibrosis in CCl4-induced wt, but not in LratΔPPARγ mice, suggesting that PPARγ signaling in HSCs is critical for HSC inactivation and regression of liver fibrosis. Common transcription factors determine the phenotype of mouse and human HSCs. Similar to murine HSCs, human aHSCs can revert into a quiescent-like, inactivated phenotype. GATA4, GATA6, and PPARγwere identified as an important driver of human HSC inactivation.
2020-01-27 | GSE140641 | GEO
Project description:Phenotypic changes induced by extracellular vesicles (EVs) have been implicated in the recovery of acute kidney injury (AKI) induced by mesenchymal stromal cells (MSCs). miRNAs are potential candidates for cell reprogramming towards a pro-regenerative phenotype. The aim of the present study was to evaluate whether miRNA de-regulation inhibits the regenerative potential of MSCs and derived-EVs in a model of glycerol-induced AKI in SCID mice. For this purpose, we generated MSCs depleted of Drosha, a critical enzyme of miRNA maturation, to alter miRNA expression within MSCs and EVs. Drosha knock-down MSCs (MSC-Dsh) maintained the phenotype and differentiation capacity. They produced EVs that did not differ from those of wild type cells in quantity, surface molecule expression and internalization within renal tubular epithelial cells. However, EVs derived from MSC-Dsh (EV-Dsh) showed global down-regulation of miRNAs. Whereas, wild type MSCs and derived EVs were able to induce morphological and functional recovery in AKI, MSC-Dsh and EV-Dsh were ineffective. RNA sequencing analysis showed that genes deregulated in the kidney of AKI mice were restored by treatment with MSCs and EVs but not by MSC-Dsh and EV-Dsh. Gene Ontology analysis showed that down-regulated genes in AKI were associated with fatty acid metabolism. The up-regulated genes in AKI were involved in inflammation, ECM-receptor interaction and cell adhesion molecules. These alterations were reverted by treatment with wild type MSCs and EVs, but not by the Drosha counterparts. In conclusion, miRNA depletion in MSCs and EVs significantly reduced their intrinsic regenerative potential in AKI, suggesting a critical role of miRNAs. RNA-seq
2015-05-07 | E-GEOD-59958 | biostudies-arrayexpress
Project description:BACKGROUND AND AIM: We previously established that endoplasmic reticulum stress leads to unfolded protein response (UPR) that promotes liver fibrosis by activating hepatic stellate cell (HSC). We aimed to determine the role of X-box binding protein 1 (XBP1), one of three UPR effector pathways, in the fibrogenic HSC activation. METHODS: XBP1 expression was measured by qPCR in tunicamycin-treated human HSC lines (TWNT4 and LX2) and culture-activated human primary HSCs. XBP1 was overexpressed in primary human HSCs and the HSC lines, and fibrogenic genes (COL1A1, ACTA2, PDGFRB, MMP2, TIMP1) and encoded proteins were assessed by qPCR and Western blotting, respectively. Autophagy was inhibited by knockdown of ATG7. Genome-wide transcriptome profiling was performed to determine modulated molecular pathway by XBP1. In vivo XBP1 activation was assessed in transcriptome datasets of fibrotic mouse models and immunohistochemistry of human liver tissues. RESULTS: XBP1 overexpression accompanied both tunicamycin- and culture-based HSC activation. Ectopic overexpression of XBP1 induced fibrogenic gene expression in the HSC lines, which was inhibited by knockdown of ATG7. UPR pathway together with PDGFRB pathway, a hallmark of HSC activation, were induced in transcriptome profiles of XBP1-transduced HSC lines. Some known fibrogenic pathways such as transforming growth factor (TGF)-beta pathway were not induced, suggesting partial contribution of XBP1 to the entire fibrogenic HSC activation machinery. XBP1 target gene signature defined in our XBP1-overexpressing HSC lines was significantly induced in liver tissue transcriptome profiles of carbon tetrachloride-treated or bile duct-ligated mouse models, and XBP1 and alpha-smooth muscle actin (encoded by ACTA2, another hallmark of HSC activation) were co-localized in human fibrotic liver tissues, collectively supporting involvement of XBP1 in HSC activation and fibrogesis in vivo. CONCLUSIONS: XBP1-mediated UPR is at least partially responsible for fibrogenic HSC activation via autophagy.
2016-12-21 | GSE78070 | GEO
Project description:Background: Acute coronary syndromes (ACS) are associated with aberrant gene expression and epigenetic mechanisms. In particular, de novo DNA methylation is typically linked to gene silencing, but its role in heart disease remains not fully understood. Extracellular vesicles (EVs) are active components in cellular communication for their ability to carry a plethora of signalling biomolecules, thus representing a promising new diagnostic/therapeutic approach in cardiovascular diseases (CVDs). Indeed, there is the need of novel biomarkers for ACS prediction and timely detection. Purpose: We hypothesized that specific epigenetic signals can be carried by EVs. In this regard, we isolated and characterized circulating EVs from ACS patients and evaluated their potential role to influence DNA methylation in target cells. Methods: Circulating EVs were recovered, by ultracentrifugation, from plasma samples of 19 ACS patients and 50 healthy subjects (HS). Nanoparticle tracking analysis (NTA) and western blot (WB) were used to confirm the EVs integrity and purity. Peripheral blood mononuclear cells (PBMCs) of volunteer donors were treated with both ACS and HS derived EVs and genomic DNA was extracted to perform epigenome wide analysis through Reduced Representation Bisulfite Sequencing. ShinyGO, PANTHER, and STRING tools were interrogated to perform GO and PPI network analyses. Flow Cytometry, qRT-PCR, and WB analysis were also performed to evaluate and validate both intra-vesicular and intra-cellular signals. Results: Plasma ACS-derived EVs showed a significant up-regulation of DNA methyltransferases (DNMTs) gene expression levels as compared to HS (P<0.001). Specifically, de novo methylation transcripts, as DNMT3A and DNMT3B were significantly increased in plasma ACS-EVs. DNA methylation analysis of PBMCs from volunteer donors treated with HS- and ACS-derived EVs showed that RNF166 and CCND3 genes resulted the most hyper- and hypo-methylated, respectively, after by ACS-EV treatment. In addition, PPI network analysis specifically evidenced the subnetwork with SRC, as a hub gene, connecting it to NOTCH1, FOXO3, CDC42, IKBKG, RXRA, DGKG, known as important genes already involved in the onset of CVDs. Surprising, other novel genes, BAIAP2, SYP, CHL1, and SHB, which were hypomethylated, were found significantly overexpressed in PBMCs (P<0.005), underlying the fundamental modulating properties of EV cargo in atherosclerosis. Conclusions: These findings support the significant role of ACS plasma-derived EVs, inducing de novo DNA methylation signals, and modulating specific signaling pathways in target cells.
2023-04-03 | E-MTAB-11641 | biostudies-arrayexpress