Effect of Combinatorial Extracellular Matrix and Substrate Stiffness on Gene Expression of Activated Hepatic Stellate Cells
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ABSTRACT:
PROVIDER: PRJNA868328 | ENA |
REPOSITORIES: ENA
Dataset's files
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| SRR21006700.fastq.gz | Fastqsanger.gz | |||
| SRR21006701.fastq.gz | Fastqsanger.gz | |||
| SRR21006702.fastq.gz | Fastqsanger.gz | |||
| SRR21006703.fastq.gz | Fastqsanger.gz | |||
| SRR21006704.fastq.gz | Fastqsanger.gz |
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Project description:Effect of Combinatorial Extracellular Matrix and Substrate Stiffness on Chromatin Accessibility of Activated Hepatic Stellate Cells
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Project description:Treating recurrent GBM is a clinical challenge due to its highly resistant and aggressive nature. In order to develop new therapeutic targets for recurrent GBM a better understanding of its molecular landscape is necessary. Here we used a cellular model, developed in our lab which generates paired primary and recurrent samples from GBM cell lines and primary patient samples hence allowing us to compare the molecular differences between the two populations. Total RNA seq analysis of parent and recurrent population of two cell lines and one patient sample revealed a significant upregulation of Extracellular matrix interaction in recurrent population. Since matrix stiffness plays a pivotal role in cell-ECM interaction and downstream signaling, we developed a system that mimicked the brain like substrate stiffness by using collagen coated polyacrylamide-based substrate whose stiffness can be modified from normal brain (0.5kPa) to tumorigenic (10kPa). Using these substrates, we were able to capture the morphological and physiological differences between parent and recurrent GBM which were not evident on plastic surfaces (~1 GPa). On 0.5kPa, unlike circular parent cells, recurrent GBM cells showed two morphologies (circular and elongated). The recurrent cells growing on 0.5kPa also showed higher proliferation, invasion, migration and in-vivo tumorigenicity in orthotropic GBM mouse model, compared to parent cells. Furthermore, recurrent cells exhibited elevated velocity irrespective of substrate stiffness, which indicated that recurrent cells may possess inherent differential mechanosignalling ability which was reflected by higher expression of ECM proteins like Collagen IVA, MMP2 and MMP9. Moreover, mice brain injected with recurrent cells grown on 0.5kPa substrate showed higher Young’s modulus values suggesting that recurrent cells conditioned on 0.5kPa make the surrounding ECM stiffer. Importantly, inhibition of EGFR signaling, that is amplified with tissue stiffening in GBM resulted in decreased invasion, migration and proliferation in 0.5kPa recurrent cells, but interestingly survival remained unaffected, highlighting the importance of mimicking the physiological stiffness of the brain mimicking clinical scenario. Total RNA seq analysis of parent and recurrent cells grown on plastic and 0.5kPa substrate identified PLEKHA7 as significantly upregulated gene specifically in 0.5kPa recurrent sample. Higher protein expression of PLEKHA7 in recurrent GBM as compared to primary GBM was validated in patient biopsies. Accordingly, PLEKHA7 knockdown reduced invasion and survival of recurrent GBM cells. Together, these data provides a model system that captures the differential mechanosensing signals of primary and recurrent GBM cells and identifies a novel potential target specific for recurrent GBM.
2023-06-29 | E-MTAB-11293 | biostudies-arrayexpress
Project description:Extracellular matrix proteomic profiling Mass spectrometry characterization illustrated a reprogrammed proteome in response to matrix stiffness, including a core subset of 84 ECM-specific proteins, including various ECM regulators and tissue-associated ECM-related proteins. Further, we observe remodeling of ECM proteome with softer substrates associated with metabolic/mitochondrial network (178 proteins), while with 40 kPa substrate (92 proteins enriched) resulting in networks associated with enrichment of collagen biosynthesis, integrin cell surface interactions, and extracellular matrix organization networks.
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Project description:In vitro cultures of primary cardiac fibroblasts (CFs), the major extracellular matrix (ECM)-producing cells of the heart, are used to determine molecular mechanisms of cardiac fibrosis. However, the supraphysiologic stiffness of tissue culture polystyrene (TCPS) automatically triggers the conversion of CFs into an activated myofibroblast-like state, and serial passage of the cells results in the induction of replicative senescence. These dramatic phenotypic switches confound interpretation of experimental data obtained with cultured CFs. In an attempt to circumvent TCPS-induced activation and senescence of CFs, we utilized poly (ethylene glycol) (PEG) hydrogels as cell culture platforms with low and high stiffness formulations to mimic healthy and fibrotic cardiac ECM, respectively. As hypothesized, low hydrogel stiffness converted activated CFs into a quiescent state with reduced abundance of a-smooth muscle actin (a-SMA)-containing stress fibers. Unexpectedly, lower substrate stiffness concomitantly augmented CF senescence, marked by elevated senescence-associated b-galactosidase (SA-b-Gal) activity and increased expression of p16 and p21, which are cyclin-dependent kinase (CDK) inhibitors and markers of senescence. Using dynamically stiffening hydrogels with phototunable crosslinking capabilities, we demonstrate that substrate-induced CF senescence is partially reversible. RNA-sequencing analysis revealed widespread transcriptional reprogramming of CFs cultured on low stiffness hydrogels, with a dramatic reduction in the expression of profibrotic genes encoding ECM proteins, and an attendant increase in expression of NF-kB-responsive inflammatory genes that typify the senescence-associated secretory phenotype (SASP). Our findings further demonstrate that alterations in matrix stiffness profoundly impact CF cell state transitions, and suggest mechanisms by which CFs change phenotype in vivo depending on the stiffness of the myocardial microenvironment to which they are exposed.
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Project description:Matrix stiffness has significant effects on cell behavior, however, less is known regarding the epigenomic and transcriptional regulation underling the effect of matrix stiffness on cells. In this study, we use an in vitro system to assess the phenotypic shifts of hepatic stellate cells (HSCs) following changes in matrix stiffness, and integrate multi-omics with imaging and biochemical assays to investigate the molecular mechanisms. We show that cells cultured on a stiff matrix display more accessible chromatin sites, which consist of primed chromatin regions that become more accessible prior to the upregulation of nearby genes. These regions are enriched in fibrosis-associated genes that function in cytoskeletal organization and response to mechanical stimuli. We also identify activation of p-JUN in response to the stiff matrix and promoting phenotypic shifts. The identified chromatin accessibility-dependent effect of matrix stiffness may be responsible for various fibrotic diseases and provide insight into
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Project description:Elevated intraocular pressure, a major risk factor of glaucoma, is caused by the abnormal function of trabecular outflow pathways. Human trabecular meshwork (HTM) tissue plays an important role in the outflow pathways. However, the molecular mechanisms that how TM cells respond to the elevated IOP are largely unknown. We cultured primary HTM cells on polyacrylamide gels with tunable stiffness corresponding to Young's moduli ranging from 1.1 to 50 kPa. Then next‐generation RNA sequencing (RNA‐seq) was performed to obtain the transcriptomic profiles of HTM cells. Bioinformatics analysis revealed that genes related to glaucoma including DCN, SPARC, and CTGF, were significantly increased with elevated substrate stiffness, as well as the global alteration of HTM transcriptome. Extracellular matrix (ECM)‐related genes were selectively activated in response to the elevated substrate stiffness, consistent with the known molecular alteration in glaucoma. Human normal and glaucomatous TM tissues were also obtained to perform RNA‐seq experiments and supported the substrate stiffness‐altered transcriptome profiles from the in vitro cell model. The current study profiled the transcriptomic changes in human TM cells upon increasing substrate stiffness. Global change of ECM‐related genes indicates that the in vitro substrate stiffness could greatly affect the biological processes of HTM cells. The in vitro HTM cell model could efficiently capture the main pathogenetic process in glaucoma patients, and provide a powerful method to investigate the underlying molecular mechanisms.
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Project description:Matrix stiffness modulates fibrogenesis in hepatic stellate cells through AP-1-induced chromatin priming
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Project description:The increased stiffness of the extracellular matrix is a key driver of liver fibrosis. The activated hepatic stellate cell (HSC) is the major producer of extracellular matrix (ECM) components. While little is known about the epigenomic changes that underlie the fibrogenic impact of ECM mechanics. In this study, we utilized a reliable in vitro system to mimic liver cirrhosis and integrated multi-omics analysis, which includes time-series RNA-seq and ATAC-seq as well as histone modification Cut&Tag, with imaging and biochemical essays to study the mechanism underlying the biomechanics function on fibrotic phenotype. We found that cells cultured in stiff matrix displayed more accessible chromatin sites, consisting of amount regions became accessible before stable fibrotic phenotype. We defined these regions as primed chromatin that chromatin accessibility foreshadows changes in gene expression. This kind of chromatin enriched in cytoskeleton organization and responding to mechanical stimulus biological process. Here, we depicted the AP-1 transcription factor family as being responsible for driving the construction of primed chromatin. Among AP-1 transcription factors, we confirmed JUN was critical to reconstruct chromatin accessibility to promote fibrogenic genotype. In addition, we described ERK contribute to the activation of JUN resulting in its binding on chromatin. Our results profiled a dynamic landscape of chromatin accessibility and defined the primed chromatin that contribute to fibrosis during responding to stiff matrix. We identified that AP-1 was capable of reorganizing the chromatin accessibility in mechanotransduction.
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2022-12-03 | GSE217379 | GEO