Substrate stiffness modulates cardiac fibroblast activation, senescence and proinflammatory secretory phenotype
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ABSTRACT:
PROVIDER: PRJNA1023891 | ENA |
REPOSITORIES: ENA
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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.
2023-10-24 | GSE244614 | GEO
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:The substrate stiffness plays an important role in mediating the cellular behavior. Neutrophils predominate the early inflammatory response and initiate the regeneration. The neutrophil activation can be regulated by physical cues. However, it is not known how neutrophils respond to substrate stiffness, which is of significant importance in determining the outcomes of engineered tissue mimics. Herein, a three-dimensional culture system made of hydrogel for bone marrow-derived neutrophils was developed to explore the effects of varying stiffness (1.5, 2.6, and 5.7 kPa) on neutrophil phenotype and polarization states.
2022-12-03 | GSE217379 | GEO
Project description:Purpose: Identify the effect of substrate stiffness on gene expression Methods:Evaluating for differentially expressed mRNAs in the SKOV-3 cells grown on the different substrates via High-throughput sequence Results: We found that the general direction of changes in gene expression of cells grown on the different substrates and the most significant signalling pathways and the expression of gene orthologs broadly involved in platinum drug resistance, apoptosis, cell cycle. Conclusions: Our study represents the first detailed analysis of the effects of substrate stiffness on gene expression of ovarian cancer cells.
2021-06-26 | GSE178888 | GEO
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.
2019-12-16 | GSE123100 | GEO
Project description:Cardiomyocytes develop abnormal beating patterns and dedifferentiate on substrate with stiffness other than the physiological heart properties. This effect can be replicated in vitro with primary cardiomyocyte. To get a better idea of pathways involved and to identify new target genes, we aimed to compare mRNA and lncRNA of embryonic cardiomyocytes plated on either soft or stiff substrates
2020-04-18 | GSE109405 | GEO
Project description:Cellular senescence is a stable cell growth arrest that is implicated in tissue aging and cancer. Senescent cells are characterized by an upregulation of proinflammatory and immunosuppressive cytokines and chemokines, which is termed as senescence-associated secretory phenotype (SASP). NAD+ metabolism plays a critical role in both tissue aging and cancer. However, the role of NAD+ metabolism in regulating the SASP is not well understood. Here we show that nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme of the NAD+ salvage pathway, governs the strengths of proinflammatory SASP during senescence. In contrast to downregulation of NAMPT during replicative senescence, NAMPT is upregulated during oncogene-induced senescence. NAMPT selectively regulates proinflammatory, but not immunosuppressive, SASP. NAMPT is regulated by HMGA1 through a distal enhancer element during senescence. HMGA1/NAMPT/NAD+ signaling axis promotes proinflammatory SASP through enhancing glycolysis and mitochondria respiration. Mechanistically, HMGA1/NAMPT promotes proinflammatory SASP through NAD+-mediated suppression of AMPK kinase, which suppresses p53-mediated inhibition of p38MAPK to enhance NFb activity. SASP regulation by NAD+ metabolism is independent of senescence-associated cell growth arrest. An increase in NAD+ levels is sufficient to convert SASP from low to high levels during replicative senescence. Together, we conclude that NAD+ metabolism governs the strengths of proinflammatory SASP. Given the tumor promoting effects of proinflammatory SASP, our results suggest that anti-ageing dietary NAD+ augmentation should be administered with precision.
2019-03-14 | GSE111841 | GEO
Project description:We undertook mRNA microarray and gene ontology analyses to screen out substrate stiffness-dependent genes. Total mRNA were extracted from E17 cortical neurons grown on soft or stiff substrates at 5 or 16 hr time points. We identified 114 differentially-expressed mRNA transcripts in cells grown on 0.1 kPa and 20 kPa gels at the 5 hr time-point. Among them, 66 were upregulated in 0.1 kPa gel cultures and the remainder were downregulated (compared to cells grown on stiffer substrates). The expressions of three endocytic genes (Cltc, Dab2, and Myo6) and four adhesion genes(Vcl, Robo2, Nrcam, and Cad11) were confirmed by QGP and smRNA FISH.
2018-01-08 | GSE102350 | GEO
Project description:Influence of substrate stiffness on chemotherapeutic response in breast cancer
| PRJNA418945 | ENA
Project description:Extracellular matrix (ECM) regulates carcinogenesis. As a major ECM component, collagen interacts with integrin and a non-typical receptor discoidin domain receptor tyrosine kinase 2 (DDR2), but how DDR2 regulates cancer progression is poorly understood. Apart from its signaling function, ECM provides a mechanical environment for cancer, but the impact of biomechanics on cancer remains enigmatic. Here, we performed RNA-seq of a human neuroblastoma cell line SH-SY5Y. We found that DDR2 knockdown, but not increasing substrate stiffness, upregulated pro-proliferative genes and down-regulated axonogenesis genes. Surprisingly, despite no transcriptome changes, increasing of stiffness attenuates SH-SY5Y cell proliferation, an effect also observed after DDR2 knockdown. Our results indicate that two ECM effectors, DDR2 and biomechanics, could control cancer cell proliferation through different mechanisms.
2024-04-19 | GSE246550 | GEO