Role of ZNF281 in intestinal fibroblast activation: new insights in IBD-related fibrosis
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE204868 | GEO | 2022/09/02
REPOSITORIES: GEO
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Project description:Background: Mechanistic target of rapamycin (mTOR) is a nodal serine/threonine protein kinase critical for the control of fundamental cellular processes. Dysregulated mTOR signalling has been shown in cancer, COPD and idiopathic pulmonary fibrosis (IPF). mTOR forms the catalytic subunit of 2 protein complexes: mTORC1 and mTORC2 which differ in upstream inputs, downstream effects and sensitivity to the allosteric inhibitor rapamycin. Aim: To compare the effects of the mTORC1 inhibitor rapamycin with an ATP-competitive inhibitor of mTORC1/2, AZD8055, on TGFβ1 induced transcriptional responses during myofibroblast differentiation by RNA-Seq analysis. Methods: Primary human lung fibroblasts were treated with TGFβ1 for 24h to induce myofibroblast differentiation and extra cellular matrix (ECM) production in the presence of rapamycin or AZD8055. Fibroblasts were also treated with rapamycin or AZD8055 without TGFβ1. Total mRNA was analysed by RNA-Seq. Results: On a global transcriptomic level, TGFβ1 significantly affected the expression of ~4,200 genes by more than 1.5 fold, of which ~1,100 changes were reversed by pan-mTORC1/2 inhibition, while being insensitive to rapamycin. Only 15 gene changes were reversed exclusively by rapamycin. Analysis of the rapamycin-insensitive, mTOR sensitive myofibroblast transcriptome revealed ECM-receptor interaction, metabolism and actin cytoskeleton regulation as major affected pathways. Conclusions: Our data support the conclusion that therapeutic approaches aimed at interfering with TGFb1 induced myofibroblast differentiation and ECM production require dual mTOR1/2 inhibition and may, in part, explain the lack of clinical efficacy of everolimus in the context of IPF.
2019-05-01 | GSE102674 | GEO
Project description:Fibrotic disease is caused by persistently activated fibroblasts, known as myofibroblasts, that continuously deposit extracellular matrix and fail to de-activate after injury resolution. There are currently no treatments for fibrotic disease; our study addresses the mechanisms whereby myofibroblasts persist in fibrotic tissues such as diseased cardiac valves. We exploit photo-softening hydrogels as synthetic valve tissue mimics and valve fibroblasts as a model to study how stiffness controls pathological myofibroblast activation and their persistence. We show that persistent myofibroblasts have condensed chromatin structure with genome-wide alterations and that this is associated with stabilization of the actin cytoskeleton. Disconnecting the nucleus from the cytoskeleton prevents chromatin condensation and myofibroblast persistence. Notably, myofibroblasts in patients with aortic valve stenosis display a condensed chromatin structure compared to myofibroblasts in a healthy patient, similar to the difference observed between cultured persistent myofibroblasts and transient myofibroblasts. Collectively, our results reveal that nuclear mechanosensing leads to distinct chromatin signatures in persistent myofibroblasts and that this novel cellular mechanism is likely relevant to human fibrotic disease.
2021-03-31 | GSE167892 | GEO
Project description:Background and aims: Intestinal fibrosis is a common complication of Crohn’s disease (CD). It is characterised by an excessive accumulation of fibroblasts differentiating into activated myofibroblasts secreting excessive extracellular matrix. The potential role of the intestinal epithelium in this fibrotic process remains poorly defined. Methods: A total of 113 CD and control subjects were studied. We performed a pilot proteomic study comparing the proteome of surface epithelium isolated by laser capture microdissection in normal and fibrotic zones of resected ileal CD strictures (n=5). Selected proteins were validated by immunohistochemistry (IHC) in colonic and ileal samples of stricturing CD patients (n=44), pure inflammatory CD (n=29) and control subjects (n=40). Functional assays with cell lines cultures and a fibroblast to myofibroblast differentiation model were used to assess the role of the highlighted epithelial proteins in CD fibrosis. Results: Proteomic study revealed an endoplasmic reticulum (ER) stress and unfolded protein response (UPR) markers increase in the epithelium overlying ileal fibrotic strictures, involving Anterior gradient protein 2 homolog (AGR2) and Binding immunoglobulin protein (BiP). This was confirmed by IHC. The ER stress induction in intestinal epithelial cells increased AGR2 as well as BiP expression and led to an extracellular secreted AGR2. A fibroblast to myofibroblast differentiation was obtained with the supernatant of intestinal epithelial cells pre-conditioned by ER stress and with recombinant AGR2. Conclusions: AGR2 and other ER stress markers are increased within the intestinal epithelium overlying fibrotic strictures and might contribute to profibrotic signals involved in CD fibrosis.
2022-04-04 | PXD022214 | Pride
Project description:Despite recent advances in understanding skin scarring, mechanisms triggering hypertrophic scar formation are still poorly understood. In the present study we performed single-cell sequencing of mature human hypertrophic scars and developing scars in mice. Compared to normal skin, we found significant differences in gene expression in most cell types present in scar tissue. Fibroblasts (FBs) showed the most prominent alterations in gene expression, displaying a distinct fibrotic signature. By comparing genes upregulated in murine FBs during scar development with genes highly expressed in mature human hypertrophic scars, we identified a group of serine proteases, tentatively involved in scar formation. Two of them, dipeptidyl-peptidase 4 (DPP4) and urokinase (PLAU), were further analyzed in functional assays, revealing a role in TGFβ1-mediated myofibroblast differentiation and over-production of components of the extracellular matrix (ECM) without interfering with the canonical TGFβ1 -signaling pathway. In this study, we delineate the genetic landscape of hypertrophic scars and present new insights into mechanisms involved in hypertrophic scar formation. Our data suggest the use of serine protease inhibitors for the treatment of skin fibrosis.
2021-05-31 | GSE156326 | GEO
Project description:After skin injury fibroblasts migrate into the wound and transform into contractile extracellular matrix-producing myofibroblasts to promote skin repair. Persistent activation of myofibroblasts can cause excessive fibrotic reactions, but the underlying mechanisms are not fully understood. We used SMA-GFP transgenic mice to study myofibroblast recruitment and activation in skin wounds. Myofibroblasts were initially recruited to wounds three days post injury reached a maximum after seven days and subsequently declined. Expression profiling showed that 1749 genes were differentially expressed in sorted myofibroblasts from wounds seven days post injury. GO-Term analysis associated most of these genes with the extracellular region and cell periphery including upregulated genes encoding for ECM proteins, integrins and focal adhesion proteins and a specific set of growth factors, cytokines and chemokines promoting the healing response. In contrast, cell adhesion molecules involved in cell-cell interaction were downregulated. Novel genes not yet known to be expressed in myofibroblast were found. Hence, this first characterization of a myofibroblast specific expression network at the peak of granulation tissue formation in situ provides important insights into myofibroblast activation in skin wounds and identifies novel target genes to control excessive ECM deposition during fibrotic reactions.
2017-12-30 | GSE95786 | GEO
Project description:The differentiation of fibroblasts into myofibroblasts mediates tissue wound healing and fibrotic remodeling. To better understand this process we performed a genome-wide screen in fibroblasts, which identified the RNA-binding protein muscleblind-like 1 (MBNL1) as a potent regulator of myofibroblast differentiation. MBNL1 promoted transformation of fibroblasts into myofibroblasts, while loss of Mbnl1 abrogated myofibroblast differentiation and impaired the fibrotic phase of wound healing in mouse models of myocardial infarction and dermal injury. Conditional fibroblast-specific MBNL1 transgenic mice showed a fibrotic phenotype in the absence of injury. Mechanistically, MBNL1 directly bound to and regulated the alternative splicing and stability of a network of myofibroblast differentiation specific genes, such as the nodal transcriptional regulator serum response factor (SRF). CRISPR-Cas9 mediated gene editing of the MBNL1 binding site in Srf impaired myofibroblast differentiation. These data establish a new RNA-dependent paradigm through MBNL1 that underlies global myofibroblast differentiation, the fibrotic response, and tissue wound healing.
2015-10-20 | GSE74185 | GEO
Project description:Background: Fibrosis is a common pathology in many cardiac disorders and is driven by the activation of resident fibroblasts. The global post-transcriptional mechanisms underlying fibroblast-to-myofibroblast conversion in the heart have not been explored. Methods: Genome-wide changes of RNA transcription and translation during human cardiac fibroblast activation were monitored with RNA sequencing and ribosome profiling. We then used a RNA-binding protein-based analyses to identify translational regulators of fibrogenic genes. The integration with cardiac ribosome occupancy levels of 30 dilated cardiomyopathy patients demonstrates that these post-transcriptional mechanisms are also active in the diseased fibrotic human heart. Results: We generated nucleotide-resolution translatome data during the TGFβ1-driven cellular transition of human cardiac fibroblasts to myofibroblasts. This identified dynamic changes of RNA transcription and translation at several time points during the fibrotic response, revealing transient and early-responder genes. Remarkably, about one-third of all changes in gene expression in activated fibroblasts are subject to translational regulation and dynamic variation in ribosome occupancy affects protein abundance independent of RNA levels. Targets of RNA-binding proteins were strongly enriched in post-transcriptionally regulated genes, suggesting genes such as MBNL2 can act as translational activators or repressors. Ribosome occupancy in the hearts of patients with dilated cardiomyopathy suggested the same post-transcriptional regulatory network was underlying cardiac fibrosis. Key network hubs include RNA-binding proteins such as PUM2 and QKI that work in concert to regulate the translation of target transcripts in human diseased hearts. Furthermore, silencing of both PUM2 and QKI inhibits the transition of fibroblasts toward pro-fibrotic myofibroblasts in response to TGFβ1. Conclusions: We reveal widespread translational effects of TGFβ1 and define novel post-transcriptional regulatory networks that control the fibroblast-to-myofibroblast transition. These networks are active in human heart disease and silencing of hub genes limits fibroblast activation. Our findings show the central importance of translational control in fibrosis and highlight novel pathogenic mechanisms in heart failure.
2019-10-03 | GSE131111 | GEO
Project description:Arthrofibrosis is characterized by excessive extracellular matrix (ECM) deposition that results in restricted joint motion after total knee arthroplasty (TKA). Current surgical and pharmacologic treatment options are limited. Therefore, an in vitro model for joint myofibroblastogenesis is valuable to investigate the arthrofibrotic process and identify diagnostic biomarkers and treatment options. In this study, we obtained intraoperative posterior capsule (PC), quadriceps tendon (QT), and suprapatellar pouch (SP) tissue from knees of four patients undergoing primary TKA for osteoarthritis and characterized primary outgrowth cells from these tissues in the absence and presence of transforming growth factor beta 1 (TGFβ1), a pro-myofibroblastic cytokine. Light microscopy of knee outgrowth cells revealed spindle-shaped cells while immunofluorescence (IF) established staining for the fibroblast-specific antigen TE-7 and Vimentin, which are characteristics of fibroblasts. These fibroblasts differentiate readily into myofibroblasts as highlighted by enhanced alpha smooth muscle actin (ACTA2) mRNA and protein expression and increased collagen mRNA (i.e., collagen type 1 (COL1A1) and collagen type 3 (COL3A1)) expression and collagenous matrix deposition in the presence of TGFβ1. Of note, these studies also revealed that knee-derived fibroblasts are more sensitive to TGFβ1-mediated myofibroblastogenesis than adipose-derived mesenchymal stem cells. Importantly, while outgrowth fibroblasts isolated from four patients and three anatomical regions exhibit similar gene expression profiles, these knee fibroblasts form a unique gene expression cluster within the fibroblast niche as revealed by RNA-sequencing analysis. In conclusion, our study provides a fibroblast/myofibroblast model of outgrowth knee cells derived from patients undergoing primary TKA that can be employed to assess myofibroblast-related processes and test novel pharmacological strategies in vitro for arthrofibrosis.
2022-03-01 | GSE185333 | GEO
Project description:Vascular inflammation and myofibroblast activation by TGF-β1 are key, yet-to-be-fully-understood pathological processes in fibrosis. Here we report the development of a novel human Tendon-on-a-Chip (hToC) to elucidate the role of TGF-β1 in peritendinous adhesions, a debilitating fibrosis condition affecting flexor tendon, which currently lacks biological therapies. The hToC allows the crosstalk between a vascular compartment harboring endothelial cells and monocytes with a tissue hydrogel compartment containing tendon fibroblasts and macrophages. We find that the hToC replicates in vivo inflammatory and fibrotic phenotypes in preclinical and clinical samples, including myofibroblast differentiation and tissue contraction, excessive ECM deposition, and inflammatory cytokines secretion. We further show the fibrotic phenotypes are driven by the interactions between the vascular and tissue compartments, mediating the transmigration of monocytes. We demonstrate significant overlap in fibrotic transcriptional signatures in the hToC with human tenolysis samples, including the mTOR pathway, a regulatory nexus of fibrosis across various organs. Treatment with Rapamycin suppressed the fibrotic phenotype on the hToC, which validates the hToC as a preclinical alternative for investigating fibrosis and testing therapeutics.
2024-01-10 | GSE246391 | GEO
Project description:Background: The differentiation of pericytes into myofibroblasts causes microvascular degeneration, extracellular matrix (ECM) accumulation, and tissue stiffening, characteristics of fibrotic diseases. It is unclear how pericyte-myofibroblast differentiation is regulated in the microvascular environment. Our previous study established a novel two-dimensional platform for coculturing microvascular endothelial cells (ECs) and pericytes derived from the same tissue. This study investigated how ECM stiffness regulated microvascular ECs, pericytes, and their interactions. Methods: Primary microvessels were cultured in the TGM2D medium. Stiff ECM was prepared by incubating ECM solution in regular culture dishes for one hour followed by PBS wash. Soft ECM with Young’s modulus of approximately 6 kPa was used unless otherwise noted. Bone grafts were prepared from the rat skull. Immunostaining, RNA sequencing, qRT-PCR, western blotting, and knockdown experiments were performed on the cells. Results: Primary microvascular pericytes differentiated into myofibroblasts (NG2+αSMA+) on stiff ECM, even with the TGFβ signaling inhibitor A83-01. Soft ECM and A83-01 cooperatively maintained microvascular stability while inhibiting pericyte-myofibroblast differentiation (NG2+αSMA-/low). We thus defined two pericyte subpopulations: primary (NG2+αSMA-/low) and activated (NG2+αSMA+) pericytes. Soft ECM promoted microvascular regeneration and inhibited fibrosis in bone graft transplantation in vivo. As Integrins are the major mechanosensor, we performed qRT-PCR screening of Integrin family members selected from RNA sequencing data. We found that Integrin β1 (Itgb1) was the major subunit downregulated by soft ECM and A83-01 treatment. Knocking down Itgb1 suppressed myofibroblast differentiation on stiff ECM. Interestingly, ITGB1 phosphorylation (Y783) was mainly located on microvascular ECs on stiff ECM, which promoted EC secretion of paracrine factors, including CTGF, to induce pericyte-myofibroblast differentiation. CTGF knockdown or monoclonal antibody treatment partially reduced myofibroblast differentiation, implying the participation of multiple pathways in fibrosis formation. Conclusions: Microvascular ECs mediate ECM stiffness-induced pericyte-myofibroblast differentiation through paracrine signaling.
2023-07-18 | GSE237069 | GEO