TGF-β modulation of cell-cell and cell-extracellular matrix interactions in post-confluent corneal endothelial cells
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ABSTRACT:
PROVIDER: PRJNA723605 | ENA |
REPOSITORIES: ENA
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Project description:We isolated healthy human corneal endothelial cells from EyeBank research-grade corneas. Passage 3 cells were grown to confluency and exposed to TGF-b2 for 7 days.
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Project description:Fuchs endothelial corneal dystrophy FECD is a progressive corneal disease that impacts the structure and stiffness of the Descemets membrane DM, a protein layer that serves as a matrix for the corneal endothelial cells CECs. These structural alterations of the DM contribute to the loss of the CECs resulting in corneal edema and blindness. Oxidative stress and TGF-b pathways have been implicated in endothelial cell loss and endothelial to mesenchymal transition of CECs in FECD. Ascorbic acid AA is found at high concentrations in FECD and its impacts on CECs survival has been investigated. However, TGF-b and AA effects on the composition and rigidity of the CECs matrix remain unknown. In this study, we investigated the effect of AA, TGF-b1 and TGF-b3 on the deposition, ultrastructure, stiffness, and composition of the extracellular matrix ECM secreted by primary bovine corneal endothelial cells BCECs. Immunofluorescence and electron microscopy post-decellularization demonstrated a robust deposition and distinct structure of ECM in response to treatments. AFM measurements showed that the modulus of the matrix in BCECs treated with TGF-b1 and TGF-b3 was significantly lower than the controls. There was no difference in the stiffness of the matrix between the AA-treated cell and controls. Gene Ontology analysis of the proteomics results revealed that AA modulates the oxidative stress pathway in the matrix while TGF-b induces the expression of matrix proteins collagen IV, laminin and lysyl oxidase homolog 1. Molecular pathways identified in this study demonstrate the differential role of soluble factors in pathogenesis of the FECD.
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Project description:Dr. Panjwani's laboratory is focusing on the mechanism by which galectins-3 and 7 mediate corneal epithelial cell migration. We are currently performing studies to: (i) identify and characterize the corneal epithelial cell surface and extracellular matrix (ECM) molecules which serve as counterreceptors of galectin-3 and -7, to establish whether the lectins modulate corneal epithelial cell migration by binding to well known integrins, growth factor receptors, and/or ECM molecules and (ii) determine whether galectin-3 mediates corneal epithelial cell migration indirectly by modulating the expression of key adhesion and/or signal transduction molecules by using small interfering RNA, cDNA microarrays and glycogene arrays. We have prepared three independent preparations of total RNA of corneal epithelial cells from WT mice (total six samples) for analysis of glycogene expression. Samples are Normal Cornea (Left eye) and Laser ablation + 16-18 hours healing (right eye)
2010-12-11 | E-GEOD-26009 | biostudies-arrayexpress
Project description:Corneal injuries remain a major cause of consultation in the ophthalmology clinics worldwide. Repair of corneal wounds is a complex mechanism that involves cell death, migration, proliferation, differentiation, and extracellular matrix (ECM) remodeling. In the present study, we used a tissue-engineered, two-layers (epithelium and stroma) human cornea as a biomaterial to study both the cellular and molecular mechanisms of wound healing. Gene profiling on microarrays revealed important alterations in the pattern of genes expressed by tissue-engineered corneas in response to wound healing. Expression of many MMPs-encoding genes was shown by microarray and qPCR analyses to increase in the migrating epithelium of wounded corneas. Many of these enzymes were converted into their enzymatically active form as wound closure proceeded. In addition, expression of MMPs by human corneal epithelial cells (HCECs) was affected both by the stromal fibroblasts and the collagen-enriched ECM they produce. Most of all, results from mass spectrometry analyses provided evidence that a fully stratified epithelium is required for proper synthesis and organization of the ECM on which the epithelial cells adhere. In conclusion, and because of the many characteristics it shares with the native cornea, this human two layers corneal substitute may prove particularly useful to decipher the mechanistic details of corneal wound healing. Primary cultures of human corneal epithelial cells cultivated on BSA (number of replicates: 7), Collagen type I (number of replicates: 2), Collagen type IV (number of replicates: 2), Fibronectin (number of replicates: 2), Tenascin C (number of replicates: 2) and Laminin (number of replicates: 2) matrix. Central, internal and external ring of wounded Tissue-engineered human cornea.
2015-11-25 | E-GEOD-75336 | biostudies-arrayexpress
Project description:We report a novel licensing strategy to improve the immunosuppressive capacity of MSCs. Licensing murine MSCs with TGF-β1 (TGF-β MSC) significantly improved their ability to modulate both the phenotype and secretome of inflammatory bone marrow-derived macrophages and significantly increased the numbers of regulatory T lymphocytes (Tregs) following co-culture assays. These TGF-β MSC-expanded Tregs also expressed significantly higher levels of PD-L1 and CD73, indicating enhanced suppressive potential. Detailed analysis of T lymphocyte co-cultures revealed modulation of secreted factors, most notably, elevated prostaglandin E2 (PGE2). Furthermore, TGF-β MSCs could significantly prolong rejection-free survival (69.2% acceptance rate compared to 21.4% for un-licensed MSC treated recipients) in a murine corneal allograft model. Mechanistic studies revealed that (i) therapeutic efficacy of TGF-β MSCs is Smad2/3-dependent; (ii) TGF-β MSC’s enhanced immunosuppressive capacity is contact-dependent and (iii) enhanced secretion of PGE2 (via prostaglandin EP4 receptor) by TGF-β MSCs is the predominant mediator of Treg expansion and T cell activation and is associated with corneal allograft survival. Collectively, we provide compelling evidence for the use of TGF-β1 licensing as an unconventional strategy for enhancing MSC immunosuppressive capacity.
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Project description:To elucidate biological processes underlying the keratocyte, fibroblast, and myofibroblast phenotypes of corneal stromal cells, the gene expression patterns of these primary cultures from mouse cornea were compared with those of the adult and 10-day postnatal mouse cornea. Keywords = corneal stroma maturation keratocyte fibroblast myofibroblast TGF beta 1 Keywords: other
2004-10-27 | GSE1867 | GEO
Project description:Corneal injuries remain a major cause of consultation in the ophthalmology clinics worldwide. Repair of corneal wounds is a complex mechanism that involves cell death, migration, proliferation, differentiation, and extracellular matrix (ECM) remodeling. In the present study, we used a tissue-engineered, two-layers (epithelium and stroma) human cornea as a biomaterial to study both the cellular and molecular mechanisms of wound healing. Gene profiling on microarrays revealed important alterations in the pattern of genes expressed by tissue-engineered corneas in response to wound healing. Expression of many MMPs-encoding genes was shown by microarray and qPCR analyses to increase in the migrating epithelium of wounded corneas. Many of these enzymes were converted into their enzymatically active form as wound closure proceeded. In addition, expression of MMPs by human corneal epithelial cells (HCECs) was affected both by the stromal fibroblasts and the collagen-enriched ECM they produce. Most of all, results from mass spectrometry analyses provided evidence that a fully stratified epithelium is required for proper synthesis and organization of the ECM on which the epithelial cells adhere. In conclusion, and because of the many characteristics it shares with the native cornea, this human two layers corneal substitute may prove particularly useful to decipher the mechanistic details of corneal wound healing.
2015-11-25 | GSE75336 | GEO
Project description:The advent of cell culture-based methods for the establishment and expansion of human corneal endothelial cells (CEnC) has provided an available source of transplantable corneal endothelium, with a significant potential to challenge the one donor-one recipient paradigm. However, concerns over cell state identity remain, and a comprehensive characterization of the cultured CEnC has not been performed. To this end, we compared two established CEnC culture methods by assessing cell function and characterizing the transcriptomic changes that occur during in vitro expansion. In confluent monolayers, low mitogenic culture conditions preserved corneal endothelial cell state identity better than culture in high mitogenic conditions. Expansion by continuous passaging induced replicative cell senescence. Transcriptomic analysis of the senescent phenotype identified a cell senescence signature distinct for CEnC. We identified activation of both classic and new cell signaling pathways that may be targeted to prevent senescence, a significant barrier to realizing the potential clinical utility of in vitro expansion.
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Project description:Fuchs’ endothelial corneal dystrophy is major corneal disorder in the western world affecting the innermost part of the cornea, which leads to visual impairment. The morphological changes observed in Fuchs’ endothelial corneal dystrophy is well described, however, much less in known of the pathology at the molecular level. As the morphological changes observed in the cornea is profound in the extracellular matrix we sought to determine in protein profiles and changes herein in the Descement’s membrane and endothelium layer of Fuchs’ endothelial conrneal dystrophy patients when compared to healthy control tissue. Using the extracted ion chromatogram label-free MS based quantification method we quantified approximately the 50 most abundant proteins of the Descemet’s membrane and endothelial layer in in patient and control tissue. In addition, using the isobaric tag for relative and absolute quantification MS method resulted in a total of 22 regulated proteins of which the majority were extracellular proteins known to be involved in proper assembly and modulation of the basement membrane in other tissues. Many of the regulated proteins were furthermore among the most abundant proteins quantified. The two MS methods performed here suggest altered arrangement of the extracellular matrix in Fuchs’ endothelial corneal dystrophy and provide new candidate proteins that may be involved in molecular mechanism of this disease.
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Project description:Transplantation of amniotic membrane-expanded limbal epithelium (AMLE) in place of donor tissue grafts results in significantly improved outcomes for patients suffering from severe limbal stem cell deficiency; however the reasons for such superior results are unclear. The purpose of this study was to identify transcriptional gene profiles specific to AMLE and donor central corneal epithelium (CE), which may contribute to the divergent clinical outcomes observed following transplant. Limbal fibroblasts which underlie the epithelium and secrete extracellular matrix proteins following injury/surgery were also profiled. Using cell culture, immunofluorescence, microarray gene expression profiling and qRT-PCR validation; this study aims to identify enriched biological processes and pathways which characterise AMLE and CE tissues. We hope the study outcomes will shed light onto the factors which contribute to provide the improved clinical outcomes associated with AMLE transplantation. Gene expression profiling of three central corneal buttons, three amniotic membrane-expanded limbal epithelial cultures and three limbal fibroblast cultures
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