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The tissue-engineered human cornea as a model to study expression of matrix metalloproteinases during corneal wound healing

ABSTRACT: 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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE75336 | GEO | 2015/11/25

SECONDARY ACCESSION(S): PRJNA304092

REPOSITORIES: GEO

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The tissue-engineered human cornea as a model to study expression of matrix metalloproteinases during corneal wound healing

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

Differentially Expressed Wound Healing-Related microRNAs in the Human Diabetic Cornea

Project description:MicroRNAs are powerful gene expression regulators, but their corneal repertoire and potential changes in corneal diseases remain unknown. Our purpose was to identify miRNAs altered in the human diabetic cornea by microarray analysis, and to examine their effects on wound healing in cultured telomerase-immortalized human corneal epithelial cells (HCEC) in vitro. Using microarrays, 29 miRNAs were identified as differentially expressed in diabetic samples. Two miRNA candidates showing the highest fold increased in expression in the diabetic cornea were confirmed by Q-PCR and further characterized. HCEC transfection with h-miR-146a or h-miR-424 significantly retarded wound closure, but their respective antagomirs significantly enhanced wound healing vs. controls. Cells treated with h-miR-146a or h-miR-424 had decreased p-p38 and p-EGFR staining, but these increased over control levels close to the wound edge upon antagomir treatment. In conclusion, several miRNAs with increased expression in human diabetic central corneas were found. Two such miRNAs inhibited cultured corneal epithelial cell wound healing. Dysregulation of miRNA expression in human diabetic cornea may be an important mediator of abnormal wound healing. Total RNA was extracted from age-matched human autopsy normal (n=6) and diabetic (n=6) central corneas, Flash Tag end-labeled, and hybridized to Affymetrix® GeneChip® miRNA Arrays. Select miRNAs associated with diabetic cornea were validated by quantitative RT-PCR (Q-PCR) and by in situ hybridization (ISH) in independent samples.

2013-11-09 | E-GEOD-52233 | biostudies-arrayexpress

Differentially Expressed Wound Healing-Related microRNAs in the Human Diabetic Cornea

2013-11-09 | GSE52233 | GEO

Analysis of gene expression in zebrafish cornea during wound healing

Project description:The cornea, transparent and outermost structure of camera-type eyes, is prone to environmental challenges, but has remarkable wound healing capabilities which enables to preserve vision. The manner in which cell plasticity impacts wound healing remains to be determined. In this study, we report rapid wound closure after zebrafish corneal epithelium abrasion. Furthermore, by investigating the cellular and molecular events taking place during corneal epithelial closure, we show the induction of a bilateral response to a unilateral wound. Our transcriptomic results, together with our TGF-beta receptor inhibition experiments, demonstrate conclusively the crucial role of TGF-beta signaling in corneal wound healing. Finally, our results on Pax6 expression and bilateral wound healing, demonstrate the decisive impact of epithelial cell plasticity on the pace of healing. Altogether, our study describes terminally differentiated cell competencies in the healing of an injured cornea. These findings will enhance the translation of research on cell plasticity to organ regeneration.

2022-01-20 | GSE193784 | GEO

Human Corneal microRNA Expression Profile in Fungal Keratitis

Project description:MicroRNAs (miRNAs) are small, stable non-coding RNA molecules with regulatory function and marked tissue specificity that post-transcriptionally regulate gene expression, however their role in fungal keratitis remain unknown. Our purpose was to identify the miRNAs in human cornea from fungal keratitis patients and understand their key role in regulation of pathogenesis. Corneal samples from normal cadaver (n=3) and fungal keratitis (n=5) patients were pooled separately and total RNA was extracted. Deep sequencing was done using Illumina HiSeq1000 platform to identify miRNA profile. We identified seventy five differentially expressed miRNAs in fungal keratitis corneas. Select miRNAs were validated by real-time RT-PCR (Q-PCR). We predicted their role in regulating target genes in several pathways by combining miRNA target genes and pathway analysis, and mRNA expression of select target genes were further analysed by Q-PCR. MiR-21-5p, miR-223-3p, miR-146b-5p, miR-155-5p, miR-511-5p were found to be involved in inflammatory and immune responses, regulating Toll like receptor signaling pathways, which is of particular interest. MiR-451a with an increased expression in keratitis may have a role in wound healing by targeting Macrophage Migration Inhibitory Factor (MIF). Further, we highlighted that Neurotrophin signaling pathway may play a role in wound healing process. One novel miRNA was also detected in cornea. In conclusion, several miRNAs with high expression in fungal keratitis corneas point towards their role in regulation of pathogenesis. Further insights in understanding miRNAs role in wound healing and inflammation may help design new therapeutic strategies. Control and fungal keratitis, human corneal miRNA profiles were generated using IlluminaHiseq1000 platform

2016-07-28 | E-GEOD-64843 | biostudies-arrayexpress

Enhanced wound healing of tissue-engineered human corneas through altered phosphorylation of the CREB and AKT signal transduction pathways

Project description:The cornea is a transparent organ, highly specialized and unique that is continually subjected to abrasive forces and occasional mechanical or chemical trauma because of its anatomical localization. Upon injury, the extracellular matrix (ECM) rapidly changes to promote wound healing through integrin-dependent activation of specific signal transduction mediators whose contribution is to favor faster closure of the wound by altering the adhesive and migratory properties of the cells surrounding the damaged area. In this study, we exploited the human tissue-engineered cornea (hTECs) as a model to study the signal transduction pathways that participate to corneal wound healing. By exploiting both gene profiling and activated kinases arrays, we could demonstrate the occurrence of important alterations in the level of expression and activation of a few mediators from the PI3K/Akt and CREB pathways in response to the ECM remodeling taking place during wound healing of damaged hTECs. Pharmacological inhibition of CREB with C646 considerably accelerated wound closure compared to controls. This process was considerably accelerated further when both C646 and SC79, an Akt agonist, were added together to wounded hTECs. Therefore, our study demonstrate that proper corneal wound healing requires the activation of Akt together with the inhibition of CREB and that wound healing in vitro can be altered by the use of pharmacological inhibitors (such as C646) or agonists (such as SC79) of these mediators.

2018-04-21 | GSE113438 | GEO

dsRNA induced IFNβ-MMP13 axis drives corneal wound healing

Project description:Cornea, the outermost transparent layer of the eye, is the first line of defense against external threats. Following injury, the wound healing response is crucial to corneal repair and regeneration, yet its underlying mechanism is poorly understood. Our study was designed to investigate the role of dsRNA and its regulatory network in corneal wound healing.

2022-02-05 | GSE191228 | GEO

Transcription profiling of mouse corneas from wildtype vs. corneas under the influence of high doses of TGF-beta

Project description:Characteristic structural details of the cornea are transparency, the absence of blood vessels, and the presence of numerous sensory nerve endings. The corneal epithelium is one of the most densely innervated tissues of the body. The characteristics of the cornea are established during fetal development, and are lost in adult life when the cornea regenerates after injury. The common reaction of the cornea to injury is the formation of opaque scars, the ingrowth of blood vessels, and distinct changes in the innervation pattern. Scar formation of the cornea is critically modulated by the expression of transforming growth factor-beta (TGF-beta). To identify genes that are important for corneal transparency, dense innervation and absence of blood vessels by comparing corneas from wildtype mice with those that are under the influence of high doses of TGF-beta. Transparency, dense innervation, and absence of blood vessels in the cornea all depend on the expression of a critical set of genes that are not expressed when TGF-beta is present. Mice were generated that overexpress TGF-beta under control of a strong lens-specific promoter. These mice developed opaque corneas that are vascularized and lack sensory nerves. In addition, these corneas were densely populated with cells expressing neural cell adhesion protein. RNA was isolated from corneas of transgenic animals and wildtype littermates in order to analyze differentially expressed genes and to identify those that are only expressed in transparent, avascular, and densely innervated wildtype corneas.

2008-06-15 | E-GEOD-6676 | biostudies-arrayexpress

RNA-Seq Analysis Unraveling Novel Genes and Pathways Influencing Corneal Wound Healing

Project description:Transparent avascular cornea providing two third refraction to the eye. Restoration of corneal transparency and clear vision in a traumatic eye involves the action of many cytokines and signaling pathways. Out of several factors, stromal keratocytes/fibroblasts (CSFs) play a central role in corneal repair and wound healing. Post trauma, keratocytes/fibroblasts produce myofibroblasts to facilitate wound repair by synthesizing and secreting large extracellular matrix components, collagens, and alpha-smooth muscle actin stress fibers. This study aimed to perform RNASeq data analysis and pathway enrichments to gain a better understanding of gene regulation in corneal fibroblasts and myofibroblasts in corneal wound repair.

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