ReLEx SMILE Method to Expansion and Create Scaffolds from Corneal Fibroblasts

Background: Exosomes secreted by corneal fibroblasts contain matrix metalloproteinase (MMP) 14, which is known to influence pro-MMP2 accumulation on exosomes. Accordingly, we hypothesized that the enzymatic activity of MMP14 may alter the protein content of corneal fibroblast- secreted exosomes. Objective: The aim of this study was to investigate the effects of MMP14 on the composition and biological activity of corneal fibroblast-derived exosomes. Methods: Knock out of the catalytic domain (ΔExon4) of MMP14 in corneal fibroblasts was used to determine the effect of MMP14 expression on the characteristics of fibroblast-secreted exosomes. The amount of secreted proteins and their size distribution were measured using Nano Tracking Analysis. Proteins within exosomes from wild-type (WT) and ΔExon4-deficient fibroblasts were identified by liquid chromatography-tandem mass spectrometry (MS/MS) proteomics analysis. The proteolytic effects of MMP14 were evaluated in vitro via MS identification of eliminated proteins. The biological functions of MMP14-carrying exosomes were investigated via fusion to endothelial cells and flow cytometric assays. Results: Exosomes isolated from WT and ΔExon4-deficient fibroblasts exhibited similar size distributions and morphologies, although WT fibroblasts secreted a greater amount of exosomes. The protein content, however, was higher in ΔExon4-deficient fibroblast-derived exosomes than in WT fibroblast-derived exosomes. Proteomics analysis revealed that WT-derived exosomes included proteins that regulated cell migration, and ΔExon4 fibroblast-derived exosomes contained additional proteins that were cleaved by MMP14. Conclusion: Our findings suggest that MMP14 expression influences the protein composition of exosomes secreted by corneal fibroblasts, and through those biological components, MMP14 in corneal fibroblasts derived-exosomes may regulate corneal angiogenesis.

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Inhibition of Interleukin-1β-Induced Matrix Metalloproteinase Expression in Human Corneal Fibroblasts by Tranilast

Current Eye Research ◽

10.3109/02713683.2014.884598 ◽

2014 ◽

Vol 39 (9) ◽

pp. 885-893 ◽

Cited By ~ 4

Author(s):

Ye Liu ◽

Dan Xu ◽

Jing Li ◽

Yang Liu

Keyword(s):

Matrix Metalloproteinase ◽

Interleukin 1Β ◽

Corneal Fibroblasts

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Chloroquine Protects Human Corneal Epithelial Cells from Desiccation Stress Induced Inflammation without Altering the Autophagy Flux

BioMed Research International ◽

10.1155/2018/7627329 ◽

2018 ◽

Vol 2018 ◽

pp. 1-13 ◽

Cited By ~ 8

Author(s):

Shivapriya Shivakumar ◽

Trailokyanath Panigrahi ◽

Rohit Shetty ◽

Murali Subramani ◽

Arkasubhra Ghosh ◽

...

Keyword(s):

T Cells ◽

Dry Eye ◽

Desiccation Stress ◽

Autophagy Flux ◽

Corneal Epithelial ◽

Corneal Fibroblasts ◽

Gene And Protein Expression ◽

Human Corneal Epithelial Cells ◽

Anti Inflammatory

Dry eye disease (DED) is a multifactorial ocular surface disorder affecting millions of individuals worldwide. Inflammation has been associated with dry eye and anti-inflammatory drugs are now being targeted as the alternate therapeutic approach for dry eye condition. In this study, we have explored the anti-inflammatory and autophagy modulating effect of chloroquine (CQ) in human corneal epithelial and human corneal fibroblasts cells exposed to desiccation stress, (anin-vitromodel for DED). Gene and protein expression profiling of inflammatory and autophagy related molecular factors were analyzed in HCE-T and primary HCF cells exposed to desiccation stress with and without CQ treatment. HCE-T and HCF cells exposed to desiccation stress exhibited increased levels of activated p65, TNF-α, MCP-1, MMP-9, and IL-6. Further, treatment with CQ decreased the levels of active p65, TNF-α, MCP-1, and MMP-9 in cells underdesiccation stress. Increased levels of LC3B and LAMP1 markers in HCE-T cells exposed to desiccation stress suggest activation of autophagy and the addition of CQ did not alter these levels. Changes in the phosphorylation levels of MAPKinase and mTOR pathway proteins were found in HCE-T cells under desiccation stress with or without CQ treatment. Taken together, the data suggests that HCE-T cells under desiccation stress showed NFκB mediated inflammation, which was rescued through the anti-inflammatory effect of CQ without altering the autophagy flux. Therefore, CQ may be used as an alternate therapeutic management for dry eye condition.

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Effect of cyclic AMP on collagen production by corneal fibroblasts

Current Eye Research ◽

10.3109/02713688609020040 ◽

1986 ◽

Vol 5 (3) ◽

pp. 177-182 ◽

Cited By ~ 4

Author(s):

Wan-Tsih H. Chao ◽

Ronald J. Walkenbach

Keyword(s):

Cyclic Amp ◽

Collagen Production ◽

Corneal Fibroblasts

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PDGFRα Is a Key Regulator of T1 and T3's Differential Effect on SMA Expression in Human Corneal Fibroblasts

Investigative Opthalmology & Visual Science ◽

10.1167/iovs.16-20016 ◽

2017 ◽

Vol 58 (2) ◽

pp. 1179 ◽

Cited By ~ 3

Author(s):

Sriniwas Sriram ◽

Jennifer A. Tran ◽

Xiaoqing Guo ◽

Audrey E. K. Hutcheon ◽

Hetian Lei ◽

...

Keyword(s):

Differential Effect ◽

Corneal Fibroblasts

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FAK Inhibition Attenuates Corneal Fibroblast Differentiation In Vitro

Biomolecules ◽

10.3390/biom11111682 ◽

2021 ◽

Vol 11 (11) ◽

pp. 1682

Author(s):

Vincent Yeung ◽

Sriniwas Sriram ◽

Jennifer A. Tran ◽

Xiaoqing Guo ◽

Audrey E. K. Hutcheon ◽

...

Keyword(s):

Wound Healing ◽

Transforming Growth Factor ◽

Smooth Muscle Actin ◽

Myofibroblast Differentiation ◽

Corneal Scarring ◽

Corneal Fibroblast ◽

Corneal Fibroblasts ◽

Corneal Wound ◽

Tgf Β1

Corneal fibrosis (or scarring) occurs in response to ocular trauma or infection, and by reducing corneal transparency, it can lead to visual impairment and blindness. Studies highlight important roles for transforming growth factor (TGF)-β1 and -β3 as modulators in corneal wound healing and fibrosis, leading to increased extracellular matrix (ECM) components and expression of α-smooth muscle actin (αSMA), a myofibroblast marker. In this study, human corneal fibroblasts (hCF) were cultured as a monolayer culture (2D) or on poly-transwell membranes to generate corneal stromal constructs (3D) that were treated with TGF-β1, TGF-β3, or TGF-β1 + FAK inhibitor (FAKi). Results show that hCF 3D constructs treated with TGF-β1 or TGF-β3 impart distinct effects on genes involved in wound healing and fibrosis—ITGAV, ITGB1, SRC and ACTA2. Notably, in the 3D construct model, TGF-β1 enhanced αSMA and focal adhesion kinase (FAK) protein expression, whereas TGF-β3 did not. In addition, in both the hCF 2D cell and 3D construct models, we found that TGF-β1 + FAKi attenuated TGF-β1-mediated myofibroblast differentiation, as shown by abrogated αSMA expression. This study concludes that FAK signaling is important for the onset of TGF-β1-mediated myofibroblast differentiation, and FAK inhibition may provide a novel beneficial therapeutic avenue to reduce corneal scarring.

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Microbial Warfare on Three Fronts: Mixed Biofilm of Aspergillus fumigatus and Staphylococcus aureus on Primary Cultures of Human Limbo-Corneal Fibroblasts

Frontiers in Cellular and Infection Microbiology ◽

10.3389/fcimb.2021.646054 ◽

2021 ◽

Vol 11 ◽

Author(s):

Adrián Ramírez-Granillo ◽

Luis Antonio Bautista-Hernández ◽

Víctor Manuel Bautista-De Lucío ◽

Fátima Sofía Magaña-Guerrero ◽

Alfredo Domínguez-López ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Aspergillus Fumigatus ◽

Biofilm Formation ◽

Primary Cultures ◽

Morphological Characterization ◽

Fibroblast Culture ◽

Bacterial Biofilm ◽

Corneal Fibroblast ◽

Corneal Fibroblasts ◽

Fibroblast Monolayer

BackgroundCoinfections with fungi and bacteria in ocular pathologies are increasing at an alarming rate. Two of the main etiologic agents of infections on the corneal surface, such as Aspergillus fumigatus and Staphylococcus aureus, can form a biofilm. However, mixed fungal–bacterial biofilms are rarely reported in ocular infections. The implementation of cell cultures as a study model related to biofilm microbial keratitis will allow understanding the pathogenesis in the cornea. The cornea maintains a pathogen-free ocular surface in which human limbo-corneal fibroblast cells are part of its cell regeneration process. There are no reports of biofilm formation assays on limbo-corneal fibroblasts, as well as their behavior with a polymicrobial infection.ObjectiveTo determine the capacity of biofilm formation during this fungal–bacterial interaction on primary limbo-corneal fibroblast monolayers.ResultsThe biofilm on the limbo-corneal fibroblast culture was analyzed by assessing biomass production and determining metabolic activity. Furthermore, the mixed biofilm effect on this cell culture was observed with several microscopy techniques. The single and mixed biofilm was higher on the limbo-corneal fibroblast monolayer than on abiotic surfaces. The A. fumigatus biofilm on the human limbo-corneal fibroblast culture showed a considerable decrease compared to the S. aureus biofilm on the limbo-corneal fibroblast monolayer. Moreover, the mixed biofilm had a lower density than that of the single biofilm. Antibiosis between A. fumigatus and S. aureus persisted during the challenge to limbo-corneal fibroblasts, but it seems that the fungus was more effectively inhibited.ConclusionThis is the first report of mixed fungal–bacterial biofilm production and morphological characterization on the limbo-corneal fibroblast monolayer. Three antibiosis behaviors were observed between fungi, bacteria, and limbo-corneal fibroblasts. The mycophagy effect over A. fumigatus by S. aureus was exacerbated on the limbo-corneal fibroblast monolayer. During fungal–bacterial interactions, it appears that limbo-corneal fibroblasts showed some phagocytic activity, demonstrating tripartite relationships during coinfection.

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