46 research outputs found
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Cornea As a Model for Testing CTGF-Based Antiscarring Drugs
Scarring remains a serious complication of the wound healing process that can lead to the formation of excessive fibrous connective tissue in an organ or tissue leading to pain and loss of function. This process is mainly regulated by Transforming growth factor β1 (TGF-β1), which binds to receptors and induces its downstream mediator, Connective tissue growth factor (CTGF). The number of drugs targeting CTGF for treating scars has been on the rise in the past few years. The purpose of this article is to suggest the possibility of using cornea as a model for testing anti-CTGF therapies for scarring
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A Role for Topographic Cues in the Organization of Collagenous Matrix by Corneal Fibroblasts and Stem Cells
Human corneal fibroblasts (HCF) and corneal stromal stem cells (CSSC) each secrete and organize a thick stroma-like extracellular matrix in response to different substrata, but neither cell type organizes matrix on tissue-culture polystyrene. This study compared cell differentiation and extracellular matrix secreted by these two cell types when they were cultured on identical substrata, polycarbonate Transwell filters. After 4 weeks in culture, both cell types upregulated expression of genes marking differentiated keratocytes (KERA, CHST6, AQP1, B3GNT7). Absolute expression levels of these genes and secretion of keratan sulfate proteoglycans were significantly greater in CSSC than HCF. Both cultures produced extensive extracellular matrix of aligned collagen fibrils types I and V, exhibiting cornea-like lamellar structure. Unlike HCF, CSSC produced little matrix in the presence of serum. Construct thickness and collagen organization was enhanced by TGF-ß3. Scanning electron microscopic examination of the polycarbonate membrane revealed shallow parallel grooves with spacing of 200–300 nm, similar to the topography of aligned nanofiber substratum which we previously showed to induce matrix organization by CSSC. These results demonstrate that both corneal fibroblasts and stromal stem cells respond to a specific pattern of topographical cues by secreting highly organized extracellular matrix typical of corneal stroma. The data also suggest that the potential for matrix secretion and organization may not be directly related to the expression of molecular markers used to identify differentiated keratocytes
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Self-Assembled Matrix by Umbilical Cord Stem Cells
Corneal integrity is critical for vision. Corneal wounds frequently heal with scarring that impairs vision. Recently, human umbilical cord mesenchymal stem cells (cord stem cells) have been investigated for tissue engineering and therapy due to their availability and differentiation potential. In this study, we used cord stem cells in a 3-dimensional (3D) stroma-like model to observe extracellular matrix organization, with human corneal fibroblasts acting as a control. For 4 weeks, the cells were stimulated with a stable Vitamin C (VitC) derivative ±TGF-β1. After 4 weeks, the mean thickness of the constructs was ∼30 μm; however, cord stem cell constructs had 50% less cells per unit volume, indicating the formation of a dense matrix. We found minimal change in decorin and lumican mRNA, and a significant increase in perlecan mRNA in the presence of TGF-β1. Keratocan on the other hand decreased with TGF-β1 in both cell lineages. With both cell types, the constructs possessed aligned collagen fibrils and associated glycosaminoglycans. Fibril diameters did not change with TGF-β1 stimulation or cell lineage; however, highly sulfated glycosaminoglycans associated with the collagen fibrils significantly increased with TGF-β1. Overall, we have shown that cord stem cells can secrete their own extracellular matrix and promote the deposition and sulfation of various proteoglycans. Furthermore, these cells are at least comparable to commonly used corneal fibroblasts and present an alternative for the 3D in vitro tissue engineered model
Development of Conjunctival Goblet Cells and Their Neuroreceptor Subtype Expression
PURPOSE. To investigate expression of muscarinic, cholinergic, and adrenergic receptors on developing conjunctival goblet cells. METHODS. Eyes were removed from rats 9 to 60 days old, fixed, and used for microscopy. For glycoconjugate expression, sections were stained with Alcian blue/periodic acid-Schiff's reagent (AB/PAS) and with the lectins Ulex europeus agglutinin I (UEA-I) and Helix pomatia agglutinin (HPA). Goblet cell bodies were identified using anti-cytokeratin 7 (CK7). Nerve fibers were localized using anti-protein gene product 9.5. Location of muscarinic and adrenergic receptors was investigated using anti-muscarinic and -adrenergic receptors. RESULTS. At days 9 and 13, single apical cells in conjunctival epithelium stained with AB/PAS, UEA-I, and CK7. At days 17 and 60, increasing numbers of goblet cells were identified by AB/PAS, UEA-I, HPA, and CK7. Nerve fibers were localized around stratified squamous cells and at the epithelial base at days 9 and 13, and around goblet cells and at the epithelial base at days 17 and 60. At days 9 and 13, M 2 -and M 3 -muscarinic and  2 -adrenergic receptors were found in stratified squamous cells, but M 1 -muscarinic and  1 -adrenergic receptors were not detected. At days 17 and 60, M 2 -and M 3 -muscarinic receptors were found in goblet cells, whereas M 1 -muscarinic receptors were in stratified squamous cells.  1 -and  2 -Adrenergic receptors were found on both cell types.  3 -Adrenergic receptors were not detected. CONCLUSIONS. In conjunctiva, nerves, M 2 -and M 3 -muscarinic, and  1 -and  2 -adrenergic receptors are present on developing goblet cells and could regulate secretion as eyelids open. (Invest Ophthalmol Vis Sci. 2000;41:2127-2137 T he tear film mucus layer consists of high molecular weight glycoconjugates including mucins, which are secreted mainly by conjunctival goblet cells. This layer plays an important role in protecting the ocular surface from exogenous agents (bacterial or chemical) and provides lubrication during all types of eye movements. 1 Goblet cells can release their secretory granules in a reflex response mediated by the activation of either parasympathetic or sympathetic nerves that surround them. 2,3 Previous reports from this laboratory showed the localization of nerve fibers adjacent to goblet cells in rat conjunctiva. 5 Use of immunofluorescence techniques demonstrated that M 2 -and M 3 -, but not M 1 -muscarinic acetylcholine receptors (MAchRs), are present on goblet cells and are located on membranes subjacent to secretory granules. VIP type 2 receptors (VIPR2s) are located in the basolateral membranes of goblet cells. 3 Although the role of the sympathetic agonists in stimulating goblet cell secretion is unknown,  1 -and  2 -adrenergic receptor (AR) subtypes appear to be present in goblet cells as well as in stratified squamous cells. Morphologic studies in developing conjunctiva suggest that based on changes in the acidity of glycoproteins in the secretory granules, goblet cells may differentiate from basal epithelial cells in the forniceal zone. 7 Watanabe et al
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PDGFRα Is a Key Regulator of T1 and T3's Differential Effect on SMA Expression in Human Corneal Fibroblasts
Purpose The goal of this study was to examine the mechanism behind the unique differential action of transforming growth factor β3 (TGF-β3) and TGF-β1 on SMA expression. It was our hypothesis that platelet-derived growth factor receptor α (PDGFRα) played a key role in determining TGF-β3's response to wounding. Methods: A stable cell line, human corneal fibroblast (HCF)-P, was created from HCFs by knocking down PDGFRα expression using a lentivirus-delivered shRNA sequence. A three-dimensional (3D) in vitro model was constructed by culturing HCF or HCF-P on poly-transwell membranes for 4 weeks in the presence and absence of 0.1 ng/mL TGF-β1 or -β3. At the end of 4 weeks, the constructs were processed for immunofluorescence and reverse transcription–quantitative polymerase chain reaction (RT-qPCR). In addition, HCF and HCF-P cell migration was evaluated. Results: In HCF, TGF-β3 treatment resulted in significantly lower α-smooth muscle actin (SMA) mRNA expression and immunolocalization when compared to TGF-β1, while in HCF-P, both TGF-β1 and -β3 treatment increased the SMA mRNA expression and immunolocalization compared to both the untreated HCF-P control and TGF-β3-treated HCF. Human corneal fibroblast-P also had a lower migration rate and construct thickness when compared to HCF. Conclusions: These results show that TGF-β3 decreases SMA in HCF, while remarkably increasing SMA in HCF-P, thus indicating that the presence or absence of PDGFRα elicits contrasting responses to the same TGF-β3 treatment. Understanding the role of PDGFRα in TGF-β3's ability to stimulate SMA may potentially help in understanding the differential functions of TGF-β1 and TGF-β3 in corneal wound healing
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Spontaneous Bacterial Keratitis in CD36 Knockout Mice
Purpose: CD36 is a Class B scavenger receptor that is constitutively expressed in the corneal epithelium and has been implicated in many homeostatic functions, including the homeostasis of the epidermal barrier. The aim of this study is to determine (1) whether CD36 is required for the maintenance of the corneal epithelial barrier to infection, and (2) whether CD36-deficient mice present with an increased susceptibility to bacterial keratitis. Methods: The corneas of CD36−/−, TSP1−/−, TLR2−/−, and C57BL/6 WT mice were screened via slit lamp microscopy or ex vivo analysis. The epithelial tight junctions and mucin layer were assessed via LC-biotin and Rose Bengal staining, respectively. Bacterial quantification was performed on corneal buttons and GFP-expressing Staphylococcus aureus was used to study bacterial binding. Results: CD36−/− mice develop spontaneous corneal defects that increased in frequency and severity with age. The mild corneal defects were characterized by a disruption in epithelial tight junctions and the mucin layer, an infiltrate of macrophages, and increased bacterial binding. Bacterial quantification revealed high levels of Staphylococcus xylosus in the corneas of CD36−/− mice with severe defects, but not in wild-type controls. Conclusions: CD36−/− mice develop spontaneous bacterial keratitis independent of TLR2 and TSP1. The authors conclude that CD36 is a critical component of the corneal epithelial barrier, and in the absence of CD36 the barrier breaks down, allowing bacteria to bind to the corneal epithelium and resulting in spontaneous keratitis. This is the first report of spontaneous bacterial keratitis in mice
TAT-Mediated Protein Transduction into Human Corneal Epithelial Cells: p15 INK4b Inhibits Cell Proliferation and Stimulates Cell Migration
PURPOSE. The cell cycle inhibitor p15 INK4b has been localized in migrating corneal epithelial cells. In this study, TAT-fusion protein technology was used to transduce p15 INK4b into human corneal epithelial cells to examine the effect on cell proliferation and migration. METHODS. Human p15 INK4b , obtained by RT-PCR, was cloned into a TAT-HA vector, and the fusion protein was purified from bacteria transformed with the TAT-HA-p15 construct. Various dilutions of TAT-HA-p15 were applied to primary human corneal epithelial cells to test potency. In addition, the effect of exposure time was examined. Cells were labeled with bromodeoxyuridine to detect proliferation, and indirect immunofluorescence was performed. Ki67 expression was also examined. To assay cell migration, human corneal epithelial cells were plated inside a cylinder and exposed to TAT-HA-p15. The cylinder was removed, the cells were allowed to spread for 2 days, and the area of cell coverage was calculated. TAT-HA--galactosidase served as the control in all experiments. Finally, the extent of retinoblastoma protein phosphorylation was assayed by Western blot in cells cultured with and without TAT-HA-p15. RESULTS. TAT-HA-p15 was successfully transduced into primary human corneal epithelial cells. TAT-HA-p15 decreased proliferation in a concentration-and time-dependent manner. The migration assay showed that TAT-HA-p15 stimulated cell migration 1.8-fold. TAT-HA--galactosidase had no effect on proliferation or migration. Finally, TAT-HA-p15 decreased the level of phosphorylated retinoblastoma protein by 4.9-fold. CONCLUSIONS. Active p15 INK4b can be efficiently transduced into primary human corneal epithelial cells using TAT-fusion protein technology. p15 INK4b appears to be sufficient to inhibit corneal epithelial cell proliferation and to stimulate cell migration. (Invest Ophthalmol Vis Sci