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Latrunculin B and substratum stiffness regulate corneal fibroblast to myofibroblast transformation.
Thomasy, Sara M; Raghunathan, Vijay Krishna; Miyagi, Hidetaka; Evashenk, Alexander T; Sermeno, Jasmyne C; Tripp, Geneva K; Morgan, Joshua T; Murphy, Christopher J.
Afiliación
  • Thomasy SM; Department of Surgical and Radiological Sciences, School of Veterinary Medicine, University of California, Davis, Davis, CA, USA; Department of Ophthalmology & Vision Science, School of Medicine, University of California, Davis, Davis, CA, USA. Electronic address: smthomasy@ucdavis.edu.
  • Raghunathan VK; Department of Surgical and Radiological Sciences, School of Veterinary Medicine, University of California, Davis, Davis, CA, USA; Department of Basic Sciences, The Ocular Surface Institute, College of Optometry, University of Houston, Houston, TX, USA.
  • Miyagi H; Department of Surgical and Radiological Sciences, School of Veterinary Medicine, University of California, Davis, Davis, CA, USA; Department of Ophthalmology and Visual Sciences, Graduate School of Biomedical Sciences, Hiroshima University, Minami-ku, Kasumi 1-2-3, Hiroshima, 7348551, Japan.
  • Evashenk AT; Department of Surgical and Radiological Sciences, School of Veterinary Medicine, University of California, Davis, Davis, CA, USA.
  • Sermeno JC; Department of Surgical and Radiological Sciences, School of Veterinary Medicine, University of California, Davis, Davis, CA, USA.
  • Tripp GK; Department of Surgical and Radiological Sciences, School of Veterinary Medicine, University of California, Davis, Davis, CA, USA.
  • Morgan JT; Department of Surgical and Radiological Sciences, School of Veterinary Medicine, University of California, Davis, Davis, CA, USA.
  • Murphy CJ; Department of Surgical and Radiological Sciences, School of Veterinary Medicine, University of California, Davis, Davis, CA, USA; Department of Ophthalmology & Vision Science, School of Medicine, University of California, Davis, Davis, CA, USA.
Exp Eye Res ; 170: 101-107, 2018 05.
Article en En | MEDLINE | ID: mdl-29421383
The transformation of keratocytes and fibroblasts to myofibroblasts is important to corneal wound healing as well as formation of stromal haze. The purpose of this study was to determine the effect of latrunculin B, an actin cytoskeleton disruptor in conjunction with a fundamental biophysical cue, substrate stiffness, on myofibroblast transformation in vitro and in vivo. Rabbit corneal fibroblasts were cultured on substrates of differing compliance (1.5, 22, and 71 kPa) and tissue culture plastic (TCP; > 1 GPa) in media containing 0 or 10 ng/ml TGFß1 for 72 h. Cells were treated with 0.4 µM Lat-B or DMSO for 30 min every 24 h for 72 h. RNA was collected from cells and expression of alpha-smooth muscle actin (α-SMA), keratocan, and ALDH1A1 determined using qPCR; immunocytochemistry was used to assess α-SMA protein expression. A rabbit phototherapeutic keratectomy (PTK) model was used to assess the impact of 0.1% Lat-B (n = 3) or 25% DMSO (vehicle control, n = 3) on corneal wound healing by assessment of epithelial wound size with fluorescein stain and semi-quantitative stromal haze scoring by an observer masked to treatment group as well as Fourier-domain optical coherence tomography (FD-OCT) at set time points. Statistical analysis was completed using one-way or two-way analysis of variance. Treatment with Lat-B versus DMSO resulted in significantly less αSMA mRNA (P ≤ 0.007) for RCF cells grown on 22 and 71 kPa substrates as well as TCP without or with TGFß1, and significantly decreased α-SMA protein expression in RCFs cultured on the intermediate (22 kPa) stiffness in the absence (P = 0.028) or presence (P = 0.018) of TGFß1. Treatment with Lat-B versus DMSO but did not significantly alter expression of keratocan or ALDH1A1 mRNA in RCFs (P > 0.05) in the absence or presence of TGFß1, but RCFs grown on stiff hydrogels (71 kPa) had significantly more keratocan mRNA expression versus the 22 kPa hydrogel or TCP (P < 0.001) without TGFß1. Administration of topical Lat-B BID was well tolerated by rabbits post-PTK but did not significantly alter epithelial wound closure, stromal haze score, stromal haze thickness as measured by FD-OCT in comparison to DMSO-treated rabbits. When corneal stromal cells are cultured on substrates possessing biologically relevant substratum stiffnesses, Lat-B modulates mRNA and protein expression of α-SMA and thus modulates myofibroblast transformation. At a dose and dose-frequency that reduced IOP in human glaucoma patients, Lat-B treatment did not substantially impact corneal epithelial or stromal wound healing in a rabbit PTK model. While a significant impact on wound healing was observed at the concentration and dose frequency reported here was not found, encouraging in vitro data support further investigations of topically applied Lat-B to determine if this compound can reduce stromal fibrosis.
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Compuestos Bicíclicos Heterocíclicos con Puentes / Elasticidad / Tiazolidinas / Transdiferenciación Celular / Miofibroblastos / Queratocitos de la Córnea Tipo de estudio: Prognostic_studies Límite: Animals Idioma: En Revista: Exp Eye Res Año: 2018 Tipo del documento: Article Pais de publicación: Reino Unido

Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Compuestos Bicíclicos Heterocíclicos con Puentes / Elasticidad / Tiazolidinas / Transdiferenciación Celular / Miofibroblastos / Queratocitos de la Córnea Tipo de estudio: Prognostic_studies Límite: Animals Idioma: En Revista: Exp Eye Res Año: 2018 Tipo del documento: Article Pais de publicación: Reino Unido