Summary

En vitro y en modelos in vivo para estudiar la transición del endotelio corneal-mesenquimal

Published: August 20, 2016
doi:

Summary

Un cultivo primario de células endoteliales corneales bovinas se utilizó para investigar el mecanismo de la transición endotelial-mesenquimal corneal. Además, se utilizó un modelo de endotelio corneal criolesión rata para demostrar transición endotelial-mesenquimal corneal in vivo.

Abstract

Corneal endothelial cells (CECs) play a crucial role in maintaining corneal clarity through active pumping. A reduced CEC count may lead to corneal edema and diminished visual acuity. However, human CECs are prone to compromised proliferative potential. Furthermore, stimulation of cell growth is often complicated by gradual endothelial-mesenchymal transition (EnMT). Therefore, understanding the mechanism of EnMT is necessary for facilitating the regeneration of CECs with competent function. In this study, we prepared a primary culture of bovine CECs by peeling the CECs with Descemet’s membrane from the corneal button and demonstrated that bovine CECs exhibited the EnMT process, including phenotypic change, nuclear translocation of β-catenin, and EMT regulators snail and slug, in the in vitro culture. Furthermore, we used a rat corneal endothelium cryoinjury model to demonstrate the EnMT process in vivo. Collectively, the in vitro primary culture of bovine CECs and in vivo rat corneal endothelium cryoinjury models offers useful platforms for investigating the mechanism of EnMT.

Introduction

Corneal endothelial cells (CECs) play a vital role in maintaining corneal clarity and thus visual acuity by regulating the hydration status of the corneal stroma through active pumping1. Because of the limited proliferative potential of human CECs, the cell number decreases with age, and the repair of corneal endothelial wounds following injury is usually achieved through cell enlargement and migration, rather than cell mitosis2. When the CEC count decreases below a threshold of approximately 500 cells/mm2, the dehydration status of the corneal stroma cannot be maintained, leading to bullous keratopathy and vision impairment3,4.

The limited proliferative potential of human CECs has been attributed to several factors, including reduced expression of the epidermal growth factor and its receptor in aging cells5, antiproliferative TGFβ2 in the aqueous humor6, and contact inhibition2,7. Although some growth factors, such as basic fibroblast growth factor (bFGF), can increase proliferation in a cultured human corneal endothelium, the culture efficiency remains limited8,9. Furthermore, CECs may undergo a phenotypic change during ex vivo expansion, resembling epithelial-mesenchymal transition (EMT)10-13. Endothelial-mesenchymal transition (EnMT) is characterized by cell junction destabilization, apical-basal polarity loss, cytoskeletal rearrangement, alpha smooth muscle actin expression, and type I collagen secretion14. All of these characteristics may abrogate the normal function of CECs, hampering the use of ex vivo cultured CECs in tissue engineering. Moreover, EnMT has been associated with the pathogenesis of several corneal endothelial diseases, including Fuchs endothelial corneal dystrophy and retrocorneal membrane formation15,16. Therefore, understanding the mechanism of EnMT may aid in manipulating the EnMT process and facilitate the regeneration of CECs to enable competent function.

In this study, we described a method for isolating bovine CECs from the corneal button. In the primary culture in vitro, the EnMT process, including a phenotypic change, the nuclear translocation of β-catenin, and EMT regulators snail and slug, was observed. We further described a method for demonstrating EnMT in vivo by using a rat corneal endothelium cryoinjury model. Using these 2 models, we demonstrated that marimastat, a broad-spectrum matrix metalloproteinase (MMP) inhibitor, can suppress the EnMT process. The described protocols facilitate the detailed analysis of the EnMT mechanism and the development of strategies for manipulating the EnMT process for further clinical application.

Protocol

Todos los procedimientos seguidos en este estudio estaba de acuerdo con la Asociación para la Investigación en Visión y Oftalmología para el Uso de Animales en Investigación Oftálmica y Visión y fueron aprobados por el Comité de Cuidado y Uso de Animales institucional del Hospital de la Universidad Nacional de Taiwán. 1. Aislamiento, cultivo primario de preparación, y la inmunotinción de bovino CEC Adquirir nuevos ojos bovinos de un matadero local. Desinfectar los ojos en una solu…

Representative Results

Después del aislamiento de CECs de la especie bovina, las células se cultivaron in vitro. La Figura 1 presenta las imágenes de contraste de fase de los países de Europa central de la especie bovina. La forma hexagonal de las células en confluencia indica que las células no estaban contaminadas por los fibroblastos del estroma corneal durante el aislamiento celular. Figura 2 representa la inmunotinción que se realizó utilizando anticuerpo…

Discussion

CECs son conocidos por su propensión a sufrir EnMT durante la proliferación celular. Para desarrollar estrategias para suprimir el proceso EnMT con fines terapéuticos, es necesario un conocimiento profundo del mecanismo EnMT. Describimos 2 modelos para investigar EnMT, a saber, la CCA bovina en el modelo de cultivo in vitro y la rata modelo de lesión criogénica endotelio corneal. Nuestros resultados demuestran el proceso EnMT en ambos modelos. Además, el efecto de supresión de EnMT de m…

Disclosures

The authors have nothing to disclose.

Acknowledgements

We thank the staff of the Second Core Lab, Department of Medical Research, National Taiwan University Hospital for their technical support.

Materials

trypsin ThermoFisher Scientific 12604-013
Dulbecco’s modified Eagle medium and Ham's F12 medium ThermoFisher Scientific 11330
fetal bovine serum ThermoFisher Scientific 26140-079
dimethyl sulfoxide Sigma D2650
human epidermal growth factor ThermoFisher Scientific PHG0311
insulin, transferrin, selenium  ThermoFisher Scientific 41400-045
cholera toxin Sigma C8052-1MG
gentamicin ThermoFisher Scientific 15750-060
amphotericin B ThermoFisher Scientific 15290-026
paraformaldehyde Electron Microscopy Sciences 111219
Triton X-100 Sigma T8787 
bovine serum albumin Sigma A7906
marimastat Sigma M2699-25MG
anti-active beta-catenin antibody Millpore 05-665
anti-snail antibody Santa cruz sc28199
anti-slug antibody Santa cruz sc15391
goat anti-mouse IgG (H+L) secondary antibody ThermoFisher Scientific A-11001 for staining of ABC of bovine CECs
goat anti-mouse IgG (H+L) secondary antibody ThermoFisher Scientific A-11003 for staining of ABC of rat corneal endothelium
goat anti-rabbit IgG (H+L) secondary antibody ThermoFisher Scientific A-11008 for staining of snail and slug of bovine CECs
antibody diluent Genemed Biotechnologies 10-0001
4',6-diamidino-2-phenylindole ThermoFisher Scientific D1306
mounting medium Vector Laboratories H-1000
laser scanning confocal microscope ZEISS LSM510
xylazine  Bayer N/A
tiletamine plus zolazepam Virbac N/A veterinary drug
proparacaine hydrochloride ophthalmic solution Alcon N/A veterinary drug
0.1% atropine Wu-Fu Laboratories Co., Ltd N/A clinical drug 
0.3% gentamicin sulfate Sinphar Group N/A clinical drug 
basic fibroblast growth factor ThermoFisher Scientific PHG0024 clinical drug 

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Cite This Article
Ho, W., Su, C., Chang, J., Chang, S., Hu, F., Jou, T., Wang, I. In Vitro and In Vivo Models to Study Corneal Endothelial-mesenchymal Transition. J. Vis. Exp. (114), e54329, doi:10.3791/54329 (2016).

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