In Vitro and In Vivo Models to Study Corneal Endothelial-mesenchymal Transition

Summary

A primary culture of bovine corneal endothelial cells was used to investigate the mechanism of corneal endothelial-mesenchymal transition. Furthermore, a rat corneal endothelium cryoinjury model was used to demonstrate corneal endothelial-mesenchymal transition 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 impairment^3,4.

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 inhibition^2,7. Although some growth factors, such as basic fibroblast growth factor (bFGF), can increase proliferation in a cultured human corneal endothelium, the culture efficiency remains limited^8,9. 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 formation^15,16. 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

All the procedures followed in this study accorded with the Association for Research in Vision and Ophthalmology Statement for Use of Animals in Ophthalmic and Vision Research and were approved by the Institutional Animal Care and Use Committee of National Taiwan University Hospital.

1. Isolation, Primary Culture Preparation, and Immunostaining of Bovine CECs

1. Acquire fresh bovine eyes from a local abattoir.
2. Disinfect the eyes in a 10% w/v povidone-iodine solution for 3 min. Wash them with phosphate-buffered saline (PBS) solution.
3. Harvest the corneal button with a scalpel and scissors under sterile conditions. Peel the Descemet's membrane with forceps under a dissecting microscope (note that in this study, the bovine eyes were enucleated by a local abattoir; therefore, the first study procedure was the disinfection of the preenucleated eyes in the laboratory).
4. Incubate the Descemet's membrane in 1 ml of trypsin at 37 °C for 30 min. Collect the bovine CECs by centrifugation at 112 x g for 5 min. Resuspend the cells in 1 ml of supplemented hormonal epithelial medium (SHEM) containing equal volumes of HEPES-buffered Dulbecco's modified Eagle medium and Ham's F12 medium, supplemented with 5% fetal bovine serum, 0.5% dimethyl sulfoxide, 2 ng/ml of human epidermal growth factor, 5 mg/ml of insulin, 5 mg/ml of transferrin, 5 ng/ml of selenium, 1 nmol/l of cholera toxin, 50 mg/ml of gentamicin, and 1.25 mg/ml of amphotericin B.
5. Seed the cells (approximately 1 x 10⁵ cells per eye) into a 6 cm dish. Culture them in the SHEM. Incubate the dish at 37 °C in an atmosphere of 5% CO₂ in air. Change the culture medium every 3 days.
6. When the cells reach confluence, wash them in PBS and incubate them in 1 ml of trypsin at 37 °C for 5 min. Collect them by centrifugation at 112 x g for 5 min. Re-suspend the cell pellet in 1 ml of the SHEM.
   1. Count the cells in a hemocytometer. Seed the cells on cover slides at a density of 1 x 10⁴ per well in a 24-well plate, and culture the cells in the SHEM. For investigating the EnMT-suppressing effect of marimastat, incubate the cells in SHEM with 10 µM of marimastat added into the culture medium, and change the culture medium every 3 days.
7. Fix the cells at an indicated time point with 250 µl of 4% paraformaldehyde, pH 7.4, for 30 min at room temperature. Permeabilize with 250 µl of 0.5% Triton X-100 for 5 min. Block with 10% bovine serum albumin for 30 min.
8. Incubate the cells with primary antibodies against active beta-catenin (ABC), snail, and slug overnight at 4 °C. The dilution of the primary antibodies used is 1:200. The antibodies are diluted in antibody diluent composed of 10 mM PBS, 1% w/v bovine serum albumin, and 0.09% w/v sodium azide.
9. Wash the cells twice with PBS for 15 min, and incubate them with the Alexa Fluor-conjugated secondary antibody (1:100 in antibody diluent) at room temperature for 1 hr.
10. Counterstain the cell nucleus by covering the cells with 2 µg/ml of 4',6-diamidino-2-phenylindole, wash the cells twice with PBS for 15 min, and mount them with anti-fading mounting solution.
11. Obtain immunofluorescent images at the excitation wavelengths of 405 and 488 nm by using a laser scanning confocal microscope with 20X objective magnification.

2. Rat Corneal Endothelium Cryoinjury Model and Intracameral Injection

1. Anesthetize 12 week-old male Sprague-Dawley rats with intramuscular injections of 2% xylazine (5.6 mg/kg) and tiletamine plus zolazepam (18 mg/kg). Gently pinch the skin of the animals to confirm proper anesthesia in the absence of skin twitching.
2. Instill one drop of 0.5% proparacaine hydrochloride to the right eye of each rat to minimize eye pain and the blink reflex. Instill tetracycline ointment to the left eye to prevent corneal dryness.
3. Cool a stainless steel probe (diameter = 3 mm) in liquid nitrogen. Apply the stainless steel probe to the central cornea of the right eye for 30 sec. Frequently instill PBS to the right eye during the procedure to prevent corneal dryness.
4. Instill 0.1% atropine and 0.3% gentamicin sulfate immediately after cryoinjury and once daily to relieve ocular pain resulting from ciliary spasm and to prevent infection.
   1. After the procedure, keep the rats warm using a heat lamp, and observe their recovery every 15 min after anesthesia until they regain motor control. Additionally, apply tetracycline ointment to the right eye to prevent corneal dryness during the recovery period.
5. Repeat the corneal cryoinjury for 3 consecutive days.
6. For delivering marimastat or bFGF into the anterior chamber of the rat eyes, anesthetize the rats as described previously. Instill one drop of 0.5% proparacaine hydrochloride into the right eye to minimize eye pain and the blink reflex.
7. Irrigate the ocular surface with sterile PBS. Perform anterior chamber paracentesis under an operating microscope by inserting a 30 G needle attached to a 1 ml syringe at the paralimbal clear cornea in a plane above and parallel to the iris.
8. Turn the needle bevel up, and slightly depress the corneal wound to drain some aqueous humor and reduce the intraocular pressure. Inject 0.02 ml of the drug intracamerally. Gently compress the needle tract with a cotton tip during the needle withdrawal.
9. Photograph the external eye at an indicated time point under the operating microscope.

3. Harvesting the Rat Corneal Button and Immunostaining

1. To euthanize the rats, place them in a euthanasia chamber and infuse 100% CO₂ at a fill rate of 10-30% of the chamber volume per minute. Maintain CO₂ infusion for an additional minute after lack of respiration and faded eye color.
2. Penetrate the rat eye at the limbus with a sharp blade. Cut the corneas with corneal scissors along the limbus. Flatten the corneas on a slide. Make additional radial incisions if the corneas curl up.
3. Fix the corneas with 250 µl of 4% paraformaldehyde, pH 7.4, for 30 min at room temperature. Permeabilize with 250 µl of 0.5% Triton X-100 for 5 min, and block with 10% bovine serum albumin for 30 min.
4. Incubate the corneas with primary antibodies against ABC overnight at 4 °C with a dilution of 1:200 in antibody diluent. Wash the corneas twice with PBS for 15 min, and incubate them with the secondary antibody (1:100 in antibody diluent) at room temperature for 1 hr. Counterstain the cell nucleus by covering the cells with 2 µg/ml of 4',6-diamidino-2-phenylindole, and wash the cells twice with PBS for 15 min.
5. Mount the corneas in anti-fading mounting solution. Obtain the immunofluorescent images at excitation wavelengths of 405 and 561 nm with a laser scanning confocal microscope at 20X objective magnification.

Results

After the isolation of bovine CECs, the cells were cultured in vitro. Figure 1 presents the phase contrast images of the bovine CECs. The hexagonal shape of the cells at confluence indicated that the cells were not contaminated by corneal stromal fibroblast during cell isolation. Figure 2 depicts the immunostaining that was performed using antibodies against ABC, snail, and slug at an indicated time point. Apart from phenotypic changes in the

Discussion

CECs are known for their propensity to undergo EnMT during cell proliferation. To develop strategies for suppressing the EnMT process for therapeutic purposes, a thorough understanding of the EnMT mechanism is necessary. We described 2 models to investigate EnMT, namely the bovine CEC in vitro culture model and rat corneal endothelium cryoinjury model. Our results demonstrated the EnMT process in both models. Furthermore, the EnMT-suppressing effect of marimastat was reproduced in both models, suggesting that th...

Materials

Name	Company	Catalog Number	Comments
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