J Korean Ophthalmol Soc.  2007 Oct;48(10):1399-1409. 10.3341/jkos.2007.48.10.1399.

Effect of Cyclosporine A 0.05% on Human Corneal Epithelial Cells

• Lee JE
• Shin WB
• Lee JS

• KMID: 2210995
• DOI: http://doi.org/10.3341/jkos.2007.48.10.1399

Abstract

PURPOSE: To evaluate the toxic effect of topical cyclosporine on cultured human corneal epithelium and to investigate the apoptotic response and cellular morphologic changes associated with cyclosporine in vitro.
METHODS
Human corneal epithelial cells were exposed to a concentration of cyclosporine A (0.05%) for a period of 3, 5, and 10 minutes. MTT-based calorimetric assay was performed to assess the metabolic activity of cellular proliferation and lactate dehydrogenase (LDH) leakage assay for cytotoxicity. Apoptotic response was evaluated with flow cytometric analysis and fluorescence staining with Annexin V and propiodium iodide. Cellular morphology was evaluated by inverted phase-contrast light microscopy and electron microscopy.
RESULTS
The inhibitory effect of human corneal epithelial cell proliferation and cytotoxicity showed a time-dependent response and had a significant effect when exposed for 10 minutes (P=0.04). The maximun response did not reach the leathal dose (LD)50. Apoptosis was seen in flow cytometry and apoptotic cells were demonstrated in fluorescent micrograph after being treated treating with cyclosporine A (0.05%). Human corneal epithelial cells were more detached from the bottom of the dish and damaged cells show degenerative changes like microvilli disappearance, vacuoles formation, and chromatin of the nuclear remnant condensed along the nuclear periphery.
CONCLUSIONS
Cyclosporine A (0.05%) could be used without any significant toxic effect on human corneal epithelial cells except for exposure times longer than 10 minutes. Induction of apoptosis modulation may be one of the cyclosporine's mechanism for inhibiting cellular proliferation.

Keyword

Apoptosis; Corneal epithelium; Cyclosporoine; Toxicity

MeSH Terms

Annexin A5
Apoptosis
Cell Proliferation
Chromatin
Cyclosporine*
Epithelial Cells*
Epithelium, Corneal
Flow Cytometry
Fluorescence
Humans*
L-Lactate Dehydrogenase
Microscopy
Microscopy, Electron
Microvilli
Vacuoles
Annexin A5
Chromatin
Cyclosporine
L-Lactate Dehydrogenase

Figure

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  Figure 1. Metabolic activities of cultured human corneal epithelial cells measured with MTT assays showed the time dependant response by the exposure time of cyclosporine. There is significant decrease in the proliferation of corneal epithelial cells comparing with control when exposed to cyclosporine A 0.05% for 10 minutes (p=0.04).

• 

  Figure 2. With time, the LDH acitivity of cultured human corneal epithelial cells in cyclosporine A 0.05% showed time dependant response for cytotoxicity. There is significant increase in the cytotoxicity of corneal epithelail cells comparing with control when exposed to cyclosporine A 0.05% for 10 minutes (p=0.03).

• 

  Figure 3. In the immunoassay with extracellular matrix, the synthesis of type I collagen synthesis (PIP) (A) and the production of laminin (B) by cyclosporine A 0.05% showed a time dependent response in human corneal epithelium.

• 

  Figure 4. Flow cytometric analysis of apoptotic cells using Annexin V FITC after exposure to cyclosporine. Human corneal epithelial cells were normal as control (A) or were treated with cyclosporine A 0.05% for 3 minutes (B), 5 minutes (C), and 10 minutes (D). Most normal epithelial cells were primarily Annexin V and PI negative (A). After treatment with cyclosporine A 0.05%, a significant number of cells are positive Annexin V and negative PI (B, C and D) indicating that the cells were in the stage of apoptosis.

• 

  Figure 5. Fluorescent micrograph of human corneal epithelial cells after exposure to cyclosporine A 0.05% (x 400): (A) control, (B) for 3 minutes, (C) for 5 minutes, (D) for 10 minutes. After treating with cyclosporine A 0.05%, apoptotic cells were visible, which represent fluorescence after binding DNA flowing into cytoplasm due to disruption of nuclear membrane. There were visible apoptotic bodies (white arrow) in extra cytoplasmic portion (B & C).

• 

  Figure 6. Inverted phase contrast micrographs of human corneal epithelial cells after exposure to cyclosporine A 0.05% (*200): (A) control, (B) for 3 minutes, (C) for 5 minutes, (D) for 10 minutes. Many epithelial cells are visible in control culture media. There are less epithelial cells which are more detached from the culture media, in the case of exposure to cyclosporine A 0.05% compared with control.

• 

  Figure 7. Transmission electron micrographs of human corneal epithelail cells after exposure to cyclosporine A 0.05% (bar length 2 um, A, B, C, & D : x3,000—4000); (A) control, (B) for 3 minutes, (C) for 5 minutes, (D) for 10 minutes. A normal corneal epithelial cell showed microvilli, homogenous cytoplasm and intact cell and nuclear membrane. The longer the exposure time of cyclosporine A 0.05%, the more decreased number of microvilli, well developed vacuole formation (white arrows), damaged mitochondria and rough endoplasmic reticulum (black arrows), and margination of chromatin in the nucleus (white arrowheads) the corneal epithelial cells revealed.

Reference

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