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Molecular mechanism of ocular surface damage: application to an in vitro dry eye model on human corneal epithelium.

Meloni M, De Servi B, Marasco D, Del Prete S - Mol. Vis. (2011)

Bottom Line: The effects of different commercially available tear substitutes on the induced dry eye condition were tested.The transcriptional study of selected biomarkers showed an increase in MUC4, MMP9, TNF-α, and hBD-2 (DEFB2) gene expression.By using a dynamic approach, we were able to define a biomarker gene signature of dry eye-induced effects that could be predictive of corneal damage in vivo and to discriminate the efficacy among different commercial artificial tears.

View Article: PubMed Central - PubMed

ABSTRACT

Purpose: The present study was concerned with the development of a new experimental model of dry eye using human reconstructed in vitro corneal epithelium (HCE). The model is based on the use of adapted culture conditions that induce relevant modifications at the cellular and molecular level thus mimicking dry eye.

Methods: The HCE model was maintained in a controlled environmental setting (relative humidity <40% and 40 °C temperature) for 24 h and up to 72 h to induce dry eye. The evolution of the dry eye condition was assessed by histology, immunohistochemistry staining, scanning electron microscopy, and gene expression by using TaqMan gene assay technology (mucin-4 [MUC4], matrix metallopeptidase-9 [MMP9], tumor necrosis factor-α [TNF-α], and defensin β-2 [DEFB2). The effects of different commercially available tear substitutes on the induced dry eye condition were tested.

Results: This in vitro dry eye HCE model, that was well established within 24 h, has the characteristic features of a dry eye epithelium and could be satisfactorily used for preliminary assessment of the protective activity of some artificial tears. The transcriptional study of selected biomarkers showed an increase in MUC4, MMP9, TNF-α, and hBD-2 (DEFB2) gene expression.

Conclusions: By using a dynamic approach, we were able to define a biomarker gene signature of dry eye-induced effects that could be predictive of corneal damage in vivo and to discriminate the efficacy among different commercial artificial tears.

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Related in: MedlinePlus

Results of EDEV biomarkers gene expression study. Real-time PCR analysis showing the relative changes of TNF-α (A), MMP9 (B), MUC4 (C), and DEFB2 (D) mRNA after 24 h and 48 h treatment with different tear substitutes simultaneously the induction of EDEV. A twofold change of Relative Quantification (RQ) is usually considered significant in comparison to the calibrator (CONTROL-HCE at each time point). A two-tailed Student’s t-test was used to determine statistical significance for real-time PCR. Data were considered significant at p<0.05 (*), p<0.01 (**), and p<0.001 (***).
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f7: Results of EDEV biomarkers gene expression study. Real-time PCR analysis showing the relative changes of TNF-α (A), MMP9 (B), MUC4 (C), and DEFB2 (D) mRNA after 24 h and 48 h treatment with different tear substitutes simultaneously the induction of EDEV. A twofold change of Relative Quantification (RQ) is usually considered significant in comparison to the calibrator (CONTROL-HCE at each time point). A two-tailed Student’s t-test was used to determine statistical significance for real-time PCR. Data were considered significant at p<0.05 (*), p<0.01 (**), and p<0.001 (***).

Mentions: All tear substitutes had a significant stronger effect on TNF-α mRNA increase after 48 h of treatment compared to 24 h. After 24 h of treatment, all of the tear substitutes tested induced an overexpression of TNF-α. Etacortilen®, a well known anti-inflammatory drug, was the only agent able to control and block the expression of the inflammatory genes to the same RQ values as those observed in the EDEV-HCE (Figure 7A). The upregulation of MMP9 observed at 24 h in the EDEV model was controlled by treatment with all tear substitutes except for Xiloial® and Optive®. At 48 h all the tears except Hyalistil® were able to counteract the increased expression of MMP9 (Figure 7B).


Molecular mechanism of ocular surface damage: application to an in vitro dry eye model on human corneal epithelium.

Meloni M, De Servi B, Marasco D, Del Prete S - Mol. Vis. (2011)

Results of EDEV biomarkers gene expression study. Real-time PCR analysis showing the relative changes of TNF-α (A), MMP9 (B), MUC4 (C), and DEFB2 (D) mRNA after 24 h and 48 h treatment with different tear substitutes simultaneously the induction of EDEV. A twofold change of Relative Quantification (RQ) is usually considered significant in comparison to the calibrator (CONTROL-HCE at each time point). A two-tailed Student’s t-test was used to determine statistical significance for real-time PCR. Data were considered significant at p<0.05 (*), p<0.01 (**), and p<0.001 (***).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3021568&req=5

f7: Results of EDEV biomarkers gene expression study. Real-time PCR analysis showing the relative changes of TNF-α (A), MMP9 (B), MUC4 (C), and DEFB2 (D) mRNA after 24 h and 48 h treatment with different tear substitutes simultaneously the induction of EDEV. A twofold change of Relative Quantification (RQ) is usually considered significant in comparison to the calibrator (CONTROL-HCE at each time point). A two-tailed Student’s t-test was used to determine statistical significance for real-time PCR. Data were considered significant at p<0.05 (*), p<0.01 (**), and p<0.001 (***).
Mentions: All tear substitutes had a significant stronger effect on TNF-α mRNA increase after 48 h of treatment compared to 24 h. After 24 h of treatment, all of the tear substitutes tested induced an overexpression of TNF-α. Etacortilen®, a well known anti-inflammatory drug, was the only agent able to control and block the expression of the inflammatory genes to the same RQ values as those observed in the EDEV-HCE (Figure 7A). The upregulation of MMP9 observed at 24 h in the EDEV model was controlled by treatment with all tear substitutes except for Xiloial® and Optive®. At 48 h all the tears except Hyalistil® were able to counteract the increased expression of MMP9 (Figure 7B).

Bottom Line: The effects of different commercially available tear substitutes on the induced dry eye condition were tested.The transcriptional study of selected biomarkers showed an increase in MUC4, MMP9, TNF-α, and hBD-2 (DEFB2) gene expression.By using a dynamic approach, we were able to define a biomarker gene signature of dry eye-induced effects that could be predictive of corneal damage in vivo and to discriminate the efficacy among different commercial artificial tears.

View Article: PubMed Central - PubMed

ABSTRACT

Purpose: The present study was concerned with the development of a new experimental model of dry eye using human reconstructed in vitro corneal epithelium (HCE). The model is based on the use of adapted culture conditions that induce relevant modifications at the cellular and molecular level thus mimicking dry eye.

Methods: The HCE model was maintained in a controlled environmental setting (relative humidity <40% and 40 °C temperature) for 24 h and up to 72 h to induce dry eye. The evolution of the dry eye condition was assessed by histology, immunohistochemistry staining, scanning electron microscopy, and gene expression by using TaqMan gene assay technology (mucin-4 [MUC4], matrix metallopeptidase-9 [MMP9], tumor necrosis factor-α [TNF-α], and defensin β-2 [DEFB2). The effects of different commercially available tear substitutes on the induced dry eye condition were tested.

Results: This in vitro dry eye HCE model, that was well established within 24 h, has the characteristic features of a dry eye epithelium and could be satisfactorily used for preliminary assessment of the protective activity of some artificial tears. The transcriptional study of selected biomarkers showed an increase in MUC4, MMP9, TNF-α, and hBD-2 (DEFB2) gene expression.

Conclusions: By using a dynamic approach, we were able to define a biomarker gene signature of dry eye-induced effects that could be predictive of corneal damage in vivo and to discriminate the efficacy among different commercial artificial tears.

Show MeSH
Related in: MedlinePlus