VIP and VIP Gene Silencing Modulate Differentiation Marker N-Cadherin and Cell Shape of Corneal Endothelium in Human Corneas Ex Vivo
Shay-Whey M. Koh 1*,
Krish Chandrasekara 2,
Cara J Abbondandolo 3,
Timothy J Coll 3,
and
Allan R Rutzen 3
1 Departments of Ophthalmology & Visual Science and Physiology, University of Maryland Baltimore, Baltimore, Maryland, United States
2 Transgenetech, Columbia, Maryland, United States
3 Department of Ophthalmology & Visual Sciences, University of Maryland Baltimore, Baltimore, Maryland, United States
* To whom correspondence should be addressed. E-mail: skoh{at}umaryland.edu.
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Abstract |
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Purpose: Vasoactive intestinal peptide (VIP) is expressed by corneal endothelial (CE) cells and present in the aqueous humor, which bathes CE cells in vivo. This study demonstrated the role of CE cell VIP in maintaining the expression level of a CE differentiation marker N-cadherin and the hexagonal cell shape. Methods: To determine the most effective VIP concentration, bovine corneoscleral explants were treated with 0 (control) and 10-12-10-6 M VIP. Paired human corneas (from nine donors) previously stored in the eye bank were used as control vs VIP-treated. To silence endogenous VIP, paired fresh human donor corneas (from seven cadavers) were transduced with either VIP shRNA or the control lentiviral particles and then bisected/quartered for quantitative analysis by semi-quantitative RT-PCR (mRNA) and Western analysis/immunocytochemistry (protein), while alizarin red S staining revealed CE cell shape. Results: VIP concentration-dependently increased bovine CE cell N-cadherin mRNA level, with the maximal effect observed between 10-10 (1.47+/-0.06-fold; p=0.002) and 10-8 M VIP (1.48+/-0.18-fold; p=0.012). VIP (10-8 M) treatment increased N-cadherin protein levels in bovine and human CE cells to 1.98+/-0.28- (p=0.005) and 1.17+/-0.10 (range 0.91-1.87)-fold (p=0.050) of their respective controls, respectively. VIP antagonist (SN)VIPhyb diminished the VIP effect. VIP silencing resulted in deterioration of the hexagonal cell shape, decreased levels of VIP protein and mRNA, N-cadherin (but not connexin-43) mRNA and protein, and the anti-apoptotic Bcl-2 protein. Conclusion:Through its autocrine VIP, CE cells play an active role in maintaining the differentiated state and suppressing apoptosis in the corneal endothelium, in situ.
Key Words:
corneal endothelial cells, neuropeptides, gene expression, cell adhesion
