VIP and VIP gene silencing modulation of differentiation marker N-cadherin and cell shape of corneal endothelium in human corneas ex vivo.
OBJECTIVE
Vasoactive intestinal peptide (VIP) is expressed by corneal endothelial (CE) cells and is 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) to 10(-6) M VIP. Paired human corneas (nine donors) from an eye bank were used as control; the other corneas were treated with VIP. To silence endogenous VIP, paired fresh human donor corneas (from seven cadavers) were transduced with VIP shRNA or the control lentiviral particles and then bisected/quartered for quantitative analysis by semiquantitative RT-PCR (for mRNA) and Western blot analysis/immunocytochemistry (for protein), whereas alizarin red S staining revealed CE cell shape.


RESULTS
VIP concentration dependently increased bovine CE cell N-cadherin mRNA levels, 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-fold (P = 0.005) and 1.17 +/- 0.10 (range, 0.91-1.87)-fold (P = 0.050) of their respective controls. VIP antagonist (SN)VIPhyb diminished the VIP effect. VIP silencing resulted in deterioration of the hexagonal cell shape and decreased levels of VIP protein and mRNA, N-cadherin (but not connexin-43) mRNA and protein, and the antiapoptotic Bcl-2 protein.


CONCLUSIONS
Through its autocrine VIP, CE cells play an active role in maintaining the differentiated state and suppressing apoptosis in the corneal endothelium in situ.
Koh
Shay-Whey M
SW
Department of Ophthalmology and Visual Sciences, University of Maryland, Baltimore, Maryland 21201, USA. skoh@umaryland.edu
Chandrasekara
Krish
K
Abbondandolo
Cara J
CJ
Coll
Timothy J
TJ
Rutzen
Allan R
AR
D000818:Animals; D015415:Biomarkers / Q000378:metabolism*; D015153:Blotting, Western; D015820:Cadherins / Q000235:genetics*; D002417:Cattle; D002454:Cell Differentiation; D048430:Cell Shape / Q000502:physiology*; D003315:Cornea / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D004728:Endothelium, Corneal / Q000166:cytology* / Q000378:metabolism; D019084:Fluorescent Antibody Technique, Indirect; D015870:Gene Expression / Q000187:drug effects; D020868:Gene Silencing / Q000502:physiology*; D005822:Genetic Vectors; D006801:Humans; D009924:Organ Culture Techniques; D034622:RNA Interference; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012590:Sclera / Q000187:drug effects; D014660:Vasoactive Intestinal Peptide / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000494:pharmacology*
D015415:Biomarkers; D015820:Cadherins; D012333:RNA, Messenger; D014660:Vasoactive Intestinal Peptide