VIP and VIP gene silencing modulation of differentiation marker N-cadherin and cell shape of corneal endothelium in human corneas ex vivo
VIP and VIP gene silencing modulation of differentiation marker N-cadherin and cell shape of corneal endothelium in human corneas ex vivo
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DOI：
10.1167/iovs.07-1543
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发表时间：
2008-08-01
影响因子：
4.4
通讯作者：
Rutzen, Allan R.
中科院分区：
文献类型：
--
作者：
Koh, Shay-Whey M.;Chandrasekara, Krish;Rutzen, Allan R.
目的。血管活性肠肽（VIP）由角膜内皮细胞（CE）表达，存在于角膜内皮细胞体内的房水中。本研究证实了CE细胞VIP在维持CE分化标志物N-cadherin的表达水平和六边形细胞形状中的作用。为了确定最有效的VIP浓度，分别用0（对照）和10(-12)~ 10(-6)M VIP处理牛角膜巩膜外植体。对照使用来自眼库的配对人角膜（9名供体）；其余角膜用VIP治疗。为了沉默内源性VIP，用VIP shRNA或对照慢病毒颗粒转导配对的新鲜人供体角膜（来自7具尸体），然后通过半定量RT-PCR （mRNA）和Western blot分析/免疫细胞化学（蛋白质）进行定量分析，茜素红S染色显示CE细胞形状。VIP浓度依赖性地提高牛CE细胞N-cadherin mRNA水平，最大影响在10(-10)（1.47 +/- 0.06倍，P = 0.002）和10(-8)M VIP（1.48 +/- 0.18倍，P = 0.012）之间。VIP （10(-8) M）处理使牛CE细胞和人CE细胞中的N-cadherin蛋白水平分别达到对照组的1.98 +/- 0.28倍（P = 0.005）和1.17 +/- 0.10倍（范围0.91 -1.87）（P = 0.050）。VIP拮抗剂（SN） VIPhyb降低VIP效应。VIP沉默导致六角形细胞形态恶化，VIP蛋白和mRNA、N-cadherin（不包括connexin-43） mRNA和蛋白水平下降，抗凋亡Bcl-2蛋白水平下降。CE细胞通过其自分泌的VIP，在维持角膜内皮原位分化状态和抑制细胞凋亡中发挥积极作用。
PURPOSE. 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.