Xie Leike, Santhoshkumar Puttur, Reneker Lixing W, Sharma K Krishna
Department of Ophthalmology, University of Missouri-Columbia School of Medicine, Columbia, Missouri, United States.
Department of Ophthalmology, University of Missouri-Columbia School of Medicine, Columbia, Missouri, United States Department of Biochemistry, University of Missouri-Columbia School of Medicine, Columbia, Missouri, United States.
Invest Ophthalmol Vis Sci. 2014 Jul 3;55(8):4731-40. doi: 10.1167/iovs.14-14109.
Posterior capsule opacification (PCO) after cataract surgery is due in part to proliferation of the adhering lens epithelial cells and transdifferentiation into mesenchymal cells. The histone deacetylase (HDAC) inhibitors, trichostatin A (TSA) and vorinostat (suberoylanilidehydroxamic acid [SAHA]) are known to modulate cell proliferation and epithelial-mesenchymal transition (EMT). Studies have shown that TGFβ2 can induce EMT similar to that seen during PCO. This study evaluated the effects of TSA and SAHA on TGFβ2-induced EMT in lens epithelial explants.
Epithelial cells adherent to lens capsules were isolated from fresh pig lenses and human donor lenses and cultured for 12 hours. Explants were pretreated with TSA or SAHA for 1 hour and then treated with TGFβ2 for up to 3 days. Scratch wound healing assay was used to determine epithelial cell proliferation and migration in the samples. The effects of TSA and SAHA on histone acetylation and HDAC 1 to 6 levels were analyzed by Western blotting.
Western blotting and immunocytochemistry demonstrated high expression of α-SMA in lens epithelial cells treated with TGFβ2. The HDAC inhibitors exerted dose-dependent inhibition of α-SMA expression, with complete inhibition occurring with 0.5 μM of TSA and 2.5 μM of SAHA. Transforming growth factor β2-induced EMT was suppressed by TSA and SAHA. Histone deacetylase inhibition in pig lens epithelia led to increased acetylation of histone 3 and 4 at multiple sites.
Histone deacetylase inhibitors, TSA, and SAHA prevent EMT in lens epithelial explants. The results also suggest that the epigenetic modifiers are the potential targets to control PCO after cataract surgery.
白内障手术后后囊膜混浊（PCO）部分归因于黏附的晶状体上皮细胞增殖并转分化为间充质细胞。已知组蛋白脱乙酰酶（HDAC）抑制剂曲古抑菌素A（TSA）和伏立诺他（辛二酰苯胺异羟肟酸[SAHA]）可调节细胞增殖和上皮-间充质转化（EMT）。研究表明，TGFβ2可诱导与PCO期间相似的EMT。本研究评估了TSA和SAHA对TGFβ2诱导的晶状体上皮外植体EMT的影响。
从新鲜猪晶状体和人类供体晶状体中分离出黏附于晶状体囊膜的上皮细胞，并培养12小时。外植体先用TSA或SAHA预处理1小时，然后用TGFβ2处理长达3天。采用划痕伤口愈合试验来确定样品中上皮细胞的增殖和迁移。通过蛋白质印迹分析TSA和SAHA对组蛋白乙酰化和HDAC 1至6水平的影响。
蛋白质印迹和免疫细胞化学显示，用TGFβ2处理的晶状体上皮细胞中α-SMA高表达。HDAC抑制剂对α-SMA表达具有剂量依赖性抑制作用，0.5 μM的TSA和2.5 μM的SAHA可完全抑制。TSA和SAHA抑制了转化生长因子β2诱导的EMT。猪晶状体上皮中的组蛋白脱乙酰酶抑制导致组蛋白3和4在多个位点的乙酰化增加。
组蛋白脱乙酰酶抑制剂TSA和SAHA可预防晶状体上皮外植体中的EMT。结果还表明，表观遗传修饰剂是控制白内障手术后PCO的潜在靶点。
