Suberoylanilide hydroxamic acid (vorinostat): its role on equine corneal fibrosis and matrix metalloproteinase activity
Suberoylanilide hydroxamic acid (vorinostat): its role on equine corneal fibrosis and matrix metalloproteinase activity
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DOI：
10.1111/vop.12129
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发表时间：
2014-07-01
影响因子：
1.6
通讯作者：
Mohan, Rajiv R.
中科院分区：
文献类型：
--
作者：
Donnelly, Kevin S.;Giuliano, Elizabeth A.;Mohan, Rajiv R.
目的利用体外马角膜纤维化模型，探讨琥珀酰苯胺异羟肟酸(SAHA)(I)对角膜成纤维细胞分化、形态和活性的影响，以及(II)对基质金属蛋白酶(MMPs)2和9表达水平的影响。用5 ng/mLTGF-β1诱导成纤维细胞形成肌成纤维细胞。用5mU M或10 mU M SAHA处理72h，同时加入转化生长因子β1。实时荧光定量聚合酶链式反应和免疫细胞化学方法检测α-平滑肌肌动蛋白(α-SMA)的表达，以确定SAHA的抗纤维化作用。实时荧光定量聚合酶链式反应检测SAHA对MMP2和MMP9表达的影响。结果SAHA可显著抑制转化生长因子β1诱导的马成纤维细胞向肌成纤维细胞的分化，αSMA基因表达减少3~3.5%(P<0.001)，αSMA+免疫细胞化学染色减少86~88%(P<0.001)。SAHA治疗还导致MMP9表达下降4.5-5.5倍(P<；0.01)。SAHA对MMP2表达的影响呈剂量依赖性的双峰效应(5 mU M剂量时增加3.5倍，10 mU M剂量时减少0.5倍)。5 mU M的SAHA剂量对成纤维细胞活力无明显影响，10 mU M的SAHA可使细胞存活率下降17%。结论SAHA能有效抑制转化生长因子β诱导的马角膜成纤维细胞向肌成纤维细胞的分化，并调节基质金属蛋白酶的产生。
Objective To explore the effect of suberoylanilide hydroxamic acid (SAHA) (i) on corneal fibroblast differentiation, morphology, and viability; and (ii) on the expression levels of matrix metalloproteinases (MMPs) 2 and 9 using an in vitro model of equine corneal fibrosis.Procedure Healthy donor corneas were used to generate primary cultures of equine corneal fibroblasts. The fibroblasts were exposed to 5 ng/mL TGF beta 1 to induce myofibroblast formation. The cultures were treated with either 5 mu M or 10 mu M SAHA for 72 h in the presence of TGF beta 1. Real-time PCR and immunocytochemistry were used to determine the antifibrotic efficacy of SAHA by quantifying alpha-smooth muscle actin (alpha SMA), a marker of myofibroblast formation and fibrosis. Real-time PCR was used to determine the effects of SAHA on MMP2 and MMP9 expression. Cytotoxicity of SAHA was evaluated with phase contrast microscopy and trypan blue exclusion assays.Results Suberoylanilide hydroxamic acid (SAHA) significantly attenuated TGF beta 1-induced differentiation of equine fibroblasts to myofibroblasts as indicated by 3- to 3.5-fold (P < 0.001) decrease in alpha SMA mRNA and 86-88% (P < 0.001) decrease in alpha SMA+immunocytochemical staining. SAHA treatment also resulted in 4.5-to 5.5-fold (P < 0.01) decrease in MMP9 expression. A dose-dependent bimodal effect of SAHA on MMP2 expression was noted (3.5-fold increase with 5 mu M dose; 0.5-fold decrease with 10 mu M dose). No change in fibroblast viability was observed with a 5 mu M SAHA dose, whereas a 10 rho M dose resulted in a moderate 17% decrease in cell viability.Conclusions Suberoylanilide hydroxamic acid (SAHA) can effectively inhibit TGF beta-induced differentiation of equine corneal fibroblasts to myofibroblasts and modulates MMP production in vitro.