Department of Cell and Molecular Biology, Iladevi Cataract and IOL Research Centre, Ahmedabad, Gujarat, India; Manipal Academy of Higher Education, Manipal, Karnataka, India.
Department of Cell and Molecular Biology, Iladevi Cataract and IOL Research Centre, Ahmedabad, Gujarat, India.
Exp Eye Res. 2020 Aug;197:108072. doi: 10.1016/j.exer.2020.108072. Epub 2020 May 27.
Proliferative retinopathies are associated with formation of fibrous epiretinal membranes. At present, there is no pharmacological intervention for the treatment of retinopathies. Cytokines such as TGFβ are elevated in the vitreous humor of the patients with proliferative vitro-retinopathy, diabetic retinopathy and age-related macular degeneration. TGFβ isoforms lead to epithelial-mesenchymal transition (EMT) or trans-differentiation of the retinal pigment epithelial (RPE) cells. PI3K/Akt and MAPK/Erk pathways play important roles in the EMT of RPE cells. Therefore, inhibition of EMT by pharmacological agents is an important therapeutic strategy in retinopathy. Dichloroacetate (DCA) is shown to prevent proliferation and EMT of cancer cell lines but its effects are not explored on the prevention of EMT of RPE cells. In the present study, we have investigated the role of DCA in preventing TGFβ2 induced EMT of RPE cell line, ARPE-19. A wound-healing assay was utilized to detect the anti-EMT effect of DCA. The expressions of EMT and cell adhesion markers were carried out by immunofluorescence, western blotting, and quantitative real-time PCR. The expression of MAPK/Erk and PI3K/Akt pathway members was carried out using western blotting. We found that TGFβ2 exposure leads to an increase in the wound healing response, expression of EMT markers (Fibronectin, Collagen I, N-cadherin, MMP9, S100A4, α-SMA, Snai1, Slug) and a decrease in the expression of cell adhesion/epithelial markers (ZO-1, Connexin 43, E-cadherin). These changes were accompanied by the activation of PI3K/Akt and MAPK/Erk pathways. Simultaneous exposure of DCA along with TGFβ2 significantly inhibited wound healing response, expression of EMT markers and cell adhesion/epithelial markers. Furthermore, DCA and TGFβ2 effectively attenuated the activation of MAPK/Erk/JNK and PI3K/Akt/GSK3β pathways. Our results demonstrate that DCA has a strong anti-EMT effect on the ARPE-19 cells and hence can be utilized as a therapeutic agent in the prevention of proliferative retinopathies.
增生性视网膜病变与纤维性视网膜前膜的形成有关。目前，对于增生性玻璃体视网膜病变、糖尿病性视网膜病变和年龄相关性黄斑变性等视网膜病变，尚无药物干预治疗方法。转化生长因子β（TGFβ）等细胞因子在增生性玻璃体视网膜病变、糖尿病性视网膜病变和年龄相关性黄斑变性患者的玻璃体中升高。TGFβ同工型导致视网膜色素上皮（RPE）细胞的上皮-间充质转化（EMT）或转分化。PI3K/Akt 和 MAPK/Erk 通路在 RPE 细胞的 EMT 中起重要作用。因此，通过药物抑制 EMT 是治疗视网膜病变的重要策略。二氯乙酸（DCA）已被证明可预防癌细胞系的增殖和 EMT，但尚未探讨其对预防 RPE 细胞 EMT 的作用。在本研究中，我们研究了 DCA 在预防 TGFβ2 诱导的 RPE 细胞系 ARPE-19 发生 EMT 中的作用。利用划痕愈合实验检测 DCA 的抗 EMT 作用。通过免疫荧光、Western blot 和实时定量 PCR 检测 EMT 和细胞黏附标志物的表达。通过 Western blot 检测 MAPK/Erk 和 PI3K/Akt 通路成员的表达。我们发现，TGFβ2 暴露会导致伤口愈合反应增加，EMT 标志物（纤连蛋白、I 型胶原、N-钙黏蛋白、MMP9、S100A4、α-SMA、Snai1、Slug）的表达增加，细胞黏附/上皮标志物（ZO-1、连接蛋白 43、E-钙黏蛋白）的表达减少。这些变化伴随着 PI3K/Akt 和 MAPK/Erk 通路的激活。同时暴露于 DCA 和 TGFβ2 可显著抑制伤口愈合反应、EMT 标志物和细胞黏附/上皮标志物的表达。此外，DCA 和 TGFβ2 可有效抑制 MAPK/Erk/JNK 和 PI3K/Akt/GSK3β 通路的激活。我们的结果表明，DCA 对 ARPE-19 细胞具有很强的抗 EMT 作用，因此可作为预防增生性视网膜病变的治疗药物。
