Department of Dermatology, Zhongshan Hospital, Fudan University, Shanghai, China.
Rheumatology (Oxford). 2019 Nov 1;58(11):1966-1975. doi: 10.1093/rheumatology/kez159.
To investigate whether autophagy mediates 2-methoxyestradiol (2-ME)-inhibited hypoxia-induced fibrosis and endothelial-to-mesenchymal transition (endoMT) in SSc.
Autophagy in the skin of SSc patients was assessed by transmission electron microscopy. SSc skin fibroblasts and human umbilical vein endothelial cells (HUVECs) were cultured under hypoxic (1% O2) conditions with 2-ME or autophagy inhibitor. Collagen I and connective tissue growth factor (CTGF) in fibroblasts and vascular endothelial (VE)-cadherin, CD31, vimentin and α-smooth muscle actin (α-SMA) in HUVECs were examined by western blotting. Autophagic markers were evaluated by confocal microscopy and immunofluorescence.
SSc skins presented increased autolysosomes, LC3-II, collagen I and CTGF. Hypoxia-challenged fibroblasts and HUVECs formed more autophagosomes and autolysosomes, with increased LC3 and decreased P62. Meanwhile, hypoxia increased collagen I and CTGF in fibroblasts and increased vimentin and α-SMA but decreased VE-cadherin and CD31 in HUVECs. Bafilomycin A1 increased LC3-II and P62 in fibroblasts and HUVECs and decreased collagen I and CTGF in fibroblasts and vimentin and α-SMA in HUVECs, while upregulating VE-cadherin and CD31. 3-methyladenine decreased autophagy and fibrosis in fibroblasts and endothelial-to-mesenchymal transition in HUVECs. 2-ME-treated HUVECs showed more autophagosomes and fewer autolysosomes while 2-ME-treated fibroblasts showed fewer of both. Moreover, 2-ME decreased LC3-II and increased P62 in fibroblasts and increased both in HUVECs. Inhibition of autophagy by 2-ME showed the same effect with bafilomycin A1 on fibroblast collagen synthesis as well as endothelial and mesenchymal markers in HUVECs.
Autophagy mediated hypoxia-induced fibroblast collagen synthesis and endoMT in SSc, which could be reversed by 2-ME.
研究自噬是否介导 2-甲氧基雌二醇（2-ME）抑制 SSc 中的缺氧诱导的纤维化和内皮-间质转化（endoMT）。
通过透射电子显微镜评估 SSc 患者皮肤中的自噬。在缺氧（1%O2）条件下，用 2-ME 或自噬抑制剂培养 SSc 皮肤成纤维细胞和人脐静脉内皮细胞（HUVEC）。通过 Western blot 检测成纤维细胞中的胶原 I 和结缔组织生长因子（CTGF）以及 HUVEC 中的血管内皮（VE）-钙粘蛋白、CD31、波形蛋白和α-平滑肌肌动蛋白（α-SMA）。通过共聚焦显微镜和免疫荧光评估自噬标志物。
SSc 皮肤中出现了更多的自噬溶酶体、LC3-II、胶原 I 和 CTGF。缺氧刺激的成纤维细胞和 HUVEC 形成了更多的自噬体和自噬溶酶体，LC3 增加，P62 减少。同时，缺氧增加了成纤维细胞中的胶原 I 和 CTGF，以及 HUVEC 中的波形蛋白和α-SMA，而降低了 VE-钙粘蛋白和 CD31。巴弗洛霉素 A1 增加了成纤维细胞和 HUVEC 中的 LC3-II 和 P62，减少了成纤维细胞中的胶原 I 和 CTGF 以及 HUVEC 中的波形蛋白和α-SMA，同时上调了 VE-钙粘蛋白和 CD31。3-甲基腺嘌呤减少了成纤维细胞中的自噬和纤维化以及 HUVEC 中的内皮-间质转化。2-ME 处理的 HUVEC 显示出更多的自噬体和更少的自噬溶酶体，而 2-ME 处理的成纤维细胞则显示出更少的自噬体和自噬溶酶体。此外，2-ME 降低了成纤维细胞中的 LC3-II 和增加了 P62，同时增加了 HUVEC 中的两者。2-ME 抑制自噬对成纤维细胞胶原合成以及 HUVEC 内皮和间充质标志物的影响与巴弗洛霉素 A1 相同。
自噬介导了 SSc 中的缺氧诱导的成纤维细胞胶原合成和 endoMT，2-ME 可逆转这一过程。
