Research and Development Laboratory for Biochemical, Molecular and Cellular Applications in Ophthalmological Sciences, IRCCS-Fondazione Bietti, Rome, Italy.
Fondazione Policlinico Universitario A. Gemelli, IRCCS, 00168, Rome, Italy.
Sci Rep. 2021 Jan 18;11(1):1672. doi: 10.1038/s41598-021-81040-x.
We previously described the profibrogenic effect of NGF on conjunctival Fibroblasts (FBs) and its ability to trigger apoptosis in TGFβ1-induced myofibroblasts (myoFBs). Herein, cell apoptosis/signalling, cytokines' signature in conditioned media and inflammatory as well as angiogenic pathway were investigated. Experimental myoFBs were exposed to NGF (0.1-100 ng/mL), at defined time-point for confocal and biomolecular analysis. Cells were analysed for apoptotic and cell signalling activation in cell extracts and for some inflammatory and proinflammatory/angiogenic factors' activations. NGF triggered cJun overexpression and phospho-p65-NFkB nuclear translocation. A decreased Bcl2:Bax ratio and a significant expression of smad7 were confirmed in early AnnexinV-positive myoFBs. A specific protein signature characterised the conditioned media: a dose dependent decrease occurred for IL8, IL6 while a selective increase was observed for VEGF and cyr61 (protein/mRNA). TIMP1 levels were unaffected. Herein, NGF modulation of smad7, the specific IL8 and IL6 as well as VEGF and cyr61 modulation deserve more attention as opening to alternative approaches to counteract fibrosis.
我们之前描述了 NGF 对结膜成纤维细胞 (FB) 的促纤维化作用及其在 TGFβ1 诱导的肌成纤维细胞 (myoFB) 中触发细胞凋亡的能力。在此，研究了细胞凋亡/信号转导、条件培养基中的细胞因子特征以及炎症和血管生成途径。将实验性 myoFB 暴露于 NGF（0.1-100ng/mL），在特定时间点进行共聚焦和生物分子分析。分析细胞提取物中的细胞凋亡和细胞信号转导激活，并分析一些炎症和促炎/血管生成因子的激活。NGF 触发 cJun 过表达和磷酸化 p65-NFkB 核易位。早期 AnnexinV 阳性 myoFB 中证实了 Bcl2:Bax 比值降低和 smad7 表达增加。特征性的蛋白质特征描述了条件培养基：IL8 和 IL6 的浓度依赖性降低，而 VEGF 和 Cyr61（蛋白质/信使 RNA）的选择性增加。TIMP1 水平不受影响。在此，需要更多关注 NGF 对 smad7 的调节、IL8 和 IL6 的特异性调节以及 VEGF 和 Cyr61 的调节，为对抗纤维化开辟了新的途径。
