An L-type calcium channel blocker nimodipine exerts anti-fibrotic effects by attenuating TGF-[beta]1 induced calcium response in an in vitro model of thyroid eye disease

• Published in: Eye and vision (Novato, Calif.) Calif.), 2024-09, Vol.11 (1)
• Main Authors: Chen, Qian, Pan, Yuan, Hu, Yunwei, Chen, Guanyu, Chen, Xiaoqing, Xie, Yanyan, Wang, Minzhen, Li, Zhuang, Huang, Jun, Shi, Yuxun, Huang, Haixiang, Zhang, Te, Wang, Mei, Zeng, Peng, Wang, Sha, Chen, Rongxin, Zheng, Yongxin, Zhong, Liuxueying, Yang, Huasheng, Liang, Dan
• Format: Article
• Language: English
• Subjects: Analysis; Calmodulin; Collagen; Gene expression; Genes; Health aspects; Immunohistochemistry; Muscle proteins; Nimodipine; RNA; RNA sequencing; Thyroid eye disease; Transforming growth factors
• ISSN: 2326-0254
• DOI: 10.1186/s40662-024-00401-5

Background Thyroid eye disease (TED) is a vision-threatening autoimmune disorder. Orbital tissue fibrosis leading to intractable complications remains a troublesome issue in TED management. Exploration of novel therapeutic targets and agents to ameliorate tissue fibrosis is crucial for TED. Recent work suggests that Ca.sup.2+ signaling participates in tissue fibrosis. However, whether an alteration of Ca.sup.2+ signaling has a role in fibrogenesis during TED remains unclear. In this study, we aimed to investigate the role of Ca.sup.2+ signaling in the fibrogenesis process during TED and the potential therapeutic effects of a highly selective inhibitor of the L-type calcium channel (LTCC), nimodipine, through a TGF-[beta]1 induced in vitro TED model. Methods Primary culture of orbital fibroblasts (OFs) were established from orbital adipose connective tissues of patients with TED and healthy control donors. Real-time quantitative polymerase chain reaction (RT-qPCR) and RNA sequencing were used to assess the genes expression associated with LTCC in OFs. Flow cytometry, RT-qPCR, 5-ethynyl-2'-deoxyuridine (EdU) proliferation assay, wound healing assay and Western blot (WB) were used to assess the intracellular Ca.sup.2+ response on TGF-[beta]1 stimulation, and to evaluate the potential therapeutic effects of nimodipine in the TGF-[beta]1 induced in vitro TED model. The roles of Ca.sup.2+/calmodulin-dependent protein kinase II (CaMKII) and signal transducer and activator of transcription 1 (STAT1) in fibrogenesis during TED were determined by immunohistochemistry, WB, flow cytometry and co-immunoprecipitation assay. Selective inhibitors were used to explore the downstream signaling pathways. Results LTCC inhibitor nimodipine blocked the TGF-[beta]1 induced intracellular Ca.sup.2+ response and further reduced the expression of alpha-smooth muscle actin ([alpha]-SMA), collagen type I alpha 1 (Col1A1) and collagen type I alpha 2 (Col1A2) in OFs. Besides, nimodipine inhibited cell proliferation and migration of OFs. Moreover, our results provided evidence that activation of the CaMKII/STAT1 signaling pathway was involved in fibrogenesis during TED, and nimodipine inhibited the pro-fibrotic functions of OFs by down-regulating the CaMKII/STAT1 signaling pathway. Conclusions TGF-[beta]1 induces an LTCC-mediated Ca.sup.2+ response, followed by activation of CaMKII/STAT1 signaling pathway, which promotes the pro-fibrotic functions of OFs and participates in fibrogenesis