Data_Sheet_1_A novel histone deacetylase inhibitor W2A-16 improves the barrier integrity in brain vascular endothelial cells.docx

The maturation of brain microvascular endothelial cells leads to the formation of a tightly sealed monolayer, known as the blood–brain barrier (BBB). The BBB damage is associated with the pathogenesis of age-related neurodegenerative diseases including vascular cognitive impairment and Alzheimer’s disease. Growing knowledge in the field of epigenetics can enhance the understanding of molecular profile of the BBB and has great potential for the development of novel therapeutic strategies or targets to repair a disrupted BBB. Histone deacetylases (HDACs) inhibitors are epigenetic regulators that can induce acetylation of histones and induce open chromatin conformation, promoting gene expression by enhancing the binding of DNA with transcription factors. We investigated how HDAC inhibition influences the barrier integrity using immortalized human endothelial cells (HCMEC/D3) and the human induced pluripotent stem cell (iPSC)-derived brain vascular endothelial cells. The endothelial cells were treated with or without a novel compound named W2A-16. W2A-16 not only activates Wnt/β-catenin signaling but also functions as a class I HDAC inhibitor. We demonstrated that the administration with W2A-16 sustained barrier properties of the monolayer of endothelial cells, as evidenced by increased trans-endothelial electrical resistance (TEER). The BBB-related genes and protein expression were also increased compared with non-treated controls. Analysis of transcript profiles through RNA-sequencing in hCMEC/D3 cells indicated that W2A-16 potentially enhances BBB integrity by influencing genes associated with the regulation of the extracellular microenvironment. These findings collectively propose that the HDAC inhibition by W2A-16 plays a facilitating role in the formation of the BBB. Pharmacological approaches to inhibit HDAC may be a potential therapeutic strategy to boost and/or restore BBB integrity.

脑微血管内皮细胞的成熟过程会形成紧密密封的单层结构，即血脑屏障（blood–brain barrier, BBB）。血脑屏障损伤与多种年龄相关性神经退行性疾病的发病机制密切相关，包括血管性认知障碍与阿尔茨海默病。表观遗传学领域的研究进展可加深对血脑屏障分子特征的理解，在开发修复受损血脑屏障的新型治疗策略或靶点方面具备巨大应用潜力。组蛋白去乙酰化酶（Histone deacetylases, HDACs）抑制剂属于表观遗传调控因子，能够诱导组蛋白乙酰化并开放染色质构象，通过增强DNA与转录因子的结合来促进基因表达。本研究采用永生化人内皮细胞（HCMEC/D3）以及人诱导多能干细胞（induced pluripotent stem cell, iPSC）分化得到的脑血管内皮细胞，探究了HDAC抑制对屏障完整性的影响。实验中，内皮细胞分别接受新型化合物W2A-16处理与不做处理两组。W2A-16不仅可激活Wnt/β-连环蛋白（Wnt/β-catenin）信号通路，同时兼具I型HDAC抑制剂的功能。研究证实，W2A-16给药可维持内皮细胞单层的屏障功能，该结论可通过跨内皮细胞电阻（trans-endothelial electrical resistance, TEER）的升高得到验证。与未处理的对照组相比，血脑屏障相关基因及蛋白的表达水平也显著上调。通过对hCMEC/D3细胞进行RNA测序（RNA-sequencing）分析转录组特征，结果表明W2A-16可能通过调控细胞外微环境相关基因来增强血脑屏障的完整性。综上，上述研究结果提示W2A-16介导的HDAC抑制在血脑屏障的形成中发挥促进作用。通过药理学手段抑制HDAC或许是增强乃至恢复血脑屏障完整性的潜在治疗策略。