Jagiellonian University, Jagiellonian Centre for Experimental Therapeutics (JCET), Bobrzynskiego 14, 30-348, Krakow, Poland.
Jagiellonian University, Centre for Nanometer-Scale Science and Advanced Materials, NANOSAM, Faculty of Physics, Astronomy, and Applied Computer Science, Lojasiewicza 11, 30-348, Krakow, Poland.
Redox Biol. 2024 Jun;72:103162. doi: 10.1016/j.redox.2024.103162. Epub 2024 Apr 16.
Protein disulfide isomerases (PDIs) are involved in many intracellular and extracellular processes, including cell adhesion and cytoskeletal reorganisation, but their contribution to the regulation of fenestrations in liver sinusoidal endothelial cells (LSECs) remains unknown. Given that fenestrations are supported on a cytoskeleton scaffold, this study aimed to investigate whether endothelial PDIs regulate fenestration dynamics in primary mouse LSECs. PDIA3 and PDIA1 were found to be the most abundant among PDI isoforms in LSECs. Taking advantage of atomic force microscopy, the effects of PDIA1 or PDIA3 inhibition on the fenestrations in LSECs were investigated using a classic PDIA1 inhibitor (bepristat) and novel aromatic N-sulfonamides of aziridine-2-carboxylic acid derivatives as PDIA1 (C-3389) or PDIA3 (C-3399) inhibitors. The effect of PDIA1 inhibition on liver perfusion was studied in vivo using dynamic contrast-enhanced magnetic resonance imaging. Additionally, PDIA1 inhibitors were examined in vitro in LSECs for effects on adhesion, cytoskeleton organisation, bioenergetics, and viability. Inhibition of PDIA1 with bepristat or C-3389 significantly reduced the number of fenestrations in LSECs, while inhibition of PDIA3 with C-3399 had no effect. Moreover, the blocking of free thiols by the cell-penetrating N-ethylmaleimide, but not by the non-cell-penetrating 4-chloromercuribenzenesulfonate, resulted in LSEC defenestration. Inhibition of PDIA1 did not affect LSEC adhesion, viability, and bioenergetics, nor did it induce a clear-cut rearrangement of the cytoskeleton. However, PDIA1-dependent defenestration was reversed by cytochalasin B, a known fenestration stimulator, pointing to the preserved ability of LSECs to form new pores. Importantly, systemic inhibition of PDIA1 in vivo affected intra-parenchymal uptake of contrast agent in mice consistent with LSEC defenestration. These results revealed the role of intracellular PDIA1 in the regulation of fenestration dynamics in LSECs, and in maintaining hepatic sinusoid homeostasis.
蛋白质二硫键异构酶（PDI）参与许多细胞内和细胞外过程，包括细胞黏附和细胞骨架重组，但它们在调节肝窦内皮细胞（LSEC）窗孔方面的作用仍不清楚。鉴于窗孔是由细胞骨架支架支撑的，本研究旨在探讨内皮 PDIs 是否调节原代小鼠 LSEC 的窗孔动力学。研究发现，PDIA3 和 PDIA1 是 LSEC 中最丰富的 PDI 同工型。利用原子力显微镜，使用经典的 PDIA1 抑制剂（bepristat）和新型芳香族氮磺酰胺类氮杂环丙烷-2-羧酸衍生物 PDIA1（C-3389）或 PDIA3（C-3399）抑制剂研究 PDIA1 或 PDIA3 抑制对 LSEC 窗孔的影响。体内使用动态对比增强磁共振成像研究 PDIA1 抑制对肝脏灌注的影响。此外，还在 LSEC 中体外研究 PDIA1 抑制剂对黏附、细胞骨架组织、生物能量和活力的影响。用 bepristat 或 C-3389 抑制 PDIA1 显著减少 LSEC 中的窗孔数量，而用 C-3399 抑制 PDIA3 则没有影响。此外，细胞穿透性 N-乙基马来酰亚胺封闭自由巯基，但非细胞穿透性 4-氯汞苯磺酸盐则导致 LSEC 去窗孔化。PDIA1 抑制不影响 LSEC 黏附、活力和生物能量，也不会引起细胞骨架明显重排。然而，细胞松弛素 B 可逆转 PDIA1 依赖性去窗孔化，细胞松弛素 B 是已知的窗孔刺激物，表明 LSEC 形成新孔的能力得以保留。重要的是，体内 PDIA1 的系统性抑制影响了小鼠的对比剂在肝实质内的摄取，与 LSEC 去窗孔化一致。这些结果揭示了细胞内 PDIA1 在调节 LSEC 窗孔动力学和维持肝窦内稳态中的作用。
