Department of Medical Biology, UiT-The Arctic University of Norway, Tromsø, Norway.
Department of Clinical Medicine, UiT-The Arctic University of Norway, Tromsø, Norway.
PLoS One. 2022 Sep 2;17(9):e0273843. doi: 10.1371/journal.pone.0273843. eCollection 2022.
Liver sinusoidal endothelial cells (LSECs) are specialized fenestrated scavenger endothelial cells involved in the elimination of modified plasma proteins and tissue turnover waste macromolecules from blood. LSECs also participate in liver immune responses. A challenge when studying LSEC biology is the rapid loss of the in vivo phenotype in culture. In this study, we have examined biological processes and pathways affected during early-stage primary culture of rat LSECs and checked for cell responses to the pro-inflammatory cytokine interleukin (IL)-1β and the anti-inflammatory drug dexamethasone.
LSECs from male Sprague Dawley rats were cultured on type I collagen in 5% oxygen atmosphere in DMEM with serum-free supplements for 2 and 24 h. Quantitative proteomics using tandem mass tag technology was used to examine proteins in cells and supernatants. Validation was done with qPCR, ELISA, multiplex immunoassay, and caspase 3/7 assay. Cell ultrastructure was examined by scanning electron microscopy, and scavenger function by quantitative endocytosis assays.
LSECs cultured for 24 h showed a characteristic pro-inflammatory phenotype both in the presence and absence of IL-1β, with upregulation of cellular responses to cytokines and interferon-γ, cell-cell adhesion, and glycolysis, increased expression of fatty acid binding proteins (FABP4, FABP5), and downregulation of several membrane receptors (STAB1, STAB2, LYVE1, CLEC4G) and proteins in pyruvate metabolism, citric acid cycle, fatty acid elongation, amino acid metabolism, and oxidation-reduction processes. Dexamethasone inhibited apoptosis and improved LSEC viability in culture, repressed inflammatory and immune regulatory pathways and secretion of IL-1β and IL-6, and further upregulated FABP4 and FABP5 compared to time-matched controls. The LSEC porosity and endocytic activity were reduced at 24 h both with and without dexamethasone but the dexamethasone-treated cells showed a less stressed phenotype.
Rat LSECs become activated towards a pro-inflammatory phenotype during early culture. Dexamethasone represses LSEC activation, inhibits apoptosis, and improves cell viability.
肝窦内皮细胞（LSEC）是一种特化的有窗孔的吞噬内皮细胞，参与从血液中清除修饰的血浆蛋白和组织更新废物大分子。LSEC 还参与肝脏免疫反应。研究 LSEC 生物学的一个挑战是，在培养过程中体内表型迅速丧失。在这项研究中，我们检查了大鼠 LSEC 早期原代培养过程中受影响的生物学过程和途径，并检查了细胞对促炎细胞因子白细胞介素（IL）-1β和抗炎药物地塞米松的反应。
在 5%氧气气氛下，将雄性 Sprague Dawley 大鼠的 LSEC 培养在 I 型胶原上，在含有无血清补充剂的 DMEM 中培养 2 和 24 小时。使用串联质量标签技术进行定量蛋白质组学研究，以检测细胞和上清液中的蛋白质。使用 qPCR、ELISA、多重免疫分析和 caspase 3/7 测定进行验证。通过扫描电子显微镜检查细胞超微结构，通过定量内吞作用测定检查吞噬功能。
培养 24 小时的 LSEC 表现出特征性的促炎表型，无论是在存在还是不存在 IL-1β的情况下，细胞对细胞因子和干扰素-γ的反应、细胞-细胞黏附以及糖酵解均上调，脂肪酸结合蛋白（FABP4、FABP5）的表达增加，而几种膜受体（STAB1、STAB2、LYVE1、CLEC4G）和丙酮酸代谢、柠檬酸循环、脂肪酸延长、氨基酸代谢和氧化还原过程中的蛋白质表达下调。与时间匹配的对照相比，地塞米松抑制凋亡并改善培养物中的 LSEC 活力，抑制炎症和免疫调节途径以及 IL-1β和 IL-6 的分泌，并进一步上调 FABP4 和 FABP5。无论是否使用地塞米松，24 小时时 LSEC 的孔隙率和内吞活性均降低，但地塞米松处理的细胞表现出应激程度较低的表型。
大鼠 LSEC 在早期培养过程中向促炎表型激活。地塞米松抑制 LSEC 激活、抑制凋亡并提高细胞活力。
