Data_Sheet_1_Identification of LPS-Activated Endothelial Subpopulations With Distinct Inflammatory Phenotypes and Regulatory Signaling Mechanisms.PDF

Sepsis is a life-threatening condition caused by a dysregulated host response to infection. Endothelial cells (EC) are actively involved in sepsis-associated (micro)vascular disturbances and subsequent organ dysfunction. Lipopolysaccharide (LPS), a Gram-negative bacterial product, can activate EC leading to the expression of pro-inflammatory molecules. This process is molecularly regulated by specific receptors and distinct, yet poorly understood intracellular signaling pathways. LPS-induced expression of endothelial adhesion molecules E-selectin and VCAM-1 in mice was previously shown to be organ- and microvascular-specific. Here we report that also within renal microvascular beds the endothelium expresses different extents of E-selectin and VCAM-1. This heterogeneity was recapitulated in vitro in LPS-activated human umbilical vein EC (HUVEC). Within 2 h after LPS exposure, four distinct HUVEC subpopulations were visible by flow cytometric analysis detecting E-selectin and VCAM-1 protein. These encompassed E-selectin−/VCAM-1− (–/–), E-selectin+/VCAM-1− (E-sel+), E-selectin+/VCAM-1+ (+/+), and E-selectin−/VCAM-1+ (VCAM-1+) subpopulations. The formation of subpopulations was a common response of endothelial cells to LPS challenge. Using fluorescence-activated cell sorting (FACS) we demonstrated that the +/+ subpopulation also expressed the highest levels of inflammatory cytokines and chemokines. The differences in responsiveness of EC subpopulations could not be explained by differential expression of LPS receptors TLR4 and RIG-I. Functional studies, however, demonstrated that the formation of the E-sel+ subpopulation was mainly TLR4-mediated, while the formation of the +/+ subpopulation was mediated by both TLR4 and RIG-I. Pharmacological blockade of NF-κB and p38 MAPK furthermore revealed a prominent role of their signaling cascades in E-sel+ and +/+ subpopulation formation. In contrast, the VCAM-1+ subpopulation was not controlled by any of these signaling pathways. Noteworthy is the existence of a “quiescent” subpopulation that was devoid of the two adhesion molecules and did not express cytokines or chemokines despite LPS exposure. Summarizing, our findings suggest that LPS activates different signaling mechanisms in EC that drive heterogeneous expression of EC inflammatory molecules. Further characterization of the signaling pathways involved will enhance our understanding of endothelial heterogeneous responses to sepsis related stimuli and enable the future design of effective therapeutic strategies to interfere in these processes to counteract sepsis-associated organ dysfunction.

脓毒症（Sepsis）是一种由宿主对感染的应答失调所引发的危及生命的病症。内皮细胞（Endothelial Cells, EC）积极参与脓毒症相关的（微）血管紊乱及后续的器官功能障碍。脂多糖（Lipopolysaccharide, LPS）作为革兰氏阴性菌的产物，可激活内皮细胞，诱导其表达促炎分子。该分子过程由特定受体及独特但尚未完全阐明的细胞内信号通路所调控。此前已有研究证实，小鼠体内脂多糖诱导的内皮黏附分子E-选择素（E-selectin）与血管细胞黏附分子1（VCAM-1）的表达，具有器官特异性与微血管特异性。本研究发现，在肾脏微血管床中，内皮细胞对E-选择素与VCAM-1的表达同样存在异质性。这种异质性在脂多糖激活的人脐静脉内皮细胞（Human Umbilical Vein Endothelial Cells, HUVEC）的体外实验中得以重现。在脂多糖暴露后的2小时内，通过流式细胞术分析（flow cytometric analysis）检测E-选择素与VCAM-1蛋白的表达，可观察到四种明确的HUVEC亚群：分别为E-选择素⁻/VCAM-1⁻（–/–）、E-选择素⁺/VCAM-1⁻（E-sel⁺）、E-选择素⁺/VCAM-1⁺（+/+）以及E-选择素⁻/VCAM-1⁺（VCAM-1⁺）亚群。亚群的形成是内皮细胞应对脂多糖刺激的普遍应答。通过荧光激活细胞分选（Fluorescence-Activated Cell Sorting, FACS）技术，我们证实+/+亚群同时表达水平最高的炎症细胞因子与趋化因子。内皮细胞亚群的应答差异无法通过脂多糖受体Toll样受体4（TLR4）与视黄酸诱导基因I蛋白（RIG-I）的差异表达来解释。然而功能实验表明，E-sel⁺亚群的形成主要由TLR4介导，而+/+亚群的形成则同时依赖TLR4与RIG-I。对核因子κB（NF-κB）及p38丝裂原活化蛋白激酶（p38 MAPK）的药理学阻断实验进一步揭示，这两条信号通路在E-sel⁺与+/+亚群的形成中发挥关键作用。与之相反，VCAM-1⁺亚群不受上述任一信号通路调控。值得注意的是，存在一类“静息”亚群：尽管暴露于脂多糖，该亚群既不表达两种黏附分子，也不产生细胞因子或趋化因子。综上，本研究结果表明，脂多糖可在内皮细胞中激活不同的信号机制，从而驱动内皮细胞炎症分子表达的异质性。对相关信号通路的进一步解析，将加深我们对内皮细胞针对脓毒症相关刺激的异质性应答的理解，并为未来设计有效治疗策略以干预这些过程、缓解脓毒症相关器官功能障碍提供理论依据。