Department of Pathology and Medical Biology, Medical Biology Section, University of Groningen, University Medical Center Groningen, Groningen, the Netherlands.
Department of Pathology and Medical Biology, Medical Biology Section, University of Groningen, University Medical Center Groningen, Groningen, the Netherlands; Department of Critical Care, University of Groningen, University Medical Center Groningen, Groningen, the Netherlands.
Biomed Pharmacother. 2021 Jan;133:111073. doi: 10.1016/j.biopha.2020.111073. Epub 2020 Dec 13.
Sepsis is a life-threatening condition often leading to multiple organ failure for which currently no pharmacological treatment is available. Endothelial cells (EC) are among the first cells to respond to pathogens and inflammatory mediators in sepsis and might be a sentinel target to prevent the occurrence of multiple organ failure. Lipopolysaccharide (LPS) is a Gram-negative bacterial component that induces endothelial expression of inflammatory adhesion molecules, cytokines, and chemokines. This expression is regulated by a network of kinases, the result of which in vivo enables leukocytes to transmigrate from the blood into the underlying tissue, causing organ damage. We hypothesised that besides the known kinase pathways, other kinases are involved in the regulation of EC in response to LPS, and that these can be pharmacologically targeted to inhibit cell activation. Using kinome profiling, we identified 58 tyrosine kinases (TKs) that were active in human umbilical vein endothelial cells (HUVEC) at various timepoints after stimulation with LPS. These included AXL tyrosine kinase (Axl), focal adhesion kinase 1 (FAK1), and anaplastic lymphoma kinase (ALK). Using siRNA-based gene knock down, we confirmed that these three TKs mediate LPS-induced endothelial inflammatory activation. Pharmacological inhibition with FAK1 inhibitor FAK14 attenuated LPS-induced endothelial inflammatory activation and leukocyte adhesion partly via blockade of NF-κB activity. Administration of FAK14 after EC exposure to LPS also resulted in inhibition of inflammatory molecule expression. In contrast, inhibition of ALK with FDA-approved inhibitor Ceritinib attenuated LPS-induced endothelial inflammatory activation via a pathway that was independent of NF-κB signalling while it did not affect leukocyte adhesion. Furthermore, Ceritinib administration after start of EC exposure to LPS did not inhibit inflammatory activation. Combined FAK1 and ALK inhibition attenuated LPS-induced endothelial activation in an additive manner, without affecting leukocyte adhesion. Summarising, our findings suggest the involvement of FAK1 and ALK in mediating LPS-induced inflammatory activation of EC. Since pharmacological inhibition of FAK1 attenuated endothelial inflammatory activation after the cells were exposed to LPS, FAK1 represents a promising target for follow up studies.
脓毒症是一种危及生命的疾病，常导致多器官衰竭，目前尚无有效的药物治疗方法。内皮细胞（EC）是对脓毒症中的病原体和炎症介质最早作出反应的细胞之一，可能是预防多器官衰竭发生的一个有希望的靶点。脂多糖（LPS）是革兰氏阴性细菌的一个组成部分，可诱导内皮细胞表达炎症性黏附分子、细胞因子和趋化因子。这种表达受激酶网络的调节，其结果是使白细胞能够从血液中迁移到下面的组织中，从而导致器官损伤。我们假设，除了已知的激酶途径外，其他激酶也参与了 LPS 诱导的 EC 调节，并且可以通过药理学靶向这些激酶来抑制细胞激活。通过激酶组谱分析，我们在 LPS 刺激后不同时间点的人脐静脉内皮细胞（HUVEC）中鉴定出 58 种酪氨酸激酶（TKs）处于活性状态。其中包括 AXL 酪氨酸激酶（Axl）、粘着斑激酶 1（FAK1）和间变性淋巴瘤激酶（ALK）。通过 siRNA 基因敲低，我们证实这三种 TK 介导了 LPS 诱导的内皮炎症激活。FAK1 抑制剂 FAK14 的药理学抑制部分通过阻断 NF-κB 活性减弱了 LPS 诱导的内皮炎症激活和白细胞黏附。在 EC 暴露于 LPS 后给予 FAK14 也导致炎症分子表达的抑制。相比之下，用美国食品和药物管理局批准的抑制剂色瑞替尼抑制 ALK 减弱了 LPS 诱导的内皮炎症激活，其途径独立于 NF-κB 信号，但不影响白细胞黏附。此外，在 EC 暴露于 LPS 后开始给予色瑞替尼并不能抑制炎症激活。FAK1 和 ALK 的联合抑制以相加的方式减弱了 LPS 诱导的内皮激活，而不影响白细胞黏附。总之，我们的研究结果表明，FAK1 和 ALK 参与了 LPS 诱导的 EC 炎症激活的介导。由于 FAK1 的药理学抑制减弱了细胞暴露于 LPS 后的内皮炎症激活，因此 FAK1 代表了一个有前途的后续研究靶点。
