α1AMP-Activated Protein Kinase Protects against Lipopolysaccharide-Induced Endothelial Barrier Disruption via Junctional Reinforcement and Activation of the p38 MAPK/HSP27 Pathway
α1AMP-Activated Protein Kinase Protects against Lipopolysaccharide-Induced Endothelial Barrier Disruption via Junctional Reinforcement and Activation of the p38 MAPK/HSP27 Pathway
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DOI：
10.3390/ijms21155581
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发表时间：
2020-08-01
影响因子：
5.6
通讯作者：
Horman, Sandrine
中科院分区：
文献类型：
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作者：
Ange, Marine;Castanares-Zapatero, Diego;Horman, Sandrine
血管通透性增高是脓毒症病理生理学的决定性因素。而已知AMP活化蛋白激酶（AMPK）在这种情况下在维持内皮屏障功能中起作用。因此，我们研究了这种保护作用的潜在分子机制。在脂多糖（LPS）处理之前，使用α 1 AMPK靶向小干扰RNA或直接药理学AMPK激活剂991调节人真皮微血管内皮细胞中α 1 AMPK的表达和/或活性。蛋白质印迹法用于分析组成细胞连接（闭锁小带-1（ZO-1）、血管内皮钙粘蛋白（VE-Cad）、连接蛋白43（Cx43））或调节肌动蛋白细胞骨架（p38 MAPK;热休克蛋白27（HSP 27））的蛋白质的表达和/或磷酸化。通过体外Transwell试验评估功能性内皮渗透性，并通过免疫荧光评估质膜中细胞连接的定量。通过肌动蛋白荧光染色评价肌动蛋白细胞骨架重塑。因此，我们证明α 1AMPK缺乏与CX43、ZO-1和VE-Cad的表达减少有关，并且CX43的急剧丧失可能是随后ZO-1和VE-Cad在质膜中的表达和定位减少的原因。此外，α 1AMPK激活991保护免受LPS诱导的内皮屏障破坏，加强皮质肌动蛋白细胞骨架。这是由于涉及p38 MAPK和HSP 27的磷酸化的机制，尽管如此，它仍然独立于小的GTdR Rac 1。这导致LPS诱导的高渗透性急剧降低。我们的结论是，适合临床使用的α 1AMPK激活剂可能提供一种特定的治疗干预，限制败血症引起的血管渗漏。
Vascular hyperpermeability is a determinant factor in the pathophysiology of sepsis. While, AMP-activated protein kinase (AMPK) is known to play a role in maintaining endothelial barrier function in this condition. Therefore, we investigated the underlying molecular mechanisms of this protective effect. alpha 1AMPK expression and/or activity was modulated in human dermal microvascular endothelial cells using either alpha 1AMPK-targeting small interfering RNA or the direct pharmacological AMPK activator 991, prior to lipopolysaccharide (LPS) treatment. Western blotting was used to analyze the expression and/or phosphorylation of proteins that compose cellular junctions (zonula occludens-1 (ZO-1), vascular endothelial cadherin (VE-Cad), connexin 43 (Cx43)) or that regulate actin cytoskeleton (p38 MAPK; heat shock protein 27 (HSP27)). Functional endothelial permeability was assessed by in vitro Transwell assays, and quantification of cellular junctions in the plasma membrane was assessed by immunofluorescence. Actin cytoskeleton remodeling was evaluated through actin fluorescent staining. We consequently demonstrate that alpha 1AMPK deficiency is associated with reduced expression of CX43, ZO-1, and VE-Cad, and that the drastic loss of CX43 is likely responsible for the subsequent decreased expression and localization of ZO-1 and VE-Cad in the plasma membrane. Moreover, alpha 1AMPK activation by 991 protects against LPS-induced endothelial barrier disruption by reinforcing cortical actin cytoskeleton. This is due to a mechanism that involves the phosphorylation of p38 MAPK and HSP27, which is nonetheless independent of the small GTPase Rac1. This results in a drastic decrease of LPS-induced hyperpermeability. We conclude that alpha 1AMPK activators that are suitable for clinical use may provide a specific therapeutic intervention that limits sepsis-induced vascular leakage.