α1AMP-Activated Protein Kinase Protects against Lipopolysaccharide-Induced Endothelial Barrier Disruption via Junctional Reinforcement and Activation of the p38 MAPK/HSP27 Pathway

Abstract

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. α1AMPK expression and/or activity was modulated in human dermal microvascular endothelial cells using either α1AMPK-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 α1AMPK 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, α1AMPK 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 α1AMPK activators that are suitable for clinical use may provide a specific therapeutic intervention that limits sepsis-induced vascular leakage.

AMP-activated protein kinase is a key regulator of acute neurovascular permeability

“…AMPK is primarily known to regulate the energy requirements of the cell, but has also been implicated in other seemingly unrelated cellular processes such as migration, cell growth and apoptosis ( Hardie, 2011 ). This protein kinase has been studied previously in relation to its protective role at the BBB, in particular when challenged by LPS ( Takata et al, 2013 ; Zhao et al, 2014 ; Ange et al, 2020 ), whereas in the retinal pigment epithelium it has been shown to be responsible for permeability induced by IL-1β ( Villarroel et al, 2011 ). However, all these studies address chronic changes and do not focus on the role of AMPK for acute permeability.…”

“…AMP-activated protein kinase (AMPK) is a phylogenetically conserved energy sensor that regulates energy homeostasis by coordinating metabolic pathways and thus balancing energy requirement with nutrient supply ( Hardie, 2018 ). Previous studies suggest that AMPK acts as a protector of BBB integrity, for instance by preventing lipopolysaccharide (LPS)-enhanced NAD(P)H oxidase expression in ECs and the consequent barrier dysfunction and enhanced permeability ( Zhao et al, 2014 ; Ange et al, 2020 ). Moreover, AMPK mediates upregulation of BBB functions induced in vitro by metformin, a drug used for the treatment of diabetes ( Takata et al, 2013 ).…”

Canagliflozin protects against sepsis capillary leak syndrome by activating endothelial α1AMPK

“…Our team and others have previously demonstrated the pivotal role of α1AMPK in the maintenance of endothelial barrier function, in models of endotoxemia 5 , 26 , 27 . In mechanistic terms, we demonstrated that endothelial barrier protection by α1AMPK was mediated by p38MAPK/HSP27-dependent enhancement of VE-Cad stability 25 . Nevertheless, none of the AMPK activators used in the different studies can be safely employed in vivo or securely administered to septic patients 5 , 8 , 27 , 28 .…”

“…The catalytic subunit of AMP-activated protein kinase (AMPK) is primarily expressed under its α1-isoform within the microvascular endothelium; there, it acts as a major regulator of the actin cytoskeleton and IEJs 23 – 25 . Our team and others have previously demonstrated the pivotal role of α1AMPK in the maintenance of endothelial barrier function, in models of endotoxemia 5 , 26 , 27 .…”

Atypical p38 Signaling, Activation, and Implications for Disease

“…Similarly, TAB1-p38 drives T-cell senescence via an AMPK-dependent regulatory pathway, resulting in downregulation of TCR signalosome [ 197 ]. AMPK also plays an essential role in the TAB1-p38 activation of HSP27 in simulated sepsis, maintaining vascular integrity [ 191 ]. Intracellular infection leading to TAB1-p38 activity was first shown in macrophages in mice infected with Toxoplasma gondii, resulting in pro-inflammatory IL-12 production specific to atypical signaling [ 189 ].…”