Autophagy inhibitor 3-methyladenine protects against endothelial cell barrier dysfunction in acute lung injury

Abstract

Autophagy is an evolutionarily conserved cellular process that facilitates the continuous recycling of intracellular components (organelles and proteins) and provides an alternative source of energy when nutrients are scarce. Recent studies have implicated autophagy in many disorders, including pulmonary diseases. However, the role of autophagy in endothelial cell (EC) barrier dysfunction and its relevance in the context of acute lung injury (ALI) remain uncertain. Here, we provide evidence that autophagy is a critical component of EC barrier disruption in ALI. Using an aerosolized bacterial lipopolysaccharide (LPS) inhalation mouse model of ALI, we found that administration of the autophagy inhibitor 3-methyladenine (3-MA), either prophylactically or therapeutically, markedly reduced lung vascular leakage and tissue edema. 3-MA was also effective in reducing the levels of proinflammatory mediators and lung neutrophil sequestration induced by LPS. To test the possibility that autophagy in EC could contribute to lung vascular injury, we addressed its role in the mechanism of EC barrier disruption. Knockdown of ATG5, an essential regulator of autophagy, attenuated thrombin-induced EC barrier disruption, confirming the involvement of autophagy in the response. Similarly, exposure of cells to 3-MA, either before or after thrombin, protected against EC barrier dysfunction by inhibiting the cleavage and loss of vascular endothelial cadherin at adherens junctions, as well as formation of actin stress fibers. 3-MA also reversed LPS-induced EC barrier disruption. Together, these data imply a role of autophagy in lung vascular injury and reveal the protective and therapeutic utility of 3-MA against ALI.

The Glycoprotein and Nucleocapsid Protein of Hantaviruses Manipulate Autophagy Flux to Restrain Host Innate Immune Responses

“…As HTNV Gn could suppress host type I IFN responses by inducing mitophagy at the early infection stage, we want to know whether blocking Gn-stimulated complete autophagy upon infection could inhibit HTNV replication in vitro and in vivo. To determine whether blocking HTNV-induced complete autophagy at the early infection stage could inhibit viral replication, we pretreated HUVECs with autophagy inhibitors, including 3-methyladenine (3-MA), which blocks PI3K and suppresses autophagosome formation, and then infected cells with HTNV at an MOI of 1 or used CQ and BAFA1 at 4 hpi, which can sufficiently block the HTNV-triggered complete autophagy but not affect viral entry (Maclean et al, 2008;Slavin et al, 2018;Yeganeh et al, 2015;Zang et al, 2016). In previous studies we have established high throughput methods named ICW assays, which could precisely and quantificationally measure the expression of HTNV NP (Ma et al, 2017a(Ma et al, , 2017b.…”

SENCRstabilizes vascular endothelial cell adherens junctions through interaction with CKAP4

“…2B). To further confirm these findings, a transendothelial electrical resistance (TEER) assay was performed (21,22). Results showed a lower electrical resistance at baseline in HUVEC with SENCR shRNA knockdown compared with control ( Fig.…”

“…TEER Assay. TEER was used to define EC monolayer integrity using an ECIS (electric-substrate impedance sensing) system (Applied BioPhysics), as previously described (22,45). Cells were seeded in eight-chambered electrode arrays (8W10E+) pretreated with cysteine and gelatin.…”

Ubiquitin-based modifications in endothelial cell–cell contact and inflammation

“…If so, do these show vascular-bedspecific distribution and how might they be targeted to preserve vascular integrity and limit inflammation? Another interesting aspect is that autophagy, which comprises lysosomal degradation and recycling of proteins, is vascular barrier-protective in both the brain and the lung (Slavin et al, 2018;Yang et al, 2019). Since K48 ubiquitylation drives lysosomal degradation, this type of ubiquitin modification may directly link cellular homeostasis, controlled by autophagy, to endothelial integrity.…”