Cells

Acute lung injury (ALI) is a clinically severe respiratory disorder, of which autophagy is the crucial mechanism. Exosomes have the potential to treat ALI, but the role of adipose-derived exosomes (ADEs) in the autophagy of ALI remains unclear. Using an LPS-induced ALI model, the effects of ADE isolated from a lean or diet-induced-obese (DIO) mouse and ADE-carried miRNAs were investigated. After administration of ADEs, the levels of autophagy-related molecules were determined by qRT-PCR, Western blotting, and immunohistochemical staining. Then, a miRNA targeting HMGB1 was screened by bioinformatic analysis and a dual-luciferase reporter assay, and its effect on the HMGB1-driven autophagy in an ALI mouse was investigated as ADEs. The data showed that LPS caused lung injury and activated HMGB1-driven autophagy. The ADEs from a lean mouse or DIO mouse significantly alleviated histopathological lesions, and they inhibited HMGB1-driven autophagy by down-regulating LC3, Beclin-1, and Atg5; the effects of ADEs were not significantly different between a lean and DIO mouse. Of the miRNAs carried by ADE, moreover, miR-142a-3p could specifically bind to HMGB1 mRNA, and up-regulation of pulmonary miR-142a-3p suppressed HMGB1-driven autophagy and relieved lung injuries. Our results indicated that miR-142a-3p and ADEs mitigate LPS-induced ALI by inhibiting HMGB1-driven autophagy, providing new insights on the prevention and treatment of ALI.

In the original publication [...]

Endothelial cell (EC) barrier integrity is tightly regulated by the activity of the non-muscle myosin light chain kinase (nmMLCK) under diverse pathological inflammatory conditions (pneumonia, sepsis) and exposure to mechanical stress. Inflammatory stimuli, including lipopolysaccharide (LPS), cytokines, and damage-associated molecular patterns (DAMPs), increase EC permeability through nmMLCK-dependent EC paracellular gap formation. However, the exact mechanisms by which nmMLCK regulates vascular barrier dysfunction in acute lung injury (ALI) remain incompletely understood. We hypothesized that inflammation-induced ROS results in the peroxynitrite-mediated nitration of nmMLCK that contributes to EC barrier disruption. Human lung EC exposure to either the peroxynitrite donor, SIN-1, or to LPS, triggered significant nmMLCK nitration, which was abolished by the oxidant scavenger, MnTMPyP. Mass spectrometry of SIN-1-treated nmMLCK identified multiple nitrated tyrosines. Nitration of Y1410 proved a critical PTM as site-directed substitution with alanine (Y1410A) abolished both SIN-1- and LPS-induced nmMLCK nitration. nmMLCK nitration disrupts wild-type nmMLCK interaction with Kindlin-2, a cytoskeletal regulator of vascular barrier stability, whereas EC transfected with the Y1410A nmMLCK mutant exhibited preserved Kindlin-2 binding, reflected by alterations in trans-EC electrical resistance (TEER). Consistent with these observations, LPS-challenged murine lungs displayed enhanced nmMLCK nitration and diminished nmMLCK-Kindlin-2 association. Functionally, SIN-1 markedly impaired EC barrier integrity (TEER), which was not observed in ECs expressing the Y1410A mutant. Together, these findings suggest that nmMLCK nitration at Y1410 is a critical molecular mechanism contributing to vascular leakage, highlighting this modification as a potential therapeutic target to reduce inflammation-induced vascular permeability. Given nmMLCK’s established role in barrier regulation, we hypothesized that LPS-induced peroxynitrite formation may promote the nitration of nmMLCK tyrosine residues: a PTM that potentially contribute to nmMLCK’s regulation of EC barrier integrity.