ANGPT2, ICAM-1, and VWF promote endothelial barrier dysfunction in an in vitro model of ARDS-associated endothelial injury

Acute respiratory distress syndrome (ARDS) is defined by alterations in the permeability and function of pulmonary endothelium, accompanied by inflammation that ultimately results in respiratory failure and increased mortality. Angiopoietin-2 (ANGPT2), intercellular adhesion molecule-1 (ICAM-1), and von Willebrand factor (VWF), among other endothelial injury biomarkers, have been associated with the development of ARDS; however, the exact role of each protein in ARDS pathology remains poorly understood. Here, we investigated the effects of ANGPT2, ICAM-1, and VWF on human pulmonary microvascular endothelial cells (HPMECs) using molecular, functional, and biophysical assays. Exposure to individual endothelial injury proteins, the ANGPT2 + ICAM-1 + VWF combination, or inflammatory cytokines was associated with disrupted VE-cadherin organization, increased F-actin stress-fiber formation, and impaired endothelial barrier integrity. The role of ANGPT2, ICAM-1, and VWF was confirmed by their knockdown using siRNAs. The barrier disruption was restored by recombinant proteins and neutralizing antibodies. At the signaling level, decreased total Tie2, phospho-Tie2, and phospho-AKT abundance occurred together with ICAM-1 upregulation, VWF-associated endothelial activation, NF-κB activation, ROS production, and increased inflammatory signaling, leading to apoptosis and impaired endothelial proliferation, migration, and tube formation. By AFM technique, there was an increase in endothelial cell stiffness, while by Luminex/TMT protein assay, there was cytokine secretion. Overall, these findings demonstrate that ANGPT2, ICAM-1, and VWF can promote barrier disruption, cytoskeletal remodeling, inflammatory signaling, and impaired endothelial function in cultured pulmonary microvascular endothelial cells. These in vitro mechanisms may contribute to endothelial dysfunction associated with ARDS, but their role in pulmonary vascular leakage in vivo requires further validation. Clinical trial number: None