Modulation of the hippo-YAP pathway by cyclic stretch in rat type 2 alveolar epithelial cells—a proof-of-concept study

Abstract

Background: Mechanical ventilation (MV) is a life supporting therapy but may also cause lung damage. This phenomenon is known as ventilator-induced lung injury (VILI). A potential pathomechanisms of ventilator-induced lung injury may be the stretch-induced production and release of cytokines and pro-inflammatory molecules from the alveolar epithelium. Yes-associated protein (YAP) might be regulated by mechanical forces and involved in the inflammation cascade. However, its role in stretch-induced damage of alveolar cells remains poorly understood. In this study, we explored the role of YAP in the response of alveolar epithelial type II cells (AEC II) to elevated cyclic stretch in vitro. We hypothesize that Yes-associated protein activates its downstream targets and regulates the interleukin-6 (IL-6) expression in response to 30% cyclic stretch in AEC II.Methods: The rat lung L2 cell line was exposed to 30% cyclic equibiaxial stretch for 1 or 4 h. Non-stretched conditions served as controls. The cytoskeleton remodeling and cell junction integrity were evaluated by F-actin and Pan-cadherin immunofluorescence, respectively. The gene expression and protein levels of IL-6, Yes-associated protein, Cysteine-rich angiogenic inducer 61 (Cyr61/CCN1), and connective tissue growth factor (CTGF/CCN2) were studied by real-time polymerase chain reaction (RT-qPCR) and Western blot, respectively. Verteporfin (VP) was used to inhibit Yes-associated protein activation. The effects of 30% cyclic stretch were assessed by two-way ANOVA. Statistical significance as accepted at p < 0.05.Results: Cyclic stretch of 30% induced YAP nuclear accumulation, activated the transcription of Yes-associated protein downstream targets Cyr61/CCN1 and CTGF/CCN2 and elevated IL-6 expression in AEC II after 1 hour, compared to static control. VP (2 µM) inhibited Yes-associated protein activation in response to 30% cyclic stretch and reduced IL-6 protein levels.Conclusion: In rat lung L2 AEC II, 30% cyclic stretch activated YAP, and its downstream targets Cyr61/CCN1 and CTGF/CCN2 and proinflammatory IL-6 expression. Target activation was blocked by a Yes-associated protein inhibitor. This novel YAP-dependent pathway could be involved in stretch-induced damage of alveolar cells.

Effect of antibody-mediated connective tissue growth factor neutralization on lung edema in ventilator-induced lung injury in rats

“…CTGF modulates inflammatory responses, which are not only precursors to the fibrotic process, but also play a role in the regulation of vascular permeability. Indeed, increased CTGF levels were paralleled by increased paracellular permeability in cultured epithelial cells exposed to cyclic stretch (Ran et al 2023 ) or following thrombin administration (Akter et al 2022 ). In an experimental animal model of lung injury, increased CTGF levels were paralleled by pulmonary edema (Yang et al 2015 ) and blocking of CTGF reduced pulmonary interstitial edema in an experimental fibrosis model (Bickelhaupt et al 2017 ).…”

“…Probably, vascular permeability is due to disruption of epithelial barrier function during (injurious) mechanical ventilation settings (Wu et al 2008 ; Frank et al 2006 ; Eyal et al 2007 ). In vitro cyclic stretch of alveolar epithelial cells, representing excessive stretch during injurious ventilation, indeed increased CTGF levels and paracellular permeability (Ran et al 2023 ).…”