Myosin Light Chain Kinase–Mediated Endothelial Hyperpermeability Underlies Temsirolimus-Induced Lung Injury

Abstract

Pulmonary toxicity is a common adverse effect of temsirolimus (a first-generation mammalian target of rapamycin (mTOR) inhibitor), but its mechanisms are unclear. Disruption of endothelial-barrier integrity is key in the pathophysiology of lung injury. We investigated the role and mechanisms of endothelial-barrier dysfunction in the pathogenesis of temsirolimus-induced lung injury. We evaluated the impact of temsirolimus on the permeability of human pulmonary microvascular endothelial cells (HPMECs) using transendothelial electrical resistance and albumin leakage while simultaneously investigating its effects on Ca²⁺ release via ryanodine receptors (RyRs) in endothelial cells (ECs). The roles of myosin light chain kinase (MLCK) in endothelial-barrier permeability were studied in MLCK small interfering (si)RNA-transfected HPMECs. In addition, we established a mouse model subjected to intraperitoneal injections of temsirolimus to explore its effects on pulmonary vascular permeability and lung injury. We highlighted the contribution of the MLCK to temsirolimus-induced vascular hyperpermeability and lung injury, supported by studies in two lines of transgenic mice with knocking out MLCK or conditional deletion of MLCK in the endothelium. Temsirolimus increased the permeability of HPMECs, which was correlated with the phosphorylation of myosin light chain (MLC), MLCK activation, and the formation of F-actin stress fibers. Temsirolimus caused a rise in intracellular Ca²⁺ leakage within HPMECs, an effect that was reversed by pretreatment with ryanodine. The latter diminished the phosphorylation of MLCK/MLC induced by temsirolimus, which subsequently led to disruption of the endothelial barrier in HPMECs. Aligning with these in vitro findings, temsirolimus administration resulted in dysfunction of the lung–vascular barrier, characterized by increased protein levels in bronchoalveolar lavage fluid (BALF) and increased permeability of the lung capillary endothelium. Mice with systemic and EC-specific MLCK knockout exhibited reduced temsirolimus-induced pulmonary microvascular hyperpermeability and lung injury. Temsirolimus induced pulmonary endothelial hyperpermeability mediated (at least in part) by the Ca²⁺-dependent MLCK/p-MLC pathway caused EC contraction and contributed to lung injury through mTOR-independent mechanisms.