Compromised endothelial-cell (EC) barrier function is a hallmark of inflammatory diseases. mTOR inhibitors, widely applied as clinical therapies, cause pneumonitis through mechanisms that are not yet fully understood. This study aimed to elucidate the EC mechanisms underlying the pathogenesis of pneumonitis caused by mTOR inhibition (mTORi). Mice with EC-specific deletion of mTOR complex components (Mtor, Rptor or Rictor) were administered LPS to induce pulmonary injury. Cultured ECs were treated with pharmacologic inhibitors, siRNA, or overexpression plasmids. EC barrier function was evaluated in vivo with Evans blue assay and in vitro by measurement of transendothelial electrical resistance and albumin flux. mTORi increased basal and TNFα-induced EC permeability, which was caused by myosin light chain (MLC) phosphorylation–dependent cell contraction. Inactivation of mTOR kinase activity by mTORi triggered PKCδ/p38/NF-κB signaling that significantly upregulated TNFα-induced MLCK (MLC kinase) expression, whereas Raptor promoted the phosphorylation of PKCα/MYPT1 independently of its interaction with mTOR, leading to suppression of MLCP (MLC phosphatase) activity. EC-specific deficiency in mTOR, Raptor or Rictor aggravated lung inflammation in LPS-treated mice. These findings reveal that mTORi induces PKC-dependent endothelial MLC phosphorylation, contraction, and hyperpermeability that promote pneumonitis.

受损的内皮细胞 (EC) 屏障功能是炎症性疾病的标志。mTOR 抑制剂广泛应用于临床治疗，通过尚未完全了解的机制引起肺炎。本研究旨在阐明 mTOR 抑制 (mTORi) 引起的肺炎发病机制的 EC 机制。对具有 mTOR 复合成分（Mtor、Rptor或Rictor）的EC 特异性缺失的小鼠施用 LPS 以诱导肺损伤。培养的 ECs 用药物抑制剂、siRNA 或过表达质粒进行处理。使用伊文思蓝测定在体内和体外评估 EC 屏障功能通过测量跨内皮电阻和白蛋白通量。mTORi 增加了基础和 TNFα 诱导的 EC 通透性，这是由肌球蛋白轻链 (MLC) 磷酸化依赖性细胞收缩引起的。mTORi 对 mTOR 激酶活性的失活触发了 PKCδ/p38/NF-κB 信号，显着上调了 TNFα 诱导的 MLCK（MLC 激酶）表达，而 Raptor 促进了 PKCα/MYPT1 的磷酸化，而与其与 mTOR 的相互作用无关，导致 MLCP 的抑制(MLC 磷酸酶) 活性。mTOR、Raptor 或 Rictor 的 EC 特异性缺陷加剧了 LPS 治疗小鼠的肺部炎症。这些发现表明，mTORi 诱导 PKC 依赖性内皮 MLC 磷酸化、收缩和渗透性过高，从而促进肺炎。