Everolimus-induced hyperpermeability of endothelial cells causes lung injury.

The mammalian target of rapamycin (mTOR) inhibitors, everolimus (but not dactolisib), is frequently associated with lung injury in clinical therapies. However, the underlying mechanisms remain unclear. Endothelial cell barrier dysfunction plays a major role in the pathogenesis of the lung injury. This study hypothesizes that everolimus increases pulmonary endothelial permeability, which leads to lung injury. We tested the effects of everolimus on human pulmonary microvascular endothelial cell (HPMEC) permeability and a mouse model of intraperitoneal injection of everolimus was established to investigate the effect of everolimus on pulmonary vascular permeability. Our data showed that everolimus increased human pulmonary microvascular endothelial cell (HPMEC) permeability which was associated with MLC phosphorylation and F-actin stress fiber formation. Furthermore, everolimus induced an increasing concentration of intracellular calcium Ca2+ leakage in HPMECs and this was normalized with ryanodine pretreatment. In addition, ryanodine decreased everolimus-induced phosphorylation of PKCα and MLC, and barrier disruption in HPMECs. Consistent with in vitro data, everolimus treatment caused a visible lung-vascular barrier dysfunction, including an increase in protein in BALF and lung capillary-endothelial permeability, which was significantly attenuated by pretreatment with an inhibitor of PKCα, MLCK, and ryanodine. This study shows that everolimus induced pulmonary endothelial hyper-permeability, at least partly, in an MLC phosphorylation-mediated EC contraction which is influenced in a Ca2+-dependent manner and can lead to lung injury through mTOR-independent mechanisms.

PECAM-1 mediates temsirolimus-induced increase in neutrophil transendothelial migration that leads to lung injury.

Temsirolimus is a first-generation mTOR inhibitor commonly used in the clinical treatment of cancers that is associated with lung injury. However, the mechanism underlying this adverse effect remains elusive. Endothelial barrier dysfunction plays a pivotal role in the infiltration of neutrophils into the pulmonary alveoli, which eventually induces lung injury. The present study demonstrates that temsirolimus induces the aberrant expression of adhesion molecules in endothelial cells, leading to enhanced neutrophil infiltration and subsequent lung injury. Results of a mouse model revealed that temsirolimus disrupted capillary-alveolar barrier function and facilitated neutrophil transmigration across the endothelium within the alveolar space. Consistent with our in vivo observations, temsirolimus impaired intercellular barrier function within monolayers of human lung endothelial cells, resulting in increased neutrophil infiltration. Furthermore, we demonstrated that temsirolimus-induced neutrophil transendothelial migration was mediated by platelet endothelial cell adhesion molecule-1 (PECAM-1) in both in vitro and in vivo experiments. Collectively, these findings highlight that temsirolimus induces endothelial barrier dysfunction via PECAM-1-dependent pathway both in vitro and in vivo, ultimately leading to neutrophil infiltration and subsequent pulmonary injury.

Activation of mTOR mediates hyperglycemia-induced renal glomerular endothelial hyperpermeability via the RhoA/ROCK/pMLC signaling pathway.

OBJECTIVE: Hyperglycemia is associated with albuminuria and renal glomerular endothelial dysfunction in patients with diabetic nephropathy. The mTOR and RhoA/ROCK signaling pathways are involved in glomerular filtration barrier (GFB) regulation, but their role in high glucose (HG)-induced GFB dysfunction in human renal glomerular endothelial cells (HRGECs) has not been investigated. This study aimed to investigate the mechanisms of HG-induced GFB dysfunction in vitro. MATERIALS AND METHODS: HRGECs were cultured in vitro and exposed to HG. The horseradish peroxidase-albumin leakage and transendothelial electrical resistance of the endothelial monolayer were measured after HG treatment with or without rapamycin preincubation. A fluorescence probe was used to study the distribution of F-actin reorganization. The phosphorylation levels of myosin light chain (MLC) and mTOR were measured via western blotting. RhoA activity was evaluated via GTPase activation assay. The effects of blocking mTOR or the RhoA/ROCK pathway on endothelial permeability and MLC phosphorylation under HG conditions were observed. RESULTS: HG exposure induced F-actin reorganization and increased MLC phosphorylation, leading to EC barrier disruption. This effect was attenuated by treatment with rapamycin or Y-27632. Phospho-MLC (pMLC) activation in HRGECs was mediated by RhoA/ROCK signaling. mTOR and RhoA/ROCK inhibition or knockdown attenuated pMLC activation, F-actin reorganization and barrier disruption that occurred in response to HG exposure. CONCLUSIONS: Our results revealed that HG stimulation upregulated RhoA expression and activity through an mTOR-dependent pathway, leading to MLC-mediated endothelial cell cytoskeleton rearrangement and glomerular endothelial barrier dysfunction.

[Effects of Straw Returning with Chemical Fertilizer on Soil Enzyme Activities and Microbial Community Structure in Rice-Rape Rotation].

Straw returning is an effective technique for improving soil fertility and maintaining crop productivity in agro-ecosystems. The effects of straw returning, when combined with chemical fertilizer, on soil nutrients, enzyme activity, and microbial community were explored in rice-rape rotation farmland in the Chaohu Area. We carried out a 4-year field experiment (2016-2020) and set up four treatments (no straw+no fertilization, CK; conventional fertilization, F; straw returning+conventional fertilization, SF; and straw returning+conventional fertilization minus 20%, SDF) to explore the key environmental factors affecting soil enzyme activity and microbial and fungal communities. The results showed that straw returning combined with chemical fertilizer could improve soil nutrient content, with the SF treatment resulting in the highest soil nutrient content. Compared with F, the SF treatment significantly increased the organic matter (OM) and total phosphorus (TP) content of the soil, by 7.94% and 24.07%, respectively, in rice seasons (P<0.05), while the alkaline nitrogen (AN) content was significantly increased by 13.62% in rape seasons (P<0.05). Compared with F, the SF treatment also significantly increased soil phosphatase and urease, by 28.54% and 24.13% in rice seasons and 38.97% and 30.70% in rape seasons, respectively (P<0.05). Compared with F, SDF treatments significantly increased urease activity by 20.31% in rice seasons and 24.33% in rape seasons (P<0.05). The results indicated that straw returning increased both the Chao1 and Shannon indices of soil bacteria in rice seasons, whereas decreased these indices in rape seasons. However, the Chao1 and Shannon index of the fungal community increased after straw returning. In terms of microbial community structure, the relative abundance of Proteobacteria in SF and SDF treatments increased by 8.22% and 7.88% in rice seasons and 18.53% and 5.68% in rape seasons, respectively, compared with the F treatment. Compared with F, the relative abundance of Chloroflexi in SF and SDF treatments increased by 12.00% and 11.25% in rice seasons and 15.02% and 8.43% in rape seasons, respectively. Compared with F, the relative abundance of Basidiomycota in SF and SDF treatments in rice seasons increased by 70% and 43.42% (P<0.05), respectively, while ascomycetes in rape seasons increased by 69.79% and 43.72% (P<0.05), respectively. In conclusion, straw returning combined with chemical fertilizer can improve soil nutrient content. Soil urease and phosphatase were more sensitive to straw returning. The compositional changes in the bacterial community of the soil were mainly affected by soil TP and available phosphorus (AP), whereas OM, AN, and pH were the main environmental factors causing changes in the fungal community composition. Consequently, straw returning can improve soil fertility and maintain ecosystem health.