MPs play an important role in pulmonary vascular leakage and lung injury after I/R.
To explore the relationship between MPs and vascular permeability, I/R and hemorrhage-transfusion (Hemo/Trans) rats were utilized, and flow cytometry was used to analyze MP concentrations in blood. The results showed that the concentration of MP in blood was significantly increased in Hemo/Trans and I/R rats as compared to sham groups (Fig 1A-D). Simultaneously, pulmonary vascular permeability was increased (Fig 1E, F). In addition, large amount of inflammatory cells leaked into the interstitial space of lung (Fig 1G, H) and the protein concentration in bronchoalveolar lavage fluid (BALF) was significantly increased in I/R rats (Suppl Fig 1B). Furthermore, H/R-treated pulmonary VECs were used to confirm the relationship between MPs and vascular permeability. The results showed that H/R induced MP production (Suppl Fig 1C) and caused the increase of monolayer pulmonary VECs permeability with the decrease of trans-endothelial resistance (TER) of monolayer pulmonary VECs (Suppl Fig 1D). The results showed that the the change of MP concentration in blood is positively related to the change of vascular permeability after I/R.
To evaluate the role of MPs in pulmonary vascular leakage and lung injury after I/R, MPs from normal rats (NC-MPs) and I/R rats (I/R-MPs) were obtained and administered to the normal rats (1×108/kg, iv), then the pulmonary vascular leakage and lung injury were observed. The results showed that I/R-MPs induced Evans blue and FITC-labeled albumin leakage to lung tissue, while NC-MPs had no effect (Fig 1I, J, Suppl Fig 1E). In the microvasculature, I/R-MPs also induced the leakage of FITC-labeled albumin into the extra-vascular space (Suppl Fig 1F). Additionally, I/R-MPs induced inflammatory cells infiltration to lung tissue and led to interstitial edema (Fig 1K, L) and also increased the protein concentration in BALF (Suppl Fig 1G). Moreover, in vitro I/R-MPs decreased the TER of monolayer pulmonary VECs (Suppl Fig 1H). These results demonstrated that MPs played a vital role in pulmonary vascular leakage and lung injury after I/R.
EMPs and PMPs have synergistic effect on pulmonary vascular leakage and lung injury after I/R.
Blood MPs can be derived from platelets, red blood cells, endothelial cells and leukocytes. But it is not known which one takes part in vascular leakage after I/R. Thus, we firstly observed the change of EMPs, LMPs and PMPs in blood after I/R. The results indicated that the concentrations of EMPs (Fig 2A, B, Suppl Fig 2A, B), LMPs (Fig 2C, D) and PMPs (Fig 2E, F) in blood were significantly increased after I/R. In vitro, H/R stimulation also induced EMP, LMP and PMP production in VECs, leukocytes and platelets respectively (Suppl Fig2C). Further, administration of H/R-PMPs and H/R-EMPs (Produced in H/R treated platelets and VECs) significantly increased the vascular permeability of normal rats, including a striking pulmonary vascular leakage (Fig 2G, H) and microvasculature leakage (Fig 2I), while H/R-LMPs had no effect. Additionally, H/R-PMPs and H/R-EMPs also induced inflammatory cells infiltration into the pulmonary interstitial space (Fig 2J, K) and increased the protein concentration in BALF (Suppl Fig 2D). In vitro, H/R-PMP and H/R-EMP stimulation led to the decrease of the TER of monolayer pulmonary VECs (Suppl Fig 2E). These results suggest that EMPs and PMPs are the main MPs that participate in pulmonary vascular leakage and lung injury after I/R.
To evaluate the synergistic effect of EMPs and PMPs in the regulation of pulmonary permeability, EMPs and PMPs were infused to the normal rats through the veins, and then the concentrations of the MPs in blood and BALF were analyzed. The results showed that H/R-EMPs increased EMPs in blood as expected, and meanwhile the PMPs both in blood and BALF were also increased (Fig 2L, M). And interestingly, the PMPs in BALF was higher than that in blood (Fig 2L, M). Inversely, H/R-PMPs only increased PMPs in blood and BALF but not EMPs or LMPs in blood and BALF (Suppl Fig 2F, G). These results suggest that H/R-EMPs can induce PMPs production. The further study found that H/R-EMPs induced obvious pulmonary sequestration of platelets, while H/R-PMPs and H/R-LMPs did not had such effect (Fig 2N). These results suggested that in addition to directly regulating pulmonary vascular permeability, EMPs may exacerbate pulmonary leakage and lung injury after I/R through inducing pulmonary sequestration of platelets and PMP production, EMPs and PMPs play a synergistic effect on pulmonary vascular leakage and lung injury.
EMPs and PMPs transport miR-155 and miR-126 respectively to regulate pulmonary vascular permeability.
To confirm the direct interaction between MPs and VECs, PKH26-labeled EMPs, PMPs and LMPs were administered intravenously to normal rats. The results showed that PKH26-labeled EMPs, PMPs and LMPs massively located in the endothelium of the pulmonary vasculature (Fig 3A) and microvasculature (Suppl Fig 3A). In addition, in vitro the EMPs, PMPs and LMPs were absorbed by pulmonary VECs after coculture for 30 min (Suppl Fig 3B, C).
To investigate the mechanism of EMP and PMP regulation of vascular permeability, 17 miRNAs that are closely related to vascular permeability were screened, including miR-141, miR-133, miR-126, miR-129, miR-1, miR-181, miR-125 and so on. The results showed that the amount of miR-1, miR-155, and miR-542 in H/R-EMP were significantly higher than those in NC-EMP (Fig 3B), while the amount of miR-29 and miR-126 in H/R-PMP were significantly higher than those in NC-PMP (Fig 3C). Further experiments showed that in H/R-EMP treated pulmonary VECs, the expression of miR-1, miR-155 and miR-542 was increased(Fig 3D), while in H/R-PMPs treated VECs, the expression of miR-126 and miR-29 was increased(Fig 3E), These results suggested that H/R-EMPs and H/R-PMPs may transport different miRNAs to pulmonary VECs to regulate permeability. To confirm this result, red fluorescence-labeled miR-155 was transfected into the pulmonary VECs, which were then used to produce EMPs. The EMPs were cocultured with VECs for 30 min. Expectedly, The red fluorescence labeled miR-155 was observed entering into the VECs (Suppl Fig 3D), demonstrating that MPs transport miRNAs to the target cells.
To unveil the role of miRNAs in EMPs and PMPs in regulation of vascular permeability, high and low levels of miRNAs containing EMPs and PMPs were used to stimulate corresponding miRNA-inhibited monolayer pulmonary VECs. The results showed that inhibition of miR-1, miR-29, miR-126, miR-155 and miR-542 had no effects on the TER of monolayer VECs, as compared to the normal groups; however, miR-155-high containing EMPs and miR-126-high containing PMPs significantly decreased the TER of monolayer pulmonary VECs (Fig 3F, G). The other miRNA-high-containing EMPs and PMPs had no influence on TER of pulmonary VEC (Fig 3F, G). These results suggest that EMPs carrying miR-155 and PMPs carrying miR-126 participate in regulation of vascular permeability.
EMPs transporting miR-155 that targets ZO-1 and claudin-5, and PMPs transporting miR-126 that targets Cav-1, synergistically regulate pulmonary vascular permeability after I/R.
To investigate how miR-155 transported by EMPs and miR-126 transported by PMPs regulate pulmonary vascular permeability, the effects of H/R-EMPs and H/R-PMPs on the expression of ZO-1, cluadin-5, occludin and VE-cad, and Cav-1 were observed both in vivo and in vitro. The results showed that intravenous administration of H/R-EMPs significantly inhibited the expression of ZO-1, claudin-5 and occludin in the pulmonary vasculature (Fig 4A, B). Moreover, H/R-EMPs inhibited the expression of ZO-1, claudin-5 and occludin in the microvasculature (Fig 4C). These results were confirmed by western blot and confocal microscopy imaging in vitro in pulmonary VECs (Suppl Fig 4A-D). H/R-PMPs induced the high expression of Cav-1 and VE-cad in pulmonary vasculature of rats (Fig 4D, E) and also in pulmonary VECs, H/R-PMP only induced the expression of Cav-1 (Suppl Fig 4, E).
Furthermore, the effects of miR-155-high containing EMPs and miR-126-high containing PMPs on the expression of vascular permeability proteins were observed. The results showed that miR-155-highcontaining EMPs significantly decreased the expression of ZO -1 and claudin-5 (Fig 4F, G) but had no effect on occludin, Cav-1 or VE-Cad. MiR-126-high containing PMPs increased the expression of Cav-1 but had no effect on ZO-1, claudin-5, occludin, or VE-Cad (Fig 4H, I). These results suggest that miR-155 decreases the expression of ZO-1 and claudin-5, miR-126 increases the expression of Cav-1. To elucidate the mechanism by which miR-155 regulates the expression of ZO-1 and claudin-5, a dual-luciferase reporter system was used. The results showed that upregulation of miR-155 significantly reduced the translation of target gene (Fig 4J, K). These results suggest that EMPs transporting miR-155 to down-regulate ZO-1 and claudin-5 and PMPs transporting miR-126 to up-regulate Cav-1 synergistically induce pulmonary vascular leakage via both para-cellular and trans-cellular pathway.
Inhibition of EMP and PMP production benefits pulmonary vascular permeability and lung injury after I/R.
To verify the role of EMPs and PMPs in pulmonary vascular leakage and lung injury after I/R, the effect of EMP inhibitor blebbistatin (BLE)(18) and PMP inhibitor amitriptyline (AMI)(19) on pulmonary vascular permeability and lung injury were observed. The results showed that BLE (10 mg/kg, iv) and AMI (10 mg/kg, iv) markedly reduced the leakage of Evans blues and FITC-labeled albumin into pulmonary interstitial space and improved vascular permeability (Fig 5A, B). Moreover, BLE and AMI treatment significantly alleviated inflammatory cells infiltration (Fig 5C, D) and reduced the concentration of proteins in BALF (Fig 5E). Notably, the combined treatment of BLE (10mg/kg) and AMI (5mg/kg) significantly improved pulmonary vascular permeability and lung injury (Fig 5A-E), exhibiting a good synergistic effect. These results suggest that inhibition of EMP and PMP production benefits pulmonary vascular permeability and lung injury and may be a potential therapeutic strategy.