MPs played an important role in pulmonary vascular leakage and lung injury after I/R.
To explore the link 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 the blood. The results showed that the MP concentration was significantly increased in the blood in I/R and Hemo/Trans rats, as compared to sham groups (Fig. 1A, B, Suppl Fig. 1A). Simultaneously, pulmonary vascular permeability was also increased, with a large amount of Evans Blue leakage to the pulmonary interstitial space (Fig. 1C). Additionally, large amount of neutrophils were found infiltrating into the interstitial space (Fig. 1D, Suppl Fig. 1B) and the protein concentration in Bronchoalveolar Lavage Fluid (BALF) was significantly increased (Suppl Fig. 1C). Furthermore, hypoxia/reoxygenation (H/R)-treated pulmonary VECs were used to investigate the role of MPs on vascular permeability in vitro. The results showed that H/R treatment induced MP production (Suppl Fig. 1D) and caused the TER of pulmonary VEC significantly decreased (Suppl Fig. 1E). These results showed that the MP concentration in blood after I/R is positively related to vascular permeability.
To verify the role of MPs in pulmonary vascular leakage and lung injury after I/R, MPs from normal rats (NC-MPs) and from I/R rats (I/R-MPs) were obtained and administered to rats (1 × 108/kg, iv), the pulmonary vascular leakage and lung injury were observed. The results showed that I/R-MPs induced Evans blue and FITC-labeled albumin leakage into the pulmonary interstitial space, while NC-MPs had no effect (Fig. 1E, Suppl Fig. 1F). In the microvasculature, I/R-MPs also induced the leakage of FITC-labeled albumin into the extra-vascular space (Suppl Fig. 1G). Moreover, I/R-MPs decreased the TER of pulmonary VEC (Suppl Fig. 1H). Additionally, I/R-MPs induced neutrophil infiltration into pulmonary interstitial space (Fig. 1F, Suppl Fig. 1I) and increased the protein concentration in BALF (Suppl Fig. 1J). These results demonstrate that MPs play a vital role in pulmonary vascular leakage and lung injury after I/R.
EMPs and PMPs had synergistic effect in pulmonary vascular leakage and lung injury after I/R.
Blood MPs can be derived from platelets, red blood cells, endothelial cells and neutrophils. But it is not known which one takes part in vascular leakage after I/R. Our study showed that EMP and PMP played an important effect. The results indicated that the concentrations of EMPs (Fig. 2A), PMPs (Fig. 2B) and NMPs in blood (Fig. 2C, Suppl Fig. 2A) were significantly increased after I/R. Administration of H/R-PMPs and H/R-EMPs (Produced in H/R treated platelets and VECs) could significantly increase the permeability of pulmonary vasculature (Fig. 2D) as well as microvasculature in rats (Suppl Fig. 2B), while H/R-NMPs had no effect. H/R-PMP and H/R-EMP stimulation could significantly decrease the TER of pulmonary VEC monolayers (Suppl Fig. 2C). Moreover, H/R-PMPs and H/R-EMPs induced neutrophil infiltration into the pulmonary interstitial space (Fig. 2E) and increased the protein concentration in BALF (Suppl Fig. 2D). These results suggest that EMPs and PMPs are the main MPs that participate in pulmonary vascular leakage and lung injury after I/R.
To examine the synergistic effect of EMPs and PMPs in the regulation of pulmonary permeability, EMPs and PMPs were infused to the rats, and then the concentrations of the MPs in blood and BALF were analyzed. The results showed that H/R-EMPs increased the blood EMPs as expected, and meanwhile the PMP concentration both in blood and BALF were also increased. And interestingly, the PMP concentration in BALF was higher than that in blood (Suppl Fig. 2E, F). Inversely, H/R-PMPs only increased the blood and BALF PMP concentration but not increased the blood and BALF EMPs or NMPs concentration (Suppl Fig. 2G, H). These results suggest that H/R-EMPs can induce the production of PMPs. Our further study found that H/R-EMPs induced obvious pulmonary sequestration of platelets, while H/R-PMPs and H/R-NMPs did not had such effect (Suppl Fig. 2K). These results suggest that in addition to directly regulating pulmonary vascular permeability, EMPs may be through inducing pulmonary sequestration of platelets and PMP production to exacerbate pulmonary leakage and lung injury after I/R, EMPs and PMPs had a synergistic effect on pulmonary vascular leakage and lung injury.
EMP and PMP transport miR-155 and miR-126, respectively, and participate in the regulation of pulmonary vascular permeability.
To confirm the direct interaction between MPs and the endothelium, PKH26-labeled EMPs, PMPs and NMPs were administered intravenously to rats. The results showed that PKH26-labeled EMPs, PMPs and NMPs were massively located in the endodermis of the pulmonary vasculature (Fig. 3A) and microvasculature (Suppl Fig. 3A). In addition, the EMPs, PMPs and NMPs were absorbed by pulmonary VECs after coculture for 30 min (Suppl Fig. 3B).
To investigate the mechanism of EMP and PMP regulation of vascular permeability, 17 miRNAs that are closely related to vascular permeability were studied, including miR-141, miR-133, miR-126, miR-129, miR-1, miR-181, miR-125 and so on. The results showed that the expression of miR-1, miR-155, and miR-542 in the H/R-EMP group were significantly higher than those in the NC-EMP group (Fig. 3B); the expression of miR-29 and miR-126 in the H/R-PMP group were significantly higher than those in the NC-PMP group (Fig. 3C). The further results showed that H/R-EMP treatment could increase the expression of miR-1, miR-155 and miR-542 in pulmonary VEC (Fig. 3D), while H/R-PMPs could increase the expression of miR-126 and miR-29 in pulmonary VECs (Fig. 3E), suggesting that H/R-EMPs and H/R-PMPs may transport different miRNAs to pulmonary VECs. To confirm this conclusion, red fluorescence-labeled miR-155 was transfected into the pulmonary VECs, which were then used to produce EMPs. The EMPs were cocultured with pulmonary VECs for 30 min. The results found red fluorescence was observed in the VECs (Suppl Fig. 3D), demonstrating that MPs may transport miRNAs to the target cells.
To investigate the role of miRNAs in EMP and PMP in regulation of vascular permeability, EMPs and PMPs expressing high and low levels of miRNAs were used to stimulate miRNA-inhibited pulmonary VEC. The results showed that inhibition of miR-1, miR-29, miR-126, miR-155 and miR-542 had no influence on the VEC TER compared to those of the normal groups; however, miR-155-high-expression EMPs significantly decreased the TER of pulmonary VEC (Fig. 3F), and miR-126-high-expressed PMPs significantly decreased the TER pulmonary VEC (Fig. 3G). The other miRNA-high-expressed 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 regulated pulmonary vascular permeability after I/R.
To investigate the mechanism of miR-155 transported by EMPs and miR-126 transported by PMPs regulating 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. 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, Suppl Fig. 4A). The same results were obtained in H/R-EMP-treated pulmonary VECs (Suppl Fig. 4C, D). Moreover, H/R-EMPs inhibited the expression of ZO-1, claudin-5 and occludin in the microvasculature (Fig. 4B). H/R-PMPs increased the expression of Cav-1 both in the pulmonary vasculature of rats and in pulmonary VECs (Fig. 4C, Suppl Fig. 4B, E).
Furthermore, the effects of miR-155-high-expressed EMPs and miR-126-high-expressed PMPs on the expression of vascular permeability proteins were observed. The results showed that miR-155-high-expressed EMPs significantly decreased the expression of ZO -1 and claudin-5 (Fig. 4D, Suppl Fig. 4F) but had no influence on occludin, Cav-1 or VE-Cad. MiR-126-high-expressed PMPs increased the expression of Cav-1 but had no influence on ZO-1, claudin-5, occludin, or VE-Cad (Fig. 4E, Suppl Fig. 4G). These results suggest that miR-155 decreases the expression of ZO-1 and claudin-5, miR-126 increases the expression of Cav-1. To investigate 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 high expression of miR-155 significantly reduced the transcriptional activity of the promoters of ZO-1 and claudin-5 (Fig. 4F, G). These results suggest that EMPs transporting miR-155 to target ZO-1 and claudin-5 and PMPs transporting miR-126 to up-regulate Cav-1 synergistically regulate pulmonary vascular permeability via both para-cellular and trans-cellular pathway.
The inhibition of EMP and PMP production benefited pulmonary vascular permeability and lung injury after I/R.
To verify that EMPs and PMPs play a vital role in pulmonary vascular leakage and lung injury after I/R, the EMP inhibitor blebbistatin (BLE)(18) and PMP inhibitor amitriptyline (AMI)(19) were administered intravenously to rats after I/R, and then 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 the pulmonary interstitial space and improved vascular permeability (Fig. 5A, B). Moreover, BLE and AMI treatment significantly alleviated pulmonary neutrophils infiltration (Fig. 5C, D) and reduced the concentration of proteins in BALF (Fig. 5E). These results suggest that inhibition of EMP and PMP production benefits pulmonary vascular permeability and lung injury and could be a potential therapeutic strategy.