Pulmonary Permeability Research Articles

Rhei Radix et Rhizoma in Xuanbai-Chengqi decoction strengthens the intestinal barrier function and promotes lung barrier repair in preventing severe viral pneumonia induced by influenza A virus

Ethnopharmacological relevanceXuanbai-Chengqi decoction (XCD) is a traditional prescription for treating multiple organ injuries, which has been used to manage pneumonia caused by various pathogens. However, the effects of XCD on repairing pulmonary/intestinal barrier damage remain unclear, and there is a need to understand the compatibility mechanism of rhubarb. Aim of the studyThis work aims to investigate the protective effect and mechanism of XCD on the pulmonary/intestinal barrier guided by the theory of “gut-lung concurrent treatment”. Moreover, we elucidate the compatibility mechanism of rhubarb in XCD. Materials and methodsAn H1N1 virus-infected mouse model was adopted to investigate the reparative effects of XCD on the lung-intestinal barrier by assessing lung-intestinal permeability. Additionally, the characterization of type I alveolar epithelial cells (AT1) and type II alveolar epithelial cells (AT2) was performed to evaluate the damage to the alveolar epithelial barrier. The specific barrier-protective mechanisms of XCD were elucidated by detecting tight junction proteins and the epithelial cell repair factor IL-22. The role of rhubarb in XCD to pneumonia treatment was investigated through lung tissue transcriptome sequencing and flow cytometry. ResultsXCD significantly improved lung tissue edema, inflammation, and alveolar epithelial barrier damage by regulating IL-6, IL-10, and IL-22, which, could further improve pulmonary barrier permeability when combined with the protection of alveolar epithelial cells (AT1 and AT2) as well as inhibition of H1N1 virus replication. Simultaneously, XCD significantly reduced intestinal inflammation and barrier damage by regulating IL-6, IL-1β, and tight junction protein levels (Claudin-1 and ZO-1), improving intestinal barrier permeability. The role of rhubarb in the treatment of pneumonia is clarified for the first time. In the progression of severe pneumonia, rhubarb can significantly protect the intestinal barrier, promote the repair of AT2 cells, and inhibit the accumulation of CD11b+Ly6Gvariable aberrant neutrophils by regulating the S100A8 protein. ConclusionIn summary, our findings suggest that rhubarb in XCD plays a critical role in protecting intestinal barrier function and promoting lung barrier repair in preventing severe viral pneumonia caused by influenza A virus.

Allergic Airway Inflammation Emerges from Gut Inflammation and Leakage in Mouse Model of Asthma.

Asthma is an allergic airway inflammatory disease characterized by type 2 immune responses. Growing evidence suggests an association between allergic airways and intestinal diseases. However, the primary site of disease origin and initial mechanisms involved in the development of allergic airway inflammation (AAI) is not yet understood. Therefore, the initial contributing organs and mechanisms involved in the development of AAI are investigated using a mouse model of asthma. This study, without a local allergen challenge into the lungs, demonstrates a significant increase in intestinal inflammation with signature type-2 mediators including IL-4, IL-13, STAT6, eosinophils, and Th2 cells. In addition, gut leakage and mRNA expressions of gut leakage markers significantly increase in the intestine. Moreover, reduced mRNA expressions of tight junction proteins are observed in gut and interestingly, in lung tissues. Furthermore, in lung tissues, an increased pulmonary barrier permeability and IL-4 and IL-13 levels associated with significant increase of lipopolysaccharide-binding protein (LBP-gut leakage marker) and eosinophils are observed. However, with local allergen challenges into the lungs, these mechanisms are further enhanced in both gut and lungs. In conclusion, the primary gut originated inflammatory responses translocates into the lungs to orchestrate AAI in a mouse model of asthma.

Pulmonary, circulatory and renal considerations in the early postoperative management of the lung transplant recipient.

Lung transplantation volumes and survival rates continue to increase worldwide. Primary graft dysfunction (PGD) and acute kidney injury (AKI) are common early postoperative complications that significantly affect short-term mortality and long-term outcomes. These conditions share overlapping risk factors and are driven, in part, by circulatory derangements. The prevalence of severe PGD is up to 20% and is the leading cause of early death. Patients with pulmonary hypertension are at a higher risk. Prevention and management are based on principles learned from acute lung injury of other causes. Targeting the lowest effective cardiac filling pressure will reduce alveolar edema formation in the setting of increased pulmonary capillary permeability. AKI is reported in up to one-half of lung transplant recipients and is strongly associated with one-year mortality as well as long-term chronic kidney disease. Optimization of renal perfusion is critical to reduce the incidence and severity of AKI. In this review, we highlight key early post-transplant pulmonary, circulatory, and renal perturbations and our center's management approach.

Ganoderma lucidum polysaccharide peptides GL-PPSQ2 alleviate intestinal ischemia-reperfusion injury via inhibiting cytotoxic neutrophil extracellular traps

Ganoderma lucidum polysaccharides peptides (GLPP) are the main effective ingredients from G. lucidum (Leyss. ex Fr.) Karst with anti-inflammatory, antioxidant, and immunoregulatory activities. We extracted and characterized a novel GLPP, named GL-PPSQ2, which were found to have 18 amino acids and 48 proteins, connected by O-glycosidic bonds. The monosaccharide composition of GL-PPSQ2 was determined to be composed of fucose, mannose, galactose and glucose with a molar ratio of 1:1.45:2.37:16.46. By using asymmetric field-flow separation technique, GL-PPSQ2 were found to have a highly branched structure. Moreover, in an intestinal ischemia-reperfusion (I/R) mouse model, GL-PPSQ2 significantly increased the survival rate and alleviated intestinal mucosal hemorrhage, pulmonary permeability, and pulmonary edema. Meanwhile, GL-PPSQ2 significantly promoted intestinal tight junction, decreased inflammation, oxidative stress and cellular apoptosis in the ileum and lung. Analysis with Gene Expression Omnibus series indicates that neutrophil extracellular trap (NET) formation plays an important role in intestinal I/R injury. GL-PPSQ2 remarkedly inhibited NETs-related protein myeloperoxidase (MPO) and citrulline-Histone H3 (citH3) expression. GL-PPSQ2 could alleviate intestinal I/R and its induced lung injury via inhibiting oxidative stress, inflammation, cellular apoptosis, and cytotoxic NETs formation. This study proves that GL-PPSQ2 is a novel drug candidate for preventing and treating intestinal I/R injury.

Silica nanoparticles induce pulmonary damage in rats via VEGFC/D–VEGFR3 signaling-mediated lymphangiogenesis and remodeling

Silica nanoparticles (SiNPs) are widely used as drug carriers for improving drug delivery and retention. The lungs are highly sensitive to the toxicity of SiNPs entering the respiratory tract. Furthermore, pulmonary lymphangiogenesis, which is the growth of lymphatic vessels observed during multiple pulmonary diseases, plays a vital role in promoting the lymphatic transport of silica in the lungs. However, more research is required on the effects of SiNPs on pulmonary lymphangiogenesis. We investigated the effect of SiNP-induced pulmonary toxicity on lymphatic vessel formation in rats and evaluated the toxicity and possible molecular mechanisms of 20-nm SiNPs. Saline containing 3.0, 6.0, and 12.0 mg/kg of SiNPs was instilled intrathecally into female Wistar rats once a day for five days, then sacrificed on day seven. Lung histopathology, pulmonary permeability, pulmonary lymphatic vessel density changes, and the ultrastructure of the lymph trunk were investigated using light microscopy, spectrophotometry, immunofluorescence, and transmission electron microscopy. CD45 expression in lung tissues was determined using immunohistochemical staining, and protein expression in the lung and lymph trunk was quantified using western blotting. We observed increased pulmonary inflammation and permeability, lymphatic endothelial cell damage, pulmonary lymphangiogenesis, and remodeling with increasing SiNP concentration. Moreover, SiNPs activated the VEGFC/D–VEGFR3 signaling pathway in the lung and lymphatic vessel tissues. SiNPs caused pulmonary damage, increased permeability and resulted in inflammation-associated lymphangiogenesis and remodeling by activating VEGFC/D–VEGFR3 signaling. Our findings provide evidence for SiNP-induced pulmonary damage and a new perspective for the prevention and treatment of occupational exposure to SiNPs.

Schisandrin A ameliorates increased pulmonary capillary endothelial permeability accompanied with sepsis through inhibition of RhoA/ROCK1/MLC pathways.

Sepsis is a systemic inflammatory response, and vascular leakage associated with acute lung injury (ALI) is an important pathophysiological process during sepsis. Schisandrin A (SchA) is a bioactive lignan which has been reported to have the anti-inflammatory effects in many studies, while whether SchA can ameliorate ALI-related vascular leakage caused by sepsis is unknown. To evaluate the role and the underlying mechanism of SchA in increase of pulmonary vascular permeability induced by sepsis. The effect of SchA on pulmonary vascular permeability was examined in rat acute lung injury model. The effect of SchA on skin vascular permeability of mice was investigated through Miles assay. MTT assay was performed to detect the cell activity, and transwell assay was used to detect the effect of SchA on cell permeability. The effects of SchA on junction proteins and RhoA/ROCK1/MLC signaling pathway were manifested by immunofluorescence staining and western blot. The administration of SchA alleviated rat pulmonary endothelial dysfunction, relieved increased permeability in the mouse skin and HUVECs induced by lipopolysaccharide (LPS). Meanwhile, SchA inhibited the formation of stress fibers, reversed the decrease of expression of ZO-1 and VE-cadherin. Subsequent experiments confirmed that SchA inhibited RhoA/ROCK1/MLC canonical pathway in rat lungs and HUVECs induced by LPS. Moreover, overexpression of RhoA reversed the inhibitory effect of SchA in HUVECs, which suggested that SchA protected the pulmonary endothelial barrier by inhibiting RhoA/ROCK1/MLC pathway. In summary, our results indicate that SchA ameliorates the increase of pulmonary endothelial permeability induced by sepsis through inhibition of RhoA/ROCK1/MLC pathway, providing a potentially effective therapeutic strategy for sepsis.

Nicotine promotes e-cigarette vapour-induced lung inflammation and structural alterations.

Electronic cigarette (e-cigarette) vapour is gaining popularity as an alternative to tobacco smoking and can induce acute lung injury. However, the specific role of nicotine in e-cigarette vapour and its long-term effects on the airways, lung parenchyma and vasculature remain unclear. In vitro exposure to nicotine-containing e-cigarette vapour extract (ECVE) or to nicotine-free e-cigarette vapour extract (NF ECVE) induced changes in gene expression of epithelial cells and pulmonary arterial smooth muscle cells (PASMCs), but ECVE in particular caused functional alterations (e.g. a decrease in human and mouse PASMC proliferation by 29.3±5.3% and 44.3±8.4%, respectively). Additionally, acute inhalation of nicotine-containing e-cigarette vapour (ECV) but not nicotine-free e-cigarette vapour (NF ECV) increased pulmonary endothelial permeability in isolated lungs. Long-term in vivo exposure of mice to ECV for 8 months significantly increased the number of inflammatory cells, in particular lymphocytes, compared to control and NF ECV in the bronchoalveolar fluid (BALF) (ECV: 853.4±150.8 cells·mL-1; control: 37.0±21.1 cells·mL-1; NF ECV: 198.6±94.9 cells·mL-1) and in lung tissue (ECV: 25.7±3.3 cells·mm-3; control: 4.8±1.1 cells·mm-3; NF ECV: 14.1±2.2 cells·mm-3). BALF cytokines were predominantly increased by ECV. Moreover, ECV caused significant changes in lung structure and function (e.g. increase in airspace by 17.5±1.4% compared to control), similar to mild tobacco smoke-induced alterations, which also could be detected in the NF ECV group, albeit to a lesser degree. In contrast, the pulmonary vasculature was not significantly affected by ECV or NF ECV. NF ECV components induce cell type-specific effects and mild pulmonary alterations, while inclusion of nicotine induces significant endothelial damage, inflammation and parenchymal alterations.

Low temperature reduces occludin expression in bronchial epithelial cells: Implications in cold-induced asthma

BackgroundCold exposure is a common factor to trigger asthma attacks. However, the underlying mechanism has not been thoroughly elucidated. We aimed to investigate the hypothesis that low temperature reduces occludin expression and compromises epithelial barrier function in airways, which in turn, results in asthma exacerbation. MethodsWe examined occludin expression in human bronchial epithelial cell line (Beas-2B) cells exposed to either 29 °C or 37 °C. The following drugs were administered prior to cold treatment: MG132 (a proteasome inhibitor), cycloheximide (a protein synthesis inhibitor), HC-067047 plus GSK2193874 (transient receptor potential vanilloid 4 [TRPV4] antagonists), or C4-ceramide (a glucocorticoid-inducible kinase [SGK1] activator). siNedd4–2 was transfected into Beas-2B cells to investigate the role that Nedd4–2 plays in mediating occludin instability induced by cold. In animal experiments, we treated ovalbumin (OVA)-induced asthmatic mice with a thermoneutral temperature of 30 °C or cold exposure (10 °C, 6 h/day) for 2 weeks. GSK2193874 or C4-ceramide was administered during the cold treatment. Occludin expression of the lung, pulmonary permeability, serum IgE levels, and lung inflammation were assessed. ResultsLow temperature treatment (29 °C) significantly reduced the expression of occludin in Beas-2B cells from 1 to 9 h, which was rescued upon treatment with MG132, HC-067047 plus GSK2193874, C4-ceramide, or Nedd4–2 knockdown. Low temperatures affected occludin stability through SGK1/Nedd4–2-dependent proteolysis. In vivo mice data revealed that cold exposure compromised the airway epithelial barrier function, decreased occludin expression, and exacerbated lung inflammation, which was attenuated by the GSK2193874 or C4-ceramide injection. ConclusionWe identified a potential mechanism underlying cold-induced asthma exacerbation involving Nedd4–2-mediated occludin proteolysis and airway epithelial barrier disruption.

The Wnt/β-catenin pathway regulates inflammation and apoptosis in ventilator-induced lung injury.

Ventilator-induced lung injury (VILI) may be caused by incorrect mechanical ventilation (MV), and its progression is mainly related to inflammatory reaction, apoptosis, and oxidative stress. The Wnt/β-catenin pathway can modulate inflammation and apoptosis; however, its role in VILI is unknown. This research aims to explore the role of the Wnt/β-catenin pathway in VILI. VILI models were established using rats and type II alveolar epithelial (ATII) cells. Glycogen synthase kinase 3β (GSK-3β), β-catenin, and cyclin D1 were determined using western blotting and immunofluorescence. Apoptosis of lung tissues was evaluated using TUNEL, flow cytometry, Bax, and Bcl2 protein. Interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) were detected via enzyme-linked immunosorbent assay (ELISA). Lung pathological injury was evaluated through hematoxylin and eosin (H&E) staining. Lung permeability was evaluated by the ratio of dry to wet weight of lung tissue and the total protein level of bronchoalveolar lavage fluid (BALF). The results showed that GSK-3β expression was enhanced and β-catenin expression was diminished in lung tissue under MV. SB216763 increased β-catenin and cyclin D1 expression by inhibiting GSK-3β expression and inhibited the inflammatory response and apoptosis of lung, alleviated pulmonary edema and lung tissue permeability, and significantly mitigated lung injury. However, inhibition of β-catenin expression by MSAB attenuated the anti-inflammatory and antiapoptotic effects of SB216763 in VILI. Overall, the present study demonstrates that the Wnt/β-catenin pathway activation in MV may play an anti-inflammatory and antiapoptotic role, thereby alleviating lung injury and delaying VILI progression, which may be a key point of intervention in VILI.

Bone mesenchymal stromal cell-derived small extracellular vesicles inhibit inflammation and ameliorate sepsis via delivery of microRNA-21a-5p

Background aimsSepsis is a potentially life-threatening disease that results from a severe systemic inflammatory response due to infection. Mesenchymal stromal cell-derived small extracellular vesicles (MSC sEVs) are able to transfer bioactive molecules and have been demonstrated to play an important role in the pathophysiological process of sepsis. Herein the authors aimed to investigate the potential role and downstream molecular mechanism of MSC sEVs in sepsis. MethodsMSC sEVs were acquired by ultracentrifugation and then injected into a cecal ligation and puncture mouse model. The efficacy of MSC sEVs in both in vitro and in vivo models of sepsis was evaluated. ResultsMSC sEV therapy improved survival, reduced sepsis-induced inflammation, attenuated pulmonary capillary permeability and improved liver and kidney function in septic mice. In addition, the authors found that microRNA-21a-5p (miR-21a-5p) was highly enriched in MSC sEVs, could be transferred to recipient cells, inhibited inflammation and increased survival in septic mice. Furthermore, the authors demonstrated that MSC sEV miR-21a-5p suppressed inflammation by targeting toll-like receptor 4 and programmed cell death 4. The therapeutic efficacy of MSC sEVs was partially abrogated by transfection with miR-21a-5p inhibitors. ConclusionsCollectively, the authors’ data suggest that miR-21a-5p-bearing MSC sEVs may be a prospective and effective sepsis therapeutic strategy.

Integrated Proteomics and Metabolomics Analysis to Explore the Amelioration Mechanisms of Rosa roxburghii Tratt Fruit Polyphenols on Lipopolysaccharide-Induced Acute Lung Injury Mice

Acute lung injury (ALI) is the main cause of death for the elderly and children due to its high morbidity and mortality rates. Plant-derived functional foods are becoming increasingly important to the healthcare and food industries for adjunctive and alternative treatments of ALI. Polyphenols have been regarded to be beneficial to the prevention and amelioration of ALI. Rosa roxburghii Tratt fruit polyphenols (RRTP) has potential to prevent ALI, but mechanism remains unclear. This study was set up to systematically analyze the RRTP extract active ingredients, comprehensively evaluate its protective effects via lung histopathological examination, protein concentration, and cytokines production in ALI mice induced by lipopolysaccharide (LPS), and finally revealed alleviation mechanisms of the regulatory effects of RRTP by proteomics and metabolomics approach. The results demonstrated RRTP could synergistically exert significant preventive effects against ALI by notably ameliorating lung histopathological damage and pulmonary capillary permeability in ALI mice, inhibiting lung tissue inflammatory response and acute phase proteins and S-100 calcium binding proteins, suppressing excessive activation of complement and coagulation cascades, and regulating disordered lipids metabolism and amino acid metabolism. This study illustrated that RRTP has obvious advantages in ALI adjunctive therapy and revealed the complicated amelioration mechanisms, which provides a breakthrough for the development and demonstration of RRTP as a nutritional compound additive for complementary therapy of ALI.

Down-regulation of connexin 43 contributes to structure and function of pulmonary artery in nicotine-administered mice

Dysregulated connexin signaling is implicated in the pathophysiology of pulmonary artery hypertension (PAH). Nicotine affects pulmonary vascular remodeling. However, the potential mechanistic link between connexin signaling and nicotine-induced pulmonary artery remodeling remains unclear. We aimed to investigate the role of connexin 43 (Cx43) in pulmonary artery remodeling in nicotine-administered C57BL/6 J wild-type (WT) and Cx43 heterozygous (Cx43+/−) mice. Hemodynamic parameters and right ventricle pathology were assessed in the mice. Serum biochemical indices of hepatic and renal function were measured. The RT-PCR, immunofluorescence, and western blotting were conducted to evaluate Cx43 mRNA and protein levels. We performed histological staining to identify pulmonary arteries. Wire myography was used to examine contraction and relaxation responses in the pulmonary arteries. Pulmonary vascular permeability was assessed through Evans blue staining. Compared with the WT group, the Cx43+/- group showed lower Cx43 mRNA and protein expression in the pulmonary arteries (P < 0.01). Nicotine treatment significantly increased Cx43 expression (P < 0.01) and induced morphological changes in the pulmonary arteries (P < 0.01). Our findings suggest that Cx43 plays a crucial role in pulmonary artery reactivity and permeability in mice. Furthermore, downregulation of Cx43 expression may contribute to alterations in pulmonary artery structure and function.

Ventilator-induced Lung Injury Is Modulated by the Circadian Clock.

Rationale: Mechanical ventilation (MV) is life-saving but may evoke ventilator-induced lung injury (VILI). Objectives: To explore how the circadian clock modulates severity of murine VILI via the core clock component BMAL1 (basic helix-loop-helix ARNT like 1) in myeloid cells. Methods: Myeloid cell BMAL1-deficient (LysM (lysozyme 2 promoter/enhancer driving cre recombinase expression)Bmal1-/-) or wild-type control (LysMBmal1+/+) mice were subjected to 4 hours MV (34 ml/kg body weight) to induce lung injury. Ventilation was initiated at dawn or dusk or in complete darkness (circadian time [CT] 0 or CT12) to determine diurnal and circadian effects. Lung injury was quantified by lung function, pulmonary permeability, blood gas analysis, neutrophil recruitment, inflammatory markers, and histology. Neutrophil activation and oxidative burst were analyzed ex vivo. Measurements and Main Results: In diurnal experiments, mice ventilated at dawn exhibited higher permeability and neutrophil recruitment compared with dusk. Experiments at CT showed deterioration of pulmonary function, worsening of oxygenation, and increased mortality at CT0 compared with CT12. Wild-type neutrophils isolated at dawn showed higher activation and reactive oxygen species production compared with dusk, whereas these day-night differences were dampened in LysMBmal1-/- neutrophils. In LysMBmal1-/- mice, circadian variations in VILI severity were dampened and VILI-induced mortality at CT0 was reduced compared with LysMBmal1+/+ mice. Conclusions: Inflammatory response and lung barrier dysfunction upon MV exhibit diurnal variations, regulated by the circadian clock. LysMBmal1-/- mice are less susceptible to ventilation-induced pathology and lack circadian variation of severity compared with LysMBmal1+/+ mice. Our data suggest that the internal clock in myeloid cells is an important modulator of VILI.

Challenges and Strategies to Enhance the Systemic Absorption of Inhaled Peptides and Proteins.

Proteins and peptides-based therapeutics are making substantial access to the market due to their obvious advantages of strong potency, high specificity and desirable safety profile. However, most clinical products are mainly delivered via parenteral route with inferior convenience. Lung is an attractive non-invasive alternative passage for systemic administration of biologics with numerous outstanding features, as examples of large absorptive surface area, extensive vascularization and mild local microenvironment. Even so, mucociliary clearance, alveolar macrophage phagocytosis, enzymatic metabolism, pulmonary surfactant adsorption and limited epithelium permeability constitute major obstacles affecting the systemic absorption of inhaled proteins and peptides. This article begins by giving a brief overview of challenges for the systemic absorption of inhaled proteins and peptides, and then goes on to a comprehensive review of possible strategies for enhanced pulmonary absorption, including chemical modification, addition of protease inhibitors, incorporation of absorption enhancers, modification with fusion proteins and development of particulate-based drug delivery systems. These strategies can provide enhanced transmembrane absorption capacity while avoiding pulmonary clearance, offering a valuable reference for designing pulmonary delivery systems of protein and peptide drugs.

Xuanfei Baidu formula alleviates impaired mitochondrial dynamics and activated NLRP3 inflammasome by repressing NF-κB and MAPK pathways in LPS-induced ALI and inflammation models

BackgroundXuanfei Baidu Formula (XBF) is an effective traditional Chinese medicine (TCM) remedy for treating coronavirus disease 2019 (COVID-19) in China. This herbal medicine has shown effects in reducing clinical symptoms and shortening the average length of hospital stay for COVID-19 patients. Previous studies have demonstrated that XBF alleviates acute lung injury (ALI) by regulating macrophage-mediated immune inflammation, but the mechanisms of action remain elusive. PurposeThis study aimed to evaluate the lung-protective and anti-inflammatory effects of XBF and its underlying mechanisms. MethodsHere, XBF's effects were investigated in an ALI mouse model induced by inhalation of atomized lipopolysaccharide (LPS). Besides, the LPS-induced inflammation model in RAW264.7 cells was used to clarify the underlying mechanisms of XBF against ALI. ResultsOur results showed that XBF treatment alleviated LPS-induced lung injury, as evidenced by reduced histopathological changes, pulmonary alveoli permeability, fibrosis, and apoptosis in the lung tissues. In addition, inflammation was alleviated as shown by decreased levels of tumor necrosis factor (TNF)-α, interleukin (IL)-6, IL-1β in serum and bronchoalveolar lavage fluid (BALF), and reduced white blood cell (WBC) count in BALF. Furthermore, consistent with the in vivo assay, XBF inhibited LPS-induced inflammatory cytokines release and pro-inflammatory polarization in RAW264.7 cells. Mechanistically, XBF increased mitochondrial fusion by upregulating Mfn1 and attenuated NLRP3 inflammasome activation by repressing Casp11, respectively, to inhibit NF-κB and MAPK pathways, thus repressing pro-inflammatory macrophage polarization. ConclusionIn this study, we demonstrate that XBF exerts anti-ALI and -inflammatory effects by recovering mitochondrial dynamics and reducing inflammasome activation, providing a biological illustration of the clinical efficacy of XBF in treating COVID-19 patients.

Abstract 15352: Lung Endothelial Transport in Glypican 1 Knockout Mice

Introduction: Pulmonary edema is a life-threatening condition that can result from the acute increase in hemodynamic forces in the lung vasculature. The mechanisms whereby pulmonary edema develops are unresolved but increased lung endothelial permeability may be important. The endothelial glycocalyx is a surface layer known to mediate the effects of mechanical forces in the lung vasculature by eNOS-dependent mechanisms. Among the components of the glycocalyx, glypican 1 (Gpc1) is considered a potential mechano-transducer due to its physical interaction with the cellular membrane. Herein we sought to investigate the mechanisms whereby Gpc1 regulates pulmonary permeability. Hypothesis: We hypothesize that Gpc1 regulates endothelial transport by eNOS-dependent mechanisms. Methods: Mouse lung endothelial cells (MLEC) isolated from male and female wild-type (WT) and Gpc1 knockout mice (Gpc1KO, 6-8 weeks old) were used. Endothelial transport was assessed by dextran permeability in vitro, lung barrier stability was assessed by TEER, eNOS pathway was assessed by immunoblotting. To assess the role of eNOS to Gpc1 effects on endothelial transport, MLEC were exposed to an eNOS decoy peptide. Results: Gpc1KO MLEC formed a tighter barrier in vitro when compared to WT MLEC (3.18±0.11 [Gpc1KO] vs 2.34±0.11 [WT], normalized TEER, n=3/group). This was preserved during high pressure conditions. High pressure increased transcellular but not paracellular transport in both WT and Gpc1KO MLEC (1.8 fold in WT and 1.2 fold in Gpc1KO, n=6/group). Gpc1KO showed decreased eNOS expression (0.3 fold, n=4/group ) and activity during (1.947±0.32 [WT] vs 1.316±0.13 [Gpc1KO], n=4/group). The eNOS decoy peptide increased transcellular transport only in Gpc1KO MLEC (3 fold, n=5/group). Conclusions: In conclusion, these data suggest that intracellular transport may be the main mechanism for pressure-induced changes in endothelial permeability in MLEC . The mechanisms whereby Gpc1 regulates transcellular transport seem to be associated with eNOS signaling. This may be important for the management of acute changes in pressure seen in clinical settings when NO donors are used as vasodilators to decrease blood pressure.

Computational prediction of Calu-3-based in vitro pulmonary permeability of chemicals

Pulmonary is a potential route for drug delivery and exposure to toxic chemicals. The human bronchial epithelial cell line Calu-3 is generally considered to be a useful in vitro model of pulmonary permeability by calculating the apparent permeability coefficient (Papp) values. Since in vitro experiments are time-consuming and labor-intensive, computational models for pulmonary permeability are desirable for accelerating drug design and toxic chemical assessment. This study presents the first attempt for developing quantitative structure-activity relationship (QSAR) models for addressing this goal. A total of 57 chemicals with Papp values based on Calu-3 experiments was first curated from literature for model development and testing. Subsequently, eleven descriptors were identified by a sequential forward feature selection algorithm to maximize the cross-validation performance of a voting regression model integrating linear regression and nonlinear random forest algorithms. With applicability domain adjustment, the developed model achieved high performance with correlation coefficient values of 0.935 and 0.824 for cross-validation and independent test, respectively. The preliminary results showed that computational models could be helpful for predicting Calu-3-based in vitro pulmonary permeability of chemicals. Future works include the collection of more data for further validating and improving the model.

The impact of platelets on pulmonary microcirculation throughout COVID-19 and its persistent activating factors.

Patients with COVID-19 often have hypoxemia, impaired lung function, and abnormal imaging manifestations in acute and convalescent stages. Alveolar inflammation, pulmonary vasculitis, and thromboembolism synergistically damage the blood-air barrier, resulting in increased pulmonary permeability and gas exchange disorders. The incidence of low platelet counts correlates with disease severity. Platelets are also involved in the impairment of pulmonary microcirculation leading to abnormal lung function at different phases of COVID-19. Activated platelets lose the ability to protect the integrity of blood vessel walls, increasing the permeability of pulmonary microvasculature. High levels of platelet activation markers are observed in both mild and severe cases, short and long term. Therefore, the risk of thrombotic events may always be present. Vascular endothelial injury, immune cells, inflammatory mediators, and hypoxia participate in the high reactivity and aggregation of platelets in various ways. Microvesicles, phosphatidylserine (PS), platelets, and coagulation factors are closely related. The release of various cell-derived microvesicles can be detected in COVID-19 patients. In addition to providing a phospholipid surface for the synthesis of intrinsic factor Xase complex and prothrombinase complex, exposed PS also promotes the decryption of tissue factor (TF) which then promotes coagulant activity by complexing with factor VIIa to activate factor X. The treatment of COVID-19 hypercoagulability and thrombosis still focuses on early intervention. Antiplatelet therapy plays a role in relieving the disease, inhibiting the formation of the hypercoagulable state, reducing thrombotic events and mortality, and improving sequelae. PS can be another potential target for the inhibition of hypercoagulable states.

Adjunctive therapy with the Tie2 agonist Vasculotide reduces pulmonary permeability in Streptococcus pneumoniae infected and mechanically ventilated mice

Community acquired pneumonia, mainly caused by Streptococcus pneumoniae (S.pn.), is a common cause of death worldwide. Despite adequate antibiotic therapy, pneumococcal pneumonia can induce pulmonary endothelial hyperpermeability leading to acute lung injury, which often requires mechanical ventilation (MV) causing ventilator-induced lung injury (VILI). Endothelial stabilization is mediated by angiopoietin-1 induced Tie2 activation. PEGylated (polyethylene glycol) Tie2-agonist Vasculotide (VT) mimics Angiopietin-1 effects. Recently, VT has been shown to reduce pulmonary hyperpermeability in murine pneumococcal pneumonia. The aim of this study was to determine whether VT reduces lung damage in S.pn. infected and mechanically ventilated mice. Pulmonary hyperpermeability, immune response and bacterial load were quantified in S.pn. infected mice treated with Ampicillin + /−VT and undergoing six hours of MV 24 h post infection. Histopathological lung changes, Tie2-expression and -phosphorylation were evaluated. VT did not alter immune response or bacterial burden, but interestingly combination treatment with ampicillin significantly reduced pulmonary hyperpermeability, histological lung damage and edema formation. Tie2-mRNA expression was reduced by S.pn. infection and/or MV but not restored by VT. Moreover, Tie2 phosphorylation was not affected by VT. These findings indicate that VT may be a promising adjunctive treatment option for prevention of VILI in severe pneumococcal pneumonia.

MILD TRAUMATIC BRAIN INJURY ATTENUATES PNEUMONIA-INDUCED LUNG INJURY BY MODULATIONS OF ALVEOLAR MACROPHAGE BACTERICIDAL ACTIVITY AND M1 POLARIZATION.

Traumatic brain injury is one of the main causes of death and disability worldwide, and results in multisystem complications. However, the mechanism of mild traumatic brain injury (MTBI) on lung injury remains unclear. In this study, we used a murine model of MTBI and pneumonia ( Pseudomonas aeruginosa ;) to explore the relationship between these conditions and the underlying mechanism. Methods: Mice (n = 104) were divided into control, MTBI, pneumonia, and MTBI + pneumonia groups. MTBI was induced by the weight-drop method. Pneumonia was induced by intratracheal injection with P. aeruginosa Xen5 strain. Animals were killed 24 h after bacterial challenging. Histological, cellular, and molecular indices of brain and lung injury were assessed using various methods. Results: Mice in both the MTBI and pneumonia groups had more Fluoro-Jade C-positive neurons than did the controls ( P < 0.01), but mice in the MTBI + pneumonia group had fewer Fluoro-Jade C-positive cells than did the pneumonia group ( P < 0.01). The MTBI + pneumonia mice showed decreased bacterial load ( P < 0.05), reduced lung injury score and pulmonary permeability ( P < 0.01), less inflammatory cells, and lower levels of proinflammatory cytokines (TNF-α and IL-1β; P < 0.01) when compared with the pneumonia group. Molecular analysis indicated lower levels of phosphorylated nuclear factor-κB in the lung of MTBI + pneumonia mice compared with the pneumonia group ( P < 0.01). Furthermore, alveolar macrophages from MTBI mice exhibited enhanced bactericidal capacity compared with those from controls ( P < 0.01). Moreover, MTBI + pneumonia mice exhibited less CD86-positive M1 macrophages compared with the pneumonia group ( P < 0.01). Conclusions: MTBI attenuates pneumonia-induced acute lung injury through the modulation of alveolar macrophage bactericidal capacity and M1 polarization in bacterial pneumonia model.