Lung Permeability Research Articles

COVID-19-RELATED ARDS PHENOTYPES AND PROGRESSION BETWEEN THEM: A CASE REPORT

SESSION TITLE: Fellows' COVID-19 SESSION TYPE: Fellow Case Reports PRESENTED ON: October 18-21, 2020 INTRODUCTION: Since January 20th, 2020 when the first case of the novel coronavirus SARS-CoV-2 was confirmed in the United States [1], there have been several reports of the disease caused by the virus known as COVID19. In the recent literature there have been descriptions of two different phenotypes of the virus presentation among hospitalized patients and particularly those requiring mechanical ventilation. These phenotypes, known as “L type” and “H type”, report what appear to be two distinct presentations with drastically different management which a patient can transition between [2]. We present a case of a patient with confirmed COVID19 who progressed through both phenotypes during his treatment consistent with these theorized phenotypes. CASE PRESENTATION: A 40 year old male presented to the hospital with shortness of breath and cough. Medical and social history was unremarkable. Workup initially showed patchy bilateral infiltrates, negative procalcitonin, fever of 103, and sats of 93% on 4L. The patient’s respiratory status quickly degraded and he required mechanical ventilation. He tested positive for SARS-CoV-2 via PCR. Initially the patient had subpleural ground glass opacities and low peep requirements before stress index became >1. He developed dyssynchrony and required more peep causing higher pressure with worse compliance. Standard supportive care for ARDS was followed. After several days the peak and plateau pressures continued to be elevated. The patient was started on nitric oxide and flolan with paralytics. He returned to a high compliance state and was ultimately extubated and discharged. DISCUSSION: There are two phenotypes with COVID-19 ARDS. The first, L-type, has high compliance, low V/Q ratio, and low lung recruitability. It has been theorized that viral infection leads to interstitial edema with vasoplegia accounting for the severe hypoxemia. This leads to increased minute volume and a more negative intrathoracic pressure with little dyspnea. The increased negative intrathoracic pressure and high tidal volumes likely lead to edema due to inflammation and increased lung permeability. This leads to dependent atelectasis and increased work of breathing. It is unclear if this is from the virus itself versus and/or high-stress ventilation. The second, H-type, has decreased compliance and high recruitability. One of the crucial things to prevent progression from L to H type is controlling work of breathing. In community hospital settings this is hard to monitor. The patient's dyssynchrony caused a significantly negative intrathoracic pressure and resulted in a form of patient-self inflicted lung injury that caused the transition from L to H phenotype. Once this was controlled with paralytics, the patient returned to L-type and recovered. CONCLUSIONS: Control of the work of breathing in a patient with COVID-19 ARDS is critical in preventing progression between L and H phenotypes. Reference #1: 1. Holshue LH, DeBolt C, Lindquist S, et al. First Case of 2019 Novel Coronavirus in the United States. N Engl J Med. 2020; 382:929-936. Reference #2: 2. Gattinoni DC, Caironi P, Busana M, et al. COVID-19 pneumonia: different respiratory treatment for different phenotypes?. J Intens Care Med. 2020; DOI: 10.1007/s00134-020-06033-2. Reference #3: 3. Yoshida T, Grieco DL, Brochard L, Fujino Y. Patient self inflicted lung injury and positive end-expiratory pressure for safe spontaneous breathing. Curr Opin Crit Care. 2020; 26(1):59-65. DISCLOSURES: No relevant relationships by Muhammad Ahmed, source=Admin input no disclosure on file for Nisarfathima Kazimuddin; No relevant relationships by Ahmed Qadir, source=Web Response No relevant relationships by karan Singh, source=Web Response No relevant relationships by Rodney Steff, source=Web Response

Gut-lymph-lung pathway mediates sepsis-induced acute lung injury.

This review attempts to unveil the possible mechanisms underlying how gut lymph affects lung and further gives rise to acute respiratory distress syndrome, as well as potential interventional targets under the condition of ischemia-reperfusion injury. We searched electronic databases including PubMed, MEDLINE, Cochrane Central Register of Controlled Trials, Google Scholar, Web of Science, and Embase to identify relevant literatures published up to December 2019. We enrolled the literatures including the Mesh Terms of “gut lymph or intestinal lymph and acute lung injury or acute respiratory distress syndrome.” Gut is considered to be the origin of systemic inflammation and the engine of multiple organ distress syndrome in the field of critical care medicine, whereas gut lymph plays a pivotal role in initiation of ischemia-reperfusion injury-induced acute respiratory distress syndrome. In fact, in the having been established pathologic model of sepsis leading to multiple organ dysfunction named by Gut Lymph theory, a variety of literatures showed the position and role of changes in gut lymph components in the initiation of systemic inflammatory response, which allows us to screen out potential intervention targets to pave the way for future clinic and basic research.

Protectin DX ameliorates inflammation in sepsis-induced acute lung injury through mediating PPARγ/NF-κB pathway.

Previous reports have demonstrated that the newly identified lipid mediator protectin DX (PDX) could effectively attenuate multiple organ injuries in sepsis. The aim of our study was to clarify whether PDX could improve acute lung injury (ALI) induced by sepsis and elucidate the relevant potential mechanism. After inducing sepsis by the cecal ligation and puncture approach, mice were treated with a high or low dose of PDX. Pathological changes in the pulmonary tissue were analyzed by hematoxylin-eosin staining, and lung injury score was evaluated. Lung permeability and edema were assessed by lung wet/dry ratio, and protein and cellular load of the bronchoalveolar lavage fluid (BALF). Inflammatory cytokine levels in BALF were measured by ELISA and the expression of PPARγ in the lung tissue was analyzed by immunoblotting. The results suggested that PDX could diminish the inflammatory response in lung tissue after sepsis by upregulating PPARγ and inhibiting the phosphorylation and activation of NF-κB p65. PDX treatment lowered the levels of pro-inflammation cytokines IL-1β, IL-6, TNF-α, and MCP-1, and the levels of anti-inflammatory cytokine IL-10 was increased in the BALF. It also improved lung permeability and reduced lung injury. Furthermore, the protective effect of PDX on lung tissue could be reversed by GW9662, a specific PPAR-γ antagonist. Taken together, our study indicated that PDX could ameliorate the inflammatory response in ALI by activating the PPARγ/NF-κB pathway in a mouse model of sepsis.

Role of microRNA-15a-5p/TNFAIP3-interacting protein 2 axis in acute lung injury induced by traumatic hemorrhagic shock.

The present study aimed to investigate the role of microRNA (miR)-15a-5p in the pathogenesis of acute lung injury induced by traumatic hemorrhagic shock (THS), and to explore the underlying molecular mechanism. The expression level of miR-15a-5p was detected using reverse transcription-quantitative (RT-qPCR) and the association between miR-15a-5p and TNFAIP3-interacting protein 2 (TNIP2) was revealed using TargetScan and dual luciferase reporter assays. To investigate the effect of miR-15a-5p on THS-induced acute lung injury, a THS rat model was established. Lung capillary permeability and lung edema were then determined. Moreover, proinflammatory factors in the bronchoalveolar lavage fluid (BALF) and serum of the THS rat model were detected using ELISA. In addition, protein levels in the current study were measured via western blotting. It was revealed that miR-15a-5p was significantly upregulated in both patients with THS and samples from the THS rat model. TNIP2 represents a direct target of miR-15a-5p, and it was downregulated in both patients with THS and the THS rat model. Further analyses indicated that downregulation of miR-15a-5p significantly relieved acute lung injury induced by THS, evidenced by a decreased ratio of Evan's blue dye (EBD) in the BALF to EBD in plasma of THS rats, decreased lung permeability index and reduced lung wet/dry ratio. Inhibition of miR-15a-5p also decreased THS-induced upregulation of pro-inflammatory factors. Furthermore, the data revealed that THS-induced NF-κB activation in the lung tissues of rats was inhibited by miR-15a-5p knockdown. Moreover, it was demonstrated that all the effects of miR-15a-5p on THS rats were ablated following TNIP2 silencing. Taken together, the data of the current study indicate that miR-15a-5p downregulation serves a protective role in THS-induced acute lung injury via directly targeting TNIP2.

Role and Mechanism of Maresin-1 in Acute Lung Injury Induced by Trauma-Hemorrhagic Shock.

BackgroundIt is reported that trauma hemorrhagic shock (THS) could resulted in organ injury and is related to a high mortality rate. Maresin-1 (MaR1), a derived medium through biosynthesis, is involved in inflammatory responses. However, the mechanism of MaR1 against acute lung injury needs to be further understood. This report aimed to explore whether MaR1 had a protective effect on lung injury.Material/MethodsWe constructed a THS-induced acute lung damage rat model and then treated the rats with MaR1. We determined Evan’s blue dye (EBD) lung permeability, lung permeability index, wet/dry (W/D) weight ratio, nitric oxide (NO) concentration and inducible nitric oxide synthase (iNOS) expression in lung tissue samples. The inflammation-related cytokines levels in the bronchoalveolar lavage fluid (BALF) and serum of rats were determined by enzyme-linked immunosorbent assay (ELISA). Finally, the TLR4/p38MAPK/NF-κB pathway was analyzed by quantitative real-time polymerase chain reaction and western blot assay.ResultsThe increased EBD ratio, lung permeability index and W/D weight ratio, NO concentration and iNOS levels were suppressed by MaR1 treatment. THS-induced over-production of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) in BALF and serum was suppressed by MaR1. Besides, the TLR4/p38MAPK/NF-κB pathway activation in THS-induced rats were inhibited by MaR1 treatment.ConclusionsOur study showed that MaR1 could effectively alleviated THS-induced lung injury via inhibiting the excitation of the TLR4/p38MAPK/NF-κB pathway in THS-induced rats, suggesting that MaR1 might be a novel agent for lung damage treatment.

α-Tocopherol Attenuates the Severity of Pseudomonas aeruginosa-induced Pneumonia.

Pseudomonas aeruginosa is a lethal pathogen that causes high mortality and morbidity in immunocompromised and critically ill patients. The type III secretion system (T3SS) of P. aeruginosa mediates many of the adverse effects of infection with this pathogen, including increased lung permeability in a Toll-like receptor 4/RhoA/PAI-1 (plasminogen activator inhibitor-1)-dependent manner. α-Tocopherol has antiinflammatory properties that may make it a useful adjunct in treatment of this moribund infection. We measured transendothelial and transepithelial resistance, RhoA and PAI-1 activation, stress fiber formation, P. aeruginosa T3SS exoenzyme (ExoY) intoxication into host cells, and survival in a murine model of pneumonia in the presence of P. aeruginosa and pretreatment with α-tocopherol. We found that α-tocopherol alleviated P. aeruginosa-mediated alveolar endothelial and epithelial paracellular permeability by inhibiting RhoA, in part, via PAI-1 activation, and increased survival in a mouse model of P. aeruginosa pneumonia. Furthermore, we found that α-tocopherol decreased the activation of RhoA and PAI-1 by blocking the injection of T3SS exoenzymes into alveolar epithelial cells. P. aeruginosa is becoming increasingly antibiotic resistant. We provide evidence that α-tocopherol could be a useful therapeutic agent for individuals who are susceptible to infection with P. aeruginosa, such as those who are immunocompromised or critically ill.

The effects of human prothrombin complex concentrate on hemorrhagic shock-induced lung injury in rats: Implications for testing human blood products in rodents.

Hemorrhagic shock (HS) and trauma can result in an endotheliopathy of trauma, characterized by endothelial compromise, inflammation, and aberrant coagulation. Kcentra, a prothrombin concentrate, has been demonstrated to mitigate pulmonary vascular leak in a murine model of HS. We investigated the effects of Kcentra in a rat model of HS, to achieve physiologic endpoints of relevance. Rats subjected to a grade intravenous splenic injury and controlled hemorrhage for 60 minutes were resuscitated with shed volumes of (1) Lactated Ringer's (LR) solution, (2) LR + 20 IU/kg Kcentra, (3) LR + 50 IU/kg Kcentra, (4) rat fresh frozen plasma (RFFP), or (5) human fresh frozen plasma (HFFP). Blood was harvested for monitoring metabolic and coagulation function. Rat lungs were evaluated for lung injury and permeability. Animals resuscitated with LR displayed a significant increase in pulmonary vascular permeability (sham, 407.9 ± 122.4; shock + LR, 2040 ± 1462). Resuscitation with RFFP (606.5 ± 169.3) reduced leak; however, treatment with Kcentra (HS + Kcentra [20 IU/kg]: 1792 ± 903.4, HS + Kcentra [50 IU/kg]: 1876 ± 1103), and HFFP (1450 ± 533.2) had no significant effect on permeability. Kcentra modestly altered clotting parameters. Metabolic measures, such as lactate, pH, and base deficit, were restored to baseline levels by both RFFP and HFFP, but not Kcentra or LR. Kcentra did not alter pulmonary vascular permeability, but modestly increased clotting potential in injured rats. This suggests that there may be a xenogenic reaction of human products in rats and that the effects of Kcentra on vascular stability may be distinct from its ability to modulate clotting. Our data indicate that the species chosen and utilized for in vivo preclinical testing of human derived blood products is of critical importance in determining their efficacy in animal models and is the primary impetus to communicate these results.

IL-6 Deficiency Exacerbates Allergic Asthma and Abrogates the Protective Effect of Allergic Inflammation against Streptococcus pneumoniae Pathogenesis.

Allergic asthma (AA) is characterized as a Th2-biased airway inflammation that can develop lung inflammation and remodeling of the respiratory tract. Streptococcus pneumoniae is a major respiratory pathogen, causing noninvasive (otitis media and pneumonia) and invasive diseases (sepsis) in humans. We sought to determine the role of IL-6 in the regulation of lung inflammation in murine AA caused by Aspergillus fumigatus as well as its consequence on the regulation of airway barrier integrity and S. pneumoniae disease. In an AA model, IL-6 deficiency led to increased lung inflammation, eosinophil recruitment, tissue pathology, and collagen deposition. Additionally, IL-6-deficient asthmatic mice exhibited reduced goblet cell hyperplasia and increased TGF-β production. These key changes in the lungs of IL-6-deficient asthmatic mice resulted in dysregulated tight junction proteins and increased lung permeability. Whereas the host response to AA protected against S. pneumoniae lung disease, the IL-6 deficiency abrogated the protective effect of allergic inflammation against S. pneumoniae pathogenesis. Consistent with in vivo data, IL-6 knockdown by small interfering RNA or the blockade of IL-6R signaling exacerbated the TGF-β-induced dysregulation of tight junction proteins, E-cadherin and N-cadherin expression, and STAT3 phosphorylation in MLE-12 epithelial cells. Our findings demonstrate a previously unrecognized role of host IL-6 response in the regulation of lung inflammation during AA and the control of S. pneumoniae bacterial disease. A better understanding of the interactions between lung inflammation and barrier framework could lead to the development of therapies to control asthma inflammation and preserve barrier integrity.

Haem oxygenase protects against thrombocytopaenia and malaria-associated lung injury

BackgroundMalaria-triggered lung injury can occur in both severe and non-severe cases. Platelets may interact with parasitized erythrocytes, leukocytes and endothelium. These interactions can lead to microvessel obstructions and induce release of inflammatory mediators. Induction of the haem oxygenase enzyme is important in the host’s response to free haem and to several other molecules generated by infectious or non-infectious diseases. In addition, an important role for the haem oxygenase-1 isotype has been demonstrated in experimental cerebral malaria and in clinical cases. Therefore, the present work aims to determine the influence of haem oxygenase in thrombocytopaenia and acute pulmonary injury during infection with Plasmodium berghei strain NK65.MethodsC57BL/6 mice were infected with P. berghei and analysed 7-10 days post-infection. For each experiment, Cobalt Protoporphyrin IX/CoPPIX or saline were administered. Bronchoalveolar lavage fluid was used for total and differential leukocyte count and for protein measurement. Lungs were used for histological analyses or for analysis of cytokines and western blotting. The lung permeability was analysed by Evans blue dye concentration. Platelet-leukocyte aggregate formation was assayed using the flow cytometer.ResultsPlasmodium berghei NK65 infection generated an intense lung injury, with increased levels of inflammatory mediators, oedema, and cell migration into the lung. Plasmodium berghei infection was also accompanied by marked thrombocytopaenia and formation of platelet-leukocyte aggregates in peripheral blood. Treatment with the HO-1 inducer cobalt protoporphyrin IX (CoPPIX) modified the inflammatory response but did not affect the evolution of parasitaemia. Animals treated with CoPPIX showed an improvement in lung injury, with decreased inflammatory infiltrate in the lung parenchyma, oedema and reduced thrombocytopaenia.ConclusionData here presented suggest that treatment with CoPPIX inducer leads to less severe pulmonary lung injury and thrombocytopaenia during malaria infection, thus increasing animal survival.

Aerosol vaccines – perspectives and therapeutic impact

Introduction: Aerosol vaccination is a promising non-injectable method that provides immune resistance to pathogens since it follows the natural route of transmission of many infectious agents. The immune response, occurring after intranasal or respiratory vaccine administration, provides credible protection due to the common mucosal immune system, excellent blood supply, and lung permeability. Aim: The study aims to highlight the unconventional and perspective application use of aerosol dosage forms as a technological approach for vaccine drug delivery. Materials and Methods: A detailed literature survey in scientific databases such as PubMed, ScienceDirect, ResearchGate has been conducted, and the relevant information has been summarized and interpreted. Results: The aerosol vaccination method, as an alternative to the subcutaneous, intramuscular or intradermal application route, ensures defense against the inhaled pathogens, avoiding at the same time drawbacks associated with the injection administration such as the risk of reuse of disposable medical consumables, potential spreading of blood-borne diseases and the necessity of qualified medical personnel. Additionally, aerosol vaccination is an easier and more practical approach for patients, leading to improved compliance. Vaccines applied by the inhalation or nasal route of administration could be a successful approach for the treatment of diseases such as measles, tuberculosis, or influenza A, that although preventable, remain a global challenge. Conclusion: The aerosol method is promising for vaccine delivery with the potential to be fully evaluated in the upcoming years.

A nebulised antitumour necrosis factor receptor-1 domain antibody in patients at risk of postoperative lung injury: A randomised, placebo-controlled pilot study.

BACKGROUNDTumour necrosis factor receptor 1 (TNFR1) signalling mediates the cell death and inflammatory effects of TNF-α.OBJECTIVEThe current clinical trial investigated the effects of a nebulised TNFR1 antagonist (GSK2862277) on signs of lung injury in patients undergoing oesophagectomy.DESIGNRandomised double-blind (sponsor unblind), placebo-controlled, parallel group study.SETTINGEight secondary care centres, the United Kingdom between April 2015 and June 2017.PATIENTSThirty-three patients undergoing elective transthoracic oesophagectomy.INTERVENTIONSPatients randomly received a single nebulised dose (26 mg) of GSK2862277 (n = 17) or placebo (n = 16), given 1 to 5 h before surgery; 14 and 16, respectively competed the study.MAIN OUTCOME MEASUREMENTSPhysiological and biochemical markers of lung injury, pharmacokinetic and safety endpoints were measured. The primary endpoint was the change from baseline in pulmonary vascular permeability index (PVPI) at completion of surgery, measured using single-indicator transpulmonary thermodilution. Adjusted point estimates and 95% credible intervals (analogous to conventional confidence intervals) were constructed for each treatment using Bayesian statistical models.RESULTSThe mean change (with 95% credible intervals) from baseline in PVPI on completion of surgery was 0.00 (−0.23, 0.39) in the placebo and 0.00 (−0.24, 0.37) in the GSK2862277 treatment groups. There were no significant treatment-related differences in PaO2/FiO2 or Sequential Organ Failure Assessment score. Levels of free soluble TNFR1, Macrophage Inflammatory Protein-1 alpha and total protein were significantly reduced in the bronchoalveolar lavage fluid of patients treated with GSK2862277 (posterior probability of decrease with GSK2862277 vs. placebo:≥0.977; equivalent to P < 0.05). The frequency of adverse events and serious adverse events were distributed evenly across the two treatment arms.CONCLUSIONPre-operative treatment with a single 26 mg inhaled dose of GSK2862277 did not result in significantly lower postoperative alveolar capillary leak or extra vascular lung water. Unexpectedly small increases in transpulmonary thermodilution-measured PVPI and extra vascular lung water index at completion of surgery suggest less postoperative lung injury than historically reported, which may have also compromised a clear assessment of efficacy in this trial. GSK2862277 was well tolerated, resulted in expected lung exposure and reduced biomarkers of lung permeability and inflammation.TRIAL REGISTRATIONclinicaltrials.gov: NCT02221037.

MicroRNA‑93 contributes to the suppression of lung inflammatory responses in LPS‑induced acute lung injury in mice via the TLR4/MyD88/NF‑κB signaling pathway.

Acute lung injury (ALI) is a severe inflammatory lung disease with a rapid onset. The anti-inflammatory functions of microRNA-93 (miRNA/miR-93) have been described in various types of tissue injury and disease. However, the biological role of miR-93 and its molecular mechanisms underlying the initiation and progression of ALI have not yet been reported, at least to the best of our knowledge. The present study aimed to investigate the regulatory effects exerted by miR-93 in ALI. Using an in vivo murine model of ALI induced by lipopolysaccharide (LPS), miR-93 expression was found to be downregulated in the lung tissues and bronchoalveolar lavage fluid (BALF) compared with the control group. Following agomiR-93 injection, it was observed that agomiR-93 attenuated lung injury, as evidenced by decreased lung permeability, a reduced lung wet/dry weight ratio and an increased survival rate of the mice. Concomitantly, agomiR-93 significantly reduced LPS-induced the interleukin (IL)-6, IL-1β, and tumor necrosis factor (TNF)-α levels in BALF. Of note, Toll-like receptor 4 (TLR4), an upstream regulator of the nuclear factor (NF)-κB signaling pathway, was directly suppressed by miR-93 in RAW 264.7 cells. Importantly, agomiR-93 induced a significant suppression of the TLR4/myeloid differentiation primary response 88 (MyD88)/NF-κB signaling pathway, as demonstrated by the downregulation of MyD88, and the phosphorylation of IκB-α and p65 in the lung tissues of mice with ALI. Taken together, the findings of the present study indicate that miR-93 attenutes LPS-induced lung injury by regulating the TLR4/MyD88/NF-κB signaling pathway, suggesting that miR-93 may prove to be a potential therapeutic target for ALI.

MicroRNA‑17 contributes to the suppression of the inflammatory response in lipopolysaccharide‑induced acute lung injury in mice via targeting the toll‑like receptor 4/nuclear factor‑κB pathway.

Acute lung injury (ALI) is a common lung disease with a high mortality rate, which is characterized by an excessive uncontrolled inflammatory response. MicroRNA (miR)-17 has previously emerged as a novel regulatory molecule of inflammatory response in various complex diseases; however, the anti-inflammatory action and associated molecular mechanisms of miR-17 in ALI have not been fully elucidated. The aim of the present study was to investigate the role of miR-17 in the inflammatory response in ALI and to elucidate the potential underlying mechanism. Using a lipopolysaccharide (LPS)-induced ALI mouse model, it was observed that miR-17 was significantly downregulated in lung tissues compared with the control group. In this model, ectopic expression of miR-17 attenuated lung pathological damage, reduced lung wet/dry ratio and lung permeability, and increased survival rate in ALI mice. In addition, agomiR-17 injection significantly suppressed LPS-induced inflammation, as evidenced by a reduction in the activity of myeloperoxidase and the production of interleukin (IL)-6, IL-1β and tumor necrosis factor-α in lung tissues. Of note, toll-like receptor (TLR) 4, an upstream regulator of the nuclear factor (NF)-κB inflammatory signaling pathway, was directly targeted by miR-17, and its translation was suppressed by miR-17 in vitro and in vivo. Using an LPS-induced RAW264.1 macrophage injury model, it was observed that miR-17 overexpression suppressed the pro-inflammatory effect of LPS, while these inhibitory effects were markedly abrogated by TLR4 overexpression. In addition, TLR4 knockdown by si-TLR4 mimicked the effects of miR-17 overexpression on LPS-induced cytokine secretion in the in vitro model. Further experiments revealed that miR-17 significantly reduced the expression of key proteins in the NF-κB pathway, including IKKβ, p-IκBα and nuclear p-p65, and suppressed the NF-κB activity in ALI mice. Collectively, these results indicated that miR-17 protected mice against LPS-induced lung injury via inhibiting inflammation by targeting the TLR4/NF-κB pathway; therefore, miR-17 may serve as a potential therapeutic target for ALI.

MiR-342 attenuates lipopolysaccharide-induced acute lung injury via inhibiting MAPK1 expression.

Micro RNA (miRNA) and mitogen-activated protein kinase (MAPK) are reported as the crucial regulators of inflammatory responses in acute lung injury (ALI). This study will explore the role of the miR-342/MAPK1 axis in regulation of lipopolysaccharide (LPS)-induced ALI. We found that miR-342 was down-regulated in LPS-induced A549 cells compared with the control group with DMSO, accompanied by elevated inflammatory cytokines and apoptosis. Over-expression of miR-342 reduced LPS-induced inflammatory responses and apoptosis in LPS-stimulated A549 cells, and had a protective role in LPS-treated mice with ALI by decreasing levels of inflammatory cytokines, improving survival of mice with ALI, and ameliorating the lung permeability. Dual-luciferase reporter gene assay demonstrated that miR-342 regulated the expression of MAPK1 by directly targeting its 3' untranslated region (3'-UTR). Mechanistically, MAPK1 silencing abrogated LPS-induced inflammatory injury in A549 cells, and partially enhanced the protective effect of miR-342. Therefore, miR-342 attenuates LPS-induced ALI by targeting MAPK1 expression, thereby protecting against A549 cell injury induced by LPS and lung injury of mice with ALI.

Vascular permeability in the fibrotic lung.

Idiopathic pulmonary fibrosis (IPF) is thought to result from aberrant tissue repair processes in response to chronic or repetitive lung injury. The origin and nature of the injury, as well as its cellular and molecular targets, are likely heterogeneous, which complicates accurate pre-clinical modelling of the disease and makes therapeutic targeting a challenge. Efforts are underway to identify central pathways in fibrogenesis which may allow targeting of aberrant repair processes regardless of the initial injury stimulus. Dysregulated endothelial permeability and vascular leak have long been studied for their role in acute lung injury and repair. Evidence that these processes are of importance to the pathogenesis of fibrotic lung disease is growing. Endothelial permeability is increased in non-fibrosing lung diseases, but it resolves in a self-limited fashion in conditions such as bacterial pneumonia and acute respiratory distress syndrome. In progressive fibrosing diseases such as IPF, permeability appears to persist, however, and may also predict mortality. In this hypothesis-generating review, we summarise available data on the role of endothelial permeability in IPF and focus on the deleterious consequences of sustained endothelial hyperpermeability in response to and during pulmonary inflammation and fibrosis. We propose that persistent permeability and vascular leak in the lung have the potential to establish and amplify the pro-fibrotic environment. Therapeutic interventions aimed at recognising and "plugging" the leak may therefore be of significant benefit for preventing the transition from lung injury to fibrosis and should be areas for future research.

Zonulin antagonist protects against tight junction disruption by Pseudomonas aeruginosa in airway epithelial cells

Zonulin is known as a regulator of barrier function in both epithelial and endothelial cells in the intestine and kidney by acting through EGFR (epidermal growth factor receptor) and PAR2 (Proteinase activated receptors) to cause the dissociation of ZO‐1 from occludin and claudin‐1 from the cell junctions. Respiratory infection is characterized by increased lung permeability caused by the reduction in the abundance and/or distribution of proteins at the tight junction. Here the effect of the zonulin antagonist, larazotide acetate (LA) on airway epithelial cell barrier function after the addition of lipopolysaccharide (LPS) from Pseudomonas aeruginosa or undiluted P.aeruginosa filtrate isolated from an overnight culture was investigated. Two human airway epithelial cell models were used, H441 cells grown at air‐liquid interface and 16HBE14o‐cells grown as submerged monolayers. Cells were incubated with 10 μg/ml LPS and 12 mM larazotide acetate. Transepithelial electrical resistance (TEER) and permeability was measured after 1h and 6h. TEER was decreased with LPS which was prevented by LA, (p&lt;0.05, n=3) LPS vs LPS/LA at 1 hour. Permeability was increased with LPS after 6h (p&lt;0.05, n=3) LPS vs control. Cells were immunostained for ZO‐1 and E‐cadherin, LPS caused the disappearance of ZO‐1 staining at the apical surface which was conserved with LA. E‐cadherin localization did not change with either LPS or LPS/LA treatment. Total protein abundance assessed by western blot showed no changes. Exposure of 16HBE14o‐ cells to P.aeruginosa filtrate for 24 h lead to a 61% decrease in TEER compared to control (p&lt;0.0001, n=3). P.aeruginosa filtrate/LA increased TEER by 39% (p&lt;0.02, n=3). Tight junction localization of E‐cadherin and claudin‐1 were both disrupted with P.aeruginosa filtrate while incubation with LA attenuated this rearrangement. This study shows that larazotide acetate helped to counteract the damage to tight junctions caused by LPS and P.aeruginosa filtrate, this supports a role for zonulin as a regulator of lung epithelial barrier function.Support or Funding InformationFunded by Astra Zeneca

Evaluation of Lung Quality by Near-Infrared Fluorescent Imaging during Ex Vivo Lung Perfusion

Improved real-time assessment of donor lung quality during Ex Vivo Lung Perfusion (EVLP) could enhance safe utilization of lungs for transplantation. Near-infrared (NIR) fluorescent imaging with indocyanine green (ICG) has been used in various clinical intraoperative applications to evaluate tissue perfusion. We investigated the feasibility of NIR imaging during EVLP and aimed to detect and quantify lung perfusion and permeability. Pig lungs with extended 24-hour cold ischemia (n=4) and human lungs rejected from clinical transplantation (n=2) were assessed with NIR imaging during EVLP. Serial NIR imaging was performed with SPY Elite imaging system after ICG (0.3, 0.6 or 1.0 mg) administration through pulmonary artery (Fig A). Tissue perfusion was quantified using SPYQ analysis software, and tissue ICG accumulation was correlated with lung damage. Strong NIR signal was detected immediately after ICG administration with 0.6 mg (pig lungs) or 1.0 mg (human lungs) doses resulting in optimal fluorescence for tissue perfusion evaluation. In contrast to the transient kinetics and fast liver metabolism during clinical in vivo ICG administration, a single ICG dose during EVLP resulted in stable NIR signal for over 10 hours. Perfusion quantification of pig lungs with extended cold preservation revealed peripheral areas with compromised tissue perfusion that gradually recovered over time with recruitment of additional pulmonary vascular beds (Fig B). ICG accumulation was detected in sites with damage and increased vascular permeability in pig and human lungs (Fig C and D). NIR imaging with a clinically-approved intravascularly-delivered ICG fluorescent probe can dynamically quantify tissue perfusion and detect damage-induced vascular permeability. This novel and promising imaging modality could be used during EVLP to assess lung quality, or to determine effects of therapeutics that target tissue perfusion and vascular permeability.

Alpha 1 Antitrypsin Treatment during Human Ex Vivo Lung Perfusion Improves Lung Function by Protecting Lung Endothelium

The majority of potential donor lungs are not being used. The primary reason for underutilization is the concern for primary graft dysfunction (PGD). There is currently no clinically available therapy for PGD. Alpha 1 antitrypsin (A1AT) is a serine protease inhibitor with anti-inflammatory and cytoprotective properties. We and others have shown benefits of A1AT in small and large animal lung transplant studies. Before performing clinical trials, evidence of therapeutic efficacy in human lungs would be valuable. We tested the effect of A1AT given during ex vivo lung perfusion (EVLP) to human lungs rejected for transplantation. Double lung blocks rejected for transplantation (n=8) were divided and placed on separate EVLP circuits for 12h. Lungs were randomly assigned to receive A1AT or placebo, with the contralateral lung serving as control for the treated lung. Outcome measures included: hourly physiologic lung function, perfusate loss, wet-dry weight ratio, inflammatory mediators, endothelin-1 (ET-1), and zonula occludens tight junction protein-1 (ZO-1, immunofluorescence staining). The A1AT-treated group demonstrated significantly better lung function: higher pO2 and compliance (Fig A), lower pulmonary artery pressure (-0.4 mmHg p= 0.04) and vascular resistance (-30.4 dynes·s cm-5 p= 0.01). Perfusate loss, a surrogate for lung permeability, was lower in the A1AT group, as was the wet-dry ratio (Fig B). A1AT also decreased ET-1 levels in perfusate (Fig C) and increased ZO-1 expression on endothelial cells (Fig D). In this study we demonstrated that human A1AT was able to improve the quality of severly injuried human lungs, likely through endothelial cell protection. The encouraging results justify a clinical trial, in order to improve donor lung quality and clinical outcomes in lung transplantation. Our study also illustrates that testing selected drugs on injured human lungs on EVLP is a viable strategy prior to clinical application.

Propofol alleviates ventilator-induced lung injury through regulating the Nrf2/NLRP3 signaling pathway

Ventilator-induced lung injury (VILI) causes problems during acute lung injury treatment, and propofol is a well-known drug to prevent VILI. Herein, we discussed how propofol protects against VILI-induced inflammation with the interaction of nuclear factor E2-related factor 2 (Nrf2)/NOD-like receptor protein 3 (NLRP3). We established VILI mouse models for collecting lung tissues, and these mice were later treated with propofol and Nrf2/NLRP3 activator or inhibitor to observe their effects on VILI with inflammatory factors, 8-hydroxy-2 deoxyguanosine, malondialchehyche level, mitochondrial reactive oxygen species production rate, lung wet/dry weight ratio, lung permeability index measured. Propofol treatment improved VILI, alleviated pulmonary inflammation induced by mechanical ventilation. Propofol up-regulated Nrf2 and down-regulated NLRP3 in VILI model. Activating Nrf2 or inhibiting NLRP3 downregulated pro-inflammatory factors in lung tissues in VILI mice. Above all, we can conclude that propofol exerts it protective function against VILI and the subsequent inflammatory responses through activating Nrf2 and inhibiting NLRP3 expression. Therefore, Nrf2 activator and NLRP3 inhibitor might be latent targets in the VILI prevention.

Early progression of pulmonary hypertension in the monocrotaline model in males is associated with increased lung permeability

BackgroundThe mechanisms involved in pulmonary hypertension (PH) development in patients and pre-clinical models are poorly understood. PH has a well-established sex dimorphism in patients with increased frequency of PH in females, and more severe disease with poor survival prognosis in males. Previously, we found that heme signaling plays an essential role in the development phase of the Sugen/Hypoxia (SU/Hx) model. This study is focused on the elucidation of sex differences in mechanisms of PH development related to heme action at the early stage of the monocrotaline (MCT) PH model.MethodsRats received MCT injection (60 mg/kg, i.p.) and followed for 14 days to investigate early disease changes. Hemodynamic parameters were recorded at the end of the study; plasma, lung homogenates, and nuclear fractions were used for the evaluation of protein levels.ResultsOur data indicate that on day 14, rats did not show any significant increase in the Fulton index due to the early disease phase. However, the right ventricular systolic pressure was significantly increased in male rats, while female rats showed only a trend. Interestingly, only males demonstrated an increased lung-to-bodyweight ratio that indicated lung edema. Indeed, lung histology confirmed severe perivascular edema in males. Previously, we have reported that the increased perivascular edema in SU/Hx model correlated with intravascular hemolysis and activated heme signaling. Here, we found that elevated free hemoglobin levels and perivascular edema were increased, specifically in males showing more rapid progress of PH. A high level of heme carrier protein 1 (HCP-1), which is involved in heme uptake from the bloodstream into the cells, was also found elevated in the lungs of males. The upregulation of heme oxygenase in males indicated increased intracellular heme catabolism. Increased heme signaling resulted in the activation of heme-mediated barrier-disruptive mechanisms. Thus, hemolysis in males can be responsible for increased permeability of the lungs and early disease development.ConclusionsOur study indicates the importance of barrier-disruptive mechanisms as an earlier event in the induction of pulmonary hypertension. Importantly, males are more susceptible to hemolysis and develop PH earlier than females.