Extracellular vesicle-encapsulated CC16 as novel nanotherapeutics for treatment of acute lung injury.

Acute lung injury (ALI) is still associated with high mortality. Growing evidence suggests that Club Cell Protein 16 (CC16) plays a protective role against ALI. However, the doses of recombinant CC16 (rCC16) used in preclinical studies are supraphysiological for clinical applications. Extracellular vesicles (EVs) are nanovesicles endogenously generated by mammalian cells. Our study demonstrated that CC16 is released via small EVs and EV-encapsulated CC16 (sEV-CC16) and has anti-inflammatory activities, which protect mice from lipopolysaccharide (LPS) or bacteria-induced ALI. Additionally, sEV-CC16 can activate the DNA damage repair signaling pathways. Consistent with this activity, we observed more severe DNA damage in lungs from Cc16 knockout (KO) than wild-type (WT) mice. Mechanistically, we elucidated that CC16 suppresses nuclear factor κB (NF-κB) signaling activation by binding to heat shock protein 60 (HSP60). We concluded that sEV-CC16 could be a potential therapeutic agent for ALI by inhibiting the inflammatory and DNA damage responses by reducing NF-κB signaling.

A proinflammatory long noncoding RNA Lncenc1 regulates inflammasome activation in macrophage.

Mammalian genomes encode thousands of long noncoding RNAs (lncRNAs). LncRNAs are extensively expressed in various immune cells. The lncRNAs have been reported to be involved in diverse biological processes, including the regulation of gene expression, dosage compensation, and genomic imprinting. However, very little research has been conducted to explore how they alter innate immune responses during host-pathogen interactions. In this study, we found that a lncRNA, named long noncoding RNA, embryonic stem cells expressed 1 (Lncenc1), was strikingly increased in mouse lungs after gram-negative (G-) bacterial infection or exposure to lipopolysaccharides (LPS). Interestingly, our data indicated that Lncenc1 was upregulated in macrophages but not in primary epithelial cells (PECs) or polymorphonuclear leukocytes (PMN). The upregulation was also observed in human THP-1 and U937 macrophages. Besides, Lncenc1 was highly induced during ATP-induced inflammasome activation. Functionally, Lncenc1 showed proinflammatory effects in macrophages as demonstrated by increased expressions of cytokine and chemokines, as well as enhanced NF-κB promoter activity. Overexpression of Lncenc1 promoted the releases of IL-1ß and IL-18, and Caspase-1 activity in macrophages, suggesting a role in inflammasome activation. Consistently, knockdown of Lncenc1 inhibited inflammasome activation in LPS-treated macrophages. Moreover, knockdown of Lncenc1 using antisense oligo (ASO)-loaded exosomes (EXO) attenuated LPS-induced lung inflammation in mice. Similarly, Lncenc1 deficiency protects mice from bacteria-induced lung injury and inflammasome activation. Taken together, our work identified Lncenc1 as a modulator of inflammasome activation in macrophages during bacterial infection. Our study suggested that Lncenc1 could serve as a therapeutic target for lung inflammation and injury.

Plasma matrix metalloproteinase-3 predicts mortality in acute respiratory distress syndrome: a biomarker analysis of a randomized controlled trial.

BACKGROUND: Matrix metalloproteinase-3 (MMP-3) is a proteolytic enzyme involved in acute respiratory distress syndrome (ARDS) pathophysiology that may serve as a lung-specific biomarker in ARDS. METHODS: This study was a secondary biomarker analysis of a subset of Albuterol for the Treatment of Acute Lung Injury (ALTA) trial patients to determine the prognostic value of MMP-3. Plasma sample MMP-3 was measured by enzyme-linked immunosorbent assay. The primary outcome was the area under the receiver operating characteristic (AUROC) of MMP-3 at day 3 for the prediction of 90-day mortality. RESULTS: A total of 100 unique patient samples were evaluated and the AUROC analysis of day three MMP-3 showed an AUROC of 0.77 for the prediction of 90-day mortality (95% confidence interval: 0.67-0.87), corresponding to a sensitivity of 92% and specificity of 63% and an optimal cutoff value of 18.4 ng/mL. Patients in the high MMP-3 group (≥ 18.4 ng/mL) showed higher mortality compared to the non-elevated MMP-3 group (< 18.4 ng/mL) (47% vs. 4%, p < 0.001). A positive difference in day zero and day three MMP-3 concentration was predictive of mortality with an AUROC of 0.74 correlating to 73% sensitivity, 81% specificity, and an optimal cutoff value of + 9.5 ng/mL. CONCLUSIONS: Day three MMP-3 concentration and difference in day zero and three MMP-3 concentrations demonstrated acceptable AUROCs for predicting 90-day mortality with a cut-point of 18.4 ng/mL and + 9.5 ng/mL, respectively. These results suggest a prognostic role of MMP-3 in ARDS.

The Potential Synergistic Risk of Albuterol and Vasoactives in Acute Lung Injury Trials.

BACKGROUND: Critically ill patients are often prescribed both inhaled beta-agonists and intravenous vasoactive; however, the interaction of the additive beta-agonist effects of these 2 agents remains largely uncharacterized. OBJECTIVE: The purpose of this study was to evaluate how concomitant use of albuterol and vasoactive or inotropes affected ventilator-free days (VFDs) by re-analyzing the data from the Albuterol to Treat Acute Lung Injury (ALTA) trial. METHODS: In this study, subjects were grouped to albuterol-vasoactive (n = 84) versus (vs) placebo-vasoactive (n = 62). Ventilator-free days, intensive care unit (ICU)-free days, organ failure-free days, cardiovascular adverse events, and 90-day mortality were compared. The primary outcome was VFDs. RESULTS: Patients in the albuterol-vasoactive group had significantly fewer VFDs than patients in the placebo-vasoactive group (11 vs 19, P = 0.05). Patients in the albuterol-vasoactive group also had significantly fewer ICU-free days (9.5 vs 18.5, P = .006). The 90-day mortality was similar between groups (36.9% vs 27.4%, P = .20). Similarly, no significant difference in cardiac adverse events between the groups (14.3% vs 11.3%, P = 0.59). CONCLUSION AND RELEVANCE: This study has shown fewer VFDs for patients who received both vasoactive and albuterol. There were also fewer ICU-free days when compared to those on vasoactive only. Given the common use of both agents, a prospective evaluation of the additive adverse effects of beta-agonism is warranted.

Dysregulation of miR-103a Mediates Cigarette Smoking-induced Lipid-laden Macrophage Formation.

Cigarette smoke (CS) is considered a major risk factor for chronic obstructive pulmonary disease (COPD) that is currently the third leading cause of death in the United States. Studies have indicated that patients with COPD have elevated blood low-density lipoprotein levels, which may contribute to the dysregulation of lipid metabolism. Accumulating data show that microRNAs (miRNAs) are involved in various human diseases. However, the role of microRNAs in the pathogenesis of COPD remains poorly defined. In this study, we found that miR-103a expression was significantly reduced in alveolar macrophages from smokers and patients with COPD versus that in alveolar macrophages from nonsmokers. Our data indicated that reactive oxygen species negatively regulate miR-103a in macrophages. Functionally, miR-103a modulates the expressions of genes involved in lipid metabolism and directly targets low-density lipoprotein receptors in macrophages. Furthermore, overexpression of miR-103a suppressed the accumulation of lipid droplets and reduced the reactive oxygen species, both in vitro and in vivo. Taken together, our findings indicate that downregulation of miR-103a contributes to cigarette smoke-induced lipid-laden macrophage formation and plays a critical role in lipid homeostasis in lung macrophages in the pathogenesis of COPD.

CC16 Regulates Inflammation, ROS Generation and Apoptosis in Bronchial Epithelial Cells during Klebsiella pneumoniae Infection.

Gram-negative (G-) bacteria are the leading cause of hospital-acquired pneumonia in the United States. The devastating damage caused by G- bacteria results from the imbalance of bactericidal effects and overwhelming inflammation. Despite decades of research, the underlying mechanisms by which runaway inflammation is developed remain incompletely understood. Clara Cell Protein 16 (CC16), also known as uteroglobin, is the major protein secreted by Clara cells and the most abundant protein in bronchoalveolar lavage fluid (BALF). However, the regulation and functions of CC16 during G- bacterial infection are unknown. In this study, we aimed to assess the regulation of CC16 in response to Klebsiella pneumoniae (K. pneu) and to investigate the role of CC16 in bronchial epithelial cells. After K. pneu infection, we found that CC16 mRNA expression was significantly decreased in bronchial epithelial cells. Our data also showed that K. pneu infection upregulated cytokine and chemokine genes, including IL-1ß, IL-6, and IL-8 in BEAS-2B cells. Endogenously overexpressed CC16 in BEAS-2B cells provided an anti-inflammatory effect by reducing these markers. We also observed that endogenous CC16 can repress NF-κB reporter activity. In contrast, the recombinant CC16 (rCC16) did not show an anti-inflammatory effect in K. pneu-infected cells or suppression of NF-κB promoter activity. Moreover, the overexpression of CC16 reduced reactive oxygen species (ROS) levels and protected BEAS-2B cells from K. pneu-induced apoptosis.

The Roles of CCN1/CYR61 in Pulmonary Diseases.

CCN1 (cysteine-rich 61, connective tissue growth factor, and nephroblastoma-1), previously named CYR61 (cysteine-rich angiogenic inducer 61) belongs to the CCN family of matricellular proteins. CCN1 plays critical roles in the regulation of proliferation, differentiation, apoptosis, angiogenesis, and fibrosis. Recent studies have extensively characterized the important physiological and pathological roles of CCN1 in various tissues and organs. In this review, we summarize both basic and clinical aspects of CCN1 in pulmonary diseases, including acute lung injury (ALI), chronic obstructive pulmonary disease (COPD), lung fibrosis, pulmonary arterial hypertension (PAH), lung infection, and lung cancer. We also emphasize the important challenges for future investigations to better understand the CCN1 and its role in physiology and pathology, as well as the questions that need to be addressed for the therapeutic development of CCN1 antagonists in various lung diseases.

Secondary Analysis of Fluids and Catheters Treatment Trial (FACTT) data reveal poor clinical outcomes in acute respiratory distress syndrome patients with diabetes.

OBJECTIVES: Conflicting reports exist about the link between diabetes mellitus (DM) and acute respiratory distress syndrome (ARDS). Our study examines the impact of pre-existing DM on ARDS patients within the Fluid and Catheter Treatment Trial (FACTT). DESIGN: Conducting a secondary analysis of FACTT data, we incorporated 967 participants with identified DM status (173 with DM, 794 without DM) and examined outcomes like 90-day mortality, hospital and ICU stays, and ventilator days until unassisted breathing. The primary outcome of hospital mortality at day 90 was evaluated through logistic regression using IBM SPSS software. Additionally, we assessed plasma cytokines and chemokines utilizing a human magnetic bead-based multiplex assay. RESULTS: Patients with pre-existing DM exhibited a lower survival rate compared to non-DM patients (61.3 vs. 72.3 %, p = 0.006). Subjects with DM experienced significantly longer hospital lengths of stay (24.5 vs. 19.7 days; p = 0.008) and prolonged ICU stays (14.8 vs. 12.4 days; p = 0.029). No significant difference was found in ventilator days until unassisted breathing between the two groups (11.7 vs. 10; p = 0.1). Cytokine/chemokine analyses indicated a non-significant trend toward heightened levels of cytokines (TNF-α, IL-10, and IL-6) and chemokines (CRP, MCP-1) in DM patients compared to non-DM on both days 0 and 1. Notably, lipopolysaccharide-binding protein (LBP) exhibited significantly higher levels in DM compared to non-DM individuals. CONCLUSIONS: ARDS patients with DM suffered worse clinical outcomes compared to non-DM patients, indicating that DM may negatively affect the respiratory functions in these subjects. Further comprehensive clinical and pre-clinical studies will strengthen this relationship.

Club Cell Secretory Protein-16 (CC16) as a Prognostic Biomarker for COVID-19 and H1N1 Viral Infections.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) and H1N1 viruses are inflammatory lung pathogens that can lead to acute lung injury (ALI) and acute respiratory distress syndrome (ARDS). ALI/ARDS are still life-threatening diseases in critically ill patients with 30-40% mortality in the last decade. Currently, there are no laboratory tests for the early diagnosis or prognosis of ALI/ARDS. Club cell secretory protein (CC16) has been investigated as a potential biomarker of lung epithelial damage in various lung diseases. In this study, we evaluated whether plasma CC16 reflects the severity of COVID-19 and H1N1 infections. The plasma CC16 levels showed no significant differences between H1N1 and COVID-19 groups (p = 0.09). Among all subjects, CC16 levels were significantly higher in non-survivors than in survivors (p = 0.001). Upon the area under the receiver operating characteristic (AUROC) analysis, CC16 had an acceptable value to distinguish survivors and non-survivors (p = 0.002). In the COVID-19 group, plasma CC16 levels moderately correlated with the Acute Physiology and Chronic Health Evaluation II (APACHE II) score (r = 0.374, p = 0.003) and Sequential Organ Failure Assessment (SOFA) score (r = 0.474, p < 0.001). In the H1N1 group, a positive correlation was observed between the CC16 levels and hospital length of stay (r = 0.311, p = 0.022). Among all the patients, weak correlations between plasma CC16 levels with the SOFA score (r = 0.328, p < 0.001) and hospital length of stay (r = 0.310, p < 0.001) were observed. Thus, circulating CC16 might reflect the severity of COVID-19 and H1N1 infections.

Estrogen-dependent gene regulation: Molecular basis of TIMP-1 as a sex-specific biomarker for acute lung injury.

Increased circulating tissue inhibitor of metalloproteinases-1 (TIMP-1) levels have been observed in patients with acute lung injury (ALI). However, the sex-specific regulation of TIMP-1 and the underlying molecular mechanisms have not been well elucidated. In this study, we found that plasma TIMP-1 levels were significantly higher in COVID-19 and H1N1 patients compared with those in healthy subjects (n = 25). TIMP-1 concentrations were significantly different between males and females in each disease group. Among female but not male patients, TIMP-1 levels significantly correlated with the PaO2/FiO2 ratio and hospital length of stay. Using the mouse model of ALI induced by the H1N1 virus, we found that TIMP-1 is strikingly induced in PDGFRα-positive cells in the murine lungs. Moreover, female mice showed a higher Timp-1 expression in the lungs on day 3 postinfection. Mechanistically, we observed that estrogen can upregulate TIMP-1 expression in lung fibroblasts, not epithelial cells. In addition, overexpression of estrogen receptor α (ERα) increased the TIMP-1 promoter activity. In summary, TIMP-1 is an estrogen-responsive gene, and its promoter activity is regulated by ERα. Circulating TIMP-1 may serve as a sex-specific marker, reflecting the severity and worst outcomes in female patients with SARS-CoV2- and IAV-related ALI.

Validation of Prognostic Club Cell Secretory Protein (CC16) Cut-point in an Independent ALTA Cohort.

Background: Club cell secretory protein (CC16) has demonstrated utility as a lung-specific biomarker in predicting mortality in acute respiratory distress syndrome (ARDS). These findings have been observed in pre-clinical trials and a re-analysis of a large, randomized controlled trial of ARDS (Fluid and Catheter Treatment Trial (FACTT)). Objectives: The purpose of this study was to validate previous findings by evaluating CC16 level as a mortality predictor in patients from the albuterol to treat acute lung injury (ALTA) trial. Design and Method: In this secondary biomarker analysis, plasma CC16 level was measured from 100 ALTA subjects using enzyme-linked immunosorbent assay (ELISA). The rate of mortality was assessed in patients with high (⩾45 ng/mL) versus low CC16 (<45 ng/mL) levels. This cut-off level was applied based on our previous analysis from FACTT trial. Significance was assessed using Kaplan-Meier curves and a log-rank test. Results: Subjects were an average of 50 years old and 46% of them were females. Patients with high CC16 levels had higher 90-day mortality compared to those with low CC16 levels, (37.73% vs 8.95%, P < .001). Other clinical outcomes including ICU-free days, ventilator-free days, and organ failure free days were significantly different between the groups (All P < .05). Conclusion: In this validation study, we demonstrated that ARDS patients with high plasma CC16 concentration had a higher mortality rate than those with low CC16 levels, confirming previous findings that CC16 levels are associated with ARDS mortality.

MMP3 in Severe COVID-19: A Biomarker or Therapeutic Target?

Identifying novel therapies is a critical need in the treatment of coronavirus disease-19 (COVID-19) and acute respiratory distress syndrome (ARDS). Stromelysin-1, also known as matrixmetalloproteinase 3 (MMP3), has been investigated as a diagnostic biomarker and a potential pharmacological target. Here, we discuss the recent findings of Gelzo et al. in the context of additional MMP3 investigations to delineate its exact role in diagnosis, prognostication, and phenotyping, in addition to its promising role as a therapeutic target in COVID-19-associated respiratory failure.

Long Noncoding RNA: A Novel Insight into the Pathogenesis of Acute Lung Injury.

Acute lung injury (ALI) and its severe form, acute respiratory distress syndrome (ARDS), represent an acute stage of lung inflammation where the alveolar epithelium loses its functionality. ALI has a devastating impact on the population as it not only has a high rate of incidence, but also has high rates of morbidity and mortality. Due to the involvement of multiple factors, the pathogenesis of ALI is complex and is not fully understood yet. Long noncoding RNAs (lncRNAs) are a group of non-protein-coding transcripts longer than 200 nucleotides. Growing evidence has shown that lncRNAs have a decisive role in the pathogenesis of ALI. LncRNAs can either promote or hinder the development of ALI in various cell types in the lungs. Mechanistically, current studies have found that lncRNAs play crucial roles in the pathogenesis of ALI via the regulation of small RNAs (e.g., microRNAs) or downstream proteins. Undoubtedly, lncRNAs not only have the potential to reveal the underlying mechanisms of ALI pathogenesis but also serve as diagnostic and therapeutic targets for the therapy of ALI.

Circulating Surfactant Protein D: A Biomarker for Acute Lung Injury?

Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are life-threatening lung diseases in critically ill patients. The lack of prognostic biomarkers has halted detection methods and effective therapy development. Quantitative biomarker-based approaches in the systemic circulation have been proposed as a means of enhancing diagnostic strategies as well as pharmacotherapy in a patient-specific manner. Pulmonary surfactants are complex mixtures made up of lipids and proteins, which are secreted into the alveolar space by epithelial type II cells under normal and pathological conditions. In this review, we summarize the current knowledge of SP-D in lung injury from both preclinical and clinical studies. Among surfactant proteins, surfactant protein-D (SP-D) has been more widely studied in ALI and ARDS. Recent studies have reported that SP-D has a superior discriminatory ability compared to other lung epithelial proteins for the diagnosis of ARDS, which could reflect the severity of lung injury. Furthermore, we shed light on recombinant SP-D treatment and its benefits as a potential drug for ALI, and we encourage further studies to translate SP-D into clinical use for diagnosis and treatment.

TIMP-1 and its potential diagnostic and prognostic value in pulmonary diseases.

Tissue inhibitors of metalloproteases (TIMPs) have caught the attention of many scientists due to their role in various physiological and pathological processes. TIMP-1, 2, 3, and 4 are known members of the TIMPs family. TIMPs exert their biological effects by, but are not limited to, inhibiting the activity of metalloproteases (MMPs). The balance between MMPs and TIMPs is critical for maintaining homeostasis of the extracellular matrix (ECM), while the imbalance between MMPs and TIMPs can lead to pathological changes, such as cancer. In this review, we summarized the current knowledge of TIMP-1 in several pulmonary diseases namely, acute lung injury (ALI)/acute respiratory distress syndrome (ARDS), pneumonia, asthma, chronic obstructive pulmonary disease (COPD), cystic fibrosis, and pulmonary fibrosis. Considering the potential of TIMP-1 serving as a non-invasive diagnostic and/or prognostic biomarker, we also reviewed the circulating TIMP-1 levels in translational and clinical studies.

Club Cell Secretory Protein-Derived Acute Respiratory Distress Syndrome Phenotypes Predict 90-Day Mortality: A Reanalysis of the Fluids and Catheter Treatment Trial.

Club cell secretory protein (CC16) is a protein with potential utility as a lung-specific biomarker for acute respiratory distress syndrome. The purpose of this study was to characterize CC16 in plasma from patients enrolled in the Fluid and Catheter Treatment Trial (FACTT) to determine the prognostic value for patient outcomes in our subgroup of FACTT patients. DESIGN: A secondary biomarker analysis of a prospective randomized-controlled trial. The primary outcome was area under the receiver operating characteristic (AUROC) of CC16 for prediction of 90-day mortality. Secondary outcomes included differences in mortality, length of stay, and ventilator-free days (VFDs) between patients with high and low CC16. Statistical analyses were performed with IBM SPSS Statistics. SETTING: Single-center laboratory analysis. SUBJECTS: Plasma samples from 68 FACTT subjects and 20 healthy controls. INTERVENTIONS: CC16 was measured in patient plasma samples by enzyme-linked immunosorbent assay. MEASUREMENTS AND MAIN RESULTS: Subjects were an average of 48 years old (sd, 16.7 yr old) and 51.5% male. AUROC analysis of CC16 on day 1 showed an area under the ROC curve of 0.78 for prediction of mortality (odds ratio, 1.011; 95% CI, 1.003-1.021) with an optimal cutoff value of 45 ng/mL. Patients in the low CC16 group (<45 ng/mL) had lower mortality (7.5 vs 50.0%; p < 0.001) and similar VFD (11.9 vs 13.2; p = 0.638). When stratified by CC16 concentration, there was no difference between mortality in the fluid liberal (36.4 vs 58.8%; p = 0.256) or conservative (4.3 vs 11.8%; p = 0.366) groups. CONCLUSIONS: CC16 demonstrated an acceptable AUROC for prediction of patient mortality with a cut point of 45 ng/mL. Patients with high CC16 on day 1 had worse outcomes compared with those with low CC16, suggesting a prognostic role for this lung-specific biomarker.

Plasma TIMP-1 as a sex-specific biomarker for acute lung injury.

BACKGROUND: Acute respiratory distress syndrome (ARDS) confers high morbidity and mortality, with a death rate reaching 40%. Pre-clinical and clinical studies have cited sex-specific sex hormones as a critical contributor to divergent immunologic responses. Therefore, exploration of sex and sex hormone roles following lung injury and ARDS development is needed. Tissue inhibitor of metalloproteinase-1 (TIMP-1) was the first-discovered natural collagenase inhibitor and is located exclusively on the X chromosome. This study aimed to evaluate the prognostic role of circulating TIMP-1, and if concentration differences between males and females correlate with the mortality of ARDS patients. METHODS: Human plasma samples from 100 ARDS patients enrolled in Albuterol to Treat Acute Lung Injury (ALTA) trial on the day of randomization were evaluated. The amount of TIMP-1 was measured using an enzyme-linked immunoassay (ELISA). Area under the receiver operating characteristic (AUROC) was computed to assess the predictive power of TIMP-1 for 30 and 90-day mortality. Chi-squared tests and Kaplan-Meier curves were computed to assess different variables and survival. RESULTS: AUROC analysis of TIMP-1 and 30-day mortality among females showed that TIMP-1 exhibited an AUC of 0.87 (95% confidence interval [CI] 0.78 to 0.97; P = 0.0014) with an optimal cut-off value of 159.7 ng/mL producing a 100% sensitivity and 74% specificity. For 90-day mortality, AUROC analysis showed an AUC of 0.82 (95% confidence interval [CI] 0.67 to 0.97; P = 0.0016) with a similar cut-off value producing a 90% sensitivity and 76.47% specificity. Stratifying subjects by TIMP-1 concentration as high (≥ 159.7 ng/mL) or low (< 159.7 ng/mL) indicated that high TIMP-1 was associated with increased 30 and 90-day mortality rates (all P < 0.0001). Lastly, high TIMP-1 group was associated with worse other outcomes including ventilator-free days (VFDs) and ICU-free days (all P < 0.05). CONCLUSION: Circulating TIMP-1 appeared to be a promising biomarker for mortality among females with ARDS. The high TIMP-1 group showed worse VFDs and ICU-free days. Circulating TIMP-1 may be a sex-specific biomarker in the setting of ARDS and could improve ARDS phenotyping as well as provide a novel therapeutic target in females.

Nebulization of extracellular vesicles: A promising small RNA delivery approach for lung diseases.

Small extracellular vesicles (sEVs) are a group of cell-secreted nanovesicles with a diameter up to 200 nm. A growing number of studies have indicated that sEVs can reflect the pathogenesis of human diseases and mediate intercellular communications. Recently, sEV research has drastically increased due to their drug delivery property. However, a comprehensive method of delivering exogenous small RNAs-loaded sEVs through nebulization has not been reported. The methodology is complicated by uncertainty regarding the integrity of sEVs after nebulization, the delivery efficiency of aerosolized sEVs, their deposition in the lungs/cells, etc. This study demonstrates that sEVs can be delivered to murine lungs through a vibrating mesh nebulizer (VMN). In vivo sEV tracking indicated that inhaled sEVs were distributed exclusively in the lung and localized primarily in lung macrophages and airway epithelial cells. Additionally, sEVs loaded with small RNAs were successfully delivered into the lungs. The administration of siMyd88-loaded sEVs through inhalation reduced lipopolysaccharide (LPS)-induced lung injury in mice, supporting an application of this nebulization methodology to deliver functional small RNAs. Collectively, our study proposes a novel method of sEVs-mediated small RNA delivery into the murine lung through nebulization and presents a potential sEV-based therapeutic strategy for human lung diseases.

Identification of circulating microvesicle-encapsulated miR-223 as a potential novel biomarker for ARDS.

Acute respiratory distress syndrome (ARDS) is a lethal disease with severe forms conferring a mortality rate approaching 40%. The initial phase of ARDS results in acute lung injury (ALI) characterized by a severe inflammatory response and exudative alveolar flooding due to pulmonary capillary leak. Timely therapies to reduce ARDS mortality are limited by the lack of laboratory-guided diagnostic biomarkers for ARDS. The purpose of this study was to evaluate the prognostic role of circulating microvesicles (MVs)-containing miR-223 (MV-miR-223) if indicate more severe lung injury and worse outcomes in ARDS patients. Human plasma samples from one hundred ARDS patients enrolled in Albuterol to Treat Acute Lung Injury (ALTA) trial were compared to a control group of twenty normal human plasma specimens. The amount of MV-miR-223 was measured using absolute real-time polymerase chain reaction (PCR) with a standard curve. Mann-Whitney-Wilcoxon, Spearman correlation, Chi-squared tests, and Kaplan-Meier curves were computed to assess different variables and survival. Plasma levels of MV-miR-223 were significantly higher in ARDS patients compared to normal control subjects. Upon receiver operator characteristic (ROC) analysis of MV-miR-223 in relation to 30-day mortality, MV-miR-223 had an area under the curve (AUC) of 0.7021 with an optimal cut-off value of 2.413 pg/ml. Patients with high MV-miR-223 had higher 30-day mortality than subjects with low MV-miR-223 levels. MV-miR-223 was negatively correlated with ICU-free days, ventilator-free days, and organ failure-free days. Patients with high MV-miR-223 levels had higher 30 and 90-day mortality. MV-miR-223 was associated with 28-day clinical outcomes of ALTA trial including ICU-free days, ventilator-free days, and organ failure-free days. Thus, circulating MV-miR-223 may be a potential biomarker in prognosticating patient-centered outcomes and predicting mortality in ARDS.

The Potential of Lung Epithelium Specific Proteins as Biomarkers for COVID-19-Associated Lung Injury.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection was first reported in Wuhan, China, and was declared a pandemic by the World Health Organization (WHO) on 20 March 2020. The respiratory system is the major organ system affected by COVID-19. Numerous studies have found lung abnormalities in patients with COVID-19, including shortness of breath, respiratory failure, and acute respiratory distress syndrome. The identification of lung-specific biomarkers that are easily measurable in serum would be valuable for both clinicians and patients with such conditions. This review is focused on the pneumoproteins and their potential to serve as biomarkers for COVID-19-associated lung injury, including Krebs von den Lungen-6 (KL-6), surfactant proteins (SP-A, SP-B, SP-C, SP-D), and Clara cell secretory protein (CC16). The current findings indicate the aforementioned pneumoproteins may reflect the severity of pulmonary manifestations and could serve as potential biomarkers in COVID-19-related lung injury.