A potential role of microvesicle-containing miR-223/142 in lung inflammation.

BACKGROUND: Uncontrolled lung inflammation is one of the prominent features in the pathogenesis of lung infection- associated acute lung injury (ALI). Microvesicles (MVs) are extracellular nanovesicles that are generated via direct membrane budding. METHODS: Bronchoalveolar lavage fluid (BALF) samples were collected from mice with or without intratracheal lipopolysaccharide (LPS) instillation. BALF MVs were characterised and MV-containing microRNA (miRNA) profiles were assessed and confirmed. Secretion and function of MV-containing miR-223/142 (MV-miR-223/142) were analysed in vivo. RESULTS: In BALF, MVs are mainly derived from macrophages in response to LPS. After intratracheal instillation (i.t.) of LPS or Klebsiella pneumoniae, MV-containing miR-223/142 are dramatically induced in both BALF and serum. Mechanistically, miRNA 3' end uridylation mediates the packing of miR-223/142 into MVs. To investigate the functional role of MV-miR-223/142, we loaded miR-223/142 mimics into unstimulated MVs and delivered them into the murine lungs via i.t. The miR-223/142 mimics-enriched MVs selectively targeted lung macrophages and suppressed the inflammatory lung responses that were triggered by LPS or K. pneumoniae. Mechanistically, miR-223 and miR-142 synergistically suppress Nlrp3 inflammasome activation in macrophages via inhibition of Nlrp3 and Asc, respectively. CONCLUSIONS: In the pathogenesis of lung macrophage-mediated inflammatory responses, MV-miR-223/142 secretion is robustly enhanced and detectable in BALF and serum. Furthermore, restoration of intracellular miR-223/142 via vesicle-mediated delivery suppresses macrophage activation and lung inflammation via inhibition of Nlrp3 inflammasome activation.

Exosome-Mediated Small RNA Delivery: A Novel Therapeutic Approach for Inflammatory Lung Responses.

Exosomes (EXOs) are a type of extracellular nanovesicles released from living cells. Accumulating evidence suggests that EXOs are involved in the pathogenesis of human diseases, including lung conditions. In recent years, the potential of EXO-mediated drug delivery has gained increasing interest. In this report, we investigated whether inhaled EXOs serve as an efficient and practical delivery vehicle to activate or inhibit alveolar macrophages (AMs), subsequently modulating pulmonary immune responses. We first identified the recipient cells of the inhaled EXOs, which were labeled with PKH26. We found that only lung macrophages efficiently take up intratracheally instilled EXOs in vivo. Using modified calcium chloride-mediated transformation, we manipulated small RNA molecules in serum-derived EXOs, including siRNAs, microRNA (miRNA) mimics, and miRNA inhibitors. Via intratracheal instillation, we successfully delivered siRNA and miRNA mimics or inhibitors into lung macrophages using the serum-derived EXOs as vehicles. Furthermore, EXO siRNA or miRNA molecules are functional in modulating LPS-induced lung inflammation in vivo. Beneficially, serum-derived EXOs themselves do not trigger lung immune responses, adding more favorable features to serve as drug delivery agents. Collectively, we developed a novel protocol using serum-derived EXOs to deliver designated small RNA molecules into lung macrophages in vivo, potentially shedding light on future gene therapy of human lung diseases.

Sorting Mechanisms for MicroRNAs into Extracellular Vesicles and Their Associated Diseases.

Extracellular vesicles (EV) are secretory membranous elements used by cells to transport proteins, lipids, mRNAs, and microRNAs (miRNAs). While their existence has been known for many years, only recently has research begun to identify their function in intercellular communication and gene regulation. Importantly, cells have the ability to selectively sort miRNA into EVs for secretion to nearby or distant targets. These mechanisms broadly include RNA-binding proteins such as hnRNPA2B1 and Argonaute-2, but also membranous proteins involved in EV biogenesis such as Caveolin-1 and Neural Sphingomyelinase 2. Moreover, certain disease states have also identified dysregulated EV-miRNA content, shedding light on the potential role of selective sorting in pathogenesis. These pathologies include chronic lung disease, immune response, neuroinflammation, diabetes mellitus, cancer, and heart disease. In this review, we will overview the mechanisms whereby cells selectively sort miRNA into EVs and also outline disease states where EV-miRNAs become dysregulated.

Extracellular vesicle-cargo miR-185-5p reflects type II alveolar cell death after oxidative stress.

Acute respiratory distress syndrome (ARDS) is a devastating syndrome responsible for significant morbidity and mortality. Diffuse alveolar epithelial cell death, including but not limited to apoptosis and necroptosis, is one of the hallmarks of ARDS. Currently, no detectable markers can reflect this feature of ARDS. Hyperoxia-induced lung injury is a well-established murine model that mimics human ARDS. We found that hyperoxia and its derivative, reactive oxygen species (ROS), upregulate miR-185-5p, but not miR-185-3p, in alveolar cells. This observation is particularly more significant in alveolar type II (ATII) than alveolar type I (ATI) cells. Functionally, miR-185-5p promotes expression and activation of both receptor-interacting kinase I (RIPK1) and receptor-interacting kinase III (RIPK3), leading to phosphorylation of mixed lineage kinase domain-like (MLKL) and necroptosis. MiR-185-5p regulates this process probably via suppressing FADD and caspase-8 which are both necroptosis inhibitors. Furthermore, miR-185-5p also promotes intrinsic apoptosis, reflected by enhancing caspase-3/7 and 9 activity. Importantly, extracellular vesicle (EV)-containing miR-185-5p, but not free miR-185-5p, is detectable and significantly elevated after hyperoxia-induced cell death, both in vitro and in vivo. Collectively, hyperoxia-induced miR-185-5p regulates both necroptosis and apoptosis in ATII cells. The extracellular level of EV-cargo miR-185-5p is elevated in the setting of profound epithelial cell death.

Identification of miRNA-rich vesicles in bronchoalveolar lavage fluid: Insights into the function and heterogeneity of extracellular vesicles.

Despite emerging interest in the role of extracellular vesicle (EV)-containing microRNAs (EV-miRNAs), the existence of functional EV-miRNAs under patho-physiological conditions has been viewed with skepticism. Due to the heterogenicity of EVs, several barriers related to EV-miRNA research are to be explored before the in vivo function of EV-miRNAs can be thoroughly delineated. For example, it has been reported that far less than one copy of a given miRNA can be detected per exosome. In this study, we demonstrated that miRNA-rich-EVs exist and can be consistently isolated using differential centrifugation & density-gradient fractionation from bronchoalveolar lavage fluid (BALF) in vivo. The absolute number of this 'miRNA-rich'-EV population is only about 7.05 × 109 per mouse (6% of total EVs). However, the RNA amount detected in this population of EVs represents approximately 39% of the total EV RNAs in the BALF. In contrast, the remaining populations of BALF EVs (76% of total EVs) contain extremely low concentrations of RNAs and miRNAs. The miRNA-rich-EVs in BALF are likely derived from alveolar epithelial type-I cells (ATIs). Notably, caveolin-1, a lipid raft protein, is exclusively detected in the miRNA-rich-EVs, suggesting the lipid raft protein as a biomarker of EV-miRNA enrichment. We further demonstrated that miRNAs contained in the ATI-EVs are actively delivered into alveolar macrophages, subsequently promoting inflammasome activation, neutrophil recruitment, and M1-macrophage polarization in response to P. aeruginosa pneumonia in vitro and in vivo. Collectively, we are the first to identify and characterize the miRNA-rich-EVs in BALF. These miRNA-rich EVs endorse pro-inflammatory responses in bacterial lung infection. Our study provides a novel insight into the development of biomarkers, therapeutic strategies and underlying mechanisms for lung pathology.

Long Non-Coding RNA Review and Implications in Lung Diseases.

Non-coding genes occupy the majority of the human genome and have recently garnered increased attention for their implications in a range of diseases. This review illustrates the current scientific landscape concerning long non-coding RNA biogenesis, regulation, and degradation, as well as their functional roles in lung pathogenesis. LncRNAs share many similar biogenesis and regulatory processes with mRNA, such as capping, polyadenylation, post-transcriptional modifications, and exonuclease degradation. Evidence suggests that these lncRNAs become dysregulated in lung diseases such as Acute Lung Injury, Idiopathic Pulmonary Fibrosis, COPD, Lung Cancer, and Pulmonary Arterial Fiypertension. Some lncRNAs have known functions, but the overwhelming majority requires further research to completely understand.

Early prediction of functional outcome after stroke.

OBJECTIVES: (I) To obtain biomechanical parameters and assessment scores applied at a very early stage after stroke that predict best the functional outcome after rehabilitation. (II) To evaluate the predictive value of changes (i.e. increase or decrease) of these parameters during the first week in relation to the predictive value of their absolute scores. DESIGN: Prospective outcome study. SUBJECTS: Forty-one stroke patients, admitted to the stroke unit within 24 hours. MAIN OUTCOME MEASURES: Barthel Index, Rivermead Motor Assessment, Motor Club Assessment and Functional movement activities, NIH-Stroke scale (NIH-SS), Grip strength. RESULTS: Parameters assessed within the first hours after stroke correlated only weakly with the outcome. The best model predicting functional outcome and independence in activities of daily living of stroke patients after 6 months was that including NIH-SS, grip strength, age and previous stroke explaining 79% of the variance. These parameters assessed on day 7 post-stroke are more predictive than the difference between stroke onset and day 7 post-stroke. CONCLUSION: Parameters for predicting outcome should not be assessed before day 7 post-stroke.

Treatment of alcohol withdrawal syndrome with a combination of tiapride/carbamazepine: results of a pooled analysis in 540 patients.

This was a retrospective study to examine the efficacy, practicability and medical safety of a combination of tiapride and unretarded (fast acting formula) carbamazepine in the treatment of alcohol withdrawal syndrome. In five hospitals using this combination for treatment of alcohol withdrawal, 540 patients who had been treated with this combination were identified. An intensive evaluation of patients files and charts was performed. Details of alcohol history and comorbid disorders were extracted from patient files. Severity of alcohol withdrawal had been assessed using the CIWA-A-Score. Gender differences and differences between patients in their first and at least second withdrawal were computed by means of variance analyses (GLM). At baseline (day 1) mean dosage given was 796 for tiapride and 543 mg for carbamazepine. A pooled analysis of the results showed that, in general, medication was well tolerated. Withdrawal symptomatology as indicated by CIWA-A scores clearly decreased over time. Although a significant number of patients had a history of alcohol withdrawal delirium (103) and epileptic seizures (151), few patients suffered from them during treatment (8 and 5, respectively). Only 24 (4.4%) patients dropped out because of lack of efficacy or change of medication, 15 (2.8%) because of side effects. No case of malignant neuroleptic syndrome was recorded. Data analysis showed gender differences and differences between patients in their first and at least second withdrawal for side effects, complications, and in some CIWA-A-scores. In general, severe complications of withdrawal syndrome were more frequent in men compared to women and in patients with repeated inpatient treatment. In line with previous research, the results from this study give further evidence that a combination of the anticonvulsant carbamazepine and tiapride is an effective and safe treatment for alcohol withdrawal treatment.