In response to vascular injury, vascular smooth muscle cells (VSMCs) may switch from a contractile to a proliferative phenotype thereby contributing to neointima formation. Previous studies showed that the long noncoding RNA (lncRNA) is critical for paraspeckle formation and tumorigenesis by promoting cell proliferation and migration. However, the role of in VSMC phenotypic modulation is unknown. Herein we showed that expression was induced in VSMCs during phenotypic switching in vivo and in vitro. Silencing in VSMCs resulted in enhanced expression of SM-specific genes while attenuating VSMC proliferation and migration. Conversely, overexpression of in VSMCs had opposite effects. These in vitro findings were further supported by in vivo studies in which knockout mice exhibited significantly decreased neointima formation following vascular injury, due to attenuated VSMC proliferation. Mechanistic studies demonstrated that sequesters the key chromatin modifier WDR5 (WD Repeat Domain 5) from SM-specific gene loci, thereby initiating an epigenetic "off" state, resulting in down-regulation of SM-specific gene expression. Taken together, we demonstrated an unexpected role of the lncRNA in regulating phenotypic switching by repressing SM-contractile gene expression through an epigenetic regulatory mechanism. Our data suggest that is a therapeutic target for treating occlusive vascular diseases.

Objective To investigate the functional role of the miR-15b/16 in vascular smooth muscle phenotypic modulation. Approach and Results We found that miR-15b/16 is the one of most abundant microRNAs expressed in contractile vascular smooth muscle cells (VSMCs). However, when contractile VSMCs convert to a synthetic phenotype miR-15b/16 expression is significantly reduced. Knocking-down endogenous miR-15b/16 in VSMCs attenuates smooth muscle-specific gene expression but promotes VSMC proliferation and migration. Conversely, over-expression of miR-15b/16 promotes smooth muscle contractile gene expression while attenuating VSMC migration and proliferation. Consistent with this, over-expression of miR-15b/16 in a rat carotid balloon injury model markedly attenuates injury-induced smooth muscle de-differentiation and neointima formation. Mechanistically, we identified the potent oncoprotein yes-associated protein (YAP) as a downstream target of miR-15b/16 in VSMCs. Reporter assays validated that miR-15b/16 targets YAP’s 3′-untranslated region. Moreover, overexpression of miR-15b/16 significantly represses YAP expression, whereas conversely, depletion of endogenous miR-15b/16 results in up-regulation of YAP expression. Conclusions These results indicate that miR-15b/16 plays a critical role in smooth muscle phenotypic modulation at least partly through targeting YAP. Restoring expression of miR-15b/16 would be a potential therapeutic approach for treatment of proliferative vascular diseases.

Background Previous reports have identified that human bone marrow mesenchymal stem cell-derived extracellular vesicles (BMSC-EVs) with their cargo microRNAs (miRNAs) are a promising therapeutic approach for the treatment of idiopathic pulmonary fibrosis (IPF). Therefore, we explored whether delivery of microRNA-186 (miR-186), a downregulated miRNA in IPF, by BMSC EVs could interfere with the progression of IPF in a murine model. Methods In a co-culture system, we assessed whether BMSC-EVs modulated the activation of fibroblasts. We established a mouse model of PF to evaluate the in vivo therapeutic effects of BMSC-EVs and determined miR-186 expression in BMSC-EVs by polymerase chain reaction. Using a loss-of-function approach, we examined how miR-186 delivered by BMSC-EVs affected fibroblasts. The putative relationship between miR-186 and SRY-related HMG box transcription factor 4 (SOX4) was tested using luciferase assay. Next, we investigated whether EV-miR-186 affected fibroblast activation and PF by targeting SOX4 and its downstream gene, Dickkopf-1 (DKK1). Results BMSC-EVs suppressed lung fibroblast activation and delayed IPF progression in mice. miR-186 was downregulated in IPF but enriched in the BMSC-EVs. miR-186 delivered by BMSC-EVs could suppress fibroblast activation. Furthermore, miR-186 reduced the expression of SOX4, a target gene of miR-186, and hence suppressed the expression of DKK1. Finally, EV-delivered miR-186 impaired fibroblast activation and alleviated PF via downregulation of SOX4 and DKK1. Conclusion In conclusion, miR-186 delivered by BMSC-EVs suppressed SOX4 and DKK1 expression, thereby blocking fibroblast activation and ameliorating IPF, thus presenting a novel therapeutic target for IPF.

As a comprehensive analysis of all metabolites in a biological system, metabolomics is being widely applied in various clinical/health areas for disease prediction, diagnosis, and prognosis. However, challenges remain in dealing with the metabolomic complexity, massive data, metabolite identification, intra- and inter-individual variation, and reproducibility, which largely limit its widespread implementation. This study provided a comprehensive workflow for clinical metabolomics, including sample collection and preparation, mass spectrometry (MS) data acquisition, and data processing and analysis. Sample collection from multiple clinical sites was strictly carried out with standardized operation procedures (SOP). During data acquisition, three types of quality control (QC) samples were set for respective MS platforms (GC-MS, LC-MS polar, and LC-MS lipid) to assess the MS performance, facilitate metabolite identification, and eliminate contamination. Compounds annotation and identification were implemented with commercial software and in-house-developed PAppLineTM and UlibMS library. The batch effects were removed using a deep learning model method (NormAE). Potential biomarkers identification was performed with tree-based modeling algorithms including random forest, AdaBoost, and XGBoost. The modeling performance was evaluated using the F1 score based on a 10-times repeated trial for each. Finally, a sub-cohort case study validated the reliability of the entire workflow.

Infection and inflammation serve an important role in tumor development. Toll-like receptor 4 (TLR4) is a pivotal component of the innate and adaptive immune response during infection and inflammation. Programmed-death ligand 1 (PD-L1) is hypothesized as an important factor for non-small cell lung cancer (NSCLC) immune escape. In the present study, the relationship between TLR4 and PD-L1, in addition to the associated molecular mechanism, were investigated. TLR4 and PD-L1 expression in lung cancer tissues were detected using immunohistochemistry, whilst overall patient survival was measured using the Kaplan-Meier method. The A549 cell line stimulated using lipopolysaccharide (LPS) was applied as the in vitro inflammatory NSCLC model. Associated factors were investigated using reverse transcription-quantitative PCR and western blotting. Lung cancer tissues exhibited increased PD-L1 and TLR4 levels compared with those of adjacent para-cancerous tissues, where there was a positive correlation between TLR4 and PD-L1 expression. In addition, increased expression of these two proteins was found to be linked with poorer prognoses. Following the stimulation of A549 cells with LPS, TLR4 and PD-L1 expression levels were revealed to be upregulated in a dose-dependent manner, where the ERK and PI3K/AKT signaling pathways were found to be activated. Interestingly, in the presence of inhibitors of these two pathways aforementioned, upregulation of PD-L1 expression was only inhibited by the MEK inhibitor PD98059, which can inhibit ERK activity. These data suggested that the ERK signaling pathway is necessary for the TLR4/PD-L1 axis. In conclusion, data from the present study suggest that TLR4 and PD-L1 expression can serve as important prognostic factors for NSCLC, where TLR4 activation may induce PD-L1 expression through the ERK signaling pathway.

Background/aim N-Acetylcysteine (NAC) demonstrates applications in the prevention of exacerbation of chronic obstructive pulmonary disease (COPD). COPD is often characterized by fibrosis of the small airways. This study aims at investigating the physiological mechanisms by which NAC might mediate the pulmonary fibrosis in COPD. Methods A total of 10 non-smokers without COPD and 10 smokers with COPD were recruited in this study, and COPD rat models were established. Cigarette smoke extract (CSE) cell models were constructed. The gain- or loss-of-function experiments were adopted to determine the expression of VWF and the extent of p38 MAPK phosphorylation, levels of interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and immunoglobulins (IgG, IgM and IgA) in the serum of COPD rats and supernatant of alveolar epithelial cells and to detect cell invasion and migration and the ratio of CD3+, CD4+, CD8+ and CD4+/CD8+T lymphocytes. Results Expression of VWF and the extent of p38 MAPK phosphorylation were increased in COPD. NAC inhibited p38 MAPK phosphorylation by reducing the VWF expression. NAC could inhibit cell migration and invasion, elevate E-cadherin expression, the ratio of CD3+, CD4+, CD8+ and CD4+/CD8+T lymphocytes, and levels of IgG, IgA, and IgM, and reduce N-cadherin expression and levels of IL-6 and TNF-α in CSE cells and serum of COPD rats. NAC promoted immune response and suppressed epithelial-mesenchymal transformation (EMT) to relieve COPD-induced pulmonary fibrosis in vitro and in vivo by inhibiting the VWF/p38 MAPK axis. Conclusions Collectively, NAC could ameliorate COPD-induced pulmonary fibrosis by promoting immune response and inhibiting EMT process via the VWF/p38 MAPK axis, therefore providing us with a potential therapeutic target for treating COPD.

Sepsis is a major cause of mortality in intensive care units, which results from a severely dysregulated inflammatory response that ultimately leads to organ failure. While antibiotics can help in the early stages, effective strategies to curtail inflammation remain limited. The high mobility group (HMG) proteins are chromosomal proteins with important roles in regulating gene transcription. While HMGB1 has been shown to play a role in sepsis, the role of other family members including HMGXB4 remains unknown. We found that expression of HMGXB4 is strongly induced in response to lipopolysaccharide (LPS)-elicited inflammation in murine peritoneal macrophages. Genetic deletion of Hmgxb4 protected against LPS-induced lung injury and lethality and cecal ligation and puncture (CLP)-induced lethality in mice, and attenuated LPS-induced proinflammatory gene expression in cultured macrophages. By integrating genome-wide transcriptome profiling and a publicly available ChIP-seq dataset, we identified HMGXB4 as a transcriptional activator that regulates the expression of the proinflammatory gene, Nos2 (inducible nitric oxide synthase 2) by binding to its promoter region, leading to NOS2 induction and excessive NO production and tissue damage. Similar to Hmgxb4 ablation in mice, administration of a pharmacological inhibitor of NOS2 robustly decreased LPS-induced pulmonary vascular permeability and lethality in mice. Additionally, we identified the cell adhesion molecule, ICAM1, as a target of HMGXB4 in endothelial cells that facilitates inflammation by promoting monocyte attachment. In summary, our study reveals a critical role of HMGXB4 in exacerbating endotoxemia via transcriptional induction of Nos2 and Icam1 gene expression and thus targeting HMGXB4 may be an effective therapeutic strategy for the treatment of sepsis.

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