Construction of a novel cell-free tracheal scaffold promoting vascularization for repairing tracheal defects.

Functional vascularization is crucial for maintaining the long-term patency of tissue-engineered trachea and repairing defective trachea. Herein, we report the construction and evaluation of a novel cell-free tissue-engineered tracheal scaffold that effectively promotes vascularization of the graft. Our findings demonstrated that exosomes derived from endothelial progenitor cells (EPC-Exos) enhance the proliferation, migration, and tube formation of endothelial cells. Taking advantage of the angiogenic properties of EPC-Exos, we utilized methacrylate gelatin (GelMA) as a carrier for endothelial progenitor cell exosomes and encapsulated them within a 3D-printed polycaprolactone (PCL) scaffold to fabricate a composite tracheal scaffold. The results demonstrated the excellent angiogenic potential of the methacrylate gelatin/vascular endothelial progenitor cell exosome/polycaprolactone tracheal scaffold. Furthermore, in vivo reconstruction of tracheal defects revealed the capacity of this composite tracheal stent to remodel vasculature. In conclusion, we have successfully developed a novel tracheal stent composed of methacrylate gelatin/vascular endothelial progenitor exosome/polycaprolactone, which effectively promotes angiogenesis for tracheal repair, thereby offering significant prospects for clinical and translational medicine.

Biomimetic in situ tracheal microvascularization for segmental tracheal reconstruction in one-step.

Formation of functional and perfusable vascular network is critical to ensure the long-term survival and functionality of the engineered tissue tracheae after transplantation. However, the greatest challenge in tracheal-replacement therapy is the promotion of tissue regeneration by rapid graft vascularization. Traditional prevascularization methods for tracheal grafts typically utilize omentum or muscle flap wrapping, which requires a second operation; vascularized segment tracheal orthotopic transplantation in one step remains difficult. This study proposes a method to construct a tissue-engineered tracheal graft, which directly forms the microvascular network after orthotopic transplantation in vivo. The focus of this study was the preparation of a hybrid tracheal graft that is non-immunogenic, has good biomechanical properties, supports cell proliferation, and quickly vascularizes. The results showed that vacuum-assisted decellularized trachea-polycaprolactone hybrid scaffold could match most of the above requirements as closely as possible. Furthermore, endothelial progenitor cells (EPCs) were extracted and used as vascularized seed cells and seeded on the surfaces of hybrid grafts before and during the tracheal orthotopic transplantation. The results showed that the microvascularized tracheal grafts formed maintained the survival of the recipient, showing a satisfactory therapeutic outcome. This is the first study to utilize EPCs for microvascular construction of long-segment trachea in one-step; the approach represents a promising method for microvascular tracheal reconstruction.

A novel decellularized trachea preparation method for the rapid construction of a functional tissue engineered trachea to repair tracheal defects.

Long segment trachea defects are repaired by tracheal substitution, while decellularized technology has been effectively employed to prepare tissue engineering trachea (TET). However, its clinical application is restricted by the long preparation cycle, while poor vascularization is associated with the transplantation failure. In the present study, we used sodium lauryl ether sulfate (SLES) to develop a novel rapid decellularized tracheal preparation method, then constructed a TET with revascularization functions. Summarily, we decellularized rabbit trachea using various SLES concentrations. Results from histological analysis, immunohistochemical and DAPI staining, as well as DNA quantitative assay, revealed that 1-0.1% (v/v) SLES treatment not only entirely removed cellular components to reduce its immunogenicity, but also retained the tracheal matrix's gross structure. SEM images, safranine O-fast green staining, total collagen content assay and collagen II immunofluorescence revealed that low SLES concentrations preserved the bioactive components of the decellularized tracheal matrix. Next, we performed cytobiocompatible and cytotoxin assays to verify biocompatibility of the decellularized tracheal matrix, and is confirmed by the omentum transplantation of rats. Results from omentum transplantation revealed that the decellularized tracheal matrix had low immunogenicity and excellent biocompatibility. Its revascularization capacity was confirmed by histologic appearance and CD31 immunofluorescence. Based on these findings, we selected 0.1% (v/v) as the optimal SLES concentration for preparing a decellularized tracheal matrix. Next, we seeded allogeneic bone marrow stem cells (BMSC) onto the matrix to construct TET patches. In vivo tracheal defect reconstruction confirmed the biocompatibility and revascularization capacity of this novel TET, and the formation of a vascular network around the patch promoted submucosa and mucosa regeneration without significant stenosis, 4 weeks post-surgery. In conclusion, we used SLES to successfully develop a novel decellularized approach for the preparation of TET, which has low immunogenic and inflammatory responses, as well as excellent biocompatibility, and revascularization ability in vivo without additional exogenous cytokines.

Directly construct microvascularization of tissue engineering trachea in orthotopic transplantation.

Tissue engineering technology provides effective alternative treatments for tracheal reconstruction. The formation of a functional microvascular network is essential to support cell metabolism and ensure the long-term survival of grafts. However, given the lack of an identifiable vascular pedicle of the trachea that could be anastomosed to the blood vessels directly in the recipient's neck, successful tracheal transplantation faces significant challenges in rebuilding the adequate blood supply of the graft. Herein, we describe a one-step method to construct microvascularization of tissue-engineered trachea in orthotopic transplantation. Forty rabbit tracheae were decellularized using a vacuum-assisted decellularization (VAD) method. Histological appearance and immunohistochemical (IHC) analysis demonstrated efficient removal of cellular components and nuclear material from natural tissue, which was also confirmed by 4'-6-diamidino-2-phenylindole(DAPI) staining and DNA quantitative analysis, thus significantly reducing the antigenicity. Scanning electron microscopy (SEM), immunofluorescence (IF) analysis, GAG and collagen quantitative analysis showed that the hierarchical structures, composition and integrity of the extracellular matrix (ECM) were protected. IF analysis also demonstrated that basic fibroblast growth factor (b-FGF) was preserved during the decellularization process, and also exerted biocompatibility and proangiogenic properties by the chick chorioallantoic membrane(CAM) assay. Xenotransplantation assays indicated that the VAD tracheal matrix would no longer induced inflammatory reactions implanted in the body for 4 weeks after treated by VAD more than 16 h. Furthermore, we seeded the matrix with bone marrow-derived endothelial cells (BMECs) in vitro and performed in vivo tracheal patch repair assays to prove the biocompatibility and neovascularization of VAD-treated tracheal matrix, and the formation of a vascular network around the patch promoted the crawling of surrounding ciliated epithelial cells to the surface of the graft. We conclude that this natural VAD tracheal matrix is non-immunogenic and no inflammatory reactions in vivo transplantation. Seeding with BMECs on the grafts and then performing orthotopic transplantation can effectively promote the microvascularization and accelerate the native epithelium cells crawling to the lumen of the tracheal graft.

Rapid Preparation Method for Preparing Tracheal Decellularized Scaffolds: Vacuum Assistance and Optimization of DNase I.

Decellularized scaffolds are an effective way for tracheal tissue engineering to perform alternative treatments. However, clinically used decellularized tracheal scaffolds have a long preparation cycle. The purpose of this study is to improve the efficiency of decellularization by vacuum assistance and optimizing the concentration of DNase I in the decellularization process and to quickly obtain tracheal decellularized scaffolds. The trachea of New Zealand white rabbits was decellularized with 2, 4, 6, and 8 KU/mL DNase I under vacuum. The performance of the decellularized tracheal scaffold was evaluated through histological analysis, immunohistochemical staining, DNA residue, extracellular matrix composition, scanning electron microscopy, mechanical properties, cell compatibility, and in vivo experiments. Histological analysis and immunohistochemical staining showed that compared with the native trachea, the hierarchical structure of the decellularized trachea remained unchanged after decellularization, nonchondrocytes were effectively removed, and the antigenicity of the scaffold was significantly weakened. Deoxyribonucleic acid (DNA) quantitative analysis showed that the amount of residual DNA in the 6-KU group was significantly decreased. Scanning electron microscopy and mechanical tests showed that small gaps appeared in the basement membrane of the 6-KU group, and the mechanical properties decreased. The CCK-8 test results and in vivo experiments showed that the 6-KU group's acellular scaffold had good cell compatibility and new blood vessels were visible on the surface. Taken together, the 6-KU group could quickly prepare rabbit tracheal scaffolds with good decellularization effects in only 2 days, which significantly shortened the preparation cycle reducing the required cost.

Exosomal Delivery of FTO Confers Gefitinib Resistance to Recipient Cells through ABCC10 Regulation in an m6A-dependent Manner.

Gefitinib is suitable for the treatment of locally advanced or metastatic non-small cell lung cancer. However, the development of acquired resistance limits its long-term efficacy in regardless of significant clinical benefit to patients. Therefore, to elucidate the mechanism of gefitinib resistance in addition to target gene mutation may greatly increase its clinical efficacy. It was found first that N 6-methyladenosine RNA demethylase FTO was significantly enriched in serum exosomes of gefitinib-resistant (GR) patients compared with that of gefitinib-sensitive (GS) patients through exosomal RNA sequencing. Meanwhile, the average m6A proportion in GR patients was significantly lower when compared with that in GS patients. Besides, GR cell-derived exosome internalization attenuated the total m6A abundance and gefitinib sensitivity of PC9 cells. Not only FTO knockdown enhanced the gefitinib sensitivity of GR cells but also FTO reduction in donor exosomes alleviated the acquired resistance of recipient PC9 cells. GR cell-derived exosomal-FTO promoted ABCC10 of recipient cells in a m6A-dependent manner. FTO/YTHDF2/ABCC10 axis played a role in intercellular transmission of GR cell-derived exosome-mediated gefitinib resistance both in vitro and in vivo. In general, this research showed that m6A modification was involved in the decrease of gefitinib sensitivity. GR cell-derived exosomes could decrease gefitinib sensitivity of recipient cells in exosomal delivery of FTO-dependent manner. FTO/YTHDF2/ABCC10 axis played a role in intercellular transmission of GR cell-derived exosome-mediated gefitinib resistance. IMPLICATIONS: Our results elucidated another potential molecular mechanism of gefitinib resistance in non-small cell lung cancer besides secondary EGFR mutations.

Primary malignant peripheral nerve sheath tumour of the trachea: a case report and literature review.

BACKGROUND: Malignant peripheral nerve sheath tumours (MPNSTs) of the trachea are extremely uncommon neoplasms with unknown genetic and clinical profiles. Only individual cases have been reported in the literature to date. CASE PRESENTATION: Here, we present a rare case of a 61-year-old female patient with a primary MPNST of the trachea who complained of irritating cough and progressively increasing breathlessness for 4 weeks. This patient initially underwent intraluminal resection of the mass and was misdiagnosed with clear cell sarcoma. Less than a year later, the mass relapsed, and the obstructive symptoms reappeared and gradually worsened. Debulking of the endotracheal tumour mass was performed once again, and an MPNST was definitively diagnosed. Open sleeve tracheal resection and tracheoplasty were later performed with curative intent. This patient was alive without recurrence at her six-month postoperative follow-up. We also compared the clinical outcomes of previously reported cases of MPNSTs and our case. CONCLUSIONS: This paper emphasizes that thoracic surgeons should be aware that malignant peripheral nerve sheath tumours of the trachea can be misdiagnosed in clinical practice and must be included in the differential diagnosis of tracheal neoplasms.

Autophagy decreases alveolar epithelial cell injury by regulating the release of inflammatory mediators.

To research the impact of autophagy on alveolar epithelial cell inflammation and its possible mechanism in the early stages of hypoxia, we established a cell hypoxia-reoxygenation model and orthotopic left lung ischemia-reperfusion model. Rat alveolar epithelial cells stably expressing GFP-LC3 were treated with an autophagy inhibitor (3-MA) or an autophagy promoter (rapamycin), followed by hypoxia-reoxygenation treatment for 2, 4, and 6 hr in vitro. In vivo, 20 male Sprague Dawley rats were randomly divided into four groups (model group: No blocking of the hilum in the left lung; control group: Blocking of the hilum in the left lung for 1 hr with dimethyl sulfoxide lavage; 3-MA group: Blocking of the hilum in the left lung for 1 hr with 100 ml/kg of 3-MA (5 µmol/L) solution lavage; and rapamycin group: Blocking of the hilum in the left lung for 1 hr with 100 ml/kg of rapamycin (250 nmol/L) solution lavage) to establish an orthotopic left lung ischemia model. This study demonstrated that rapamycin significantly suppressed the nuclear factor kappa B signaling pathway and limited the expression of proinflammatory factors. A contrary result was found after the 3-MA pretreatment. These findings indicate that autophagy reduces ischemia-reperfusion injury by repressing inflammatory signaling pathways in the early stages of hypoxia in vitro and in vivo. Autophagy could be a new protective method for application in lung ischemia-reperfusion injury.

Proteasome inhibition promotes autophagy and protects from endoplasmic reticulum stress in rat alveolar macrophages exposed to hypoxia-reoxygenation injury.

Alveolar macrophages play vital roles in acute lung injury, and macrophage response to hypoxia play relevant roles to disease mechanisms. There is growing evidence that cell death pathways play crucial roles in physiological and pathological settings and that the ubiquitin-proteasome system is involved in the regulation of these processes. However, the functional role of proteasome in alveolar macrophages exposed to hypoxia-reoxygenation (H/R) injury is unknown. We aimed to investigate the function of proteasome on alveolar macrophages exposed to H/R and the underlying mechanisms. NR8383 cells were pretreated with proteasome activator sulforaphane (SFN) or inhibitor MG-132 for 1 hr, and then submitted to 2/6 hr, 4/6 hr, and 6/6 hr H/R treatment. Cell viability was assessed with MTT assay. Autophagy was monitored using electron transmission microscope and flow cytometry and western blotting. The endoplasmic reticulum (ER) stress and unfolded protein response (UPR) pathways were equally analyzed by western blotting. Cell apoptosis was detected by immunohistochemistry, caspase3/7 activity, and western blotting. The viability of NR8383 cells exposed to H/R was affected by proteasome activity and proteasome inhibition significantly inhibited cell death. Treatment with MG-132 led to autophagy activation and induced the survival of NR8383 cells exposed to H/R. Pretreatment with SFN significantly decreased cell autophagy and induced cell death. ER stress was activated in H/R-treated NR8383 cells, and SFN further promoted ER stress whereas proteasome inhibition led to contrary results. Proteasome inhibtion hindered cell apoptosis as demonstrated by decreased caspase-3/7 activity, immunolabelling, and western blot results. Proteasome inhibition might be a promising approach for treating H/R injury-related lung diseases.

Autophagy Activation by Rapamycin Before Hypoxia-Reoxygenation Reduces Endoplasmic Reticulum Stress in Alveolar Epithelial Cells.

BACKGROUND: To determine potential effects of autophagy activation on hypoxia-reoxygenation (H/R) induced damage of a rat alveolar epithelial cell line. METHODS: CCL149 cells were subjected to autophagy agonist (rapamycin, Rap), autophagy inhibitor (3-methyladenine, 3-MA) or PBS for 1 h before H/R treatment for 2 h, 4 h and 6 h. The optimal concentration of Rap (150 nM, 200 nM and 250 nM) or 3-MA (5 mM, 10 mM and 15 mM) was obtained from MTT assay. Autophagy was determined by fluorescence microscopy of eRFP-LC3 positive cells, transmission electron microscopy of autophagosome, western blot of LC3, AMPK, Beclin-1, HDAC6 and p62 proteins. Endoplasmatic reticulum stress was indicated by detecting expressions of BIP, XBP-1 and CHOP via western blot. RESULTS: Rap at concentration of 250 nM before H/R increased the autophagy formation with more eRFP-LC3 positive cells and higher expressions of LC3-II, Beclin-1, HDAC6 and p62, but lower expressions of BIP, XBP-1 and CHOP in H/R treated CCL149. This effect seemed to be still obvious after H/R exposure for 6 h. The contrary results were obtained by treatment with 5 mM 3-MA. CONCLUSION: Rap might be a promising agent before mechanical ventilation or reperfusion to prevent re-damage in hypoxia related lung diseases.

Autophagy decreases alveolar macrophage apoptosis by attenuating endoplasmic reticulum stress and oxidative stress.

To study the impact of autophagy on alveolar macrophage apoptosis and its mechanism in the early stages of hypoxia, we established a cell hypoxia-reoxygenation model and orthotopic left lung ischemia-reperfusion model. Rat alveolar macrophages stably expressing RFP-LC3 were treated with autophagy inhibitor (3-methyladenine, 3-MA) or autophagy promoter (rapamycin), followed by hypoxia-reoxygenation treatment 2 h, 4 h or 6 h later. Twenty Sprague-Dawley male rats were randomly divided into four different groups: no blocking of left lung hilum (model group), left lung hilum blocked for 1h with DMSO lavage (control group), left lung hilum blocked for 1 h with 100 ml/kg 3-MA (5 µmol/L) lavage (3-MA group), and left lung hilum blocked for 1 h with 100 ml/kg rapamycin (250 nmol/L) lavage (rapamycin group). Rapamycin decreased the unfolded protein response, which reduced endoplasmic reticulum stress-mediated apoptosis in the presence of oxygen deficiency. Rapamycin increased superoxide dismutase activities and decreased malondialdehyde levels, whereas 3-MA decreased superoxide dismutase activities and increased malondialdehyde levels. Thus, autophagy decreases alveolar macrophage apoptosis by attenuating endoplasmic reticulum stress and oxidative stress in the early stage of hypoxia in vitro and in vivo. This could represent a new approach to protecting against lung ischemia-reperfusion injury.

Regulatory mechanisms of microRNAs in lung cancer stem cells.

Increasing evidence suggests that cancer stem cells (CSCs) are a key occurrence in the process of many human cancers. Lung cancer is the most common aggressive malignancy and cause of cancer death worldwide. The research on lung cancer stem cells has been highlighted for many years. Lung CSCs seem to play a major role in lung cancer metastasis, drug resistance and tumour-self-renewal. MicroRNAs (miRNAs), a class of newly emerging small noncoding RNAs that act as post-transcriptional regulators of gene expression, have been demonstrated to serve as a vital player in fine-tuning a number of biological activities ranging from embryogenesis to programmed cell death as well as tumourigenesis. In recent years, several miRNAs have been highlighted to be specifically expressed in CSCs. The miRNA profile of CSCs is remarkably different from non-stem cancer cells. As such, many miRNAs have been shown to regulate self-renewal and differentiation properties of CSCs. In this review, we present the latest findings on miRNAs that regulate the tumour microenvironment of lung CSCs with the goal to prompt the development of novel therapeutic strategies for patients with lung cancer.

Associations between autophagy, the ubiquitin-proteasome system and endoplasmic reticulum stress in hypoxia-deoxygenation or ischemia-reperfusion.

The activation of autophagy has been demonstrated to exert protective roles during hypoxia-reoxygenation (H/R)-induced brain injuries. This study aimed to investigate whether and how preconditioning with a proteasome inhibitor (MG-132), a proteasome promoter (Adriamycin, ADM), an autophagy inhibitor (3-methyladenine, 3-MA) and an autophagy promoter (Rapamycin, Rap) affected endoplasmic reticulum stress (ERS), the ubiquitin-proteasome system (UPS), autophagy, inflammation and apoptosis. Ubiquitin protein and 26S proteasome activity levels were decreased by MG-132 pretreatment but increased by ADM pretreatment at 2h, 4h and 6h following H/R treatment. MG-132 pretreatment led to the increased expression of autophagy-related genes, ER stress-associated genes and IκB but decreased the expression levels of NF-κB and caspase-3. ADM pretreatment led to the decreased expression of autophagy-related genes, ERS-associated genes and IκB but increased the expression of NF-κB and caspase-3. Pretreatment with 3-MA reduced the expression of autophagy-related genes, autophagy and UPS co-related genes, as well as apoptosis-related although the latter was increased by Rap pretreatment at 2h, 4h and 6h following H/R treatment. In vivo, pretreatment of rats with ADM, MG-132, 3-MA or Rap followed by ischemia-reperfusion (I/R) treatment resulted in similar changes. Proteasome inhibition preconditioning strengthened autophagy and ER stress but decreased apoptosis and inflammation. Autophagy promotion preconditioning exhibited similar changes. The combination of a proteasome inhibitor and an autophagy promoter might represent a new possible therapy to treat H/R or I/R injury-related diseases.

Five microRNAs in plasma as novel biomarkers for screening of early-stage non-small cell lung cancer.

BACKGROUND: In order to find novel noninvasive biomarkers with high accuracy for the screening of early-stage non-small cell lung cancer (NSCLC), we investigate the predictive power of 5 microRNAs (miR-20a, miR-145, miR-21, miR223 and miR-221) as potential biomarkers in early-stage NSCLC. METHODS: In training set, 25 early-stage NSCLC patients and 25 matched healthy controls are included to assess the miRNA expression profile between early-stage NSCLC patients and healthy controls by real-time RT-PCR. We found that five of these miRNAs (miR-20a, miR-223, miR-21, miR-221 and miR-145) levels in NSCLC patients were significantly dysregulated compared with the healthy groups and thus were selected to validation set. Therefore, a validation experiment was further performed to investigate the potential predictive power of these five miRNAs based on 126 early-stage NSCLC patients, 42 NCPD patients and 60 healthy controls. The receiver operating characteristic (ROC) curves were generated for the five miRNAs. RESULTS: ROC curve analyses suggested that these five plasma miRNAs could be promising biomarkers for NSCLC, with relatively high AUC values as follows: miR-20a, 0.89 with 95% CI of [0.85-0.93]; miR-223, 0.94 with 95% CI of [0.91-0.96]; miR-21, 0.77 with 95% CI of [0.71-0.83]; miR-155, 0.92 with 95% CI of [0.89-0.96]; miR-145, 0.77 with 95% CI of [0.71-0.83]. Stratified analyses indicated that plasma miR-20a, miR-223, miR-21 and miR-145 showed better predictive value in smokers than in non-smokers, while miR-155 might be more suitable for non-smokers. In addition, all of these five miRNAs could differentiate NSCLC from controls with a higher accuracy in advanced stage and squamous carcinoma subgroups. CONCLUSIONS: In conclusion, our study suggested that five plasma miRNAs (miR-20a, miR-145, miR-21, miR-223 and miR-221) can be used as promising biomarkers in early screening of NSCLC. Nevertheless, further validation and optimizing improvement should be performed on larger sample to confirm our results.

Indoor radon levels in selected hot spring hotels in Guangdong, China.

Guangdong is one of the provinces that have most hot springs in China, and many hotels have been set up near hot springs, with spring water introduced into the bath inside each hotel room for hot spring bathing to attract tourists. In the present study, we measured radon in indoor and outdoor air, as well as in hot spring waters, in four hot spring hotels in Guangdong by using NR-667A (III) continuous radon detector. Radon concentrations ranged 53.4-292.5 Bq L(-1) in the hot spring water and 17.2-190.9 Bq m(-3) in outdoor air. Soil gas intrusion, indoor hot spring water use and inefficient ventilation all contributed to the elevated indoor radon levels in the hotel rooms. From the variation of radon levels in closed unoccupied hotel rooms, soil gas intrusion was found to be a very important source of indoor radon in hotel rooms with floors in contact with soils. When there was spring water bathing in the bathes, average radon levels were 10.9-813% higher in the hotel rooms and 13.8-489% higher in bathes compared to their corresponding average levels when there was no spring water use. Spring water use in the hotel rooms had radon transfer coefficients from 1.6x10(-4) to 5.0x10(-3). Radon in some hotel rooms maintained in concentrations much higher than guideline levels might thus have potential health risks to the hotel workers, and technical and management measures should be taken to lower their exposure of radon through inhalation.