A simple optical tissue clearing pipeline for 3D vasculature imaging of the mediastinal organs in mice.

Optical tissue clearing (OTC) methods render tissue transparent by matching the refractive index within a sample to enable three-dimensional (3D) imaging with advanced microscopes. The application of OTC method in mediastinal organs in mice remains poorly understand. Our aim was to establish a simple protocol pipeline for 3D imaging of the mediastinal organs in mice. Trachea, oesophagus, thymus and heart were harvested from mice after retrograde perfusion via the abdominal aorta. We combined and optimized antibody labelling of thick tissue samples, OTC with cheap and non-toxic solvent ethyl cinnamate (ECi), and light-sheet fluorescence microscopy (LSFM) or laser confocal fluorescence microscopy (LCFM) to visualize the vasculature of those tissues. A high degree of optical transparency of trachea, oesophagus, thymus and heart was achieved after ECi-based OTC. With anti-CD31 antibody immunofluorescence labelling before ECi-based OTC, the vasculature of these tissues with their natural morphology, location and organizational network was imaged using LSFM or LCFM. This simple protocol pipeline provides an easy-to-setup and comprehensive way to study the vasculature of mediastinal organs in 3D without any special equipment. We anticipate that it will facilitate diverse applications in biomedical research of thoracic diseases and even other organs.

Application of ethyl cinnamate based optical tissue clearing and expansion microscopy combined with retrograde perfusion for 3D lung imaging.

PURPOSE: 3 D imaging of the lung is not a trivial undertaking as during preparation the lung may collapse. Also serial sections and scans followed by 3 D reconstruction may lead to artifacts. The present study aims to figure out the best way to perform 3 D imaging in lung research. MATERIALS AND METHODS: We applied an optical tissue clearing (OTC) method, which uses ethyl cinnamate (ECi) as a fast, nontoxic and cheap clearing solvent, for 3 D imaging of retrograde perfused lungs by laser confocal fluorescence microscopy and light sheet fluorescence microscopy. We also introduced expansion microscopy (ExM), a cutting-edge technique, in 3 D imaging of lungs. We examined and compared the usefulness of these techniques for 3 D lung imaging. The ExM protocol was further extended to paraffin-embedded lung metastases blocks. RESULTS: The MHI148-PEI labeled lung vasculature was visualized by retrograde perfusion combined with trachea ligation and ECi based OTC. As compared with trans-cardiac perfusion, the retrograde perfusion results in a better maintenance of lung morphology. 3 D structure of alveoli, vascular branches and cilia in lung were revealed by immunofluorescence staining after ExM. 3 D distribution of microvasculature and neutrophil cells in 10 years old paraffin-embedded lung metastases were analyzed by ExM. CONCLUSIONS: The retrograde perfusion combined with trachea ligation technique could be applied in the lung research in mice. 3 D structure of lung vasculature can be visualized by MHI148-PEI perfusion and ECi based OTC in an efficient way. ExM and immunofluorescence staining protocol is highly recommended to perform 3 D imaging of fresh fixed lung as well as paraffin-embedded lung blocks.

Iron Overload-induced Ferroptosis as a Target for Protection against Obliterative Bronchiolitis after Orthotopic Tracheal Transplantation in Mice.

INTRODUCTION: The major complication of Obliterative Bronchiolitis (OB) is characterized by epithelial cell loss, fibrosis, and luminal occlusion of the terminal small airways, which limits the long-term survival of the recipient after lung transplantation. However, the underlying mechanisms are still not fully clarified. This research aims to investigate whether iron overload-induced ferroptosis is involved in OB development and provide a new target for OB prevention. MATERIALS AND METHODS: Allograft orthotopic tracheal transplantation in mice was applied in our study. Ferrostatin-1 and deferoxamine were administrated to inhibit ferroptosis and get rid of ferric iron, while iron dextran was used to induce an iron overload condition in the recipient. The histological examination, luminal occlusion rate, collagen deposition, iron level, ferroptosis marker (GPX4, PTGS2), and mitochondrial morphological changes of the graft were evaluated in mice. RESULTS: Our research indicated that ferroptosis and iron overload contribute to OB development, while ferroptosis inhibition and iron chelator could reverse the changes. Iron overload exacerbated OB development after orthotopic tracheal transplantation via promoting ferroptosis. CONCLUSION: Overall, this research demonstrated that iron overload-induced ferroptosis is involved in OB, which may be a potential therapeutic target for OB after lung transplantation.

LncRNA HOXC-AS3 increases non-small cell lung cancer cell migration and invasion by sponging premature miR-96.

BACKGROUND: Long noncoding RNA (lncRNA) HOXC cluster antisense RNA 3 (HOXC-AS3) has been involved in breast cancer and gastric cancer, while its role in non-small cell lung cancer (NSCLC) is unknown. METHODS: The expression of HOXC-AS3 and miR-96 (both mature and premature) were detected using RT-qPCR. Nuclear fractionation assay and RNA pull-down assay were performed to detect the subcellular location of HOXC-AS3 and potential interaction with premature miR-96, respectively. Overexpression assays were performed to determine the role of HOXC-AS3 in the maturation of miR-96. Transwell assays were performed to explore the role of HOXC-AS3 and miR-96 in NSCLC cell invasion and migration. RESULTS: NSCLC tissues exhibited significantly increased expression levels of HOXC-AS3 and premature miR-96. HOXC-AS3 was localized to both nucleus and cytoplasm, and a direct interaction between HOXC-AS3 and premature miR-96 was observed. In NSCLC cells, HOXC-AS3 upregulated the expression of premature miR-96 but downregulated the expression of mature miR-96. Moreover, HOXC-AS3 suppressed the role of miR-96 in inhibiting NSCLC cell invasion and migration. CONCLUSION: HOXC-AS3 may increase NSCLC cell growth and invasion by sponging premature miR-96 to suppress its maturation.

New technical approaches for 3D morphological imaging and quantification of measurements.

3D imaging is becoming more and more popular, as it allows us to identify interactions between structures in organs. Furthermore, it gives the possibility to quantify and size these structures. To allow 3D imaging, the tissue sample has to be transparent. This is usually achieved by using optical tissue clearing protocols. Although using optical tissue clearing often results in perfect 3D images, these protocols have some pitfalls, like long duration of sample preparation (up to several weeks), use of toxic substances, damage to antibody staining, fluorescent proteins or dyes, high refractive indices, and high costs of sample processing.Recently we described [Huang et al., Scientific Reports 9(1): 521 (2019)] a fast, safe, and inexpensive ethyl cinnamate (ECi) based optical tissue clearing protocol. Here, we present extensions of our protocol with respect to the deparaffinization of old paraffin-embedded samples allowing 3D imaging of the blocks. In addition, we learned to remove ECi from the samples allowing the use of routine immunolabeling protocols. Furthermore, we demonstrate new pictures of lungs after expansion microscopy and adaptation of already existing protocols. The aim of our work is, in summary, to describe the advances in these methodologies, focusing on the morphological imaging of kidneys and lungs.

Maresin 1 Ameliorates Lung Ischemia/Reperfusion Injury by Suppressing Oxidative Stress via Activation of the Nrf-2-Mediated HO-1 Signaling Pathway.

Lung ischemia/reperfusion (I/R) injury occurs in various clinical conditions and heavily damaged lung function. Oxidative stress reaction and antioxidant enzymes play a pivotal role in the etiopathogenesis of lung I/R injury. In the current study, we investigated the impact of Maresin 1 on lung I/R injury and explored the possible mechanism involved in this process. MaR 1 ameliorated I/R-induced lung injury score, wet/dry weight ratio, myeloperoxidase, tumor necrosis factor, bronchoalveolar lavage fluid (BALF) leukocyte count, BALF neutrophil ratio, and pulmonary permeability index levels in lung tissue. MaR 1 significantly reduced ROS, methane dicarboxylic aldehyde, and 15-F2t-isoprostane generation and restored antioxidative enzyme (superoxide dismutase, glutathione peroxidase, and catalase) activities. Administration of MaR 1 improved the expression of nuclear Nrf-2 and cytosolic HO-1 in I/R-treated lung tissue. Furthermore, we also found that the protective effects of MaR 1 on lung tissue injury and oxidative stress were reversed by HO-1 activity inhibitor, Znpp-IX. Nrf-2 transcription factor inhibitor, brusatol, significantly decreased MaR 1-induced nuclear Nrf-2 and cytosolic HO-1 expression. In conclusion, these results indicate that MaR 1 protects against lung I/R injury through suppressing oxidative stress. The mechanism is partially explained by activation of the Nrf-2-mediated HO-1 signaling pathway.

Maresin 1 inhibits transforming growth factor-ß1-induced proliferation, migration and differentiation in human lung fibroblasts.

The myofibroblast has been implicated to be an important pathogenic cell in all fibrotic diseases, through synthesis of excess extracellular matrix. Lung fibroblast migration, proliferation and differentiation into a myofibroblast­like cell type are regarded as important steps in the formation of lung fibrosis. In the present study, the effect of maresin 1 (MaR 1), a pro­resolving lipid mediator, on transforming growth factor (TGF)­ß1­stimulated lung fibroblasts was investigated, and the underlying molecular mechanisms were examined. The results of the present study demonstrated that MaR 1 inhibited TGF­ß1­induced proliferative and migratory ability, assessed using MTT and scratch wound healing assays. The TGF­ß1­induced expression of α­smooth muscle actin (α­SMA) and collagen type I, the hallmarks of myofibroblast differentiation, was decreased by MaR 1 at the mRNA and protein levels, determined using the reverse transcription­quantitative polymerase chain reaction and western blot analysis, respectively. Immunofluorescence demonstrated that MaR 1 downregulated the TGF­ß1­induced expression of α­SMA. In addition, phosphorylated mothers against decapentaplegic homolog 2/3 (Smad2/3) and extracellular signal­related kinases (ERK) 1/2 were upregulated in TGF-ß1-induced lung fibroblasts, and these effects were attenuated by MaR 1 administration. In conclusion, the results of the present study demonstrated that MaR 1 inhibited the TGF­ß1­induced proliferation, migration and differentiation of human lung fibroblasts. These observed effects may be mediated in part by decreased phosphorylation of Smad2/3 and ERK1/2 signaling pathways. Therefore, MaR 1 may be a potential therapeutic approach to lung fibrotic diseases.

Knockdown of Long Noncoding RNA PCAT6 Inhibits Proliferation and Invasion in Lung Cancer Cells.

As a newly identified oncogenic long noncoding RNA (lncRNA), prostate cancer-associated transcript 6 (PCAT6) promoted cellular proliferation and colony formation of prostate cancer. However, the biological function of PCAT6 in lung cancer is still largely unknown. In this study, we found that PCAT6 is significantly increased in cancer tissues compared to normal tissues and positively correlates with metastasis of lung cancer in patients. We then examined PCAT6 expression in lung cancer cell lines and identified that PCAT6 expression was significantly elevated in lung cancer cells compared to normal human bronchial epithelial (NHBE) cells, especially in CL1-5 and H446 cells. PCAT6 knockdown significantly inhibited cellular proliferation and metastasis, as well as induced early apoptosis of lung cancer cells. Molecular analysis revealed that PCAT6 regulated the expression of two pivotal cancer-related proteins, c-Myc and p53, in lung cancer cells. However, PCAT6 was not directly combined with c-Myc and p53 as confirmed by RNA immunoprecipitation. Finally, a retrospective study further revealed that PCAT6 negatively correlates with overall survival of lung cancer patients. In conclusion, these results suggest that PCAT6 could play an oncogenic role in lung cancer progression and may serve as a biomarker for prognosis of lung cancer patients.

Circular RNA-ITCH Suppresses Lung Cancer Proliferation via Inhibiting the Wnt/ß-Catenin Pathway.

As a special form of noncoding RNAs, circular RNAs (circRNAs) played important roles in regulating cancer progression mainly by functioning as miRNA sponge. While the function of circular RNA-ITCH (cir-ITCH) in lung cancer is still less reported, in this study, we firstly detected the expression of cir-ITCH in tumor tissues and paired adjacent noncancer tissues of 78 patients with lung cancer using a TaqMan-based quantitative real-time PCR (qRT-PCR). The results showed that the expression of cir-ITCH was significantly decreased in lung cancer tissues. In cellular studies, cir-ITCH was also enhanced in different lung cancer cell lines, A549 and NIC-H460. Ectopic expression of cir-ITCH markedly elevated its parental cancer-suppressive gene, ITCH, expression and inhibited proliferation of lung cancer cells. Molecular analysis further revealed that cir-ITCH acted as sponge of oncogenic miR-7 and miR-214 to enhance ITCH expression and thus suppressed the activation of Wnt/ß-catenin signaling. Altogether, our results suggested that cir-ITCH may play an inhibitory role in lung cancer progression by enhancing its parental gene, ITCH, expression.