culturing-freezing-processing-imaging-entire-organoids-spheroids

Culturing, Freezing, Processing, and Imaging of Entire Organoids and Spheroids While Still in a Hydrogel

Organoids and spheroids, three-dimensional growing structures in cell culture labs, are becoming increasingly recognized as superior models compared to ...

Koc University

Idiopathic pulmonary fibrosis (IPF) is a complex lung disease, whose build-up scar tissue is induced by several molecules. Gastrin-releasing peptide (GRP) is released from pulmonary neuroendocrine cells, alveolar macrophages, and some nerve endings in the lung. A possible role of GRP in IPF is unclear. We aimed to investigate the fibrotic response to GRP, at the cellular level in MRC5 and A549 cell lines. The proliferative and fibrotic effects of GRP on these cells were evaluated by using BrdU, immunoblotting, immunofluorescence and qRT-PCR for molecules associated with myofibroblast differentiation, TGF-β and Wnt signalling. All doses of GRP increased the amount of BrdU incorporation in A549 cells. In contrast, the amount of BrdU increased in MRC5 cells in the first 24 h, though progressively decreased by 72 h. GRP did not stimulate epithelial-mesenchymal transition in A549 cells, rather, it stimulated the differentiation of MRC5 cells into myofibroblasts. Furthermore, GRP induced gene and protein expressions of p-Smad2/3 and Smad4, and reduced the levels of Smad7 in MRC5 cells. In addition, GRP decreased Wnt5a protein levels and stimulated β-catenin activation by increasing Wnt4, Wnt7a and β-catenin protein levels. GRP caused myofibroblast differentiation by inducing TGF-βand Wnt pathways via paracrine and autocrine signalling in MRC5 cells. In conclusion, GRP may lead to pulmonary fibrosis due to its proliferative and fibrotic effects on lung fibroblasts. The abrogation of GRP-mediated signal activation might be considered as a treatment modality for fibrotic lung diseases. Video Abstract.

Gastrin-releasing peptide induces fibrotic response in MRC5s and proliferation in A549s.

Vitamin U (Vit U) is a novel free-radical scavenger. The protective effect of Vit U on valproic acid (VPA)-induced lung damage was examined. Rats were divided into four groups: control rats; rats given Vit U (50 mg/kg/d, by gavage) for 15 days; rats treated with VPA (500 mg/kg/d, intraperitoneally) for 15 days; and rats were given VPA + Vit U (in same dose and time). On the 16th day of the experiment, the lungs were collected from rats. Lung structure, pulmonary oxidant/antioxidant parameters and Nrf2, α-SMA, and collagen-1 were evaluated by microscopic and biochemical analysis. Additionally, it was determined the interactions of Vit U with Nrf2 and Keap1 by in silico analysis. VPA administration increased lipid peroxidation and the activity of lactate dehydrogenase and myeloperoxidase. However, it decreased the glutathione level, and the activities of glutathione peroxidase, glutathione-S-transferase, catalase, and superoxide dismutase. VPA-mediated oxidative stress prompted structural distortion and fibrotic alterations in the lung. Vit U supplementation reversed structural and biochemical alterations, induced antioxidant system through Nrf2 activation, and attenuated fibrosis by reducing collagen expression in VPA-administered rats. However, Vit U pretreatment was unable to reduce α-SMA levels in the lung of VPA-treated rats. Molecular docking analysis showed the binding of Vit U to ETGE motif leads to dissociation of Nrf2 from the Nrf2/Keap1 complex and its transfer to nuclei. In conclusion, Vit U attenuated VPA-induced tissue damage by restoring antioxidative systems through amelioration of Nrf2 activity in the lung under oxidative stress.

The protective effect of vitamin U on valproic acid-induced lung toxicity in rats via amelioration of oxidative stress.

Long noncoding RNAs (LncRNAs) regulate epithelial-mesenchymal transition (EMT). EMT involves myofibroblast differentiation and pulmonary fibrosis (PF). We aimed to determine the expression profiles of HOTAIR, CARLo-5, and CD99P1 LncRNAs in EMT-mediated myofibroblast differentiation in A549 cells and fibrotic human lungs and to explain their roles. A group of A549s was stimulated with transforming growth factor β (TGF-β; 5 ng/ml) to induce EMT. The remaining A549s were incubated with 20 μM FH535 after 24 h of TGF-β treatment to inhibit EMT. A549s were collected at 0, 24, 36, and 48 h. Expressions of three LncRNAs and protein/genes related to EMT, myofibroblast differentiation, and PF were assayed by quantitative reverse-transcription polymerase chain reaction and Western blot analysis in A549s and fibrotic human lungs. The targets of three LncRNAs were investigated by bioinformatics methods. TGF-β stimulation resulted in increased expressions of three LncRNAs, ACTA2, COL1A1, SNAI1, CTNNB1, TCF4, LEF1, α-SMA, and active-β-catenin, and decreased E-cadherin at 24, 36, and 48 h in A549s. FH535 treatment regressed these alterations. But it increased HOTAIR expression at 36 h and did not increase E-cadherin at 48 h. Fibrotic human lungs were characterized by increased expressions of HOTAIR, CARLo-5, CD99P1, and miR-214, decreased expressions of miR-148b, miR-218-1, miR-7-1, and the presence of CARLo-5 and CD99P1 in HDAC1-LncRNAs coprecipitation products, but not HOTAIR. Bioinformatic analysis showed the interactions of three LncRNAs with both proteins and at least 13 microRNAs related to EMT and PF. In conclusion, HOTAIR, CARLo-5, and CD99P1 can regulate EMT-mediated myofibroblast differentiation through interacting with proteins and miRNAs associated with EMT and PF. These LncRNAs can be considered as potential targets to decrease EMT for treating PF.

Effect of long noncoding RNAs on epithelial-mesenchymal transition in A549 cells and fibrotic human lungs.

Coronavirus disease 2019 (COVID-19) is caused by the SARS-CoV-2 virus and has been affecting the world since the end of 2019. The disease led to significant mortality and morbidity in Turkey, since the first case was reported on March 11th, 2020. Studies suggest a positive association between air pollution and SARS-CoV-2 infection. The aim of the present study was to investigate the role of ambient particulate matters (PM), as potential carriers for SARS-CoV-2. Ambient PM samples in various size ranges were collected from 13 sites including urban and urban-background locations and hospital gardens in 10 cities across Turkey between 13th of May and 14th of June 2020 to investigate the possible presence of SARS-CoV-2 RNA on ambient PM. A total of 203 daily samples (TSP, n = 80; PM, n = 33; PM, n = 23; PMμm, n = 19; and 6 size segregated PM, n = 48) were collected using various samplers. The N1 gene and RdRP gene expressions were analyzed for the presence of SARS-CoV-2, as suggested by the Centers for Disease Control and Prevention (CDC). According to real time (RT)-PCR and three-dimensional (3D) digital (d) PCR analysis, dual RdRP and N1 gene positivity were detected in 20 (9.8%) samples. Ambient PM-bound SARS-CoV-2 was analyzed quantitatively and the air concentrations of the virus ranged from 0.1 copies/m to 23 copies/m. The highest percentages of virus detection on PM samples were from hospital gardens in Tekirdağ, Zonguldak, and Istanbul, especially in PM mode. Findings of this study have suggested that SARS-CoV-2 may be transported by ambient particles, especially at sites close to the infection hot-spots. However, whether this has an impact on the spread of the virus infection remains to be determined.

Existence of SARS-CoV-2 RNA on ambient particulate matter samples: A nationwide study in Turkey.

Malignant pleural mesothelioma (MPM) arises from mesothelial cells lining the pleural cavity of asbestos-exposed individuals and rapidly leads to death. MPM harbors loss-of-function mutations in BAP1, NF2, CDKN2A, and TP53, but isolated deletion of these genes alone in mice does not cause MPM and mouse models of the disease are sparse. Here, we show that a proportion of human MPM harbor point mutations, copy number alterations, and overexpression of KRAS with or without TP53 changes. These are likely pathogenic, since ectopic expression of mutant KRAS in the pleural mesothelium of conditional mice causes epithelioid MPM and cooperates with TP53 deletion to drive a more aggressive disease form with biphasic features and pleural effusions. Murine MPM cell lines derived from these tumors carry the initiating KRAS lesions, secondary Bap1 alterations, and human MPM-like gene expression profiles. Moreover, they are transplantable and actionable by KRAS inhibition. Our results indicate that KRAS alterations alone or in accomplice with TP53 alterations likely play an important and underestimated role in a proportion of patients with MPM, which warrants further exploration.

KRAS signaling in malignant pleural mesothelioma.

Extravasation of monocytes into tissue and to the site of injury is a fundamental immunological process, which requires rapid responses via post translational modifications (PTM) of proteins. Protein arginine methyltransferase 7 (PRMT7) is an epigenetic factor that has the capacity to mono-methylate histones on arginine residues. Here we show that in chronic obstructive pulmonary disease (COPD) patients, PRMT7 expression is elevated in the lung tissue and localized to the macrophages. In mouse models of COPD, lung fibrosis and skin injury, reduced expression of PRMT7 associates with decreased recruitment of monocytes to the site of injury and hence less severe symptoms. Mechanistically, activation of NF-κB/RelA in monocytes induces PRMT7 transcription and consequential mono-methylation of histones at the regulatory elements of RAP1A, which leads to increased transcription of this gene that is responsible for adhesion and migration of monocytes. Persistent monocyte-derived macrophage accumulation leads to ALOX5 over-expression and accumulation of its metabolite LTB4, which triggers expression of ACSL4 a ferroptosis promoting gene in lung epithelial cells. Conclusively, inhibition of arginine mono-methylation might offer targeted intervention in monocyte-driven inflammatory conditions that lead to extensive tissue damage if left untreated.

The arginine methyltransferase PRMT7 promotes extravasation of monocytes resulting in tissue injury in COPD.

Chronic obstructive pulmonary disease (COPD) is known as the third leading cause of human death globally. Enhanced chronic inflammation and pathological remodeling are the main consequences of COPD, leading to decreased life span. Histological and molecular investigations revealed that prominent immune cell infiltration and release of several cytokines contribute to progressive chronic remodeling. Recent investigations have revealed that exosomes belonging to extracellular vesicles are involved in the pathogenesis of COPD. It has been elucidated that exosomes secreted from immune cells are eligible to carry numerous pro-inflammatory factors exacerbating the pathological conditions. Here, in this review article, we have summarized various and reliable information about the negative role of immune cell-derived exosomes in the remodeling of pulmonary tissue and airways destruction in COPD patients.

Emerging role of exosomes in the pathology of chronic obstructive pulmonary diseases; destructive and therapeutic properties.

Statins have anti-inflammatory and antifibrotic effects in addition to cholesterol-lowering effect. We aimed to investigate the effect of atorvastatin (ATR) in fibrotic mouse lung and human lung fibroblasts (MRC5s). Pulmonary fibrosis was induced by a single dose of bleomycin by intratracheal instillation in adult mice. ATR was administered (20 mg/kg ip) to mice with healthy and pulmonary fibrosis for 10 days from Day 7 of the experiment. Mice were dissected on the 21st day. The levels of alpha-smooth muscle actin (α-SMA), pSMAD2/3, LOXL2, and p-Src were determined by Western blot analysis in the lungs. Furthermore, a group of MRC5 was differentiated into myofibroblasts by transforming growth factor-beta (TGF-β). Another group of MRC5s was treated with 10 µM ATR at 24 h after TGF-β stimulation. Cells were collected at 0, 24, 48, and 72 h. The effects of ATR on myofibroblast differentiation, apoptosis, and TGF-β and Wnt/β-catenin signaling activations were examined by Western blot analysis and flow cytometry in MRC5s. ATR attenuated pulmonary fibrosis by regulating myofibroblast differentiation and interstitial accumulation of collagen, by acting on LOXL2, p-Src, and pSMAD2/3 in mice lungs. Additionally, it blocked myofibroblast differentiation via reduced TGF-β and Wnt/β-catenin signaling and decreased α-SMA in MRC5s stimulated with TGF-β. Moreover, ATR caused myofibroblast apoptosis via caspase-3 activation. ATR treatment attenuates pulmonary fibrosis in mice treated with bleomycin. It also inhibits fibroblast/myofibroblast activation, by both reducing myofibroblasts differentiation and inducing myofibroblast apoptosis.

Atorvastatin attenuates pulmonary fibrosis in mice and human lung fibroblasts, by the regulation of myofibroblast differentiation and apoptosis.

Air pollution, especially at chronic exposure to high concentrations, is a respiratory risk factor for the development of chronic obstructive pulmonary disease (COPD). E-cadherin, a cell-cell adhesion protein, is involved in the integrity of the alveolar epithelium. Causes of E-cadherin decreases in emphysematous areas with pulmonary cell damage related to COPD are not well understood. We aimed to determine the molecules causing the decrease of E-cadherin and interactions between these molecules. In emphysematous and non-emphysematous areas of lungs from COPD patients (n = 35), levels of E-cadherin, HDACs, Snail, Zeb1, active-β-catenin, p120ctn, and Kaiso were determined by using Western Blot. The interactions of HDAC1, HDAC2, and p120ctn with transcription co-activators and Kaiso were examined by co-immunoprecipitation experiments. The methylation status of the  promoter was investigated. E-cadherin, Zeb1, Kaiso, and active-β-catenin were decreased in emphysema, while HDAC1, HDAC2, and p120ctn2 were increased. Snail, Zeb1, Twist, active-β-catenin, Kaiso, and p120ctn co-precipitated with HDAC1 and HDAC2. E-cadherin, Kaiso, and active-β-catenin co-precipitated with p120ctn. HDAC1-Snail and HDAC2-Kaiso interactions were increased in emphysema, but p120ctn-E-cadherin interaction was decreased. The results show that HDAC1-Snail and HDAC2-Kaiso interactions are capable of decreasing the E-cadherin in emphysema. The decreased interaction of p120ctn/E-cadherin leads to E-cadherin destruction. The decreased E-cadherin and its induced degradation in pneumocytes cause impaired repair and disintegrity of the epithelium. Approaches to suppress HDAC1-Snail and HDAC2-Kaiso interactions may help the protection of alveolar epithelial integrity by increasing the E-cadherin stability in pneumocytes.

Dysregulation of E-cadherin in pulmonary cell damage related with COPD contributes to emphysema.

There are contradictory views on which calcitonin gene-related peptide (CGRP) causes pulmonary fibrosis. Fibrotic potency of CGRP was tested and compared to that of transforming growth factor-β (TGF-β). Myofibroblast differentiation, cell proliferation, and activations of TGF-β and Wnt pathways were examined for 24, 48, and 72 h in A549 and MRC5 cell lines stimulated with CGRP and TGF-β. CGRP-induced cell proliferation in MRC5s early on while cell proliferation in A549 occurred progressively. CGRP promoted fibroblast-myofibroblast differentiation by inducing the transcription of ACTA2, COL1A1, SMAD2/3, and SMAD4 genes, the production of collagen, fibronectin, α-smooth muscle actin, and activation of TGF-β signaling starting from 24 h. Additionally, TGF-β signaling induced by CGRP decreased the DKK1 level and activated the Wnt signaling in MRC5s. After CGRP stimulation, Wnt7a levels were increased from 24 to 72 h, while Wnt5a levels were elevated at 72 h in MRC5s. CGRP did not induce epithelial-mesenchymal transition in A549s, unlike TGF-β. A comparison of fibrotic potency of CGRP and TGF-β showed that TGF-β is a powerful profibrotic molecule and induces earlier myofibroblast differentiation. Even so, CGRP promotes myofibroblast differentiation and extracellular matrix production by inducing Smad-dependent-TGF-β and Wnt signalings via autocrine and paracrine signalings in MRC5s.

CGRP induces myofibroblast differentiation and the production of extracellular matrix in MRC5s via autocrine and paracrine signalings.

Infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been growing swiftly worldwide. Patients with background chronic pulmonary inflammations such as asthma or chronic obstructive pulmonary diseases (COPD) are likely to be infected with this virus. Of note, there is an argument that COVID-19 can remain with serious complications like fibrosis or other pathological changes in the pulmonary tissue of patients with chronic diseases. Along with conventional medications, regenerative medicine, and cell-based therapy could be alternative approaches to compensate for organ loss or restore injured sites using different stem cell types. Owing to unique differentiation capacity and paracrine activity, these cells can accelerate the healing procedure. In this review article, we have tried to scrutinize different reports related to the harmful effects of SARS-CoV-2 on patients with asthma and COPD, as well as the possible therapeutic effects of stem cells in the alleviation of post-COVID-19 complications. Video abstract.

Ozgecan Kayalar

School of Medicine

Koc University

Culturing, Freezing, Processing, and Imaging of Entire Organoids and Spheroids While Still in a Hydrogel

Ozgecan Kayalar

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}School of Medicine

Koc University

Culturing, Freezing, Processing, and Imaging of Entire Organoids and Spheroids While Still in a Hydrogel

School of Medicine