| 1. |
- Almendros, Isaac, et al.
(author)
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Early Career Members at the ERS Lung Science Conference: cell-matrix interactions in lung disease and regeneration: Early career forum
- 2018
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In: Breathe. - : European Respiratory Society (ERS). - 1810-6838 .- 2073-4735. ; 14:2, s. 78-83
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Journal article (peer-reviewed)abstract
- The 16th ERS Lung Science Conference (LSC) took place on March 8–11, 2018, in Estoril, Portugal, with around 200 delegates from all over the world. This year’s topic was “Cell-matrix interactions in lung disease and regeneration” and involved excellent presentations by leading experts in the field covering everything from exploratory studies on how the matrix functions, matrix remodelling and biomarkers in disease, to more technical knowledge described in the field of lung bioengineering. As in previous years, the Saturday afternoon was reserved for a programme dedicated to early career delegates, which this year focussed on “Maximising your publication output”. In this article, we summarise the Early Career Member highlights of this year’s LSC.
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| 2. |
- Alsafadi, Hani N, et al.
(author)
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Applications and Approaches for Three-Dimensional Precision-Cut Lung Slices. Disease Modeling and Drug Discovery
- 2020
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In: American Journal of Respiratory Cell and Molecular Biology. - 1044-1549. ; 62:6, s. 681-691
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Research review (peer-reviewed)abstract
- Chronic lung diseases (CLDs), such as chronic obstructive pulmonary disease, interstitial lung disease, and lung cancer, are among the leading causes of morbidity globally and impose major health and financial burdens on patients and society. Effective treatments are scarce, and relevant human model systems to effectively study CLD pathomechanisms and thus discover and validate potential new targets and therapies are needed. Precision-cut lung slices (PCLS) from healthy and diseased human tissue represent one promising tool that can closely recapitulate the complexity of the lung's native environment, and recently, improved methodologies and accessibility to human tissue have led to an increased use of PCLS in CLD research. Here, we discuss approaches that use human PCLS to advance our understanding of CLD development, as well as drug discovery and validation for CLDs. PCLS enable investigators to study complex interactions among different cell types and the extracellular matrix in the native three-dimensional architecture of the lung. PCLS further allow for high-resolution (live) imaging of cellular functions in several dimensions. Importantly, PCLS can be derived from diseased lung tissue upon lung surgery or transplantation, thus allowing the study of CLDs in living human tissue. Moreover, CLDs can be modeled in PCLS derived from normal lung tissue to mimic the onset and progression of CLDs, complementing studies in end-stage diseased tissue. Altogether, PCLS are emerging as a remarkable tool to further bridge the gap between target identification and translation into clinical studies, and thus open novel avenues for future precision medicine approaches.
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| 3. |
- Alsafadi, Hani N.
(author)
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Role of the co-transcriptional regulators Yap/Taz in the normal and fibrotic lung epithelia
- 2023
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Doctoral thesis (other academic/artistic)abstract
- Idiopathic pulmonary fibrosis (IPF) is a fatal disease that exhibits patterns of usual interstitial pneumonia with honeycombing. IPF is characterized by damaged distal lung epithelium with excessive tissue scarring and extracellular matrix remodeling. The etiology of IPF is unknown and current therapies cannot end or reverse disease progression. Aberrant reactivation of developmental pathways is evident in IPF. Among these developmental actors are the co-transcriptional regulators Yap and Taz (YT). YT modulate processes such as proliferation, differentiation, and organ size and are regulated by the Hippo pathway. YT do not have a DNA binding domain but act through interaction with other transcription factors (TFs). YT play a role in fibrotic fibroblasts, but their role is not yet known in the fibrotic lung epithelium. The aim of this thesis project is to develop the tools needed to explore the role of Hippo-YT in fibrotic lung epithelium and to identify the TFs that YT interact with to exert their various functions.We first developed a method to simultaneously isolate proximal and distal lung progenitor cells from an individual mouse with the aid of a 3D printed surgical guide and found that the precision of dissecting the lung lobes affects the purity of the isolated distal progenitors and how they behave in organoid assays. We further found the Hippo pathway to be dysregulated in the fibrotic lung epithelium which led to increases in nuclear YT as well as known downstream targets. Interestingly, we found epithelial YT signaling to be actively involved in extracellular matrix remodeling in the fibrotic lung epithelium through modulation of lysyl oxidase expression, a collagen crosslinking enzyme. Targeting YT in vivo using an FDA approved drug ameliorated the fibrotic phenotype, indicating that YT targeting may be an option to treat fibrosis. We further used cleavage under target and release using nuclease (CUT&RUN) to identify the exact motif sequences on the genome where complexes containing YT bind in the normal and fibrotic lung epithelial. We further identified putative TFs that are known to bind the motif sequences identified. We found that YT have different interaction partners in the proximal and distal lung epithelium and further identified specific YT interactions in the human fibrotic lung epithelium.This current research project sets the basis for the identification of exact targeting mechanisms for finding therapeutics for IPF. YT are known to be responsible for a wide range of biologic processes and targeting YT’s profibrotic activity and promoting their pro-regenerative activities may result in beneficial effects for IPF patients.
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| 4. |
- Alsafadi, Hani N, et al.
(author)
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Simultaneous isolation of proximal and distal lung progenitor cells from individual mice using a 3D printed guide reduces proximal cell contamination of distal lung epithelial cell isolations
- 2022
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In: Stem Cell Reports. - : Elsevier BV. - 2213-6711. ; 17:12, s. 2718-2731
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Journal article (peer-reviewed)abstract
- The respiratory epithelium consists of multiple, functionally distinct cell types and is maintained by regionally specific progenitor populations that repair the epithelium following injury. Several in vitro methods exist for studying lung epithelial repair using primary murine lung cells, but isolation methods are hampered by a lack of surface markers distinguishing epithelial progenitors along the respiratory epithelium. Here, we developed a 3D printed lobe divider (3DLD) to aid in simultaneous isolation of proximal versus distal lung epithelial progenitors from individual mice that give rise to differentiated epithelia in multiple in vitro assays. In contrast to 3DLD-isolated distal progenitor cells, commonly used manual tracheal ligation methods followed by lobe removal resulted in co-isolation of rare proximal cells with distal cells, which altered the transcriptional landscape and size distribution of distal organoids. The 3DLD aids in reproducible isolation of distal versus proximal progenitor populations and minimizes the potential for contaminating populations to confound in vitro assays.
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| 5. |
- Bölükbas, Deniz A., et al.
(author)
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The Preparation of Decellularized Mouse Lung Matrix Scaffolds for Analysis of Lung Regenerative Cell Potential
- 2019
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In: Methods in molecular biology (Clifton, N.J.). - New York, NY : Springer New York. - 1940-6029. ; 1940, s. 275-295
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Journal article (peer-reviewed)abstract
- Lung transplantation is the only option for patients with end-stage lung disease, but there is a shortage of available lung donors. Furthermore, efficiency of lung transplantation has been limited due to primary graft dysfunction. Recent mouse models mimicking lung disease in humans have allowed for deepening our understanding of disease pathomechanisms. Moreover, new techniques such as decellularization and recellularization have opened up new possibilities to contribute to our understanding of the regenerative mechanisms involved in the lung. Stripping the lung of its native cells allows for unprecedented analyses of extracellular matrix and sets a physiologic platform to study the regenerative potential of seeded cells. A comprehensive understanding of the molecular pathways involved for lung development and regeneration in mouse models can be translated to regeneration strategies in higher organisms, including humans. Here we describe and discuss several techniques used for murine lung de- and recellularization, methods for evaluation of efficacy including histology, protein/RNA isolation at the whole lung, as well as lung slices level.
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| 6. |
- De Santis, Martina M, et al.
(author)
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Extracellular-Matrix-Reinforced Bioinks for 3D Bioprinting Human Tissue
- 2021
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In: Advanced Materials. - : Wiley. - 1521-4095 .- 0935-9648. ; 33:3
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Journal article (peer-reviewed)abstract
- Recent advances in 3D bioprinting allow for generating intricate structures with dimensions relevant for human tissue, but suitable bioinks for producing translationally relevant tissue with complex geometries remain unidentified. Here, a tissue-specific hybrid bioink is described, composed of a natural polymer, alginate, reinforced with extracellular matrix derived from decellularized tissue (rECM). rECM has rheological and gelation properties beneficial for 3D bioprinting while retaining biologically inductive properties supporting tissue maturation ex vivo and in vivo. These bioinks are shear thinning, resist cell sedimentation, improve viability of multiple cell types, and enhance mechanical stability in hydrogels derived from them. 3D printed constructs generated from rECM bioinks suppress the foreign body response, are pro-angiogenic and support recipient-derived de novo blood vessel formation across the entire graft thickness in a murine model of transplant immunosuppression. Their proof-of-principle for generating human tissue is demonstrated by 3D bioprinting human airways composed of regionally specified primary human airway epithelial progenitor and smooth muscle cells. Airway lumens remained patent with viable cells for one month in vitro with evidence of differentiation into mature epithelial cell types found in native human airways. rECM bioinks are a promising new approach for generating functional human tissue using 3D bioprinting.
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| 7. |
- Gerckens, Michael, et al.
(author)
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Generation of Human 3D Lung Tissue Cultures (3D-LTCs) for Disease Modeling
- 2019
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In: Journal of visualized experiments : JoVE. - : MyJove Corporation. - 1940-087X. ; :144
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Journal article (peer-reviewed)abstract
- Translation of novel discoveries to human disease is limited by the availability of human tissue-based models of disease. Precision-cut lung slices (PCLS) used as 3D lung tissue cultures (3D-LTCs) represent an elegant and biologically highly relevant 3D cell culture model, which highly resemble in situ tissue due to their complexity, biomechanics and molecular composition. Tissue slicing is widely applied in various animal models. 3D-LTCs derived from human PCLS can be used to analyze responses to novel drugs, which might further help to better understand the mechanisms and functional effects of drugs in human tissue. The preparation of PCLS from surgically resected lung tissue samples of patients, who experienced lung lobectomy, increases the accessibility of diseased and peritumoral tissue. Here, we describe a detailed protocol for the generation of human PCLS from surgically resected soft-elastic patient lung tissue. Agarose was introduced into the bronchoalveolar space of the resectates, thus preserving lung structure and increasing the tissue's stiffness, which is crucial for subsequent slicing. 500 µm thick slices were prepared from the tissue block with a vibratome. Biopsy punches taken from PCLS ensure comparable tissue sample sizes and further increase the amount of tissue samples. The generated lung tissue cultures can be applied in a variety of studies in human lung biology, including the pathophysiology and mechanisms of different diseases, such as fibrotic processes at its best at (sub-)cellular levels. The highest benefit of the 3D-LTC ex vivo model is its close representation of the in situ human lung in respect of 3D tissue architecture, cell type diversity and lung anatomy as well as the potential for assessment of tissue from individual patients, which is relevant to further develop novel strategies for precision medicine.
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| 8. |
- Gerckens, Michael, et al.
(author)
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Phenotypic drug screening in a human fibrosis model identified a novel class of antifibrotic therapeutics
- 2021
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In: Science Advances. - : American Association for the Advancement of Science (AAAS). - 2375-2548. ; 7:52, s. 1-19
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Journal article (peer-reviewed)abstract
- Fibrogenic processes instigate fatal chronic diseases leading to organ failure and death. Underlying biological processes involve induced massive deposition of extracellular matrix (ECM) by aberrant fibroblasts. We subjected diseased primary human lung fibroblasts to an advanced three-dimensional phenotypic high-content assay and screened a repurposing drug library of small molecules for inhibiting ECM deposition. Fibrotic Pattern Detection by Artificial Intelligence identified tranilast as an effective inhibitor. Structure-activity relationship studies confirmed N-(2-butoxyphenyl)-3-(phenyl)acrylamides (N23Ps) as a novel and highly potent compound class. N23Ps suppressed myofibroblast transdifferentiation, ECM deposition, cellular contractility, and altered cell shapes, thus advocating a unique mode of action. Mechanistically, transcriptomics identified SMURF2 as a potential therapeutic target network. Antifibrotic activity of N23Ps was verified by proteomics in a human ex vivo tissue fibrosis disease model, suppressing profibrotic markers SERPINE1 and CXCL8. Conclusively, N23Ps are a novel class of highly potent compounds inhibiting organ fibrosis in patients.
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| 9. |
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| 10. |
- Langwiński, Wojciech, et al.
(author)
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Allergic inflammation in lungs and nasal epithelium of rat model is regulated by tissue-specific miRNA expression
- 2022
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In: Molecular Immunology. - : Elsevier BV. - 0161-5890. ; 147, s. 115-125
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Journal article (peer-reviewed)abstract
- Introduction: Atopic asthma and allergic rhinitis are common chronic inflammatory diseases affecting lower airways and nasal mucosa, respectively. Several reports demonstrated frequent co-occurrence of these two diseases, however, the exact molecular mechanism has not been described. The present study aimed to investigate if small non-coding RNA might be responsible for the co-occurrence of asthma and allergic rhinitis in an animal model of allergic airway inflammation. Materials and methods: As an in vivo model of allergic airway inflammation, we used Brown Norway rats exposed intranasally to house dust mite (HDM). Histological analysis, total IgE concentration, eosinophil counts and iNOS gene expression were determined to confirm inflammatory changes. Small RNA sequencing in the lung tissue and nasal epithelium was performed with TruSeq Small RNA Library Preparation Kit and analyzed using the BaseSpace tool. Validation of sequencing results was performed using qPCR. To assess the functional role of hsa-miR-223–3p, we transfected normal human bronchial epithelial (NHBE) cells with specific LNA-inhibitor and measured phosphorylated protein level of NF-kB with ELISA. Expression analysis of NF-kB pathway-related genes was performed using qPCR with SYBR Green and analyzed in DataAssist v3.01. Statistical analysis were done with STATISTICA version 13. Results: We found 9 miRNA genes differentially expressed in the lungs of allergic rats. In nasal epithelium, only rno-miR-184 was upregulated in animals exposed to HDM. Validation with qPCR confirmed increased expression only for rno-miR-223–3p in the lungs from allergic rats. The expression of this miRNA was also increased in normal bronchial epithelial ALI cell culture stimulated with IL-13, but not in cells cultured in monolayer due to the low mRNA level of IL13RA1 and IL13RA2. Transfecting NHBE cells with hsa-miR-223–3p inhibitor increased the amount of phosphorylated NF-kB protein level and expression of MUC5AC, CCL24 and TSLP genes. Conclusions: These findings suggest that miRNAs that regulate allergic inflammation in the lungs and nasal epithelium are specific for upper and lower airways. Furthermore, our study provides new insight on the role of hsa-miR-223–3p, that via targeting NF-kB signaling pathway, regulates the expression of MUC5AC, CCL24 and TSLP. Taken together, our study suggests that miR-223–3p is a regulator of allergic inflammation and could potentially be used to develop novel and targeted therapy for asthma.
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