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Hammoud S, Kern J, Mukherjee S, Lutkewitte AJ, Singh P, Newberry K, Finck BN, Gewin LS. Assays to enhance metabolic phenotyping in the kidney. Am J Physiol Renal Physiol 2025; 328:F563-F577. [PMID: 39819047 PMCID: PMC12145867 DOI: 10.1152/ajprenal.00232.2024] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/07/2024] [Revised: 08/22/2024] [Accepted: 01/06/2025] [Indexed: 01/19/2025] Open
Abstract
The kidney is highly metabolically active, and injury induces changes in metabolism that can impact repair and fibrosis progression. Changes in the expression of metabolism-related genes and proteins provide valuable data, but functional metabolic assays are critical to confirm changes in metabolic activity. Stable isotope metabolomics is the gold standard, but these involve considerable cost and specialized expertise. Both the Seahorse bioflux assays and substrate oxidation assays in tissues ex vivo are two relatively cost-effective assays for interrogating metabolism. Many institutions have access to Seahorse bioflux analyzers, which can easily and quickly generate data, but guidelines to enhance reproducibility are lacking. We investigate how variables (e.g. primary vs. immortalized cells, time in culture) impact the data generated by Seahorse bioflux analyzers. In addition, we show the utility of 3H-palmitate, a new approach for assessing fatty acid oxidation (FAO) in the kidney, in uninjured and injured kidney cortices. The 3H-palmitate substrate oxidation assays also demonstrate significant sex-dependent and strain-dependent differences in rates of fatty acid oxidation. These data should facilitate metabolic interrogation in the kidney field with enhanced reproducibility.NEW & NOTEWORTHY We show significant metabolic differences between both primary and immortalized cells, and among primary cells with different durations in cell culture. In addition, 3H-palmitate oxidation in tissue ex vivo is described as a method novel to the kidney for assessing the complete oxidation of long-chain fatty acids. This method shows that female mice have significantly increased fatty acid oxidation across two different strains of mice and significant strain-specific effects on metabolism.
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Affiliation(s)
- Safaa Hammoud
- Division of Nephrology, Department of Medicine, Washington University in St. Louis, St. Louis, Missouri, United States
| | - Justin Kern
- Division of Nephrology, Department of Medicine, Washington University in St. Louis, St. Louis, Missouri, United States
| | - Sandip Mukherjee
- Division of Nutritional Sciences and Obesity Medicine, Department of Medicine, Washington University in St. Louis, St. Louis, Missouri, United States
| | - Andrew J Lutkewitte
- Division of Nutritional Sciences and Obesity Medicine, Department of Medicine, Washington University in St. Louis, St. Louis, Missouri, United States
| | - Prabhleen Singh
- Division of Nephrology and Hypertension, University of California San Diego, San Diego, California, United States
- Department of Medicine, VA San Diego Healthcare System, San Diego, California, United States
| | - Kate Newberry
- Division of Nephrology, Department of Medicine, Washington University in St. Louis, St. Louis, Missouri, United States
| | - Brian N Finck
- Division of Nutritional Sciences and Obesity Medicine, Department of Medicine, Washington University in St. Louis, St. Louis, Missouri, United States
| | - Leslie S Gewin
- Division of Nephrology, Department of Medicine, Washington University in St. Louis, St. Louis, Missouri, United States
- Department of Medicine, Veterans Affairs Hospital, St. Louis VA Health Care System, St. Louis, Missouri, United States
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Mertlitz S, Riesner K, Kalupa M, Uhlig N, Cordes S, Verlaat L, Jamali M, Li N, Mohamed HMER, Bullinger L, Moss S, Greenwood J, Jatzlau J, Knaus P, Vallecillo-Garcia P, Penack O. Leucine-rich α-2 glycoprotein 1 (LRG1) during inflammatory complications after allogeneic stem cell transplantation and CAR-T cell therapy. J Immunother Cancer 2025; 13:e009372. [PMID: 40118496 PMCID: PMC11934407 DOI: 10.1136/jitc-2024-009372] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/03/2024] [Accepted: 03/10/2025] [Indexed: 03/23/2025] Open
Abstract
BACKGROUND Previous data indicated that the leucine-rich α-2 glycoprotein 1 (LRG1) pathway contributes to vascular dysfunction during cancer growth. Therapeutic targeting of LRG1 normalized tumor vessel dysfunction and enhanced the efficacy of anti-cancer adoptive T cell therapy. A major clinical problem after allogeneic hematopoietic stem cell transplantation (alloHSCT) and after chimeric antigen receptor (CAR) T-cell therapy is the induction of hyperinflammatory side effects, which are typically associated with severe endothelial dysfunction. METHODS We investigated LRG1 in preclinical models and in patient samples. RESULTS In prospective studies, we found elevated LRG1 serum levels in patients with cytokine release syndrome and immune effector cell-associated neurotoxicity syndrome after CAR-T-cell therapy as well as in patients with acute graft-versus-host disease (aGVHD) after alloHSCT.In preclinical models of aGVHD, we found vasculature-associated LRG1 upregulation as well as LRG1 pathway gene upregulation. The genetic deletion of LRG1 in alloHSCT donors and in alloHSCT recipients led to reduced clinical and histological aGVHD. In line with this, LRG1 deletion led to clinically and histologically reduced disease severity in experimental inflammatory models of colitis (dextran sulfate sodium colitis) and paw edema. LRG1 deletion reduced inflammation-related vascular leakiness, endothelial cell proliferation, and migration. CONCLUSIONS The current data support the hypothesis that LRG1 is an attractive therapeutic target after alloHSCT and after CAR-T cell therapy for cancer because of its role in dysfunctional tumor vessels as well as in inflammatory complications.
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Affiliation(s)
- Sarah Mertlitz
- Department of Hematology, Oncology and Tumorimmunology, Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin and Humboldt-Universität zu Berlin, Berlin, Germany
- German Cancer Consortium (Deutsches Konsortium Für Translationale Krebsforschung, DKTK), Partner Site, Berlin, Germany
- National Center for Tumor Diseases (NCT), Berlin, Germany
- Berlin Center for Translational Vascular Biomedicine, Berlin Institute of Health at Charité - Universitätsmedizin Berlin, Berlin, Germany
| | - Katarina Riesner
- Department of Hematology, Oncology and Tumorimmunology, Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin and Humboldt-Universität zu Berlin, Berlin, Germany
- German Cancer Consortium (Deutsches Konsortium Für Translationale Krebsforschung, DKTK), Partner Site, Berlin, Germany
- National Center for Tumor Diseases (NCT), Berlin, Germany
- Berlin Center for Translational Vascular Biomedicine, Berlin Institute of Health at Charité - Universitätsmedizin Berlin, Berlin, Germany
| | - Martina Kalupa
- Department of Hematology, Oncology and Tumorimmunology, Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin and Humboldt-Universität zu Berlin, Berlin, Germany
- German Cancer Consortium (Deutsches Konsortium Für Translationale Krebsforschung, DKTK), Partner Site, Berlin, Germany
- National Center for Tumor Diseases (NCT), Berlin, Germany
- Berlin Center for Translational Vascular Biomedicine, Berlin Institute of Health at Charité - Universitätsmedizin Berlin, Berlin, Germany
| | - Nora Uhlig
- Department of Hematology, Oncology and Tumorimmunology, Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin and Humboldt-Universität zu Berlin, Berlin, Germany
- German Cancer Consortium (Deutsches Konsortium Für Translationale Krebsforschung, DKTK), Partner Site, Berlin, Germany
- National Center for Tumor Diseases (NCT), Berlin, Germany
- Berlin Center for Translational Vascular Biomedicine, Berlin Institute of Health at Charité - Universitätsmedizin Berlin, Berlin, Germany
| | - Steffen Cordes
- Department of Hematology, Oncology and Tumorimmunology, Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin and Humboldt-Universität zu Berlin, Berlin, Germany
- German Cancer Consortium (Deutsches Konsortium Für Translationale Krebsforschung, DKTK), Partner Site, Berlin, Germany
- National Center for Tumor Diseases (NCT), Berlin, Germany
- Berlin Center for Translational Vascular Biomedicine, Berlin Institute of Health at Charité - Universitätsmedizin Berlin, Berlin, Germany
| | - Lydia Verlaat
- Department of Hematology, Oncology and Tumorimmunology, Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin and Humboldt-Universität zu Berlin, Berlin, Germany
- German Cancer Consortium (Deutsches Konsortium Für Translationale Krebsforschung, DKTK), Partner Site, Berlin, Germany
- National Center for Tumor Diseases (NCT), Berlin, Germany
- Berlin Center for Translational Vascular Biomedicine, Berlin Institute of Health at Charité - Universitätsmedizin Berlin, Berlin, Germany
| | - Mina Jamali
- Department of Hematology, Oncology and Tumorimmunology, Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin and Humboldt-Universität zu Berlin, Berlin, Germany
- German Cancer Consortium (Deutsches Konsortium Für Translationale Krebsforschung, DKTK), Partner Site, Berlin, Germany
- National Center for Tumor Diseases (NCT), Berlin, Germany
- Berlin Center for Translational Vascular Biomedicine, Berlin Institute of Health at Charité - Universitätsmedizin Berlin, Berlin, Germany
| | - Ningyu Li
- Department of Hematology, Oncology and Tumorimmunology, Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin and Humboldt-Universität zu Berlin, Berlin, Germany
- German Cancer Consortium (Deutsches Konsortium Für Translationale Krebsforschung, DKTK), Partner Site, Berlin, Germany
- National Center for Tumor Diseases (NCT), Berlin, Germany
- Berlin Center for Translational Vascular Biomedicine, Berlin Institute of Health at Charité - Universitätsmedizin Berlin, Berlin, Germany
| | - Hadeer Mohamed Elsayed Rasheed Mohamed
- Department of Hematology, Oncology and Tumorimmunology, Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin and Humboldt-Universität zu Berlin, Berlin, Germany
- German Cancer Consortium (Deutsches Konsortium Für Translationale Krebsforschung, DKTK), Partner Site, Berlin, Germany
- National Center for Tumor Diseases (NCT), Berlin, Germany
- Berlin Center for Translational Vascular Biomedicine, Berlin Institute of Health at Charité - Universitätsmedizin Berlin, Berlin, Germany
| | - Lars Bullinger
- Department of Hematology, Oncology and Tumorimmunology, Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin and Humboldt-Universität zu Berlin, Berlin, Germany
- German Cancer Consortium (Deutsches Konsortium Für Translationale Krebsforschung, DKTK), Partner Site, Berlin, Germany
- National Center for Tumor Diseases (NCT), Berlin, Germany
| | - Stephen Moss
- Institute of Ophthalmology, University College London, London, UK
| | - John Greenwood
- Institute of Ophthalmology, University College London, London, UK
| | - Jerome Jatzlau
- Institute of Chemistry and Biochemistry, Freie Universitaet Berlin, Berlin, Germany
| | - Petra Knaus
- Institute of Chemistry and Biochemistry, Freie Universitaet Berlin, Berlin, Germany
| | - Pedro Vallecillo-Garcia
- Department of Hematology, Oncology and Tumorimmunology, Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin and Humboldt-Universität zu Berlin, Berlin, Germany
- German Cancer Consortium (Deutsches Konsortium Für Translationale Krebsforschung, DKTK), Partner Site, Berlin, Germany
- National Center for Tumor Diseases (NCT), Berlin, Germany
- Berlin Center for Translational Vascular Biomedicine, Berlin Institute of Health at Charité - Universitätsmedizin Berlin, Berlin, Germany
| | - Olaf Penack
- Department of Hematology, Oncology and Tumorimmunology, Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin and Humboldt-Universität zu Berlin, Berlin, Germany
- German Cancer Consortium (Deutsches Konsortium Für Translationale Krebsforschung, DKTK), Partner Site, Berlin, Germany
- National Center for Tumor Diseases (NCT), Berlin, Germany
- Berlin Center for Translational Vascular Biomedicine, Berlin Institute of Health at Charité - Universitätsmedizin Berlin, Berlin, Germany
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Nakagaki R, Mukaibo T, Monir A, Gao X, Munemasa T, Nodai T, Tamura A, Obikane YH, Kondo Y, Masaki C, Hosokawa R. Simulated microgravity environment inhibits matrix mineralization during the osteoblast to osteocyte differentiation. Biochem Biophys Res Commun 2024; 739:150963. [PMID: 39550861 DOI: 10.1016/j.bbrc.2024.150963] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/21/2024] [Revised: 10/16/2024] [Accepted: 11/06/2024] [Indexed: 11/19/2024]
Abstract
This study investigates the effects of microgravity on the differentiation and mineralization of IDG-SW3 osteocyte-like cells to understand the response of bone cells to microgravity and develop strategies to mitigate bone loss in astronauts. IDG-SW3 cells were cultured in collagen-coated dishes and subjected to a 3D clinostat to simulate microgravity 14 days after initiating differentiation. The static group remained under normal gravity. Cells were analyzed on days 14, 18, 22, and 26. Alizarin red staining demonstrated a substantial and time-dependent increase in mineralization in the static group, whereas the microgravity group exhibited little detectable mineralization throughout the experimental period. Quantitative RT-PCR revealed significant upregulation of Rankl, Alpl, Dmp1, and Fgf23 and downregulation of Sost and Phex in the microgravity group. RNA sequencing on day 26 showed distinct gene expression profiles between conditions. Heatmaps highlighted upregulated genes (Ptgs2, Alpl, Comp, Atf4, Lox) and downregulated genes (Rspo2, Ank, Ptn, Mmp13, Aspn, Spp1) under microgravity. Gene ontology (GO) enrichment analysis indicated that upregulated genes were associated with cytoskeletal organization and receptor activities, while downregulated genes were linked to extracellular matrix components and immune response. These findings provide insights into the molecular mechanisms of bone loss in space and emphasize the importance of gravity in bone remodeling. Future studies should validate these genes' functions in osteocyte biology under microgravity.
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Affiliation(s)
- Ryutaro Nakagaki
- Division of Oral Reconstruction and Rehabilitation, Kyushu Dental University, Kitakyushu, Fukuoka, Japan
| | - Taro Mukaibo
- Division of Oral Reconstruction and Rehabilitation, Kyushu Dental University, Kitakyushu, Fukuoka, Japan.
| | - Ahmed Monir
- Division of Oral Reconstruction and Rehabilitation, Kyushu Dental University, Kitakyushu, Fukuoka, Japan; Department of Surgery, Anesthesiology, and Radiology, Faculty of Veterinary Medicine, Zagazig University, Sharkia, Egypt
| | - Xin Gao
- Lister Hill National Center for Biomedical Communication, National Library of Medicine, NIH, Bethesda, MD, USA
| | - Takashi Munemasa
- Division of Oral Reconstruction and Rehabilitation, Kyushu Dental University, Kitakyushu, Fukuoka, Japan
| | - Tomotaka Nodai
- Division of Oral Reconstruction and Rehabilitation, Kyushu Dental University, Kitakyushu, Fukuoka, Japan
| | - Akiko Tamura
- Division of Oral Reconstruction and Rehabilitation, Kyushu Dental University, Kitakyushu, Fukuoka, Japan
| | - Yui Hirata Obikane
- Division of Oral Reconstruction and Rehabilitation, Kyushu Dental University, Kitakyushu, Fukuoka, Japan
| | - Yusuke Kondo
- Division of Oral Reconstruction and Rehabilitation, Kyushu Dental University, Kitakyushu, Fukuoka, Japan
| | - Chihiro Masaki
- Division of Oral Reconstruction and Rehabilitation, Kyushu Dental University, Kitakyushu, Fukuoka, Japan
| | - Ryuji Hosokawa
- Division of Oral Reconstruction and Rehabilitation, Kyushu Dental University, Kitakyushu, Fukuoka, Japan
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Yamaguchi N, Horio E, Sonoda J, Yamagishi M, Miyakawa S, Murakami F, Hasegawa H, Katahira Y, Mizoguchi I, Fujii Y, Chikazu D, Yoshimoto T. Immortalization of Mesenchymal Stem Cells for Application in Regenerative Medicine and Their Potential Risks of Tumorigenesis. Int J Mol Sci 2024; 25:13562. [PMID: 39769322 PMCID: PMC11676347 DOI: 10.3390/ijms252413562] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/02/2024] [Revised: 12/13/2024] [Accepted: 12/16/2024] [Indexed: 01/11/2025] Open
Abstract
Regenerative medicine utilizes stem cells to repair damaged tissues by replacing them with their differentiated cells and activating the body's inherent regenerative abilities. Mesenchymal stem cells (MSCs) are adult stem cells that possess tissue repair and regenerative capabilities and immunomodulatory properties with a much lower risk of tumorigenicity, making them a focus of numerous clinical trials worldwide. MSCs primarily exert their therapeutic effects through paracrine effects via secreted factors, such as cytokines and exosomes. This has led to increasing interest in cell-free therapy, where only the conditioned medium (also called secretome) from MSC cultures is used for regenerative applications. However, MSCs face certain limitations, including cellular senescence, scarcity, donor heterogeneity, complexity, short survival post-implantation, and regulatory and ethics hurdles. To address these challenges, various types of immortalized MSCs (ImMSCs) capable of indefinite expansion have been developed. These cells offer significant promise and essential tools as a reliable source for both cell-based and cell-free therapies with the aim of translating them into practical medicine. However, the process of immortalization, often involving the transduction of immortalizing genes, poses potential risks of genetic instability and resultant malignant transformation. Cell-free therapy is particularly attractive, as it circumvents the risks of tumorigenicity and ethical concerns associated with live cell therapies. Rigorous safety tests, such as monitoring chromosomal abnormalities, are critical to ensure safety. Technologies like inducible or suicide genes may allow for the controlled proliferation of MSCs and induce apoptosis after their therapeutic task is completed. This review highlights recent advancements in the immortalization of MSCs and the associated risks of tumorigenesis.
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Affiliation(s)
- Natsuki Yamaguchi
- Department of Immunoregulation, Institute of Medical Science, Tokyo Medical University, 6-1-1 Shinjuku, Shinjuku-ku, Tokyo 160-8402, Japan
| | - Eri Horio
- Department of Immunoregulation, Institute of Medical Science, Tokyo Medical University, 6-1-1 Shinjuku, Shinjuku-ku, Tokyo 160-8402, Japan
| | - Jukito Sonoda
- Department of Immunoregulation, Institute of Medical Science, Tokyo Medical University, 6-1-1 Shinjuku, Shinjuku-ku, Tokyo 160-8402, Japan
| | - Miu Yamagishi
- Department of Immunoregulation, Institute of Medical Science, Tokyo Medical University, 6-1-1 Shinjuku, Shinjuku-ku, Tokyo 160-8402, Japan
| | - Satomi Miyakawa
- Department of Immunoregulation, Institute of Medical Science, Tokyo Medical University, 6-1-1 Shinjuku, Shinjuku-ku, Tokyo 160-8402, Japan
| | - Fumihiro Murakami
- Department of Immunoregulation, Institute of Medical Science, Tokyo Medical University, 6-1-1 Shinjuku, Shinjuku-ku, Tokyo 160-8402, Japan
| | - Hideaki Hasegawa
- Department of Immunoregulation, Institute of Medical Science, Tokyo Medical University, 6-1-1 Shinjuku, Shinjuku-ku, Tokyo 160-8402, Japan
| | - Yasuhiro Katahira
- Department of Immunoregulation, Institute of Medical Science, Tokyo Medical University, 6-1-1 Shinjuku, Shinjuku-ku, Tokyo 160-8402, Japan
| | - Izuru Mizoguchi
- Department of Immunoregulation, Institute of Medical Science, Tokyo Medical University, 6-1-1 Shinjuku, Shinjuku-ku, Tokyo 160-8402, Japan
| | - Yasuyuki Fujii
- Department of Oral and Maxillofacial Surgery, Tokyo Medical University, 6-7-1 Nishishinjuku, Shinjuku-ku, Tokyo 160-0023, Japan
| | - Daichi Chikazu
- Department of Oral and Maxillofacial Surgery, Tokyo Medical University, 6-7-1 Nishishinjuku, Shinjuku-ku, Tokyo 160-0023, Japan
| | - Takayuki Yoshimoto
- Department of Immunoregulation, Institute of Medical Science, Tokyo Medical University, 6-1-1 Shinjuku, Shinjuku-ku, Tokyo 160-8402, Japan
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Kim JJ, Yang EJ, Molina David J, Cho S, Ficarella M, Pape N, Schiffer JE, Njeim R, Kim SS, Lo Re C, Fontanella A, Kaber M, Sloan A, Merscher S, Fornoni A. Ezetimibe Enhances Lipid Droplet and Mitochondria Contact Formation, Improving Fatty Acid Transfer and Reducing Lipotoxicity in Alport Syndrome Podocytes. Int J Mol Sci 2024; 25:13134. [PMID: 39684843 DOI: 10.3390/ijms252313134] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/29/2024] [Revised: 11/28/2024] [Accepted: 12/03/2024] [Indexed: 12/18/2024] Open
Abstract
Mitochondrial dysfunction is a critical factor in the pathogenesis of Alport syndrome (AS), contributing to podocyte injury and disease progression. Ezetimibe, a lipid-lowering drug, is known to inhibit cholesterol and fatty acid uptake and to reduce triglyceride content in the kidney cortex of mice with AS. However, its effects on lipid droplet (LD) utilization by mitochondria have not been explored. Transmission electron microscopy (TEM) and mitochondrial functional assays (ATP production, mitochondrial membrane potential, and citrate synthase activity) were used to investigate the impact of ezetimibe on LD-mitochondria contact formation and mitochondrial function in Col4a3KO (AS) and wildtype (WT) podocytes. TEM analysis revealed significant mitochondrial abnormalities in AS podocytes, including swollen mitochondria and reduced cristae density, while mitochondrial function assays showed decreased ATP production and lowered mitochondrial membrane potential. AS podocytes also demonstrated a higher content of LD but with reduced LD-mitochondria contact sites. Ezetimibe treatment significantly increased the number of LD-mitochondria contact sites, enhanced fatty acid transfer efficiency, and reduced intracellular lipid accumulation. These changes were associated with a marked reduction in the markers of lipotoxicity, such as apoptosis and oxidative stress. Mitochondrial function was significantly improved, evidenced by increased basal respiration, ATP production, maximal respiration capacity, and the restoration of mitochondrial membrane potential. Additionally, mitochondrial swelling was significantly reduced in ezetimibe-treated AS podocytes. Our findings reveal a novel role for ezetimibe in enhancing LD-mitochondria contact formation, leading to more efficient fatty acid transfer, reduced lipotoxicity, and improved mitochondrial function in AS podocytes. These results suggest that ezetimibe could be a promising therapeutic agent for treating mitochondrial dysfunction and lipid metabolism abnormalities in AS.
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Affiliation(s)
- Jin-Ju Kim
- Katz Family Division of Nephrology and Hypertension, Department of Medicine, University of Miami Miller School of Medicine, Miami, FL 33136, USA
- Peggy and Harold Katz Family Drug Discovery Center, University of Miami Miller School of Medicine, Miami, FL 33136, USA
| | - Eun-Jeong Yang
- Institute for Immunology and Immunological Diseases, Yonsei University College of Medicine, Seoul 03722, Republic of Korea
| | - Judith Molina David
- Katz Family Division of Nephrology and Hypertension, Department of Medicine, University of Miami Miller School of Medicine, Miami, FL 33136, USA
- Peggy and Harold Katz Family Drug Discovery Center, University of Miami Miller School of Medicine, Miami, FL 33136, USA
| | - Sunjoo Cho
- Katz Family Division of Nephrology and Hypertension, Department of Medicine, University of Miami Miller School of Medicine, Miami, FL 33136, USA
- Peggy and Harold Katz Family Drug Discovery Center, University of Miami Miller School of Medicine, Miami, FL 33136, USA
| | - Maria Ficarella
- Katz Family Division of Nephrology and Hypertension, Department of Medicine, University of Miami Miller School of Medicine, Miami, FL 33136, USA
- Peggy and Harold Katz Family Drug Discovery Center, University of Miami Miller School of Medicine, Miami, FL 33136, USA
| | - Nils Pape
- Katz Family Division of Nephrology and Hypertension, Department of Medicine, University of Miami Miller School of Medicine, Miami, FL 33136, USA
- Peggy and Harold Katz Family Drug Discovery Center, University of Miami Miller School of Medicine, Miami, FL 33136, USA
| | - Josephin Elizabeth Schiffer
- Katz Family Division of Nephrology and Hypertension, Department of Medicine, University of Miami Miller School of Medicine, Miami, FL 33136, USA
- Peggy and Harold Katz Family Drug Discovery Center, University of Miami Miller School of Medicine, Miami, FL 33136, USA
| | - Rachel Njeim
- Katz Family Division of Nephrology and Hypertension, Department of Medicine, University of Miami Miller School of Medicine, Miami, FL 33136, USA
- Peggy and Harold Katz Family Drug Discovery Center, University of Miami Miller School of Medicine, Miami, FL 33136, USA
| | - Stephanie S Kim
- Katz Family Division of Nephrology and Hypertension, Department of Medicine, University of Miami Miller School of Medicine, Miami, FL 33136, USA
- Peggy and Harold Katz Family Drug Discovery Center, University of Miami Miller School of Medicine, Miami, FL 33136, USA
| | - Claudia Lo Re
- Katz Family Division of Nephrology and Hypertension, Department of Medicine, University of Miami Miller School of Medicine, Miami, FL 33136, USA
- Peggy and Harold Katz Family Drug Discovery Center, University of Miami Miller School of Medicine, Miami, FL 33136, USA
- Unit of Nephrology and Dialysis, Department of Clinical and Experimental Medicine, A.O.U "G. Martino", University of Messina, 98122 Messina, Italy
| | - Antonio Fontanella
- Katz Family Division of Nephrology and Hypertension, Department of Medicine, University of Miami Miller School of Medicine, Miami, FL 33136, USA
- Peggy and Harold Katz Family Drug Discovery Center, University of Miami Miller School of Medicine, Miami, FL 33136, USA
| | - Maria Kaber
- Katz Family Division of Nephrology and Hypertension, Department of Medicine, University of Miami Miller School of Medicine, Miami, FL 33136, USA
- Peggy and Harold Katz Family Drug Discovery Center, University of Miami Miller School of Medicine, Miami, FL 33136, USA
| | - Alexis Sloan
- Katz Family Division of Nephrology and Hypertension, Department of Medicine, University of Miami Miller School of Medicine, Miami, FL 33136, USA
- Peggy and Harold Katz Family Drug Discovery Center, University of Miami Miller School of Medicine, Miami, FL 33136, USA
| | - Sandra Merscher
- Katz Family Division of Nephrology and Hypertension, Department of Medicine, University of Miami Miller School of Medicine, Miami, FL 33136, USA
- Peggy and Harold Katz Family Drug Discovery Center, University of Miami Miller School of Medicine, Miami, FL 33136, USA
| | - Alessia Fornoni
- Katz Family Division of Nephrology and Hypertension, Department of Medicine, University of Miami Miller School of Medicine, Miami, FL 33136, USA
- Peggy and Harold Katz Family Drug Discovery Center, University of Miami Miller School of Medicine, Miami, FL 33136, USA
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Cederroth CR, Dyhrfjeld-Johnsen J, Canlon B. Pharmacological Approaches to Hearing Loss. Pharmacol Rev 2024; 76:1063-1088. [PMID: 39164117 PMCID: PMC11549935 DOI: 10.1124/pharmrev.124.001195] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/14/2024] [Revised: 07/08/2024] [Accepted: 07/16/2024] [Indexed: 08/22/2024] Open
Abstract
Hearing disorders pose significant challenges to individuals experiencing them and their overall quality of life, emphasizing the critical need for advanced pharmacological approaches to address these conditions. Current treatment options often focus on amplification devices, cochlear implants, or other rehabilitative therapies, leaving a substantial gap regarding effective pharmacological interventions. Advancements in our understanding of the molecular and cellular mechanisms involved in hearing disorders induced by noise, aging, and ototoxicity have opened new avenues for drug development, some of which have led to numerous clinical trials, with promising results. The development of optimal drug delivery solutions in animals and humans can also enhance the targeted delivery of medications to the ear. Moreover, large genome studies contributing to a genetic understanding of hearing loss in humans combined with advanced molecular technologies in animal studies have shown a great potential to increase our understanding of the etiologies of hearing loss. The auditory system exhibits circadian rhythms and temporal variations in its physiology, its vulnerability to auditory insults, and its responsiveness to drug treatments. The cochlear clock rhythms are under the control of the glucocorticoid system, and preclinical evidence suggests that the risk/benefit profile of hearing disorder treatments using chronopharmacological approaches would be beneficial. If translatable to the bedside, such approaches may improve the outcome of clinical trials. Ongoing research into the molecular and genetic basis of auditory disorders, coupled with advancements in drug formulation and delivery as well as optimized timing of drug administration, holds great promise of more effective treatments. SIGNIFICANCE STATEMENT: Hearing disorders pose significant challenges to individuals and their overall quality of life, emphasizing the critical need for advanced pharmacological approaches to address these conditions. Ongoing research into the molecular and genetic basis of auditory disorders, coupled with advancements in drug delivery procedures and optimized timing of drug administration, holds the promise of more effective treatments.
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Affiliation(s)
- Christopher R Cederroth
- Department of Physiology and Pharmacology, Karolinska Institute, Stockholm, Sweden (C.R.C., B.C.); Translational Hearing Research, Tübingen Hearing Research Center, Department of Otolaryngology, Head and Neck Surgery, University of Tübingen, Tübingen, Germany (C.R.C.); and Acousia Therapeutics GmbH, Tübingen, Germany (J.D.-J.)
| | - Jonas Dyhrfjeld-Johnsen
- Department of Physiology and Pharmacology, Karolinska Institute, Stockholm, Sweden (C.R.C., B.C.); Translational Hearing Research, Tübingen Hearing Research Center, Department of Otolaryngology, Head and Neck Surgery, University of Tübingen, Tübingen, Germany (C.R.C.); and Acousia Therapeutics GmbH, Tübingen, Germany (J.D.-J.)
| | - Barbara Canlon
- Department of Physiology and Pharmacology, Karolinska Institute, Stockholm, Sweden (C.R.C., B.C.); Translational Hearing Research, Tübingen Hearing Research Center, Department of Otolaryngology, Head and Neck Surgery, University of Tübingen, Tübingen, Germany (C.R.C.); and Acousia Therapeutics GmbH, Tübingen, Germany (J.D.-J.)
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7
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Lekkos K, Bhuiyan AA, Albloshi AMK, Brooks PM, Coate TM, Lionikas A. Validation of positional candidates Rps6ka6 and Pou3f4 for a locus associated with skeletal muscle mass variability. G3 (BETHESDA, MD.) 2024; 14:jkae046. [PMID: 38577978 PMCID: PMC11075558 DOI: 10.1093/g3journal/jkae046] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 01/18/2024] [Accepted: 02/17/2024] [Indexed: 04/06/2024]
Abstract
Genetic variability significantly contributes to individual differences in skeletal muscle mass; however, the specific genes involved in that process remain elusive. In this study, we examined the role of positional candidates, Rps6ka6 and Pou3f4, of a chromosome X locus, implicated in muscle mass variability in CFW laboratory mice. Histology of hindlimb muscles was studied in CFW male mice carrying the muscle "increasing" allele C (n = 15) or "decreasing" allele T (n = 15) at the peak marker of the locus, rs31308852, and in the Pou3f4y/- and their wild-type male littermates. To study the role of the Rps6ka6 gene, we deleted exon 7 (Rps6ka6-ΔE7) using clustered regularly interspaced palindromic repeats-Cas9 based method in H2Kb myogenic cells creating a severely truncated RSK4 protein. We then tested whether that mutation affected myoblast proliferation, migration, and/or differentiation. The extensor digitorum longus muscle was 7% larger (P < 0.0001) due to 10% more muscle fibers (P = 0.0176) in the carriers of the "increasing" compared with the "decreasing" CFW allele. The number of fibers was reduced by 15% (P = 0.0268) in the slow-twitch soleus but not in the fast-twitch extensor digitorum longus (P = 0.2947) of Pou3f4y/- mice. The proliferation and migration did not differ between the Rps6ka6-ΔE7 and wild-type H2Kb myoblasts. However, indices of differentiation (myosin expression, P < 0.0001; size of myosin-expressing cells, P < 0.0001; and fusion index, P = 0.0013) were significantly reduced in Rps6ka6-ΔE7 cells. This study suggests that the effect of the X chromosome locus on muscle fiber numbers in the fast-twitch extensor digitorum longus is mediated by the Rps6ka6 gene, whereas the Pou3f4 gene affects fiber number in slow-twitch soleus.
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Affiliation(s)
- Konstantinos Lekkos
- School of Medicine, Medical Sciences and Nutrition, University of Aberdeen, Aberdeen AB25 2ZD, UK
| | - Afra A Bhuiyan
- School of Medicine, Medical Sciences and Nutrition, University of Aberdeen, Aberdeen AB25 2ZD, UK
| | - Abdullah M K Albloshi
- School of Medicine, Medical Sciences and Nutrition, University of Aberdeen, Aberdeen AB25 2ZD, UK
- Department of Anatomy and Histology, School of Medicine, University of Albaha, Alaqiq 65779, Saudi Arabia
| | - Paige M Brooks
- Department of Biology, Georgetown University, Washington, DC 20057, USA
| | - Thomas M Coate
- Department of Biology, Georgetown University, Washington, DC 20057, USA
| | - Arimantas Lionikas
- School of Medicine, Medical Sciences and Nutrition, University of Aberdeen, Aberdeen AB25 2ZD, UK
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8
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Haghayegh Jahromi N, Gkountidi AO, Collado-Diaz V, Blatter K, Bauer A, Zambounis L, Medina-Sanchez JD, Russo E, Runge P, Restivo G, Gousopoulos E, Lindenblatt N, Levesque MP, Halin C. CD112 Supports Lymphatic Migration of Human Dermal Dendritic Cells. Cells 2024; 13:424. [PMID: 38474388 PMCID: PMC10931060 DOI: 10.3390/cells13050424] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/30/2023] [Revised: 02/02/2024] [Accepted: 02/22/2024] [Indexed: 03/14/2024] Open
Abstract
Dendritic cell (DC) migration from peripheral tissues via afferent lymphatic vessels to draining lymph nodes (dLNs) is important for the organism's immune regulation and immune protection. Several lymphatic endothelial cell (LEC)-expressed adhesion molecules have thus far been found to support transmigration and movement within the lymphatic vasculature. In this study, we investigated the contribution of CD112, an adhesion molecule that we recently found to be highly expressed in murine LECs, to this process. Performing in vitro assays in the murine system, we found that transmigration of bone marrow-derived dendritic cells (BM-DCs) across or adhesion to murine LEC monolayers was reduced when CD112 was absent on LECs, DCs, or both cell types, suggesting the involvement of homophilic CD112-CD112 interactions. While CD112 was highly expressed in murine dermal LECs, CD112 levels were low in endogenous murine dermal DCs and BM-DCs. This might explain why we observed no defect in the in vivo lymphatic migration of adoptively transferred BM-DCs or endogenous DCs from the skin to dLNs. Compared to murine DCs, human monocyte-derived DCs expressed higher CD112 levels, and their migration across human CD112-expressing LECs was significantly reduced upon CD112 blockade. CD112 expression was also readily detected in endogenous human dermal DCs and LECs by flow cytometry and immunofluorescence. Upon incubating human skin punch biopsies in the presence of CD112-blocking antibodies, DC emigration from the tissue into the culture medium was significantly reduced, indicating impaired lymphatic migration. Overall, our data reveal a contribution of CD112 to human DC migration.
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Affiliation(s)
- Neda Haghayegh Jahromi
- Institute of Pharmaceutical Sciences, ETH Zurich, Vladimir-Prelog-Weg 1-5/10, 8093 Zurich, Switzerland; (N.H.J.); (A.-O.G.); (V.C.-D.); (K.B.); (L.Z.); (J.D.M.-S.); (E.R.); (P.R.)
| | - Anastasia-Olga Gkountidi
- Institute of Pharmaceutical Sciences, ETH Zurich, Vladimir-Prelog-Weg 1-5/10, 8093 Zurich, Switzerland; (N.H.J.); (A.-O.G.); (V.C.-D.); (K.B.); (L.Z.); (J.D.M.-S.); (E.R.); (P.R.)
| | - Victor Collado-Diaz
- Institute of Pharmaceutical Sciences, ETH Zurich, Vladimir-Prelog-Weg 1-5/10, 8093 Zurich, Switzerland; (N.H.J.); (A.-O.G.); (V.C.-D.); (K.B.); (L.Z.); (J.D.M.-S.); (E.R.); (P.R.)
| | - Katharina Blatter
- Institute of Pharmaceutical Sciences, ETH Zurich, Vladimir-Prelog-Weg 1-5/10, 8093 Zurich, Switzerland; (N.H.J.); (A.-O.G.); (V.C.-D.); (K.B.); (L.Z.); (J.D.M.-S.); (E.R.); (P.R.)
| | - Aline Bauer
- Institute of Pharmaceutical Sciences, ETH Zurich, Vladimir-Prelog-Weg 1-5/10, 8093 Zurich, Switzerland; (N.H.J.); (A.-O.G.); (V.C.-D.); (K.B.); (L.Z.); (J.D.M.-S.); (E.R.); (P.R.)
| | - Lito Zambounis
- Institute of Pharmaceutical Sciences, ETH Zurich, Vladimir-Prelog-Weg 1-5/10, 8093 Zurich, Switzerland; (N.H.J.); (A.-O.G.); (V.C.-D.); (K.B.); (L.Z.); (J.D.M.-S.); (E.R.); (P.R.)
| | - Jessica Danielly Medina-Sanchez
- Institute of Pharmaceutical Sciences, ETH Zurich, Vladimir-Prelog-Weg 1-5/10, 8093 Zurich, Switzerland; (N.H.J.); (A.-O.G.); (V.C.-D.); (K.B.); (L.Z.); (J.D.M.-S.); (E.R.); (P.R.)
| | - Erica Russo
- Institute of Pharmaceutical Sciences, ETH Zurich, Vladimir-Prelog-Weg 1-5/10, 8093 Zurich, Switzerland; (N.H.J.); (A.-O.G.); (V.C.-D.); (K.B.); (L.Z.); (J.D.M.-S.); (E.R.); (P.R.)
| | - Peter Runge
- Institute of Pharmaceutical Sciences, ETH Zurich, Vladimir-Prelog-Weg 1-5/10, 8093 Zurich, Switzerland; (N.H.J.); (A.-O.G.); (V.C.-D.); (K.B.); (L.Z.); (J.D.M.-S.); (E.R.); (P.R.)
| | - Gaetana Restivo
- Department of Dermatology, University Hospital Zurich, University of Zurich, 8091 Zurich, Switzerland; (G.R.); (M.P.L.)
| | - Epameinondas Gousopoulos
- Department of Plastic Surgery and Hand Surgery, University Hospital Zurich, Raemistrasse 100, 8091 Zurich, Switzerland; (E.G.); (N.L.)
| | - Nicole Lindenblatt
- Department of Plastic Surgery and Hand Surgery, University Hospital Zurich, Raemistrasse 100, 8091 Zurich, Switzerland; (E.G.); (N.L.)
| | - Mitchell P. Levesque
- Department of Dermatology, University Hospital Zurich, University of Zurich, 8091 Zurich, Switzerland; (G.R.); (M.P.L.)
| | - Cornelia Halin
- Institute of Pharmaceutical Sciences, ETH Zurich, Vladimir-Prelog-Weg 1-5/10, 8093 Zurich, Switzerland; (N.H.J.); (A.-O.G.); (V.C.-D.); (K.B.); (L.Z.); (J.D.M.-S.); (E.R.); (P.R.)
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9
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Jaime D, Fish LA, Madigan LA, Xi C, Piccoli G, Ewing MD, Blaauw B, Fallon JR. The MuSK-BMP pathway maintains myofiber size in slow muscle through regulation of Akt-mTOR signaling. Skelet Muscle 2024; 14:1. [PMID: 38172960 PMCID: PMC10763067 DOI: 10.1186/s13395-023-00329-9] [Citation(s) in RCA: 5] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/21/2023] [Accepted: 10/19/2023] [Indexed: 01/05/2024] Open
Abstract
Myofiber size regulation is critical in health, disease, and aging. MuSK (muscle-specific kinase) is a BMP (bone morphogenetic protein) co-receptor that promotes and shapes BMP signaling. MuSK is expressed at all neuromuscular junctions and is also present extrasynaptically in the mouse soleus, whose predominantly oxidative fiber composition is akin to that of human muscle. To investigate the role of the MuSK-BMP pathway in vivo, we generated mice lacking the BMP-binding MuSK Ig3 domain. These ∆Ig3-MuSK mice are viable and fertile with innervation levels comparable to wild type. In 3-month-old mice, myofibers are smaller in the slow soleus, but not in the fast tibialis anterior (TA). Transcriptomic analysis revealed soleus-selective decreases in RNA metabolism and protein synthesis pathways as well as dysregulation of IGF1-Akt-mTOR pathway components. Biochemical analysis showed that Akt-mTOR signaling is reduced in soleus but not TA. We propose that the MuSK-BMP pathway acts extrasynaptically to maintain myofiber size in slow muscle by promoting protein synthetic pathways including IGF1-Akt-mTOR signaling. These results reveal a novel mechanism for regulating myofiber size in slow muscle and introduce the MuSK-BMP pathway as a target for promoting muscle growth and combatting atrophy.
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Grants
- R41 AG073144 NIA NIH HHS
- T32 MH020068 NIMH NIH HHS
- U01 NS064295, R41 AG073144, R21 NS112743, R21 AG073743, P30 GM103410, P30 RR031153, P20 RR018728, S10 RR02763, R25GM083270, 2T32AG041688, and T32 MH20068 NIH HHS
- P30 GM103410 NIGMS NIH HHS
- T32 AG041688 NIA NIH HHS
- P30 RR031153 NCRR NIH HHS
- U01 NS064295 NINDS NIH HHS
- R21 NS112743 NINDS NIH HHS
- P20 RR018728 NCRR NIH HHS
- R21 AG073743 NIA NIH HHS
- R25 GM083270 NIGMS NIH HHS
- National Institutes of Health
- Carney Institute for Brain Sciences
- ALS Finding a Cure
- AFM-Téléthon
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Affiliation(s)
- Diego Jaime
- Department of Molecular Biology, Cell Biology, and Biochemistry, Brown University, Providence, RI, USA
| | - Lauren A Fish
- Department of Neuroscience, Brown University, Providence, RI, 02912, USA
| | - Laura A Madigan
- Department of Molecular Biology, Cell Biology, and Biochemistry, Brown University, Providence, RI, USA
| | - Chengjie Xi
- Department of Neuroscience, Brown University, Providence, RI, 02912, USA
| | - Giorgia Piccoli
- Veneto Institute of Molecular Medicine (VIMM), Padua, Italy
- Department of Biomedical Sciences, University of Padua, Padua, Italy
| | - Madison D Ewing
- Department of Neuroscience, Brown University, Providence, RI, 02912, USA
| | - Bert Blaauw
- Veneto Institute of Molecular Medicine (VIMM), Padua, Italy
- Department of Biomedical Sciences, University of Padua, Padua, Italy
| | - Justin R Fallon
- Department of Neuroscience, Brown University, Providence, RI, 02912, USA.
- Carney Institute for Neuroscience, Brown University, Providence, RI, USA.
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10
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Jung HJ, Dixon EE, Coleman R, Watnick T, Reiter JF, Outeda P, Cebotaru V, Woodward OM, Welling PA. Polycystin-2-dependent transcriptome reveals early response of autosomal dominant polycystic kidney disease. Physiol Genomics 2023; 55:565-577. [PMID: 37720991 PMCID: PMC11178268 DOI: 10.1152/physiolgenomics.00040.2023] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/15/2023] [Revised: 09/12/2023] [Accepted: 09/13/2023] [Indexed: 09/19/2023] Open
Abstract
Autosomal dominant polycystic kidney disease (ADPKD) is caused by mutations in polycystin genes, Pkd1 and Pkd2, but the underlying pathogenic mechanisms are poorly understood. To identify genes and pathways that operate downstream of polycystin-2 (PC2), a comprehensive gene expression database was created, cataloging changes in the transcriptome immediately following PC2 protein depletion. To explore cyst initiation processes, an immortalized mouse inner medullary collecting duct line was developed with the ability to knock out the Pkd2 gene conditionally. Genome-wide transcriptome profiling was performed using RNA sequencing in the cells immediately after PC2 was depleted and compared with isogenic control cells. Differentially expressed genes were identified, and a bioinformatic analysis pipeline was implemented. Altered expression of candidate cystogenic genes was validated in Pkd2 knockout mice. The expression of nearly 900 genes changed upon PC2 depletion. Differentially expressed genes were enriched for genes encoding components of the primary cilia, the canonical Wnt pathway, and MAPK signaling. Among the PC2-dependent ciliary genes, the transcription factor Glis3 was significantly downregulated. MAPK signaling formed a key node at the epicenter of PC2-dependent signaling networks. Activation of Wnt and MAPK signaling, concomitant with the downregulation of Glis3, was corroborated in Pkd2 knockout mice. The data identify a PC2 cilia-to-nucleus signaling axis and dysregulation of the Gli-similar subfamily of transcription factors as a potential initiator of cyst formation in ADPKD. The catalog of PC2-regulated genes should provide a valuable resource for future ADPKD research and new opportunities for drug development.NEW & NOTEWORTHY Autosomal dominant polycystic kidney disease (ADPKD) is the most common inherited kidney disease. Mutations in polycystin genes cause the disease, but the underlying mechanisms of cystogenesis are unknown. To help fill this knowledge gap, we created an inducible cell model of ADPKD and assembled a catalog of genes that respond in immediate proximity to polycystin-2 depletion using transcriptomic profiling. The catalog unveils a ciliary signaling-to-nucleus axis proximal to polycystin-2 dysfunction, highlighting Glis, Wnt, and MAPK signaling.
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Affiliation(s)
- Hyun Jun Jung
- Division of Nephrology, Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States
| | - Eryn E Dixon
- Department of Physiology, University of Maryland School of Medicine, Baltimore, Maryland, United States
| | - Richard Coleman
- Division of Nephrology, Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States
| | - Terry Watnick
- Division of Nephrology, Department of Medicine, University of Maryland School of Medicine, Baltimore, Maryland, United States
| | - Jeremy F Reiter
- Department of Biochemistry and Biophysics, University of California, San Francisco, California, United States
- Chan Zuckerberg Biohub, San Francisco, California, United States
| | - Patricia Outeda
- Division of Nephrology, Department of Medicine, University of Maryland School of Medicine, Baltimore, Maryland, United States
| | - Valeriu Cebotaru
- Division of Nephrology, Department of Medicine, University of Maryland School of Medicine, Baltimore, Maryland, United States
| | - Owen M Woodward
- Department of Physiology, University of Maryland School of Medicine, Baltimore, Maryland, United States
| | - Paul A Welling
- Division of Nephrology, Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States
- Department of Physiology, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States
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11
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Ferreira RM, de Almeida R, Culp C, Witzmann F, Wang M, Kher R, Nagami GT, Mohallem R, Andolino CJ, Aryal UK, Eadon MT, Bacallao RL. Proteomic analysis of murine kidney proximal tubule sub-segment derived cell lines reveals preferences in mitochondrial pathway activity. J Proteomics 2023; 289:104998. [PMID: 37657718 PMCID: PMC10843797 DOI: 10.1016/j.jprot.2023.104998] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/19/2023] [Revised: 07/16/2023] [Accepted: 08/28/2023] [Indexed: 09/03/2023]
Abstract
The proximal tubule (PT) is a nephron segment that is responsible for the majority of solute and water reabsorption in the kidney. Each of its sub-segments have specialized functions; however, little is known about the genes and proteins that determine the oxidative phosphorylation capacity of the PT sub-segments. This information is critical to understanding kidney function and will provide a comprehensive landscape of renal cell adaptations to injury, physiologic stressors, and development. This study analyzed three immortalized murine renal cell lines (PT S1, S2, and S3 segments) for protein content and compared them to a murine fibroblast cell line. All three proximal tubule cell lines generate ATP predominantly by oxidative phosphorylation while the fibroblast cell line is glycolytic. The proteomic data demonstrates that the most significant difference in proteomic signatures between the cell lines are proteins known to be localized in the nucleus followed by mitochondrial proteins. Mitochondrial metabolic substrate utilization assays were performed using the proximal tubule cell lines to determine substrate utilization kinetics thereby providing a physiologic context to the proteomic dataset. This data will allow researchers to study differences in nephron-specific cell lines, between epithelial and fibroblast cells, and between actively respiring cells and glycolytic cells. SIGNIFICANCE: Proteomic analysis of proteins expressed in immortalized murine renal proximal tubule cells was compared to a murine fibroblast cell line proteome. The proximal tubule segment specific cell lines: S1, S2 and S3 are all grown under conditions whereby the cells generate ATP by oxidative phosphorylation while the fibroblast cell line utilizes anaerobic glycolysis for ATP generation. The proteomic studies allow for the following queries: 1) comparisons between the proximal tubule segment specific cell lines, 2) comparisons between polarized epithelia and fibroblasts, 3) comparison between cells employing oxidative phosphorylation versus anaerobic glycolysis and 4) comparisons between cells grown on clear versus opaque membrane supports. The data finds major differences in nuclear protein expression and mitochondrial proteins. This proteomic data set will be an important baseline dataset for investigators who need immortalized renal proximal tubule epithelial cells for their research.
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Affiliation(s)
- Ricardo Melo Ferreira
- Division of Nephrology, Indiana University School of Medicine, Indianapolis, IN 46202, USA.
| | - Rita de Almeida
- Instituto de Física and Instituto Nacional de Ciência e Tecnologia, Universidade Federal do Rio Grande do Sul, 91501-970 Porto Alegre, RS, Brazil.
| | - Clayton Culp
- Division of Nephrology, Indiana University School of Medicine, Indianapolis, IN 46202, USA.
| | - Frank Witzmann
- Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, IN 46202, USA.
| | - Mu Wang
- Division of Nephrology, Indiana University School of Medicine, Indianapolis, IN 46202, USA.
| | - Rajesh Kher
- Division of Nephrology, Indiana University School of Medicine, Indianapolis, IN 46202, USA.
| | - Glenn T Nagami
- Division of Nephrology, VA Greater Los Angeles Healthcare System, David Geffen School of Medicine, UCLA, Los Angeles, CA, USA.
| | - Rodrigo Mohallem
- Department of Comparative Pathobiology, Purdue University, West Lafayette, IN 47907, USA; Purdue Proteomics Facility, Bindley Bioscience Center, Purdue University, West Lafayette, IN 47907, USA.
| | - Chaylen Jade Andolino
- Purdue Proteomics Facility, Bindley Bioscience Center, Purdue University, West Lafayette, IN 47907, USA.
| | - Uma K Aryal
- Department of Comparative Pathobiology, Purdue University, West Lafayette, IN 47907, USA; Purdue Proteomics Facility, Bindley Bioscience Center, Purdue University, West Lafayette, IN 47907, USA.
| | - Michael T Eadon
- Division of Nephrology, Indiana University School of Medicine, Indianapolis, IN 46202, USA.
| | - Robert L Bacallao
- Division of Nephrology, Indiana University School of Medicine, Indianapolis, IN 46202, USA.
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12
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Wu F, Hu R, Huang X, Lou J, Cai Z, Chen G, Zhao W, Xiong H, Sha SH, Zheng Y. CFTR potentiator ivacaftor protects against noise-induced hair cell loss by increasing Nrf2 and reducing oxidative stress. Biomed Pharmacother 2023; 166:115399. [PMID: 37657258 PMCID: PMC10528730 DOI: 10.1016/j.biopha.2023.115399] [Citation(s) in RCA: 7] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/12/2023] [Revised: 08/23/2023] [Accepted: 08/26/2023] [Indexed: 09/03/2023] Open
Abstract
Over-production of reactive oxygen species (ROS) in the inner ear can be triggered by a variety of pathological events identified in animal models after traumatic noise exposure. Our previous research found that inhibition of the AMP-activated protein kinase alpha subunit (AMPKα) protects against noise-induced cochlear hair cell loss and hearing loss by reducing ROS accumulation. However, the molecular pathway through which AMPKα exerts its antioxidative effect is still unclear. In this study, we have investigated a potential target of AMPKα and ROS, cystic fibrosis transmembrane conductance regulator (CFTR), and the protective effect against noise-induced hair cell loss of an FDA-approved CFTR potentiator, ivacaftor, in FVB/NJ mice, mouse explant cultures, and HEI-OC1 cells. We found that noise exposure increases phosphorylation of CFTR at serine 737 (p-CFTR, S737), which reduces wildtype CFTR function, resulting in oxidative stress in cochlear sensory hair cells. Pretreatment with a single dose of ivacaftor maintains CFTR function by preventing noise-increased p-CFTR (S737). Furthermore, ivacaftor treatment increases nuclear factor E2-related factor 2 (Nrf2) expression, diminishes ROS formation, and attenuates noise-induced hair cell loss and hearing loss. Additionally, inhibition of noise-induced AMPKα activation by compound C also diminishes p-CFTR (S737) expression. In line with these in-vivo results, administration of hydrogen peroxide to cochlear explants or HEI-OC1 cells increases p-CFTR (S737) expression and induces sensory hair cell or HEI-OC1 cell damage, while application of ivacaftor halts these effects. Although ivacaftor increases Nrf2 expression and reduces ROS accumulation, cotreatment with ML385, an Nrf2 inhibitor, abolishes the protective effects of ivacaftor against hydrogen-peroxide-induced HEI-OC1 cell death. Our results indicate that noise-induced sensory hair cell damage is associated with p-CFTR. Ivacaftor has potential for treatment of noise-induced hearing loss by maintaining CFTR function and increasing Nrf2 expression for support of redox homeostasis in sensory hair cells.
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Affiliation(s)
- Fan Wu
- Department of Otolaryngology, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangzhou, Guangdong, China; Guangdong Provincial Key Laboratory of Malignant Tumor Epigenetics and Gene Regulation, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangzhou, Guangdong, China; Institute of Hearing and Speech-Language Science, Sun Yat-sen University, Guangzhou 510120, China; Department of Pathology and Laboratory Medicine, The Medical University of South Carolina, Charleston, SC, USA
| | - Rui Hu
- Department of Otolaryngology, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangzhou, Guangdong, China; Shenshan Medical Center, Memorial Hospital of Sun Yat-sen University, Shanwei, Guangdong, China; Institute of Hearing and Speech-Language Science, Sun Yat-sen University, Guangzhou 510120, China
| | - Xueping Huang
- Department of Otolaryngology, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangzhou, Guangdong, China; Guangdong Provincial Key Laboratory of Malignant Tumor Epigenetics and Gene Regulation, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangzhou, Guangdong, China; Institute of Hearing and Speech-Language Science, Sun Yat-sen University, Guangzhou 510120, China
| | - Jintao Lou
- Department of Otolaryngology, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangzhou, Guangdong, China; Guangdong Provincial Key Laboratory of Malignant Tumor Epigenetics and Gene Regulation, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangzhou, Guangdong, China; Institute of Hearing and Speech-Language Science, Sun Yat-sen University, Guangzhou 510120, China
| | - Ziyi Cai
- Department of Otolaryngology, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangzhou, Guangdong, China; Guangdong Provincial Key Laboratory of Malignant Tumor Epigenetics and Gene Regulation, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangzhou, Guangdong, China; Institute of Hearing and Speech-Language Science, Sun Yat-sen University, Guangzhou 510120, China
| | - Guisheng Chen
- Department of Otolaryngology, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangzhou, Guangdong, China; Guangdong Provincial Key Laboratory of Malignant Tumor Epigenetics and Gene Regulation, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangzhou, Guangdong, China; Institute of Hearing and Speech-Language Science, Sun Yat-sen University, Guangzhou 510120, China
| | - Wenji Zhao
- Department of Otolaryngology, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangzhou, Guangdong, China; Guangdong Provincial Key Laboratory of Malignant Tumor Epigenetics and Gene Regulation, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangzhou, Guangdong, China; Institute of Hearing and Speech-Language Science, Sun Yat-sen University, Guangzhou 510120, China
| | - Hao Xiong
- Department of Otolaryngology, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangzhou, Guangdong, China; Guangdong Provincial Key Laboratory of Malignant Tumor Epigenetics and Gene Regulation, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangzhou, Guangdong, China; Institute of Hearing and Speech-Language Science, Sun Yat-sen University, Guangzhou 510120, China
| | - Su-Hua Sha
- Department of Pathology and Laboratory Medicine, The Medical University of South Carolina, Charleston, SC, USA.
| | - Yiqing Zheng
- Department of Otolaryngology, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangzhou, Guangdong, China; Shenshan Medical Center, Memorial Hospital of Sun Yat-sen University, Shanwei, Guangdong, China; Guangdong Provincial Key Laboratory of Malignant Tumor Epigenetics and Gene Regulation, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangzhou, Guangdong, China; Institute of Hearing and Speech-Language Science, Sun Yat-sen University, Guangzhou 510120, China.
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13
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Huang M, Zou M, Mao S, Xu W, Hong Y, Wang H, Gui F, Yang L, Lian F, Chen R. 3,5,6-Trichloro-2-pyridinol confirms ototoxicity in mouse cochlear organotypic cultures and induces cytotoxicity in HEI-OC1 cells. Toxicol Appl Pharmacol 2023; 475:116612. [PMID: 37463651 DOI: 10.1016/j.taap.2023.116612] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/23/2023] [Revised: 06/12/2023] [Accepted: 06/30/2023] [Indexed: 07/20/2023]
Abstract
The metabolite of organophosphate pesticide chlorpyrifos (CPF), 3,5,6-Trichloro-2-pyridinol (TCP), is persistent and mobile toxic substance in soil and water environments, exhibiting cytotoxic, genotoxic, and neurotoxic properties. However, little is known about its effects on the peripheral auditory system. Herein, we investigated the effects of TCP exposure on mouse postnatal day 3 (P3) cochlear culture and an auditory cell line HEI-OC1 to elucidate the underlying molecular mechanisms of ototoxicity. The damage of TCP to outer hair cells (OHC) and support cells (SC) was observed in a dose and time-dependent manner. OHC and SC were a significant loss from basal to apical turn of the cochlea under exposure over 800 μM TCP for 96 h. As TCP concentrations increased, cell viability was reduced whereas reactive oxygen species (ROS) generation, apoptotic cells, and the extent of DNA damage were increased, accordingly. TCP-induced phosphorylation of the p38 and JNK MAPK are the downstream effectors of ROS. The antioxidant agent, N-acetylcysteine (NAC), could reverse TCP-mediated intracellular ROS generation, inhibit the expressive level of cleaved-caspase 3 and block phosphorylation of p38/JNK. Overall, this is the first demonstration of TCP damaging to peripheral sensory HCs and SC in organotypic cultures from the postnatal cochlea. Data also showed that TCP exposure induced oxidase stress, cell apoptosis and DNA damage in the HEI-OC1 cells. These findings serve as an important reference for assessing the risk of TCP exposure.
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Affiliation(s)
- Mao Huang
- School of Public Health, Hangzhou Normal University, Hangzhou, Zhejiang 311121, China
| | - Mingshan Zou
- School of Public Health, Hangzhou Normal University, Hangzhou, Zhejiang 311121, China
| | - Shuangshuang Mao
- School of Public Health, Hangzhou Normal University, Hangzhou, Zhejiang 311121, China
| | - Wenqi Xu
- School of Public Health, Hangzhou Normal University, Hangzhou, Zhejiang 311121, China
| | - Yu Hong
- School of Public Health, Hangzhou Normal University, Hangzhou, Zhejiang 311121, China
| | - Haiyan Wang
- School of Public Health, Hangzhou Normal University, Hangzhou, Zhejiang 311121, China
| | - Fei Gui
- School of Public Health, Hangzhou Normal University, Hangzhou, Zhejiang 311121, China
| | - Lei Yang
- School of Public Health, Hangzhou Normal University, Hangzhou, Zhejiang 311121, China
| | - Fuzhi Lian
- School of Public Health, Hangzhou Normal University, Hangzhou, Zhejiang 311121, China.
| | - Rong Chen
- School of Public Health, Hangzhou Normal University, Hangzhou, Zhejiang 311121, China.
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14
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Ge M, Molina J, Kim JJ, Mallela SK, Ahmad A, Varona Santos J, Al-Ali H, Mitrofanova A, Sharma K, Fontanesi F, Merscher S, Fornoni A. Empagliflozin reduces podocyte lipotoxicity in experimental Alport syndrome. eLife 2023; 12:e83353. [PMID: 37129368 PMCID: PMC10185338 DOI: 10.7554/elife.83353] [Citation(s) in RCA: 30] [Impact Index Per Article: 15.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/08/2022] [Accepted: 04/26/2023] [Indexed: 05/03/2023] Open
Abstract
Sodium-glucose cotransporter-2 inhibitors (SGLT2i) are anti-hyperglycemic agents that prevent glucose reabsorption in proximal tubular cells. SGLT2i improves renal outcomes in both diabetic and non-diabetic patients, indicating it may have beneficial effects beyond glycemic control. Here, we demonstrate that SGLT2i affects energy metabolism and podocyte lipotoxicity in experimental Alport syndrome (AS). In vitro, we found that the SGLT2 protein was expressed in human and mouse podocytes to a similar extent in tubular cells. Newly established immortalized podocytes from Col4a3 knockout mice (AS podocytes) accumulate lipid droplets along with increased apoptosis when compared to wild-type podocytes. Treatment with SGLT2i empagliflozin reduces lipid droplet accumulation and apoptosis in AS podocytes. Empagliflozin inhibits the utilization of glucose/pyruvate as a metabolic substrate in AS podocytes but not in AS tubular cells. In vivo, we demonstrate that empagliflozin reduces albuminuria and prolongs the survival of AS mice. Empagliflozin-treated AS mice show decreased serum blood urea nitrogen and creatinine levels in association with reduced triglyceride and cholesterol ester content in kidney cortices when compared to AS mice. Lipid accumulation in kidney cortices correlates with a decline in renal function. In summary, empagliflozin reduces podocyte lipotoxicity and improves kidney function in experimental AS in association with the energy substrates switch from glucose to fatty acids in podocytes.
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Affiliation(s)
- Mengyuan Ge
- Katz Family Division of Nephrology and Hypertension, Department of Medicine, University of Miami Miller School of MedicineMiamiUnited States
- Peggy and Harold Katz Family Drug Discovery Center, University of Miami Miller School of MedicineMiamiUnited States
| | - Judith Molina
- Katz Family Division of Nephrology and Hypertension, Department of Medicine, University of Miami Miller School of MedicineMiamiUnited States
- Peggy and Harold Katz Family Drug Discovery Center, University of Miami Miller School of MedicineMiamiUnited States
| | - Jin-Ju Kim
- Katz Family Division of Nephrology and Hypertension, Department of Medicine, University of Miami Miller School of MedicineMiamiUnited States
- Peggy and Harold Katz Family Drug Discovery Center, University of Miami Miller School of MedicineMiamiUnited States
| | - Shamroop K Mallela
- Katz Family Division of Nephrology and Hypertension, Department of Medicine, University of Miami Miller School of MedicineMiamiUnited States
- Peggy and Harold Katz Family Drug Discovery Center, University of Miami Miller School of MedicineMiamiUnited States
| | - Anis Ahmad
- Department of Radiation Oncology, University of Miami Miller School of MedicineMiamiUnited States
| | - Javier Varona Santos
- Katz Family Division of Nephrology and Hypertension, Department of Medicine, University of Miami Miller School of MedicineMiamiUnited States
- Peggy and Harold Katz Family Drug Discovery Center, University of Miami Miller School of MedicineMiamiUnited States
| | - Hassan Al-Ali
- Katz Family Division of Nephrology and Hypertension, Department of Medicine, University of Miami Miller School of MedicineMiamiUnited States
- Peggy and Harold Katz Family Drug Discovery Center, University of Miami Miller School of MedicineMiamiUnited States
| | - Alla Mitrofanova
- Katz Family Division of Nephrology and Hypertension, Department of Medicine, University of Miami Miller School of MedicineMiamiUnited States
- Peggy and Harold Katz Family Drug Discovery Center, University of Miami Miller School of MedicineMiamiUnited States
| | - Kumar Sharma
- Center for Precision Medicine, School of Medicine, University of Texas Health San AntonioSan AntonioUnited States
| | - Flavia Fontanesi
- Department of Biochemistry and Molecular Biology, University of MiamiMiamiUnited States
| | - Sandra Merscher
- Katz Family Division of Nephrology and Hypertension, Department of Medicine, University of Miami Miller School of MedicineMiamiUnited States
- Peggy and Harold Katz Family Drug Discovery Center, University of Miami Miller School of MedicineMiamiUnited States
| | - Alessia Fornoni
- Katz Family Division of Nephrology and Hypertension, Department of Medicine, University of Miami Miller School of MedicineMiamiUnited States
- Peggy and Harold Katz Family Drug Discovery Center, University of Miami Miller School of MedicineMiamiUnited States
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15
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Sutyagina OI, Beilin AK, Vorotelyak EA, Vasiliev AV. Immortalization Reversibility in the Context of Cell Therapy Biosafety. Int J Mol Sci 2023; 24:7738. [PMID: 37175444 PMCID: PMC10178325 DOI: 10.3390/ijms24097738] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/09/2023] [Revised: 04/18/2023] [Accepted: 04/20/2023] [Indexed: 05/15/2023] Open
Abstract
Immortalization (genetically induced prevention of replicative senescence) is a promising approach to obtain cellular material for cell therapy or for bio-artificial organs aimed at overcoming the problem of donor material shortage. Immortalization is reversed before cells are used in vivo to allow cell differentiation into the mature phenotype and avoid tumorigenic effects of unlimited cell proliferation. However, there is no certainty that the process of de-immortalization is 100% effective and that it does not cause unwanted changes in the cell. In this review, we discuss various approaches to reversible immortalization, emphasizing their advantages and disadvantages in terms of biosafety. We describe the most promising approaches in improving the biosafety of reversibly immortalized cells: CRISPR/Cas9-mediated immortogene insertion, tamoxifen-mediated self-recombination, tools for selection of successfully immortalized cells, using a decellularized extracellular matrix, and ensuring post-transplant safety with the use of suicide genes. The last process may be used as an add-on for previously existing reversible immortalized cell lines.
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Affiliation(s)
- Oksana I. Sutyagina
- N.K. Koltzov Institute of Developmental Biology of Russian Academy of Sciences, Laboratory of Cell Biology, Vavilov Str. 26, 119334 Moscow, Russia
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16
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Chiereghin C, Robusto M, Lewis MA, Caetano S, Massa V, Castorina P, Ambrosetti U, Steel KP, Duga S, Asselta R, Soldà G. In-depth genetic and molecular characterization of diaphanous related formin 2 (DIAPH2) and its role in the inner ear. PLoS One 2023; 18:e0273586. [PMID: 36689403 PMCID: PMC9870134 DOI: 10.1371/journal.pone.0273586] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/08/2022] [Accepted: 01/09/2023] [Indexed: 01/24/2023] Open
Abstract
Diaphanous related formins are regulatory cytoskeletal protein involved in actin elongation and microtubule stabilization. In humans, defects in two of the three diaphanous genes (DIAPH1 and DIAPH3) have been associated with different types of hearing loss. Here, we investigate the role of the third member of the family, DIAPH2, in nonsyndromic hearing loss, prompted by the identification, by exome sequencing, of a predicted pathogenic missense variant in DIAPH2. This variant occurs at a conserved site and segregated with nonsyndromic X-linked hearing loss in an Italian family. Our immunohistochemical studies indicated that the mouse ortholog protein Diaph2 is expressed during development in the cochlea, specifically in the actin-rich stereocilia of the sensory outer hair cells. In-vitro studies showed a functional impairment of the mutant DIAPH2 protein upon RhoA-dependent activation. Finally, Diaph2 knock-out and knock-in mice were generated by CRISPR/Cas9 technology and auditory brainstem response measurements performed at 4, 8 and 14 weeks. However, no hearing impairment was detected. Our findings indicate that DIAPH2 may play a role in the inner ear; further studies are however needed to clarify the contribution of DIAPH2 to deafness.
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Affiliation(s)
| | - Michela Robusto
- Experimental Therapeutics Program, IFOM ETS -The AIRC Institute of Molecular Oncology, Milan, Italy
| | - Morag A. Lewis
- Wolfson Centre for Age-Related Diseases, King’s College London, London, United Kingdom
| | - Susana Caetano
- Wolfson Centre for Age-Related Diseases, King’s College London, London, United Kingdom
| | - Valentina Massa
- Dipartimento di Scienze della Salute, Università degli Studi di Milano, Milano, Italy
| | | | - Umberto Ambrosetti
- Dipartimento di Scienze Cliniche e di Comunità, Università degli Studi di Milano and Fondazione IRCCS Cà Granda Ospedale Maggiore Policlinico, UO Audiologia, Milano, Italy
| | - Karen P. Steel
- Wolfson Centre for Age-Related Diseases, King’s College London, London, United Kingdom
| | - Stefano Duga
- IRCCS Humanitas Research Hospital, Rozzano, Milan, Italy
- Humanitas University, Department of Biomedical Sciences, Pieve Emanuele, Milan, Italy
| | - Rosanna Asselta
- IRCCS Humanitas Research Hospital, Rozzano, Milan, Italy
- Humanitas University, Department of Biomedical Sciences, Pieve Emanuele, Milan, Italy
| | - Giulia Soldà
- IRCCS Humanitas Research Hospital, Rozzano, Milan, Italy
- Humanitas University, Department of Biomedical Sciences, Pieve Emanuele, Milan, Italy
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17
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McMurray HR, Stern HA, Ambeskovic A, Land H, McCall MN. Protocol to use TopNet for gene regulatory network modeling using gene expression data from perturbation experiments. STAR Protoc 2022; 3:101737. [PMID: 36181678 PMCID: PMC9529586 DOI: 10.1016/j.xpro.2022.101737] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/02/2022] [Revised: 06/28/2022] [Accepted: 09/07/2022] [Indexed: 01/26/2023] Open
Abstract
Inference of gene regulatory networks from gene perturbation experiments is the most reliable approach for investigating interdependence between genes. Here, we describe the initial gene perturbations, expression measurements, and preparation steps, followed by network modeling using TopNet. Summarization and visualization of the estimated networks and optional genetic testing of dependencies revealed by the network model are demonstrated. While developed for gene perturbation experiments, TopNet models data in which nodes are both perturbed and measured. For complete details on the use and execution of this protocol, please refer to McMurray et al. (2021).
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Affiliation(s)
- Helene R McMurray
- Department of Biomedical Genetics, University of Rochester Medical Center, 601 Elmwood Avenue, Rochester, NY 14642, USA; Department of Pathology and Laboratory Medicine, University of Rochester Medical Center, 601 Elmwood Avenue, Rochester, NY 14642, USA
| | - Harry A Stern
- Center for Integrated Research Computing, University of Rochester Medical Center, 601 Elmwood Avenue, Rochester, NY 14642, USA
| | - Aslihan Ambeskovic
- Department of Biomedical Genetics, University of Rochester Medical Center, 601 Elmwood Avenue, Rochester, NY 14642, USA; Wilmot Cancer Institute, University of Rochester Medical Center, 601 Elmwood Avenue, Rochester, NY 14642, USA
| | - Hartmut Land
- Department of Biomedical Genetics, University of Rochester Medical Center, 601 Elmwood Avenue, Rochester, NY 14642, USA; Wilmot Cancer Institute, University of Rochester Medical Center, 601 Elmwood Avenue, Rochester, NY 14642, USA
| | - Matthew N McCall
- Department of Biomedical Genetics, University of Rochester Medical Center, 601 Elmwood Avenue, Rochester, NY 14642, USA; Wilmot Cancer Institute, University of Rochester Medical Center, 601 Elmwood Avenue, Rochester, NY 14642, USA; Department of Biostatistics and Computational Biology, University of Rochester Medical Center, 601 Elmwood Avenue, Rochester, NY 14642, USA.
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18
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Liu PJ, Gunther LK, Garone ME, Zhang C, Perez D, Bi-Karchin J, Pellenz CD, Chase SE, Presti MF, Plante EL, Martin CE, Lovric S, Yengo CM, Hildebrandt F, Krendel M. Steroid-Resistant Nephrotic Syndrome-Associated MYO1E Mutations Have Differential Effects on Myosin 1e Localization, Dynamics, and Activity. J Am Soc Nephrol 2022; 33:1989-2007. [PMID: 36316095 PMCID: PMC9678034 DOI: 10.1681/asn.2021111505] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/23/2021] [Accepted: 08/22/2022] [Indexed: 12/14/2022] Open
Abstract
BACKGROUND Myo1e is a nonmuscle motor protein enriched in podocytes. Mutations in MYO1E are associated with steroid-resistant nephrotic syndrome (SRNS). Most of the MYO1E variants identified by genomic sequencing have not been functionally characterized. Here, we set out to analyze two mutations in the Myo1e motor domain, T119I and D388H, which were selected on the basis of protein sequence conservation. METHODS EGFP-tagged human Myo1e constructs were delivered into the Myo1e-KO mouse podocyte-derived cells via adenoviral infection to analyze Myo1e protein stability, Myo1e localization, and clathrin-dependent endocytosis, which is known to involve Myo1e activity. Furthermore, truncated Myo1e constructs were expressed using the baculovirus expression system and used to measure Myo1e ATPase and motor activity in vitro. RESULTS Both mutants were expressed as full-length proteins in the Myo1e-KO cells. However, unlike wild-type (WT) Myo1e, the T119I variant was not enriched at the cell junctions or clathrin-coated vesicles (CCVs). In contrast, D388H variant localization was similar to that of WT. The rate of dissociation of the D388H variant from cell-cell junctions and CCVs was decreased, suggesting this mutation affects Myo1e interactions with binding partners. ATPase activity and ability to translocate actin filaments were drastically reduced for the D388H mutant, supporting findings from cell-based experiments. CONCLUSIONS T119I and D388H mutations are deleterious to Myo1e functions. The experimental approaches used in this study can be applied to future characterization of novel MYO1E variants associated with SRNS.
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Affiliation(s)
- Pei-Ju Liu
- Department of Cell and Developmental Biology, State University of New York Upstate Medical University, Syracuse, New York
| | - Laura K. Gunther
- Department of Cellular and Molecular Physiology, College of Medicine, Pennsylvania State University, Hershey, Pennsylvania
| | - Michael E. Garone
- Department of Cell and Developmental Biology, State University of New York Upstate Medical University, Syracuse, New York
| | - Chunling Zhang
- Department of Neuroscience and Physiology, State University of New York Upstate Medical University, Syracuse, New York
| | - Diana Perez
- Department of Cell and Developmental Biology, State University of New York Upstate Medical University, Syracuse, New York
| | - Jing Bi-Karchin
- Department of Cell and Developmental Biology, State University of New York Upstate Medical University, Syracuse, New York
| | - Christopher D. Pellenz
- Department of Cell and Developmental Biology, State University of New York Upstate Medical University, Syracuse, New York
| | - Sharon E. Chase
- Department of Cell and Developmental Biology, State University of New York Upstate Medical University, Syracuse, New York
| | - Maria F. Presti
- Department of Cell and Developmental Biology, State University of New York Upstate Medical University, Syracuse, New York
| | - Eric L. Plante
- Department of Cell and Developmental Biology, State University of New York Upstate Medical University, Syracuse, New York
| | - Claire E. Martin
- Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario, Canada
- Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Sinai Health System, Toronto, Ontario, Canada
| | - Svjetlana Lovric
- Divison of Nephrology, Department of Pediatrics, Boston Children’s Hospital, Harvard Medical School, Boston, Massachusetts
| | - Christopher M. Yengo
- Department of Cellular and Molecular Physiology, College of Medicine, Pennsylvania State University, Hershey, Pennsylvania
| | - Friedhelm Hildebrandt
- Divison of Nephrology, Department of Pediatrics, Boston Children’s Hospital, Harvard Medical School, Boston, Massachusetts
| | - Mira Krendel
- Department of Cell and Developmental Biology, State University of New York Upstate Medical University, Syracuse, New York
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19
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Bouchareb R, Yu L, Lassen E, Daehn IS. Isolation of Conditionally Immortalized Mouse Glomerular Endothelial Cells with Fluorescent Mitochondria. J Vis Exp 2022:10.3791/64147. [PMID: 36190268 PMCID: PMC10840453 DOI: 10.3791/64147] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2024] Open
Abstract
Glomerular endothelial cell (GEC) dysfunction can initiate and contribute to glomerular filtration barrier breakdown. Increased mitochondrial oxidative stress has been suggested as a mechanism resulting in GEC dysfunction in the pathogenesis of some glomerular diseases. Historically the isolation of GECs from in vivo models has been notoriously challenging due to difficulties in isolating pure cultures from glomeruli. GECs have complex growth requirements in vitro and a very limited lifespan. Here, we describe the procedure for isolating and culturing conditionally immortalized GECs with fluorescent mitochondria, enabling the tracking of mitochondrial fission and fusion events. GECs were isolated from the kidneys of a double transgenic mouse expressing the thermolabile SV40 TAg (from the Immortomouse), conditionally promoting proliferation and suppressing cell differentiation, and a photo-convertible fluorescent protein (Dendra2) in all mitochondria (from the photo-activatable mitochondria [PhAMexcised] mouse). The stable cell line generated allows for cell differentiation after inactivation of the immortalizing SV40 TAg gene and photo-activation of a subset of mitochondria causing a switch in fluorescence from green to red. The use of mitoDendra2-GECs allows for live imaging of fluorescent mitochondria's distribution, fusion, and fission events without staining the cells.
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Affiliation(s)
- Rihab Bouchareb
- Department of Medicine, Division of Nephrology, The Icahn School of Medicine at Mount Sinai;
| | - Liping Yu
- Department of Medicine, Division of Nephrology, The Icahn School of Medicine at Mount Sinai
| | - Emelie Lassen
- Department of Medicine, Division of Nephrology, The Icahn School of Medicine at Mount Sinai
| | - Ilse S Daehn
- Department of Medicine, Division of Nephrology, The Icahn School of Medicine at Mount Sinai;
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20
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Metabolic Abnormalities Linked to Auditory Pathways in ApoE-Knockout HEI-OC1 Cells: A Transcription-Metabolism Co-Analysis. Biomolecules 2022; 12:biom12091217. [PMID: 36139057 PMCID: PMC9496352 DOI: 10.3390/biom12091217] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/14/2022] [Revised: 08/26/2022] [Accepted: 08/26/2022] [Indexed: 11/16/2022] Open
Abstract
Lipid transporter protein apolipoprotein E (APOE) has contributed to functional studies of various organ functions. Animals with ApoE knockout (KO) have been used to study atherosclerosis and hyperlipidemia while an increasing number of researchers have recently focused on the association of ApoE with hearing loss. A study found that ApoE KO mice experience sensorineural hearing loss and hair cell loss, but the exact mechanism is unclear. To explore the potential relationship between ApoE and hearing loss, we used HEI-OC1 cells (House Ear Institute-Organ of Corti) with Corti apparatus properties to reveal cell changes after ApoE knockout by combined transcriptome and metabolomic analysis. We found that glutamate deficiency, caused by reduced expression of glutamine transporter proteins, was a key correlate of basal metabolism and that inadequate glutamate causes apoptosis by reducing the cells’ resistance to external damage. Our study provides a reference mechanism for hearing loss due to ApoE KO.
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21
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Flierl A, Schriner SE, Hancock S, Coskun PE, Wallace DC. The mitochondrial adenine nucleotide transporters in myogenesis. Free Radic Biol Med 2022; 188:312-327. [PMID: 35714845 DOI: 10.1016/j.freeradbiomed.2022.05.022] [Citation(s) in RCA: 7] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 11/01/2021] [Revised: 05/21/2022] [Accepted: 05/25/2022] [Indexed: 01/06/2023]
Abstract
Adenine Nucleotide Translocator isoforms (ANTs) exchange ADP/ATP across the inner mitochondrial membrane, are also voltage-activated proton channels and regulate mitophagy and apoptosis. The ANT1 isoform predominates in heart and muscle while ANT2 is systemic. Here, we report the creation of Ant mutant mouse myoblast cell lines with normal Ant1 and Ant2 genes, deficient in either Ant1 or Ant2, and deficient in both the Ant1 and Ant2 genes. These cell lines are immortal under permissive conditions (IFN-γ + serum at 32 °C) permitting expansion but return to normal myoblasts that can be differentiated into myotubes at 37 °C. With this system we were able to complement our Ant1 mutant studies by demonstrating that ANT2 is important for myoblast to myotube differentiation and myotube mitochondrial respiration. ANT2 is also important in the regulation of mitochondrial biogenesis and antioxidant defenses. ANT2 is also associated with increased oxidative stress response and modulation for Ca++ sequestration and activation of the mitochondrial permeability transition (mtPTP) pore during cell differentiation.
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Affiliation(s)
- Adrian Flierl
- Center for Molecular and Mitochondrial Medicine and Genetics and the Department of Biological Chemistry, University of California, Irvine, CA, USA
| | - Samuel E Schriner
- Center for Molecular and Mitochondrial Medicine and Genetics and the Department of Biological Chemistry, University of California, Irvine, CA, USA
| | - Saege Hancock
- Center for Molecular and Mitochondrial Medicine and Genetics and the Department of Biological Chemistry, University of California, Irvine, CA, USA; Center for Mitochondrial and Epigenomic Medicine, Department of Pediatrics, Division of Human Genetics, Children's Hospital of Philadelphia and The Perelman School of Medicine, University of Pennsylvania, PA, USA
| | - Pinar E Coskun
- Center for Molecular and Mitochondrial Medicine and Genetics and the Department of Biological Chemistry, University of California, Irvine, CA, USA
| | - Douglas C Wallace
- Center for Molecular and Mitochondrial Medicine and Genetics and the Department of Biological Chemistry, University of California, Irvine, CA, USA; Center for Mitochondrial and Epigenomic Medicine, Department of Pediatrics, Division of Human Genetics, Children's Hospital of Philadelphia and The Perelman School of Medicine, University of Pennsylvania, PA, USA.
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22
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Huang H, Wang Z, Zhang Y, Pradhan RN, Ganguly D, Chandra R, Murimwa G, Wright S, Gu X, Maddipati R, Müller S, Turley SJ, Brekken RA. Mesothelial cell-derived antigen-presenting cancer-associated fibroblasts induce expansion of regulatory T cells in pancreatic cancer. Cancer Cell 2022; 40:656-673.e7. [PMID: 35523176 PMCID: PMC9197998 DOI: 10.1016/j.ccell.2022.04.011] [Citation(s) in RCA: 289] [Impact Index Per Article: 96.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/04/2021] [Revised: 02/25/2022] [Accepted: 04/14/2022] [Indexed: 12/11/2022]
Abstract
Recent studies have identified a unique cancer-associated fibroblast (CAF) population termed antigen-presenting CAFs (apCAFs), characterized by the expression of major histocompatibility complex class II molecules, suggesting a function in regulating tumor immunity. Here, by integrating multiple single-cell RNA-sequencing studies and performing robust lineage-tracing assays, we find that apCAFs are derived from mesothelial cells. During pancreatic cancer progression, mesothelial cells form apCAFs by downregulating mesothelial features and gaining fibroblastic features, a process induced by interleukin-1 and transforming growth factor β. apCAFs directly ligate and induce naive CD4+ T cells into regulatory T cells (Tregs) in an antigen-specific manner. Moreover, treatment with an antibody targeting the mesothelial cell marker mesothelin can effectively inhibit mesothelial cell to apCAF transition and Treg formation induced by apCAFs. Taken together, our study elucidates how mesothelial cells may contribute to immune evasion in pancreatic cancer and provides insight on strategies to enhance cancer immune therapy.
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Affiliation(s)
- Huocong Huang
- Department of Surgery, University of Texas Southwestern Medical Center, 6000 Harry Hines Blvd, Dallas, TX 75390, USA; Hamon Center for Therapeutic Oncology Research, University of Texas Southwestern Medical Center, 6000 Harry Hines Blvd, Dallas, TX 75390, USA.
| | - Zhaoning Wang
- Department of Cellular and Molecular Medicine, University of California San Diego, La Jolla, CA 92093, USA
| | - Yuqing Zhang
- Hamon Center for Therapeutic Oncology Research, University of Texas Southwestern Medical Center, 6000 Harry Hines Blvd, Dallas, TX 75390, USA; Cancer Biology Graduate Program, University of Texas Southwestern Medical Center, 6000 Harry Hines Blvd, Dallas, TX 75390, USA
| | | | - Debolina Ganguly
- Hamon Center for Therapeutic Oncology Research, University of Texas Southwestern Medical Center, 6000 Harry Hines Blvd, Dallas, TX 75390, USA; Cancer Biology Graduate Program, University of Texas Southwestern Medical Center, 6000 Harry Hines Blvd, Dallas, TX 75390, USA
| | - Raghav Chandra
- Department of Surgery, University of Texas Southwestern Medical Center, 6000 Harry Hines Blvd, Dallas, TX 75390, USA; Hamon Center for Therapeutic Oncology Research, University of Texas Southwestern Medical Center, 6000 Harry Hines Blvd, Dallas, TX 75390, USA
| | - Gilbert Murimwa
- Department of Surgery, University of Texas Southwestern Medical Center, 6000 Harry Hines Blvd, Dallas, TX 75390, USA; Hamon Center for Therapeutic Oncology Research, University of Texas Southwestern Medical Center, 6000 Harry Hines Blvd, Dallas, TX 75390, USA
| | - Steven Wright
- Hamon Center for Therapeutic Oncology Research, University of Texas Southwestern Medical Center, 6000 Harry Hines Blvd, Dallas, TX 75390, USA
| | - Xiaowu Gu
- Department of Molecular Biology, University of Texas Southwestern Medical Center, 6000 Harry Hines Blvd, Dallas, TX 75390, USA
| | - Ravikanth Maddipati
- Hamon Center for Therapeutic Oncology Research, University of Texas Southwestern Medical Center, 6000 Harry Hines Blvd, Dallas, TX 75390, USA; Department of Internal Medicine, University of Texas Southwestern Medical Center, 6000 Harry Hines Blvd, Dallas, TX 75390, USA
| | | | | | - Rolf A Brekken
- Department of Surgery, University of Texas Southwestern Medical Center, 6000 Harry Hines Blvd, Dallas, TX 75390, USA; Hamon Center for Therapeutic Oncology Research, University of Texas Southwestern Medical Center, 6000 Harry Hines Blvd, Dallas, TX 75390, USA; Cancer Biology Graduate Program, University of Texas Southwestern Medical Center, 6000 Harry Hines Blvd, Dallas, TX 75390, USA; Department of Pharmacology, University of Texas Southwestern Medical Center, 6000 Harry Hines Blvd, Dallas, TX 75390, USA.
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23
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Shi X, Wang Z, Ren W, Chen L, Xu C, Li M, Fan S, Xu Y, Chen M, Zheng F, Zhang W, Zhou X, Zhang Y, Qiu S, Wu L, Zhou P, Lv X, Cui T, Qiao Y, Zhao H, Guo W, Chen W, Li S, Zhong W, Lin J, Yang S. LDL receptor-related protein 1 (LRP1), a novel target for opening the blood-labyrinth barrier (BLB). Signal Transduct Target Ther 2022; 7:175. [PMID: 35680846 PMCID: PMC9184653 DOI: 10.1038/s41392-022-00995-z] [Citation(s) in RCA: 14] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/21/2021] [Revised: 03/16/2022] [Accepted: 04/07/2022] [Indexed: 11/18/2022] Open
Abstract
Inner ear disorders are a cluster of diseases that cause hearing loss in more than 1.5 billion people worldwide. However, the presence of the blood-labyrinth barrier (BLB) on the surface of the inner ear capillaries greatly hinders the effectiveness of systemic drugs for prevention and intervention due to the low permeability, which restricts the entry of most drug compounds from the bloodstream into the inner ear tissue. Here, we report the finding of a novel receptor, low-density lipoprotein receptor-related protein 1 (LRP1), that is expressed on the BLB, as a potential target for shuttling therapeutics across this barrier. As a proof-of-concept, we developed an LRP1-binding peptide, IETP2, and covalently conjugated a series of model small-molecule compounds to it, including potential drugs and imaging agents. All compounds were successfully delivered into the inner ear and inner ear lymph, indicating that targeting the receptor LRP1 is a promising strategy to enhance the permeability of the BLB. The discovery of the receptor LRP1 will illuminate developing strategies for crossing the BLB and for improving systemic drug delivery for inner ear disorders.
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Affiliation(s)
- Xi Shi
- Department of Pharmacy, Peking University Third Hospital, Beijing, China.,Artificial Auditory Laboratory of Jiangsu Province, Xuzhou Medical University, Xuzhou, China
| | - Zihao Wang
- National Engineering Research Center for the Emergency Drug, Beijing Institute of Pharmacology and Toxicology, Beijing, China
| | - Wei Ren
- College of Otolaryngology Head and Neck Surgery, Chinese PLA General Hospital, Beijing, China.,National Clinical Research Center for Otolaryngologic Diseases, Beijing, China.,Key Lab of Hearing Science, Ministry of Education, Beijing, China.,Beijing Key Lab of Hearing Impairment for Prevention and Treatment, Beijing, China
| | - Long Chen
- Department of Pharmacy, Peking University Third Hospital, Beijing, China.,Synthetic and Functional Biomolecules Center, Beijing National Laboratory for Molecular Sciences, Key Laboratory of Bioorganic Chemistry and Molecular Engineering of Ministry of Education, College of Chemistry and Molecular Engineering, Innovation Center for Genomics, Peking University, Beijing, China
| | - Cong Xu
- College of Otolaryngology Head and Neck Surgery, Chinese PLA General Hospital, Beijing, China.,National Clinical Research Center for Otolaryngologic Diseases, Beijing, China.,Key Lab of Hearing Science, Ministry of Education, Beijing, China.,Beijing Key Lab of Hearing Impairment for Prevention and Treatment, Beijing, China
| | - Menghua Li
- Artificial Auditory Laboratory of Jiangsu Province, Xuzhou Medical University, Xuzhou, China
| | - Shiyong Fan
- National Engineering Research Center for the Emergency Drug, Beijing Institute of Pharmacology and Toxicology, Beijing, China
| | - Yuru Xu
- National Engineering Research Center for the Emergency Drug, Beijing Institute of Pharmacology and Toxicology, Beijing, China
| | - Mengbing Chen
- College of Otolaryngology Head and Neck Surgery, Chinese PLA General Hospital, Beijing, China.,National Clinical Research Center for Otolaryngologic Diseases, Beijing, China.,Key Lab of Hearing Science, Ministry of Education, Beijing, China.,Beijing Key Lab of Hearing Impairment for Prevention and Treatment, Beijing, China
| | - Fanjun Zheng
- College of Otolaryngology Head and Neck Surgery, Chinese PLA General Hospital, Beijing, China.,National Clinical Research Center for Otolaryngologic Diseases, Beijing, China.,Key Lab of Hearing Science, Ministry of Education, Beijing, China.,Beijing Key Lab of Hearing Impairment for Prevention and Treatment, Beijing, China
| | - Wenyuan Zhang
- Synthetic and Functional Biomolecules Center, Beijing National Laboratory for Molecular Sciences, Key Laboratory of Bioorganic Chemistry and Molecular Engineering of Ministry of Education, College of Chemistry and Molecular Engineering, Innovation Center for Genomics, Peking University, Beijing, China
| | - Xinbo Zhou
- National Engineering Research Center for the Emergency Drug, Beijing Institute of Pharmacology and Toxicology, Beijing, China
| | - Yue Zhang
- College of Otolaryngology Head and Neck Surgery, Chinese PLA General Hospital, Beijing, China.,National Clinical Research Center for Otolaryngologic Diseases, Beijing, China.,Key Lab of Hearing Science, Ministry of Education, Beijing, China.,Beijing Key Lab of Hearing Impairment for Prevention and Treatment, Beijing, China
| | - Shiwei Qiu
- Artificial Auditory Laboratory of Jiangsu Province, Xuzhou Medical University, Xuzhou, China
| | - Liyuan Wu
- Artificial Auditory Laboratory of Jiangsu Province, Xuzhou Medical University, Xuzhou, China
| | - Peng Zhou
- Synthetic and Functional Biomolecules Center, Beijing National Laboratory for Molecular Sciences, Key Laboratory of Bioorganic Chemistry and Molecular Engineering of Ministry of Education, College of Chemistry and Molecular Engineering, Innovation Center for Genomics, Peking University, Beijing, China
| | - Xinze Lv
- Artificial Auditory Laboratory of Jiangsu Province, Xuzhou Medical University, Xuzhou, China
| | - Tianyu Cui
- Artificial Auditory Laboratory of Jiangsu Province, Xuzhou Medical University, Xuzhou, China
| | - Yuehua Qiao
- Artificial Auditory Laboratory of Jiangsu Province, Xuzhou Medical University, Xuzhou, China
| | - Hui Zhao
- College of Otolaryngology Head and Neck Surgery, Chinese PLA General Hospital, Beijing, China.,National Clinical Research Center for Otolaryngologic Diseases, Beijing, China.,Key Lab of Hearing Science, Ministry of Education, Beijing, China.,Beijing Key Lab of Hearing Impairment for Prevention and Treatment, Beijing, China
| | - Weiwei Guo
- College of Otolaryngology Head and Neck Surgery, Chinese PLA General Hospital, Beijing, China.,National Clinical Research Center for Otolaryngologic Diseases, Beijing, China.,Key Lab of Hearing Science, Ministry of Education, Beijing, China.,Beijing Key Lab of Hearing Impairment for Prevention and Treatment, Beijing, China
| | - Wei Chen
- College of Otolaryngology Head and Neck Surgery, Chinese PLA General Hospital, Beijing, China.,National Clinical Research Center for Otolaryngologic Diseases, Beijing, China.,Key Lab of Hearing Science, Ministry of Education, Beijing, China.,Beijing Key Lab of Hearing Impairment for Prevention and Treatment, Beijing, China
| | - Song Li
- National Engineering Research Center for the Emergency Drug, Beijing Institute of Pharmacology and Toxicology, Beijing, China
| | - Wu Zhong
- National Engineering Research Center for the Emergency Drug, Beijing Institute of Pharmacology and Toxicology, Beijing, China.
| | - Jian Lin
- Department of Pharmacy, Peking University Third Hospital, Beijing, China. .,Synthetic and Functional Biomolecules Center, Beijing National Laboratory for Molecular Sciences, Key Laboratory of Bioorganic Chemistry and Molecular Engineering of Ministry of Education, College of Chemistry and Molecular Engineering, Innovation Center for Genomics, Peking University, Beijing, China.
| | - Shiming Yang
- College of Otolaryngology Head and Neck Surgery, Chinese PLA General Hospital, Beijing, China. .,National Clinical Research Center for Otolaryngologic Diseases, Beijing, China. .,Key Lab of Hearing Science, Ministry of Education, Beijing, China. .,Beijing Key Lab of Hearing Impairment for Prevention and Treatment, Beijing, China.
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24
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Aguilar-González A, González-Correa JE, Barriocanal-Casado E, Ramos-Hernández I, Lerma-Juárez MA, Greco S, Rodríguez-Sevilla JJ, Molina-Estévez FJ, Montalvo-Romeral V, Ronzitti G, Sánchez-Martín RM, Martín F, Muñoz P. Isogenic GAA-KO Murine Muscle Cell Lines Mimicking Severe Pompe Mutations as Preclinical Models for the Screening of Potential Gene Therapy Strategies. Int J Mol Sci 2022; 23:6298. [PMID: 35682977 PMCID: PMC9181599 DOI: 10.3390/ijms23116298] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/09/2022] [Revised: 05/30/2022] [Accepted: 06/01/2022] [Indexed: 11/17/2022] Open
Abstract
Pompe disease (PD) is a rare disorder caused by mutations in the acid alpha-glucosidase (GAA) gene. Most gene therapies (GT) partially rely on the cross-correction of unmodified cells through the uptake of the GAA enzyme secreted by corrected cells. In the present study, we generated isogenic murine GAA-KO cell lines resembling severe mutations from Pompe patients. All of the generated GAA-KO cells lacked GAA activity and presented an increased autophagy and increased glycogen content by means of myotube differentiation as well as the downregulation of mannose 6-phosphate receptors (CI-MPRs), validating them as models for PD. Additionally, different chimeric murine GAA proteins (IFG, IFLG and 2G) were designed with the aim to improve their therapeutic activity. Phenotypic rescue analyses using lentiviral vectors point to IFG chimera as the best candidate in restoring GAA activity, normalising the autophagic marker p62 and surface levels of CI-MPRs. Interestingly, in vivo administration of liver-directed AAVs expressing the chimeras further confirmed the good behaviour of IFG, achieving cross-correction in heart tissue. In summary, we generated different isogenic murine muscle cell lines mimicking the severe PD phenotype, as well as validating their applicability as preclinical models in order to reduce animal experimentation.
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Affiliation(s)
- Araceli Aguilar-González
- GENYO, Centre for Genomics and Oncological Research: Pfizer, University of Granada, Andalusian Regional Government PTS Granada-Avenida de la Ilustración 114, 18016 Granada, Spain; (A.A.-G.); (J.E.G.-C.); (E.B.-C.); (I.R.-H.); (S.G.); (J.J.R.-S.); (F.J.M.-E.); (R.M.S.-M.)
- Department of Medicinal & Organic Chemistry and Excellence Research Unit of “Chemistry Applied to Biomedicine and the Environment”, Faculty of Pharmacy, University of Granada, Campus de Cartuja s/n, 18071 Granada, Spain
| | - Juan Elías González-Correa
- GENYO, Centre for Genomics and Oncological Research: Pfizer, University of Granada, Andalusian Regional Government PTS Granada-Avenida de la Ilustración 114, 18016 Granada, Spain; (A.A.-G.); (J.E.G.-C.); (E.B.-C.); (I.R.-H.); (S.G.); (J.J.R.-S.); (F.J.M.-E.); (R.M.S.-M.)
| | - Eliana Barriocanal-Casado
- GENYO, Centre for Genomics and Oncological Research: Pfizer, University of Granada, Andalusian Regional Government PTS Granada-Avenida de la Ilustración 114, 18016 Granada, Spain; (A.A.-G.); (J.E.G.-C.); (E.B.-C.); (I.R.-H.); (S.G.); (J.J.R.-S.); (F.J.M.-E.); (R.M.S.-M.)
| | - Iris Ramos-Hernández
- GENYO, Centre for Genomics and Oncological Research: Pfizer, University of Granada, Andalusian Regional Government PTS Granada-Avenida de la Ilustración 114, 18016 Granada, Spain; (A.A.-G.); (J.E.G.-C.); (E.B.-C.); (I.R.-H.); (S.G.); (J.J.R.-S.); (F.J.M.-E.); (R.M.S.-M.)
| | - Miguel A. Lerma-Juárez
- Instituto de Investigación del Hospital Universitario La Paz, IdiPAZ, 28029 Madrid, Spain;
| | - Sara Greco
- GENYO, Centre for Genomics and Oncological Research: Pfizer, University of Granada, Andalusian Regional Government PTS Granada-Avenida de la Ilustración 114, 18016 Granada, Spain; (A.A.-G.); (J.E.G.-C.); (E.B.-C.); (I.R.-H.); (S.G.); (J.J.R.-S.); (F.J.M.-E.); (R.M.S.-M.)
| | - Juan José Rodríguez-Sevilla
- GENYO, Centre for Genomics and Oncological Research: Pfizer, University of Granada, Andalusian Regional Government PTS Granada-Avenida de la Ilustración 114, 18016 Granada, Spain; (A.A.-G.); (J.E.G.-C.); (E.B.-C.); (I.R.-H.); (S.G.); (J.J.R.-S.); (F.J.M.-E.); (R.M.S.-M.)
| | - Francisco Javier Molina-Estévez
- GENYO, Centre for Genomics and Oncological Research: Pfizer, University of Granada, Andalusian Regional Government PTS Granada-Avenida de la Ilustración 114, 18016 Granada, Spain; (A.A.-G.); (J.E.G.-C.); (E.B.-C.); (I.R.-H.); (S.G.); (J.J.R.-S.); (F.J.M.-E.); (R.M.S.-M.)
- Fundación para la Investigación Biosanitaria de Andalucía Oriental-Alejandro Otero (FIBAO), 18012 Granada, Spain
| | - Valle Montalvo-Romeral
- Généthon, Integrare Research Unit UMR_S951, INSERM, Université Paris-Saclay, Univ Evry, 91002 Evry, France; (V.M.-R.); (G.R.)
| | - Giuseppe Ronzitti
- Généthon, Integrare Research Unit UMR_S951, INSERM, Université Paris-Saclay, Univ Evry, 91002 Evry, France; (V.M.-R.); (G.R.)
| | - Rosario María Sánchez-Martín
- GENYO, Centre for Genomics and Oncological Research: Pfizer, University of Granada, Andalusian Regional Government PTS Granada-Avenida de la Ilustración 114, 18016 Granada, Spain; (A.A.-G.); (J.E.G.-C.); (E.B.-C.); (I.R.-H.); (S.G.); (J.J.R.-S.); (F.J.M.-E.); (R.M.S.-M.)
- Department of Medicinal & Organic Chemistry and Excellence Research Unit of “Chemistry Applied to Biomedicine and the Environment”, Faculty of Pharmacy, University of Granada, Campus de Cartuja s/n, 18071 Granada, Spain
| | - Francisco Martín
- GENYO, Centre for Genomics and Oncological Research: Pfizer, University of Granada, Andalusian Regional Government PTS Granada-Avenida de la Ilustración 114, 18016 Granada, Spain; (A.A.-G.); (J.E.G.-C.); (E.B.-C.); (I.R.-H.); (S.G.); (J.J.R.-S.); (F.J.M.-E.); (R.M.S.-M.)
- Departamento de Bioquímica y Biología Molecular 3 e Inmunología, Facultad de Medicina, Universidad de Granada, Avda. de la Investigación 11, 18071 Granada, Spain
| | - Pilar Muñoz
- GENYO, Centre for Genomics and Oncological Research: Pfizer, University of Granada, Andalusian Regional Government PTS Granada-Avenida de la Ilustración 114, 18016 Granada, Spain; (A.A.-G.); (J.E.G.-C.); (E.B.-C.); (I.R.-H.); (S.G.); (J.J.R.-S.); (F.J.M.-E.); (R.M.S.-M.)
- Departmento de Biología Celular, Facultad de Ciencias, Universidad de Granada, Campus Fuentenueva, 18071 Granada, Spain
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25
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Hua R, Gu S, Jiang JX. Connexin 43 Hemichannels Regulate Osteoblast to Osteocyte Differentiation. Front Cell Dev Biol 2022; 10:892229. [PMID: 35693933 PMCID: PMC9184820 DOI: 10.3389/fcell.2022.892229] [Citation(s) in RCA: 18] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/08/2022] [Accepted: 04/18/2022] [Indexed: 11/13/2022] Open
Abstract
Connexin 43 (Cx43) is the predominant connexin subtype expressed in osteocytes. Osteocytes, accounting for 90%-95% of total bone cells, function as orchestrators coordinating balanced activity between bone-resorbing osteoclasts and bone-forming osteoblasts. In this study, two newly developed osteocytic cell lines, OCY454 and IDG-SW3, were used to determine the role of Cx43 gap junctions and hemichannels (HCs) in the regulation of osteoblast to osteocyte differentiation. We found that the Cx43 level was substantially increased during the differentiation of IDG-SW3 cells and is also much higher than that of OCY454 cells. We knocked down Cx43 expression using the lentiviral CRISPR/Cas9 approach and inhibition of Cx43 HCs using Cx43 (E2) antibody in IDG-SW3 cells. Cx43 knockdown (KD) or Cx43 HC inhibition decreased gene expression for osteoblast and osteocyte markers, including alkaline phosphatase, type I collagen, dentin matrix protein 1, sclerostin, and fibroblast growth factor 23, whereas increasing the osteoclastogenesis indicator and the receptor activator of nuclear factor kappa-B ligand (RANKL)/osteoprotegerin (OPG) ratio at early and late differentiation stages. Moreover, mineralization was remarkably attenuated in differentiated Cx43-deficient IDG-SW3 cells compared to ROSA26 control. The conditioned medium collected from fully differentiated IDG-SW3 cells with Cx43 KD promoted osteoclastogenesis of RAW264.7 osteoclast precursors. Our results demonstrated that Cx43 HCs play critical roles in osteoblast to osteocyte differentiation process and regulate osteoclast differentiation via secreted factors.
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Affiliation(s)
| | | | - Jean X. Jiang
- Department of Biochemistry and Structural Biology, University of Texas Health Science Center, San Antonio, TX, United States
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26
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Lee MP, Waldhaus J. In vitro and in vivo models: What have we learnt about inner ear regeneration and treatment for hearing loss? Mol Cell Neurosci 2022; 120:103736. [PMID: 35577314 PMCID: PMC9551661 DOI: 10.1016/j.mcn.2022.103736] [Citation(s) in RCA: 7] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/31/2021] [Revised: 04/29/2022] [Accepted: 05/10/2022] [Indexed: 01/07/2023] Open
Abstract
The sensory cells of the inner ear, called hair cells, do not regenerate spontaneously and therefore, hair cell loss and subsequent hearing loss are permanent in humans. Conversely, functional hair cell regeneration can be observed in non-mammalian vertebrate species like birds and fish. Also, during postnatal development in mice, limited regenerative capacity and the potential to isolate stem cells were reported. Together, these findings spurred the interest of current research aiming to investigate the endogenous regenerative potential in mammals. In this review, we summarize current in vitro based approaches and briefly introduce different in vivo model organisms utilized to study hair cell regeneration. Furthermore, we present an overview of the findings that were made synergistically using both, the in vitro and in vivo based tools.
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Affiliation(s)
- Mary P Lee
- Department of Otolaryngology-Head and Neck Surgery, Kresge Hearing Research Institute, University of Michigan, Ann Arbor, MI 48109, USA
| | - Joerg Waldhaus
- Department of Otolaryngology-Head and Neck Surgery, Kresge Hearing Research Institute, University of Michigan, Ann Arbor, MI 48109, USA.
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27
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MicroRNA Signature and Cellular Characterization of Undifferentiated and Differentiated House Ear Institute-Organ of Corti 1 (HEI-OC1) Cells. J Assoc Res Otolaryngol 2022; 23:467-489. [PMID: 35546217 PMCID: PMC9094604 DOI: 10.1007/s10162-022-00850-6] [Citation(s) in RCA: 6] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/13/2020] [Accepted: 04/20/2022] [Indexed: 11/29/2022] Open
Abstract
MicroRNAs (miRNAs) regulate gene expressions and control a wide variety of cellular functions. House Ear Institute-Organ of Corti 1 (HEI-OC1) cells are widely used to screen ototoxic drugs and to investigate cellular and genetic alterations in response to various conditions. HEI-OC1 cells are almost exclusively studied under permissive conditions that promote cell replication at the expense of differentiation. Many researchers suggest that permissive culture condition findings are relevant to understanding human hearing disorders. The mature human cochlea however consists of differentiated cells and lacks proliferative capacity. This study therefore aimed to compare the miRNA profiles and cellular characteristics of HEI-OC1 cells cultured under permissive (P-HEI-OC1) and non-permissive (NP-HEI-OC1) conditions. A significant increase in the level of expression of tubulin β1 class VI (Tubb1), e-cadherin (Cdh1), espin (Espn), and SRY (sex determining region Y)-box2 (Sox2) mRNAs was identified in non-permissive cells compared with permissive cells (P < 0.05, Kruskal–Wallis H test, 2-sided). miR-200 family, miR-34b/c, and miR-449a/b functionally related cluster miRNAs, rodent-specific maternally imprinted gene Sfmbt2 intron 10th cluster miRNAs (-466a/ -467a), and miR-17 family were significantly (P < 0.05, Welch’s t-test, 2-tailed) differentially expressed in non-permissive cells when compared with permissive cells. Putative target genes were significantly predominantly enriched in mitogen-activated protein kinase (MAPK), epidermal growth factor family of receptor tyrosine kinases (ErbB), and Ras signaling pathways in non-permissive cells compared with permissive cells. This distinct miRNA signature of differentiated HEI-OC1 cells could help in understanding miRNA-mediated cellular responses in the adult cochlea.
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28
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Lua I, Balog S, Asahina K. TAZ/WWTR1 mediates liver mesothelial-mesenchymal transition induced by stiff extracellular environment, TGF-β1, and lysophosphatidic acid. J Cell Physiol 2022; 237:2561-2573. [PMID: 35445400 DOI: 10.1002/jcp.30750] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/28/2021] [Revised: 02/14/2022] [Accepted: 03/04/2022] [Indexed: 11/08/2022]
Abstract
Mesothelial cells cover the surface of the internal organs and the walls of body cavities, facilitating the movement between organs by secretion of a lubricating fluid. Upon injury, mesothelial cells undergo a mesothelial-mesenchymal transition (MMT) and give rise to myofibroblasts during organ fibrosis, including in the liver. Although transforming growth factor-β1 (TGF-β1) was shown to induce MMT, molecular and cellular mechanisms underlying MMT remain to be clarified. In the present study, we examined how the extracellular environment, soluble factors, and cell density control the phenotype of liver mesothelial cells by culturing them at different cell densities or on hydrogels of different stiffness. We found that TGF-β1 does not fully induce MMT in mesothelial cells cultured at high cell density or in the absence of fetal bovine serum. Extracellular lysophosphatidic acid (LPA) synergistically induced MMT in the presence of TGF-β1 in mesothelial cells. LPA induced nuclear localization of WW domain-containing transcription regulator1 (WWTR1/TAZ) and knockdown of Taz, which suppressed LPA-induced MMT. Mesothelial cells cultured on stiff hydrogels upregulated nuclear localization of TAZ and myofibroblastic differentiation. Knockdown of Taz suppressed MMT of mesothelial cells cultured on stiff hydrogels, but inhibition of TGF-β1 signaling failed to suppress MMT. Our data indicate that TAZ mediates MMT induced by TGF-β1, LPA, and a stiff matrix. The microenvironment of a stiff extracellular matrix is a strong inducer of MMT.
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Affiliation(s)
- Ingrid Lua
- Department of Pathology, Keck School of Medicine, University of Southern California, Los Angeles, California, USA
| | - Steven Balog
- Department of Pathology, Keck School of Medicine, University of Southern California, Los Angeles, California, USA
| | - Kinji Asahina
- Department of Pathology, Keck School of Medicine, University of Southern California, Los Angeles, California, USA.,Central Research Laboratory, Shiga University of Medical Science, Shiga, Japan
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29
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Chen J, Wang X, He Q, Harris RC. TAZ is important for maintenance of the integrity of podocytes. Am J Physiol Renal Physiol 2022; 322:F419-F428. [PMID: 35157550 PMCID: PMC8934679 DOI: 10.1152/ajprenal.00426.2021] [Citation(s) in RCA: 11] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/23/2021] [Revised: 02/01/2022] [Accepted: 02/07/2022] [Indexed: 11/22/2022] Open
Abstract
The podocyte is an important component of the glomerular filtration barrier, and maintenance of the integrity of its highly specified structure and function is critical for normal kidney function. Yes-associated protein (YAP)/transcriptional coactivator with PDZ-binding motif (TAZ) are two crucial effectors of the Hippo signaling pathway, and recent studies have shown that podocyte-specific YAP deletion causes podocyte apoptosis and the development of focal segmental glomerulosclerosis followed by progressive renal failure. In the present study, we investigated a potential role of the YAP paralog TAZ in podocytes. TAZ was found to be constitutively active in podocytes, and mice with podocyte-specific deletion of TAZ (TazpodKO) developed proteinuria starting at 4 wk of age and had increased podocyte apoptosis. Using primary cultured podocytes or immortalized mouse podocytes from Tazflox/flox mice, we found that TAZ is a transcriptional activator for TEAD-dependent expression of synaptopodin, zonula occludens-1, and zonula occludens-2. This is the first study to determine that TAZ plays an important role in the maintenance of the structure and function of podocytes.NEW & NOTEWORTHY Podocytes play an important role in maintaining the integrity of the structure and function of the kidney. We observed that mice with selective deletion of transcriptional coactivator with PDZ-binding motif (TAZ) in podocytes developed proteinuria. TAZ is constitutively active and critical for expression of synaptopodin, zonula occludens-1, and zonula occludens-2 in podocytes. The findings of this study implicate TAZ as an important mediator of podocyte structural integrity and provide further insights into the role of Hippo-Yes-associated protein/TAZ in podocyte biology.
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Affiliation(s)
- Jianchun Chen
- United States Department of Veterans Affairs, Nashville, Tennessee
- Department of Medicine, Vanderbilt University Medical Center, Nashville, Tennessee
- Vanderbilt Center for Kidney Disease, Vanderbilt University Medical Center, Nashville, Tennessee
| | - Xiaoyong Wang
- Department of Medicine, Vanderbilt University Medical Center, Nashville, Tennessee
| | - Qian He
- Department of Medicine, Vanderbilt University Medical Center, Nashville, Tennessee
| | - Raymond C Harris
- United States Department of Veterans Affairs, Nashville, Tennessee
- Department of Medicine, Vanderbilt University Medical Center, Nashville, Tennessee
- Vanderbilt Center for Kidney Disease, Vanderbilt University Medical Center, Nashville, Tennessee
- Department of Molecular Physiology and Biophysics, Vanderbilt University Medical Center, Nashville, Tennessee
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30
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Vallecillo-Zúniga ML, Rathgeber M, Poulson D, Kartchner B, Luddington J, Gill H, Hayes S, Teynor M, Stowell CS, Arthur CM, Stowell SR, Van Ry PM. Evaluating Therapeutic Activity of Galectin-1 in Sarcolemma Repair of Skeletal Muscle. Methods Mol Biol 2022; 2442:663-683. [PMID: 35320552 DOI: 10.1007/978-1-0716-2055-7_36] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 06/14/2023]
Abstract
Galectin-1 is a small (14.5 kDa) multifunctional protein with cell-cell and cell-ECM adhesion due to interactions with the carbohydrate recognition domain (CRD). In two types of muscular dystrophies, this lectin protein has shown therapeutic properties, including positive regulation of skeletal muscle differentiation and regeneration. Both Duchenne and limb-girdle muscular dystrophy 2B (LGMD2B) are subtypes of muscular dystrophies characterized by deficient membrane repair, muscle weakness, and eventual loss of ambulation. This chapter explains confocal techniques such as laser injury, calcium imaging, and galectin-1 localization to examine the effects of galectin-1 on membrane repair in injured LGMD2B models.
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Affiliation(s)
| | - Matthew Rathgeber
- Department of Chemistry and Biochemistry, Brigham Young University, Provo, UT, USA
| | - Daniel Poulson
- Department of Chemistry and Biochemistry, Brigham Young University, Provo, UT, USA
| | - Braden Kartchner
- Department of Chemistry and Biochemistry, Brigham Young University, Provo, UT, USA
| | - Jacob Luddington
- Department of Chemistry and Biochemistry, Brigham Young University, Provo, UT, USA
| | - Hailie Gill
- Department of Chemistry and Biochemistry, Brigham Young University, Provo, UT, USA
| | - Spencer Hayes
- Department of Chemistry and Biochemistry, Brigham Young University, Provo, UT, USA
| | - Matthew Teynor
- Department of Chemistry and Biochemistry, Brigham Young University, Provo, UT, USA
| | - Caleb S Stowell
- Department of Chemistry and Biochemistry, Brigham Young University, Provo, UT, USA
| | - Connie M Arthur
- Joint Program in Transfusion Medicine, Department of Pathology, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA
- Harvard Glycomics Center, Harvard Medical School, Boston, MA, USA
| | - Sean R Stowell
- Joint Program in Transfusion Medicine, Department of Pathology, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA
- Harvard Glycomics Center, Harvard Medical School, Boston, MA, USA
| | - Pam M Van Ry
- Department of Chemistry and Biochemistry, Brigham Young University, Provo, UT, USA.
- Department of Biochemistry, Brigham Young University, Provo, UT, USA.
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Rousset F, Schmidbauer D, Fink S, Adel Y, Obexer B, Müller M, Glueckert R, Löwenheim H, Senn P. Phoenix auditory neurons as 3R cell model for high throughput screening of neurogenic compounds. Hear Res 2021; 414:108391. [PMID: 34844170 DOI: 10.1016/j.heares.2021.108391] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/14/2021] [Revised: 10/25/2021] [Accepted: 10/27/2021] [Indexed: 11/25/2022]
Abstract
Auditory neurons connect the sensory hair cells from the inner ear to the brainstem. These bipolar neurons are relevant targets for pharmacological intervention aiming at protecting or improving the hearing function in various forms of sensorineural hearing loss. In the research laboratory, neurotrophic compounds are commonly used to improve survival and to promote regeneration of auditory neurons. One important roadblock delaying eventual clinical applications of these strategies in humans is the lack of powerful in vitro models allowing high throughput screening of otoprotective and regenerative compounds. The recently discovered auditory neuroprogenitors (ANPGs) derived from the A/J mouse with an unprecedented capacity to self-renew and to provide mature auditory neurons offer the possibility to overcome this bottleneck. In the present study, we further characterized the new phoenix ANPGs model and compared it to the current gold-standard spiral ganglion organotypic explant (SGE) model to assay neurite outgrowth, neurite length and glutamate-induced Ca2+ response in response to neurotrophin-3 (NT-3) and brain derived neurotrophic factor (BDNF) treatment. Whereas both, SGEs and phoenix ANPGs exhibited a robust and sensitive response to neurotrophins, the phoenix ANPGs offer a considerable range of advantages including high throughput suitability, lower experimental variability, single cell resolution and an important reduction of animal numbers. The phoenix ANPGs in vitro model therefore provides a robust high-throughput platform to screen for otoprotective and regenerative neurotrophic compounds in line with 3R principles and is of interest for the field of auditory neuroscience.
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Affiliation(s)
- Francis Rousset
- The Inner Ear & Olfaction Lab, Department of Pathology and Immunology, Faculty of Medicine, University of Geneva, Switzerland.
| | - Dominik Schmidbauer
- Inner Ear Laboratory, Department of Otolaryngology, Medical University of Innsbruck, Austria
| | - Stefan Fink
- Translational Hearing Research, Tübingen Hearing Research Center, Department of Otolaryngology, Head & Neck Surgery, University of Tübingen, Germany
| | - Youssef Adel
- Translational Hearing Research, Tübingen Hearing Research Center, Department of Otolaryngology, Head & Neck Surgery, University of Tübingen, Germany
| | - Benjamin Obexer
- Inner Ear Laboratory, Department of Otolaryngology, Medical University of Innsbruck, Austria
| | - Marcus Müller
- Translational Hearing Research, Tübingen Hearing Research Center, Department of Otolaryngology, Head & Neck Surgery, University of Tübingen, Germany
| | - Rudolf Glueckert
- Inner Ear Laboratory, Department of Otolaryngology, Medical University of Innsbruck, Austria.
| | - Hubert Löwenheim
- Translational Hearing Research, Tübingen Hearing Research Center, Department of Otolaryngology, Head & Neck Surgery, University of Tübingen, Germany
| | - Pascal Senn
- The Inner Ear & Olfaction Lab, Department of Pathology and Immunology, Faculty of Medicine, University of Geneva, Switzerland; Department of Clinical Neurosciences, Service of ORL & Head and Neck Surgery, University Hospital of Geneva, Switzerland
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West J, Rathinasabapathy A, Chen X, Shay S, Gladson S, Talati M. Overexpression of Msx1 in Mouse Lung Leads to Loss of Pulmonary Vessels Following Vascular Hypoxic Injury. Cells 2021; 10:cells10092306. [PMID: 34571956 PMCID: PMC8471093 DOI: 10.3390/cells10092306] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/02/2021] [Revised: 08/11/2021] [Accepted: 08/19/2021] [Indexed: 11/16/2022] Open
Abstract
Pulmonary arterial hypertension (PAH) is a progressive lung disease caused by thickening of the pulmonary arterial wall and luminal obliteration of the small peripheral arteries leading to increase in vascular resistance which elevates pulmonary artery pressure that eventually causes right heart failure and death. We have previously shown that transcription factor Msx1 (mainly expressed during embryogenesis) is strongly upregulated in transformed lymphocytes obtained from PAH patients, especially IPAH. Under pathological conditions, Msx1 overexpression can cause cell dedifferentiation or cell apoptosis. We hypothesized that Msx1 overexpression contributes to loss of small pulmonary vessels in PAH. In IPAH lung, MSX1 protein localization was strikingly increased in muscularized remodeled pulmonary vessels, whereas it was undetectable in control pulmonary arteries. We developed a transgenic mouse model overexpressing MSX1 (MSX1OE) by about 4-fold and exposed these mice to normoxic, sugen hypoxic (3 weeks) or hyperoxic (100% 02 for 3 weeks) conditions. Under normoxic conditions, compared to controls, MSX1OE mice demonstrated a 30-fold and 2-fold increase in lung Msx1 mRNA and protein expression, respectively. There was a significant retinal capillary dropout (p < 0.01) in MSX1OE mice, which was increased further (p < 0.03) with sugen hypoxia. At baseline, the number of pulmonary vessels in MSX1OE mice was similar to controls. In sugen-hypoxia-treated MSX1OE mice, the number of small (0-25 uM) and medium (25-50 uM) size muscularized vessels increased approximately 2-fold (p < 0.01) compared to baseline controls; however, they were strikingly lower (p < 0.001) in number than in sugen-hypoxia-treated control mice. In MSX1OE mouse lung, 104 genes were upregulated and 67 genes were downregulated compared to controls. Similarly, in PVECs, 156 genes were upregulated and 320 genes were downregulated from siRNA to MSX1OE, and in PVSMCs, 65 genes were upregulated and 321 genes were downregulated from siRNA to MSX1OE (with control in the middle). Many of the statistically significant GO groups associated with MSX1 expression in lung, PVECs, and PVSMCs were similar, and were involved in cell cycle, cytoskeletal and macromolecule organization, and programmed cell death. Overexpression of MSX1 suppresses many cell-cycle-related genes in PVSMCs but induces them in PVECs. In conclusion, overexpression of Msx1 leads to loss of pulmonary vessels, which is exacerbated by sugen hypoxia, and functional consequences of Msx1 overexpression are cell-dependent.
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Affiliation(s)
| | | | | | | | | | - Megha Talati
- Correspondence: ; Tel.: +1-615-322-8095; Fax: +1-615-343-7448
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Ge Y, Smits AM, Liu J, Zhang J, van Brakel TJ, Goumans MJTH, Jongbloed MRM, de Vries AAF. Generation, Characterization, and Application of Inducible Proliferative Adult Human Epicardium-Derived Cells. Cells 2021; 10:2064. [PMID: 34440833 PMCID: PMC8391799 DOI: 10.3390/cells10082064] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/10/2021] [Revised: 08/02/2021] [Accepted: 08/04/2021] [Indexed: 12/20/2022] Open
Abstract
RATIONALE In recent decades, the great potential of human epicardium-derived cells (EPDCs) as an endogenous cell source for cardiac regeneration has been recognized. The limited availability and low proliferation capacity of primary human EPDCs and phenotypic differences between EPDCs obtained from different individuals hampers their reproducible use for experimental studies. AIM To generate and characterize inducible proliferative adult human EPDCs for use in fundamental and applied research. METHODS AND RESULTS Inducible proliferation of human EPDCs was achieved by doxycycline-controlled expression of simian virus 40 large T antigen (LT) with a repressor-based lentiviral Tet-On system. In the presence of doxycycline, these inducible EPDCs (iEPDCs) displayed high and long-term proliferation capacity. After doxycycline removal, LT expression ceased and the iEPDCs regained their cuboidal epithelial morphology. Similar to primary EPDCs, iEPDCs underwent an epithelial-to-mesenchymal transition (EMT) after stimulation with transforming growth factor β3. This was confirmed by reverse transcription-quantitative polymerase chain reaction analysis of epithelial and mesenchymal marker gene expression and (immuno) cytochemical staining. Collagen gel-based cell invasion assays demonstrated that mesenchymal iEPDCs, like primary EPDCs, possess increased invasion and migration capacities as compared to their epithelial counterparts. Mesenchymal iEPDCs co-cultured with sympathetic ganglia stimulated neurite outgrowth similarly to primary EPDCs. CONCLUSION Using an inducible LT expression system, inducible proliferative adult human EPDCs were generated displaying high proliferative capacity in the presence of doxycycline. These iEPDCs maintain essential epicardial characteristics with respect to morphology, EMT ability, and paracrine signaling following doxycycline removal. This renders iEPDCs a highly useful new in vitro model for studying human epicardial properties.
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Affiliation(s)
- Yang Ge
- Department of Anatomy & Embryology, Leiden University Medical Center, Einthovenweg 20, 2333 ZC Leiden, The Netherlands; (Y.G.); (M.R.M.J.)
- Department of Cardiology, Leiden University Medical Center, Albinusdreef 2, 2333 ZA Leiden, The Netherlands; (J.L.); (J.Z.); (A.A.F.d.V.)
| | - Anke M. Smits
- Department of Cell and Chemical Biology, Leiden University Medical Center, Einthovenweg 20, 2333 ZC Leiden, The Netherlands;
| | - Jia Liu
- Department of Cardiology, Leiden University Medical Center, Albinusdreef 2, 2333 ZA Leiden, The Netherlands; (J.L.); (J.Z.); (A.A.F.d.V.)
- Central Laboratory, Longgang District People’s Hospital of Shenzhen & The Third Affiliated Hospital of The Chinese University of Hong Kong, Shenzhen 518172, China
| | - Juan Zhang
- Department of Cardiology, Leiden University Medical Center, Albinusdreef 2, 2333 ZA Leiden, The Netherlands; (J.L.); (J.Z.); (A.A.F.d.V.)
| | - Thomas J. van Brakel
- Department of Cardiothoracic Surgery, Leiden University Medical Center, Albinusdreef 2, 2333 ZC Leiden, The Netherlands;
| | - Marie José T. H. Goumans
- Department of Cell and Chemical Biology, Leiden University Medical Center, Einthovenweg 20, 2333 ZC Leiden, The Netherlands;
| | - Monique R. M. Jongbloed
- Department of Anatomy & Embryology, Leiden University Medical Center, Einthovenweg 20, 2333 ZC Leiden, The Netherlands; (Y.G.); (M.R.M.J.)
- Department of Cardiology, Leiden University Medical Center, Albinusdreef 2, 2333 ZA Leiden, The Netherlands; (J.L.); (J.Z.); (A.A.F.d.V.)
| | - Antoine A. F. de Vries
- Department of Cardiology, Leiden University Medical Center, Albinusdreef 2, 2333 ZA Leiden, The Netherlands; (J.L.); (J.Z.); (A.A.F.d.V.)
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Daly AZ, Mortensen AH, Bando H, Camper SA. Pituitary Tumors and Immortalized Cell Lines Generated by Cre-Inducible Expression of SV40 T Antigen. Endocrinology 2021; 162:6219492. [PMID: 33837405 PMCID: PMC8183496 DOI: 10.1210/endocr/bqab073] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/27/2020] [Indexed: 02/07/2023]
Abstract
Targeted oncogenesis is the process of driving tumor formation by engineering transgenic mice that express an oncogene under the control of a cell-type specific promoter. Such tumors can be adapted to cell culture, providing immortalized cell lines. To make it feasible to follow the process of tumorigenesis and increase the opportunity for generating cell lines, we developed a mouse strain that expresses SV40 T antigens in response to Cre-recombinase. Using CRISPR/Cas9 we inserted a cassette with coding sequences for SV40 T antigens and an internal ribosome entry site with green fluorescent protein cassette (IRES-GFP) into the Rosa26 locus, downstream from a stop sequence flanked by loxP sites: Rosa26LSL-SV40-GFP. These mice were mated with previously established Prop1-cre and Tshb-cre transgenic lines. Both the Rosa26LSL-SV40-GFP/+; Prop1-cre and Rosa26LSL-SV40-GFP/+; Tshb-cre mice developed fully penetrant dwarfism and large tumors by 4 weeks. Tumors from both of these mouse lines were adapted to growth in cell culture. We have established a progenitor-like cell line (PIT-P1) that expresses Sox2 and Pitx1, and a thyrotrope-like cell line (PIT-T1) that expresses Pou1f1 and Cga. These studies demonstrate the utility of the novel, Rosa26LSL-SV40-GFP mouse line for reliable targeted oncogenesis and development of unique cell lines.
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Affiliation(s)
| | | | - Hironori Bando
- University of Michigan Medical School, Ann Arbor, MI 48109, USA
| | - Sally A Camper
- University of Michigan Medical School, Ann Arbor, MI 48109, USA
- Correspondence: Sally A. Camper, Ph.D., 5704 Medical Science Building II, 1301 Catherine St, Department of Human Genetics, University of Michigan Medical School, Ann Arbor, MI 48109, USA.
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Jiang H, Song S, Li J, Yin Q, Hu S, Nie Y. Establishment and characterization of an immortalized epicardial cell line. J Cell Mol Med 2021; 25:6070-6081. [PMID: 33822475 PMCID: PMC8406488 DOI: 10.1111/jcmm.16496] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/05/2020] [Revised: 03/03/2021] [Accepted: 03/06/2021] [Indexed: 12/11/2022] Open
Abstract
Recently, the increasing significance of the epicardium in cardiac development and regeneration is beginning to be recognized. However, because of the small proportion of primary epicardial cells and the limited cell culture time, further research on the mechanism of epicardial cells is hindered. Here, we transfected simian virus 40 Large T (SV40-LT) into primary epicardial cells to establish an immortalized cell line, named EpiSV40. We further demonstrated that EpiSV40 can be easy to culture and has the proliferation, migration and differentiation capacities comparable to primary epicardial cells. EpiSV40 can serve as an ideal in vitro model for epicardial cell research, which will booster the study of the epicardium in cardiac development and heart regeneration.
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Affiliation(s)
- Haobin Jiang
- State Key Laboratory of Cardiovascular DiseaseFuwai HospitalNational Center for Cardiovascular DiseaseChinese Academy of Medical Sciences and Peking Union Medical CollegeBeijingChina
| | - Shen Song
- State Key Laboratory of Cardiovascular DiseaseFuwai HospitalNational Center for Cardiovascular DiseaseChinese Academy of Medical Sciences and Peking Union Medical CollegeBeijingChina
| | - Jiacheng Li
- Biodynamic Optical Imaging Center and Center for Reproductive MedicineCollege of Life SciencesThird HospitalPeking UniversityBeijingChina
| | - Qianqian Yin
- State Key Laboratory of Cardiovascular DiseaseFuwai HospitalNational Center for Cardiovascular DiseaseChinese Academy of Medical Sciences and Peking Union Medical CollegeBeijingChina
| | - Shengshou Hu
- State Key Laboratory of Cardiovascular DiseaseFuwai HospitalNational Center for Cardiovascular DiseaseChinese Academy of Medical Sciences and Peking Union Medical CollegeBeijingChina
| | - Yu Nie
- State Key Laboratory of Cardiovascular DiseaseFuwai HospitalNational Center for Cardiovascular DiseaseChinese Academy of Medical Sciences and Peking Union Medical CollegeBeijingChina
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Morita M, Toida A, Horiuchi Y, Watanabe S, Sasahara M, Kawaguchi K, So T, Imanaka T. Generation of an immortalized astrocytic cell line from Abcd1-deficient H-2K btsA58 mice to facilitate the study of the role of astrocytes in X-linked adrenoleukodystrophy. Heliyon 2021; 7:e06228. [PMID: 33659749 PMCID: PMC7892932 DOI: 10.1016/j.heliyon.2021.e06228] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/19/2020] [Revised: 11/16/2020] [Accepted: 02/04/2021] [Indexed: 12/27/2022] Open
Abstract
X-linked adrenoleukodystrophy (X-ALD) is an inherited metabolic disease characterized by inflammatory demyelination, and activated astrocytes as well as microglia are thought to be involved in its pathogenesis. Conditionally immortalized astrocytic cell clones were prepared from wild-type or Abcd1-deficient H-2KbtsA58 transgenic mice to study the involvement of astrocytes in the pathogenesis of X-ALD. The established astrocyte clones expressed astrocyte-specific molecules such as Vimentin, S100β, Aldh1L1 and Glast. The conditionally immortalized astrocytes proliferated vigorously and exhibited a compact cell body under a permissive condition at 33 °C in the presence of IFN-γ, whereas they became quiescent and exhibited substantial cell enlargement under a non-permissive condition at 37 °C in the absence of IFN-γ. An Abcd1-deficient astrocyte clone exhibited a decrease in the β-oxidation of very long chain fatty acid (VLCFA) and an increase in cellular levels of VLCFA, typical features of Abcd1-deficiency. Upon stimulation with LPS, the Abcd1-deficient astrocyte clone expressed higher levels of pro-inflammatory genes, such as Il6, Nos2, Ccl2 and Cxcl10, compared to wild-type (WT) astrocytes. Furthermore, the Abcd1-deficient astrocytes produced higher amounts of chondroitin sulfate, a marker of reactive astrocytes. These results suggest that dysfunction of Abcd1 renders astrocytes highly responsive to innate immune stimuli. Conditionally immortalized cell clones which preserve astrocyte properties are a useful tool for analyzing the cellular and molecular pathology of ALD.
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Affiliation(s)
- Masashi Morita
- Department of Biological Chemistry, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama, 930-0194, Japan
| | - Ai Toida
- Department of Biological Chemistry, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama, 930-0194, Japan
| | - Yuki Horiuchi
- Department of Biological Chemistry, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama, 930-0194, Japan
| | - Shiro Watanabe
- Division of Nutritional Biochemistry, Institute of Natural Medicine, University of Toyama, Toyama, 930-0194, Japan
| | - Masakiyo Sasahara
- Department of Pathology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama, 930-0194, Japan
| | - Kosuke Kawaguchi
- Department of Biological Chemistry, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama, 930-0194, Japan
| | - Takanori So
- Department of Biological Chemistry, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama, 930-0194, Japan
| | - Tsuneo Imanaka
- Faculty of Pharmaceutical Sciences, Hiroshima International University, Kure, Hiroshima, 737-0112, Japan
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CD112 Regulates Angiogenesis and T Cell Entry into the Spleen. Cells 2021; 10:cells10010169. [PMID: 33467729 PMCID: PMC7830896 DOI: 10.3390/cells10010169] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/04/2020] [Revised: 01/08/2021] [Accepted: 01/11/2021] [Indexed: 12/19/2022] Open
Abstract
Junctional adhesion proteins play important roles in controlling angiogenesis, vascular permeability and leukocyte trafficking. CD112 (nectin-2) belongs to the immunoglobulin superfamily and was shown to engage in homophilic and heterophilic interactions with a variety of binding partners expressed on endothelial cells and on leukocytes. Recent in vitro studies suggested that CD112 regulates human endothelial cell migration and proliferation as well as transendothelial migration of leukocytes. However, so far, the role of CD112 in endothelial cell biology and in leukocyte trafficking has not been elucidated in vivo. We found CD112 to be expressed by lymphatic and blood endothelial cells in different murine tissues. In CD112-deficient mice, the blood vessel coverage in the retina and spleen was significantly enhanced. In functional in vitro studies, a blockade of CD112 modulated endothelial cell migration and significantly enhanced endothelial tube formation. An antibody-based blockade of CD112 also significantly reduced T cell transmigration across endothelial monolayers in vitro. Moreover, T cell homing to the spleen was significantly reduced in CD112-deficient mice. Overall, our results identify CD112 as a regulator of angiogenic processes in vivo and demonstrate a novel role for CD112 in T cell entry into the spleen.
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Agarwal S, Sudhini YR, Reiser J, Altintas MM. From Infancy to Fancy: A Glimpse into the Evolutionary Journey of Podocytes in Culture. KIDNEY360 2020; 2:385-397. [PMID: 35373019 PMCID: PMC8740988 DOI: 10.34067/kid.0006492020] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 10/30/2020] [Accepted: 12/22/2020] [Indexed: 02/04/2023]
Abstract
Podocytes are critical components of the filtration barrier and responsible for maintaining healthy kidney function. An assault on podocytes is generally associated with progression of chronic glomerular diseases. Therefore, podocyte pathophysiology is a favorite research subject for nephrologists. Despite this, podocyte research has lagged because of the unavailability of techniques for culturing such specialized cells ex vivo in quantities that are adequate for mechanistic studies. In recent years, this problem was circumvented by the efforts of researchers, who successfully developed several in vitro podocyte cell culture model systems that paved the way for incredible discoveries in the field of nephrology. This review sets us on a journey that provides a comprehensive insight into the groundbreaking breakthroughs and novel technologic advances made in the field of podocyte cell culture so far, beginning from its inception, evolution, and progression. In this study, we also describe in detail the pros and cons of different models that are being used to culture podocytes. Our extensive and exhaustive deliberation on the status of podocyte cell culture will facilitate researchers to choose wisely an appropriate model for their own research to avoid potential pitfalls in the future.
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Kim JJ, David JM, Wilbon SS, Santos JV, Patel DM, Ahmad A, Mitrofanova A, Liu X, Mallela SK, Ducasa GM, Ge M, Sloan AJ, Al-Ali H, Boulina M, Mendez AJ, Contreras GN, Prunotto M, Sohail A, Fridman R, Miner JH, Merscher S, Fornoni A. Discoidin domain receptor 1 activation links extracellular matrix to podocyte lipotoxicity in Alport syndrome. EBioMedicine 2020; 63:103162. [PMID: 33340991 PMCID: PMC7750578 DOI: 10.1016/j.ebiom.2020.103162] [Citation(s) in RCA: 35] [Impact Index Per Article: 7.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/15/2020] [Revised: 11/21/2020] [Accepted: 11/24/2020] [Indexed: 12/11/2022] Open
Abstract
Background Discoidin domain receptor 1 (DDR1) is a receptor tyrosine kinase that is activated by collagens that is involved in the pathogenesis of fibrotic disorders. Interestingly, de novo production of the collagen type I (Col I) has been observed in Col4a3 knockout mice, a mouse model of Alport Syndrome (AS mice). Deletion of the DDR1 in AS mice was shown to improve survival and renal function. However, the mechanisms driving DDR1-dependent fibrosis remain largely unknown. Methods Podocyte pDDR1 levels, Collagen and cluster of differentiation 36 (CD36) expression was analyzed by Real-time PCR and Western blot. Lipid droplet accumulation and content was determined using Bodipy staining and enzymatic analysis. CD36 and DDR1 interaction was determined by co-immunoprecipitation. Creatinine, BUN, albuminuria, lipid content, and histological and morphological assessment of kidneys harvested from AS mice treated with Ezetimibe and/or Ramipril or vehicle was performed. Findings We demonstrate that Col I-mediated DDR1 activation induces CD36-mediated podocyte lipotoxic injury. We show that Ezetimibe interferes with the CD36/DDR1 interaction in vitro and prevents lipotoxicity in AS mice thus preserving renal function similarly to ramipril. Interpretation Our study suggests that Col I/DDR1-mediated lipotoxicity contributes to renal failure in AS and that targeting this pathway may represent a new therapeutic strategy for patients with AS and with chronic kidney diseases (CKD) associated with Col4 mutations. Funding This study is supported by the NIH grants R01DK117599, R01DK104753, R01CA227493, U54DK083912, UM1DK100846, U01DK116101, UL1TR000460 (Miami Clinical Translational Science Institute, National Center for Advancing Translational Sciences and the National Institute on Minority Health and Health Disparities), F32DK115109, Hoffmann-La Roche and Alport Syndrome Foundation.
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Affiliation(s)
- Jin-Ju Kim
- Katz Family Division of Nephrology and Hypertension, Drug Discovery center, Department of Medicine, University of Miami Miller School of Medicine, 1580 NW 10th Ave, Miami, FL 33136, United States.
| | - Judith M David
- Katz Family Division of Nephrology and Hypertension, Drug Discovery center, Department of Medicine, University of Miami Miller School of Medicine, 1580 NW 10th Ave, Miami, FL 33136, United States
| | - Sydney S Wilbon
- Katz Family Division of Nephrology and Hypertension, Drug Discovery center, Department of Medicine, University of Miami Miller School of Medicine, 1580 NW 10th Ave, Miami, FL 33136, United States
| | - Javier V Santos
- Katz Family Division of Nephrology and Hypertension, Drug Discovery center, Department of Medicine, University of Miami Miller School of Medicine, 1580 NW 10th Ave, Miami, FL 33136, United States
| | - Devang M Patel
- Department of Diabetes, Central Clinical School, Monash University, Melbourne, Australia
| | - Anis Ahmad
- Department of Radiation Oncology, University of Miami, FL 33136, United States
| | - Alla Mitrofanova
- Katz Family Division of Nephrology and Hypertension, Drug Discovery center, Department of Medicine, University of Miami Miller School of Medicine, 1580 NW 10th Ave, Miami, FL 33136, United States; Department of Surgery, University of Miami Miller School of Medicine, Miami, FL 33136, United States
| | - Xiaochen Liu
- Katz Family Division of Nephrology and Hypertension, Drug Discovery center, Department of Medicine, University of Miami Miller School of Medicine, 1580 NW 10th Ave, Miami, FL 33136, United States
| | - Shamroop K Mallela
- Katz Family Division of Nephrology and Hypertension, Drug Discovery center, Department of Medicine, University of Miami Miller School of Medicine, 1580 NW 10th Ave, Miami, FL 33136, United States
| | - Gloria M Ducasa
- Katz Family Division of Nephrology and Hypertension, Drug Discovery center, Department of Medicine, University of Miami Miller School of Medicine, 1580 NW 10th Ave, Miami, FL 33136, United States
| | - Mengyuan Ge
- Katz Family Division of Nephrology and Hypertension, Drug Discovery center, Department of Medicine, University of Miami Miller School of Medicine, 1580 NW 10th Ave, Miami, FL 33136, United States
| | - Alexis J Sloan
- Katz Family Division of Nephrology and Hypertension, Drug Discovery center, Department of Medicine, University of Miami Miller School of Medicine, 1580 NW 10th Ave, Miami, FL 33136, United States
| | - Hassan Al-Ali
- Katz Family Division of Nephrology and Hypertension, Drug Discovery center, Department of Medicine, University of Miami Miller School of Medicine, 1580 NW 10th Ave, Miami, FL 33136, United States
| | - Marcia Boulina
- Diabetes Research Institute, University of Miami Miller School of Medicine, Miami, FL 33136, United States
| | - Armando J Mendez
- Diabetes Research Institute, University of Miami Miller School of Medicine, Miami, FL 33136, United States
| | - Gabriel N Contreras
- Katz Family Division of Nephrology and Hypertension, Drug Discovery center, Department of Medicine, University of Miami Miller School of Medicine, 1580 NW 10th Ave, Miami, FL 33136, United States
| | - Marco Prunotto
- Roche Pharma Research and Early Development, Roche Innovation Center, Basel, Switzerland; School of Pharmaceutical Sciences, University of Geneva, Geneva, Switzerland
| | - Anjum Sohail
- Department of Pathology, Wayne State University School of Medicine, Detroit, Michigan 48201, United States
| | - Rafael Fridman
- Department of Pathology, Wayne State University School of Medicine, Detroit, Michigan 48201, United States
| | - Jeffrey H Miner
- Division of Nephrology, Department of Medicine, Washington University School of Medicine, St. Louis, Missouri 63110, United States
| | - Sandra Merscher
- Katz Family Division of Nephrology and Hypertension, Drug Discovery center, Department of Medicine, University of Miami Miller School of Medicine, 1580 NW 10th Ave, Miami, FL 33136, United States
| | - Alessia Fornoni
- Katz Family Division of Nephrology and Hypertension, Drug Discovery center, Department of Medicine, University of Miami Miller School of Medicine, 1580 NW 10th Ave, Miami, FL 33136, United States.
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The nSMase2/Smpd3 gene modulates the severity of muscular dystrophy and the emotional stress response in mdx mice. BMC Med 2020; 18:343. [PMID: 33208172 PMCID: PMC7677854 DOI: 10.1186/s12916-020-01805-5] [Citation(s) in RCA: 14] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/28/2020] [Accepted: 10/01/2020] [Indexed: 01/07/2023] Open
Abstract
BACKGROUND Duchenne muscular dystrophy (DMD) is a progressive, degenerative muscular disorder and cognitive dysfunction caused by mutations in the dystrophin gene. It is characterized by excess inflammatory responses in the muscle and repeated degeneration and regeneration cycles. Neutral sphingomyelinase 2/sphingomyelin phosphodiesterase 3 (nSMase2/Smpd3) hydrolyzes sphingomyelin in lipid rafts. This protein thus modulates inflammatory responses, cell survival or apoptosis pathways, and the secretion of extracellular vesicles in a Ca2+-dependent manner. However, its roles in dystrophic pathology have not yet been clarified. METHODS To investigate the effects of the loss of nSMase2/Smpd3 on dystrophic muscles and its role in the abnormal behavior observed in DMD patients, we generated mdx mice lacking the nSMase2/Smpd3 gene (mdx:Smpd3 double knockout [DKO] mice). RESULTS Young mdx:Smpd3 DKO mice exhibited reduced muscular degeneration and decreased inflammation responses, but later on they showed exacerbated muscular necrosis. In addition, the abnormal stress response displayed by mdx mice was improved in the mdx:Smpd3 DKO mice, with the recovery of brain-derived neurotrophic factor (Bdnf) expression in the hippocampus. CONCLUSIONS nSMase2/Smpd3-modulated lipid raft integrity is a potential therapeutic target for DMD.
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Senthilkumar S, Venugopal C, Parveen S, K S, Rai KS, Kutty BM, Dhanushkodi A. Remarkable migration propensity of dental pulp stem cells towards neurodegenerative milieu: An in vitro analysis. Neurotoxicology 2020; 81:89-100. [PMID: 32905802 DOI: 10.1016/j.neuro.2020.08.006] [Citation(s) in RCA: 16] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/29/2020] [Revised: 07/25/2020] [Accepted: 08/30/2020] [Indexed: 02/07/2023]
Abstract
Stem cell therapy provides a ray of hope for treating neurodegenerative diseases (ND). Bone marrow mesenchymal stem cells (BM-MSC) were extensively investigated for their role in neuroregeneration. However, drawbacks like painful bone marrow extraction, less proliferation and poor CNS engraftment following systemic injections of BM-MSC prompt us to search for alternate/appropriate source of MSC for treating ND. In this context, dental pulp stem cells (DPSC) could be an alternative to BM-MSC as it possess both mesenchymal and neural characteristic features due to its origin from ectoderm, ease of isolation, higher proliferation index and better neuroprotection. A study on the migration potential of DPSC compared to BM-MSC in a neurodegenerative condition is warranted. Given the neural crest origin, we hypothesize that DPSC possess better migration towards neurodegenerative milieu as compared to BM-MSC. In this prospect, we investigated the migration potential of DPSC in an in vitro neurodegenerative condition. Towards this, transwell, Matrigel and chorioallantoic membrane (CAM) migration assays were carried-out by seeding hippocampal neurons in the lower chamber and treated with 300 μM kainic acid (KA) for 6 h to induce neurodegeneration. Subsequently, the upper chamber of transwell was loaded with DPSC/BM-MSC and their migration potential was assessed following 24 h of incubation. Our results revealed that the migration potential of DPSC/BM-MSC was comparable in non-degenerative condition. However, following injury the migration potential of DPSC towards the degenerating site was significantly higher as compared to BM-MSC. Furthermore, upon exposure of naïve DPSC/BM-MSCs to culture medium derived from neurodegenerative milieu resulted in significant upregulation of homing factors like SDF-1alpha, CXCR-4, VCAM-1, VLA-4, CD44, MMP-2 suggesting that the superior migration potential of DPSC might be due to prompt expression of homing factors in DPSC compared to BM-MSCs.
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Affiliation(s)
- Sivapriya Senthilkumar
- Manipal Institute of Regenerative Medicine, Manipal Academy of Higher Education, Bangalore, Karnataka, India
| | - Chaitra Venugopal
- Manipal Institute of Regenerative Medicine, Manipal Academy of Higher Education, Bangalore, Karnataka, India
| | - Shagufta Parveen
- Manipal Institute of Regenerative Medicine, Manipal Academy of Higher Education, Bangalore, Karnataka, India
| | - Shobha K
- Manipal Institute of Regenerative Medicine, Manipal Academy of Higher Education, Bangalore, Karnataka, India
| | - Kiranmai S Rai
- Dept. of Physiology, Melaka Manipal Medical College, Manipal Academy of Higher Education, Manipal, Karnataka, India
| | - Bindu M Kutty
- Department of Neurophysiology, National Institute of Mental Health and Neurosciences, Bangalore, Karnataka, India
| | - Anandh Dhanushkodi
- Manipal Institute of Regenerative Medicine, Manipal Academy of Higher Education, Bangalore, Karnataka, India.
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Vallecillo-Zúniga ML, Rathgeber MF, Poulson PD, Hayes S, Luddington JS, Gill HN, Teynor M, Kartchner BC, Valdoz J, Stowell C, Markham AR, Arthur C, Stowell S, Van Ry PM. Treatment with galectin-1 improves myogenic potential and membrane repair in dysferlin-deficient models. PLoS One 2020; 15:e0238441. [PMID: 32881965 PMCID: PMC7470338 DOI: 10.1371/journal.pone.0238441] [Citation(s) in RCA: 14] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/25/2020] [Accepted: 08/17/2020] [Indexed: 11/18/2022] Open
Abstract
Limb-girdle muscular dystrophy type 2B (LGMD2B) is caused by mutations in the dysferlin gene, resulting in non-functional dysferlin, a key protein found in muscle membrane. Treatment options available for patients are chiefly palliative in nature and focus on maintaining ambulation. Our hypothesis is that galectin-1 (Gal-1), a soluble carbohydrate binding protein, increases membrane repair capacity and myogenic potential of dysferlin-deficient muscle cells and muscle fibers. To test this hypothesis, we used recombinant human galectin-1 (rHsGal-1) to treat dysferlin-deficient models. We show that rHsGal-1 treatments of 48 h-72 h promotes myogenic maturation as indicated through improvements in size, myotube alignment, myoblast migration, and membrane repair capacity in dysferlin-deficient myotubes and myofibers. Furthermore, increased membrane repair capacity of dysferlin-deficient myotubes, independent of increased myogenic maturation is apparent and co-localizes on the membrane of myotubes after a brief 10min treatment with labeled rHsGal-1. We show the carbohydrate recognition domain of Gal-1 is necessary for observed membrane repair. Improvements in membrane repair after only a 10 min rHsGal-1treatment suggest mechanical stabilization of the membrane due to interaction with glycosylated membrane bound, ECM or yet to be identified ligands through the CDR domain of Gal-1. rHsGal-1 shows calcium-independent membrane repair in dysferlin-deficient and wild-type myotubes and myofibers. Together our novel results reveal Gal-1 mediates disease pathologies through both changes in integral myogenic protein expression and mechanical membrane stabilization.
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Affiliation(s)
- Mary L. Vallecillo-Zúniga
- Department of Chemistry & Biochemistry, Brigham Young University, Provo, UT, United States of America
| | - Matthew F. Rathgeber
- Department of Chemistry & Biochemistry, Brigham Young University, Provo, UT, United States of America
| | - P. Daniel Poulson
- Department of Chemistry & Biochemistry, Brigham Young University, Provo, UT, United States of America
| | - Spencer Hayes
- Department of Chemistry & Biochemistry, Brigham Young University, Provo, UT, United States of America
| | - Jacob S. Luddington
- Department of Chemistry & Biochemistry, Brigham Young University, Provo, UT, United States of America
| | - Hailie N. Gill
- Department of Chemistry & Biochemistry, Brigham Young University, Provo, UT, United States of America
| | - Matthew Teynor
- Department of Chemistry & Biochemistry, Brigham Young University, Provo, UT, United States of America
| | - Braden C. Kartchner
- Department of Chemistry & Biochemistry, Brigham Young University, Provo, UT, United States of America
| | - Jonard Valdoz
- Department of Chemistry & Biochemistry, Brigham Young University, Provo, UT, United States of America
| | - Caleb Stowell
- Department of Chemistry & Biochemistry, Brigham Young University, Provo, UT, United States of America
| | - Ashley R. Markham
- Department of Chemistry & Biochemistry, Brigham Young University, Provo, UT, United States of America
| | - Connie Arthur
- Center for Apheresis, Emory Hospital, Laboratory and Blood Bank, Emory Orthopaedics and Spine Hospital, Center for Transfusion and Cellular Therapies, School of Medicine, Emory University, Atlanta, GA, United States of America
| | - Sean Stowell
- Center for Apheresis, Emory Hospital, Laboratory and Blood Bank, Emory Orthopaedics and Spine Hospital, Center for Transfusion and Cellular Therapies, School of Medicine, Emory University, Atlanta, GA, United States of America
| | - Pam M. Van Ry
- Department of Chemistry & Biochemistry, Brigham Young University, Provo, UT, United States of America
- * E-mail:
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Li S, Liu Y, He Y, Rong W, Zhang M, Li L, Liu Z, Zen K. Podocytes present antigen to activate specific T cell immune responses in inflammatory renal disease. J Pathol 2020; 252:165-177. [PMID: 32686090 DOI: 10.1002/path.5508] [Citation(s) in RCA: 26] [Impact Index Per Article: 5.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/31/2020] [Revised: 06/21/2020] [Accepted: 07/07/2020] [Indexed: 12/15/2022]
Abstract
Infiltration of activated T cells into renal tissue plays an essential role in inflammatory nephropathy. However, the mechanism enabling the renal recruitment and activation of T cells remains elusive. Here we report that inflammatory cytokine-promoted antigen presentation by podocytes is a key for recruiting and activating specific T cells. Our results showed that diabetes-associated inflammatory cytokines IFNγ and IL-17 all upregulated expression of MHC-I, MHC-II, CD80 and CD86 on the podocyte surface. Both IFNγ and IL-17 stimulated the uptake and processing of ovalbumin (OVA) by mouse podocytes, resulting in presentation of OVA antigen peptide on the cell surface. OVA antigen presentation by podocytes was also validated using human podocytes. Furthermore, OVA antigen-presenting mouse podocytes were able to activate OT-I mouse T cell proliferation and inflammatory cytokine secretion, which in turn caused podocyte injury and apoptosis. Finally, OT-I mice subjected to direct renal injection of OVA plus IFNγ/IL-17 but not OVA alone exhibited OVA antigen presentation by podocytes and developed nephropathy in 4 weeks. In conclusion, antigen presentation by podocytes under inflammatory conditions plays an important role in activating T cell immune responses and facilitating immune-mediated glomerular disease development. © 2020 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
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Affiliation(s)
- Shan Li
- National Clinical Research Center of Kidney Diseases, Jinling Hospital, Nanjing University School of Medicine, Nanjing, PR China.,State Key Laboratory of Pharmaceutical Biotechnology, Jiangsu Engineering Research Center for MicroRNA Biology and Biotechnology, Nanjing University School of Life Sciences, Nanjing, PR China
| | - Ying Liu
- National Clinical Research Center of Kidney Diseases, Jinling Hospital, Nanjing University School of Medicine, Nanjing, PR China.,State Key Laboratory of Pharmaceutical Biotechnology, Jiangsu Engineering Research Center for MicroRNA Biology and Biotechnology, Nanjing University School of Life Sciences, Nanjing, PR China
| | - Yueqin He
- National Clinical Research Center of Kidney Diseases, Jinling Hospital, Nanjing University School of Medicine, Nanjing, PR China.,State Key Laboratory of Pharmaceutical Biotechnology, Jiangsu Engineering Research Center for MicroRNA Biology and Biotechnology, Nanjing University School of Life Sciences, Nanjing, PR China
| | - Weiwei Rong
- National Clinical Research Center of Kidney Diseases, Jinling Hospital, Nanjing University School of Medicine, Nanjing, PR China.,State Key Laboratory of Pharmaceutical Biotechnology, Jiangsu Engineering Research Center for MicroRNA Biology and Biotechnology, Nanjing University School of Life Sciences, Nanjing, PR China
| | - Mingchao Zhang
- National Clinical Research Center of Kidney Diseases, Jinling Hospital, Nanjing University School of Medicine, Nanjing, PR China
| | - Limin Li
- National Clinical Research Center of Kidney Diseases, Jinling Hospital, Nanjing University School of Medicine, Nanjing, PR China.,State Key Laboratory of Pharmaceutical Biotechnology, Jiangsu Engineering Research Center for MicroRNA Biology and Biotechnology, Nanjing University School of Life Sciences, Nanjing, PR China
| | - Zhihong Liu
- National Clinical Research Center of Kidney Diseases, Jinling Hospital, Nanjing University School of Medicine, Nanjing, PR China
| | - Ke Zen
- National Clinical Research Center of Kidney Diseases, Jinling Hospital, Nanjing University School of Medicine, Nanjing, PR China.,State Key Laboratory of Pharmaceutical Biotechnology, Jiangsu Engineering Research Center for MicroRNA Biology and Biotechnology, Nanjing University School of Life Sciences, Nanjing, PR China
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Vaishnavi A, Scherzer MT, Kinsey CG, Parkman GL, Truong A, Ghazi P, Schuman S, Battistone B, Garrido-Laguna I, McMahon M. Inhibition of MEK1/2 Forestalls the Onset of Acquired Resistance to Entrectinib in Multiple Models of NTRK1-Driven Cancer. Cell Rep 2020; 32:107994. [PMID: 32755586 PMCID: PMC7478141 DOI: 10.1016/j.celrep.2020.107994] [Citation(s) in RCA: 18] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/05/2019] [Revised: 05/11/2020] [Accepted: 07/15/2020] [Indexed: 12/15/2022] Open
Abstract
NTRK1 gene fusions are actionable drivers of numerous human malignancies. Here, we show that expression of the TPR-NTRK1 fusion kinase in immortalized mouse pancreatic ductal epithelial (IMPE) (pancreas) or mouse lung epithelial (MLE-12) cells is sufficient to promote rapidly growing tumors in mice. Both tumor models are exquisitely sensitive to targeted inhibition with entrectinib, a tropomyosin-related kinase A (TRKA) inhibitor. Initial regression of NTRK1-driven tumors is driven by induced expression of BIM, such that BIM silencing leads to a diminished response to entrectinib in vivo. However, the emergence of drug-resistant disease limits the long-term durability of responses. Based on the reactivation of RAF>MEK>ERK signaling observed in entrectinib-treated tumors, we show that the combination of entrectinib plus the MEK1/2 inhibitor cobimetinib dramatically forestalls the onset of drug resistance in vivo. Collectively, these data provide a mechanistic rationale for rapid clinical deployment of combined inhibition of TRKA plus MEK1/2 in NTRK1-driven cancers.
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Affiliation(s)
- Aria Vaishnavi
- Huntsman Cancer Institute, University of Utah, Salt Lake City, UT 84112, USA
| | - Michael T Scherzer
- Huntsman Cancer Institute, University of Utah, Salt Lake City, UT 84112, USA; Department of Oncological Sciences, University of Utah, Salt Lake City, UT 84112, USA
| | - Conan G Kinsey
- Huntsman Cancer Institute, University of Utah, Salt Lake City, UT 84112, USA; Department of Internal Medicine, Division of Oncology, University of Utah, Salt Lake City, UT 84112, USA
| | - Gennie L Parkman
- Huntsman Cancer Institute, University of Utah, Salt Lake City, UT 84112, USA; Department of Oncological Sciences, University of Utah, Salt Lake City, UT 84112, USA
| | - Amanda Truong
- Huntsman Cancer Institute, University of Utah, Salt Lake City, UT 84112, USA; Department of Oncological Sciences, University of Utah, Salt Lake City, UT 84112, USA
| | - Phaedra Ghazi
- Huntsman Cancer Institute, University of Utah, Salt Lake City, UT 84112, USA; Department of Oncological Sciences, University of Utah, Salt Lake City, UT 84112, USA
| | - Sophia Schuman
- Huntsman Cancer Institute, University of Utah, Salt Lake City, UT 84112, USA
| | - Benjamin Battistone
- Huntsman Cancer Institute, University of Utah, Salt Lake City, UT 84112, USA
| | - Ignacio Garrido-Laguna
- Huntsman Cancer Institute, University of Utah, Salt Lake City, UT 84112, USA; Department of Internal Medicine, Division of Oncology, University of Utah, Salt Lake City, UT 84112, USA
| | - Martin McMahon
- Huntsman Cancer Institute, University of Utah, Salt Lake City, UT 84112, USA; Department of Oncological Sciences, University of Utah, Salt Lake City, UT 84112, USA; Department of Dermatology, University of Utah, Salt Lake City, UT 84112, USA.
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46
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Parray HA, Shukla S, Samal S, Shrivastava T, Ahmed S, Sharma C, Kumar R. Hybridoma technology a versatile method for isolation of monoclonal antibodies, its applicability across species, limitations, advancement and future perspectives. Int Immunopharmacol 2020; 85:106639. [PMID: 32473573 PMCID: PMC7255167 DOI: 10.1016/j.intimp.2020.106639] [Citation(s) in RCA: 124] [Impact Index Per Article: 24.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/11/2020] [Revised: 05/06/2020] [Accepted: 05/22/2020] [Indexed: 02/06/2023]
Abstract
The advancements in technology and manufacturing processes have allowed the development of new derivatives, biosimilar or advanced improved versions for approved antibodies each year for treatment regimen. There are more than 700 antibody-based molecules that are in different stages of phase I/II/ III clinical trials targeting new unique targets. To date, approximately more than 80 monoclonal antibodies (mAbs) have been approved. A total of 7 novel antibody therapeutics had been granted the first approval either in the United States or European Union in the year 2019, representing approximately 20% of the total number of approved drugs. Most of these licenced mAbs or their derivatives are either of hybridoma origin or their improvised engineered versions. Even with the recent development of high throughput mAb generation technologies, hybridoma is the most favoured method due to its indigenous nature to preserve natural cognate antibody pairing information and preserves innate functions of immune cells. The recent advent of antibody engineering technology has superseded the species level barriers and has shown success in isolation of hybridoma across phylogenetically distinct species. This has led to the isolation of monoclonal antibodies against human targets that are conserved and non-immunogenic in the rodent. In this review, we have discussed in detail about hybridoma technology, its expansion towards different animal species, the importance of antibodies isolated from different animal sources that are useful in biological applications, advantages, and limitations. This review also summarizes the challenges and recent progress associated with hybridoma development, and how it has been overcome in these years to provide new insights for the isolation of mAbs.
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Affiliation(s)
- Hilal Ahmed Parray
- Translational Health Science & Technology Institute, NCR Biotech Science Cluster, Faridabad, Haryana 121001, India
| | - Shivangi Shukla
- Translational Health Science & Technology Institute, NCR Biotech Science Cluster, Faridabad, Haryana 121001, India
| | - Sweety Samal
- Translational Health Science & Technology Institute, NCR Biotech Science Cluster, Faridabad, Haryana 121001, India
| | - Tripti Shrivastava
- Translational Health Science & Technology Institute, NCR Biotech Science Cluster, Faridabad, Haryana 121001, India
| | - Shubbir Ahmed
- Translational Health Science & Technology Institute, NCR Biotech Science Cluster, Faridabad, Haryana 121001, India
| | - Chandresh Sharma
- Translational Health Science & Technology Institute, NCR Biotech Science Cluster, Faridabad, Haryana 121001, India.
| | - Rajesh Kumar
- Translational Health Science & Technology Institute, NCR Biotech Science Cluster, Faridabad, Haryana 121001, India.
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Bossone KA, Ellis JA, Holaska JM. Histone acetyltransferase inhibition rescues differentiation of emerin-deficient myogenic progenitors. Muscle Nerve 2020; 62:128-136. [PMID: 32304242 DOI: 10.1002/mus.26892] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/23/2019] [Revised: 03/05/2020] [Accepted: 04/07/2020] [Indexed: 12/20/2022]
Abstract
INTRODUCTION Emery-Dreifuss muscular dystrophy (EDMD) is a disease characterized by skeletal muscle wasting, major tendon contractures, and cardiac conduction defects. Mutations in the gene encoding emerin cause EDMD1. Our previous studies suggested that emerin activation of histone deacetylase 3 (HDAC3) to reduce histone 4-lysine 5 (H4K5) acetylation (ac) is important for myogenic differentiation. METHODS Pharmacological inhibitors (Nu9056, L002) of histone acetyltransferases targeting acetylated H4K5 were used to test whether increased acetylated H4K5 was responsible for the impaired differentiation seen in emerin-deficient myogenic progenitors. RESULTS Nu9056 and L002 rescued impaired differentiation in emerin deficiency. SRT1720, which inhibits the nicotinamide adenine dinucleotide (NAD)+ -dependent deacetylase sirtuin 1 (SIRT1), failed to rescue myotube formation. DISCUSSION We conclude that emerin regulation of HDAC3 activity to affect H4K5 acetylation dynamics is important for myogenic differentiation. Targeting H4K5ac dynamics represents a potential new strategy for ameliorating the skeletal muscle wasting seen in EDMD1.
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Affiliation(s)
- Katherine A Bossone
- Department of Biomedical Sciences, Cooper Medical School of Rowan University, Camden, New Jersey, United States.,Department of Pharmaceutical Sciences, University of the Sciences, Philadelphia, Pennsylvania, United States
| | - Joseph A Ellis
- Department of Pharmaceutical Sciences, University of the Sciences, Philadelphia, Pennsylvania, United States
| | - James M Holaska
- Department of Biomedical Sciences, Cooper Medical School of Rowan University, Camden, New Jersey, United States.,Department of Pharmaceutical Sciences, University of the Sciences, Philadelphia, Pennsylvania, United States
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van Gorp PRR, Trines SA, Pijnappels DA, de Vries AAF. Multicellular In vitro Models of Cardiac Arrhythmias: Focus on Atrial Fibrillation. Front Cardiovasc Med 2020; 7:43. [PMID: 32296716 PMCID: PMC7138102 DOI: 10.3389/fcvm.2020.00043] [Citation(s) in RCA: 22] [Impact Index Per Article: 4.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/23/2019] [Accepted: 03/06/2020] [Indexed: 12/13/2022] Open
Abstract
Atrial fibrillation (AF) is the most common cardiac arrhythmia in clinical practice with a large socioeconomic impact due to its associated morbidity, mortality, reduction in quality of life and health care costs. Currently, antiarrhythmic drug therapy is the first line of treatment for most symptomatic AF patients, despite its limited efficacy, the risk of inducing potentially life-threating ventricular tachyarrhythmias as well as other side effects. Alternative, in-hospital treatment modalities consisting of electrical cardioversion and invasive catheter ablation improve patients' symptoms, but often have to be repeated and are still associated with serious complications and only suitable for specific subgroups of AF patients. The development and progression of AF generally results from the interplay of multiple disease pathways and is accompanied by structural and functional (e.g., electrical) tissue remodeling. Rational development of novel treatment modalities for AF, with its many different etiologies, requires a comprehensive insight into the complex pathophysiological mechanisms. Monolayers of atrial cells represent a simplified surrogate of atrial tissue well-suited to investigate atrial arrhythmia mechanisms, since they can easily be used in a standardized, systematic and controllable manner to study the role of specific pathways and processes in the genesis, perpetuation and termination of atrial arrhythmias. In this review, we provide an overview of the currently available two- and three-dimensional multicellular in vitro systems for investigating the initiation, maintenance and termination of atrial arrhythmias and AF. This encompasses cultures of primary (animal-derived) atrial cardiomyocytes (CMs), pluripotent stem cell-derived atrial-like CMs and (conditionally) immortalized atrial CMs. The strengths and weaknesses of each of these model systems for studying atrial arrhythmias will be discussed as well as their implications for future studies.
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Affiliation(s)
| | | | | | - Antoine A. F. de Vries
- Laboratory of Experimental Cardiology, Department of Cardiology, Leiden University Medical Center, Leiden, Netherlands
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49
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Belizário J, Destro Rodrigues MF. Checkpoint inhibitor blockade and epigenetic reprogrammability in CD8 + T-cell activation and exhaustion. Ther Adv Vaccines Immunother 2020; 8:2515135520904238. [PMID: 32206744 PMCID: PMC7074507 DOI: 10.1177/2515135520904238] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/13/2019] [Accepted: 12/19/2019] [Indexed: 11/17/2022] Open
Abstract
CD8+ T-cell exhaustion is a dysfunctional state that is regulated through the expression of inhibitory checkpoint receptor genes including the cytotoxic T-lymphocyte–associated antigen 4, programmed death 1, and DNA methylation of effector genes interferon-γ, perforin, and granzyme B. Different strategies have been used to reverse T-cell exhaustion, which is an adverse event of checkpoint inhibitor blockade. Here, we present the mechanisms by which DNA methyltransferase inhibitors and Simian virus 40 large T antigen through viral mimicry can promote the reversion of exhausted CD8+ T cells. We examine how these pharmacological strategies can work together to improve the clinical efficacy of immunotherapies.
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Affiliation(s)
- José Belizário
- Department of Pharmacology, Institute Biomedical Sciences of the University of Sao Paulo, Avenida Lineu Prestes, 1524, São Paulo, CEP 05508-900, Brazil
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50
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Dou X, Tong P, Huang H, Zellmer L, He Y, Jia Q, Zhang D, Peng J, Wang C, Xu N, Liao DJ. Evidence for immortality and autonomy in animal cancer models is often not provided, which causes confusion on key issues of cancer biology. J Cancer 2020; 11:2887-2920. [PMID: 32226506 PMCID: PMC7086263 DOI: 10.7150/jca.41324] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/20/2019] [Accepted: 02/08/2020] [Indexed: 11/08/2022] Open
Abstract
Modern research into carcinogenesis has undergone three phases. Surgeons and pathologists started the first phase roughly 250 years ago, establishing morphological traits of tumors for pathologic diagnosis, and setting immortality and autonomy as indispensable criteria for neoplasms. A century ago, medical doctors, biologists and chemists started to enhance "experimental cancer research" by establishing many animal models of chemical-induced carcinogenesis for studies of cellular mechanisms. In this second phase, the two-hit theory and stepwise carcinogenesis of "initiation-promotion" or "initiation-promotion-progression" were established, with an illustrious finding that outgrowths induced in animals depend on the inducers, and thus are not authentically neoplastic, until late stages. The last 40 years are the third incarnation, molecular biologists have gradually dominated the carcinogenesis research fraternity and have established numerous genetically-modified animal models of carcinogenesis. However, evidence has not been provided for immortality and autonomy of the lesions from most of these models. Probably, many lesions had already been collected from animals for analyses of molecular mechanisms of "cancer" before the lesions became autonomous. We herein review the monumental work of many predecessors to reinforce that evidence for immortality and autonomy is essential for confirming a neoplastic nature. We extrapolate that immortality and autonomy are established early during sporadic human carcinogenesis, unlike the late establishment in most animal models. It is imperative to resume many forerunners' work by determining the genetic bases for initiation, promotion and progression, the genetic bases for immortality and autonomy, and which animal models are, in fact, good for identifying such genetic bases.
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Affiliation(s)
- Xixi Dou
- Shandong Provincial Key Laboratory of Transmucosal and Transdermal Drug Delivery, Shandong Freda Pharmaceutical Group Co., Ltd., Jinan 250101, Shandong Province, P.R. China
| | - Pingzhen Tong
- Department of Pathology, The Second Hospital of Guizhou University of Traditional Chinese Medicine, Guiyang 550001, Guizhou Province, P.R. China
| | - Hai Huang
- Center for Clinical Laboratories, The Affiliated Hospital of Guizhou Medical University, Guiyang 550004, Guizhou Province, P.R. China
| | - Lucas Zellmer
- Masonic Cancer Center, University of Minnesota, 435 E. River Road, Minneapolis, MN 55455, USA
| | - Yan He
- Key Lab of Endemic and Ethnic Diseases of The Ministry of Education of China in Guizhou Medical University, Guiyang, Guizhou Province 550004, P. R. China
| | - Qingwen Jia
- Shandong Provincial Key Laboratory of Transmucosal and Transdermal Drug Delivery, Shandong Freda Pharmaceutical Group Co., Ltd., Jinan 250101, Shandong Province, P.R. China
| | - Daizhou Zhang
- Shandong Provincial Key Laboratory of Transmucosal and Transdermal Drug Delivery, Shandong Freda Pharmaceutical Group Co., Ltd., Jinan 250101, Shandong Province, P.R. China
| | - Jiang Peng
- Department of Orthopaedics, Shandong Provincial Hospital Affiliated to Shandong University, Jinan 250021, Shandong Province, P.R. China
| | - Chenguang Wang
- Department of Orthopaedics, Shandong Provincial Hospital Affiliated to Shandong University, Jinan 250021, Shandong Province, P.R. China
| | - Ningzhi Xu
- Tianjin LIPOGEN Gene Technology Ltd., #238 Baidi Road, Nankai District, Tianjin 300192, P.R. China
| | - Dezhong Joshua Liao
- Department of Pathology, The Second Hospital of Guizhou University of Traditional Chinese Medicine, Guiyang 550001, Guizhou Province, P.R. China
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