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Fujihara K, Yoneda T, Sugidono A, Okada Y, Hiyama S, Kajikawa S, Fukunaga Y, Koch M, Izu Y. Collagen XII deficiency promotes ligament-specific heterotopic ossification via fibrochondrocyte differentiation. Biochem Biophys Res Commun 2025; 757:151621. [PMID: 40088675 DOI: 10.1016/j.bbrc.2025.151621] [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: 03/04/2025] [Revised: 03/05/2025] [Accepted: 03/10/2025] [Indexed: 03/17/2025]
Abstract
Heterotopic ossification of tendons and ligaments causes pain and dysfunction, significantly reducing quality of life. However, its underlying mechanisms remain elusive. In addition to injury, tissue organization and stiffness have been implicated in heterotopic ossification. Collagen XII, a member of the fibril-associated collagens with interrupted triple helices (FACIT) family, plays a crucial role in maintaining the structural integrity and function of tendons and ligaments. Its deficiency alters tissue stiffness and predisposes ligaments to rupture. In this study, we investigated whether collagen XII contributes to the development of heterotopic ossification. Three-dimensional microcomputed tomography (3D-μCT) and X-ray analyses revealed heterotopic bone formation in the knee and ankle ligaments, but not in tendons, of Col12a1-deficient mice, with a 100 % incidence in mice older than 19 weeks. Histological analysis showed the presence of Alcian blue- and Toluidine blue-positive fibrochondrocyte-like cells in Col12a1-deficient ligaments, which were subsequently replaced by bone tissue, as indicated by Alizarin red staining. Real-time qPCR analysis of knee ligaments demonstrated a slight increase in chondrogenic markers and a significant upregulation of osteogenic markers in Col12a1-deficient mice compared with wild-type controls. In vitro chondrogenesis and osteogenesis assays using primary tenocytes from wild-type and Col12a1-deficient mice revealed that collagen XII deficiency enhanced osteogenic potential, whereas chondrogenic potential remained comparable. Our findings indicate that collagen XII deficiency specifically induces heterotopic bone formation in knee and ankle ligaments, occurring via fibrochondrocytes rather than through endochondral or intramembranous ossification.
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Affiliation(s)
- Kei Fujihara
- Graduate School of Veterinary Medicine, Nippon Veterinary and Life Science University, 1-7-1 Kyounan, Musashino, Tokyo, 180-8602, Japan; Department of Comparative Cell Biology, Faculty of Veterinary Medicine, Nippon Veterinary and Life Science University, 1-7-1 Kyounan, Musashino, Tokyo, 180-8602, Japan; Department of Laboratory Animal Science, Faculty of Veterinary Medicine, Okayama University of Science, 1-3 Ikoinooka, Imabari, Ehime, 794-8555, Japan
| | - Taiju Yoneda
- Department of Laboratory Animal Science, Faculty of Veterinary Medicine, Okayama University of Science, 1-3 Ikoinooka, Imabari, Ehime, 794-8555, Japan
| | - Akira Sugidono
- Department of Laboratory Animal Science, Faculty of Veterinary Medicine, Okayama University of Science, 1-3 Ikoinooka, Imabari, Ehime, 794-8555, Japan
| | - Yukina Okada
- Department of Animal Risk Management, Faculty of Risk and Crisis Management, Chiba Institute of Science, 15-8 Shiomi, Choshi, Chiba, 288-0025, Japan
| | - Sakura Hiyama
- Department of Animal Risk Management, Faculty of Risk and Crisis Management, Chiba Institute of Science, 15-8 Shiomi, Choshi, Chiba, 288-0025, Japan
| | - Shuhei Kajikawa
- Department of Laboratory Animal Science, Faculty of Veterinary Medicine, Okayama University of Science, 1-3 Ikoinooka, Imabari, Ehime, 794-8555, Japan
| | - Yuko Fukunaga
- Department of Animal Risk Management, Faculty of Risk and Crisis Management, Chiba Institute of Science, 15-8 Shiomi, Choshi, Chiba, 288-0025, Japan
| | - Manuel Koch
- Institute for Dental Research and Oral Musculoskeletal Biology, Faculty of Medicine and University Hospital Cologne, University of Cologne, D-50931, Cologne, Germany
| | - Yayoi Izu
- Graduate School of Veterinary Medicine, Nippon Veterinary and Life Science University, 1-7-1 Kyounan, Musashino, Tokyo, 180-8602, Japan; Department of Comparative Cell Biology, Faculty of Veterinary Medicine, Nippon Veterinary and Life Science University, 1-7-1 Kyounan, Musashino, Tokyo, 180-8602, Japan; Department of Laboratory Animal Science, Faculty of Veterinary Medicine, Okayama University of Science, 1-3 Ikoinooka, Imabari, Ehime, 794-8555, Japan.
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Lu C, Jin A, Liu H, Gao C, Sun W, Zhang Y, Dai Q, Liu Y. Advancing tissue engineering through vascularized cell spheroids: building blocks of the future. Biomater Sci 2025; 13:1901-1922. [PMID: 40067332 DOI: 10.1039/d4bm01206b] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 04/09/2025]
Abstract
Vascularization is a crucial aspect of biofabrication, as the development of vascular networks is essential for tissue survival and the optimization of cellular functions. Spheroids have emerged as versatile units for vascularization, demonstrating significant potential in angiogenesis and prevascularization for tissue engineering and regenerative medicine. However, a major challenge in creating customized vascularized spheroids is the construction of a biomimetic extracellular matrix (ECM) microenvironment. This process requires careful regulation of environmental factors, including the modulation of growth factors, the selection of culture media, and the co-culture of diverse cell types. Recent advancements in biofabrication have expanded the potential applications of vascularized spheroids. The integration of microfluidic technology with bioprinting offers promising solutions to existing challenges in regenerative medicine. Spheroids have been widely studied for their ability to promote vascularization in in vitro models. This review highlights the latest developments in vascularized biofabrication, and systematically explores strategies for constructing vascularized spheroids. We provide a comprehensive analysis of spheroid applications in specific tissues, including skin, liver, bone, cardiac, and tumor models. Finally, the review addresses the major challenges and future directions in the field.
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Affiliation(s)
- Chunxiang Lu
- School of Mechatronic Engineering and Automation, Shanghai University, Shanghai 200444, China.
| | - Aoxiang Jin
- School of Mechatronic Engineering and Automation, Shanghai University, Shanghai 200444, China.
| | - Huazhen Liu
- School of Medicine, Shanghai University, Shanghai 200444, China
| | - Chuang Gao
- School of Mechatronic Engineering and Automation, Shanghai University, Shanghai 200444, China.
| | - Wenbin Sun
- School of Mechatronic Engineering and Automation, Shanghai University, Shanghai 200444, China.
| | - Yi Zhang
- School of Mechatronic Engineering and Automation, Shanghai University, Shanghai 200444, China.
| | - Qiqi Dai
- School of Medicine, Shanghai University, Shanghai 200444, China
| | - Yuanyuan Liu
- School of Mechatronic Engineering and Automation, Shanghai University, Shanghai 200444, China.
- National Center for Translational Medicine (Shanghai) SHU Branch, Shanghai, 200444, China
- Wenzhou Institute of Shanghai University, Wenzhou, 325000, China
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Nguyen A, Heim JB, Cordara G, Chan MC, Johannesen H, Charlesworth C, Li M, Azumaya CM, Madden B, Krengel U, Meves A, Campbell MG. Structural and functional characterization of integrin α5-targeting antibodies for anti-angiogenic therapy. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2025:2025.01.08.631572. [PMID: 39829743 PMCID: PMC11741253 DOI: 10.1101/2025.01.08.631572] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Download PDF] [Subscribe] [Scholar Register] [Indexed: 01/22/2025]
Abstract
Integrins are a large family of heterodimeric receptors important for cell adhesion and signaling. Integrin α5β1, also known as the fibronectin receptor, is a key mediator of angiogenesis and its dysregulation is associated with tumor proliferation, progression, and metastasis. Despite numerous efforts, α5β1-targeting therapeutics have been unsuccessful in large part due to efficacy and off-target effects. To mediate activation and signaling, integrins undergo drastic conformational changes. However, how therapeutics influence or are affected by integrin conformation remains incompletely characterized. Using cell biology, biophysics, and electron microscopy, we shed light on these relationships by characterizing two potentially therapeutic anti-α5β1 antibodies, BIIG2 and MINT1526A. We show that both antibodies bind α5β1 with nanomolar affinity and reduce angiogenesis in vitro. We demonstrate BIIG2 reduces tumor growth in two human xenograft mouse models and exhibits a strong specificity for connective tissue-resident fibroblasts and melanoma cells. Using electron microscopy, we map out the molecular interfaces mediating the integrin-antibody interactions and reveal that although both antibodies have overlapping epitopes and block fibronectin binding via steric hindrance, the effect on the conformational equilibrium is drastically different. While MINT1526A constricts α5β1's range of flexibility, BIIG2 binds without restricting the available conformational states. These mechanistic insights, coupled with the functional analysis, guide which aspects should be prioritized to avoid off-target effects or partial agonism in the design of future integrin-targeted therapeutics.
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Affiliation(s)
- Adam Nguyen
- Basic Sciences Division, Fred Hutchinson Cancer Center, Seattle, Washington 98109, USA
- Biological Physics Structure and Design Program, University of Washington, Seattle, Washington 98195, USA
| | - Joel B. Heim
- Department of Dermatology, Mayo Clinic, Rochester, Minnesota 55905, USA
- Department of Chemistry, University of Oslo, NO-0315 Oslo, Norway
- Current Address: Nykode Therapeutics, Oslo Science Park, 0349 Oslo, Norway
| | - Gabriele Cordara
- Department of Chemistry, University of Oslo, NO-0315 Oslo, Norway
| | - Matthew C. Chan
- Basic Sciences Division, Fred Hutchinson Cancer Center, Seattle, Washington 98109, USA
| | - Hedda Johannesen
- Department of Chemistry, University of Oslo, NO-0315 Oslo, Norway
| | - Cristine Charlesworth
- Medical Genome Facility, Proteomics Core, Mayo Clinic, Rochester, Minnesota 55905, USA
| | - Ming Li
- Department of Dermatology, Mayo Clinic, Rochester, Minnesota 55905, USA
| | - Caleigh M. Azumaya
- Basic Sciences Division, Fred Hutchinson Cancer Center, Seattle, Washington 98109, USA
- Current Address: Genentech, South San Francisco, California 94080, USA
| | - Benjamin Madden
- Medical Genome Facility, Proteomics Core, Mayo Clinic, Rochester, Minnesota 55905, USA
| | - Ute Krengel
- Department of Chemistry, University of Oslo, NO-0315 Oslo, Norway
| | - Alexander Meves
- Department of Dermatology, Mayo Clinic, Rochester, Minnesota 55905, USA
| | - Melody G. Campbell
- Basic Sciences Division, Fred Hutchinson Cancer Center, Seattle, Washington 98109, USA
- Biological Physics Structure and Design Program, University of Washington, Seattle, Washington 98195, USA
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Zhong C, Shi Z, Liu CY, Binzel DW, Jin K, Li X, Guo P, Li SK. Inhibition of Endothelial Cell Tube Formation by Anti-Vascular Endothelial Growth Factor/Anti-Angiopoietin-2 RNA Nanoparticles. Pharmaceutics 2025; 17:55. [PMID: 39861703 PMCID: PMC11769471 DOI: 10.3390/pharmaceutics17010055] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/16/2024] [Revised: 12/13/2024] [Accepted: 12/27/2024] [Indexed: 01/27/2025] Open
Abstract
RNA nanoparticles, derived from the packaging RNA three-way junction motif (pRNA-3WJ) of the bacteriophage phi29 DNA packaging motor, have been demonstrated to be thermodynamically and chemically stable, with promise as a nanodelivery system. Background/Objectives: A previous study showed that RNA nanoparticles with antiangiogenic aptamers (anti-vascular endothelial growth factor (VEGF) and anti-angiopoietin-2 (Ang2) aptamers) inhibited cell proliferation via WST-1 assay. To further investigate the antiangiogenic potential of these RNA nanoparticles, a modified three-dimensional (3D) spheroid sprouting assay model of human umbilical vein endothelial cells was utilized in the present study. Methods: Three groups of RNA nanoparticles were evaluated, namely, pRNA-3WJ series, RNA square series (polygon-type RNA nanoparticles), and 8WJ series (multiple-way junction RNA nanoparticles), which were conjugated with a single anti-VEGF, the combination of one anti-VEGF and one anti-Ang2, or multiple anti-VEGF aptamers. The core scaffold RNA nanoparticles (without aptamers) were used as the references, and bevacizumab was used as the positive control. Results: The results demonstrated the inhibition effects of the RNA nanoparticles on endothelial cell tube formation at 67 nM in a 3D spheroid sprouting model. The results in the 3D spheroid sprouting assay are consistent with those of the WST-1 proliferation assays. Conclusions: Among the RNA nanoparticles evaluated, 3WJ-3VEGF and SQR-VEGF-Ang2 had inhibition effects equivalent to bevacizumab and were promising for anti-angiogenesis treatment.
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Affiliation(s)
- Cheng Zhong
- Division of Pharmaceutical Sciences, James L Winkle College of Pharmacy, University of Cincinnati, Cincinnati, OH 45267, USA
| | - Zhanquan Shi
- Division of Pharmaceutical Sciences, James L Winkle College of Pharmacy, University of Cincinnati, Cincinnati, OH 45267, USA
| | - Chia-Yang Liu
- Department of Ophthalmology, College of Medicine, University of Cincinnati, Cincinnati, OH 45267, USA
| | - Daniel W. Binzel
- Center for RNA Nanobiotechnology and Nanomedicine, College of Pharmacy, James Comprehensive Cancer Center, The Ohio State University, Columbus, OH 43210, USA
| | - Kai Jin
- Center for RNA Nanobiotechnology and Nanomedicine, College of Pharmacy, James Comprehensive Cancer Center, The Ohio State University, Columbus, OH 43210, USA
| | - Xin Li
- Center for RNA Nanobiotechnology and Nanomedicine, College of Pharmacy, James Comprehensive Cancer Center, The Ohio State University, Columbus, OH 43210, USA
| | - Peixuan Guo
- Center for RNA Nanobiotechnology and Nanomedicine, College of Pharmacy, James Comprehensive Cancer Center, The Ohio State University, Columbus, OH 43210, USA
- College of Medicine, The Ohio State University, Columbus, OH 43210, USA
| | - S. Kevin Li
- Division of Pharmaceutical Sciences, James L Winkle College of Pharmacy, University of Cincinnati, Cincinnati, OH 45267, USA
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Ribeiro VL, Dernowsek JA, Fernandes RR, Pitol DL, Issa JPM, Mazzi-Chaves JF, Bombonato-Prado KF, Sousa-Neto MD, Passos GA. Repopulation of a 3D simulated periapical lesion cavity with dental pulp stem cell spheroids with triggered osteoblastic differentiation. Braz Dent J 2024; 35:e235847. [PMID: 39699491 DOI: 10.1590/0103-644020235847] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/24/2024] [Accepted: 09/02/2024] [Indexed: 12/20/2024] Open
Abstract
We established a proof-of-concept model system for the biological healing of periapical lesions using stem cell spheroids. Mesenchymal stem cells from human exfoliated deciduous teeth (SHED) were cultured in a 2D monolayer and then as 3D multicellular spheroids. An image of a periapical lesion of an upper lateral incisor tooth was obtained by computed tomography and was used as a model for photopolymer resin 3D printing to generate a negative frame of the lesion. The negative model served to prepare a positive model of the periapical lesion cavity in an agarose gel. SHED that were cultured in monolayers or as spheroids were seeded in the positive lesion mold before or after osteoblastic differentiation. The results showed that compared to cells cultured in monolayers, spheroids exhibited uniform cellularity and a greater viability within the lesion cavity, which was accompanied by a temporal reduction in the expression of CD13, CD29, CD44, CD73, and CD90 mRNAs that are typically expressed by stem cells. Concomitantly, the expression of markers that characterize osteoblastic differentiation (RUNX2, ALP, and BGLAP) increased. These results provide a new perspective for regenerative endodontics with the use of SHED-derived spheroids to repair periapical lesions.
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Affiliation(s)
- Vítor Luís Ribeiro
- Program in Restorative Dentistry, Department of Restorative Dentistry, School of Dentistry of Ribeirão Preto, University of São Paulo(USP), Ribeirão Preto, SP, Brazil
- Center for Cell-Based Therapy in Dentistry, School of Dentistry of Ribeirão Preto, University of São Paulo(USP), Ribeirão Preto, SP, Brazil
| | - Janaína A Dernowsek
- Biotechnology Center, Energy and Nuclear Research Institute (IPEN), University of São Paulo, São Paulo, SP, Brazil
| | - Roger R Fernandes
- Department of Bucomaxilofacial Surgery, School of Dentistry of Ribeirão Preto, University of São Paulo (USP), Ribeirão Preto, SP, Brazil
| | - Dimitrius L Pitol
- Department of Basic and Oral Biology, School of Dentistry of Ribeirão Preto, University of São Paulo(USP), Ribeirão Preto, SP, Brazil
| | - João Paulo Mardegan Issa
- Department of Basic and Oral Biology, School of Dentistry of Ribeirão Preto, University of São Paulo(USP), Ribeirão Preto, SP, Brazil
| | - Jardel F Mazzi-Chaves
- Program in Restorative Dentistry, Department of Restorative Dentistry, School of Dentistry of Ribeirão Preto, University of São Paulo(USP), Ribeirão Preto, SP, Brazil
- Center for Cell-Based Therapy in Dentistry, School of Dentistry of Ribeirão Preto, University of São Paulo(USP), Ribeirão Preto, SP, Brazil
| | - Karina Fittipaldi Bombonato-Prado
- Department of Basic and Oral Biology, School of Dentistry of Ribeirão Preto, University of São Paulo(USP), Ribeirão Preto, SP, Brazil
- Center for Cell-Based Therapy in Dentistry, School of Dentistry of Ribeirão Preto, University of São Paulo(USP), Ribeirão Preto, SP, Brazil
| | - Manoel Damião Sousa-Neto
- Program in Restorative Dentistry, Department of Restorative Dentistry, School of Dentistry of Ribeirão Preto, University of São Paulo(USP), Ribeirão Preto, SP, Brazil
- Center for Cell-Based Therapy in Dentistry, School of Dentistry of Ribeirão Preto, University of São Paulo(USP), Ribeirão Preto, SP, Brazil
| | - Geraldo Aleixo Passos
- Program in Restorative Dentistry, Department of Restorative Dentistry, School of Dentistry of Ribeirão Preto, University of São Paulo(USP), Ribeirão Preto, SP, Brazil
- Department of Basic and Oral Biology, School of Dentistry of Ribeirão Preto, University of São Paulo(USP), Ribeirão Preto, SP, Brazil
- Center for Cell-Based Therapy in Dentistry, School of Dentistry of Ribeirão Preto, University of São Paulo(USP), Ribeirão Preto, SP, Brazil
- Laboratory of Genetics and Molecular Biology, Department of Basic and Oral Biology, School of Dentistry of Ribeirão Preto, University of São Paulo(USP), Ribeirão Preto, SP, Brazil
- Molecular Immunogenetics Group, Department of Genetics, Ribeirão Preto Medical School, USP, Ribeirão Preto, SP, Brazil
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McRobb LS, Lee VS, Faqihi F, Stoodley MA. A Simple Model to Study Mosaic Gene Expression in 3D Endothelial Spheroids. J Cardiovasc Dev Dis 2024; 11:305. [PMID: 39452276 PMCID: PMC11508842 DOI: 10.3390/jcdd11100305] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/07/2024] [Revised: 09/13/2024] [Accepted: 09/30/2024] [Indexed: 10/26/2024] Open
Abstract
AIMS The goal of this study was to establish a simple model of 3D endothelial spheroids with mosaic gene expression using adeno-associated virus (AAV) transduction, with a future aim being to study the activity of post-zygotic mutations common to vascular malformations. METHODS In this study, 96-well U-bottom plates coated with a commercial repellent were seeded with two immortalized human endothelial cell lines and aggregation monitored using standard microscopy or live-cell analysis. The eGFP expression was used to monitor the AAV transduction. RESULTS HUVEC-TERT2 could not form spheroids spontaneously. The inclusion of collagen I in the growth medium could stimulate cell aggregation; however, these spheroids were not stable. In contrast, the hCMEC/D3 cells aggregated spontaneously and formed reproducible, robust 3D spheroids within 3 days, growing steadily for at least 4 weeks without the need for media refreshment. The hCMEC/D3 spheroids spontaneously developed a basement membrane, including collagen I, and expressed endothelial-specific CD31 at the spheroid surface. Serotypes AAV1 and AAV2QUADYF transduced these spheroids without toxicity and established sustained, mosaic eGFP expression. CONCLUSIONS In the future, this simple approach to endothelial spheroid formation combined with live-cell imaging could be used to rapidly assess the 3D phenotypes and drug and radiation sensitivities arising from mosaic mutations common to brain vascular malformations.
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Affiliation(s)
- Lucinda S. McRobb
- Macquarie Medical School, Faculty of Medicine, Health, and Human Sciences, Macquarie University, Sydney, NSW 2109, Australia (M.A.S.)
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Wu Y, Wang J, Zhao J, Su Y, Li X, Chen Z, Wu X, Huang S, He X, Liang L. LTR retrotransposon-derived LncRNA LINC01446 promotes hepatocellular carcinoma progression and angiogenesis by regulating the SRPK2/SRSF1/VEGF axis. Cancer Lett 2024; 598:217088. [PMID: 38945203 DOI: 10.1016/j.canlet.2024.217088] [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: 01/08/2024] [Revised: 06/15/2024] [Accepted: 06/25/2024] [Indexed: 07/02/2024]
Abstract
The causal link between long terminal repeat (LTR) retrotransposon-derived lncRNAs and hepatocellular carcinoma (HCC) remains elusive and whether these cancer-exclusive lncRNAs contribute to the effectiveness of current HCC therapies is yet to explore. Here, we investigated the activation of LTR retrotransposon-derived lncRNAs in a broad range of liver diseases. We found that LTR retrotransposon-derived lncRNAs are mainly activated in HCC and is correlated with the proliferation status of HCC. Furthermore, we discovered that an LTR retrotransposon-derived lncRNA, LINC01446, exhibits specific expression in HCC. HCC patients with higher LINC01446 expression had shorter overall survival times. In vitro and in vivo assays showed that LINC01446 promoted HCC growth and angiogenesis. Mechanistically, LINC01446 bound to serine/arginine protein kinase 2 (SRPK2) and activated its downstream target, serine/arginine splicing factor 1 (SRSF1). Furthermore, activation of the SRPK2-SRSF1 axis increased the splicing and expression of VEGF isoform A165 (VEGFA165). Notably, inhibiting LINC01446 expression dramatically impaired tumor growth in vivo and resulted in better therapeutic outcomes when combined with antiangiogenic agents. In addition, we found that the transcription factor MESI2 bound to the cryptic MLT2B3 LTR promoter and drove LINC01446 transcription in HCC cells. Taken together, our findings demonstrate that LTR retrotransposon-derived LINC01446 promotes the progression of HCC by activating the SRPK2/SRSF1/VEGFA165 axis and highlight targeting LINC01446 as a potential therapeutic strategy for HCC patients.
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MESH Headings
- Animals
- Female
- Humans
- Male
- Mice
- Carcinoma, Hepatocellular/genetics
- Carcinoma, Hepatocellular/pathology
- Carcinoma, Hepatocellular/metabolism
- Cell Line, Tumor
- Cell Proliferation/genetics
- Disease Progression
- Gene Expression Regulation, Neoplastic
- Liver Neoplasms/genetics
- Liver Neoplasms/pathology
- Liver Neoplasms/metabolism
- Mice, Inbred BALB C
- Mice, Nude
- Neovascularization, Pathologic/genetics
- Protein Serine-Threonine Kinases/genetics
- Protein Serine-Threonine Kinases/metabolism
- Retroelements/genetics
- RNA, Long Noncoding/genetics
- RNA, Long Noncoding/metabolism
- Serine-Arginine Splicing Factors/genetics
- Serine-Arginine Splicing Factors/metabolism
- Signal Transduction
- Terminal Repeat Sequences/genetics
- Vascular Endothelial Growth Factor A/genetics
- Vascular Endothelial Growth Factor A/metabolism
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Affiliation(s)
- Yangjun Wu
- Fudan University Shanghai Cancer Center and Institutes of Biomedical Sciences, Shanghai Medical College, Fudan University, Shanghai, China; Department of Gynecologic Oncology, Fudan University Shanghai Cancer Center, Fudan University, Shanghai, China; Department of Oncology, Shanghai Medical College, Fudan University, Shanghai, China
| | - Jiajia Wang
- Fudan University Shanghai Cancer Center and Institutes of Biomedical Sciences, Shanghai Medical College, Fudan University, Shanghai, China
| | - Jingjing Zhao
- Fudan University Shanghai Cancer Center and Institutes of Biomedical Sciences, Shanghai Medical College, Fudan University, Shanghai, China
| | - Yue Su
- Fudan University Shanghai Cancer Center and Institutes of Biomedical Sciences, Shanghai Medical College, Fudan University, Shanghai, China
| | - Xinrong Li
- Fudan University Shanghai Cancer Center and Institutes of Biomedical Sciences, Shanghai Medical College, Fudan University, Shanghai, China
| | - Zhiao Chen
- Fudan University Shanghai Cancer Center and Institutes of Biomedical Sciences, Shanghai Medical College, Fudan University, Shanghai, China
| | - Xiaohua Wu
- Department of Gynecologic Oncology, Fudan University Shanghai Cancer Center, Fudan University, Shanghai, China; Department of Oncology, Shanghai Medical College, Fudan University, Shanghai, China
| | - Shenglin Huang
- Fudan University Shanghai Cancer Center and Institutes of Biomedical Sciences, Shanghai Medical College, Fudan University, Shanghai, China; Department of Integrative Oncology, Fudan University Shanghai Cancer Center, China
| | - Xianghuo He
- Fudan University Shanghai Cancer Center and Institutes of Biomedical Sciences, Shanghai Medical College, Fudan University, Shanghai, China.
| | - Linhui Liang
- Fudan University Shanghai Cancer Center and Institutes of Biomedical Sciences, Shanghai Medical College, Fudan University, Shanghai, China.
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8
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Pellowe AS, Wu MJ, Kang TY, Chung TD, Ledesma-Mendoza A, Herzog E, Levchenko A, Odell I, Varga J, Gonzalez AL. TGF-β1 Drives Integrin-Dependent Pericyte Migration and Microvascular Destabilization in Fibrotic Disease. THE AMERICAN JOURNAL OF PATHOLOGY 2024; 194:1171-1184. [PMID: 38548268 PMCID: PMC11220919 DOI: 10.1016/j.ajpath.2024.02.021] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 11/27/2023] [Revised: 02/08/2024] [Accepted: 02/21/2024] [Indexed: 04/10/2024]
Abstract
Interactions between endothelial cells (ECs) and mural pericytes (PCs) are critical in maintaining the stability and function of the microvascular wall. Abnormal interactions between these two cell types are a hallmark of progressive fibrotic diseases such as systemic sclerosis (also known as scleroderma). However, the role of PCs in signaling microvascular dysfunction remains underexplored. We hypothesized that integrin-matrix interactions contribute to PC migration from the vascular wall and conversion into interstitial myofibroblasts. Herein, pro-inflammatory tumor necrosis factor α (TNFα) or a fibrotic growth factor [transforming growth factor β1 (TGF-β1)] were used to evaluate human PC inflammatory and fibrotic phenotypes by assessing their migration, matrix deposition, integrin expression, and subsequent effects on endothelial dysfunction. Both TNFα and TGF-β1 treatment altered integrin expression and matrix protein deposition, but only fibrotic TGF-β1 drove PC migration in an integrin-dependent manner. In addition, integrin-dependent PC migration was correlated to changes in EC angiopoietin-2 levels, a marker of vascular instability. Finally, there was evidence of changes in vascular stability corresponding to disease state in human systemic sclerosis skin. This work shows that TNFα and TGF-β1 induce changes in PC integrin expression and matrix deposition that facilitate migration and reduce vascular stability, providing evidence that microvascular destabilization can be an early indicator of tissue fibrosis.
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Affiliation(s)
- Amanda S Pellowe
- Department of Biomedical Engineering, Yale University, New Haven, Connecticut
| | - Michelle J Wu
- Department of Biomedical Engineering, Yale University, New Haven, Connecticut
| | - Tae-Yun Kang
- Department of Biomedical Engineering, Yale University, New Haven, Connecticut
| | - Tracy D Chung
- Department of Biomedical Engineering, Yale University, New Haven, Connecticut
| | | | - Erica Herzog
- Section of Pulmonary, Critical Care and Sleep Medicine, Yale University School of Medicine, New Haven, Connecticut
| | - Andre Levchenko
- Department of Biomedical Engineering, Yale University, New Haven, Connecticut
| | - Ian Odell
- Department of Dermatology, Yale University School of Medicine, New Haven, Connecticut
| | - John Varga
- Michigan Scleroderma Program, Division of Rheumatology, Department of Internal Medicine, University of Michigan, Ann Arbor, Michigan
| | - Anjelica L Gonzalez
- Department of Biomedical Engineering, Yale University, New Haven, Connecticut.
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Davoodi P, Rezaei N, Hassan M, Hay DC, Vosough M. Bioengineering vascularized liver tissue for biomedical research and application. Scand J Gastroenterol 2024; 59:623-629. [PMID: 38319110 DOI: 10.1080/00365521.2024.2310172] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/30/2023] [Revised: 01/18/2024] [Accepted: 01/20/2024] [Indexed: 02/07/2024]
Abstract
The liver performs a wide range of biological functions that are essential to body homeostasis. Damage to liver tissue can result in reduced organ function, and if chronic in nature can lead to organ scarring and progressive disease. Currently, donor liver transplantation is the only longterm treatment for end-stage liver disease. However, orthotopic organ transplantation suffers from several drawbacks that include organ scarcity and lifelong immunosuppression. Therefore, new therapeutic strategies are required. One promising strategy is the engineering of implantable and vascularized liver tissue. This resource could also be used to build the next generation of liver tissue models to better understand human health, disease and aging in vitro. This article reviews recent progress in the field of liver tissue bioengineering, including microfluidic-based systems, bio-printed vascularized tissue, liver spheroids and organoid models, and the induction of angiogenesis in vivo.
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Affiliation(s)
- Parsa Davoodi
- Department of Regenerative Medicine, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran
| | - Niloofar Rezaei
- Department of Regenerative Medicine, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran
| | - Moustapha Hassan
- Experimental Cancer Medicine, Institution for Laboratory Medicine, Karolinska Institutet, Stockholm, Sweden
| | - David C Hay
- Centre for Regenerative Medicine, Institute for Repair and Regeneration, University of Edinburgh, Edinburgh, UK
| | - Massoud Vosough
- Department of Regenerative Medicine, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran
- Experimental Cancer Medicine, Institution for Laboratory Medicine, Karolinska Institutet, Stockholm, Sweden
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10
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Kukita K, Matsuzaka N, Takai M, Imamura Y, Shin Y. Notch signaling pathway induces expression of type IV collagen in angiogenesis. J Biochem 2024; 175:539-549. [PMID: 38167713 DOI: 10.1093/jb/mvad120] [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: 09/04/2023] [Accepted: 12/16/2023] [Indexed: 01/05/2024] Open
Abstract
Mural cell adhesion is important for the localization of basement membrane components during angiogenesis, and cell-cell interactions are thought to be critical for basement membrane formation. Type IV collagen, a component of the basement membrane, and non-triple helical type IV collagen α1 chain (NTH α1(IV)) co-localize in the basement membrane of neovascular vessels. However, it remains unclear how type IV collagen and NTH α1(IV) are produced around the basement membrane. In the present study, we developed a de novo angiogenesis model using human umbilical vein endothelial cell spheroids and TIG-1 fibroblast cells and demonstrated that NTH α1(IV), probably with α1(IV) chain before forming triple helix molecule, was localized in the fibroblasts in contact with vascular endothelial cells. This localization was disrupted by DAPT, a Notch signaling inhibitor. DAPT treatment also reduced type IV collagen and NTH α1(IV) secretion in TIG-1 fibroblasts, along with diminished COL4A1 and COL4A2 gene expression. Downregulation of Notch3 in TIG-1 fibroblasts decreased the secretion of type IV collagen and NTH α1(IV). Taken together, these findings suggest that heterogeneous and homogeneous intercellular Notch signaling via Notch3 induces type IV collagen and NTH α1(IV) expression in fibroblasts and contributes to basement membrane formation in neovascular vessels.
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Affiliation(s)
- Kazuki Kukita
- Graduate School of Engineering, Kogakuin University, 2665-1, Nakanomachi, Tokyo 1920015, Japan
| | - Nanaka Matsuzaka
- Department of Chemistry and Life Science, School of Advanced Engineering, Kogakuin University, Tokyo, Japan
| | - Mikihisa Takai
- Department of Chemistry and Life Science, School of Advanced Engineering, Kogakuin University, Tokyo, Japan
| | - Yasutada Imamura
- Graduate School of Engineering, Kogakuin University, 2665-1, Nakanomachi, Tokyo 1920015, Japan
- Department of Chemistry and Life Science, School of Advanced Engineering, Kogakuin University, Tokyo, Japan
| | - Yongchol Shin
- Graduate School of Engineering, Kogakuin University, 2665-1, Nakanomachi, Tokyo 1920015, Japan
- Department of Chemistry and Life Science, School of Advanced Engineering, Kogakuin University, Tokyo, Japan
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11
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Liu XT, Huang Y, Liu D, Jiang YC, Zhao M, Chung LH, Han XD, Zhao Y, Chen J, Coleman P, Ting KK, Tran C, Su Y, Dennis CV, Bhatnagar A, Liu K, Don AS, Vadas MA, Gorrell MD, Zhang S, Murray M, Kavurma MM, McCaughan GW, Gamble JR, Qi Y. Targeting the SphK1/S1P/PFKFB3 axis suppresses hepatocellular carcinoma progression by disrupting glycolytic energy supply that drives tumor angiogenesis. J Transl Med 2024; 22:43. [PMID: 38200582 PMCID: PMC10782643 DOI: 10.1186/s12967-023-04830-z] [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: 12/22/2023] [Accepted: 12/24/2023] [Indexed: 01/12/2024] Open
Abstract
BACKGROUND Hepatocellular carcinoma (HCC) remains a leading life-threatening health challenge worldwide, with pressing needs for novel therapeutic strategies. Sphingosine kinase 1 (SphK1), a well-established pro-cancer enzyme, is aberrantly overexpressed in a multitude of malignancies, including HCC. Our previous research has shown that genetic ablation of Sphk1 mitigates HCC progression in mice. Therefore, the development of PF-543, a highly selective SphK1 inhibitor, opens a new avenue for HCC treatment. However, the anti-cancer efficacy of PF-543 has not yet been investigated in primary cancer models in vivo, thereby limiting its further translation. METHODS Building upon the identification of the active form of SphK1 as a viable therapeutic target in human HCC specimens, we assessed the capacity of PF-543 in suppressing tumor progression using a diethylnitrosamine-induced mouse model of primary HCC. We further delineated its underlying mechanisms in both HCC and endothelial cells. Key findings were validated in Sphk1 knockout mice and lentiviral-mediated SphK1 knockdown cells. RESULTS SphK1 activity was found to be elevated in human HCC tissues. Administration of PF-543 effectively abrogated hepatic SphK1 activity and significantly suppressed HCC progression in diethylnitrosamine-treated mice. The primary mechanism of action was through the inhibition of tumor neovascularization, as PF-543 disrupted endothelial cell angiogenesis even in a pro-angiogenic milieu. Mechanistically, PF-543 induced proteasomal degradation of the critical glycolytic enzyme 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3, thus restricting the energy supply essential for tumor angiogenesis. These effects of PF-543 could be reversed upon S1P supplementation in an S1P receptor-dependent manner. CONCLUSIONS This study provides the first in vivo evidence supporting the potential of PF-543 as an effective anti-HCC agent. It also uncovers previously undescribed links between the pro-cancer, pro-angiogenic and pro-glycolytic roles of the SphK1/S1P/S1P receptor axis. Importantly, unlike conventional anti-HCC drugs that target individual pro-angiogenic drivers, PF-543 impairs the PFKFB3-dictated glycolytic energy engine that fuels tumor angiogenesis, representing a novel and potentially safer therapeutic strategy for HCC.
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Affiliation(s)
- Xin Tracy Liu
- Centenary Institute of Cancer Medicine and Cell Biology, The University of Sydney, Sydney, NSW, 2050, Australia
| | - Yu Huang
- Centenary Institute of Cancer Medicine and Cell Biology, The University of Sydney, Sydney, NSW, 2050, Australia
| | - Da Liu
- Centenary Institute of Cancer Medicine and Cell Biology, The University of Sydney, Sydney, NSW, 2050, Australia
| | - Yingxin Celia Jiang
- Centenary Institute of Cancer Medicine and Cell Biology, The University of Sydney, Sydney, NSW, 2050, Australia
| | - Min Zhao
- School of Science, Technology and Engineering, University of the Sunshine Coast, Maroochydore DC, QLD, 4558, Australia
| | - Long Hoa Chung
- Centenary Institute of Cancer Medicine and Cell Biology, The University of Sydney, Sydney, NSW, 2050, Australia
| | - Xingxing Daisy Han
- Centenary Institute of Cancer Medicine and Cell Biology, The University of Sydney, Sydney, NSW, 2050, Australia
| | - Yinan Zhao
- Key Laboratory of Biotechnology and Bioresources Utilization of Ministry of Education, Dalian Minzu University, Dalian, 116600, Liaoning, China
| | - Jinbiao Chen
- Centenary Institute of Cancer Medicine and Cell Biology, The University of Sydney, Sydney, NSW, 2050, Australia
| | - Paul Coleman
- Centenary Institute of Cancer Medicine and Cell Biology, The University of Sydney, Sydney, NSW, 2050, Australia
| | - Ka Ka Ting
- Centenary Institute of Cancer Medicine and Cell Biology, The University of Sydney, Sydney, NSW, 2050, Australia
| | - Collin Tran
- School of Medical Sciences, Faculty of Medicine and Health, The University of Sydney, Sydney, NSW, 2006, Australia
| | - Yingying Su
- Sydney Microscopy and Microanalysis, The University of Sydney, Sydney, NSW, 2006, Australia
| | - Claude Vincent Dennis
- AW Morrow Gastroenterology and Liver Centre, Royal Prince Alfred Hospital, Sydney Local Health District, Sydney, NSW, 2050, Australia
| | - Atul Bhatnagar
- Sydney Mass Spectrometry, The University of Sydney, Sydney, NSW, 2006, Australia
| | - Ken Liu
- AW Morrow Gastroenterology and Liver Centre, Royal Prince Alfred Hospital, Sydney Local Health District, Sydney, NSW, 2050, Australia
| | - Anthony Simon Don
- School of Medical Sciences, Faculty of Medicine and Health, The University of Sydney, Sydney, NSW, 2006, Australia
| | - Mathew Alexander Vadas
- Centenary Institute of Cancer Medicine and Cell Biology, The University of Sydney, Sydney, NSW, 2050, Australia
| | - Mark Douglas Gorrell
- Centenary Institute of Cancer Medicine and Cell Biology, The University of Sydney, Sydney, NSW, 2050, Australia
- AW Morrow Gastroenterology and Liver Centre, Royal Prince Alfred Hospital, Sydney Local Health District, Sydney, NSW, 2050, Australia
| | - Shubiao Zhang
- Key Laboratory of Biotechnology and Bioresources Utilization of Ministry of Education, Dalian Minzu University, Dalian, 116600, Liaoning, China
| | - Michael Murray
- Sydney Pharmacy School, Faculty of Medicine and Health, The University of Sydney, Sydney, NSW, 2006, Australia
| | | | - Geoffrey William McCaughan
- Centenary Institute of Cancer Medicine and Cell Biology, The University of Sydney, Sydney, NSW, 2050, Australia
- AW Morrow Gastroenterology and Liver Centre, Royal Prince Alfred Hospital, Sydney Local Health District, Sydney, NSW, 2050, Australia
| | - Jennifer Ruth Gamble
- Centenary Institute of Cancer Medicine and Cell Biology, The University of Sydney, Sydney, NSW, 2050, Australia
| | - Yanfei Qi
- Centenary Institute of Cancer Medicine and Cell Biology, The University of Sydney, Sydney, NSW, 2050, Australia.
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12
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Alve S, Gramolelli S, Jukonen J, Juteau S, Pink A, Manninen AA, Hänninen S, Monto E, Lackman MH, Carpén O, Saharinen P, Karaman S, Vaahtomeri K, Ojala PM. DLL4/Notch3/WNT5B axis mediates bidirectional prometastatic crosstalk between melanoma and lymphatic endothelial cells. JCI Insight 2024; 9:e171821. [PMID: 37971882 PMCID: PMC10906450 DOI: 10.1172/jci.insight.171821] [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: 04/27/2023] [Accepted: 11/15/2023] [Indexed: 11/19/2023] Open
Abstract
Despite strong indications that interactions between melanoma and lymphatic vessels actively promote melanoma progression, the molecular mechanisms are not yet completely understood. To characterize molecular factors of this crosstalk, we established human primary lymphatic endothelial cell (LEC) cocultures with human melanoma cell lines. Here, we show that coculture with melanoma cells induced transcriptomic changes in LECs and led to multiple changes in their function. WNT5B, a paracrine signaling molecule upregulated in melanoma cells upon LEC interaction, was found to contribute to the functional changes in LECs. Moreover, WNT5B transcription was regulated by Notch3 in melanoma cells following the coculture with LECs, and Notch3 and WNT5B were coexpressed in melanoma patient primary tumor and metastasis samples. Moreover, melanoma cells derived from LEC coculture escaped efficiently from the primary site to the proximal tumor-draining lymph nodes, which was impaired upon WNT5B depletion. This supported the role of WNT5B in promoting the metastatic potential of melanoma cells through its effects on LECs. Finally, DLL4, a Notch ligand expressed in LECs, was identified as an upstream inducer of the Notch3/WNT5B axis in melanoma. This study elucidated WNT5B as a key molecular factor mediating bidirectional crosstalk between melanoma cells and lymphatic endothelium and promoting melanoma metastasis.
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Affiliation(s)
- Sanni Alve
- Translational Cancer Medicine Research Program, Research Programs Unit, Faculty of Medicine, University of Helsinki, Helsinki, Finland
| | - Silvia Gramolelli
- Faculty of Science and Engineering, Cell Biology, Åbo Akademi University, Turku, Finland
- Turku Bioscience Centre, University of Turku and Åbo Akademi University, Turku, Finland
| | - Joonas Jukonen
- Translational Cancer Medicine Research Program, Research Programs Unit, Faculty of Medicine, University of Helsinki, Helsinki, Finland
| | - Susanna Juteau
- Department of Pathology, Helsinki University Hospital (HUS), University of Helsinki, Helsinki, Finland
| | - Anne Pink
- Translational Cancer Medicine Research Program, Research Programs Unit, Faculty of Medicine, University of Helsinki, Helsinki, Finland
| | - Atte A. Manninen
- Department of Plastic Surgery, Park Hospital, Helsinki University Hospital (HUS), and
| | - Satu Hänninen
- Department of Pathology, Helsinki University Hospital (HUS), University of Helsinki, Helsinki, Finland
| | - Elisa Monto
- Translational Cancer Medicine Research Program, Research Programs Unit, Faculty of Medicine, University of Helsinki, Helsinki, Finland
| | - Madeleine H. Lackman
- Individualized Drug Therapy Research Program, Faculty of Medicine, University of Helsinki, Helsinki, Finland
| | - Olli Carpén
- Helsinki Biobank, and
- Department of Pathology and Research Program in Systems Oncology, University of Helsinki, HUS Diagnostic Center, Helsinki University Hospital, Finland
| | - Pipsa Saharinen
- Translational Cancer Medicine Research Program, Research Programs Unit, Faculty of Medicine, University of Helsinki, Helsinki, Finland
- Wihuri Research Institute, Biomedicum, Helsinki, Finland
- Department of Biochemistry and Developmental Biology, Faculty of Medicine, University of Helsinki, Helsinki, Finland
| | - Sinem Karaman
- Individualized Drug Therapy Research Program, Faculty of Medicine, University of Helsinki, Helsinki, Finland
- Wihuri Research Institute, Biomedicum, Helsinki, Finland
| | - Kari Vaahtomeri
- Translational Cancer Medicine Research Program, Research Programs Unit, Faculty of Medicine, University of Helsinki, Helsinki, Finland
- Wihuri Research Institute, Biomedicum, Helsinki, Finland
| | - Päivi M. Ojala
- Translational Cancer Medicine Research Program, Research Programs Unit, Faculty of Medicine, University of Helsinki, Helsinki, Finland
- Department of Pathology, Helsinki University Hospital (HUS), University of Helsinki, Helsinki, Finland
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13
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Beunk L, Wen N, van Helvert S, Bekker B, Ran L, Kang R, Paulat T, Syga S, Deutsch A, Friedl P, Wolf K. Cell jamming in a collagen-based interface assay is tuned by collagen density and proteolysis. J Cell Sci 2023; 136:jcs260207. [PMID: 37987169 PMCID: PMC10753497 DOI: 10.1242/jcs.260207] [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: 07/01/2022] [Accepted: 11/15/2023] [Indexed: 11/22/2023] Open
Abstract
Tumor cell invasion into heterogenous interstitial tissues consisting of network-, channel- or rift-like architectures involves both matrix metalloproteinase (MMP)-mediated tissue remodeling and cell shape adaptation to tissue geometry. Three-dimensional (3D) models composed of either porous or linearly aligned architectures have added to the understanding of how physical spacing principles affect migration efficacy; however, the relative contribution of each architecture to decision making in the presence of varying MMP availability is not known. Here, we developed an interface assay containing a cleft between two high-density collagen lattices, and we used this assay to probe tumor cell invasion efficacy, invasion mode and MMP dependence in concert. In silico modeling predicted facilitated cell migration into confining clefts independently of MMP activity, whereas migration into dense porous matrix was predicted to require matrix degradation. This prediction was verified experimentally, where inhibition of collagen degradation was found to strongly compromise migration into 3D collagen in a density-dependent manner, but interface-guided migration remained effective, occurring by cell jamming. The 3D interface assay reported here may serve as a suitable model to better understand the impact of in vivo-relevant interstitial tissue topologies on tumor invasion patterning and responses to molecular interventions.
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Affiliation(s)
- Lianne Beunk
- Department of Medical BioSciences, Radboud University Medical Center, Nijmegen, GA 6525, The Netherlands
| | - Nan Wen
- Department of Medical BioSciences, Radboud University Medical Center, Nijmegen, GA 6525, The Netherlands
| | - Sjoerd van Helvert
- Department of Medical BioSciences, Radboud University Medical Center, Nijmegen, GA 6525, The Netherlands
| | - Bram Bekker
- Department of Mathematics, Faculty of Natural Science, Mathematics and Informatics, Radboud University, 6500 GL Nijmegen, The Netherlands
| | - Lars Ran
- Department of Mathematics, Faculty of Natural Science, Mathematics and Informatics, Radboud University, 6500 GL Nijmegen, The Netherlands
| | - Ross Kang
- Department of Mathematics, Faculty of Natural Science, Mathematics and Informatics, Radboud University, 6500 GL Nijmegen, The Netherlands
| | - Tom Paulat
- Department of Innovative Computing, Centre for Information Services and High Performance Computing, Technical University Dresden, 01062 Dresden, Germany
| | - Simon Syga
- Department of Innovative Computing, Centre for Information Services and High Performance Computing, Technical University Dresden, 01062 Dresden, Germany
| | - Andreas Deutsch
- Department of Innovative Computing, Centre for Information Services and High Performance Computing, Technical University Dresden, 01062 Dresden, Germany
| | - Peter Friedl
- Department of Medical BioSciences, Radboud University Medical Center, Nijmegen, GA 6525, The Netherlands
- David H. Koch Center for Applied Genitourinary Cancers, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA
| | - Katarina Wolf
- Department of Medical BioSciences, Radboud University Medical Center, Nijmegen, GA 6525, The Netherlands
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14
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Sporkova A, Nahar T, Cao M, Ghosh S, Sens-Albert C, Friede PAP, Nagel A, Al-Hasani J, Hecker M. Characterisation of Lipoma-Preferred Partner as a Novel Mechanotransducer in Vascular Smooth Muscle Cells. Cells 2023; 12:2315. [PMID: 37759537 PMCID: PMC10529303 DOI: 10.3390/cells12182315] [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: 08/01/2023] [Revised: 08/29/2023] [Accepted: 09/15/2023] [Indexed: 09/29/2023] Open
Abstract
In arteries and arterioles, a chronic increase in blood pressure raises wall tension. This continuous biomechanical strain causes a change in gene expression in vascular smooth muscle cells (VSMCs) that may lead to pathological changes. Here we have characterised the functional properties of lipoma-preferred partner (LPP), a Lin11-Isl1-Mec3 (LIM)-domain protein, which is most closely related to the mechanotransducer zyxin but selectively expressed by smooth muscle cells, including VSMCs in adult mice. VSMCs isolated from the aorta of LPP knockout (LPP-KO) mice displayed a higher rate of proliferation than their wildtype (WT) counterparts, and when cultured as three-dimensional spheroids, they revealed a higher expression of the proliferation marker Ki 67 and showed greater invasion into a collagen gel. Accordingly, the gelatinase activity was increased in LPP-KO but not WT spheroids. The LPP-KO spheroids adhering to the collagen gel responded with decreased contraction to potassium chloride. The relaxation response to caffeine and norepinephrine was also smaller in the LPP-KO spheroids than in their WT counterparts. The overexpression of zyxin in LPP-KO VSMCs resulted in a reversal to a more quiescent differentiated phenotype. In native VSMCs, i.e., in isolated perfused segments of the mesenteric artery (MA), the contractile responses of LPP-KO segments to potassium chloride, phenylephrine or endothelin-1 did not vary from those in isolated perfused WT segments. In contrast, the myogenic response of LPP-KO MA segments was significantly attenuated while zyxin-deficient MA segments displayed a normal myogenic response. We propose that LPP, which we found to be expressed solely in the medial layer of different arteries from adult mice, may play an important role in controlling the quiescent contractile phenotype of VSMCs.
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Affiliation(s)
| | | | | | | | | | | | | | | | - Markus Hecker
- Department of Cardiovascular Physiology, Heidelberg University, Im Neuenheimer Feld 326, 69120 Heidelberg, Germany; (A.S.)
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15
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Capik O, Gumus R, Karatas OF. Hypoxia-induced tumor exosomes promote angiogenesis through miR-1825/TSC2/mTOR axis in oral squamous cell carcinoma. Head Neck 2023; 45:2259-2273. [PMID: 37449548 DOI: 10.1002/hed.27460] [Citation(s) in RCA: 16] [Impact Index Per Article: 8.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/04/2023] [Revised: 06/21/2023] [Accepted: 07/04/2023] [Indexed: 07/18/2023] Open
Abstract
BACKGROUND Oral squamous cell carcinoma (OSCC) is characterized by enhanced angiogenesis resulting in poor prognosis despite improvements in diagnostic/therapeutic techniques. Here, we aimed at investigating potential roles of miR-1825 enclosed in OSCC-derived exosomes on angiogenesis under hypoxic conditions. METHODS Effects of miR-1825 mimic/inhibitor as well as hypoxia-induced tumor derived exosomes on human umbilical vein endothelial cells (HUVECs) were evaluated using cell viability, migration/invasion, tube formation, and spheroid-based 3D angiogenesis assays. RESULTS Hypoxic conditions caused significant increase in miR-1825 levels in OSCC cells and hiTDEs. miR-1825 alone and within hiTDEs promoted endothelial cell viability, migration, invasion, and angiogenic potential, which is reversed via inhibition of miR-1825 expression. miR-1825 within hiTDEs altered the angiogenesis potential of HUVEC cells via deregulation of TSC2/mTOR axis. CONCLUSIONS We showed that hypoxia led to OSCC-derived exosome mediated transfer of miR-1825 to HUVECs and enhanced angiogenesis in OSCC in vitro.
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Affiliation(s)
- Ozel Capik
- Department of Molecular Biology and Genetics, Erzurum Technical University, Erzurum, Turkey
- Molecular Cancer Biology Laboratory, High Technology Application and Research Center, Erzurum Technical University, Erzurum, Turkey
| | - Rasim Gumus
- Department of Molecular Biology and Genetics, Erzurum Technical University, Erzurum, Turkey
- Molecular Cancer Biology Laboratory, High Technology Application and Research Center, Erzurum Technical University, Erzurum, Turkey
| | - Omer Faruk Karatas
- Department of Molecular Biology and Genetics, Erzurum Technical University, Erzurum, Turkey
- Molecular Cancer Biology Laboratory, High Technology Application and Research Center, Erzurum Technical University, Erzurum, Turkey
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16
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Schulik J, Salehi S, Boccaccini AR, Schrüfer S, Schubert DW, Arkudas A, Kengelbach-Weigand A, Horch RE, Schmid R. Comparison of the Behavior of 3D-Printed Endothelial Cells in Different Bioinks. Bioengineering (Basel) 2023; 10:751. [PMID: 37508778 PMCID: PMC10376299 DOI: 10.3390/bioengineering10070751] [Citation(s) in RCA: 7] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/26/2023] [Revised: 06/19/2023] [Accepted: 06/21/2023] [Indexed: 07/30/2023] Open
Abstract
Biomaterials with characteristics similar to extracellular matrix and with suitable bioprinting properties are essential for vascular tissue engineering. In search for suitable biomaterials, this study investigated the three hydrogels alginate/hyaluronic acid/gelatin (Alg/HA/Gel), pre-crosslinked alginate di-aldehyde with gelatin (ADA-GEL), and gelatin methacryloyl (GelMA) with respect to their mechanical properties and to the survival, migration, and proliferation of human umbilical vein endothelial cells (HUVECs). In addition, the behavior of HUVECs was compared with their behavior in Matrigel. For this purpose, HUVECs were mixed with the inks both as single cells and as cell spheroids and printed using extrusion-based bioprinting. Good printability with shape fidelity was determined for all inks. The rheological measurements demonstrated the gelling consistency of the inks and shear-thinning behavior. Different Young's moduli of the hydrogels were determined. However, all measured values where within the range defined in the literature, leading to migration and sprouting, as well as reconciling migration with adhesion. Cell survival and proliferation in ADA-GEL and GelMA hydrogels were demonstrated for 14 days. In the Alg/HA/Gel bioink, cell death occurred within 7 days for single cells. Sprouting and migration of the HUVEC spheroids were observed in ADA-GEL and GelMA. Similar behavior of the spheroids was seen in Matrigel. In contrast, the spheroids in the Alg/HA/Gel ink died over the time studied. It has been shown that Alg/HA/Gel does not provide a good environment for long-term survival of HUVECs. In conclusion, ADA-GEL and GelMA are promising inks for vascular tissue engineering.
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Affiliation(s)
- Jana Schulik
- Laboratory for Tissue-Engineering and Regenerative Medicine, Department of Plastic and Hand Surgery University Hospital of Erlangen, Krankenhausstraße 12, 91054 Erlangen, Germany
| | - Sahar Salehi
- Chair of Biomaterials, University of Bayreuth, Prof.-Rüdiger-Bormann-Str. 1, 95447 Bayreuth, Germany
| | - Aldo R Boccaccini
- Institute of Biomaterials, Friedrich-Alexander University Erlangen-Nürnberg, Cauerstrasse 6, 91058 Erlangen, Germany
| | - Stefan Schrüfer
- Institute of Polymer Materials, Friedrich-Alexander University Erlangen-Nürnberg, Martensstraße 7, 91058 Erlangen, Germany
| | - Dirk W Schubert
- Institute of Polymer Materials, Friedrich-Alexander University Erlangen-Nürnberg, Martensstraße 7, 91058 Erlangen, Germany
| | - Andreas Arkudas
- Laboratory for Tissue-Engineering and Regenerative Medicine, Department of Plastic and Hand Surgery University Hospital of Erlangen, Krankenhausstraße 12, 91054 Erlangen, Germany
| | - Annika Kengelbach-Weigand
- Laboratory for Tissue-Engineering and Regenerative Medicine, Department of Plastic and Hand Surgery University Hospital of Erlangen, Krankenhausstraße 12, 91054 Erlangen, Germany
| | - Raymund E Horch
- Laboratory for Tissue-Engineering and Regenerative Medicine, Department of Plastic and Hand Surgery University Hospital of Erlangen, Krankenhausstraße 12, 91054 Erlangen, Germany
| | - Rafael Schmid
- Laboratory for Tissue-Engineering and Regenerative Medicine, Department of Plastic and Hand Surgery University Hospital of Erlangen, Krankenhausstraße 12, 91054 Erlangen, Germany
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17
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Ahmed TA, Eldaly B, Eldosuky S, Elkhenany H, El-Derby AM, Elshazly MF, El-Badri N. The interplay of cells, polymers, and vascularization in three-dimensional lung models and their applications in COVID-19 research and therapy. Stem Cell Res Ther 2023; 14:114. [PMID: 37118810 PMCID: PMC10144893 DOI: 10.1186/s13287-023-03341-4] [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: 10/15/2022] [Accepted: 04/14/2023] [Indexed: 04/30/2023] Open
Abstract
Millions of people have been affected ever since the emergence of the corona virus disease of 2019 (COVID-19) outbreak, leading to an urgent need for antiviral drug and vaccine development. Current experimentation on traditional two-dimensional culture (2D) fails to accurately mimic the in vivo microenvironment for the disease, while in vivo animal model testing does not faithfully replicate human COVID-19 infection. Human-based three-dimensional (3D) cell culture models such as spheroids, organoids, and organ-on-a-chip present a promising solution to these challenges. In this report, we review the recent 3D in vitro lung models used in COVID-19 infection and drug screening studies and highlight the most common types of natural and synthetic polymers used to generate 3D lung models.
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Affiliation(s)
- Toka A Ahmed
- Center of Excellence for Stem Cells and Regenerative Medicine (CESC), Zewail City of Science and Technology, October Gardens, 6th of October City, Giza, 12582, Egypt
- Egypt Center for Research and Regenerative Medicine (ECRRM), Cairo, Egypt
| | - Bassant Eldaly
- Center of Excellence for Stem Cells and Regenerative Medicine (CESC), Zewail City of Science and Technology, October Gardens, 6th of October City, Giza, 12582, Egypt
| | - Shadwa Eldosuky
- Center of Excellence for Stem Cells and Regenerative Medicine (CESC), Zewail City of Science and Technology, October Gardens, 6th of October City, Giza, 12582, Egypt
| | - Hoda Elkhenany
- Department of Surgery, Faculty of Veterinary Medicine, Alexandria University, Alexandria, 22785, Egypt
| | - Azza M El-Derby
- Center of Excellence for Stem Cells and Regenerative Medicine (CESC), Zewail City of Science and Technology, October Gardens, 6th of October City, Giza, 12582, Egypt
| | - Muhamed F Elshazly
- Center of Excellence for Stem Cells and Regenerative Medicine (CESC), Zewail City of Science and Technology, October Gardens, 6th of October City, Giza, 12582, Egypt
| | - Nagwa El-Badri
- Center of Excellence for Stem Cells and Regenerative Medicine (CESC), Zewail City of Science and Technology, October Gardens, 6th of October City, Giza, 12582, Egypt.
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Weidemann H, Feger D, Ehlert JE, Menger MM, Krempien RC. Markedly divergent effects of Ouabain on a Temozolomide-resistant (T98G) vs. a Temozolomide-sensitive (LN229) Glioblastoma cell line. Discov Oncol 2023; 14:27. [PMID: 36840822 PMCID: PMC9968366 DOI: 10.1007/s12672-023-00633-2] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/20/2022] [Accepted: 02/17/2023] [Indexed: 02/26/2023] Open
Abstract
BACKGROUND Glioblastoma multiforme (GBM) is the most aggressive primary brain tumor with poor prognosis. GMB are highly recurrent mainly because of radio- and chemoresistance. Radiotherapy with Temozolomide (TMZ) is until today the golden standard adjuvant therapy, however, the optimal treatment of recurrent glioblastoma remains controversial. Ouabain belongs to the Cardiotonic Steroids (CTS) the natural ligands of the Na/K-ATPase (NKA). It is established that the NKA represents a signal transducer with either stimulating or inhibiting cell growth, apoptosis, migration and angiogenesis. Over the last decade evidence grew that CTS have anti-tumor properties especially in GBM. AIM Proceeding from recent studies we wanted to further demonstrate a divergent effect of Ouabain on a TMZ-resistant (T98G) as compared to a TMZ-sensitive (LN229) GBM cell line. METHODS We analyzed the effect of Ouabain on cell migration and plasma cell membrane potential (PCMP) in the LN229 and T98G GBM cell line as well as underlying mechanisms (Bcl-2 and p-Akt/pan-Akt expression). Moreover, we analyzed the anti-angiogenic effect of Ouabain on human umbilical vein endothelial cells (HUVECs). RESULTS T98G cells showed a significant inhibition of cell migration and a significant depolarization of the PCMP at similar Ouabain concentrations (IC50 = 1.67 × 10-7 M) resp. (IC50 = 2.72 × 10-7 M) with a strong inverse correlation (R2 = 0.95). In contrast, LN229 cells did not respond to Ouabain in these assays at all. Similarly, only T98G but not LN229 cells revealed Bcl-2 down-regulation at nanomolar Ouabain concentrations. This unique response to Ouabain is associated with a down-regulation of pan-Akt in T98G cells 24 h after Ouabain (1.0 × 10-6 M) treatment. For the first time, the anti-angiogenic effect of Ouabain on HUVEC cells (IC50 = 5.49 × 10-8 M) was demonstrated which correlated strongly with the anti-migratory effect (R2 = 0.85). CONCLUSION The TMZ-resistant T98G cell line as compared to the TMZ-sensitive LN229 cell line shows a high sensitivity towards Ouabain. We consider it as a promising new compound especially in recurrent GBM to overcome the resistance to TMZ and irradiation.
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Affiliation(s)
- Heidrun Weidemann
- Clinic for Radiotherapy, HELIOS Hospital Berlin-Buch, Schwanebecker Chaussee 50, 13125 Berlin, Germany
| | - Daniel Feger
- Reaction Biology Europe GmbH, Engesserstr.4, 79108 Freiburg, Germany
| | - Jan E. Ehlert
- Reaction Biology Europe GmbH, Engesserstr.4, 79108 Freiburg, Germany
| | - Marcus M. Menger
- Fraunhofer Institute for Cell Therapy and Immunology, Branch Bioanalytics and Bioprocesses (IZI-BB), Am Mühlenberg13, 14476 Potsdam, Germany
| | - Robert C. Krempien
- Clinic for Radiotherapy, HELIOS Hospital Berlin-Buch, Schwanebecker Chaussee 50, 13125 Berlin, Germany
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Bone Sialoprotein Immobilized in Collagen Type I Enhances Angiogenesis In Vitro and In Ovo. Polymers (Basel) 2023; 15:polym15041007. [PMID: 36850289 PMCID: PMC9968013 DOI: 10.3390/polym15041007] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/19/2022] [Revised: 01/24/2023] [Accepted: 02/14/2023] [Indexed: 02/22/2023] Open
Abstract
Bone fracture healing is a multistep process, including early immunological reactions, osteogenesis, and as a key factor, angiogenesis. Molecules inducing osteogenesis as well as angiogenesis are rare, but hold promise to be employed in bone tissue engineering. It has been demonstrated that the bone sialoprotein (BSP) can induce bone formation when immobilized in collagen type I, but its effect on angiogenesis still has to be characterized in detail. Therefore, the aim of this study was to analyse the effects of BSP immobilized in a collagen type I gel on angiogenesis. First, in vitro analyses with endothelial cells (HUVECs) were performed detecting enhancing effects of BSP on proliferation and gene expression of endothelial markers. A spheroid model was employed confirming these results. Finally, the inducing impact of BSP-collagen on vascular density was proved in a yolk sac membrane assay. Our results demonstrate that BSP is capable of inducing angiogenesis and confirm that collagen type I is the optimal carrier for this protein. Taking into account former results, and literature showing that BSP also induces osteogenesis, one can hypothesize that BSP couples angiogenesis and osteogenesis, making it a promising molecule to be used in bone tissue regeneration.
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20
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Boos F, Oo JA, Warwick T, Günther S, Izquierdo Ponce J, Lopez M, Rafii D, Buchmann G, Pham MD, Msheik ZS, Li T, Seredinski S, Haydar S, Kashefiolasl S, Plate KH, Behr R, Mietsch M, Krishnan J, Pullamsetti SS, Bibli SI, Hinkel R, Baker AH, Boon RA, Schulz MH, Wittig I, Miller FJ, Brandes RP, Leisegang MS. The endothelial-enriched lncRNA LINC00607 mediates angiogenic function. Basic Res Cardiol 2023; 118:5. [PMID: 36700983 PMCID: PMC9879848 DOI: 10.1007/s00395-023-00978-3] [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] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/04/2022] [Revised: 01/09/2023] [Accepted: 01/09/2023] [Indexed: 01/27/2023]
Abstract
Long non-coding RNAs (lncRNAs) can act as regulatory RNAs which, by altering the expression of target genes, impact on the cellular phenotype and cardiovascular disease development. Endothelial lncRNAs and their vascular functions are largely undefined. Deep RNA-Seq and FANTOM5 CAGE analysis revealed the lncRNA LINC00607 to be highly enriched in human endothelial cells. LINC00607 was induced in response to hypoxia, arteriosclerosis regression in non-human primates, post-atherosclerotic cultured endothelial cells from patients and also in response to propranolol used to induce regression of human arteriovenous malformations. siRNA knockdown or CRISPR/Cas9 knockout of LINC00607 attenuated VEGF-A-induced angiogenic sprouting. LINC00607 knockout in endothelial cells also integrated less into newly formed vascular networks in an in vivo assay in SCID mice. Overexpression of LINC00607 in CRISPR knockout cells restored normal endothelial function. RNA- and ATAC-Seq after LINC00607 knockout revealed changes in the transcription of endothelial gene sets linked to the endothelial phenotype and in chromatin accessibility around ERG-binding sites. Mechanistically, LINC00607 interacted with the SWI/SNF chromatin remodeling protein BRG1. CRISPR/Cas9-mediated knockout of BRG1 in HUVEC followed by CUT&RUN revealed that BRG1 is required to secure a stable chromatin state, mainly on ERG-binding sites. In conclusion, LINC00607 is an endothelial-enriched lncRNA that maintains ERG target gene transcription by interacting with the chromatin remodeler BRG1 to ultimately mediate angiogenesis.
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Affiliation(s)
- Frederike Boos
- Institut für Kardiovaskuläre Physiologie, Fachbereich Medizin der Goethe-Universität, Theodor-Stern-Kai 7, 60590, Frankfurt am Main, Germany
- German Center of Cardiovascular Research (DZHK), Partner Site RheinMain, Frankfurt, Germany
| | - James A Oo
- Institut für Kardiovaskuläre Physiologie, Fachbereich Medizin der Goethe-Universität, Theodor-Stern-Kai 7, 60590, Frankfurt am Main, Germany
- German Center of Cardiovascular Research (DZHK), Partner Site RheinMain, Frankfurt, Germany
| | - Timothy Warwick
- Institut für Kardiovaskuläre Physiologie, Fachbereich Medizin der Goethe-Universität, Theodor-Stern-Kai 7, 60590, Frankfurt am Main, Germany
- German Center of Cardiovascular Research (DZHK), Partner Site RheinMain, Frankfurt, Germany
| | - Stefan Günther
- Max-Planck-Institute for Heart and Lung Research, Bad Nauheim, Germany
| | - Judit Izquierdo Ponce
- Institut für Kardiovaskuläre Physiologie, Fachbereich Medizin der Goethe-Universität, Theodor-Stern-Kai 7, 60590, Frankfurt am Main, Germany
| | - Melina Lopez
- Institut für Kardiovaskuläre Physiologie, Fachbereich Medizin der Goethe-Universität, Theodor-Stern-Kai 7, 60590, Frankfurt am Main, Germany
- German Center of Cardiovascular Research (DZHK), Partner Site RheinMain, Frankfurt, Germany
| | - Diba Rafii
- Institut für Kardiovaskuläre Physiologie, Fachbereich Medizin der Goethe-Universität, Theodor-Stern-Kai 7, 60590, Frankfurt am Main, Germany
- German Center of Cardiovascular Research (DZHK), Partner Site RheinMain, Frankfurt, Germany
| | - Giulia Buchmann
- Institut für Kardiovaskuläre Physiologie, Fachbereich Medizin der Goethe-Universität, Theodor-Stern-Kai 7, 60590, Frankfurt am Main, Germany
- German Center of Cardiovascular Research (DZHK), Partner Site RheinMain, Frankfurt, Germany
| | - Minh Duc Pham
- Genome Biologics, Frankfurt, Germany
- Institute for Cardiovascular Regeneration, Goethe University, Frankfurt, Germany
| | - Zahraa S Msheik
- Max-Planck-Institute for Heart and Lung Research, Bad Nauheim, Germany
- Department of Internal Medicine, Member of the DZL, Member of Cardio-Pulmonary Institute (CPI), Justus Liebig University, Giessen, Germany
| | - Tianfu Li
- Institut für Kardiovaskuläre Physiologie, Fachbereich Medizin der Goethe-Universität, Theodor-Stern-Kai 7, 60590, Frankfurt am Main, Germany
- German Center of Cardiovascular Research (DZHK), Partner Site RheinMain, Frankfurt, Germany
| | - Sandra Seredinski
- Institut für Kardiovaskuläre Physiologie, Fachbereich Medizin der Goethe-Universität, Theodor-Stern-Kai 7, 60590, Frankfurt am Main, Germany
- German Center of Cardiovascular Research (DZHK), Partner Site RheinMain, Frankfurt, Germany
| | - Shaza Haydar
- Institut für Kardiovaskuläre Physiologie, Fachbereich Medizin der Goethe-Universität, Theodor-Stern-Kai 7, 60590, Frankfurt am Main, Germany
- German Center of Cardiovascular Research (DZHK), Partner Site RheinMain, Frankfurt, Germany
| | - Sepide Kashefiolasl
- Department of Neurosurgery, University Hospital Frankfurt, Frankfurt, Germany
| | - Karl H Plate
- Institute of Neurology (Edinger Institute), Neuroscience Center, Goethe University, Frankfurt, Germany
- Frankfurt Cancer Institute, University Hospital, Goethe University, Frankfurt, Germany
- German Cancer Consortium (DKTK), Partner Site Frankfurt/Mainz, Frankfurt, Germany
- German Cancer Research Centre (DKFZ), Heidelberg, Germany
| | - Rüdiger Behr
- DZHK (German Center for Cardiovascular Research), Partner Site Göttingen, Göttingen, Germany
- Platform Degenerative Diseases, German Primate Center-Leibniz Institute for Primate Research, Göttingen, Germany
| | - Matthias Mietsch
- DZHK (German Center for Cardiovascular Research), Partner Site Göttingen, Göttingen, Germany
- Laboratory Animal Science Unit, German Primate Center, Leibniz Institute for Primate Research, Göttingen, Germany
| | - Jaya Krishnan
- German Center of Cardiovascular Research (DZHK), Partner Site RheinMain, Frankfurt, Germany
- Institute for Cardiovascular Regeneration, Goethe University, Frankfurt, Germany
- Cardio-Pulmonary Institute, Giessen, Germany
- Department of Medicine III, Cardiology/Angiology/Nephrology, Goethe University Hospital, Frankfurt am Main, Germany
| | - Soni S Pullamsetti
- Max-Planck-Institute for Heart and Lung Research, Bad Nauheim, Germany
- Department of Internal Medicine, Member of the DZL, Member of Cardio-Pulmonary Institute (CPI), Justus Liebig University, Giessen, Germany
- Cardio-Pulmonary Institute, Giessen, Germany
| | - Sofia-Iris Bibli
- German Center of Cardiovascular Research (DZHK), Partner Site RheinMain, Frankfurt, Germany
- Institute for Vascular Signalling, Goethe University, Frankfurt, Germany
| | - Rabea Hinkel
- DZHK (German Center for Cardiovascular Research), Partner Site Göttingen, Göttingen, Germany
- Laboratory Animal Science Unit, German Primate Center, Leibniz Institute for Primate Research, Göttingen, Germany
- Institute for Animal Hygiene, Animal Welfare and Farm Animal Behavior, University of Veterinary Medicine, Hannover, Germany
| | - Andrew H Baker
- Centre for Cardiovascular Science, The Queen's Medical Research Institute, University of Edinburgh, Edinburgh, Scotland
- CARIM Institute, University of Maastricht, Maastricht, The Netherlands
| | - Reinier A Boon
- German Center of Cardiovascular Research (DZHK), Partner Site RheinMain, Frankfurt, Germany
- Institute for Cardiovascular Regeneration, Goethe University, Frankfurt, Germany
- Department of Physiology, Amsterdam Cardiovascular Sciences, VU Medical Center, Amsterdam UMC, Amsterdam, The Netherlands
| | - Marcel H Schulz
- German Center of Cardiovascular Research (DZHK), Partner Site RheinMain, Frankfurt, Germany
- Institute for Cardiovascular Regeneration, Goethe University, Frankfurt, Germany
| | - Ilka Wittig
- Institut für Kardiovaskuläre Physiologie, Fachbereich Medizin der Goethe-Universität, Theodor-Stern-Kai 7, 60590, Frankfurt am Main, Germany
- German Center of Cardiovascular Research (DZHK), Partner Site RheinMain, Frankfurt, Germany
| | - Francis J Miller
- Department of Medicine, Vanderbilt University Medical Center, Nashville, USA
- Veterans Affairs Medical Center, Nashville, TN, USA
| | - Ralf P Brandes
- Institut für Kardiovaskuläre Physiologie, Fachbereich Medizin der Goethe-Universität, Theodor-Stern-Kai 7, 60590, Frankfurt am Main, Germany.
- German Center of Cardiovascular Research (DZHK), Partner Site RheinMain, Frankfurt, Germany.
| | - Matthias S Leisegang
- Institut für Kardiovaskuläre Physiologie, Fachbereich Medizin der Goethe-Universität, Theodor-Stern-Kai 7, 60590, Frankfurt am Main, Germany.
- German Center of Cardiovascular Research (DZHK), Partner Site RheinMain, Frankfurt, Germany.
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21
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Mesenchymal Stem/Stromal Cells in Three-Dimensional Cell Culture: Ion Homeostasis and Ouabain-Induced Apoptosis. Biomedicines 2023; 11:biomedicines11020301. [PMID: 36830836 PMCID: PMC9953635 DOI: 10.3390/biomedicines11020301] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/18/2022] [Revised: 01/15/2023] [Accepted: 01/16/2023] [Indexed: 01/24/2023] Open
Abstract
This study describes the changes in ion homeostasis of human endometrial mesenchymal stem/stromal cells (eMSCs) during the formation of three-dimensional (3D) cell structures (spheroids) and investigates the conditions for apoptosis induction in 3D eMSCs. Detached from the monolayer culture, (2D) eMSCs accumulate Na+ and have dissipated transmembrane ion gradients, while in compact spheroids, eMSCs restore the lower Na+ content and the high K/Na ratio characteristic of functionally active cells. Organized as spheroids, eMSCs are non-proliferating cells with an active Na/K pump and a lower K+ content per g cell protein, which is typical for quiescent cells and a mean lower water content (lower hydration) in 3D eMSCs. Further, eMSCs in spheroids were used to evaluate the role of K+ depletion and cellular signaling context in the induction of apoptosis. In both 2D and 3D eMSCs, treatment with ouabain (1 µM) results in inhibition of pump-mediated K+ uptake and severe K+ depletion as well as disruption of the mitochondrial membrane potential. In 3D eMSCs (but not in 2D eMSCs), ouabain initiates apoptosis via the mitochondrial pathway. It is concluded that, when blocking the Na/K pump, cardiac glycosides prime mitochondria to apoptosis, and whether a cell enters the apoptotic pathway depends on the cell-specific signaling context, which includes the type of apoptotic protein expressed.
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22
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Tutty MA, Prina-Mello A. Three-Dimensional Spheroids for Cancer Research. Methods Mol Biol 2023; 2645:65-103. [PMID: 37202612 DOI: 10.1007/978-1-0716-3056-3_3] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 05/20/2023]
Abstract
In vitro cell culture is one of the most widely used tools used today for increasing our understanding of various things such as protein production, mechanisms of drug action, tissue engineering, and overall cellular biology. For the past decades, however, cancer researchers have relied heavily on conventional two-dimensional (2D) monolayer culture techniques to test a variety of aspects of cancer research ranging from the cytotoxic effects of antitumor drugs to the toxicity of diagnostic dyes and contact tracers. However, many promising cancer therapies have either weak or no efficacy in real-life conditions, therefore delaying or stopping altogether their translating to the clinic. This is, in part, due to the reductionist 2D cultures used to test these materials, which lack appropriate cell-cell contacts, have altered signaling, do not represent the natural tumor microenvironment, and have different drug responses, due to their reduced malignant phenotype when compared to real in vivo tumors. With the most recent advances, cancer research has moved into 3D biological investigation. Three-dimensional (3D) cultures of cancer cells not only recapitulate the in vivo environment better than their 2D counterparts, but they have, in recent years, emerged as a relatively low-cost and scientifically accurate methodology for studying cancer. In this chapter, we highlight the importance of 3D culture, specifically 3D spheroid culture, reviewing some key methodologies for forming 3D spheroids, discussing the experimental tools that can be used in conjunction with 3D spheroids and finally their applications in cancer research.
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Affiliation(s)
- Melissa Anne Tutty
- Laboratory for Biological Characterization of Advanced Materials (LBCAM), Trinity Translational Medicine Institute, Trinity Centre for Health Sciences, Trinity College Dublin, Dublin, Ireland.
| | - Adriele Prina-Mello
- Laboratory for Biological Characterization of Advanced Materials (LBCAM), Trinity Translational Medicine Institute, Trinity Centre for Health Sciences, Trinity College Dublin, Dublin, Ireland
- Nanomedicine and Molecular Imaging Group, Trinity Translational Medicine Institute, (TTMI), School of Medicine, Trinity College Dublin, Dublin, Ireland
- Trinity St. James's Cancer Institute, St. James's Hospital, Trinity College Dublin, Dublin, Ireland
- Advanced Materials and Bioengineering Research (AMBER) Centre, CRANN Institute, Trinity College Dublin, Dublin, Ireland
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23
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Griffin KH, Fok SW, Kent Leach J. Strategies to capitalize on cell spheroid therapeutic potential for tissue repair and disease modeling. NPJ Regen Med 2022; 7:70. [PMID: 36494368 PMCID: PMC9734656 DOI: 10.1038/s41536-022-00266-z] [Citation(s) in RCA: 32] [Impact Index Per Article: 10.7] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/10/2022] [Accepted: 11/29/2022] [Indexed: 12/13/2022] Open
Abstract
Cell therapies offer a tailorable, personalized treatment for use in tissue engineering to address defects arising from trauma, inefficient wound repair, or congenital malformation. However, most cell therapies have achieved limited success to date. Typically injected in solution as monodispersed cells, transplanted cells exhibit rapid cell death or insufficient retention at the site, thereby limiting their intended effects to only a few days. Spheroids, which are dense, three-dimensional (3D) aggregates of cells, enhance the beneficial effects of cell therapies by increasing and prolonging cell-cell and cell-matrix signaling. The use of spheroids is currently under investigation for many cell types. Among cells under evaluation, spheroids formed of mesenchymal stromal cells (MSCs) are particularly promising. MSC spheroids not only exhibit increased cell survival and retained differentiation, but they also secrete a potent secretome that promotes angiogenesis, reduces inflammation, and attracts endogenous host cells to promote tissue regeneration and repair. However, the clinical translation of spheroids has lagged behind promising preclinical outcomes due to hurdles in their formation, instruction, and use that have yet to be overcome. This review will describe the current state of preclinical spheroid research and highlight two key examples of spheroid use in clinically relevant disease modeling. It will highlight techniques used to instruct the phenotype and function of spheroids, describe current limitations to their use, and offer suggestions for the effective translation of cell spheroids for therapeutic treatments.
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Affiliation(s)
- Katherine H Griffin
- Department of Orthopaedic Surgery, UC Davis Health, Sacramento, CA, 95817, USA
- School of Veterinary Medicine, University of California, Davis, Davis, CA, 95616, USA
| | - Shierly W Fok
- Department of Orthopaedic Surgery, UC Davis Health, Sacramento, CA, 95817, USA
| | - J Kent Leach
- Department of Orthopaedic Surgery, UC Davis Health, Sacramento, CA, 95817, USA.
- Department of Biomedical Engineering, University of California, Davis, Davis, CA, 95616, USA.
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24
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Gerwin N, Scotti C, Halleux C, Fornaro M, Elliott J, Zhang Y, Johnson K, Shi J, Walter S, Li Y, Jacobi C, Laplanche N, Belaud M, Paul J, Glowacki G, Peters T, Wharton KA, Vostiar I, Polus F, Kramer I, Guth S, Seroutou A, Choudhury S, Laurent D, Gimbel J, Goldhahn J, Schieker M, Brachat S, Roubenoff R, Kneissel M. Angiopoietin-like 3-derivative LNA043 for cartilage regeneration in osteoarthritis: a randomized phase 1 trial. Nat Med 2022; 28:2633-2645. [PMID: 36456835 PMCID: PMC9800282 DOI: 10.1038/s41591-022-02059-9] [Citation(s) in RCA: 25] [Impact Index Per Article: 8.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/06/2021] [Accepted: 09/28/2022] [Indexed: 12/02/2022]
Abstract
Osteoarthritis (OA) is a common, debilitating, chronic disease with no disease-modifying drug approved to date. We discovered LNA043-a derivative of angiopoietin-like 3 (ANGPTL3)-as a potent chondrogenesis inducer using a phenotypic screen with human mesenchymal stem cells. We show that LNA043 promotes chondrogenesis and cartilage matrix synthesis in vitro and regenerates hyaline articular cartilage in preclinical OA and cartilage injury models in vivo. LNA043 exerts at least part of these effects through binding to the fibronectin receptor, integrin α5β1 on mesenchymal stem cells and chondrocytes. In a first-in-human (phase 1), randomized, double-blinded, placebo-controlled, single ascending dose, single-center trial ( NCT02491281 ; sponsored by Novartis Pharmaceuticals), 28 patients with knee OA were injected intra-articularly with LNA043 or placebo (3:1 ratio) either 2 h, 7 d or 21 d before total knee replacement. LNA043 met its primary safety endpoint and showed short serum pharmacokinetics, cartilage penetration and a lack of immunogenicity (secondary endpoints). Post-hoc transcriptomics profiling of cartilage revealed that a single LNA043 injection reverses the OA transcriptome signature over at least 21 d, inducing the expression of hyaline cartilage matrix components and anabolic signaling pathways, while suppressing mediators of OA progression. LNA043 is a novel disease-modifying OA drug candidate that is currently in a phase 2b trial ( NCT04864392 ) in patients with knee OA.
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Affiliation(s)
- Nicole Gerwin
- Novartis Institutes for BioMedical Research, Basel, Switzerland.
| | | | | | - Mara Fornaro
- Novartis Institutes for BioMedical Research, Basel, Switzerland
| | - Jimmy Elliott
- Novartis Institutes for BioMedical Research, San Diego, CA, USA
| | - Yunyu Zhang
- Novartis Institutes for BioMedical Research, Cambridge, MA, USA
| | | | - Jian Shi
- Novartis Institutes for BioMedical Research, San Diego, CA, USA
| | - Sandra Walter
- Novartis Institutes for BioMedical Research, Basel, Switzerland
| | - Yufei Li
- Novartis Institutes for BioMedical Research, Basel, Switzerland
| | - Carsten Jacobi
- Novartis Institutes for BioMedical Research, Basel, Switzerland
| | - Nelly Laplanche
- Novartis Institutes for BioMedical Research, Basel, Switzerland
| | - Magali Belaud
- Novartis Institutes for BioMedical Research, Basel, Switzerland
| | | | | | - Thomas Peters
- Novartis Institutes for BioMedical Research, Basel, Switzerland
| | | | - Igor Vostiar
- Novartis Institutes for BioMedical Research, Basel, Switzerland
| | - Florine Polus
- Novartis Institutes for BioMedical Research, Basel, Switzerland
| | - Ina Kramer
- Novartis Institutes for BioMedical Research, Basel, Switzerland
| | - Sabine Guth
- Novartis Institutes for BioMedical Research, Basel, Switzerland
| | | | | | - Didier Laurent
- Novartis Institutes for BioMedical Research, Basel, Switzerland
| | | | - Jörg Goldhahn
- Institute for Translational Medicine, ETH Zürich, Zürich, Switzerland
| | | | - Sophie Brachat
- Novartis Institutes for BioMedical Research, Basel, Switzerland
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25
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Oo JA, Pálfi K, Warwick T, Wittig I, Prieto-Garcia C, Matkovic V, Tomašković I, Boos F, Izquierdo Ponce J, Teichmann T, Petriukov K, Haydar S, Maegdefessel L, Wu Z, Pham MD, Krishnan J, Baker AH, Günther S, Ulrich HD, Dikic I, Leisegang MS, Brandes RP. Long non-coding RNA PCAT19 safeguards DNA in quiescent endothelial cells by preventing uncontrolled phosphorylation of RPA2. Cell Rep 2022; 41:111670. [PMID: 36384122 PMCID: PMC9681662 DOI: 10.1016/j.celrep.2022.111670] [Citation(s) in RCA: 13] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/29/2022] [Revised: 08/18/2022] [Accepted: 09/24/2022] [Indexed: 11/17/2022] Open
Abstract
In healthy vessels, endothelial cells maintain a stable, differentiated, and growth-arrested phenotype for years. Upon injury, a rapid phenotypic switch facilitates proliferation to restore tissue perfusion. Here we report the identification of the endothelial cell-enriched long non-coding RNA (lncRNA) PCAT19, which contributes to the proliferative switch and acts as a safeguard for the endothelial genome. PCAT19 is enriched in confluent, quiescent endothelial cells and binds to the full replication protein A (RPA) complex in a DNA damage- and cell-cycle-related manner. Our results suggest that PCAT19 limits the phosphorylation of RPA2, primarily on the serine 33 (S33) residue, and thereby facilitates an appropriate DNA damage response while slowing cell cycle progression. Reduction in PCAT19 levels in response to either loss of cell contacts or knockdown promotes endothelial proliferation and angiogenesis. Collectively, PCAT19 acts as a dynamic guardian of the endothelial genome and facilitates rapid switching from quiescence to proliferation.
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Affiliation(s)
- James A Oo
- Institute for Cardiovascular Physiology, Goethe University, Theodor-Stern-Kai 7, 60596 Frankfurt, Germany; German Center of Cardiovascular Research (DZHK), Partner Site RheinMain, Frankfurt, Germany
| | - Katalin Pálfi
- Institute for Cardiovascular Physiology, Goethe University, Theodor-Stern-Kai 7, 60596 Frankfurt, Germany; German Center of Cardiovascular Research (DZHK), Partner Site RheinMain, Frankfurt, Germany
| | - Timothy Warwick
- Institute for Cardiovascular Physiology, Goethe University, Theodor-Stern-Kai 7, 60596 Frankfurt, Germany; German Center of Cardiovascular Research (DZHK), Partner Site RheinMain, Frankfurt, Germany
| | - Ilka Wittig
- Institute for Cardiovascular Physiology, Goethe University, Theodor-Stern-Kai 7, 60596 Frankfurt, Germany; German Center of Cardiovascular Research (DZHK), Partner Site RheinMain, Frankfurt, Germany; Functional Proteomics, Institute for Cardiovascular Physiology, Goethe University, 60596 Frankfurt, Germany
| | - Cristian Prieto-Garcia
- Institute of Biochemistry II, Faculty of Medicine, Goethe University, 60596 Frankfurt, Germany
| | - Vigor Matkovic
- Institute of Biochemistry II, Faculty of Medicine, Goethe University, 60596 Frankfurt, Germany; Buchmann Institute for Molecular Life Sciences, Goethe University, 60438 Frankfurt, Germany
| | - Ines Tomašković
- Institute of Biochemistry II, Faculty of Medicine, Goethe University, 60596 Frankfurt, Germany
| | - Frederike Boos
- Institute for Cardiovascular Physiology, Goethe University, Theodor-Stern-Kai 7, 60596 Frankfurt, Germany; German Center of Cardiovascular Research (DZHK), Partner Site RheinMain, Frankfurt, Germany
| | - Judit Izquierdo Ponce
- Institute for Cardiovascular Physiology, Goethe University, Theodor-Stern-Kai 7, 60596 Frankfurt, Germany
| | - Tom Teichmann
- Institute for Cardiovascular Physiology, Goethe University, Theodor-Stern-Kai 7, 60596 Frankfurt, Germany; German Center of Cardiovascular Research (DZHK), Partner Site RheinMain, Frankfurt, Germany
| | | | - Shaza Haydar
- Institute for Cardiovascular Physiology, Goethe University, Theodor-Stern-Kai 7, 60596 Frankfurt, Germany; German Center of Cardiovascular Research (DZHK), Partner Site RheinMain, Frankfurt, Germany
| | - Lars Maegdefessel
- Department of Vascular and Endovascular Surgery, Klinikum rechts der Isar-Technical University Munich, 81675 Munich, Germany; German Center of Cardiovascular Research (DZHK), Partner Site Munich, Munich, Germany
| | - Zhiyuan Wu
- Department of Vascular and Endovascular Surgery, Klinikum rechts der Isar-Technical University Munich, 81675 Munich, Germany; German Center of Cardiovascular Research (DZHK), Partner Site Munich, Munich, Germany
| | - Minh Duc Pham
- Institute of Cardiovascular Regeneration, Center for Molecular Medicine, Goethe University, 60596 Frankfurt, Germany; Genome Biologics, Theodor-Stern-Kai 7, 60596 Frankfurt, Germany
| | - Jaya Krishnan
- German Center of Cardiovascular Research (DZHK), Partner Site RheinMain, Frankfurt, Germany; Institute of Cardiovascular Regeneration, Center for Molecular Medicine, Goethe University, 60596 Frankfurt, Germany; Cardio-Pulmonary Institute, Giessen, Germany
| | - Andrew H Baker
- The Queen's Medical Research Institute, Centre for Cardiovascular Science, University of Edinburgh, Edinburgh EH16 4TJ, Scotland; CARIM Institute, University of Maastricht, Universiteitssingel 50, 6200 Maastricht, the Netherlands
| | - Stefan Günther
- Max Planck Institute for Heart and Lung Research, 61231 Bad Nauheim, Germany
| | - Helle D Ulrich
- Institute of Molecular Biology (IMB), 55128 Mainz, Germany
| | - Ivan Dikic
- Institute of Biochemistry II, Faculty of Medicine, Goethe University, 60596 Frankfurt, Germany; Buchmann Institute for Molecular Life Sciences, Goethe University, 60438 Frankfurt, Germany; Max Planck Institute of Biophysics, Max-von-Laue Straße 3, 60438 Frankfurt, Germany
| | - Matthias S Leisegang
- Institute for Cardiovascular Physiology, Goethe University, Theodor-Stern-Kai 7, 60596 Frankfurt, Germany; German Center of Cardiovascular Research (DZHK), Partner Site RheinMain, Frankfurt, Germany.
| | - Ralf P Brandes
- Institute for Cardiovascular Physiology, Goethe University, Theodor-Stern-Kai 7, 60596 Frankfurt, Germany; German Center of Cardiovascular Research (DZHK), Partner Site RheinMain, Frankfurt, Germany.
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HIF1α-AS1 is a DNA:DNA:RNA triplex-forming lncRNA interacting with the HUSH complex. Nat Commun 2022; 13:6563. [PMID: 36323673 PMCID: PMC9630315 DOI: 10.1038/s41467-022-34252-2] [Citation(s) in RCA: 22] [Impact Index Per Article: 7.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/29/2021] [Accepted: 10/19/2022] [Indexed: 11/07/2022] Open
Abstract
DNA:DNA:RNA triplexes that are formed through Hoogsteen base-pairing of the RNA in the major groove of the DNA duplex have been observed in vitro, but the extent to which these interactions occur in cells and how they impact cellular functions remains elusive. Using a combination of bioinformatic techniques, RNA/DNA pulldown and biophysical studies, we set out to identify functionally important DNA:DNA:RNA triplex-forming long non-coding RNAs (lncRNA) in human endothelial cells. The lncRNA HIF1α-AS1 was retrieved as a top hit. Endogenous HIF1α-AS1 reduces the expression of numerous genes, including EPH Receptor A2 and Adrenomedullin through DNA:DNA:RNA triplex formation by acting as an adapter for the repressive human silencing hub complex (HUSH). Moreover, the oxygen-sensitive HIF1α-AS1 is down-regulated in pulmonary hypertension and loss-of-function approaches not only result in gene de-repression but also enhance angiogenic capacity. As exemplified here with HIF1α-AS1, DNA:DNA:RNA triplex formation is a functionally important mechanism of trans-acting gene expression control.
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Koivunotko E, Snirvi J, Merivaara A, Harjumäki R, Rautiainen S, Kelloniemi M, Kuismanen K, Miettinen S, Yliperttula M, Koivuniemi R. Angiogenic Potential of Human Adipose-Derived Mesenchymal Stromal Cells in Nanofibrillated Cellulose Hydrogel. Biomedicines 2022; 10:2584. [PMID: 36289846 PMCID: PMC9599553 DOI: 10.3390/biomedicines10102584] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/11/2022] [Revised: 10/05/2022] [Accepted: 10/13/2022] [Indexed: 11/16/2022] Open
Abstract
Adipose-derived mesenchymal stromal cells (ASCs) hold great potential for cellular therapies by having immunomodulatory behavior and tissue regenerative properties. Due to the capability of ASCs to differentiate into endothelial cells (ECs) and other angiogenic cell types, such as pericytes, ASCs are a highly valuable source for stimulating angiogenesis. However, cellular therapies in tissue engineering have faced challenges in poor survival of the cells after transplantation, which is why a protective biomaterial scaffold is required. In this work, we studied the potential of nanofibrillated cellulose (NFC) hydrogel to be utilized as a suitable matrix for three-dimensional (3D) cell culturing of human-derived ASCs (hASCs) and studied their angiogenic properties and differentiation potential in ECs and pericytes. In addition, we tested the effect of hASC-conditioned medium and stimulation with angiopoietin-1 (Ang-1) on human umbilical vein endothelial cells (HUVECs) to induce blood vessel-type tube formation in NFC hydrogel. The hASCs were successfully 3D cell cultured in NFC hydrogel as they formed spheroids and had high cell viability with angiogenic features. Most importantly, they showed angiogenic potential by having pericyte-like characteristics when differentiated in EC medium, and their conditioned medium improved HUVEC viability and tube formation, which recalls the active paracrine properties. This study recommends NFC hydrogel for future use as an animal-free biomaterial scaffold for hASCs in therapeutic angiogenesis and other cell therapy purposes.
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Affiliation(s)
- Elle Koivunotko
- Drug Research Program, Division of Pharmaceutical Biosciences, Faculty of Pharmacy, University of Helsinki, 00790 Helsinki, Finland
| | - Jasmi Snirvi
- Drug Research Program, Division of Pharmaceutical Biosciences, Faculty of Pharmacy, University of Helsinki, 00790 Helsinki, Finland
| | - Arto Merivaara
- Drug Research Program, Division of Pharmaceutical Biosciences, Faculty of Pharmacy, University of Helsinki, 00790 Helsinki, Finland
| | - Riina Harjumäki
- Drug Research Program, Division of Pharmaceutical Biosciences, Faculty of Pharmacy, University of Helsinki, 00790 Helsinki, Finland
| | - Swarna Rautiainen
- Drug Research Program, Division of Pharmaceutical Biosciences, Faculty of Pharmacy, University of Helsinki, 00790 Helsinki, Finland
| | - Minna Kelloniemi
- Department of Plastic and Reconstructive Surgery, Tampere University Hospital, 33520 Tampere, Finland
| | - Kirsi Kuismanen
- Department of Obstetrics and Gynecology, Tampere University Hospital, 33520 Tampere, Finland
| | - Susanna Miettinen
- Faculty of Medicine and Health Technologies, University of Tampere, 33520 Tampere, Finland
- Research, Development and Innovation Centre, Tampere University Hospital, 33520 Tampere, Finland
| | - Marjo Yliperttula
- Drug Research Program, Division of Pharmaceutical Biosciences, Faculty of Pharmacy, University of Helsinki, 00790 Helsinki, Finland
| | - Raili Koivuniemi
- Drug Research Program, Division of Pharmaceutical Biosciences, Faculty of Pharmacy, University of Helsinki, 00790 Helsinki, Finland
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Laschke MW, Gu Y, Menger MD. Replacement in angiogenesis research: Studying mechanisms of blood vessel development by animal-free in vitro, in vivo and in silico approaches. Front Physiol 2022; 13:981161. [PMID: 36060683 PMCID: PMC9428454 DOI: 10.3389/fphys.2022.981161] [Citation(s) in RCA: 6] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/29/2022] [Accepted: 07/21/2022] [Indexed: 01/10/2023] Open
Abstract
Angiogenesis, the development of new blood vessels from pre-existing ones, is an essential process determining numerous physiological and pathological conditions. Accordingly, there is a high demand for research approaches allowing the investigation of angiogenic mechanisms and the assessment of pro- and anti-angiogenic therapeutics. The present review provides a selective overview and critical discussion of such approaches, which, in line with the 3R principle, all share the common feature that they are not based on animal experiments. They include in vitro assays to study the viability, proliferation, migration, tube formation and sprouting activity of endothelial cells in two- and three-dimensional environments, the degradation of extracellular matrix compounds as well as the impact of hemodynamic forces on blood vessel formation. These assays can be complemented by in vivo analyses of microvascular network formation in the chorioallantoic membrane assay and early stages of zebrafish larvae. In addition, the combination of experimental data and physical laws enables the mathematical modeling of tissue-specific vascularization, blood flow patterns, interstitial fluid flow as well as oxygen, nutrient and drug distribution. All these animal-free approaches markedly contribute to an improved understanding of fundamental biological mechanisms underlying angiogenesis. Hence, they do not only represent essential tools in basic science but also in early stages of drug development. Moreover, their advancement bears the great potential to analyze angiogenesis in all its complexity and, thus, to make animal experiments superfluous in the future.
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Islam S, Parker J, Dash BC, Hsia HC. Human iPSC-Vascular smooth muscle cell spheroids demonstrate size-dependent alterations in cellular viability and secretory function. J Biomed Mater Res A 2022; 110:1813-1823. [PMID: 35815599 DOI: 10.1002/jbm.a.37423] [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: 03/31/2022] [Revised: 06/08/2022] [Accepted: 06/15/2022] [Indexed: 11/11/2022]
Abstract
Human-induced pluripotent stem cells (hiPSC) and their differentiated vascular cells have been revolutionizing the field of regenerative wound healing. These cells are shown to be rejuvenated with immense potentials in secreting paracrine factors. Recently, hiPSC-derived vascular smooth muscle cells (hiPSC-VSMC) have shown regenerative wound healing ability via their paracrine secretion. The quest to modulate the secretory function of these hiPSC-VSMC is an ongoing effort and involves the use of both biochemical and biophysical stimuli. This study explores the development and optimization of a reproducible, inexpensive protocol to form hiPSC-VSMC derived spheroids to investigate the implications of spheroid size on viability and paracrine secretion. Our data show the successful formation of different sizes of spheroids using various amount of hiPSC-VSMC. The hiPSC-VSMC spheroids formed with 10,000 cells strike an ideal balance between overall cell health and maximal paracrine secretion. The conditioned medium from these spheroids was found to be bioactive in enhancing human dermal fibroblast cell proliferation and migration. This research will inform future studies on the optimal spheroid size for regenerative wound healing applications.
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Affiliation(s)
- Sara Islam
- Section of Plastic Surgery, Department of Surgery, Yale School of Medicine, Yale University, New Haven, Connecticut, USA
| | - Jackson Parker
- Section of Plastic Surgery, Department of Surgery, Yale School of Medicine, Yale University, New Haven, Connecticut, USA
| | - Biraja C Dash
- Section of Plastic Surgery, Department of Surgery, Yale School of Medicine, Yale University, New Haven, Connecticut, USA
| | - Henry C Hsia
- Section of Plastic Surgery, Department of Surgery, Yale School of Medicine, Yale University, New Haven, Connecticut, USA.,Department of Biomedical Engineering, Yale University, New Haven, Connecticut, USA
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Hauke M, Eckenstaler R, Ripperger A, Ender A, Braun H, Benndorf RA. Active RhoA Exerts an Inhibitory Effect on the Homeostasis and Angiogenic Capacity of Human Endothelial Cells. J Am Heart Assoc 2022; 11:e025119. [PMID: 35699166 PMCID: PMC9238636 DOI: 10.1161/jaha.121.025119] [Citation(s) in RCA: 10] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/30/2022]
Abstract
Background The small GTPase RhoA (Ras homolog gene family, member A) regulates a variety of cellular processes, including cell motility, proliferation, survival, and permeability. In addition, there are reports indicating that RhoA‐ROCK (rho associated coiled‐coil containing protein kinase) activation is essential for VEGF (vascular endothelial growth factor)‐mediated angiogenesis, whereas other work suggests VEGF‐antagonistic effects of the RhoA‐ROCK axis. Methods and Results To elucidate this issue, we examined human umbilical vein endothelial cells and human coronary artery endothelial cells after stable overexpression (lentiviral transduction) of constitutively active (G14V/Q63L), dominant‐negative (T19N), or wild‐type RhoA using a series of in vitro angiogenesis assays (proliferation, migration, tube formation, angiogenic sprouting, endothelial cell viability) and a human umbilical vein endothelial cells xenograft assay in immune‐incompetent NOD scid gamma mice in vivo. Here, we report that expression of active and wild‐type RhoA but not dominant‐negative RhoA significantly inhibited endothelial cell proliferation, migration, tube formation, and angiogenic sprouting in vitro. Moreover, active RhoA increased endothelial cell death in vitro and decreased human umbilical vein endothelial cell‐related angiogenesis in vivo. Inhibition of RhoA by C3 transferase antagonized the inhibitory effects of RhoA and strongly enhanced VEGF‐induced angiogenic sprouting in control‐treated cells. In contrast, inhibition of RhoA effectors ROCK1/2 and LIMK1/2 (LIM domain kinase 1/2) did not significantly affect RhoA‐related effects, but increased angiogenic sprouting and migration of control‐treated cells. In agreement with these data, VEGF did not activate RhoA in human umbilical vein endothelial cells as measured by a Förster resonance energy transfer–based biosensor. Furthermore, global transcriptome and subsequent bioinformatic gene ontology enrichment analyses revealed that constitutively active RhoA induced a differentially expressed gene pattern that was enriched for gene ontology biological process terms associated with mitotic nuclear division, cell proliferation, cell motility, and cell adhesion, which included a significant decrease in VEGFR‐2 (vascular endothelial growth factor receptor 2) and NOS3 (nitric oxide synthase 3) expression. Conclusions Our data demonstrate that increased RhoA activity has the potential to trigger endothelial dysfunction and antiangiogenic effects independently of its well‐characterized downstream effectors ROCK and LIMK.
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Affiliation(s)
- Michael Hauke
- Department of Clinical Pharmacy and PharmacotherapyInstitute of PharmacyMartin‐Luther‐University Halle‐WittenbergHalle (Saale)Germany
| | - Robert Eckenstaler
- Department of Clinical Pharmacy and PharmacotherapyInstitute of PharmacyMartin‐Luther‐University Halle‐WittenbergHalle (Saale)Germany
| | - Anne Ripperger
- Department of Clinical Pharmacy and PharmacotherapyInstitute of PharmacyMartin‐Luther‐University Halle‐WittenbergHalle (Saale)Germany
| | - Anna Ender
- Department of Clinical Pharmacy and PharmacotherapyInstitute of PharmacyMartin‐Luther‐University Halle‐WittenbergHalle (Saale)Germany
| | - Heike Braun
- Department of Clinical Pharmacy and PharmacotherapyInstitute of PharmacyMartin‐Luther‐University Halle‐WittenbergHalle (Saale)Germany
| | - Ralf A. Benndorf
- Department of Clinical Pharmacy and PharmacotherapyInstitute of PharmacyMartin‐Luther‐University Halle‐WittenbergHalle (Saale)Germany
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Umapathy D, Karthikeyan MC, Ponnuchamy K, Kannan MK, Ganeshan M, Arockiam AJV. The absence of cellular glucose triggers oncogene AEG-1 that instigates VEGFC in HCC: A possible genetic root cause of angiogenesis. Gene X 2022; 826:146446. [PMID: 35337853 DOI: 10.1016/j.gene.2022.146446] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/20/2021] [Revised: 03/02/2022] [Accepted: 03/18/2022] [Indexed: 11/25/2022] Open
Abstract
BACKGROUND AND OBJECTIVE Astrocyte Elevated Gene-1 (AEG-1) is the master and multi-regulator of the various transcriptional factor primarily regulating chemoresistance, angiogenesis, metastasis, and invasion under the pathological condition, including liver cancer. This study was focused on investigating the process of tumor angiogenesis in liver carcinoma by studying the role of AEG-1 under GD/2DG conditions. METHOD AND RESULTS The PCR and western blot analysis revealed that glucose depletion (GD) induces the overexpression of AEG-1. Further, it leads to the constant expression of VEGFC through the activation of HIF-1α/CCR7 via the stimulations of PI3K/Akt signaling pathways. GLUT2 is the major transporter of a glucose molecule that is highly participating under GD through the expression of AEG-1 and constantly expresses glucokinase (GCK). The obtained data suggest that AEG-1 act as an angiogenesis and glycolysis regulator by modulating the expression of GCK through HIF-1α and GLUT2. 2-deoxy-D-glucose (2DG) is a glycolysis inhibitor that induces impaired glycolysis and cellular apoptosis by cellular oxidative stress. The administration of 2DG has led to the chemoresistance of AEG-1. CONCLUSION The total findings of the study judged that disruption of cellular energy metabolism induced by the absence of glucose or the presence of mutant glucose moiety (2DG) promotes the overexpression of AEG-1. The GD/2DG activates the VEGFC by inducing the HIF-1α and CCR7. Moreover, AEG-1 induces the expression of OPN, which regulates metastasis, angiogenesis, and actively participates in protective autophagy by promoting LC3 a/b.
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Affiliation(s)
- Devan Umapathy
- Department of Biochemistry, Molecular Oncology Laboratory, School of Life Sciences, Bharathidasan University, Tiruchirappalli 620 024, Tamil Nadu, India
| | - Mano Chitra Karthikeyan
- Department of Biochemistry, Molecular Oncology Laboratory, School of Life Sciences, Bharathidasan University, Tiruchirappalli 620 024, Tamil Nadu, India
| | - Kumar Ponnuchamy
- Department of Animal Health and Management, Food Chemistry and Molecular Cancer Biology Laboratory, Alagappa University, Karaikudi 630 003, Tamil Nadu, India
| | - Mahesh Kumar Kannan
- Department of Biochemistry, Molecular Oncology Laboratory, School of Life Sciences, Bharathidasan University, Tiruchirappalli 620 024, Tamil Nadu, India
| | - Mathan Ganeshan
- Cancer Biology Laboratory, Department of Biomedical Science, School of Life Sciences, Bharathidasan University, Tiruchirappalli 620 024, Tamil Nadu, India
| | - Antony Joseph Velanganni Arockiam
- Department of Biochemistry, Molecular Oncology Laboratory, School of Life Sciences, Bharathidasan University, Tiruchirappalli 620 024, Tamil Nadu, India.
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Kannan P, Schain M, Lane DP. An Automated Quantification Tool for Angiogenic Sprouting From Endothelial Spheroids. Front Pharmacol 2022; 13:883083. [PMID: 35571133 PMCID: PMC9093605 DOI: 10.3389/fphar.2022.883083] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/24/2022] [Accepted: 03/30/2022] [Indexed: 11/17/2022] Open
Abstract
The process of sprouting angiogenesis can be measured in vitro using endothelial cells in sprouting assays such as the fibrin bead assay and the spheroid-based assay. While the technical aspects of these sprouting assays have been well-optimized, the analysis aspects have been limited to manual methods, which can be time-consuming and difficult to reproduce. Here, we developed an automated analysis tool called AQuTAS to quantify sprouting parameters from the spheroid-based sprouting assay. We trained and validated the algorithm on two subsets of data, and tested its sensitivity by measuring changes in sprouting parameters over a range of concentrations of pro- and antiangiogenic compounds. Our results demonstrate that the algorithm detects known differences in sprouting parameters in endothelial spheroids treated with pro- and antiangiogenic compounds. Moreover, it is sensitive to biological changes that are ≥40%. Among the five quantified parameters, cumulative sprout length is likely the most discriminative parameter for measuring differences in sprouting behavior because it had the highest effect size (>1.5 Cohen’s d). In summary, we have generated an automated tool that quantifies sprouting parameters from the spheroid-based assay in a reproducible and sensitive manner.
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Affiliation(s)
- Pavitra Kannan
- Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Stockholm, Sweden
| | | | - David P Lane
- Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Stockholm, Sweden
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Li LK, Huang WC, Hsueh YY, Yamauchi K, Olivares N, Davila R, Fang J, Ding X, Zhao W, Soto J, Hasani M, Novitch B, Li S. Intramuscular delivery of neural crest stem cell spheroids enhances neuromuscular regeneration after denervation injury. Stem Cell Res Ther 2022; 13:205. [PMID: 35578348 PMCID: PMC9109326 DOI: 10.1186/s13287-022-02877-1] [Citation(s) in RCA: 7] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/02/2021] [Accepted: 03/28/2022] [Indexed: 11/12/2022] Open
Abstract
BACKGROUND Muscle denervation from trauma and motor neuron disease causes disabling morbidities. A limiting step in functional recovery is the regeneration of neuromuscular junctions (NMJs) for reinnervation. Stem cells have the potential to promote these regenerative processes, but current approaches have limited success, and the optimal types of stem cells remain to be determined. Neural crest stem cells (NCSCs), as the developmental precursors of the peripheral nervous system, are uniquely advantageous, but the role of NCSCs in neuromuscular regeneration is not clear. Furthermore, a cell delivery approach that can maintain NCSC survival upon transplantation is critical. METHODS We established a streamlined protocol to derive, isolate, and characterize functional p75+ NCSCs from human iPSCs without genome integration of reprogramming factors. To enhance survival rate upon delivery in vivo, NCSCs were centrifuged in microwell plates to form spheroids of desirable size by controlling suspension cell density. Human bone marrow mesenchymal stem cells (MSCs) were also studied for comparison. NCSC or MSC spheroids were injected into the gastrocnemius muscle with denervation injury, and the effects on NMJ formation and functional recovery were investigated. The spheroids were also co-cultured with engineered neuromuscular tissue to assess effects on NMJ formation in vitro. RESULTS NCSCs cultured in spheroids displayed enhanced secretion of soluble factors involved in neuromuscular regeneration. Intramuscular transplantation of spheroids enabled long-term survival and retention of NCSCs, in contrast to the transplantation of single-cell suspensions. Furthermore, NCSC spheroids significantly improved functional recovery after four weeks as shown by gait analysis, electrophysiology, and the rate of NMJ innervation. MSC spheroids, on the other hand, had insignificant effect. In vitro co-culture of NCSC or MSC spheroids with engineered myotubes and motor neurons further evidenced improved innervated NMJ formation with NCSC spheroids. CONCLUSIONS We demonstrate that stem cell type is critical for neuromuscular regeneration and that NCSCs have a distinct advantage and therapeutic potential to promote reinnervation following peripheral nerve injury. Biophysical effects of spheroidal culture, in particular, enable long-term NCSC survival following in vivo delivery. Furthermore, synthetic neuromuscular tissue, or "tissues-on-a-chip," may offer a platform to evaluate stem cells for neuromuscular regeneration.
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Affiliation(s)
- LeeAnn K Li
- Departments of Bioengineering and Department of Medicine, University of California, Los Angeles, USA
- David Geffen School of Medicine, University of California, Los Angeles, USA
| | - Wen-Chin Huang
- Departments of Bioengineering and Department of Medicine, University of California, Los Angeles, USA
| | - Yuan-Yu Hsueh
- Departments of Bioengineering and Department of Medicine, University of California, Los Angeles, USA
- Division of Plastic and Reconstructive Surgery, Department of Surgery, National Cheng Kung University Hospital, College of Medicine, National Cheng Kung University, Tainan, Taiwan
| | - Ken Yamauchi
- Department of Neurobiology, University of California, Los Angeles, USA
| | - Natalie Olivares
- Departments of Bioengineering and Department of Medicine, University of California, Los Angeles, USA
| | - Raul Davila
- Departments of Bioengineering and Department of Medicine, University of California, Los Angeles, USA
| | - Jun Fang
- Departments of Bioengineering and Department of Medicine, University of California, Los Angeles, USA
| | - Xili Ding
- Departments of Bioengineering and Department of Medicine, University of California, Los Angeles, USA
| | - Weikang Zhao
- Departments of Bioengineering and Department of Medicine, University of California, Los Angeles, USA
| | - Jennifer Soto
- Departments of Bioengineering and Department of Medicine, University of California, Los Angeles, USA
| | - Mahdi Hasani
- Departments of Bioengineering and Department of Medicine, University of California, Los Angeles, USA
| | - Bennett Novitch
- Department of Neurobiology, University of California, Los Angeles, USA
| | - Song Li
- Departments of Bioengineering and Department of Medicine, University of California, Los Angeles, USA.
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Eckenstaler R, Ripperger A, Hauke M, Braun H, Ergün S, Schwedhelm E, Benndorf RA. Thromboxane A 2 receptor activation via G α13-RhoA/C-ROCK-LIMK2-dependent signal transduction inhibits angiogenic sprouting of human endothelial cells. Biochem Pharmacol 2022; 201:115069. [PMID: 35525325 DOI: 10.1016/j.bcp.2022.115069] [Citation(s) in RCA: 6] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/08/2022] [Revised: 04/26/2022] [Accepted: 04/27/2022] [Indexed: 12/13/2022]
Abstract
We could previously show that thromboxane A2 receptor (TP) activation inhibits the angiogenic capacity of human endothelial cells, but the underlying mechanisms remained unclear. Therefore, the aim of this study was to elucidate TP signal transduction pathways relevant to angiogenic sprouting of human endothelial cells. To clarify this matter, we used RNAi-mediated gene silencing as well as pharmacological inhibition of potential TP downstream targets in human umbilical vein endothelial cells (HUVEC) and VEGF-induced angiogenic sprouting of HUVEC spheroids in vitro as a functional read-out. In this experimental set-up, the TP agonist U-46619 completely blocked VEGF-induced angiogenic sprouting of HUVEC spheroids. Moreover, in live-cell analyses TP activation induced endothelial cell contraction, sprout retraction as well as endothelial cell tension and focal adhesion dysregulation of HUVEC. These effects were reversed by pharmacological TP inhibition or TP knockdown. Moreover, we identified a TP-Gα13-RhoA/C-ROCK-LIMK2-dependent signal transduction pathway to be relevant for U-46619-induced inhibition of VEGF-mediated HUVEC sprouting. In line with these results, U-46619-mediated TP activation potently induced RhoA and RhoC activity in live HUVEC as measured by FRET biosensors. Interestingly, pharmacological inhibition of ROCK and LIMK2 also normalized U-46619-induced endothelial cell tension and focal adhesion dysregulation of HUVEC. In summary, our work reveals mechanisms by which the TP may disturb angiogenic endothelial function in disease states associated with sustained endothelial TP activation.
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Affiliation(s)
- Robert Eckenstaler
- Martin-Luther-University Halle-Wittenberg, Department of Clinical Pharmacy and Pharmacotherapy, Halle (Saale), Germany
| | - Anne Ripperger
- Martin-Luther-University Halle-Wittenberg, Department of Clinical Pharmacy and Pharmacotherapy, Halle (Saale), Germany
| | - Michael Hauke
- Martin-Luther-University Halle-Wittenberg, Department of Clinical Pharmacy and Pharmacotherapy, Halle (Saale), Germany
| | - Heike Braun
- Martin-Luther-University Halle-Wittenberg, Department of Clinical Pharmacy and Pharmacotherapy, Halle (Saale), Germany
| | - Süleyman Ergün
- Institute of Anatomy and Cell Biology, Julius-Maximilians-University, Würzburg, Germany
| | - Edzard Schwedhelm
- Institute of Clinical Pharmacology and Toxicology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
| | - Ralf A Benndorf
- Martin-Luther-University Halle-Wittenberg, Department of Clinical Pharmacy and Pharmacotherapy, Halle (Saale), Germany.
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Choi JU, Zhang X, Hasan MM, Karim M, Chung SW, Alam F, Alqahtani F, Reddy SY, Kim IS, Al-Hilal TA, Byun Y. Targeting angiogenic growth factors using therapeutic glycosaminoglycans on doppel-expressing endothelial cells for blocking angiogenic signaling in cancer. Biomaterials 2022; 283:121423. [DOI: 10.1016/j.biomaterials.2022.121423] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/24/2021] [Revised: 01/31/2022] [Accepted: 02/17/2022] [Indexed: 01/18/2023]
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Valdoz JC, Franks NA, Cribbs CG, Jacobs DJ, Dodson EL, Knight CJ, Poulson PD, Garfield SR, Johnson BC, Hemeyer BM, Sudo MT, Saunooke JA, Kartchner BC, Saxton A, Vallecillo-Zuniga ML, Santos M, Chamberlain B, Christensen KA, Nordin GP, Narayanan AS, Raghu G, Van Ry PM. Soluble ECM promotes organotypic formation in lung alveolar model. Biomaterials 2022; 283:121464. [DOI: 10.1016/j.biomaterials.2022.121464] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/16/2021] [Revised: 02/15/2022] [Accepted: 03/06/2022] [Indexed: 11/25/2022]
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Seredinski S, Boos F, Günther S, Oo JA, Warwick T, Izquierdo Ponce J, Lillich FF, Proschak E, Knapp S, Gilsbach R, Pflüger-Müller B, Brandes RP, Leisegang MS. DNA topoisomerase inhibition with the HIF inhibitor acriflavine promotes transcription of lncRNAs in endothelial cells. MOLECULAR THERAPY. NUCLEIC ACIDS 2022; 27:1023-1035. [PMID: 35228897 PMCID: PMC8844413 DOI: 10.1016/j.omtn.2022.01.016] [Citation(s) in RCA: 6] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 07/05/2021] [Accepted: 01/21/2022] [Indexed: 02/08/2023]
Abstract
The transcription factor hypoxia-inducible factor 1 (HIF1) is an important driver of cancer and is therefore an attractive drug target. Acriflavine (ACF) has been suggested to inhibit HIF1, but its mechanism of action is unknown. Here we investigated the interaction of ACF with DNA and long non-coding RNAs (lncRNAs) and its function in human endothelial cells. ACF promoted apoptosis and reduced proliferation, network formation, and angiogenic capacity. It also induced changes in gene expression, as determined by RNA sequencing (RNA-seq), which could not be attributed to specific inhibition of HIF1. A similar response was observed in murine lung endothelial cells. Although ACF increased and decreased a similar number of protein-coding genes, lncRNAs were preferentially upregulated under normoxic and hypoxic conditions. An assay for transposase accessibility with subsequent DNA sequencing (ATAC-seq) demonstrated that ACF induced strong changes in chromatin accessibility at lncRNA promoters. Immunofluorescence showed displacement of DNA:RNA hybrids. Such effects might be due to ACF-mediated topoisomerase inhibition, which was indeed the case, as reflected by DNA unwinding assays. Comparison with other acridine derivatives and topoisomerase inhibitors suggested that the specific function of ACF is an effect of acridinium-class compounds. This study demonstrates that ACF inhibits topoisomerases rather than HIF specifically and that it elicits a unique expression response of lncRNAs.
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Affiliation(s)
- Sandra Seredinski
- Institute for Cardiovascular Physiology, Goethe University, Theodor-Stern-Kai 7, 60590 Frankfurt, Germany.,German Center of Cardiovascular Research (DZHK), Partner Site RheinMain, Frankfurt, Germany
| | - Frederike Boos
- Institute for Cardiovascular Physiology, Goethe University, Theodor-Stern-Kai 7, 60590 Frankfurt, Germany.,German Center of Cardiovascular Research (DZHK), Partner Site RheinMain, Frankfurt, Germany
| | - Stefan Günther
- Max Planck Institute for Heart and Lung Research, 61231 Bad Nauheim, Germany
| | - James A Oo
- Institute for Cardiovascular Physiology, Goethe University, Theodor-Stern-Kai 7, 60590 Frankfurt, Germany.,German Center of Cardiovascular Research (DZHK), Partner Site RheinMain, Frankfurt, Germany
| | - Timothy Warwick
- Institute for Cardiovascular Physiology, Goethe University, Theodor-Stern-Kai 7, 60590 Frankfurt, Germany.,German Center of Cardiovascular Research (DZHK), Partner Site RheinMain, Frankfurt, Germany
| | - Judit Izquierdo Ponce
- Institute for Cardiovascular Physiology, Goethe University, Theodor-Stern-Kai 7, 60590 Frankfurt, Germany
| | - Felix F Lillich
- Institute of Pharmaceutical Chemistry, Goethe University, 60438 Frankfurt, Germany
| | - Ewgenij Proschak
- Institute of Pharmaceutical Chemistry, Goethe University, 60438 Frankfurt, Germany
| | - Stefan Knapp
- Institute of Pharmaceutical Chemistry, Goethe University, 60438 Frankfurt, Germany
| | - Ralf Gilsbach
- Institute for Cardiovascular Physiology, Goethe University, Theodor-Stern-Kai 7, 60590 Frankfurt, Germany.,German Center of Cardiovascular Research (DZHK), Partner Site RheinMain, Frankfurt, Germany
| | - Beatrice Pflüger-Müller
- Institute for Cardiovascular Physiology, Goethe University, Theodor-Stern-Kai 7, 60590 Frankfurt, Germany.,German Center of Cardiovascular Research (DZHK), Partner Site RheinMain, Frankfurt, Germany
| | - Ralf P Brandes
- Institute for Cardiovascular Physiology, Goethe University, Theodor-Stern-Kai 7, 60590 Frankfurt, Germany.,German Center of Cardiovascular Research (DZHK), Partner Site RheinMain, Frankfurt, Germany
| | - Matthias S Leisegang
- Institute for Cardiovascular Physiology, Goethe University, Theodor-Stern-Kai 7, 60590 Frankfurt, Germany.,German Center of Cardiovascular Research (DZHK), Partner Site RheinMain, Frankfurt, Germany
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Eckenstaler R, Ripperger A, Hauke M, Petermann M, Hemkemeyer SA, Schwedhelm E, Ergün S, Frye M, Werz O, Koeberle A, Braun H, Benndorf RA. A Thromboxane A 2 Receptor-Driven COX-2-Dependent Feedback Loop That Affects Endothelial Homeostasis and Angiogenesis. Arterioscler Thromb Vasc Biol 2022; 42:444-461. [PMID: 35236104 PMCID: PMC8939709 DOI: 10.1161/atvbaha.121.317380] [Citation(s) in RCA: 16] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/16/2023]
Abstract
BACKGROUND TP (thromboxane A2 receptor) plays an eminent role in the pathophysiology of endothelial dysfunction and cardiovascular disease. Moreover, its expression is reported to increase in the intimal layer of blood vessels of cardiovascular high-risk individuals. Yet it is unknown, whether TP upregulation per se has the potential to affect the homeostasis of the vascular endothelium. METHODS We combined global transcriptome analysis, lipid mediator profiling, functional cell analyses, and in vivo angiogenesis assays to study the effects of endothelial TP overexpression or knockdown/knockout on the angiogenic capacity of endothelial cells in vitro and in vivo. RESULTS Here we report that endothelial TP expression induces COX-2 (cyclooxygenase-2) in a Gi/o- and Gq/11-dependent manner, thereby promoting its own activation via the auto/paracrine release of TP agonists, such as PGH2 (prostaglandin H2) or prostaglandin F2 but not TxA2 (thromboxane A2). TP overexpression induces endothelial cell tension and aberrant cell morphology, affects focal adhesion dynamics, and inhibits the angiogenic capacity of human endothelial cells in vitro and in vivo, whereas TP knockdown or endothelial-specific TP knockout exerts opposing effects. Consequently, this TP-dependent feedback loop is disrupted by pharmacological TP or COX-2 inhibition and by genetic reconstitution of PGH2-metabolizing prostacyclin synthase even in the absence of functional prostacyclin receptor expression. CONCLUSIONS Our work uncovers a TP-driven COX-2-dependent feedback loop and important effector mechanisms that directly link TP upregulation to angiostatic TP signaling in endothelial cells. By these previously unrecognized mechanisms, pathological endothelial upregulation of the TP could directly foster endothelial dysfunction, microvascular rarefaction, and systemic hypertension even in the absence of exogenous sources of TP agonists.
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Affiliation(s)
- Robert Eckenstaler
- Department of Clinical Pharmacy and Pharmacotherapy, Institute of Pharmacy, Martin-Luther-University Halle-Wittenberg, Germany (R.E., A.R., M.H., M.P., H.B., R.A.B.)
| | - Anne Ripperger
- Department of Clinical Pharmacy and Pharmacotherapy, Institute of Pharmacy, Martin-Luther-University Halle-Wittenberg, Germany (R.E., A.R., M.H., M.P., H.B., R.A.B.)
| | - Michael Hauke
- Department of Clinical Pharmacy and Pharmacotherapy, Institute of Pharmacy, Martin-Luther-University Halle-Wittenberg, Germany (R.E., A.R., M.H., M.P., H.B., R.A.B.)
| | - Markus Petermann
- Department of Clinical Pharmacy and Pharmacotherapy, Institute of Pharmacy, Martin-Luther-University Halle-Wittenberg, Germany (R.E., A.R., M.H., M.P., H.B., R.A.B.)
| | - Sandra A Hemkemeyer
- Institute of Clinical Chemistry and Laboratory Medicine (S.A.H., M.F.), University Medical Center Hamburg-Eppendorf, Germany
| | - Edzard Schwedhelm
- Institute of Clinical Pharmacology and Toxicology (E.S.), University Medical Center Hamburg-Eppendorf, Germany
| | - Süleyman Ergün
- Institute of Anatomy and Cell Biology, Julius-Maximilians-University Würzburg, Germany (S.E.)
| | - Maike Frye
- Institute of Clinical Chemistry and Laboratory Medicine (S.A.H., M.F.), University Medical Center Hamburg-Eppendorf, Germany
| | - Oliver Werz
- Department of Pharmaceutical/Medicinal Chemistry, Institute of Pharmacy, Friedrich-Schiller-University Jena, Germany (O.W., A.K.)
| | - Andreas Koeberle
- Department of Pharmaceutical/Medicinal Chemistry, Institute of Pharmacy, Friedrich-Schiller-University Jena, Germany (O.W., A.K.).,Michael Popp Institute and Center for Molecular Biosciences Innsbruck, University of Innsbruck, Austria (A.K.)
| | - Heike Braun
- Department of Clinical Pharmacy and Pharmacotherapy, Institute of Pharmacy, Martin-Luther-University Halle-Wittenberg, Germany (R.E., A.R., M.H., M.P., H.B., R.A.B.)
| | - Ralf A Benndorf
- Department of Clinical Pharmacy and Pharmacotherapy, Institute of Pharmacy, Martin-Luther-University Halle-Wittenberg, Germany (R.E., A.R., M.H., M.P., H.B., R.A.B.)
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Hyun J, Lee M, Rehman J, Pajcini KV, Malik AB. Notch1 promotes ordered revascularization through Semaphorin 3g modulation of downstream vascular patterning signalling factors. J Physiol 2022; 600:509-530. [PMID: 34921404 PMCID: PMC9305962 DOI: 10.1113/jp282286] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/27/2021] [Accepted: 11/29/2021] [Indexed: 11/12/2022] Open
Abstract
Here we genetically and functionally addressed potential pathways of Notch signalling in mediating vascular regeneration in mouse models. We first used transgenic adult mice with either gain- or loss-of-function Notch signalling in vascular endothelial cells and monitored perfusion in the hindlimb following ischaemia induced by femoral artery ligation. Mice deficient in Notch signalling showed defective perfusion recovery and expansion of collateral arteries. Transcriptomics analysis of arterial endothelial cells in the Notch mutants identified the guidance factor Sema3g as a candidate gene mediating reperfusion downstream of Notch. Studies in the retinal circulation showed the central role of SEMA3G downstream of Notch signalling in the orderly regulation of vascular patterning. These studies in multiple vascular beds show the primacy of Notch signalling and downstream generation of guidance peptides such as SEMA3G in promoting well-ordered vascular regeneration. KEY POINTS: Notch signalling is a critical mediator of revascularization. Yet the cellular processes activated during recovery following vascular injury are incompletely understood. Here we used genetic and cellular approaches in two different vascular beds and cultured endothelial cells to address the generalizability of mechanisms. By utilizing a highly reproducible murine model of hindlimb ischaemia in transgenic mice in which Notch signalling was inhibited at the transcriptional level, we demonstrated the centrality of Notch signalling in perfusion recovery and revascularization. RNA-sequencing of Notch mutants identified class 3 Semaphorins regulated by Notch signalling as downstream targets. Studies in retinal vessels and endothelial cells showed an essential role of guidance peptide Sema3g as a modulator of angiogenesis and orderly vascular patterning. The Notch to Sema3g signalling axis functions as a feedback mechanism to sculpt the growing vasculature in multiple beds.
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Affiliation(s)
- James Hyun
- Department of Pharmacology and Regenerative Medicine, University of Illinois College of Medicine, Chicago, IL, USA
| | - Monica Lee
- Department of Physiology and Biophysics, University of Illinois College of Medicine, Chicago, IL, USA
| | - Jalees Rehman
- Department of Pharmacology and Regenerative Medicine, University of Illinois College of Medicine, Chicago, IL, USA
| | - Kostandin V Pajcini
- Department of Pharmacology and Regenerative Medicine, University of Illinois College of Medicine, Chicago, IL, USA
| | - Asrar B Malik
- Department of Pharmacology and Regenerative Medicine, University of Illinois College of Medicine, Chicago, IL, USA
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Te Boekhorst V, Jiang L, Mählen M, Meerlo M, Dunkel G, Durst FC, Yang Y, Levine H, Burgering BMT, Friedl P. Calpain-2 regulates hypoxia/HIF-induced plasticity toward amoeboid cancer cell migration and metastasis. Curr Biol 2022; 32:412-427.e8. [PMID: 34883047 PMCID: PMC10439789 DOI: 10.1016/j.cub.2021.11.040] [Citation(s) in RCA: 21] [Impact Index Per Article: 7.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/28/2019] [Revised: 07/05/2021] [Accepted: 11/16/2021] [Indexed: 12/29/2022]
Abstract
Hypoxia, through hypoxia inducible factor (HIF), drives cancer cell invasion and metastatic progression in various cancer types. In epithelial cancer, hypoxia induces the transition to amoeboid cancer cell dissemination, yet the molecular mechanisms, relevance for metastasis, and effective intervention to combat hypoxia-induced amoeboid reprogramming remain unclear. Here, we identify calpain-2 as a key regulator and anti-metastasis target of hypoxia-induced transition from collective to amoeboid dissemination of breast and head and neck (HN) carcinoma cells. Hypoxia-induced amoeboid dissemination occurred through low extracellular matrix (ECM)-adhesive, predominantly bleb-based amoeboid movement, which was maintained by a low-oxidative and -glycolytic energy metabolism ("eco-mode"). Hypoxia induced calpain-2-mediated amoeboid conversion by deactivating β1 integrins through enzymatic cleavage of the focal adhesion adaptor protein talin-1. Consequently, targeted downregulation or pharmacological inhibition of calpain-2 restored talin-1 integrity and β1 integrin engagement and reverted amoeboid to elongated phenotypes under hypoxia. Calpain-2 activity was required for hypoxia-induced amoeboid conversion in the orthotopic mouse dermis and upregulated in invasive HN tumor xenografts in vivo, and attenuation of calpain activity prevented hypoxia-induced metastasis to the lungs. This identifies the calpain-2/talin-1/β1 integrin axis as a druggable mechanosignaling program that conserves energy yet enables metastatic dissemination that can be reverted by interfering with calpain activity.
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Affiliation(s)
- Veronika Te Boekhorst
- David H. Koch Center for Applied Research of Genitourinary Cancers, The University of Texas MD Anderson Cancer Center, Houston, TX, USA; Department of Cell Biology, Radboud University Medical Centre, 6525 GA Nijmegen, the Netherlands
| | - Liying Jiang
- David H. Koch Center for Applied Research of Genitourinary Cancers, The University of Texas MD Anderson Cancer Center, Houston, TX, USA
| | - Marius Mählen
- Department of Cell Biology, Radboud University Medical Centre, 6525 GA Nijmegen, the Netherlands
| | - Maaike Meerlo
- Department of Molecular Cancer Research, Center for Molecular Medicine, UMC Utrecht, the Netherlands; Oncode Institute, 3521 AL Utrecht, the Netherlands
| | - Gina Dunkel
- Department of Cell Biology, Radboud University Medical Centre, 6525 GA Nijmegen, the Netherlands
| | - Franziska C Durst
- David H. Koch Center for Applied Research of Genitourinary Cancers, The University of Texas MD Anderson Cancer Center, Houston, TX, USA
| | - Yanjun Yang
- Center for Theoretical Biological Physics, Department of Applied Physics, Rice University, Houston, TX 77005, USA; Department of Physics, Northeastern University, Boston, MA 02115, USA
| | - Herbert Levine
- Center for Theoretical Biological Physics, Department of Applied Physics, Rice University, Houston, TX 77005, USA; Department of Physics, Northeastern University, Boston, MA 02115, USA
| | - Boudewijn M T Burgering
- Department of Molecular Cancer Research, Center for Molecular Medicine, UMC Utrecht, the Netherlands; Oncode Institute, 3521 AL Utrecht, the Netherlands; Cancer Genomics Center, 3584 CG Utrecht, the Netherlands
| | - Peter Friedl
- David H. Koch Center for Applied Research of Genitourinary Cancers, The University of Texas MD Anderson Cancer Center, Houston, TX, USA; Department of Cell Biology, Radboud University Medical Centre, 6525 GA Nijmegen, the Netherlands; Cancer Genomics Center, 3584 CG Utrecht, the Netherlands.
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Evaluation of the Usefulness of Human Adipose-Derived Stem Cell Spheroids Formed Using SphereRing® and the Lethal Damage Sensitivity to Synovial Fluid In Vitro. Cells 2022; 11:cells11030337. [PMID: 35159147 PMCID: PMC8834569 DOI: 10.3390/cells11030337] [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: 12/16/2021] [Revised: 01/12/2022] [Accepted: 01/13/2022] [Indexed: 11/17/2022] Open
Abstract
Osteoarthritis (OA) is an irreversible degenerative condition causing bone deformation in the joints and articular cartilage degeneration with chronic pain and impaired movement. Adipose-derived stem cell (ADSC) or crushed adipose tissue injection into the joint cavity reportedly improve knee function and symptoms, including pain. Stem cell spheroids may be promising treatment options due to their anti-inflammatory and enhanced tissue regeneration/repair effects. Herein, to form human ADSC spheroids, we used first SphereRing® (Fukoku Co., Ltd., Ageo, Japan), a newly developed rotating donut-shaped tube and determined their characteristics by DNA microarray of mRNA analysis. The variable gene expression cluster was then identified and validated by RT-PCR. Gene expression fluctuations were observed, such as COL15A1 and ANGPTL2, related to vascular endothelial cells and angiogenesis, and TNC, involved in tissue formation. In addition, multiplex cytokine analysis in the medium revealed significant cytokines and growth factors production increase of IL-6, IL-10, etc. However, ADSC administration into the joint cavity involves their contact with the synovial fluid (SF). Therefore, we examined how SF collected from OA patient joint cavities affect 2D-culture ADSCs and ADSC spheroids and observed SF induced cell death. ADSC spheroids could become promising OA treatment options, although studying the administration methods and consider their interaction with SF is essential.
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Vakhrushev IV, Nezhurina EK, Karalkin PA, Tsvetkova AV, Sergeeva NS, Majouga AG, Yarygin KN. Heterotypic Multicellular Spheroids as Experimental and Preclinical Models of Sprouting Angiogenesis. BIOLOGY 2021; 11:18. [PMID: 35053016 PMCID: PMC8772844 DOI: 10.3390/biology11010018] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 11/18/2021] [Revised: 12/18/2021] [Accepted: 12/20/2021] [Indexed: 12/12/2022]
Abstract
Sprouting angiogenesis is the common response of live tissues to physiological and pathological angiogenic stimuli. Its accurate evaluation is of utmost importance for basic research and practical medicine and pharmacology and requires adequate experimental models. A variety of assays for angiogenesis were developed, none of them perfect. In vitro approaches are generally less physiologically relevant due to the omission of essential components regulating the process. However, only in vitro models can be entirely non-xenogeneic. The limitations of the in vitro angiogenesis assays can be partially overcome using 3D models mimicking tissue O2 and nutrient gradients, the influence of the extracellular matrix (ECM), and enabling cell-cell interactions. Here we present a review of the existing models of sprouting angiogenesis that are based on the use of endothelial cells (ECs) co-cultured with perivascular or other stromal cells. This approach provides an excellent in vitro platform for further decoding of the cellular and molecular mechanisms of sprouting angiogenesis under conditions close to the in vivo conditions, as well as for preclinical drug testing and preclinical research in tissue engineering and regenerative medicine.
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Affiliation(s)
- Igor V. Vakhrushev
- Laboratory of Cell Biology, Institute of Biomedical Chemistry, 119121 Moscow, Russia;
| | - Elizaveta K. Nezhurina
- P.A. Hertsen Moscow Oncology Research Center, National Medical Research Radiological Center, 125284 Moscow, Russia;
| | - Pavel A. Karalkin
- Institute for Cluster Oncology, Sechenov University, 119435 Moscow, Russia;
| | | | - Nataliya S. Sergeeva
- Department of Biology, Pirogov Russian National Research Medical University, 117997 Moscow, Russia;
| | - Alexander G. Majouga
- Faculty of Chemical and Pharmaceutical Technologies and Biomedical Products, D. Mendeleev University of Chemical Technology of Russia, 125047 Moscow, Russia;
| | - Konstantin N. Yarygin
- Laboratory of Cell Biology, Institute of Biomedical Chemistry, 119121 Moscow, Russia;
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Shafiee S, Shariatzadeh S, Zafari A, Majd A, Niknejad H. Recent Advances on Cell-Based Co-Culture Strategies for Prevascularization in Tissue Engineering. Front Bioeng Biotechnol 2021; 9:745314. [PMID: 34900955 PMCID: PMC8655789 DOI: 10.3389/fbioe.2021.745314] [Citation(s) in RCA: 29] [Impact Index Per Article: 7.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/21/2021] [Accepted: 11/02/2021] [Indexed: 12/14/2022] Open
Abstract
Currently, the fabrication of a functional vascular network to maintain the viability of engineered tissues is a major bottleneck in the way of developing a more advanced engineered construct. Inspired by vasculogenesis during the embryonic period, the in vitro prevascularization strategies have focused on optimizing communications and interactions of cells, biomaterial and culture conditions to develop a capillary-like network to tackle the aforementioned issue. Many of these studies employ a combination of endothelial lineage cells and supporting cells such as mesenchymal stem cells, fibroblasts, and perivascular cells to create a lumenized endothelial network. These supporting cells are necessary for the stabilization of the newly developed endothelial network. Moreover, to optimize endothelial network development without impairing biomechanical properties of scaffolds or differentiation of target tissue cells, several other factors, including target tissue, endothelial cell origins, the choice of supporting cell, culture condition, incorporated pro-angiogenic factors, and choice of biomaterial must be taken into account. The prevascularization method can also influence the endothelial lineage cell/supporting cell co-culture system to vascularize the bioengineered constructs. This review aims to investigate the recent advances on standard cells used in in vitro prevascularization methods, their co-culture systems, and conditions in which they form an organized and functional vascular network.
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Affiliation(s)
- Sepehr Shafiee
- Department of Pharmacology, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran
| | - Siavash Shariatzadeh
- Department of Pharmacology, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran
| | - Ali Zafari
- Department of Pharmacology, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran
| | - Alireza Majd
- Department of Pharmacology, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran
| | - Hassan Niknejad
- Department of Pharmacology, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran
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Shanbhag S, Rashad A, Nymark EH, Suliman S, de Lange Davies C, Stavropoulos A, Bolstad AI, Mustafa K. Spheroid Coculture of Human Gingiva-Derived Progenitor Cells With Endothelial Cells in Modified Platelet Lysate Hydrogels. Front Bioeng Biotechnol 2021; 9:739225. [PMID: 34513817 PMCID: PMC8427051 DOI: 10.3389/fbioe.2021.739225] [Citation(s) in RCA: 9] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/10/2021] [Accepted: 08/12/2021] [Indexed: 01/12/2023] Open
Abstract
Cell coculture strategies can promote angiogenesis within tissue engineering constructs. This study aimed to test the angiogenic potential of human umbilical vein endothelial cells (HUVEC) cocultured with gingiva-derived progenitor cells (GPC) as spheroids in a xeno-free environment. Human platelet lysate (HPL) was used as a cell culture supplement and as a hydrogel matrix (HPLG) for spheroid encapsulation. HUVEC and HUVEC + GPC (1:1 or 5:1) spheroids were encapsulated in various HPLG formulations. Angiogenesis was assessed via in vitro sprouting and in vivo chick chorioallantoic membrane (CAM) assays. HUVEC revealed characteristic in vitro sprouting in HPL/HPLG and this was significantly enhanced in cocultures with GPC (p < 0.05). A trend for greater sprouting was observed in 5:1 vs 1:1 HUVEC + GPC spheroids and in certain HPLG formulations (p > 0.05). Both HUVEC and HUVEC + GPC spheroids in HPLG revealed abundant and comparable neoangiogenesis in the CAM assay (p > 0.05). Spheroid coculture of HUVEC + GPC in HPLG represents a promising strategy to promote angiogenesis.
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Affiliation(s)
- Siddharth Shanbhag
- Center for Translational Oral Research (TOR), Department of Clinical Dentistry, Faculty of Medicine, University of Bergen, Bergen, Norway.,Department of Immunology and Transfusion Medicine, Haukeland University Hospital, Bergen, Norway
| | - Ahmad Rashad
- Center for Translational Oral Research (TOR), Department of Clinical Dentistry, Faculty of Medicine, University of Bergen, Bergen, Norway
| | - Ellen Helgeland Nymark
- Department of Physics, Norwegian University of Science and Technology, Trondheim, Norway
| | - Salwa Suliman
- Center for Translational Oral Research (TOR), Department of Clinical Dentistry, Faculty of Medicine, University of Bergen, Bergen, Norway
| | | | - Andreas Stavropoulos
- Department of Periodontology, Faculty of Odontology, Malmö University, Malmö, Sweden.,Division of Regenerative Medicine and Periodontology, University Clinics of Dental Medicine, University of Geneva, Geneva, Switzerland
| | - Anne Isine Bolstad
- Center for Translational Oral Research (TOR), Department of Clinical Dentistry, Faculty of Medicine, University of Bergen, Bergen, Norway
| | - Kamal Mustafa
- Center for Translational Oral Research (TOR), Department of Clinical Dentistry, Faculty of Medicine, University of Bergen, Bergen, Norway
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Braun H, Hauke M, Ripperger A, Ihling C, Fuszard M, Eckenstaler R, Benndorf RA. Impact of DICER1 and DROSHA on the Angiogenic Capacity of Human Endothelial Cells. Int J Mol Sci 2021; 22:ijms22189855. [PMID: 34576018 PMCID: PMC8471234 DOI: 10.3390/ijms22189855] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/20/2021] [Revised: 09/08/2021] [Accepted: 09/10/2021] [Indexed: 12/15/2022] Open
Abstract
RNAi-mediated knockdown of DICER1 and DROSHA, enzymes critically involved in miRNA biogenesis, has been postulated to affect the homeostasis and the angiogenic capacity of human endothelial cells. To re-evaluate this issue, we reduced the expression of DICER1 or DROSHA by RNAi-mediated knockdown and subsequently investigated the effect of these interventions on the angiogenic capacity of human umbilical vein endothelial cells (HUVEC) in vitro (proliferation, migration, tube formation, endothelial cell spheroid sprouting) and in a HUVEC xenograft assay in immune incompetent NSGTM mice in vivo. In contrast to previous reports, neither knockdown of DICER1 nor knockdown of DROSHA profoundly affected migration or tube formation of HUVEC or the angiogenic capacity of HUVEC in vivo. Furthermore, knockdown of DICER1 and the combined knockdown of DICER1 and DROSHA tended to increase VEGF-induced BrdU incorporation and induced angiogenic sprouting from HUVEC spheroids. Consistent with these observations, global proteomic analyses showed that knockdown of DICER1 or DROSHA only moderately altered HUVEC protein expression profiles but additively reduced, for example, expression of the angiogenesis inhibitor thrombospondin-1. In conclusion, global reduction of miRNA biogenesis by knockdown of DICER1 or DROSHA does not inhibit the angiogenic capacity of HUVEC. Further studies are therefore needed to elucidate the influence of these enzymes in the context of human endothelial cell-related angiogenesis.
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Affiliation(s)
- Heike Braun
- Department of Clinical Pharmacy and Pharmacotherapy, Institute of Pharmacy, Martin-Luther-University Halle-Wittenberg, 06120 Halle (Saale), Germany; (H.B.); (M.H.); (A.R.); (R.E.)
| | - Michael Hauke
- Department of Clinical Pharmacy and Pharmacotherapy, Institute of Pharmacy, Martin-Luther-University Halle-Wittenberg, 06120 Halle (Saale), Germany; (H.B.); (M.H.); (A.R.); (R.E.)
| | - Anne Ripperger
- Department of Clinical Pharmacy and Pharmacotherapy, Institute of Pharmacy, Martin-Luther-University Halle-Wittenberg, 06120 Halle (Saale), Germany; (H.B.); (M.H.); (A.R.); (R.E.)
| | - Christian Ihling
- Department of Pharmaceutical Chemistry and Bioanalytics, Institute of Pharmacy, Charles Tanford Center, Martin Luther University Halle-Wittenberg, 06120 Halle (Saale), Germany;
| | - Matthew Fuszard
- Core Facility—Proteomics Mass Spectrometry, Charles Tanford Centre, Martin-Luther-University Halle-Wittenberg, 06120 Halle (Saale), Germany;
| | - Robert Eckenstaler
- Department of Clinical Pharmacy and Pharmacotherapy, Institute of Pharmacy, Martin-Luther-University Halle-Wittenberg, 06120 Halle (Saale), Germany; (H.B.); (M.H.); (A.R.); (R.E.)
| | - Ralf A. Benndorf
- Department of Clinical Pharmacy and Pharmacotherapy, Institute of Pharmacy, Martin-Luther-University Halle-Wittenberg, 06120 Halle (Saale), Germany; (H.B.); (M.H.); (A.R.); (R.E.)
- Correspondence: ; Tel.: +49-345-55-25150
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Skowronek D, Pilz RA, Schwefel K, Much CD, Felbor U, Rath M. Bringing CCM into a dish: cell culture models for cerebral cavernous malformations. MED GENET-BERLIN 2021; 33:251-259. [PMID: 38835694 PMCID: PMC11006332 DOI: 10.1515/medgen-2021-2091] [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: 06/23/2021] [Accepted: 10/21/2021] [Indexed: 06/06/2024]
Abstract
Cerebral cavernous malformations (CCMs) are vascular lesions that can cause severe neurological complications due to intracranial hemorrhage. Although the CCM disease genes, CCM1, CCM2, and CCM3, have been known for more than 15 years now, our understanding of CCM pathogenesis is still incomplete. CCM research currently focuses on three main disease mechanisms: (1) clonal expansion of endothelial cells with biallelic inactivation of CCM1, CCM2, or CCM3, (2) recruitment of cells with preserved CCM protein expression into the growing lesion, and (3) disruption of endothelial cell-cell junctions in CCMs. We here describe novel CRISPR/Cas9-based in vitro models of CCM and discuss their strengths and limitations in the context of high-throughput drug screening and repurposing approaches.
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Affiliation(s)
- Dariush Skowronek
- Department of Human Genetics, University Medicine Greifswald, Greifswald, Germany
- Interfaculty Institute of Genetics and Functional Genomics, University of Greifswald, Greifswald, Germany
| | - Robin A Pilz
- Department of Human Genetics, University Medicine Greifswald, Greifswald, Germany
- Interfaculty Institute of Genetics and Functional Genomics, University of Greifswald, Greifswald, Germany
| | - Konrad Schwefel
- Department of Human Genetics, University Medicine Greifswald, Greifswald, Germany
- Interfaculty Institute of Genetics and Functional Genomics, University of Greifswald, Greifswald, Germany
| | - Christiane D Much
- Department of Human Genetics, University Medicine Greifswald, Greifswald, Germany
- Interfaculty Institute of Genetics and Functional Genomics, University of Greifswald, Greifswald, Germany
| | - Ute Felbor
- Department of Human Genetics, University Medicine Greifswald, Greifswald, Germany
- Interfaculty Institute of Genetics and Functional Genomics, University of Greifswald, Greifswald, Germany
| | - Matthias Rath
- Department of Human Genetics, University Medicine Greifswald, Fleischmannstraße 43, D-17475 Greifswald, Germany
- Interfaculty Institute of Genetics and Functional Genomics, University of Greifswald, Greifswald, Germany
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Habibi M, Chehelcheraghi F. Effect of Bone Marrow Mesenchymal Stem Cell Sheets on Skin Capillary Parameters in a diabetic wound model: A Novel Preliminary Study. IRANIAN BIOMEDICAL JOURNAL 2021; 25:334-42. [PMID: 34481425 PMCID: PMC8487679 DOI: 10.52547/ibj.25.5.334] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 09/10/2020] [Accepted: 01/18/2021] [Indexed: 01/04/2023]
Abstract
Background Treatment with BMMSCs has anti-inflammatory, tissue regenerative, angiogenic, and immune-stimulating effects. When using as sheets or accumulate, BMMSCs causes the development of neoangiogenesis in damaged skin tissue. Diabetes, a metabolic disorder, can negatively affect many physiological functions, including the process of skin injury repair. This adverse impact may increase the risk of skin surgery. RSF is commonly used in reconstructive surgery. The terminal part of the RSF is often affected by necrosis because of impaired blood flow, which is exacerbated in diabetes. This study investigated the effect of stem cells, applied as accumulated or cell sheets, along with RSF surgery on skin capillaries in STZ-induced diabetic rats. Methods Thirty male Wistar rats were divided into three groups (n = 10): diabetes-RSF control, diabetes-RSF local applied stem cells (loc-BMMSCs), diabetes-RSF applied stem cells as accumulated or cell sheets (ac-BMMSCs). Two weeks after the STZ injection, RSF surgery and stem cell therapy (6 × 109) were carried out (day zero). Furthermore, stereological methods were used to investigate the capillary patterns among the groups. Anti-CD31/PCAM1 immunohistochemistry was also used for further confirmation of changes in capillary parameters. Results The results demonstrated that capillaries were protected by MSC sheets in the flap tissue, and the thickness of the epidermal layer was improved, indicationg the possible beneficial effects of MSC sheets on diabetic wound treatment. Conclusion Stem cells, as ac-BMMSCs, may decrease the levels of wound healing complications in diabetes and can be considered as a cell therapy option in such conditions.
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Affiliation(s)
- Maryam Habibi
- Student Research Committee, School of Medicine, Lorestan University of Medical Sciences, Khorramabad, Iran
| | - Farzaneh Chehelcheraghi
- Department of Anatomical Sciences, School of Medicine, Lorestan University of Medical Sciences, Khorramabad, Iran
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Abstract
Over the past decade, 3D culture models of human and animal cells have found their way into tissue differentiation, drug development, personalized medicine and tumour behaviour studies. Embryoid bodies (EBs) are in vitro 3D cultures established from murine pluripotential stem cells, whereas tumoroids are patient-derived in vitro 3D cultures. This thesis aims to describe a new implication of an embryoid body model and to characterize the patient-specific microenvironment of the parental tumour in relation to tumoroid growth rate. In this thesis, we described a high-throughput monitoring method, where EBs are used as a dynamic angiogenesis model. In this model, digital image analysis (DIA) is implemented on immunohistochemistry (IHC) stained sections of the cultures over time. Furthermore, we have investigated the correlation between the genetic profile and inflammatory microenvironment of parental tumours on the in vitro growth rate of tumoroids. The EBs were cultured in spinner flasks. The samples were collected at days 4, 6, 9, 14, 18 and 21, dehydrated and embedded in paraffin. The histological sections were IHC stained for the endothelial marker CD31 and digitally scanned. The virtual whole-image slides were digitally analysed by Visiopharm® software. Histological evaluation showed vascular-like structures over time. The quantitative DIA was plausible to monitor significant increase in the total area of the EBs and an increase in endothelial differentiation. The tumoroids were established from 32 colorectal adenocarcinomas. The in vitro growth rate of the tumoroids was followed by automated microscopy over an 11-day period. The parental tumours were analysed by next-generation sequencing for KRAS, TP53, PIK3CA, SMAD4, MAP2K1, BRAF, FGFR3 and FBXW7 status. The tumoroids established from KRAS-mutated parental tumours showed a significantly higher growth rate compared to their wild-type counterparts. The density of CD3+ T lymphocytes and CD68+ macrophages was calculated in the centre of the tumours and at the invasive margin of the tumours. The high density of CD3+ cells and the low density of CD68+ cells showed a significant correlation with a higher growth rate of the tumoroids. In conclusion, a novel approach for histological monitoring of endothelial differentiation is presented in the stem cell-derived EBs. Furthermore, the KRAS status and density of CD3+ T cells and macrophages in the parental tumour influence the growth rate of the tumoroids. Our results indicate that these parameters should be included when tumoroids are to be implemented in personalized medicine.
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Affiliation(s)
- Nabi Mousavi
- Department of Pathology, Rigshospitalet, University of Copenhagen, Copenhagen, Denmark
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Alsaigh T, Di Bartolo BA, Mulangala J, Figtree GA, Leeper NJ. Bench-to-Bedside in Vascular Medicine: Optimizing the Translational Pipeline for Patients With Peripheral Artery Disease. Circ Res 2021; 128:1927-1943. [PMID: 34110900 PMCID: PMC8208504 DOI: 10.1161/circresaha.121.318265] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/20/2022]
Abstract
Peripheral arterial disease is a growing worldwide problem with a wide spectrum of clinical severity and is projected to consume >$21 billion per year in the United States alone. While vascular researchers have brought several therapies to the clinic in recent years, few of these approaches have leveraged advances in high-throughput discovery screens, novel translational models, or innovative trial designs. In the following review, we discuss recent advances in unbiased genomics and broader omics technology platforms, along with preclinical vascular models designed to enhance our understanding of disease pathobiology and prioritize targets for additional investigation. Furthermore, we summarize novel approaches to clinical studies in subjects with claudication and ischemic ulceration, with an emphasis on streamlining and accelerating bench-to-bedside translation. By providing a framework designed to enhance each aspect of future clinical development programs, we hope to enrich the pipeline of therapies that may prevent loss of life and limb for those with peripheral arterial disease.
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Affiliation(s)
- Tom Alsaigh
- Department of Surgery, Division of Vascular Surgery, Stanford University School of Medicine, Stanford, California, United States of America
| | - Belinda A. Di Bartolo
- Cardiothoracic and Vascular Health, Kolling Institute and Department of Cardiology, Royal North Shore Hospital, Northern Sydney Local Health District, Australia
| | | | - Gemma A. Figtree
- Cardiothoracic and Vascular Health, Kolling Institute and Department of Cardiology, Royal North Shore Hospital, Northern Sydney Local Health District, Australia
| | - Nicholas J. Leeper
- Department of Surgery, Division of Vascular Surgery, Stanford University School of Medicine, Stanford, California, United States of America
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Katsila T, Chasapi SA, Gomez Tamayo JC, Chalikiopoulou C, Siapi E, Moros G, Zoumpoulakis P, Spyroulias GA, Kardamakis D. Three-Dimensional Cell Metabolomics Deciphers the Anti-Angiogenic Properties of the Radioprotectant Amifostine. Cancers (Basel) 2021; 13:cancers13122877. [PMID: 34207535 PMCID: PMC8230228 DOI: 10.3390/cancers13122877] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/03/2021] [Revised: 05/28/2021] [Accepted: 06/04/2021] [Indexed: 12/13/2022] Open
Abstract
Simple Summary Cancer and inflammation share aberrant angiogenesis as a hallmark, and, thus, anti-angiogenetic strategies remain of key interest. Amifostine, which is already a drug on the market, may be of further benefit to patients also in the context of drug repurposing. To shed light on the anti-angiogenic properties of amifostine during human adult angiogenesis and grasp the early events of angiogenesis, we employed 3D cell untargeted metabolomics by liquid chromatography–mass spectrometry and nuclear magnetic resonance spectroscopy in the presence of vascular endothelial growth factor-A or deferoxamine (pro-angiogenic factors that exhibit distinct angiogenesis induction profiles). Our findings reveal mechanism-specific inhibitory profiles of amifostine against VEGF-A- and deferoxamine-induced angiogenesis. Amifostine may serve as a dual radioprotective and anti-angiogenic agent in radiotherapy patients. Abstract Aberrant angiogenesis is a hallmark for cancer and inflammation, a key notion in drug repurposing efforts. To delineate the anti-angiogenic properties of amifostine in a human adult angiogenesis model via 3D cell metabolomics and upon a stimulant-specific manner, a 3D cellular angiogenesis assay that recapitulates cell physiology and drug action was coupled to untargeted metabolomics by liquid chromatography–mass spectrometry and nuclear magnetic resonance spectroscopy. The early events of angiogenesis upon its most prominent stimulants (vascular endothelial growth factor-A or deferoxamine) were addressed by cell sprouting measurements. Data analyses consisted of a series of supervised and unsupervised methods as well as univariate and multivariate approaches to shed light on mechanism-specific inhibitory profiles. The 3D untargeted cell metabolomes were found to grasp the early events of angiogenesis. Evident of an initial and sharp response, the metabolites identified primarily span amino acids, sphingolipids, and nucleotides. Profiles were pathway or stimulant specific. The amifostine inhibition profile was rather similar to that of sunitinib, yet distinct, considering that the latter is a kinase inhibitor. Amifostine inhibited both. The 3D cell metabolomics shed light on the anti-angiogenic effects of amifostine against VEGF-A- and deferoxamine-induced angiogenesis. Amifostine may serve as a dual radioprotective and anti-angiogenic agent in radiotherapy patients.
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Affiliation(s)
- Theodora Katsila
- Institute of Chemical Biology, National Hellenic Research Foundation, 11635 Athens, Greece; (C.C.); (E.S.); (G.M.); (P.Z.)
- Department of Radiation Oncology, University of Patras Medical School, 26504 Patras, Greece;
- Correspondence: ; Tel.: +30-210-727-3752
| | - Styliani A. Chasapi
- Department of Pharmacy, University of Patras, 26504 Patras, Greece; (S.A.C.); (G.A.S.)
| | | | - Constantina Chalikiopoulou
- Institute of Chemical Biology, National Hellenic Research Foundation, 11635 Athens, Greece; (C.C.); (E.S.); (G.M.); (P.Z.)
| | - Eleni Siapi
- Institute of Chemical Biology, National Hellenic Research Foundation, 11635 Athens, Greece; (C.C.); (E.S.); (G.M.); (P.Z.)
| | - Giorgos Moros
- Institute of Chemical Biology, National Hellenic Research Foundation, 11635 Athens, Greece; (C.C.); (E.S.); (G.M.); (P.Z.)
| | - Panagiotis Zoumpoulakis
- Institute of Chemical Biology, National Hellenic Research Foundation, 11635 Athens, Greece; (C.C.); (E.S.); (G.M.); (P.Z.)
| | | | - Dimitrios Kardamakis
- Department of Radiation Oncology, University of Patras Medical School, 26504 Patras, Greece;
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