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1
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Brunmaier LAE, Ozdemir T, Walker TW. Angiogenesis: Biological Mechanisms and In Vitro Models. Ann Biomed Eng 2025:10.1007/s10439-025-03721-2. [PMID: 40210793 DOI: 10.1007/s10439-025-03721-2] [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: 01/10/2025] [Accepted: 03/25/2025] [Indexed: 04/12/2025]
Abstract
The translation of biomedical devices and drug research is an expensive and long process with a low probability of receiving FDA approval. Developing physiologically relevant in vitro models with human cells offers a solution to not only improving the odds of FDA approval but also to expand our ability to study complex in vivo systems in a simpler fashion. Animal models remain the standard for pre-clinical testing; however, the data from animal models is an unreliable extrapolation when anticipating a human response in clinical trials, thus contributing to the low rates of translation. In this review, we focus on in vitro vascular or angiogenic models because of the incremental role that the vascular system plays in the translation of biomedical research. The first section of this review discusses the most common angiogenic cytokines that are used in vitro to initiate angiogenesis, followed by angiogenic inhibitors where both initiators and inhibitors work to maintain vascular homeostasis. Next, we evaluate previously published in vitro models, where we evaluate capturing the physical environment for biomimetic in vitro modeling. These topics provide a foundation of parameters that must be considered to improve and achieve vascular biomimicry. Finally, we summarize these topics to suggest a path forward with the goal of engineering human in vitro models that emulate the in vivo environment and provide a platform for biomedical device and drug screening that produces data to support clinical translation.
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Affiliation(s)
- Laura A E Brunmaier
- Nanoscience and Biomedical Engineering Department, South Dakota School of Mines & Technology, 501 E St. Joseph St., Rapid City, SD, 57701, USA
| | - Tugba Ozdemir
- Nanoscience and Biomedical Engineering Department, South Dakota School of Mines & Technology, 501 E St. Joseph St., Rapid City, SD, 57701, USA
| | - Travis W Walker
- Karen M. Swindler Department of Chemical and Biological Engineering, South Dakota School of Mines & Technology, 501 E St. Joseph St., Rapid City, SD, 57701, USA.
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2
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Buranawat B, Shaalan A, Garna DF, Di Silvio L. Development of Prevascularized Synthetic Block Graft for Maxillofacial Reconstruction. J Funct Biomater 2025; 16:18. [PMID: 39852574 PMCID: PMC11766148 DOI: 10.3390/jfb16010018] [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: 11/01/2024] [Revised: 12/18/2024] [Accepted: 01/07/2025] [Indexed: 01/26/2025] Open
Abstract
Cranio-maxillofacial bone reconstruction, especially for large defects, remains challenging. Synthetic biomimetic materials are emerging as alternatives to autogenous grafts. Tissue engineering aims to create natural tissue-mimicking materials, with calcium phosphate-based scaffolds showing promise for bone regeneration applications. This study developed a porous calcium metaphosphate (CMP) scaffold with physicochemical properties mimicking natural bone, aiming to create a prevascularized synthetic bone graft. The scaffold, fabricated using sintered monocalcium phosphate with poly (vinyl alcohol) as a porogen, exhibited pore sizes ranging from 0 to 400 μm, with the highest frequency between 80 and 100 μm. The co-culture of endothelial cells (ECs) with human alveolar osteoblasts (aHOBs) on the scaffold led to the formation of tube-like structures and intrinsic VEGF release, reaching 10,455.6 pg/mL This level approached the optimal dose for vascular formation. Conversely, the co-culture with mesenchymal stem cells did not yield similar results. Combining ECs and aHOBs in the CMP scaffold offers a promising approach to developing prevascularized grafts for cranio-maxillofacial reconstruction. This innovative strategy can potentially enhance vascularization in large tissue-engineered constructs, addressing a critical limitation in current bone regeneration techniques. The prevascularized synthetic bone graft developed in this study could significantly improve the success rate of maxillofacial reconstructions, offering a viable alternative to autogenous grafts.
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Affiliation(s)
- Borvornwut Buranawat
- Center for Implant Dentistry and Periodontics, Faculty of Dentistry and Research Unit in Innovations in Periodontics, Oral Surgery and Advanced Technology in Implant Dentistry, Thammasat University, Bangkok 10200, Thailand;
| | - Abeer Shaalan
- Center for Oral, Clinical and Translational Sciences, Faculty of Dentistry, Oral & Craniofacial Sciences, King’s College London, London SE1 9RT, UK; (A.S.); (D.F.G.)
| | - Devy F. Garna
- Center for Oral, Clinical and Translational Sciences, Faculty of Dentistry, Oral & Craniofacial Sciences, King’s College London, London SE1 9RT, UK; (A.S.); (D.F.G.)
| | - Lucy Di Silvio
- Center for Oral, Clinical and Translational Sciences, Faculty of Dentistry, Oral & Craniofacial Sciences, King’s College London, London SE1 9RT, UK; (A.S.); (D.F.G.)
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3
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Loiola RA, Hachani J, Duban-Deweer S, Sevin E, Bugno P, Kowalska A, Rizzi E, Shimizu F, Kanda T, Mysiorek C, Mazurek M, Gosselet F. Secretome of brain microvascular endothelial cells promotes endothelial barrier tightness and protects against hypoxia-induced vascular leakage. Mol Med 2024; 30:132. [PMID: 39187765 PMCID: PMC11348522 DOI: 10.1186/s10020-024-00897-6] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/22/2024] [Accepted: 08/12/2024] [Indexed: 08/28/2024] Open
Abstract
Cell-based therapeutic strategies have been proposed as an alternative for brain and blood vessels repair after stroke, but their clinical application is hampered by potential adverse effects. We therefore tested the hypothesis that secretome of these cells might be used instead to still focus on cell-based therapeutic strategies. We therefore characterized the composition and the effect of the secretome of brain microvascular endothelial cells (BMECs) on primary in vitro human models of angiogenesis and vascular barrier. Two different secretome batches produced in high scale (scHSP) were analysed by mass spectrometry. Human primary CD34+-derived endothelial cells (CD34+-ECs) were used as well as in vitro models of EC monolayer (CMECs) and blood-brain barrier (BBB). Cells were also exposed to oxygen-glucose deprivation (OGD) conditions and treated with scHSP during reoxygenation. Protein yield and composition of scHSP batches showed good reproducibility. scHSP increased CD34+-EC proliferation, tubulogenesis, and migration. Proteomic analysis of scHSP revealed the presence of growth factors and proteins modulating cell metabolism and inflammatory pathways. scHSP improved the integrity of CMECs, and upregulated the expression of junctional proteins. Such effects were mediated through the activation of the interferon pathway and downregulation of Wnt signalling. Furthermore, OGD altered the permeability of both CMECs and BBB, while scHSP prevented the OGD-induced vascular leakage in both models. These effects were mediated through upregulation of junctional proteins and regulation of MAPK/VEGFR2. Finally, our results highlight the possibility of using secretome from BMECs as a therapeutic alternative to promote brain angiogenesis and to protect from ischemia-induced vascular leakage.
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Affiliation(s)
- Rodrigo Azevedo Loiola
- UR 2465, Laboratory of the Blood-Brain Barrier (LBHE), Sciences Faculty Jean Perrin, Artois University, 62300, Lens, France
| | - Johan Hachani
- UR 2465, Laboratory of the Blood-Brain Barrier (LBHE), Sciences Faculty Jean Perrin, Artois University, 62300, Lens, France
| | - Sophie Duban-Deweer
- UR 2465, Laboratory of the Blood-Brain Barrier (LBHE), Sciences Faculty Jean Perrin, Artois University, 62300, Lens, France
| | - Emmanuel Sevin
- UR 2465, Laboratory of the Blood-Brain Barrier (LBHE), Sciences Faculty Jean Perrin, Artois University, 62300, Lens, France
| | - Paulina Bugno
- Pure Biologics S.A., Duńska 11, 54-427, Wroclaw, Poland
| | | | - Eleonora Rizzi
- UR 2465, Laboratory of the Blood-Brain Barrier (LBHE), Sciences Faculty Jean Perrin, Artois University, 62300, Lens, France
| | - Fumitaka Shimizu
- Department of Neurology and Clinical Neuroscience, Graduate School of Medicine, Yamaguchi University, Ube, Japan
| | - Takashi Kanda
- Department of Neurology and Clinical Neuroscience, Graduate School of Medicine, Yamaguchi University, Ube, Japan
| | - Caroline Mysiorek
- UR 2465, Laboratory of the Blood-Brain Barrier (LBHE), Sciences Faculty Jean Perrin, Artois University, 62300, Lens, France
| | | | - Fabien Gosselet
- UR 2465, Laboratory of the Blood-Brain Barrier (LBHE), Sciences Faculty Jean Perrin, Artois University, 62300, Lens, France.
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4
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Vo Q, Benam KH. Advancements in preclinical human-relevant modeling of pulmonary vasculature on-chip. Eur J Pharm Sci 2024; 195:106709. [PMID: 38246431 PMCID: PMC10939731 DOI: 10.1016/j.ejps.2024.106709] [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: 11/05/2023] [Revised: 01/05/2024] [Accepted: 01/18/2024] [Indexed: 01/23/2024]
Abstract
Preclinical human-relevant modeling of organ-specific vasculature offers a unique opportunity to recreate pathophysiological intercellular, tissue-tissue, and cell-matrix interactions for a broad range of applications. Lung vasculature is particularly important due to its involvement in genesis and progression of rare, debilitating disorders as well as common chronic pathologies. Here, we provide an overview of the latest advances in the development of pulmonary vascular (PV) models using emerging microfluidic tissue engineering technology Organs-on-Chips (so-called PV-Chips). We first review the currently reported PV-Chip systems and their key features, and then critically discuss their major limitations in reproducing in vivo-seen and disease-relevant cellularity, localization, and microstructure. We conclude by presenting latest efforts to overcome such technical and biological limitations and future directions.
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Affiliation(s)
- Quoc Vo
- Division of Pulmonary, Allergy, Critical Care, and Sleep Medicine, Department of Medicine, University of Pittsburgh, Pittsburgh, PA 15213, USA
| | - Kambez H Benam
- Division of Pulmonary, Allergy, Critical Care, and Sleep Medicine, Department of Medicine, University of Pittsburgh, Pittsburgh, PA 15213, USA; Department of Bioengineering, University of Pittsburgh, Pittsburgh, PA 15219, USA; Vascular Medicine Institute, University of Pittsburgh, Pittsburgh, PA 15213, USA.
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5
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Vo Q, Carlson KA, Chiknas PM, Brocker CN, DaSilva L, Clark E, Park SK, Ajiboye AS, Wier EM, Benam KH. On-Chip Reconstitution of Uniformly Shear-Sensing 3D Matrix-embedded Multicellular Blood Microvessel. ADVANCED FUNCTIONAL MATERIALS 2024; 34:2304630. [PMID: 38465199 PMCID: PMC10923530 DOI: 10.1002/adfm.202304630] [Citation(s) in RCA: 1] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 04/26/2023] [Indexed: 03/12/2024]
Abstract
Preclinical human-relevant modeling of organ-specific vasculature offers a unique opportunity to recreate pathophysiological intercellular, tissue-tissue, and cell-matrix interactions for a broad range of applications. Here, we present a reliable, and simply reproducible process for constructing user-controlled long rounded extracellular matrix (ECM)-embedded vascular microlumens on-chip for endothelization and co-culture with stromal cells obtained from human lung. We demonstrate the critical impact of microchannel cross-sectional geometry and length on uniform distribution and magnitude of vascular wall shear stress, which is key when emulating in vivo-observed blood flow biomechanics in health and disease. In addition, we provide an optimization protocol for multicellular culture and functional validation of the system. Moreover, we show the ability to finely tune rheology of the three-dimensional natural matrix surrounding the vascular microchannel to match pathophysiological stiffness. In summary, we provide the scientific community with a matrix-embedded microvasculature on-chip populated with all-primary human-derived pulmonary endothelial cells and fibroblasts to recapitulate and interrogate lung parenchymal biology, physiological responses, vascular biomechanics, and disease biogenesis in vitro. Such a mix-and-match synthetic platform can be feasibly adapted to study blood vessels, matrix, and ECM-embedded cells in other organs and be cellularized with additional stromal cells.
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Affiliation(s)
- Quoc Vo
- Division of Pulmonary, Allergy and Critical Care Medicine, Department of Medicine, University of Pittsburgh, Pittsburgh, PA 15213, USA
| | - Kaely A. Carlson
- Department of Bioengineering, University of Pittsburgh, Pittsburgh, PA 15219, USA
| | - Peter M. Chiknas
- Division of Pulmonary, Allergy and Critical Care Medicine, Department of Medicine, University of Pittsburgh, Pittsburgh, PA 15213, USA
| | - Chad N. Brocker
- Center for Tobacco Products, U.S. Food and Drug Administration, Silver Spring, MD 20993, USA
| | - Luis DaSilva
- Center for Tobacco Products, U.S. Food and Drug Administration, Silver Spring, MD 20993, USA
| | - Erica Clark
- Center for Tobacco Products, U.S. Food and Drug Administration, Silver Spring, MD 20993, USA
| | - Sang Ki Park
- Center for Tobacco Products, U.S. Food and Drug Administration, Silver Spring, MD 20993, USA
| | - A. Seun Ajiboye
- Center for Tobacco Products, U.S. Food and Drug Administration, Silver Spring, MD 20993, USA
| | - Eric M. Wier
- Center for Tobacco Products, U.S. Food and Drug Administration, Silver Spring, MD 20993, USA
| | - Kambez H. Benam
- Division of Pulmonary, Allergy and Critical Care Medicine, Department of Medicine, University of Pittsburgh, Pittsburgh, PA 15213, USA
- Department of Bioengineering, University of Pittsburgh, Pittsburgh, PA 15219, USA
- Vascular Medicine Institute, University of Pittsburgh, Pittsburgh, PA 15213, USA
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Hashimoto A, Hashimoto S. ADP-Ribosylation Factor 6 Pathway Acts as a Key Executor of Mesenchymal Tumor Plasticity. Int J Mol Sci 2023; 24:14934. [PMID: 37834383 PMCID: PMC10573442 DOI: 10.3390/ijms241914934] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/31/2023] [Revised: 10/02/2023] [Accepted: 10/04/2023] [Indexed: 10/15/2023] Open
Abstract
Despite the "big data" on cancer from recent breakthroughs in high-throughput technology and the development of new therapeutic modalities, it remains unclear as to how intra-tumor heterogeneity and phenotypic plasticity created by various somatic abnormalities and epigenetic and metabolic adaptations orchestrate therapy resistance, immune evasiveness, and metastatic ability. Tumors are formed by various cells, including immune cells, cancer-associated fibroblasts, and endothelial cells, and their tumor microenvironment (TME) plays a crucial role in malignant tumor progression and responses to therapy. ADP-ribosylation factor 6 (ARF6) and AMAP1 are often overexpressed in cancers, which statistically correlates with poor outcomes. The ARF6-AMAP1 pathway promotes the intracellular dynamics and cell-surface expression of various proteins. This pathway is also a major target for KRAS/TP53 mutations to cooperatively promote malignancy in pancreatic ductal adenocarcinoma (PDAC), and is closely associated with immune evasion. Additionally, this pathway is important in angiogenesis, acidosis, and fibrosis associated with tumor malignancy in the TME, and its inhibition in PDAC cells results in therapeutic synergy with an anti-PD-1 antibody in vivo. Thus, the ARF6-based pathway affects the TME and the intrinsic function of tumors, leading to malignancy. Here, we discuss the potential mechanisms of this ARF6-based pathway in tumorigenesis, and novel therapeutic strategies.
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Affiliation(s)
- Ari Hashimoto
- Department of Molecular Biology, Graduate School of Medicine, Hokkaido University, Sapporo 060-8638, Japan
| | - Shigeru Hashimoto
- Division of Molecular Psychoimmunology, Institute for Genetic Medicine, Hokkaido University, Sapporo 060-0815, Japan
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7
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de Silva L, Bernal PN, Rosenberg A, Malda J, Levato R, Gawlitta D. Biofabricating the vascular tree in engineered bone tissue. Acta Biomater 2023; 156:250-268. [PMID: 36041651 DOI: 10.1016/j.actbio.2022.08.051] [Citation(s) in RCA: 19] [Impact Index Per Article: 9.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/28/2022] [Revised: 08/22/2022] [Accepted: 08/23/2022] [Indexed: 01/18/2023]
Abstract
The development of tissue engineering strategies for treatment of large bone defects has become increasingly relevant, given the growing demand for bone substitutes. Native bone is composed of a dense vascular network necessary for the regulation of bone development, regeneration and homeostasis. A major obstacle in fabricating living, clinically relevant-sized bone mimics (1-10 cm3) is the limited supply of nutrients, including oxygen to the core of the construct. Therefore, strategies to support vascularization are pivotal for the development of tissue engineered bone constructs. Creating a functional bone construct integrated with a vascular network, capable of delivering the necessary nutrients for optimal tissue development is imperative for translation into the clinics. The vascular system is composed of a complex network that runs throughout the body in a tree-like hierarchical branching fashion. A significant challenge for tissue engineering approaches lies in mimicking the intricate, multi-scale structures consisting of larger vessels (macro-vessels) which interconnect with multiple sprouting vessels (microvessels) in a closed network. The advent of biofabrication has enabled complex, out of plane channels to be generated and has laid the groundwork for the creation of multi-scale vasculature in recent years. This review highlights the key state-of-the-art achievements for the development of vascular networks of varying scales in the field of biofabrication with a particular focus for its application in developing a functional tissue engineered bone construct. STATEMENT OF SIGNIFICANCE: There is a growing need for bone substitutes to overcome the limited supply of patient-derived bone. Bone tissue engineering aims to overcome this by combining stem cells with scaffolds to restore missing bone. The current bottleneck in upscaling is the lack of an integrated vascular network, required for the delivery of nutrients to cells. 3D bioprinting techniques has enabled the creation of complex hollow structures of varying dimensions that resemble native blood vessels. The convergence of multiple materials, cell types and fabrication approaches, opens the possibility of developing clinically-relevant sized vascularized bone constructs. This review provides an up-to-date insight of the technologies currently available for the generation of complex vascular networks, with a focus on their application in bone tissue engineering.
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Affiliation(s)
- Leanne de Silva
- Department of Oral and Maxillofacial Surgery & Special Dental Care, University Medical Center Utrecht, Utrecht University, Utrecht, 3508 GA, the Netherlands; Regenerative Medicine Center Utrecht, Utrecht, 3584 CT, the Netherlands.
| | - Paulina N Bernal
- Regenerative Medicine Center Utrecht, Utrecht, 3584 CT, the Netherlands; Department of Orthopaedics, University Medical Center Utrecht, Utrecht University, Utrecht, 3508 GA, the Netherlands
| | - Ajw Rosenberg
- Department of Oral and Maxillofacial Surgery & Special Dental Care, University Medical Center Utrecht, Utrecht University, Utrecht, 3508 GA, the Netherlands
| | - Jos Malda
- Regenerative Medicine Center Utrecht, Utrecht, 3584 CT, the Netherlands; Department of Orthopaedics, University Medical Center Utrecht, Utrecht University, Utrecht, 3508 GA, the Netherlands; Department of Clinical Sciences, Faculty of Veterinary Medicine, Utrecht University, Utrecht, 3584 CT, the Netherlands
| | - Riccardo Levato
- Regenerative Medicine Center Utrecht, Utrecht, 3584 CT, the Netherlands; Department of Orthopaedics, University Medical Center Utrecht, Utrecht University, Utrecht, 3508 GA, the Netherlands; Department of Clinical Sciences, Faculty of Veterinary Medicine, Utrecht University, Utrecht, 3584 CT, the Netherlands
| | - Debby Gawlitta
- Department of Oral and Maxillofacial Surgery & Special Dental Care, University Medical Center Utrecht, Utrecht University, Utrecht, 3508 GA, the Netherlands; Regenerative Medicine Center Utrecht, Utrecht, 3584 CT, the Netherlands
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Abstract
The angiogenesis process was described in its basic concepts in the works of the Scottish surgeon John Hunter and terminologically assessed in the early twentieth century. An aberrant angiogenesis is a prerequisite for cancer cells in solid tumors to grow and metastasize. The sprouting of new blood vessels is one of the major characteristics of cancer and represents a gateway for tumor cells to enter both the blood and lymphatic circulation systems. In vivo, ex vivo, and in vitro models of angiogenesis have provided essential tools for cancer research and antiangiogenic drug screening. Several in vivo studies have been performed to investigate the various steps of tumor angiogenesis and in vitro experiments contributed to dissecting the molecular bases of this phenomenon. Moreover, coculture of cancer and endothelial cells in 2D and 3D matrices have contributed to improve the recapitulation of the complex process of tumor angiogenesis, including the peculiar conditions of tumor microenvironment.
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Affiliation(s)
- Gianfranco Natale
- Department of Translational Research and New Technologies in Medicine and Surgery, School of Medicine, University of Pisa, Pisa, Italy
- Museum of Human Anatomy "Filippo Civinini", School of Medicine, University of Pisa, Pisa, Italy
| | - Guido Bocci
- Department of Clinical and Experimental Medicine, School of Medicine, University of Pisa, Pisa, Italy.
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Piquet L, Coutant K, Mitchell A, Ben Anes A, Bollmann E, Schoonjans N, Bérubé J, Bordeleau F, Brisson A, Landreville S. Extracellular Vesicles from Ocular Melanoma Have Pro-Fibrotic and Pro-Angiogenic Properties on the Tumor Microenvironment. Cells 2022; 11:cells11233828. [PMID: 36497088 PMCID: PMC9736613 DOI: 10.3390/cells11233828] [Citation(s) in RCA: 9] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/06/2022] [Revised: 11/19/2022] [Accepted: 11/25/2022] [Indexed: 12/05/2022] Open
Abstract
Uveal melanoma (UM) is the most common primary intraocular tumor and often spreads to the liver. Intercellular communication though extracellular vesicles (EVs) plays an important role in several oncogenic processes, including metastasis, therapeutic resistance, and immune escape. This study examines how EVs released by UM cells modify stellate and endothelial cells in the tumor microenvironment. The surface markers, and the concentration and size of EVs derived from UM cells or choroidal melanocytes were characterized by high-resolution flow cytometry, electron microscopy, and Western blotting. The selective biodistribution of EVs was studied in mice by fluorescence imaging. The activation/contractility of stellate cells and the tubular organization of endothelial cells after exposure to melanomic EVs were determined by traction force microscopy, collagen gel contraction, or endothelial tube formation assays. We showed that large EVs from UM cells and healthy melanocytes are heterogenous in size, as well as their expression of phosphatidylserine, tetraspanins, and Tsg101. Melanomic EVs mainly accumulated in the liver and lungs of mice. Hepatic stellate cells with internalized melanomic EVs had increased contractility, whereas EV-treated endothelial cells developed more capillary-like networks. Our study demonstrates that the transfer of EVs from UM cells leads to a pro-fibrotic and pro-angiogenic phenotype in hepatic stellate and endothelial cells.
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Affiliation(s)
- Léo Piquet
- Faculté de Médecine, Université Laval, Quebec City, QC G1V 0A6, Canada
- Centre de Recherche du CHU de Québec-Université Laval, Quebec City, QC G1S 4L8, Canada
- Centre de Recherche sur le Cancer de l’Université Laval, Quebec City, QC G1R 3S3, Canada
- Centre de Recherche en Organogénèse Expérimentale de l’Université Laval/LOEX, Quebec City, QC G1J 1Z4, Canada
| | - Kelly Coutant
- Faculté de Médecine, Université Laval, Quebec City, QC G1V 0A6, Canada
- Centre de Recherche du CHU de Québec-Université Laval, Quebec City, QC G1S 4L8, Canada
- Centre de Recherche sur le Cancer de l’Université Laval, Quebec City, QC G1R 3S3, Canada
- Centre de Recherche en Organogénèse Expérimentale de l’Université Laval/LOEX, Quebec City, QC G1J 1Z4, Canada
| | - Andrew Mitchell
- Centre de Recherche du CHU de Québec-Université Laval, Quebec City, QC G1S 4L8, Canada
- Centre de Recherche sur le Cancer de l’Université Laval, Quebec City, QC G1R 3S3, Canada
- Centre de Recherche en Organogénèse Expérimentale de l’Université Laval/LOEX, Quebec City, QC G1J 1Z4, Canada
| | - Amel Ben Anes
- Faculté de Médecine, Université Laval, Quebec City, QC G1V 0A6, Canada
| | - Enola Bollmann
- Faculté de Médecine, Université Laval, Quebec City, QC G1V 0A6, Canada
- Centre de Recherche du CHU de Québec-Université Laval, Quebec City, QC G1S 4L8, Canada
- Centre de Recherche sur le Cancer de l’Université Laval, Quebec City, QC G1R 3S3, Canada
- Centre de Recherche en Organogénèse Expérimentale de l’Université Laval/LOEX, Quebec City, QC G1J 1Z4, Canada
| | - Nathan Schoonjans
- Faculté de Médecine, Université Laval, Quebec City, QC G1V 0A6, Canada
| | - Julie Bérubé
- Centre de Recherche du CHU de Québec-Université Laval, Quebec City, QC G1S 4L8, Canada
- Centre de Recherche sur le Cancer de l’Université Laval, Quebec City, QC G1R 3S3, Canada
- Centre de Recherche en Organogénèse Expérimentale de l’Université Laval/LOEX, Quebec City, QC G1J 1Z4, Canada
| | - François Bordeleau
- Faculté de Médecine, Université Laval, Quebec City, QC G1V 0A6, Canada
- Centre de Recherche du CHU de Québec-Université Laval, Quebec City, QC G1S 4L8, Canada
- Centre de Recherche sur le Cancer de l’Université Laval, Quebec City, QC G1R 3S3, Canada
- Centre de Recherche en Organogénèse Expérimentale de l’Université Laval/LOEX, Quebec City, QC G1J 1Z4, Canada
| | - Alain Brisson
- UMR-CBMN, CNRS-Université de Bordeaux-IPB, 33600 Pessac, France
| | - Solange Landreville
- Faculté de Médecine, Université Laval, Quebec City, QC G1V 0A6, Canada
- Centre de Recherche du CHU de Québec-Université Laval, Quebec City, QC G1S 4L8, Canada
- Centre de Recherche sur le Cancer de l’Université Laval, Quebec City, QC G1R 3S3, Canada
- Centre de Recherche en Organogénèse Expérimentale de l’Université Laval/LOEX, Quebec City, QC G1J 1Z4, Canada
- Correspondence: ; Tel.: +1-418-682-7693
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Human endothelial cells form an endothelium in freestanding collagen hollow filaments fabricated by direct extrusion printing. BIOMATERIALS AND BIOSYSTEMS 2022; 8:100067. [PMID: 36824376 PMCID: PMC9934428 DOI: 10.1016/j.bbiosy.2022.100067] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/30/2021] [Revised: 10/08/2022] [Accepted: 10/09/2022] [Indexed: 11/05/2022] Open
Abstract
Fiber-shaped materials have great potential for tissue engineering applications as they provide structural support and spatial patterns within a three-dimensional construct. Here we demonstrate the fabrication of mechanically stable, meter-long collagen hollow filaments by a direct extrusion printing process. The fibres are permeable for oxygen and proteins and allow cultivation of primary human endothelial cells (ECs) at the inner surface under perfused conditions. The cells show typical characteristics of a well-organized EC lining including VE-cadherin expression, cellular response to flow and ECM production. The results demonstrate that the collagen tubes are capable of creating robust soft tissue filaments. The mechanical properties and the biofunctionality of these collagen hollow filaments facilitate the engineering of prevascularised tissue engineering constructs.
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Freitas-Ribeiro S, Diogo GS, Oliveira C, Martins A, Silva TH, Jarnalo M, Horta R, Reis RL, Pirraco RP. Growth Factor-Free Vascularization of Marine-Origin Collagen Sponges Using Cryopreserved Stromal Vascular Fractions from Human Adipose Tissue. Mar Drugs 2022; 20:md20100623. [PMID: 36286447 PMCID: PMC9604698 DOI: 10.3390/md20100623] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/31/2022] [Revised: 09/19/2022] [Accepted: 09/28/2022] [Indexed: 11/05/2022] Open
Abstract
The successful integration of transplanted three-dimensional tissue engineering (TE) constructs depends greatly on their rapid vascularization. Therefore, it is essential to address this vascularization issue in the initial design of constructs for perfused tissues. Two of the most important variables in this regard are scaffold composition and cell sourcing. Collagens with marine origins overcome some issues associated with mammal-derived collagen while maintaining their advantages in terms of biocompatibility. Concurrently, the freshly isolated stromal vascular fraction (SVF) of adipose tissue has been proposed as an advantageous cell fraction for vascularization purposes due to its highly angiogenic properties, allowing extrinsic angiogenic growth factor-free vascularization strategies for TE applications. In this study, we aimed at understanding whether marine collagen 3D matrices could support cryopreserved human SVF in maintaining intrinsic angiogenic properties observed for fresh SVF. For this, cryopreserved human SVF was seeded on blue shark collagen sponges and cultured up to 7 days in a basal medium. The secretome profile of several angiogenesis-related factors was studied throughout culture times and correlated with the expression pattern of CD31 and CD146, which showed the formation of a prevascular network. Upon in ovo implantation, increased vessel recruitment was observed in prevascularized sponges when compared with sponges without SVF cells. Immunohistochemistry for CD31 demonstrated the improved integration of prevascularized sponges within chick chorioalantoic membrane (CAM) tissues, while in situ hybridization showed human cells lining blood vessels. These results demonstrate the potential of using cryopreserved SVF combined with marine collagen as a streamlined approach to improve the vascularization of TE constructs.
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Affiliation(s)
- Sara Freitas-Ribeiro
- 3B’s Research Group, I3Bs—Research Institute on Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, Barco, 4805-017 Guimarães, Portugal
- ICVS/3B’s—PT Government Associate Laboratory, Braga, 4710-057 Guimarães, Portugal
| | - Gabriela S. Diogo
- 3B’s Research Group, I3Bs—Research Institute on Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, Barco, 4805-017 Guimarães, Portugal
- ICVS/3B’s—PT Government Associate Laboratory, Braga, 4710-057 Guimarães, Portugal
| | - Catarina Oliveira
- 3B’s Research Group, I3Bs—Research Institute on Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, Barco, 4805-017 Guimarães, Portugal
- ICVS/3B’s—PT Government Associate Laboratory, Braga, 4710-057 Guimarães, Portugal
| | - Albino Martins
- 3B’s Research Group, I3Bs—Research Institute on Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, Barco, 4805-017 Guimarães, Portugal
- ICVS/3B’s—PT Government Associate Laboratory, Braga, 4710-057 Guimarães, Portugal
| | - Tiago H. Silva
- 3B’s Research Group, I3Bs—Research Institute on Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, Barco, 4805-017 Guimarães, Portugal
- ICVS/3B’s—PT Government Associate Laboratory, Braga, 4710-057 Guimarães, Portugal
| | - Mariana Jarnalo
- Department of Plastic and Reconstructive Surgery, and Burn Unity, Centro Hospitalar de São João, 4200-319 Porto, Portugal
- Faculty of Medicine, University of Porto, 4200-319 Porto, Portugal
| | - Ricardo Horta
- Department of Plastic and Reconstructive Surgery, and Burn Unity, Centro Hospitalar de São João, 4200-319 Porto, Portugal
- Faculty of Medicine, University of Porto, 4200-319 Porto, Portugal
| | - Rui L. Reis
- 3B’s Research Group, I3Bs—Research Institute on Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, Barco, 4805-017 Guimarães, Portugal
- ICVS/3B’s—PT Government Associate Laboratory, Braga, 4710-057 Guimarães, Portugal
| | - Rogério P. Pirraco
- 3B’s Research Group, I3Bs—Research Institute on Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, Barco, 4805-017 Guimarães, Portugal
- ICVS/3B’s—PT Government Associate Laboratory, Braga, 4710-057 Guimarães, Portugal
- Correspondence:
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12
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Sentoku M, Iida K, Hashimoto H, Yasuda K. Dominant geometrical factors of collective cell migration in flexible 3D gelatin tube structures. BIOPHYSICAL REPORTS 2022; 2:100063. [PMID: 36425328 PMCID: PMC9680702 DOI: 10.1016/j.bpr.2022.100063] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 07/05/2022] [Accepted: 07/15/2022] [Indexed: 06/16/2023]
Abstract
Collective cell migration is a dynamic and interactive behavior of cell cohorts essential for diverse physiological developments in living organisms. Recent studies have revealed the importance of three-dimensional (3D) topographical confinements to regulate the migration modes of cell cohorts in tubular confinement. However, conventional in vitro assays fail to observe cells' behavior in response to 3D structural changes, which is necessary for examining the geometric regulation factors of collective migration. Here, we introduce a newly developed assay for fabricating flexible 3D structures of capillary microtunnels to examine the behavior of vascular endothelial cells (ECs) as they progress through the successive transition across wide or narrow tube structures. The microtunnels with altered diameters were formed inside gelatin-gel blocks by photo-thermal etching with micrometer-sized spot heating of the focused infrared laser absorption. The ECs migrated and spread two-dimensionally on the inner surface of gelatin capillary microtunnels as a monolayer instead of filling the entire capillary. In the straight cylindrical topographical constraint, leading ECs exhibited no apparent diameter dependence for the maximum peak migration velocity. However, widening the diameter in the narrow-wide structures caused a decrease in migration velocity following in direct proportion to the diameter increase ratio, whereas narrowing the diameter in wide-narrow microtunnels increased the speed without obvious correlation between velocity change and diameter change. The results demonstrated the ability of the newly developed flexible 3D gelatin tube structures for collective cell migration, and the findings provide insights into the dominant geometric factor of the emerging migratory modes for endothelial migration as asymmetric fluid flow-like behavior in the borderless cylindrical cell sheets.
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Affiliation(s)
- Mitsuru Sentoku
- Department of Pure and Applied Physics, Graduate School of Advanced Science and Engineering, Waseda University, Shinjuku, Tokyo, Japan
| | - Kento Iida
- Department of Pure and Applied Physics, Graduate School of Advanced Science and Engineering, Waseda University, Shinjuku, Tokyo, Japan
| | - Hiromichi Hashimoto
- Department of Pure and Applied Physics, Graduate School of Advanced Science and Engineering, Waseda University, Shinjuku, Tokyo, Japan
| | - Kenji Yasuda
- Department of Pure and Applied Physics, Graduate School of Advanced Science and Engineering, Waseda University, Shinjuku, Tokyo, Japan
- Department of Physics, School of Advanced Science and Engineering, Waseda University, Shinjuku, Tokyo, Japan
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13
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Yavvari P, Laporte A, Elomaa L, Schraufstetter F, Pacharzina I, Daberkow AD, Hoppensack A, Weinhart M. 3D-Cultured Vascular-Like Networks Enable Validation of Vascular Disruption Properties of Drugs In Vitro. Front Bioeng Biotechnol 2022; 10:888492. [PMID: 35769106 PMCID: PMC9234334 DOI: 10.3389/fbioe.2022.888492] [Citation(s) in RCA: 6] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/02/2022] [Accepted: 04/13/2022] [Indexed: 02/06/2023] Open
Abstract
Vascular-disrupting agents are an interesting class of anticancer compounds because of their combined mode of action in preventing new blood vessel formation and disruption of already existing vasculature in the immediate microenvironment of solid tumors. The validation of vascular disruption properties of these drugs in vitro is rarely addressed due to the lack of proper in vitro angiogenesis models comprising mature and long-lived vascular-like networks. We herein report an indirect coculture model of human umbilical vein endothelial cells (HUVECs) and human dermal fibroblasts (HDFs) to form three-dimensional profuse vascular-like networks. HUVECs embedded and sandwiched in the collagen scaffold were cocultured with HDFs located outside the scaffold. The indirect coculture approach with the vascular endothelial growth factor (VEGF) producing HDFs triggered the formation of progressively maturing lumenized vascular-like networks of endothelial cells within less than 7 days, which have proven to be viably maintained in culture beyond day 21. Molecular weight-dependent Texas red-dextran permeability studies indicated high vascular barrier function of the generated networks. Their longevity allowed us to study the dose-dependent response upon treatment with the three known antiangiogenic and/or vascular disrupting agents brivanib, combretastatin A4 phosphate (CA4P), and 6´-sialylgalactose (SG) via semi-quantitative brightfield and qualitative confocal laser scanning microscopic (CLSM) image analysis. Compared to the reported data on in vivo efficacy of these drugs in terms of antiangiogenic and vascular disrupting effects, we observed similar trends with our 3D model, which are not reflected in conventional in vitro angiogenesis assays. High-vascular disruption under continuous treatment of the matured vascular-like network was observed at concentrations ≥3.5 ng·ml−1 for CA4P and ≥300 nM for brivanib. In contrast, SG failed to induce any significant vascular disruption in vitro. This advanced model of a 3D vascular-like network allows for testing single and combinational antiangiogenic and vascular disrupting effects with optimized dosing and may thus bridge the gap between the in vitro and in vivo experiments in validating hits from high-throughput screening. Moreover, the physiological 3D environment mimicking in vitro assay is not only highly relevant to in vivo studies linked to cancer but also to the field of tissue regeneration.
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Affiliation(s)
| | - Anna Laporte
- Institute of Physical Chemistry and Electrochemistry, Leibniz Universität Hannover, Hannover, Germany
| | - Laura Elomaa
- Institute of Chemistry and Biochemistry, Freie Universität Berlin, Berlin, Germany
| | | | - Inga Pacharzina
- Institute of Chemistry and Biochemistry, Freie Universität Berlin, Berlin, Germany
| | | | - Anke Hoppensack
- Institute of Chemistry and Biochemistry, Freie Universität Berlin, Berlin, Germany
| | - Marie Weinhart
- Institute of Chemistry and Biochemistry, Freie Universität Berlin, Berlin, Germany
- Institute of Physical Chemistry and Electrochemistry, Leibniz Universität Hannover, Hannover, Germany
- *Correspondence: Marie Weinhart, ,
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14
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Rathod L, Bhowmick S, Patel P, Sawant K. Calendula flower extract loaded collagen film exhibits superior wound healing potential: Preparation, evaluation, in-vitro & in-vivo wound healing study. J Drug Deliv Sci Technol 2022. [DOI: 10.1016/j.jddst.2022.103363] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/18/2022]
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15
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Nawrot DA, Ozer LY, Al Haj Zen A. A Novel High Content Angiogenesis Assay Reveals That Lacidipine, L-Type Calcium Channel Blocker, Induces In Vitro Vascular Lumen Expansion. Int J Mol Sci 2022; 23:ijms23094891. [PMID: 35563280 PMCID: PMC9100973 DOI: 10.3390/ijms23094891] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/01/2022] [Revised: 04/23/2022] [Accepted: 04/26/2022] [Indexed: 11/21/2022] Open
Abstract
Angiogenesis is a critical cellular process toward establishing a functional circulatory system capable of delivering oxygen and nutrients to the tissue in demand. In vitro angiogenesis assays represent an important tool for elucidating the biology of blood vessel formation and for drug discovery applications. Herein, we developed a novel, high content 2D angiogenesis assay that captures endothelial morphogenesis’s cellular processes, including lumen formation. In this assay, endothelial cells form luminized vascular-like structures in 48 h. The assay was validated for its specificity and performance. Using the optimized assay, we conducted a phenotypic screen of a library containing 150 FDA-approved cardiovascular drugs to identify modulators of lumen formation. The screening resulted in several L-type calcium channel blockers being able to expand the lumen space compared to controls. Among these blockers, Lacidipine was selected for follow-up studies. We found that the endothelial cells treated with Lacidipine showed enhanced activity of caspase-3 in the luminal space. Pharmacological inhibition of caspase activity abolished the Lacidipine-enhancing effect on lumen formation, suggesting the involvement of apoptosis. Using a Ca2+ biosensor, we found that Lacipidine reduces the intracellular Ca2+ oscillations amplitude in the endothelial cells at the early stage, whereas Lacidipine blocks these Ca2+ oscillations completely at the late stage. The inhibition of MLCK exhibits a phenotype of lumen expansion similar to that of Lacidipine. In conclusion, this study describes a novel high-throughput phenotypic assay to study angiogenesis. Our findings suggest that calcium signalling plays an essential role during lumen morphogenesis. L-type Ca2+ channel blockers could be used for more efficient angiogenesis-mediated therapies.
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Affiliation(s)
- Dorota A. Nawrot
- BHF Centre of Research Excellence, Division of Cardiovascular Medicine, Radcliffe Department of Medicine, University of Oxford, Oxford OX3 9DU, UK;
- Alzheimer’s Research UK, Oxford Drug Discovery Institute, Target Discovery Institute, Nuffield Department of Medicine, University of Oxford, Oxford OX3 7FZ, UK
| | - Lutfiye Yildiz Ozer
- College of Health and Life Sciences, Hamad Bin Khalifa University, Education City, Doha P.O. Box 34110, Qatar;
| | - Ayman Al Haj Zen
- BHF Centre of Research Excellence, Division of Cardiovascular Medicine, Radcliffe Department of Medicine, University of Oxford, Oxford OX3 9DU, UK;
- College of Health and Life Sciences, Hamad Bin Khalifa University, Education City, Doha P.O. Box 34110, Qatar;
- Correspondence: ; Tel.: +974-4454-6352
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16
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Massimini M, Romanucci M, Maria RD, Della Salda L. Histological Evaluation of Long-Term Collagen Type I Culture. Methods Mol Biol 2022; 2514:95-105. [PMID: 35771422 DOI: 10.1007/978-1-0716-2403-6_10] [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] [Indexed: 06/15/2023]
Abstract
Histological approach to long-term culture on collagen type I permits the evaluation of vasculogenic mimicry morphological features and the identification of endothelial-like cell-specific antigens. Here, we show the preparation of collagen type I solution, the embedding and the sections cutting of D17 osteosarcoma cells long-term culture, and then the hematoxylin and eosin (H&E) staining to identify endothelial-like structure. Moreover, we provide the protocols for periodic acid-Schiff (PAS) staining to evidence glycoproteins and CD31 immunohistochemistry to exclude the presence of this endothelial marker, as per definition by vasculogenic mimicry concept. This method allows to consider long-term culture as tissue, promoting the deeper study of vascular-like structures in vitro.
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17
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Kemp SS, Lin PK, Sun Z, Castaño MA, Yrigoin K, Penn MR, Davis GE. Molecular basis for pericyte-induced capillary tube network assembly and maturation. Front Cell Dev Biol 2022; 10:943533. [PMID: 36072343 PMCID: PMC9441561 DOI: 10.3389/fcell.2022.943533] [Citation(s) in RCA: 7] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/13/2022] [Accepted: 07/25/2022] [Indexed: 11/13/2022] Open
Abstract
Here we address the functional importance and role of pericytes in capillary tube network assembly, an essential process that is required for vascularized tissue development, maintenance, and health. Healthy capillaries may be directly capable of suppressing human disease. Considerable advances have occurred in our understanding of the molecular and signaling requirements controlling EC lumen and tube formation in 3D extracellular matrices. A combination of SCF, IL-3, SDF-1α, FGF-2 and insulin ("Factors") in conjunction with integrin- and MT1-MMP-induced signaling are required for EC sprouting behavior and tube formation under serum-free defined conditions. Pericyte recruitment to the abluminal EC tube surface results in elongated and narrow tube diameters and deposition of the vascular basement membrane. In contrast, EC tubes in the absence of pericytes continue to widen and shorten over time and fail to deposit basement membranes. Pericyte invasion, recruitment and proliferation in 3D matrices requires the presence of ECs. A detailed analysis identified that EC-derived PDGF-BB, PDGF-DD, ET-1, HB-EGF, and TGFβ1 are necessary for pericyte recruitment, proliferation, and basement membrane deposition. Blockade of these individual factors causes significant pericyte inhibition, but combined blockade profoundly interferes with these events, resulting in markedly widened EC tubes without basement membranes, like when pericytes are absent.
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Affiliation(s)
- Scott S Kemp
- Department of Molecular Pharmacology and Physiology, Morsani College of Medicine, University of South Florida School of Medicine, Tampa, FL, United States
| | - Prisca K Lin
- Department of Molecular Pharmacology and Physiology, Morsani College of Medicine, University of South Florida School of Medicine, Tampa, FL, United States
| | - Zheying Sun
- Department of Molecular Pharmacology and Physiology, Morsani College of Medicine, University of South Florida School of Medicine, Tampa, FL, United States
| | - Maria A Castaño
- Department of Molecular Pharmacology and Physiology, Morsani College of Medicine, University of South Florida School of Medicine, Tampa, FL, United States
| | - Ksenia Yrigoin
- Department of Molecular Pharmacology and Physiology, Morsani College of Medicine, University of South Florida School of Medicine, Tampa, FL, United States
| | - Marlena R Penn
- Department of Molecular Pharmacology and Physiology, Morsani College of Medicine, University of South Florida School of Medicine, Tampa, FL, United States
| | - George E Davis
- Department of Molecular Pharmacology and Physiology, Morsani College of Medicine, University of South Florida School of Medicine, Tampa, FL, United States
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18
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Santiago FS, Li Y, Khachigian LM. Serine 26 in Early Growth Response-1 Is Critical for Endothelial Proliferation, Migration, and Network Formation. J Am Heart Assoc 2021; 10:e020521. [PMID: 34476983 PMCID: PMC8649526 DOI: 10.1161/jaha.120.020521] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/18/2022]
Abstract
Background Vascular endothelial cell proliferation, migration, and network formation are key proangiogenic processes involving the prototypic immediate early gene product, Egr‐1 (early growth response‐1). Egr‐1 undergoes phosphorylation at a conserved Ser26 but its function is completely unknown in endothelial cells or any other cell type. Methods and Results A CRISPR/Cas9 strategy was used to introduce a homozygous Ser26>Ala mutation into endogenous Egr‐1 in human microvascular endothelial cells. In the course of generating mutant cells, we produced cells with homozygous deletion in Egr‐1 caused by frameshift and premature termination. We found that Ser26 mutation in Egr‐1, or Egr‐1 deletion, perturbed endothelial cell proliferation in models of cell counting or real‐time growth using the xCELLigence System. We found that Ser26 mutation or Egr‐1 deletion ameliorated endothelial cell migration toward VEGF‐A165 (vascular endothelial growth factor‐A) in a dual‐chamber model. On solubilized basement membrane preparations, Ser26 mutation or Egr‐1 deletion prevented endothelial network (or tubule) formation, an in vitro model of angiogenesis. Flow cytometry further revealed that Ser26 mutation or Egr‐1 deletion elevated early and late apoptosis. Finally, we demonstrated that Ser26 mutation or Egr‐1 deletion increased VE‐cadherin (vascular endothelial cadherin) expression, a regulator of endothelial adhesion and signaling, permeability, and angiogenesis. Conclusions These findings not only indicate that Egr‐1 is essential for endothelial cell proliferation, migration, and network formation, but also show that point mutation in Ser26 is sufficient to impair each of these processes and trigger apoptosis as effectively as the absence of Egr‐1. This highlights the importance of Ser26 in Egr‐1 for a range of proangiogenic processes.
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Affiliation(s)
- Fernando S Santiago
- Vascular Biology and Translational Research School of Medical Sciences UNSW Medicine and HealthUniversity of New South Wales Sydney NSW Australia
| | - Yue Li
- Vascular Biology and Translational Research School of Medical Sciences UNSW Medicine and HealthUniversity of New South Wales Sydney NSW Australia
| | - Levon M Khachigian
- Vascular Biology and Translational Research School of Medical Sciences UNSW Medicine and HealthUniversity of New South Wales Sydney NSW Australia
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19
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Groger A, Megas IF, Noah EM, Pallua N, Grieb G. Proliferation of endothelial cells (HUVEC) on specific-modified collagen sponges loaded with different growth factors. Int J Artif Organs 2021; 44:880-886. [PMID: 34496659 DOI: 10.1177/03913988211043198] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022]
Abstract
In general, matrices for tissue engineering must maintain structural integrity during the process of tissue formation and promote vascularization of developing tissue. Therefore, collagen sponges, manufactured by an approach that offers the potential of unidirectional pore size, were seeded with human umbilical vein endothelial cells (HUVEC) to demonstrate a positive effect on cell proliferation. In addition, vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) have been used to promote proliferation of HUVEC on optimized collagen sponges. Growth and viability of the cells were evaluated. Potential unidirectional pore structure demonstrated an improvement of both, endothelial cell growth and viability. Supplementation of growth factors showed an additional increase of endothelial cell growth on collagen sponges, which confirmed the high potential of combining this biomaterial with growth factors. The results suggest that a collagen sponge with a potential specific pore size could be a suitable scaffold for endothelial cells and might be a promising implantable biomaterial with enhanced angiogenic capabilities for future clinical applications.
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Affiliation(s)
- Andreas Groger
- Department of Plastic Surgery and Hand Surgery, Burn Center, Medical Faculty, RWTH Aachen University, Aachen, Germany
| | - Ioannis-Fivos Megas
- Department of Plastic Surgery and Hand Surgery, Gemeinschaftskrankenhaus Havelhöhe, Berlin, Germany
| | - Ernst Magnus Noah
- Department of Plastic Surgery and Hand Surgery, Burn Center, Medical Faculty, RWTH Aachen University, Aachen, Germany.,Department of Plastic, Reconstructive, Aesthetic and Hand Surgery, Rotes Kreuz Krankenhaus Kassel, Kassel, Germany
| | - Norbert Pallua
- Department of Plastic Surgery and Hand Surgery, Burn Center, Medical Faculty, RWTH Aachen University, Aachen, Germany
| | - Gerrit Grieb
- Department of Plastic Surgery and Hand Surgery, Burn Center, Medical Faculty, RWTH Aachen University, Aachen, Germany.,Department of Plastic Surgery and Hand Surgery, Gemeinschaftskrankenhaus Havelhöhe, Berlin, Germany
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20
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Xin Y, Roh K, Cho E, Park D, Whang W, Jung E. Isookanin Inhibits PGE 2-Mediated Angiogenesis by Inducing Cell Arrest through Inhibiting the Phosphorylation of ERK1/2 and CREB in HMEC-1 Cells. Int J Mol Sci 2021; 22:ijms22126466. [PMID: 34208772 PMCID: PMC8234715 DOI: 10.3390/ijms22126466] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/13/2021] [Revised: 06/09/2021] [Accepted: 06/14/2021] [Indexed: 11/16/2022] Open
Abstract
Inflammation is increasingly recognized as a critical mediator of angiogenesis, and unregulated angiogenic responses often involve human diseases. The importance of regulating angiogenesis in inflammatory diseases has been demonstrated through some successful cases of anti-angiogenesis therapies in related diseases, including arthritis, but it has been reported that some synthetic types of antiangiogenic drugs have potential side effects. In recent years, the importance of finding alternative strategies for regulating angiogenesis has begun to attract the attention of researchers. Therefore, identification of natural ingredients used to prevent or treat angiogenesis-related diseases will play a greater role. Isookanin is a phenolic flavonoid presented in Bidens extract, and it has been reported that isookanin possesses some biological properties, including antioxidative and anti-inflammatory effects, anti-diabetic properties, and an ability to inhibit α-amylase. However, its antiangiogenic effects and mechanism thereof have not been studied yet. In this study, our results indicate that isookanin has an effective inhibitory effect on the angiogenic properties of microvascular endothelial cells. Isookanin shows inhibitory effects in multiple stages of PGE2-induced angiogenesis, including the growth, proliferation, migration, and tube formation of microvascular endothelial cells. In addition, isookanin induces cell cycle arrest in S phase, which is also the reason for subsequent inhibition of cell proliferation. The mechanism of inhibiting angiogenesis by isookanin is related to the inhibition of PGE2-mediated ERK1/2 and CREB phosphorylation. These findings make isookanin a potential candidate for the treatment of angiogenesis-related diseases.
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Affiliation(s)
- Yingji Xin
- Biospectrum Life Science Institute, Yongin 16827, Korea; (Y.X.); (K.R.); (E.C.); (D.P.)
- Department of Global Innovative Drug, Graduate School, College of Pharmacy, Chung-Ang University, Heukseok-dong, Dongjak-gu, Seoul 156756, Korea
| | - Kyungbaeg Roh
- Biospectrum Life Science Institute, Yongin 16827, Korea; (Y.X.); (K.R.); (E.C.); (D.P.)
| | - Eunae Cho
- Biospectrum Life Science Institute, Yongin 16827, Korea; (Y.X.); (K.R.); (E.C.); (D.P.)
| | - Deokhoon Park
- Biospectrum Life Science Institute, Yongin 16827, Korea; (Y.X.); (K.R.); (E.C.); (D.P.)
| | - Wankyunn Whang
- Department of Global Innovative Drug, Graduate School, College of Pharmacy, Chung-Ang University, Heukseok-dong, Dongjak-gu, Seoul 156756, Korea
- Correspondence: (W.W.); (E.J.); Tel.: +82-70-5117-0043 (E.J.)
| | - Eunsun Jung
- Biospectrum Life Science Institute, Yongin 16827, Korea; (Y.X.); (K.R.); (E.C.); (D.P.)
- Correspondence: (W.W.); (E.J.); Tel.: +82-70-5117-0043 (E.J.)
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Pouw AE, Greiner MA, Coussa RG, Jiao C, Han IC, Skeie JM, Fingert JH, Mullins RF, Sohn EH. Cell-Matrix Interactions in the Eye: From Cornea to Choroid. Cells 2021; 10:687. [PMID: 33804633 PMCID: PMC8003714 DOI: 10.3390/cells10030687] [Citation(s) in RCA: 50] [Impact Index Per Article: 12.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/14/2021] [Revised: 03/03/2021] [Accepted: 03/16/2021] [Indexed: 02/07/2023] Open
Abstract
The extracellular matrix (ECM) plays a crucial role in all parts of the eye, from maintaining clarity and hydration of the cornea and vitreous to regulating angiogenesis, intraocular pressure maintenance, and vascular signaling. This review focuses on the interactions of the ECM for homeostasis of normal physiologic functions of the cornea, vitreous, retina, retinal pigment epithelium, Bruch's membrane, and choroid as well as trabecular meshwork, optic nerve, conjunctiva and tenon's layer as it relates to glaucoma. A variety of pathways and key factors related to ECM in the eye are discussed, including but not limited to those related to transforming growth factor-β, vascular endothelial growth factor, basic-fibroblastic growth factor, connective tissue growth factor, matrix metalloproteinases (including MMP-2 and MMP-9, and MMP-14), collagen IV, fibronectin, elastin, canonical signaling, integrins, and endothelial morphogenesis consistent of cellular activation-tubulogenesis and cellular differentiation-stabilization. Alterations contributing to disease states such as wound healing, diabetes-related complications, Fuchs endothelial corneal dystrophy, angiogenesis, fibrosis, age-related macular degeneration, retinal detachment, and posteriorly inserted vitreous base are also reviewed.
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Affiliation(s)
- Andrew E. Pouw
- Department of Ophthalmology and Visual Sciences, Carver College of Medicine, University of Iowa Hospitals & Clinics, Iowa City, IA 52242, USA; (A.E.P.); (M.A.G.); (R.G.C.); (C.J.); (I.C.H.); (J.M.S.); (J.H.F.); (R.F.M.)
- Institute for Vision Research, University of Iowa, Iowa City, IA 52242, USA
| | - Mark A. Greiner
- Department of Ophthalmology and Visual Sciences, Carver College of Medicine, University of Iowa Hospitals & Clinics, Iowa City, IA 52242, USA; (A.E.P.); (M.A.G.); (R.G.C.); (C.J.); (I.C.H.); (J.M.S.); (J.H.F.); (R.F.M.)
- Institute for Vision Research, University of Iowa, Iowa City, IA 52242, USA
| | - Razek G. Coussa
- Department of Ophthalmology and Visual Sciences, Carver College of Medicine, University of Iowa Hospitals & Clinics, Iowa City, IA 52242, USA; (A.E.P.); (M.A.G.); (R.G.C.); (C.J.); (I.C.H.); (J.M.S.); (J.H.F.); (R.F.M.)
- Institute for Vision Research, University of Iowa, Iowa City, IA 52242, USA
| | - Chunhua Jiao
- Department of Ophthalmology and Visual Sciences, Carver College of Medicine, University of Iowa Hospitals & Clinics, Iowa City, IA 52242, USA; (A.E.P.); (M.A.G.); (R.G.C.); (C.J.); (I.C.H.); (J.M.S.); (J.H.F.); (R.F.M.)
- Institute for Vision Research, University of Iowa, Iowa City, IA 52242, USA
| | - Ian C. Han
- Department of Ophthalmology and Visual Sciences, Carver College of Medicine, University of Iowa Hospitals & Clinics, Iowa City, IA 52242, USA; (A.E.P.); (M.A.G.); (R.G.C.); (C.J.); (I.C.H.); (J.M.S.); (J.H.F.); (R.F.M.)
- Institute for Vision Research, University of Iowa, Iowa City, IA 52242, USA
| | - Jessica M. Skeie
- Department of Ophthalmology and Visual Sciences, Carver College of Medicine, University of Iowa Hospitals & Clinics, Iowa City, IA 52242, USA; (A.E.P.); (M.A.G.); (R.G.C.); (C.J.); (I.C.H.); (J.M.S.); (J.H.F.); (R.F.M.)
| | - John H. Fingert
- Department of Ophthalmology and Visual Sciences, Carver College of Medicine, University of Iowa Hospitals & Clinics, Iowa City, IA 52242, USA; (A.E.P.); (M.A.G.); (R.G.C.); (C.J.); (I.C.H.); (J.M.S.); (J.H.F.); (R.F.M.)
- Institute for Vision Research, University of Iowa, Iowa City, IA 52242, USA
| | - Robert F. Mullins
- Department of Ophthalmology and Visual Sciences, Carver College of Medicine, University of Iowa Hospitals & Clinics, Iowa City, IA 52242, USA; (A.E.P.); (M.A.G.); (R.G.C.); (C.J.); (I.C.H.); (J.M.S.); (J.H.F.); (R.F.M.)
- Institute for Vision Research, University of Iowa, Iowa City, IA 52242, USA
| | - Elliott H. Sohn
- Department of Ophthalmology and Visual Sciences, Carver College of Medicine, University of Iowa Hospitals & Clinics, Iowa City, IA 52242, USA; (A.E.P.); (M.A.G.); (R.G.C.); (C.J.); (I.C.H.); (J.M.S.); (J.H.F.); (R.F.M.)
- Institute for Vision Research, University of Iowa, Iowa City, IA 52242, USA
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Evaluation of Wound-Healing and Antioxidant Effects of Marantodes pumilum (Blume) Kuntze in an Excision Wound Model. Molecules 2021; 26:molecules26010228. [PMID: 33466302 PMCID: PMC7795968 DOI: 10.3390/molecules26010228] [Citation(s) in RCA: 16] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/13/2020] [Revised: 12/27/2020] [Accepted: 12/29/2020] [Indexed: 11/17/2022] Open
Abstract
Marantodes pumilum (MP) is a great source of herbal medicine used traditionally by both men and women for various purposes. MP may have potential wound-healing effects due to its diverse biological properties. An extensive study was conducted in a normal male rat model for determining the effects of MP var. pumila (MPvp) and var. alata (MPva) on the wound healing process. Here, 126 male Sprague-Dawley rats were divided randomly into seven groups as follows: sham-operated (SH), vehicle dressing (VD), flavine dressing (FD), MPvp leaves (PL), MPvp roots (PR), MPva leaves (AL), and MPva roots (AR). The parameters studied were the percentage of wound contraction, histomorphology study by hematoxylin and eosin (H&E), Masson–Goldner trichrome (MGT), and immunohistochemistry (IHC) staining. In addition, the levels of enzymatic antioxidants and malondialdehyde were also measured in the wound tissue homogenates. Wounds treated with extracts (PL, PR, AL, and AR) showed significantly faster healing (p < 0.05) compared to untreated and control groups (SH, VD, and FD). Histological analysis among MP-treated groups revealed better re-epithelialization, higher collagen deposition, enhanced fibronectin content and fibroblast cells, and higher fiber transformation from collagen-III to collagen-I, accompanied with a significant surge in enzymatic antioxidant activities and a decline in lipid peroxidation. MP has antioxidant effects that may enhance wound healing in the rat model.
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Bansal RK, Gupta MK, Gupta VK, Kaur G, Seth AK. Endoscopic Treatment of Upper Gastrointestinal Bleeding Using Haemoseal Spray: A Retrospective, Observational Study from a Tertiary Center in North India. JOURNAL OF DIGESTIVE ENDOSCOPY 2020. [DOI: 10.1055/s-0040-1722387] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/25/2022] Open
Abstract
Abstract
Introduction United States Food and Drug Administration recently approved use of Hemospray for the management of gastrointestinal (GI) Bleeding. We report our experience with Haemoseal Spray (HS, Shaili Endoscopy) for the treatment of upper GI bleeding (UGIB).
Methods Records of patients who received HS for UGIB from January 2013 to June 2018 were studied retrospectively. Patients with UGIB from focal lesions refractory to conventional endotherapy or those with diffuse/multiple lesions not amenable to conventional endotherapy received 5cc HS spray. Primary end-point studied was clinical success, defined as control of bleeding over 24 hours. Secondary end-points evaluated included recurrence of bleeding within 7 days, in-hospital mortality, and complications secondary to HS.
Results Thirty-eight patients were treated with HS. The median age was 57 (range: 5–87) years with 27 males and 11 females. In 24 patients, HS was used as monotherapy, while it was combined with Injection/Clip/Argon Plasma Coagulation in 14. Etiology of bleeding was ulcers or erosions in 22, malignancy in 10, portal hypertensive gastropathy/gastric antral vascular ectasia in 4, and radiation gastropathy in 2. Clinical success was achieved in 32/38 (84%). All six nonresponders had coagulopathy related to chemotherapy/bone marrow transplant. Recurrent bleeding within 7 days was observed in four patients (gastric malignancy 2, radiation gastropathy 2). In-hospital mortality was seen in 8/38 (21%) of which 2(4.8%) were directly related to ongoing GI bleeding. There was no procedure-related complication.
Conclusion HS is an effective and safe tool in the endoscopic management of UGIB due to diffuse or multiple focal lesions or focal lesions refractory to conventional endotherapy.
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Affiliation(s)
- Rinkesh Kumar Bansal
- Department of Gastroenterology and Hepatobiliary Sciences, Fortis Memorial Research Institute, Gurugram, Haryana, India
| | - Mahesh Kumar Gupta
- Department of Gastroenterology and Hepatobiliary Sciences, Fortis Memorial Research Institute, Gurugram, Haryana, India
| | - Varun Kumar Gupta
- Department of Gastroenterology and Hepatobiliary Sciences, Fortis Memorial Research Institute, Gurugram, Haryana, India
| | - Gursimran Kaur
- Department of Gastroenterology and Hepatobiliary Sciences, Fortis Memorial Research Institute, Gurugram, Haryana, India
| | - Avnish Kumar Seth
- Department of Gastroenterology and Hepatobiliary Sciences, Fortis Memorial Research Institute, Gurugram, Haryana, India
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24
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Rujirachotiwat A, Suttamanatwong S. Curcumin Promotes Collagen Type I, Keratinocyte Growth Factor-1, and Epidermal Growth Factor Receptor Expressions in the In Vitro Wound Healing Model of Human Gingival Fibroblasts. Eur J Dent 2020; 15:63-70. [PMID: 33003239 PMCID: PMC7902102 DOI: 10.1055/s-0040-1715781] [Citation(s) in RCA: 14] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/24/2022] Open
Abstract
OBJECTIVE Curcumin promotes oral wound healing; however, the underlying mechanism remains unknown. We hypothesized that curcumin may regulate gene expression in human gingival fibroblasts (hGFs). This study investigated the effect of curcumin on the expression of wound healing-related genes, collagen type I (COL1), keratinocyte growth factor (KGF)-1, and epidermal growth factor receptor (EGFR), in the in vitro wound healing model of hGFs, as well as the signaling pathway involved in the regulation of these genes by curcumin. MATERIALS AND METHODS The hGFs were treated with curcumin in the unwounded condition and in the in vitro wound healing model (scratch assay). Gene expression was determined by quantitative polymerase chain reaction. PD98059 was used to elucidate whether extracellular signal regulated kinase (ERK) signaling is involved in the curcumin-regulated gene expression in hGFs. Cell migration was also analyzed by the scratch assay. STATISTICAL ANALYSIS Data were analyzed by independent t-test or one-way analysis of variance (ANOVA) followed by Tukey's Honestly Significant Difference ( HSD) test. RESULTS In unwounded hGFs, curcumin significantly increased KGF-1 and EGFR expressions but not COL1 mRNA expression. Interestingly, curcumin significantly upregulated COL1, KGF-1, and EGFR expressions in the in vitro wound healing model. Furthermore, PD98059 significantly decreased the curcumin-induced COL1 and EGFR expressions, but did not significantly affect KGF-1 upregulation by curcumin. However, hGF migration was not affected by curcumin treatment. CONCLUSION Curcumin induced KGF-1 and EGFR expressions in unwounded hGFs. In the in vitro wound healing model, curcumin upregulated COL1 and EGFR expression via the ERK pathway and increased KGF-1 expression, possibly by an ERK-independent mechanism.
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Affiliation(s)
- Auspreeya Rujirachotiwat
- Department of Pediatric Dentistry, Faculty of Dentistry, Chulalongkorn University, Bangkok, Thailand.,Division of Dentistry, Banphue Hospital, Banphue District, Udonthani, Thailand
| | - Supaporn Suttamanatwong
- Department of Physiology, Faculty of Dentistry, Chulalongkorn University, Bangkok, Thailand.,Research Unit of Herbal Medicine and Natural Product for Dental Treatment, Faculty of Dentistry, Chulalongkorn University, Bangkok, Thailand
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25
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The Role of Methionine Aminopeptidase 2 in Lymphangiogenesis. Int J Mol Sci 2020; 21:ijms21145148. [PMID: 32708166 PMCID: PMC7403956 DOI: 10.3390/ijms21145148] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/18/2020] [Revised: 07/14/2020] [Accepted: 07/16/2020] [Indexed: 11/17/2022] Open
Abstract
During the metastasis process, tumor cells invade the blood circulatory system directly from venous capillaries or indirectly via lymphatic vessels. Understanding the relative contribution of each pathway and identifying the molecular targets that affect both processes is critical for reducing cancer spread. Methionine aminopeptidase 2 (MetAp2) is an intracellular enzyme known to modulate angiogenesis. In this study, we investigated the additional role of MetAp2 in lymphangiogenesis. A histological staining of tumors from human breast-cancer donors was performed in order to detect the level and the localization of MetAp2 and lymphatic capillaries. The basal enzymatic level and activity in vascular and lymphatic endothelial cells were compared, followed by loss of function studies determining the role of MetAp2 in lymphangiogenesis in vitro and in vivo. The results from the histological analyses of the tumor tissues revealed a high MetAp2 expression, with detectable sites of co-localization with lymphatic capillaries. We showed slightly reduced levels of the MetAp2 enzyme and MetAp2 mRNA expression and activity in primary lymphatic cells when compared to the vascular endothelial cells. The genetic and biochemical manipulation of MetAp2 confirmed the dual activity of the enzyme in both vascular and lymphatic remodulation in cell function assays and in a zebrafish model. We found that cancer-related lymphangiogenesis is inhibited in murine models following MetAp2 inhibition treatment. Taken together, our study provides an indication that MetAp2 is a significant contributor to lymphangiogenesis and carries a dual role in both vascular and lymphatic capillary formation. Our data suggests that MetAp2 inhibitors can be effectively used as anti-metastatic broad-spectrum drugs.
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26
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Angiogenesis Analyzer for ImageJ - A comparative morphometric analysis of "Endothelial Tube Formation Assay" and "Fibrin Bead Assay". Sci Rep 2020; 10:11568. [PMID: 32665552 PMCID: PMC7360583 DOI: 10.1038/s41598-020-67289-8] [Citation(s) in RCA: 315] [Impact Index Per Article: 63.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/13/2020] [Accepted: 06/02/2020] [Indexed: 12/31/2022] Open
Abstract
Angiogenesis assays based on in vitro capillary-like growth of endothelial cells (EC) are widely used, either to evaluate the effect of anti- and pro-angiogenesis drugs of interest, or to test and compare the functional capacities of various types of EC and progenitor cells. Among the different methods applied to study angiogenesis, the most commonly used is the "Endothelial Tube Formation Assay" (ETFA). In suitable culture conditions, EC form two-dimensional (2D) branched structures that can lead to a meshed pseudo-capillary network. An alternative approach to ETFA is the "Fibrin Bead Assay" (FBA), based on the use of Cytodex 3 microspheres, which promote the growth of 3D capillary-like patterns from coated EC, suitable for high throughput in vitro angiogenesis studies. The analytical evaluation of these two widely used assays still remains challenging in terms of observation method and image analysis. We previously developed the "Angiogenesis Analyzer" for ImageJ (AA), a tool allowing analysis of ETFA-derived images, according to characteristics of the pseudo-capillary networks. In this work, we developed and implemented a new algorithm for AA able to recognize microspheres and to analyze the attached capillary-like structures from the FBA model. Such a method is presented for the first time in fully automated mode and using non-destructive image acquisition. We detailed these two algorithms and used the new AA version to compare both methods (i.e. ETFA and FBA) in their efficiency, accuracy and statistical relevance to model angiogenesis patterns of Human Umbilical Vein EC (HUVEC). Although the two methods do not assess the same biological step, our data suggest that they display specific and complementary information on the angiogenesis processes analysis.
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27
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Brown A, He H, Trumper E, Valdez J, Hammond P, Griffith LG. Engineering PEG-based hydrogels to foster efficient endothelial network formation in free-swelling and confined microenvironments. Biomaterials 2020; 243:119921. [PMID: 32172030 PMCID: PMC7203641 DOI: 10.1016/j.biomaterials.2020.119921] [Citation(s) in RCA: 54] [Impact Index Per Article: 10.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/03/2019] [Revised: 02/22/2020] [Accepted: 02/25/2020] [Indexed: 02/07/2023]
Abstract
In vitro tissue engineered models are poised to have significant impact on disease modeling and preclinical drug development. Reliable methods to induce microvascular networks in such microphysiological systems are needed to improve the size and physiological function of these models. By systematically engineering several physical and biomolecular properties of the cellular microenvironment (including crosslinking density, polymer density, adhesion ligand concentration, and degradability), we establish design principles that describe how synthetic matrix properties influence vascular morphogenesis in modular and tunable hydrogels based on commercial 8-arm poly (ethylene glycol) (PEG8a) macromers. We apply these design principles to generate endothelial networks that exhibit consistent morphology throughout depths of hydrogel greater than 1 mm. These PEG8a-based hydrogels have relatively high volumetric swelling ratios (>1.5), which limits their utility in confined environments such as microfluidic devices. To overcome this limitation, we mitigate swelling by incorporating a highly functional PEG-grafted alpha-helical poly (propargyl-l-glutamate) (PPLGgPEG) macromer along with the canonical 8-arm PEG8a macromer in gel formation. This hydrogel platform supports enhanced endothelial morphogenesis in neutral-swelling environments. Finally, we incorporate PEG8a-PPLGgPEG gels into microfluidic devices and demonstrate improved diffusion kinetics and microvascular network formation in situ compared to PEG8a-based gels.
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Affiliation(s)
- Alexander Brown
- Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, 02139, USA
| | - Hongkun He
- Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA, 02139, USA
| | - Ella Trumper
- Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, 02139, USA
| | - Jorge Valdez
- Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, 02139, USA
| | - Paula Hammond
- Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA, 02139, USA; Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, 02139, USA
| | - Linda G Griffith
- Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, 02139, USA; Center for Gynepathology Research, Massachusetts Institute of Technology, Cambridge, MA, 02139, USA.
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28
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UV irradiation of Type I collagen gels changed the morphology of the interconnected brain capillary endothelial cells on them. MATERIALS SCIENCE & ENGINEERING. C, MATERIALS FOR BIOLOGICAL APPLICATIONS 2020; 112:110907. [PMID: 32409061 DOI: 10.1016/j.msec.2020.110907] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 07/02/2019] [Revised: 03/07/2020] [Accepted: 03/27/2020] [Indexed: 12/13/2022]
Abstract
We cultured mouse brain capillary endothelial cell line bEnd.3 on the UV-irradiated Type I collagen gel. Morphology of bEnd.3 cells on the Type I collagen gel was drastically changed if the gel was crosslinked by UV irradiation. The interconnecting network of bEnd.3 cells which have cord-like morphology on the soft collagen gels was converted to the monolayer of the flat cells, tightly-bound each other covering the gel surface, in a confluent state. The collagen gels were mechanically stiffened by UV irradiation for 15 min with UV light at 254 nm showing approximately two times higher value of Young's modulus E (1.51 ± 0.58 kPa) than the control gel (3.17 ± 1.17 kPa). AFM images of the collagen fibrils were not severely changed after irradiation. Collagen subunit proteins were crosslinked and degraded simultaneously under UV irradiation proved by results of SDS-PAGE and separation by centrifugation. Expression of Integrin gene was measured by quantitative real-time PCR. Expression of the integrin α2 gene, tight junction protein 1 gene, and claudin 5 gene were down-regulated in cells on the UV irradiated collagen gel in comparison with the unirradiated one while expression of the integrin β1 gene and Integrin α1 gene did not significantly change. Thick actin filaments were more clearly observed in the cells on the UV-irradiated collagen gel than the unirradiated one by fluorescent microscopy. We conclude that UV irradiation made the collagen gel stiffened and changed the physiological state of bEnd.3 cells including their adhesion, extension, and proliferation.
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29
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Zucchelli E, Majid QA, Foldes G. New artery of knowledge: 3D models of angiogenesis. VASCULAR BIOLOGY 2019; 1:H135-H143. [PMID: 32923965 PMCID: PMC7439835 DOI: 10.1530/vb-19-0026] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 11/27/2019] [Accepted: 12/03/2019] [Indexed: 12/13/2022]
Abstract
Angiogenesis and vasculogenesis are complex processes by which new blood vessels are formed and expanded. They play a pivotal role not only in physiological development and growth and tissue and organ repair, but also in a range of pathological conditions, from tumour formation to chronic inflammation and atherosclerosis. Understanding the multistep cell-differentiation programmes and identifying the key molecular players of physiological angiogenesis/vasculogenesis are critical to tackle pathological mechanisms. While many questions are yet to be answered, increasingly sophisticated in vitro, in vivo and ex vivo models of angiogenesis/vasculogenesis, together with cutting-edge imaging techniques, allowed for recent major advances in the field. This review aims to summarise the three-dimensional models available to study vascular network formation and to discuss advantages and limitations of the current systems.
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Affiliation(s)
| | - Qasim A Majid
- National Heart and Lung Institute, Imperial College London, London, UK
| | - Gabor Foldes
- National Heart and Lung Institute, Imperial College London, London, UK.,Heart and Vascular Center, Semmelweis University, Budapest, Hungary
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30
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Turner KR, Adams C, Staelens S, Deckmyn H, San Antonio J. Crucial Role for Endothelial Cell α2β1 Integrin Receptor Clustering in Collagen-Induced Angiogenesis. Anat Rec (Hoboken) 2019; 303:1604-1618. [PMID: 31581346 DOI: 10.1002/ar.24277] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/31/2018] [Revised: 04/11/2019] [Accepted: 04/26/2019] [Indexed: 11/07/2022]
Abstract
Angiogenesis is a crucial mechanism of vascular growth and regeneration that requires biosynthesis and cross-linking of collagens in vivo and is induced by collagen in vitro. Here, we use an in vitro model in which apical Type I collagen gels rapidly induce angiogenesis in endothelial monolayers. We extend previous studies demonstrating the importance of the endothelial α2β1 integrin, a key collagen receptor, in angiogenesis by investigating the roles of receptor clustering and conformational activation. Immunocytochemical localization of α2β1 integrins in endothelial monolayers showed a concentration of integrins along cell-cell borders. After inducing angiogenesis with collagen, the receptors redistributed to apical cell surfaces, aligning with collagen fibers, which were also redistributed during angiogenesis. Levels of conformationally activated α2β1 integrins were unchanged during angiogenesis and undetected on endothelial cells binding collagen in suspension. We mimicked the polyvalency of collagen fibrils using antibody-coated polystyrene beads to cluster endothelial cell surface α2β1 integrins, which induced rapid angiogenesis in the absence of collagen gels. Clustering of αvβ3 integrins and PECAM-1 but not of α1 integrins also induced angiogenesis. Soluble antibodies alone had no effect. Thus, the angiogenic property of collagen may reside in its ability to ligate and cluster cell surface receptors such as α2β1 integrins. Furthermore, synthetic substrates that promote the clustering of select endothelial cell surface receptors mimic the angiogenic properties of Type I collagen and may have applications in promoting vascularization of engineered tissues. Anat Rec, 2019. © 2019 American Association for Anatomy.
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Affiliation(s)
- Kevin R Turner
- Cardeza Foundation for Hematologic Research, Department of Medicine, Thomas Jefferson University, Philadelphia, Pennsylvania.,Department of Pathology, Oregon Health and Science University, Portland, Oregon
| | - Christopher Adams
- Department of Anatomy, Philadelphia College of Osteopathic Medicine, Philadelphia, Pennsylvania
| | - Stephanie Staelens
- Agrosavfe NV, Ghent, Zwijnaarde, Belgium.,Laboratory for Thrombosis Research, KU Leuven Campus Kulak Kortrijk, Kortrijk, Belgium
| | - Hans Deckmyn
- Laboratory for Thrombosis Research, KU Leuven Campus Kulak Kortrijk, Kortrijk, Belgium
| | - James San Antonio
- Cardeza Foundation for Hematologic Research, Department of Medicine, Thomas Jefferson University, Philadelphia, Pennsylvania
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31
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Alternating Differentiation and Dedifferentiation between Mature Osteoblasts and Osteocytes. Sci Rep 2019; 9:13842. [PMID: 31554848 PMCID: PMC6761144 DOI: 10.1038/s41598-019-50236-7] [Citation(s) in RCA: 28] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/27/2019] [Accepted: 09/09/2019] [Indexed: 12/22/2022] Open
Abstract
Osteocytes are terminally differentiated osteoblasts embedded in the bone matrix. Evidence indicates that cells in the mesenchymal lineage possess plasticity. However, whether or not osteocytes have the capacity to dedifferentiate back into osteoblasts is unclear. This study aimed to clarify the dedifferentiation potential of osteocytes. Mouse calvarial osteoblasts were isolated and maintained in normal two-dimensional (2D) or collagen gel three-dimensional (3D) cultures. In 2D cultures, osteoblasts exhibited a typical fibroblast-like shape with high Alpl and minimal Sost, Fgf23, and Dmp1 expression and osteoblasts formed mineralised nodules. When these osteoblasts were transferred into 3D cultures, they showed a stellate shape with diminished cytoplasm and numerous long processes and expression of Alpl decreased while Sost, Fgf23, and Dmp1 were significantly increased. These cells were in cell cycle arrest and showed suppressed mineralisation, indicating that they were osteocytes. When these osteocytes were recovered from 3D cultures and cultured two-dimensionally again, they regained adequate cytoplasm and lost the long processes, resulting in a fibroblast-like shape. These cells showed high Alpl and low Sost, Fgf23, and Dmp1 expression with a high mineralisation capability, indicating that they were osteoblasts. This report shows that osteocytes possess the capacity to dedifferentiate back into mature osteoblasts without gene manipulation.
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32
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Varol C. Tumorigenic Interplay Between Macrophages and Collagenous Matrix in the Tumor Microenvironment. Methods Mol Biol 2019; 1944:203-220. [PMID: 30840245 DOI: 10.1007/978-1-4939-9095-5_15] [Citation(s) in RCA: 15] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
Abstract
The tumor microenvironment is a heterogeneous tissue that in addition to tumor cells, contain tumor-associated cell types such as immune cells, fibroblasts, and endothelial cells. Considerably important in the tumor microenvironment is its noncellular component, namely, the extracellular matrix (ECM). In particular, the collagenous matrix is subjected to significant alterations in its composition and structure that create a permissive environment for tumor growth, invasion, and dissemination. Among tumor-infiltrating immune cells, tumor-associated macrophages (TAMs) are numerous in the tumor stroma and are locally educated to mediate important biological functions that profoundly affect tumor initiation, growth, and dissemination. While the influence of TAMs and mechanical properties of the collagenous matrix on tumor invasion and progression have been comprehensively investigated individually, their interaction within the complex tumor microenvironment was overlooked. This review summarizes accumulating evidence that indicate the existence of an intricate tumorigenic crosstalk between TAMs and collagenous matrix. A better mechanistic comprehension of this reciprocal interplay may open a novel arena for cancer therapeutics.
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Affiliation(s)
- Chen Varol
- The Research Center for Digestive Tract and Liver Diseases, Sourasky Medical Center, Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel. .,Department of Clinical Microbiology and Immunology, Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel.
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33
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Sun M, Puri S, Mutoji KN, Coulson-Thomas YM, Hascall VC, Jackson DG, Gesteira TF, Coulson-Thomas VJ. Hyaluronan Derived From the Limbus is a Key Regulator of Corneal Lymphangiogenesis. Invest Ophthalmol Vis Sci 2019; 60:1050-1062. [PMID: 30897620 PMCID: PMC6432804 DOI: 10.1167/iovs.18-25920] [Citation(s) in RCA: 22] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/22/2022] Open
Abstract
Purpose We recently reported that the glycosaminoglycan hyaluronan (HA), which promotes inflammatory angiogenesis in other vascular beds, is an abundant component of the limbal extracellular matrix. Consequently, we have explored the possibility that HA contributes to lymphangiogenesis in the inflamed cornea. Methods To study the role of HA on lymphangiogenesis, we used mice lacking the hyaluronan synthases and injury models that induce lymphangiogenesis. Results Here we report that HA regulates corneal lymphangiogenesis, both during post-natal development and in response to adult corneal injury. Furthermore, we show that injury to the cornea by alkali burn upregulates both HA production and lymphangiogenesis and that these processes are ablated in HA synthase 2 deficient mice. Conclusion These findings raise the possibility that therapeutic blockade of HA-mediated lymphangiogenesis might prevent the corneal scarring and rejection that frequently results from corneal transplantation.
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Affiliation(s)
- Mingxia Sun
- College of Optometry, University of Houston, Houston, Texas, United States
| | - Sudan Puri
- College of Optometry, University of Houston, Houston, Texas, United States
| | - Kazadi N Mutoji
- College of Optometry, University of Houston, Houston, Texas, United States
| | | | | | - David G Jackson
- MRC Human Immunology Unit, University of Oxford, Oxford, United Kingdom
| | - Tarsis F Gesteira
- College of Optometry, University of Houston, Houston, Texas, United States.,Universidade Federal de São Paulo, São Paulo, Brazil
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Min S, Ko IK, Yoo JJ. State-of-the-Art Strategies for the Vascularization of Three-Dimensional Engineered Organs. Vasc Specialist Int 2019; 35:77-89. [PMID: 31297357 PMCID: PMC6609020 DOI: 10.5758/vsi.2019.35.2.77] [Citation(s) in RCA: 21] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/23/2019] [Revised: 06/03/2019] [Accepted: 06/05/2019] [Indexed: 02/07/2023] Open
Abstract
Engineering three-dimensional (3D) implantable tissue constructs is a promising strategy for replacing damaged or diseased tissues and organs with functional replacements. However, the efficient vascularization of new 3D organs is a major scientific and technical challenge since large tissue constructs or organs require a constant blood supply to survive in vivo. Current approaches to solving this problem generally fall into the following three major categories: (a) cell-based, (b) angiogenic factor-based, and (c) scaffold-based. In this review, we summarize state-of-the-art technologies that are used to develop complex, stable, and functional vasculature for engineered 3D tissue constructs and organs; additionally, we have suggested directions for future research.
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Affiliation(s)
- Sangil Min
- Wake Forest Institute for Regenerative Medicine, Wake Forest School of Medicine, Winston-Salem, NC, USA.,Department of Surgery, Seoul National University College of Medicine, Seoul, Korea
| | - In Kap Ko
- Wake Forest Institute for Regenerative Medicine, Wake Forest School of Medicine, Winston-Salem, NC, USA
| | - James J Yoo
- Wake Forest Institute for Regenerative Medicine, Wake Forest School of Medicine, Winston-Salem, NC, USA
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35
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Massimini M, De Maria R, Malatesta D, Romanucci M, D'Anselmo A, Della Salda L. Establishment of three-dimensional canine osteosarcoma cell lines showing vasculogenic mimicry and evaluation of biological properties after treatment with 17-AAG. Vet Comp Oncol 2019; 17:376-384. [PMID: 31006970 DOI: 10.1111/vco.12482] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/21/2018] [Revised: 04/11/2019] [Accepted: 04/14/2019] [Indexed: 12/17/2022]
Abstract
Vasculogenic mimicry (VM) is an alternative type of blood perfusion characterized by formation of non-endothelial cell-lined microcirculatory channels and is responsible for aggressive tumour biology and increased tumour-related mortality. VM-correlated genes are associated with vascular endothelial grown factor receptor 1 (VEGFR1), and hypoxia-related (hypoxia inducible factor 1 α-HIF1α) signalling pathways, whose molecules are client proteins of Hsp90 (heat shock protein 90) and are potential therapeutic targets. This pilot study was aimed to investigate vasculogenic mimicry in a three-dimensional (3D) cell culture system of two aggressive canine osteosarcoma (OSA) cell lines (D22 and D17), and to evaluate the response of these cells to 17-AAG (17-N-allylamino-17-demethoxygeldanamycin) treatment in relation to tubular-like structure formation in vitro. Only D17 cell line formed hollow matrix channels in long-term 3D cultures and assumed endothelial morphology, with cells expressing both Hsp90 and VEGFR1, but lacking expression of endothelial marker CD31. 17-AAG treatment inhibited migration of D17 OSA cells, also decreasing VM markers in vitro and inducing a reduction of HIF1α transcript and protein in this cell line. Taken together, these preliminary data indicate that the biological effects of 17-AAG on D17 3D culture and on HIF1α regulation can provide interesting information to translate these findings from the basic research to clinical approach for the treatment of canine OSA as a model in comparative oncology.
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Affiliation(s)
| | - Raffaella De Maria
- Department of Veterinary Sciences, University of Turin, Grugliasco, Italy
| | | | | | - Angela D'Anselmo
- Faculty of Veterinary Medicine, University of Teramo, Teramo, Italy
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Abstract
The ability to generate new microvessels in desired numbers and at desired locations has been a long-sought goal in vascular medicine, engineering, and biology. Historically, the need to revascularize ischemic tissues nonsurgically (so-called therapeutic vascularization) served as the main driving force for the development of new methods of vascular growth. More recently, vascularization of engineered tissues and the generation of vascularized microphysiological systems have provided additional targets for these methods, and have required adaptation of therapeutic vascularization to biomaterial scaffolds and to microscale devices. Three complementary strategies have been investigated to engineer microvasculature: angiogenesis (the sprouting of existing vessels), vasculogenesis (the coalescence of adult or progenitor cells into vessels), and microfluidics (the vascularization of scaffolds that possess the open geometry of microvascular networks). Over the past several decades, vascularization techniques have grown tremendously in sophistication, from the crude implantation of arteries into myocardial tunnels by Vineberg in the 1940s, to the current use of micropatterning techniques to control the exact shape and placement of vessels within a scaffold. This review provides a broad historical view of methods to engineer the microvasculature, and offers a common framework for organizing and analyzing the numerous studies in this area of tissue engineering and regenerative medicine. © 2019 American Physiological Society. Compr Physiol 9:1155-1212, 2019.
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Affiliation(s)
- Joe Tien
- Department of Biomedical Engineering, Boston University, Boston, Massachusetts, USA
- Division of Materials Science and Engineering, Boston University, Brookline, Massachusetts, USA
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Morss Clyne A, Swaminathan S, Díaz Lantada A. Biofabrication strategies for creating microvascular complexity. Biofabrication 2019; 11:032001. [PMID: 30743247 DOI: 10.1088/1758-5090/ab0621] [Citation(s) in RCA: 26] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/28/2022]
Abstract
Design and fabrication of effective biomimetic vasculatures constitutes a relevant and yet unsolved challenge, lying at the heart of tissue repair and regeneration strategies. Even if cell growth is achieved in 3D tissue scaffolds or advanced implants, tissue viability inevitably requires vascularization, as diffusion can only transport nutrients and eliminate debris within a few hundred microns. This engineered vasculature may need to mimic the intricate branching geometry of native microvasculature, referred to herein as vascular complexity, to efficiently deliver blood and recreate critical interactions between the vascular and perivascular cells as well as parenchymal tissues. This review first describes the importance of vascular complexity in labs- and organs-on-chips, the biomechanical and biochemical signals needed to create and maintain a complex vasculature, and the limitations of current 2D, 2.5D, and 3D culture systems in recreating vascular complexity. We then critically review available strategies for design and biofabrication of complex vasculatures in cell culture platforms, labs- and organs-on-chips, and tissue engineering scaffolds, highlighting their advantages and disadvantages. Finally, challenges and future directions are outlined with the hope of inspiring researchers to create the reliable, efficient and sustainable tools needed for design and biofabrication of complex vasculatures.
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Affiliation(s)
- Alisa Morss Clyne
- Vascular Kinetics Laboratory, Mechanical Engineering & Mechanics, Drexel University, 3141 Chestnut Street, Philadelphia, PA 19104, United States of America
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38
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Ramakrishnan VM, Boyd NL. The Adipose Stromal Vascular Fraction as a Complex Cellular Source for Tissue Engineering Applications. TISSUE ENGINEERING. PART B, REVIEWS 2018; 24:289-299. [PMID: 28316259 PMCID: PMC6080106 DOI: 10.1089/ten.teb.2017.0061] [Citation(s) in RCA: 101] [Impact Index Per Article: 14.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 02/06/2017] [Accepted: 03/17/2017] [Indexed: 12/27/2022]
Abstract
A major challenge in tissue engineering is the generation of sufficient volumes of viable tissue for organ transplant. The development of a stable, mature vasculature is required to sustain the metabolic and functional activities of engineered tissues. Adipose stromal vascular fraction (SVF) cells are an easily accessible, heterogeneous cell system comprised of endothelial cells, macrophages, pericytes, and various stem cell populations. Collectively, SVF has been shown to spontaneously form vessel-like networks in vitro and robust, patent, and functional vasculatures in vivo. Capitalizing on this ability, we and others have demonstrated adipose SVF's utility in generating and augmenting engineered liver, cardiac, and vascular tissues, to name a few. This review highlights the scientific origins of SVF, the use of SVF as a clinically relevant vascular source, various SVF constituents and their roles, and practical considerations associated with isolating SVF for various tissue engineering applications.
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Affiliation(s)
- Venkat M. Ramakrishnan
- Cardiovascular Innovation Institute, Department of Physiology, University of Louisville School of Medicine, Louisville, Kentucky
| | - Nolan L. Boyd
- Cardiovascular Innovation Institute, Department of Physiology, University of Louisville School of Medicine, Louisville, Kentucky
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39
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Nowak-Sliwinska P, Alitalo K, Allen E, Anisimov A, Aplin AC, Auerbach R, Augustin HG, Bates DO, van Beijnum JR, Bender RHF, Bergers G, Bikfalvi A, Bischoff J, Böck BC, Brooks PC, Bussolino F, Cakir B, Carmeliet P, Castranova D, Cimpean AM, Cleaver O, Coukos G, Davis GE, De Palma M, Dimberg A, Dings RPM, Djonov V, Dudley AC, Dufton NP, Fendt SM, Ferrara N, Fruttiger M, Fukumura D, Ghesquière B, Gong Y, Griffin RJ, Harris AL, Hughes CCW, Hultgren NW, Iruela-Arispe ML, Irving M, Jain RK, Kalluri R, Kalucka J, Kerbel RS, Kitajewski J, Klaassen I, Kleinmann HK, Koolwijk P, Kuczynski E, Kwak BR, Marien K, Melero-Martin JM, Munn LL, Nicosia RF, Noel A, Nurro J, Olsson AK, Petrova TV, Pietras K, Pili R, Pollard JW, Post MJ, Quax PHA, Rabinovich GA, Raica M, Randi AM, Ribatti D, Ruegg C, Schlingemann RO, Schulte-Merker S, Smith LEH, Song JW, Stacker SA, Stalin J, Stratman AN, Van de Velde M, van Hinsbergh VWM, Vermeulen PB, Waltenberger J, Weinstein BM, Xin H, Yetkin-Arik B, Yla-Herttuala S, Yoder MC, Griffioen AW. Consensus guidelines for the use and interpretation of angiogenesis assays. Angiogenesis 2018; 21:425-532. [PMID: 29766399 PMCID: PMC6237663 DOI: 10.1007/s10456-018-9613-x] [Citation(s) in RCA: 457] [Impact Index Per Article: 65.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
Abstract
The formation of new blood vessels, or angiogenesis, is a complex process that plays important roles in growth and development, tissue and organ regeneration, as well as numerous pathological conditions. Angiogenesis undergoes multiple discrete steps that can be individually evaluated and quantified by a large number of bioassays. These independent assessments hold advantages but also have limitations. This article describes in vivo, ex vivo, and in vitro bioassays that are available for the evaluation of angiogenesis and highlights critical aspects that are relevant for their execution and proper interpretation. As such, this collaborative work is the first edition of consensus guidelines on angiogenesis bioassays to serve for current and future reference.
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Affiliation(s)
- Patrycja Nowak-Sliwinska
- Molecular Pharmacology Group, School of Pharmaceutical Sciences, Faculty of Sciences, University of Geneva, University of Lausanne, Rue Michel-Servet 1, CMU, 1211, Geneva 4, Switzerland.
- Translational Research Center in Oncohaematology, University of Geneva, Geneva, Switzerland.
| | - Kari Alitalo
- Wihuri Research Institute and Translational Cancer Biology Program, University of Helsinki, Helsinki, Finland
| | - Elizabeth Allen
- Laboratory of Tumor Microenvironment and Therapeutic Resistance, Department of Oncology, VIB-Center for Cancer Biology, KU Leuven, Louvain, Belgium
| | - Andrey Anisimov
- Wihuri Research Institute and Translational Cancer Biology Program, University of Helsinki, Helsinki, Finland
| | - Alfred C Aplin
- Department of Pathology, University of Washington, Seattle, WA, USA
| | | | - Hellmut G Augustin
- European Center for Angioscience, Medical Faculty Mannheim, Heidelberg University, Heidelberg, Germany
- Division of Vascular Oncology and Metastasis Research, German Cancer Research Center, Heidelberg, Germany
- German Cancer Consortium, Heidelberg, Germany
| | - David O Bates
- Division of Cancer and Stem Cells, School of Medicine, University of Nottingham, Nottingham, UK
| | - Judy R van Beijnum
- Angiogenesis Laboratory, Department of Medical Oncology, VU University Medical Center, Cancer Center Amsterdam, De Boelelaan 1117, 1081 HV, Amsterdam, The Netherlands
| | - R Hugh F Bender
- Department of Molecular Biology and Biochemistry, University of California, Irvine, CA, USA
| | - Gabriele Bergers
- Laboratory of Tumor Microenvironment and Therapeutic Resistance, Department of Oncology, VIB-Center for Cancer Biology, KU Leuven, Louvain, Belgium
- Department of Neurological Surgery, Brain Tumor Research Center, Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, CA, USA
| | - Andreas Bikfalvi
- Angiogenesis and Tumor Microenvironment Laboratory (INSERM U1029), University Bordeaux, Pessac, France
| | - Joyce Bischoff
- Vascular Biology Program and Department of Surgery, Harvard Medical School, Boston Children's Hospital, Boston, MA, USA
| | - Barbara C Böck
- European Center for Angioscience, Medical Faculty Mannheim, Heidelberg University, Heidelberg, Germany
- Division of Vascular Oncology and Metastasis Research, German Cancer Research Center, Heidelberg, Germany
- German Cancer Consortium, Heidelberg, Germany
| | - Peter C Brooks
- Center for Molecular Medicine, Maine Medical Center Research Institute, Scarborough, ME, USA
| | - Federico Bussolino
- Department of Oncology, University of Torino, Turin, Italy
- Candiolo Cancer Institute-FPO-IRCCS, 10060, Candiolo, Italy
| | - Bertan Cakir
- Department of Ophthalmology, Harvard Medical School, Boston Children's Hospital, Boston, MA, USA
| | - Peter Carmeliet
- Laboratory of Angiogenesis and Vascular Metabolism, Department of Oncology and Leuven Cancer Institute (LKI), KU Leuven, Leuven, Belgium
- Laboratory of Angiogenesis and Vascular Metabolism, Center for Cancer Biology, VIB, Leuven, Belgium
| | - Daniel Castranova
- Division of Developmental Biology, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD, USA
| | - Anca M Cimpean
- Department of Microscopic Morphology/Histology, Angiogenesis Research Center, Victor Babes University of Medicine and Pharmacy, Timisoara, Romania
| | - Ondine Cleaver
- Department of Molecular Biology, Center for Regenerative Science and Medicine, University of Texas Southwestern Medical Center, Dallas, TX, USA
| | - George Coukos
- Ludwig Institute for Cancer Research, Department of Oncology, University of Lausanne, Lausanne, Switzerland
| | - George E Davis
- Department of Medical Pharmacology and Physiology, University of Missouri, School of Medicine and Dalton Cardiovascular Center, Columbia, MO, USA
| | - Michele De Palma
- School of Life Sciences, Swiss Federal Institute of Technology, Lausanne, Switzerland
| | - Anna Dimberg
- Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden
| | - Ruud P M Dings
- Department of Radiation Oncology, University of Arkansas for Medical Sciences, Little Rock, AR, USA
| | | | - Andrew C Dudley
- Department of Microbiology, Immunology, and Cancer Biology, University of Virginia, Charlottesville, VA, USA
- Emily Couric Cancer Center, The University of Virginia, Charlottesville, VA, USA
| | - Neil P Dufton
- Vascular Sciences, Imperial Centre for Translational and Experimental Medicine, National Heart and Lung Institute, Imperial College London, London, UK
| | - Sarah-Maria Fendt
- Laboratory of Cellular Metabolism and Metabolic Regulation, VIB Center for Cancer Biology, Leuven, Belgium
- Laboratory of Cellular Metabolism and Metabolic Regulation, Department of Oncology, KU Leuven and Leuven Cancer Institute, Leuven, Belgium
| | | | - Marcus Fruttiger
- Institute of Ophthalmology, University College London, London, UK
| | - Dai Fukumura
- Edwin L. Steele Laboratories, Department of Radiation Oncology, Massachusetts General Hospital and Harvard Medical School, Boston, MA, USA
| | - Bart Ghesquière
- Metabolomics Expertise Center, VIB Center for Cancer Biology, VIB, Leuven, Belgium
- Department of Oncology, Metabolomics Expertise Center, KU Leuven, Leuven, Belgium
| | - Yan Gong
- Department of Ophthalmology, Harvard Medical School, Boston Children's Hospital, Boston, MA, USA
| | - Robert J Griffin
- Department of Radiation Oncology, University of Arkansas for Medical Sciences, Little Rock, AR, USA
| | - Adrian L Harris
- Molecular Oncology Laboratories, Oxford University Department of Oncology, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, Oxford, UK
| | - Christopher C W Hughes
- Department of Molecular Biology and Biochemistry, University of California, Irvine, CA, USA
| | - Nan W Hultgren
- Department of Molecular Biology and Biochemistry, University of California, Irvine, CA, USA
| | | | - Melita Irving
- Ludwig Institute for Cancer Research, Department of Oncology, University of Lausanne, Lausanne, Switzerland
| | - Rakesh K Jain
- Edwin L. Steele Laboratories, Department of Radiation Oncology, Massachusetts General Hospital and Harvard Medical School, Boston, MA, USA
| | - Raghu Kalluri
- Department of Cancer Biology, Metastasis Research Center, The University of Texas MD Anderson Cancer Center, Houston, TX, USA
| | - Joanna Kalucka
- Laboratory of Angiogenesis and Vascular Metabolism, Department of Oncology and Leuven Cancer Institute (LKI), KU Leuven, Leuven, Belgium
- Laboratory of Angiogenesis and Vascular Metabolism, Center for Cancer Biology, VIB, Leuven, Belgium
| | - Robert S Kerbel
- Department of Medical Biophysics, Biological Sciences Platform, Sunnybrook Research Institute, University of Toronto, Toronto, ON, Canada
| | - Jan Kitajewski
- Department of Physiology and Biophysics, University of Illinois, Chicago, IL, USA
| | - Ingeborg Klaassen
- Ocular Angiogenesis Group, Departments of Ophthalmology and Medical Biology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands
| | - Hynda K Kleinmann
- The George Washington University School of Medicine, Washington, DC, USA
| | - Pieter Koolwijk
- Department of Ophthalmology, University of Lausanne, Jules-Gonin Eye Hospital, Fondation Asile des Aveugles, Lausanne, Switzerland
| | - Elisabeth Kuczynski
- Department of Medical Biophysics, Biological Sciences Platform, Sunnybrook Research Institute, University of Toronto, Toronto, ON, Canada
| | - Brenda R Kwak
- Department of Pathology and Immunology, University of Geneva, Geneva, Switzerland
| | | | - Juan M Melero-Martin
- Department of Cardiac Surgery, Harvard Medical School, Boston Children's Hospital, Boston, MA, USA
| | - Lance L Munn
- Edwin L. Steele Laboratories, Department of Radiation Oncology, Massachusetts General Hospital and Harvard Medical School, Boston, MA, USA
| | - Roberto F Nicosia
- Department of Pathology, University of Washington, Seattle, WA, USA
- Pathology and Laboratory Medicine Service, VA Puget Sound Health Care System, Seattle, WA, USA
| | - Agnes Noel
- Laboratory of Tumor and Developmental Biology, GIGA-Cancer, University of Liège, Liège, Belgium
| | - Jussi Nurro
- Department of Biotechnology and Molecular Medicine, University of Eastern Finland, Kuopio, Finland
| | - Anna-Karin Olsson
- Department of Medical Biochemistry and Microbiology, Science for Life Laboratory, Uppsala Biomedical Center, Uppsala University, Uppsala, Sweden
| | - Tatiana V Petrova
- Department of oncology UNIL-CHUV, Ludwig Institute for Cancer Research Lausanne, Lausanne, Switzerland
| | - Kristian Pietras
- Division of Translational Cancer Research, Department of Laboratory Medicine, Lund, Sweden
| | - Roberto Pili
- Genitourinary Program, Indiana University-Simon Cancer Center, Indianapolis, IN, USA
| | - Jeffrey W Pollard
- Medical Research Council Centre for Reproductive Health, College of Medicine and Veterinary Medicine, University of Edinburgh, Edinburgh, UK
| | - Mark J Post
- Department of Physiology, Maastricht University, Maastricht, The Netherlands
| | - Paul H A Quax
- Einthoven Laboratory for Experimental Vascular Medicine, Department Surgery, LUMC, Leiden, The Netherlands
| | - Gabriel A Rabinovich
- Laboratory of Immunopathology, Institute of Biology and Experimental Medicine, National Council of Scientific and Technical Investigations (CONICET), Buenos Aires, Argentina
| | - Marius Raica
- Department of Microscopic Morphology/Histology, Angiogenesis Research Center, Victor Babes University of Medicine and Pharmacy, Timisoara, Romania
| | - Anna M Randi
- Vascular Sciences, Imperial Centre for Translational and Experimental Medicine, National Heart and Lung Institute, Imperial College London, London, UK
| | - Domenico Ribatti
- Department of Basic Medical Sciences, Neurosciences and Sensory Organs, University of Bari Medical School, Bari, Italy
- National Cancer Institute "Giovanni Paolo II", Bari, Italy
| | - Curzio Ruegg
- Department of Oncology, Microbiology and Immunology, Faculty of Science and Medicine, University of Fribourg, Fribourg, Switzerland
| | - Reinier O Schlingemann
- Ocular Angiogenesis Group, Departments of Ophthalmology and Medical Biology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands
- Department of Ophthalmology, University of Lausanne, Jules-Gonin Eye Hospital, Fondation Asile des Aveugles, Lausanne, Switzerland
| | - Stefan Schulte-Merker
- Institute of Cardiovascular Organogenesis and Regeneration, Faculty of Medicine, WWU, Münster, Germany
| | - Lois E H Smith
- Department of Ophthalmology, Harvard Medical School, Boston Children's Hospital, Boston, MA, USA
| | - Jonathan W Song
- Department of Mechanical and Aerospace Engineering, The Ohio State University, Columbus, OH, USA
- Comprehensive Cancer Center, The Ohio State University, Columbus, OH, USA
| | - Steven A Stacker
- Tumour Angiogenesis and Microenvironment Program, Peter MacCallum Cancer Centre and The Sir Peter MacCallum, Department of Oncology, University of Melbourne, Melbourne, VIC, Australia
| | - Jimmy Stalin
- Institute of Cardiovascular Organogenesis and Regeneration, Faculty of Medicine, WWU, Münster, Germany
| | - Amber N Stratman
- Division of Developmental Biology, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD, USA
| | - Maureen Van de Velde
- Laboratory of Tumor and Developmental Biology, GIGA-Cancer, University of Liège, Liège, Belgium
| | - Victor W M van Hinsbergh
- Department of Ophthalmology, University of Lausanne, Jules-Gonin Eye Hospital, Fondation Asile des Aveugles, Lausanne, Switzerland
| | - Peter B Vermeulen
- HistoGeneX, Antwerp, Belgium
- Translational Cancer Research Unit, GZA Hospitals, Sint-Augustinus & University of Antwerp, Antwerp, Belgium
| | - Johannes Waltenberger
- Medical Faculty, University of Münster, Albert-Schweitzer-Campus 1, Münster, Germany
| | - Brant M Weinstein
- Division of Developmental Biology, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD, USA
| | - Hong Xin
- University of California, San Diego, La Jolla, CA, USA
| | - Bahar Yetkin-Arik
- Ocular Angiogenesis Group, Departments of Ophthalmology and Medical Biology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands
| | - Seppo Yla-Herttuala
- Department of Biotechnology and Molecular Medicine, University of Eastern Finland, Kuopio, Finland
| | - Mervin C Yoder
- Department of Pediatrics, Indiana University School of Medicine, Indianapolis, IN, USA
| | - Arjan W Griffioen
- Angiogenesis Laboratory, Department of Medical Oncology, VU University Medical Center, Cancer Center Amsterdam, De Boelelaan 1117, 1081 HV, Amsterdam, The Netherlands.
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40
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Abstract
Tumor-induced angiogenesis is a key event for neoplastic progression. In vitro assays are important for identification of potential angiogenic agents and rapid sceening for pharmacological inhibitors. The increased interest in this field of study has generated several in vitro assays that recapitulate the steps of endothelial cell activation and differentiation. In this short report we emphasize the utility of Matrigel, a reconstituted basement membrane, to define two different steps in the angiogenic process: invasion in response to growth factors and organization of microvessels into a network with branching morphology on a Matrigel substrate.
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Affiliation(s)
- R Benelli
- Molecular Oncology Service, Advanced Biotechnologies Center, Genova, Italy
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41
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Chang P, Guo B, Hui Q, Liu X, Tao K. A bioartificial dermal regeneration template promotes skin cell proliferation in vitro and enhances large skin wound healing in vivo. Oncotarget 2018; 8:25226-25241. [PMID: 28423680 PMCID: PMC5421924 DOI: 10.18632/oncotarget.16005] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/30/2017] [Accepted: 02/22/2017] [Indexed: 12/30/2022] Open
Abstract
A novel bioartificial dermal regeneration template has been developed using platelet-rich plasma and acellular animal skin collagen sponge for the treatment of larger area and full thickness skin wounds. This platelet-rich plasma-collagen sponge keeps native skin structure and contains huge amounts of growth factors. The effect of this bioartificial dermal regeneration template was tested in vitro and in vivo via a mimic poor wound healing process by adding collagenase I into cell culture medium or the wound area. The in vitro experimental results indicated that the rat skin cells grew faster and produced more collagen in platelet-rich plasma-collagen sponge with collagenase than those treated either with collagen sponge plus collagenase, or collagenase, or control group without treatment. The in vivo experiments were performed by large rat skin wounds, 1.5 cm diameter, treated either with collagenase, or collagenase plus collagen sponge, or collagenase plus platelet-rich plasma-collagen sponge. The wound without treatment was used as a control. The wounds treated with collagenase-containing platelet-rich plasma-collagen sponge healed 4 times faster than the untreated wounds, 6 times faster than the collagenase treated wounds, 2.4 times faster than collagenase-containing collagen sponge treated wounds. The immunostaining indicated that the healed tissues in the wound areas treated with collagenase-containing platelet-rich plasma-collagen sponge were composed of collagen type I and collagen III with blood vessels and hair follicles. The results demonstrated that this collagenase-containing platelet-rich plasma-collagen sponge works as a bioartificial dermal regeneration template. The application of this collagenase-containing platelet-rich plasma-collagen sponge promotes the traumatic skin wound healing and permits the reconstitution of the inherent barrier functions of the skin.
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Affiliation(s)
- Peng Chang
- Department of Plastic and Reconstructive Surgery, General Hospital of Shenyang Military Area Command, Shenyang, 110840, Liaoning, P.R. China
| | - Bingyu Guo
- Department of Plastic and Reconstructive Surgery, General Hospital of Shenyang Military Area Command, Shenyang, 110840, Liaoning, P.R. China
| | - Qiang Hui
- Department of Plastic and Reconstructive Surgery, General Hospital of Shenyang Military Area Command, Shenyang, 110840, Liaoning, P.R. China
| | - Xiaoyan Liu
- Department of Plastic and Reconstructive Surgery, General Hospital of Shenyang Military Area Command, Shenyang, 110840, Liaoning, P.R. China
| | - Kai Tao
- Department of Plastic and Reconstructive Surgery, General Hospital of Shenyang Military Area Command, Shenyang, 110840, Liaoning, P.R. China
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Messina V, Valtieri M, Rubio M, Falchi M, Mancini F, Mayor A, Alano P, Silvestrini F. Gametocytes of the Malaria Parasite Plasmodium falciparum Interact With and Stimulate Bone Marrow Mesenchymal Cells to Secrete Angiogenetic Factors. Front Cell Infect Microbiol 2018; 8:50. [PMID: 29546035 PMCID: PMC5838020 DOI: 10.3389/fcimb.2018.00050] [Citation(s) in RCA: 25] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/18/2017] [Accepted: 02/09/2018] [Indexed: 12/31/2022] Open
Abstract
The gametocytes of Plasmodium falciparum, responsible for the transmission of this malaria parasite from humans to mosquitoes, accumulate and mature preferentially in the human bone marrow. In the 10 day long sexual development of P. falciparum, the immature gametocytes reach and localize in the extravascular compartment of this organ, in contact with several bone marrow stroma cell types, prior to traversing the endothelial lining and re-entering in circulation at maturity. To investigate the host parasite interplay underlying this still obscure process, we developed an in vitro tridimensional co-culture system in a Matrigel scaffold with P. falciparum gametocytes and self-assembling spheroids of human bone marrow mesenchymal cells (hBM-MSCs). Here we show that this co-culture system sustains the full maturation of the gametocytes and that the immature, but not the mature, gametocytes adhere to hBM-MSCs via trypsin-sensitive parasite ligands exposed on the erythrocyte surface. Analysis of a time course of gametocytogenesis in the co-culture system revealed that gametocyte maturation is accompanied by the parasite induced stimulation of hBM-MSCs to secrete a panel of 14 cytokines and growth factors, 13 of which have been described to play a role in angiogenesis. Functional in vitro assays on human bone marrow endothelial cells showed that supernatants from the gametocyte mesenchymal cell co-culture system enhance ability of endothelial cells to form vascular tubes. These results altogether suggest that the interplay between immature gametocytes and hBM-MSCs may induce functional and structural alterations in the endothelial lining of the human bone marrow hosting the P. falciparum transmission stages.
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Affiliation(s)
- Valeria Messina
- Dipartimento di Malattie Infettive, Istituto Superiore di Sanità, Rome, Italy
| | - Mauro Valtieri
- Dipartimento di Oncologia e Medicina Molecolare, Istituto Superiore di Sanità, Rome, Italy
| | - Mercedes Rubio
- ISGlobal, Barcelona Ctr. Int. Health Res, Hospital Clínic - Universitat de Barcelona, Barcelona, Spain
| | - Mario Falchi
- AIDS National Center, Istituto Superiore di Sanità, Rome, Italy
| | - Francesca Mancini
- Dipartimento di Biotecnologie Cellulari ed Ematologia, Umberto I - Policlinico di Roma, Rome, Italy
| | - Alfredo Mayor
- ISGlobal, Barcelona Ctr. Int. Health Res, Hospital Clínic - Universitat de Barcelona, Barcelona, Spain.,Centro de Investigação em Saúde da Manhiça, Maputo, Mozambique
| | - Pietro Alano
- Dipartimento di Malattie Infettive, Istituto Superiore di Sanità, Rome, Italy
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Desgranges P, Barritault D, Caruelle J, Tardieu M. Transmural Endothelialization of Vascular Prostheses is Regulated in Vitro by Fibroblast Growth Factor 2 and Heparan-Like Molecule. Int J Artif Organs 2018. [DOI: 10.1177/039139889702001009] [Citation(s) in RCA: 16] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/17/2022]
Abstract
Endothelialization of vascular prostheses may result from transmural migration of endothelial cells. Angiogenesis is controlled by growth factors like Fibroblast Growth Factor 2 (FGF2) and regulators like heparan-like molecules. To that end, we used heparan-like molecules named RGTA for ReGeneraTing Agent. The RGTA11 used was a chemically derived dextran obtained by successive substitutions with carboxymethyl, benzylamide, and benzylamide sulfonate groups on glucose residues. This agent was further selected for its ability to bind, stabilize and protect FGF2. We defined firstly the angiogenic capability of FGF2 in combination with RGTA 11 on bovine aortic endothelial cells (BAEC) cultured on collagen I gels. Secondly, the role of FGF2 and RGTA 11 in transmural endothelialization was assessed in a three-dimensional in vitro model using a polyethylene terephtalate prosthesis included in collagen gel. BAEC seeded on the external face can migrate to the luminal face of the prosthesis. Microscopic and histological evaluations were performed at 4 and 7 days. Results showed that the addition of RGTA 11 alone did not promote angiogenesis while FGF2 alone did. However, RGTA11 combined with FGF2 produced a significant acceleration in angiogenesis compared to FGF2 alone. This combination magnifies and enhances the angiogenic processes leading to endothelialization of luminal face through transmural cellular migration. Our data demonstrates that in vitro transmural endothelialization of porous vascular prostheses by BAEC cultured on collagen I gels is upregulated by RGTA 11 combined with FGF2.
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Affiliation(s)
- P. Desgranges
- Centre de Recherches Chirurgicales, CNRS URA 1431, Hopital Henri Mondor, Creteil
| | - D. Barritault
- Laboratoire de Recherche sur la Croissance Cellulaire, la Regénération et la Réparation Tissulaires, Université Paris XII Val de Marne, CNRS URA 1813, Creteil - France
| | - J.P. Caruelle
- Laboratoire de Recherche sur la Croissance Cellulaire, la Regénération et la Réparation Tissulaires, Université Paris XII Val de Marne, CNRS URA 1813, Creteil - France
| | - M. Tardieu
- Laboratoire de Recherche sur la Croissance Cellulaire, la Regénération et la Réparation Tissulaires, Université Paris XII Val de Marne, CNRS URA 1813, Creteil - France
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Duran CL, Howell DW, Dave JM, Smith RL, Torrie ME, Essner JJ, Bayless KJ. Molecular Regulation of Sprouting Angiogenesis. Compr Physiol 2017; 8:153-235. [PMID: 29357127 DOI: 10.1002/cphy.c160048] [Citation(s) in RCA: 46] [Impact Index Per Article: 5.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022]
Abstract
The term angiogenesis arose in the 18th century. Several studies over the next 100 years laid the groundwork for initial studies performed by the Folkman laboratory, which were at first met with some opposition. Once overcome, the angiogenesis field has flourished due to studies on tumor angiogenesis and various developmental models that can be genetically manipulated, including mice and zebrafish. In addition, new discoveries have been aided by the ability to isolate primary endothelial cells, which has allowed dissection of various steps within angiogenesis. This review will summarize the molecular events that control angiogenesis downstream of biochemical factors such as growth factors, cytokines, chemokines, hypoxia-inducible factors (HIFs), and lipids. These and other stimuli have been linked to regulation of junctional molecules and cell surface receptors. In addition, the contribution of cytoskeletal elements and regulatory proteins has revealed an intricate role for mobilization of actin, microtubules, and intermediate filaments in response to cues that activate the endothelium. Activating stimuli also affect various focal adhesion proteins, scaffold proteins, intracellular kinases, and second messengers. Finally, metalloproteinases, which facilitate matrix degradation and the formation of new blood vessels, are discussed, along with our knowledge of crosstalk between the various subclasses of these molecules throughout the text. Compr Physiol 8:153-235, 2018.
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Affiliation(s)
- Camille L Duran
- Department of Molecular and Cellular Medicine, Texas A&M Health Science Center, College Station, Texas, USA
| | - David W Howell
- Department of Molecular and Cellular Medicine, Texas A&M Health Science Center, College Station, Texas, USA
| | - Jui M Dave
- Department of Molecular and Cellular Medicine, Texas A&M Health Science Center, College Station, Texas, USA
| | - Rebecca L Smith
- Department of Molecular and Cellular Medicine, Texas A&M Health Science Center, College Station, Texas, USA
| | - Melanie E Torrie
- Department of Genetics, Development and Cell Biology, Iowa State University, Ames, Iowa, USA
| | - Jeffrey J Essner
- Department of Genetics, Development and Cell Biology, Iowa State University, Ames, Iowa, USA
| | - Kayla J Bayless
- Department of Molecular and Cellular Medicine, Texas A&M Health Science Center, College Station, Texas, USA
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45
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Abraham VC, Miller LN, Pratt SD, Putman B, Kim L, Gopalakrishnan SM, King A. Implementation of a human podocyte injury model of chronic kidney disease for profiling of renoprotective compounds. Eur J Pharmacol 2017; 815:219-232. [DOI: 10.1016/j.ejphar.2017.09.006] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/03/2017] [Revised: 09/01/2017] [Accepted: 09/04/2017] [Indexed: 12/15/2022]
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Abstract
During vascular development, endothelial cells (ECs) and neighboring stromal cells interact and communicate through autocrine and paracrine signaling mechanisms involving extracellular matrix (ECM) proteins and their cell surface integrin adhesion receptors. Integrin-mediated adhesion and signaling pathways are crucial for normal vascular development and physiology, and alterations in integrin expression and/or function drive several vascular-related pathologies including thrombosis, autoimmune disorders, and cancer. The purpose of this chapter is to discuss integrin adhesion and signaling pathways important for EC growth, survival, and migration. Integrin-mediated paracrine links between ECs and surrounding stromal cells in the organ microenvironment will also be discussed. Lastly, we will review roles for integrins in vascular pathologies and discuss possible targets for therapeutic intervention.
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Affiliation(s)
- Paola A Guerrero
- University of Texas MD Anderson Cancer Center, Houston, TX, United States
| | - Joseph H McCarty
- University of Texas MD Anderson Cancer Center, Houston, TX, United States.
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47
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Chen S, Kawazoe N, Chen G. Biomimetic Assembly of Vascular Endothelial Cells and Muscle Cells in Microgrooved Collagen Porous Scaffolds. Tissue Eng Part C Methods 2017; 23:367-376. [DOI: 10.1089/ten.tec.2017.0088] [Citation(s) in RCA: 22] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/25/2023] Open
Affiliation(s)
- Shangwu Chen
- International Center for Materials Nanoarchitectonics, National Institute for Materials Science, Tsukuba, Japan
- Department of Materials Science and Engineering, Graduate School of Pure and Applied Sciences, University of Tsukuba, Tsukuba, Japan
| | - Naoki Kawazoe
- Department of Materials Science and Engineering, Graduate School of Pure and Applied Sciences, University of Tsukuba, Tsukuba, Japan
- Research Center for Functional Materials, National Institute for Materials Science, Tsukuba, Japan
| | - Guoping Chen
- International Center for Materials Nanoarchitectonics, National Institute for Materials Science, Tsukuba, Japan
- Department of Materials Science and Engineering, Graduate School of Pure and Applied Sciences, University of Tsukuba, Tsukuba, Japan
- Research Center for Functional Materials, National Institute for Materials Science, Tsukuba, Japan
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48
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Zhang J, Schwartz MP, Hou Z, Bai Y, Ardalani H, Swanson S, Steill J, Ruotti V, Elwell A, Nguyen BK, Bolin J, Stewart R, Thomson JA, Murphy WL. A Genome-wide Analysis of Human Pluripotent Stem Cell-Derived Endothelial Cells in 2D or 3D Culture. Stem Cell Reports 2017; 8:907-918. [PMID: 28343999 PMCID: PMC5390115 DOI: 10.1016/j.stemcr.2017.02.014] [Citation(s) in RCA: 37] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/07/2016] [Revised: 02/15/2017] [Accepted: 02/16/2017] [Indexed: 02/07/2023] Open
Abstract
A defined protocol for efficiently deriving endothelial cells from human pluripotent stem cells was established and vascular morphogenesis was used as a model system to understand how synthetic hydrogels influence global biological function compared with common 2D and 3D culture platforms. RNA sequencing demonstrated that gene expression profiles were similar for endothelial cells and pericytes cocultured in polyethylene glycol (PEG) hydrogels or Matrigel, while monoculture comparisons identified distinct vascular signatures for each cell type. Endothelial cells cultured on tissue-culture polystyrene adopted a proliferative phenotype compared with cells cultured on or encapsulated in PEG hydrogels. The proliferative phenotype correlated to increased FAK-ERK activity, and knockdown or inhibition of ERK signaling reduced proliferation and expression for cell-cycle genes while increasing expression for “3D-like” vasculature development genes. Our results provide insight into the influence of 2D and 3D culture formats on global biological processes that regulate cell function.
Defined, high-efficiency differentiation of human PSCs to endothelial cell Comprehensive genome-wide comparisons of 2D and 3D cell-culture formats Gene expression profiles for endothelial cells and pericytes in 3D cell culture Highly proliferative phenotypes on tissue-culture polystyrene surfaces
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Affiliation(s)
- Jue Zhang
- Regenerative Biology, Morgridge Institute for Research, Madison, WI 53715, USA
| | - Michael P Schwartz
- Department of Biomedical Engineering, University of Wisconsin-Madison, Wisconsin Institute for Medical Research, Room 5405, Madison, WI 53706, USA.
| | - Zhonggang Hou
- Regenerative Biology, Morgridge Institute for Research, Madison, WI 53715, USA
| | - Yongsheng Bai
- Regenerative Biology, Morgridge Institute for Research, Madison, WI 53715, USA
| | - Hamisha Ardalani
- Department of Biomedical Engineering, University of Wisconsin-Madison, Wisconsin Institute for Medical Research, Room 5405, Madison, WI 53706, USA
| | - Scott Swanson
- Regenerative Biology, Morgridge Institute for Research, Madison, WI 53715, USA
| | - John Steill
- Regenerative Biology, Morgridge Institute for Research, Madison, WI 53715, USA
| | - Victor Ruotti
- Regenerative Biology, Morgridge Institute for Research, Madison, WI 53715, USA
| | - Angela Elwell
- Regenerative Biology, Morgridge Institute for Research, Madison, WI 53715, USA
| | - Bao Kim Nguyen
- Regenerative Biology, Morgridge Institute for Research, Madison, WI 53715, USA
| | - Jennifer Bolin
- Regenerative Biology, Morgridge Institute for Research, Madison, WI 53715, USA
| | - Ron Stewart
- Regenerative Biology, Morgridge Institute for Research, Madison, WI 53715, USA
| | - James A Thomson
- Regenerative Biology, Morgridge Institute for Research, Madison, WI 53715, USA; Department of Cell & Regenerative Biology, University of Wisconsin-Madison, Madison, WI 53705, USA; Department of Molecular, Cellular, & Developmental Biology, University of California, Santa Barbara, CA 93117, USA
| | - William L Murphy
- Department of Biomedical Engineering, University of Wisconsin-Madison, Wisconsin Institute for Medical Research, Room 5405, Madison, WI 53706, USA; Materials Science Program, University of Wisconsin-Madison, Madison, WI 53706, USA; Department of Orthopedics and Rehabilitation, University of Wisconsin-Madison, Madison, WI 53705, USA.
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49
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Takei T, Sakai S, Yoshida M. In vitro formation of vascular-like networks using hydrogels. J Biosci Bioeng 2016; 122:519-527. [DOI: 10.1016/j.jbiosc.2016.03.023] [Citation(s) in RCA: 18] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/14/2016] [Revised: 03/22/2016] [Accepted: 03/29/2016] [Indexed: 01/19/2023]
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50
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Wang S, Sun J, Zhang DD, Wong PK. A nanobiosensor for dynamic single cell analysis during microvascular self-organization. NANOSCALE 2016; 8:16894-901. [PMID: 27547924 PMCID: PMC5042875 DOI: 10.1039/c6nr03907c] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/24/2023]
Abstract
The formation of microvascular networks plays essential roles in regenerative medicine and tissue engineering. Nevertheless, the self-organization mechanisms underlying the dynamic morphogenic process are poorly understood due to a paucity of effective tools for mapping the spatiotemporal dynamics of single cell behaviors. By establishing a single cell nanobiosensor along with live cell imaging, we perform dynamic single cell analysis of the morphology, displacement, and gene expression during microvascular self-organization. Dynamic single cell analysis reveals that endothelial cells self-organize into subpopulations with specialized phenotypes to form microvascular networks and identifies the involvement of Notch1-Dll4 signaling in regulating the cell subpopulations. The cell phenotype correlates with the initial Dll4 mRNA expression level and each subpopulation displays a unique dynamic Dll4 mRNA expression profile. Pharmacological perturbations and RNA interference of Notch1-Dll4 signaling modulate the cell subpopulations and modify the morphology of the microvascular network. Taken together, a nanobiosensor enables a dynamic single cell analysis approach underscoring the importance of Notch1-Dll4 signaling in microvascular self-organization.
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Affiliation(s)
- S. Wang
- Department of Mechanical Engineering, University of Michigan, Ann Arbor, MI 48109, USA
- Department of Aerospace and Mechanical Engineering, University of Arizona, Tucson, AZ 85721, USA
| | - J. Sun
- Department of Aerospace and Mechanical Engineering, University of Arizona, Tucson, AZ 85721, USA
- Department of Mechanical Science and Engineering, University of Illinois at Urbana-Champaign, Urbana, IL 61801 USA
| | - D. D. Zhang
- Department of Pharmacology and Toxicology, University of Arizona, Tucson, AZ 85724, USA
| | - P. K. Wong
- Department of Aerospace and Mechanical Engineering, University of Arizona, Tucson, AZ 85721, USA
- Departments of Biomedical Engineering, Mechanical Engineering and Surgery, The Pennsylvania State University, University Park, PA 16802, USA
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