scholarly journals Carbonic Anhydrases as Potential Targets Against Neurovascular Unit Dysfunction in Alzheimer’s Disease and Stroke

2021 ◽  
Vol 13 ◽  
Author(s):  
Nicole Lemon ◽  
Elisa Canepa ◽  
Marc A. Ilies ◽  
Silvia Fossati
Keyword(s):  
Genetic Manipulation ◽  
Neurovascular Unit ◽  
Distribution Patterns ◽  
Cell Types ◽  
Carbonic Anhydrases ◽  
Ph Homeostasis ◽  
Cerebrovascular Pathology ◽  
Blood Brain Barrier Disruption

The Neurovascular Unit (NVU) is an important multicellular structure of the central nervous system (CNS), which participates in the regulation of cerebral blood flow (CBF), delivery of oxygen and nutrients, immunological surveillance, clearance, barrier functions, and CNS homeostasis. Stroke and Alzheimer Disease (AD) are two pathologies with extensive NVU dysfunction. The cell types of the NVU change in both structure and function following an ischemic insult and during the development of AD pathology. Stroke and AD share common risk factors such as cardiovascular disease, and also share similarities at a molecular level. In both diseases, disruption of metabolic support, mitochondrial dysfunction, increase in oxidative stress, release of inflammatory signaling molecules, and blood brain barrier disruption result in NVU dysfunction, leading to cell death and neurodegeneration. Improved therapeutic strategies for both AD and stroke are needed. Carbonic anhydrases (CAs) are well-known targets for other diseases and are being recently investigated for their function in the development of cerebrovascular pathology. CAs catalyze the hydration of CO2 to produce bicarbonate and a proton. This reaction is important for pH homeostasis, overturn of cerebrospinal fluid, regulation of CBF, and other physiological functions. Humans express 15 CA isoforms with different distribution patterns. Recent studies provide evidence that CA inhibition is protective to NVU cells in vitro and in vivo, in models of stroke and AD pathology. CA inhibitors are FDA-approved for treatment of glaucoma, high-altitude sickness, and other indications. Most FDA-approved CA inhibitors are pan-CA inhibitors; however, specific CA isoforms are likely to modulate the NVU function. This review will summarize the literature regarding the use of pan-CA and specific CA inhibitors along with genetic manipulation of specific CA isoforms in stroke and AD models, to bring light into the functions of CAs in the NVU. Although pan-CA inhibitors are protective and safe, we hypothesize that targeting specific CA isoforms will increase the efficacy of CA inhibition and reduce side effects. More studies to further determine specific CA isoforms functions and changes in disease states are essential to the development of novel therapies for cerebrovascular pathology, occurring in both stroke and AD.

scholarly journals Induction and Maturation of Hepatocyte-Like Cells In Vitro: Focus on Technological Advances and Challenges

2021 ◽  
Vol 9 ◽  
Author(s):  
Ye Xie ◽  
Jia Yao ◽  
Weilin Jin ◽  
Longfei Ren ◽  
Xun Li
Keyword(s):  
Genetic Manipulation ◽  
Toxicity Testing ◽  
Basic Research ◽  
Drug Approval ◽  
Cell Types ◽  
Comprehensive Overview ◽  
Technological Advances ◽  
Degree Of Differentiation

Limited by the poor proliferation and restricted sources of adult hepatocytes, there is an urgent need to find substitutes for proliferation and cultivation of mature hepatocytes in vitro for use in disease treatment, drug approval, and toxicity testing. Hepatocyte-like cells (HLCs), which originate from undifferentiated stem cells or modified adult cells, are considered good candidates because of their advantages in terms of cell source and in vitro expansion ability. However, the majority of induced HLCs are in an immature state, and their degree of differentiation is heterogeneous, diminishing their usability in basic research and limiting their clinical application. Therefore, various methods have been developed to promote the maturation of HLCs, including chemical approaches, alteration of cell culture systems, and genetic manipulation, to meet the needs of in vivo transplantation and in vitro model establishment. This review proposes different cell types for the induction of HLCs, and provide a comprehensive overview of various techniques to promote the generation and maturation of HLCs in vitro.

The BH3 Mimetic, ABT-737, Is Effective Against Bcl-2 Overexpressing Lymphoid Tumors.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 826-826 ◽  
Author(s):  
Kylie D. Mason ◽  
Cassandra J. Vandenberg ◽  
Mark F. van Delft ◽  
Andrew H. Wei ◽  
Suzanne Cory ◽  
...  
Keyword(s):  
Cell Lines ◽  
Genetic Manipulation ◽  
Single Agent ◽  
B Cell Lymphoma ◽  
Cell Types ◽  
Clinical Samples ◽  
Bh3 Mimetic ◽  
Mouse Lymphoma

Abstract Lymphoid tumors often respond poorly to conventional cytotoxics, a common cause being their impaired sensitivity to apoptosis, such as that caused by Bcl-2 overexpression. A strategy to overcoming this is to use mimics of the natural antagonists of pro-survival Bcl-2, the BH3 only proteins. A promising BH3 mimetic is ABT-737, which targets Bcl-2 and closely related pro-survival proteins. We evaluated its potential utility by testing it on cell lines, clinical samples and on a relevant mouse lymphoma model. We assessed the sensitivity of B cell lymphoma cell lines and primary CLL samples to ABT-737, either alone or in combination. To ascertain its efficacy in vivo, we utilized a mouse model based on the Eμ-myc tumor that is readily transplantable and amenable to genetic manipulation. When syngeneic recipient mice were inoculated with tumors, they develop widespread lymphoma, fatal unless treated by agents such as cyclophosphamide. We found that ABT-737, on its own, was cytotoxic only to a subset of cell lines and primary CLL samples. However, it can synergize potently with agents such as dexamethasone, suggesting that this agent might be useful in combination with currently used chemotherapeutics. In the Eμ myc mouse lymphoma model, treatment with ABT-737 alone did not control the disease as multiple independently derived tumors proved refractory to treatment with this agent. However, ABT-737 was partially effective as a single agent for treating bitransgenic tumors derived from crosses of the Eμmyc and Eμ-bcl-2 transgenic mice. ABT-737 therapy prolonged the survival of recipient mice transplanted with tumors from 30 to 60 days. When combined with a low dose of cyclophosphamide (50mg/kg), long term stable remissions were achieved, which were sustained even longer than control mice treated with much higher doses of cyclophosphamide (300mg/kg). We found that ABT-737 was well tolerated as a single agent and when combined with low doses of cytotoxics such as cyclophosphamide. Thus, ABT-737 may prove to be efficacious for those tumors highly dependent on Bcl-2 for their survival. We found that despite its high affinity for Bcl-2, Bcl-xL and Bcl-w, many cell types proved refractory to ABT-737 as a single agent. We show that this resistance reflects its inability to target another pro-survival relative Mcl-1. Down-regulation of Mcl-1 by several strategies conferred sensitivity to ABT-737. Furthermore, enforced Mcl-1 expression in the Eμmyc/bcl-2 bitransgenic mouse lymphoma model conferred marked resistance as mice transplanted with such tumors died as rapidly as the untreated counterparts. However, enhanced Bcl-2 overexpression on these tumors had little impact on the in vivo response, suggesting that ABT-737 can be utilized even when Bcl-2 is markedly overexpressed. ABT-737 appears to be a promising agent for the clinic. It potently sensitizes certain lymphoid tumors to conventional cytotxics in vitro. The synergy observed between dexamethasone and ABT-737 on some lymphoid lines in culture suggests that it is attractive for clinical testing. Encouragingly, ABT-737 appeared efficacious in vivo against Bcl-2 overexpressing tumors when combined with a reduced dose of cyclophosphamide, suggesting that it will be useful for treating even those Bcl-2-overexpressing tumors that are normally highly chemoresistant.

scholarly journals Studying the Neurovascular Unit: An Improved Blood–Brain Barrier Model

2009 ◽  
Vol 29 (12) ◽  
pp. 1879-1884 ◽  
Author(s):  
Christoph M Zehendner ◽  
Heiko J Luhmann ◽  
Christoph RW Kuhlmann
Keyword(s):  
Blood Brain Barrier ◽  
Laser Scanning ◽  
Neurovascular Unit ◽  
Cell Types ◽  
Laser Scanning Confocal Microscopy ◽  
Brain Barrier ◽  
Scanning Confocal Microscopy ◽  
Blood Brain

The blood–brain barrier (BBB) closely interacts with the neuronal parenchyma in vivo. To replicate this interdependence in vitro, we established a murine coculture model composed of brain endothelial cell (BEC) monolayers with cortical organotypic slice cultures. The morphology of cell types, expression of tight junctions, formation of reactive oxygen species, caspase-3 activity in BECs, and alterations of electrical resistance under physiologic and pathophysiological conditions were investigated. This new BBB model allows the application of techniques such as laser scanning confocal microscopy, immunohistochemistry, fluorescent live cell imaging, and electrical cell substrate impedance sensing in real time for studying the dynamics of BBB function under defined conditions.

scholarly journals Are In Vitro Human Blood–Brain–Tumor-Barriers Suitable Replacements for In Vivo Models of Brain Permeability for Novel Therapeutics?

Cancers ◽  
2021 ◽  
Vol 13 (5) ◽  
pp. 955
Author(s):  
Archana Prashanth ◽  
Heather Donaghy ◽  
Shihani P. Stoner ◽  
Amanda L. Hudson ◽  
Helen R. Wheeler ◽  
...  
Keyword(s):  
Neurovascular Unit ◽  
Experimental Studies ◽  
Cell Types ◽  
In Vivo Models ◽  
Advantages And Disadvantages ◽  
Blood Brain ◽  
Animal Use ◽  
Permeability Studies

Background: High grade gliomas (HGG) are incapacitating and prematurely fatal diseases. To overcome the poor prognosis, novel therapies must overcome the selective and restricted permeability of the blood–brain barrier (BBB). This study critically evaluated whether in vitro human normal BBB and tumor BBB (BBTB) are suitable alternatives to “gold standard” in vivo models to determine brain permeability. Methods: A systematic review utilizing the PRISMA guidelines used English and full-text articles from the past 5 years in the PubMed, Embase, Medline and Scopus databases. Experimental studies employing human cell lines were included. Results: Of 1335 articles, the search identified 24 articles for evaluation after duplicates were removed. Eight in vitro and five in vivo models were identified with the advantages and disadvantages compared within and between models, and against patient clinical data where available. The greatest in vitro barrier integrity and stability, comparable to in vivo and clinical permeability data, were achieved in the presence of all cell types of the neurovascular unit: endothelial cells, astrocytes/glioma cells, pericytes and neurons. Conclusions: In vitro co-culture BBB models utilizing stem cell-derived or primary cells are a suitable proxy for brain permeability studies in order to reduce animal use in medical research.

scholarly journals Review of Design Considerations for Brain-on-a-Chip Models

Micromachines ◽  
2021 ◽  
Vol 12 (4) ◽  
pp. 441
Author(s):  
Tiffany Cameron ◽  
Tanya Bennet ◽  
Elyn Rowe ◽  
Mehwish Anwer ◽  
Cheryl Wellington ◽  
...  
Keyword(s):  
Brain Research ◽  
Neurovascular Unit ◽  
Cell Monolayer ◽  
Cell Types ◽  
In Vitro Models ◽  
Research Field ◽  
Design Development ◽  
Organ On A Chip

In recent years, the need for sophisticated human in vitro models for integrative biology has motivated the development of organ-on-a-chip platforms. Organ-on-a-chip devices are engineered to mimic the mechanical, biochemical and physiological properties of human organs; however, there are many important considerations when selecting or designing an appropriate device for investigating a specific scientific question. Building microfluidic Brain-on-a-Chip (BoC) models from the ground-up will allow for research questions to be answered more thoroughly in the brain research field, but the design of these devices requires several choices to be made throughout the design development phase. These considerations include the cell types, extracellular matrix (ECM) material(s), and perfusion/flow considerations. Choices made early in the design cycle will dictate the limitations of the device and influence the end-point results such as the permeability of the endothelial cell monolayer, and the expression of cell type-specific markers. To better understand why the engineering aspects of a microfluidic BoC need to be influenced by the desired biological environment, recent progress in microfluidic BoC technology is compared. This review focuses on perfusable blood–brain barrier (BBB) and neurovascular unit (NVU) models with discussions about the chip architecture, the ECM used, and how they relate to the in vivo human brain. With increased knowledge on how to make informed choices when selecting or designing BoC models, the scientific community will benefit from shorter development phases and platforms curated for their application.

scholarly journals 3D hydrogel models of the neurovascular unit to investigate blood-brain barrier dysfunction

2021 ◽  
Author(s):  
Geoffrey Potjewyd ◽  
Katherine Kellett ◽  
Nigel M Hooper
Keyword(s):  
Blood Brain Barrier ◽  
Neurovascular Unit ◽  
Cell Types ◽  
Brain Parenchyma ◽  
Physical Models ◽  
Brain Barrier ◽  
Barrier Dysfunction ◽  
Blood Brain

The neurovascular unit (NVU), consisting of neurons, glial cells, vascular cells (endothelial cells, pericytes and vascular smooth muscle cells) together with the surrounding extracellular matrix (ECM), is an important interface between the peripheral blood and the brain parenchyma. Disruption of the NVU impacts on blood-brain barrier (BBB) regulation and underlies the development and pathology of multiple neurological disorders, including stroke and Alzheimer’s disease. The ability to differentiate induced pluripotent stem cells (iPSCs) to the different cell types of the NVU and incorporate them into physical models provides a reverse engineering approach to generate human NVU models to study BBB function. To recapitulate the in vivo situation such NVU models must also incorporate the ECM to provide a 3D environment with appropriate mechanical and biochemical cues for the cells of the NVU. In this review we provide an overview of the cells of the NVU and the surrounding ECM, before discussing the characteristics (stiffness, functionality and porosity) required of hydrogels to mimic the ECM when incorporated into in vitro NVU models. We summarise the approaches available to measure BBB functionality and present the techniques in use to develop robust and translatable models of the NVU, including transwell models, hydrogel models, 3D-bioprinting, microfluidic models and organoids. The incorporation of iPSCs either without or with disease-specific genetic mutations into these NVU models provides a platform in which to study normal and disease mechanisms, test BBB permeability to drugs, screen for new therapeutic targets and drugs, or to design cell-based therapies.

Ultrastructural observations on the in vitro cytoadherence of Plasmodium falciparum infected red blood cells

1990 ◽  
Vol 48 (3) ◽  
pp. 914-915
Author(s):  
D.J.P. Ferguson ◽  
A.R. Berendt ◽  
J. Tansey ◽  
K. Marsh ◽  
C.I. Newbold
Keyword(s):  
Plasmodium Falciparum ◽  
Red Blood Cells ◽  
Blood Cells ◽  
Umbilical Vein ◽  
Cell Types ◽  
Amelanotic Melanoma ◽  
Cytoplasmic Process ◽  
Umbilical Vein Endothelial Cells

In human malaria, the most serious clinical manifestation is cerebral malaria (CM) due to infection with Plasmodium falciparum. The pathology of CM is thought to relate to the fact that red blood cells containing mature forms of the parasite (PRBC) cytoadhere or sequester to post capillary venules of various tissues including the brain. This in vivo phenomenon has been studied in vitro by examining the cytoadherence of PRBCs to various cell types and purified proteins. To date, three Ijiost receptor molecules have been identified; CD36, ICAM-1 and thrombospondin. The specific changes in the PRBC membrane which mediate cytoadherence are less well understood, but they include the sub-membranous deposition of electron-dense material resulting in surface deformations called knobs. Knobs were thought to be essential for cytoadherence, lput recent work has shown that certain knob-negative (K-) lines can cytoadhere. In the present study, we have used electron microscopy to re-examine the interactions between K+ PRBCs and both C32 amelanotic melanoma cells and human umbilical vein endothelial cells (HUVEC).We confirm previous data demonstrating that C32 cells possess numerous microvilli which adhere to the PRBC, mainly via the knobs (Fig. 1). In contrast, the HUVEC were relatively smooth and the PRBCs appeared partially flattened onto the cell surface (Fig. 2). Furthermore, many of the PRBCs exhibited an invagination of the limiting membrane in the attachment zone, often containing a cytoplasmic process from the endothelial cell (Fig. 2).

Melatonin modifies tumor hypoxia and metabolism by inhibiting HIF-1α and energy metabolic pathway in the in vitro and in vivo models of breast cancer

2019 ◽  
Vol 2 (4) ◽  
pp. 83-98 ◽  
Author(s):  
André De Lima Mota ◽  
Bruna Vitorasso Jardim-Perassi ◽  
Tialfi Bergamin De Castro ◽  
Jucimara Colombo ◽  
Nathália Martins Sonehara ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Glucose Metabolism ◽  
Tumor Growth ◽  
Tumor Cells ◽  
Carbonic Anhydrases ◽  
In Vivo Models ◽  
Ca Ix ◽  
Gene And Protein Expression

Breast cancer is the most common cancer among women and has a high mortality rate. Adverse conditions in the tumor microenvironment, such as hypoxia and acidosis, may exert selective pressure on the tumor, selecting subpopulations of tumor cells with advantages for survival in this environment. In this context, therapeutic agents that can modify these conditions, and consequently the intratumoral heterogeneity need to be explored. Melatonin, in addition to its physiological effects, exhibits important anti-tumor actions which may associate with modification of hypoxia and Warburg effect. In this study, we have evaluated the action of melatonin on tumor growth and tumor metabolism by different markers of hypoxia and glucose metabolism (HIF-1α, glucose transporters GLUT1 and GLUT3 and carbonic anhydrases CA-IX and CA-XII) in triple negative breast cancer model. In an in vitro study, gene and protein expressions of these markers were evaluated by quantitative real-time PCR and immunocytochemistry, respectively. The effects of melatonin were also tested in a MDA-MB-231 xenograft animal model. Results showed that melatonin treatment reduced the viability of MDA-MB-231 cells and tumor growth in Balb/c nude mice (p <0.05). The treatment significantly decreased HIF-1α gene and protein expression concomitantly with the expression of GLUT1, GLUT3, CA-IX and CA-XII (p <0.05). These results strongly suggest that melatonin down-regulates HIF-1α expression and regulates glucose metabolism in breast tumor cells, therefore, controlling hypoxia and tumor progression. 

Improvement of effectiveness in maize breeding

2006 ◽  
Vol 54 (3) ◽  
pp. 351-358 ◽  
Author(s):  
P. Pepó
Keyword(s):  
Recurrent Selection ◽  
Genetic Manipulation ◽  
Field Testing ◽  
Tissue Cultures ◽  
Maize Breeding ◽  
Breeding Programmes ◽  
Speed Up ◽  
Selection For

Plant regeneration via tissue culture is becoming increasingly more common in monocots such as maize (Zea mays L.). Pollen (gametophytic) selection for resistance to aflatoxin in maize can greatly facilitate recurrent selection and the screening of germplasm for resistance at much less cost and in a shorter time than field testing. In vivo and in vitro techniques have been integrated in maize breeding programmes to obtain desirable agronomic attributes, enhance the genes responsible for them and speed up the breeding process. The efficiency of anther and tissue cultures in maize and wheat has reached the stage where they can be used in breeding programmes to some extent and many new cultivars produced by genetic manipulation have now reached the market.

Epithelial Organotypic Cultures: A Viable Model to Address Mechanisms of Carcinogenesis by Epitheliotropic Viruses

2018 ◽  
Vol 18 (4) ◽  
pp. 246-255 ◽  
Author(s):  
Lara Termini ◽  
Enrique Boccardo
Keyword(s):  
Three Dimensional ◽  
Cell Types ◽  
Experimental Models ◽  
Biological Research ◽  
Specific Gene ◽  
Specific Cell ◽  
Organotypic Cultures ◽  
Skin Physiology

In vitro culture of primary or established cell lines is one of the leading techniques in many areas of basic biological research. The use of pure or highly enriched cultures of specific cell types obtained from different tissues and genetics backgrounds has greatly contributed to our current understanding of normal and pathological cellular processes. Cells in culture are easily propagated generating an almost endless source of material for experimentation. Besides, they can be manipulated to achieve gene silencing, gene overexpression and genome editing turning possible the dissection of specific gene functions and signaling pathways. However, monolayer and suspension cultures of cells do not reproduce the cell type diversity, cell-cell contacts, cell-matrix interactions and differentiation pathways typical of the three-dimensional environment of tissues and organs from where they were originated. Therefore, different experimental animal models have been developed and applied to address these and other complex issues in vivo. However, these systems are costly and time consuming. Most importantly the use of animals in scientific research poses moral and ethical concerns facing a steadily increasing opposition from different sectors of the society. Therefore, there is an urgent need for the development of alternative in vitro experimental models that accurately reproduce the events observed in vivo to reduce the use of animals. Organotypic cultures combine the flexibility of traditional culture systems with the possibility of culturing different cell types in a 3D environment that reproduces both the structure and the physiology of the parental organ. Here we present a summarized description of the use of epithelial organotypic for the study of skin physiology, human papillomavirus biology and associated tumorigenesis.

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