Development of a Cell Co-Culture Model to Mimic Cardiac Ischemia/Reperfusion In Vitro

10.3791/62913 ◽  
2021 ◽  
Author(s):  
Zhu Li ◽  
Matthew J. W. Hampton ◽  
Matthew B. Barajas ◽  
Matthias L. Riess
Keyword(s):  
Ischemia Reperfusion ◽  
Cardiac Ischemia ◽  
Culture Model ◽  
A Cell

scholarly journals Regulation of cilia abundance in multiciliated cells

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Rashmi Nanjundappa ◽  
Dong Kong ◽  
Kyuhwan Shim ◽  
Tim Stearns ◽  
Steven L Brody ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Surface Area ◽  
Ex Vivo ◽  
Compensatory Mechanism ◽  
Multiciliated Cells ◽  
Culture Model ◽  
A Cell ◽  
Dependent Mechanism

Multiciliated cells (MCC) contain hundreds of motile cilia used to propel fluid over their surface. To template these cilia, each MCC produces between 100-600 centrioles by a process termed centriole amplification. Yet, how MCC regulate the precise number of centrioles and cilia remains unknown. Airway progenitor cells contain two parental centrioles (PC) and form structures called deuterosomes that nucleate centrioles during amplification. Using an ex vivo airway culture model, we show that ablation of PC does not perturb deuterosome formation and centriole amplification. In contrast, loss of PC caused an increase in deuterosome and centriole abundance, highlighting the presence of a compensatory mechanism. Quantification of centriole abundance in vitro and in vivo identified a linear relationship between surface area and centriole number. By manipulating cell size, we discovered that centriole number scales with surface area. Our results demonstrate that a cell-intrinsic surface area-dependent mechanism controls centriole and cilia abundance in multiciliated cells.

scholarly journals Assessing the Efficacy of Dietary Selenomethionine Supplementation in the Setting of Cardiac Ischemia/Reperfusion Injury

Antioxidants ◽  
2019 ◽  
Vol 8 (11) ◽  
pp. 546 ◽  
Author(s):  
Leila Reyes ◽  
David P. Bishop ◽  
Clare L. Hawkins ◽  
Benjamin S. Rayner
Keyword(s):  
Cardiac Myocyte ◽  
Hypochlorous Acid ◽  
Ischemia Reperfusion Injury ◽  
Heart Function ◽  
Ischemia Reperfusion ◽  
Cytosolic Calcium ◽  
Cardiac Ischemia ◽  
Cellular Damage

Oxidative stress is a major hallmark of cardiac ischemia/reperfusion (I/R) injury. This partly arises from the presence of activated phagocytes releasing myeloperoxidase (MPO) and its production of hypochlorous acid (HOCl). The dietary supplement selenomethionine (SeMet) has been shown to bolster endogenous antioxidant processes as well as readily react with MPO-derived oxidants. The aim of this study was to assess whether supplementation with SeMet could modulate the extent of cellular damage observed in an in vitro cardiac myocyte model exposed to (patho)-physiological levels of HOCl and an in vivo rat model of cardiac I/R injury. Exposure of the H9c2 cardiac myoblast cell line to HOCl resulted in a dose-dependent increase in necrotic cell death, which could be prevented by SeMet supplementation and was attributed to SeMet preventing the HOCl-induced loss of mitochondrial inner trans-membrane potential, and the associated cytosolic calcium accumulation. This protection was credited primarily to the direct oxidant scavenging ability of SeMet, with a minor contribution arising from the ability of SeMet to bolster cardiac myoblast glutathione peroxidase (GPx) activity. In vivo, a significant increase in selenium levels in the plasma and heart tissue were seen in male Wistar rats fed a diet supplemented with 2 mg kg−1 SeMet compared to controls. However, SeMet-supplementation demonstrated only limited improvement in heart function and did not result in better heart remodelling following I/R injury. These data indicate that SeMet supplementation is of potential benefit within pathological settings where excessive HOCl is known to be generated but has limited efficacy as a therapeutic agent for the treatment of heart attack.

scholarly journals Gp91phox (NOX2) in Activated Microglia Exacerbates Neuronal Damage Induced by Oxygen Glucose Deprivation and Hyperglycemia in an in Vitro Model

2018 ◽  
Vol 50 (2) ◽  
pp. 783-797 ◽  
Author(s):  
Xianzhang Zeng ◽  
Hongliang Ren ◽  
Yana Zhu ◽  
Ruru Zhang ◽  
Xinxin Xue ◽  
...  
Keyword(s):  
Ischemia Reperfusion Injury ◽  
Neuronal Damage ◽  
Neuronal Survival ◽  
Ischemia Reperfusion ◽  
Survival Rates ◽  
Glucose Deprivation ◽  
Oxygen Glucose Deprivation ◽  
Sirna Transfection ◽  
Culture Model

Background/Aims: Peri-operative cerebral ischemia reperfusion injury is one of the most serious peri-operative complications that can be aggravated in patients with diabetes. A previous study showed that microglia NOX2 (a NADPH oxidase enzyme) may play an important role in this process. Here, we investigated whether increased microglial derived gp91phox, also known as NOX2, reduced oxygen glucose deprivation (OGD) after induction of hyperglycemia (HG). Methods: A rat neuronal-microglial in vitro co-culture model was used to determine the effects of gp91phox knockdown on OGD after HG using six treatment groups: A rat microglia and neuron co-culture model was established and divided into the following six groups: high glucose + scrambled siRNA transfection (HG, n = 5); HG + gp91phoxsiRNA transfection (HG-gp91siRNA, n = 5); oxygen glucose deprivation + scrambled siRNA transfection (OGD, n = 5); OGD + gp91phoxsiRNA transfection (OGD-gp91siRNA, n = 5); HG + OGD + scrambled siRNA transfection (HG-OGD, n = 5); and HG + OGD + gp91phoxsiRNA transfection (HG-OGD-gp91siRNA, n = 5). The neuronal survival rate was measured by the MTT assay, while western blotting was used to determine gp91phox expression. Microglial derived ROS and neuronal apoptosis rates were analyzed by flow cytometry. Finally, the secretion of cytokines, including IL-6, IL-8, TNF-α, and 8-iso-PGF2α was determined using an ELISA kit. Results: Neuronal survival rates were significantly decreased by HG and OGD, while knockdown of gp91phox reversed these rates. ROS production and cytokine secretion were also significantly increased by HG and OGD but were significantly inhibited by knockdown of gp91phoxsiRNA. Conclusion: Knockdown of gp91phoxsiRNA significantly reduced oxidative stress and the inflammatory response, and alleviated neuronal damage after HG and OGD treatment in a rat neuronal-microglial co-culture model.

scholarly journals Adhesion, Invasion, and Translocation Characteristics of Listeria monocytogenes Serotypes in Caco-2 Cell and Mouse Models

2003 ◽  
Vol 69 (6) ◽  
pp. 3640-3645 ◽  
Author(s):  
Ziad W. Jaradat ◽  
Arun K. Bhunia
Keyword(s):  
Listeria Monocytogenes ◽  
Mouse Model ◽  
Cell Line ◽  
Mouse Bioassay ◽  
Adhesion Properties ◽  
Culture Model ◽  
A Cell ◽  
In Vitro Adhesion ◽  
The Brain

ABSTRACT Adhesion is a crucial first step in Listeria monocytogenes pathogenesis. In this study, we examined how the adhesion properties of serotypes correlate with their invasion efficiencies in a cell culture model (Caco-2) and in a mouse model. Adhesion characteristics of all 13 serotypes of L. monocytogenes (25 strains) were analyzed, which yielded three distinct groups (P < 0.05) with high-, medium-, and low-level-adhesion profiles. The efficiency of these strains in invading the Caco-2 cell line was analyzed, which produced two groups; however, the overall correlation (R 2) was only 0.1236. In the mouse bioassay, all selected strains, irrespective of their adhesion profiles, translocated to the liver and the spleen with almost equal frequencies that did not show any clear relationship with adhesion profiles. However, the serotypes with increased adhesion showed a slightly increased translocation to the brain (R 2 = 0.3371). Collectively, these results indicate that an in vitro adhesion profile might not be an accurate assessment of a strain's ability to invade a cultured cell line or organs or tissues in a mouse model.

scholarly journals Extensive transcriptional and chromatin changes underlie astrocyte maturation in vivo and in culture

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Michael Lattke ◽  
Robert Goldstone ◽  
James K. Ellis ◽  
Stefan Boeing ◽  
Jerónimo Jurado-Arjona ◽  
...  
Keyword(s):  
Three Dimensional ◽  
Cell Culture Model ◽  
Functional Maturation ◽  
Culture Model ◽  
Mature Astrocyte ◽  
Transcriptional Changes ◽  
A Cell ◽  
Brain Homeostasis

AbstractAstrocytes have essential functions in brain homeostasis that are established late in differentiation, but the mechanisms underlying the functional maturation of astrocytes are not well understood. Here we identify extensive transcriptional changes that occur during murine astrocyte maturation in vivo that are accompanied by chromatin remodelling at enhancer elements. Investigating astrocyte maturation in a cell culture model revealed that in vitro-differentiated astrocytes lack expression of many mature astrocyte-specific genes, including genes for the transcription factors Rorb, Dbx2, Lhx2 and Fezf2. Forced expression of these factors in vitro induces distinct sets of mature astrocyte-specific transcripts. Culturing astrocytes in a three-dimensional matrix containing FGF2 induces expression of Rorb, Dbx2 and Lhx2 and improves astrocyte maturity based on transcriptional and chromatin profiles. Therefore, extrinsic signals orchestrate the expression of multiple intrinsic regulators, which in turn induce in a modular manner the transcriptional and chromatin changes underlying astrocyte maturation.

scholarly journals Extensive transcriptional and chromatin changes underlie astrocyte maturation in vivo and in culture

2020 ◽  
Author(s):  
Michael Lattke ◽  
Robert Goldstone ◽  
Francois Guillemot
Keyword(s):  
Three Dimensional ◽  
Cell Culture Model ◽  
Culture Model ◽  
Mature Astrocyte ◽  
Transcriptional Changes ◽  
A Cell ◽  
Late Stages ◽  
Brain Homeostasis

SummaryAstrocytes have diverse functions in brain homeostasis. Many of these functions are acquired during late stages of differentiation when astrocytes become fully mature. The mechanisms underlying astrocyte maturation are not well understood. Here we identified extensive transcriptional changes that occur during astrocyte maturation and are accompanied by chromatin remodelling at enhancer elements. Investigating astrocyte maturation in a cell culture model revealed that in vitro-differentiated astrocytes lacked expression of many mature astrocyte-specific genes, including genes for the transcription factors Rorb, Dbx2, Lhx2 and Fezf2. Forced expression of these factors in vitro induced distinct sets of mature astrocytes-specific transcripts. Culturing astrocytes with FGF2 in a three-dimensional gel induced expression of Rorb, Dbx2 and Lhx2 and improved their maturity based on transcriptional and chromatin profiles. Therefore extrinsic signals orchestrate the expression of multiple intrinsic regulators, which in turn induce in a modular manner the transcriptional and chromatin changes underlying astrocyte maturation.

scholarly journals The long noncoding RNA lncCIRBIL disrupts the nuclear translocation of Bclaf1 alleviating cardiac ischemia–reperfusion injury

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Yang Zhang ◽  
Xiaofang Zhang ◽  
Benzhi Cai ◽  
Ying Li ◽  
Yuan Jiang ◽  
...  
Keyword(s):  
Long Noncoding Rna ◽  
Noncoding Rna ◽  
Nuclear Translocation ◽  
Ischemia Reperfusion Injury ◽  
Ischemia Reperfusion ◽  
Cardiac Injury ◽  
Pathological Process ◽  
Cardiac Ischemia

AbstractCardiac ischemia–reperfusion (I/R) injury is a pathological process resulting in cardiomyocyte death. The present study aims to evaluate the role of the long noncoding RNA Cardiac Injury-Related Bclaf1-Inhibiting LncRNA (lncCIRBIL) on cardiac I/R injury and delineate its mechanism of action. The level of lncCIRBIL is reduced in I/R hearts. Cardiomyocyte-specific transgenic overexpression of lncCIRBIL reduces infarct area following I/R injury. Knockout of lncCIRBIL in mice exacerbates cardiac I/R injury. Qualitatively, the same results are observed in vitro. LncCIRBIL directly binds to BCL2-associated transcription factor 1 (Bclaf1), to inhibit its nuclear translocation. Cardiomyocyte-specific transgenic overexpression of Bclaf1 worsens, while partial knockout of Bclaf1 mitigates cardiac I/R injury. Meanwhile, partial knockout of Bclaf1 abrogates the detrimental effects of lncCIRBIL knockout on cardiac I/R injury. Collectively, the protective effect of lncCIRBIL on I/R injury is accomplished by inhibiting the nuclear translocation of Bclaf1. LncCIRBIL and Bclaf1 are potential therapeutic targets for ischemic cardiac disease.

scholarly journals A Cell Culture Model of BK Polyomavirus Persistence, Genome Recombination, and Reactivation

2021 ◽  
Author(s):  
Michael Imperiale ◽  
Linbo Zhao
Keyword(s):  
Persistent Infection ◽  
Hemorrhagic Cystitis ◽  
Dna Amplification ◽  
Transplant Recipients ◽  
Genome Recombination ◽  
Bk Polyomavirus ◽  
Culture Model ◽  
A Cell ◽  
Vitro Model

BK Polyomavirus (BKPyV) is a small non-enveloped DNA virus that establishes a ubiquitous, asymptomatic, and lifelong persistent infection in at least 80% of the world's population. In some immunosuppressed transplant recipients, BKPyV reactivation causes polyomavirus-associated nephropathy and hemorrhagic cystitis. We report a novel in vitro model of BKPyV persistence and reactivation using a BKPyV natural host cell line. In this system, viral genome loads remain constant for various times post-establishment of persistent infection, during which BKPyV undergoes extensive random genome recombination. Certain recombination events result in viral DNA amplification and protein expression, resulting in production of viruses with enhanced replication ability.

scholarly journals Multidrug treatment with nelfinavir and cepharanthine against COVID-19

2020 ◽  
Author(s):  
Hirofumi Ohashi ◽  
Koichi Watashi ◽  
Wakana Saso ◽  
Kaho Shionoya ◽  
Shoya Iwanami ◽  
...  
Keyword(s):  
Combined Treatment ◽  
Antiviral Agents ◽  
Hiv Protease ◽  
Viral Attachment ◽  
Main Protease ◽  
Culture Model ◽  
Progression Of Disease ◽  
A Cell ◽  
Approved Drugs

SummaryAntiviral treatments targeting the emerging coronavirus disease 2019 (COVID-19) are urgently required. We screened a panel of already-approved drugs in a cell culture model of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and identified two new antiviral agents: the HIV protease inhibitor Nelfinavir and the anti-inflammatory drug Cepharanthine. In silico modeling shows Nelfinavir binds the SARS-CoV-2 main protease consistent with its inhibition of viral replication, whilst Cepharanthine inhibits viral attachment and entry into cells. Consistent with their different modes of action, in vitro assays highlight a synergistic effect of this combined treatment to limit SARS-CoV-2 proliferation. Mathematical modeling in vitro antiviral activity coupled with the known pharmacokinetics for these drugs predicts that Nelfinavir will facilitate viral clearance. Combining Nelfinavir/Cepharanthine enhanced their predicted efficacy to control viral proliferation, to ameliorate both the progression of disease and risk of transmission. In summary, this study identifies a new multidrug combination treatment for COVID-19.

scholarly journals A Cell Culture Model of BK Polyomavirus Persistence, Genome Recombination, and Reactivation

mBio ◽  
2021 ◽  
Author(s):  
Linbo Zhao ◽  
Michael J. Imperiale
Keyword(s):  
Severe Disease ◽  
Transplant Recipients ◽  
Healthy Individuals ◽  
Cell Culture Model ◽  
Genome Recombination ◽  
Bk Polyomavirus ◽  
Culture Model ◽  
A Cell ◽  
Practical Model

BK polyomavirus (BKPyV) generally establishes a persistent subclinical infection in healthy individuals but can cause severe disease in transplant recipients. While an in vitro model to study acute replication exists, no practical model with which to study BKPyV persistence is currently available.

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