Molecular effects of rotenone exposure and wash-out in an in vitro 3D dopaminergic cell model

Abstract Background: Pulmonary artery endothelial cell (PAEC) inflammation is a critical event in the development of pulmonary arterial hypertension (PAH). However, the pathogenesis of PAEC inflammation remains unclear. Methods: Purified recombinant human inhibitor of κB kinase subunit β (IKKβ) protein, human PAECs and monocrotaline-induced pulmonary hypertensive rats were employed in the study. Site-directed mutagenesis, gene knockdown or overexpression were conducted to manipulate the expression or activity of a target protein. Results: We showed that hydrogen sulfide (H2S) inhibited IKKβ activation in the cell model of human PAEC inflammation induced by monocrotaline pyrrole-stimulation or knockdown of cystathionine γ-lyase (CSE), an H2S generating enzyme. Mechanistically, H2S was proved to inhibit IKKβ activity directly via sulfhydrating IKKβ at cysteinyl residue 179 (C179) in purified recombinant IKKβ protein in vitro, whereas thiol reductant dithiothreitol (DTT) reversed H2S-induced IKKβ inactivation. Furthermore, to demonstrate the significance of IKKβ sulfhydration by H2S in the development of PAEC inflammation, we mutated C179 to serine (C179S) in IKKβ. In purified IKKβ protein, C179S mutation of IKKβ abolished H2S-induced IKKβ sulfhydration and the subsequent IKKβ inactivation. In human PAECs, C179S mutation of IKKβ blocked H2S-inhibited IKKβ activation and PAEC inflammatory response. In pulmonary hypertensive rats, C179S mutation of IKKβ abolished the inhibitory effect of H2S on IKKβ activation and pulmonary vascular inflammation and remodeling. Conclusion: Collectively, our in vivo and in vitro findings demonstrated, for the first time, that endogenous H2S directly inactivated IKKβ via sulfhydrating IKKβ at Cys179 to inhibit nuclear factor-κB (NF-κB) pathway activation and thereby control PAEC inflammation in PAH.

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Preincubation with Sn-complexes causes intensive intracellular retention of 99mTc in thyroid cells in vitro

Nuklearmedizin ◽

10.3413/nukmed-0450-11-12 ◽

2012 ◽

Vol 51 (05) ◽

pp. 179-185 ◽

Cited By ~ 3

Author(s):

M. Wendisch ◽

D. Aurich ◽

R. Runge ◽

R. Freudenberg ◽

J. Kotzerke ◽

...

Keyword(s):

Cell Model ◽

Low Energy ◽

Thyroid Cells ◽

Low Energy Electrons ◽

A Cell ◽

Vitro Model ◽

Uptake Studies ◽

Radiobiological Effects ◽

Radioactivity Retention

SummaryTechnetium radiopharmaceuticals are well established in nuclear medicine. Besides its well-known gamma radiation, 99mTc emits an average of five Auger and internal conversion electrons per decay. The biological toxicity of these low-energy, high-LET (linear energy transfer) emissions is a controversial subject. One aim of this study was to estimate in a cell model how much 99mTc can be present in exposed cells and which radiobiological effects could be estimated in 99mTc-overloaded cells. Methods: Sodium iodine symporter (NIS)- positive thyroid cells were used. 99mTc-uptake studies were performed after preincubation with a non-radioactive (cold) stannous pyro - phosphate kit solution or as a standard 99mTc pyrophosphate kit preparation or with pure pertechnetate solution. Survival curves were analyzed from colony-forming assays. Results: Preincubation with stannous complexes causes irreversible intracellular radioactivity retention of 99mTc and is followed by further pertechnetate influx to an unexpectedly high 99mTc level. The uptake of 99mTc pertechnetate in NIS-positive cells can be modified using stannous pyrophosphate from 3–5% to >80%. The maximum possible cellular uptake of 99mTc was 90 Bq/cell. Compared with nearly pure extracellular irradiation from routine 99mTc complexes, cell survival was reduced by 3–4 orders of magnitude after preincubation with stannous pyrophosphate. Conclusions: Intra cellular 99mTc retention is related to reduced survival, which is most likely mediated by the emission of low-energy electrons. Our findings show that the described experiments constitute a simple and useful in vitro model for radiobiological investigations in a cell model.

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The TGFβ Family in Human Placental Development at the Fetal-Maternal Interface

Biomolecules ◽

10.3390/biom10030453 ◽

2020 ◽

Vol 10 (3) ◽

pp. 453

Author(s):

Susana M. Chuva de Sousa Lopes ◽

Marta S. Alexdottir ◽

Gudrun Valdimarsdottir

Keyword(s):

Stem Cell ◽

Transforming Growth Factor Beta ◽

Molecular Mechanisms ◽

Transforming Growth Factor ◽

Cell Model ◽

Embryonic Stem ◽

Model Systems ◽

Special Focus ◽

Placental Development

Emerging data suggest that a trophoblast stem cell (TSC) population exists in the early human placenta. However, in vitro stem cell culture models are still in development and it remains under debate how well they reflect primary trophoblast (TB) cells. The absence of robust protocols to generate TSCs from humans has resulted in limited knowledge of the molecular mechanisms that regulate human placental development and TB lineage specification when compared to other human embryonic stem cells (hESCs). As placentation in mouse and human differ considerably, it is only with the development of human-based disease models using TSCs that we will be able to understand the various diseases caused by abnormal placentation in humans, such as preeclampsia. In this review, we summarize the knowledge on normal human placental development, the placental disease preeclampsia, and current stem cell model systems used to mimic TB differentiation. A special focus is given to the transforming growth factor-beta (TGFβ) family as it has been shown that the TGFβ family has an important role in human placental development and disease.

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MMP-9-mediated regulation of hypoxia-reperfusion injury-related neutrophil inflammation in an in vitro proximal tubular cell model

Renal Failure ◽

10.1080/0886022x.2021.1930558 ◽

2021 ◽

Vol 43 (1) ◽

pp. 900-910

Author(s):

Yan Dong ◽

Hong Zhao ◽

Jiangwei Man ◽

Shengjun Fu ◽

Li Yang

Keyword(s):

Reperfusion Injury ◽

Cell Model ◽

Proximal Tubular Cell ◽

Tubular Cell ◽

Proximal Tubular

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SIRT1 attenuates sepsis-induced acute kidney injury via Beclin1 deacetylation-mediated autophagy activation

Cell Death and Disease ◽

10.1038/s41419-021-03508-y ◽

2021 ◽

Vol 12 (2) ◽

Author(s):

Zhiya Deng ◽

Maomao Sun ◽

Jie Wu ◽

Haihong Fang ◽

Shumin Cai ◽

...

Keyword(s):

Acute Kidney Injury ◽

Protective Effect ◽

Cell Model ◽

Kidney Injury ◽

Underlying Mechanism ◽

Autophagy Induction ◽

Promising Therapeutic Approach ◽

Related Proteins ◽

Caecal Ligation And Puncture

AbstractOur previous studies showed that silent mating-type information regulation 2 homologue-1 (SIRT1, a deacetylase) upregulation could attenuate sepsis-induced acute kidney injury (SAKI). Upregulated SIRT1 can deacetylate certain autophagy-related proteins (Beclin1, Atg5, Atg7 and LC3) in vitro. However, it remains unclear whether the beneficial effect of SIRT1 is related to autophagy induction and the underlying mechanism of this effect is also unknown. In the present study, caecal ligation and puncture (CLP)-induced mice, and an LPS-challenged HK-2 cell line were established to mimic a SAKI animal model and a SAKI cell model, respectively. Our results demonstrated that SIRT1 activation promoted autophagy and attenuated SAKI. SIRT1 deacetylated only Beclin1 but not the other autophagy-related proteins in SAKI. SIRT1-induced autophagy and its protective effect against SAKI were mediated by the deacetylation of Beclin1 at K430 and K437. Moreover, two SIRT1 activators, resveratrol and polydatin, attenuated SAKI in CLP-induced septic mice. Our study was the first to demonstrate the important role of SIRT1-induced Beclin1 deacetylation in autophagy and its protective effect against SAKI. These findings suggest that pharmacologic induction of autophagy via SIRT1-mediated Beclin1 deacetylation may be a promising therapeutic approach for future SAKI treatment.

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Localization matters: nuclear-trapped Survivin sensitizes glioblastoma cells to temozolomide by elevating cellular senescence and impairing homologous recombination

Cellular and Molecular Life Sciences ◽

10.1007/s00018-021-03864-0 ◽

2021 ◽

Author(s):

Thomas R. Reich ◽

Christian Schwarzenbach ◽

Juliana Brandstetter Vilar ◽

Sven Unger ◽

Fabian Mühlhäusler ◽

...

Keyword(s):

Homologous Recombination ◽

Nuclear Export ◽

Cell Model ◽

Chromosome Instability ◽

Direct Interaction ◽

High Grade Glioma ◽

Strand Break ◽

Γh2ax Foci

AbstractTo clarify whether differential compartmentalization of Survivin impacts temozolomide (TMZ)-triggered end points, we established a well-defined glioblastoma cell model in vitro (LN229 and A172) and in vivo, distinguishing between its nuclear and cytoplasmic localization. Expression of nuclear export sequence (NES)-mutated Survivin (SurvNESmut-GFP) led to impaired colony formation upon TMZ. This was not due to enhanced cell death but rather due to increased senescence. Nuclear-trapped Survivin reduced homologous recombination (HR)-mediated double-strand break (DSB) repair, as evaluated by γH2AX foci formation and qPCR-based HR assay leading to pronounced induction of chromosome aberrations. Opposite, clones, expressing free-shuttling cytoplasmic but not nuclear-trapped Survivin, could repair TMZ-induced DSBs and evaded senescence. Mass spectrometry-based interactomics revealed, however, no direct interaction of Survivin with any of the repair factors. The improved TMZ-triggered HR activity in Surv-GFP was associated with enhanced mRNA and stabilized RAD51 protein expression, opposite to diminished RAD51 expression in SurvNESmut cells. Notably, cytoplasmic Survivin could significantly compensate for the viability under RAD51 knockdown. Differential Survivin localization also resulted in distinctive TMZ-triggered transcriptional pathways, associated with senescence and chromosome instability as shown by global transcriptome analysis. Orthotopic LN229 xenografts, expressing SurvNESmut exhibited diminished growth and increased DNA damage upon TMZ, as manifested by PCNA and γH2AX foci expression, respectively, in brain tissue sections. Consequently, those mice lived longer. Although tumors of high-grade glioma patients expressed majorly nuclear Survivin, they exhibited rarely NES mutations which did not correlate with survival. Based on our in vitro and xenograft data, Survivin nuclear trapping would facilitate glioma response to TMZ.

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β-elemene alleviates airway stenosis via the ILK/Akt pathway modulated by MIR143HG sponging miR-1275

Cellular & Molecular Biology Letters ◽

10.1186/s11658-021-00261-0 ◽

2021 ◽

Vol 26 (1) ◽

Author(s):

Guoying Zhang ◽

Cheng Xue ◽

Yiming Zeng

Keyword(s):

Cell Cycle ◽

Cell Viability ◽

Cell Model ◽

Protective Efficacy ◽

Rabbit Model ◽

Akt Pathway ◽

Loss Of Function ◽

Airway Stenosis

Abstract Background We have previously found that β-elemene could inhibit the viability of airway granulation fibroblasts and prevent airway hyperplastic stenosis. This study aimed to elucidate the underlying mechanism and protective efficacy of β-elemene in vitro and in vivo. Methods Microarray and bioinformatic analysis were used to identify altered pathways related to cell viability in a β-elemene-treated primary cell model and to construct a β-elemene-altered ceRNA network modulating the target pathway. Loss of function and gain of function approaches were performed to examine the role of the ceRNA axis in β-elemene's regulation of the target pathway and cell viability. Additionally, in a β-elemene-treated rabbit model of airway stenosis, endoscopic and histological examinations were used to evaluate its therapeutic efficacy and further verify its mechanism of action. Results The hyperactive ILK/Akt pathway and dysregulated LncRNA-MIR143HG, which acted as a miR-1275 ceRNA to modulate ILK expression, were suppressed in β-elemene-treated airway granulation fibroblasts; β-elemene suppressed the ILK/Akt pathway via the MIR143HG/miR-1275/ILK axis. Additionally, the cell cycle and apoptotic phenotypes of granulation fibroblasts were altered, consistent with ILK/Akt pathway activity. In vivo application of β-elemene attenuated airway granulation hyperplasia and alleviated scar stricture, and histological detections suggested that β-elemene's effects on the MIR143HG/miR-1275/ILK axis and ILK/Akt pathway were in line with in vitro findings. Conclusions MIR143HG and ILK may act as ceRNA to sponge miR-1275. The MIR143HG/miR-1275/ILK axis mediates β-elemene-induced cell cycle arrest and apoptosis of airway granulation fibroblasts by modulating the ILK/Akt pathway, thereby inhibiting airway granulation proliferation and ultimately alleviating airway stenosis.

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Influences of advanced glycosylation end products on the inner blood–retinal barrier in a co-culture cell model in vitro

Open Life Sciences ◽

10.1515/biol-2020-0067 ◽

2020 ◽

Vol 15 (1) ◽

pp. 619-628

Author(s):

Chen Yuan ◽

Ya Mo ◽

Jie Yang ◽

Mei Zhang ◽

Xuejun Xie

Keyword(s):

Electrical Resistance ◽

Cell Model ◽

Polyclonal Antibodies ◽

Time Dependent ◽

Dependent Manner ◽

Blood Retinal Barrier ◽

Advanced Glycosylation End Products ◽

End Products ◽

Advanced Glycosylation

AbstractAdvanced glycosylation end products (AGEs) are harmful factors that can damage the inner blood–retinal barrier (iBRB). Rat retinal microvascular endothelial cells (RMECs) were isolated and cultured, and identified by anti-CD31 and von Willebrand factor polyclonal antibodies. Similarly, rat retinal Müller glial cells (RMGCs) were identified by H&E staining and with antibodies of glial fibrillary acidic protein and glutamine synthetase. The transepithelial electrical resistance (TEER) value was measured with a Millicell electrical resistance system to observe the leakage of the barrier. Transwell cell plates for co-culturing RMECs with RMGCs were used to construct an iBRB model, which was then tested with the addition of AGEs at final concentrations of 50 and 100 mg/L for 24, 48, and 72 h. AGEs in the in vitro iBRB model constructed by RMEC and RMGC co-culture led to the imbalance of the vascular endothelial growth factor (VEGF) and pigment epithelial derivative factor (PEDF), and the permeability of the RMEC layer increased because the TEER decreased in a dose- and time-dependent manner. AGEs increased VEGF but lowered PEDF in a dose- and time-dependent manner. The intervention with AGEs led to the change of the transendothelial resistance of the RMEC layer likely caused by the increased ratio of VEGF/PEDF.

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Generation of tetracycline-controllable CYP3A4-expressing Caco-2 cells by the piggyBac transposon system

Scientific Reports ◽

10.1038/s41598-021-91160-z ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Moe Ichikawa ◽

Hiroki Akamine ◽

Michika Murata ◽

Sumito Ito ◽

Kazuo Takayama ◽

...

Keyword(s):

Small Intestine ◽

Drug Absorption ◽

Cell Model ◽

Negative Effects ◽

Piggybac Transposon ◽

Drug Metabolizing Enzyme ◽

Metabolizing Enzyme ◽

Cyp3a4 Expression

AbstractCaco-2 cells are widely used as an in vitro intestinal epithelial cell model because they can form a monolayer and predict drug absorption with high accuracy. However, Caco-2 cells hardly express cytochrome P450 (CYP), a drug-metabolizing enzyme. It is known that CYP3A4 is the dominant drug-metabolizing enzyme in human small intestine. In this study, we generated CYP3A4-expressing Caco-2 (CYP3A4-Caco-2) cells and attempted to establish a model that can simultaneously evaluate drug absorption and metabolism. CYP3A4-Caco-2 cells were generated by piggyBac transposon vectors. A tetracycline-controllable CYP3A4 expression cassette (tet-on system) was stably transduced into Caco-2 cells, thus regulating the levels of CYP3A4 expression depending on the doxycycline concentration. The CYP3A4 expression levels in CYP3A4-Caco-2 cells cultured in the presence of doxycycline were similar to or higher than those of adult small intestine. The CYP3A4-Caco-2 cells had enough ability to metabolize midazolam, a substrate of CYP3A4. CYP3A4 overexpression had no negative effects on cell proliferation, barrier function, and P-glycoprotein activity in Caco-2 cells. Thus, we succeeded in establishing Caco-2 cells with CYP3A4 metabolizing activity comparable to in vivo human intestinal tissue. This cell line would be useful in pharmaceutical studies as a model that can simultaneously evaluate drug absorption and metabolism.

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Positively Charged Lipid as Potential Tool to Influence the Fate of Ethosomes

Applied Sciences ◽

10.3390/app11157060 ◽

2021 ◽

Vol 11 (15) ◽

pp. 7060

Author(s):

Antonia Mancuso ◽

Maria Chiara Cristiano ◽

Massimo Fresta ◽

Daniele Torella ◽

Donatella Paolino

Keyword(s):

Cell Model ◽

Human Keratinocytes ◽

Skin Delivery ◽

Physico Chemical ◽

Cell Mortality ◽

Mean Size ◽

A Cell ◽

Negative Surface ◽

Cytotoxic Studies

Ethosomes® are one of the main deformable vesicles proposed to overcome the stratum corneum. They are composed of lecithin, ethanol and water, resulting in round vesicles characterized by a narrow size distribution and a negative surface charge. Taking into account their efficiency to deliver drugs into deeper skin layers, the current study was designed to evaluate the influence of different lipids on the physico-chemical features of traditional ethosomes in the attempt to influence their fate. Three lipids (DOPE, DSPE and DOTAP) were used for the study, but only DOTAP conferred a net positive charge to ethosomes, maintaining a narrow mean size lower than 300 nm and a good polydispersity index. Stability and in vitro cytotoxic studies have been performed using Turbiscan Lab analysis and MTT dye exclusion assay, respectively. Data recorded demonstrated the good stability of modified ethosomes and a reasonable absence of cell mortality when applied to human keratinocytes, NCTC 2544, which are used as a cell model. Finally, the best formulations were selected to evaluate their ability to encapsulate drugs, through the use of model compounds. Cationic ethosomes encapsulated oil red o and rhodamine b in amounts comparable to those recorded from conventional ethosomes (over 50%). Results recorded from this study are encouraging as cationic ethosomes may open new opportunities for skin delivery.

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