Melatonin Alleviates LPS-Induced Pyroptotic Cell Death in Human Stem Cell-Derived Cardiomyocytes by Activating Autophagy

Endotoxemia in sepsis remains a problem due to a lack of effective strategies. Our previous studies have demonstrated that melatonin (Mel) protects against ischemic heart injury and arteriosclerosis. However, its role in endotoxemia-exposed cardiomyocytes remains poorly understood. This study explored, for the first time, the protective effect of Mel on the pyroptosis of human stem cell-derived cardiomyocytes (hiPSC-CMs) exposed to lipopolysaccharide (LPS). Our results showed that treatment with 1 μM or 10 μM Mel for 12 h significantly improved 1 μg/ml LPS-induced hiPSC-CM injuries, as reflected by drastically decreased LDH release and increased cell viability, which was accompanied by the overt induction of autophagy. Specifically, Mel profoundly alleviated LPS-induced cell pyroptosis, as evidenced by decreased propidium iodide (PI) and active caspase-1 double-positive cell rates; suppressed the expression of NLRP3, cleaved caspase-1 (activated form of caspase-1), and GSDMD-NT (functional N-terminal fragment of GSDMD) expression; and inhibited the production of the cleaved IL-1β and cleaved IL-18 cytokines. Additionally, double-membrane autophagosomes were observed in LPS-injured hiPSC-CMs treated with 1 μM or 10 μM Mel. The hiPSC-CMs treated with LPS exhibited considerably fewer acidic vesicles (as revealed by LAMP1 staining) and autophagosomes (as revealed by LC3-II staining); however, Mel reversed this outcome in a dose-dependent manner. Furthermore, coincubation with rapamycin (an autophagy activator) or 3-MA (an autophagy inhibitor) accentuated and attenuated the antipyroptotic actions of Mel, respectively. Collectively, our findings demonstrate that Mel shields hiPSC-CMs against pyroptosis during endotoxemia by activating autophagy.

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Abstract 142: Modeling Drug-Induced Long QT Syndrome with Patient-Specific Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Circulation Research ◽

10.1161/res.113.suppl_1.a142 ◽

2013 ◽

Vol 113 (suppl_1) ◽

Author(s):

Francesca Stillitano ◽

Ioannis Karakikes ◽

Chi-wai Kong ◽

Brett Martinelli ◽

Ronald Li ◽

...

Keyword(s):

Stem Cell ◽

Pluripotent Stem Cell ◽

Induced Pluripotent Stem Cell ◽

Patient Specific ◽

Dependent Manner ◽

Long Qt ◽

Drug Induced ◽

Qt Syndrome ◽

Induced Pluripotent ◽

Dose Dependent

Long QT syndrome (LQTS) is characterized by prolonged cardiac repolarization time and increased risk of ventricular arrhythmia. LQTS can be either inherited or induced notably after drugs intake. Mutations in genes encoding cardiac ion channels have been reported to underlie inherited LQTS. In contrast, drug-induced LQTS (diLQTS) most frequently arises from altered function of the hERG channel; the risk of developing diLQTS varies largely between subjects and most people who have life-threatening diLQTS have no known genetic risk factors. We investigated whether the susceptibility to develop diLQTS observed in vivo can be recapitulated in vitro using patient-specific induced pluripotent stem cell (iPSC) technology. We collected skin fibroblasts from ten subjects who developed significant diLQTS after administration of Sotalol and/or Erythromycin. Ten other individuals who displayed no changes in QT interval after administration of the same drugs, were selected. iPSC were generated by retroviral delivery of Oct4, Sox2, Nanog and Klf4 in 17 of the 20 individuals. We report preliminary results obtained from iPSC-derived cardiomyocytes (iPSC-CMs) of two subjects. All experiments were performed in a blinded fashion without knowledge of the associated clinical phenotype. Cardiac differentiation of iPSC resulted in the generation of spontaneously beating embryoid bodies. iPSC-CMs showed positive staining for TNNT2, ACTN2 and Cx43. Gene expression analysis confirmed the expression of NKX2.5, MLC2v, MYH6 and MYH7, and of the relevant KCNH2 gene. The two lines had similar basal electrophysiological properties as assessed by measurements of action potential (AP) by patch-clamp technique and extracellular field potentials (FP) using micro-electrode array (MEA). E4031, a classical HERG blocker, significantly prolonged the FP duration (FPD) in a dose-dependent manner in both lines (EC50: 30.19 and 51.57 respectively). When both Sotalol and Erythromicin were used, FPD was prolonged in one of the two samples in a dose-dependent manner (EC50Sotalol: 100; EC50Erythr: 9.64) while drug response was blunted in the other cell line. This study suggests that patient-specific iPSC can be used to model the functional abnormalities observed in acquired diLQTS.

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PI3K in Stemness Regulation: From Development to Cancer

10.20944/preprints201911.0170.v1 ◽

2019 ◽

Author(s):

Ralitsa Madsen

Keyword(s):

Stem Cell ◽

Pluripotent Stem Cells ◽

Cancer Biology ◽

Pi3k Pathway ◽

Dependent Manner ◽

Pten Loss ◽

Pi3k Activation ◽

Developmental Signalling ◽

Dose Dependent

The PI3K/AKT pathway is a key target in oncology where most efforts are focussed on phenotypes such as cell proliferation and survival. Comparatively little attention has been paid to PI3K in stemness regulation, despite the emerging link between acquisition of stem cell-like features and therapeutic failure in cancer. The aim of this review is to summarise current known and unknowns of PI3K-dependent stemness regulation, by integrating knowledge from the fields of developmental, signalling and cancer biology. Particular attention is given to the role of the PI3K pathway in pluripotent stem cells (PSCs) and the emerging parallels to dedifferentiated cancer cells with stem cell-like features. Compelling evidence suggests that PI3K/AKT signalling forms part of a ‘core molecular stemness programme’ in both mouse and human PSCs. In cancer, the oncogenic PIK3CAH1047R variant causes constitutive activation of the PI3K pathway and has recently been linked to increased stemness in a dose-dependent manner, similar to observations in mouse PSCs with heterozygous versus homozygous Pten loss. There is also evidence that the stemness phenotype may become ‘locked’ and thus independent of the original PI3K activation, posing limitations for the success of PI3K monotherapy in cancer.Ongoing therapeutic developments for PI3K-associated cancers may therefore benefit from a better understanding of the pathway’s two-layered and highly context-dependent regulation of cell growth versus stemness.

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Mode of Action of the Hypertrehalosaemic Peptides from the American Cockroach

Zeitschrift für Naturforschung C ◽

10.1515/znc-1985-9-1015 ◽

1985 ◽

Vol 40 (9-10) ◽

pp. 670-676 ◽

Cited By ~ 16

Author(s):

Gerd Gäde

Keyword(s):

Cyclic Amp ◽

Adenylate Cyclase ◽

Mode Of Action ◽

Glycogen Phosphorylase ◽

Fat Body ◽

American Cockroach ◽

Dependent Manner ◽

Low Concentrations ◽

First Time ◽

Dose Dependent

Abstract Although crude extracts of cockroach (Periplaneta amencana) corpora cardiaca have been shown previously to affect the activity of adenylate cyclase and phosphorylase, we demonstrate in the present study for the first time that low concentrations (0.5 to 5 pmol) of the synthetic myoactive peptides. M I and M II, also affect these systems; these myoactive peptides are identical to the hypertrehalosaemic hormones I and II, and cause an increase in the concentration of the second messenger cyclic AMP in the fat body.In addition, both octapeptides activate fat body glycogen phosphorylase and promote breakdown of fat body glycogen. Both peptides increase the levels to haemolymph carbohydrate in a dose-dependent manner.

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The Influence of Bee Venom Melittin on the Functioning of the Immune System and the Contractile Activity of the Insect Heart—A Preliminary Study

Toxins ◽

10.3390/toxins11090494 ◽

2019 ◽

Vol 11 (9) ◽

pp. 494 ◽

Cited By ~ 5

Author(s):

Jan Lubawy ◽

Arkadiusz Urbański ◽

Lucyna Mrówczyńska ◽

Eliza Matuszewska ◽

Agata Światły-Błaszkiewicz ◽

...

Keyword(s):

Immune System ◽

Contractile Activity ◽

Model Organism ◽

Dependent Manner ◽

Physiological Studies ◽

Almost All ◽

First Time ◽

Dose Dependent ◽

Positive Effect

Melittin (MEL) is a basic polypeptide originally purified from honeybee venom. MEL exhibits a broad spectrum of biological activity. However, almost all studies on MEL activity have been carried out on vertebrate models or cell lines. Recently, due to cheap breeding and the possibility of extrapolating the results of the research to vertebrates, insects have been used for various bioassays and comparative physiological studies. For these reasons, it is valuable to examine the influence of melittin on insect physiology. Here, for the first time, we report the immunotropic and cardiotropic effects of melittin on the beetle Tenebrio molitor as a model insect. After melittin injection at 10−7 M and 10−3 M, the number of apoptotic cells in the haemolymph increased in a dose-dependent manner. The pro-apoptotic action of MEL was likely compensated by increasing the total number of haemocytes. However, the injection of MEL did not cause any changes in the percent of phagocytic haemocytes or in the phenoloxidase activity. In an in vitro bioassay with a semi-isolated Tenebrio heart, MEL induced a slight chronotropic-positive effect only at a higher concentration (10−4 M). Preliminary results indicated that melittin exerts pleiotropic effects on the functioning of the immune system and the endogenous contractile activity of the heart. Some of the induced responses in T. molitor resemble the reactions observed in vertebrate models. Therefore, the T. molitor beetle may be a convenient invertebrate model organism for comparative physiological studies and for the identification of new properties and mechanisms of action of melittin and related compounds.

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Neural stem cell conditioned medium alleviates Aβ25-35 damage to SH-SY5Y cells through the PCMT1/MST1 pathway

European Journal of Histochemistry ◽

10.4081/ejh.2020.3135 ◽

2020 ◽

Author(s):

Guoyong Jia ◽

Hongna Yang ◽

Zengyan Diao ◽

Ying Liu ◽

Congcong Sun

Keyword(s):

Stem Cell ◽

Cell Viability ◽

Conditioned Medium ◽

Neural Stem Cell ◽

Inhibitory Effect ◽

Complete Medium ◽

Dependent Manner ◽

Induced Apoptosis ◽

Dose Dependent ◽

Cell Conditioned Medium

Alzheimer’s disease (AD) is a progressive, neurodegenerative disease. Accumulating evidence suggests that protein isoaspartate methyltransferase 1 (PCMT1) is highly expressed in brain tissue (substantia nigra, blue plaque, paraventricular nucleus). In this study, we investigated the effect of neural stem cell conditioned medium alleviates Aβ25-35 damage to SH-SY5Y cells by PCMT1/MST1 pathway. Results demonstrated that Aβ25-35 significantly decreased the cell viability in time and dose dependent manner. However, Neural stem cell-complete medium (NSC-CPM) or NSC-CDM had inhibitory effect on toxicity when fibrillation of Aβ25-35 occurred in their presence and NSC-CDM had a better inhibitor result. An increase of the PCMT1 expression levels was found in Aβ25-35 + NSC-CDM group. sh-PCMT1 significantly reduced the PCMT1, the cell viability and inhibited the protective effect; induced apoptosis and increased the expression of p-MST1. Overexpression of PCMT1 group reversed the effect of Aβ25-35 inhibited the cell viability and Aβ25-35 induced the apoptosis. In conclusion, NSC-CDM corrects the damage of Aβ25-35 to cells by increasing PCMT1, reducing MST phosphorylation.

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Interferon gamma selectively inhibits very primitive CD342+CD38- and not more mature CD34+CD38+ human hematopoietic progenitor cells.

Journal of Experimental Medicine ◽

10.1084/jem.180.3.1177 ◽

1994 ◽

Vol 180 (3) ◽

pp. 1177-1182 ◽

Cited By ~ 47

Author(s):

H W Snoeck ◽

D R Van Bockstaele ◽

G Nys ◽

M Lenjou ◽

F Lardon ◽

...

Keyword(s):

Stem Cell ◽

Progenitor Cells ◽

Interferon Gamma ◽

Bone Marrow Cells ◽

Hematopoietic Progenitor Cells ◽

Hematopoietic Progenitor ◽

Ifn Gamma ◽

Hematopoietic Stem ◽

First Time ◽

Dose Dependent

To assess the effects of interferon gamma (IFN-gamma) on very primitive hematopoietic progenitor cells, CD34(2+)CD38- human bone marrow cells were isolated and cultured in a two-stage culture system, consisting of a primary liquid culture phase followed by a secondary semisolid colony assay. CD34(2+)CD38- cells needed at least the presence of interleukin 3 (IL-3) and kit ligand (KL) together with either IL-1, IL-6, or granulocyte-colony-stimulating factor (G-CSF) in the primary liquid phase in order to proliferate and differentiate into secondary colony-forming cells (CFC). Addition of IFN-gamma to the primary liquid cultures inhibited cell proliferation and generation of secondary CFC in a dose-dependent way. This was a direct effect since it was also seen in primary single cell cultures of CD34(2+)CD38- cells. The proliferation of more mature CD34+CD38+ cells, however, was not inhibited by IFN-gamma, demonstrating for the first time that IFN-gamma is a specific and direct hematopoietic stem cell inhibitor. IFN-gamma, moreover, preserves the viability of CD34(2+)CD38- cells in the absence of other cytokines. IFN-gamma could, therefore, play a role in the protection of the stem cell compartment from exhaustion in situations of hematopoietic stress and may be useful as stem cell protecting agent against chemotherapy for cancer.

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MSI1 Promotes the Expression of the GBM Stem Cell Marker CD44 by Impairing miRNA-Dependent Degradation

Cancers ◽

10.3390/cancers12123654 ◽

2020 ◽

Vol 12 (12) ◽

pp. 3654

Author(s):

Rebecca Pötschke ◽

Jacob Haase ◽

Markus Glaß ◽

Sebastian Simmermacher ◽

Claudia Misiak ◽

...

Keyword(s):

Stem Cell ◽

Stem Cell Marker ◽

Mrna Turnover ◽

Stem Cell Markers ◽

Dependent Manner ◽

Cell Marker ◽

Cell Models ◽

Comprehensive Understanding ◽

Self Renewal ◽

First Time

The stem cell marker Musashi1 (MSI1) is highly expressed during neurogenesis and in glioblastoma (GBM). MSI1 promotes self-renewal and impairs differentiation in cancer and non-malignant progenitor cells. However, a comprehensive understanding of its role in promoting GBM-driving networks remains to be deciphered. We demonstrate that MSI1 is highly expressed in GBM recurrences, an oncologist’s major defiance. For the first time, we provide evidence that MSI1 promotes the expression of stem cell markers like CD44, co-expressed with MSI1 within recurrence-promoting cells at the migrating front of primary GBM samples. With GBM cell models of pediatric and adult origin, including isolated primary tumorspheres, we show that MSI1 promotes stem cell-like characteristics. Importantly, it impairs CD44 downregulation in a 3′UTR- and miRNA-dependent manner by controlling mRNA turnover. This regulation is disturbed by the previously reported MSI1 inhibitor luteolin, providing further evidence for a therapeutic target potential of MSI1 in GBM treatment.

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Torque Teno Virus (SANBAN Isolate) ORF2 Protein Suppresses NF-κB Pathways via Interaction with IκB Kinases

Journal of Virology ◽

10.1128/jvi.01101-07 ◽

2007 ◽

Vol 81 (21) ◽

pp. 11917-11924 ◽

Cited By ~ 46

Author(s):

Hong Zheng ◽

Linbai Ye ◽

Xiaonan Fang ◽

Baozong Li ◽

Yuhua Wang ◽

...

Keyword(s):

Western Blot ◽

Nuclear Factor Κb ◽

Torque Teno Virus ◽

Dependent Manner ◽

Nuclear Factor ◽

Innate And Adaptive Immunity ◽

Reading Frame ◽

Orf2 Protein ◽

First Time ◽

Dose Dependent

ABSTRACT Since the first discovery of Torque teno virus (TTV) in 1997, many researchers focused on its epidemiology and transcriptional regulation, but the function of TTV-encoded proteins remained unknown. The function of the TTV open reading frame (ORF) in the nuclear factor κB (NF-κB) pathway has not yet been established. In this study, we found for the first time that the TTV ORF2 protein could suppress NF-κB activity in a dose-dependent manner in the canonical NF-κB pathway. By Western blot analysis, we proved that the TTV ORF2 protein did not alter the level of NF-κB expression but prevented the p50 and p65 subunits from entering the nucleus due to the inhibition of IκBα protein degradation. Further immunoprecipitation assays showed that the TTV ORF2 protein could physically interact with IKKβ as well as IKKα, but not IKKγ. Luciferase assays and Western blot experiments showed that the TTV ORF2 protein could also suppress NF-κB activity in the noncanonical NF-κB pathway and block the activation and translocation of p52. Finally, we found that the TTV ORF2 protein inhibited the transcription of NF-κB-mediated downstream genes (interleukin 6 [IL-6], IL-8, and COX-2) through down-regulation of NF-κB. Together, these data indicate that the TTV ORF2 protein suppresses the canonical and noncanonical NF-κB pathways, suggesting that the TTV ORF2 protein may be involved in regulating the innate and adaptive immunity of organisms, contributing to TTV pathogenesis, and even be related to some diseases.

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Wnt/beta-catenin signalling is dispensable for adult neural stem cell homeostasis and activation

Development ◽

10.1242/dev.199629 ◽

2021 ◽

Author(s):

Sophie H. L. Austin ◽

Rut Gabarró-Solanas ◽

Piero Rigo ◽

Oana Paun ◽

Lachlan Harris ◽

...

Keyword(s):

Stem Cell ◽

In Vitro Models ◽

Beta Catenin ◽

Dependent Manner ◽

Direct Role ◽

Adult Nscs ◽

Hippocampal Networks ◽

Dose Dependent

Adult mouse hippocampal neural stem cells (NSCs) generate new neurons that integrate into existing hippocampal networks and modulate mood and memory. These NSCs are largely quiescent and are stimulated by niche signals to activate and produce neurons. Wnt/β-catenin signalling acts at different steps along the hippocampal neurogenic lineage, but whether it has a direct role in the regulation of NSCs remains unclear. Here we used Wnt/β-catenin reporters and transcriptomic data from in vivo and in vitro models to show that adult NSCs respond to Wnt/β-catenin signalling. Wnt/β-catenin stimulation instructed neuronal differentiation of NSCs in an active state and promoted the activation or differentiation of quiescent NSCs in a dose-dependent manner. However, we found that deletion of β-catenin in NSCs did not affect their activation or maintenance of their stem cell characteristics. Together, our results indicate that whilst NSCs do respond to Wnt/β-catenin stimulation in a dose-dependent and state-specific manner, Wnt/β-catenin signalling is not cell-autonomously required to maintain NSC homeostasis, which reconciles some of the contradictions in the literature as to the role of Wnt/β-catenin signalling in adult hippocampal NSCs.

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Wnt/beta-catenin signalling is dispensable for adult neural stem cell homeostasis and activation

10.1101/2020.12.29.424694 ◽

2020 ◽

Author(s):

Sophie H. L. Austin ◽

Lachlan Harris ◽

Oana Paun ◽

Piero Rigo ◽

François Guillemot ◽

...

Keyword(s):

Stem Cell ◽

Beta Catenin ◽

Dependent Manner ◽

Direct Role ◽

Adult Nscs ◽

Hippocampal Networks ◽

Dose Dependent

AbstractAdult mouse hippocampal neural stem cells (NSCs) generate new neurons that integrate into existing hippocampal networks and modulate mood and memory. These NSCs are largely quiescent and are stimulated by niche signals to activate and produce neurons. Wnt/β-catenin signalling acts at different steps along the hippocampal neurogenic lineage and has been shown to promote the proliferation of intermediate progenitor cells. However, whether it has a direct role in the regulation of NSCs still remains unclear. Here we used Wnt/β-catenin reporters and transcriptomic data from in vivo and in vitro models to show that both active and quiescent adult NSCs respond to Wnt/β-catenin signalling. Wnt/β-catenin stimulation instructed neuronal differentiation of active NSCs and promoted the activation or differentiation of quiescent NSCs in a dose-dependent manner. However, we found that inhibiting NSCs response to Wnt, by conditionally deleting β-catenin, did not affect their activation or maintenance of their stem cell characteristics. Together, our results indicate that whilst NSCs do respond to Wnt/β-catenin stimulation in a dose-dependent and state-specific manner, Wnt/β-catenin signalling is not cell-autonomously required to maintain NSC homeostasis, which could reconcile some of the contradictions in the literature as to the role of Wnt/β-catenin signalling in adult hippocampal NSCs.

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