scholarly journals Zebrafish Kit ligands cooperate with erythropoietin to promote erythroid cell expansion

2020 ◽  
Author(s):  
Jana Oltova ◽  
Ondrej Svoboda ◽  
Olga Machonova ◽  
Petra Svatonova ◽  
David Traver ◽  
...  
Keyword(s):  
Suspension Culture ◽  
Cell Expansion ◽  
Ex Vivo ◽  
Culture Conditions ◽  
Erythroid Cell ◽  
List Type ◽  
Loss Of Function ◽  
Erythroid Progenitors

AbstractKit ligand (Kitlg) is pleiotropic cytokine with a prominent role in vertebrate erythropoiesis. Although the role of Kitlg in this process has not yet been reported in Danio rerio (zebrafish), in the present study, we show that its function is evolutionary conserved. Zebrafish possess two copies of Kitlg genes (Kitlga and Kitlgb) due to whole genome duplication. To determine the role of each ligand in zebrafish, we performed a series of ex vivo and in vivo gain- and loss-of-function experiments. First, we tested the biological activity of recombinant Kitlg proteins in suspension culture from zebrafish whole kidney marrow and we demonstrate that Kitlga is necessary for expansion of erythroid progenitors ex vivo. To further address the role of kitlga and kitlgb in hematopoietic development in vivo, we performed gain-of-function experiments in zebrafish embryos, showing that both ligands cooperate with erythropoietin (Epo) to promote erythroid cell expansion. Finally, using the kita mutant (kitab5/b5 or sparse), we show that Kita receptor is crucial for Kitlga/b cooperation with Epo in erythroid cells. In summary, using optimized suspension culture conditions with recombinant cytokines (Epo, Kitlga), we are reporting for the first time ex vivo suspension cultures of zebrafish hematopoietic progenitor cells, which can serve as an indispensable tool to study normal and aberrant hematopoiesis in zebrafish. Furthermore, we conclude that although partial functional diversification of Kit ligands has been described in other processes, in erythroid development, both paralogs play a similar role and their function is evolutionary conserved.Key points:Kit signaling contributes to erythroid cell development and is conserved from fish to manEx vivo expansion and self-renewal of zebrafish erythroid progenitors requires addition of recombinant Kitlga

scholarly journals Zebrafish Kit ligands cooperate with erythropoietin to promote erythroid cell expansion

2020 ◽  
Vol 4 (23) ◽  
pp. 5915-5924
Author(s):  
Jana Oltova ◽  
Ondrej Svoboda ◽  
Olga Machonova ◽  
Petra Svatonova ◽  
David Traver ◽  
...  
Keyword(s):  
Suspension Culture ◽  
Cell Expansion ◽  
Ex Vivo ◽  
Culture Conditions ◽  
Erythroid Cell ◽  
Loss Of Function ◽  
Erythroid Progenitors ◽  
Evolutionarily Conserved

Abstract Kit ligand (Kitlg) is pleiotropic cytokine with a prominent role in vertebrate erythropoiesis. Although the role of Kitlg in this process has not been reported in Danio rerio (zebrafish), in the present study we show that its function is evolutionarily conserved. Zebrafish possess 2 copies of Kitlg genes (Kitlga and Kitlgb) as a result of whole-genome duplication. To determine the role of each ligand in zebrafish, we performed a series of ex vivo and in vivo gain- and loss-of-function experiments. First, we tested the biological activity of recombinant Kitlg proteins in suspension culture from zebrafish whole-kidney marrow, and we demonstrate that Kitlga is necessary for expansion of erythroid progenitors ex vivo. To further address the role of kitlga and kitlgb in hematopoietic development in vivo, we performed gain-of-function experiments in zebrafish embryos, showing that both ligands cooperate with erythropoietin (Epo) to promote erythroid cell expansion. Finally, using the kita mutant (kitab5/b5 or sparse), we show that the Kita receptor is crucial for Kitlga/b cooperation with Epo in erythroid cells. In summary, using optimized suspension culture conditions with recombinant cytokines (Epo, Kitlga), we report, for the first time, ex vivo suspension cultures of zebrafish hematopoietic progenitor cells that can serve as an indispensable tool to study normal and aberrant hematopoiesis in zebrafish. Furthermore, we conclude that, although partial functional diversification of Kit ligands has been described in other processes, in erythroid development, both paralogs play a similar role, and their function is evolutionarily conserved.

scholarly journals Exposure to Mites Sensitizes Intestinal Stem Cell Maintenance, Splenic Marginal Zone B Cell Homeostasis, And Heart Development to Notch Dosage and Cooperativity

2020 ◽  
Author(s):  
Francis M. Kobia ◽  
Kristina Preusse ◽  
Quanhui Dai ◽  
Nicholas Weaver ◽  
Praneet Chaturvedi ◽  
...  
Keyword(s):  
B Cell ◽  
Heart Development ◽  
Marginal Zone ◽  
B Cell Lymphoma ◽  
Mite Infestation ◽  
List Type ◽  
Loss Of Function ◽  
B Cell Homeostasis

AbstractCooperative DNA binding is a key feature of transcriptional regulation. Here we examined the role of cooperativity in Notch signaling by CRISPR-mediated engineering of mice in which neither Notch1 nor Notch2 can homo- or heterodimerize, essential for cooperative binding to sequence paired sites (SPS) located near many Notch-regulated genes. While most known Notch-dependent phenotypes were unaffected in Notch1/2 dimer-deficient mice, a subset of tissues proved highly sensitive to loss of cooperativity. These phenotypes include heart development, compromising viability in combination with low gene dose, and the gut, developing ulcerative colitis in response to 1% DSS. The most striking phenotypes – gender imbalance and splenic marginal zone B cell lymphoma – emerged in combination with dose reduction or when challenged by chronic fur mite infestation. This study highlights the role of the environment in malignancy and colitis, and is consistent with Notch-dependent anti-parasite immune responses being compromised in the dimer deficient animals.HighlightsNotch dimerization has an in vivo role in contributing to intestinal homeostasisLoss of cooperativity can manifest as Notch gain or loss of function phenotypesMite infestation exacerbates all phenotypes, triggers MZB hyperproliferation in mutant animalsMite-infested mutant mice develop SMZL with age

The Role of Polyphenols in the Modulation of Plasma Non-Enzymatic Antioxidant Capacity (NEAC)

2012 ◽  
Vol 82 (3) ◽  
pp. 228-232 ◽  
Author(s):  
Mauro Serafini ◽  
Giuseppa Morabito
Keyword(s):  
Antioxidant Capacity ◽  
Dietary Intervention ◽  
Ex Vivo ◽  
The Body ◽  
Dietary Polyphenols ◽  
Enzymatic Antioxidant ◽  
Endogenous Antioxidant

Dietary polyphenols have been shown to scavenge free radicals, modulating cellular redox transcription factors in different in vitro and ex vivo models. Dietary intervention studies have shown that consumption of plant foods modulates plasma Non-Enzymatic Antioxidant Capacity (NEAC), a biomarker of the endogenous antioxidant network, in human subjects. However, the identification of the molecules responsible for this effect are yet to be obtained and evidences of an antioxidant in vivo action of polyphenols are conflicting. There is a clear discrepancy between polyphenols (PP) concentration in body fluids and the extent of increase of plasma NEAC. The low degree of absorption and the extensive metabolism of PP within the body have raised questions about their contribution to the endogenous antioxidant network. This work will discuss the role of polyphenols from galenic preparation, food extracts, and selected dietary sources as modulators of plasma NEAC in humans.

scholarly journals Heparan sulfate and heparin interactions with proteins

2015 ◽  
Vol 12 (110) ◽  
pp. 20150589 ◽  
Author(s):  
Maria C. Z. Meneghetti ◽  
Ashley J. Hughes ◽  
Timothy R. Rudd ◽  
Helena B. Nader ◽  
Andrew K. Powell ◽  
...  
Keyword(s):  
Heparan Sulfate ◽  
Ex Vivo ◽  
Sequence Specificity ◽  
Structure Activity ◽  
Multiple Binding Sites ◽  
Multiple Binding ◽  
Heparin Activity

Heparan sulfate (HS) polysaccharides are ubiquitous components of the cell surface and extracellular matrix of all multicellular animals, whereas heparin is present within mast cells and can be viewed as a more sulfated, tissue-specific, HS variant. HS and heparin regulate biological processes through interactions with a large repertoire of proteins. Owing to these interactions and diverse effects observed during in vitro , ex vivo and in vivo experiments, manifold biological/pharmacological activities have been attributed to them. The properties that have been thought to bestow protein binding and biological activity upon HS and heparin vary from high levels of sequence specificity to a dependence on charge. In contrast to these opposing opinions, we will argue that the evidence supports both a level of redundancy and a degree of selectivity in the structure–activity relationship. The relationship between this apparent redundancy, the multi-dentate nature of heparin and HS polysaccharide chains, their involvement in protein networks and the multiple binding sites on proteins, each possessing different properties, will also be considered. Finally, the role of cations in modulating HS/heparin activity will be reviewed and some of the implications for structure–activity relationships and regulation will be discussed.

scholarly journals Pivotal role of Acitretin nanovesicular gel for effective treatment of psoriasis: ex vivo–in vivo evaluation study

2018 ◽  
Vol Volume 13 ◽  
pp. 1059-1079 ◽  
Author(s):  
Irhan Abu Hashim ◽  
Noha Abo El-Magd ◽  
Ahmed El-Sheakh ◽  
Mohammed Hamed ◽  
Abd El-Gawad Abd El-Gawad
Keyword(s):  
Effective Treatment ◽  
Ex Vivo ◽  
Evaluation Study ◽  
Pivotal Role ◽  
In Vivo Evaluation

scholarly journals The pathophysiologic role of the protein kinase Cδ pathway in the intervertebral discs of rabbits and mice: In vitro, ex vivo, and in vivo studies

2012 ◽  
Vol 64 (6) ◽  
pp. 1950-1959 ◽  
Author(s):  
Michael B. Ellman ◽  
Jae-Sung Kim ◽  
Howard S. An ◽  
Jeffrey S. Kroin ◽  
Xin Li ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Ex Vivo ◽  
Intervertebral Discs ◽  
In Vivo Studies ◽  
Protein Kinase Cδ

Abstract 5858: Role of Oxygen Tension and Oxidative Stress in Human Saphenous Vein Remodeling

Circulation ◽  
2008 ◽  
Vol 118 (suppl_18) ◽  
Author(s):  
Binata Joddar ◽  
Rashmeet K Reen ◽  
Michael Firstenberg ◽  
Keith J Gooch
Keyword(s):  
Oxidative Stress ◽  
Oxygen Tension ◽  
Ex Vivo ◽  
Neointimal Hyperplasia ◽  
Fold Increase ◽  
List Type ◽  
Saphenous Veins ◽  
Arterial Po2 ◽  
And Oxidative Stress

Vessels cultured ex vivo maintain viability and vasoactivity for weeks and can remodel in response to mechanical cues. When cultured in the presence of 5% CO2/balance air veins develop neointimal hyperplasia (IH) while arteries do not suggesting that exposure to significant increases in pO2 levels might stimulate IH. Neointimal hyperplasia (IH) is a known mechanism by which saphenous veins have a decreased patency compared to arterial conduits when used for coronary artery bypass. We sought to explore the role of oxygen tension and oxidative stress in IH. Test the hypothesis that exposure of human saphenous veins (HSV) to arterial pO2 stimulates IH via ROS-mediated pathways. Almost 40 HSV remnants acquired following CABG were cultured ex vivo with arterial (~95mmHg) pO2 or venous (~40mmHg) pO2 for 14 days. All differences reported have a p<0.05 via Student’s t-test. Results: HSV cultured at arterial pO2 exhibited significant IH as evidenced by disruption of the IEL, invasion of cells from the media, and a 2.8-fold greater intimal area than fresh HSV, a 5.8-fold increase in cell proliferation compared to fresh HSV, increased ROS levels and oxidative stress as evidenced by 4-fold increase in 4-HNE level (a marker of oxidative stress), increased DHE staining (indicative of superoxide generation), and a progressive increase in total ROS levels with time as assessed by DCF fluorescence, and a 3-fold increase in phosphorylated p38-MAPK, which is implicated in SMC proliferation. In stark contrast vessels culture at arterial pO2, HSV cultured with venous pO2 did not develop increased IH and were indistinguishable from fresh vessels with respect to proliferation, markers of oxidative stress, and MAPK expression levels. Supplementing culture medium with antioxidants including Tiron or NAC blocked the pO2-induced changes. These data indicate that exposure to arterial pO2 increases cellular proliferation and stimulates IH, potentially via oxidative stress or ROS signaling and also suggest that exposure to elevated arterial pO2 might stimulate pathological remodeling of veins grafted into the arterial circulation. This research has received full or partial funding support from the American Heart Association, AHA Great Rivers Affiliate (Delaware, Kentucky, Ohio, Pennsylvania & West Virginia).

Abstract P283: Parkin Mediates Mitophagy in Cardioprotection

2011 ◽  
Vol 109 (suppl_1) ◽  
Author(s):  
Allen M Andres ◽  
Chengqun Huang ◽  
Eric P Ratliff ◽  
Genaro Hernandez ◽  
Pamela Lee ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Wild Type ◽  
Simulated Ischemia ◽  
Selective Targeting ◽  
Cardioprotective Effects ◽  
Mitochondrial Turnover ◽  
First Time

Autophagy-dependent mitochondrial turnover in response to cellular stress is necessary for maintaining cellular homeostasis. However, the mechanisms that govern the selective targeting of damaged mitochondria are poorly understood. Parkin, an E3 ubiquitin ligase, has been shown to be essential for the selective clearance of damaged mitochondria. Parkin is expressed in the heart, yet its function has not been investigated in the context of cardioprotection. We previously reported that autophagy is required for cardioprotection by ischemic preconditioning (IPC). In the present study, we used simulated ischemia in vitro and IPC in hearts (in vivo and ex vivo) to investigate the role of Parkin in mediating cardioprotection. In HL-1 cells, simulated ischemia induced Parkin translocation to mitochondria and mitochondrial elimination. Mitochondrial loss was blunted in Atg5-deficient cells, revealing the requirement for autophagy in mitochondrial elimination. Consistent with previous reports implicating p62/SQSTM1 in mitophagy, we found that downregulation of p62 attenuated mitophagy and exacerbated cell death in HL-1 cardiomyocytes subjected to simulated ischemia. While wild type mice showed p62 translocation to mitochondria after IPC, Parkin knockout mice exhibited attenuated translocation of p62 to mitochondria. Importantly, ablation of Parkin in mice abolished the cardioprotective effects of IPC. These results reveal for the first time the crucial role of Parkin and mitophagy in cardioprotection.

scholarly journals ROLE OF NITRIC OXIDE (NO) IN CAPSAICIN MEDIATED ANTI-PLATELET ACTIVITY IN IN VITRO, IN VIVO, EX-VIVO MODEL OF PLATELET AGGREGATION ASSAY AND ARTERIAL THROMBOSIS IN RAT: POTENTIAL THERAPEUTIC TARGET?

2018 ◽  
Vol 10 (4) ◽  
pp. 44
Author(s):  
Mihir K Patel ◽  
Kiranj K. Chaudagar ◽  
Anita A. Mehta
Keyword(s):  
Platelet Aggregation ◽  
Arterial Thrombosis ◽  
Ex Vivo ◽  
Platelet Activity ◽  
Aggregation Assay ◽  
Thrombosis Model ◽  
Platelet Aggregation Assay

Objective: Although recent advances in the treatment of congestive heart disease, mortality among patients’ remains a questionable remark. Therefore, we evaluated the role of capsaicin on in vitro and ex vivo platelet aggregation induced by Adenosine Di-Phosphate (ADP) as well as in in vivo thrombosis models and role of NO, KATP was also identified in the capsaicin-induced anti-platelet animal model as well as in vivo model of arterial thrombosis.Methods: According to body weight wistar rats were divided into five groups. Group I and Group II was treated with saline and capsaicin (3 mg/kg, i. v), while animals from Group III were treated with N(ω)-nitro-L-arginine methyl ester (L-NAME) (30 mg/kg, i. v) 30 min before administration of capsaicin (3 mg/kg, i. v). Group IV animals were treated with glibenclamide (10 mg/kg,i. v) 30 min before administration of capsaicin (3 mg/kg, i. v). Group V was considered as a positive control and administered clopidogrel (30 mg/kg, p. o). Animals were subjected for in vitro, ex-vivo platelet aggregation assay. ADP (30µM) was utilized as an aggregating agent in these experiments. After these assays; animals of each group were subjected for subaqueous tail bleeding time in a rat model and FeCl3-induced arterial thrombosis model in rats.Results: In ADP-induced in vitro platelet aggregation, a significant reduction in % platelet aggregation was observed at 50µM (64.35±4.641) and 100µM (52.72±4.192) concentration of capsaicin as compared to vehicle control (85.82±3.716). Capsaicin (3 mg/kg, i. v) also showed a significant reduction (49.53±4.075) in ex-vivo ADP-induced platelet aggregation as compared to vehicle control (89.38±2.057). In FeCl3 induced arterial thrombosis model, Capsaicin (3 mg/kg, i. v) exhibited an increase in time to occlusion in this rodent model and presence of the L-NAME and glibenclamide had inhibited the activity of capsaicin.Conclusion: In our study, capsaicin (50 µM, 100µM) exhibited potent anti-platelet activity in ADP-induced platelet aggregation, similarly capsaicin exhibited significant anti-platelet action in the ex-vivo study. Moreover, the presence of L-NAME and glibenclamide inhibited the anti-thrombotic and anti-platelet action of capsaicin. Therefore, it was concluded that NO and KATP may be involved in the anti-thrombotic action of capsaicin.

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