scholarly journals P1238EVALUATION OF AN IN VITRO CO-CULTURE MODEL FOR TESTING EFFECTS OF CYTOPROTECTIVE ADDITIVES IN PERITONEAL DIALYSIS FLUIDS ON CARDIOVASCULAR OUTCOME

2020 ◽  
Vol 35 (Supplement_3) ◽  
Author(s):  
Juan Manuel Sacnun ◽  
Rebecca Herzog ◽  
Maria Bartosova ◽  
Claus Schmitt ◽  
Klaus Kratochwill
Keyword(s):  
Endothelial Cells ◽  
Cell Death ◽  
Peritoneal Dialysis ◽  
Cardiovascular Risk ◽  
Cell Damage ◽  
Peritoneal Membrane ◽  
Cytoskeleton Reorganization ◽  
Harmful Effects

Abstract Background and Aims The composition of all currently available peritoneal dialysis (PD) fluids triggers morphological and functional changes in the peritoneal membrane. Periodic exposure leads to vasculopathy, hypervascularization, and diabetes-like damage of vessels, eventually leading to failure of the technique. Patients undergoing dialysis generally, have a high risk of cardiovascular events. It is currently unclear if there is a mechanistic link between peritoneal membrane failure and cardiovascular risk. In vitro and in vivo studies have shown that cytoprotective additives (e.g. dipeptide alanyl-glutamine (AlaGln) or kinase inhibitor lithium chloride (LiCl)) to PDF reduce peritoneal damage. Here, we developed an experimental model for investigating effects of these cytoprotective additives in PDF in the cardiovascular context. Method For modelling the peritoneal membrane in vitro, mesothelial and endothelial cells were co-cultured in transwell plates. Mesothelial cells were grown in the upper compartment and primary human umbilical vein endothelial cells (HUVEc) or primary microvascular cells were grown in the lower compartment. PDF with or without cytoprotective compounds, was added to the upper compartment to only expose mesothelial cells directly to different dilutions of the fluid. Effects on cell damage was assessed by quantification of lactate-dehydrogenase (LDH) release and live-dead staining of cells. Proteome profiles were analysed for both cell-types separately and in combination using two-dimensional difference gel electrophoresis (2D-DiGE) and liquid chromatography coupled to mass spectrometry (LC-MS). In vitro findings were related to PD-induced arteriolar changes based on abundance profiles of micro-dissected omental arterioles of children treated with conventional PD-fluids and age-matched controls with normal renal function. Results Marked cellular injury of HUVEc after PD-fluid exposure was associated with a molecular landscape of the enriched biological process clusters ‘glucose catabolic process’, ‘cell redox homeostasis’, ‘RNA metabolic process’, ‘protein folding’, ‘regulation of cell death’, and ‘actin cytoskeleton reorganization’ that characterize PD-fluid cytotoxicity and counteracting cellular repair process respectively. PDF-induced cell damage was reduced by AlaGln and LiCl both in mesothelial and endothelial cells. Proteome analysis revealed perturbation of major cellular processes including regulation of cell death and cytoskeleton reorganization. Selected markers of angiogenesis, oxidative stress, cell junctions and transdifferentiation were counter-regulated by the additives. Co-cultured cells yielded differently regulated pathways following PDF exposure compared to separate culture. Comparison to human arterioles confirmed overlapping protein regulation between endothelial cells in vitro and in vivo, proving harmful effects of PD-fluids on endothelial cells leading to drastic changes of the cellular process landscape. Conclusion In summary, this study shows harmful effects of PD-fluids also effecting endothelial cells and elucidates potential mechanisms by which cytoprotective additives may counteract the signalling axis between local peritoneal damage and systemic vasculopathy. An in vitro co-culture system may be an attractive approach to simulate the peritoneal membrane for testing direct and indirect effects of cytoprotective additives in PDF. When cultured and stressed in close proximity cells may respond differently. Characterisation of PD-induced perturbations will allow identifying molecular mechanisms linking the peritoneal and cardiovascular context, offering therapeutic targets to reduce current limitations of PD and ultimately decreasing cardiovascular risk of dialysis patients.

MO684A CO-CULTURE MODEL FOR TESTING EFFECTS OF CYTOPROTECTIVE ADDITIVES IN PD FLUIDS ON THE SECRETOME OF MESOTHELIAL AND ENDOTHELIAL CELLS

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
Juan Manuel Sacnun ◽  
Rebecca Herzog ◽  
Klaus Kratochwill
Keyword(s):  
Endothelial Cells ◽  
Cardiovascular Risk ◽  
Culture System ◽  
Molecular Mechanisms ◽  
Cell Types ◽  
Cell Junctions ◽  
Peptide Ligand ◽  
Peritoneal Membrane

Abstract Background and Aims The composition of all currently available peritoneal dialysis fluids (PDF) triggers morphological and functional changes in the peritoneal membrane. Periodic exposure leads to vasculopathy, hypervascularization, and diabetes-like damage of vessels of the peritoneal membrane, eventually leading to technique failure. Patients undergoing dialysis generally, have a high risk of cardiovascular events. It is currently unclear if there is a mechanistic link between peritoneal membrane failure and cardiovascular risk. In vitro and in vivo studies have shown that cytoprotective additives (e.g. dipeptide alanyl-glutamine (AlaGln) or kinase inhibitor lithium chloride (LiCl)) to PDF reduce peritoneal damage. Here, we developed an experimental model for investigating effects of such additives on secretome-mediated signalling between cell-types of the peritoneal membrane which are relevant in the cardiovascular context. Method For modelling the peritoneal membrane in vitro, mesothelial (MC) and endothelial cells (EC) were co-cultured in transwell plates. MC were grown in the upper compartment and primary microvascular cells were grown in the lower compartment. MC were exposed to PDF with or without cytoprotective compounds (8 mM AlaGln in glucose-based PDF 3.86% or 10 mM LiCl in icodextrin-based PDF), while EC below were kept in medium. Cell damage was assessed by quantification of lactate-dehydrogenase (LDH) release, neutral red uptake and cell morphology. Proteome and secretome profiles were analysed for both cell-types in co-culture or separately with an isobaric-tag labelling approach with a multiplexed liquid chromatography/mass spectrometry (LC-MS) approach. Prior to analysis of the secretome a bead-based equalizer approach based on a combinatorial peptide ligand library (CPLL) was performed to enrich low abundant proteins. Results EC injury after PD-fluid exposure of MC was decreased with the addition of AlaGln or LiCl, showing a link between the individual cell outcomes. Proteome analysis revealed perturbation of major cellular processes including regulation of cell death and cytoskeleton re-organization, which characterize PDF cytotoxicity. Selected markers of angiogenesis, oxidative stress, cell junctions and transdifferentiation were counter-regulated by the additives. Co-cultured cells yielded differently regulated pathways following PDF exposure compared to separate culture. We were able to identify and quantify 334 secreted proteins in the co-culture system. The secretome analysis showed variation in several clinically relevant proteins and important extracellular processes such as extracellular matrix reorganization, vesicle transport or collagen deposition. Comparison to previously published abundance profiles of omental arteriolar proteins from paediatric PD patient and age-matched controls confirmed overlapping protein regulation between endothelial cells in vitro and in vivo. Conclusion This study shows that harmful effects of PDF-stressed MC also affect EC and elucidates potential mechanisms by which cytoprotective additives may counteract the signalling axis between local peritoneal damage and systemic vasculopathy. An in vitro co-culture system may be an attractive approach to simulate the close proximity of different cell types in the peritoneal membrane for testing direct and indirect effects of cytoprotective additives. Characterisation of PD-induced perturbations may allow identifying molecular mechanisms linking the peritoneal and cardiovascular context, offering therapeutic targets to reduce current limitations of PD and ultimately decreasing cardiovascular risk of dialysis patients.

scholarly journals Regulation of the Mitochondrial BKCa Channel by the Citrus Flavonoid Naringenin as a Potential Means of Preventing Cell Damage

Molecules ◽  
2020 ◽  
Vol 25 (13) ◽  
pp. 3010 ◽  
Author(s):  
Anna Kicinska ◽  
Rafał P. Kampa ◽  
Jan Daniluk ◽  
Aleksandra Sek ◽  
Wieslawa Jarmuszkiewicz ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Cell Damage ◽  
Herbal Medicines ◽  
Pharmacological Action ◽  
Bkca Channel ◽  
Inner Mitochondrial Membrane ◽  
Beneficial Effects ◽  
Factor Α

Naringenin, a flavanone obtained from citrus fruits and present in many traditional Chinese herbal medicines, has been shown to have various beneficial effects on cells both in vitro and in vivo. Although the antioxidant activity of naringenin has long been believed to be crucial for its effects on cells, mitochondrial pathways (including mitochondrial ion channels) are emerging as potential targets for the specific pharmacological action of naringenin in cardioprotective strategies. In the present study, we describe interactions between the mitochondrial large-conductance calcium-regulated potassium channel (mitoBKCa channel) and naringenin. Using the patch-clamp method, we showed that 10 µM naringenin activated the mitoBKCa channel present in endothelial cells. In the presence of 30 µM Ca2+, the increase in the mitoBKCa channel probability of opening from approximately 0.25 to 0.50 at −40 mV was observed. In addition, regulation of the mitoBKCa channel by naringenin was dependent on the concentration of calcium ions. To confirm our data, physiological studies on the mitochondria were performed. An increase in oxygen consumption and a decrease in membrane potential was observed after naringenin treatment. In addition, contributions of the mitoBKCa channel to apoptosis and necrosis were investigated. Naringenin protected cells against damage induced by tumor necrosis factor α (TNF-α) in combination with cycloheximide. In this study, we demonstrated that the flavonoid naringenin can activate the mitoBKCa channel present in the inner mitochondrial membrane of endothelial cells. Our studies describing the regulation of the mitoBKCa channel by this natural, plant-derived substance may help to elucidate flavonoid-induced cytoprotective mechanisms.

Preclinical photodynamic therapy research in Spain 3: Localization of photosensitizers and mechanisms of cell deathin vitro

2009 ◽  
Vol 13 (04n05) ◽  
pp. 544-551 ◽  
Author(s):  
Magdalena Cañete ◽  
Juan C. Stockert ◽  
Angeles Villanueva
Keyword(s):  
Cell Death ◽  
Photodynamic Therapy ◽  
Cell Cultures ◽  
Cellular Localization ◽  
Cell Damage ◽  
Therapeutic Modality ◽  
Action Mechanisms ◽  
Cell Death Mechanisms

Photodynamic therapy (PDT) is a subject of increasing biomedical research and represents a very promising therapeutic modality for palliative or even curative treatment of some superficial or endoscopically accessible tumors. In addition to the first photosensitizers (PSs) applied (hematoporphyrin-based drugs), second generation PSs with improved photophysical and photobiological properties are now studied using cell cultures, experimental tumors and clinical trials. On the other hand, there is a growing interest in the analysis of cell death mechanisms by apoptosis, which is especially relevant in oncology, because many anticancer drugs work, at least in part, by triggering apoptosis in neoplastic cells both in vitro and in vivo. The evaluation of cell death mechanisms is an important parameter to determine the efficacy and the potential toxicity of a treatment, allowing better adjustment of protocol. Using cell cultures, our research team has studied the mechanisms of cell damage and death implicated in the photodynamic processes, as well as the relationship between the cellular localization of the PS and the organelle damage during photosensitization. The results obtained in our laboratory provide a deeper understanding on the action mechanisms that lead to cell inactivation by PDT, and also allow selection of PSs with higher potential for clinical application than those currently in use.

scholarly journals Mouse Hippocampal Organotypic Tissue Cultures Exposed to In Vitro “Ischemia” Show Selective and Delayed CA1 Damage that is Aggravated by Glucose

2003 ◽  
Vol 23 (1) ◽  
pp. 23-33 ◽  
Author(s):  
Anna Rytter ◽  
Tobias Cronberg ◽  
Fredrik Asztély ◽  
Sailasree Nemali ◽  
Tadeusz Wieloch
Keyword(s):  
Cell Death ◽  
Cerebrospinal Fluid ◽  
Cell Damage ◽  
Extracellular Fluid ◽  
Oxygen Deprivation ◽  
Tissue Cultures ◽  
Ion Composition ◽  
In Vitro Ischemia

Oxygen and glucose deprivation (OGD) in cell cultures is generally studied in a medium, such as artificial cerebrospinal fluid (CSF), with an ion composition similar to that of the extracellular fluid of the normal brain (2 to 4 mmol/L K+, 2 to 3 mmol/L Ca2+; pH 7.4). Because the distribution of ions across cell membranes dramatically shifts during ischemia, the authors exposed mouse organotypic hippocampal tissue cultures to OGD in a medium, an ischemic cerebrospinal fluid, with an ion composition similar to the extracellular fluid of the brain during ischemia in vivo (70 mmol/L K+, 0.3 mmol/L Ca2+; pH 6.8). In ischemic CSF, OGD induced a selective and delayed cell death in the CA1 region, as assessed by propidium iodide uptake. Cell death was glutamate receptor dependent since blockade of the N-methyl-D-aspartate and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors mitigated cell damage. Hyperglycemia aggravates ischemic brain damage in vivo, whereas in vitro glucose in artificial CSF prevents oxygen deprivation-induced damage. The authors demonstrate that glucose in ischemic CSF significantly exacerbates cell damage after oxygen deprivation. This new model of in vitro “ischemia” can be useful in future studies of the mechanisms and treatment of ischemic cell death, including studies using genetically modified mice.

Inositol pyrophosphates modulate hydrogen peroxide signalling

2009 ◽  
Vol 423 (1) ◽  
pp. 109-118 ◽  
Author(s):  
Sara Maria Nancy Onnebo ◽  
Adolfo Saiardi
Keyword(s):  
Dna Damage ◽  
Cell Death ◽  
Actin Polymerization ◽  
Cell Damage ◽  
Inhibitory Effect ◽  
Inositol Pyrophosphate ◽  
Direct Modification ◽  
Inositol Pyrophosphates

Inositol pyrophosphates are involved in a variety of cellular functions, but the specific pathways and/or downstream targets remain poorly characterized. In the present study we use Saccharomyces cerevisiae mutants to examine the potential roles of inositol pyrophosphates in responding to cell damage caused by ROS (reactive oxygen species). Yeast lacking kcs1 [the S. cerevisiae IP6K (inositol hexakisphosphate kinase)] have greatly reduced IP7 (diphosphoinositol pentakisphosphate) and IP8 (bisdiphosphoinositol tetrakisphosphate) levels, and display increased resistance to cell death caused by H2O2, consistent with a sustained activation of DNA repair mechanisms controlled by the Rad53 pathway. Other Rad53-controlled functions, such as actin polymerization, appear unaffected by inositol pyrophosphates. Yeast lacking vip1 [the S. cerevisiae PP-IP5K (also known as IP7K, IP7 kinase)] accumulate large amounts of the inositol pyrophosphate IP7, but have no detectable IP8, indicating that this enzyme represents the physiological IP7 kinase. Similar to kcs1Δ yeast, vip1Δ cells showed an increased resistance to cell death caused by H2O2, indicating that it is probably the double-pyrophosphorylated form of IP8 [(PP)2-IP4] which mediates the H2O2 response. However, these inositol pyrophosphates are not involved in directly sensing DNA damage, as kcs1Δ cells are more responsive to DNA damage caused by phleomycin. We observe in vivo a rapid decrease in cellular inositol pyrophosphate levels following exposure to H2O2, and an inhibitory effect of H2O2 on the enzymatic activity of Kcs1 in vitro. Furthermore, parallel cysteine mutagenesis studies performed on mammalian IP6K1 are suggestive that the ROS signal might be transduced by the direct modification of this evolutionarily conserved class of enzymes.

Mechanism of Phosphatidylcholine Action during Peritoneal Dialysis

1987 ◽  
Vol 7 (1) ◽  
pp. 6-9 ◽  
Author(s):  
Andrzej Breborowicz ◽  
Kostas Sombolos ◽  
Helen Rodela ◽  
Raymond Ogilvie ◽  
Joanne Bargman ◽  
...  
Keyword(s):  
Peritoneal Dialysis ◽  
Absorption Rate ◽  
Glucose Absorption ◽  
In Vivo Studies ◽  
Peritoneal Membrane ◽  
Anionic Sites ◽  
Fluid Layers ◽  
Positively Charged

We studied the effect of phosphatidyl- choline (PDC) (50 mg/L) on the peritoneal ultrafiltration and permeability in vivo and in vitro. Our in vivo studies with normal rabbits confirmed previous observations of increased ultrafiltration mainly by decreasing the reabsorption phase. We observed no effect on glucose absorption rate. In in vitro studies, using isolated section of rabbit's mesentery, phosphatidylcholine increased the permeability of the mesothelium to water, urea and glucose from the vascular to the mesothelial side but not in the opposite direction. Following exposure of the peritoneal membrane to Alcian blue, a positively charged dye, phosphatidylcholine had no effect on mesothelial permeability. Our observations suggest that necessary for the action of phosphatidylcholine is its attachment to the anionic sites of the mesothelium. We speculate that improvement in UF is achieved by diminishing the thickness of the stagnant fluid layers trapped between the microvilli.

Schistosoma mansoni: differential cell death associated with in vitro culture and treatment with Astiban (Roche)

Parasitology ◽  
1977 ◽  
Vol 75 (1) ◽  
pp. 101-109 ◽  
Author(s):  
J. R. Shaw ◽  
D. A. Erasmus
Keyword(s):  
Cell Death ◽  
Schistosoma Mansoni ◽  
Cell Damage ◽  
Ultrastructural Level ◽  
Egg Laying ◽  
Biochemical Status ◽  
Differential Cell ◽  
Vitelline Cells

A simple technique for the maintenance in vitro of mature Schistosoma mansoni is described and critically assessed at the ultrastructural level. Females were cultured for 4–6 days with no apparent ultrastructural change, but after this period changes appeared in the cells of the ovary and vitelline gland. At a later stage (10–12 days) lipid bodies appeared in the parenchyma cells. These changes occurred in worms which were active, paired with males and were egg–laying. Thus the activity, pairing behaviour and egg–laying characteristics are not adequate to reveal the true morphological condition and presumably the physiological and biochemical status of cultured worms.This technique was used to study the effect of Astiban on females and the results were compared with worms treated in vivo. Astiban concentrations greater than 30 µg/ml killed worms within 7–20 h and acted non–selectively. Astiban at low concentrations (10µg/ml) during short–term culture (1–3 h) resulted in a selective action of the drug on maturing vitelline cells. Thus, although the degree of cell damage caused by drug treatment was more severe and occurred earlier than the effects observed in worms cultured in vitro without drugs, both treatments resulted in differential cell death.

scholarly journals Cell Ferroptosis: New Mechanism and New Hope for Retinitis Pigmentosa

Cells ◽  
2021 ◽  
Vol 10 (8) ◽  
pp. 2153
Author(s):  
Ming Yang ◽  
Kwok-Fai So ◽  
Wai-Ching Lam ◽  
Amy Cheuk Yin Lo
Keyword(s):  
Cell Death ◽  
Retinitis Pigmentosa ◽  
Photoreceptor Cell ◽  
Vision Loss ◽  
Peripheral Vision ◽  
Cell Damage ◽  
Research Area ◽  
Night Vision

Retinitis pigmentosa (RP) is a leading cause of inherited retinal degeneration, with more than 60 gene mutations. Despite the genetic heterogenicity, photoreceptor cell damage remains the hallmark of RP pathology. As a result, RP patients usually suffer from reduced night vision, loss of peripheral vision, decreased visual acuity, and impaired color perception. Although photoreceptor cell death is the primary outcome of RP, the underlying mechanisms are not completely elucidated. Ferroptosis is a novel programmed cell death, with characteristic iron overload and lipid peroxidation. Recent studies, using in vitro and in vivo RP models, discovered the involvement of ferroptosis-associated cell death, suggesting a possible new mechanism for RP pathogenesis. In this review, we discuss the association between ferroptosis and photoreceptor cell damage, and its implication in the pathogenesis of RP. We propose that ferroptotic cell death not only opens up a new research area in RP, but may also serve as a novel therapeutic target for RP.

Expression of Defensin Antimicrobial Peptides in the Peritoneal Cavity of Patients on Peritoneal Dialysis

2001 ◽  
Vol 21 (5) ◽  
pp. 501-508 ◽  
Author(s):  
Krystyna H. Zarrinkalam ◽  
David I. Leavesley ◽  
Jodie M. Stanley ◽  
Gerald J. Atkins ◽  
Randall J. Faull
Keyword(s):  
Peritoneal Dialysis ◽  
Peritoneal Cavity ◽  
Messenger Rna ◽  
Mesothelial Cells ◽  
Peritoneal Membrane ◽  
Rt Pcr ◽  
Necrosis Factor Alpha ◽  
Peritoneal Mesothelial Cells

Objective To investigate the expression and regulation of defensins in the peritoneal cavity of peritoneal dialysis (PD) patients. Design The presence of defensins in the peritoneal cavity was assessed using reverse transcription polymerase chain reaction (RT-PCR). In vivo defensin expression was analyzed in human peritoneal membrane biopsies and in peritoneal cavity leukocytes isolated from spent dialysate. Defensin expression in vitro was assessed in cultured human peritoneal mesothelial cells (HPMC) and confirmed with PCR Southern blot and DNA sequencing. The effect of tumor necrosis factor alpha (TNFa) and epidermal growth factor (EGF) on b2 defensin expression in HPMC was analyzed by Northern blot analysis and RT-PCR respectively. Results Both a and b classes of defensins are expressed in the peritoneal cavity of PD patients. Messenger RNA for the a-defensin human neutrophil peptide 3 and for b-defensin-1 (hbD-1) were found in preparations containing predominantly peritoneal leukocytes, whereas b-defensin-2 (hbD-2) is expressed by HPMC. HPMC isolated from different individuals displayed variability in both basal hbD-2 expression and in response to stimulation by TNFa. Conversely, EGF consistently downregulated the level of hbD-2 message in HPMC. Conclusion a- and b-defensins are expressed in the peritoneal cavity, and hbD-2 is the main defensin present in the peritoneal membrane. Variable levels of expression of hbD-2 by mesothelial cells were seen, with evidence of regulation by cytokines and growth factors. This provides evidence for a previously unknown mechanism of innate immunity at that site.

scholarly journals Matrix Attachment Regulates FAS-Induced Apoptosis in Endothelial Cells

2001 ◽  
Vol 152 (3) ◽  
pp. 633-644 ◽  
Author(s):  
Fawzi Aoudjit ◽  
Kristiina Vuori
Keyword(s):  
Endothelial Cells ◽  
Cell Death ◽  
Molecular Mechanisms ◽  
Cell Attachment ◽  
Expression Level ◽  
Caspase 8 ◽  
Fas L ◽  
Induced Apoptosis

Survival of endothelial cells is critical for cellular processes such as angiogenesis. Cell attachment to extracellular matrix inhibits apoptosis in endothelial cells both in vitro and in vivo, but the molecular mechanisms underlying matrix-induced survival signals or detachment-induced apoptotic signals are unknown. We demonstrate here that matrix attachment is an efficient regulator of Fas-mediated apoptosis in endothelial cells. Thus, matrix attachment protects cells from Fas-induced apoptosis, whereas matrix detachment results in susceptibility to Fas-mediated cell death. Matrix attachment modulates Fas-mediated apoptosis at two different levels: by regulating the expression level of Fas, and by regulating the expression level of c-Flip, an endogenous antagonist of caspase-8. The extracellular signal–regulated kinase (Erk) cascade functions as a survival pathway in adherent cells by regulating c-Flip expression. We further show that detachment-induced cell death, or anoikis, itself results from activation of the Fas pathway by its ligand, Fas-L. Fas-L/Fas interaction, Fas–FADD complex formation, and caspase-8 activation precede the bulk of anoikis in endothelial cells, and inhibition of any of these events blocks anoikis. These studies identify matrix attachment as a survival factor against death receptor–mediated apoptosis and provide a molecular mechanism for anoikis and previously observed Fas resistance in endothelial cells.

Sign in / Sign up

Export Citation Format

Share Document