Abstract 1440: Endothelial And Cardiomyocyte Toxicity Of Endogenously Produced Nucleoside: 4-pyridone-3-carboxamide-1-β-d-riboside

Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
Ewa M Slominska ◽  
Czeslawa Orlewska ◽  
Cesare M Terracciano ◽  
Ada H Yuen ◽  
Urszula Siedlecka ◽  
...  
Keyword(s):  
Renal Failure ◽  
Endothelial Cells ◽  
Chronic Renal Failure ◽  
Cell Line ◽  
Cell Function ◽  
Cell Types ◽  
Human Erythrocytes ◽  
Rat Hearts ◽  
Prolongation Of Action Potential ◽  
Ribonucleoside Triphosphate

Our recent studies identified hitherto unknown, naturally occurring nucleotide: 4-pyridone-3-carboxamide-1-β-D-ribonucleoside triphosphate (4PYTP). This nucleotide was found to accumulate in the erythrocytes of patients with chronic renal failure, a condition that precipitates heart failure and atherosclerosis. We found also increase in plasma concentration of nucleoside precursor of 4PYTP: 4-pyridone-3-carboxamide-1-β-D-ribonucleoside (4PYR) in patients with renal failure. This study was aimed to evaluate metabolism and potential toxic effect of this nucleoside in cardiomyocytes and endothelial cells. We used human erythrocytes isolated from peripheral blood, human endothelial cell line HMEC-1 cell line and cardiomyocytes isolated from rat hearts. Cells were incubated with chemically synthesized 4PYR. Metabolic assessments were performed with HPLC or liquid chromatography/mass spectrometry. We demonstrated formation of 4PYTP in human erythrocytes during incubation with 4PYR. We noted however, preferential accumulation of monophosphate of 4PYR (4PYMP) over 4PYTP. Concentration of 4PYMP increased in the erythrocytes from below 5 μM to 76.9±7.1, 254.7±13.9 and 674.3±34.3 μM after 6h incubation with 0.1, 0.3 and 1 mM 4PYR. 4PYMP progressively accumulated in the cultured endothelial cells during incubation with 4PYR up to 72 h. Cardiomyocytes were also shown to accumulate 4PYMP. In all cell types formation of 4PYR nucleotides was accompanied by decrease in cellular ATP concentration. Furthermore, treatment with 4PYR and formation of its nucleotides in endothelial cells reduced NO synthase pathway while in cardiac myocytes resulted in prolongation of action potential. We conclude that endogenously formed 4PYR is effectively metabolized to nucleotide derivatives in the erythrocytes, cardiomyocytes and endothelium. Depletion of ATP in these cells accompanying 4PYR metabolism and deleterious effects on cell function suggests that accumulation of 4PYR and its enhanced metabolism to nucleotides in endothelium and cardiomyocyted may contribute to cardiovascular pathology observed in chronic renal failure.

scholarly journals A Novel Nucleotide Found in Human Erythrocytes, 4-Pyridone-3-carboxamide-1-β-d-ribonucleoside Triphosphate

2006 ◽  
Vol 281 (43) ◽  
pp. 32057-32064 ◽  
Author(s):  
Ewa M. Slominska ◽  
Elizabeth A. Carrey ◽  
Henryk Foks ◽  
Czeslawa Orlewska ◽  
Ewa Wieczerzak ◽  
...  
Keyword(s):  
Renal Failure ◽  
Chronic Renal Failure ◽  
Plasma Concentration ◽  
Healthy Subjects ◽  
Human Erythrocytes ◽  
Adenosine Kinase ◽  
Atp Depletion ◽  
Mass Detection ◽  
Atp Concentration ◽  
Ribonucleoside Triphosphate

We report the identification of a hitherto unknown nucleotide that is present in micromolar concentrations in the erythrocytes of healthy subjects and accumulates at levels comparable with the ATP concentration in erythrocytes of patients with chronic renal failure. The unknown nucleotide was isolated and identified by liquid chromatography with UV and tandem mass detection, 1H nuclear magnetic resonance and infrared spectroscopy as 4-pyridone-3-carboxamide-1-β-d-ribonucleoside triphosphate (4PYTP), a structure indicating association with metabolism of the oxidized nicotinamide compounds. Subsequently, we demonstrated formation of 4PYTP in intact human erythrocytes during incubation with the chemically synthesized nucleoside precursor 4-pyridone-3-carboxamide-1-β-d-ribonucleoside (4PYR). We noted preferential accumulation of monophosphate of 4PYR (4PYMP) over 4PYTP as well as a decrease in erythrocyte ATP concentration during incubation with 4PYR. Both the 4PYR phosphorylation and ATP depletion were blocked by an inhibitor of adenosine kinase. Plasma concentration of 4PYR was detectable but very low (0.013 ± 0.006 μm) in contrast with the high daily urine excretion of this compound (26.7 ± 18.2 μmol/24 h) in healthy subjects, indicating much greater renal clearance than other nicotinamide metabolites, nucleosides, or creatinine. We also noted a 40-fold increase in 4PYR plasma concentration in patients with chronic renal failure (0.563 ± 0.321 μm). We suggest that 4PYTP formation in the erythrocytes is a hitherto unknown process aimed at sequestering potentially toxic 4PYR in a form that could be safely transported and subsequently released and excreted during passage of erythrocytes through the kidney.

Identification of human thrombin-activatable fibrinolysis inhibitor in vascular and inflammatory cells

2011 ◽  
Vol 105 (06) ◽  
pp. 999-1009 ◽  
Author(s):  
Joellen Lin ◽  
Mathieu Garand ◽  
Branislava Zagorac ◽  
Steven Schadinger ◽  
Corey Scipione ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Cell Line ◽  
Cell Lines ◽  
Umbilical Vein ◽  
Inflammatory Cells ◽  
Cell Types ◽  
Rt Pcr ◽  
Thrombin Activatable Fibrinolysis Inhibitor ◽  
Gene Encoding ◽  
Fibrinolysis Inhibitor

SummaryTAFI (thrombin-activatable fibrinolysis inhibitor) is a carboxypeptidase zymogen originally identified in plasma. The TAFI pathway helps to regulate the balance between the coagulation and fibrinolytic cascades. Activated TAFI (TAFIa) can also inactivate certain pro-inflammatory mediators, suggesting that the TAFI pathway may also regulate communication between coagulation and inflammation. Expression in the liver is considered to be the source of plasma TAFI. TAFI has also been identified in platelets and CPB2 (the gene encoding TAFI) mRNA has been detected in megakaryocytic cell lines as well as in endothelial cells. We have undertaken a quantitative analysis of CPB2 mRNA and TAFI protein in extrahepatic cell types relevant to vascular disease. Using RT-PCR and quantitative RT-PCR, we detected CPB2 mRNA in the human megakaryoblastic cell lines MEG-01 and Dami, the human monocytoid cell line THP-1 as well as THP-1 cells differentiated into a macrophage-like phenotype, and in primary human umbilical vein and coronary artery endothelial cells. CPB2 mRNA abundance in MEG-01, Dami, and THP-1 cells was modulated by the state of differentiation of these cells. Using a recently developed TAFIa assay, we detected TAFI protein in the lysates of the human hepatocellular carcinoma cell line HepG2 as well as in MEG-01 and Dami cells and in the conditioned medium of HepG2 cells, differentiated Dami cells, and THP-1 macrophages. We have obtained clear evidence for extrahepatic expression of TAFI, which has clear implications for the physiological and pathophysiological functions of the TAFI pathway.

Glucose and Insulin Modulate the Capacity of Endothelial Cells (HUVEC) to Express P-selectin and Bind a Monocytic Cell Line (U937)

2001 ◽  
Vol 86 (08) ◽  
pp. 680-685 ◽  
Author(s):  
Kamal Chettab ◽  
Jacques Duhault ◽  
Elisabeth Koenig-Berard ◽  
John McGregor ◽  
Marta Puente Navazo
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Cell Line ◽  
High Glucose ◽  
Cell Function ◽  
Leukocyte Adhesion ◽  
Human Umbilical Cord ◽  
Monocytic Cell ◽  
Monocytic Cell Line ◽  
Endothelial Cell Function

SummaryDiabetes mellitus is associated with increased prevalence of endothelial cell dysfunction and vascular diseases. Mechanisms leading to alterations in endothelial cell function are poorly understood. We report here that hyperglycaemia results in the expression of endothelial adhesion molecules involved in leukocyte adhesion and extravasation. Incubation of human umbilical cord endothelial cells (HUVEC) with 25 mM glucose induced the expression of P-selectin. This effect was reversed by the addition of 1 nM insulin. Moreover, increased ICAM-1 expression was observed upon HUVEC incubation with 25 mM glucose. Increased adhesion of U937 cells (a monocytic cell line) to endothelial cells cultured with 25 mM glucose was observed. High glucose-induced monocytes cell adhesion was inhibited by an anti-P-selectin monoclonal antibody (LYP20). These results show that high glucose concentration activates endothelial cells leading to monocytes adhesion providing further evidence that hyperglycaemia might be implicated in vessel wall lesions contributing to diabetic vascular disease.Present address: Dr. M. D. Puente Navazo, Centre Pluridisciplinaire d’Oncologie, ISREC, Epalinges, Switzerland

scholarly journals Differences between studying an islet β cell and studying whole pancreatic islets: immunological implications

2016 ◽  
Vol 1 (2) ◽  
Keyword(s):  
Endothelial Cells ◽  
Cell Function ◽  
Cell Types ◽  
Main Role ◽  
Β Cells ◽  
Β Cell ◽  
Exciting Field ◽  
Patients With Diabetes ◽  
Β Cell Function ◽  
Paracrine Interaction

The main role of the pancreatic islet β cell is to release the appropriate amount of insulin upon glucose stimulation. For this reason, islet transplantation has been advancing in the past few years as a therapeutic alternative for patients with diabetes, alongside the exciting field of manipulating β cell differentiation for the sake of β cell transplantation. However, do isolated β cells function the same as β cells within an intact islet? Within islets, β cells are surrounded by other cell types, including endocrine cells, endothelial cells and immune cells, a proximity which appears to be relevant for proper glucose homeostasis. Although insulin and glucose are the main regulators in this scenario, other factors, such as angiogenesis, local anti-inflammatory components and the activity profile of resident macrophages, have a profound effect on the function and fate of β cells. A paracrine interaction between β cells and α cell holds a dramatic effect on β cell function, which is additionally dependent on blood flow through the islet. Another important intercellular communication exists between β cells and endothelial cells, in this case a bidirectional interface. Moreover, β cell survival and proliferation is dependent on the potency of ECM proteins. Further parameters distinguish functionally between the isolated β cell and the intact islet, including the deposition of Zinc by β cells, synchronicity by electrical and calcium routes, the physical innervation of islets and more. In this review, we explore major parameters that relate to differences between the function of the isolated β cell and that of the β cell within an intact islet. These and some yet to be investigated aspects of β cell function should be included in the list of considerations when examining therapeutic targets for β cell–related pathologies and for the prospect of effective β cell replacement therapy

Influence of microvascular endothelial cells on transcriptional regulation of proximal tubular epithelial cells

2008 ◽  
Vol 294 (2) ◽  
pp. C543-C554 ◽  
Author(s):  
Sonia Aydin ◽  
Sara Signorelli ◽  
Thomas Lechleitner ◽  
Michael Joannidis ◽  
Clara Pleban ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Endothelial Cells ◽  
Epithelial Cell ◽  
Cell Line ◽  
Cell Function ◽  
Microvascular Endothelial Cells ◽  
Tubular Epithelial Cells ◽  
Proximal Tubular Epithelial Cells ◽  
Proximal Tubular ◽  
Microvascular Endothelial

In the renal cortex the peritubular capillary network and the proximal tubular epithelium cooperate in solute and water reabsorption, secretion, and inflammation. However, the mechanisms by which these two cell types coordinate such diverse functions remain to be characterized. Here we investigated the influence of microvascular endothelial cells on proximal tubule cells, using a filter-based, noncontact, close-proximity coculture of the human microvascular endothelial cell line HMEC-1 and the human proximal tubular epithelial cell line HK-2. With the use of DNA microarrays the transcriptomes of HK-2 cells cultured in mono- and coculture were compared. HK-2 cells in coculture exhibited a differential expression of 99 genes involved in pathways such as extracellular matrix (e.g., lysyl oxidase), cell-cell communication (e.g., IL-6 and IL-1β), and transport (e.g., GLUT3 and lipocalin 2). HK-2 cells also exhibited an enhanced paracellular gating function in coculture, which was dependent on HMEC-1-derived extracellular matrix. We identified a number of HMEC-1-enriched genes that are potential regulators of epithelial cell function such as extracellular matrix proteins (e.g., collagen I, III, IV, and V, laminin-α IV) and cytokines/growth factors (e.g., hepatocyte growth factor, endothelin-1, VEGF-C). This study demonstrates a complex network of communication between microvascular endothelial cells and proximal tubular epithelial cells that ultimately affects proximal tubular cell function. This coculture model and the data described will be important in the further elucidation of microvascular endothelial and proximal tubular epithelial cross talk mechanisms.

scholarly journals iTIME.219: An Immortalized KSHV Infected Endothelial Cell Line Inducible by a KSHV-Specific Stimulus to Transition From Latency to Lytic Replication and Infectious Virus Release

2021 ◽  
Vol 11 ◽  
Author(s):  
Stephen J. Dollery ◽  
Tania D. Maldonado ◽  
Eric A. Brenner ◽  
Edward A. Berger
Keyword(s):  
Endothelial Cells ◽  
Infected Cell ◽  
Cell Line ◽  
Kaposi's Sarcoma ◽  
Infectious Virus ◽  
Kaposi’S Sarcoma ◽  
Cell Types ◽  
Lytic Replication ◽  
Transcription Activator ◽  
Specific Stimulus

Kaposi’s sarcoma-associated herpesvirus (KSHV/HHV-8) is the causative agent of Kaposi’s sarcoma and two B cell lymphoproliferative disorders: primary effusion lymphoma and KSHV-associated multicentric Castleman’s disease. These distinct pathologies involve different infected cell types. In Kaposi’s sarcoma, the virus is harbored in spindle-like tumor cells of endothelial origin, in contrast with the two pathologies of B cells. These distinctions highlight the importance of elucidating potential differences in the mechanisms of infection for these alternate target cell types and in the properties of virus generated from each. To date there is no available chronically KSHV-infected cell line of endothelial phenotype that can be activated by the viral lytic switch protein to transition from latency to lytic replication and production of infectious virus. To advance these efforts, we engineered a novel KSHV chronically infected derivative of TIME (telomerase immortalized endothelial) cells harboring a previously reported recombinant virus (rKSHV.219) and the viral replication and transcription activator (RTA) gene under the control of a doxycycline-inducible system. The resulting cells (designated iTIME.219) maintained latent virus as indicated by expression of constitutively expressed (eGFP) but not a lytic phase (RFP) reporter gene and can be sustained under long term selection. When exposed to either sodium butyrate or doxycycline, the cells were activated to lytic replication as evidenced by the expression of RFP and KSHV lytic genes and release of large quantities of infectious virus. The identity of the iTIME.219 cells was confirmed both phenotypically (specific antigen expression) and genetically (short tandem repeat analysis), and cell stability was maintained following repeated serial passage. These results suggest the potential utility of the iTime.219 cells in future studies of the KSHV replication in endothelial cells, properties of virus generated from this biologically relevant cell type and mechanisms underlying KSHV tropism and pathogenesis.

scholarly journals Increased Erythrocyte Phosphatidylserine Exposure in Chronic Renal Failure

1999 ◽  
Vol 10 (9) ◽  
pp. 1982-1990
Author(s):  
MARIO BONOMINI ◽  
VITTORIO SIROLLI ◽  
NICOLA SETTEFRATI ◽  
SECONDO DOTTORI ◽  
LORENZO DI LIBERATO ◽  
...  
Keyword(s):  
Renal Failure ◽  
Chronic Renal Failure ◽  
Cell Function ◽  
Red Cell ◽  
Creatinine Concentration ◽  
Red Cell Membrane ◽  
Phospholipid Asymmetry ◽  
Outer Leaflet ◽  
Phosphatidylserine Exposure

Abstract. The appearance of phosphatidylserine, an aminophospholipid normally confined to the inner monolayer, at the outer leaflet of red cell membrane may have several pathophysiologic implications. This study examines erythrocyte phosphatidylserine exposure in chronic renal failure (CRF) patients on conservative treatment or on dialysis, to assess possible alterations to phospholipid asymmetry in a condition associated with a state of deranged red cell function. A significant increase in phosphatidylserine-expressing erythrocytes was found in undialyzed patients with CRF (2.32%) and patients on hemodialysis (3.06%) and on peritoneal dialysis (2.14%) compared with control subjects (0.68%). In undialyzed CRF patients, a strong correlation (r = 0.903) was found between the percentage of phosphatidylserine-expressing red cells and the serum creatinine concentration. The increased exposure of phosphatidylserine in uremic erythrocytes may be due to inhibition of phosphatidylserine transport from the outer to the inner leaflet of plasma membrane and may promote an increased erythrophagocytosis. In reconstitution experiments, normal erythrocytes showed an increase in phosphatidylserine-expressing cells when incubated in uremic plasma (3.2% after 2 h versus 1.1% at beginning of incubation), whereas phosphatidylserine-positive uremic erythrocytes decreased when resuspended in normal plasma (2.03% after 2 h and 1.65% after 8 h versus 2.9% at beginning of incubation). Preliminary characterization of the putative uremic compound(s) indicates a molecular weight between 10,000 and 20,000, as well as heat instability. These findings show an impairment of erythrocyte membrane phospholipid asymmetry in CRF patients, regardless of the dialysis treatment. Such abnormality seems related to the uremic state and could contribute to the red cell pathology present in CRF.

scholarly journals Endothelial cells downregulate apolipoprotein D expression in mural cells through paracrine secretion and Notch signaling

2011 ◽  
Vol 301 (3) ◽  
pp. H784-H793 ◽  
Author(s):  
Mohanasundari Pajaniappan ◽  
Nancy K. Glober ◽  
Simone Kennard ◽  
Hua Liu ◽  
Ning Zhao ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Notch Signaling ◽  
Cell Function ◽  
Cell Types ◽  
Fiber Formation ◽  
Cell Contact ◽  
Mural Cell ◽  
Mural Cells ◽  
Paracrine Secretion ◽  
Apolipoprotein D

Endothelial and mural cell interactions are vitally important for proper formation and function of blood vessels. These two cell types communicate to regulate multiple aspects of vessel function. In studying genes regulated by this interaction, we identified apolipoprotein D (APOD) as one gene that is downregulated in mural cells by coculture with endothelial cells. APOD is a secreted glycoprotein that has been implicated in governing stress response, lipid metabolism, and aging. Moreover, APOD is known to regulate smooth muscle cells and is found in abundance within atherosclerotic lesions. Our data show that the regulation of APOD in mural cells is bimodal. Paracrine secretion by endothelial cells causes partial downregulation of APOD expression. Additionally, cell contact-dependent Notch signaling plays a role. NOTCH3 on mural cells promotes the downregulation of APOD, possibly through interaction with the JAGGED-1 ligand on endothelial cells. Our results show that NOTCH3 contributes to the downregulation of APOD and by itself is sufficient to attenuate APOD transcript expression. In examining the consequence of decreased APOD expression in mural cells, we show that APOD negatively regulates cell adhesion. APOD attenuates adhesion by reducing focal contacts; however, it has no effect on stress fiber formation. These data reveal a novel mechanism in which endothelial cells control neighboring mural cells through the downregulation of APOD, which, in turn, influences mural cell function by modulating adhesion.

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