scholarly journals Activation of group VI phospholipase A2 isoforms in cardiac endothelial cells

2011 ◽  
Vol 300 (4) ◽  
pp. C872-C879 ◽  
Author(s):  
Janhavi Sharma ◽  
John Turk ◽  
David J. Mancuso ◽  
Harold F. Sims ◽  
Richard W. Gross ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Arachidonic Acid ◽  
Platelet Activating Factor ◽  
Selective Inhibition ◽  
Wild Type ◽  
Human Coronary Artery ◽  
Group Vi ◽  
Bromoenol Lactone ◽  
Protease Activated Receptor 1 ◽  
Stimulation Of

The endothelium comprises a cellular barrier between the circulation and tissues. We have previously shown that activation of protease-activated receptor 1 (PAR-1) and PAR-2 on the surface of human coronary artery endothelial cells by tryptase or thrombin increases group VIA phospholipase A2 (iPLA2β) activity and results in production of multiple phospholipid-derived inflammatory metabolites. We isolated cardiac endothelial cells from hearts of iPLA2β-knockout (iPLA2β-KO) and wild-type (WT) mice and measured arachidonic acid (AA), prostaglandin I2 (PGI2), and platelet-activating factor (PAF) production in response to PAR stimulation. Thrombin (0.1 IU/ml) or tryptase (20 ng/ml) stimulation of WT endothelial cells rapidly increased AA and PGI2 release and increased PAF production. Selective inhibition of iPLA2β with ( S)-bromoenol lactone (5 μM, 10 min) completely inhibited thrombin- and tryptase-stimulated responses. Thrombin or tryptase stimulation of iPLA2β-KO endothelial cells did not result in significant PAF production and inhibited AA and PGI2 release. Stimulation of cardiac endothelial cells from group VIB (iPLA2γ)-KO mice increased PAF production to levels similar to those of WT cells but significantly attenuated PGI2 release. These results indicate that cardiac endothelial cell PAF production is dependent on iPLA2β activation and that both iPLA2β and iPLA2γ may be involved in PGI2 release.

Calcium-independent phospholipase A2-catalyzed plasmalogen hydrolysis in hypoxic human coronary artery endothelial cells

2007 ◽  
Vol 292 (1) ◽  
pp. C251-C258 ◽  
Author(s):  
Maureen C. Meyer ◽  
Jane McHowat
Keyword(s):  
Endothelial Cells ◽  
Coronary Artery ◽  
Biologically Active ◽  
Membrane Phospholipid ◽  
Human Coronary Artery ◽  
Ischemic Event ◽  
Phospholipid Hydrolysis ◽  
Bromoenol Lactone ◽  
Calcium Independent Phospholipase A2 ◽  
Stimulation Of

Thrombin stimulation of human coronary artery endothelial cells (HCAEC) results in activation of a membrane-associated, calcium-independent phospholipase A2 (iPLA2) that selectively hydrolyzes membrane plasmalogen phospholipids. Rupture of an atherosclerotic plaque and occlusion of the coronary vasculature results in a coronary ischemic event in which HCAEC in the ischemic area would be exposed to dramatic decreases in oxygen tension in addition to thrombin exposure. We exposed HCAEC to hypoxia in the presence or absence of thrombin stimulation and measured iPLA2 activation, membrane phospholipid hydrolysis, and the accumulation of biologically active phospholipid metabolites. HCAEC exposed to hypoxia, thrombin stimulation, or a combination of the two conditions demonstrated an increase in iPLA2 activity and an increase in arachidonic acid release from plasmenylcholine. Thrombin stimulation of normoxic HCAEC did not result in an accumulation of choline lysophospholipids, but hypoxia alone and in combination with thrombin stimulation led to a significant accumulation of lysoplasmenylcholine (LPlsCho). We propose that the presence of hypoxia inhibits LPlsCho catabolism, at least in part, as a result of the accumulation of long-chain acylcarnitines. The combination of increased production and decreased catabolism of LPlsCho is necessary for its accumulation. Pretreatment with bromoenol lactone to inhibit iPLA2 blocked membrane phospholipid hydrolysis and production of membrane phospholipid-derived metabolites. The increase in iPLA2 activity and the subsequent accumulation of membrane phospholipid-derived metabolites in HCAEC exposed to hypoxia or thrombin stimulation alone, and particularly in combination, have important implications in inflammation and arrhythmogenesis in atherosclerosis/thrombosis and subsequent myocardial ischemia.

Phospholipid metabolite production in human urothelial cells after protease-activated receptor cleavage

2002 ◽  
Vol 283 (5) ◽  
pp. F944-F951 ◽  
Author(s):  
Alice Rickard ◽  
Jane McHowat
Keyword(s):  
Arachidonic Acid ◽  
Platelet Activating Factor ◽  
Urothelial Cells ◽  
Protease Activated Receptors ◽  
Phospholipid Hydrolysis ◽  
Bromoenol Lactone ◽  
Normal Human ◽  
Urothelial Carcinoma Cell ◽  
Mediate Inflammation ◽  
Stimulation Of

Our laboratory demonstrated previously that stimulation of protease-activated receptors (PARs) on the human urothelial carcinoma cell line RT4 results in activation of a calcium-independent phospholipase A2 (iPLA2), leading to arachidonic acid and PGE2 release. In this study, we have examined PAR activation in normal human urothelial cells (HUR) leading to the production of inflammatory or cytoprotective phospholipid metabolites. The presence of both PAR-1 and PAR-2 on HUR was confirmed by immunoblotting. Stimulation of PAR-1 with thrombin or PAR-2 by tryptase leads to activation of a membrane-associated iPLA2 and the production of platelet-activating factor, arachidonic acid, and PGE2. These responses were all blocked by pretreatment with the iPLA2-selective inhibitor bromoenol lactone. Thus stimulation of PAR-1 or PAR-2 on HUR leads to iPLA2-catalyzed phospholipid hydrolysis, resulting in the production of metabolites that may mediate inflammation or provide cytoprotection to the bladder.

scholarly journals Mice with Genetic Deletion of Group VIA Phospholipase A2β Exhibit Impaired Macrophage Function and Increased Parasite Load in Trypanosoma cruzi-Induced Myocarditis

2016 ◽  
Vol 84 (4) ◽  
pp. 1137-1142 ◽  
Author(s):  
Janhavi Sharma ◽  
Jennifer R. Blase ◽  
Daniel F. Hoft ◽  
John O. Marentette ◽  
John Turk ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Trypanosoma Cruzi ◽  
Chagas Disease ◽  
Platelet Activating Factor ◽  
Parasite Load ◽  
Inflammatory Cells ◽  
Myocardial Inflammation ◽  
Wild Type ◽  
Human Coronary Artery ◽  
Content Type

Trypanosoma cruziinfection, which is the etiological agent of Chagas disease, is associated with intense inflammation during the acute and chronic phases. The pathological progression of Chagas disease is influenced by the infiltration and transmigration of inflammatory cells across the endothelium to infected tissues, which are carefully regulated processes involving several molecular mediators, including adhesion molecules and platelet-activating factor (PAF). We have shown that PAF production is dependent upon calcium-independent group VIA phospholipase A2β (iPLA2β) following infection of human coronary artery endothelial cells (HCAECs) withT. cruzi, suggesting that the absence of iPLA2β may decrease the recruitment of inflammatory cells to the heart to manage parasite accumulation. Cardiac endothelial cells isolated from iPLA2β-knockout (iPLA2β-KO) mice infected withT. cruzidemonstrated decreased PAF production compared to that by cells isolated from wild-type (WT) mice but demonstrated increases in adhesion molecule expression similar to those seen in WT mice. Myocardial inflammation in iPLA2β-KO mice infected withT. cruziwas similar in severity to that in WT mice, but the iPLA2β-KO mouse myocardium contained more parasite pseudocysts. Upon activation, macrophages from iPLA2β-KO mice produced significantly less nitric oxide (NO) and caused lessT. cruziinhibition than macrophages from wild-type mice. Thus, the absence of iPLA2β activity does not influence myocardial inflammation, but iPLA2β is essential forT. cruziclearance.

scholarly journals Accumulation of tissue factor in endothelial cells promotes cellular apoptosis through over-activation of Src1 and involves β1-integrin signalling

APOPTOSIS ◽  
2019 ◽  
Vol 25 (1-2) ◽  
pp. 29-41 ◽  
Author(s):  
Ali M. Ethaeb ◽  
Mohammad A. Mohammad ◽  
Yahya Madkhali ◽  
Sophie Featherby ◽  
Anthony Maraveyas ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Tissue Factor ◽  
Kinase Activity ◽  
Β1 Integrin ◽  
Wild Type ◽  
Human Coronary Artery ◽  
Src Inhibitor ◽  
Cellular Apoptosis ◽  
Measured Activation ◽  
First Time

Abstract Accumulation of tissue factor (TF) within cells leads to cellular apoptosis mediated through p38 and p53 pathways. In this study, the involvement of Src1 in the induction of TF-mediated cell apoptosis, and the mechanisms of Src1 activation were investigated. Human coronary artery endothelial cell (HCAEC) were transfected with plasmids to express the wild-type TF (TFWt-tGFP), or a mutant (Ser253 → Ala) which is incapable of being released from cells (TFAla253-tGFP). The cells were then activated with PAR2-agonist peptide (SLIGKV-NH) and the phosphorylation of Src and Rac, and also the kinase activity of Src were assessed. Transfected cells were also pre-incubated with pp60c Src inhibitor, FAK inhibitor-14, or a blocking anti-β1-integrin antibody prior to activation and the phosphorylation of p38 as well as cellular apoptosis was examined. Finally, cells were co-transfected with the plasmids, together with a Src1-specific siRNA, activated as above and the cellular apoptosis measured. Activation of PAR2 lead to the phosphorylation of Src1 and Rac1 proteins at 60 min regardless of TF expression. Moreover, Src phosphorylation and kinase activity was prolonged up to 100 min in the presence of TF, with a significantly higher magnitude when the non-releasable TFAla253-tGFP was expressed in HCAEC. Inhibition of Src with pp60c, or suppression of Src1 expression in cells, reduced p38 phosphorylation and prevented cellular apoptosis. In contrast, inhibition of FAK had no significant influence on Src kinase activity or cellular apoptosis. Finally, pre-incubation of cells with an inhibitory anti-β1-integrin antibody reduced both Src1 activation and cellular apoptosis. Our data show for the first time that the over-activation of Src1 is a mediator of TF-induced cellular apoptosis in endothelial cells through a mechanism that is dependent on its interaction with β1-integrin.

Selective plasmalogen substrate utilization by thrombin-stimulated Ca2+-independent PLA2in cardiomyocytes

2000 ◽  
Vol 278 (6) ◽  
pp. H1933-H1940 ◽  
Author(s):  
Jane McHowat ◽  
Michael H. Creer
Keyword(s):  
Arachidonic Acid ◽  
Ventricular Myocytes ◽  
Platelet Activating Factor ◽  
Substrate Utilization ◽  
Phospholipase A ◽  
Concomitant Change ◽  
Choline Phospholipid ◽  
Rabbit Ventricular Myocytes ◽  
Stimulation Of

Thrombin stimulation of rabbit ventricular myocytes activates a membrane-associated, Ca2+-independent phospholipase A2(PLA2) capable of hydrolyzing plasmenylcholine (choline plasmalogen), plasmanylcholine (alkylacyl choline phospholipid), and phosphatidylcholine substrates. To identify the endogenous phospholipid substrates, we quantified the effects of thrombin stimulation on diradyl phospholipid mass and arachidonic acid and lysophospholipid production. Thrombin stimulation resulted in a selective decrease in arachidonylated plasmenylcholine, with no change in arachidonylated phosphatidylcholine. The decrease in arachidonylated plasmenylcholine was accompanied by an increase in plasmenylcholine species containing linoleic and linolenic acids at the sn-2 position. A decrease in arachidonylated plasmenylethanolamine was also observed after thrombin stimulation, with no concomitant change in arachidonylated phosphatidylethanolamine. Thrombin stimulation resulted in the selective production of lysoplasmenylcholine, with no increase in lysophosphatidylcholine content. There was no evidence for significant acetylation of lysophospholipids to form platelet-activating factor. Arachidonic acid released after thrombin stimulation was rapidly oxidized to prostacyclin. Thus thrombin-stimulated Ca2+-independent PLA2selectively hydrolyzes arachidonylated plasmalogen substrates, resulting in production of lysoplasmalogens and prostacyclin as the principal bioactive products.

Histamine stimulation of prostaglandin and HETE synthesis in human endothelial cells

1988 ◽  
Vol 255 (2) ◽  
pp. C214-C225 ◽  
Author(s):  
G. E. Revtyak ◽  
M. J. Hughes ◽  
A. R. Johnson ◽  
W. B. Campbell
Keyword(s):  
Endothelial Cells ◽  
Arachidonic Acid ◽  
Umbilical Artery ◽  
Epoxyeicosatrienoic Acids ◽  
Human Endothelial Cells ◽  
Intact Cells ◽  
Histamine Stimulation ◽  
Human Umbilical Artery ◽  
A Minor ◽  
Stimulation Of

Endothelial cells (EC) cultured from human umbilical artery (UA) and vein (UV) metabolized [14C]arachidonic acid to prostaglandins (PGs), monohydroxyeicosatetraenoic acids (HETEs), and epoxyeicosatrienoic acids (EETs). Major radioactive products were identified as 6-keto-PGF1 alpha, PGE2, PGF2 alpha, 12-hydroxy heptadecatrienoic acid, 15-HETE, and 11-HETE. In addition, extracts from UV ECs contained 12-HETE, 5-HETE, 14,15-EET, and 5,6-EET as minor products, whereas extracts from UA ECs contained only 12-HETE as a minor product. UA ECs also produced metabolites comigrating with 14,15-EET, 11,12-EET, 8,9-EET, and 5,6-EET. Histamine increased the release of [14C]PGs and [14C]HETEs from [14C]arachidonic acid-labeled ECs. Indomethacin, aspirin, and nordihydroguauretic acid completely inhibited synthesis of both [14C]PGs and [14C]HETEs from exogenous [14C]arachidonic acid in these cells. Microsomes metabolized [14C]arachidonic acid to the same [14C]PGs and [14C]HETEs as intact cells. Pretreatment of microsomes with indomethacin completely inhibited formation of these products. These data indicate that UA ECs and UV ECs metabolize endogenous and exogenous arachidonic acid to both PGs and HETEs. Also 15-HETE and 11-HETE appear to be synthesized by a microsomal enzyme with the properties of cyclooxygenase.

scholarly journals Factor VIIa Induces Biased Cytoprotective Signaling through the Cleavage of Protease Activated Receptor 1 at Canonical Arg41 Site

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 481-481
Author(s):  
Vijay Kumar Reddy Kondreddy ◽  
Usha R. Pendurthi ◽  
Xiao Xu ◽  
John H. Griffin ◽  
L. Vijaya Mohan Rao
Keyword(s):  
Endothelial Cells ◽  
Protein C ◽  
Factor Viia ◽  
Brain Endothelial Cells ◽  
Wild Type ◽  
Barrier Disruption ◽  
Low Concentrations ◽  
Biased Signaling ◽  
Protease Activated Receptor 1

Endothelial cell protein C receptor (EPCR) interacts with diverse ligands, in addition to its known ligands protein C and activated protein C (APC). We reported earlier that procoagulant clotting factor VIIa (FVIIa) binds EPCR with the same affinity as APC. FVIIa binding to EPCR leads to the down regulation of the EPCR-mediated anticoagulation pathway. Our recent studies showed that FVIIa, like APC, induces EPCR-dependent cytoprotective signaling through activation of protease activated receptor 1 (PAR1). Recent studies of Griffin, Mosnier and their colleagues revealed that APC noncanonical cleavage of PAR1 at Arg46 site that generates a novel tethered ligand is responsible for APC-induced β-arrestin2-dependent PAR1 biased signaling. It is unknown at present whether FVIIa follows a similar mechanism as APC in inducing PAR1 biased signaling. PAR1 reporter constructs - wild-type and cleavage site-specific mutants - were routinely used to investigate PAR1 cleavage by thrombin, APC, or other proteases, and to determine protease-specific cleavage sites in PAR1. Unfortunately, this approach was not useful in determining any FVIIa cleavage site in PAR1. In contrast to thrombin or APC, FVIIa treatment failed to show a detectable cleavage (over the background) of transfected wild-type PAR1 reporter constructs expressed in cultured endothelial cells. However, in other studies, FVIIa was shown to cleave endogenous PAR1 in endothelial cells as assessed by the loss of cleavage-specific PAR1 mAb binding. The recent generation of transgenic mice strains carrying R41Q or R46Q homozygous point mutations in PAR1 has allowed us in the present study to investigate in vivo mechanisms for PAR1-dependent cytoprotective signaling of FVIIa. We employed two murine injury models, LPS-induced inflammation and VEGF-induced barrier disruption. Murine brain endothelial cells isolated from the PAR1 mutated strains and primary human endothelial cells were used to validate in vivo findings and extend the mechanistic studies. Our studies show that administration of rFVIIa (250 µg/kg body weight) reduced LPS-induced cytokine elaboration and neutrophil infiltration in the lung tissues of wild-type (WT) PAR1 and QQ46-PAR1 mice but not in QQ41-PAR1 mice. Similarly, FVIIa suppression of the VEGF-induced barrier disruption was abolished in the QQ41-PAR1 mice but not in WT and the QQ46-PAR1 mice. Parallel experiments conducted with APC showed, as expected, that it protected WT and QQ41-PAR1 mice but not QQ46-PAR1 mice against LPS-induced inflammation and VEGF-mediated barrier destabilization. In vitro signaling studies performed with brain endothelial cells isolated from WT, QQ41-PAR1 and QQ46-PAR1 mice showed that FVIIa activation of Akt in endothelial cells required Arg41 in PAR1. Additional studies showed that FVIIa-cleaved endogenous PAR1 was readily internalized, whereas APC-cleaved PAR1 remained on the cell surface. Very low concentrations of thrombin (< 1 nM) mimicked FVIIa in inducing PAR1-dependent cytoprotective signaling. However, very low concentration thrombin-induced cytoprotective signaling differed from EPCR-FVIIa-induced cytoprotective signaling in the isoform of ß-arrestin required for the protective effect. EPCR-FVIIa-induced PAR1-mediated cytoprotective signaling was soley mediated via the β-arrestin1-dependent pathway whereas very low dose thrombin-induced cytoprotective effects appear to be mediated by either β-arrestin1 or β-arrestin2. Silencing of ß-arrestin1 or ß-arrestin2 alone did not affect the protective effects of very low doses of thrombin, but the silencing both ß-arrestin1 and ß-arrestin2 together completely prevented a low dose of thrombin-induced protective effect. In summary, our data strongly support the hypothesis that the in vivo mechanism of action for FVIIa's pharmacologic benefits in curbing inflammation and endothelial barrier disruption involves biased signaling of PAR1 due to cleavage at Arg41. Moreover, our studies demonstrate that very low concentrations of thrombin are also capable of inducing PAR1 biased beneficial -cytoprotective signaling by coupling to either ß-arrestin1 or 2. These results emphasize the striking diversity of PAR1's conformational states and interactomes that initiate canonical and biased signaling. Disclosures No relevant conflicts of interest to declare.

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