scholarly journals Molecular Signature of Mineralocorticoid Receptor Signaling in Cardiomyocytes: From Cultured Cells to Mouse Heart

Endocrinology ◽  
2010 ◽  
Vol 151 (9) ◽  
pp. 4467-4476 ◽  
Author(s):  
Celine Latouche ◽  
Yannis Sainte-Marie ◽  
Marja Steenman ◽  
Paulo Castro Chaves ◽  
Aniko Naray-Fejes-Toth ◽  
...  
Keyword(s):  
Signaling Pathways ◽  
Mineralocorticoid Receptor ◽  
Cultured Cells ◽  
Ex Vivo ◽  
Plasminogen Activator Inhibitor ◽  
Molecular Signature ◽  
H9c2 Cell ◽  
Mouse Heart ◽  
Plasminogen Activator Inhibitor 1

Excess mineralocorticoid signaling is deleterious for cardiovascular functions, as demonstrated by the beneficial effects of mineralocorticoid receptor (MR) antagonism on morbidity and mortality in patients with heart failure. However, the understanding of signaling pathways after MR activation in the heart remains limited. We performed transcriptomic analyses in the heart of double-transgenic mice with conditional, cardiomyocyte-specific, overexpression of the MR (MRcardio mice) or the glucocorticoid receptor (GR; GRcardio mice). Some of the genes induced in MRcardio mice were selected for comparative evaluation (real time PCR) in vivo in the heart of mice and ex vivo in the MR-expressing cardiomyocyte H9C2 cell line after aldosterone or corticosterone treatment. We demonstrate that chronic MR overexpression in the heart results in a limited number of induced (n = 24) and repressed (n = 22) genes compared with their control littermates. These genes are specifically modulated by MR because there is limited overlap (three induced, four repressed) with the genes that are regulated in the heart of GRcardio mice (compared with control mice: 70 induced, 73 repressed). Interestingly, some MR-induced genes that are up-regulated in vivo in mice are also induced by 24-h aldosterone treatment in H9C2 cells, such as plasminogen activator inhibitor 1 and Serpina-3 (α1-antichymotrypsin). The signaling pathways that are affected by long-term activation of MR may be of particular interest to design novel therapeutic targets in cardiac diseases.

scholarly journals Role of Nongenomic Signaling Pathways Activated by Aldosterone During Cardiac Reperfusion Injury

2015 ◽  
Vol 29 (8) ◽  
pp. 1144-1155 ◽  
Author(s):  
Anthony W. Ashton ◽  
Thi Y. L. Le ◽  
Celso E. Gomez-Sanchez ◽  
Marie-Christine Morel-Kopp ◽  
Brett McWhinney ◽  
...  
Keyword(s):  
Reperfusion Injury ◽  
Signaling Pathways ◽  
Transcriptional Activation ◽  
Ex Vivo ◽  
Plasminogen Activator Inhibitor ◽  
H9c2 Cells ◽  
Plasminogen Activator Inhibitor 1 ◽  
Experimental Rat Model ◽  
First Time ◽  
G Protein Coupled

Abstract Aldosterone (Aldo) activates both genomic and nongenomic signaling pathways in the cardiovascular system. Activation of genomic signaling pathways contributes to the adverse cardiac actions of Aldo during reperfusion injury; however, the extent nongenomic signaling pathways contribute has been difficult to identify due to lack of a specific ligand that activates only nongenomic signaling pathways. Using a pegylated aldosterone analog, aldosterone-3-carboxymethoxylamine-TFP ester conjugated to methoxypegylated amine (Aldo-PEG), we are able for the first time to distinguish between nongenomic and genomic cardiac actions of Aldo. We confirm Aldo-PEG activates phosphorylation of ERK1/2 in rat cardiomyocyte H9c2 cells similar to Aldo and G protein-coupled receptor 30 (GPR30 or GPER) agonist G1. GPER antagonist, G36, but not mineralocorticoid receptor (MR) antagonist spironolactone, prevented ERK1/2 phosphorylation by Aldo, Aldo-PEG, and G1. The selective nongenomic actions of Aldo-PEG are confirmed, with Aldo-PEG increasing superoxide production in H9c2 cells to similar levels as Aldo but having no effect on subcellular localization of MR. Striatin serves as a scaffold for GPER and MR, with GPER antagonist G36, but not spironolactone, restoring MR-striatin complexes. Aldo-PEG had no effect on MR-dependent transcriptional activation, whereas Aldo increased transcript levels of serum-regulated kinase 1 and plasminogen activator inhibitor-1. Using our ex vivo experimental rat model of myocardial infarction, we found aggravated infarct size and apoptosis by Aldo but not Aldo-PEG. Our studies confirm that in the heart, activation of nongenomic signaling pathways alone are not sufficient to trigger the deleterious effects of aldosterone during myocardial reperfusion injury.

Anti-thrombotic Effect of a PAI-1 Inhibitor in Rats Given Endotoxin

1996 ◽  
Vol 75 (01) ◽  
pp. 118-126 ◽  
Author(s):  
T Abrahamsson ◽  
V Nerme ◽  
M Strömqvist ◽  
B Åkerblom ◽  
A Legnehed ◽  
...  
Keyword(s):  
Plasminogen Activator Inhibitor ◽  
Rat Plasma ◽  
Clot Lysis ◽  
Fibrin Deposition ◽  
Plasminogen Activator Inhibitor 1 ◽  
Fab Fragment ◽  
Dose Dependent Decrease ◽  
Pai 1

SummaryThe aim of this study was to investigate the anti-thrombotic effects of an inhibitor of the plasminogen activator inhibitor-1 (PAI-1) in rats given endotoxin. In studies in vitro, PRAP-1, a Fab-fragment of a polyclonal antibody against human PAI-1, was shown to inhibit PAI-1 activity in rat plasma as well as to stimulate clot-lysis of the euglobulin fraction derived from rat plasma. Endotoxin administered to anaesthetised rats produced a marked increase in plasma PAI-1 activity. To study fibrin formation and lysis in vivo after intravenous (i. v.) injection of the coagulant enzyme batroxobin, 125I-fibrinogen was administered to the animals. The thrombi formed by batroxobin were rapidly lysed in control animals, while the rate of lysis was markedly attenuated in rats given endotoxin. PRAP-1 was administered i.v. (bolus + infusion) to rats given endotoxin and batroxobin and the PAI-1 inhibitor caused a dose-dependent decrease in the 125I-fibrin deposition in the lungs. An immunohistochemical technique was used to confirm this decrease in density of fibrin clots in the tissue. Furthermore, PRAP-1 decreased plasma PAI-1 activity in the rats and this reduction was correlated to the decrease in lung 125I-fibrin deposition at the corresponding time point. It is concluded that in this experimental model the PAI-1 antibody PRAP-1 may indeed inhibit thrombosis in animals exposed to endotoxin.

The Roles of α2-Antiplasmin and Plasminogen Activator Inhibitor 1 (PAI-1) in the Inhibition of Clot Lysis

1993 ◽  
Vol 70 (02) ◽  
pp. 301-306 ◽  
Author(s):  
Linda A Robbie ◽  
Nuala A Booth ◽  
Alison M Croll ◽  
Bruce Bennett
Keyword(s):  
Plasminogen Activator ◽  
Plasminogen Activator Inhibitor ◽  
Fibrin Clot ◽  
Clot Lysis ◽  
Plasminogen Activator Inhibitor 1 ◽  
Dependent Inhibition ◽  
Activator Inhibitor ◽  
Pai 1

SummaryThe relative importance of the two major inhibitors of fibrinolysis, α2-antiplasmin (α2-AP) and plasminogen activator inhibitor (PAI-1), were investigated using a simple microtitre plate system to study fibrin clot lysis in vitro. Cross-linked fibrin clots contained plasminogen and tissue plasminogen activator (t-PA) at concentrations close to physiological. Purified α2-AP and PAI-1 caused dose-dependent inhibition. All the inhibition due to normal plasma, either platelet-rich or poor, was neutralised only by antibodies to α2-AP. Isolated platelets, at a final concentration similar to that in blood, 2.5 × 108/ml, markedly inhibited clot lysis. This inhibition was neutralised only by antibodies to PAI-1. At the normal circulating ratio of plasma to platelets, α2-AP was the dominant inhibitor. When the platelet:plasma ratio was raised some 20-fold, platelet PAI-1 provided a significant contribution. High local concentrations of PAI-1 do occur in thrombi in vivo, indicating a role for PAI-1, complementary to that of α2-AP, in such situations.

scholarly journals Plasma tissue plasminogen activator and plasminogen activator inhibitor-1 in hospitalized COVID-19 patients

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Yu Zuo ◽  
Mark Warnock ◽  
Alyssa Harbaugh ◽  
Srilakshmi Yalavarthi ◽  
Kelsey Gockman ◽  
...  
Keyword(s):  
Plasminogen Activator ◽  
Ex Vivo ◽  
Plasminogen Activator Inhibitor ◽  
Tissue Type ◽  
Bleeding Complications ◽  
Clot Lysis ◽  
Thrombosis Prophylaxis ◽  
Plasminogen Activator Inhibitor 1 ◽  
Activator Inhibitor ◽  
Pai 1

AbstractPatients with coronavirus disease-19 (COVID-19) are at high risk for thrombotic arterial and venous occlusions. However, bleeding complications have also been observed in some patients. Understanding the balance between coagulation and fibrinolysis will help inform optimal approaches to thrombosis prophylaxis and potential utility of fibrinolytic-targeted therapies. 118 hospitalized COVID-19 patients and 30 healthy controls were included in the study. We measured plasma antigen levels of tissue-type plasminogen activator (tPA) and plasminogen activator inhibitor-1 (PAI-1) and performed spontaneous clot-lysis assays. We found markedly elevated tPA and PAI-1 levels in patients hospitalized with COVID-19. Both factors demonstrated strong correlations with neutrophil counts and markers of neutrophil activation. High levels of tPA and PAI-1 were associated with worse respiratory status. High levels of tPA, in particular, were strongly correlated with mortality and a significant enhancement in spontaneous ex vivo clot-lysis. While both tPA and PAI-1 are elevated among COVID-19 patients, extremely high levels of tPA enhance spontaneous fibrinolysis and are significantly associated with mortality in some patients. These data indicate that fibrinolytic homeostasis in COVID-19 is complex with a subset of patients expressing a balance of factors that may favor fibrinolysis. Further study of tPA as a biomarker is warranted.

scholarly journals Structural Insight into the Two-Step Mechanism of PAI-1 Inhibition by Small Molecule TM5484

2021 ◽  
Vol 22 (3) ◽  
pp. 1482 ◽  
Author(s):  
Machteld Sillen ◽  
Toshio Miyata ◽  
Douglas E. Vaughan ◽  
Sergei V. Strelkov ◽  
Paul J. Declerck
Keyword(s):  
Binding Site ◽  
Small Molecule ◽  
Intracellular Signaling ◽  
Plasminogen Activator Inhibitor ◽  
Plasminogen Activator Inhibitor 1 ◽  
Joint Region ◽  
Flexible Joint ◽  
Step Mechanism ◽  
Pai 1

Plasminogen activator inhibitor-1 (PAI-1), a key regulator of the fibrinolytic system, is the main physiological inhibitor of plasminogen activators. By interacting with matrix components, including vitronectin (Vn), PAI-1 plays a regulatory role in tissue remodeling, cell migration, and intracellular signaling. Emerging evidence points to a role for PAI-1 in various pathological conditions, including cardiovascular diseases, cancer, and fibrosis. Targeting PAI-1 is therefore a promising therapeutic strategy in PAI-1-related pathologies. A class of small molecule inhibitors including TM5441 and TM5484, designed to bind the cleft in the central β-sheet A of PAI-1, showed to be potent PAI-1 inhibitors in vivo. However, their binding site has not yet been confirmed. Here, we report two X-ray crystallographic structures of PAI-1 in complex with TM5484. The structures revealed a binding site at the flexible joint region, which is distinct from the presumed binding site. Based on the structural analysis and biochemical data we propose a mechanism for the observed dose-dependent two-step mechanism of PAI-1 inhibition. By binding to the flexible joint region in PAI-1, TM5484 might restrict the structural flexibility of this region, thereby inducing a substrate form of PAI-1 followed by a conversion to an inert form.

Abstract 290: Haemogenic Endocardium Contribute To Definitive Hematopoiesis During Cardiogenesis

2013 ◽  
Vol 113 (suppl_1) ◽  
Author(s):  
Haruko Nakano ◽  
Xiaoqian Liu ◽  
Armin Arshi ◽  
Ben van Handel ◽  
Rajkumar Sasidharan ◽  
...  
Keyword(s):  
De Novo ◽  
Ex Vivo ◽  
Embryonic Stem ◽  
Circulatory System ◽  
Lineage Tracing ◽  
Mouse Heart ◽  
Heart Tube ◽  
Cardiac Progenitors ◽  
Definitive Hematopoiesis

The circulatory system is the first functional organ system that develops during mammalian life. Accumulating evidences suggest that cardiac and endocardial cells can arise from a single common progenitor cell during mammalian cardiogenesis. Notably, these early cardiac progenitors express multiple hematopoietic transcription factors, consistent with previous reports. Indeed, a close relationship among cardiac, endocardial and hematopoietic lineages has been suggested in fly, zebrafish, and embryonic stem cell in vitro differentiation models. However, it is unclear when, where and how this hematopoietic gene program is in operation during in vivo mammalian cardiogenesis. Hematopoietic colony assay suggests that mouse heart explants generate myeloids and erythroids in the absence of circulation, suggesting that the heart tube is a de novo site for the definitive hematopoiesis. Lineage tracing revealed that putative cardiac-derived Nkx2-5+/Isl1+ endocardial cells give rise to CD41+ hematopoietic progenitors that contribute to definitive hematopoiesis in vivo and ex vivo during embryogenesis earlier than in the AGM region. Furthermore, Nkx2-5 and Isl1 are both required for the hemogenic activity of the endocardium. Together, identification of Nkx2-5/Isl1-dependent hemogenic endocardial cells (1) adds hematopoietic component in the cardiogenesis lineage tree, (2) changes the long-held dogma that AGM is the only major source of definitive hematopoiesis in the embryo proper, and (3) represents phylogenetically conserved fundamental mechanism of cardio-vasculo-hematopoietic differentiation pathway during the development of circulatory system.

scholarly journals Water-Pipe Smoke Exposure-Induced Circulatory Disturbances in Mice, and the Influence of Betaine Supplementation Thereon

2017 ◽  
Vol 41 (3) ◽  
pp. 1098-1112 ◽  
Author(s):  
Abderrahim Nemmar ◽  
Suhail Al-Salam ◽  
Sumaya Beegam ◽  
Priya Yuvaraju ◽  
Abderrahim Oulhaj ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Plasma Concentrations ◽  
Immunohistochemical Analysis ◽  
Plasminogen Activator Inhibitor ◽  
Water Pipe ◽  
Plasminogen Activator Inhibitor 1 ◽  
Factor Α ◽  
Air Control

Background/Aims: It has been shown, both experimentally and clinically, that water-pipe smoke (WPS) exposure adversely affects the cardiovascular system (CVS) through the generation of oxidative stress and inflammation. Betaine, a naturally occurring compound in common foods, has antioxidant and anti-inflammatory actions. However, its potential to mitigate the adverse effect of WPS on the CVS has never been reported before. This is the subject of this study in mice. Methods: Mice were exposed daily for 30 min to either normal air (control), or to WPS for two consecutive weeks. Betaine was administered daily by gavage at a dose of 10mg/kg, 1h before either air or WPS exposure. Results: Betaine mitigated the in vivo prothrombotic effect of WPS in pial arterioles and venules. Moreover, it reversed the WPS-induced decrease in circulating platelets. Likewise, betaine alleviated platelet aggregation in vitro, and the shortening of activated partial thromboplastin time and prothrombin time induced by WPS. Betaine reduced the increase of plasminogen activator inhibitor-1 and fibrinogen concentrations in plasma induced by WPS. Betaine also diminished the WPS-induced increase of plasma concentrations of interleukin 6 and tumor necrosis factor α, and attenuated the increase of lipid peroxidation and superoxide dismutase. Immunohistochemical analysis of the heart revealed an increase in the expression of inducible nitric oxide synthase and cytochrome C by cardiomyocytes of the WPS-exposed mice. These effects were averted by betaine. Conclusion: Our findings suggest that betaine treatment significantly mitigated WPS-induced hypercoagulability, and inflammation, as well as systemic and cardiac oxidative stress.

Abstract 294: Hematopoietic Cells Are Required For Proper Development Of Coronary Vasculature

2013 ◽  
Vol 113 (suppl_1) ◽  
Author(s):  
Gentian Lluri ◽  
Xiaoqian Liu ◽  
Atsushi N
Keyword(s):  
Ex Vivo ◽  
Hematopoietic Cells ◽  
Coronary Vessels ◽  
Explant Culture ◽  
Hematopoietic Cell ◽  
Mouse Heart ◽  
Blood Island ◽  
Epicardial Cells ◽  
Whole Mount

Objective: We examined whether the hematopoietic cells induce the coronary artery formation using genetically modified mouse models of hematopoietic ablation in vivo and ex vivo . Methods: As a model of for hematopoietic cell deficient animals, we used Runx1 (a transcription factor required for definitive hematopoiesis) knockout embryos and Vav1-cre; R26-DTA embryos, which ablates 2/3 of CD45+ hematopoietic cells. The coronary growth and the hematopoietic cells were evaluated in whole-mount, section and ex vivo explant culture. Results: The developing coronary endothelial cells form blood-island-like structure at around E12.5 in the subepicardial region. Interestingly, however, the histological analyses suggest that the first Ter119+ and CD45+ blood cells appear in the subendocardial area at E10.5, even before the formation of coronary channels. These initial hematopoietic cells in the heart are not likely derived from the epicardium, as the sorted epicardial cells yielded no hematopoietic cell in colony formation assay. These observations raised a question whether these heart-resident hematopoietic cells rather play an inductive role during coronary formation. To examine this possibility, we analyzed two hematopoietic ablation models. Both Runx1 knockout embryos and Vav1-cre; R26-DTA embryos revealed disorganized, hypoplastic microvasculature of coronary vessels on section and whole-mount stainings. Furthermore, coronary explant experiments showed that the mouse heart explants from Runx1 knockout embryos and Vav1-cre; R26-DTA embryos exhibited impaired coronary formation ex vivo. Conclusion: Hematopoietic cells are not merely transported via coronary vessels, but substantially involved in the induction of the coronary vessels during cardiogenesis.

scholarly journals Protease-Activated Receptors 1 and 3 are Differentially Expressed on Human Monocyte Subsets and are Upregulated by Lipopolysaccharide Ex Vivo and In Vivo

2019 ◽  
Vol 119 (09) ◽  
pp. 1394-1402 ◽  
Author(s):  
Barbara Thaler ◽  
Philipp J. Hohensinner ◽  
Johanna Baumgartner ◽  
Patrick Haider ◽  
Konstantin A. Krychtiuk ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Critical Role ◽  
Human Monocyte ◽  
In Vivo Model ◽  
Monocyte Subsets ◽  
Plasminogen Activator Inhibitor 1 ◽  
Protease Activated Receptors ◽  
Messenger Ribonucleic Acid ◽  
Pai 1

AbstractMonocytes are activated in inflammatory conditions via a variety of cytokine receptors as well as in a procoagulatory setting through thrombin, acting upon protease-activated receptors (PARs). This study investigated the expression pattern of PAR1 and PAR3 on human monocyte subsets. Furthermore, a possible regulation of the expression of PAR1 and PAR3 in these cells by inflammatory activation were studied. CD16+ monocytes showed significantly higher levels of PAR1 and PAR3 as compared with CD16− monocytes. Ex vivo treatment of whole blood with lipopolysaccharide (LPS) increased PAR1 and PAR3 messenger ribonucleic acid (mRNA) in human monocytes. In addition, increase of PAR1 was seen in all three subsets upon LPS treatment, whereas PAR3 increased significantly only in CD16− monocytes and nonclassical CD16+ monocytes. Protein levels of PAR1 and PAR3 significantly increased on monocytes in vivo in human endotoxemia 1 hour after LPS infusion. PAR1 increased significantly in CD16− monocytes and nonclassical CD16+ monocytes. In this in vivo model, PAR3 was also significantly elevated in CD16− monocytes and increased slightly albeit not significantly in CD16+ monocytes. Endotoxemia increased plasminogen activator inhibitor-1 (PAI-1) and tissue factor (TF) expression in monocytes in humans. Pretreatment of healthy volunteers with the PAR1 antagonist vorapaxar blocked the increase in PAI-1 but not the increase in TF. We here provide new evidence for a critical role for monocytes as cellular mediators that contribute to the activation of coagulation in diseases characterized by an inflammatory state.

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