Discovery and Preclinical Characterization of Fulacimstat (BAY 1142524), a Potent and Selective Chymase Inhibitor As a New Profibrinolytic Approach for Safe Thrombus Resolution.

Chymase is a serine-protease produced by mast cells. In the past few decades, its role in fibrotic diseases triggered the search for orally available chymase inhibitors. Aiming at reducing adverse cardiac remodeling after myocardial infarction, our research efforts resulted in the discovery of fulacimstat (BAY 1142524).

Enhanced Extraction of Blood and Tissue Time-Activity Curves in Cardiac Mouse FDG PET Imaging by Means of Constrained Nonnegative Matrix Factorization.

We propose an enhanced method to accurately retrieve time-activity curves (TACs) of blood and tissue from dynamic 2-deoxy-2-[F]fluoro-D-glucose ([F]FDG) positron emission tomography (PET) cardiac images of mice. The method is noninvasive and consists of using a constrained nonnegative matrix factorization algorithm (CNMF) applied to the matrix () containing the intensity values of the voxels of the left ventricle (LV) PET image. CNMF factorizes into nonnegative matrices and , respectively, representing the physiological factors (blood and tissue) and their associated weights, by minimizing an extended cost function.

Chymase Inhibition Resolves and Prevents Deep Vein Thrombosis Without Increasing Bleeding Time in the Mouse Model.

Background Deep vein thrombosis (DVT) is the primary cause of pulmonary embolism and the third most life-threatening cardiovascular disease in North America. Post-DVT anticoagulants, such as warfarin, heparin, and direct oral anticoagulants, reduce the incidence of subsequent venous thrombi. However, all currently used anticoagulants affect bleeding time at various degrees, and there is therefore a need for improved therapeutic regimens in DVT.

Mast Cell Degranulation Increases Mouse Mast Cell Protease 4-Dependent Vasopressor Responses to Big Endothelin-1 But Not Angiotensin I.

Mouse mast cell protease 4 (mMCP-4), the murine functional analog to the human chymase, is a serine protease synthesized and stored in mast cell secretory granules. Our previous studies reported physiologic and pathologic roles for mMCP-4 in the maturation and synthesis of the vasoactive peptide endothelin-1 (ET-1) from its precursor, big ET-1. The aim of this study was to investigate the impact of mast cell degranulation or stabilization on mMCP-4-dependent pressor responses after the administration of big ET-1 or angiotensin I (Ang I).

Experimental Autoimmune Encephalomyelitis Potentiates Mouse Mast Cell Protease 4-Dependent Pressor Responses to Centrally or Systemically Administered Big Endothelin-1.

Multiple sclerosis is a neurodegenerative disease affecting predominantly female patients between 20 and 45 years of age. We previously reported the significant contribution of mouse mast cell protease 4 (mMCP-4) in the synthesis of endothelin-1 (ET-1) in healthy mice and in a murine model of experimental autoimmune encephalomyelitis (EAE). In the current study, the cardiovascular effects of ET-1 and big endothelin-1 (big-ET-1) administered systemically or intrathecally were assessed in the early preclinical phase of EAE in telemetry instrumented/conscious mice.

Endothelins in inflammatory neurological diseases.

Endothelins were discovered more than thirty years ago as potent vasoactive compounds. Beyond their well-documented cardiovascular properties, however, the contributions of the endothelin pathway have been demonstrated in several neuroinflammatory processes and the peptides have been reported as clinically relevant biomarkers in neurodegenerative diseases. Several studies report that endothelin-1 significantly contributes to the progression of neuroinflammatory processes, particularly during infections in the central nervous system (CNS), and is associated with a loss of endothelial integrity at the blood brain barrier level.

Mouse Mast Cell Protease 4 Deletion Protects Heart Function and Survival After Permanent Myocardial Infarction.

Chymase, a mast cell serine protease involved in the generation of multiple cardiovascular factors, such as angiotensin II and endothelin-1 (ET-1), is elevated and participates in tissue degeneration after permanent myocardial infarction (PMI). Anesthetized 4-month old male wild-type (WT) C57BL/6J mice and mouse mast cell protease-4 knockout (mMCP-4 KO) congeners were subjected to ligation of the left anterior descending (LAD) coronary artery. A group of mice was then subjected to Kaplan-Meier 28-day survival analysis.

Physical contact between human vascular endothelial and smooth muscle cells modulates cytosolic and nuclear calcium homeostasis.

The interaction between vascular endothelial cells (VECs) and vascular smooth muscle cells (VSMCs) plays an important role in the modulation of vascular tone. There is, however, no information on whether direct physical communication regulates the intracellular calcium levels of human VECs (hVECs) and (or) human VSMCs (hVSMCs). Thus, the objective of the study is to verify whether co-culture of hVECs and hVSMCs modulates cytosolic ([Ca]) and nuclear calcium ([Ca]) levels via physical contact and (or) factors released by both cell types.

Neuropeptide Y and its receptors in ventricular endocardial endothelial cells.

Endocardial endothelial cells (EECs) constitute an important component of the heart. These cells form a monolayer that covers the cavities of the right (EECRs) and left (EECLs) ventricles. They play an important role in cardiac excitation-contraction coupling via their secretion of cardioactive factors such as neuropeptide Y (NPY).

Role of endothelin-1 and its receptors, ET and ET, in the survival of human vascular endothelial cells.

Our previous work showed the presence of endothelin-1 (ET-1) receptors, ET and ET, in human vascular endothelial cells (hVECs). In this study, we wanted to verify whether ET-1 plays a role in the survival of hVECs via the activation of its receptors ET and (or) ET (ETR and ETR, respectively). Our results showed that treatment of hVECs with ET-1 prevented apoptosis induced by genistein, an effect that was mimicked by treatment with ETR-specific agonist IRL1620.

Inhibition of platelet aggregation ex vivo is repressed in apolipoprotein E deficient mice.

In the present study, we assessed whether the endogenous platelet inhibitory mechanisms are altered in the early to moderate stages of the atherosclerotic process. Apolipoprotein E deficient mice (ApoE-/-), a mouse model of atherosclerosis, and their wild-type (WT) counterparts were used to assess agonist-stimulated synthesis of prostacyclin (PGI), inhibition of platelet aggregation ex vivo, and intra-platelet cAMP levels. Basal U46619 and ADP -induced platelet aggregation in vitro were increased in ApoE-/- mice at 18-20 weeks in comparison with 8-10 weeks of age.

Significant Contribution of Mouse Mast Cell Protease 4 in Early Phases of Experimental Autoimmune Encephalomyelitis.

Experimental autoimmune encephalomyelitis (EAE) is a mouse model that reproduces cardinal signs of clinical, histopathological, and immunological features found in Multiple Sclerosis (MS). Mast cells are suggested to be involved in the main inflammatory phases occurring during EAE development, possibly by secreting several autacoids and proteases. Among the latter, the chymase mouse mast cell protease 4 (mMCP-4) can contribute to the inflammatory response by producing endothelin-1 (ET-1).

Endothelin-1: Biosynthesis, Signaling and Vasoreactivity.

Endothelin-1 (ET-1) is an extremely potent vasoconstrictor peptide originally isolated from endothelial cells. Its synthesis, mainly regulated at the gene transcription level, involves processing of a precursor by a furin-type proprotein convertase to an inactive intermediate, big ET-1. The latter peptide can then be cleaved directly by an endothelin-converting enzyme (ECE) into ET-1 or reach the active metabolite through a two-step process involving chymase hydrolyzing big ET-1 to ET-1 (1-31), itself needing conversion to ET-1 by neprilysin (NEP) to exert physiological activity.

Endothelin receptor antagonist macitentan or deletion of mouse mast cell protease 4 delays lesion development in atherosclerotic mice.

Aims: To determine the impact of mixed endothelin receptor antagonist and mouse mast cell protease-4 (mMCP-4) in the development of atherosclerosis in the mouse model.

Materials And Methods: Apolipoprotein E (ApoE) KO mice were crossed with mMCP-4 KO mice to generate ApoE/mMCP-4 double KO mice. Atherosclerosis was induced with a normal- or high-fat diet for 12, 27 or 52weeks.

Receptor for Advanced Glycation End-Products Signaling Interferes with the Vascular Smooth Muscle Cell Contractile Phenotype and Function.

Increased blood glucose concentrations promote reactions between glucose and proteins to form advanced glycation end-products (AGE). Circulating AGE in the blood plasma can activate the receptor for advanced end-products (RAGE), which is present on both endothelial and vascular smooth muscle cells (VSMC). RAGE exhibits a complex signaling that involves small G-proteins and mitogen activated protein kinases (MAPK), which lead to increased nuclear factor kappa B (NF-κB) activity.

Contribution of oxidative stress and prostanoids in endothelial dysfunction induced by chronic fluoxetine treatment.

Objectives: The effects of chronic fluoxetine treatment were investigated on blood pressure and on vascular reactivity in the isolated rat aorta.

Methods And Results: Male Wistar rats were treated with fluoxetine (10 mg/kg/day) for 21 days. Fluoxetine increased systolic blood pressure.

Using carboxyfluorescein diacetate succinimidyl ester to monitor intracellular protein glycation.

Protein glycation is a ubiquitous process involved in vascular complications observed in diabetes. Glyoxal (GO), an intracellular reactive oxoaldehyde that is one of the most potent glycation agents, readily reacts with amines present on proteins to produce the lysine-derived adduct carboxymethyllysine, which is a prevalent advanced glycation end-product (AGE). Our group previously showed that cell exposure to GO leads to an alteration in the cell contractile activity that could occur as a result of the glycation of various proteins regulating the cell contractile machinery.

Nuclear membrane R-type calcium channels mediate cytosolic ET-1-induced increase of nuclear calcium in human vascular smooth muscle cells.

The objective of this work was to verify whether, as in the case of the plasma membrane of human vascular smooth muscle cells (hVSMCs), cytosolic ET-1-induced increase of nuclear calcium is mediated via the activation of calcium influx through the steady-state R-type calcium channel. Pharmacological tools to identify the R-type calcium channels, as well as real 3-D confocal microscopy imaging techniques coupled to calcium fluorescent probes, were used to study the effect of cytosolic ET-1 on nuclear calcium in isolated nuclei of human hepatocytes and plasma membrane perforated hVSMCs. Our results showed that pre-treatment with pertussis toxin (PTX) or cholera toxin (CTX) prevented cytosolic ET-1 (10(-9) mol/L) from inducing a sustained increase in nuclear calcium.

Chymase inhibitor-sensitive synthesis of endothelin-1 (1-31) by recombinant mouse mast cell protease 4 and human chymase.

Important structural differences imply that human and mouse mast cell chymases may differ with respect to their enzymatic properties. We compared in this study the catalytic efficiencies of recombinant human chymase (rCMA1) and its functional murine homologue recombinant mouse mast cell protease-4 (rmMCP-4) toward a fluorogenic chymase substrate (Suc-Ala-Ala-Pro-Phe-7-amino-4-methylcoumarin (AMC) and by their ability to convert Big-endothelin (ET)-1 into ET-1 (1-31) using a LC/MS/MS system. Activities toward a fluorogenic substrate (Suc-Leu-Leu-Val-Tyr-AMC) and Big ET-1 were also measured in extracts from mouse peritoneal mast cells, LUVA human mast cell-like cells and human aortas.

High salt-induced hypertension in B2 knockout mice is corrected by the ETA antagonist, A127722.

Background And Purpose: The contribution of endothelin-1 (ET-1) in a B2KO mouse model of a high salt-induced arterial hypertension was investigated.

Experimental Approach: Wild-type (WT) or B2KO mice receiving a normal diet (ND) or a high-salt diet (HSD) were monitored by radiotelemetry up to a maximum of 18 weeks. At the 12th week of diet, subgroups under ND or HSD received by gavage the ETA antagonist A127722 during 5 days.

Pivotal role of mouse mast cell protease 4 in the conversion and pressor properties of Big-endothelin-1.

The serine protease chymase has been reported to generate intracardiac angiotensin-II (Ang-II) from Ang-I as well as an intermediate precursor of endothelin-1 (ET-1), ET-1 (1-31) from Big-ET-1. Although humans possess only one chymase, several murine isoforms are documented, each with its own specific catalytic activity. Among these, mouse mast cell protease 4 (mMCP-4) is the isoform most similar to the human chymase for its activity.

Endothelin-2-mediated protection of mutant photoreceptors in inherited photoreceptor degeneration.

Expression of the Endothelin-2 (Edn2) mRNA is greatly increased in the photoreceptors (PRs) of mouse models of inherited PR degeneration (IPD). To examine the role of Edn2 in mutant PR survival, we generated Edn2(-/-) mice carrying homozygous Pde6b(rd1) alleles or the Tg(RHO P347S) transgene. In the Edn2(-/-) background, PR survival increased 110% in Pde6b(rd1/rd1) mice at post-natal (PN) day 15, and 60% in Tg(RHO P347S) mice at PN40.

8-BuS-ATP derivatives as specific NTPDase1 inhibitors.

Background And Purpose: Ectonucleotidases control extracellular nucleotide levels and consequently, their (patho)physiological responses. Among these enzymes, nucleoside triphosphate diphosphohydrolase-1 (NTPDase1), -2, -3 and -8 are the major ectonucleotidases responsible for nucleotide hydrolysis at the cell surface under physiological conditions, and NTPDase1 is predominantly located at the surface of vascular endothelial cells and leukocytes. Efficacious inhibitors of NTPDase1 are required to modulate responses induced by nucleotides in a number of pathological situations such as thrombosis, inflammation and cancer.

Further pharmacological evaluation of a novel synthetic peptide bradykinin B2 receptor agonist.

We recently identified a novel human B2 receptor (B2R) agonist [Hyp(3),Thi(5),(N)Chg(7),Thi(8)]-bradykinin (NG291) with greater in vitro and in vivo potency and duration of action than natural bradykinin (BK). Here, we further examined its stability and selectivity toward B2R. The hypotensive, antithrombotic, and profibrinolytic functions of NG291 relative to BK and its analogue ([Hyp(3),Thi(5),(4-Me)Tyr(8)(ΨCH(2)NH)Arg(9)]-BK) (RMP-7) were also tested.