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BACKGROUND: Thromboembolic events, including myocardial infarction (MI) or stroke, caused by the rupture or erosion of unstable atherosclerotic plaques are the leading cause of death worldwide. Although most mouse models of atherosclerosis develop lesions in the aorta and carotid arteries, they do not develop advanced coronary artery lesions. Moreover, they do not undergo spontaneous plaque rupture with MI and stroke or do so at such a low frequency that they are not viable experimental models to study late-stage thrombotic events or to identify novel therapeutic approaches for treating atherosclerotic disease. This has stymied the development of more effective therapeutic approaches for reducing these events beyond what has been achieved with aggressive lipid lowering. Here, we describe a diet-inducible mouse model that develops widespread advanced atherosclerosis in coronary, brachiocephalic, and carotid arteries with plaque rupture, MI, and stroke. METHODS: We characterized a novel mouse model with a C-terminal mutation in the scavenger receptor class B, type 1 (SR-BI), combined with Ldlr knockout (designated SR-BI∆CT/∆CT/Ldlr-/-). Mice were fed Western diet (WD) for 26 weeks and analyzed for MI and stroke. Coronary, brachiocephalic, and carotid arteries were analyzed for atherosclerotic lesions and indices of plaque stability. To validate the utility of this model, SR-BI∆CT/∆CT/Ldlr-/- mice were treated with the drug candidate AZM198, which inhibits myeloperoxidase, an enzyme produced by activated neutrophils that predicts rupture of human atherosclerotic lesions. RESULTS: SR-BI∆CT/∆CT/Ldlr-/- mice show high (>80%) mortality rates after 26 weeks of WD feeding because of major adverse cardiovascular events, including spontaneous plaque rupture with MI and stroke. Moreover, WD-fed SR-BI∆CT/∆CT/Ldlr-/- mice displayed elevated circulating high-sensitivity cardiac troponin I and increased neutrophil extracellular trap formation within lesions compared with control mice. Treatment of WD-fed SR-BI∆CT/∆CT/Ldlr-/- mice with AZM198 showed remarkable benefits, including >90% improvement in survival and >60% decrease in the incidence of plaque rupture, MI, and stroke, in conjunction with decreased circulating high-sensitivity cardiac troponin I and reduced neutrophil extracellular trap formation within lesions. CONCLUSIONS: WD-fed SR-BI∆CT/∆CT/Ldlr-/- mice more closely replicate late-stage clinical events of advanced human atherosclerotic disease than previous models and can be used to identify and test potential new therapeutic agents to prevent major adverse cardiac events.
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BACKGROUND: Myeloperoxidase released after neutrophil and monocyte activation can generate reactive oxygen species, leading to host tissue damage. Extracellular glomerular myeloperoxidase deposition, seen in ANCA-associated vasculitis, may enhance crescentic GN through antigen-specific T and B cell activation. Myeloperoxidase-deficient animals have attenuated GN early on, but augmented T cell responses. We investigated the effect of myeloperoxidase inhibition, using the myeloperoxidase inhibitor AZM198, to understand its potential role in treating crescentic GN. METHODS: We evaluated renal biopsy samples from patients with various forms of crescentic GN for myeloperoxidase and neutrophils, measured serum myeloperoxidase concentration in patients with ANCA-associated vasculitis and controls, and assessed neutrophil extracellular trap formation, reactive oxygen species production, and neutrophil degranulation in ANCA-stimulated neutrophils in the absence and presence of AZM198. We also tested the effect of AZM198 on ANCA-stimulated neutrophil-mediated endothelial cell damage in vitro, as well as on crescentic GN severity and antigen-specific T cell reactivity in the murine model of nephrotoxic nephritis. RESULTS: All biopsy specimens with crescentic GN had extracellular glomerular myeloperoxidase deposition that correlated significantly with eGFR and crescent formation. In vitro, AZM198 led to a significant reduction in neutrophil extracellular trap formation, reactive oxygen species production, and released human neutrophil peptide levels, and attenuated neutrophil-mediated endothelial cell damage. In vivo, delayed AZM198 treatment significantly reduced proteinuria, glomerular thrombosis, serum creatinine, and glomerular macrophage infiltration, without increasing adaptive T cell responses. CONCLUSIONS: Myeloperoxidase inhibition reduced neutrophil degranulation and neutrophil-mediated endothelial cell damage in patients with ANCA-associated vasculitis. In preclinical crescentic GN, delayed myeloperoxidase inhibition suppressed kidney damage without augmenting adaptive immune responses, suggesting it might offer a novel adjunctive therapeutic approach in crescentic GN.
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Anticuerpos Anticitoplasma de Neutrófilos/inmunología , Células Endoteliales/patología , Glomerulonefritis/tratamiento farmacológico , Activación Neutrófila/efectos de los fármacos , Peroxidasa/antagonistas & inhibidores , Inmunidad Adaptativa/efectos de los fármacos , Animales , Vasculitis Asociada a Anticuerpos Citoplasmáticos Antineutrófilos/tratamiento farmacológico , Degranulación de la Célula/efectos de los fármacos , Trampas Extracelulares/efectos de los fármacos , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Peroxidasa/sangre , Peroxidasa/metabolismoRESUMEN
Objective- Inflammation-driven endothelial dysfunction initiates and contributes to the progression of atherosclerosis, and MPO (myeloperoxidase) has been implicated as a potential culprit. On release by circulating phagocytes, MPO is thought to contribute to endothelial dysfunction by limiting NO bioavailability via formation of reactive oxidants including hypochlorous acid. However, it remains largely untested whether specific pharmacological inhibition of MPO attenuates endothelial dysfunction. We, therefore, tested the ability of a mechanism-based MPO inhibitor, AZM198, to inhibit endothelial dysfunction in models of vascular inflammation. Approach and Results- Three models of inflammation were used: femoral cuff, the tandem stenosis model of plaque rupture in Apoe-/- mice, and C57BL/6J mice fed a high-fat, high-carbohydrate diet as a model of insulin resistance. Endothelial dysfunction was observed in all 3 models, and oral administration of AZM198 significantly improved endothelial function in the femoral cuff and tandem stenosis models only. Improvement in endothelial function was associated with decreased arterial MPO activity, determined by the in vivo conversion of hydroethidine to 2-chloroethidium, without affecting circulating inflammatory cytokines or arterial MPO content. Mechanistic studies in Mpo-/- mice confirmed the contribution of MPO to endothelial dysfunction and revealed oxidation of sGC (soluble guanylyl cyclase) as the underlying cause of the observed limited NO bioavailability. Conclusions- Pharmacological inhibition of MPO is a potential strategy to limit endothelial dysfunction in vascular inflammation. Visual Overview- An online visual overview is available for this article.
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Aterosclerosis/tratamiento farmacológico , Células Endoteliales/efectos de los fármacos , Inflamación/tratamiento farmacológico , Peroxidasa/antagonistas & inhibidores , Enfermedades Vasculares/tratamiento farmacológico , Animales , Apolipoproteínas E/fisiología , Aterosclerosis/fisiopatología , Modelos Animales de Enfermedad , Células Endoteliales/fisiología , Inhibidores Enzimáticos/farmacología , Inflamación/fisiopatología , Masculino , Ratones , Ratones Endogámicos C57BL , Peroxidasa/fisiología , Enfermedades Vasculares/fisiopatologíaRESUMEN
A high-fat diet (HFD) can rapidly recruit neutrophils to insulin target tissues and within days induce microvascular insulin resistance (IR). Myeloperoxidase (MPO) is highly enriched in neutrophils, can inhibit nitric oxide-mediated vasorelaxation in vitro and is associated with increased cardiovascular disease risk. AZD5904 irreversibly inhibits MPO and in human clinical trials. MPO knockout, or chemical inhibition, blunts HFD-induced metabolic IR in mice. Whether MPO affects microvascular IR or muscle metabolic insulin sensitivity in vivo is unknown. We used contrast-enhanced ultrasound and the euglycemic insulin clamp to test whether inhibiting MPO could prevent the development or reverse established HFD-induced metabolic and/or microvascular IR in Sprague-Dawley rats. Two weeks of HFD feeding blocked insulin-mediated skeletal muscle capillary recruitment, inhibited glucose utilization, and insulin signaling to muscle. Continuous subcutaneous AZD5904 infusion during the 2 wk selectively blocked HFD's microvascular effect. Furthermore, AZD5904 infusion during the last 2 of 4 wk of HFD feeding restored microvascular insulin sensitivity but not metabolic IR. We conclude that inhibiting MPO selectively improves vascular IR. This selective microvascular effect may connote a therapeutic potential for MPO inhibition in the prevention of vascular disease/dysfunction seen in IR humans.
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Aorta/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Resistencia a la Insulina , Microvasos/efectos de los fármacos , Músculo Esquelético/efectos de los fármacos , Peroxidasa/antagonistas & inhibidores , Animales , Aorta/metabolismo , Velocidad del Flujo Sanguíneo/efectos de los fármacos , Dieta Alta en Grasa , Técnica de Clampeo de la Glucosa , Masculino , Microcirculación/efectos de los fármacos , Microvasos/metabolismo , Proteína Quinasa 1 Activada por Mitógenos/efectos de los fármacos , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/efectos de los fármacos , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Músculo Esquelético/irrigación sanguínea , Músculo Esquelético/metabolismo , Óxido Nítrico Sintasa de Tipo III/efectos de los fármacos , Óxido Nítrico Sintasa de Tipo III/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
AIMS: Myeloperoxidase activity can contribute to impaired vascular endothelial function and fibrosis in chronic inflammation-related cardiovascular disease. Here, we investigated the safety, tolerability and pharmacokinetics of the myeloperoxidase inhibitor, AZD4831. METHODS: In this randomized, single-blind, placebo-controlled, phase I, first-in-human study, healthy men in five sequential cohorts were randomized 3:1 to receive a single oral dose of AZD4831 (5, 15, 45, 135 or 405 mg) or placebo, after overnight fasting. After at least 7 days' washout, one cohort additionally received AZD4831 45 mg after a high-calorie meal. RESULTS: Forty men participated in the study (eight per cohort: AZD4831, n = 6; placebo, n = 2). AZD4831 distributed rapidly into plasma, with a half-life of 38.2-50.0 hours. The area under the plasma concentration-time curve (AUC) increased proportionally with dose (AUC0-â slope estimate 1.060; 95% confidence interval [CI] 0.9943, 1.127). Increases in maximum plasma concentration were slightly more than dose proportional (slope estimate 1.201; 95% CI 1.071, 1.332). Food intake reduced AZD4831 absorption rate but did not substantially affect overall exposure or plasma half-life (n = 4). Serum uric acid concentrations decreased by 71.77 (95% CI 29.15, 114.39) and 84.42 (58.90, 109.94) µmol L-1 with AZD4831 135 mg and 405 mg, respectively. Maculopapular rash (moderate intensity) occurred in 4/30 participants receiving AZD4831 (13.3%). No other safety concerns were identified. CONCLUSIONS: AZD4831 was generally well tolerated, rapidly absorbed, had a long plasma half-life and lowered uric acid concentrations after single oral doses in healthy men. These findings support the further clinical development of AZD4831.
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Inhibidores Enzimáticos/administración & dosificación , Peroxidasa/antagonistas & inhibidores , Pirimidinas/administración & dosificación , Pirroles/administración & dosificación , Ácido Úrico/sangre , Administración Oral , Adulto , Área Bajo la Curva , Enfermedades Cardiovasculares/prevención & control , Esquema de Medicación , Inhibidores Enzimáticos/efectos adversos , Inhibidores Enzimáticos/farmacocinética , Semivida , Voluntarios Sanos , Humanos , Masculino , Persona de Mediana Edad , Placebos , Pirimidinas/efectos adversos , Pirimidinas/farmacocinética , Pirroles/efectos adversos , Pirroles/farmacocinética , Método Simple Ciego , Adulto JovenRESUMEN
Aims: As the inflammatory enzyme myeloperoxidase (MPO) is abundant in ruptured human atherosclerotic plaques, we aimed to investigate the role of MPO as a potential diagnostic and therapeutic target for high-risk plaque. Methods and results: We employed the tandem stenosis model of atherosclerotic plaque instability in apolipoprotein E gene knockout (Apoe-/-) mice. To test the role of MPO, we used Mpo-/-Apoe-/- mice and the 2-thioxanthine MPO inhibitor AZM198. In vivo MPO activity was assessed by liquid chromatography-tandem mass spectrometry detection of 2-chloroethidium generation from hydroethidine and by bis-5HT-DTPA-Gd (MPO-Gd) molecular magnetic resonance imaging (MRI), while plaque phenotype was verified histologically. Myeloperoxidase activity was two-fold greater in plaque with unstable compared with stable phenotype. Genetic deletion of MPO significantly increased fibrous cap thickness, and decreased plaque fibrin and haemosiderin content in plaque with unstable phenotype. AZM198 inhibited MPO activity and it also increased fibrous cap thickness and decreased fibrin and haemosiderin in plaque with unstable phenotype, without affecting lesion monocytes and red blood cell markers or circulating leukocytes and lipids. MPO-Gd MRI demonstrated sustained enhancement of plaque with unstable phenotype on T1-weighted imaging that was two-fold greater than stable plaque and was significantly attenuated by both AZM198 treatment and deletion of the Mpo gene. Conclusion: Our data implicate MPO in atherosclerotic plaque instability and suggest that non-invasive imaging and pharmacological inhibition of plaque MPO activity hold promise for clinical translation in the management of high-risk coronary artery disease.
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Aterosclerosis/diagnóstico por imagen , Aterosclerosis/enzimología , Imagen por Resonancia Magnética/métodos , Imagen Molecular , Peroxidasa/metabolismo , Placa Aterosclerótica/diagnóstico por imagen , Placa Aterosclerótica/enzimología , Animales , Modelos Animales de Enfermedad , Fibrina/metabolismo , Hemosiderina/metabolismo , Espectrometría de Masas , Ratones Noqueados , Peroxidasa/antagonistas & inhibidores , Tioxantenos/farmacologíaRESUMEN
Liposome-based drug formulations represent an exciting avenue of research as they increase efficacy to toxicity ratios. Current formulations rely on passive accumulation to the disease site where drug is taken up by the cells. Ligand mediated targeting increases the net accumulation of liposomes, however, an unexplored benefit is to potentially refine pharmacodynamics (PD) of a drug specifically to different cell types within diseased tissue. As a model system, we engineered cardiomyocyte- (I-1) and endothelial-targeted (B-40) liposomes to carry a VEGFR2 inhibitor (PTK787), and examined the effect of cell type-specific delivery on both pharmacokinetics (PK) and PD. Neovascularization in post-myocardial infarction was significantly reduced by B-40 liposomes loaded with PTK787 as compared to animals injected with I-1 liposomes, and profoundly more as compared to free PTK787. This study thus shows that the intraorgan targeting of drugs through cell type-specific delivery holds substantial promise towards lowering the minimal efficacious dose administered systemically.
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Liposomas/química , Péptidos/química , Ftalazinas/administración & dosificación , Inhibidores de Proteínas Quinasas/administración & dosificación , Piridinas/administración & dosificación , Animales , Sistemas de Liberación de Medicamentos , Ratones , Infarto del Miocardio/complicaciones , Neovascularización Patológica/tratamiento farmacológico , Neovascularización Patológica/etiología , Biblioteca de Péptidos , Ftalazinas/farmacocinética , Ftalazinas/uso terapéutico , Inhibidores de Proteínas Quinasas/farmacocinética , Inhibidores de Proteínas Quinasas/uso terapéutico , Piridinas/farmacocinética , Piridinas/uso terapéutico , Receptor 2 de Factores de Crecimiento Endotelial Vascular/antagonistas & inhibidoresRESUMEN
AIMS: Mitiperstat (formerly AZD4831) is a novel selective myeloperoxidase inhibitor. Currently, no effective therapies target comorbidity-induced systemic inflammation, which may be a key mechanism underlying heart failure with preserved or mildly reduced ejection fraction (HFpEF/HFmrEF). Circulating neutrophils secrete myeloperoxidase, causing oxidative stress, microvascular endothelial dysfunction, interstitial fibrosis, cardiomyocyte remodelling and diastolic dysfunction. Mitiperstat may therefore improve function of the heart and other organs, and ameliorate heart failure symptoms and exercise intolerance. ENDEAVOR is a combined, seamless phase 2b-3 study of the efficacy and safety of mitiperstat in patients with HFpEF/HFmrEF. METHODS: In phase 2b, approximately 660 patients with heart failure and ejection fraction >40% are being randomized 1:1:1 to mitiperstat 2.5 mg, 5 mg or placebo for 48 weeks. Eligible patients have baseline 6-min walk distance (6MWD) of 30-400 m with a <50 m difference between screening and randomization and Kansas City Cardiomyopathy Questionnaire total symptom score (KCCQ-TSS) ≤90 points at screening and randomization. The dual primary endpoints are change from baseline to week 16 in 6MWD and KCCQ-TSS. The sample size provides 85% power to detect placebo-adjusted improvements of 21 m in 6MWD and 6.0 points in KCCQ-TSS at overall two-sided alpha of 0.05. Safety is monitored throughout treatment, with a focus on maculopapular rash. In phase 3 of ENDEAVOR, approximately 820 patients will be randomized 1:1 to mitiperstat or placebo. CONCLUSION: ENDEAVOR is the first phase 2b-3 study to evaluate whether myeloperoxidase inhibition can improve symptoms and exercise capacity in patients with HFpEF/HFmrEF.
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Cardiopatías , Insuficiencia Cardíaca , Humanos , Insuficiencia Cardíaca/tratamiento farmacológico , Volumen Sistólico/fisiología , Tolerancia al Ejercicio/fisiología , Peroxidasa/farmacología , Peroxidasa/uso terapéutico , ComorbilidadRESUMEN
BACKGROUND: Systemic microvascular dysfunction and inflammation are postulated to play a pathophysiologic role in heart failure with preserved ejection fraction (HFpEF). OBJECTIVES: This study aimed to identify biomarker profiles associated with clinical outcomes in HFpEF and investigate how inhibition of the neutrophil-derived reactive oxygen species-producing enzyme, myeloperoxidase, affects these biomarkers. METHODS: Using supervised principal component analyses, the investigators assessed the associations between baseline plasma proteomic Olink biomarkers and clinical outcomes in 3 independent observational HFpEF cohorts (n = 86, n = 216, and n = 242). These profiles were then compared with the biomarker profiles discriminating patients treated with active drug vs placebo in SATELLITE (Safety and Tolerability Study of AZD4831 in Patients With Heart Failure), a double-blind randomized 3-month trial evaluating safety and tolerability of the myeloperoxidase inhibitor AZD4831 in HFpEF (n = 41). Pathophysiological pathways were inferred from the biomarker profiles by interrogation of the Ingenuity Knowledge Database. RESULTS: TNF-R1, TRAIL-R2, GDF15, U-PAR, and ADM were the top individual biomarkers associated with heart failure hospitalization or death, and FABP4, HGF, RARRES2, CSTB, and FGF23 were associated with lower functional capacity and poorer quality of life. AZD4831 downregulated many markers (most significantly CDCP1, PRELP, CX3CL1, LIFR, VSIG2). There was remarkable consistency among pathways associated with clinical outcomes in the observational HFpEF cohorts, the top canonical pathways being associated with tumor microenvironments, wound healing signaling, and cardiac hypertrophy signaling. These pathways were predicted to be downregulated in AZD4831 relative to placebo-treated patients. CONCLUSIONS: Biomarker pathways that were most strongly associated with clinical outcomes were also the ones reduced by AZD4831. These results support the further investigation of myeloperoxidase inhibition in HFpEF.
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Insuficiencia Cardíaca , Humanos , Antígenos de Neoplasias/uso terapéutico , Biomarcadores , Moléculas de Adhesión Celular/uso terapéutico , Peroxidasa/uso terapéutico , Proteómica , Calidad de Vida , Volumen Sistólico/fisiologíaRESUMEN
Currently there are no established therapies to treat high-risk patients with unstable atherosclerotic lesions that are prone to rupture and can result in thrombosis, abrupt arterial occlusion, and a precipitous infarction. Rather than being stenotic, rupture-prone non-occlusive plaques are commonly enriched with inflammatory cells and have a thin fibrous cap. We reported previously that inhibition of the pro-inflammatory enzyme myeloperoxidase (MPO) with the suicide inhibitor AZM198 prevents formation of unstable plaque in the Tandem Stenosis (TS) mouse model of plaque instability. However, in our previous study AZM198 was administered to animals before unstable plaque was present and hence it did not test the significant unmet clinical need present in high-risk patients with vulnerable atherosclerosis. In the present study we therefore asked whether pharmacological inhibition of MPO with AZM198 can stabilize pre-existing unstable lesions in an interventional setting using the mouse model of plaque instability. In vivo molecular magnetic resonance imaging of arterial MPO activity using bis-5-hydroxytryptamide-DTPA-Gd and histological analyses revealed that arterial MPO activity was elevated one week after TS surgery, prior to the presence of unstable lesions observed two weeks after TS surgery. Animals with pre-existing unstable plaque were treated with AZM198 for one or five weeks. Both short- and long-term intervention effectively inhibited arterial MPO activity and increased fibrous cap thickness, indicative of a more stable plaque phenotype. Plaque stabilization was observed without AZM198 affecting the arterial content of Ly6B.2+- and CD68+-cells and MPO protein. These findings demonstrate that inhibition of arterial MPO activity converts unstable into stable atherosclerotic lesions in a preclinical model of plaque instability and highlight the potential therapeutic potency of MPO inhibition for the management of high-risk patients and the development of novel protective strategies against cardiovascular diseases.
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Aterosclerosis , Enfermedades Cardiovasculares , Peroxidasa , Placa Aterosclerótica , Animales , Ratones , Aterosclerosis/tratamiento farmacológico , Enfermedades Cardiovasculares/prevención & control , Modelos Animales de Enfermedad , Peroxidasa/antagonistas & inhibidores , Placa Aterosclerótica/tratamiento farmacológico , Placa Aterosclerótica/patologíaRESUMEN
Myeloperoxidase is a promising therapeutic target for treatment of patients suffering from heart failure with preserved ejection fraction (HFpEF). We aimed to discover a covalent myeloperoxidase inhibitor with high selectivity for myeloperoxidase over thyroid peroxidase, limited penetration of the blood-brain barrier, and pharmacokinetics suitable for once-daily oral administration at low dose. Structure-activity relationship, biophysical, and structural studies led to prioritization of four compounds for in-depth safety and pharmacokinetic studies in animal models. One compound (AZD4831) progressed to clinical studies on grounds of high potency (IC50, 1.5 nM in vitro) and selectivity (>450-fold vs thyroid peroxidase in vitro), the mechanism of irreversible inhibition, and the safety profile. Following phase 1 studies in healthy volunteers and a phase 2a study in patients with HFpEF, a phase 2b/3 efficacy study of AZD4831 in patients with HFpEF started in 2021.
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Insuficiencia Cardíaca , Animales , Insuficiencia Cardíaca/tratamiento farmacológico , Humanos , Yoduro Peroxidasa/uso terapéutico , Peroxidasa , Pirimidinas , Pirroles , Volumen Sistólico/fisiologíaRESUMEN
AIM: Epicardial adipose tissue (EAT) may play a role in the pathophysiology of heart failure with preserved ejection fraction (HFpEF). We investigated associations of EAT with proteomics, coronary flow reserve (CFR), cardiac structure and function, and quality of life (QoL) in the prospective multinational PROMIS-HFpEF cohort. METHODS AND RESULTS: Epicardial adipose tissue was measured by echocardiography in 182 patients and defined as increased if ≥9 mm. Proteins were measured using high-throughput proximity extension assays. Microvascular dysfunction was evaluated with Doppler-based CFR, cardiac structural and functional indices with echocardiography and QoL by Kansas City Cardiomyopathy Questionnaire (KCCQ). Patients with increased EAT (n = 54; 30%) had higher body mass index (32 [28-40] vs. 27 [23-30] kg/m2 ; p < 0.001), lower N-terminal pro-B-type natriuretic peptide (466 [193-1133] vs. 1120 [494-1990] pg/ml; p < 0.001), smaller indexed left ventricular (LV) end-diastolic and left atrial (LA) volumes and tendency to lower KCCQ score. Non-indexed LV/LA volumes did not differ between groups. When adjusted for body mass index, EAT remained associated with LV septal wall thickness (coefficient 1.02, 95% confidence interval [CI] 1.00-1.04; p = 0.018) and mitral E wave deceleration time (coefficient 1.03, 95% CI 1.01-1.05; p = 0.005). Increased EAT was associated with proteomic markers of adipose biology and inflammation, insulin resistance, endothelial dysfunction, and dyslipidaemia but not significantly with CFR. CONCLUSION: Increased EAT was associated with cardiac structural alterations and proteins expressing adiposity, inflammation, lower insulin sensitivity and endothelial dysfunction related to HFpEF pathology, probably driven by general obesity. Potential local mechanical or paracrine effects mediated by EAT remain to be elucidated.
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Insuficiencia Cardíaca , Humanos , Volumen Sistólico/fisiología , Calidad de Vida , Función Ventricular Izquierda/fisiología , Estudios Prospectivos , Proteómica , Tejido Adiposo/diagnóstico por imagen , Inflamación/patologíaRESUMEN
The clinical heterogeneity of heart failure has challenged our understanding of the underlying genetic mechanisms of this disease. In this respect, large-scale patient DNA sequencing studies have become an invaluable strategy for identifying potential genetic contributing factors. The complex aetiology of heart failure, however, also means that in vivo models are vital to understand the links between genetic perturbations and functional impacts as part of the process for validating potential new drug targets. Traditional approaches (e.g., genetically-modified mice) are optimal for assessing small numbers of genes, but less practical when multiple genes are identified. The zebrafish, in contrast, offers great potential for higher throughput in vivo gene functional assessment to aid target prioritisation, by providing more confidence in target relevance and facilitating gene selection for definitive loss of function studies undertaken in mice. Here we used whole-exome sequencing and bioinformatics on human patient data to identify 3 genes (API5, HSPB7, and LMO2) suggestively associated with heart failure that were also predicted to play a broader role in disease aetiology. The role of these genes in cardiovascular system development and function was then further investigated using in vivo CRISPR/Cas9-mediated gene mutation analysis in zebrafish. We observed multiple impacts in F0 knockout zebrafish embryos (crispants) following effective somatic mutation, including changes in ventricle size, pericardial oedema, and chamber malformation. In the case of lmo2, there was also a significant impact on cardiovascular function as well as an expected reduction in erythropoiesis. The data generated from both the human in silico and zebrafish in vivo assessments undertaken supports further investigation of the potential roles of API5, HSPB7, and LMO2 in human cardiovascular disease. The data presented also supports the use of human in silico genetic variant analysis, in combination with zebrafish crispant phenotyping, as a powerful approach for assessing gene function as part of an integrated multi-level drug target validation strategy.
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Myocardial infarction (MI) is a leading cause of morbidity and mortality worldwide. Improved survival has led to an increasing incidence of ischemic cardiomyopathy, making it a major reason for hospitalization in the western world. The inflammatory response in the ischemic myocardium determines the extent of structural remodeling and functional deterioration, with neutrophils (PMN) being a key modulator of the propagation and resolution of inflammation. The heme enzyme myeloperoxidase (MPO) is abundantly expressed in PMN and is an important mediator of their inflammatory capacities. Here, we examine the effects of PMN reduction, MPO deficiency and MPO inhibition in two murine models of MI. Reduction in PMN count resulted in less scar formation and improved cardiac function. Similar results were obtained in genetically MPO deficient mice, suggesting that MPO is a critical factor in PMN-mediated cardiac remodeling. To test our findings in a therapeutic approach, we orally administered the MPO inhibitor AZM198 in the context of MI and could demonstrate improved cardiac function and reduced structural remodeling. Therefore, MPO appears to be a favorable pharmacological target for the prevention of long-term morbidity after MI.
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AIMS: Hypertrophic cardiomyopathy (HCM) is characterized by cardiomyocyte hypertrophy and disarray, and myocardial stiffness due to interstitial fibrosis, which result in impaired left ventricular filling and diastolic dysfunction. The latter manifests as exercise intolerance, angina, and dyspnoea. There is currently no specific treatment for improving diastolic function in HCM. Here, we investigated whether myeloperoxidase (MPO) is expressed in cardiomyocytes and provides a novel therapeutic target for alleviating diastolic dysfunction in HCM. METHODS AND RESULTS: Human cardiomyocytes derived from control-induced pluripotent stem cells (iPSC-CMs) were shown to express MPO, with MPO levels being increased in iPSC-CMs generated from two HCM patients harbouring sarcomeric mutations in the MYBPC3 and MYH7 genes. The presence of cardiomyocyte MPO was associated with higher chlorination and peroxidation activity, increased levels of 3-chlorotyrosine-modified cardiac myosin binding protein-C (MYBPC3), attenuated phosphorylation of MYBPC3 at Ser-282, perturbed calcium signalling, and impaired cardiomyocyte relaxation. Interestingly, treatment with the MPO inhibitor, AZD5904, reduced 3-chlorotyrosine-modified MYBPC3 levels, restored MYBPC3 phosphorylation, and alleviated the calcium signalling and relaxation defects. Finally, we found that MPO protein was expressed in healthy adult murine and human cardiomyocytes, and MPO levels were increased in diseased hearts with left ventricular hypertrophy. CONCLUSION: This study demonstrates that MPO inhibition alleviates the relaxation defect in hypertrophic iPSC-CMs through MYBPC3 phosphorylation. These findings highlight cardiomyocyte MPO as a novel therapeutic target for improving myocardial relaxation associated with HCM, a treatment strategy which can be readily investigated in the clinical setting, given that MPO inhibitors are already available for clinical testing.
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Cardiomiopatía Hipertrófica/tratamiento farmacológico , Inhibidores Enzimáticos/farmacología , Hipertrofia Ventricular Izquierda/tratamiento farmacológico , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Contracción Miocárdica/efectos de los fármacos , Miocitos Cardíacos/efectos de los fármacos , Peroxidasa/antagonistas & inhibidores , Función Ventricular Izquierda/efectos de los fármacos , Animales , Miosinas Cardíacas/genética , Miosinas Cardíacas/metabolismo , Cardiomiopatía Hipertrófica/enzimología , Cardiomiopatía Hipertrófica/genética , Cardiomiopatía Hipertrófica/fisiopatología , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Línea Celular , Modelos Animales de Enfermedad , Humanos , Hipertrofia Ventricular Izquierda/enzimología , Hipertrofia Ventricular Izquierda/genética , Hipertrofia Ventricular Izquierda/fisiopatología , Células Madre Pluripotentes Inducidas/enzimología , Células Madre Pluripotentes Inducidas/patología , Masculino , Ratones Endogámicos C57BL , Mutación Missense , Miocitos Cardíacos/enzimología , Miocitos Cardíacos/patología , Cadenas Pesadas de Miosina/genética , Cadenas Pesadas de Miosina/metabolismo , Peroxidasa/metabolismo , Fosforilación , Especies Reactivas de Oxígeno/metabolismo , Tirosina/análogos & derivados , Tirosina/metabolismoRESUMEN
We evaluated safety, tolerability, pharmacokinetics (PKs), and pharmacodynamics of AZD4831, a novel oral myeloperoxidase (MPO) inhibitor, in a randomized, single-blind, placebo-controlled study, following once-daily multiple ascending dosing to steady-state in healthy subjects. Target engagement was measured as specific MPO activity in plasma following ex vivo zymosan stimulation of whole blood. Except for generalized maculopapular rash in 4 of 13 subjects receiving the 2 highest doses, 15 and 45 mg AZD4831, no clinically relevant safety and tolerability findings were observed. AZD4831 was rapidly absorbed and plasma concentrations declined slowly with an elimination half-life of ~ 60 hours. A dose/concentration-effect relationship between MPO inhibition vs. AZD4831 exposure was established with > 50% MPO inhibition in plasma at concentrations in the low nanomolar range. Steady-state levels were achieved within 10 days. Taken together, the PK profile, the sustained dose/concentration-dependent MPO inhibition, and available clinical data support further clinical development of AZD4831 in patients with heart failure with preserved ejection fraction.
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Insuficiencia Cardíaca/tratamiento farmacológico , Peroxidasa/antagonistas & inhibidores , Pirimidinas/efectos adversos , Pirroles/efectos adversos , Administración Oral , Adulto , Relación Dosis-Respuesta a Droga , Método Doble Ciego , Voluntarios Sanos , Humanos , Masculino , Persona de Mediana Edad , Pirimidinas/administración & dosificación , Pirimidinas/farmacocinética , Pirroles/administración & dosificación , Pirroles/farmacocinética , Método Simple Ciego , Volumen Sistólico , Función Ventricular Izquierda , Adulto JovenRESUMEN
BACKGROUND: Cystic fibrosis (CF) lung disease is characterized by severe bacterial infections, excessive neutrophilic inflammation and oxidative stress. The neutrophil enzyme myeloperoxidase (MPO), which produces hypochlorous acid, is associated with worse disease outcomes. Therefore, pharmacological inhibition of MPO in the airways has therapeutic potential. We investigated whether treating mice with an MPO inhibitor during pulmonary infection decreases oxidative stress and improves infection outcomes in mice with CF-like lung inflammation without impacting on bacterial clearance. METHODS: Transgenic ß-epithelial sodium channel (ßENaC)-overexpressing mice (n = 10) were infected with Burkholderia multivorans and treated twice daily with the MPO inhibitor AZM198 (125 µmol/kg) or vehicle administered by oral gavage for two days. Bodyweight was recorded daily. MPO activity, markers of oxidative stress, inflammatory cytokines and leukocytes numbers were measured in bronchoalveolar lavage fluid (BALF). Bacterial burden was determined in lung tissue homogenates. RESULTS: During the course of infection, mice treated with AZM198 lost less weight than vehicle-treated mice (p < 0.01). MPO activity and glutathione sulfonamide, a hypochlorous acid-specific glutathione oxidation product, were significantly lower in BALF from AZM198-treated mice (p < 0.05). The inflammatory cytokines CXCL1 and TNF-α in BALF and bacterial burden in the lung were not significantly different between treated and control mice. CONCLUSIONS: Orally administered AZM198 inhibits MPO activity in epithelial lining fluid. Blocking hypochlorous acid production in epithelial lining fluid during pulmonary infections through inhibition of MPO improves morbidity in mice with CF-like lung inflammation without interfering with clearance of bacteria. Pharmacological inhibition of MPO is an approach to limit destructive oxidative stress in cystic fibrosis lung disease in humans.
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Fibrosis Quística , Neumonía , Animales , Líquido del Lavado Bronquioalveolar , Burkholderia , Fibrosis Quística/tratamiento farmacológico , Inflamación , Pulmón/metabolismo , Ratones , Morbilidad , Estrés Oxidativo , Peroxidasa/metabolismo , Neumonía/tratamiento farmacológicoRESUMEN
AIMS: In heart failure (HF) with preserved ejection fraction (HFpEF), microvascular inflammation is proposed as an underlying mechanism. Myeloperoxidase (MPO) is associated with vascular dysfunction and prognosis in congestive HF. METHODS AND RESULTS: MPO, MPO-related biomarkers, and echocardiography were assessed in 86 patients, 4-8 weeks after presentation with acute HF (EF ≥ 45%), and in 46 healthy controls. Patients were followed up for median 579 days (Q1;Q3 276;1178) regarding the composite endpoint all-cause mortality or HF hospitalization. Patients were 73 years old, 51% were female, EF was 64% (Q1;Q3 58;68), E/e' was ratio 10.8 (8.3;14.0), and left atrial volume index (LAVI) was 43 mL/m2 (38;52). Controls were 60 (57;62) years old (vs. patients; P < 0.001), 24% were female (P = 0.005), and left ventricular EF was 63% (59;66; P = 0.790). MPO was increased in HFpEF compared with controls, 101 (81;132) vs. 86 (74;101 ng/mL, P = 0.015), as was uric acid 369 (314;439) vs. 289 (252;328 µmol/L, P < 0.001), calprotectin, asymmetric dimethyl arginine (ADMA), and symmetric dimethyl arginine (SDMA), while arginine was decreased. MPO correlated with uric acid (r = 0.26; P = 0.016). In patients with E/e' > 14, uric acid and SDMA were elevated (421 vs. 344 µM, P = 0.012; 0.54 vs. 0.47 µM, P = 0.039, respectively), and MPO was 121 vs. 98 ng/mL (P = 0.090). The ratios of arginine/ADMA (112 vs. 162; P < 0.001) and ADMA/SDMA (1.36 vs. 1.17; P = 0.002) were decreased in HFpEF patients, suggesting reduced NO availability and increased enzymatic clearance of ADMA, respectively. Uric acid independently predicted the endpoint [hazard ratio (HR) 3.76 (95% CI 1.19-11.85; P = 0.024)] but not MPO [HR 1.48 (95% CI 0.70-3.14; P = 0.304)] or the other biomarkers. CONCLUSIONS: In HFpEF, MPO-dependent oxidative stress reflected by uric acid and calprotectin is increased, and SDMA is associated with diastolic dysfunction and uric acid with outcome. This suggests microvascular neutrophil involvement mirroring endothelial dysfunction, a central component of the HFpEF syndrome and a potential treatment target.
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Insuficiencia Cardíaca , Peroxidasa , Anciano , Biomarcadores , Femenino , Insuficiencia Cardíaca/diagnóstico , Humanos , Inflamación , Masculino , Persona de Mediana Edad , Volumen SistólicoRESUMEN
Myeloperoxidase (MPO) activity has been associated with the metabolic syndrome, cardiovascular and liver disease. Here, we evaluate the therapeutic potential of MPO inhibition on nonalcoholic steatohepatitis (NASH) and NASH-induced fibrosis, the main determinant of outcomes. MPO plasma levels were elevated in patients with nonalcoholic fatty liver disease (NAFLD) compared with healthy controls. In a second cohort, hepatic MPO messenger RNA expression correlated with higher body mass index and hemoglobin A1c, both being risk factors for NAFLD. We could establish by immunohistochemistry that MPO-positive cells were recruited to the liver in various mouse models of fibrogenic liver injury, including bile duct ligation, carbon tetrachloride (CCl4) treatment, spontaneous liver fibrogenesis in multidrug resistance 2 knockout (MDR2 KO) mice, and NASH-inducing diet. Comparison of MPO-deficient mice and their wild-type littermates exposed to a high-caloric diet revealed that MPO deficiency protects against NASH-related liver injury and fibrosis. In line with this, hepatic gene expression analysis demonstrated a MPO-dependent activation of pathways relevant for wound healing, inflammation, and cell death in NASH. MPO deficiency did not affect NAFLD-independent liver injury and fibrosis in MDR2 KO or CCl4-treated mice. Finally, we treated wild-type mice exposed to NASH-inducing diet with an oral MPO inhibitor. Pharmacological MPO inhibition not only reduced markers of MPO-mediated liver damage, serum alanine aminotransferase levels, and hepatic steatosis, but also significantly decreased NASH-induced liver fibrosis. MPO inhibitor treatment, but not MPO deficiency, significantly altered gut microbiota including a significant expansion of Akkermansia muciniphila. Conclusions: MPO specifically promotes NASH-induced liver fibrosis. Pharmacological MPO inhibition attenuates NASH progression and NASH-induced liver fibrosis in mice and is associated with beneficial changes of intestinal microbiota.
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This manuscript is a companion paper to Ulleryd M.U. et al., "Stimulation of alpha 7 nicotinic acetylcholine receptor (α7nAChR) inhibits atherosclerosis via immunomodulatory effects on myeloid cells" Atherosclerosis, 2019 [1]. Data shown here include RNA sequencing data from whole aorta of ApoE-/- mice fed high fat diet and treated with the alpha 7 nicotinic acetylcholine receptor (α7nAChR) agonist AZ6983 for 8 weeks using subcutaneously implanted osmotic minipumps. Here we present the top gene networks affected by treatment with AZ6983, as well as the up- and down-regulated genes in aorta after treatment. Further, a URL link to the RNA sequencing datasets submitted to GEO is included.