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1.
Physiol Genomics ; 56(10): 649-660, 2024 Oct 01.
Artículo en Inglés | MEDLINE | ID: mdl-39007510

RESUMEN

The prevalence of metabolic syndrome in cardiac diseases such as heart failure with preserved ejection fraction (HFpEF) prompts the scientific community to investigate its adverse effects on cardiac function and remodeling. However, the selection of a preclinical model of obesity-induced cardiac remodeling has proven more challenging with inconsistencies often found in very similar mouse models. Here, we investigated the implication of genetic background as well as diet composition to identify a suitable model of diet-induced cardiac alterations. C57Bl/6J and C57Bl/6N male mice were subjected to distinct obesogenic diets consisting of high-fat and moderate sucrose content (HF-S) or high-sucrose and moderate lipid content (F-HS) versus matching control diets. Five-month dietary intervention with obesogenic diets induced weight gain, adipocyte hypertrophy, and increased visceral and subcutaneous fat mass in both substrains. Obese mice showed similar impairment of glucose disposition and insulin tolerance, with both strains developing insulin resistance within 2 mo. However, echocardiographic follow-up and histological analysis confirmed that the HF-S diet increased cardiac hypertrophy, interstitial fibrosis, and left atrial area in the C57Bl/6J strain only. In contrast, the C57Bl/6N strain exhibited cardiac eccentric remodeling under control diets, possibly owing to a genetic mutation in the myosin light chain kinase 3 (Mylk3) gene, specific to this substrain, which was not further enhanced under obesogenic diets. Altogether, the present results highlight the importance of carefully selecting the suitable mouse strain and diets to model diet-induced cardiac remodeling. In this regard, C57Bl/6J mice develop significant cardiac remodeling in response to HF-S and seem to be a suitable model for cardiometabolic disease.NEW & NOTEWORTHY Metabolic syndrome is highly prevalent in cardiac pathologies. Underlying mechanisms have not been thoroughly investigated, owing to the lack of reliable preclinical model of diet-induced cardiac remodeling. Our work demonstrates that genetic variants in inbred strains influence the response to metabolic stress and identifies C57Bl/6J mice as a suitable model for cardiometabolic disease in response to high-fat diet. These findings reinforce the need to carefully select the mouse strain in relation to the imposed pathophysiologic stress.


Asunto(s)
Dieta Alta en Grasa , Ratones Endogámicos C57BL , Obesidad , Remodelación Ventricular , Animales , Masculino , Obesidad/patología , Dieta Alta en Grasa/efectos adversos , Ratones , Resistencia a la Insulina , Modelos Animales de Enfermedad
2.
Clin Sci (Lond) ; 138(18): 1151-1171, 2024 Sep 18.
Artículo en Inglés | MEDLINE | ID: mdl-39206703

RESUMEN

Metabolic dysfunction-associated steatohepatitis (MASH) confers a risk for cardiovascular diseases in patients. Animal models may help exploring the mechanisms linking liver and heart diseases. Hence, we explored the cardiac phenotype in two MASH mouse models: foz/foz mice fed a high-fat diet (HFD) for 24 or 60 weeks and C57BL/6J mice fed a high-fat-, high-cholesterol-, and high-fructose diet for 60 weeks. Angiotensin II (AngII) was used as an additional cardiovascular stressor for 4 weeks in 10 weeks HFD-fed foz/foz mice. Foz/foz mice with fibrosing MASH developed cardiac hypertrophy with adverse cardiac remodelling not seen in WT similarly fed the HFD. AngII caused hypertension and up-regulated the expression of genes contributing to pathological cardiac hypertrophy (Nppa, Myh7) more severely so in foz/foz mice than in controls. After 60 weeks of HFD, while liver disease had progressed to burn-out non steatotic MASH with hepatocellular carcinoma in 50% of the animals, the cardiomyopathy did not. In an independent model (C57BL/6J mice fed a fat-, cholesterol- and fructose-rich diet), moderate fibrosing MASH is associated with cardiac fibrosis and dysregulation of genes involved in pathological remodelling (Col1a1, Col3a1, Vim, Myh6, Slc2a1). Thus, animals with MASH present consistent adverse structural changes in the heart with no patent alteration of cardiac function even when stressed with exogenous AngII. Liver disease, and likely not overfeeding or aging alone, is associated with this cardiac phenotype. Our findings support foz/foz mice as suitable for studying links between MASH and heart structural changes ahead of heart failure.


Asunto(s)
Cardiomegalia , Dieta Alta en Grasa , Modelos Animales de Enfermedad , Ratones Endogámicos C57BL , Remodelación Ventricular , Animales , Dieta Alta en Grasa/efectos adversos , Cardiomegalia/fisiopatología , Cardiomegalia/patología , Cardiomegalia/etiología , Cardiomegalia/metabolismo , Angiotensina II/metabolismo , Hígado/metabolismo , Hígado/patología , Masculino , Ratones , Hígado Graso/patología , Hígado Graso/fisiopatología , Hígado Graso/metabolismo
3.
Part Fibre Toxicol ; 18(1): 7, 2021 02 09.
Artículo en Inglés | MEDLINE | ID: mdl-33563307

RESUMEN

BACKGROUND: Ambient air pollution by particulate matters, including diesel exhaust particles (DEP), is a major cause of cardiovascular and metabolic mortality worldwide. The mechanisms by which DEP cause these adverse outcomes are not completely understood. Because the gut microbiota controls cardiovascular and metabolic health, we hypothesized that the fraction of inhaled DEP which reach the gut after mucociliary clearance and swallowing might induce gut dysbiosis and, in turn, contribute to aggravate or induce cardiovascular and metabolic diseases. RESULTS: Female ApoE-/- mice fed a Western diet, and wild-type (C57Bl/6) mice fed standard diet were gavaged with DEP (SRM2975) doses corresponding to mucociliary clearance from inhalation exposure (200 or 1000 ng/day, 3 times a week for 3 months; and 40, 200 or 1000 ng/day, 3 times a week for 6 months, respectively). No mortality, overt systemic or digestive toxicity was observed. A dose-dependent alteration of the gut microbiota was recorded in both strains. In ApoE-/-, ß-diversity was modified by DEP, but no significant modification of the relative abundance of the phyla, families or genera was identified. In C57BL/6 mice, DEP reduced α-diversity (Shannon and Simpson indices), and modified ß-diversity, including a reduction of the Proteobacteria and Patescibacteria phyla, and an increase of the Campylobacterota phylum. In both mouse models, perturbation of the gut microbiota composition was associated with a dose-dependent reduction of bacterial short chain fatty acids (butyrate and propionate) in cecal content. However, DEP ingestion did not aggravate (ApoE-/-), or induce (C57BL/6 mice) atherosclerotic plaques, and no metabolic alteration (glucose tolerance, resistance to insulin, or lipidemia) was recorded. CONCLUSIONS: We show here that oral exposure to DEP, at doses relevant for human health, changes the composition and function of the gut microbiota. These modifications were, however, not translated into ultimate atherosclerotic or metabolic outcomes.


Asunto(s)
Microbioma Gastrointestinal , Administración Oral , Animales , Femenino , Ratones , Ratones Endogámicos C57BL , Material Particulado , Emisiones de Vehículos
4.
Int J Mol Sci ; 23(1)2021 Dec 23.
Artículo en Inglés | MEDLINE | ID: mdl-35008579

RESUMEN

Graves' disease (GD) is an autoimmune thyroiditis often associated with Graves' orbitopathy (GO). GD thyroid and GO orbital fat share high oxidative stress (OS) and hypervascularization. We investigated the metabolic pathways leading to OS and angiogenesis, aiming to further decipher the link between local and systemic GD manifestations. Plasma and thyroid samples were obtained from patients operated on for multinodular goiters (controls) or GD. Orbital fats were from GO or control patients. The NADPH-oxidase-4 (NOX4)/HIF-1α/VEGF-A signaling pathway was investigated by Western blotting and immunostaining. miR-199a family expression was evaluated following quantitative real-time PCR and/or in situ hybridization. In GD thyroids and GO orbital fats, NOX4 was upregulated and correlated with HIF-1α stabilization and VEGF-A overexpression. The biotin assay identified NOX4, HIF-1α and VEGF-A as direct targets of miR-199a-5p in cultured thyrocytes. Interestingly, GD thyroids, GD plasmas and GO orbital fats showed a downregulation of miR-199a-3p/-5p. Our results also highlighted an activation of STAT-3 signaling in GD thyroids and GO orbital fats, a transcription factor known to negatively regulate miR-199a expression. We identified NOX4/HIF-1α/VEGF-A as critical actors in GD and GO. STAT-3-dependent regulation of miR-199a is proposed as a common driver leading to these events in GD thyroids and GO orbital fats.


Asunto(s)
Tejido Adiposo/metabolismo , Regulación hacia Abajo/genética , Enfermedad de Graves/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , MicroARNs/genética , NADPH Oxidasa 4/genética , Glándula Tiroides/metabolismo , Factor A de Crecimiento Endotelial Vascular/genética , Adulto , Femenino , Oftalmopatía de Graves/genética , Oftalmopatía de Graves/metabolismo , Humanos , Masculino , Persona de Mediana Edad , Transducción de Señal/genética
5.
Am J Transplant ; 20(8): 2030-2043, 2020 08.
Artículo en Inglés | MEDLINE | ID: mdl-32012434

RESUMEN

With oxygenation proposed as a resuscitative measure during hypothermic models of preservation, the aim of this study was to evaluate the optimal start time of oxygenation during continuous hypothermic machine perfusion (HMP). In this porcine ischemia-reperfusion autotransplant model, the left kidney of a ±40 kg pig was exposed to 30 minutes of warm ischemia prior to 22 hours of HMP and autotransplantation. Kidneys were randomized to receive 2 hours of oxygenation during HMP either at the start (n = 6), or end of the perfusion (n = 5) and outcomes were compared to standard, nonoxygenated HMP (n = 6) and continuous oxygenated HMP (n = 8). The brief initial and continuous oxygenated HMP groups were associated with superior graft recovery compared to either standard, nonoxygenated HMP or kidneys oxygenated at the end of HMP. This correlated with significant metabolic differences in perfusate (eg, lactate, succinate, flavin mononucleotide) and tissues (eg, succinate, adenosine triphosphate, hypoxia-inducible factor-1α, nuclear factor erythroid 2-related factor 2) suggesting superior mitochondrial preservation with initial oxygenation. Brief initial O2 uploading during HMP at procurement site might be an easy and effective preservation strategy to maintain aerobic metabolism, protect mitochondria, and achieve an improved early renal graft function compared with standard HMP or oxygen supply shortly at the end of HMP preservation.


Asunto(s)
Hipotermia Inducida , Preservación de Órganos , Animales , Autoinjertos , Riñón , Perfusión , Porcinos , Trasplante Autólogo
6.
Arterioscler Thromb Vasc Biol ; 38(10): 2345-2357, 2018 10.
Artículo en Inglés | MEDLINE | ID: mdl-29976767

RESUMEN

Objective- Members of the microRNA (miR)-199a family, namely miR-199a-5p and miR-199a-3p, have been recently identified as potential regulators of cardiac homeostasis. Also, upregulation of miR-199a expression in cardiomyocytes was reported to influence endothelial cells. Whether miR-199a is expressed by endothelial cells and, if so, whether it directly regulates endothelial function remains unknown. We investigate the implication of miR-199a products on endothelial function by focusing on the NOS (nitric oxide synthase)/NO pathway. Approach and Results- Bovine aortic endothelial cells were transfected with specific miRNA inhibitors (locked-nucleic acids), and potential molecular targets identified with prediction algorithms were evaluated by Western blot or immunofluorescence. Ex vivo experiments were performed with mice treated with antagomiRs targeting miR-199a-3p or -5p. Isolated vessels and blood were used for electron paramagnetic resonance or myograph experiments. eNOS (endothelial NO synthase) activity (through phosphorylations Ser1177/Thr495) is increased by miR-199a-3p/-5p inhibition through an upregulation of the PI3K (phosphoinositide 3-kinase)/Akt (protein kinase B) and calcineurin pathways. SOD1 (superoxide dismutase 1) and PRDX1 (peroxiredoxin 1) upregulation was also observed in locked-nucleic acid-treated cells. Moreover, miR-199a-5p controls angiogenesis and VEGFA (vascular endothelial growth factor A) production and upregulation of NO-dependent relaxation were observed in vessels from antagomiR-treated mice. This was correlated with increased circulated hemoglobin-NO levels and decreased superoxide production. Angiotensin infusion for 2 weeks also revealed an upregulation of miR-199a-3p/-5p in vascular tissues. Conclusions- Our study reveals that miR-199a-3p and miR-199a-5p participate in a redundant network of regulation of the NOS/NO pathway in the endothelium. We highlighted that inhibition of miR-199a-3p and -5p independently increases NO bioavailability by promoting eNOS activity and reducing its degradation, thereby supporting VEGF-induced endothelial tubulogenesis and modulating vessel contractile tone.


Asunto(s)
Células Endoteliales/enzimología , Endotelio Vascular/enzimología , MicroARNs/metabolismo , Neovascularización Fisiológica , Óxido Nítrico Sintasa de Tipo III/metabolismo , Óxido Nítrico/metabolismo , Vasodilatación , Inhibidores de la Angiogénesis/farmacología , Animales , Antagomirs/genética , Antagomirs/metabolismo , Bovinos , Células Cultivadas , Modelos Animales de Enfermedad , Células Endoteliales/efectos de los fármacos , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/fisiopatología , Estabilidad de Enzimas , Regulación Neoplásica de la Expresión Génica , Hipertensión/enzimología , Hipertensión/genética , Hipertensión/fisiopatología , Masculino , Ratones Endogámicos C57BL , MicroARNs/genética , Neovascularización Fisiológica/efectos de los fármacos , Óxido Nítrico Sintasa de Tipo III/genética , Oligonucleótidos/genética , Oligonucleótidos/metabolismo , Peroxirredoxinas/metabolismo , Fosfatidilinositol 3-Quinasa/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Fosforilación , Inhibidores de Proteínas Quinasas/farmacología , Proteolisis , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal , Superóxido Dismutasa-1/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Vasodilatación/efectos de los fármacos
7.
Gut ; 67(2): 271-283, 2018 02.
Artículo en Inglés | MEDLINE | ID: mdl-28377388

RESUMEN

OBJECTIVE: To investigate the beneficial role of prebiotics on endothelial dysfunction, an early key marker of cardiovascular diseases, in an original mouse model linking steatosis and endothelial dysfunction. DESIGN: We examined the contribution of the gut microbiota to vascular dysfunction observed in apolipoprotein E knockout (Apoe-/-) mice fed an n-3 polyunsaturated fatty acid (PUFA)-depleted diet for 12 weeks with or without inulin-type fructans (ITFs) supplementation for the last 15 days. Mesenteric and carotid arteries were isolated to evaluate endothelium-dependent relaxation ex vivo. Caecal microbiota composition (Illumina Sequencing of the 16S rRNA gene) and key pathways/mediators involved in the control of vascular function, including bile acid (BA) profiling, gut and liver key gene expression, nitric oxide and gut hormones production were also assessed. RESULTS: ITF supplementation totally reverses endothelial dysfunction in mesenteric and carotid arteries of n-3 PUFA-depleted Apoe-/- mice via activation of the nitric oxide (NO) synthase/NO pathway. Gut microbiota changes induced by prebiotic treatment consist in increased NO-producing bacteria, replenishment of abundance in Akkermansia and decreased abundance in bacterial taxa involved in secondary BA synthesis. Changes in gut and liver gene expression also occur upon ITFs suggesting increased glucagon-like peptide 1 production and BA turnover as drivers of endothelium function preservation. CONCLUSIONS: We demonstrate for the first time that ITF improve endothelial dysfunction, implicating a short-term adaptation of both gut microbiota and key gut peptides. If confirmed in humans, prebiotics could be proposed as a novel approach in the prevention of metabolic disorders-related cardiovascular diseases.


Asunto(s)
Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/fisiopatología , Fructanos/farmacología , Microbioma Gastrointestinal/efectos de los fármacos , Prebióticos , Aminopeptidasas/genética , Animales , Péptidos Catiónicos Antimicrobianos/genética , Bacterias/efectos de los fármacos , Ácidos y Sales Biliares/biosíntesis , Ácidos y Sales Biliares/sangre , Arterias Carótidas/fisiología , Ciego/microbiología , Suplementos Dietéticos , Modelos Animales de Enfermedad , Ácidos Grasos Omega-3/administración & dosificación , Ácidos Grasos Omega-3/deficiencia , Expresión Génica/efectos de los fármacos , Péptido 1 Similar al Glucagón/biosíntesis , Masculino , Arterias Mesentéricas/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados para ApoE , Neurotensina/genética , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa/metabolismo , Transportadores de Anión Orgánico Sodio-Dependiente/genética , Proglucagón/genética , Simportadores/genética , Vasodilatación
8.
Ann Surg ; 266(5): 754-764, 2017 11.
Artículo en Inglés | MEDLINE | ID: mdl-28742686

RESUMEN

OBJECTIVE: During the last decade, face allotransplantation has been shown to be a revolutionary reconstructive procedure for severe disfigurements. However, offer to patients remains limited due to lifelong immunosuppression. To move forward in the field, a new pathway in tissue engineering is proposed. BACKGROUND: Our previously reported technique of matrix production of a porcine auricular subunit graft has been translated to a human face model. METHODS: 5 partial and 1 total face grafts were procured from human fresh cadavers. After arterial cannulation, the specimens were perfused using a combined detergent/polar solvent decellularization protocol. Preservation of vascular patency was assessed by imaging, cell and antigen removal by DNA quantification and histology. The main extracellular matrix proteins and associated cytokines were evaluated. Lip scaffolds were cultivated with dermal, muscle progenitor and endothelial cells, either on discs or in a bioreactor. RESULTS: Decellularization was successful in all facial grafts within 12 days revealing acellular scaffolds with full preservation of innate morphology. Imaging demonstrated a preservation of the entire vascular tree patency. Removal of cells and antigens was confirmed by reduction of DNA and antigen markers negativation. Microscopic evaluation revealed preservation of tissue structures as well as of major proteins. Seeded cells were viable and well distributed within all scaffolds. CONCLUSIONS: Complex acellular facial scaffolds were obtained, preserving simultaneously a cell-friendly extracellular matrix and a perfusable vascular tree. This step will enable further engineering of postmortem facial grafts, thereby offering new perspectives in composite tissue allotransplantation.


Asunto(s)
Trasplante Facial , Ingeniería de Tejidos/métodos , Biomarcadores/metabolismo , Reactores Biológicos , Citocinas/metabolismo , Matriz Extracelular/metabolismo , Humanos , Inmunohistoquímica , Técnicas In Vitro , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Perfusión/métodos , Reperfusión/métodos , Andamios del Tejido
9.
J Immunol ; 194(4): 1874-81, 2015 Feb 15.
Artículo en Inglés | MEDLINE | ID: mdl-25595790

RESUMEN

Nucleotides are released in the heart under pathological conditions, but little is known about their contribution to cardiac inflammation. The present study defines the P2Y4 nucleotide receptor, expressed on cardiac microvascular endothelial cells and involved in postnatal heart development, as an important regulator of the inflammatory response to cardiac ischemia. P2Y4-null mice displayed smaller infarcts in the left descending artery ligation model, as well as reduced neutrophil infiltration and fibrosis. Gene profiling identified inter alia endothelin-1 (ET-1) as one of the target genes of P2Y4 in ischemic heart. The reduced level of ET-1 was correlated with reduction of microvascular hyperpermeability, neutrophil infiltration, and endothelial adhesion molecule expression, and it could be explained by the decreased number of endothelial cells in P2Y4-null mice. Expression analysis of metalloproteinases and their tissue inhibitors in ischemic heart revealed reduced expression of matrix metalloproteinase (MMP)-9, reported to be potentially regulated by ET-1, and MMP-8, considered as neutrophil collagenase, as well as reduction of tissue inhibitor of MMP-1 and tissue inhibitor of MMP-4 in P2Y4-null mice. Reduction of cardiac permeability and neutrophil infiltration was also observed in P2Y4-null mice in LPS-induced inflammation model. Protection against infarction resulting from loss of P2Y4 brings new therapeutic perspectives for cardiac ischemia and remodeling.


Asunto(s)
Endotelina-1/biosíntesis , Infarto del Miocardio/metabolismo , Infarto del Miocardio/patología , Receptores Purinérgicos P2/deficiencia , Animales , Modelos Animales de Enfermedad , Regulación hacia Abajo , Ensayo de Inmunoadsorción Enzimática , Femenino , Inmunohistoquímica , Ratones , Ratones Noqueados , Infarto del Miocardio/fisiopatología , Reacción en Cadena en Tiempo Real de la Polimerasa , Transcriptoma
10.
J Pharmacol Exp Ther ; 358(1): 151-63, 2016 07.
Artículo en Inglés | MEDLINE | ID: mdl-27189971

RESUMEN

Activation of peroxisome proliferator-activated receptor-ß/δ (PPARß) lowers blood pressure in genetic and mineralocorticoid-induced hypertension. Regulator of G-protein-coupled receptor signaling 5 (RGS5) protein, which interferes in angiotensin II (AngII) signaling, is a target gene to PPARß The aim of the present study was to examine whether PPARß activation in resistance arteries and brain tissues prevents the elevated blood pressure in AngII-induced hypertension and evaluate the role of RGS5 in this effect. C57BL/6J male mice were divided into five groups (control mice, PPARß agonist [4-[[[2-[3-Fluoro-4-(trifluoromethyl)phenyl]-4-methyl-5-thiazolyl]methyl]thio]-2-methylphenoxy]acetic acid (GW0742)-treated mice AngII-infused mice, GW0742-treated AngII-infused mice, and AngII-infused mice treated with GW0742 plus PPARß antagonist 3-[[[2-Methoxy-4-(phenylamino)phenyl]amino]sulfonyl]-2-thiophenecarboxylic acid methyl ester (GSK0660)) and were followed for 3 weeks. GW0742 prevented the increase in both arterial blood pressure and plasma noradrenaline levels and the higher reduction of blood pressure after ganglionic blockade, whereas it reduced the mesenteric arterial remodeling and the hyper-responsiveness to vasoconstrictors (AngII and endothelin-1) in AngII-infused mice. These effects were accompanied by an inhibition of NADPH oxidase expression and activity in the brain. Gene expression profiling revealed a marked loss of brainstem and vascular RGS5 in AngII-infused mice, which was restored by GW0742. GW0742-induced effects were abolished by GSK0660. Small interfering RNA targeting RGS5 caused augmented contractile response to AngII in resistance mesenteric arteries and blunted the inhibitory effect of GW0742 on this response. In conclusion, GW0742 exerted antihypertensive effects, restoring sympathetic tone and vascular structure and function in AngII-infused mice by PPARß activation in brain and vessels inhibiting AngII signaling as a result of RGS5 upregulation.


Asunto(s)
Angiotensina II/farmacología , Antihipertensivos/uso terapéutico , Hipertensión/tratamiento farmacológico , PPAR-beta/agonistas , Proteínas RGS/metabolismo , Tiazoles/uso terapéutico , Animales , Antihipertensivos/administración & dosificación , Presión Sanguínea/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Frecuencia Cardíaca/efectos de los fármacos , Hipertensión/inducido químicamente , Hipertensión/metabolismo , Hipertensión/patología , Masculino , Arterias Mesentéricas/efectos de los fármacos , Arterias Mesentéricas/patología , Arterias Mesentéricas/fisiopatología , Ratones Endogámicos C57BL , Norepinefrina/sangre , PPAR-beta/antagonistas & inhibidores , Proteínas RGS/genética , Sulfonas/farmacología , Tiazoles/administración & dosificación , Tiofenos/farmacología , Resistencia Vascular/efectos de los fármacos , Vasoconstricción/efectos de los fármacos
11.
Circulation ; 129(4): 451-62, 2014 Jan 28.
Artículo en Inglés | MEDLINE | ID: mdl-24190960

RESUMEN

BACKGROUND: ß1-2-adrenergic receptors (AR) are key regulators of cardiac contractility and remodeling in response to catecholamines. ß3-AR expression is enhanced in diseased human myocardium, but its impact on remodeling is unknown. METHODS AND RESULTS: Mice with cardiac myocyte-specific expression of human ß3-AR (ß3-TG) and wild-type (WT) littermates were used to compare myocardial remodeling in response to isoproterenol (Iso) or Angiotensin II (Ang II). ß3-TG and WT had similar morphometric and hemodynamic parameters at baseline. ß3-AR colocalized with caveolin-3, endothelial nitric oxide synthase (NOS) and neuronal NOS in adult transgenic myocytes, which constitutively produced more cyclic GMP, detected with a new transgenic FRET sensor. Iso and Ang II produced hypertrophy and fibrosis in WT mice, but not in ß3-TG mice, which also had less re-expression of fetal genes and transforming growth factor ß1. Protection from Iso-induced hypertrophy was reversed by nonspecific NOS inhibition at low dose Iso, and by preferential neuronal NOS inhibition at high-dose Iso. Adenoviral overexpression of ß3-AR in isolated cardiac myocytes also increased NO production and attenuated hypertrophy to Iso and phenylephrine. Hypertrophy was restored on NOS or protein kinase G inhibition. Mechanistically, ß3-AR overexpression inhibited phenylephrine-induced nuclear factor of activated T-cell activation. CONCLUSIONS: Cardiac-specific overexpression of ß3-AR does not affect cardiac morphology at baseline but inhibits the hypertrophic response to neurohormonal stimulation in vivo and in vitro, through a NOS-mediated mechanism. Activation of the cardiac ß3-AR pathway may provide future therapeutic avenues for the modulation of hypertrophic remodeling.


Asunto(s)
Ventrículos Cardíacos/patología , Miocitos Cardíacos/metabolismo , Neurotransmisores/farmacología , Óxido Nítrico Sintasa/fisiología , Receptores Adrenérgicos beta 3/metabolismo , Remodelación Ventricular/efectos de los fármacos , Angiotensina II/efectos adversos , Angiotensina II/farmacología , Animales , Células Cultivadas , GMP Cíclico/fisiología , Proteínas Quinasas Dependientes de GMP Cíclico/fisiología , Modelos Animales de Enfermedad , Ventrículos Cardíacos/fisiopatología , Humanos , Hipertrofia/inducido químicamente , Hipertrofia/patología , Hipertrofia/fisiopatología , Técnicas In Vitro , Isoproterenol/efectos adversos , Isoproterenol/farmacología , Masculino , Ratones , Ratones Transgénicos , Miocitos Cardíacos/patología , Miocitos Cardíacos/fisiología , Neurotransmisores/efectos adversos , Receptores Adrenérgicos beta 3/genética , Transducción de Señal/fisiología , Remodelación Ventricular/fisiología
12.
Proc Natl Acad Sci U S A ; 109(31): 12526-31, 2012 Jul 31.
Artículo en Inglés | MEDLINE | ID: mdl-22802674

RESUMEN

We report the structure-based design and synthesis of a unique NOS inhibitor, called nanoshutter NS1, with two-photon absorption properties. NS1 targets the NADPH site of NOS by a nucleotide moiety mimicking NADPH linked to a conjugated push-pull chromophore with nonlinear absorption properties. Because NS1 could not provide reducing equivalents to the protein and competed with NADPH binding, it efficiently inhibited NOS catalysis. NS1 became fluorescent once bound to NOS with an excellent signal-to-noise ratio because of two-photon excitation avoiding interference from the flavin-autofluorescence and because free NS1 was not fluorescent in aqueous solutions. NS1 fluorescence enhancement was selective for constitutive NOS in vitro, in particular for endothelial NOS (eNOS). Molecular dynamics simulations suggested that two variable residues among NOS isoforms induced differences in binding of NS1 and in local solvation around NS1 nitro group, consistent with changes of NS1 fluorescence yield. NS1 colocalized with eNOS in living human umbilical vein endothelial cells. Thus, NS1 constitutes a unique class of eNOS probe with two-photon excitation in the 800-950-nm range, with great perspectives for eNOS imaging in living tissues.


Asunto(s)
Colorantes Fluorescentes , Células Endoteliales de la Vena Umbilical Humana/enzimología , Microscopía de Fluorescencia por Excitación Multifotónica/métodos , NADP , Óxido Nítrico Sintasa de Tipo III , Catálisis , Colorantes Fluorescentes/síntesis química , Colorantes Fluorescentes/química , Colorantes Fluorescentes/farmacología , Células Endoteliales de la Vena Umbilical Humana/química , Células Endoteliales de la Vena Umbilical Humana/citología , Humanos , Isoenzimas/química , Isoenzimas/metabolismo , Simulación de Dinámica Molecular , NADP/análogos & derivados , NADP/síntesis química , NADP/química , NADP/farmacología , Óxido Nítrico/biosíntesis , Óxido Nítrico/química , Óxido Nítrico Sintasa de Tipo III/química , Óxido Nítrico Sintasa de Tipo III/metabolismo
13.
Am J Physiol Heart Circ Physiol ; 307(8): H1120-33, 2014 Oct 15.
Artículo en Inglés | MEDLINE | ID: mdl-25128166

RESUMEN

Exposure of cardiomyocytes to high glucose concentrations (HG) stimulates reactive oxygen species (ROS) production by NADPH oxidase (NOX2). NOX2 activation is triggered by enhanced glucose transport through a sodium-glucose cotransporter (SGLT) but not by a stimulation of glucose metabolism. The aim of this work was to identify potential therapeutic approaches to counteract this glucotoxicity. In cultured adult rat cardiomyocytes incubated with 21 mM glucose (HG), AMP-activated protein kinase (AMPK) activation by A769662 or phenformin nearly suppressed ROS production. Interestingly, glucagon-like peptide 1 (GLP-1), a new antidiabetic drug, concomitantly induced AMPK activation and prevented the HG-mediated ROS production (maximal effect at 100 nM). α2-AMPK, the major isoform expressed in cardiomyocytes (but not α1-AMPK), was activated in response to GLP-1. Anti-ROS properties of AMPK activators were not related to changes in glucose uptake or glycolysis. Using in situ proximity ligation assay, we demonstrated that AMPK activation prevented the HG-induced p47phox translocation to caveolae, whatever the AMPK activators used. NOX2 activation by either α-methyl-d-glucopyranoside, a glucose analog transported through SGLT, or angiotensin II was also counteracted by GLP-1. The crucial role of AMPK in limiting HG-mediated NOX2 activation was demonstrated by overexpressing a constitutively active form of α2-AMPK using adenoviral infection. This overexpression prevented NOX2 activation in response to HG, whereas GLP-1 lost its protective action in α2-AMPK-deficient mouse cardiomyocytes. Under HG, the GLP-1/AMPK pathway inhibited PKC-ß2 phosphorylation, a key element mediating p47phox translocation. In conclusion, GLP-1 induces α2-AMPK activation and blocks HG-induced p47phox translocation to the plasma membrane, thereby preventing glucotoxicity.


Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Péptido 1 Similar al Glucagón/farmacología , Glucosa/farmacología , Hipoglucemiantes/farmacología , Miocitos Cardíacos/metabolismo , NADPH Oxidasas/metabolismo , Proteínas Quinasas Activadas por AMP/genética , Animales , Compuestos de Bifenilo , Células Cultivadas , Masculino , Glicoproteínas de Membrana/metabolismo , Metilglucósidos/farmacología , Miocitos Cardíacos/efectos de los fármacos , NADPH Oxidasa 2 , NADPH Oxidasas/genética , Fenformina/farmacología , Proteína Quinasa C/metabolismo , Transporte de Proteínas , Pironas/farmacología , Ratas , Ratas Wistar , Especies Reactivas de Oxígeno/metabolismo , Tiofenos/farmacología
14.
Arterioscler Thromb Vasc Biol ; 32(9): 2241-9, 2012 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-22814746

RESUMEN

OBJECTIVE: We investigated the impact of hypoxia-reoxygenation on endothelial relaxation and aimed to clarify the role of transient receptor potential cation channels V4 (TRPV4) and gap junctions in the protective effect associated with hypoxic preconditioning on the vascular function. METHODS AND RESULTS: By mimicking ischemia-reperfusion in C57BL/6 male mice in vivo, we documented a reduced NO-mediated relaxation and an increased endothelium-derived hyperpolarization (EDH[F])-mediated relaxation. Hypoxic preconditioning, however, restored NO relaxation and further improved the EDH(F) response. We also examined specifically 2 major effectors of the EDH(F) pathway, transient receptor potential cation channels V4 and connexins. We found that in endothelial cells, expression and activity of transient receptor potential cation channels V4 were increased by hypoxic stimuli independently of preconditioning which was interestingly associated with an increase of structural caveolar component caveolin-1 at membrane locations. Gap junctions, however, seemed to directly support EDH(F)-driven preconditioning as connexin 40 and connexin 43 expression increased and as in vivo carbenoxolone treatment completely inhibited the EDH(F) pathway and significantly reduced the protection afforded by preconditioning for the concomitant NO-mediated relaxation. CONCLUSIONS: Our work provides evidence on how transient receptor potential cation channels V4 and connexins might participate in preserving vasorelaxation under hypoxia and restoring the NO-mediated pathway in hypoxic preconditioning conditions pointing out caveolae as a common signaling location.


Asunto(s)
Señalización del Calcio , Comunicación Celular , Endotelio Vascular/metabolismo , Uniones Comunicantes/metabolismo , Hipoxia/metabolismo , Daño por Reperfusión Miocárdica/prevención & control , Canales Catiónicos TRPV/metabolismo , Vasodilatación , Animales , Factores Biológicos/metabolismo , Señalización del Calcio/efectos de los fármacos , Caveolas/metabolismo , Caveolina 1/metabolismo , Comunicación Celular/efectos de los fármacos , Células Cultivadas , Conexinas/metabolismo , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/fisiopatología , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Hipoxia/genética , Hipoxia/fisiopatología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Daño por Reperfusión Miocárdica/genética , Daño por Reperfusión Miocárdica/metabolismo , Daño por Reperfusión Miocárdica/fisiopatología , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa de Tipo III/metabolismo , Canales Catiónicos TRPV/deficiencia , Canales Catiónicos TRPV/genética , Factores de Tiempo , Vasodilatación/efectos de los fármacos , Vasodilatadores/farmacología
15.
Antioxidants (Basel) ; 12(2)2023 Feb 10.
Artículo en Inglés | MEDLINE | ID: mdl-36830003

RESUMEN

ROS in cancer cells play a key role in pathways regulating cell death, stemness maintenance, and metabolic reprogramming, all of which have been implicated in resistance to chemo/ immunotherapy. Adjusting ROS levels to reverse the resistance of cancer cells without impairing normal cell functions is a new therapeutic avenue. In this paper, we describe new inhibitors of NADPH oxidase (NOX), a key enzyme in many cells of the tumor microenvironment. The first inhibitor, called Nanoshutter-1, NS1, decreased the level of tumor-promoting "M2" macrophages differentiated from human blood monocytes. NS1 disrupted the active NADPH oxidase-2 (NOX2) complex at the membrane and in the mitochondria of the macrophages, as shown by confocal microscopy. As one of the characteristics of tumor invasion is hypoxia, we tested whether NS1 would affect vascular reactivity by reducing ROS or NO levels in wire and pressure myograph experiments on isolated blood vessels. The results show that NS1 vasodilated blood vessels and would likely reduce hypoxia. Finally, as both NOX2 and NOX4 are key proteins in tumors and their microenvironment, we investigated whether NS1 would probe these proteins differently. Models of NOX2 and NOX4 were generated by homology modeling, showing structural differences at their C-terminal NADPH site, in particular in their last Phe. Thus, the NADPH site presents an unexploited chemical space for addressing ligand specificity, which we exploited to design a novel NOX2-specific inhibitor targeting variable NOX2 residues. With the proper smart vehicle to target specific cells of the microenvironment as TAMs, NOX2-specific inhibitors could open the way to new precision therapies.

16.
Angiogenesis ; 15(3): 349-60, 2012 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-22437266

RESUMEN

Communication between endothelial cells and cardiomyocytes is critical for cardiac development and regeneration. However the mechanisms involved in these endothelial-cardiomyocyte interactions remain poorly understood. Nucleotides are released within the heart, especially under ischemia or pressure overload. The function of P2Y nucleotide receptors in cardiac development has never been investigated. Here we show that adult P2Y(4)-null mice display microcardia. P2Y(4) nucleotide receptor is expressed in cardiac endothelial cells but not in cardiomyocytes. Loss of P2Y(4) in cardiac endothelial cells strongly inhibits their growth, migration and PDGF-B secretion in response to UTP. Proliferation of microvessels and cardiomyocytes is reduced in P2Y(4)-null hearts early after birth, resulting in reduced heart growth. Our study uncovers mouse P2Y(4) receptor as an essential regulator of cardiac endothelial cell function, and illustrates the involvement of endothelial-cardiomyocyte interactions in post-natal heart development. We also detected P2Y(4) expression in human cardiac microvessels. P2Y(4) receptor could constitute a therapeutic target to regulate cardiac remodelling and post-ischemic revascularisation.


Asunto(s)
Corazón/crecimiento & desarrollo , Receptores Purinérgicos P2/fisiología , Animales , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Humanos , Inmunohistoquímica , Ratones , Ratones Noqueados , Neovascularización Fisiológica , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa
17.
Am J Physiol Heart Circ Physiol ; 303(7): H835-43, 2012 Oct 01.
Artículo en Inglés | MEDLINE | ID: mdl-22865387

RESUMEN

Nucleotides released within the heart under pathological conditions can be involved in cardioprotection or cardiac fibrosis through the activation purinergic P2Y(2) and P2Y(6) receptors, respectively. We previously demonstrated that adult P2Y(4)-null mice display a microcardia phenotype related to a cardiac angiogenic defect. To evaluate the functional consequences of this defect, we performed here a combination of cardiac monitoring and exercise tests. We investigated the exercise capacity of P2Y(4) wild-type and P2Y(4)-null mice in forced swimming and running tests. Analysis of their stress, locomotion, and resignation was realized in open field, black and white box, and tail suspension experiments. Exercise-induced cardiac hypertrophy was evaluated after repeated and prolonged exercise in P2Y(4) wild-type and P2Y(4)-null hearts. We showed that P2Y(4)-null mice have a lower exercise capacity in both swimming and treadmill tests. This was not related to decreased motivation or increased stress, since open field, white and black box, and mouse tail suspension tests gave comparable results in P2Y(4) wild-type and P2Y(4)-null mice. Heart rate and blood pressure rose normally in P2Y(4)-null swimming mice equipped with a telemetric implant. On the contrary, we observed a delayed recovery of postexercise blood pressure after exercise in P2Y(4)-null mice. The heart rate increment in response to catecholamines was also similar in P2Y(4) wild-type and P2Y(4)-null implanted mice, which is consistent with a similar level of cardiac ß-receptor expression. Interestingly, the heart of P2Y(4)-null mice displayed a reduced sympathetic innervation associated with a decreased norepinephrine level. We also demonstrated that exercise-induced cardiac hypertrophy was lower in P2Y(4)-null mice after repeated and prolonged exercise. This was associated with a lower increase in cardiomyocyte size and microvessel density. In conclusion, besides its role in cardiac development, P2Y(4) receptor could constitute an important regulator of acute and chronic response to exercise.


Asunto(s)
Cardiomegalia Inducida por el Ejercicio/genética , Cardiomegalia/prevención & control , Tolerancia al Ejercicio/genética , Eliminación de Gen , Corazón/fisiopatología , Miocardio/metabolismo , Receptores Purinérgicos P2/deficiencia , Natación , Fibras Adrenérgicas/metabolismo , Agonistas Adrenérgicos beta/farmacología , Animales , Conducta Animal , Presión Sanguínea/genética , Monitoreo Ambulatorio de la Presión Arterial , Cardiomegalia/genética , Cardiomegalia/metabolismo , Cardiomegalia/patología , Cardiomegalia/fisiopatología , Catecolaminas/metabolismo , Modelos Animales de Enfermedad , Dobutamina/farmacología , Prueba de Esfuerzo , Tolerancia al Ejercicio/efectos de los fármacos , Genotipo , Corazón/inervación , Frecuencia Cardíaca/genética , Hipotermia/genética , Hipotermia/metabolismo , Hipotermia/fisiopatología , Locomoción , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Miocardio/patología , Fenotipo , Receptores Adrenérgicos beta/efectos de los fármacos , Receptores Adrenérgicos beta/metabolismo , Receptores Purinérgicos P2/genética , Recuperación de la Función , Telemetría , Factores de Tiempo
18.
J Vasc Res ; 49(6): 522-33, 2012.
Artículo en Inglés | MEDLINE | ID: mdl-22948674

RESUMEN

In addition to its role in the regulation of artery contraction, Rho kinase (ROCK) was reported to be involved in the cytosolic calcium response to vasoconstrictor agonists in rat aorta and superior mesenteric artery (SMA). However, it remains to be determined whether ROCK also contributes to calcium signaling in resistance arteries, which play a major role in blood pressure regulation. The investigation of the effect of ROCK inhibition on the calcium and contractile responses of rat resistance mesenteric artery (RMA), in comparison with aorta and SMA, indicated that the calcium response to noradrenaline was inhibited by the ROCK inhibitor Y-27632 in aorta and SMA but not in RMA. The effect of Y-27632 on the calcium signal was unaffected by cytochalasin-D. ROCK activation in noradrenaline-stimulated arteries was confirmed by the inhibition of myosin light chain phosphorylation by Y-27632. Moreover, noradrenaline-induced calcium signaling was similarly inhibited by nimodipine in aorta, SMA and RMA, but nimodipine sensitivity of the contraction increased from the aorta to the RMA, suggesting that the contraction was controlled by different sources of calcium. In pressurized RMA, Y-27632 and H-1152 depressed pressure-induced calcium responses and abolished myogenic contraction. These results stress the important differences in calcium signaling between conductance and resistance arteries.


Asunto(s)
Amidas/farmacología , Aorta/efectos de los fármacos , Señalización del Calcio/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Arterias Mesentéricas/efectos de los fármacos , Piridinas/farmacología , Quinasas Asociadas a rho/antagonistas & inhibidores , Animales , Aorta/metabolismo , Masculino , Arteria Mesentérica Superior/efectos de los fármacos , Norepinefrina/farmacología , Ratas , Ratas Wistar , Vasoconstricción/efectos de los fármacos , Vasoconstrictores/farmacología
19.
Curr Hypertens Rep ; 14(4): 310-7, 2012 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-22639015

RESUMEN

Beta-blockers are widely prescribed for the treatment of a variety of cardiovascular pathologies. Compared to traditional beta-adrenergic antagonists, beta-blockers of the new generation exhibit ancillary properties such as vasodilation through different mechanisms. This translates into a more favorable hemodynamic profile. The relative affinities of beta-adrenoreceptor antagonists towards the three beta-adrenoreceptor isotypes matter for predicting their functional impact on vasomotor control. This review will focus on the mechanisms underlying beta-blocker-evoked vasorelaxation with a specific emphasis on agonist properties of beta(3)-adrenergic receptors.


Asunto(s)
Antagonistas Adrenérgicos beta/farmacología , Antihipertensivos/farmacología , Endotelio Vascular/efectos de los fármacos , Hipertensión/tratamiento farmacológico , Receptores Adrenérgicos beta/efectos de los fármacos , Vasodilatación/efectos de los fármacos , Antagonistas Adrenérgicos beta/uso terapéutico , Antihipertensivos/uso terapéutico , Benzopiranos/farmacología , Etanolaminas/farmacología , Hemodinámica/efectos de los fármacos , Humanos , Hipertensión/patología , Nebivolol
20.
Arterioscler Thromb Vasc Biol ; 31(9): 2098-105, 2011 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-21659644

RESUMEN

OBJECTIVE: We analyzed the role of caveolin-1 (Cav-1) in the cross-talk between NADPH oxidase and endothelial nitric oxide synthase (eNOS) signaling in endothelial caveolae. METHODS AND RESULTS: In intact endothelial cells, angiotensin II (AII) concurrently increased NO and O(2)(-·) production (to 158±12% and 209±5% of control). NO production was sensitive to inhibition of NADPH oxidase and small interfering RNA downregulation of nonreceptor tyrosine kinase cAbl. Reciprocally, N-nitro-l-arginine methyl ester, a NOS inhibitor, partly inhibited O(2)(-·) stimulated by AII (by 47±11%), indicating eNOS uncoupling, as confirmed by increased eNOS monomer/dimer ratio (by 35%). In endothelial cell fractions separated by isopycnic ultracentrifugation, AII promoted colocalization of cAbl and the NADPH oxidase subunit p47phox with eNOS to Cav-1-enriched fractions, as confirmed by proximity ligation assay. Downregulation of Cav-1 by small interfering RNA (to 50%), although it preserved eNOS confinement, inhibited AII-stimulated p47phox translocation and NADPH oxidase activity in Cav-1-enriched fractions and reversed eNOS uncoupling. AII infusion produced hypertension and decreased blood hemoglobin-NO in Cav-1(+/+) mice but not in heterozygote Cav-1(+/-) mice with similar Cav-1 reduction. CONCLUSIONS: Cav-1 critically regulates reactive oxygen species-dependent eNOS activation but also eNOS uncoupling in response to AII, underlining the possibility to treat endothelial dysfunction by modulating Cav-1 abundance.


Asunto(s)
Angiotensina II/farmacología , Caveolina 1/fisiología , Células Endoteliales/metabolismo , NADPH Oxidasas/fisiología , Óxido Nítrico Sintasa de Tipo III/fisiología , Animales , Células Cultivadas , Regulación hacia Abajo , Células Endoteliales/efectos de los fármacos , Hemoglobinas/metabolismo , Humanos , Hipertensión/prevención & control , Masculino , Ratones , Óxido Nítrico/biosíntesis , Proteínas Proto-Oncogénicas c-abl/fisiología , Especies Reactivas de Oxígeno/metabolismo , Superóxidos/metabolismo
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