RESUMEN
INTRODUCTION: Evidences have been suggested that phosphodiesterase type 5 (PDE5) inhibition promotes vasculoprotective benefits in patients with cardiovascular diseases. AIM: The aim of this study is to analyze the systemic effect of PDE5 inhibition in type 2 diabetes mellitus patients with erectile dysfunction (ED) determining changes in the expression levels of plasma proteins. METHODS: Seventeen patients with controlled type 2 diabetes mellitus and ED were included in the study. Patients received vardenafil hydrochloride 20 mg on demand during 12 weeks. At the beginning and 12 weeks after vardenafil administration, plasma samples were collected and analyzed using proteomics. MAIN OUTCOME MEASURES: International Index of Erectile Function-Erectile Function Domain (IIEF-EFD) and plasma protein expression before and after vardenafil administration. Nitrate/nitrite release, PDE5, and soluble guanylate cyclase (sGC) expression and cyclic guanosine monophosphate (cGMP) content in cultured bovine aortic endothelial cells (BAECs). RESULTS: The IIEF-EFD score was markedly improved after 12 weeks of vardenafil administration. Plasma levels of alpha 1-antitrypsin isotypes 4 and 6 and ß-tropomyosin were decreased, whereas apolipoprotein AI isoype 5 was increased 12 weeks after vardenafil administration. Only ß-tropomyosin plasma levels were inversely correlated with IIEF-EFD score. Tropomyosin has been added to cultured BAECs and after 24 hours reduced the protein expression level of sGC-ß1 subunit and decreased the cGMP content. Tropomyosin did not modify PDE5 expression and nitric oxide release in BAECs as compared with control BAECs. Vardenafil (10 µg/mL) did not modify sGC-ß1 subunit expression in tropomyosin + vardenafil-incubated BAECs; however, vardenafil significantly reversed the reduction of cGMP content induced by tropomyosin. CONCLUSION: Vardenafil administration improved erectile functionality in controlled type 2 diabetes mellitus patients with ED, which was associated with reduction of circulating plasma ß-tropomyosin levels. Tropomyosin affected by itself the cGMP generating system suggesting a possible new mechanism involved in ED. Vardenafil reversed the reduction effect of cGMP content elicited by tropomyosin in BAECs.
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Diabetes Mellitus Tipo 2/complicaciones , Disfunción Eréctil/tratamiento farmacológico , Imidazoles/uso terapéutico , Erección Peniana/efectos de los fármacos , Inhibidores de Fosfodiesterasa 5/uso terapéutico , Piperazinas/uso terapéutico , Tropomiosina/fisiología , Animales , Bovinos , GMP Cíclico/metabolismo , Disfunción Eréctil/sangre , Disfunción Eréctil/etiología , Guanilato Ciclasa/metabolismo , Humanos , Imidazoles/administración & dosificación , Masculino , Persona de Mediana Edad , Óxido Nítrico/metabolismo , Óxido Nítrico/farmacología , Inhibidores de Fosfodiesterasa 5/administración & dosificación , Hidrolasas Diéster Fosfóricas/metabolismo , Piperazinas/administración & dosificación , Receptores Citoplasmáticos y Nucleares/metabolismo , Guanilil Ciclasa Soluble , Sulfonas/administración & dosificación , Sulfonas/uso terapéutico , Triazinas/administración & dosificación , Triazinas/uso terapéutico , Tropomiosina/sangre , Diclorhidrato de VardenafilRESUMEN
BACKGROUND: Heart produces ATP through long-chain fatty acids beta oxidation. PURPOSE: To analyze whether in ventricular myocardium, high-fat diet may modify the expression of proteins associated with energy metabolism before myocardial function was affected. METHODS: Wistar Kyoto rats were divided into two groups: (a) rats fed standard diet (control; n = 6) and (b) rats fed high-fat diet (HFD; n = 6). Proteins from left ventricles were analyzed by two-dimensional electrophoresis, mass spectrometry and Western blotting. RESULTS: Rats fed with HFD showed higher body weight, insulin, glucose, leptin and total cholesterol plasma levels as compared with those fed with standard diet. However, myocardial functional parameters were not different between them. The protein expression of 3-ketoacyl-CoA thiolase, acyl-CoA hydrolase mitochondrial precursor and enoyl-CoA hydratase, three long-chain fatty acid ß-oxidation-related enzymes, and carnitine-O-palmitoyltransferase I was significantly higher in left ventricles from HFD rats. Protein expression of triosephosphate isomerase was higher in left ventricles from HFD rats than in those from control. Two α/ß-enolase isotypes and glyceraldehyde-3-phosphate isomerase were significantly increased in HFD rats as compared with control. Pyruvate and lactate contents were similar in HFD and control groups. Expression of proteins associated with Krebs cycle and mitochondrial oxidative phosphorylation was higher in HFD rats. CONCLUSIONS: Expression of proteins involved in left ventricle metabolic energy was enhanced before myocardial functionality was affected in rats fed with HFD. These findings may probably indicate higher cardiac energy requirement due to weight increase by HFD.
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Dieta Alta en Grasa , Metabolismo Energético , Redes y Vías Metabólicas/fisiología , Miocardio/metabolismo , Sobrepeso/metabolismo , Acetil-CoA C-Aciltransferasa/genética , Acetil-CoA C-Aciltransferasa/metabolismo , Animales , Western Blotting , Peso Corporal , Carnitina O-Palmitoiltransferasa/genética , Carnitina O-Palmitoiltransferasa/metabolismo , Colesterol/sangre , Enoil-CoA Hidratasa/genética , Enoil-CoA Hidratasa/metabolismo , Ácidos Grasos/metabolismo , Gliceraldehído 3-Fosfato/genética , Gliceraldehído 3-Fosfato/metabolismo , Procesamiento de Imagen Asistido por Computador , Insulina/sangre , Ácido Láctico/análisis , Leptina/sangre , Masculino , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Fosforilación Oxidativa , Palmitoil-CoA Hidrolasa/genética , Palmitoil-CoA Hidrolasa/metabolismo , Fosfopiruvato Hidratasa/genética , Fosfopiruvato Hidratasa/metabolismo , Ácido Pirúvico/análisis , Ratas , Ratas Endogámicas WKY , Triglicéridos/sangre , Triosa-Fosfato Isomerasa/genética , Triosa-Fosfato Isomerasa/metabolismoRESUMEN
Stroke patients have a high risk of vascular recurrence. Biomarkers related to vascular recurrence, however, remain to be identified. The aim of the study was to identify, through proteomic analysis, plasma biomarkers associated with vascular recurrence within one year after the first ischemic stroke. This is a substudy (n = 134) of a large prospective multicenter study of post-stroke patients with an ischemic stroke. Plasma samples were obtained at inclusion. Among the identified proteins, only plasma levels of desmoplakin I were associated with protection against a new vascular event (Odds ratio: 0.64; 95% CI: 0.46-0.89; p = 0.009) after adjustment for hypercholesterolemia, statins and previous atherothrombotic stroke subtype. A greater number of patients without vascular recurrence had been treated with statins within three months of the recent ischemic stroke. Only patients who had been taking statins for 3 months after the ischemic stroke and did not suffer vascular recurrence over a follow-up year, have higher levels of desmoplakin I at the time of inclusion (Odds ratio 0.49; 95% CI: 0.28-0.86; p = 0.013). Increased desmoplakin I levels, determined within 1-3 months of the first ischemic stroke, could be a biomarker for statin responsiveness against a new vascular event in post-ischemic stroke patients taking statins early (1-3 months) after the ischemic stroke.
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Biomarcadores/sangre , Isquemia Encefálica/sangre , Desmoplaquinas/sangre , Accidente Cerebrovascular/sangre , 1-Alquil-2-acetilglicerofosfocolina Esterasa/análisis , Anciano , Secuencia de Aminoácidos , Western Blotting , Isquemia Encefálica/complicaciones , Proteína C-Reactiva/análisis , Enfermedades Cardiovasculares/complicaciones , Electroforesis en Gel Bidimensional , Femenino , Estudios de Seguimiento , Humanos , Inhibidores de Hidroximetilglutaril-CoA Reductasas/uso terapéutico , Procesamiento de Imagen Asistido por Computador , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Enfermedades del Sistema Nervioso/etiología , Estudios Prospectivos , Proteómica , Recurrencia , Accidente Cerebrovascular/etiología , Espectrometría de Masas en TándemRESUMEN
Acute coronary syndromes (ACS) are associated with platelet activation. The aim of the present study was to study the protein expression level associated with glycolysis, oxidative stress, cytoskeleton and cell survival in platelets obtained during an ACS. Platelets from 42 coronary ischemic patients, divided into patients admitted within 24 h after the onset of chest pain (ACS group; n=16) and patients with stable coronary ischemic disease (CAD, n=26), were analyzed using proteomics. The expression levels of proteins involved in cellular cytoskeleton (F-actin capping, ß-tubulin, α-tubulin isotypes 1 and 2, vinculin, vimentin and two Ras-related protein Rab-7b isotypes), glycolysis pathway (glyceraldehyde-3-phosphate dehydrogenase, lactate dehydrogenase and two pyruvate kinase isotypes) and cellular-related antioxidant system (manganese superoxide dismutase) and even the expression and activity of glutathione-S-transferase were significantly reduced in platelets from ACS patients compared to CAD patients. Moreover, reduction in the expression of proteins associated with cell survival such as proteasome subunit ß type 1 was also observed in ACS platelets compared with CAD platelets. Principal component and logistic regression analysis suggested the existence of factors (proteins) expressed in the platelets inversely associated with acute coronary ischemia. In summary, these results suggest the existence of circulating antioxidant, cytoskeleton and glycolytic-"bewildered" platelets during the acute phase of a coronary event.
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Síndrome Coronario Agudo/metabolismo , Plaquetas/metabolismo , Proteínas Sanguíneas/metabolismo , Proteoma/metabolismo , Proteómica/métodos , Síndrome Coronario Agudo/sangre , Anciano , Secuencia de Aminoácidos , Biomarcadores/sangre , Biomarcadores/metabolismo , Plaquetas/química , Supervivencia Celular/fisiología , Proteínas del Citoesqueleto/metabolismo , Electroforesis en Gel Bidimensional , Femenino , Citometría de Flujo , Glucólisis , Humanos , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Miocardio/metabolismo , Estrés Oxidativo , Activación Plaquetaria , Análisis de Componente Principal , Estadísticas no Paramétricas , Espectrometría de Masas en TándemRESUMEN
The purpose of the study was to determine if an intensive pre- season training program modifies the inflammatory status in professional soccer players and if this inflammatory profile may be associated with the physical state. We compared plasma protein biomarkers, using proteomics, and the physiological state and cardiac function in 12 professional soccer players and 9 recreational soccer players. Reduced cardiac low frequency [LF] after the pre- season training program previous competition with respect to recreational soccer players was found. No differences were found in cardiac high frequency, cardiac high frequency/low frequency ratio, tension index and oxygen volume consumption. Alpha-1-antitrypsin isotype-3, fibrinogen-gamma isotypes-1, 2 and 3 and vitamin-D-binding protein isotype-1 were reduced in professionals players compared with those in recreational players. However, an increased content of alpha-1-antitrypsin isotype-6 and alpha-1-antichymotrypsin 1 and 4 were found in professional soccer players. Spearman's analysis showed a positive correlation between LF and fibrinogen-gamma chain isotype 3; but LF was negatively correlated with alpha-antichymotrypsin isotype 4. Professional soccer players submitted to an intensive training showed differences in the content of plasma proteins associated with inflammatory/oxidative stress and thrombosis with respect to recreational soccer players. Proteomics analysis in combination with the analysis of cardiac function assessment may be useful to know more in depth molecular processes associated with sport and intensive exercise. Key pointsProteomics allow us to find differences in the plasma protein content in sportsmen.Just after pre-season training program, professional soccer players showed lower content of circulating proteins associated with inflammation compared to recreational soccer players.Proteomic analysis in combination with the analysis of cardiac function may be useful to know more in depth molecular inflammatory and oxidative processes associated with the sport.
RESUMEN
It is well known the effects of the vascular wall on platelet activity but little is known about the effects of platelets on the proteins expression in the vascular wall. We analyzed whether platelets may modify the protein expression in the vascular wall. We used an in vitro model coincubating human platelet rich plasma (PRP) with control and 10 ng/ml tumor necrosis factor-α (TNF-α)-preincubated bovine aortic segments. 2DE, mass spectrometry and Western blot analysis were used to determine changes in the expression of proteins associated with the cytoskeleton and energetic metabolism in the aortic segments. In control healthy vascular wall, only the cytoskeleton-related proteins expression was modified by PRP. However, when PRP was coincubated with TNF-α pre-stimulated aortic segments lesser number of cytoskeleton-related proteins were modified. With respect to energetic metabolism, in control segments, PRP failed to modify any of the analyzed energetic-related proteins. However, in TNF-α-preincubated segments the presence of PRP upexpressed glyceraldehyde-3-phosphate dehydrogenase. Moreover, by western blot experiments it was observed that in TNF-α-preincubated segments the expression of fructose 1,6-bisphosphate aldolase was downregulated by platelets. However, no differences were found in the expression of triosephosphate isomerase and ATP synthase α-chain. In addition, the activity of fructose 1,6-bisphosphate aldolase and piruvate content was significantly reduced without modification on triosephosphate isomerase activity. In conclusion, the crosstalk between platelets and vascular wall is bidirectional and platelets regulated in the vascular wall the expression of proteins associated with the cytoskeleton and energetic metabolism, particularly in the healthy vascular wall.
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Aorta/metabolismo , Plaquetas/metabolismo , Proteínas/metabolismo , Proteómica/métodos , Adulto , Secuencia de Aminoácidos , Animales , Western Blotting , Bovinos , Proteínas del Citoesqueleto/química , Proteínas del Citoesqueleto/metabolismo , Citoesqueleto/metabolismo , Electroforesis en Gel Bidimensional , Metabolismo Energético , Humanos , Técnicas In Vitro , Datos de Secuencia Molecular , Plasma Rico en Plaquetas/metabolismo , Proteínas/químicaRESUMEN
BACKGROUND: Different works have suggested that in the hypertrophied heart the energy metabolic pathway shifts to glycolysis. Our aim was to evaluate using proteomics the expression of proteins associated with different energetic metabolic pathways in hypertrophied left ventricles of spontaneously hypertensive rats (SHR). METHODS: 24-weeks-old SHR with stable hypertension and established left ventricle hypertrophy were used. Normotensive Wistar Kyoto rats were used as control. Proteins from left ventricles were analyzed by 2-dimensional electrophoresis and identified by comparison with a virtual rat heart proteomic map and mass spectrometry. RESULTS: Enoyl-CoA hydratase expression, an enzyme involved in fatty acid beta-oxidation, was reduced whereas the expression of other beta-oxidation enzymes, 3-ketoacyl-CoA thiolase and the mitochondrial precursor of acyl-CoA thioester hydrolase, was increased in the hypertrophied left ventricles. The expression of two enzymes involved in the first steps of glycolysis, fructose bisphosphate aldolase and triosephosphate isomerase, was reduced in the left ventricle of SHR. Pyruvate dehydrogenase expression, enzyme involved in glucose oxidation, was enhanced in the hypertrophied ventricles whereas proteins of the tricarboxylic acid cycle were not modified. Proteins involved in the mitochondrial oxidative phosphorylation were overexpressed whereas the alpha-subunit of the mitochondrial precursor of ATP synthase was downexpressed. CONCLUSIONS: Several proteins involved in the main energy metabolic pathways were up and downexpressed. Moreover, our results seem to suggest that probably neither fatty acid beta-oxidation nor glycolysis are the only sources for energy in the hypertrophied left ventricle.
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Hipertensión/complicaciones , Hipertrofia Ventricular Izquierda/metabolismo , Miocardio/metabolismo , Proteómica/métodos , Acetil-CoA C-Aciltransferasa/metabolismo , Animales , Creatina Quinasa/metabolismo , Electroforesis en Gel Bidimensional , Metabolismo Energético , Enoil-CoA Hidratasa/metabolismo , Fructosa-Bifosfato Aldolasa/metabolismo , Glucólisis , Hipertrofia Ventricular Izquierda/etiología , Espectrometría de Masas , Proteína Trifuncional Mitocondrial , Complejos Multienzimáticos/metabolismo , Fosforilación Oxidativa , Ratas , Ratas Endogámicas SHR , Ratas Endogámicas WKY , Triosa-Fosfato Isomerasa/metabolismoRESUMEN
Current available data show that about 5 to 40% of coronary patients treated with conventional doses of antithrombotic drugs do not display adequate antiplatelet response. Nowadays, aspirin remains the main antiplatelet therapy. However, a significant number of patients show platelet resistance to aspirin therapy, and recurrent thrombotic events occur. Combined antithrombotic therapies with thienopyridines, such as clopidogrel have been used to resolve this problem. However, clopidogrel treatment has been also associated with wide response variability, and non-responsiveness to clopidogrel also occurs in some patients. Therefore, the main question arising about the antithrombotic therapy is why particular patients do not benefit from the therapy and how they might be identified to improve their treatment. Different hypotheses have been suggested, including genetic factors, platelet heterogeneity, non-compliance and others. However, it is probably that many molecular mechanisms involved in platelet resistance to antithrombotic therapies still remains unknown. New technologies, such as proteomics and genetic, are beginning to show new unknown biological biomarkers and molecular mechanisms which may be associated with platelet antithrombotic drug resistance.
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Antitrombinas/farmacología , Plaquetas/efectos de los fármacos , Aspirina/farmacología , Clopidogrel , Humanos , Cooperación del Paciente , Ticlopidina/análogos & derivados , Ticlopidina/farmacologíaRESUMEN
Although many uremic patients show platelet dysfunctionality, there are others with normal platelet functionality and even with thrombotic tendencies. Our aim was to evaluate changes in the expression of proteins in functional and dysfunctional uremic platelets. Using the platelet function analyzer (PFA-100) assay, uremic patients were divided according to their platelet functionality into normal (n=7) and dysfunctional (n=8). There were no significant differences in the number of circulating platelets and hematocrit and hemoglobin levels. Two-dimensional electrophoresis and mass spectrometry were used to determine and identify changes in protein expression. The closure time (CT) in the PFA-100 assay was significantly prolonged in the dysfunctional uremic platelets. In the dysfunctional platelets, actin-interacting protein-1 isotype 1 was down-regulated, while integrin IIb was up-regulated. Glutathione-S-transferase isotypes 1 and 2 and peroxiredoxin VI were up-regulated in the dysfunctional platelets. Pearson analysis showed a negative correlation between the platelet expression of integrin IIb and creatinine clearance. A positive correlation was found between creatinine clearance and glutathione-S-transferase isotype 2. Serum uric acid concentration was positively correlated with CT values and glutathione-S-transferase isotype 1. In conclusion, the analysis of the protein expression in uremic platelets with normal and dysfunctional activity revealed differences which may occur at the megakaryocyte level.
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Plaquetas/metabolismo , Proteínas Sanguíneas/metabolismo , Proteómica , Uremia/sangre , Uremia/fisiopatología , Adulto , Anciano , Anciano de 80 o más Años , Antígenos CD18/sangre , Comunicación Celular/fisiología , Creatinina/sangre , Citoesqueleto/fisiología , Metabolismo Energético/fisiología , Femenino , Glutatión Transferasa/sangre , Humanos , Masculino , Proteínas de Microfilamentos/sangre , Persona de Mediana Edad , Estrés Oxidativo/fisiología , Peroxiredoxina VI/sangre , Ácido Úrico/sangreRESUMEN
Evidence suggests that PTHrP [PTH (parathyroid hormone)-related protein] can act as an inflammatory mediator in several pathological settings including cardiovascular disease. The aim of the present study was to determine whether PTHrP might be involved in human platelet activation. We used a turbidimetric method to determine platelet aggregation. The expression of PTH1R (PTH type 1 receptor) in human platelets was analysed by Western blot and flow cytometry analyses. PTHrP-(1-36) (10(-7) mol/l) by itself failed to modify the activation of platelets. However, it significantly enhanced ADP-induced platelet activation, and also increased the ability of other agonists (thrombin, collagen and arachidonic acid) to induce platelet aggregation. H89 (10(-6) mol/l) and 25 x 10(-6) mol/l Rp-cAMPS (adenosine 3',5'-cyclic monophosphorothioate Rp-isomer), two protein kinase A inhibitors, and 25 x 10(-9) mol/l bisindolylmaleimide I, a protein kinase C inhibitor, partially decreased the enhancing effect of PTHrP-(1-36) on ADP-induced platelet activation. Meanwhile, 10(-6) mol/l PTHrP-(7-34), a PTH1R antagonist, as well as 10(-5) mol/l PD098059, a MAPK (mitogen-activated protein kinase) inhibitor, or a farnesyltransferase inhibitor abolished this effect of PTHrP-(1-36). Moreover, 10(-7) mol/l PTHrP-(1-36) increased (2-fold over control) MAPK activation in human platelets. PTH1R was detected in platelets, and the number of platelets expressing it on their surface in patients during AMI (acute myocardial infarction) was not different from that in a group of patients with similar cardiovascular risk factors without AMI. Western blot analysis showed that total PTH1R protein levels were markedly higher in platelets from control than those from AMI patients. PTH1R was found in plasma, where its levels were increased in AMI patients compared with controls. In conclusion, human platelets express the PTH1R. PTHrP can interact with this receptor to enhance human platelet activation induced by several agonists through a MAPK-dependent mechanism.
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Infarto del Miocardio/fisiopatología , Proteína Relacionada con la Hormona Paratiroidea/farmacología , Activación Plaquetaria/efectos de los fármacos , Anciano , Anciano de 80 o más Años , Plaquetas/metabolismo , Western Blotting , Citometría de Flujo , Humanos , Indoles/metabolismo , Maleimidas/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteína Relacionada con la Hormona Paratiroidea/fisiología , Agregación Plaquetaria/efectos de los fármacosRESUMEN
OBJECTIVES: The aim of this study was to analyze modifications in the plasma protein map during an acute coronary syndrome (ACS) using proteomics. BACKGROUND: Proteomics is a new technology that allows the detection and identification of several proteins at a given time in a sample. METHODS: Plasma from 19 patients, 11 with acute myocardial infarction (AMI) and 8 with unstable angina (UA), was investigated. The control group included nine age-matched volunteers. RESULTS: In two-dimensional electrophoresis using a pH range of 4 to 7, constant differences were found in at least four different areas within the plasma protein map. In area 1, we identified the presence of seven alpha(1)-antitrypsin (AAT) isoforms in plasma from control subjects. alpha(1)-antitrypsin isoform 1 was undetectable in plasma from UA and AMI patients. The AAT isoforms 5, 6, and 7 were reduced in plasma from AMI patients when compared with UA patients. Three fibrinogen gamma chain isoforms were identified in area 2. Fibrinogen gamma chain isoforms 1 and 2 were increased in AMI patients with respect to UA patients. Five apolipoprotein A-I isoforms were identified in area 3. All of them were reduced in plasma from AMI patients with respect to UA patients. In area 4, the gamma-immunoglobulin heavy chains were detected and were found increased in plasma from ACS patients. CONCLUSIONS: Plasma proteomic analysis makes it possible to develop a map of the protein isoforms that are expressed in plasma during an ACS.
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Angina Inestable/sangre , Proteínas Sanguíneas/análisis , Infarto del Miocardio/sangre , Proteómica , Enfermedad Aguda , Anciano , Apolipoproteína A-I/sangre , Femenino , Fibrinógeno/análisis , Humanos , Masculino , Persona de Mediana Edad , Isoformas de Proteínas/sangre , Valores de Referencia , Factores de Riesgo , Síndrome , alfa 1-Antitripsina/análisisRESUMEN
Inflammation and platelet activation are critical phenomena in the setting of acute coronary syndromes. Platelets may contribute to increase ischemic injury by enhancing the inflammatory response of leukocytes and endothelial myocardial cells. Pharmacological inhibition of platelet activation prevents ischemic complications in patients with coronary diseases. Agents directed against the integrin glycoprotein IIb/IIIa (GP IIb/IIIa) receptor not only inhibit platelet aggregation but also have been demonstrated to limit the inflammatory response in acute coronary syndromes. The question then raised is if the inhibition of platelet activation by other mechanisms than the blockade of GP IIb/IIIa may also exert anti-inflammatory effects. The aim of the present study was to analyze if clopidogrel may exert anti-inflammatory effects during the acute phase of myocardial infarction. A ligature was placed around the left anterior descending coronary artery of New Zealand White rabbits. After 15 min of ischemia, the myocardium was reperfused and the ischemic coronary artery was isolated 24 h after the ischemia. A group of ischemic rabbits was given a single oral dose of clopidogrel (20 mg kg(-1)) just after the arterial occlusion and the animal was recovered. Sham-operated animals served as control. P-selectin expression was significantly increased in infarcted rabbits with respect to control rabbits. Clopidogrel administration reduced P-selectin expression with respect to untreated infarcted rabbits. CD40 ligand and tissue factor expression was increased in the ischemic coronary artery and reduced after clopidogrel administration. Clopidogrel also protected endothelial nitric oxide synthase protein expression in the ischemic coronary artery, a protein that has been found downregulated under inflammatory conditions. In conclusion, inhibition of platelet activation by clopidogrel exerted anti-inflammatory effects on the ischemic coronary artery.
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Antiinflamatorios/uso terapéutico , Vasos Coronarios/efectos de los fármacos , Isquemia Miocárdica/tratamiento farmacológico , Inhibidores de Agregación Plaquetaria/uso terapéutico , Ticlopidina/análogos & derivados , Animales , Ligando de CD40/metabolismo , Clopidogrel , Vasos Coronarios/metabolismo , Modelos Animales de Enfermedad , Masculino , Selectina-P/metabolismo , Conejos , Tromboplastina/metabolismo , Ticlopidina/uso terapéuticoRESUMEN
Hypertension is a widely prevalent and important risk factor for cardiovascular diseases that increase with aging. The hallmark of hypertension in the elderly is increased vascular dysfunction. However, the molecular mechanisms by which increased blood pressure leads to vascular injury and impaired endothelial function are not well defined. In the present paper, we will analyze several mechanisms described in the scientific literature involved in hypertension in the elderly as endothelial dysfunction, increased oxygen delivery to tissues, inflammation, cellular apoptosis, and increased concentration of active metabolites. Also, we will focus on new molecular mechanisms involved in hypertension such as telomeres shortening, progenitor cells, circulating microparticles, and epigenetic factors that have appeared as possible causes of hypertension in the elderly. These molecular mechanisms may elucidate different origin for hypertension in the elderly and provide us with new targets for hypertension treatment.
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PURPOSE: Although BRCA1 gene mutations have been associated with breast cancer, BRCA1 mutations have been also involved in other functions. Thrombosis and coagulation are novel mechanisms recently associated with cancer. The aims of the present study were (a) to evaluate, using proteomics, if BRCA1 mutation carriers have a different plasma proteins expression related to thrombosis and coagulation profile than non-mutant BRCA1 women and (b) to analyze if the expression of these proteins may be different among BRCA1 mutation carriers with and without breast cancer. METHODS: Proteomic study was based on 2-dimensional electrophoresis and mass spectrometry. The study was performed in 10 BRCA1 non-mutant controls and 21 women with BRCA1 mutations (with breast cancer (n = 8) and breast cancer-free (n = 13)), all of them free of family history or diagnosis of ovarian cancer. RESULTS: Proteomic study showed that fibrinogen gamma chain isotypes 2 and 3, serotransferrin isotype 4, and convertase C3/C5 isotypes 1-5 were significantly increased in plasma from BRCA1 mutation carriers with respect to BRCA1 non-mutant controls. Plasma levels of alpha-1 antitrypsin isotypes 2-5, apolipoprotein A-IV, and vitamin D-binding protein isotypes 1 and 2 were significantly reduced in BRCA1 mutation carriers with respect to non-mutant controls. Only apolipoprotein A-IV plasma levels were significantly higher in cancer-free BRCA1 mutations carriers compared with BRCA1 mutations carriers who developed breast cancer. CONCLUSION: It is suggested that independently of breast cancer generation, BRCA1-encoded gene alterations are associated with changes in the expression of circulating proteins associated with thrombosis and coagulation.
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Proteínas Sanguíneas/metabolismo , Neoplasias de la Mama/genética , Carcinoma/genética , Genes BRCA1 , Trombosis/metabolismo , Adulto , Coagulación Sanguínea/genética , Proteínas Sanguíneas/genética , Neoplasias de la Mama/sangre , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Carcinoma/sangre , Carcinoma/metabolismo , Carcinoma/patología , Estudios de Casos y Controles , Análisis Mutacional de ADN , Femenino , Regulación Neoplásica de la Expresión Génica , Heterocigoto , Humanos , Persona de Mediana Edad , Mutación/fisiología , Proteoma/análisis , Proteoma/genética , Proteoma/metabolismo , Trombosis/sangre , Trombosis/genéticaRESUMEN
The aim of the present study was to analyse differences in the protein expression profile between platelets from aspirin (ASA)-resistant patients and ASA-sensitive patients. We analysed platelets from 51 clinically stable coronary ischaemic patients taking ASA (100 mg/day) divided into ASA-resistant (n=25) and ASA-sensitive (n=26) based on a platelet functionality test (PFA-100). Proteins associated with cytoskeleton, energetic metabolism, oxidative stress, inflammation and cell survival were analysed by two-dimensional electrophoresis and mass spectrometry. The expression of two gelsolin precursor isotypes and one F-acting capping protein isotype was decreased in ASA-resistant platelets (p<0.05). The expression of glyceraldehyde 3-phosphate dehydrogenase was increased in the ASA-resistant platelets (1751.1 + or - 220.6 vs. 4273.3 + or - 971.7, 95% confidence interval [CI] 1815.11 to 4061.2, p=0.001). It was accompanied by a reduced expression and activity of 1,6-bisphosphate aldolase in platelets without changes in the content of pyruvate. A reduced expression of gluthathione-S-transferase and the protein disulfide isomerase isotype 1 was found in ASA-resistant platelets. The protein expression of the chloride intracellular channel isotype 1 was increased in ASA-resistant platelets (21.3 + or - 3.8 vs. 48.8 + or - 6.0, CI 29.5 to 45.95, p=0.03) while the expression of two HSP60 and two HSP71 isotypes was decreased. No changes were observed in proteins associated with inflammation. In conclusion, ASA-resistant and ASA-sensitive platelets are different in terms of the level of expression of proteins associated with mechanisms such as energetic metabolism, cytoskeleton, oxidative stress and cell survival which may be associated with their different ability to respond to ASA.
Asunto(s)
Aspirina/uso terapéutico , Plaquetas/efectos de los fármacos , Proteínas Sanguíneas/análisis , Resistencia a Medicamentos , Isquemia Miocárdica/tratamiento farmacológico , Inhibidores de Agregación Plaquetaria/uso terapéutico , Anciano , Biomarcadores/sangre , Plaquetas/metabolismo , Western Blotting , Supervivencia Celular , Proteínas del Citoesqueleto/sangre , Electroforesis en Gel Bidimensional , Metabolismo Energético , Femenino , Humanos , Mediadores de Inflamación/sangre , Masculino , Espectrometría de Masas , Persona de Mediana Edad , Isquemia Miocárdica/sangre , Estrés Oxidativo , Mapeo Peptídico , Pruebas de Función Plaquetaria , Proteómica/métodos , Resultado del TratamientoRESUMEN
BACKGROUND: The aim of the present study was to identify, using proteomics, changes in the expression of proteins and protein isoforms mainly associated with inflammation and vascular damage in the coronary sinus of patients undergoing off-pump coronary artery bypass (OPCAB) surgery. METHODS: Plasma from the coronary sinus of 18 patients undergoing OPCAB was obtained before the ischemic procedure and after revascularization of the left anterior descending artery (LAD) with an internal thoracic artery graft. The mean LAD ischemic time was 11.1+/-0.7 min. For the proteomic study, two-dimensional gel electrophoresis was performed. RESULTS: A significant decrease in the expression of three fibrinogen gamma-chain (FGC) isoforms, three vitamin D-binding protein (DBP) isoforms, six haptoglobin (HPT) isoforms, two apolipoprotein (apo)-AI isoforms, one ceruloplasmin (CP) isoform and apoAIV was found after revascularization. Before anastomosis, negative correlations between blood flow and FGC isoforms 2 and 3 and positive correlations between blood flow and apoAI isoform 5 were observed. After anastomosis, we observed positive correlations between haptoglobin isoform 3 and DBP isoform 2 and blood flow. Before anastomosis, positive correlation between DBP isoform 2 and troponin I was observed. After LAD grafting, positive correlations between troponin I and HPT isoform 6, CP isoform 1 and apoAI isoforms 2 and 4 were observed. After the procedure, positive correlations between creatine kinase-MB and coronary sinus expression of FGC isoforms 1, 2 and 3 and HPT isoforms 1 and 2 were also observed. conclusions: In blood from coronary sinus the expression of a number of proteins and protein isoforms associated with inflammation and vascular protection was modified after OPCAB.
Asunto(s)
Proteínas Sanguíneas/metabolismo , Puente de Arteria Coronaria Off-Pump/efectos adversos , Proteoma/metabolismo , Secuencia de Aminoácidos , Biomarcadores/sangre , Biomarcadores/química , Proteínas Sanguíneas/química , Seno Coronario/metabolismo , Seno Coronario/patología , Electroforesis en Gel Bidimensional , Femenino , Regulación de la Expresión Génica , Humanos , Inflamación/sangre , Inflamación/complicaciones , Inflamación/etiología , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Isoformas de Proteínas/sangre , Isoformas de Proteínas/química , Proteoma/química , Enfermedades Vasculares/sangre , Enfermedades Vasculares/complicaciones , Enfermedades Vasculares/etiologíaRESUMEN
The effect of pravastatin on matrix metalloproteinase-9 (MMP-9) and the level of tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2 was studied in explants of human abdominal aortic aneurysm (AAA) obtained from 13 patients. The effect of pravastatin on the apoptotic status of human AAA explants was also examined. Total MMP-9 content did not differ in human AAA explants incubated in vitro in the presence or absence of pravastatin (10-6 mol/L) for 48 h. TIMP-1 levels were significantly increased in pravastatin-incubated AAA explants, but TIMP-2 production was not modified by pravastatin. Western blot experiments showed that, whereas Bax expression was increased in pravastatin-incubated AAA explants, the expression of Bcl-2 was not modified. On the other hand, the ratio of the expression of Bax to Bcl-2, an apoptotic index, was not modified by pravastatin. In the human AAA explants, the increase in Bax expression, but not the increase in TIMP-1 expression elicited by pravastatin, was reversed by L-mevalonate, a downstream HMG-CoA reductase metabolite, suggesting that the expression of Bax and TIMP-1 followed HMG-CoA reductase-dependent and -independent pathways, respectively. In conclusion, pravastatin increases both TIMP-1 and Bax expression in human AAA explants without changes in either MMP-9 activity or the apoptotic status.
Asunto(s)
Aneurisma de la Aorta Abdominal/metabolismo , Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacología , Pravastatina/farmacología , Inhibidor Tisular de Metaloproteinasa-1/biosíntesis , Proteína X Asociada a bcl-2/biosíntesis , Anciano , Apoptosis/efectos de los fármacos , Western Blotting , Femenino , Humanos , Masculino , Metaloproteinasa 9 de la Matriz/biosíntesis , Persona de Mediana Edad , Proteínas Proto-Oncogénicas c-bcl-2/biosíntesis , Estimulación Química , Inhibidor Tisular de Metaloproteinasa-2/biosíntesisRESUMEN
Our aim was to analyze the plasma proteome in aspirin (acetylsalicylic acid [ASA])-sensitive and ASA-resistant coronary ischemic patients. Plasma from 19 ASA-sensitive and 19 ASA-resistant patients was analyzed. For the proteomic study, two-dimensional electrophoresis was performed. The expression of one isotype of the fibrinogen gamma chain and three isotypes of haptoglobin was increased in ASA-resistant patients. Three vitamin D binding protein isotypes were increased in ASA-resistant patients. In vitro incubation of vitamin D binding protein (DBP) with blood from healthy volunteers reduced the inhibitory effect of ASA on thromboxane A2 production. DBP may be a new regulator of the inhibitory effect of ASA on platelets.
Asunto(s)
Aspirina/uso terapéutico , Enfermedad Coronaria/sangre , Resistencia a Medicamentos , Proteoma/análisis , Proteína de Unión a Vitamina D/sangre , Anciano , Aspirina/farmacología , Proteínas Sanguíneas/análisis , Proteínas Sanguíneas/metabolismo , Proteínas Sanguíneas/farmacología , Enfermedad Coronaria/tratamiento farmacológico , Enfermedad Coronaria/metabolismo , Electroforesis en Gel Bidimensional , Femenino , Humanos , Masculino , Persona de Mediana Edad , Agregación Plaquetaria/efectos de los fármacos , Inhibidores de Agregación Plaquetaria/farmacología , Isoformas de Proteínas/sangre , Proteoma/metabolismo , Proteómica , Tromboxano A2/antagonistas & inhibidores , Tromboxano A2/metabolismo , Proteína de Unión a Vitamina D/metabolismo , Proteína de Unión a Vitamina D/farmacologíaRESUMEN
Proteomics is a technology to detect and identify several proteins and their isoforms in a single sample. We used proteomics to analyze modifications in the protein map of plasma after simvastatin treatment of moderate hypercholesterolemic patients. Plasma from hypercholesterolemic patients (n = 9) was compared before and after 12 weeks of simvastatin treatment (40 mg/day). Patients with similar cardiovascular risk factors were used as controls (CR group). By using two-dimensional electrophoresis and mass spectrometry, we identified the different protein isoforms. The plasma expression of three fibrinogen gamma chain isoforms (FGG) was enhanced, whereas the expression of two isoforms of the fibrinogen beta chain (FGB) was reduced in the hypercholesterolemic patients compared with the CR group. The expression of apolipoprotein A-IV and three haptoglobin isoforms was higher in hypercholesterolemic patients. Simvastatin treatment modified the plasma expression of FGG chain isoform 1, FGB chain isoforms 1 and 2, vitamin D binding protein isoform 3, apo A-IV, and haptoglobin isoform 2. The modification of FGG chain isoform 1 and FGB chain isoforms 1 and 2 was positively correlated with total plasma cholesterol level. Proteomic analysis of plasma may help to know more in depth the molecular mechanism modified by simvastatin treatment.
Asunto(s)
Proteínas Sanguíneas/análisis , Regulación de la Expresión Génica/efectos de los fármacos , Hipercolesterolemia/sangre , Proteómica/métodos , Secuencia de Aminoácidos , Apolipoproteínas/sangre , Proteínas Sanguíneas/genética , Electroforesis en Gel Bidimensional , Fibrinógeno/análisis , Humanos , Hipercolesterolemia/tratamiento farmacológico , Espectrometría de Masas , Datos de Secuencia Molecular , Simvastatina/farmacología , Simvastatina/uso terapéuticoRESUMEN
F12511(S)-2',3',5'-trimethyl-4'-hydroxy-alpha-dodecylthio-alpha-phenylacetanilide (F12511) is a new Acyl-CoA cholesterol acyltransferase (ACAT) inhibitor that not only reduces the plasma cholesterol levels but also has anti-atherosclerotic actions in animals models. The study's aim was to analyze if F12511 may directly modify the ability of tumor necrosis factor--alpha (TNF-alpha)-incubated bovine aortic endothelial cells (BAEC) to express endothelial nitric oxide synthase (eNOS) protein and inflammatory-related proteins such as platelet endothelial cell adhesion molecule (PECAM) and CD40 ligand (CD40L). The addition of increasing concentrations of F12511 (10 to 10 mol/L) failed to modify the level of eNOS protein expressed in control BAEC. TNF-alpha (10 ng/mL) reduced the expression of eNOS protein. In TNF-alpha--incubated BAEC, F12511 protected eNOS expression in a concentration-dependent manner. TNF-alpha stimulated the expression of both CD40L and PECAM in cultured BAEC. F12511 (10 mol/L) failed to modify the expression of CD40L and PECAM in control and TNF-alpha-incubated BAEC. Reverse transcriptase polymerase chain reaction showed a marked expression of the ACAT-2 isoform and absent of expression of the ACAT-1 isoform in BAEC. The presence of ACAT-2 isoform in BAEC was further confirmed by Western blot. F12511 failed to modify the expression of the proinflammatory associated proteins PECAM and CD40L in the endothelium but protected eNOS expression in the endothelial cells exposed to inflammatory conditions.