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Perirenal adipose tissue (PRAT) surrounding the kidney is emerging as a player and novel independent risk factor in diabetic kidney disease (DKD); DKD is a complication of diabetes and is a major cause of increased cardiovascular (CV) risk and CV mortality in affected patients. We determined the effect of diabetes induction on (i) kidney and CV damage and (ii) on the expression of proinflammatory and profibrotic factors in both the PRAT and the mesenteric adipose tissue (MAT) of Munich Wistar Frömter (MWF) rats. The 16-week-old male MWF rats (n = 10 rats/group) were fed standard chow (MWF-C) or a high-fat/high-sucrose diet for 6 weeks together with low-dose streptozotocin (15 mg/kg i.p.) at the start of dietary exposure (MWF-D). Phenotyping was performed at the end of treatment through determining water intake, urine excretion, and oral glucose tolerance; use of the homeostatic model assessment-insulin resistance index (HOMA-IR) evidenced the development of overt diabetes manifestation in MWF-D rats. The kidney damage markers Kim-1 and Ngal were significantly higher in MWF-D rats, as were the amounts of PRAT and MAT. A diabetes-induced upregulation in IL-1, IL-6, Tnf-α, and Tgf-ß was observed in both the PRAT and the MAT. Col1A1 was increased in the PRAT but not in the MAT of MWF-D, whereas IL-10 was lower and higher in the PRAT and the MAT, respectively. Urinary albumin excretion and blood pressure were not further increased by diabetes induction, while heart weight was higher in the MWF-D. In conclusion, our results show a proinflammatory and profibrotic in vivo environment in PRAT induced by diabetes which might be associated with kidney damage progression in the MWF strain.
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Diabetes Mellitus , Enfermedades Renales , Humanos , Ratas , Masculino , Animales , Ratas Wistar , Albuminuria , Regulación hacia Arriba , Inflamación , Colágeno , Tejido AdiposoRESUMEN
Arterial stiffness is a major vascular complication of chronic kidney disease (CKD). The development of renal damage, hypertension, and increased pulse wave velocity (PWV) in CKD might be associated with an imbalance in bone morphogenetic proteins (BMP)-2 and BMP-7. Plasma BMP-2 and BMP-7 were determined by ELISA in CKD patients (stages I-III; n = 95) and Munich Wistar Frömter (MWF) rats. Age-matched Wistar rats were used as a control. The expression of BMP-2, BMP-7, and profibrotic and calcification factors was determined in kidney and perivascular adipose tissues (PVAT). BMP-2 was higher in stage III CKD patients compared to control subjects. BMP-7 was lower at any CKD stage compared to controls, with a significant further reduction in stage III patients. A similar imbalance was observed in MWF rats together with the increase in systolic (SBP) and diastolic blood pressure (DBP), or pulse wave velocity (PWV). MWF exhibited elevated urinary albumin excretion (UAE) and renal expression of BMP-2 or kidney damage markers, Kim-1 and Ngal, whereas renal BMP-7 was significantly lower than in Wistar rats. SBP, DBP, PWV, UAE, and plasma creatinine positively correlated with the plasma BMP-2/BMP-7 ratio. Periaortic and mesenteric PVAT from MWF rats showed an increased expression of BMP-2 and profibrotic and calcification markers compared to Wistar rats, together with a reduced BMP-7 expression. BMP-2 and BMP-7 imbalance in plasma, kidney, and PVATs is associated with vascular damage, suggesting a profibrotic/pro-calcifying propensity associated with progressive CKD. Thus, their combined analysis stratified by CKD stages might be of clinical interest to provide information about the degree of renal and vascular damage in CKD.
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Insuficiencia Renal Crónica , Rigidez Vascular , Animales , Ratas , Proteína Morfogenética Ósea 7 , Riñón , Análisis de la Onda del Pulso , Ratas Wistar , Insuficiencia Renal Crónica/complicacionesRESUMEN
Background Obesity is a risk factor for cardiovascular disease, the leading cause of death. Health education, nutritional follow-up, and life style habits modification are key for cardiovascular risk reduction in obese patients. Objective To measure the impact of pharmacist's intervention on cardiovascular risk in obese patients. Setting A Spanish community pharmacy. Method Obese patients (BMI ≥ 30) with (group A, n = 30) and without (group B, n = 14) comorbidities were selected. Variables determined in first visit on-site: anthropometric values (weight, height, waist circumference), blood pressure, glycemic (glucose, HbA1c) and lipid parameters (total cholesterol, HDL-c, LDL-c, triglycerides). The PharmaFit Protocol consisted in a 24-month follow-up focusing (i) monthly on adherence to nutritional guidelines and modification of life style habits, and (ii) bi-monthly on anthropometric variables, blood pressure, and biochemical determinations. Feedback was provided to the primary care physician or specialist. Main ouitcome measure Cardiovascular risk estimated by REGICOR score. Results Anthropometric variables significantly decreased in all groups. Plasma glucose levels were significantly reduced in group A without changes in HbA1c. Lipid parameters significantly improved in group A, whereas HDL-c significantly raised in all groups. REGICOR score was significantly reduced in group A female (13.8 ± 1.6 vs. 5.8 ± 1, p < 0.0001) and male (12.7 ± 1.7 vs. 4.4. ± 0.6, p < 0.005) patients, and in group B female patients (3.5 ± 0.7 vs. 1.9 ± 0.4, p < 0.001). Conclusion Community pharmacist intervention, delivered as a 24-month follow-up and combining health and dietary education, has a highly positive impact on the reduction of cardiovascular risk in obese patients.
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Enfermedades Cardiovasculares/epidemiología , Obesidad/epidemiología , Farmacéuticos , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Antropometría , Glucemia/metabolismo , Presión Sanguínea/fisiología , Estudios de Casos y Controles , Servicios Comunitarios de Farmacia , Comorbilidad , Femenino , Hemoglobina Glucada/metabolismo , Adhesión a Directriz , Evaluación del Impacto en la Salud , Humanos , Estilo de Vida , Lípidos/sangre , Masculino , Persona de Mediana Edad , Factores de Riesgo , Conducta de Reducción del Riesgo , Factores Sexuales , España/epidemiología , Adulto JovenRESUMEN
Caloric restriction (CR) improves endothelial function through the upregulation of adenosine monophosphate-activated protein kinase (AMPK) and endothelial nitric oxide synthase (eNOS). Moreover, hydrogen peroxide (H2O2) is upregulated in yeast subjected to CR. Our aim was to assess if mild short-term CR increases vascular H2O2 formation as a link with AMPK and eNOS activation. Twelve-week old Zucker obese (fa/fa) and control Zucker lean male rats were fed a standard chow either ad libitum (AL, n=10) or with a 20% CR (CR, n=10) for two weeks. CR significantly improved relaxation to ACh in fa/fa rats because of an enhanced endogenous production of H2O2 in aortic rings (H2O2 levels fa/faAL=0.5⯱â¯0.05â¯nmol/mg vs. H2O2 levels fa/faCR=0.76⯱â¯0.07â¯nmol/mg protein; p<0.05). Expression of mitochondrial superoxide dismutase (Mn-SOD) and total SOD activity were increased in aorta from fa/fa animals after CR. In cultured aortic endothelial cells, serum deprivation or 2-deoxy-d-glucose induced a significant increase in: i) superoxide anion and H2O2 levels, ii) p-AMPK/AMPK and p-eNOS/eNOS expression and iii) nitric oxide levels. This effect was reduced by catalase and strongly inhibited by Ca2+/calmodulin-dependent kinase II (CamkII) silencing. In conclusion, we propose that mild short-term CR might be a trigger of mechanisms aimed at protecting the vascular wall by the increase of H2O2, which then activates AMPK and nitric oxide release, thus improving endothelium-dependent relaxation. In addition, we demonstrate that CAMKII plays a key role in mediating CR-induced AMPK activation through H2O2 increase.
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Proteínas Quinasas Activadas por AMP/genética , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/genética , Restricción Calórica , Peróxido de Hidrógeno/metabolismo , Óxido Nítrico Sintasa de Tipo III/genética , Obesidad/genética , Proteínas Quinasas Activadas por AMP/metabolismo , Animales , Aorta Torácica/metabolismo , Aorta Torácica/patología , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Catalasa/genética , Catalasa/metabolismo , Desoxiglucosa/farmacología , Células Endoteliales/citología , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Regulación de la Expresión Génica , Masculino , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa de Tipo III/metabolismo , Obesidad/metabolismo , Obesidad/patología , Ratas , Ratas Zucker , Transducción de Señal , Superóxido Dismutasa/genética , Superóxido Dismutasa/metabolismo , Superóxidos/metabolismo , VasodilataciónRESUMEN
Resistant albuminuria, developed under adequate chronic blockade of the renin-angiotensin system, is a clinical problem present in a small number of patients with chronic kidney disease (CKD). The mechanism underlying this resistant albuminuria remains unknown. Matrix metalloproteinases (MMPs) are involved in the pathophysiology of cardiovascular and renal diseases. In the present study we tested the role of MMPs in resistant albuminuria. First we evaluated gelatinase MMP-2 and MMP-9 activity by zymography in the Munich Wistar Frömter (MWF) rat, a model of progressive albuminuria, and subsequently in patients with resistant albuminuria. Markers of oxidative stress were observed in the kidneys of MWF rats, together with a significant increase in pro-MMP-2 and active MMP-9 forms. These changes were normalized together with reduced albuminuria in consomic MWF-8(SHR) rats, in which chromosome 8 of MWF was replaced with the respective chromosome from spontaneously hypertensive rats. The MMP-2 and MMP-9 protein levels were similar in patients with normal and resistant albuminuria; however, high circulating levels of collagen IV, a specific biomarker of tissue collagen IV degradation, were observed in patients with resistant albuminuria. These patients showed a significant increase in gelatinase MMP-2 and MMP-9 activity, but only a significant increase in the active MMP-9 form quantified by ELISA, which correlated significantly with the degree of albuminuria. Although the expression of the tissue inhibitor of MMP-9 (TIMP)-1 was similar, a novel AlphaLISA assay demonstrated that the MMP-9-TIMP-1 interaction was reduced in patients with resistant albuminuria. It is of interest that oxidized TIMP-1 expression was higher in patients with resistant albuminuria. Therefore, increased circulating MMP-9 activity is associated with resistant albuminuria and a deleterious oxidative stress environment appears to be the underlying mechanism. These changes might contribute to the progression of CKD in these patients.
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Albuminuria/enzimología , Riñón/enzimología , Metaloproteinasa 9 de la Matriz/sangre , Insuficiencia Renal Crónica/enzimología , Anciano , Albuminuria/sangre , Albuminuria/diagnóstico , Albuminuria/genética , Albuminuria/prevención & control , Bloqueadores del Receptor Tipo 1 de Angiotensina II/uso terapéutico , Inhibidores de la Enzima Convertidora de Angiotensina/uso terapéutico , Animales , Antihipertensivos/uso terapéutico , Modelos Animales de Enfermedad , Resistencia a Medicamentos , Femenino , Humanos , Hipertensión/diagnóstico , Hipertensión/tratamiento farmacológico , Hipertensión/fisiopatología , Riñón/efectos de los fármacos , Masculino , Metaloproteinasa 2 de la Matriz/sangre , Persona de Mediana Edad , Oxidación-Reducción , Estrés Oxidativo , Unión Proteica , Ratas Wistar , Insuficiencia Renal Crónica/sangre , Insuficiencia Renal Crónica/diagnóstico , Insuficiencia Renal Crónica/genética , Insuficiencia Renal Crónica/prevención & control , Transducción de Señal , Inhibidor Tisular de Metaloproteinasa-1/sangreRESUMEN
The aim of this study was to characterize alterations in vascular structure and mechanics in murine mesenteric arteries from obese non-hypertensive mice, as well as their relationship with adipokines. Four-week old C57BL/6J male mice were assigned either to a control (C, 10% kcal from fat) or a high-fat diet (HFD, 45% kcal from fat) for 32weeks. HFD animals weighed 30% more than controls (p<0.001), exhibited similar blood pressure, increased leptin, insulin and superoxide anion (O2(-)) levels, and reduced adiponectin levels and nitric oxide (NO) bioavailability. Arterial structure showed an outward remodeling with an increase in total number of both adventitial and smooth muscle cells in HFD. Moreover, HFD mice exhibited an increased arterial stiffness assessed by ß-values (C=2.4±0.5 vs HFD=5.3±0.8; p<0.05) and aortic pulse wave velocity (PWV, C=3.4±0.1 vs HFD=3.9±0.1; p<0.05). ß-Values and PWV positively correlated with leptin, insulin or O2(-) levels, whereas they negatively correlated with adiponectin levels and NO bioavailability (p<0.01). A reduction in fenestrae number together with an increase in type-I collagen amount (p<0.05) were observed in HFD. These data demonstrate that HFD accounts for the development of vascular remodeling and arterial stiffness associated with adipokine dysregulation and oxidative stress, independently of hypertension development.
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Adipoquinas/sangre , Arterias Mesentéricas/fisiopatología , Obesidad/inmunología , Rigidez Vascular/fisiología , Adipoquinas/inmunología , Animales , Dieta Alta en Grasa/efectos adversos , Endotelio Vascular/inmunología , Masculino , Arterias Mesentéricas/inmunología , Arterias Mesentéricas/metabolismo , Arterias Mesentéricas/patología , Ratones Endogámicos C57BL , Microscopía Confocal , Obesidad/metabolismo , Obesidad/patología , Obesidad/fisiopatología , Estrés Oxidativo/inmunología , Análisis de la Onda del Pulso , Remodelación Vascular/fisiología , Rigidez Vascular/inmunologíaRESUMEN
SCOPE: Activation of endothelial adenosine monophosphate-activated protein kinase (AMPK) contributes to increase nitric oxide (NO) availability. The aim of this study was to assess if high-fat diet (HFD)-induced endothelial dysfunction is linked to AMPK deregulation. METHODS AND RESULTS: Twelve-week-old Sprague Dawley male rats were assigned either to control (10 kcal % from fat) or to HFD (45 kcal % from fat) for 8 wk. HFD rats segregated in obesity-prone (OP) or obesity-resistant (OR) rats according to body weight. HFD triggered an impaired glucose management together with impaired endothelium-dependent relaxation, reduced endothelial AMPK activity and lower NO availability in aortic rings of OP and OR cohorts. Relaxation evoked by AMPK activator, 5-aminoimidazole-4-carboxamide-1-ß-d-ribofuranoside (AICAR) was reduced in both OP and OR rings, which exhibited lower p-AMPKα-Thr(172) /AMPKα ratios that negatively correlated with plasma non-esterified fatty acids (NEFA) and triglycerides (TG). Inhibition of PI3K (wortmannin, 10(-7) M) or Akt (triciribine, 10(-5) M) reduced relaxation to AICAR only in the control group (p < 0.001). Akt (p-Akt-Ser(473) ) and eNOS phosphorylation (p-eNOS-Ser(1177) ) were significantly reduced in OP and OR (p < 0.01). CONCLUSION: Endothelial dysfunction caused by HFD is related to a dysfunctional endothelial AMPK-PI3K-Akt-eNOS pathway correlating with the increase of plasma NEFA, TG, and an impaired glucose management.
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Dieta Alta en Grasa/efectos adversos , Endotelio Vascular/metabolismo , Endotelio Vascular/fisiopatología , Quinasas de la Proteína-Quinasa Activada por el AMP , Proteínas Quinasas Activadas por AMP/metabolismo , Animales , Peso Corporal/efectos de los fármacos , Regulación hacia Abajo , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Endotelio Vascular/efectos de los fármacos , Masculino , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Óxido Nítrico Sintasa de Tipo III/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ratas Sprague-Dawley , Transducción de Señal , Superóxido Dismutasa/metabolismoRESUMEN
OBJECTIVE: Albuminuria has been recently described in hypertensive patients under chronic renin-angiotensin system (RAS) suppression. We investigated whether this fact could be related to an increase in oxidative stress. METHODS: We examined normoalbuminuric and albuminuric patients in stage 2 chronic kidney disease, both with more than 2 years of RAS blockade. The relationship between albuminuria and circulating biomarkers for both oxidative damage, that is carbonyl and malondialdehyde, as well as antioxidant defense, that is reduced glutathione, thiol groups, uric acid, bilirubin, or catalase, and superoxide scavenging activity, was assessed. RESULTS: We found that only patients with albuminuria showed an important increase in carbonyls (Pâ<â0.001) and malondialdehyde (Pâ<â0.05) compared to normoalbuminuric patients. This increase in oxidative damage was also accompanied by a rise in catalase activity (Pâ<â0.05) and low-molecular-weight antioxidants only when they were measured as total antioxidant capacity (Pâ<â0.01). In order to establish the specific oxidative status of each group, new indexes of oxidative damage and antioxidant defense were calculated with all these markers following a mathematical and statistical approach. Although both pro-oxidant and antioxidant indexes were significantly increased in patients with albuminuria, only the oxidative damage index positively correlated with the increase of albumin/creatinine ratio (Pâ=â0.0024). CONCLUSIONS: We conclude that albuminuria is accompanied by an amplified oxidative damage in patients in early stages of chronic kidney disease. These results indicate that chronic RAS protection must be directed to avoid development of albuminuria and oxidative damage.
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Albuminuria/diagnóstico , Antagonistas de Receptores de Angiotensina/uso terapéutico , Inhibidores de la Enzima Convertidora de Angiotensina/uso terapéutico , Hipertensión/tratamiento farmacológico , Estrés Oxidativo , Sistema Renina-Angiotensina/efectos de los fármacos , Anciano , Albuminuria/etiología , Biomarcadores/sangre , Catalasa/sangre , Femenino , Humanos , Hipertensión/complicaciones , Masculino , Malondialdehído/sangre , Persona de Mediana Edad , Especies Reactivas de Oxígeno , Insuficiencia Renal Crónica/complicaciones , Ácido Úrico/sangreRESUMEN
BACKGROUND: The hypothesis of this study is that long-term high-fat diets (HFD) induce perivascular adipose tissue (PVAT) dysfunction characterized by a redox imbalance, which might contribute to aggravate endothelial dysfunction in obesity. METHODS AND RESULTS: C57BL/6J mice were fed either control or HFD (45% kcal from fat) for 32 weeks. Body weight, lumbar and mesenteric adipose tissue weights were significantly higher in HFD animals compared to controls. The anticontractile effect of PVAT in mesenteric arteries (MA) was lost after 32 week HFD and mesenteric endothelial-dependent relaxation was significantly impaired in presence of PVAT in HFD mice (Emaxâ=â71.0±5.1 vs Emaxâ=â58.5±4.2, p<0.001). The inhibitory effect of L-NAME on Ach-induced relaxation was less intense in the HFD group compared with controls suggesting a reduction of endothelial NO availability. Expression of eNOS and NO bioavailability were reduced in MA and almost undetectable in mesenteric PVAT of the HFD group. Superoxide levels and NOX activity were higher in PVAT of HFD mice. Apocynin only reduced contractile responses to NA in HFD animals. Expression of ec-SOD and total SOD activity were significantly reduced in PVAT of HFD mice. No changes were observed in Mn-SOD, Cu/Zn-SOD or catalase. The ratio [GSSG]/([GSH]+[GSSG]) was 2-fold higher in the mesenteric PVAT from HFD animals compared to controls. CONCLUSIONS: We suggest that the imbalance between pro-oxidant (NOX, superoxide anions, hydrogen peroxide) and anti-oxidant (eNOS, NO, ecSOD, GSSG) mechanisms in PVAT after long-term HFD might contribute to the aggravation of endothelial dysfunction.
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Tejido Adiposo/metabolismo , Dieta Alta en Grasa/efectos adversos , Animales , Peso Corporal/fisiología , Peróxido de Hidrógeno/metabolismo , Masculino , Arterias Mesentéricas/metabolismo , Arterias Mesentéricas/fisiopatología , Ratones , Ratones Endogámicos C57BL , Microscopía Confocal , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa de Tipo III/metabolismo , Oxidación-Reducción , Superóxidos/metabolismoRESUMEN
Dietary treatment with high-fat diets (HFD) triggers diabetes and hyperleptinemia, concomitantly with a partial state of leptin resistance that affects hepatic and adipose tissue but not the heart. In this context, characterized by widespread steatosis, cardiac lipid content remains unchanged. As previously reported, HFD-evoked hyperleptinemia could be a pivotal element contributing to increase fatty-acid (FA) metabolism in the heart and to prevent cardiac steatosis. This metabolic adaptation might theoretically reduce energy efficiency in cardiomyocytes and lead to cardiac electrophysiological remodeling. Therefore the aim of the current study has been to investigate the impact of long-term HFD on cardiac metabolism and electrophysiological properties of the principal ionic currents responsible of the action potential duration in mouse cardiomyocytes. Male C57BL/6J mice were fed a control (10 kcal% from fat) or HFD (45 kcal% from fat) during 32 weeks. Quantification of enzymatic activities regulating mitochondrial uptake of pyruvate and FA showed an increase of both carnitine-palmitoyltransferase and citrate synthase activities together with a decrease of lactate dehydrogenase and pyruvate dehydrogenase activities. Increased expression of uncoupling protein-3, Mn-, and Cu/Zn-superoxide dismutases and catalase were also detected. Total glutathione/oxidized glutathione ratios were unaffected by HFD. These data suggest that HFD triggers adaptive mechanisms aimed at (i) facilitating FA catabolism, and (ii) preventing oxidative stress. All these changes did not affect the duration of action potentials in cardiomyocytes and only slightly modified electrocardiographic parameters.
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Most blood vessels are surrounded by adipose tissue. Similarly to the adventitia, perivascular adipose tissue (PVAT) was considered only as a passive structural support for the vasculature, and it was routinely removed for isolated blood vessel studies. In 1991, Soltis and Cassis demonstrated for the first time that PVAT reduced contractions to noradrenaline in rat aorta. Since then, an important number of adipocyte-derived factors with physiological and pathophysiological paracrine vasoactive effects have been identified. PVAT undergoes structural and functional changes in obesity. During early diet-induced obesity, an adaptative overproduction of vasodilator factors occurs in PVAT, probably aimed at protecting vascular function. However, in established obesity, PVAT loses its anticontractile properties by an increase of contractile, oxidative, and inflammatory factors, leading to endothelial dysfunction and vascular disease. The aim of this review is to focus on PVAT dysfunction mechanisms in obesity.
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OBJECTIVE: The Munich Wistar Frömter (MWF) rat develops progressive spontaneous albuminuria largely attributable to quantitative trait loci on rat chromosome (RNO)6 and RNO8, respectively. We tested the hypothesis whether quantitative trait loci on these chromosomes are linked to both albuminuria and endothelial dysfunction. METHODS: Experiments were performed in male 12-week-old MWF, Wistar Kyoto (WKY), spontaneously hypertensive rat (SHR) and consomic MWF-6 and MWF-8 rats, in which RNO6 or RNO8 was replaced by the respective SHR chromosome (nâ=â10 per strain). Vascular function was assessed in aorta and vascular superoxide anion production was determined by confocal microscopy. RESULTS: Acetylcholine potency to induce relaxation was significantly reduced in MWF (6.2â±â0.1) compared with WKY (7.1â±â0.1) or SHR (7.3â±â0.1; Pâ<â0.01). N-nitro-L-arginine methyl ester abolished relaxation to acetylcholine in all three strains, whereas indomethacin exhibited no effect in WKY and MWF. Contractions to noradrenaline and superoxide production were significantly higher in MWF compared with SHR and WKY (Pâ<â0.05, respectively). In consomic MWF-6 and MWF-8 rats albuminuria was markedly suppressed (-85 and -92%, Pâ<â0.005 compared with MWF, respectively). Interestingly, relaxation to acetylcholine and contraction to noradrenaline were restored to normal WKY levels only in MWF-8, but not in MWF-6, due to an increase in stimulated and basal nitric oxide availability, respectively. CONCLUSION: These data demonstrate the partial genetic independence of albuminuria and endothelial dysfunction in this model. From two known albuminuria quantitative trait loci one locus affects both albuminuria and endothelial dysfunction. Whether this observation is based on a common genetic mechanism related to NO availability warrants further investigation.
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Albuminuria/genética , Predisposición Genética a la Enfermedad , Sitios de Carácter Cuantitativo , Acetilcolina/antagonistas & inhibidores , Acetilcolina/farmacología , Albuminuria/fisiopatología , Animales , Aorta/fisiopatología , Arginina/análogos & derivados , Arginina/farmacología , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/metabolismo , Endotelio Vascular/fisiopatología , Indometacina/farmacología , Masculino , Óxido Nítrico/genética , Ratas , Ratas Endogámicas SHR , Ratas Endogámicas WKY , Superóxidos/metabolismo , Enfermedades VascularesRESUMEN
Leptin causes vasodilatation both by endothelium-dependent and -independent mechanisms. Leptin is synthesized by perivascular adipose tissue (PVAT). The hypothesis of this study is that a decrease of leptin production in PVAT of spontaneously hypertensive rats (SHR) might contribute to a diminished paracrine anticontractile effect of the hormone. We have determined in aorta from Wistar-Kyoto (WKY) and SHR (i) leptin mRNA and protein levels in PVAT, (ii) the effect of leptin and PVAT on contractile responses, and (iii) leptin-induced relaxation and nitric oxide (NO) production. Leptin mRNA and protein expression were significantly lower in PVAT from SHR. Concentration-response curves to angiotensin II were significantly blunted in presence of PVAT as well as by exogenous leptin (10(-9) M) only in WKY. This anticontractile effect was endothelium-dependent. Vasodilatation induced by leptin was smaller in SHR than in WKY, and was also endothelium-dependent. Moreover, release of endothelial NO in response to acute leptin was higher in WKY compared to SHR, but completely abolished in the absence of endothelium. In conclusion, the reduced anticontractile effect of PVAT in SHR might be attributed to a reduced PVAT-derived leptin and to an abrogated effect of leptin on endothelial NO release probably due to an impaired activation of endothelial NO synthase.
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The aim of this study was to determine whether alterations in periadventitial adipose tissue and its anti-contractile effect precede hypertension development. We used 4-week-old male Wistar Kyoto (WKY) and spontaneously hypertensive rats (SHR), which were pre-hypertensive. Vascular function was studied in the perfused mesenteric bed (MB, 1.5 mL/min). MB weight was lower in SHR (8.0 +/- 0.3 mg/g body weight) than in WKY (9.0 +/- 0.3 mg/g body weight) rats. Concentration-response curves to KCI (6 to 75 mmol/L) and to acetylcholine (10(-9) to 10(-5) mol/L) were similar between groups. Contractile responses to serotonin (10(-9) to 10(-5) mol/L) were significantly higher in SHR compared to WKY. 4-Aminopyridine (4-AP, 2 mmol/L), a blocker of Kv channels, induced a similar increase in perfusion pressure in both strains. However, 4-AP (2 mmol/L) significantly increased the contractile response to serotonin (10(-9) to 10(-5) mol/L) only in WKY. The anti-contractile effect of fat was confirmed by a comparison of (+) fat and (-) fat mesenteric arteries, which revealed that 4-AP significantly enhanced contractions only in (+) fat rings from WKY. These results show that alterations in visceral periadventitial fat mass and function in SHR precede hypertension, suggesting a constitutive mechanism independent of age and the hypertensive state.
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Tejido Adiposo/fisiopatología , Hipertensión/fisiopatología , Arterias Mesentéricas/fisiopatología , Vasoconstricción/fisiología , 4-Aminopiridina/farmacología , Animales , Masculino , Óxido Nítrico/fisiología , Norepinefrina/metabolismo , Ratas , Ratas Endogámicas SHR , Ratas Endogámicas WKY , Circulación EsplácnicaRESUMEN
Intracerebral infection with Theiler's murine encephalomyelitis virus (TMEV) induces a demyelinating disease that resembles human multiple sclerosis. In order to delineate the early events in this virus-induced neuroinflammatory disease, we have analyzed global GTPases gene activation following TMEV infection of murine brain astrocytes. DNA hybridization microchip analysis demonstrated that 10 sequences described as GTPbinding proteins and GTPases in different protein databases were over-expressed, in response to this infectious agent in astroglial cells. We have first characterized both the GTP-binding and GTPase activities in uninfected astrocyte membranes from a biochemical point of view. The increase in such activities was further validated in TMEV-infected astrocytes, peaking 2-4 h after infection. Over-expression is also induced by the inflammation-related chemokines interleukin-6 and interferon-gamma but not by interleukin-1alpha or tumor necrosis factor-alpha. From the many GTPases that could be over-expressed we have studied two, because of its biological significance; Ras p21 and the subunit alphai2 of G proteins. Western blots revealed increases in both proteins after infection with TMEV, in accordance with the previous enzymologic results. An increase in the active form of Ras (the GTP bound form) in cell lysates was also confirmed by affinity binding to a glutathione-S-transferase-fusion protein, following TMEV infection. A final demonstration of physiological up-regulation is provided by UV cross-linking of membrane proteins with the hydrolysis-resistant GTP agonist GTP [gamma-(35)S]. This technique allow us to detect, after SDS-PAGE, the increase of two further majoritary GTPbinding proteins with MW of 62 and 49 KDa. A quantitative analysis of four selected genes coding for p21 ras, Galphai2 subunit of protein G, Munc-18 and protein interacting with C kinase 1, was performed by real-time RT-PCR to verify the microarray results. The study of GTPase activity and of the above genes by RT-PCR in brains of sick mice, demonstrated a significative increase in mRNA coding for p21ras and protein interacting with C kinase 1 in vivo. Here we demonstrate that one of the mechanisms triggered by TMEV infection of astrocytes is the up-regulation of proteins related to GTP metabolism, one important signal transduction system in mammalian cells.
Asunto(s)
Astrocitos/ultraestructura , Membrana Celular/metabolismo , Membrana Celular/virología , GTP Fosfohidrolasas/metabolismo , Proteínas de Unión al GTP/metabolismo , Theilovirus/fisiología , Animales , Animales Recién Nacidos , Astrocitos/metabolismo , Astrocitos/virología , Membrana Celular/efectos de los fármacos , Células Cultivadas , Corteza Cerebral/citología , Quimiocinas/farmacología , Cricetinae , Regulación de la Expresión Génica/efectos de los fármacos , Ratones , Ratones Endogámicos , Activación TranscripcionalRESUMEN
Young shoots of Sambucus ebulus L. contain a monomeric d-galactose binding lectin (SELlm), which disappears upon shoot development, and was previously undetected since it co-purifies with the non-toxic type 2 ribosome-inactivating protein ebulin l and the dimeric lectin SELld. Molecular cloning of cDNA coding for SELlm and mass spectrometry analysis revealed a protein with a molecular mass of 34,239 Da, which displays 80%, 77% and 45% of amino acid sequence identity with the ebulin l-B chain, SELld and ricin-B chain, respectively. Furthermore, the cloned precursor, with respect to the ebulin l precursor is truncated and contains the signal peptide, a piece of the A chain, a piece of the connecting peptide and the B chain. Further processing yields the lectin protein, which contains only the B chain. Despite the fact that SELlm displays the same d-galactose-binding sites than ricin, it was found that the lectin has different binding properties to D-galactose-containing matrix than ricin. Notably, and unlike ricin, the binding of SELlm and other Sambucus lectins to such matrix was maximum in range of 0-10 degrees C and abolished at 20 degrees C.
Asunto(s)
Galectinas/metabolismo , Lectinas de Plantas/metabolismo , Brotes de la Planta/metabolismo , Sambucus/metabolismo , Secuencia de Aminoácidos , Cromatografía de Afinidad , Simulación por Computador , Galectinas/química , Galectinas/genética , Modelos Genéticos , Modelos Moleculares , Datos de Secuencia Molecular , Hojas de la Planta/genética , Hojas de la Planta/metabolismo , Lectinas de Plantas/química , Lectinas de Plantas/genética , Brotes de la Planta/genética , Unión Proteica , Estructura Secundaria de Proteína , Ricina/metabolismo , Sambucus/genética , Homología de Secuencia de Aminoácido , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
OBJECTIVE: Perivascular adipose tissue of normotensive rats releases a transferable factor that induces relaxation by opening voltage-dependent K+ (Kv) channels. The relevance of these observations to hypertension is unknown. METHODS AND RESULTS: We characterized mesenteric perivascular adipose tissue from 3-month-old Wistar Kyoto rats (WKY) and aged-matched spontaneously hypertensive rats (SHR). Mesenteric bed (MB) weight and MB total lipid content were lower in SHR than in WKY. Freshly isolated MB adipocytes were smaller in SHR. Plasma triglycerides, glycerol, nonesterified free-fatty acids, and cholesterol were also lower in SHR. Plasma and mesenteric leptin were correlated with the quantity of mesenteric fat. To study vascular function, the MB was cannulated and perfused at a constant 2 mL/min flow. The Kv channel blocker 4-aminopyridine (4-AP; 2 mmol/L) increased perfusion pressure less in SHR MB than WKY and was directly correlated with the mesenteric fat amount. In isolated mesenteric artery rings, 4-AP (2 mmol/L) induced a contractile effect that was attenuated in SHR compared with WKY. The anticontractile effects of perivascular fat were reduced in SHR mesenteric artery rings compared with WKY. CONCLUSIONS: Differences in visceral perivascular adipose tissue mass and function may contribute to the increased vascular resistance observed in SHR.