Cardioimplante autólogo de células madre de médula ósea en territorios miocárdicos necróticos / Cardiac implant autologous bone marrow stem cells in necrotic myocardial territories / Implante de células-tronco cardíacas autólogas da medula óssea em áreas de necrose miocárdica

Introducción. El concepto clásico de que el corazón era un órgano no regenerativo ha cambiado en la actualidad, por el de ser un órgano en regeneración continua, constituyendo una evidencia sólida de que el tejido cardíaco se encuentra en un proceso continuo de crecimiento, muerte y renovación. Material y método. Se protocolizaron doce pacientes desde el 29 de Mayo de 2004 al 30 de Agosto de 2007. Se excluyeron cinco y de los 7 restantes, cinco fueron evaluados. Se extrajeron por punción de la cresta ilíaca de cada paciente en condiciones estériles 60 cm³ de médula ósea, y se obtuvieron por sedimentación células madre que se identificaron por inmunomarcación con anticuerpo monoclonal anti-CD34 en citómetro de flujo.La evaluación de la viabilidad miocárdica pre y post implante se realizó con PET-FDG. Finalizadas las anastomosis, se implantaron las células madre directamente por punción en territorios no viables. Resultados. Se expresan como porcentajes (variables categóricas) y como media con su desvío estándar (variables continuas). Para evaluar significancia en el cambio de la fracción de eyección se utilizó el test de Wilcoxon. Se consideró significativa una p<0,05. De los 15 segmentos no viables, se implantaron 11 (73%) y se recuperaron 4 (36,36%). La fracción de eyección de ventrículo izquierdo media evaluada por SPECT gatillado fue del 30,2±4,9% en el pre procedimiento y del 34,8±9,5% en el post procedimiento, la mejoría fue de 4,6 puntos (p=0,34), el 60% de los pacientes mejoraron. Conclusiones. Hay una relación entre la concentración de CD34+ (que fue baja: 0,76% de media) y la viabilidad miocárdica. La aparición de viabilidad miocárdica en el 36,36% de los segmentos implantados fue el hallazgo más importante. El método demostró ser seguro, efectivo y reproducible. La utilización de PET-FDG en todos los pacientes para evaluación de viabilidad pre y post implante como método gold standard fue evidencia de regeneración tisular.

Introduction. Currently, the classical concept that the heart was a non-regenerative organ has changed, by being an organ in continuous regeneration constitute strong evidence that cardiac tissue is in a continuous process of growth, death and renewal. Materials and methods. Twelve patients were protocolized from May 29, 2004 to August 30, 2007. We excluded five. Of the 7 remaining, five were evaluated by puncturing of the iliac crest from each patient under sterile conditions 60 cm³ extracted bone marrow and stem cells were obtained by sedimentation and identified by immunostaining with anti-CD34 monoclonal antibody in the flow cytometer. The assessment of myocardial viability before and after implantation was performed with PET-FDG. After the completion anastomosis, the stem cells were implanted directly by puncture nonviable territories. Results. They are expressed as percentages (categorical variables) and as mean with standard deviation (continuous variables). To assess significance in changing ejection fraction was used the Wilcoxon test. Was considered significant at p <0.05. We implanted 11 (73%) of the 15 segments non-viable were recovered 4 (36.36%). The ejection fraction of the left ventricle assessed by SPECT triggered average was 30.2 ± 4.9% in the pre procedure and 34.8 ± 9.5% in the post process, the improvement was 4.6 points (p=0.34), 60% of the patients improved. Conclusions. There is a relationship between the concentration of CD34 + (which was low 0.76% on average) and myocardial viability. The appearance of myocardial viability in 36.36% of the implanted segments was the most important finding. The method proved to be safe, effective and reproducible. The use of PET-FDG in all patients for viability assessment before and after implantation as gold standard method was evidence of tissue regeneration.

Introdução.O conceito clássico que o coração era um órgão não regenerativo mudou agora, por ser um órgão em regeneração contínua constituem fortes provas de que o tecido cardíaco é um processo contínuo de crescimento, morte e renovação. Material e método.Doze pacientes foram incluídos em um protocolo de pesquisa a partir de 29 de Maio de 2004 a 30 de Agosto de 2007. Cinco excluídos. Dos restantes sete, cinco foram avaliados por punção da crista ilíaca do paciente em condições estéreis, foram obtidos 60 cm³ de medula óssea. As células-tronco foram recolhidos por sedimentação e identificados por imunomarcação com o anticorpo monoclonal anti-CD34 no citómetro de fluxo. A avaliação da viabilidade miocárdica antes e após a implantação foi realizada com PET- FDG. Após o término das anastomoses, as células-tronco foram implantadas diretamente por punção em territórios não viáveis. Resultados. Eles são expressos como porcentagens (variáveis categóricas) e média com desvio padrão (variáveis contínuas). Para avaliar a importância na mudança de fração de ejeção foi utilizado o teste de Wilcoxon. Considerou-se significante p<0,05. Dos 15 segmentos não viáveis,foram implantados 11 (73%) e quatro (36,36%) foram recuperados. A fração de ejeção do ventrículo esquerdo avaliada pelo SPECT foi de 30,2±4,9% na fase pré e de 34,8±9,5% no processo de pós, a melhoria foi de 4,6 pontos (p=0,34), 60% dos pacientes melhoraram. Conclusões. Existe uma relação entre a concentração de células CD34+ (baixa: 0,76 %, em média) e a viabilidade do miocárdio. A incidência da viabilidade do miocárdio em 36,36% dos segmentos implantados foi o achado mais importante. O método demonstrou ser seguro, eficaz e reprodutível. O uso de PET-FDG em todos os pacientes para avaliação de viabilidade, antes e depois da implantação como método padrão-ouro, foi evidência de regeneração de tecidos.

Cardioimplante autólogo de células madre de médula ósea en territorios miocárdicos necróticos / Cardiac implant autologous bone marrow stem cells in necrotic myocardial territories / Implante de células-tronco cardíacas autólogas da medula óssea em áreas de necrose miocárdica

Introducción. El concepto clásico de que el corazón era un órgano no regenerativo ha cambiado en la actualidad, por el de ser un órgano en regeneración continua, constituyendo una evidencia sólida de que el tejido cardíaco se encuentra en un proceso continuo de crecimiento, muerte y renovación. Material y método. Se protocolizaron doce pacientes desde el 29 de Mayo de 2004 al 30 de Agosto de 2007. Se excluyeron cinco y de los 7 restantes, cinco fueron evaluados. Se extrajeron por punción de la cresta ilíaca de cada paciente en condiciones estériles 60 cm³ de médula ósea, y se obtuvieron por sedimentación células madre que se identificaron por inmunomarcación con anticuerpo monoclonal anti-CD34 en citómetro de flujo.La evaluación de la viabilidad miocárdica pre y post implante se realizó con PET-FDG. Finalizadas las anastomosis, se implantaron las células madre directamente por punción en territorios no viables. Resultados. Se expresan como porcentajes (variables categóricas) y como media con su desvío estándar (variables continuas). Para evaluar significancia en el cambio de la fracción de eyección se utilizó el test de Wilcoxon. Se consideró significativa una p<0,05. De los 15 segmentos no viables, se implantaron 11 (73%) y se recuperaron 4 (36,36%). La fracción de eyección de ventrículo izquierdo media evaluada por SPECT gatillado fue del 30,2±4,9% en el pre procedimiento y del 34,8±9,5% en el post procedimiento, la mejoría fue de 4,6 puntos (p=0,34), el 60% de los pacientes mejoraron. Conclusiones. Hay una relación entre la concentración de CD34+ (que fue baja: 0,76% de media) y la viabilidad miocárdica. La aparición de viabilidad miocárdica en el 36,36% de los segmentos implantados fue el hallazgo más importante. El método demostró ser seguro, efectivo y reproducible. La utilización de PET-FDG en todos los pacientes para evaluación de viabilidad pre y post implante como método gold standard fue evidencia de regeneración tisular.(AU)

Introduction. Currently, the classical concept that the heart was a non-regenerative organ has changed, by being an organ in continuous regeneration constitute strong evidence that cardiac tissue is in a continuous process of growth, death and renewal. Materials and methods. Twelve patients were protocolized from May 29, 2004 to August 30, 2007. We excluded five. Of the 7 remaining, five were evaluated by puncturing of the iliac crest from each patient under sterile conditions 60 cm³ extracted bone marrow and stem cells were obtained by sedimentation and identified by immunostaining with anti-CD34 monoclonal antibody in the flow cytometer. The assessment of myocardial viability before and after implantation was performed with PET-FDG. After the completion anastomosis, the stem cells were implanted directly by puncture nonviable territories. Results. They are expressed as percentages (categorical variables) and as mean with standard deviation (continuous variables). To assess significance in changing ejection fraction was used the Wilcoxon test. Was considered significant at p <0.05. We implanted 11 (73%) of the 15 segments non-viable were recovered 4 (36.36%). The ejection fraction of the left ventricle assessed by SPECT triggered average was 30.2 ± 4.9% in the pre procedure and 34.8 ± 9.5% in the post process, the improvement was 4.6 points (p=0.34), 60% of the patients improved. Conclusions. There is a relationship between the concentration of CD34 + (which was low 0.76% on average) and myocardial viability. The appearance of myocardial viability in 36.36% of the implanted segments was the most important finding. The method proved to be safe, effective and reproducible. The use of PET-FDG in all patients for viability assessment before and after implantation as gold standard method was evidence of tissue regeneration.(AU)

IntroduþÒo.O conceito clássico que o coraþÒo era um órgÒo nÒo regenerativo mudou agora, por ser um órgÒo em regeneraþÒo contínua constituem fortes provas de que o tecido cardíaco é um processo contínuo de crescimento, morte e renovaþÒo. Material e método.Doze pacientes foram incluídos em um protocolo de pesquisa a partir de 29 de Maio de 2004 a 30 de Agosto de 2007. Cinco excluídos. Dos restantes sete, cinco foram avaliados por punþÒo da crista ilíaca do paciente em condiþ§es estéreis, foram obtidos 60 cm³ de medula óssea. As células-tronco foram recolhidos por sedimentaþÒo e identificados por imunomarcaþÒo com o anticorpo monoclonal anti-CD34 no citómetro de fluxo. A avaliaþÒo da viabilidade miocárdica antes e após a implantaþÒo foi realizada com PET- FDG. Após o término das anastomoses, as células-tronco foram implantadas diretamente por punþÒo em territórios nÒo viáveis. Resultados. Eles sÒo expressos como porcentagens (variáveis categóricas) e média com desvio padrÒo (variáveis contínuas). Para avaliar a importÔncia na mudanþa de fraþÒo de ejeþÒo foi utilizado o teste de Wilcoxon. Considerou-se significante p<0,05. Dos 15 segmentos nÒo viáveis,foram implantados 11 (73%) e quatro (36,36%) foram recuperados. A fraþÒo de ejeþÒo do ventrículo esquerdo avaliada pelo SPECT foi de 30,2±4,9% na fase pré e de 34,8±9,5% no processo de pós, a melhoria foi de 4,6 pontos (p=0,34), 60% dos pacientes melhoraram. Conclus§es. Existe uma relaþÒo entre a concentraþÒo de células CD34+ (baixa: 0,76 %, em média) e a viabilidade do miocárdio. A incidÛncia da viabilidade do miocárdio em 36,36% dos segmentos implantados foi o achado mais importante. O método demonstrou ser seguro, eficaz e reprodutível. O uso de PET-FDG em todos os pacientes para avaliaþÒo de viabilidade, antes e depois da implantaþÒo como método padrÒo-ouro, foi evidÛncia de regeneraþÒo de tecidos.(AU)

Insulin resistance promotes early atherosclerosis via increased proinflammatory proteins and oxidative stress in fructose-fed ApoE-KO mice.

High fructose intake induces an insulin resistance state associated with metabolic syndrome (MS). The effect of vascular inflammation in this model is not completely addressed. The aim of this study was to evaluate vascular remodeling, inflammatory and oxidative stress markers, and atheroma development in high-fructose diet-induced insulin resistance of ApoE-deficient mice (ApoE-KO). Mice were fed with either a normal chow or a 10% w/v fructose (HF) in drinking water over a period of 8 weeks. Thereafter, plasma metabolic parameters, vascular remodeling, atheroma lesion size, inflammatory markers, and NAD(P)H oxidase activity in the arteries were determined. HF diet induced a marked increase in plasma glucose, insulin, and triglycerides in ApoE-KO mice, provoked vascular remodeling, enhanced expression of vascular cell-adhesion molecule-1 (VCAM-1) and matrix metalloprotease 9 (MMP-9) and enlarged atherosclerotic lesion in aortic and carotid arteries. NAD(P)H oxidase activity was enhanced by fructose intake, and this effect was attenuated by tempol, a superoxide dismutase mimetic, and losartan, an Angiotensin II receptor antagonist. Our study results show that high-fructose-induced insulin resistance promotes a proinflammatory and prooxidant state which accelerates atherosclerotic plaque formation in ApoE-KO mice.

Quercetin and catechin synergistically inhibit angiotensin II-induced redox-dependent signalling pathways in vascular smooth muscle cells from hypertensive rats.

Dietary flavonoids, present in different amount in foods, are associated with the prevention of hypertension, but little is known about the interactions between them. The aim of this study was to explore the effect of quercetin (Q), catechin (C) and the mixture, on Angiotensin II (AngII)-induced redox-dependent signalling pathways and cell behaviour. Mesenteric smooth muscle cells (MesSMC) from spontaneously hypertensive rats (SHR) were incubated with AngII (0.1 µmol/L) alone, or with the mixture of low concentrations of Q and C. AngII-increased ROS production was reduced by the mixture of separately ineffective low concentration of Q (15 µmol/L) plus C (20 µmol/L). This mixture reduced AngII-stimulated NAD(P)H oxidase activation and p47phox translocation to the cell membrane, without affecting Nox2 expression. Co-incubation of Q + C significantly inhibited AngII-induced migration and proliferation, and these effects were independent of p-ERK1/2 and related with reduced p38MAPK phosphorylation. These findings demonstrated that low concentrations of singly non-effective flavonoids when are combined exert a synergistic effect in inhibiting AngII-induced redox-sensitive signalling pathways.

Garlic components inhibit angiotensin II-induced cell-cycle progression and migration: Involvement of cell-cycle inhibitor p27(Kip1) and mitogen-activated protein kinase.

Garlic has been used for prevention and treatment of hypertension; however, the molecular mechanisms of garlic's effects remain to be elucidated. In this study, the mechanisms of the in vitro effect of organosulphur compounds derived from garlic on growth and migration of cultured aortic smooth muscle cells isolated from spontaneously hypertensive rats were investigated. We demonstrated that allyl methyl sulphide (AMS) and diallyl sulphide (DAS) inhibited aortic smooth muscle cell angiotensin II-stimulated cell-cycle progression and migration. Neither cell viability nor annexin-V-binding analysis revealed cytotoxic effects of both organosulphur compounds at the used concentrations. Instead, their inhibitory effects were associated to the prevention of the cell-cycle inhibitor p27(Kip1) (p27) downregulation and the reduction of extracellular signal-regulated kinase 1/2 phosphorylation. When we assessed the antioxidant activity of AMS and DAS, we found that both organosulphur compounds inhibited angiotensin II-reactive oxygen species generation. Our findings show that AMS and DAS, compounds derivate from garlic, could be effective antioxidants targeted at the arterial remodelling seen in hypertension.

Expresión vascular de factores de transcripción proinflamatorios en un modelo de síndrome metabólico / Vascular expression of proinflammatory transcription factors in a model of metabolic syndrome

Con el objetivo de estudiar la expresión de factores de transcripción sensibles a redox en las arterias en un modelo experimental de hipertensión arterial asociada con síndrome metabólico (FFHR), ratas Wistar Kyoto (WKY) y espontáneamente hipertensas (SHR) macho de 30 días fueron distribuidas en forma aleatoria en cuatro grupos (n = 8 c/u): 1) WKY (control), 2) FFR: administración de fructosa 10 por ciento P/V en el agua de bebida durante un período de 10 semanas, 3) SHR y 4) FFHR: ídem 3 + 2.Los grupos FFR y FFHR presentaron valores de HOMA y área bajo la curva en la prueba de tolerancia característicos de resistencia a la insulina. También mostraron diferencias significativas en los niveles de triglicéridos y colesterol HDL respecto de sus controles y aumentaron su presión arterial sistólica. El estado de estrés oxidativo, demostrado por la actividad de NAD(P)H oxidasa y TBARS fue significativamente mayor en FFR y FFHR, en tanto que en estos mismos grupos disminuyó significativamente la actividad de eNOS. El peso cardíaco relativo aumentó en FFR y FFHR, con mayor área de los miocitos de la pared libre ventricular. Los cortes de arteria carótida izquierda mostraron crecimiento de su capa media de tipo eutróico en FFHR. La densidad óptica media para los anticuerpos anti-c-fos, anti-NF-kB y anti-VCAM-1 fue mayor en las arterias renales de resistencia y en la carótida de los grupos FFHR y FFR. Los datos confirman el desarrollo del modelo experimental patológico y sugieren que el estrés oxidativo y la consecuente activación de genes que participan en el proceso inflamatorio intervienen activamente en el desarrollo de remodelación vascular.

With the goal of assessing the expression of redox-sensitive transcription factors in the arteries of an experimental model of hypertension associated to metabolic syndrome (FFHR), we studied Wistar Kyoto rats (WKY) and spontaneously hypertensive 30-day male rats (SHR), which were randomly distributed in 4 groups (n=8 in each group). Group 1: WKY (control rats), Group 2: FFR: rats that received 10 per cent W/V fructose in drinking water during a 10-week period, Group 3: SHR rats, and Group 4: FFHR: 2+3, i.e., SHR rats treated like Group 2. With the glucose tolerance test, groups FFR and FFHR had HOMA (homeostasis assessment model) and area under the curve (AUC) values that were characteristic of insulinresistance. They also showed significant differences in triglyceride and HDL cholesterol levels compared to controls, and increased their systolic blood pressure. Oxidative stress, as assessed by NAD(P)H oxidase and TBARS (thiobarbituric acid reactive substances) activity was significantly higher in FFR and FFHR groups, whereas in these same groups eNOS activity decreased markedly. Relative cardiac weight increased in FFR and FFHR groups, with a larger myocyte area in the left ventricular free wall. Sections of the left carotid artery exhibited eutrophic growth of the middle layer in FFHR. Mean optical density for anti-c-fos, anti-NF-kB and anti-VCAM-1 antibodies was greater in resistance renal arteries and in the carotid artery of FFHR and FFR groups. The data confirm the findings of the pathological experimental model and suggest that oxidative stress and the subsequent activation of genes that participate in the inflammatory process are actively involved in the development of vascular remodeling.

Expresión vascular de factores de transcripción proinflamatorios en un modelo de síndrome metabólico / Vascular expression of proinflammatory transcription factors in a model of metabolic syndrome

Con el objetivo de estudiar la expresión de factores de transcripción sensibles a redox en las arterias en un modelo experimental de hipertensión arterial asociada con síndrome metabólico (FFHR), ratas Wistar Kyoto (WKY) y espontáneamente hipertensas (SHR) macho de 30 días fueron distribuidas en forma aleatoria en cuatro grupos (n = 8 c/u): 1) WKY (control), 2) FFR: administración de fructosa 10 por ciento P/V en el agua de bebida durante un período de 10 semanas, 3) SHR y 4) FFHR: ídem 3 + 2.Los grupos FFR y FFHR presentaron valores de HOMA y área bajo la curva en la prueba de tolerancia característicos de resistencia a la insulina. También mostraron diferencias significativas en los niveles de triglicéridos y colesterol HDL respecto de sus controles y aumentaron su presión arterial sistólica. El estado de estrés oxidativo, demostrado por la actividad de NAD(P)H oxidasa y TBARS fue significativamente mayor en FFR y FFHR, en tanto que en estos mismos grupos disminuyó significativamente la actividad de eNOS. El peso cardíaco relativo aumentó en FFR y FFHR, con mayor área de los miocitos de la pared libre ventricular. Los cortes de arteria carótida izquierda mostraron crecimiento de su capa media de tipo eutróico en FFHR. La densidad óptica media para los anticuerpos anti-c-fos, anti-NF-kB y anti-VCAM-1 fue mayor en las arterias renales de resistencia y en la carótida de los grupos FFHR y FFR. Los datos confirman el desarrollo del modelo experimental patológico y sugieren que el estrés oxidativo y la consecuente activación de genes que participan en el proceso inflamatorio intervienen activamente en el desarrollo de remodelación vascular.(AU)

With the goal of assessing the expression of redox-sensitive transcription factors in the arteries of an experimental model of hypertension associated to metabolic syndrome (FFHR), we studied Wistar Kyoto rats (WKY) and spontaneously hypertensive 30-day male rats (SHR), which were randomly distributed in 4 groups (n=8 in each group). Group 1: WKY (control rats), Group 2: FFR: rats that received 10 per cent W/V fructose in drinking water during a 10-week period, Group 3: SHR rats, and Group 4: FFHR: 2+3, i.e., SHR rats treated like Group 2. With the glucose tolerance test, groups FFR and FFHR had HOMA (homeostasis assessment model) and area under the curve (AUC) values that were characteristic of insulinresistance. They also showed significant differences in triglyceride and HDL cholesterol levels compared to controls, and increased their systolic blood pressure. Oxidative stress, as assessed by NAD(P)H oxidase and TBARS (thiobarbituric acid reactive substances) activity was significantly higher in FFR and FFHR groups, whereas in these same groups eNOS activity decreased markedly. Relative cardiac weight increased in FFR and FFHR groups, with a larger myocyte area in the left ventricular free wall. Sections of the left carotid artery exhibited eutrophic growth of the middle layer in FFHR. Mean optical density for anti-c-fos, anti-NF-kB and anti-VCAM-1 antibodies was greater in resistance renal arteries and in the carotid artery of FFHR and FFR groups. The data confirm the findings of the pathological experimental model and suggest that oxidative stress and the subsequent activation of genes that participate in the inflammatory process are actively involved in the development of vascular remodeling.(AU)

11,13-dihydro-dehydroleucodine, a derivative of dehydroleucodine with an inactivated alkylating function conserves the anti-proliferative activity in G2 but does not cause cytotoxicity.

Modulation of vascular smooth muscle cell (VSMC) proliferation has critical therapeutic implications for vascular disease. Recently, we demonstrated that the sesquiterpene lactone dehydroleucodine (DhL) inhibited the proliferation of VSMCs in G2 phase. It is known that the alpha,beta-unsaturated carbonyl group of the sesquiterpene lactone has a nonspecific alkylating activity that inhibits a large number of enzymes or factors involved in key biological processes. We analyzed whether the DhL alpha-methylene-gamma-lactone function is directly involved in cell proliferation arrest in G2 and in cell toxicity. To this end, the effects of both DhL and 11,13-dihydro-dehydroleucodine (2H-DhL), a derivative of DhL with inactivated alpha-methylenelactone function, on cultured VSMC viability and proliferation were assessed. We found that both DhL and 2H-DhL inhibited the proliferation of VSMCs in a dose-dependent manner, inducing a transient arrest in G2 phase. DhL, but not 2H-DhL, had a cytotoxic effect at concentrations up to 12 microM, indicating that cell proliferation arrest and cytotoxicity are mediated by different cellular targets. From these results we infer that only 2H-DhL is able to arrest cell proliferation in G2 without affecting cell viability at any concentration.

Vascular remodeling in experimental hypertension.

The basic hemodynamic abnormality in hypertension is an increased peripheral resistance that is due mainly to a decreased vascular lumen derived from structural changes in the small arteries wall, named (as a whole) vascular remodeling. The vascular wall is an active, flexible, and integrated organ made up of cellular (endothelial cells, smooth muscle cells, adventitia cells, and fibroblasts) and noncellular (extracellular matrix) components, which in a dynamic way change shape or number, or reorganize in response to physiological and pathological stimuli, maintaining the integrity of the vessel wall in physiological conditions or participating in the vascular changes in cardiovascular diseases such as hypertension. Research focused on new signaling pathways and molecules that can participate in the mechanisms of vascular remodeling has provided evidence showing that vascular structure is not only affected by blood pressure, but also by mechanisms that are independent of the increased pressure. This review will provide an overview of the evidence, explaining some of the pathophysiologic mechanisms participating in the development of the vascular remodeling, in experimental models of hypertension, with special reference to the findings in spontaneously hypertensive rats as a model of essential hypertension, and in fructose-fed rats as a model of secondary hypertension, in the context of the metabolic syndrome. The understanding of the mechanisms producing the vascular alterations will allow the development of novel pharmacological tools for vascular protection in hypertensive disease.

Chronic administration of resveratrol prevents biochemical cardiovascular changes in fructose-fed rats.

BACKGROUND: There seems to be a link between the cluster of risk factors known as insulin resistance syndrome with endothelial dysfunction. Resveratrol (3,4,5-trihydroxyestilbene) (RV), an antioxidant found in many components of the human diet, has been proposed as an effective agent in the prevention of several pathologic processes. This study examined the effect of chronic administration of RV on endothelial nitric oxide synthase (eNOS) activity in cardiovascular tissues and on plasma lipid peroxidation in fructose-fed rats (FFR), an experimental model of this syndrome. METHODS: Male Sprague Dawley rats were separated into four groups: Control, Control + RV, FFR, and FFR + RV (n = 8 in each group). The RV (10 mg/kg/d by gavage) and fructose (10% in drinking water) were administered for 45 days. Metabolic variables and systolic blood pressure (BP) were measured. The eNOS activity was estimated in the mesenteric arterial bed and cardiac tissue homogenates by conversion of (3)H-arginine to (3)H-citrulline. Lipid peroxidation was estimated through the measurement of plasmatic thiobarbituric acid-reactive substances (TBARS). RESULTS: The RV chronic treatment prevented the increase in systolic BP and cardiac hypertrophy, restored FFR mesenteric and cardiac eNOS activities, and decreased the elevated TBARS levels that characterize FFR, without an effect on other metabolic variables. CONCLUSIONS: In concert with other effects, the increase in eNOS activity may contribute to the protective properties attributed to RV and, thus, to its beneficial effects on the cardiovascular system. These results suggest that an adequate supplementation of RV might help to prevent or delay the occurrence of atherogenic cardiovascular diseases associated to insulin-resistant states.

Vascular smooth muscle cell NAD(P)H oxidase activity during the development of hypertension: Effect of angiotensin II and role of insulinlike growth factor-1 receptor transactivation.

OBJECTIVE: We investigated whether angiotensin II (Ang II)-induced reactive oxygen species (ROS) generation is altered in vascular smooth muscle cells (VSMCs) from spontaneously hypertensive rats (SHR) during the phases of prehypertension, developing hypertension, and established hypertension and assessed the putative role of insulinlike growth factor-1 receptor (IGF-1R) in Ang II-mediated actions. METHODS: The VSMCs from SHR and Wistar-Kyoto rats (WKY) aged 4 (prehypertensive), 9 (developing hypertension), and 16 (established hypertension) weeks were studied. The ROS production and NAD(P)H oxidase activation were determined by fluorescence and chemiluminescence, respectively. The role of IGF-1R was assessed with the selective inhibitor AG1024. The ROS bioavailability was manipulated with Tiron (10(-5) mol/L) and diphenylene iodonium (DPI) (10(-6) mol/L). RESULTS: Angiotensin II dose dependently increased ROS production in WKY and SHR at all ages. The Ang II-induced responses were greater in SHR versus WKY at 9 and 16 weeks (P < .05). The Ang II-stimulated ROS increase was greater in 9- and 16-week-old SHR versus 4-week SHR (P < .05). These effects were reduced by AG 1024. Basal NAD(P)H oxidase activity was higher in VSMCs from 9-week-old SHR versus 4-week-old rats (P < .05). Angiotensin II induced a significant increase in oxidase activity in VSMCs from 9- and 16-week-old SHR (P < .001), without influencing responses in cells from 4-week-old SHR. Pretreatment of 9- and 16-week-old SHR cells with AG1024 reduced Ang II-mediated NAD(P)H oxidase activation (P < .05). CONCLUSIONS: Basal and Ang II-induced NAD(P)H-driven ROS generation are enhanced in VSMCs from SHR during development of hypertension, but not in cells from prehypertensive rats. Transactivation of IGF-1R by Ang II may be important in vascular oxidative excess in the development of hypertension in SHR.

Mechanisms of cardiovascular changes in an experimental model of syndrome X and pharmacological intervention on the renin-angiotensin-system.

Various cardiovascular risk factors and disease states similar to those present in type 2 diabetic patients also seem to be present in non-diabetic individuals. This cluster of risk factors has been called syndrome X, also known as metabolic cardiovascular syndrome or insulin resistance syndrome. Vascular wall components changes, including endothelial dysfunction and vascular smooth muscle cell (VSMC) migration and proliferation, could be involved in the cardiovascular alterations associated with this state. Fructose fed rats (FFR) provide a model of dietary-induced insulin resistance, which has been used to assess the pathophysiological mechanisms of the metabolic and cardiovascular changes associated to the syndrome X. FFR have hyperinsulinemia, insulin resistance (altered glucose tolerance test) and hypertriglyceridemia; they also develop moderate hypertension and cardiac hypertrophy. This has been confirmed in male rats of different strains, such as Wistar and Sprague-Dawley, chronically fed with a 60% fructose-chow or 10% fructose in the drinking water. At different levels of the cardiovascular system, FFR exhibit changes in the nitric oxide generation system and in primary cultured proliferation of VSMC from conduit and resistance arteries. These abnormalities were normalized by long-term treatment with pharmacological agents acting on the renin-angiotensin system (RAS), such as angiotensin converting-enzyme inhibitors or angiotensin-AT(1) receptor antagonists, that also lowered blood pressure to control levels and reversed cardiac hypertrophy. Evidence suggests an important role for the RAS in the pathogenic mechanisms involved in this model of syndrome X. Furthermore, beneficial pharmacological intervention seems to be mediated by AT(2) receptors and kinins.

Proteoglycans production by aortic vascular smooth muscle cells from hypertensive rats.

Remodeling of large and small arteries contributes to the development and complications of hypertension. Artery structural changes in chronic sustained hypertension include vascular smooth muscle cells (VSMC) proliferation and extracellular matrix (ECM) modifications. Extracellular constituents such as proteoglycans (PGs), may modulate vascular stiffness and VSMC growth and differentiation. We examined the effect of growth factors on secreted and membrane-bound PGs synthesis by cultured aortic smooth muscle cells (SMC) from 12- to 14- week-old spontaneously hypertensive rats (SHR) and age-matched Wistar rats. After stimulation with platelet-derived growth factor (PDGF-BB), 10% fetal calf serum (FCS) or 0.1% FCS as control, PGs synthesis (dpm/ng DNA) was evaluated in the medium (M-ECM) and in the cell layer (P-ECM) by a double-isotopic label method using both [3H]-glucosamine and [35S]-sodium sulfate which are incorporated into all complex carbohydrates or only into sulfated dysaccharides, respectively. Data are presented as percent of the control (0.1% FCS). SHR VSMC displayed a significantly greater synthesis of M-ECM [3H]-PGs than Wistar rat cells, with both treatments, but no differences in M-ECM [35S] uptake were found in any case. In the P-ECM, both PDGF-BB and 10% FCS produced a greater effect on [3H]-PGs and sulfated PGs synthesis in VSMC from SHR. An important change seen in SHR cells was a significant decreased sulfation, assessed by [35S]/[3H] ratio, in basal and stimulation conditions. Present results indicate the existence of changes in PGS synthesis and modulation in VSMC from a conduit-artery of SHR and support the pathophysiological role proposed for matrix proteoglycans in the vascular wall changes associated to hypertension and related vascular diseases as atherosclerosis.

Redox-dependent MAP kinase signaling by Ang II in vascular smooth muscle cells: role of receptor tyrosine kinase transactivation.

We investigated the role of receptor tyrosine kinases in Ang II-stimulated generation of reactive oxygen species (ROS) and assessed whether MAP kinase signaling by Ang II is mediated via redox-sensitive pathways. Production of ROS and activation of NADPH oxidase were determined by DCFDA (dichlorodihydrofluorescein diacetate; 2 micromol/L) fluorescence and lucigenin (5 micromol/L) chemiluminescence, respectively, in rat vascular smooth muscle cells (VSMC). Phosphorylation of ERK1/2, p38MAP kinase and ERK5 was determined by immunoblotting. The role of insulin-like growth factor-1 receptor (IGF-1R) and epidermal growth factor receptor (EGFR) was assessed with the antagonists AG1024 and AG1478, respectively. ROS bioavailability was manipulated with Tiron (10(-5) mol/L), an intracellular scavenger, and diphenylene iodinium (DPI; 10(-6) mol/L), an NADPH oxidase inhibitor. Ang II stimulated NADPH oxidase activity and dose-dependently increased ROS production (p < 0.05). These actions were reduced by AG1024 and AG1478. Ang II-induced ERK1/2 phosphorylation (276% of control) was decreased by AG1478 and AG1024. Neither DPI nor tiron influenced Ang II-stimulated ERK1/2 activity. Ang II increased phosphorylation of p38 MAP kinase (204% of control) and ERK5 (278% of control). These effects were reduced by AG1024 and AG1478 and almost abolished by DPI and tiron. Thus Ang II stimulates production of NADPH-inducible ROS partially through transactivation of IGF-1R and EGFR. Inhibition of receptor tyrosine kinases and reduced ROS bioavaliability attenuated Ang II-induced phosphorylation of p38 MAP kinase and ERK5, but not of ERK1/2. These findings suggest that Ang II activates p38MAP kinase and ERK5 via redox-dependent cascades that are regulated by IGF-1R and EGFR transactivation. ERK1/2 regulation by Ang II is via redox-insensitive pathways.

Proteoglycans production by aortic vascular smooth muscle cells from hypertensive rats.

Remodeling of large and small arteries contributes to the development and complications of hypertension. Artery structural changes in chronic sustained hypertension include vascular smooth muscle cells (VSMC) proliferation and extracellular matrix (ECM) modifications. Extracellular constituents such as proteoglycans (PGs), may modulate vascular stiffness and VSMC growth and differentiation. We examined the effect of growth factors on secreted and membrane-bound PGs synthesis by cultured aortic smooth muscle cells (SMC) from 12- to 14- week-old spontaneously hypertensive rats (SHR) and age-matched Wistar rats. After stimulation with platelet-derived growth factor (PDGF-BB), 10

fetal calf serum (FCS) or 0.1

FCS as control, PGs synthesis (dpm/ng DNA) was evaluated in the medium (M-ECM) and in the cell layer (P-ECM) by a double-isotopic label method using both [3H]-glucosamine and [35S]-sodium sulfate which are incorporated into all complex carbohydrates or only into sulfated dysaccharides, respectively. Data are presented as percent of the control (0.1

FCS). SHR VSMC displayed a significantly greater synthesis of M-ECM [3H]-PGs than Wistar rat cells, with both treatments, but no differences in M-ECM [35S] uptake were found in any case. In the P-ECM, both PDGF-BB and 10

FCS produced a greater effect on [3H]-PGs and sulfated PGs synthesis in VSMC from SHR. An important change seen in SHR cells was a significant decreased sulfation, assessed by [35S]/[3H] ratio, in basal and stimulation conditions. Present results indicate the existence of changes in PGS synthesis and modulation in VSMC from a conduit-artery of SHR and support the pathophysiological role proposed for matrix proteoglycans in the vascular wall changes associated to hypertension and related vascular diseases as atherosclerosis.

Effects of enalapril on the vascular wall in an experimental model of syndrome X.

Evidence links the insulin resistance syndrome with endothelial dysfunction. Previously, we have described a decreased endothelial nitric oxide synthase (eNOS) activity in both aortic endothelium and cardiac tissue, and an increased proliferation of aortic primary cultured vascular smooth muscle cells (pC-VSMCs), obtained from fructose-fed rats (FFR), an experimental model of syndrome X. Because the participation of the renin-angiotensin system (RAS) in this model is still unclear, the present study examined the effect of chronic administration of an angiotensin converting enzyme (ACE) inhibitor enalapril (E) on pC-VSMCs proliferation and eNOS activity in a conduit artery (aorta) and in resistance vessels (mesenteric vascular bed) from fructose-fed rats. Male Wistar rats were used: Control, FFR, Control + E, and FFR + E (n = 8 in each group). After 8 weeks, tissue samples were obtained and 10% fetal calf serum (FCS) proliferative effect was examined in pC-SMCs of aortic and mesenteric arteries by [(3)H]thymidine incorporation. The eNOS activity was estimated in endothelial lining from both origins by conversion of [(3)H]arginine into [(3)H]citrulline. The FFR aortic and mesenteric pC-VSMCs showed a significantly increased 10% FCS-induced [(3)H]thymidine incorporation compared to controls. The FFR aortic and mesenteric endothelium eNOS activity was significantly decreased. Chronic treatment with E abolished the increased proliferation and restored eNOS activity. These data confirm that changes in VSMCs proliferation and endothelial dysfunction at different levels of the vascular system are involved in syndrome X, and that the inhibition of angiotensin II production can revert those changes, suggesting an important role for RAS and possibly kinins, in the physiopathologic mechanism of this model of syndrome X.

Changes of inducible nitric oxide synthase in aortic cells during the development of hypertension: effect of angiotensin II.

Nitric oxide (NO) generation by inducible nitric oxide synthase (iNOS) in the vascular smooth muscle cells (VSMC), may play a role in blood vessel tone regulation. Lipopolysaccharide (LPS) induced iNOS activity and subsequent nitrite production by cultured aortic VSMC, from SHR with an established chronic blood pressure elevation (adult SHR) or during the period preceding the development of hypertension (young SHR) and from age-matched normotensive Wistar (W) rats were compared. Angiotensin II (Ang II) effect was also evaluated. Both basal LPS-induced iNOS activity and nitrite accumulation were significantly lower in young SHR VSMC compared to young W rat cells. In contrast, adult hypertensive and normotensive rat cells did not differ in NO generation. Besides, young SHR cells exhibited a significant smaller iNOS activity and nitrites than adult SHR cells. After 24 h-incubation with Ang II, both variables were markedly reduced in all groups. The proportional reduction of iNOS activity and nitrites by Ang II was not different between hypertensive and normotensive rat cells, at any age. However, this Ang II inhibitory effect was greater in both adult SHR and W cells than in VSMC from young rats. In conclusion, a reduced LPS-induced iNOS activity and NO generation was observed in VSMC form spontaneously hypertensive rats before the raise of blood pressure, but not in adult hypertensive rat cells. Additionally, an inhibitory effect of angiotensin II on these variables is described. We can speculate that the impairment in vascular smooth muscle NO production precedes the development of hypertension in SHR and may play a pathophysiologic role in the early blood pressure elevation in genetically hypertensive rats.

Early changes in proteoglycans production by resistance arteries smooth muscle cells of hypertensive rats.

Several functional and structural modifications at the vascular level have been described in spontaneously hypertensive rats (SHR) during the early development of hypertension. In this study, we hypothesize that changes in the extracellular matrix (ECM) could precede the development of hypertension. Synthesis of secreted and membrane-bound sulfated proteoglycans (S-PG) by cultured vascular smooth muscle cells (VSMC) obtained from young spontaneously hypertensive rats (pSHR) mesenteric resistance arteries, during the period preceding the elevation of blood pressure (BP) was tested. After 24 h of stimulation with angiotensin II (Ang II), 10% fetal calf serum (FCS), or 0.1% FCS as control, medium and cell layer S-PG synthesis was evaluated by labeling sulfated disaccharides with [35S] sodium sulfate. To relate this variable with cell proliferation, DNA synthesis was measured by incorporation of [3H]thymidine in the cell lysate. The VSMC from pSHR synthesized more secreted and membrane-bound S-PG than age-matched Wistar rat (pW) cells in the nonstimulated (0.1% FCS) and stimulated (Ang II or 10% FCS) experimental groups. When data were expressed as percent of their own control value, both Ang II and 10% FCS lowered basal secreted and cell-associated S-PG content in VSMC from pSHR, whereas in pW rat cells, these agents produced a small increase or no change. An inverse relationship between proliferation and total S-PG production (secreted plus membrane-bound) was found in pSHR cells, but not in pW cells. In conclusion, the present study demonstrates that changes in S-PG synthesis by VSMC of resistance arteries precede the vascular dysfunction associated with the development of hypertension in SHR.

Changes of inducible nitric oxide synthase in aortic cells during the development of hypertension: effect of angiotensin II

Nitric oxide (NO) generation by inducible nitric oxide synthase (iNOS) in the vascular smooth muscle cells (VSMC), may play a role in blood vessel tone regulation. Lipopolysaccharide (LPS) induced iNOS activity and subsequent nitrite production by cultured aortic VSMC, from SHR with an established chronic blood pressure elevation (adult SHR) or during the period preceding the development of hypertension (young SHR) and from age-matched normotensive Wistar (W) rats were compared. Angiotensin II (Ang II) effect was also evaluated. Both basal LPS-induced iNOS activity and nitrite accumulation were significantly lower in young SHR VSMC compared to young W rat cells. In contrast, adult hypertensive and normotensive rat cells did not differ in NO generation. Besides, young SHR cells exhibited a significant smaller iNOS activity and nitrites than adult SHR cells. After 24 h-incubation with Ang II, both variables were markedly reduced in all groups. The proportional reduction of iNOS activity and nitrites by Ang II was not different between hypertensive and normotensive rat cells, at any age. However, this Ang II inhibitory effect was greater in both adult SHR and W cells than in VSMC from young rats. In conclusion, a reduced LPS-induced iNOS activity and NO generation was observed in VSMC form spontaneously hypertensive rats before the raise of blood pressure, but not in adult hypertensive rat cells. Additionally, an inhibitory effect of angiotensin II on these variables is described. We can speculate that the impairment in vascular smooth muscle NO production precedes the development of hypertension in SHR and may play a pathophysiologic role in the early blood pressure elevation in genetically hypertensive rats.

Changes of inducible nitric oxide synthase in aortic cells during the development of hypertension: effect of angiotensin II

Nitric oxide (NO) generation by inducible nitric oxide synthase (iNOS) in the vascular smooth muscle cells (VSMC), may play a role in blood vessel tone regulation. Lipopolysaccharide (LPS) induced iNOS activity and subsequent nitrite production by cultured aortic VSMC, from SHR with an established chronic blood pressure elevation (adult SHR) or during the period preceding the development of hypertension (young SHR) and from age-matched normotensive Wistar (W) rats were compared. Angiotensin II (Ang II) effect was also evaluated. Both basal LPS-induced iNOS activity and nitrite accumulation were significantly lower in young SHR VSMC compared to young W rat cells. In contrast, adult hypertensive and normotensive rat cells did not differ in NO generation. Besides, young SHR cells exhibited a significant smaller iNOS activity and nitrites than adult SHR cells. After 24 h-incubation with Ang II, both variables were markedly reduced in all groups. The proportional reduction of iNOS activity and nitrites by Ang II was not different between hypertensive and normotensive rat cells, at any age. However, this Ang II inhibitory effect was greater in both adult SHR and W cells than in VSMC from young rats. In conclusion, a reduced LPS-induced iNOS activity and NO generation was observed in VSMC form spontaneously hypertensive rats before the raise of blood pressure, but not in adult hypertensive rat cells. Additionally, an inhibitory effect of angiotensin II on these variables is described. We can speculate that the impairment in vascular smooth muscle NO production precedes the development of hypertension in SHR and may play a pathophysiologic role in the early blood pressure elevation in genetically hypertensive rats.(AU)