Régulation épigénétique de la défense antioxydante et de l'Angiopoietin-like 2 dans le contexte du vieillissement et des maladies cardiovasculaires

Suite à l’exposition à des facteurs de risque incluant la malnutrition, la dyslipidémie, la sédentarité et les désordres métaboliques, les maladies cardiovasculaires (MCV) sont caractérisées par un état pro-oxydant et pro-inflammatoire, et une dérégulation de l’expression de divers facteurs responsables de l’homéostasie de l’environnement rédox et inflammatoire. L’implication d’enzymes antioxydantes telles que les superoxyde dismutases (SOD) et les glutathion peroxydases (Gpx), ainsi que la contribution de médiateurs pro-inflammatoires tels que l’angiopoietin-like 2 (Angptl2) ont été rapportées dans le cadre des MCV. Toutefois, les mécanismes moléculaires sensibles aux facteurs de risque et menant au développement des MCV sont peu connus. L’épigénétique est un mécanisme de régulation de l’expression génique sensible aux stimuli extracellulaires et pourrait donc contribuer au développement des MCV. La méthylation de l’ADN est un des mécanismes épigénétiques pouvant varier tant de manière gène-spécifique qu’à l’échelle génomique, et la conséquence de tels changements sur l’expression des gènes ciblés dépend du site de méthylation. Puisqu’il a été démontré que des variations au niveau de la méthylation de l’ADN peuvent être associées à divers contextes pathologiques incluant les MCV, le but de nos travaux était d’étudier le lien entre la méthylation de gènes antioxydants et pro-inflammatoires avec leurs répercussions fonctionnelles biologiques en présence de facteurs de risques associés aux MCV, tels que le vieillissement, la dyslipidémie et la sédentarité. Dans la première étude, nous avons observé que dans l’artère fémorale de souris vieillissantes, la méthylation au niveau du promoteur du gène Sod2, codant pour l’enzyme antioxydante superoxyde dismutase de type 2 (SOD2 ou MnSOD), diminue avec l’âge. Ceci serait associé à l’induction de l’expression de MnSOD, renforçant ainsi la défense antioxydante endogène. Le vieillissement étant associé à une accumulation de la production de radicaux libres, nous avons étudié la vasodilatation dépendante de l’endothélium qui est sensible au stress oxydant. Nous avons observé que la capacité vasodilatatrice globale a été maintenue chez les souris âgées, aux dépens d’une diminution des facteurs hyperpolarisants dérivés de l’endothélium (EDHF) et d’une contribution accentuée de la voie du monoxyde d’azote (NO). Nous avons ensuite utilisé deux approches visant à réduire les niveaux de stress oxydant in vivo, soit la supplémentation avec un antioxydant, la catéchine, et l’exposition chronique à de l’exercice physique volontaire. Ces interventions ont permis de prévenir à la fois les changements au niveau de la fonction endothéliale et de l’hypométhylation de Sod2. Cette première étude démontre donc la sensibilité de la méthylation de l’ADN à l’environnement rédox. Dans la deuxième étude, nous avons démontré une régulation de l’expression de l’enzyme antioxydante glutathion peroxydase 1 (Gpx1) en lien avec la méthylation de son gène codant, Gpx1, dans un contexte de dyslipidémie sévère. Nos résultats démontrent que dans le muscle squelettique de souris transgéniques sévèrement dyslipidémiques (LDLr-/-; hApoB+/+), Gpx1 est hyperméthylé, ce qui diminue l’expression de Gpx1 et affaiblit la défense antioxydante endogène. Chez ces souris, l’exercice physique chronique a permis d’augmenter l’expression de Gpx1 en lien avec une hypométhylation transitoire de son gène. Cette étude démontre que le stress oxydant associé à la dyslipidémie sévère altère les mécanismes de défense antioxydante, en partie via un mécanisme épigénétique. De plus, on observe également que l’exercice physique permet de renverser ces effets et peut induire des changements épigénétiques, mais de manière transitoire. La troisième étude avait pour but d’étudier la régulation de l’Angptl2, une protéine circulante pro-inflammatoire, dans le contexte des MCV. Nous avons observé que chez des patients coronariens, la concentration circulante d’Angptl2 est significativement plus élevée que chez des sujets sains et ce, en lien avec une hypométhylation de son gène, ANGPTL2, mesurée dans les leucocytes circulants. Nous sommes les premiers à démontrer qu’en réponse à l’environnement pro-inflammatoire associé à une MCV, l’expression de l’Angptl2 est stimulée par un mécanisme épigénétique. Nos études ont permis d’identifier des nouvelles régions régulatrices différentiellement méthylées situées dans les gènes impliqués dans la défense antioxydante, soit Sod2 en lien avec le vieillissement et Gpx1 en lien avec la dyslipidémie et l’exercice. Nous avons également démontré un mécanisme de régulation de l’Angptl2 dépendant de la méthylation d’ANGPTL2 et ce, pour la première fois dans un contexte de MCV. Ces observations illustrent la nature dynamique de la régulation épigénétique par la méthylation de l’ADN en réponse aux stimuli environnementaux. Nos études contribuent ainsi à la compréhension et l’identification de mécanismes moléculaires impliqués dans le développement du phénotype pathologique suite à l’exposition aux facteurs de risque, ce qui ouvre la voie à de nouvelles approches thérapeutiques.

The pathophysiology of CADASIL: studies in a Scottish cohort

Since identification that mutations in NOTCH3 are responsible for cerebral autosomal dominant arteriopathy with subcortical infarcts and leucoencephalopathy (CADASIL) in the early 1990s, there has been extensive characterisation of the clinical and radiological features of the disease. However therapeutic interventions remain elusive, partly due to a limited understanding of the vascular pathophysiology and how it leads to the development of strokes, cognitive decline and disability. The apparent rarity and heterogenous natural history of CADASIL potentially make conducting any longitudinal or therapeutic trials difficult. The role of disease biomarkers is therefore of some interest. This thesis focuses on vascular function in CADASIL and how it may relate to clinical and radiological markers of disease. Establishing the prevalence of CADASIL in the West of Scotland was important to assess the impact of the disease, and how feasible a trial would be. A mutation prevalence of 10.7 per 100,000 was demonstrated, suggesting significant under diagnosis of the disease across much of Scotland. Cerebral hypoperfusion is thought to be important in CADASIL, and it has been shown that vascular abnormalities precede the development of brain pathology in mouse models. Investigation of vascular function in patients, both in the brain and systemically, requires less invasive measures. Arterial spin labelling magnetic resonance imaging (MRI) and transcranial Doppler ultrasound (TCD) can both be used to obtain non-invasive and quantifiable indices of vascular function. Monitoring patients with MRI whilst they receive different concentrations of inspired oxygen and carbon dioxide can provide information on brain function, and I reviewed the practicalities of this technique in order to guide the design of the studies in this thesis. 22 CADASIL patients were recruited to a longitudinal study. Testing included peripheral vascular assessment, assessment of disability, neurological dysfunction, mood and cognition. A CO2 reactivity challenge during both TCD and arterial spin labelling MRI, and detailed MRI sequences were obtained. I was able to demonstrate that vasoreactivity was associated with the number of lacunes and brain atrophy, as were carotid intima-media thickness, vessel stiffness, and age. Patients with greater disability, higher depressive symptoms and poorer processing speed showed a tendency to worse cerebral vasoreactivity but numbers were small. This observation suggests vasoreactivity may have potential as a therapeutic target, or a biomarker. I then wished to establish if arterial spin labelling MRI was useful for assessing change in cerebral blood flow in CADASIL patients. Cortical grey matter showed the highest blood flow, mean (SD), 55 (10) ml/100g/min and blood flow was significantly lower within hyperintensities (19 (4) ml/100g/min; p <0.001). Over one year, blood flow in both grey matter (mean -7 (10) %; p = 0.028) and deep white matter (-8 (13) %; p = 0.036) declined significantly. Cerebrovascular reactivity did not change over one year. I then investigated whether baseline vascular markers were able to predict change in radiological or neuropsychological measures of disease. Changes in brain volume, lacunes, microbleeds and normalised subcortical hyperintensity volume (increase of 0.8%) were shown over one year. Baseline vascular parameters were not able to predict these changes, or those in neuropsychological testing. NOTCH3 is found throughout the body and a systemic vasculopathy has been seen particularly affecting resistance vessels. Gluteal biopsies were obtained from 20 CADASIL patients, and ex vivo myography investigated the response to vasoactive agents. Evidence of impairment in both vasodilation and vasoconstriction was shown. The addition of antioxidants improved endothelium-dependent relaxation, indicating a role for oxidative stress in CADASIL pathology. Myography measures were not related to in vivo measures in the sub-group of patients who had taken part in both studies. The small vessels affected in CADASIL are unable to be imaged by conventional MR imaging so I aimed to establish which vessels might be responsible for lacunes with use of a microangiographic template overlaid onto brain images registered to a standard brain template. This showed most lacunes are small and associated with tertiary arterioles. On the basis of this thesis, it is concluded that vascular dysfunction plays an important role in the pathophysiology of CADASIL, and further assessment of vascular measures in longitudinal studies is needed. Arterial spin labelling MRI should be used as it is a reliable, non-invasive modality that can measure change over one year. Furthermore conventional cardiovascular risk factor prevention should be undertaken in CADASIL patients to delay the deleterious effects of the disease.

Régulation épigénétique de la défense antioxydante et de l'Angiopoietin-like 2 dans le contexte du vieillissement et des maladies cardiovasculaires

Suite à l’exposition à des facteurs de risque incluant la malnutrition, la dyslipidémie, la sédentarité et les désordres métaboliques, les maladies cardiovasculaires (MCV) sont caractérisées par un état pro-oxydant et pro-inflammatoire, et une dérégulation de l’expression de divers facteurs responsables de l’homéostasie de l’environnement rédox et inflammatoire. L’implication d’enzymes antioxydantes telles que les superoxyde dismutases (SOD) et les glutathion peroxydases (Gpx), ainsi que la contribution de médiateurs pro-inflammatoires tels que l’angiopoietin-like 2 (Angptl2) ont été rapportées dans le cadre des MCV. Toutefois, les mécanismes moléculaires sensibles aux facteurs de risque et menant au développement des MCV sont peu connus. L’épigénétique est un mécanisme de régulation de l’expression génique sensible aux stimuli extracellulaires et pourrait donc contribuer au développement des MCV. La méthylation de l’ADN est un des mécanismes épigénétiques pouvant varier tant de manière gène-spécifique qu’à l’échelle génomique, et la conséquence de tels changements sur l’expression des gènes ciblés dépend du site de méthylation. Puisqu’il a été démontré que des variations au niveau de la méthylation de l’ADN peuvent être associées à divers contextes pathologiques incluant les MCV, le but de nos travaux était d’étudier le lien entre la méthylation de gènes antioxydants et pro-inflammatoires avec leurs répercussions fonctionnelles biologiques en présence de facteurs de risques associés aux MCV, tels que le vieillissement, la dyslipidémie et la sédentarité. Dans la première étude, nous avons observé que dans l’artère fémorale de souris vieillissantes, la méthylation au niveau du promoteur du gène Sod2, codant pour l’enzyme antioxydante superoxyde dismutase de type 2 (SOD2 ou MnSOD), diminue avec l’âge. Ceci serait associé à l’induction de l’expression de MnSOD, renforçant ainsi la défense antioxydante endogène. Le vieillissement étant associé à une accumulation de la production de radicaux libres, nous avons étudié la vasodilatation dépendante de l’endothélium qui est sensible au stress oxydant. Nous avons observé que la capacité vasodilatatrice globale a été maintenue chez les souris âgées, aux dépens d’une diminution des facteurs hyperpolarisants dérivés de l’endothélium (EDHF) et d’une contribution accentuée de la voie du monoxyde d’azote (NO). Nous avons ensuite utilisé deux approches visant à réduire les niveaux de stress oxydant in vivo, soit la supplémentation avec un antioxydant, la catéchine, et l’exposition chronique à de l’exercice physique volontaire. Ces interventions ont permis de prévenir à la fois les changements au niveau de la fonction endothéliale et de l’hypométhylation de Sod2. Cette première étude démontre donc la sensibilité de la méthylation de l’ADN à l’environnement rédox. Dans la deuxième étude, nous avons démontré une régulation de l’expression de l’enzyme antioxydante glutathion peroxydase 1 (Gpx1) en lien avec la méthylation de son gène codant, Gpx1, dans un contexte de dyslipidémie sévère. Nos résultats démontrent que dans le muscle squelettique de souris transgéniques sévèrement dyslipidémiques (LDLr-/-; hApoB+/+), Gpx1 est hyperméthylé, ce qui diminue l’expression de Gpx1 et affaiblit la défense antioxydante endogène. Chez ces souris, l’exercice physique chronique a permis d’augmenter l’expression de Gpx1 en lien avec une hypométhylation transitoire de son gène. Cette étude démontre que le stress oxydant associé à la dyslipidémie sévère altère les mécanismes de défense antioxydante, en partie via un mécanisme épigénétique. De plus, on observe également que l’exercice physique permet de renverser ces effets et peut induire des changements épigénétiques, mais de manière transitoire. La troisième étude avait pour but d’étudier la régulation de l’Angptl2, une protéine circulante pro-inflammatoire, dans le contexte des MCV. Nous avons observé que chez des patients coronariens, la concentration circulante d’Angptl2 est significativement plus élevée que chez des sujets sains et ce, en lien avec une hypométhylation de son gène, ANGPTL2, mesurée dans les leucocytes circulants. Nous sommes les premiers à démontrer qu’en réponse à l’environnement pro-inflammatoire associé à une MCV, l’expression de l’Angptl2 est stimulée par un mécanisme épigénétique. Nos études ont permis d’identifier des nouvelles régions régulatrices différentiellement méthylées situées dans les gènes impliqués dans la défense antioxydante, soit Sod2 en lien avec le vieillissement et Gpx1 en lien avec la dyslipidémie et l’exercice. Nous avons également démontré un mécanisme de régulation de l’Angptl2 dépendant de la méthylation d’ANGPTL2 et ce, pour la première fois dans un contexte de MCV. Ces observations illustrent la nature dynamique de la régulation épigénétique par la méthylation de l’ADN en réponse aux stimuli environnementaux. Nos études contribuent ainsi à la compréhension et l’identification de mécanismes moléculaires impliqués dans le développement du phénotype pathologique suite à l’exposition aux facteurs de risque, ce qui ouvre la voie à de nouvelles approches thérapeutiques.

Endothelial dysfunction in cardiovascular and endocrine-metabolic diseases: an update

The endothelium plays a vital role in maintaining circulatory homeostasis by the release of relaxing and contracting factors. Any change in this balance may result in a process known as endothelial dysfunction that leads to impaired control of vascular tone and contributes to the pathogenesis of some cardiovascular and endocrine/metabolic diseases. Reduced endothelium-derived nitric oxide (NO) bioavailability and increased production of thromboxane A2, prostaglandin H2 and superoxide anion in conductance and resistance arteries are commonly associated with endothelial dysfunction in hypertensive, diabetic and obese animals, resulting in reduced endothelium-dependent vasodilatation and in increased vasoconstrictor responses. In addition, recent studies have demonstrated the role of enhanced overactivation ofβ-adrenergic receptors inducing vascular cytokine production and endothelial NO synthase (eNOS) uncoupling that seem to be the mechanisms underlying endothelial dysfunction in hypertension, heart failure and in endocrine-metabolic disorders. However, some adaptive mechanisms can occur in the initial stages of hypertension, such as increased NO production by eNOS. The present review focuses on the role of NO bioavailability, eNOS uncoupling, cyclooxygenase-derived products and pro-inflammatory factors on the endothelial dysfunction that occurs in hypertension, sympathetic hyperactivity, diabetes mellitus, and obesity. These are cardiovascular and endocrine-metabolic diseases of high incidence and mortality around the world, especially in developing countries and endothelial dysfunction contributes to triggering, maintenance and worsening of these pathological situations.

Lipid Metabolism in Subclinical Hypothyroidism: Plasma Kinetics of Triglyceride-Rich Lipoproteins and Lipid Transfers to High-Density Lipoprotein Before and After Levothyroxine Treatment

Background: Subclinical hypothyroidism (SCH) has been associated with atherosclerosis, but the abnormalities in plasma lipids that can contribute to atherogenesis are not prominent. The aim of this study was to test the hypothesis that patients with normocholesterolemic, normotriglyceridemic SCH display abnormalities in plasma lipid metabolism not detected in routine laboratory tests including abnormalities in the intravascular metabolism of triglyceride-rich lipoproteins, lipid transfers to high-density lipoprotein (HDL), and paraoxonase 1 activity. The impact of levothyroxine (LT4) treatment and euthyroidism in these parameters was also tested. Methods: The study included 12 SCH women and 10 matched controls. Plasma kinetics of an artificial triglyceride-rich emulsion labeled with radioactive triglycerides and cholesteryl esters as well as in vitro transfer of four lipids from an artificial donor nanoemulsion to HDL were determined at baseline in both groups and after 4 months of euthyroidism in the SCH group. Results: Fractional clearance rates of triglycerides (SCH 0.035 +/- 0.016 min(-1), controls 0.029 +/- 0.013 min(-1), p=0.336) and cholesteryl esters (SCH 0.009 +/- 0.007 min(-1), controls 0.009 +/- 0.009 min(-1), p=0.906) were equal in SCH and controls and were unchanged by LT4 treatment and euthyroidism in patients with SCH, suggesting that lipolysis and remnant removal of triglyceride-rich lipoproteins were normal. Transfer of triglycerides to HDL (SCH 3.6 +/- 0.48%, controls 4.7 +/- 0.63%, p=0.001) and phospholipids (SCH 16.2 +/- 3.58%, controls 21.2 +/- 3.32%, p=0.004) was reduced when compared with controls. After LT4 treatment, transfers increased and achieved normal values. Transfer of free and esterified cholesterol to HDL, HDL particle size, and paraoxonase 1 activity were similar to controls and were unchanged by treatment. Conclusions: Although intravascular metabolism of triglyceride-rich lipoproteins was normal, patients with SCH showed abnormalities in HDL metabolism that were reversed by LT4 treatment and achievement of euthyroidism.

Effect of the effluent released from the canine internal mammary artery after intraluminal and extraluminal perfusion of acetylcholine and adenosine diphosphate

Segments of the canine internal mammary artery (35 mm in length) were suspended in vitro in an organ chamber containing physiological salt solution (95% O(2)/5% CO(2), pH = 7.4, 37 degrees C). Segments were individually cannulated and perfused at 5 ml/minute using a roller pump. Vasorelaxant activity of the effluent from the perfused internal mammary arteries was bioassayed by measuring the decrease in tension induced by the effluent of the coronary artery endothelium-free ring which had been contracted with prostaglandin F(2 alpha) (2 x 10(-6) M). Intraluminal perfusion of adenosine diphosphate (10(-5) M) induced significant increase in relaxant activity in the effluent from the perfused blood vessel. However, when adenosine diphosphate (10(-5) M) was added extraluminally to the internal mammary artery, no change in relaxant activity in the effluent was noted. In contrast, acetylcholine produced significant increase in the relaxant activity on the effluent of the perfused internal mammary artery with both intraluminal and extraluminal perfusion. The intraluminal and extraluminal release of endothelium-derived relaxing factor (EDRF) by acetylcholine (10(-5) M) can be inhibited by site-specific administration of atropine (10(-5) M). These experiments indicate that certain agonists can induce the release of EDRF only by binding to intravascular receptors while other agonists can induce endothelium-dependent vasodilatation by acting on neural side receptors.

An Apparent Paradox: Attenuation of Phenylephrine-mediated Calcium Mobilization and Hyperreactivity to Phenylephrine in Contralateral Carotid After Balloon Injury

Balloon catheter injury promotes hyperreactivity to phenylephrine (Phe) in the contralateral carotid. Phe-induced contraction involves calcium mobilization, a process that may be sensitive to reactive oxygen species. In this study, we investigated whether increased reactivity to Phe in the contralateral carotid is due to alterations in calcium mobilization by Phe and reactive oxygen species signaling. Concentration-response curves to Phe were obtained in control and contralateral arteries 4 days after balloon injury. Tiron did not modify E(max) to Phe in control arteries but reduced this parameter in the contralateral carotid to control levels. Moreover, immunofluorescence to dihydroethydine showed increased basal oxidative stress in the contralateral artery compared with control artery. Intracellular calcium mobilization by Phe in the contralateral artery was not different from control, but Phe-induced extracellular calcium mobilization was reduced in the contralateral artery compared with that in the control. These data were confirmed by confocal microscopy using Fluo 3-AM. Tiron and SC-236 increased Phe-induced calcium influx in the contralateral artery, which was similar to controls in the same conditions. However, catalase did not modify this response. Taken together, our results suggest that superoxide anions and prostanoids from cyclooxygenase-2 alter pathways downstream of alpha(1)-adrenoceptor activation in the contralateral carotid in response to injury. This results in reduced Phe-induced calcium influx, despite hyperreactivity to Phe.

Chronic methionine load-induced hyperhomocysteinemia impairs the relaxation induced by bradykinin in the isolated rat carotid

This study investigates the effects of chronic methionine intake on bradykinin (BK)-relaxation. Vascular reactivity experiments were performed on carotid rings from male Wistar rats. Treatment with methionine (0.1, 1 or 2 g kg(-1) per day) for 8 and 16 weeks, but not for 2 and 4 weeks, reduced the relaxation induced by BK. Indomethacin, a non-selective cyclooxygenase (COX) inhibitor, and SQ29548, a selective thromboxane A(2) (TXA(2))/prostaglandin H(2) (PGH(2)) receptor antagonist prevented the reduction in BK-relaxation observed in the carotid from methionine-treated rats. Conversely, AH6809, a selective prostaglandin F(2 alpha) (PGF(2 alpha)) receptor antagonist did not alter BK-relaxation in the carotid from methionine-treated rats. The nitric oxide synthase (NOS) inhibitors L-NAME, L-NNA and 7-nitroindazole reduced the relaxation induced by BK in carotids from control and methionine-treated rats. In summary, we found that chronic methionine intake impairs the endothelium-dependent relaxation induced by BK and this effect is due to an increased production of endothelial vasoconstrictor prostanoids (possibly TXA(2)) that counteracts the relaxant action displayed by the peptide.

Antioxidant treatment reduces matrix metalloproteinase-2-induced vascular changes in renovascular hypertension

Mounting evidence indicates that structural and functional vascular changes associated with two-kidney, one-clip (2K-1C) hypertension result, at least in part, from altered activity of matrix metalloproteinases (MMPs). Because MMPs are upregulated by increased formation of reactive oxygen species (ROS), we hypothesized that antioxidant approaches could attenuate the increases in MMP-2 expression/activity and the vascular dysfunction and remodeling associated with 2K-1C hypertension. Sham-operated or 2K-1C hypertensive rats were treated with tempol 18 mg/kg/day or apocyanin 25 mg/kg/day (or vehicle). Systolic blood pressure was monitored weekly. After 8 weeks of treatment, aortic rings were isolated to assess endothelium-dependent and -independent relaxation. Quantitative morphometry of structural changes in the aortic wall was studied in hematoxylin/eosin sections. Aortic and systemic ROS levels were measured using dihydroethidine and thiobarbituric acid-reactive substances, respectively. Aortic MMP-2 levels and activity were determined by gelatin and in situ zymography, fluorimetry, and immunohistochemistry. Tempol and apocyanin attenuated 2K-1C hypertension (181 +/- 20.8 and 192 +/- 17.6 mm Hg, respectively, versus 213 +/- 18 mm Hg in hypertensive controls; both p<0.05) and prevented the reduction in endothelium-dependent vasorelaxation found in 2K-1C rats. Tempol, but not apocyanin (p>0.05), prevented the vascular remodeling found in 2K-1C rats (all p<0.01). Tempol was more effective than apocyanin in attenuating hypertension-induced increases in oxidative stress (both p<0.05), MMP-2 levels, and MMP-2 activity in hypertensive rats (all p<0.05). Our results suggest that antioxidant approaches decrease MMP-2 upregulation and attenuate the vascular dysfunction and remodeling during 2K-1C hypertension. (C) 2009 Elsevier Inc. All rights reserved.

A high-fat meal impairs muscle vasodilatation response to mental stress in humans with Glu27 beta(2)-adrenoceptor polymorphism

Background: Forearm blood flow responses during mental stress are greater in individuals homozygous for the Glu27 allele. A high-fat meal is associated with impaired endothelium-dependent dilatation. We investigated the impact of high-fat ingestion on the muscle vasodilatory responses during mental stress in individuals with the Glu27 allele and those with the Gln27 allele of the beta(2)-adrenoceptor gene. Methods: A total of 162 preselected individuals were genotyped for the Glu27Gln beta(2)-adrenoceptor polymorphism. Twenty-four individuals participated in the study. Fourteen were homozygous for the Gln27 allele (Gln27Gln, 40 +/- 2 years; 64 +/- 2 kg), and 10 were homozygous for the Glu27 allele (Glu27Glu, 40 +/- 3 years; 65 +/- 3 kg). Forearm blood flow was evaluated by venous occlusion plethysmography before and after ingestion of 62 g of fat. Results: The high-fat meal caused no changes in baseline forearm vascular conductance (FVC, 2.2 +/- 0.1 vs. 2.4 +/- 0.2; P = 0.27, respectively), but reduced FVC responses to mental stress (1.5 +/- 0.2 vs. 0.8 +/- 0.2 units; P = 0.04). When volunteers were divided according to their genotypes, baseline FVC was not different between groups (Glu27Glu = 2.4 +/- 0.1 vs. Gln27Gln = 2.1 +/- 0.1 units; P = 0.08), but it was significantly greater in Glu27Glu individuals during mental stress (1.9 +/- 0.4 vs. 1.0 +/- 0.3 units; P = 0.04). High-fat intake eliminated the difference in FVC responses between Glu27Glu and Gln27Gln individuals (FVC, 1.3 +/- 0.4 vs. 1.2 +/- 0.4; P = 0.66, respectively). Conclusion: These findings demonstrate that a high-fat meal impairs muscle vasodilatation responses to mental stress in humans. However, this reduction can be attributed to the presence of the homozygous Glu27 allele of the beta(2)-adrenoceptor gene.

Asymmetric dimethylarginine endogenous inhibition of nitric oxide synthase causes differential vasculature effects

Background: Asymmetric dimethylarginine (ADMA), produced during protein metabolism, is an endogenous inhibitor of nitric oxide synthase, but little is known about its direct vasoactive properties in different arterial beds. Material/Methods: Segments of canine coronary, renal, and femoral arteries were pretreated with increasing concentrations of ADMA, and endothelial function was evaluated in organ chambers. Results: In precontracted canine coronary arteries, the highest concentrations of ADMA inhibited endothelium-dependent relaxation mediated by acetylcholine (n=7), but no concentration of ADMA inhibited receptor-independent relaxation mediated by calcium ionophore (n=7) (P<.001). The effect of ADMA on acetylcholine-mediated relaxation was shown to be competitive inhibition of the nitric oxide synthase pathway, because the addition of L-arginine (10(-3) M), but not D-arginine (101 M), reversed the effect produced by 10(-5) M ADMA. Further, ADMA did not alter endothelium-independent relaxation mediated by sodium nitroprusside (10(-9) to 10(-6) M; n=7). Femoral arteries (n=7) and renal arteries (n=7) were more sensitive to ADMA than were coronary arteries, and they demonstrated significant ADMA inhibition to receptor dependent relaxation induced by acetylcholine (P=.03 and P=.01, respectively) and to receptor-independent relaxation induced by calcium ionophore (P=.02 and P=.01, respectively). Conclusions: Endothelium-dependent relaxation mediated by ADMA is more marked in femoral and renal arteries than in coronary arteries. The response in coronary arteries may be overall protective. Considering these different effects in various artery types, the role of ADMA as a confiable and specific cardiovascular risk factor is questioned.

Ethanol consumption increases blood pressure and alters the responsiveness of the mesenteric vasculature in rats

Chronic ethanol Consumption and hypertension are related. In the current study we investigated whether changes in reactivity of the mesenteric arterial bed could account for the increased blood pressure associated with chronic ethanol intake. Changes in reactivity to phenylephrine and acetylcholine were investigated in the perfused mesenteric bed from rats treated with ethanol for 2 or 6 weeks and their age-matched controls. Mild hypertension was observed in chronically ethanol-treated rats. Treatment of rats for 6 weeks induced an increase in the contractile response of endothelium-intact mesenteric bed to phenylephrine, but not denuded rat mesenteric bed. The phenylephrine-induced increase in perfusion pressure was not altered after 2 weeks` treatment with ethanol. Moreover, acetylcholine-induced endothelium-dependent relaxation was reduced by ethanol treatment for 6 weeks, but not 2 weeks. Pre-treatment with indometacin, a cyclooxygenase inhibitor, reduced the maximum effect induced by phenylephrine (E-max) in endothelium-intact mesenteric bed from both control and ethanol-treated rats. No differences in the E-max values for phenylephrine were observed between groups in the presence of indometacin. L-NNA, a nitric oxide (NO) synthase (NOS) inhibitor, increased the E-max for phenylephrine in endothelium-intact mesenteric bed from control rats but not from ethanol-treated rats. Levels of endothelial NOS (eNOS) mRNA were not altered by chronic ethanol consumption. However, chronic ethanol intake strongly reduced eNOS protein levels in the mesenteric bed. This study shows that chronic ethanol consumption increases blood pressure and alters the reactivity of the mesenteric bed. Moreover, the increased vascular response to phenylephrine observed in the mesenteric bed is maintained by two mechanisms: an increased release of endothelial-derived vasoconstrictor prostanoids and a reduced modulatory action of endothelial NO, which seems to be associated with reduced post-transcriptional expression of eNOS.

Improved Endothelial Function and Reversal of Myocardial Perfusion Defects after Aerobic Physical Training in a Patient with Microvascular Myocardial Ischemia

The objective of this report is to document the effects of an aerobic training program on myocardial perfusion, and endothelial function abnormalities, and on the relief of angina in a patient with microvascular myocardial ischemia. A 53-year-old female patient exhibited precordial pain on effort and angiographically normal coronaries. Her symptoms had been present for 4 yrs despite pharmacologic treatment for the control of risk factors, with myocardial perfusion scintigraphy revealing an extensive reversible perfusion defect. She was submitted to aerobic training for 4 mos, obtaining significant improvement of the anginal symptoms. Additionally, after the aerobic training program, scintigraphy revealed the disappearance of the myocardial perfusion defect, with a marked improvement of endothelium-dependent vasodilatory response and an improved quality-of-life score. These results suggest that aerobic training can improve endothelial function, leading to a reduction of ischemia and an improved quality-of-life in patients with microvascular myocardial ischemia.

TNF-alpha Knockout Mice Have Increased Corpora Cavernosa Relaxation

Erectile dysfunction is considered an early clinical manifestation of vascular disease and an independent risk factor for cardiovascular events associated with endothelial dysfunction and increased levels of pro-inflammatory cytokines. Tumor necrosis factor-alpha (TNF-alpha), a pro-inflammatory cytokine, suppresses endothelial nitric oxide synthase (eNOS) expression. Considering that nitric oxide (NO) is of critical importance in penile erection, we hypothesized that blockade of TNF-alpha actions would increase cavernosal smooth muscle relaxation. In vitro organ bath studies were used to measure cavernosal reactivity in wild type and TNF-alpha knockout (TNF-alpha KO) mice and NOS expression was evaluated by western blot. In addition, spontaneous erections (in vivo) were evaluated by videomonitoring the animals (30 minutes). Collagen and elastin expression were evaluated by Masson trichrome and Verhoff-van Gieson stain reaction, respectively. Corpora cavernosa from TNF-alpha KO mice exhibited increased NO-dependent relaxation, which was associated with increased eNOS and neuronal NOS (nNOS) cavernosal expression. Cavernosal strips from TNF-alpha KO mice displayed increased endothelium-dependent (97.4 +/- 5.3 vs. Control: 76.3 +/- 6.3, %) and nonadrenergic-noncholinergic (93.3 +/- 3.0 vs. Control: 67.5 +/- 16.0; 16 Hz) relaxation compared to control animals. These responses were associated with increased protein expression of eNOS and nNOS (P < 0.05). Sympathetic-mediated (0.69 +/- 0.16 vs. Control: 1.22 +/- 0.22; 16 Hz) as well as phenylephrine-induced contractile responses (1.6 +/- 0.1 vs. Control: 2.5 +/- 0.1, mN) were attenuated in cavernosal strips from TNF-alpha KO mice. Additionally, corpora cavernosa from TNF-alpha KO mice displayed increased collagen and elastin expression. In vivo experiments demonstrated that TNF-alpha KO mice display increased number of spontaneous erections. Corpora cavernosa from TNF-alpha KO mice display alterations that favor penile tumescence, indicating that TNF-alpha plays a detrimental role in erectile function. A key role for TNF-alpha in mediating endothelial dysfunction in ED is markedly relevant since we now have access to anti-TNF-alpha therapies. Carneiro FS, Sturgis LC, Giachini FRC, Carneiro ZN, Lima VV, Wynne BM, Martin SS, Brands MW, Tostes RC, and Webb RC. TNF-alpha knockout mice have increased corpora cavernosa relaxation. J Sex Med 2009;6:115-125.

Metalloproteinase inhibition ameliorates hypertension and prevents vascular dysfunction and remodeling in renovascular hypertensive rats

Altered activity of matrix metalloproteinases (MMPs) is implicated in the vascular remodeling of hypertension. We examined whether increased MMP-2 expression/activity plays a role in the vascular remodeling and dysfunction found in the two-kidney, one-clip (2K-1C) hypertension. Sham operated or 2K-1C hypertension rats were treated with doxycycline 30 mg/(kg day) (or vehicle). Systolic blood pressure was monitored weekly. After 8 weeks of treatment, aortic rings were isolated to assess endothelium-dependent and independent relaxations. Quantitative morphometry of structural changes, collagen, and elastin contents in the aortic wall were studied in hematoxylin/eosin, Sirius Red, and Orceine stained aortic sections, respectively. Aortic MMP-2 levels were determined by gelatin zymography and aortic MMP-2 proteolytic activity was measured using DQ gelatin as the substrate after MMP-2 was captured by a specific antibody and immobilized on a microplate. Aortic MMP-2/tissue inhibitor of metalloprotemases (TIMP)-2 mRNA levels were determined by real time RT-PCR. Doxycycline attenuated 2K-1C hypertension (215 +/- 8 mmHg versus 167 +/- 13 mmHg in 2K-1C rats and 2K-1C + doxy rats, respectively; P < 0.01) and prevented the 35% reduction in endothelium-dependent vasorelaxation found in 2K-1C rats. Doxycycline prevented the increases in media thickness, and was associated with lower media/lumen and cross-sectional areas (all P<0.01). Doxycycline also prevented excessive collagen and elastin deposition in the vascular wall. Increased MMP-2 and Pro-MMP-2 levels and MMP-2 activity were found in the aortas of 2K-1C rats (all P<0.05). A 21-fold increase (P<0.001) in the ratio of MMP-2/TIMP-2 mRNA expression was found in the 2K-1C group, whereas this ratio remained unaltered in 2K-1C+doxy rats. Our results suggest that MMP-2 plays a role in 2K-1C hypertension and its structural and functional vascular changes, which were attenuated by doxycycline. (C) 2007 Elsevier Ireland Ltd. All rights reserved.