L'activité physique (AP) régulière se révèle essentielle en terme de santé publique et est un déterminant important, impactant l'endothélium, pour assurer la prévention primaire et secondaire de nombreuses pathologies et facteurs de risques cardiovasculaires (HTA, IC, hypercholestérolémie, diabète, sédentarité, obésité, surpoids).Tant chez l'homme que chez l'animal, il est couramment accepté que l'AP modérée permet de restaurer la vasodilatation endothélium dépendante médiée par le NO/EDRF (VED-NO) lorsque celle ci est altérée chez des sujets diabétiques (1 et 2), obèses, en surpoids ou sédentaires. De nombreuses investigations, tant expérimentales que cliniques restent à explorer pour évaluer les paramètres (fréquence, intensité, durée) de différents types (endurance, résistance) d'exercices se révélant les plusbénéfiques pour améliorer une VED-NO détériorée dans de nombreuses pathologies et facteurs de risques cardiovasculaires. Dans un premier temps, par une approche expérimentale, nous avons eu pour objectif de déterminer les effets d'un entraînement intense sur VED-NO. L'étude a été réalisée avec des aortes isolées de rats diabétiques (type1) sédentaires et entraînés. Dans un second temps, dans une étude clinique chez des adolescents (14-16 ans), nous avons cherché à savoir si un entrainement préalable modifiait la VED-NO après un exercice isolé aigu. Ce travail a été réalisé au niveau de la microcirculation cutanée de l'avant-bras, par débitmètrie « Laser Doppler ». Dans un troisième temps, toujours par une étude clinique par débitmétrie « Laser Doppler », nous avons enregistré les effets d'un entraînement musculaire de deux mois chez des femmes obèses tant au niveau de VED-NO qu'au niveau de biomarqueurs de l'inflammation (TNF alpha, leptine, Interleukine, visfatine).Nos principaux résultats montrent que: 1) L'inactivité physique et l'état diabétique de type 1 chez le rat provoquent, sur l'aorte isolée, une augmentation de la vasoconstriction à la Phényléphrine ainsi qu'une réduction importante de la VED -NO - inhibée par le L-NAME- induite par l'Ach comme par l'ADPβS via des récepteurs muscariniques M1/3 et purinergiques P2Y, 2) L'AP intense (sur tapis roulant, 60min/j, 25m/min, 5j/semaine,8semaines) chez le rat diabétique est inefficace à rétablir une VED-NO, 3) L'entraînement préalable de jeunes hommes (15+ ou – 1ans) permet, suite à un exercice isolé et aigu, d'améliorer la VED-NO induite par une iontophorèse d'ACh au niveau de la microcirculation cutanée de l'avant-bras (flux sanguin mesuré par Laser-Doppler), 4) .L'AP (bicyclette ergométrique 1h, intensité à 50% de la FC max, 3j/semaine, 2 mois) chez des femmes obèses -IMC=38.5-, comparativement à dessujets témoins, restaure la VED-NO induite par une iontophorèse d'ACh au niveau de la microcirculation cutanée, 5) De plus, dans cette dernière étude, les capacités aérobies sont améliorées et apparaît une réduction importante des biomarqueurs de l'inflammation. En conclusion, nos travaux montrent qu'il y a encore beaucoup d'investigations à mener pour comprendre les mécanismes moléculaires conduisant à une réhabilitation de la fonction endothéliale préalablement altérée et qu'il faut continuer à préciser, pour chaque type de pathologie cardiovasculaire, les paramètres d'AP (intensité, durée, fréquence) les plus adaptés pour rétablir la VED-NO chez l'animal comme chez l'homme. / Regular physical activity appears to be essential in terms of public health. It is an important determinant, concerning the endothelium, for primary and secondary prevention of many diseases and cardiovascular risk factors (Hypertension, heart failure, hypercholesterolemia, diabetes, sedentarity,obesity, overweight).Both in man and in animals, it is well know that moderate physical activity can restore endothelium-dependent vasodilatation mediated by NO/EDRF (EDV-NO) when it was altered in diabetics (type 1 and 2), obese, overweight, sedentary. Many investigations, both experimental and clinical remain to be explored to evaluate parameters (frequency, intensity, duration) of different types of exercise (endurance, resistance) being the most beneficial for improving EDV-NO deteriorated in many diseases and cardiovascular risk factors. In a first time, by an experimental approach, we aimed to determine the effects of intense training on EDV-NO. The study was performed with isolated aortas of sedentary and trained diabetics rats (type 1). In a second time, in a clinical study in youngmen (14-16 years), we investigated whether prior training modifies EDV-NO after an acute isolated exercise. This work was performed in the cutaneous microcirculation of the forearm by « Laser Doppler » flowmetry. In a third time, in a clinical study by « Laser- Doppler »flowmetry, we recorded the effects of two months resistance training in obese women both in EDV-NO and on inflammatory biomarkers ( TNF alpha, leptin, interleukin, visfatin). Our main results show that: 1) physical inactivity and type 1 diabetic state in rats cause on the isolated aorta, increased vasoconstriction induced by phenylephrine and a significant reduction in EDV-NO(inhibited by L-NAME) induced by Ach as ADPβS via M1/3 muscarinic and P2Y purinergic receptors. 2) Intense training (treadmill, 60min/day, 25m/min, 5d/week, , 8 weeks) in diabetic rats is ineffective in restoring EDV-NO. 3) Prior training of young men, allows, after an isolated acute exercise, to improve EDV-NO induced by ACh iontophoresis in the cutaneous microcirculation of the forearm. 4) Physical activity (treadmill, walking 1 h, intensity 50% Heart Rate max, 3d/week, 2 months) in obese women (BMI=38.5) compared to control subjects, restores EDV-NO induced by Ach iontophoresis in the cutaneous microcirculation of the forearm. 5) In addition, the previous study, aerobic capacities are improved and appears a significant reduction in inflammatory biomarkers concentration. In conclusion, our researchesshow that there is still many studies to understand the molecular mechanisms leading to a restoration of impaired endothelial function. It is necessary to continue to specify, for each type of cardiovascular disease, the most appropriate parameters of physical activity adapted to restore EDV-NO in animal and man.

Background and Purpose: The endothelium is a single-cell layer positioned at the blood-vascular wall interface, where in response to blood-borne signals and hemodynamic forces, endothelial cells act as central regulators of vascular homeostatic processes including vascular tone, growth and remodeling, inflammation and adhesion, and blood fluidity and coagulation. Agonist- or flow-stimulated endothelium-dependent vasorelaxation becomes impaired in states of cardiovascular disease (CVD) risk and has been identified as a possible biomarker of overall endothelial dysfunction leading to vascular dysregulation and disease pathogenesis. Accordingly, it is important to elucidate the mechanisms accounting for this endothelial vasomotor dysfunction. Upon stimulation, endothelial cells can synthesize and release a variety of endothelium-derived relaxing factors (EDRFs), the most prominent of which is nitric oxide (NO) derived from NO synthase (NOS). In addition, under certain CVD risk conditions including hypertension and aging, stimulated endothelial cells can become a prominent source of endothelium-derived contracting factors (EDCFs) produced in a cyclooxygenase (COX)-dependent manner. Consequently, endothelial dysfunction may be caused by under-active EDRF signaling and/or competitive over-active EDCF signaling. Much attention has been given to elucidating the mechanisms of under-active EDRF signaling and its role in causing endothelial dysfunction, wherein excess reactive oxygen species (ROS) accumulation and oxidative stress under CVD risk conditions have been recognized as major factors in reducing NO bioavailability thus causing under-active EDRF signaling and endothelial dysfunction. Less attention however, has been given to elucidating the mechanisms of over-active COX-mediated EDCF signaling and its role in causing endothelial dysfunction. Moreover, while COX-mediated EDCF signaling activity has been investigated in some segments of the vasculature, most notably the aorta, it has not been well-investigated in the common carotid artery (CCA), a highly accessible cerebral blood flow conduit particularly advantageous in exploring the roles of the endothelium in vascular pathogenesis. It was the global purpose of this thesis to gain a better understanding of the cellular-molecular mechanisms accounting for endothelial dysfunction in the CCA of animal models known to exhibit COX-mediated EDCF signaling activity, in particular essential (spontaneous) hypertension and aging. Experimental Objective and Approach: This thesis comprises three studies. Study I and Study II investigated the CCA of young-adult (16-24wk old) normotensive Wistar Kyoto (WKY) and Spontaneously Hypertensive (SHR) rats. Study III investigated the CCA of Adult (25-36wks old) and Aging (60-75wks old) Sprague Dawley (SD) rats treated in vivo (or not; CON) with L-buthionine sulfoximine (BSO) to chronically deplete the cellular anti-oxidant glutathione (GSH) and increase ROS accumulation and oxidative stress. The global objective and approach across these studies was to systematically examine the relative contributions of NOS and COX signaling pathways in mediating the acetylcholine (ACh)-stimulated endothelium-dependent relaxation (EDRF) and contractile (EDCF) activities of isometrically-mounted CCA in tissue baths in vitro, with a particular focus on elucidating the mechanisms of COX-mediated EDCF signaling activity. An added objective was to examine the in vivo hemodynamic characteristics of the CCA in each animal model investigated, serving both to identify the pressure-flow environment that the CCA is exposed to in vivo and to provide assessment of potential hypertension, aging, and oxidative stress effects on large artery hemodynamics. Key Findings: Study I hemodynamic analysis confirmed a hypertensive state in young adult SHR while also exposing a reduction in mean CCA blood flow in SHR compared to WKY accompanied by a multi-faceted pressure-flow interaction across the cardiac cycle relating to flow and pressure augmentation. Study III hemodynamic analysis found that neither aging nor chronic BSO-induced GSH depletion affected CCA blood pressure or blood flow parameters in SD rats. Study I and II demonstrated that a COX-mediated EDCF response impaired ACh-stimulated endothelium-dependent vasorelaxation in pre-contracted CCA from young adult SHR, while EDRF signaling activity, predominantly mediated by NO, remained well-preserved compared to WKY. Examining ACh-stimulated contractile function specifically from a quiescent (non pre-contracted) state revealed that EDCF activity did exist in WKY CCA but could be completely suppressed by NO-mediated EDRF signaling activity, whereas the similarly robust NO-meditated EDRF signaling activity in SHR CCA could not fully suppress its >2-fold augmented EDCF activity vs. WKY CCA. Further pharmaco-dissection of ACh-stimulated contractile function in the SHR-WKY CCA model revealed that the EDCF signaling activity was completely dependent on the COX-1 (but not COX-2) isoform of COX and was almost exclusively mediated by the thromboxane-prostanoid (TP) sub-type of the prostaglandin (PG) G-protein coupled receptor family and by Rho-associated kinase (ROCK), a down-stream effector of the molecular switch RhoA. Furthermore, it was found that while exogenous ROS-stimulated CCA contractile function was similarly >2-fold augmented in SHR vs. WKY and dependent on COX-1 and TP receptor and ROCK effectors, ACh-stimulated CCA EDCF signaling activity was only minimally affected by in-bath ROS manipulating compounds. Additional biochemical and molecular analysis revealed that ACh stimulation was associated with PG over-production from an over-expressed COX-1 in SHR CCA, and with CCA plasma membrane localization and activation of RhoA. Study III demonstrated that a COX-mediated EDCF response impaired ACh-stimulated endothelium-dependent vasorelaxation in pre-contracted CCA from Aging SD rats, while EDRF signaling activity, predominantly mediated by NO, remained well-preserved compared to Adult SD rats. Specific examination of ACh-stimulated contractile function revealed that EDCF activity did exist in Adult CCA but could be completely suppressed by NO-mediated EDRF signaling activity, whereas the similarly robust NO-meditated EDRF signaling activity in Aging CCA could not fully suppress its >3-fold augmented EDCF activity vs. Adult CCA. Further pharmaco-dissection of ACh-stimulated contractile function in the Adult-Aging SD rat CCA model revealed that EDCF signaling activity was completely dependent on COX-1, but while exogenous ROS was able to elicit a COX-dependent CCA contractile response, in-bath ROS manipulating compounds were found to be without effect on ACh-stimulated CCA EDCF signaling activity. Furthermore, biochemical analysis revealed that aging was not associated with a change in tissue (liver and vascular) GSH content or ROS accumulation. Chronic in vivo BSO treatment was effective in depleting tissue GSH content and increasing ROS accumulation, to a similar extent, in both Adult and Aging SD rats. However, regardless of age, neither ACh-stimulated NO-mediated EDRF signaling activity nor COX-mediated EDCF signaling activity were affected by these BSO-induced perturbations. Conclusions and Perspective: In the CCA of animals at the early pathological stages of either essential hypertension (young adult SHR) or normotensive aging (Aging SD rats), endothelial vasomotor dysfunction can be caused solely by over-active EDCF signaling, apparently disconnected from changes in NO bioavailability or oxidative stress. While NO and ROS may act, respectively, as negative and positive modulators of the established COX-PG-TP receptor-RhoA-ROCK cell-signaling axis mediating endothelium-dependent contractile activity, these factors do not appear to be essential to the mechanism(s) underlying the development of over-active EDCF signaling. Further elucidation of the cellular-molecular causes of over-active EDCF signaling, and its patho-biological consequences, in the SHR-WKY and Adult-Aging SD rat CCA models of EDCF activity established and hemodynamically characterized in this thesis, may help to identify new or more effective targets to be used in prevention or treatment strategies to combat the pathogenesis of CVD.

Background and Purpose: The endothelium is a single-cell layer positioned at the blood-vascular wall interface, where in response to blood-borne signals and hemodynamic forces, endothelial cells act as central regulators of vascular homeostatic processes including vascular tone, growth and remodeling, inflammation and adhesion, and blood fluidity and coagulation. Agonist- or flow-stimulated endothelium-dependent vasorelaxation becomes impaired in states of cardiovascular disease (CVD) risk and has been identified as a possible biomarker of overall endothelial dysfunction leading to vascular dysregulation and disease pathogenesis. Accordingly, it is important to elucidate the mechanisms accounting for this endothelial vasomotor dysfunction. Upon stimulation, endothelial cells can synthesize and release a variety of endothelium-derived relaxing factors (EDRFs), the most prominent of which is nitric oxide (NO) derived from NO synthase (NOS). In addition, under certain CVD risk conditions including hypertension and aging, stimulated endothelial cells can become a prominent source of endothelium-derived contracting factors (EDCFs) produced in a cyclooxygenase (COX)-dependent manner. Consequently, endothelial dysfunction may be caused by under-active EDRF signaling and/or competitive over-active EDCF signaling. Much attention has been given to elucidating the mechanisms of under-active EDRF signaling and its role in causing endothelial dysfunction, wherein excess reactive oxygen species (ROS) accumulation and oxidative stress under CVD risk conditions have been recognized as major factors in reducing NO bioavailability thus causing under-active EDRF signaling and endothelial dysfunction. Less attention however, has been given to elucidating the mechanisms of over-active COX-mediated EDCF signaling and its role in causing endothelial dysfunction. Moreover, while COX-mediated EDCF signaling activity has been investigated in some segments of the vasculature, most notably the aorta, it has not been well-investigated in the common carotid artery (CCA), a highly accessible cerebral blood flow conduit particularly advantageous in exploring the roles of the endothelium in vascular pathogenesis. It was the global purpose of this thesis to gain a better understanding of the cellular-molecular mechanisms accounting for endothelial dysfunction in the CCA of animal models known to exhibit COX-mediated EDCF signaling activity, in particular essential (spontaneous) hypertension and aging. Experimental Objective and Approach: This thesis comprises three studies. Study I and Study II investigated the CCA of young-adult (16-24wk old) normotensive Wistar Kyoto (WKY) and Spontaneously Hypertensive (SHR) rats. Study III investigated the CCA of Adult (25-36wks old) and Aging (60-75wks old) Sprague Dawley (SD) rats treated in vivo (or not; CON) with L-buthionine sulfoximine (BSO) to chronically deplete the cellular anti-oxidant glutathione (GSH) and increase ROS accumulation and oxidative stress. The global objective and approach across these studies was to systematically examine the relative contributions of NOS and COX signaling pathways in mediating the acetylcholine (ACh)-stimulated endothelium-dependent relaxation (EDRF) and contractile (EDCF) activities of isometrically-mounted CCA in tissue baths in vitro, with a particular focus on elucidating the mechanisms of COX-mediated EDCF signaling activity. An added objective was to examine the in vivo hemodynamic characteristics of the CCA in each animal model investigated, serving both to identify the pressure-flow environment that the CCA is exposed to in vivo and to provide assessment of potential hypertension, aging, and oxidative stress effects on large artery hemodynamics. Key Findings: Study I hemodynamic analysis confirmed a hypertensive state in young adult SHR while also exposing a reduction in mean CCA blood flow in SHR compared to WKY accompanied by a multi-faceted pressure-flow interaction across the cardiac cycle relating to flow and pressure augmentation. Study III hemodynamic analysis found that neither aging nor chronic BSO-induced GSH depletion affected CCA blood pressure or blood flow parameters in SD rats. Study I and II demonstrated that a COX-mediated EDCF response impaired ACh-stimulated endothelium-dependent vasorelaxation in pre-contracted CCA from young adult SHR, while EDRF signaling activity, predominantly mediated by NO, remained well-preserved compared to WKY. Examining ACh-stimulated contractile function specifically from a quiescent (non pre-contracted) state revealed that EDCF activity did exist in WKY CCA but could be completely suppressed by NO-mediated EDRF signaling activity, whereas the similarly robust NO-meditated EDRF signaling activity in SHR CCA could not fully suppress its >2-fold augmented EDCF activity vs. WKY CCA. Further pharmaco-dissection of ACh-stimulated contractile function in the SHR-WKY CCA model revealed that the EDCF signaling activity was completely dependent on the COX-1 (but not COX-2) isoform of COX and was almost exclusively mediated by the thromboxane-prostanoid (TP) sub-type of the prostaglandin (PG) G-protein coupled receptor family and by Rho-associated kinase (ROCK), a down-stream effector of the molecular switch RhoA. Furthermore, it was found that while exogenous ROS-stimulated CCA contractile function was similarly >2-fold augmented in SHR vs. WKY and dependent on COX-1 and TP receptor and ROCK effectors, ACh-stimulated CCA EDCF signaling activity was only minimally affected by in-bath ROS manipulating compounds. Additional biochemical and molecular analysis revealed that ACh stimulation was associated with PG over-production from an over-expressed COX-1 in SHR CCA, and with CCA plasma membrane localization and activation of RhoA. Study III demonstrated that a COX-mediated EDCF response impaired ACh-stimulated endothelium-dependent vasorelaxation in pre-contracted CCA from Aging SD rats, while EDRF signaling activity, predominantly mediated by NO, remained well-preserved compared to Adult SD rats. Specific examination of ACh-stimulated contractile function revealed that EDCF activity did exist in Adult CCA but could be completely suppressed by NO-mediated EDRF signaling activity, whereas the similarly robust NO-meditated EDRF signaling activity in Aging CCA could not fully suppress its >3-fold augmented EDCF activity vs. Adult CCA. Further pharmaco-dissection of ACh-stimulated contractile function in the Adult-Aging SD rat CCA model revealed that EDCF signaling activity was completely dependent on COX-1, but while exogenous ROS was able to elicit a COX-dependent CCA contractile response, in-bath ROS manipulating compounds were found to be without effect on ACh-stimulated CCA EDCF signaling activity. Furthermore, biochemical analysis revealed that aging was not associated with a change in tissue (liver and vascular) GSH content or ROS accumulation. Chronic in vivo BSO treatment was effective in depleting tissue GSH content and increasing ROS accumulation, to a similar extent, in both Adult and Aging SD rats. However, regardless of age, neither ACh-stimulated NO-mediated EDRF signaling activity nor COX-mediated EDCF signaling activity were affected by these BSO-induced perturbations. Conclusions and Perspective: In the CCA of animals at the early pathological stages of either essential hypertension (young adult SHR) or normotensive aging (Aging SD rats), endothelial vasomotor dysfunction can be caused solely by over-active EDCF signaling, apparently disconnected from changes in NO bioavailability or oxidative stress. While NO and ROS may act, respectively, as negative and positive modulators of the established COX-PG-TP receptor-RhoA-ROCK cell-signaling axis mediating endothelium-dependent contractile activity, these factors do not appear to be essential to the mechanism(s) underlying the development of over-active EDCF signaling. Further elucidation of the cellular-molecular causes of over-active EDCF signaling, and its patho-biological consequences, in the SHR-WKY and Adult-Aging SD rat CCA models of EDCF activity established and hemodynamically characterized in this thesis, may help to identify new or more effective targets to be used in prevention or treatment strategies to combat the pathogenesis of CVD.