Aim: Endothelial microparticles (EMPs) are released from dysfunctional endothelial cells. We hypothesised that patients with unstable carotid plaque have higher levels of circulating microparticles compared to patients with stable plaques, and this may correlate with serum markers of plaque instability and inflammation. Method: Circulating EMPs and inflammatory markers were measured in twenty healthy controls and seventy patients undergoing carotid endarterectomy. EMPs were quantified using flow cytometry. Bioplex assays profiled systemic inflammatory and bone-related proteins. Immunohistological analysis detailed the contribution of differentially-regulated systemic markers to plaque pathology. Alizarin red staining showed calcification. Results: EMPs were significantly higher in patients with carotid stenosis (greater than or equal to 70%) compared to controls, with no differences between asymptomatic vs symptomatic patients. Asymptomatic patients with unstable plaques exhibited higher levels of EMPs compared to those with stable plaques, with a similar trend observed in symptomatic patients. CXCL9 and SCGF-β were significantly elevated in asymptomatic patients with unstable plaques, with IL-16 and macrophage inhibitory factor significantly elevated in the stable plaque group. CXCL9, CTACK and SCGF-β were detected within all plaques, suggesting a contribution to both localised and systemic inflammation. Osteopontin and osteoprotegerin were significantly elevated in the symptomatic vs asymptomatic group, while osteocalcin was higher in asymptomatic patients with stable plaque. All plaques exhibited calcification, which was significantly greater in asymptomatic patients. This may impact on plaque stability. Conclusions: Circulatory EMP, CXCL9 and SCGF-β levels are raised in asymp-tomatic patients with unstable plaques, which could be important in identifying patients at most benefit from intervention.

Purpose: Evidence has accumulated to show that blood pressure variability (BPV) has a striking relationship with cardiovascular (CV) risk. Despite the mounting evidence implicating BPV as a CV risk factor, scant attention has been paid to: (1) the mechanisms by which high BPV confers greater CV risk; and (2) the efficacy of non-pharmacologic treatment modalities in the attenuation of BPV. In order to address these two unresolved questions, the purpose of this dissertation was twofold. The purpose of study #1 was to investigate the association between measures of short-term BPV (24-hour BPV) and long-term BPV (visit-to-visit BPV) with markers of endothelial health in a cohort of African Americans in order to determine if increased BPV may confer greater CV risk by eliciting injury to the endothelium. The purpose of study #2 was to investigate the effects of a 6-month aerobic exercise training (AEXT) intervention on visit-to-visit BPV and 24-hour BPV in the same cohort of African Americans in order to provide the first available data on the efficacy of a non-pharmacologic treatment modality in the lowering of BPV. Methods: We recruited 72 African Americans who were sedentary, non-diabetic, non-smoking, and free of CV and renal disease. Before and after a 6-month AEXT intervention, office blood pressure (BP) was measured at 3 separate visits and 24-hour ambulatory BP monitoring (ABPM) was conducted to measure visit-to-visit BPV and 24-hour BPV, respectively. Right brachial artery diameter was assessed at rest, during flow-mediated dilation (FMD), and after nitroglycerin-mediated dilation (NMD). Peak and area under the curve (AUC) were calculated as measures of FMD and NMD, and the FMD/NMD ratio was calculated as a measure of endothelial function normalized by smooth muscle function. Fasted blood samples were obtained and were analyzed for circulating EMPs expressed as CD31+CD42- and CD62E+ EMPs. Results: In study #1, participants with higher 24-hour diastolic BPV (DBPV) had significantly lower CD31+CD42- EMPs compared to participants with lower 24-hour DBPV. When categorized according to visit-to-visit DBPV, participants with higher visit-to-visit DBPV had a significantly lower FMD/NMD ratio, and significantly higher %NMDpeak and NMDAUCs compared to participants with lower visit-to-visit DBPV. When analyzed as continuous variables, 24-hour mean arterial pressure variability (MAPV) was inversely associated with CD31+CD42- EMPs visit-to-visit DBPV was inversely associated with the FMD/NMD ratio and positively associated with %NMDpeak and NMDAUC; and 24-hour DBPV was positively associated with NMDAUC. All associations were independent of age, gender, BMI, mean BP, and pulse pressure. In study #2 investigating the effects of AEXT in 33 participants who completed the study, 24-hour DBPV and 24-hour MAPV were significantly increased after AEXT. The increase in 24-hour DBPV was independent of changes in BMI, mean BP, and self-reported sleep time. Heart rate variability (HRV) derived from ABPM was associated with the changes in 24-hour DBPV and 24-hour MAPV. There were no significant changes in visit-to-visit BPV after AEXT. Conclusions: The results from study #1 provide evidence that BPV is associated with vascular health as endothelial function was decreased in participants with high visit-to-visit DBPV, while smooth muscle function was increased in participants with higher visit-to-visit and 24-hour DBPV. The findings from study #2 show that 6-months of AEXT do not elicit beneficial changes in BPV. The finding of an association between changes in 24-hour BPV with HRV could indicate, however, that changes in activity levels during ABPM, in part, contributed to the observed changes in 24-hour BPV. / Kinesiology

Le seul traitement actuellement disponible pour les accidents vasculaires cérébraux d’origine ischémique est la thrombolyse par l’activateur tissulaire du plasminogène recombinant (rt-PA). Cependant, l’efficacité du rt-PA est souvent partielle ou absente, et des phénomènes de réocclusion du vaisseau peuvent être observés. Par ailleurs, l’administration de rt-PA est associée à un risque hémorragique. Il apparaît donc indispensable de rechercher les mécanismes à l’origine de la toxicité vasculaire du rt-PA, afin de pouvoir développer des stratégies capables de protéger le lit vasculaire. Parmi ces stratégies, notre équipe a montré dans des modèles expérimentaux que l’inhibition d’une enzyme nucléaire, la poly(ADP-ribose) polymérase ou PARP, permet de protéger la barrière hémato-encéphalique, de réduire les hémorragies et d’améliorer la reperfusion cérébrale suite à l’administration post-ischémique de rt-PA. Dans ce contexte, mon travail a consisté à étudier les mécanismes impliqués dans les altérations vasculaires associées à l’administration de rt-PA à la suite de l’ischémie. Mes travaux de recherche ont comporté un volet in vivo et un volet in vitro. Les études réalisées in vivo ont été menées dans un modèle murin d’ischémie cérébrale thrombo-embolique. Nos résultats indiquent que ni l’ischémie, ni le rt-PA, ni l’association au rt-PA d’un puissant inhibiteur de PARP, le PJ34, ne modifient à 24 heures la présence de dépôts de fibrine, marqueur d’hypoperfusion et de réocclusion. Nous nous sommes ensuite intéressés à deux marqueurs endothéliaux d’inflammation : VCAM-1 et ICAM-1, et avons montré que leur expression, qui augmente 24 heures après l’ischémie, n’est pas modifiée par le rt-PA. Enfin, l’association du PJ34 au rt-PA réduit significativement l’expression post-ischémique de VCAM-1, ce qui suggère le rôle de la PARP dans l’expression de cette molécule d’adhésion. La seconde partie de mon travail a été réalisée in vitro sur une lignée de cellules endothéliales cérébrales murines (bEnd.3). Le rt-PA est à l’origine de changements caractéristiques au niveau de l’organisation et de la morphologie de ces cellules. Ces changements ne sont pourtant associés ni à une dégradation de l’expression des molécules de jonctions inter-endothéliales (occludine, VE-cadhérine), ni à une augmentation de l’expression des marqueurs endothéliaux pro-inflammatoires (VCAM-1, ICAM-1). Nous nous sommes également intéressés à d’autres marqueurs de dysfonction endothéliale, les microparticules endothéliales (MPE). Nos résultats montrent que le rt-PA est à l’origine d’une augmentation importante de la libération des MPE. L’utilisation d’un inhibiteur de la protéine p38, le SB203580, et d’un inhibiteur de PARP, le PJ34, permet de réduire cette augmentation, ce qui suggère que p38 et la PARP pourraient être impliquées dans la production de MPE induite par le rt-PA. En conclusion, l’ensemble de ce travail contribue à préciser les effets vasculaires du rt-PA. Parmi ces effets, la mise en évidence de la production de MPE, via la PARP, est particulièrement novatrice. / Thrombolysis with recombinant tissue plasminogen activator (rt-PA) is currently the only approved pharmacological strategy for acute ischemic stroke. However, the efficacy of rt-PA is rarely complete, and arterial reocclusion can be observed. Furthermore, administration of rt-PA increases the risk of hemorrhagic transformations. Therefore, it is essential to seek mechanisms underlying the vascular toxicity of rt-PA in order to develop strategies protecting the vascular bed. Among these strategies, our laboratory has previously shown that inhibition of poly (ADP-ribose) polymerase (PARP), a nuclear enzyme, protects the blood-brain barrier, reduces hemorrhagic transformations and improves cerebral reperfusion following the post-ischemic administration of rt-PA. In this context, the aim of the present work was to establish the post-ischemic mechanisms of rt-PA-induced vascular alterations. The research was divided into (1) in vivo experiments and (2) in vitro studies to examine the effect of rt-PA on the endothelium. The in vivo studies were performed in a mouse model of thrombo-embolic stroke induced by thrombin injection in the middle cerebral artery. Our results showed that neither ischemia, nor rt-PA, nor the association to rt-PA of the potent inhibitor of PARP PJ34 alter cerebral fibrin deposits, a marker of hypoperfusion and reocclusion, at 24 hours after ischemia. We then evaluated the expression of two endothelial markers of inflammation : VCAM-1 (vascular cell adhesion molecule-1) and ICAM-1 (intercellular adhesion molecule-1). Our results showed that their expressions increase 24 hours after ischemia and are not modified by rt-PA. Finally, the association of PJ34 to rt-PA significantly reduced the post-ischemic expression of VCAM-1, suggesting a role for PARP in the expression of this adhesion molecule. The second part of my work was carried out in vitro in cultures of mouse brain-derived endothelial cells bEnd.3. In the presence of rt-PA, the organization and the morphology of the endothelial cells radically changed. However, these changes were associated neither to a degradation of endothelial junction proteins (occludin, VE-cadherin (vascular endothelial-cadherin)), nor to an increase in the expression of pro-inflammatory endothelial markers (VCAM-1, ICAM-1). We were also interested in a recently identified marker of endothelial dysfunction : endothelial microparticles (EMP). Our results showed that rt-PA induces a significant increase in the EMP released by bEnd.3 cells. The use of a p38 inhibitor, SB203580, and the PARP inhibitor, PJ34, reduced this increase, suggesting that p38 and PARP could be involved in the EMP production induced by rt-PA. In conclusion, this work helps to clarify the vascular effects of rt-PA. Among these effects, the highlight of EMP production, through PARP pathway, is particularly original.