Introduction: Hypertension is the number one risk factor for cardiovascular diseases. Total peripheral resistance (TPR) is strongly involved in blood pressure homeostasis. TPR is primarily determined by resistance arteries (RAs). Pathogenic factors which change RA structure are associated with cardiovascular disease. Despite this, methods employed in the study of RAs lack efficiency. Methods: A polymer microfluidic device (Artery-on-a-Chip Device, AoC) made from polydimethylsiloxane (PDMS) was developed. RAs from CD1 mice were measured on the device. Their responses to phenylephrine (PE), acetylcholine (Ach), FURA-2 imaging, and 24-h culture were assessed. Results: Following several modifications, vessel function on the AoC device was successfully measured. Robust PE constriction and Ach-induced vasodilation were observed. AoC arteries were viable after 24-hour culture, and FURA-2 was successfully imaged. Conclusions: The AoC device is a viable alternative to cannulation myography. The AoC can greatly increase the efficiency of RA studies, while also decreasing training time and difficulty.

Introduction: Hypertension is the number one risk factor for cardiovascular diseases. Total peripheral resistance (TPR) is strongly involved in blood pressure homeostasis. TPR is primarily determined by resistance arteries (RAs). Pathogenic factors which change RA structure are associated with cardiovascular disease. Despite this, methods employed in the study of RAs lack efficiency. Methods: A polymer microfluidic device (Artery-on-a-Chip Device, AoC) made from polydimethylsiloxane (PDMS) was developed. RAs from CD1 mice were measured on the device. Their responses to phenylephrine (PE), acetylcholine (Ach), FURA-2 imaging, and 24-h culture were assessed. Results: Following several modifications, vessel function on the AoC device was successfully measured. Robust PE constriction and Ach-induced vasodilation were observed. AoC arteries were viable after 24-hour culture, and FURA-2 was successfully imaged. Conclusions: The AoC device is a viable alternative to cannulation myography. The AoC can greatly increase the efficiency of RA studies, while also decreasing training time and difficulty.

In this thesis, I present a microfluidic platform that enables automated image-based assessment of biological structure and function. My work focuses on assessing intact resistance arteries from the mouse cerebral vascular bed with a diameter of approximately 120µm in vitro. The experimental platform consists of a microfluidic device and a world-to-chip fluidic interconnect that minimizes unwanted dead volumes and eliminates the need for any liquid-filled peripheral equipment. The integrated platform is computer controlled and capable of fully automated operation once a small blood vessel segment is loaded onto the chip. Robust operation of the platform was demonstrated through a series of case studies that assessed small artery function and changes therein induced by incubation with the drug nifedipine, a dihydropyridine calcium channel blocker. In addition artery segments were stained for L-type calcium channels, F-actin and nuclei, from which structural information about cell alignment and shape was quantified.

In this thesis, I present a microfluidic platform that enables automated image-based assessment of biological structure and function. My work focuses on assessing intact resistance arteries from the mouse cerebral vascular bed with a diameter of approximately 120µm in vitro. The experimental platform consists of a microfluidic device and a world-to-chip fluidic interconnect that minimizes unwanted dead volumes and eliminates the need for any liquid-filled peripheral equipment. The integrated platform is computer controlled and capable of fully automated operation once a small blood vessel segment is loaded onto the chip. Robust operation of the platform was demonstrated through a series of case studies that assessed small artery function and changes therein induced by incubation with the drug nifedipine, a dihydropyridine calcium channel blocker. In addition artery segments were stained for L-type calcium channels, F-actin and nuclei, from which structural information about cell alignment and shape was quantified.

In this thesis I present a microfluidic platform developed to control dissolved gases and monitor dissolved oxygen concentrations within the microenvironment of isolated small blood vessels. Dissolved gas concentrations are controlled via permeation through the device substrate material using a 3D network of gas and liquid channels. Dissolved oxygen concentrations are measured on-chip via fluorescence quenching of an oxygen sensitive probe embedded in the device. Dissolved oxygen control was validated using the on-chip sensors as well as a 3D computational model. The platform was used in a series of preliminary experiments using olfactory resistance arteries from the mouse cerebral vascular bed. The presented platform provides the unique opportunity to control dissolved oxygen concentrations at high temporal resolutions (<1 min) and monitor dissolved oxygen concentrations in the microenvironment surrounding isolated blood vessels.

Background/Rationale: In addition to their established cardioprotective role, β-adrenergic antagonists also increase the risk of stroke and mortality. We propose that a vascular mechanism could contribute to cerebral tissue ischemia in β-blocked patients. Methods: Cardiac output (CO), mean arterial pressure (MAP) and microvascular brain oxygen tension (PBrmvO2) were measured in anesthesized mice treated with metoprolol (3mg•kg-1, i.v.). Dose-response curves (DRCs) for adrenergic-agonists were generated in mesenteric resistance arteries (MRAs; isoproterenol, clenbuterol) and posterior cerebral arteries (PCAs; phenylephrine, isoproterenol) before and after metoprolol treatment. Results: Metoprolol reduced CO, maintained MAP and increased systemic vascular resistance (SVR) resulting in a decreased PBrmvO2 in mice. Metoprolol attenuated β-adrenergic mediated vasodilation in both MRAs and PCAs. Conclusions: Metoprolol reduced brain perfusion in mice. A decrease in CO contributed however, metoprolol also inhibited β-adrenergic vasodilation of mesenteric and cerebral arteries. This provides evidence in support of a vascular mechanism for cerebral ischemia in β-blocked patients.

Background/Rationale: In addition to their established cardioprotective role, β-adrenergic antagonists also increase the risk of stroke and mortality. We propose that a vascular mechanism could contribute to cerebral tissue ischemia in β-blocked patients. Methods: Cardiac output (CO), mean arterial pressure (MAP) and microvascular brain oxygen tension (PBrmvO2) were measured in anesthesized mice treated with metoprolol (3mg•kg-1, i.v.). Dose-response curves (DRCs) for adrenergic-agonists were generated in mesenteric resistance arteries (MRAs; isoproterenol, clenbuterol) and posterior cerebral arteries (PCAs; phenylephrine, isoproterenol) before and after metoprolol treatment. Results: Metoprolol reduced CO, maintained MAP and increased systemic vascular resistance (SVR) resulting in a decreased PBrmvO2 in mice. Metoprolol attenuated β-adrenergic mediated vasodilation in both MRAs and PCAs. Conclusions: Metoprolol reduced brain perfusion in mice. A decrease in CO contributed however, metoprolol also inhibited β-adrenergic vasodilation of mesenteric and cerebral arteries. This provides evidence in support of a vascular mechanism for cerebral ischemia in β-blocked patients.

In this thesis I present a microfluidic platform developed to control dissolved gases and monitor dissolved oxygen concentrations within the microenvironment of isolated small blood vessels. Dissolved gas concentrations are controlled via permeation through the device substrate material using a 3D network of gas and liquid channels. Dissolved oxygen concentrations are measured on-chip via fluorescence quenching of an oxygen sensitive probe embedded in the device. Dissolved oxygen control was validated using the on-chip sensors as well as a 3D computational model. The platform was used in a series of preliminary experiments using olfactory resistance arteries from the mouse cerebral vascular bed. The presented platform provides the unique opportunity to control dissolved oxygen concentrations at high temporal resolutions (<1 min) and monitor dissolved oxygen concentrations in the microenvironment surrounding isolated blood vessels.

With old age, blood flow to the high-oxidative red skeletal muscle is reduced and blood flow to the low-oxidative white muscle is elevated during exercise. Changes in the number of feed arteries perforating the muscle are thought to contribute to this altered hyperemic response during exercise. We tested the hypothesis that exercise training would ameliorate age-related differences in blood flow during exercise and feed artery structure in skeletal muscle. Young (6–7 mo old, n = 36) and old (24 mo old, n = 25) male Fischer 344 rats were divided into young sedentary (Sed), old Sed, young exercise-trained (ET), and old ET groups, where training consisted of 10–12 wk of treadmill exercise. In Sed and ET rats, blood flow to the red and white portions of the gastrocnemius muscle (GastRed and GastWhite) and the number and luminal cross-sectional area (CSA) of all feed arteries perforating the muscle were measured at rest and during exercise. In the old ET group, blood flow was greater to GastRed (264 ± 13 and 195 ± 9 ml·min−1·100 g−1 in old ET and old Sed, respectively) and lower to GastWhite (78 ± 5 and 120 ± 6 ml·min−1·100 g−1 in old ET and old Sed, respectively) than in the old Sed group. There was no difference in the number of feed arteries between the old ET and old Sed group, although the CSA of feed arteries from old ET rats was larger. In young ET rats, there was an increase in the number of feed arteries perforating the muscle. Exercise training mitigated old age-associated differences in blood flow during exercise within gastrocnemius muscle. However, training-induced adaptations in resistance artery morphology differed between young (increase in feed artery number) and old (increase in artery CSA) animals. The altered blood flow pattern induced by exercise training with old age would improve the local matching of O2 delivery to consumption within the skeletal muscle.

Le système endothéline (ET) est activé en condition d’hypertension pulmonaire (HTP). L’efficacité des antagonistes des récepteurs à l’ET a clairement été démontrée et a menée à l’approbation clinique de tels antagonistes dans le traitement de l’hypertension artérielle pulmonaire (HTAP). Toutefois, il existe présentement un important débat opposant l’utilisation d’un antagoniste sélectif des récepteur ETA à l’utilisation d’un antagoniste double ETA/ETB dans le traitement de cette pathologie. Bien que nous sachions que le système ET est activé et contribue à l’HTAP, les modifications locales de ce système induites par la pathologie, particulièrement au niveau des artères de résistance pulmonaires, demeurent inconnues. De plus, l’impact de ces modifications sur la réponse pharmacologique aux divers antagonistes des récepteurs à l’ET (sélectifs versus double) est d’une importance capitale. Ainsi, le but de la première étude de cette thèse était d’évaluer les modifications potentielles de la pharmacologie du système ET au niveau des artères de résistance pulmonaires induites par l’HTAP. Dans cette étude, nous avons démontré qu’en condition contrôle l’antagoniste sélectif ETA et l’antagoniste double n’ont eu aucun effet sur la réponse vasoconstrictrice à l’ET-1. Toutefois, en condition d’HTAP, les antagonistes sélectif et double ont tous deux été en mesure de réduire la vasoconstriction pulmonaire induite par l’ET-1. Une diminution importante de l’expression génique du récepteur ETB pourrait être à l’origine de cette modification du profil pharmacologique des antagonistes. Une meilleure compréhension des rôles joués par les récepteurs ETA et ETB au niveau des artères de résistance pulmonaires pourrait permettre l’optimisation des traitements de l’HTAP. Ainsi, le but de la deuxième étude était d’évaluer les effets d’un traitement antisens ex vivo dirigé contre l’ARNm des récepteurs ETA et ETB dans la vasoconstriction des artères de résistance pulmonaires induite par l’ET-1. Dans cette étude, nous avons démontré dans un premier temps que les récepteurs ETA et ETB pouvaient former des dimères au niveau des artères de résistance pulmonaires. De plus, nous avons observé qu’une réduction de l’expression protéique du R-ETA entraînait une potentialisation de la vasoconstriction ETB dépendante suggérant ainsi qu’en condition contrôle, le récepteur ETA aurait un effet inhibiteur sur la vasoconstriction pulmonaire induite par la stimulation du récepteur ETB. Les effets délétères de l’ET-1 sur la circulation pulmonaire sont bien connus, toutefois seules quelques études ont porté leur attention sur l’implication de l’ET-3 dans l’HTAP. Ainsi, le but de la troisième étude était d’évaluer l’implication potentielle de l’ET-3 dans l’HTAP. Dans cette étude, nous avons démontré qu’il était nécessaire en condition contrôle de bloquer simultanément les récepteurs ETA et ETB afin de réduire la réponse vasoconstrictrice pulmonaire à l’ET-3. En condition d’HTAP, nous avons observé une augmentation non-significative des concentrations plasmatiques d’ET-3 ainsi qu’une modification du profil pharmacologique des antagonistes des récepteurs à l’ET. En effet, l’utilisation de l’antagoniste sélectif ETA ou de l’antagoniste double était dans les deux cas en mesure de réduire la vasoconstriction pulmonaire à l’ET-3. Les résultats de ces trois études suggèrent qu’il est préférable d’utiliser un antagoniste double dans le traitement de l’HTAP. En effet, (1) en condition d’HTAP, l’utilisation d’un antagoniste double est aussi efficace que l’utilisation d’un antagoniste sélectif ETA; (2) les récepteurs ETA et ETB peuvent former des dimères au niveau des artères de résistance pulmonaires et (3) le récepteur ETB joue un rôle prédominant dans la vasoconstriction pulmonaire, il semble donc essentiel de bloquer simultanément les récepteurs ETA et ETB afin d’inhiber la réponse vasoconstrictrice induite par l’ET. Mots-clés: endothéline-1, endothéline-3, artère de résistance pulmonaire, récepteur vasculaire, antagoniste des récepteurs à l’ET, dimérisation, phosphorothioate, hypertension artérielle pulmonaire / The endothelin (ET) system is activated in pulmonary arterial hypertension (PAH); indeed, increased plasma levels of ET-1 were detected in patients with various forms of PAH and in various experimental models. The therapeutic value of pharmacological blockade of ET receptors has been demonstrated in various animal models and led to the current approval and continued development of these drugs for the therapy of human PAH. Whether the net effect of either selective ETA receptor blockade or combined ETA/ETB receptor blockade provides greater therapeutic benefit remains a subject of debate. Although the ET system contributes to PAH, we currently incompletely comprehend which local modifications of this system occur as a consequence of PAH, particularly in small resistance arteries, and how this could affect the pharmacological response to ET receptor antagonists. The purpose of the first study was therefore to evaluate potential modifications of the pharmacology of the ET system in rat pulmonary resistance arteries from monocrotaline-induced PAH. Our results reveal striking changes in pulmonary vasculature sensitivity to ET receptor antagonism in PAH that may be related to a reduction in ETB receptor expression. A better understanding of the exact role played by both ETA and ETB receptors on pulmonary resistance arteries might contribute to optimization of PAH treatments. Therefore the aim of the second study was to clarify the role played by both ETA and ETB receptors in ET-1 induced pulmonary vasoconstriction using an antisense (AS) oligonucleotide ex vivo treatment. In this study we have demonstrated that ETA and ETB receptors can form heterodimers in pulmonary resistance arteries. Furthermore, suppression of ETA receptors potentiated the response to ET-1 suggesting that in control condition the ETA receptor has an inhibitory action on the ET-1 induced pulmonary vasoconstriction induced by the stimulation of the ETB receptor. Although the deleterious effects of ET-1 on the pulmonary circulation are well established, only a few studies have focused on ET-3 in PAH. Therefore, the purpose of the last study was to evaluate the potential implication of ET-3 in MCT-induced PAH and evaluate the roles of ETA and ETB receptors on ET-3-induced pulmonary vascular reactivity. In control condition, the use of a combination of both ETA and ETB receptor antagonists is necessary to reduce the ET-3 induced pulmonary vasoconstriction. In PAH, we found an increased ET-3 plasma levels and a modification of the pharmacological profile of ET receptor antagonists. Indeed, the use of either the ETA receptor antagonist or the dual antagonist was able to reduce the ET-3 response. The results from these three studies suggest that it is preferable to use a dual antagonist in the treatment of PAH. Indeed, (1) in PAH the use of a dual antagonist is as effective as the use of a selective ETA receptor antagonist (2) ETA and ETB receptors can form heterodimers in pulmonary resistance arteries and (3) ETB receptor plays an important role in the ET-1 induced pulmonary vasoconstriction, suggesting that it is necessary to block both receptors to reduce the ET-1 induced pulmonary vasoconstriction. Keywords: endothelin-1, endothelin-3, pulmonary resistance artery, receptor, endothelin receptor antagonist, dimerisation, phosphorothioate pulmonary arterial hypertension