To determine a possible role for NCX in myogenically active smooth muscle arterioles, studies were conducted by manipulation of extracellular Na+ levels and inhibition of the exchanger. Western blotting was performed for the identification of the NCX protein. Real-time PCR was performed to demonstrate the level of expression of mRNA, for the NCX isoforms. Antisense oligonucleotides against NCX mRNA were introduced in an isolated cremaster arteriole followed by functional studies after 24 hours. Level of expression of NCX was determined by western blotting. The data are consistent with the presence of NCX1 in the cremaster arterioles.

The zebrafish (Danio rerio) has been used as a model for studying vertebrate development in the cardiovascular system. In order to monitor heart contraction and cytosolic calcium oscillations, fish were either embedded in methylcellulose or anesthetized with tricaine. Using high-resolution differential interference contrast and calcium imaging microscopy, we here show that dopamine and verapamil alter calcium signaling and muscle contraction in anesthetized zebrafish, but not in embedded zebrafish. In anesthetized fish, dopamine increases the amplitude of cytosolic calcium oscillation with a subsequent increase in heart contraction, whereas verapamil decreases the frequency of calcium oscillation and heart rate. Interestingly, verapamil also increases myocardial contraction. Our data further indicate that verapamil can increase myocardial calcium sensitivity in anesthetized fish. Taken together, our data reinforce in vivo cardiac responses to dopamine and verapamil. Furthermore, effects of dopamine and verapamil on myocardial calcium and contraction are greater in anesthetized than embedded fish. We suggest that while the zebrafish is an excellent model for a cardiovascular imaging study, the cardio-pharmacological profiles are very different between anesthetized and embedded fish.

Cerebral parenchymal arterioles (PAs) play a critical role in assuring appropriate blood flow and perfusion pressure within the brain. PAs are unique in contrast to upstream pial arteries, as defined by their critical roles in neurovascular coupling, distinct sensitivities to vasoconstrictors, and enhanced myogenic responsiveness. Dysfunction of these blood vessels is implicated in numerous cardiovascular diseases. However, treatments are limited due to incomplete understanding of the fundamental control mechanisms at this level of the circulation. One of the key elements within most vascular networks, including the cerebral circulation, is the presence of myogenic tone, an intrinsic process whereby resistance arteries constrict and reduce their diameter in response to elevated arterial pressure. This process is centrally involved in the ability of the brain to maintain nearly constant blood flow over a broad range of systemic blood pressures. The overall goal of this dissertation was to investigate the unique mechanisms of myogenic tone regulation in the cerebral microcirculation. To reveal the contributions of various signaling factors in this process, measurements of diameter, intracellular Ca2+ concentration ([Ca2+]i), membrane potential and ion channel activity were performed. Initial work determined that two purinergic G protein-coupled receptors, P2Y4 and P2Y6 receptors, play a unique role in mediating pressure-induced vasoconstriction of PAs in a ligand-independent manner. Moreover, a particular transient receptor potential (TRP) channel in the melastatin subfamily, i.e. TRPM4, was also identified as a mediator of PA myogenic responses. Notably, the observations that inhibiting TRPM4 channels substantially reduces P2Y receptor-mediated depolarization and vasoconstriction, and that P2Y receptor ligands markedly activate TRPM4 currents provide definitive evidence that this ion channel functions as an important link between mechano-sensitive P2Y receptor activation and the myogenic response in PAs. Next, the signaling cascades that mediate stretch-induced TRPM4 activation in PA myocytes were explored. Interestingly, these experiments determined that the RhoA/Rho kinase signaling pathway is involved in this mechanism by facilitating pressure-induced, P2Y receptor-mediated stimulation of TRPM4 channels, leading to subsequent smooth muscle depolarization, [Ca2+]i increase and contraction. Since Rho kinase is generally accepted as a 'Ca2+-sensitization' mediator, the present, contrasting observations point to an underappreciated role of RhoA/Rho kinase signaling in the excitation-contraction mechanisms within the cerebral microcirculation. Overall, this dissertation provides evidence that myogenic regulation of cerebral PAs is mediated by mechano-sensitive P2Y receptors, which initiate the RhoA/Rho kinase signaling pathway, subsequent TRPM4 channel opening, and concomitant depolarization and contraction of arteriolar smooth muscle cells. Revealing the unique mechanochemical coupling mechanisms in the cerebral microcirculation may lead to development of innovative therapeutic strategies for prevention and treatment of microvascular pathologies in the brain.

Traumatic brain injury (TBI) is the most frequent cause of death in children and young adults in the United States. Besides emergency neurosurgical procedures, there are few medical treatment options to improve recovery in people who have experienced a TBI. Management of patients who survive TBI is complicated by both central nervous system and peripheral systemic effects. The pathophysiology of systemic inflammation and coagulopathy following TBI has been attributed to trauma-induced endothelial cell dysfunction; however, there is little knowledge of the mechanisms by which trauma might impact the functions of the vascular endothelium at sites remote from the injury. The endothelium lining these small vessels normally produces nitric oxide (NO), arachidonic acid metabolites, and endothelial-dependent hyperpolarizing factors to relax the surrounding vascular smooth muscle. For this research study we investigated the effects of fluid-percussion-induced TBI on endothelial-dependent vasodilatory functions in a remote tissue bed (the mesenteric circulation) 24 hours after injury. We hypothesized that TBI causes changes in the mesenteric artery endothelium that result in a loss of endothelial-dependent vasodilation. We found that vasodilations induced by the muscarinic-receptor agonist, acetylcholine, are attenuated following TBI. While the endothelial-derived hyperpolarizing component of vasodilation was preserved, the NO component was severely impaired. Therefore, we tested whether the loss of NO component was due to a decrease in bioavailablity of the NO synthase (NOS) cofactor BH4, the NOS substrate L-arginine, or to changes in expression/activity of the enzyme arginase, which competes with NOS for L-arginine. We found that supplementation of L-arginine and inhibition of the enzyme arginase rescues endothelial-dependent vasodilations in TBI arteries. This study demonstrates that there are pathological systemic effects outside the point of injury following TBI leading to a dysfunctional endothelial vasodilatory pathway. These data provide insight into the pathophysiology of endothelial dysfunction after trauma and may lead to new potential targets for drug therapy.

In the past decades, tumors have progressively been perceived as highly integrated systems in which the genetically unstable tumor cells and the genetically stable host cells cooperate to promote tumor growth. This view suggests that, beside tumor cells (that are targeted by conventional anticancer treatments such as radio- and chemotherapy), host cells within the tumor microenvironment can be targeted by antitumor therapy. Such alternative strategies are strongly supported by the need to overcome several limitations of the conventional therapies targeting tumor cells, such as collateral toxicity due to lack of tumor selectivity, limited tumor accessibility, and the selection of treatment-resistant variants. By contrast to tumor cells, the genetically stable host cells should not develop resistance to treatments. In this context, the observation that tumor growth is fundamentally dependent on the onset of a private tumor neovasculature (tumor angiogenesis) has revolutionized the field of cancer research. Several treatments have been developed aimed to prevent tumor angiogenesis (anti-angiogenic strategies) or to erase the existent tumor vasculature (anti-vascular approaches) supporting the survival and growth of thousands of tumor cells. However, although such therapies achieved cancer cure in animal models, they turned out to be rather inefficient when tested in patients. This can be attributed to differences in the angiogenic status between fast-growing animal tumors and slow-growing human tumors at the time of clinical detection. Another reading of the above-mentioned observations is that anticancer treatments could benefit from interventions aimed at increasing their efficiency. For instance, radiotherapy could benefit from tumor reoxygenation while a decrease in tumor interstitial pressure could facilitate tumor accessibility to circulating agents. In this context, the mature vasculature is an attractive target since it controls tumor blood supply and is highly accessible for therapy. Therefore, strategies aimed at exploiting its functional reactivity by inducing vasorelaxation have the potential to improve tumor perfusion/drug delivery and oxygenation/radiosensitivity. To be exploited in the clinics, such pro-vascular approaches have to fulfill essential requirements. First, they need to achieve high selectivity for tumor vessels. It should prevent systemic toxicity as well as the stealing of the blood flow towards the peripheral vasculature. Second, vasodilation has to be transient, so that the tumor should not take advantage of an increased energetic supply to grow faster. Third, the therapeutic effects have to be achieved in several tumor types and in different host strains to gain a wide therapeutic range of applicability. Finally, vasomodulation has to be achieved with interventions relevant to the clinical situation, ensuring direct therapeutic significance. However, the therapeutic exploitation of agents modulating tumor perfusion was generally hampered by confounding effects on the systemic blood pressure. In our studies, we have documented that this lack of tumor selectivity can be overcome by identifying vasomodulatory pathways that are selectively altered within the tumor microenvironment, allowing selective vasomodulatory interventions. According to the criteria detailed above, to identify a differential tumor vascular reactivity, we had to work with mice models of mature tumor vascularization. We reasoned that preexisting host arterioles in mice, if coopted, should retain architectural characteristics (such as a muscular coat) necessary for functional reactivity but also be influenced by the tumor microenvironment at both molecular and functional levels. To gain in reproducibility, this model was developed by injecting syngeneic tumor cells in the vicinity of the saphenous arteriole (i.e., a collateral branch of the femoral artery) in the rear leg of mice. With tumor growth, this arteriole was progressively included in the tumor cortex (coopted), with side branches running deeply into tumors. This model was developed using several tumors and mice strains. It provides the unique advantage to allow the easy identification and isolation of mature tumor vessels from fast-growing animal tumors. To evaluate differential vasoreactivity in those tumor-coopted vessels, we adapted pressure myography, a device initially dedicated to the study of the reactivity of coronary arterioles (see annex 1). In our hands, the unprecedented application of pressure myography to the study of small tumor vessels proved to be very efficient. Indeed, this technique not only served us to confirm that arterioles remain sensitive to vasomodulation under tumor cooption, but also allowed us to evidence two major adaptations of host vessels to the tumor microenvironment: the acquisition of an ET-1-mediated basal constrictive tone and a defect in the vasodilatory NO pathway. Furthermore, we used pressure myography to identify and characterize vasomodulatory strategies exploiting these differential reactivities. More particularly, we showed that both BQ123 (an ETA inhibitor) and ionizing radiations (that restored a functional NO pathway) promoted the vasodilation of the tumor-coopted vessels. In vivo, we verified that these strategies fulfilled the essential requirements of pro-vascular approaches: tumor selectivity, transient effects, broad range of applicability, and therapeutic significance in clinically relevant regimens. This latter study led us to further explore the effects of radiotherapy on the status of the tumor vasculature. Hence, we showed that fractionated radiotherapy induced tumor angiogenesis, thereby providing a rationale to combine radiotherapy to anti-angiogenic therapies.

In vivo, la pression artérielle au niveau des artères cérébrales est pulsée, alors que ex vivo, l’étude de la fonction cérébrovasculaire est majoritairement mesurée en pression statique. L’impact de la pression pulsée sur la régulation du tonus myogénique et sur la fonction endothéliale cérébrale est inconnu. Nous avons posé l’hypothèse selon laquelle en présence d'une pression pulsée physiologique, la dilatation dépendante de l’endothélium induite par le flux et le tonus myogénique seraient optimisés. L’objectif de notre étude est d’étudier ex vivo l’impact de la pression pulsée sur le tonus myogénique et la dilatation induite par le flux dans les artères cérébrales de souris. Nous avons utilisé un artériographe pressurisé couplé à un système générant une onde pulsée de fréquence et d’amplitude réglables. Les artères cérébrales moyennes (≈160 μm de diamètre) ont été isolées de souris C57BL6 âgées de 3 mois et pressurisées à 60 mm Hg, en pression statique ou en pression pulsée. En pression statique, le tonus myogénique est faible mais est potentialisé par le L-NNA (un inhibiteur de la eNOS) et la PEG-catalase (qui dégrade le H2O2), suggérant une influence des produits dilatateurs dérivés de la eNOS sur le tonus myogénique. En présence de pression pulsée (pulse de 30 mm Hg, pression moyenne de 60 mm Hg, 550 bpm), le tonus myogénique est significativement augmenté, indépendamment du L-NNA et de la PEG-catalase, suggérant que la pression pulsée lève l’impact de la eNOS. En pression statique ou pulsée, les artères pré-contractées se dilatent de façon similaire jusqu’à une force de cisaillement de 15 dyn/cm2. Cette dilatation, dépendante de l’endothélium et de la eNOS, est augmentée en condition pulsée à une force de cisaillement de 20 dyn/cm2. En présence de PEG-catalase, la dilatation induite par le flux est diminuée en pression statique mais pas en pression pulsée, suggérant que la pression statique, mais pas la pression pulsée, favorise la production de O2 -/H2O2. En effet, la dilatation induite par le flux est associée à une production de O2 -/H2O2 par la eNOS, mesurable en pression statique, alors que la dilatation induite par le flux en pression pulsée est associée à la production de NO. Les différences de sensibilité à la dilatation induite par le flux ont été abolies après inhibition de Nox2, en condition statique ou pulsée. La pression pulsée physiologique régule donc l’activité de la eNOS cérébrale, en augmentant le tonus myogénique et, en présence de flux, permet la relâche de NO via la eNOS. / While in vivo arterial blood pressure in cerebral arteries is pulsatile, in vitro cerebral arterial function is generally assessed under a static pressure. Thus, whether pulse pressure regulates cerebral endothelial shear stress sensitivity and myogenic tone is unknown. We hypothesized that a physiological pulse pressure induces a better flow-mediated dilation and optimized myogenic tone. The aim of this study was to test in vitro the impact of pulse pressure on myogenic tone and eNOS-dependent flow-mediated dilation in mouse cerebral arteries. Using a custom computer-controlled pneumatic system generating a pulse pressure (used at 30 mm Hg, rate of 550 bpm) coupled to an arteriograph, isolated posterior cerebral arteries from 3-month old C57Bl/6J mice were pressurized at 60 mm Hg, either in static or pulse pressure conditions. Shear stress from 2 to 20 dyn/cm2 was applied and flow-mediated dilation measured. Without pulse pressure, myogenic tone was low but potentiated by both L-NNA (eNOS inhibitor) and PEG-catalase (catalyses H2O2), suggesting an influence of eNOS-derived dilator products on myogenic tone. Pulse pressure significantly increased myogenic tone, independently of L-NNA and PEG-catalase, suggesting that pulse pressure prevents the impact of eNOS. In both static and pulse pressure conditions, cerebral arteries did not dilate to shear stress in the presence of L-NNA or after endothelial denudation, confirming the endothelial origin of the dilatory response. Up to 15 dyn/cm2, shear stress elicited similar flow-mediated dilation in static and pulse pressure conditions; at 20 dyn/cm2, however, flow-mediated dilation were higher in the presence of pulse pressure. PEG-catalase reduced flow-mediated dilation in static but not in pulse pressure, suggesting that in static conditions eNOS is responsible for O2 -/H2O2 production. Indeed, eNOS-derived O2 -/H2O2 production was measured during flow-mediated dilation in static pressure, while pulse pressure promoted eNOS-derived NO production. Differences in flow-mediated dilation between static and pulse pressure conditions were abolished after Nox2 inhibition. In conclusion, pulse pressure modulates cerebrovascular eNOS activity: at rest, pulse pressure inhibits eNOS, increasing myogenic tone. In the presence of flow, pulse pressure permits a shear stress-dependent eNOS-derived NO release, leading to higher flow-mediated dilation.

Vasomotor tone is largely maintained by sympathetic nerves, myogenic reactivity and key local and circulating hormones. Acting together, these factors ensure moment-to-moment adjustments of net vascular tone required to maintain hemodynamic stability. In rat mesenteric small veins (MSV) and arteries (MSA), we investigated the contribution of the endothelium, L-type voltage operated calcium channels (L-VOCCs), PKC and Rho kinase to myogenic reactivity. The interaction of myogenic reactivity with norepinephrine (NE), endothelin-1 (ET-1), and sympathetic nerve activation was also investigated under conditions of changing transmural distending pressure. We also evaluated the relative contribution of alpha adrenergic (α-A) and endothelinergic receptors to NE and ET-1 contractile responses, respectively. Additionally, the effects of changing transmural pressure on endothelial dilator function of MSV were examined. Myogenic reactivity was not altered by nitric oxide synthase (NOS) inhibition or endothelium removal in both vessels. L-VOCCs blockade completely abolished arterial tone, while only partially reducing venous tone. PKC and Rho kinase inhibitors largely abolished venous and arterial myogenic reactivity. Increasing transmural pressure did not alter NE, ET-1, and bradykinin responses, but it significantly reduced neurogenic contractions. MSV were more sensitive to NE, ET-1 and sympathetic nerve activation compared with corresponding arteries. α-A and ET-1 receptor agonist and antagonist application revealed the participation of α1-A and ETA receptors in NE and ET-1 contractile responses, respectively. α2-A and ETB receptors appeared to mediate NE and ET-1 responses in MSV, respectively. Bradykinin induced-vasodilation was mainly reduced by NOS inhibition, and BKCa and SkCa blockade. These results suggest that myogenic factors are important contributors to net venous tone in MSV; PKC and Rho kinase activation are important to myogenic reactivity in both vessels, while L-VOCCs play a limited role in the veins versus the arteries; mesenteric veins maintain an enhanced sensitivity to NE, ET-1 and sympathetic nerve activation compared to the arteries with neurogenic contractions being affected by transmural pressure elevations; α1-ARs and ETA are the predominant receptors mediating contractile responses to NE and ET-1, respectively, with functional evidence indicating the presence of α2-ARs and ETB receptors in MSV; and venous endothelial dilator function is not affected by an elevation of transmural pressure. / Natural Sciences and Engineering Research Council of Canada (NSERC). Libyan Ministry of Education and Scientific Research.