Hélène Girouard

Angiotensin II Impairs Neurovascular Coupling in Neocortex Through NADPH Oxidase–Derived Radicals

Angiotensin II (Ang II) exerts detrimental effects on cerebral circulation, the mechanisms of which have not been elucidated. In particular, Ang II impairs the increase in cerebral blood flow (CBF) produced by neural activity, a critical mechanism that matches substrate delivery with energy demands in brain. We investigated whether Ang II exerts its deleterious actions by activating Ang II type 1 (AT1) receptors on cerebral blood vessels and producing reactive oxygen species (ROS) through NADPH oxidase. Somatosensory cortex CBF was monitored in anesthetized mice by laser-Doppler flowmetry. Ang II (0.25 &mgr;g/kg per minute IV) attenuated the CBF increase produced by mechanical stimulation of the vibrissae. The effect was blocked by the AT1 antagonist losartan and by ROS scavenger superoxide dismutase or tiron and was not observed in mice lacking the gp91phox subunit of NADPH oxidase or in wild-type mice treated with the NADPH oxidase peptide inhibitor gp91ds-tat. Ang II increased ROS production in cerebral microvessels, an effect blocked by the ROS scavenger Mn(III)tetrakis (4-benzoic acid) porphyrin and by the NADPH oxidase assembly inhibitor apocynin. Ang II did not increase ROS production in gp91-null mice. Double-label immunoelectron microscopy demonstrated that AT1 and gp91phox immunoreactivities were present in endothelium and adventitia of neocortical arterioles. Collectively, these findings suggest that Ang II impairs functional hyperemia by activating AT1 receptors and inducing ROS production via a gp91phox containing NADPH oxidase. The data provide the mechanistic basis for the cerebrovascular dysregulation induced by Ang II and suggest novel therapeutic strategies to counteract the effects of hypertension on the brain.

Nox2-Derived Reactive Oxygen Species Mediate Neurovascular Dysregulation in the Aging Mouse Brain

Aging is associated with cerebrovascular dysregulation, which may underlie the increased susceptibility to ischemic stroke and vascular cognitive impairment occurring in the elder individuals. Although it has long been known that oxidative stress is responsible for the cerebrovascular dysfunction, the enzymatic system(s) generating the reactive oxygen species (ROS) have not been identified. In this study, we investigated whether the superoxide-producing enzyme NADPH oxidase is involved in alterations of neurovascular regulation induced by aging. Cerebral blood flow (CBF) was recorded by laser-Doppler flowmetry in anesthetized C57BL/6 mice equipped with a cranial window (age = 3, 12, and 24 months). In 12-month-old mice, the CBF increases evoked by whisker stimulation or by the endothelium-dependent vasodilators acetylcholine and bradykinin were attenuated by 42, 36, and 53%, respectively (P < 0.05). In contrast, responses to the nitric oxide donor S-nitroso-D-penicillamine or adenosine were not attenuated (P > 0.05). These cerebrovascular effects were associated with increased production of ROS in neurons and cerebral blood vessels, assessed by hydroethidine microfluorography. The cerebrovascular impairment present in 12-month-old mice was reversed by the ROS scavenger Mn (III) tetrakis (4-benzoic acid) porphyrin chloride or by the NADPH oxidase peptide inhibitor gp91ds-tat, and was not observed in mice lacking the Nox2 subunit of NADPH oxidase. These findings establish Nox2 as a critical source of the neurovascular oxidative stress mediating the deleterious cerebrovascular effects associated with increasing age.

NMDA Receptor Activation Increases Free Radical Production through Nitric Oxide and NOX2

Reactive oxygen species (ROS) and nitric oxide (NO) participate in NMDA receptor signaling. However, the source(s) of the ROS and their role in the increase in cerebral blood flow (CBF) induced by NMDA receptor activation have not been firmly established. NADPH oxidase generates ROS in neurons, but there is no direct evidence that this enzyme is present in neurons containing NMDA receptors, or that is involved in NMDA receptor-dependent ROS production and CBF increase. We addressed these questions using a combination of in vivo and in vitro approaches. We found that the CBF and ROS increases elicited by topical application of NMDA to the mouse neocortex were both dependent on neuronal NO synthase (nNOS), cGMP, and the cGMP effector kinase protein kinase G (PKG). In mice lacking the NADPH oxidase subunit NOX2, the ROS increase was not observed, but the CBF increase was still present. Electron microscopy of the neocortex revealed NOX2 immunolabeling in postsynaptic somata and dendrites that also expressed the NMDA receptor NR1 subunit and nNOS. In neuronal cultures, the NMDA-induced increase in ROS was mediated by NADPH oxidase through NO, cGMP and PKG. We conclude that NADPH oxidase in postsynaptic neurons generates ROS during NMDA receptor activation. However, NMDA receptor-derived ROS do not contribute to the CBF increase. The findings establish a NOX2-containing NADPH oxidase as a major source of ROS produced by NMDA receptor activation, and identify NO as the critical link between NMDA receptor activity and NOX2-dependent ROS production.

Angiotensin II Attenuates Endothelium-Dependent Responses in the Cerebral Microcirculation Through Nox-2–Derived Radicals

Objective—Angiotensin II (Ang II) exerts deleterious effect on the cerebral circulation through production of reactive oxygen species (ROS). However, the enzymatic source of the ROS has not been defined. We tested the hypothesis that Ang II impairs endothelium-dependent responses in the cerebral microcirculation through ROS generated in cerebrovascular cells by the enzyme NADPH oxidase. Methods and Results—Cerebral blood flow (CBF) was monitored by laser Doppler flowmetry in anesthetized mice equipped with a cranial window. Ang II (0.25±0.02 &mgr;g/kg per minute for 30 to 45 minutes) attenuated the CBF increase produced by the endothelium-dependent vasodilators acetylcholine (−42±5%; P<0.05), bradykinin (−53±5%; P<0.05), and A23187 (−43±4%; P<0.05), and induced cerebrovascular ROS production, assessed by hydroethidine fluoromicrography. These actions of Ang II were prevented by losartan, by the ROS scavenger Mn(III) tetrakis (4-benzoic acid) porphyrin chloride (100 &mgr;mol/L), or by the NADPH oxidase peptide inhibitor gp91ds-tat (1 &mgr;mol/L), and were not observed in mice lacking the NADPH oxidase subunit gp91phox (nox-2). Conclusions—Ang II impairs the endothelial regulation of the cerebral microcirculation through AT1 receptor-mediated cerebrovascular oxidative stress. The source of the ROS is a nox-2-containing NADPH oxidase. These effects of Ang II could threaten the cerebral blood supply and contribute to the increased susceptibility to stroke and dementia associated with hypertension.

Cerebrovascular Nitrosative Stress Mediates Neurovascular and Endothelial Dysfunction Induced by Angiotensin II

Objective—Angiotensin II (AngII) disrupts the regulation of the cerebral circulation through superoxide, a reactive oxygen species (ROS) generated by a nox2-containing NADPH oxidase. We tested the hypothesis that AngII-derived superoxide reacts with nitric oxide (NO) to form peroxynitrite, which, in turn, contributes to the vascular dysfunction. Methods and Results—Cerebral blood flow (CBF) was monitored by laser Doppler flowmetry in the neocortex of anesthetized mice equipped with a cranial window. AngII (0.25±0.02 &mgr;g/kg/min; intravenous for 30 to 45 minutes) attenuated the cerebral blood flow (CBF) increase produced by topical application of the endothelium-dependent vasodilator acetylcholine (−43±1%) and by whisker stimulation (−47±1%). AngII also increased the nitration marker 3-nitrotyrosine (3-NT) in cerebral blood vessels, an effect dependent on NO and nox2-derived ROS. Both the cerebrovascular effects of AngII and the nitration were attenuated by pharmacological inhibition or genetic inactivation of NO synthase. The nitration inhibitor uric acid or the peroxynitrite decomposition catalyst FeTPPS abolished AngII-induced cerebrovascular nitration and prevented the cerebrovascular effects of AngII. Conclusions—These findings provide evidence that peroxynitrite, formed from NO and nox2-derived superoxide, contributes to the deleterious cerebrovascular effects of AngII. Inhibitors of peroxynitrite action may be valuable tools to counteract the deleterious cerebrovascular effects of AngII-induced hypertension.

Effects of chronic N-acetylcysteine treatment on the actions of peroxynitrite on aortic vascular reactivity in hypertensive rats.

Background Peroxynitrite (ONOO−), the product of superoxide and nitric oxide, seems to be involved in vascular alterations in hypertension. Objectives To evaluate the effects of ONOO− on endothelium-dependent and independent aortic vascular responsiveness, oxidized/reduced glutathione balance (GSSG/GSH), malondialdehyde aortic content, and the formation of 3-nitrotyrosine (3-NT), a stable marker of ONOO−, in N-acetylcysteine (NAC)-treated normotensive Wistar–Kyoto (WKY) rats and spontaneously hypertensive rats (SHR). Results In SHR only, NAC significantly reduced heart rate and systolic, but not diastolic, blood pressure. It also improved endothelium-dependent aortic relaxation in SHR, but not after exposure to ONOO−. Endothelium-dependent and independent aortic relaxations were markedly impaired by ONOO− in both strains of rat. NAC partially protected SHR against the ONOO−-induced reduction in endothelium-independent relaxation. Aortic GSSG/GSH ratio and malondialdehyde, which were higher in SHR than in WKY rats, showed a greater increase in SHR after exposure to ONOO−. NAC decreased GSSG/GSH and malondialdehyde in both strains of rat before and after exposure to ONOO−. The 3-NT concentration, which was similar in both strains of rat under basal conditions, was greater in SHR than in WKY rats after the addition of ONOO−, with a reduction only in NAC-treated SHR. Conclusions These findings suggest an increased vulnerability of SHR aortas to the effects of ONOO− as compared with those of WKY rats. The selective improvements produced by NAC, in systolic arterial pressure, heart rate, aortic endothelial function, ONOO−-induced impairment of endothelium-independent relaxation, aortic GSSG/GSH balance, malondialdehyde content and 3-NT formation in SHR suggest that chronic administration of NAC may have a protective effect against aortic vascular dysfunction in the SHR model of hypertension.

N-Acetylcysteine improves nitric oxide and α-adrenergic pathways in mesenteric beds of spontaneously hypertensive Rats

BACKGROUND The aim of this study was to assess the effects of N-acetylcysteine (NAC) on nitric oxide and adrenergic pathways in mesenteric artery from spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY). METHODS Rats were treated with 4 g x kg(-1) x day(-1) of NAC during 4 weeks or mesenteric beds were treated with 10 mmol/L of NAC during 20 min. RESULTS In conscious rats, the NAC treatment produced a significant reduction of mean arterial pressure (MAP) and heart rate in SHR (P <.001). N(omega)-nitro-L-arginine methyl ester (L-NAME) caused a MAP increase in NAC-treated SHR of magnitude similar to that in WKY, which was significantly higher than that observed in control untreated SHR (P <.05). Chronic treatments with NAC improved the maximal relaxation of mesenteric arteries to A23187 in SHR (P <.001). Acute NAC treatment in vitro induced a vasodilation in Phe preconstricted arteries (P <.001) that was stronger in SHR than in WKY (P <.05) and was not abolished by L-NAME. The vasoconstrictory response and increases in inositol phosphate production induced by superoxide anion were attenuated by NAC treatment through its superoxide scavenging properties. In contrast, chronic and acute NAC treatments did not alter the vasodilatory response to beta-adrenergic receptor stimulation. CONCLUSIONS The increase in NO-mediated vasodilator tone and the possible decrease in adrenergic vasoconstriction induced by NAC treatment in SHR could explain the hypotensive effect of NAC in this model of hypertension.

Vasorelaxant effects of the chronic treatment with melatonin on mesenteric artery and aorta of spontaneously hypertensive rats

Objective To investigate the effect of a chronic treatment with melatonin on arterial pressure and a possible improvement of the vascular muscarinic and NO synthase (NOS) pathways in spontaneously hypertensive rats (SHR) and Wistar–Kyoto (WKY) rats. Design and methods Mean arterial pressure (MAP), systolic (SBP), diastolic blood pressure (DBP), and heart rate (HR) were evaluated in conscious rats treated with 30 mg/kg per day of melatonin during 4 weeks. Changes in MAP were evaluated following an intravenous injection of the NOS inhibitor Nω-nitro-l-arginine methyl ester (l-NAME). Relaxant effects of acetylcholine (Ach), sodium nitroprusside (SNP), and the calcium ionophore A23187 were examined on mesenteric beds and aortic rings with or without treatment with melatonin. Results Melatonin produced a significant reduction of MAP, SBP, DBP and HR in SHR (P < 0.05). l-NAME increased the MAP of melatonin-treated SHR by the same magnitude as that of WKY rats which was significantly higher than that of non-treated SHR (P < 0.05). Melatonin treatment improved the maximal relaxation of mesenteric arteries to A23187 in SHR (P < 0.001) to the WKY level and caused a slight increament in Ach- and A23187-induced vasodilations in aorta from SHR and WKY rats (P < 0.05). Conclusion The present study showed that melatonin exerted a bradycardic and an antihypertensive action in SHR. The enhancement by melatonin of the endothelium-dependent vasodilation (Ach and/or A23187) in mesenteric artery and aorta from SHR and WKY rats and the higher increase in MAP following l-NAME treatment in melatonin-treated SHR suggest the contribution of an improved vascular NOS pathway activity in the hypotensive effect of melatonin.

The key role of transient receptor potential melastatin-2 channels in amyloid-β-induced neurovascular dysfunction

Alzheimers dementia is a devastating and incurable disease afflicting over 35 million people worldwide. Amyloid-β (Aβ), a key pathogenic factor in this disease, has potent cerebrovascular effects that contribute to brain dysfunction underlying dementia by limiting the delivery of oxygen and glucose to the working brain. However, the downstream pathways responsible for the vascular alterations remain unclear. Here we report that the cerebrovascular dysfunction induced by Aβ is mediated by DNA damage caused by vascular oxidative-nitrosative stress in cerebral endothelial cells, which, in turn, activates the DNA repair enzyme poly(ADP)-ribose polymerase. The resulting increase in ADP ribose opens transient receptor potential melastatin-2 (TRPM2) channels in endothelial cells leading to intracellular Ca(2+) overload and endothelial dysfunction. The findings provide evidence for a previously unrecognized mechanism by which Aβ impairs neurovascular regulation and suggest that TRPM2 channels are a potential therapeutic target to counteract cerebrovascular dysfunction in Alzheimers dementia and related pathologies.

Remote control of the permeability of the blood-brain barrier by magnetic heating of nanoparticles: A proof of concept for brain drug delivery.

Despite advances in neurology, drug delivery to the brain remains a substantial challenge. This is mainly due to the insurmountable and selective nature of the blood-brain barrier (BBB). In this study, we show that the thermal energy generated by magnetic heating (hyperthermia) of commercially available magnetic nanoparticles (MNPs) in the brain capillaries of rats can transiently increase barrier permeability. Here, the fluorescent Evans Blue (EB) dye was used to verify the BBB integrity. Results indicate a substantial but reversible opening of the BBB where hyperthermia is applied. Also, in this investigation, analysis of CD68 immunoreactivity, an indicator of inflammation, implies that this technique is not associated with any inflammation. We have previously investigated theranostic (therapeutic and diagnostic) capabilities of the MNPs, therefore, the findings presented in this investigation are particularly encouraging for a novel targeted drug delivery system to the brain.