Laibaik Park

Prostaglandin E2 EP1 receptors: downstream effectors of COX-2 neurotoxicity

Cyclooxygenase-2 (COX-2), a rate-limiting enzyme for prostanoid synthesis, has been implicated in the neurotoxicity resulting from hypoxia-ischemia, and its inhibition has therapeutic potential for ischemic stroke. However, COX-2 inhibitors increase the risk of cardiovascular complications. We therefore sought to identify the downstream effectors of COX-2 neurotoxicity, and found that prostaglandin E2 EP1 receptors are essential for the neurotoxicity mediated by COX-2–derived prostaglandin E2. EP1 receptors disrupt Ca2+ homeostasis by impairing Na+-Ca2+ exchange, a key mechanism by which neurons cope with excess Ca2+ accumulation after an excitotoxic insult. Thus, EP1 receptors contribute to neurotoxicity by augmenting the Ca2+ dysregulation underlying excitotoxic neuronal death. Pharmacological inhibition or gene inactivation of EP1 receptors ameliorates brain injury induced by excitotoxicity, oxygen glucose deprivation and middle cerebral artery (MCA) occlusion. An EP1 receptor inhibitor reduces brain injury when administered 6 hours after MCA occlusion, suggesting that EP1 receptor inhibition may be a viable therapeutic option in ischemic stroke.

Nox2-derived radicals contribute to neurovascular and behavioral dysfunction in mice overexpressing the amyloid precursor protein

Alterations in cerebrovascular regulation related to vascular oxidative stress have been implicated in the mechanisms of Alzheimers disease (AD), but their role in the amyloid deposition and cognitive impairment associated with AD remains unclear. We used mice overexpressing the Swedish mutation of the amyloid precursor protein (Tg2576) as a model of AD to examine the role of reactive oxygen species produced by NADPH oxidase in the cerebrovascular alterations, amyloid deposition, and behavioral deficits observed in these mice. We found that 12- to 15-month-old Tg2576 mice lacking the catalytic subunit Nox2 of NADPH oxidase do not develop oxidative stress, cerebrovascular dysfunction, or behavioral deficits. These improvements occurred without reductions in brain amyloid-β peptide (Aβ) levels or amyloid plaques. The findings unveil a previously unrecognized role of Nox2-derived radicals in the behavioral deficits of Tg2576 mice and provide a link between the neurovascular dysfunction and cognitive decline associated with amyloid pathology.

NADPH Oxidase-Derived Reactive Oxygen Species Mediate the Cerebrovascular Dysfunction Induced by the Amyloid β Peptide

Overproduction of the amyloid β (Aβ) peptide is a key factor in the pathogenesis of Alzheimers disease (AD), but the mechanisms of its pathogenic effects have not been defined. Patients with AD have cerebrovascular alterations attributable to the deleterious effects of Aβ on cerebral blood vessels. We report here that NADPH oxidase, the major source of free radicals in blood vessels, is responsible for the cerebrovascular dysregulation induced by Aβ. Thus, the free-radical production and the associated alterations in vasoregulation induced by Aβ are abrogated by the NADPH oxidase peptide inhibitor gp91ds-tat and are not observed in mice lacking the catalytic subunit of NADPH oxidase (gp91phox). Furthermore, oxidative stress and cerebrovascular dysfunction do not occur in transgenic mice overexpressing the amyloid precursor protein but lacking gp91phox. The mechanisms by which NADPH oxidase-derived radicals mediate the cerebrovascular dysfunction involve reduced bioavailability of nitric oxide. Thus, a gp91phox-containing NADPH oxidase is the critical link between Aβ and cerebrovascular dysfunction, which may underlie the alteration in cerebral blood flow regulation observed in AD patients.

Threats to the Mind Aging, Amyloid, and Hypertension

Aging, Alzheimer disease, and hypertension, major determinants of cognitive dysfunction, are associated with profound alterations in the structure and function of cerebral blood vessels. These vascular alterations may impair the delivery of energy substrates and nutrients to the active brain, and impede the clearance of potentially toxic metabolic byproducts. Reactive oxygen species derived form the enzyme NADPH oxidase are key pathogenic effectors of the cerebrovascular dysregulation. The resulting alterations in the homeostasis of the cerebral microenvironment may lead to cellular dysfunction and death and to cognitive impairment. The prominent role that cerebrovascular oxidative stress plays in conditions associated with cognitive impairment suggests new therapeutic opportunities to counteract and, possibly, reverse the devastating effects of cerebrovascular dysfunction 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.

The Class B Scavenger Receptor CD36 Mediates Free Radical Production and Tissue Injury in Cerebral Ischemia

The class B scavenger receptor CD36 is involved in the cytotoxicity associated with inflammation, but its role in the inflammatory reaction that accompanies cerebral ischemia has not been examined. In this study, we investigated whether CD36 contributes to the brain damage produced by cerebral ischemia. The middle cerebral artery was transiently occluded in wild-type mice and in mice deficient in CD36. In wild-type mice, CD36 protein expression was increased in the ischemic brain, such that it was located predominantly in cells expressing the microglia/macrophage marker CD11b. The infarct produced by middle cerebral artery occlusion was 49% smaller in CD36-null mice than in wild-type controls, an effect associated with improved neurological function. The attenuation in brain injury in CD36 nulls could not be attributed to differences in cerebral blood flow during ischemia-reperfusion. However, the increase in reactive oxygen species (ROS) produced by cerebral ischemia was markedly attenuated in CD36-null mice in the early stage after reperfusion. The data unveil a previously unrecognized role of CD36 in ischemia-induced ROS production and brain injury. Modulation of CD36 signaling may provide a new strategy for the treatment of ischemic stroke.

Neurovascular Protection by Ischemic Tolerance: Role of Nitric Oxide and Reactive Oxygen Species

Cerebral ischemic preconditioning or tolerance is a powerful neuroprotective phenomenon by which a sublethal injurious stimulus renders the brain resistant to a subsequent damaging ischemic insult. We used lipopolysaccharide (LPS) as a preconditioning stimulus in a mouse model of middle cerebral artery occlusion (MCAO) to examine whether improvements in cerebrovascular function contribute to the protective effect. Administration of LPS 24 h before MCAO reduced the infarct by 68% and improved ischemic cerebral blood flow (CBF) by 114% in brain areas spared from infarction. In addition, LPS prevented the dysfunction in cerebrovascular regulation induced by MCAO, as demonstrated by normalization of the increase in CBF produced by neural activity, hypercapnia, or by the endothelium-dependent vasodilator acetylcholine. These beneficial effects of LPS were not observed in mice lacking inducible nitric oxide synthase (iNOS) or the nox2 subunit of the superoxide-producing enzyme NADPH oxidase. LPS increased reactive oxygen species and the peroxynitrite marker 3-nitrotyrosine in wild-type mice but not in nox2 nulls. The peroxynitrite decomposition catalyst 5,10,15, 20-tetrakis(4-sulfonatophenyl)porphyrinato iron (III) attenuated LPS-induced nitration and counteracted the beneficial effects of LPS on infarct volume, ischemic CBF, and vascular reactivity. Thus, LPS preserves neurovascular function and ameliorates CBF in regions of the ischemic territory at risk for infarction. This effect is mediated by peroxynitrite formed from iNOS-derived NO and nox2-derived superoxide. The data indicate that preservation of cerebrovascular function is an essential component of ischemic tolerance and suggest that combining neuroprotection and vasoprotection may be a valuable strategy for treating ischemic brain injury.

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.

Aβ-Induced Vascular Oxidative Stress and Attenuation of Functional Hyperemia in Mouse Somatosensory Cortex:

We investigated the role of vascular oxidative stress in the mechanisms of the impairment in cerebrovascular regulation produced by the amyloid-β peptide (Aβ). In particular, we sought to provide evidence of vascular oxidative stress in mice overexpressing the amyloid precursor protein (APP) and to determine whether the Aβ-induced attenuation in functional hyperemia is mediated by free radical overproduction. Oxidative/nitrosative stress was assessed by 3-nitrotyrosine immunoreactivity, while free radical production was determined in cerebral microvessels by hydroethidine microfluorography. To study functional hyperemia the somatosensory cortex was activated by whisker stimulation while local blood flow was monitored by laser-Doppler flowmetry. It was found that APP mice show signs of oxidative/nitrosative stress in pial and intracerebral blood vessels well before they develop oxidative stress in neurons and glia or amyloid plaques. Treatment of cerebral microvessels isolated from wild-type mice with Aβ (1 μM) increased free radical production as assessed by the hydroethidine technique. The Aβ-induced attenuation of the increase in somatosensory cortex blood flow produced by whisker stimulation was prevented by treatment with the free radical scavengers MnTBAP or tiron. These data provide evidence that in APP mice vascular oxidative stress precedes the development of parenchymal oxidative stress, and that Aβ-produced vascular reactive oxygen species are involved in the attendant attenuation in functional hyperemia. Thus, vascular oxidative stress is an early event in the course of the brain dysfunction produced by APP overexpression and Aβ, and, as such, could be the target of early therapeutic interventions based on antioxidants.

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.