Costantino Iadecola - US grants

DESCRIPTION (provided by applicant): Alzheimer's disease (AD) is the leading cause of dementia, and there is a pressing need to develop treatments to mitigate its enormous human and public health impact. Amyloid-ß (Aß) is a key pathogenic factor in AD, but its mechanisms of action are not well understood. In addition to damaging neurons and glia, Aß also impairs vital neurovascular mechanisms that regulate the cerebral blood supply, thus increasing the brain's susceptibility to hypoxic-ischemic injury. Vascular dysfunction may also compromise brain Aß clearance, promoting the retention of the peptide and its damaging effects. During the previous funding period we have established that Aß exerts its deleterious vascular effects by engaging the Aß-binding scavenger receptor CD36. CD36, in turn, leads to the production of reactive oxygen species (ROS) by activating Nox2. However, the cells expressing CD36 on which brain Aß acts on the abluminal side of cerebral blood vessels to increase Nox2-dependent ROS production remain unknown and their elucidation may suggest novel targets for AD therapy. Resident brain macrophages, mainly perivascular macrophages (PVM), reside along the Aß clearance pathway, express CD36 and Nox2, and are in close contact with the outer vessel wall. Therefore, PVM are uniquely positioned to contribute to vascular oxidative stress and neurovascular dysfunction induced by brain Aß. The central hypothesis of this application is that CD36 in PVM participates in the deleterious vascular effects of Aß and in the long-term consequences of Aß overproduction. We will test the following specific hypotheses: (1) PVM are the cells on which brain Aß acts to induce vascular oxidative stress and dysfunction; (2) CD36 in PVM is responsible for these actions of Aß, and (3) CD36 in PVM contributes to the long-term consequences of Aß overproduction, including cognitive deficits and perivascular Aß accumulation. Our studies will use state-of-the-art methods to investigate neurovascular regulation in mice. Pharmacological approaches and bone marrow chimeras will be used to investigate the specific role of CD36 in PVM in Aß-induced oxidative stress, neurovascular dysfunction, and cognitive deficits.

[unreadable] DESCRIPTION (provided by applicant): Ischemic tolerance is a powerful cerebroprotective mechanism by which a sublethal injury protects from a subsequent lethal ischemic insult. Studies over the last funding period have revealed that the inducible or "immunological" isoform of nitric oxide synthase (iNOS; NOS II) is essential for the development of the ischemic tolerance induced by systemic administration of the proinflammatory mediator l. However, the mechanisms by which iNOS participates in ischemic preconditioning have not been defined. Preconditioning induces cellular and molecular changes that are thought to ultimately increase the resistance of brain pells to ischemia. However, preservation of brain tissue depends not only on protection of neurons and glia, but also on safeguarding cerebral blood vessels. Therefore, it is conceivable that both "cytoprotective" and "vasoprotective" mechanisms contribute to the development of ischemic tolerance. The goal of this renewal application is to test the hypothesis that INOS-derived NO contributes to ischemic tolerance by preserving post-ischemic vascular function (vasoprotection). as well as increasing the resistance of brain cells to ischemia (cvtoprotection). First, we will determine whether preconditioning with lipopolysaccharide leads to better preservation of cerebral perfusion after middle cerebral artery occlusion in mice (vasoprotection). Second, we will test the hypothesis that iNOS-derived NO is responsible for the vasoprotective component of preconditioning. Third, we will use a model of NMDA-mediated neocortical injury to test the hypothesis that iNOS is also involved in the tolerance to excitotoxicity induced by lipopolysaccharide (cytoprotection). Fourth, considering that NO exerts many of its actions through peroxynitrite, its reaction product with superoxide, we will test the hypothesis that reactive oxygen species produced by NADPH oxidase are needed for the cytoprotective and vasoprotective effects of iNOS-derived NO. The proposed studies will provide insight into poorly understood aspects of the mechanisms of ischemic preconditioning that may have important implications for the prevention and treatment of ischemic stroke. [unreadable] [unreadable] [unreadable]

DESCRIPTION (Adapted from applicant's abstract): The long-term goal of this research program is to elucidate the role of the prostanoid-synthesizing enzyme cyclooxygenase-2 (COX-2) in ischemic brain injury. Studies during the previous funding period have provided evidence that COX-2 is one of the factors by which the inflammatory process involving the ischemic brain (post-ischemic inflammation) contributes to the late progression of focal cerebral ischemic damage. These findings have indicated that COX-2 participates in the late stages of ischemic brain injury. Other evidence however suggests additional mechanisms by which COX-2 could contribute to ischemic brain injury. In particular, COX-2-derived prostanoids are potent constrictors of cerebral blood vessels and participate in pathogenic processes linked to the activation of glutamate receptors. Considering that cerebral blood flow and activation of glutamate receptors play a critical role in the initiation of ischemic damage, these data raise the possibility that COX-2 could also be involved in early events of the ischemic cascade. Therefore, in this renewal application we will continue to pursue our long-term goal by testing the hypothesis that COX-2, in addition to its role in post-ischemic inflammation, is also involved in the mechanisms initiating ischemic brain injury. As a starting point, we will focus on the role of COX-2 in critical hemodynamic changes that occur during ischemia-reperfusion and in glutamate receptor-mediated cytotoxicity. In the proposed experiment, we will use molecular and biochemical approaches, as well as methods to assess hemodynamic, histological, and neurological outcome after cerebral ischemia. Focal cerebral ischemia will be produced by transient occlusion of the middle cerebral artery in mice. The brain damage resulting from focal microinjection of N-methyl-D-aspartate or kainate in neocortex will also be investigated. The role of COX-2 will be examined using both pharmacological inhibition of COX-2, and COX-2-deficient mice. The role of COX-2 derived reactive oxygen species will be examined using mice over expressing the antioxidant superoxide dismutase. While the proposed studies will expand our understanding of the involvement of COX-2 in ischemic brain injury, they will also provide the rational basis for new neuroprotective strategies for human stroke.

DESCRIPTION (provided by applicant): The Experimental Neurogenic Hypertension Program is a long-standing multidisciplinary research program focusing on the reciprocal interaction between the brain and the circulatory system. The central theme of the program is that, while the brain exerts a powerful influence over the circulation, the circulatory system, in turn, can have profound effects on the brain. Alterations of this delicate balance can produce hypertension and disrupt brain function. The present renewal application is organized in three Projects supported by three Cores. The Projects address diverse aspects of the central theme, but share a common focus on angiotensin II and its recently-recognized signaling system NAD(P)H oxidase. Project 1 investigates the mechanisms by which angiotensin II-induced hypertension impairs critical homeostatic mechanisms that provide blood flow to active brain regions, and examines on the role of NAD(P)H oxidase in the cerebrovascular dysfunction. Project 2 focuses on angiotensin receptors in the nucleus of the solitary tract, their relationships to catecholaminergic receptors and NAD(P)H oxidase subunits, and the changes in their subcellular targeting brought about by chronic intermittent hypoxia or angiotensin II-induced hypertension. Project 3 examines the cellular mechanisms underlying the effects of estrogens, androgens and progestins on neurons in the rostroventrolateral medulla, focusing on angiotensin receptors, and NAD(P)H oxidase subunits. The Projects are supported by an Administrative Core, a Molecular Biology-Mouse Core and a Neuroanatomy-Imaging Core. All Projects combine neuroanatomical, neurophysiological and molecular approaches to test the proposed hypothesis. The Projects build on each other's strengths so that the scientific output of one Project interacts synergistically with the research proposed in other Projects. Thus, the collective scientific outcome of the Program is anticipated to be greater than the sum of its individual components. The proposed studies are relevant to human diseases, such as hypertension-induced cognitive dysfunction, cardiovascular complications of sleep apnea, and cardiovascular diseases in post-menopausal women.

[unreadable] DESCRIPTION (provided by the applicant): The amyloid precursor protein (APP) is intimately involved in the pathogenesis of Alzheimer's disease (AD). The AB peptide, a fragment of APP, accumulates in the neuropil and cerebral blood vessels of patients with AD and plays a critical role in the pathogenesis of the disease. However, the mechanisms by which AB exerts its deleterious effects have not been fully elucidated. Studies over the first funding period have established that AB causes a profound disruption of the mechanisms regulating the cerebral circulation. Thus, transgenic mice overexpressing mutated APP exhibit marked and selective alterations in endothelium-dependent vasodilation and in the cerebrovasodilation evoked by neural activity. These alterations are mediated by reactive oxygen species and occur in the absence of AB deposition in neuropil or blood vessels. In this renewal application, we will continue to study the mechanisms by which AB alters the regulation of the cerebral circulation. Three main hypotheses will be tested. First, that parenchymal and vascular deposition of AB worsens the cerebrovascular alterations observed in mice without AB deposition. Second, that cerebral blood vessels generate the free radicals mediating the dysfunction. Third, that the superoxide-producing enzyme NAD(P)H oxidase is a major source of the radicals responsible for the dysfunction. The reactivity of cerebral blood flow to pharmacological and physiological stimuli will be studied in mice equipped with cranial windows. First, we will study cerebrovascular responses in "young" and "old" APP mice to determine whether AB deposition in old mice worsens the dysfunction. Second, we will use markers of oxidative stress to pinpoint the cellular source(s) of the radicals responsible for the dysfunction. Third, we will study the cerebrovascular effects of synthetic All in mice lacking the gp9l phox subunit of NAD(P)H oxidase to determine whether this enzyme is involved. Fourth, we will study crosses between APP mice and gp9lphox-null mice to provide evidence that a NAD(P)H oxidase is also involved in the dysfunction induced by endogenous All in APP mice. These studies will continue to expand our understanding of the biological effects of AB and constitute a necessary step toward elucidating the contribution of vascular factors to the pathogenesis of AD.

DESCRIPTION (provided by applicant): NEUROVASCULAR REGULATION AND HYPERTENSION: Studies during the previous funding period have focused on the mechanisms responsible for the coupling between neural activity and cerebral blood flow in the normal state. In this renewal application we propose to study the alteration in neurovascular coupling that occur during elevations in arterial pressure. Hypertension exerts deleterious effects on the structure and function of the central nervous system, the cerebral circulation being a major target of these actions. The long-term goal of this project is to define the mechanisms by which hypertension alters the regulation of the cerebral circulation during neural activity and how these alterations affect the structure and function of the central nervous system. We will begin by testing the hypothesis that angiotensin II, a major mediator of hypertension, impairs the mechanisms responsible for the increase in cerebral blood flow induced by neural activation. In particular, the proposed studies will seek to determine whether Ang II exerts this effect by acting directly on cerebral blood vessels and impairing their ability to react to vasodilator signals generated by neural activity. The following specific hypotheses will be tested: (1) angiotensin 11 alters the "coupling" between cerebral blood flow and neural activity, (2) this effect is mediated by angiotensin II-induced production of reactive oxygen species in cerebral blood vessels, and (3) NAD(P)H oxidase is a major source of the vascular reactive oxygen species contributing to the dysfunction. Studies will be conducted in mice in which arterial pressure is elevated by acute or chronic administration of angiotensin II. The increase in cerebral blood flow produced in the somatosensory cortex by whisker stimulation will be used as a model of neural activation. Cerebrovascular responses to endothelium-dependent and independent vasodilators, and production of reactive oxygen species in cerebral blood vessels will also be studied. Mice overexpressing superoxide dismutase-1 will be used to examine the role of reactive oxygen species, and mice lacking the gp91phox subunit of NAD(P)H oxidase will be used to determine whether this enzyme is the source of the radicals. The results of these studies will enhance our understanding of the effects of hypertension on the brain and may provide new insights into treatment strategies aimed at counteracting these detrimental actions.

Imbalance within central autonomic networks may contribute to the development or maintenance of hypertension. Hypertension, in turn, exerts deleterious effects on the structure and function of the central nervous system, the cerebral circulation being a major target of these actions. The long-term goals of this project are: (a) to identify the mechanisms by which hypertension alters the regulation of the cerebral circulation during neural activity, and (b) to define how these alterations affect the structure and function of the brain. Project 1 will begin by testing the hypothesis that AngII impairs the mechanisms responsible for the increase in CBF induced by neural activation. In particular, the proposed studies will determine whether Ang II exerts this effect by acting on cerebral blood vessels and by interfering with the vascular mechanisms through which neural activity increases blood flow. The following specific hypotheses will be tested: (1) Ang II alters the "coupling" between cerebral blood flow and neural activity, (2) this effect is mediated by Ang II-induced production of reactive oxygen species in cerebral blood vessels and nitric oxide inactivation, and (3) NAD(P)H oxidase is a major source of the vascular reactive oxygen species contributing to the dysfunction. Studies will be conducted in mice in which arterial pressure is elevated by administration of Ang II. The increase in cerebral blood flow produced in the somatosensory cortex by whisker stimulation will serve as a model of neural activation. Cerebrovascular responses to endothelium-dependent and independent vasodilators, and cerebrovascular production of reactive oxygen species will be studied. The role of radicals will be examined using mice overexpressing superoxide dismutase-1. Mice lacking specific NAD(P)H oxidase subunits will be used to determine whether this enzyme is the source of oxidative stress. Transgenic mice overexpressing components of the renin-angiotensin system will also be used in some experiments. The results of these studies will enhance our understanding of the effects of hypertension on the brain and may provide insights into new treatment strategies aimed at counteracting these detrimental actions.

Cyclooxygenase-2 (COX-2), a rate-limiting enzyme for prostanoid synthesis, has emerged as a major pathogenic factor in ischemic brain injury and is a promising therapeutic target for stroke. However, recent basic and clinical findings have suggested that some COX-2 reaction products, such as prostacyclin, have beneficial cardiovascular effects. Therefore, in order to exploit the therapeutic potential of the COX-2 pathway, the reaction products involved in the toxicity need to be selectively targeted, sparing the beneficial effects of other COX-2 derived agents. The goals of this application are to identify the specific COX-2 reaction products that contribute to ischemic brain injury and to use preclinical approaches to identify their potential therapeutic value. The proposed studies will test the following hypotheses: (1) Prostanoids rather than reactive oxygen species are the main COX-2 reaction products initiating the injury;(2) Prostaglandin E2 acting through its EP1 receptor contributes to ischemic brain injury;(3) EP1 receptors are the effectors of the toxicity exerted by COX-2 in the post-ischemic brain;(4) the preclinical characteristics of the protective effect of EP1 receptor inhibitors suggest that they have promise in the treatment of stroke. Experiments will be conducted in mice in which cerebral ischemia is produced by transient occlusion of the middle cerebral artery. The role of COX-2 reaction products will be investigated using pharmacological inhibitors, transgenic mice overexpressing the antioxidant enzyme superoxide dismutase 1, or null mice lacking COX-2 or EP1 receptors. Ischemic brain injury will be assessed by histological and behavioral criteria. Molecular, biochemical and neuroanatomical techniques will be used to define the reaction products of the COX-2 pathway that contribute to brain injury. The application fulfills the requirements of the RFA HL-05-004 because it explores novel therapeutic approaches that, either alone or in combination with other treatments, could be useful in patients with ischemic stroke.

[unreadable] DESCRIPTION (provided by applicant): Studies during the previous funding period have established that cyclooxygenase-2 (COX-2), a rate-limiting enzyme for prostanoid synthesis, contributes to ischemic brain injury and glutamate excitotoxicity. However, the reaction products responsible for these deleterious effects, their receptor interactions, and the mechanisms of their action have not been established. Identifying the mediators of neurotoxicity provides the bases for treatment modalities targeting the deleterious effects of COX-2 without opposing the beneficial vascular actions of this enzyme. The long-term goals of this renewal application are to elucidate the specific COX-2 reaction products that mediate ischemic brain injury and to begin to define their mechanism of action. The proposed studies will test the following hypotheses: (1) Prostanoids rather than reactive oxygen species are the main COX-2 reaction product initiating the injury; (2) Prostaglandin E2, acting through neuronal EP1 receptors, is responsible for the deleterious effects of COX-2; (3) EP1 receptors play a role in ischemic brain injury by contributing to glutamate excitotoxicity; (4) EP1 receptors contribute to excitotoxicity by amplifying the Ca++ dysregulation produced by activation of glutamate receptors. Experiments will be conducted in mice in which cerebral ischemia is produced by transient occlusion of the middle cerebral artery. The role of COX-2 reaction products will be investigated using pharmacological inhibitors, transgenic mice overexpressing the antioxidant enzyme superoxide dismutase 1, or null mice lacking COX-2 or EP1 receptors. Ischemic brain injury will be assessed by histological and behavioral criteria. Cellular, molecular, biochemical and neuroanatomical techniques will be used to define the mechanisms by which COX-2 reaction products contribute to brain injury. The results of these studies will provide novel insights into the specific factors mediating COX-2-dependent neurotoxicity, and will offer the opportunity for new therapeutic approaches that selectively target the deleterious effects of COX-2. [unreadable] [unreadable]

The Administrative Core (Core A) is responsible for: (a) the financial management of the Program;(b) personnel issues;(c) the handling of manuscripts, reports, abstracts, correspondence, and reprints;(d) word processing and database management support;(e) coordinating statistical support with the Biostatistical Service of the Department of Public Health;(f) coordinating formal and informal reviews of the Program;(g) general office procedures. The Molecular Biologv-Mouse Core (Core B) provides support services including: (a) breeding and genotyping genetically-modified mice;(b) carrying out biochemical analyses (c) carrying out molecular analyses;(e) acquiring and maintaining equipment;(f) coordinating the purchase, use, and disposal of radiolabeled compounds;(g) supervising and enforcing lab safety policies;(h) dishwashing and autoclaving for the entire program. The Neuroanatomv-lmaging Core (Core C) provides support services including: (a) instruction and assistance with histological procedures;(b) assistance in preparation of tissue for light and electron microscopy;(c) characterization and specificity testing of antisera;(d) instruction and assistance with quantitative imaging;(e) coordinating the purchase, use, and disposal of chemicals used in neuroanatomical experiments;(f) maintenance of the facilities and equipment for histological procedures and image analysis. The Radiotelemetrv Core (Core D) provides support services including: (a) surgical implantation of telemetry devices, osmotic minipumps and data acquisition;(b) assess blood pressure, heart rate, locomotor activity and core body temperature;(c) examine spontaneous baroreflex activity;and (d) determine autonomic tone using power spectral analysis and pharmacological approaches;(e) provide assistance with data analyses and interpretation. RELEVANCE (See Instructions):

The Forebrain Plasticity in Hypertension Program is a new multidisciplinary research program focused on the fundamental biological processes governing the neurohumoral interaction between key forebrain centers and the cardiovascular system. The long-term objective of the program is to elucidate the role of these centers in the pathogenesis of hypertension and in the ensuing end-organ damage, particularly cerebrovascular damage. The central theme of the PPG focuses on the role of the subfornical organ (SFO), one of the circumventricular organs, and the hypothalamic paraventricular nucleus (PVN), the main output pathway of the SFO, in the sympathetic overactivity and hormonal release underlying the increase in blood pressure. The central hypothesis is that in slow-developing hypertension there are adaptive modifications (neuroplasticity) in the SFO-PVN axis that turn maladaptive, and set the stage for the neurohumoral dysregulation driving the hypertension and the cerebrovascular dysfunction. Each project addresses a specific facet of the central hypothesis using a clinically relevant mouse model of hypertension produced by systemic infusion of a low dose of angiotensin II (Angll). Project 1 will examine the downstream signaling mechanisms in SFO by which Angll induces the hypertension, focusing on the role of prostanoids as essential intermediaries. Project 2 will examine critical structural and functional maladaptive mechanisms in the PVN, which enable the neurohumoral dysfunction underlying the development of hypertension. Project 3 will focus on the structural and functional modifications in the PVN that are responsible for the increased susceptibility to hypertension in menopausal females. Project 4 will address the role of the SFO-PVN axis in the deleterious effects of hypertension on vital cerebrovascular regulatory mechanisms that assure an adequate blood flow delivery to the brain. The Projects are supported by an Administrative Core, a Molecular Biology-Mouse Core, a Neuroanatomv-lmaaina Core and a Radiotelemetrv Core. A major strength of the program is that each project combines molecular, neuroanatomical, neurophysiological and cardiovascular integrative approaches to achieve the stated goals. The projects are led by a highly interactive group of established investigators and build on each other's strengths so that the scientific output of one project interacts synergistically with the research proposed in other projects. Thus, the collective scientific outcome of the Program is anticipated to be greater than the sum of its individual components. Hypertension affects nearly one third of the general population and is a major cause of the disease burden afflicting men and post-menopausal women. The proposal provides an unprecedented look at the cellular and molecular underpinnings of the neurohumoral dysfunction that leads to hypertension and cerebrovascular dysfunction. The results may provide the rational bases for new treatments for hypertension and its devastating effects on the brain, such as stroke and dementia.

PROJECT 4 (ladecola): Forebrain Mechanisms of Neurovascular Dysfunction in Hypertension The brain is a major target of the end-organ damage produced by hypertension, an effect linked to the disruption of vital cerebrovascular regulatory mechanisms that threatens the cerebral blood supply and increases the brain's susceptibility to stroke and dementia. The long-term goal of Project 4 is to unravel the mechanisms by which hypertension exerts its deleterious effects on the brain. Angll plays a key role in the pathophysiology of hypertension. Systemic administration of Angll at concentrations not sufficient to elevate arterial pressure induces hypertension if the administration is sustained in time (slow pressor hypertension). Synaptic adaptations (neuroplasticity) in the subfornical organ (SFO), one of the forebrain circumventricular organs, and in the paraventricular nucleus of the hypothalamus (PVN) are critical for the development of the neurohumoral dysfunction underiying the hypertension. These adaptive changes, turned maladaptive, are triggered by Angll through activation of ATI receptors in the SFO and consequent production of reactive oxygen species (ROS). The SFO, in turn, activates NMDA receptors in the PVN, which contribute to the hypertension by increasing sympathetic outflow and releasing hormones, including vasopressin (AVP). Circulating Angll and AVP increase the vascular expression of endothelin-1 (ET1), a potent vasoconstrictor that is upregulated in arteries of patients with essential hypertension. Project 4 will test the hypothesis that slow pressor hvpertension elicits cerebrovascular dvsfunction, which is mediated by the SFO-PVN pathway through neurohumoral effects on cerebral blood vessels involving Anall, AVP and ET1. Experiments will be conducted in mice in which cerebrovascular regulation will be studied using cranial window preparations. Both pharmacological and genetic approaches will be used to achieve the stated goals. We will test the following hypotheses: (1) Slow pressor Angll administration disrupts key homeostatic responses of the cerebral circulation, an effect that may precede the development of hypertension;(2) The central signaling mechanisms of the cerebrovascular dysfunction involve AT1 receptors and ROS production in the SFO, and NMDA receptors in the PVN;(3) PVN-derived AVP and circulating Angll induce cerebrovascular dysfunction through upregulation of ET1 in cerebral blood vessels;(4) Angll and ET1 act synergistically to induce cerebrovascular dysfunction via NADPH-oxidase derived ROS. RELEVANCE (See instructions): Hypertension is the leading cause of stroke and vascular cognitive impairment. However, the mechanisms of these effects are pooriy understood. The proposed studies represent the first exploration of the role of the SFO-PVN axis in the cerebrovascular dysfunction induced by slow pressor hypertension. The results will provide novel insights into the fundamental mechanisms by which hypertension alters the cerebral circulation and may suggest new therapies to protect the brain from the devastating effects of hypertension.

The Forebrain Plasticity in l-lypertenslon Program is a new multidisciplinary research program focused on the fundamental biological processes governing the neurohumoral interaction between key forebrain centers and the cardiovascular system. The long-term objective of the program is to elucidate the role of these centers in the pathogenesis of hypertension and in the ensuing end-organ damage, particularly cerebrovascular damage. The central theme of the PPG focuses on the role of the subfornical organ (SFO), one of the circumventricular organs, and the hypothalamic paraventricular nucleus (PVN), the main output pathway of the SFO, in the sympathetic overactivity and hormonal release underlying the increase in blood pressure. The central hypothesis is that in slow-developing hypertension there are adaptive modifications (neuroplasticity) in the SFO-PVN axis that turn maladaptive, and set the stage for the neurohumoral dysregulation driving the hypertension and the cerebrovascular dysfunction. Each project addresses a specific facet of the central hypothesis using a clinically relevant mouse model of hypertension produced by systemic infusion of a low dose of angiotensin II (Angll). Project 1 will examine the downstream signaling mechanisms in SFO by which Angll induces the hypertension, focusing on the role of prostanoids as essential intermediaries. Project 2 will examine critical structural and functional maladaptive mechanisms in the PVN, which enable the neurohumoral dysfunction underlying the development of hypertension. Project 3 will focus on the structural and functional modifications in the PVN that are responsible for the increased susceptibility to hypertension in menopausal females. Project 4 will address the role of the SFO-PVN axis in the deleterious effects of hypertension on vital cerebrovascular regulatory mechanisms that assure an adequate blood flow delivery to the brain. The Projects are supported by an Administrative Core, a Molecular Biology-Mouse Core, a Neuroanatomv-lmaaina Core and a Radiotelemetrv Core. A major strength of the program is that each project combines molecular, neuroanatomical, neurophysiological and cardiovascular integrative approaches to achieve the stated goals. The projects are led by a highly interactive group of established investigators and build on each other's strengths so that the scientific output of one project interacts synergistically with the research proposed in other projects. Thus, the collective scientific outcome of the Program is anticipated to be greater than the sum of its individual components.

DESCRIPTION (provided by applicant): Sleep-disordered breathing, including sleep apnea (SA), is characterized by cyclical interruption of breathing during sleep, often caused by intermittent airway obstruction. SA is emerging as a highly prevalent cause of death and disability. In addition to hypertension and cardiac diseases, SA is an independent risk factor for stroke, and increases its incidence by 2-4 folds. The development of mechanism-based therapies has been hampered by the lack of insight into how SA increases the risk of cerebrovascular insufficiency and stroke. Although the pathophysiology of SA is likely to be multifactorial, chronic intermittent hypoxia (CIH) caused by the apneic episodes is considered a critical factor in the cardiovascular complications. In the systemic circulation, CIH, like SA, alters vascular function, but little is known about the impact of these alterations on the regulation of organ blood flow and on end-organ damage, particularly in brain. Considering the brain's unique susceptibility to vascular insufficiency, disruption of the regulation of the cerebral blood supply by CIH could compromise the delivery of adequate blood flow to the tissue and promote ischemic injury. The present proposal will test the central hypothesis that CIH exerts its deleterious effect on the brain by altering key cerebrovascular homeostatic mechanisms, reducing vascular reserves and increasing the vulnerability of the brain to ischemia. In particular, we will test the following specific hypotheses in four aims: (1) CIH disrupts the delivery of blood to the brain by altering vital regulatory mechanisms that assure adequate cerebral perfusion, such as functional hyperemia and cerebrovascular autoregulation;(2) CIH exerts its deleterious cerebrovascular effects by inducing vascular oxidative stress through the superoxide producing enzyme NADPH oxidase;(3) Upregulation of endothelin-1 in cerebral blood vessels, via ETA receptors, plays a major role in the neurovascular dysfunction;(4) The detrimental cerebrovascular effects of CIH deplete cerebrovascular reserves, aggravate the brain ischemia induced by middle cerebral artery occlusion, and increase the resulting tissue damage. These hypotheses will be tested using a mouse model of CIH and well-established approaches to examine cerebrovascular regulation and ischemic brain injury. The results of the proposed studies will provide new insights that may advance our understanding of the cerebrovascular complications of SA. PUBLIC HEALTH RELEVANCE: Disorders of breathing during sleep, including sleep apnea, have emerged as independent risk factors for stroke, especially silent strokes. The proposed studies will advance our understanding of the pathophysiological substrates underlying the increased susceptibility to ischemic injury in sleep apnea. Ultimately, the results of the proposed studies may suggest novel mechanism-based approaches to prevent or treat the deleterious effects of this highly prevalent condition.

? DESCRIPTION (provided by applicant): Hypertension is a leading risk factor for stroke and dementia, but the mechanisms mediating its deleterious effects on the brain remain poorly understood. Hypertension damages the structure and function of cerebral blood vessels increasing the susceptibility of the brain to stroke and cognitive impairment. In particular, hypertension disrupts critical homeostatic mechanisms that assure adequate cerebral perfusion, promoting vascular insufficiency and brain dysfunction. In the hypertension induced by infusion of low doses of angiotensin II (AngII), a peptide involved in human hypertension, or in mice with life-long hypertension on genetic basis (BPH mice), the cerebrovascular dysfunction is mediated by activation of AngII type 1 receptors (AT1R) resulting in vascular oxidative stress produced by a Nox2-containing NADPH oxidase. The cellular target(s) of AngII, its effectors in the vascular wall and the impact of the neurovascular dysfunction on cognition remain to be established and are of great translational relevance. Perivascular macrophages (PVM) are bone marrow derived cells residing in the perivascular space in close apposition to the cerebrovascular basement membrane and express AT1R and Nox2 and, as such, are well positioned to contribute to the cerebrovascular dysfunction induced by AngII-dependent hypertension. The central hypothesis of this application is that PVM are critical cells for the cerebrovascular and cognitive dysfunctio induced by hypertension. To this end, first we will establish whether AngII, infused for 2 weeks, crosses the blood-brain barrier and reaches the PVM in the perivascular space. Second, we will determine whether PVM are required for the cerebrovascular and cognitive alterations induced by AngII infusion. Third, we will determine whether AT1R and Nox2 in PVM are involved in the dysfunction. In parallel studies we will also examine the role of PVM in the cerebrovascular and cognitive dysfunction observed in young and aged mice with life-long hypertension (BPH mice). To achieve these goals we will use state-of-the-art approaches to study neurovascular regulation in combination with bone marrow chimeras and genetic models for cell specific conditional deletion of genes of interest. The findings derived from the proposed studies will fill an obvious gap in our understanding of the cellular mechanisms of the brain dysfunction induced by hypertension, and will provide proof-of-principle that PVM may be a therapeutic target for the devastating effects of hypertension on the brain.

Mitochondrial permeability transition (MPT) is an inner membrane permeabilization event, which can result in irreversible de-energization and swelling of mitochondria, leading to release of pro-death factors. Mitochondrial Ca2+ overload is the best-characterized trigger of MPT and has been implicated in the pathogenesis of diverse paradigms of neuronal death, such as ischemia-reperfusion injury, where a large influx of cytosolic Ca2+ triggers mitochondrial Ca2+ overload. While uncontrolled MPT can result in mitochondrial disruption, under certain conditions, MPT could provide mitochondria with a Ca2+ release outlet, allowing Ca2+ recycling and protecting mitochondria from Ca2+ overload. Estrogen receptors (ER) have been implicated in various paradigms of neuronal injury, and MPT modulation could be one of the mechanisms whereby they exert their role. Our studies revealed an unprecedented role of the ER? in modulating MPT. In mouse brain mitochondria, estrogen decreases mitochondrial Ca2+ capacity in an ER? and cyclophilin-D (CyPD, an MPT activator) dependent manner. Mitochondria from ER? knock out (ER?KO) mice have reduced sensitivity to cyclosporine A, a potent CyPD inhibitor and CyPD genetic ablation in ER?KO does not further increase Ca2+ capacity. These results point to ER? as a novel regulator of Ca2+-dependent MPT that functionally interacts with CyPD. In this application, we will test the hypothesis that ER? localized in mitochondria (mER?) regulates MPT, independently of transcriptional effects. The goals are to investigate the mechanisms of MPT modulation by mER? and to test the effects of MPT modulation by mER? in models of neuronal injury that involve mitochondrial Ca2+ toxicity, such as oxygen glucose deprivation (OGD) and glutamatergic toxicity. To this end we propose 1) to study the mechanisms of regulation of Ca2+-mediated MPT by ER?. This regulation will be investigated using a multipronged approach, involving biochemical and molecular studies. 2) To assess the role of ER? MPT regulation in neuronal Ca2+-mediated injury. Evidence suggests that Ca2+ dependent MPT and its regulator CyPD are involved in ischemic neuronal injury. We will use neuronal OGD and exposure to glutamatergic agents, both well-known paradigms of neuronal toxicity involving mitochondrial Ca2+ overload, to test the effects of genetic and pharmacological modulation of ER?. The impact of the project will be two-fold: first, it will elucidate novel mechanisms of MPT regulation; second, it will assess if MPT modulation by mER? could be protective in neuronal injury.

? DESCRIPTION (provided by applicant): Salt consumption worldwide greatly exceeds minimal requirements, and excessive dietary salt has emerged as a powerful risk factor for cardiovascular diseases, especially stroke. A high salt diet (HSD) is particularly damaging in older people. Although the deleterious effect of HSD was first attributed to the hypertension that develops in salt-sensitive individuals, increasing evidence indicates that dietary salt increases the incidence of stroke independently of whether or not the blood pressure is elevated. However, it remains to be established how HSD acts on the brain to increase cerebrovascular risk independently of hypertension. International attempts to curb salt consumption have met with limited success and there is an ongoing controversy about ideal levels of salt intake. Therefore, there have been calls for gaining a better mechanistic understanding of how dietary salt impacts the vascular health of the brain. Recent evidence indicates that HSD induces the expansion of a select population of T-helper lymphocytes in the gut (Th17 cells), which produce IL17, a cytokine well known for its damaging vascular effects in animals and humans. Indeed, diseases driven by a Th17 immune response, such as rheumatoid arthritis, psoriasis, multiple sclerosis or inflammatory bowel disease, are associated with increased risk of stroke. On these bases, we propose to test the central hypothesis that HSD exerts its deleterious cerebrovascular effects by inducing a Th17 response. The resulting increase in the vasotoxic cytokine IL17, in turn, acts on cerebral blood vessels to alter critical homeostatic responses that control cerebral perfusion and safeguard brain health. To this end, the present grant application will examine neurovascular function in a mouse model of HSD (4 or 8% NaCl for 8 weeks) to test the following hypotheses in young and aging mice: (1) HSD alters microvascular structure and function to disrupt key cerebrovascular regulatory mechanisms that assure that the brain receives sufficient blood flow well matched to its energetic needs; (2) The cerebrovascular dysfunction is mediated by Th17 lymphocytes via the cytokine IL17; (3) IL17, in turn, causes cerebrovascular dysfunction by inducing NADPH oxidase-dependent oxidative stress, as well as by modulating the phosphorylation state of endothelial nitric oxide synthase and suppressing nitric oxide production. The proposed studies fill an obvious gap in the understanding of the cerebrovascular effects of HSD across the lifespan, and open new avenues of translational research to develop rational strategies to contain the impact of salt on brain vascular health. Furthermore, the concept that Th17 responses initiated in other organs may alter cerebrovascular structure and function has far reaching consequences for Th17-dependent diseases associated with increased stroke risk.

Subcortical and periventricular white matter damage is a major cause of age-related cognitive impairment, but the mechanisms remain elusive. Although the association with small vessel disease leading to chronic ischemia is well recognized, the factors promoting white matter damage are poorly understood. Located at the borderzone between separate arterial territories and supplied by terminal arterioles, the deep white matter is highly vulnerable to hypoxia- ischemia. ApoE is a lipid transport protein enriched in brain and present in three allelic variants (?2, ?3, ?4). Homozygosity for the ?4 allele (?4/?4) is the main genetic risk factor for Alzheimer's disease, but ApoE4 carriers also have increased risk for white matter lesions in the setting of both vascular cognitive impairment and Alzheimer's disease. ApoE4 carriers have reduced cerebral blood flow raising the possibility that cerebrovascular factors contribute their increased propensity to white matter damage. However, it remains unclear whether ApoE4 disrupts vital cerebrovascular mechanisms that assure adequate cerebral perfusion thereby promoting white matter ischemic injury. Perivascular macrophages, bone marrow derived cells closely apposed to the outer wall of cerebral arterioles, are enriched in ApoE receptors and are a powerful source of reactive oxygen species and proinflammatory mediators. Therefore, we hypothesize that ApoE4 promotes white matter damage by disrupting critical neurovascular mechanisms that assure adequate cerebral perfusion, an effect mediated by perivascular macrophages through oxidative stress and inflammation. Since TRPM2 channels are involved in macrophage activation and neurovascular dysfunction, we will also examine their role. We will test the following hypotheses: (a) ApoE4 disrupts vital homeostatic mechanisms regulating the cerebral microcirculation; (b) perivascular macrophages contribute to the dysfunction through TRPM2 channels and ApoE receptors; (c) ApoE4 exacerbates hypoxic-ischemic white matter damage, an effect mediated by perivascular macrophages. Studies are conducted in young and old mice of both sexes with targeted replacement of mouse ApoE with human ApoE3 or 4. White matter injury is produced in the corpus callosum by bilateral carotid artery stenosis. State-of-the-art approaches are used to study neurovascular regulation, including a novel 3-photon imaging method enabling us, for the first time, to simultaneously assess microvascular perfusion and damage in the white matter of the corpus callosum in vivo. These studies will provide insight into the mechanisms underlying the impact of ApoE4 on white matter damage, and may unveil new therapeutic targets for a leading cause of cognitive dysfunction.

Alzheimer's disease (AD) is the leading cause of age-related cognitive impairment for which there are no treatments. The structural and functional integrity of the brain requires a continuous supply of oxygen and glucose through blood flow, well matched to its dynamic and regionally diverse energy needs. Accordingly, neural activity increases blood flow in active brain regions, a phenomenon termed functional hyperemia. Functional hyperemia depends in large part on the link between NMDA receptor activity and neuronal production of the potent vasodilator nitric oxide (NO), and requires tissue plasminogen activator (tPA) for its full expression. Increasing evidence indicates that alterations in the regulation of the cerebral microcirculation play a significant role in the pathogenesis of AD by reducing the cerebral blood supply, but the mechanisms of such neurovascular dysfunction have not been fully elucidated. Most studies have focused on the damaging cerebrovascular effects of A?. However, the role of tau, a key pathogenic factor in AD, remains virtually unexplored. There is a strong rationale for investigating tau. For example, in neurodegenerative diseases caused by tau mutations (tauopathies), in which A? is not present, there is evidence of cerebrovascular dysfunction early in the disease course, suggesting that tau exerts pathogenic vascular effects independently of A?. Furthermore, hyperphosphorylated tau alters NMDA receptor signaling, which is essential for the neuronal NO production driving functional hyperemia. Based on these scientific premises, we will test the central hypothesis that pathological tau alters neurovascular coupling by impairing the ability of NMDA receptors to trigger NO production, an effect occurring prior to neurodegeneration and cognitive deficits. To this end, we will use state-of-the-art multidisciplinary approaches to investigate neurovascular regulation in mouse models of tauopathies, and in vitro approaches to explore the neurophysiological and molecular mechanisms of the effects. We will test the following hypotheses: (a) Tau disrupts neurovascular function prior to the onset of tau pathology and neurodegeneration; (b) The neurovascular dysfunction induced by tau is due to uncoupling of NMDA receptor activity from NO production; (c) NMDA receptor uncoupling from NO production and neurovascular dysfunction result from a deficit in tPA caused by upregulation of the endogenous tPA inhibitor PAI-1. The findings will begin to shed light on the vascular effects of pathological tau, thereby filling a knowledge gap in our understanding of the neurovascular dysfunction of AD and other tauopathies.