David W. Busija - US grants

Hypoxia-ischemic injury in the neonate represents a major medical problem in the U.S.A. and results in neurological sequelae and death. Current treatment regimens are not optimal and there is a need for new therapeutic approaches. A novel experimental approach first described by us in the brain and cerebral circulation involves the initiation of immediate and delayed preconditioning against anoxic stress by the selective activation of mitochondria! ATP-sensitive potassium (mitoKATP) channels. No previous studies have systematically examined the dynamics and mechanisms of pharmacological preconditioning in brain tissue or the vasculature of neonatal or adult animals. Furthermore, use of several selective mitoKATP channel openers will clarify the mechanisms of preconditioning. We have created two specific aims to test our hypotheses and speculations in piglets: Specific Aim 1. Examination of the effects of mitoKATP channel activation without ROS production in immediate and delayed protection of brain and vasculature after IR. We will test the hypothesis that immediate and delayed preconditioning have distinct "windows" and that only mitoKATP channel activation and not linked ROS production is necessaryfor the initiation and expression of immediate and delayed neuro- and vascular-protection. First, we will define temporal windows for the immediate and delayed phases of protection against anoxic stress. Second, we will assess whether preconditioning via mitoKATP channel activation occurs in cerebral blood vessels as well as in neurons. Third, we will document that preconditioning occurs in the absence of ROS generation. Specific Aim 2. Determination of the mechanisms of mitoKATP channel activation in immediate and delayed protection of brain and vasculature after IR. We will test the hypothesis that initiating events are similar, but subsequent mechanisms underlying immediate and delayed preconditioning are different. First, we will assessthe role of protein kinase C (PKC) activation as a key component of preconditioning. Second, we will investigate the mechanisms by which mitoKATP channel activation limits calcium influx and mitochondrial swelling during immediate preconditioning. Third, we will explore the mechanisms by which mitoKATP channel activation leads to reduced ROS production during delayed preconditioning. We expect that our studies will lead to the development of therapies which will lessen the severity of neurological injury to ischemia in the neonate.

Cortical spreading depression (CSD) is a wave of depolarization that results in complex changes in pial arteriolar diameter, intense vasodilation lasting approximately 2 minutes, a return of diameter to baseline for several minutes, and then a prolonged period of vasoconstriction and reduced responsiveness to dilator and constrictor stimuli. CSD can be initiated by microapplication to the brain of K+ or excitatory amino acid neurotransmitters, electrical stimulation, trauma such as physical puncture of the brain or applied pressure, or by anoxia/asphyxia. Our preliminary observations in adult rabbits have led to the development of three hypotheses related to cerebral hemodynamic effects of CSD: a) CSD dilates pial arterioles via mechanisms intrinsic to the vessel wall--activation of perivascular fibers associated with the trigeminal nerve, or by an endothelium-dependent pial arteriolar dilation arising secondary to dilation of intracortical arterioles; b) prostaglandin endoperoxide synthase products (prostanoids and/or free radicals) limit arteriolar dilation during CSD; and c) delayed post-CSD vasoconstriction and reduced responsiveness are due to presence of prostaglandin endoperoxide synthase products. To test these hypotheses, two specific aims will be addressed in anesthetized rabbits. 1) DETERMINATION OF THE MECHANISM(S) OF CEREBROVASCULAR DILATION DURING CSD; AND 2) DETERMINATION OF THE MECHANISM(S) OF CEREBROVASCULAR CONSTRICTION AND REDUCED RESPONSIVENESS FOLLOWING CSD. We will use several complimentary methods, including: the "closed" cranial window to allow virtually continuous measurement of pial arteriolar diameter and periodic sampling of perivascular cerebrospinal fluid for subsequent prostanoid determination using radioimmunoassay; cerebral blood flow determination using radioactive microspheres; and superoxide anion detection using an assay involving superoxide dismutase inhibitable nitroblue tetrazolium reduction. These studies will expand our understanding of vascular control mechanisms in the brain and may provide information to allow development of therapeutic approaches to alleviate immediate and delayed vascular and neuronal impairments associated with CSD-Iike events.

[unreadable] DESCRIPTION (provided by applicant): Insulin Resistance (IR) is associated with vascular dysfunction and strokes. However, effects on the cerebral circulaton are completely unexplored. Our findings from isolated peripheral and cerebral arteries from IR rats have lead to the following hypothesis: IR impairs normal vascular dilator mechanisms in the cerebral circulaton and thereby exacerbates the potential for neurological damage due to stroke. We have created 3 specific aims: Specific Aim 1. Examination of effects of IR on responsiveness to dilator stimuli in isolated cerebral arteries. We will: First, examine effects of IR on endothelium-dependent dilator properties using pharmacological probes; Second, examine effects of IR on vascular smooth muscle function; Third, determine the effects of IR on arterial responsiveness to physiological stimuli; Fourth, determine whether restoration of normal cerebral vascular function is present after cessation of fructose feeding. Specific Aim 2. Elucidation of mechanisms of deranged cerebrovascular control mechanisms in IR. We will: First, examine the contributions of endothelium-derived factors to arterial dilation to pharmacological and physiological stimuli; Second, examine whether IR affects the vascular metabolism of arachidonic acid and L-arginine; Third, determine whether IR affects arterial responses to arachidonic acid or L-arginine metabolites; Fourth, examine effects of IR on vascular smooth muscle ATP-sensitive or calcium-activated potassium channels; Fifth, determine whether pharmacological augmentation of an endothelial-mediated dilator system restores normal vascular function in IR rats. Specific Aim 3. Examination of effects of IR on extent of neurological injury following experimental strokes. We will: First, examine effects of IR on brain injury after experimental strokes; Second, determine the role of impaired cerebral vascular dilator capacity in mediating enhanced infarct volume after stroke in IR animals; Third, determine whether cessation of fructose feeding improves outcome after stroke; Fourth, determine whether pharmacological augmentation of an endothelial-mediated dilator system protects against stroke in IR rats; Fifth, assess the ability of novel scavengers of oxygen free radicals to lessen neurological injury to stroke in IR rats. We believe that our studies will result in new and important findings that will lead to improved therapies to reduce morbidity and mortality in IR individuals.

DESCRIPTION (provided by applicant): Our recent studies show that insulin resistance (IR) severely impairs arterial dilator function in the cerebral circulation via mechanisms involving the sustained production and actions of reactive oxygen species (ROS). Our overall hypothesis is that: Vascular dysfunction of the cerebral circulation in IR is due to augmented ROS levels from enhanced activity of the NADPH oxidase system. Furthermore, we speculate that differences are present in ROS signaling occur in endothelium and VSM;that statins directly modulate NADPH oxidase activity or indirectly act via a reduction in vascular inflammation;that ROS scavenging by superoxide dismutase or gene transfer of EcSOD reverses vascular dysfunction;that neurological damage is enhanced in IR;and that acute administration of statins can limit IR-enhanced ischemic damage in IR. We have created 2 specific aims to test the following hypotheses and speculations in the cerebral circulation and brain in a genetic model of IR (Zucker obese rats): Specific Aim 1. Elucidation of mechanisms of deranged arterial function of the cerebral circulation in IR. We will test the hypothesis that IR impairs endothelium- and VSM potassium channel-dependent function of cerebral arteries via vascular production and actions of ROS. First, we will assess the role of ROS in vascular dysfunction in IR using pharmacological agents and gene transfer approaches. Second, we will determine the metabolic source and characteristics of ROS involved in dysfunction of cerebral vessels of IR rats. Third, we will document, using electrophysiological approaches, the effects of ROS on potassium channels-dependent membrane potential characteristics in cerebral arteries from IR rats. Fourth, we will examine the effects of IR on extent of neurological injury following experimental strokes. Specific Aim 2. Examination of mechanism of statins in reversing vascular dysfunction in IR. We will test the hypothesis that statins directly modulate NADPH oxidase activity in IR. First, we will assess the dynamics of statin effects on vascular responsiveness in IR. Second, we will determine the effects of statins on indices of vascular inflammation and ROS production in cerebral arteries. Third, we will examine the effects of statins and IR on VSM membrane characteristics. Fourth, we will examine the effects of statins on the extent of stroke damage in IR. We believe that our results will lead to new therapies that will help patients with insulin resistance and vascular dysfunction.

DESCRIPTION (Verbatim from the application): Insulin resistance is associated with an increased risk of hypertension and cardiovascular disease. Data from the applicant's laboratory suggests that endothelial dysfunction may be the mechanism that links insulin resistance to vascular disease. Previous studies demonstrate impaired endothelium dependent vasodilation in small coronary arteries from insulin resistant rats. Moreover, this dysfunction appears to be secondary to a defect in endothelium derived hyperpolarizing factor (EDHF). These data support the existence of a unique situation where the EDHF dilator component is selectively impaired and provides us a model to assess mechanisms leading to impaired EDHF function. Moreover, it also provides us a model to assess compensatory responses of other endothelium derived relaxing factors (NO, prostacyclin) to an EDHF deficit. The overall hypothesis to be examined is that impairment of endothelium-dependent dilator capacity occurs in coronary arteries during JR. Specific hypotheses to be tested are (1) Endothelial dysfunction associated with JR is due to a decreased production of EDHF. (2) Decreased substrate availability and/or abnormally low levels of CYP isoforms account for decreased EDHF production. (3) Endothelial dysfunction associated with JR is due to decreased sensitivity of potassium channels on vascular smooth muscle to EDHF. (4) The NO and prostacyclin systems do not compensate for this loss of dilator capacity. (5) Enhanced PKC activation links IR to vascular dysfunction. To test these hypotheses the following specific aim will be addressed. Specific aim. Determination of the mechanism of impaired endothelium-dependent vasodilation in insulin resistance. First, we will investigate the effect of JR on dilator responses to exogenous arachidonic acid. Second, we will explore the effect of IRon synthesis of bioactive metabolites of arachidonic acid. Third, we will examine the relationship between endothelial function and levels of CYP and NO synthase (NOS) protein. Fourth, we will determine the effect of application of exogenous arachidonic acid metabolites (epoxyeicosatrienoic acids) and direct activators of the calcium dependent potassium channel on vascular tone. Fifth, we will determine the effect of PKC inhibition on endothelium and vascular smooth muscle dependent vasodilation. Sixth, we will assess the effect of PKC activation on endothelium and vascular smooth muscle dependent vasodilation. And seventh, we will assess the effect of JR on PKC protein expression in small coronary arteries. These data will determine specific mechanisms of endothelial dysfunction in insulin resistance. In addition, the specific role of EDHF and the possible mechanisms for its dysfunction will be determined. Finally, these data will determine the role of PKC in vascular dysfunction associated with IR, which may help to define the global mechanisms linking insulin resistance to vascular disease, This project employs a unique approach, incorporating physiologic, cellular, and molecular biology techniques to address the proposed questions. Findings from this project may provide important information that can be used in future studies to design treatments to prevent or abort the cardiovascular complications of insulin resistance.

DESCRIPTION (provided by applicant): Potassium (K) channel activation in vascular smooth muscle (VSM) promotes dilation of arteries to physiological stimuli. Our new finding is that insulin resistance (IR) impairs dilator responses of cerebral arteries to stimuli, which are dependent on opening of K channels in VSM. The underlying basis of K channel dysfunction in IR may involve increased production of reactive oxygen species (ROS). This vascular impairment may account for the increased incidence of and/or impaired recovery from cerebrovascular accidents such as subarachnoid hemorrhage (SAH). However, these issues have not been adequately investigated. We have created 2 specific aims to examine these issues in the in situ basilar artery of the Zucker Obese rat model of IR: Specific Aim 1. Elucidation of mechanisms of deranged K function in VSM of the cerebral circulation. We will test the hypotheses that IR impairs K channel function of cerebral arteries in a subtype-specific fashion and that vascular production and actions of ROS mediate K channel dysfunction. First, we will examine effects of selective K channel agonists and antagonists on the basilar artery and its branches in vivo. Second, we will determine whether IR changes vascular levels of K channel subunits. Third, we will assess the role of ROS in K channel dysfunction in IR using pharmacological and gene transfer approaches. Fourth, we will determine the metabolic source of ROS. Fifth, we will determine whether impaired K channel-mediated dilation leads to enhance constrictor effects. And sixth, we will use electrophysiological approaches to characterize the relationship between VSM membrane potential and diameter in cerebral arteries from IR rats. Specific Aim 2. Examination of effects of IR on cerebral arterial function following experimental SAH. We will test the hypothesis that underlying IR will potentiate adverse effects of SAH on baseline artery diameter and reverse augmented vascular responses to K channel-dependent dilator agents. First, we will examine the effects of SAH on baseline diameter and vascular responsiveness in IR. Second, we will explore the role of K channels in impairment of arterial function following SAH in IR. Third, we will determine whether gene transfer protects vascular responses against SAH in IR animals. And fourth, we will examine the relationship between membrane potential and diameter in cerebral VSM in IR and SAH.

DESCRIPTION (provided by applicant): Limited studies indicate an important role of mitochondrial-derived factors in the control of the cerebral circulation beyond effects which are related to energy supply. Thus, factors which depolarize mitochondria and/or augment the release of ROS from mitochondria activate signaling pathways leading to net dilation of cerebral arteries. Furthermore, our preliminary data indicate that mitochondrial influences are adversely affected by insulin resistance (IR). Our overall hypothesis is that mitochondrial-derived influences are key regulators of cerebral vascular tone but are compromised by IR. Two Specific Aims will test our hypotheses and speculations in rats: Aim 1. Examination of the roles of mitochondrial-derived influences in mediating responses of cerebral arteries. We will: A) Determine the relationship among cerebral arterial vasodilation, mitochondrial depolarization, kinase activation, and mitochondrial ROS production. B) Elucidate the mechanisms of dilation due to mitochondrial depolarization, kinase activation, ROS generation, and plasmalemmal calcium-activated potassium channel opening. C) Evaluate the inter-relationships of mitochondrial-derived factors produced in endothelium and VSM cells in mediating integrated dilator responses. D) Determine whether preconditioning, induced by prior activation of mitochondrial-derived mechanisms, alters subsequent cerebrovascular responses to mitochondrial-derived products. E) Explore the relationship between physiological stimuli and mitochondrial activation. Aim 2. Investigation of the effects of IR on mitochondrial-derived influences on cerebral arteries. We will: A) Examine whether IR attenuates dilator responses of cerebral arteries dependent upon mitochondria-derived signaling pathways. B) Determine the mechanisms by which IR reduces the responsiveness of cerebral arteries to mitochondrial-derived influences. C) Examine whether treatment of animals with statins restores normal responsiveness to mitochondrial-derived stimuli in IR. We expect that our results will lead to the improved treatment of patients suffering from cerebrovascular disease. PUBLIC HEALTH RELEVANCE: Chronic cerebral vascular insufficiency, which occurs in IR, leads to neurological diseases such as Alzheimer's disease and strokes. However, the potential role of mitochondrial dysfunction has not been studied. Current treatment regimens are not optimal and we expect that the results of our studies will lead to new and improved therapies to prevent or slow the onset of neurological diseases in an aging population.

Aging is a major contributor to cerebrovascular disease and subsequent neurological sequelae. The frequency and severity of these diseases, age of onset, and underlying mechanisms differ between men and women and are augmented by age-related diseases such as type 2 diabetes (T2D). The endothelium, a principle component of the microcirculation, is particularly sensitive to the negative effects of aging or T2D and mitochondria appear to play a pivotal role. Progress in elucidating the underlying mechanisms negatively affecting endothelial mitochondria and developing beneficial therapies has been obstructed due to the inability to follow mitochondrial characteristics in the brain microcirculation in real time during the development of aging and age-associated diseases. To address this deficiency, we will use three new approaches. First, we will use a mouse model which we developed with genetic labeling of mitochondria only in endothelium with Dendra2 fluorescent protein (mitoDendr2 FP). Second, we will use a novel, high throughput method we developed that allows the determination of energy production by mitochondrial respiration and glycolysis in freshly harvested brain microvessel from the mouse. Glycolysis is a major energy producing process in the endothelium and the relative importance of oxidative phosphorylation (OXPHOS) and glycolysis changes with aging and T2D is unclear. We also will examine whether mitochondrial fuels change. Third, we will use RNA Sequencing and Proteomics to explore underlying mechanisms involving energy producing pathways. Our preliminary and published data have led to the overall hypothesis that mitochondria play key roles in adverse changes in the cerebral microcirculation during aging and T2D and that therapies targeting mitochondria are protective. Thus, we speculate that mitochondria in microvessels are adversely affected more and earlier than large arteries during aging, T2D acerbates changes in mitochondria in microvessels, glycolysis becomes a more important source of ATP during aging and T2D, alternative fuel sources for OXPHOS become important during aging and T2D, mitochondria are more resilient in female microvessels, and mitochondria represent a useful therapeutic target to protect microvessels. We have 2 aims. Aim 1: Elucidate mechanisms of mitochondrial and vascular changes during aging. We will: a) determine mitochondrial and vascular characteristics in vivo in male and female aged mice, b) determine effects of aging on glycolysis and OXPHOS, mitochondrial fuel, and mitoROS, c) elucidate mechanisms affecting mitochondrial and glycolytic dynamics, and d) explore treatment modalities. Aim 2: Determine mechanisms of mitochondrial and vasculature changes during aging and T2D. We will: a) determine mitochondrial and vascular characteristics in aged male and female mice during T2D, b) determine effects of aging on glycolysis and OXPHOS, mitochondrial fuel, and mitoROS, c) elucidate mechanisms involved in changes in mitochondrial and vascular dynamics, and d) explore therapeutic approaches to improve mitochondrial and vascular function.

Adverse changes in small cerebral blood vessels due to type 2 diabetes (T2D) lead to cognitive impairment, memory deficits and dementias, and potentiate brain injury due to cerebrovascular accidents. The mechanisms are not fully known but detrimental changes in mitochondrial in endothelium appear to play a pivotal, initiating role. We have generated pilot data and developed new models to study the cerebral microcirculation during T2D and strokes. Our studies are conceptually innovative based on discoveries by our laboratory: (1) major sex-differences in mitochondrial abundance under normal conditions, (2) preferential effects on mitochondria in microvessels compared with arteries in T2D, (3) differential expression of mitodestructive and mitoprotective proteins in male and female blood vessels, (4) sex-dependent responses of mitochondria in the cerebral vasculature following strokes, and (5) major changes in vascular mitochondrial characteristics at sites distant from brain injury. Our studies are technically innovative based on new approaches to study the cerebral microcirculation of the mouse. First, we have developed a mouse model that genetically labels mitochondria only in endothelium with Dendra2 green/red photoswitchable fluorescent protein. Mitochondrial density, locations in endothelium, vascular diameters, and numbers (Rhodamine red) in the cerebral microcirculation can be simultaneously measured, at the same sites in multiple brain areas, for up to 12 months with multiphoton microscopy in mice anesthetized for each determination. Second, we have developed a high throughput method, which allows for the first time the determination of mitochondrial respiration in freshly harvested brain microvessel preparations from the mouse. We will extend this method to compare ATP production in the same sample by OXPHOS and glycolysis or the use of alternative fuels by mitochondria. Third, we will use RNAseq and Proteomics to elucidate mechanisms underlying changes observed during aging and T2D. These approaches are providing novel information on signaling pathways. We also will examine effectiveness of mitochondria-directed therapies in limiting damage and/or improving recovery to the microcirculation in T2D and strokes. Our overall hypothesis is that mitochondria in endothelium represent novel targets for sex-specific and disease-specific therapies. We have 2 aims. Aim 1: Characterize mitochondrial dynamics and vascular architecture of male and female mice under baseline conditions and during the development of T2D. We will: a) determine mitochondrial and vascular characteristics using in vivo multiphoton imaging in mice on a low or high fat diet, b) investigate mitochondrial and vascular changes in harvested microvessels during progression of T2D, c) elucidate mechanisms affecting mitochondrial and vascular dynamics during T2D, and d) explore treatment modalities. Aim 2: Investigate mitochondrial dynamics and vasculature architecture of male and female diabetic mice following transient ischemia. We will: a) determine mitochondrial and vascular changes using in vivo and ex vivo approaches in diabetic mice following transient middle cerebral artery occlusion (tMCAO)ischemic stress, b) elucidate mechanisms involved in changes in mitochondrial and vascular dynamics, and c) explore therapeutic approaches to improve mitochondrial and vascular function after ischemia in T2D mice.