Meredith Hay - US grants

Information concerning the level of blood pressure originates from both arterial and cardiac baroreceptors. Activation of these receptors results in the generation of action potentials which ultimately results in the central release of baroreceptor neurotransmitters and the activation of central neurons involved in cardiovascular reflex control. In a number of different animal models, the reflex influences of either the arterial or cardiac baroreceptors is significantly influenced by activation the other. Few studies, however, have focused on the cellular mechanisms involved in these integrated responses. One hypothesized mechanism for baroreceptor afferent integration involves autoreceptive neurotransmission. Neurotransmitters, known to be released by baroreceptor neurons (i.e. glutamate) may feedback to modulate their own release. To gain insight into the possible baroreceptor neurotransmitter receptor systems which may be involved in baroreceptor integration, this project will examine the role of the 1-glutamate metabotropic and 1-AP4 receptors in the regulation of baroreceptor neuronal activity. The proposed studies will use primary cell culture techniques in conjunction with patch-clamp methodology and fluorescent histochemistry to examine the specific ion channels involved in metabotropic receptOr modulation of baroreceptor neurons. This model takes advantage of the fact that ion channels and metabotropic receptors present in the soma of sensory afferents are functionally similar to those present on the central terminals. Thus, the study of the metabotropic receptor modulation of baroreceptor somatic ion channels will closely reflect the nature of this receptor system at the central terminals. The specific aims of this project are: 1) To determine and characterize the voltage-gated and calcium. activated ionic currents present in cardiac baroreceptor neurons; 2) To identify the role of glutamate metabotropic receptors in the regulation of both arterial and cardiac baroreceptor voltage-gated and calcium-dependent ion currents; and 3) To determine the effects of l-AP4 receptor activation on voltage-gated and calcium- activated currents in arterial and cardiac baroreceptor neurons. These studies are expected to yield specific information concerning neurotransmitter regulation of baroreceptor ion channels and provide a mechanistic theory as to how peripheral baroreceptor afferents may modulate their own activity.

DESCRIPTION (Adapted from the applicant's abstract) The candidate's long-range career goals are to continue to strive to achieve both academic and research excellence within the field of neural control of the circulation with the focus of her research centered on the understanding of the central mechanisms involved in the regulation of cardiovascular function. The candidate strongly believes that the realization of her long-range goals will require the use of multiple experimental tools to integrate answers and observations made at the cellular and molecular levels back to the regulation of blood pressure in the intact animal. More specifically, her short range goals, interests and, hopefully, contributions will continue to focus on the integration of primary baroreceptor afferent information at the level of the nucleus of the solitary tract, and how this integration serves to regulate sympathetic outflow and ultimately arterial blood pressure. The proposed research career award will be instrumental in achieving these goals by offering her the opportunity to focus the extensive time and energy required to not only answer the specific questions of metabotropic glutamate receptor regulation of baroreceptor afferents outlined in the present proposal, but to begin to, hopefully in the future, develop hypotheses concerning the molecular mechanisms governing the expression of these receptors and how this expression may be involved in some forms of hypertension. The University of Missouri and the Dalton Cardiovascular Res. Cntr. fully supports this application and provides a rich physical and intellectual environment for the continued development of the candidate's research career. The project will examine the role of the 1-glutamate metabotropic and I-AP4 receptors in the regulation of baroreceptor neuronal activity. One hypothesized mechanism for baroreceptor afferent integration involves autoreceptive neurotransmission. Neurotransmitters known to be released by baroreceptor neurons (i.e. glutamate) may feedback to modulate their own release. The specific aims of the project are: 1) To characterize the voltage-gated and calcium-activated ionic currents present in cardiac baroreceptor neurons; 2) To identify the role of glutamate metabotropic receptors in the regulation of both arterial and cardiac baroreceptor voltage-gated Ca++ currents; and 3) To identify the role of glutamate metabotropic receptors in the regulation of both arterial and cardiac baroreceptor voltage-gated K+ currents. These studies are expected to yield specific information concerning neurotransmitter regulation of baroreceptor ion channels and provide a mechanistic theory as to how peripheral baroreceptor afferents may modulate their own activity.

[unreadable] DESCRIPTION (provided by applicant): There is both functional and anatomical evidence for the presence of several VGCC subtypes (N, P/Q, L, R) in the cell bodies of aortic baroreceptor afferents, with the N-type being the predominant current. It has been in general inferred that VGCC present at the terminals in approximately similar proportions to the soma and calcium currents of the cell bodies reflect calcium currents at the synapse. However, recent preliminary findings from our laboratory suggest that different population of the VGCCs may play specific roles during different levels and stages of synaptic activation. Using currently available fluorescent imaging techniques, the proposed studies will test the general hypothesis that extended, high frequency baroreceptor activation shifts the primary inward calcium channel flux responsible for exocytosis from the N-type calcium channel to other available calcium channels within the baroreceptor terminal. Further, we will test the hypothesis that different subsets of calcium channels are responsible for baroreceptor exocytosis from the different synaptic vesicle pools found in synaptic terminals. We will use in vitro imaging of synaptic vesicle turnover and intraterminal calcium and imaging of intraterminal calcium in brainstem slices to address are 4 major and distinct aims: 1) To evaluate the contribution of different VGCC subtypes to stimulation evoked vesicle exocytosis at different frequencies. 2) To determine the contribution of different VGCC subtypes to stimulation induced increases in intraterminal calcium. 3) To determine the role of different VGCC in vesicle pool mobilization. 4) To evaluate the effects of sustained hypertension on frequency induced increases in intraterminal calcium and the contribution of the different VGCC subtypes to these changes. Results from these studies are expected to yield novel information that will provide an important advances in our understanding of the cellular mechanisms regulating of baroreceptor neurotransmission. [unreadable] [unreadable]

It has been proposed that estrogen delays and or prevents the onset of hypertension and may function to keep women "cardiovascularly younger" than men of the same age. Similar observations have been made in experimental hypertensive animals, however, the underlying mechanisms of estrogen's protective effects are incompletely understood. Given the importance of estrogen replacement therapy in women's health, it is clear that an understanding of the cellular mechanisms underlying estrogen's cardiovascular protective effects is critical for continuing development of clinical therapies for the treatment of hypertension in women. Angiotensin II (Ang II) is an important factor in some forms of both clinical and experimental hypertension. Chronic intravenous infusions of low levels of Ang II in experimental animals result in an increase in blood pressure that involves an increased neurogenic contribution to the maintenance of blood pressure which is prevented by prior lesioning of the area postrema (3,6,33,57,95). It is thought that circulating Ang II acts on area postrema neurons to maintain the hypertension. The proposed studies will test the general hypothesis that estrogen protects against the Ang II induced hypertension by inhibiting the actions of Ang II on area postrema neurons. To test this hypothesis and to characterize the effects of estrogen (17beta-estradiol) on area postrema neurons, Ang II induced increases in blood pressure and baroreflex modulation, this proposal will utilize whole-cell patch clamp recordings from isolated area postrema neurons, in vivo single unit recordings of area postrema neurons and hemodynamic measurements in conscious animals to address the following 4 major and distinct aims. 1) To evaluate area postrema membrane properties following exposure to 17beta-estradiol. 2) To determine the effects of acute and chronic 17beta-estradiol on area postrema calcium handling. 3) To determine the effect of 17beta-estradiol on activation of area postrema neurons. 4) To evaluate the effects of acute and chronic 17beta-estradiol on Ang II hypertension. Determination of the effects of estrogen on CNS mechanisms underlying Ang II dependent hypertension will have a significant impact on our understanding of the cardiovascular benefits of estrogen replacement therapy.

0089018
Gillis
The objective of the proposed research is to replace the patch-clamp pipette with an aperture on a microchip and to replace the carbon fiber with electrochemical electrodes fabricated on microdevices. The specific objectives are to: (1) record the current through the cell membrane using microchip technology, (2) measure secretion from individual cells with high time resolution using electrochemical electrodes constructed on a microchip, and (3) automatically target individual cells to electrophysiological probes on the microchip. This award is co-funded by the Division of Engineering Education and Centers, the Division of Chemical and Transport Systems, and the Division of Bioengineering and Environmental Systems.

DESCRIPTION (provided by applicant): The conference "Neural Mechanisms in Cardiovascular Regulation" is a long-running FASEB Summer Research Conference previously held in 1982, 1984, 1987, 1990, 1994, 1996, 1999 and 2002. The previous conferences have been well attended and their value recognized by leading investigators in the field. The topic is widely recognized as an important area of research. The nervous system plays a major role in regulation of arterial pressure, modulation of cardiac and renal function, and occurrence of syncope, cardiac arrhythmias and sudden death in pathologic states. The field is of interest to basic scientists interested in fundamental neural mechanisms, integrative physiologists, and clinical investigators interested in mechanisms of cardiovascular dysregulation in disease. This proposal for the FASEB Summer Conference 2004 will focus on understanding the central mechanisms involved in the physiological and pathological responses of the cardiovascular system to challenges from the external and internal environment. As articulated in 1932 by Walter B. Cannon in "The Wisdom of the Body", the ability of systems to adapt to environmental challenges and maintain homeostasis is fundamental to sustaining life. The scientific sessions for the 2004 FASEB "Neural Mechanisms in Cardiovascular Regulation" Summer Conference are tied together under the subtitle Environmental Challenges and Cardiovascular Regulation in Health and Disease. Each session will cover a specific external or internal environmental challenges and the responses of the neural mechanisms that are engaged to regulate cardiovascular function. Speakers for each session will span each topic from the identification of the challenge to human health to the understanding of the molecular and cellular mechanisms involved. Approximately 160 conference attendees will be selected from interested applicants. The conference will include 4 plenary sessions consisting of 2-3 presentations each, extended discussion sessions, and poster presentations. The plenary sessions include: 1) Osmotic Challenge and Body Fluid Regulation; 2) Steroid Hormone Challenges and Central Regulation of Cardiovascular Function; 3) Respiratory Challenges and Neural Control of the Circulation; and 4) Exercise Training, Temperature Stress and Neural Control of the Circulation. The majority of invited speakers have already agreed to participate and participation of young investigators will be encouraged. Time will be allotted for informal discussions and social interaction. Scientists from the United States and other countries, young investigators, and women are well represented in the program.

PROJECT SUMMARY Every year, more than 500,000 patients in the US have coronary artery bypass surgery (CABG) to treat coronary artery disease. However, postoperative outcomes are complicated by a significant incidence of stroke and cognitive impairment. Postoperative cognitive impairment results in decreased quality of life for these individuals and higher hospital readmission rates. There is a clear unmet medical need to find treatments to attenuate or prevent cardiac disease and CABG induced cognitive impairment. Although the precise triggers are debated, CABG increases brain hypoxia and circulating cytokines. Increases in circulating inflammatory cytokines and brain hypoxia result in increased brain reactive oxygen species (ROS) production, activation of brain inflammatory pathways leading to neuronal dysfunction and cognitive impairment. Recent work by our group and others have shown in animals that Angiotensin-(1-7) (Ang-(1-7) can inhibit ROS production, increase nitric oxide production and reduce inflammatory cytokines in the brain, microvasculature and peripheral tissue via activation of the Mas receptor. The ideal therapeutic candidate to treat CABG induced cognitive impairment would be designed to interrupt this cascade by working at both sides of the blood-brain barrier, the brain vascular endothelium and neuronal cells. Ang-(1-7) meets these criteria because Ang-(1-7), acting at the Mas receptor, is known to have anti-inflammatory effects at both endothelial cells and neurons. In Q2 2014, we received regulatory support from the NHLBI SMARTT program to submit an IND to the FDA for the use of Ang-(1-7) to treat cognitive impairment in CABG patients and this IND was approved in August 2015. The present UO1 application is designed to evaluate the safety and efficacy of Ang-(1-7) to enhance cognitive function in participants undergoing CABG surgery. Further, by teaming with the unique capabilities of the NIH Clinical Center, these studies will measure, for the first time, post CABG surgery brain inflammation and microglia activation as measured by PET imaging of [11C]PBR28 and the test the hypothesis that Ang-(1- 7) will result in a decrease in brain inflammation and microglia activation in CABG patients. When completed, this clinical study will have advanced development of a new therapy with potential to treat cognitive impairment in CABG patients.

PROJECT SUMMARY The proposed supplement is directly related to the parent U01 and will provide the exploratory clinical evidence needed for a larger fully powered study to determine if blood Nfl values might serve as a prognostic biomarker and as a clinical trial enrichment tool in our post U01 planned clinical trials to treat and hopefully prevent the development of VCID in at-risk heart failure (HF) patients. In our U01 TPP, we planned our first patient population to include an at-risk VCID population that include Heart Failure Class II/IV patients with decreased ejection fraction and evidence of mild cognitive impairment. We believe that NfL may serve as a clinical trial enrichment tool in our planned Phase 1b/2a clinical trial to treat and hopefully prevent the development of VCID in ADRD at-risk HF patients. The timing of proposed study in this administrative supplement will position our team in an excellent position to deploy Nfl as a biomarker for enrollment enrichment for clinical trial eligibility criteria to identify which HF patients who are more likely to develop VCID/ ADRD. In the clinic, VCID diagnosis and disease monitoring is generally achieved by magnetic resonance imaging (MRI) scans and the presence of white matter hyperintensities (WMH) that are typically interpreted as a surrogate of cerebral small vessel disease (SVD). However, the assessment and diagnosis of VCID using MRI most often occurs after the development of significant clinically measured cognitive dysfunction. Neurofilament light protein (NfL), a neurofilament found in blood and cerebral spinal fluid, has been shown to increase following axonal damage and neurodegeneration and is tightly correlated with increased cognitive impairment and has been observed to be elevated in subjects with neurodegenerative diseases, hypoxic brain injury, and cardiac disease and related surgeries. It is unclear if NfL can be used to detect neuronal damage in individuals at risk for VCID such as those withs chronic inflammatory disease such as coronary heart disease or HF. Ideally, if we can predict the presence of neurological and cognitive complications in individuals with heart disease, we will be able to identify those at-risk VCID patients who would benefit from therapeutic treatment. Our proposed Context of Use is that NfL might serve as a Prognostic Biomarker in blood that can help predict clinical progression in early at-risk VCID with stage II/IV heart failure (HF). Our ultimate goal will be to use levels of Nfl in blood as an enrollment enrichment factor for our planned post U01 clinical trial to identify individuals with HF who are more likely to develop VCID or ADRD. Specific Aim: Establish a baseline and 12-month longitudinal Nfl values in HF patients at risk for VCID and determine the association between absolute levels of Nfl with measures cognitive function and MRI in subjects with stage I/II and III-IV HF patients.