Stephanie Watts - US grants

Regulation of vascular contraction and, thus, vascular resistance is generally associated with the activation of receptors coupled to guanine nucleotide associated proteins (G-proteins). Tyrosine kinase(s), enzymes critically important to vascular smooth muscle cell growth, may participate in the dynamic process of controlling vascular tone; a particularly important tyrosine kinase-dependent pathway is the mitogen activated protein kinase or MAPK pathway. The unifying hypothesis of this project is that G-protein coupled agonists, specifically 5-HT (serotonin, 5-hydroxytryptamine), stimulate vascular tyrosine kinase(s) to produce contraction. This is a novel and undescribed signal transduction pathway for the G-protein coupled agonist 5-HT; importantly, contractility to 5-HT and other agonists is dramatically increased in diseased states with documented vascular growth and remodeling (hypertension, atherosclerosis, restenosis) and contractile response to 5-HT is an excellent marker for vascular disease. Thus, tyrosine kinase(s), as activated by 5-HT, may play a significant role in the altered function of the vasculature in disease. Two specific experimental hypotheses which integrate studies from protein to the whole animal will be addressed: 1) 5-HT activates tyrosine kinase(s), in particular the tyrosine kinase MAPK kinase (MEK), to result in direct vascular smooth muscle contraction and potentiation of vasoconstrictor- induced contraction, 2) 5-HT increases tyrosine kinase activity, as measured by protein tyrosyl-phosphorylation and MEK activity, in vascular smooth muscle cells and arteries. Four experimental approaches will be employed: 1) isolated tissue bath technique to measure vascular contractility; 2) myograph technique to measure resistance vessel contractility; 3) Western analyses of vascular smooth muscle cell lysates and arteries to determine 5-HT-induced changes in protein tyrosyl-phosphorylation, especially that of the substrates of MEK (the MAPKs); 4) MEK activity assays. Based on preliminary results, it is predicted that tyrosine kinases, including MEK, mediate a portion of 5- HT-induced contraction, both direct contraction and that which potentiates responses to other cardiovascular hormones. The results of these experiments will reveal novel mechanisms for 5-HT signal transduction in vascular smooth muscle contraction. The pathways established will set the basis for a novel manner of normal cellular activation by 5-HT and most likely other G-protein coupled agonists, and can thus be an initial step in understanding how signal transduction for 5-HT and other agonists may change in diseased states associated with vascular smooth muscle growth or remodeling.

[unreadable] DESCRIPTION (provided by applicant): Modification of 5-hydroxytryptamine (5-HT, serotonin) concentration or receptor stimulation has resulted in treatments for depression, feeding disorders and obesity. 5-HT is also a vascular smooth muscle cell mitogen and vasoconstrictor but a significant amount of work has produced equivocal results in terms of 5-HT modifying arterial tone in vivo in the control of systemic blood pressure. Importantly, the relevance of 5-HT to peripheral arterial function has been questioned because mechanisms for arterial handing and metabolism of 5-HT are unknown. Our overall hypothesis is that the serotonin transporter (SERT) is present in peripheral arteries and has physiological consequence in arterial function. We will use an integrated set of techniques - isolated tissue baths and myographs, immunohistochemistry, Westerns, cell culture, transporter activity assays, and telemetry -- in rats and mice to address two specific Aims: Specific Aim #1: To test the hypothesis that 5-HT is taken up and released from arterial smooth muscle by SERT. We hypothesize that 5-HT is actively taken up, released and potentially stored in peripheral arteries, functions dependent on SERT. Such findings build a case for a local serotonergic system in the artery, a system which should be regulated and modifiable if physiologically important. Thus, work in this aim is directed towards understanding functional regulation of SERT and testing for the presence of components that would form a local 5-HT system: synthesis, uptake, storage and release. Specific Aim #2: To test the hypothesis that SERT modifies arterial function with physiological consequence. This aim builds on Aim 1 by studying two models in which SERT function has physiological relevance and thus enables illustration of the importance of 5-HT/SERT as it relates to modifying arterial contractility and blood pressure. The first model is the artery in which subthreshold concentrations of 5-HT potentiate arterial contraction to norepinephrine and endothelin, the second a model of hypertension. Collectively, these studies are important because they highlight the role of 5-HT in the peripheral cardiovascular system, and reveal a new functional role for SERT. Moreover, because SERT is a target for the commonly prescribed serotonin reuptake inhibitors such as ProzacZ, these studies provide potential insight into cardiovascular complications experienced by those taking SERT inhibitors. [unreadable] [unreadable] [unreadable]

Summary The primary purpose of the Biochemical and Molecular Biology Core (Core C) is to centralize support for techniques that are common to a majority of the individual projects with this Program Project Grant application. This core is thus an important resource for the investigators of this application, the individuals in their laboratories and is an essential part of this Program Project. This core will provide project investigators with technical support, teaching, upkeep of particular key pieces of equipment and centralize supply sources for commonly-used molecular and biochemical techniques. We have also developed a repository of methods/protocols kept electronically through the PPG website, protocols that are available to all participants in the program. We will continue to develop additional techniques for measurement of reactive oxygen species (ROS), use of HPLC, etc.

DESCRIPTION (provided by applicant): Humans have elevations in circulating free serotonin (5-hydroxytryptamine, 5-HT) in several important clinical, chronic situations. These include anaphylactic shock, cardiopulmonary bypass, carcinoid cancer and hemodialysis, all of which presents with a fall in blood pressure. Additionally, millions of individuals use medications that increase plasma 5-HT concentration, and changes in blood pressure are a side effect of these medications. Finally, the committed substrate for 5-HT synthesis, 5-hydroxytryptophan (5-HTP), is taken as an aide for sleep, mood disorders, menopause and many other conditions and causes a fall in blood pressure. We were the first to demonstrate that, in the rat, a long-term administration of 5-HT (1 week) directly reduced the blood pressure of the conscious, healthy rat through reduction of total peripheral resistance (TPR). The 5- HT-induced chronic fall in blood pressure is dependent on the activity of nitric oxide synthase (NOS) because the NOS inhibitor N-nitro-L-arginine (LNNA) abolished 5-HT-induced chronic fall in blood pressure in multiple situations. At no time has our understanding of the mechanisms of 5-HT been more important, and ours is the first to address this clinically relevant issue. The overall goal of thi project and long-term goal of our laboratory is to identify the mechanism(s) by which 5-HT elicits a fall in blood pressure in normotensive animals. Our central hypothesis is that 5-HT reduces TPR through 1) direct vascular; and 2) indirect vascular effects through removal of sympathetic tone mediated either centrally or peripherally. Preliminary experiments support the ability of 5-HT to increase flow in cutaneous and splanchnic circulations so we will focus on these two beds. An integrative approach and team with significant expertise will be used to address three specific aims. Sprague-Dawley rats will be the primary model, but we will also use the novel serotonin transporter (SERT) knockout rat. We employ a powerful technique for repeated measures of blood pressure using combined radiotelemetry and the programmable iPrecio(R) pump for drug delivery in conscious rats, as well as neural measures in anesthetized rats. Aim 1 directly addresses the controversial issue of whether 5-HT enters the central nervous system (CNS). Aim 2 will dissect whether 5-HT causes direct vascular relaxation and/or reduces sympathetic nerve activity to decrease blood pressure. Aims 1 and 2 will determine whether and where the effects of 5-HT are NOS-dependent. Aim 3 closes this proposal powerfully by testing whether activation of the 5-HT receptor implicated in Aims 1 and 2 causes a NOS- and 5-HT- receptor dependent fall in blood pressure, and whether 5-HTP-infusion reduces blood pressure chronically in a NOS- and 5-HT-receptor dependent manner. The impact and promise of this work lies in 1) addressing controversies head-on (5-HT enter the CNS? 5-HT in vitro vs 5-HT in vivo?) and 2) in discovering mechanisms of 5-HT action that may be beneficial to human regulation of blood pressure, given that chronic 5-HT nearly normalized elevated blood pressure of the conscious mineralocorticoid and spontaneously hypertensive rat. PUBLIC HEALTH RELEVANCE: A substantial number of medications (antidepressants, for example) and the over the counter supplement 5- hydroxytryptophan are taken with the intent to increase extracellular concentration of the hormone 5- hydroxytryptamine (5-HT) and improve mood, sleep and satiety. Elevations in circulating 5-HT also occur in anaphylactic shock, cardiopulmonary bypass, carcinoid cancer and hemodialysis and a decrease in blood pressure is associated with all these events. Our work investigates the mechanisms of 5-HT-induced fall in blood pressure and whether 5-HT-like compounds might be useful in controlling blood pressure.

DESCRIPTION (provided by applicant): Perivascular adipose tissue (PVAT) is fat perfectly situated to influence vascular tone. In this proposal, we provide substantial evidence for the expression and novel contractile function of a peptide that is typically discussed as being produced within visceral white adipose tissue (WAT) and the liver, but never associated with PVAT. Chemerin (tazarotene induced gene, TIG2; RARRES2) is a biomarker for adiposity. Circulating chemerin levels associate strongly with BMI, and chemerin levels are reduced with reduction of weight and fat. Importantly, disruption of the primary receptor for chemerin, ChemR23 (G protein coupled receptor), is associated with reduced adiposity and body mass in mice; the role of chemerin in blood pressure is not known. Chemerin is best known for activation of inflammatory cells, and regulation of adipocyte differentiation and production of pro-inflammatory cytokines (IL-1beta, TNF-alpha, IL-6) in the adipocyte. We have discovered the production and expression of chemerin in PVAT, the ability of chemerin to stimulate blood vessel contraction in a ChemR23-dependent manner, and that suppression of chemerin gene expression reduces blood pressure, novel and here-to undescribed actions of chemerin. Importantly, chemerin- induced contraction is significantly amplified with loss of the endothelial cell, inhibition of nitric oxide synthase or by prior contraction to an agonist. In other words, chemerin also has important cardiovascular effects in non-obese conditions. Our overall hypothesis is that chemerin is a functional connector of fat (including PVAT) and blood pressure and thus unites obesity and hypertension, so commonly comorbid. Our primary model is the rat, in which we have significant versatility in models of hypertension (deoxycorticosterone acetate, NOS-inhibited) and obesity (high fat fed). We focus on the mesenteric vasculature, because the splanchnic circulation controls a considerable portion of cardiac output and is the site at which significant fat is deposited in obesity. We will also use human mesenteric arteries to test whether the vascular chemerin axis exists and is relevant to human health/disease. A range of experimental techniques (gene, tissue and whole animal) allows us to study two Aims. In Aim 1, we test the hypothesis that Chemerin induces ChemR23 receptor-dependent contraction and is amplified by dysfunctional endothelium. This aim is dedicated to understanding the vascular mechanism(s) of chemerin-induced contraction in arteries, as well as contributions of chemerin to agonist-induced contraction. This is paired with a second aim, dedicated to testing the physiological relevance of the chemerin axis. In Aim 2, we test whether antagonism of the ChemR23 receptor, or knockdown of chemerin gene by new antisense oligodeoxynucleotides, will reduce endpoints of the obese or hypertensive phenotype, including elevated blood pressure. Such a finding would argue that endogenous chemerin plays a role in vascular tone and blood pressure. Our findings place chemerin as a critical regulator of arterial tone, poised to be a bridge between obesity and hypertension.

DESCRIPTION (provided by applicant): As a member of the Association of American Universities and the Association of Land-grant and Public Universities, graduate education and training are missions of Michigan State University (MSU). Our goal for all trainees in biomedical science is that they are not only highly skilled in their discipline, but flexible and well prepare to thrive in face an ever-changing biomedical workplace. The NIH BEST mechanism affords a valuable opportunity to formalize and improve our professional graduate/postdoctoral training programs, with benefits for all parties involved: graduate students, postdoctoral fellows, faculty, the university and science. It is imperative that those who that are trained are better prepared for the opportunities that are real in today's world. MSU BEST will integrate key components that are new to MSU [formal IDP, professional externships, Spheres of Success (SoS) workshops, team building, communication] and leverage existing resources and programs that support training in transferrable skills (Career Success; Wellness) and parallel mentoring]. The highly engaged BEST scholar will participate in these key components. We propose a comparison of outcomes between highly engaged BEST Scholars (test group) and trainees not engaged in BEST (control group) to determine if our input has causes discernable changes. We propose use of strong interventions such that outcomes are measurable and comparable, and our measures include both summative and formative outcomes. Our ultimate goal is that the practices employed by the MSU BEST program become accepted and integrated into all biomedical training programs such that training for and consideration of non-academic careers becomes natural, seamless and accepted.

Project Summary ? Core C The primary purpose of the Biochemical and Molecular Biology Core (Core C) is to centralize?both figuratively and physically- support for techniques that are common to a majority of the individual projects with this Program Project Grant application. This core is thus an important resource for the investigators of this application, the individuals in their laboratories and is an essential part of this Program Project. This core will provide project investigators with technical support, teaching, upkeep of particular key pieces of equipment and centralize supply sources for commonly-used molecular, biochemical and HPLC techniques. We have also developed a repository of methods/protocols kept electronically through the PPG website, protocols that are available to all participants in the program. Finally, we continue to develop new methods that benefit our investigators. There are two (2) distinct scientific foci of this core. Center I: The first center is support for basic molecular analyses, including real-time PCR, Western analyses, immunoprecipitation and immunohisto- and cytochemistry. A specific focus of this Center is fractionation of fat into adipose cells and stromal vascular fractions (SVF). This first core center will be responsible for providing technical help for performing all of these assays, support for use of central pieces of equipment and general supplies. Core D connects directly to Core C in that it provides the microscopic expertise for evaluation of the data produced within Core C. Center II: The second center is support for biochemical-based analyses using high pressure liquid chromatography (HPLC) and biochemical measurement of ROS. HPLC is a real strength of this core, enabling investigators to measure a variety of substances important to the PPG (e.g. NE and metabolites).

Project Summary/Abstract Perivascular adipose tissue (PVAT, the fat immediately adjacent to blood vessels) is a fat depot just beginning to be appreciated for the contributions it makes to vascular function and human health, normally viewed as being anticontractile and vasculoprotective. PVAT in the splanchnic vessels is the visceral fat that is particularly important; visceral fat is associated with the greatest risk of cardiovascular disease. We have made the novel discovery that PVAT possesses an adrenergic system, defined here as the ability to synthesize, release, take up and metabolize catecholamines. This PVAT may be directly innervated by the sympathetic nervous system (SNS), adding an unappreciated level of vascular control exerted by the SNS. The novel observation that the sympathomimetic tyramine causes PVAT-dependent contraction supports that this adrenergic system is functional. These experiments add a new dimension to the physiological importance of PVAT, which naturally lends itself to the study of obesity in which PVAT burdens are increased and the SNS is activated. We connect with every project of this PPG to investigate the overall hypothesis that mesenteric PVAT/visceral fat contains a functional, active adrenergic system. We will use two important models. First, the rodent model of high fat (HF) fed obesity is an ideal choice of obesity models because of the way that it reflects human obesity and the role of the SNS as a driver of the elevated blood pressure observed in most forms of obesity. From the HF model, we will use the superior mesenteric artery, superior mesenteric vein and small mesenteric arteries and veins (+/- PVAT) given that both circulations contribute to blood pressure determination and splanchnic function. Second, we use of PVAT of the human mesenteric vasculature (lean and obese). We use a suite of integrated techniques ? live cell imaging of NE transporter function, PVAT biochemistry, real time PCR, whole animal physiology, isometric contraction and pharmacology ? to examine three aims. Aim 1 tests the hypothesis that PVAT possesses the ability to synthesize, store, take up, functionally release and metabolize NE, and is innervated by the SNS (adrenergic systems); identifying mechanistic process is the goal. Aim 2 is devised to identify whether NE turnover is elevated in the PVAT of HF vs control such that PVAT NE content is reduced while circulating NE levels are elevated. Aim 3 tests the physiological contributions of splanchnic PVAT adrenergic systems. Collectively, this work provides powerful support for a novel mechanism by which PVAT modifies vascular tone and ultimately blood pressure: through a local and neuronal adrenergic system.

Project Summary Despite intensive investigation, disorders of blood pressure regulation -- both hypertension and hypotension -- continue to be serious, widespread and intractable medical problems. Relatively few effective approaches to management of blood pressure dysregulation have emerged over the last few decades so novel strategies are needed. Serotonin (5-hydroxytryptamine, 5-HT) was first discovered in the cardiovascular system, but the roles of endogenous 5-HT in cardiovascular physiology and pathophysiology are not well defined. This is because 5-HT has complex effects within the CV system that are mediated by several distinct receptor subtypes. This is well-illustrated when considering the endpoint of blood pressure. Depending on the site of injection, the dose administered, and the receptor targeted, serotonergic drugs can raise or elevate blood pressure. Of the 5-HT receptors expressed in the CV system (the 5-HT 1B/1D, 5-HT2A, 5-HT3, 5-HT4 and 5- HT7 receptor), 5-HT has the highest affinity for the 5-HT7 receptor. Since endogenous plasma and tissue levels of 5-HT are estimated to be in the low nM range, endogenous 5-HT likely acts primarily at 5-HT7 receptors to effect changes in blood pressure. Previous studies support this conjecture regarding the acute (seconds to minutes) effects of exogenous 5-HT. Over the last decade, we have proven this is also true for the chronic (days to weeks) blood pressure effects of exogenous 5-HT, using both traditional pharmacological approaches and a new genetic model (5-HT7 receptor knockout rat) that we developed. Therefore, we now are well-positioned to test the overall hypothesis that endogenous 5-HT modifies blood pressure in normal and pathophysiological conditions primarily by activation of 5-HT7 receptors in the peripheral vasculature, with the ultimate goal of capitalizing on 5-HT7 receptor pharmacology to treat disorders of blood pressure regulation. We propose to test this hypothesis via three Specific Aims: Aim 1: To test the hypothesis that 5-HT7 receptor activation dilates the microcirculation; Aim 2: To test the hypothesis that the vascular 5-HT7 receptor is (constitutively) activated endogenously to lower blood pressure in normal rats; and Aim 3: To test that the hypotension created in rat models with increased release or formation of 5-HT is 5-HT7 receptor-dependent. An integrated series of techniques that are the strengths of our combined laboratories are proposed. These include isolated tissue bath measurement of isometric contraction, measures of receptor activation that include the concept of constitutive activity, and radiotelemetry to measure cardiovascular parameters in conscious rats. Our established expertise is combined with newly gained abilities to image splanchnic venous and arterial diameters in the whole rat. This project is aimed to discover the unique ability of 5-HT to selectively activate the venous (and possibly micro) circulation through the 5-HT7 receptor and reduce blood pressure, a promising therapeutic lead for numerous cardiovascular disorders.

Project Summary/Abstract Population-specific differences in the presence of disease, health outcomes, or access to healthcare are termed ?health disparities? and they are a significant concern in our society. Lack of representation of the underserved groups?which includes minorities, financially disadvantaged, and disabled individuals?in the biomedical research and healthcare workforce has been identified as a critical factor underlying health disparities. Indeed, disparities in the demographics of individuals entering careers in biomedical research are well documented. Biomedical knowledge and healthcare will benefit from broader inclusion of underrepresented individuals engaged in biomedical research as inclusive research teams have broader perspectives in setting research agendas, can more effectively recruit diverse subjects into clinical research studies, and work more effectively to deliver healthcare and reduce health disparities. Experiential learning by active engagement in research is an effective way to nurture the next generation of scientists and health care researchers. Veterinarians conduct research important to human health through comparative studies and animal models; unfortunately the racial and ethnic diversity in the veterinary profession is no better than that of the biomedical research workforce in general. Thus, this proposal is designed to recruit and comprehensively immerse both undergraduate and veterinary medical students from underrepresented groups in summer research experiences on the campus of Michigan State University. These research experiences will be focused on three major areas: hypertension, airway disease and blood cells and components in tissue pathology. The objective of our education research program is to increase the number of students from underrepresented groups who pursue biomedical research-related educational goals and career paths relevant to the mission areas of the NHLBI. The strategy to meet this objective involves 1) providing student participants an immersive research experience that exposes them to the various aspects of a research career, 2) cultivating participants' science identity by facilitating the development of mentor and peer networks, and creating a community that supports the participants' research-related interests, and 3) fostering the participants' successes so as to give them the confidence needed to pursue a research-related career. This strategy will be implemented through the following specific aims: (1) engage undergraduate and veterinary medical students in high-quality mentored research in the areas of heart, lung, and blood diseases; (2) provide comprehensive exposure to the various aspects of biomedical research through an organized program of activities; (3) bolster the trainees' understanding of the critical concepts of responsible conduct of research; (4) recruit trainees from populations underrepresented in the biomedical sciences; and (5) conduct comprehensive evaluations to determine the effectiveness of our programming in enhancing diversity in health-related biomedical research.