Anthony J. Donato, Ph.D. - US grants

[unreadable] DESCRIPTION (provided by applicant): The candidate, Anthony J. Donate Ph.D., is a cardiovascular physiologist in the Department of Integrative Physiology at the University of Colorado at Boulder. Dr. Donato's research focuses on the mechanisms mediating vascular aging in humans and how habitual aerobic exercise can preserve and restore vascular health with aging. The aim of the research project is to establish the efficacy of a 12-week aerobic exercise intervention for restoring vascular endothelial function in middle-aged and older men and women by inducing reductions in oxidative stress and increases in nitric oxide bioavailability. The proposed research will use an "integrative" (system to gene) approach to identify the cellular and molecular mechanisms by which habitual exercise restores endothelial function in this group, including measurements of vascular endothelial expression of proteins influencing oxidative stress, inflammation and nitric oxide production. The expected results should provide clinically important information regarding the integrative physiological mechanisms by which habitual aerobic exercise improves vascular endothelial function in middle-aged and older adults. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Advancing age is associated with the development of arterial dysfunction characterized by endothelial dysfunction and large artery stiffness. One hypothesis is that the chronic reduction of caloric intake (Caloric Restriction; CR) can activate cellular and molecular events that prevent age-related arterial dysfunction. This proposal aims to determine if lifelong caloric restriction (40%) and/or a pharmacological caloric restriction mimetic can prevent the age-related arterial dysfunction and the cellular and molecular mechanisms by which this may occur. Specifically, we will examine the effect of lifelong CR on the regulation and modulation of nuclear transcription factors, by acetylation, involved in the regulation of arterial oxidative stress, inflammation and apoptosis in middle-aged (MA) and older (O) mice. The specific aims are (1) to measure endothelium dependent dilation (EDD), nitric oxide (NO) bioavailability and stiffness in large arteries of MA/O mice and to determine if advancing age is associated with a pro-oxidative, -inflammatory, -apoptotic phenotype, (2) to determine if CR attenuates the activation and acetylation of the pro- oxidative, -inflammatory and -apoptotic signaling molecules; nuclear factor kappa B (NFkB), p53, and forkhead foxO (FoxO3a) via an increase a nuclear deacetylase SIRT-1 in MA/O mice and (3) to determine if activation of the nuclear deacetylase SIRT-1 can prevent the aged arterial phenotype and dysfunction. To do so, we will study young (Y: 4-6 mo), MA (18-20 mo) and O (29-31 mo) male B6D2F1 mice. Endothelial function, nitric oxide bioavailability, and larger artery stiffness will be measured. Oxidative stress, inflammatory cytokines, markers of apoptosis and activation/acetylation of NFkB, p53 and FoxO3a will be assessed in aortic lysates and endothelial cells. Lastly, we will utilize pharmacological inhibition of the nuclear deacetylase SIRT-1 to determine its role in age and caloric restriction-associated arterial function, oxidative stress, inflammation and apoptosis. The expected results will provide novel insight into the cellular and molecular mechanisms by which CR and CR mimetics preserve age-associated arterial function.

DESCRIPTION (provided by applicant): Cardiovascular disease (CVD) is the leading cause of death in the United States. Aging is the major risk factor for development of CVD. The major age-related arterial phenotypes, which are thought to be responsible for the development of CVD in older adults, are reduced endothelial function and enhanced large artery stiffness. Recently, it has been demonstrated that inhibition of the mTOR signaling pathway, via rapamycin or genetic manipulation, extends lifespan and reduces age-related physiological dysfunction. The proposed studies aim to determine (a) if increased mTOR signaling is responsible for age-related arterial dysfunction and (b) whether dietary rapamycin treatment and activation of AMPK, a putative rapalog, can reverse age-related arterial dysfunction. Our laboratory has performed preliminary studies suggesting that arterial activation of mTOR is increased with advancing age and that this is concomitant with age-associated endothelial dysfunction and large artery stiffening known to result from increases in oxidative stress and inflammation. Furthermore, dietary treatment of old mice with mTOR inhibitor, rapamycin, can reverse this age-associated arterial phenotype and improve arterial function. In the present application, by utilizing dietary rapamycin treatment, we will directly assess the role that mTOR plays in modulating transcription factor activity and downstream gene/protein expression and the subsequent effects on arterial phenotype and function. In addition, we will determine if pharmacological activation of AMPK by AICAR will act as a rapalog, mimicking the effects of rapamycin-induced mTOR inhibition on arterial function and phenotype.

DESCRIPTION (provided by applicant): Candidate. The candidate, Anthony J. Donato Ph.D., is a cardiovascular physiologist in the Department of Internal Medicine at the University of Utah. Dr. Donato's research focuses on the mechanisms mediating arterial aging and the mechanisms by which caloric restriction can preserve and restore vascular health with aging. His long term goal is to direct an independent, extramurally-funded research laboratory that can integratively study vascular aging at the gene, cellular, tissue and systemic (whole-body) levels and to be recognized as an international leader in translational vascular function. The proposed K02 award will provide Dr. Donato the necessary protected career development time to achieve this goal. Career Development Plan. This award will support the further career development of Dr. Donato, allowing him to complete a well-rounded program in molecular genetics and the basic biology of aging while maintaining his training in Geriatrics. The career development plan contains several separate but coordinated efforts to enhance the expertise of the applicant including training in new experimental techniques related to endothelial activation and inducible transgenic mouse creation, didactic course work designed to facilitate better understanding of the application of molecular genetics for investigators with advanced degrees and continued attendance at regular aging and geriatric seminar series and other vascular and basic biology of aging meetings aimed to educate and encourage collaborative research between Dr. Donato and other investigators in the specialties of biology of aging and geriatric research. Research. The aim of the research project is twofold: first, determine the modulatory influence of SIRT-1 on endothelial activation with aging and the vasoprotection induced by long term caloric restriction; second, to determine if genetically augmenting endothelial NAMPT expression/activity can ameliorate age-related endothelial dysfunction. These novel and important studies will determine if, and how, NAMPT influences sirtuins and subsequent inflammation/oxidative stress- related vascular aging. The expected results will provide clinically important information regarding the integrative physiological mechanisms by which global endothelial dysfunction is expressed with aging and if NAMPT is a potential therapeutic target.

? DESCRIPTION (provided by applicant): Chronic diseases of aging account for a significant proportion of healthcare spending and result in a painful and limiting existence for many tens of millions of Americans and hundreds of millions of people around the world. Recursion Pharmaceuticals has developed a drug discovery platform that re-purposes known drugs for the treatment of such diseases. The platform consists of high content fluorescent image analysis using machine-learning algorithms to identify relevant and on-target changes induced in human cells in which diseases and treatments have been modeled. This system has been adapted to screen for drugs which may preferentially destroy senescent cells, as this may result in improved tissue function and decreases in pathology and mortality related to aging. In this SBIR fast-track proposal we will: PHASE I Identify a morphological profile of senescence in a variety of human cell types. Progression to Phase II will depend on our ability to demonstrate the power of our morphological approach to identify senescent cells independent of any known senescent markers. PHASE II Identify known drugs which preferentially destroy senescent cells, without affecting healthy cells, as identified by morphometric means. Evaluate the efficacy of identified senolytic agents in a variety of tissue function and health-span models in mice. *Evaluate the effect of identified senolytic agents in a murine model of age-associated cardiovascular disease. Recursion Pharmaceuticals has the experience, tools, and drive to execute this Phase I/Phase II Fast-track SBIR proposal, and to accelerate commercial development of any compounds we identify as a result.

? DESCRIPTION (provided by applicant): Advancing age is the primary risk factor for cardiovascular diseases, and arterial dysfunction is a critical contributor to this increased disease risk. The proposed studies will explore the novel hypothesis that age- associated arterial telomere dysfunction is an underlying mechanism for increased arterial inflammation and dysfunction with aging. We hypothesize that telomere dysfunction, characterized by telomere uncapping, triggers cell senescence via the p53/p21 pathway that results in increased inflammatory signaling and ultimately leading to augmented large artery stiffness and endothelial dysfunction. We will address these hypotheses by utilizing mouse and endothelial cell culture models of aging, as well as inducible systemic and endothelial specific models of telomere uncapping. Additionally, using a transgenic mouse model of greater systolic blood pressure and pulse pressure and in vitro arterial and tissue culture models of circumferential stress, we will determine if increases in circumferential wall stretch is a critical stimulus for te induction of telomere uncapping. These results will reveal a novel mechanism underlying age-associated arterial dysfunction and disease risk, as well as provide critical evidence for future studies to determine therapeutic targets to reduce chronic arterial inflammation. This is a clinically relevant and important goal given the prevalence of cardiovascular disease among older adults, the increasing age of our population and its associated morbidity, mortality, and health care burden.

Project Summary/Abstract Cardiovascular disease (CVD) is the leading cause of death in the United States and aging is an independent risk factor for CVD. With the expansion of the aging population, by 2030 >40% of the population is projected to have CVD. Advanced age is accompanied by blunted endothelium-dependent dilation (EDD), reductions in nitric oxide (NO) bioavailability and increased large artery stiffness, important contributors to CVD risk. Arterial inflammation plays an important role in these processes but the precise link is unclear. We will utilize a translational approach to determine whether T cells play a role in age-related chronic arterial inflammation and subsequent dysfunction. First, we hypothesize that with aging, pro-inflammatory T cells accumulate around arteries and exacerbate age-related arterial dysfunction. To test this, we will assess arterial function, immune cell infiltration and inflammatory subtypes in young and old mice with T cells intact, depleted or inhibited. In addition, we will employ adoptive transfer to determine whether age-related arterial dysfunction results from intrinsic age-related changes to T cells, increased T cell recruitment to the aged artery, or both. Second, we hypothesize that T cells from older human donors will home to the vasculature of humanized immuno-deficient mice and induce inflammation and subsequent arterial dysfunction. To test this hypothesis, we will adoptively transfer T cells from young and older healthy human donors to young and old NOD-scid/?cnull/A2 humanized mice and assess immune cell infiltration, inflammation, arterial function, and ROS. Third, we hypothesize that inhibition of T cell activation will improve arterial function in older adults. To test this hypothesis, we will assess vascular function and endothelial cell and T cell inflammatory phenotype in older humans before and after treatment with placebo or a T cell inhibitor, Abatacept. The results of these studies will provide insight into the etiology of age-related arterial dysfunction and identify previously unexplored targets for diagnostics and intervention with the significant goal of maintaining cardiovascular health in the elderly.

Project Summary/ Abstract Advanced age is a leading risk factor for cardiovascular diseases (CVD). As individuals age, they experience numerous vascular alterations which include increased arterial stiffness and reduced endothelial-dependent vasodilation. An underlying disturbance common in many CVDs is chronic inflammation; but its specific role has not been clearly elucidated. T cells play a central role in the immune response, and may be implicated in age-related arterial inflammation and the accompanying vascular dysfunction. We will employ a translational approach to examine the role of T cells in this process. We hypothesize that T cells from older human donors will home to the vasculature of humanized immuno-deficient mice and induce inflammation and subsequent arterial dysfunction. To test this, we will adoptively transfer T cells from young and older healthy human donors to young and old NOD-scid/?cnull/A2 humanized mice and assess immune cell infiltration, inflammation, arterial function, and ROS. This will allow us to assess the role of T cells per se, and discover their mechanistic function in the arterial dysfunction seen with age.