Yanbin Dong - US grants

[unreadable] DESCRIPTION (provided by applicant): We and others have shown that cardiovascular reactivity (CVR) to acute laboratory stress is a stable and heritable trait and predictive of future blood pressure (BP) levels and essential hypertension (EH). The sympathetic nervous system (SNS) has a major role in BP regulation and adrenergic receptor subtypes mediate BP responses to acute challenges. As such, our hypothesis is that individual differences in CVR are partly determined by variation in genes encoding for adrenergic receptors mediating the sympathoadrenal response to stress. There is increasing evidence that this cardiovascular response is regulated by multiple adrenoceptor subtypes with structural homology. To date, nine homologous adrenergic receptor subtypes have been described. Only one study has found a gene-gene interaction upon CV disease incorporating two adrenergic receptor subtypes. Genetic variants in all the nine adrenergic receptor subtypes have never been investigated in a single study. This R-21 application proposes to evaluate the effects of genetic variants in all of the nine adrenergic receptor subtype genes, alone or in combination, on CVR and other quantitative cardiovascular traits in a population of 1048 healthy youth. Subjects are Black and White twins that have already been comprehensively phenotyped as part of the Georgia Cardiovascular Twin Study (HL56622). Racial differences in adrenoceptor gene effects will receive special attention, because such differences may offer a partial explanation for the higher prevalence of EH in Blacks. Primary measures are systolic BP at rest and in response to two behavioral stressors. Secondary measures are diastolic BP, cardiac output and total peripheral resistance (TPR) at rest and in response to the stressors, left ventricular mass (LVM), endothelium dependent arterial dilation to reactive hyperemia (EDAD), arterial stiffness and 24-hour ambulatory BP. We propose to expand this data set through collection of buccal cell DNA from the parents of the twins, enabling us to perform (i) TDTs (transmission disequilibrium tests) and (ii) haplotype reconstruction and analyses. This candidate gene study in a large group of Black and White twins including TDT and haplotype analyses provides an innovative approach to help identify individuals at particular risk for the development of EH and improve options for primary prevention as well as individualized therapy of EH (pharmacogenetics). [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): Impaired stress-induced pressure natriuresis is approximately twice as prevalent in black as in white youth. Salt, stress and genetics are three of the factors hypothesized to account for the difference. The proposed project will extend previous investigations in this area that focused on hormonal responses by identifying genetic predispositions responsible for impaired stress-induced pressure natriuresis. The overall goal is to determine the role of genes involved in the sodium-handling cascade in the renal tubules on dynamic regulation of sodium homeostasis and blood pressure under stress in blacks. These include five genes encoding the epithelial sodium channel (alpha ENaC, beta ENaC and gamma ENaC) and its accessory regulatory proteins such as serum glucocorticoid-inducible kinase (SGK-1) and neural precursor cell-expressed developmentally down-regulated 4 (Nedd4-2). The concerted Nedd4-SGK-ENaC interaction plays an important role in ENaC activity and tubular sodium handling. Environmental stress may promote sodium retention via intrinsic ENaC activity. Therefore, the primary aims are to test the following hypotheses in black youth; individuals with unfavorable genotypes or haplotypes compared to those without will show 1) a reduced stress-induced increase in urinary sodium excretion; 2) delayed systolic blood pressure recovery following stress; and 3) greater plasma renin activity suppression (low-renin hypertension) during recovery following stress. The secondary aim is to explore gene-gene interactions among the five genes consisting of the ENaC pathway in relation to the three primary outcome variables. A total of 300 black youth will be studied which will include an equal number of boys and girls aged 15-19 yrs. All subjects will be tested with an extended stress protocol including a recovery period. Single nucleotide polymorphisms (SNPs) in the five genes will be systematically examined in these subjects using both direct (i.e., functional SNPs) and indirect (i.e., haplotype tagging SNPs) association approaches. Buccal cell DNA from the parents of these youth will be collected to facilitate (1) haplotype reconstruction and analyses and (2) transmission disequilibrium tests (TDTs). Classification and Regression Trees techniques will be used to explore possible gene-gene interactions. This proposed research will provide novel insight into the interactions between genetics, salt and environmental stress, and their contribution to the pathogenesis of essential hypertension. The project aims to investigate the influence of salt, stress and genes on the body's ability to release sodium. Understanding the connections between salt, stress and genetics will provide a fresh approach into evaluating risk factors for high blood pressure. [unreadable] [unreadable] [unreadable]

The Bioassay Core will provide biochemical assay and genotyping services for the individual projects within the Program Project. Biochemical assays that will be performed include analyses in plasma and urine for human and rat samples. The following assays will be performed: Plasma renin activity, aldosterone, angiotensin II, cortisol, corticosterone, ACTH, nitrite, nitrate, 8-isoprostane, TEARS, antioxidant capacity, hydrogen peroxide, norepinephrine, epinephrine, albumin, electrolytes, creatinine, total cholesterol, HDL, LDL, triglycerides, glucose, insulin, cystatin C, tetrahydrobiopterin, leptin, and transforming growth factor beta. Techniques will include radioimmunoassay, radioenzymatic assay, HPLC, ELISA, colorimetric, and ionselective electrodes. Genotyping of tagging single nucleotide polymorphisms of the genes of GCCR, CRHR1, p22phox, and EC-SOD will be performed for Project 2. Genotyping of the C-521T polymorphism of the angiotensin II receptor type 1 gene will be performed for Project 1. Genotyping services will be also performed for Project 3 and 4 using the tail snip DMA of the transgenic rats of the ETB receptor deficient rat and endothelial ET knockout mice. The extracted DMA will be genotyped to determine whether the rats are wildtype, heterozygous, or homozygous for the ETB receptor gene and whether the mice are wild-type, heterozygous, or homozygous for the ET gene. The core will also provide resources for development of additional assays as needed by individual investigators. The core will assure assay quality control through validation procedures.

DESCRIPTION (provided by applicant): Type 2 diabetes associated with obesity and chronic inflammation is a major public health problem, which causes morbidity and death due to vascular complications. Before people develop type 2 diabetes, they almost always have pre-diabetes blood glucose levels that are higher than normal, but not high enough to be diagnosed as diabetes. There are 79 million pre-diabetics in the US. Recent studies show that long-term damage to the heart and vascular system occurs during pre-diabetes. Mechanisms responsible for this pathology are not understood. While oxidative stress and inflammation are key factors, treatment with antioxidants/anti-inflammatory agents has little effect on disease progression. Previous studies have demonstrated that vascular endothelial dysfunction occurs in pre-diabetic patients prior to development of type 2 diabetes. Thus, there is great need to define the mechanisms and metabolic processes that cause vascular injury and develop new therapies for prevention or reversal. Our expert interdisciplinary team will examine key pathways and biomarkers leading to vascular complications of type 2 diabetes. Our working hypothesis is that increased arginase and indoleamine 2,3-dioxygenase (IDO) activity cause vascular endothelial dysfunction and inflammatory injury by a positive feedback cycle of inflammatory cytokine production and oxidative stress. We will examine potential molecular mechanisms that lead to arginase and IDO activation, oxidant, and cytokine production, chronic subclinical inflammation, vascular dysfunction and tissue damage. We will use rodent models and translational experimental approaches to assess function of vascular and bone marrow derived cells in human subjects who are insulin resistant and pre-type 2 diabetic, in mice with obesity/type 2 diabetes, and in cultured cells. Additionally, leukocytes, circulating bone marrow-derived cells and plasma levels of arginase and IDO (activity/expression, substrate and products) will be assessed in relation to production of oxidants and cytokines. Our objective is to generate evidence of novel biomarkers and targets for interrupting disease progression. PUBLIC HEALTH RELEVANCE: Understanding the molecular mechanisms responsible for development of vascular disease in type 2 diabetes is necessary for successful treatment and prevention.

? DESCRIPTION (provided by applicant): Aging poses the largest risk factor for cardiovascular disease (CVD). Supplementation of vitamin D and/or omega-3 has received attention as a potential anti-aging and cardio-protective strategy. Data from in vitro and animal models suggest that these nutrients are involved in the etiology of aging or behave like a biochemical Fountain of Youth to mediate healthful aging. However, enthusiasm has outpaced the scientific evidence in humans, which is currently based on observational studies and small randomized clinical trials (RCTs) often with small doses. Large clinical trials with a high dose, daily dosing regimen, adequate sample size and trial duration, and longitudinal assessment are urgently needed. The principal function of telomeres is to protect the genome against chromosomal aberrations. Leukocyte telomere length (LTL) is consistently shown to have a direct relationship with longevity, CVD and diabetes in epidemiologic studies, and is thus considered to be a biomarker of aging. What is more, LTL is relatively short in persons with chronic inflammation, which is considered to be part of CVD and diabetic risk. The on-going NIH funded VITamin D and OmegA-3 TriaL (VITAL) is a large, randomized, double-blind, placebo-controlled, 2 x 2 factorial trial of vitamin D3 (2,000 IU/day) and marine omega-3 fatty acid (?-3 FA, 1 g/day) supplements among a representative sample of US men aged ?50 years and women aged ?55 years, with an oversampling of blacks. A unique feature of VITAL is the establishment of a sub-cohort of 1,054 participants who are evaluated in person at the Harvard Clinical and Translational Science Centre (CTSC) in Boston, which allows for in-clinic plasma/buffy coat sample collection and in-depth CVD phenotyping at baseline and Year 2. We propose to examine the effects of dietary vitamin D and/or omega-3 supplementation on LTL attrition assessed longitudinally utilizing in-clinic buffy coat samples collected at baseline, Year 2, and Year 4. Thus, we will also investigate the effects of vitamin D and/or omega-3 supplementation on the longitudinal interrelationship of LTL trajectory, plasma inflammatory cytokines, and CVD risk factors in the VITAL trial. This proposal is in response to the information gaps/research needs identified by the most recent Institute of Medicine (IOM) Report on Dietary Reference Intakes. The IOM report identified the need for randomized clinical trials (RCTs) of vitamin D for prevention of CVD, and cellular biology of vitamin D (e.g. cellular aging) as areas of high priority. The American Heart Association and the IOM recommend that all adults eat fish regularly to reduce risk for CVD. This novel and cost-effective study is expected to provide the information necessary to answer the open question of whether dietary vitamin D and/or omega-3 supplementation are beneficial for LTL shortening. Studies of telomeres will also open new avenues for the basic understanding of vitamin D and/or omega-3 as well as their preventive and therapeutic effects of CVD.

PROJECT SUMMARY/ABSTRACT Previous studies including our own have demonstrated that levels of blood pressure (BP) track from childhood into adulthood. Early life stress such as low parental socioeconomic status (SES) and adverse childhood events (ACEs) is associated with hypertension in adulthood by hastening the trajectory of disease progression. In our well-recognized longitudinal Georgia Stress and Heart (GSH) study, we have recently found a significant association between the number of ACEs and BP trajectories from childhood to young adulthood. Early life stress can lead to a state of heightened systemic inflammation, which persists in adulthood. Stress-induced inflammation is one of the first responses of the immune system to danger. Damage associated molecular patterns (DAMPs) are released by stressed cells to signal danger, and circulating DAMPs are free to activate pattern recognition receptors, subsequently promoting and exacerbating inflammatory cascade in the periphery. However, critical knowledge gaps remain regarding whether these circulating danger molecules are involved in the psychopathological process. Studies have established a key role for low-grade systemic inflammation in the early stage of hypertension and cardiovascular disease (CVD). In spontaneously hypertensive rats, we have recently found that circulating DAMPs may lead to inflammatory activation and cause elevated arterial pressure and vascular dysfunction, and blocking DAMPs may attenuate BP increase. Thus, we propose translating our research in animal models to humans by testing our central hypothesis that is early life stress increases circulating DAMPs, and circulating DAMPs contribute to BP elevation. In this proposal, we will take advantage of our existing longitudinal GSH cohort in which 740 children (50% female and 47% African Americans) have been followed- up 17 times in 27 years (1989-2016). The longitudinal study provides multiple repeated measures of early life stress, BP and CVD risk factors as well as bio-sample collection from childhood to adulthood. DAMPs are endogenous signals that alert the body about danger, and stimulate an inflammatory response in response to environmental stressors. The objective of this cost-effective project undertaking longitudinal approaches using our unique GSH cohort is to establish the relationship of DAMPs with early life stress, and identify the contribution of DAMPs to the trajectories of BP elevation and preclinical CVD. By translating our findings in the animal models to humans, successful completion of our aims will provide both novel molecular mechanisms and biomarkers for early life stress and high BP. Deciphering new paradigms for stress and stress-related hypertension and CVD may inform new preventative strategies, and offer targeted stress management as well as new therapeutic interventions (e.g. HMGB1 blocking therapies).

PROJECT SUMMARY/ABSTRACT Cocoa products have become a widely consumed food, with growing demand across the world and steadily increasing interest in its potential health benefits and anti-aging biology. However, the role of cocoa consumption on epigenetic aging remains essentially unexplored. There has been compelling evidence for the anti- inflammatory role of cocoa and cocoa-derived flavanols based upon in vitro and animal models. However, a critical barrier of research progress remains the lack of definitive large-scale randomized controlled trials (RCTs). Accelerated epigenetic aging that comprises DNA methylation changes has recently emerged as a powerful, novel predictor for lifespan and health span, disease susceptibility, morbidity, and mortality risk. Further, inflammaging is a highly significant risk factor for both morbidity and mortality in the elderly, as many age-related diseases including cardiovascular disease (CVD) share an inflammatory pathogenesis. As such, interventions to slow or mitigate epigenetic aging and inflammaging are critical for promoting healthy aging. The COcoa Supplement and Multivitamin Outcomes Study (COSMOS) is an ongoing randomized, double- blind, placebo-controlled, 2x2 factorial RCT of a high-quality cocoa extract supplement (containing 600 mg/d flavanols, including 80 mg epicatechins), and a multivitamin supplement to reduce the risk of CVD and cancer in women aged ?65 years and men aged ?60 years. COSMOS has randomized 21,444 participants into the trial, including 2,006 participants with bio-samples already collected at baseline, year 1 and year 2 follow-up. COSMOS is the only large RCT testing the effects of cocoa extract in women and men, and for multivitamins in women. Thus, this proposed ancillary study will build upon the parent COSMOS trial to examine the effects of randomized cocoa or multivitamin supplementation on anti-aging (epigenetic aging and inflammaging) in 600 representative subjects with three bio-sample collections at baseline, year 1, and year 2, as well as measured CVD risk factors, and examining these findings in the context of CVD outcomes. We will test our central hypotheses that cocoa supplementation reduces epigenetic aging indices (e.g., AgeAccelHorvath, AgeAccelHannum, AgeAccelGrim, AgeAccelPheno, and DNAmTLadjAge) and inflammaging (serum hsCRP, IL- 1?, IL-4, IL-6, and TNF-?) over time. We will also determine to what extent the effects of cocoa supplementation on inflammaging and CVD risk factors are mediated by epigenetic aging. The purported anti-aging health benefits of cocoa-rich products such as dark chocolate have popularized and increased consumption of cocoa-rich products over the past decade. Moreover, multivitamins remain the most common dietary supplement taken by at least one-third of older US adults. It remains of critical public health importance to rigorously test the anti-aging properties of these two supplements via large RCTs for more definitive evidence of their potential efficacy, and to approve or refute their potential anti-aging benefits.