Recipient Organization
GEORGIA STATE UNIVERSITY
UNIVERSITY PLAZA
ATLANTA,GA 30303
Performing Department
(N/A)
Non Technical Summary
Hypertension (HTN), or high blood pressure, is prevalent in an estimated 45% of adults in the United States and is the major modifiable risk factor for heart disease which is the leading cause of death worldwide. HTN also represents an economic burden as the US health care system spent an estimated $79 billion in 2016 on patients with HTN and individuals using multiple antihypertensive medications pay, on average, $436 annually for medications alone. Thus, identifying early targets and alternative therapeutic approaches to prevent or delay the onset of HTN is important to reduce the potential development of more serious cardiovascular disease outcomes and improve quality of life. Recent studies have demonstrated an association between intestinal barrier dysfunction and HTN. This is important because the intestinal barrier is critical in preventing the entry of toxic compounds that are detrimental to health. It is largely unknown, however, whether preserving intestinal barrier function protects against the rise in blood pressure. Therefore, there is a need to investigate the interrelationship between intestinal barrier dysfunction, HTN, and therapeutic approaches to prevent them. Polyphenols are found naturally in a wide distribution of plant foods and are known to have both heart- and intestinal-protective effects. Blackberries, raspberries, blueberries, and strawberries are commonly consumed berries that have unique polyphenolic profiles; therefore, including mixed berries in the diet will not only expand the breadth of polyphenols obtained, but it may also help to improve health.The main goal of our project is to use a preclinical rat model to investigate whether a berry-rich diet is effective in preserving the intestinal barrier and whether this preservation contributes to preventing the onset of HTN. To achieve this, rats will be fed a diet with or without berries and implanted with angiotensin II to induce HTN. Markers of intestinal barrier function and blood pressure will be measured over time. We also aim to determine the mechanisms by which berries exert their beneficial effects using cell culture studies and measuring protein expression of markers related to intestinal integrity, inflammation, and oxidative stress. Results will be shared via presentations at local and national conferences and community speaking engagements, as well as via publications in peer-reviewed journals. Altogether, these experiments will improve our understanding of the relationship between intestinal barrier function and the development of HTN which will provide scientists, physicians, and dietitians a novel, early target to prevent HTN. It will also inform the greater population on safe, alternative methods for preventing and managing HTN by providing insight on how diets rich in berries can improve heart and intestinal health.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Goals / Objectives
Thelong-term goalof this project is to evaluate the effect of a berry-rich diet in the prevention and management of chronic disease. Themain goalof this study is to bring forth evidence that a diet rich in berries can effectively attenuate pathophysiological changes to the intestinal barrier associated with development and progression of hypertension (HTN) in an angiotensin (Ang) II-infusion model.To achieve this goal, we will complete the followingobjectives:1. Evaluate the effectiveness of a berry-rich diet in preventing morphological and functional changes to the intestinal barrier over time in an Ang II-treated rat model.2. Determine the mechanism by which berry consumption improve intestinal permeability and attenuate HTN in vitro.
Project Methods
Eight-week-old male Sprague Dawley (SD) rats will be acclimated for one week and will have free access to water and maintained on a semi-purified casein-based diet (AIN93-M) devoid of polyphenols in which soybean oil has been replaced with corn oil. After one-week of acclimation, rats will be randomized into one of five groups: Control (AIN-93M; saline; n=45), Ang II (AIN-93M; Ang II; n=37), Losartan (AIN-93M; Ang II; n=37), Divertin (AIN-93M; Ang II; n=37), and Berry (AIN-93M + 10% w/w BL, BB, RB, SB; Ang II; n=45).Rats randomized into the berry group will receive a diet supplemented with 10% freeze-dried berry (equal parts blackberry (BL), blueberry (BB), raspberry (RB), and strawberry (SB)) powder or will otherwise remain on the AIN-93M diet for a total of eight weeks. The 10% berry supplemented diet translates to a human consumption of approximately four servings (or three cups) of mixed berries per day based on body surface area, corresponding to a dose of approximately 333 mg total phenolics/kg BW/day. The control diet will be isocaloric and matched for macronutrients, fiber, and micronutrients such as potassium and sodium. At week four rats will be implanted with subcutaneous osmotic minipumps delivering either 0.9% saline (control) or angiotensin (Ang) II (270 ng/kg BW/min). We have chosen this dose of Ang II based on the literature and previous experience in our lab in which 270 ng/kg BW/min induced hypertension (HTN) within two weeks of implantation and demonstrated decreased protein expression of claudin (CLDN)-1 and occludin (OCLN). At this time, an Ang II group will receive losartan (LOS, 20 mg/kg BW/day) in their drinking water or daily intraperitoneal injections of divertin (DIV, 12.5 mg/kg BW/day), a new synthetic drug found to restore barrier function, for the remainder of the study to act as negative control groups.Food intake and body weight will be measured weekly in each individual animal to account for differences in dietary intake and weight. Tail-cuff plethysmography and radio-biotelemetry will be used to measure blood pressure (BP) throughout the animal study starting at week 4 (baseline) and weekly thereafter. Wire myography will be used to assess differences in endothelial function in the third to fifth order mesenteric arteries. Vessels will be pre-constricted with phenylephrine, an alpha-adrenergic agonist, and relaxed with cumulative addition of acetylcholine.Weekly sacrifices will be performed in each group starting at week 4 to evaluate the changes to intestinal barrier function over time and its relationship to blood pressure. Direct markers of intestinal permeability will be evaluated using FITC-dextran 4000 at weeks 4, 6, and 8 which represents baseline, midpoint, and final point, respectively. Indirect markers of intestinal permeability will be evaluated using serum markers of diamine oxidase, lipopolysaccharide binding-protein, and anti-flagellin immunoglobulin. Additionally, tight junction (TJ), a critical component of the intestinal barrier, protein markers such as occludin, claudins, and zonula occludens, will be measured in the ileum and proximal colon. The same markers will be visualized using immunofluorescence staining to evaluate their localization.Protein and gene expression as well as immunohistochemistry will be used to determine the inflammatory and oxidative stress status of the small intestine, proximal colon, and medial colon.Together, these experiments help to reach our first key milestone as we will 1) confirm whether there is an association between HTN and intestinal barrier dysfunction, 2) determine whether alterations in TJ assembly, and thus intestinal barrier function, precede Ang II-induced HTN, 3) provide insight into the role of Ang II-induced inflammation and oxidative stress in intestinal barrier dysfunction in vivo, and 4) demonstrate whether a berry-supplemented diet can effectively protect from Ang II-induced intestinal permeability and thus, rises in BP.Results of the animal study cannot, however, allow us to elucidate the mechanisms; therefore, further experiments will be conducted in in vitro.Caco-2 cells will be cultured and pretreated with berry extracts (BL, BB, RB, SB) alone and in combination for 1 hour, followed by 12 hours treatment with Ang II. The presence of superoxide anions and hydrogen peroxide will be detected using DHE and H2DCFDA, and markers of pro-oxidants, antioxidants, and inflammation will be measured using western blot analysis. Quantitative PCR will also be used to evaluate Ang II receptors.Transepithelial electrical resistance (TEER) is a noninvasive experiment that will be used to provide real-time data on TJ integrity without damaging cells. Caco-2 cells will be cultured on Transwell inserts to promote polarization. Data will be collected before berry pretreatment and 1-hour after pretreatment, and at 4-, 8-, and 12-hour treatment with Ang II to assess the potential changes in TJ permeability over time. Additionally, TJ markers will also be assessed from cell culture experiments using western blot.The in vitro experiments thus far bring us to our second key milestone as they will provide insight on whether pretreatment with berry extracts is correlated with improved barrier function, inflammation, and oxidative stress in Ang II-treated Caco-2 cells. This is important because we will need to reveal the potential pathway(s) through which berry extracts act to protect from Ang II insult. This leads to our final key milestone, which is to determine the mechanisms, and to this end, we will need to further explore potential pathways using techniques such as inhibitors and siRNAs that are yet to be determined at this time.Descriptive statistics will be performed for all variables collected and Shapiro-Wilk test will be used to analyze data for normality. Characteristics will be compared at baseline using ANOVA. If differences occur, adjustments to analyses will be made to account for variable effects. For non-repeated measures such as protein expression and immunofluorescence staining, normally distributed data will be analyzed using one-way ANOVA or independent samples t-test, otherwise, non-parametric tests such as Kruskal Wallis or Mann U-Whitney test will be used. For repeated measures in the same animal, namely BP, weight, and food intake, data will be analyzed using a 3 x 2 (group x time) repeated measures ANOVA. Multiple linear regression analyses will also be performed to assess the impact of inflammation, oxidative stress, and intestinal permeability on blood pressure response. Significance will be set at p ≤ 0.05. Data analyses will be performed using GraphPad 9 (LaJolle, Ca).All data generated from this project will be broadly disseminated to:NIFA through annual and final reports;Researchers and health professionals through presentation at local and national scientific conferences and publications in peer-reviewed journals;The general public through press releases and presentations in community and clinical centers.The success of this project will be measured by:Publication of research findings in peer-review journals;Number of citations of the scientific publication(s);Presentation of research findings at the annual meeting of the American Society of Nutrition and the Academy of Nutrition and Dietetics.Press release of research findings to non-academic audience through local and major news outlets such as National Public Radio, Healthline, and Georgia State University Research Magazine;Presentation of research findings to health professionals such as dietitians at local professional associations and workshops;Presentation of research findings to the general public in community centers and schools.