ROLE OF GARLIC IN DECREASING CARDIOVASCULAR DISEASE RISK BY PROMOTING REVERSE-CHOLESTEROL TRANSPORT

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1006429
• Project Start Date: Jul 1, 2015
• Project End Date: Sep 30, 2017
• Program Code: [(N/A)]- (N/A)

Recipient Organization
OKLAHOMA STATE UNIVERSITY
(N/A)
STILLWATER,OK 74078

Performing Department
Human Sciences

Non Technical Summary
The proposed research will enhance our fundamental understanding of the relationship between nutrients, bioactive food components, and mechanisms through which naturally occurring dietary compounds contribute to a reduced risk of developing metabolic diseases such as CVD, obesity, and diabetes. Additionally, provided the increased appreciation for the therapeutic roles of garlic in preventing disease, these studies could promote the development of cultivars with enhanced levels of organosulfur compounds. Further, these studies could encourage agriculture producers (e.g., farmers) to consider cultivating garlic crops that increase the economic development of rural communities while simultaneously providing locally-sourced produce for consumer consumption. Given the continued prevalence of CVD as the leading cause of death in Oklahoma, especially in non-urban areas, as well as Oklahoma having the third highest rate of death from CVD in the U.S., the broader impact of this research includes critical new understanding of mechanisms through which increased fruit and vegetable consumption can maintain and improve heart health and quality of life among Oklahoma citizens.

Animal Health Component

(N/A)

Research Effort Categories

Basic

100%

Applied

(N/A)

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
702	1451	1010	100%

Knowledge Area
702 - Requirements and Function of Nutrients and Other Food Components;

Subject Of Investigation
1451 - Onion, garlic, leek, shallot;

Field Of Science
1010 - Nutrition and metabolism;

Keywords

garlic

histone deacteylase inhibitor

histone modification

cholesterol

cardiovascular disease

Goals / Objectives
Our primary objective is to examine the extent to which garlic, and its bioactive components, leads to the stimulation of reverse-cholesterol transport (RCT) through the enhanced release of cholesterol from macrophages thereby decreasing cardiovascular disease (CVD) risk. Dietary approaches or mechanisms that enhance RCT are an attractive option in reducing blood cholesterol levels and improving CVD risk at a relatively low cost with minimal side effects. The central hypothesis is that the organosulfur compounds DADS, SAMC, and AM in garlic funding as histone modifiers and lead to the increased expression of genes involved in promoting RCT, enhanced macrophage cholesterol efflux, and a reduction in macrophage cholesterol biosynthesis.

Project Methods
AIM 1: To examine the extent to which ogranosulfur compounds DADS, SAMC, and AM in garlic enhance the expression of genes involved in cholesterol efflux, promote increased cholesterol release, reduce cholesterol synthesis, and decreased inflammation in macrophages.Strategy: Using primary peritoneal (thioglycollate-elicited) macrophages, we will investigate the effects of HDACi on cholesterol metabolism. The expression of genes involved in cholesterol efflux (e.g., Abca1) in response to HDACi treatment will be examined. Using a radiolabeling technique, we will assess the impact of HDACi treatment on cholesterol efflux into culture media. The rate of cholesterol synthesis, as well as the expression of genes involved in cholesterol biosynthesis and inflammation, will be determined in response to HDACi treatment. These studies will not only enhance our understanding of the role of dietary HDACi on macrophage cholesterol metabolism, but will also examine the potential for HDACi to reduce inflammation due to cholesterol loading. All studies will also include the appropriate negative control, but also positive controls using (1) a characterized synthetic HDACi and (2) a drug that is increases cholesterol efflux through a (LXR-dependent) mechanism distinct from that of HDACi.Expected Results and Interpretations:Based on our preliminary studies, we predict that HDACi-treatment will enhance the expression of genes involved in cholesterol efflux and reduce the expression of both pro-inflammatory genes and genes involved in cholesterol biosynthesis. The results obtained with the dietary HDACi will be compared to the synthetic HDACi as well as T1317 (known to induce Abca1/Abcg1 expression and reduce inflammation). We expect that 3H-cholesterol in the media will be more abundant in HDACi and T1317-treated cells (along with an overall decrease in cholesterol biosynthesis in treated cells). These results will support the hypothesis that HDACi de-repress Abca1 and Abcg1 to promote RCT and reduce the potential for macrophages to contribute to lesion development.Caveats and Alternative Approaches to Aim 1: The doses proposed in this study are based on studies found in the cancer literature using these compounds in cell culture models. If necessary, we will examine the dose-response to HDACi in an immortalized cell line. We have previously conducted preliminary studies in the laboratory using the peritoneal macrophage cell model. If this model should prove unexpectedly challenging, we could employ the use of an immortalized monocyte cell line, THP-1, than can be differentiated into macrophage by treatment with phorbol myristic acid. These cells may be more amenable to treatment optimization if necessary. Cells grown under culture conditions may not reflect the in vivo condition, though our initial studies are consistent with what has been observed in animals. One potential alternative would be to treat the A129 strain with (or without) a high-cholesterol (0.2% w/w)/high-fat (40% w/w) diet to induce hypercholesterolemia. Similar models have shown that peritoneal macrophages isolated from these animals exhibit a cholesterol-loaded phenotype. Under these conditions, it is not clear what doses of HDACi would be necessary, but this could be empirically determined if needed.AIM 2. To characterize mechanisms through which DADS, SAMC, and AM function as dietary HDACi in macrophages to increase cholesterol efflux and begin to define the role(s) of garlic consumption in reducing CVD risk.Strategy: In this aim we will determine how HDACi function to increase cholesterol efflux potentially contributing to a decrease CVD risk. Using chromatin immunoprecipitation (ChIP) assays to examine promoter occupancy of target genes (e.g., Abca1 and Abcg1), we will investigate the extent to which chromatin remodeling due to inhibition of endogenous HDAC affects cholesterol efflux. These studies will provide direct evidence that chromatin remodeling is critical for the de-repression of gene expression observed in response to HDACi treatment.Expected Results and Interpretations: We expect that treatment with HDACi will increase the amount of Abca1 and Abcg1 that is pulled down with the AcH3, AcH4, and AcH3K9 antibodies with no changes in amount pulled down by the IgG antibody. These findings would provide compelling and direct evidence that at HDACi present in garlic elicit at least part of their cardioprotective benefits by remodeling chromatin associated with Abca1 and Abcg1 to enhance gene expression and ultimately promote increased cholesterol efflux. This would provide some of the first evidence that HDACi present in foods can play a protective role in the prevention of chronic disease that is distinct from their anti-cancer activity.Caveats and Alternative Approaches to Aim 2: While ChIP assays provide direct data regarding protein:DNA interactions, there are often multiple factors contributing to regulation. We could examine other genes (e.g., Lxr-alpha) whose expression is repressed in response to HDACi treatment and related factors (e.g., Cebp-alpha) that may play a role in this regulation.Statistical and Data Analyses: SPSS software (SPSS, Inc., Chicago, IL) will be used to perform ANOVA or Student's t-tests to assess significance of treatment effects. Descriptive statistics, including mean and standard deviation (SD), will be calculated on all variables. All tests will be done at the 95% confidence level (α = 0.05). Sample sizes were chosen based on previous experience of the PD in order to reach statistical significance.

Progress 07/01/15 to 09/30/17

Outputs
Target Audience:Preliminary results of these studies have been shared with a national audience at the Experimental Biology meeting in April 2017 as well as with principle investogators, scientists, community partners, and students at Oklahoma State University through open seminar and poster presentations. An undergraduate research scholar, who was obtained funding independent of this project also worked on the project and presented results of these studies at a university-wide undergraduate research forum in October 2017. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?One undergraduate scholar and two graduate students have been trained during the completion of this project. Training included not only experimental design, data collection and analysis, and interpretation, but also the ability to read and crtitique existing research. Students also gained critical experience in disseminating experimental findings in both written and verbal formats. Students involved in the project participated and presented their research at both local and national conferences. How have the results been disseminated to communities of interest?The results from this project have been presented at university-wide research conferences as well as a national conference (annual meeting of the American Society for Nutrition/Experimental Biology 2017). Additionally, this project served as the basis of a graduate student's thesis submitted in November 2017. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? As a result of the studies conducted during the completion of this project, we gained insight into the role(s) of compounds enriched in garlic on the regulation of cholesterol metabolism in cells. In particular, we confirmed that cells treated with garlic-derived compounds increase the expression of genes involved in promoting cholesterol removal from cells. These results confirm previously published studies demonstrating the garlic consumption reduces serum cholesterol thereby reducing cardiovascular disease risk. Although we originally predicted that these garlic-derived compounds increase cholesterol removal by increasing the expression of ABCA1 (a protein that promotes cholesterol efflux from cells) through a mechanism involving chromatin remodeling, it does not appear that ABCA1 expression is increased by modification in histones and chromatin remodeling. We continue to examine other potential mechanisms through which these garlic-derived compounds may exert cardioprotective benefits by promoting cholesterol efflux and stimulating reverse-cholesterol transport. APPROACH: To examine the impact of garlic-derived compounds on ABCA1 gene expression, mouse macrophage-like cells (RAW 264.7) were treated with diallyl disulfide (DADS), allyl mercaptan (AM), and S-allyl-cysteine (SAC). Following treatment, total RNA was isolated and reverse-transcribed to examine ABCA1 gene expression by quantitative real-time PCR. Additionally, cells were treated and prepared for Chromatin-Immunoprecipitation (ChIP) analysis to examine histone modification. In addition to DADS, AM, and SAC, cells were treated with the histone deacetylase inhibitor Trichostatin A (TSA) which serves as a positive control for assessing histone modification and chromatin-remodeling. Finally, the activation of the ABCA1 promoter was assessed by heterologous luciferase reporter assays. RESULTS: The expression of ABCA1 was increased 3-7-fold in cells treated with DADS, AM, SAC, and TSA compared to control cells. These results indicate that these compounds may exhibit a cardioprotective benefit in macrophages by increasing the expression of ABCA1 - the protein responsible for cholesterol efflux in macrophages, Next, ChIP assays indicated that the abundance of modified histones (acetylated H3 and H4 compared to total H3 and H4) was unchanged in cells treated with DADS, AM, and SAC but increased in cells treated with TSA. These results suggest that the garlic-derived compounds modulate ABCA1 expression in a manner independent of histone modification. To begin to assess the regualtion of the ABCA1 promoter, reporter assays were utilized, Interestingly, treatment of cells with DADS increased reporter activity compared to control cells or cells treated with TSA suggesting that there may be a more direct effect of these compounds on increasing ABCA1 expression directly through an unknown mechanism controlling the ABCA1 promoter. IMPLICATIONS: Previous work has suggested that DADS, AM, and SAC exhibit the ability to participate in histone modification, though these studies focused primarily on some of the putative anti-cancer properties of these compounds. In our work, we did not obtain any evidence that the ability of these compounds to increase ABCA1 gene expression was mediated by histone modification or chromatin remodeling. Although we were unable to identify the moelcular mechanism through which DADS, AM, and SAC modulate ABCA1 gene expression, we again confirmed that these compounds play a significant role in increasing ABCA1 expression in cells and could play a central role in promoting reverse-cholesterol transport to reduce cardiovascular disease risk. Therefore, the use of garlic and/or its bioactive components remain an attractive target to investigate for the incorporation of garlic into the diet to reduce serum cholesterol and reduce disease risk.

Publications

• Type: Theses/Dissertations Status: Published Year Published: 2017 Citation: Madden AJ, Krehbiel MD, and Clarke SL. 2017, 'Organosulfur garlic-derived compounds induce ABCA1 gene expression in RAW 264.7 cells', M.S.in Nutritional Sciences, Oklahoma State University, Stillwater, OK.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2017 Citation: Abbie J. Madden, Madison D. Krehbiel, and Stephen L. Clarke. Nutrition - Cardiovascular Effects of Dietary Bioactive Components. Garlic-derived Compounds Increase Expression of ABCA1 mRNA in RAW 264.7 Murine Macrophages FASEB J April 2017 31:973.1

Progress 10/01/15 to 09/30/16

Outputs
Target Audience:Preliminary results of these studies have been shared with other investigators through open seminar presentations. A discussion of preliminary results has also been shared with potential students during recruiting visits. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The graduate student who has worked on the project has been presented the opportunity to apply their knowledge of nutrition and food to other fields such as epigenetics and molecular biology. How have the results been disseminated to communities of interest?Aside from preliminary findings, the results of the project have not been shared with the intended target audience. We expect this to change during the next reporting period. What do you plan to do during the next reporting period to accomplish the goals?The experiments will be completed prior to the end of the reporting period. Analyses will continue to be completed through the end of Fall 2017.

Impacts
What was accomplished under these goals? During the reporting period, a new graduate student has transitioned to lead the project. In addition to learning cell culture methods, the student has also developed an expertise in Chromatin Immunoprecipitation assays and similarly related molecular techniques. The student also received training in Responsible Conduct of Research and working with the Institutional Animal Care and Use Committee.

Publications

Progress 07/01/15 to 09/30/15

Outputs
Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Graduate students have received hands-on training related to animal care and handling. Further, they have optimized cell culture techniques that are central to the successful completion of the study. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?In the coming months the lab will perform preliminary experiments related to dosing and time of treatment. These studies will include both immortalized cell lines as well as primary cell culture.

Impacts
What was accomplished under these goals? Thre first few months of this project have focused on training graduate students to conduct studies. This training includes cell culture techniques as well as animal handling and care.

Publications