PROTECTIVE ROLE OF BLACKBERRY POLYPHENOLS IN INFLAMMATION, AGING AND DISEASE

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: AFRI COMPETITIVE GRANT
• Reporting Frequency: Annual
• Accession No.: 1014863
• Grant No.: 2018-67017-27518
• Cumulative Award Amt.: $500,000.00
• Proposal No.: 2017-06985
• Project Start Date: Mar 1, 2018
• Project End Date: Feb 28, 2022
• Grant Year: 2018
• Program Code: [A1341]- Food Safety, Nutrition, and Health: Function and Efficacy of Nutrients

Recipient Organization
FLORIDA STATE UNIVERSITY
118 N. WOODWARD AVE
TALLAHASSEE,FL 32306

Performing Department
Nutrition, Food and Exercise

Non Technical Summary
Cellular senescence influences organismal aging and increases predisposition to age-related and pro-inflammatory diseases, in particular cardiovascular disease (CVD). Angiotensin II (Ang II) is hormone implicated in inflammation, oxidative stress, and vascular aging or senescence that promote chronic diseases, including CVD. Polyphenols are bioactive compounds known for their strong antioxidant and anti-inflammatory capacity. However, It is unknown whether berry polyphenols could protect vascular smooth muscle cells (VSMCs), the cells that form blood vessels, from inflammation and senescence induced by Ang II. Our data show that blackberry reduced senescence and atherosclerosis in ApoE knockout mouse, an animal model of accelerated aging and atherosclerosis. Blackberry polyphenol extract reduced Ang II-induced oxidative stress and senescence by preventing the upregulation of the NADPH oxidase (Nox) 1, an enzyme that produces superoxide radicals inducing cellular damage. We also demonstrated that Nox1 is regulated in by the inflammatory gene NF-κB, . Thus, we will test the hypothesis that blackberry polyphenols prevent NF-κB activation by Ang II leading to reduced Nox1 expression and reactive oxygen species (ROS) levels, thus preventing inflammation, senescence and atherosclerosis. These studies align with the USDA-AFRI priority of improving function and efficacy of dietary bioactive compounds to prevent inflammation and chronic diseases. We will test our hypothesis through the following aims: 1) to identify specific phenolic compounds in blackberry responsible for the downregulation of Nox1 expression, inflammation and senescence; 2) to examine the contribution of the NF-κB/Nox1/COX-2 pathway to blackberry effects on ROS levels and senescence of VSMCs; and 3) determine the role of blackberry in reducing Nox1 expression, inflammation, vascular senescence and atherosclerosis in vivo. These studies will greatly advance our understanding of mechanisms regulating aging and will help to design nutritional therapies to delay inflammatory and age-related pathologies.

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	1129	1010	100%

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

Subject Of Investigation
1129 - Berries and cane fruits, general/other;

Field Of Science
1010 - Nutrition and metabolism;

Keywords

ang ii

berry polyphenols

inflammation

oxidative stress

senescence

vsmcs

Goals / Objectives
Cellular senescence influences organismal aging and increases predisposition to age-related and pro-inflammatory diseases, in particular cardiovascular disease (CVD). Angiotensin II (Ang II) is implicated in inflammation, oxidative stress, and senescence that promote chronic diseases, including CVD. Polyphenols are bioactive compounds known for their strong antioxidant and anti-inflammatory capacity. However, It is known whether berry polyphenols could protect vascular smooth muscle cells (VSMCs) from inflammation and senescence induced by Ang II. Our preliminary data show that blackberry reduced senescence and atherosclerosis in ApoE-/- mice. Blackberry polyphenol extract reduced Ang II-induced oxidative stress and senescence by preventing the upregulation of the NADPH oxidase (Nox) 1 in VSMCs, which is regulated in by NF-κB. Thus, we will test the hypothesis that blackberry polyphenols prevent NF-κB activation by Ang II leading to reduced Nox1 expression and ROS levels, thus preventing inflammation, senescence and atherosclerosis. These studies align with the USDA-AFRI priority of improving function and efficacy of dietary bioactive compounds to prevent inflammation and chronic diseases. . The major goal of this proposal is to examine the protective role of functional foods, in particular blackberries, which are rich in polyphenols, in reducing vascular senescence and atherosclerosis in vivo. The objectives of this proposal are: 1) to identify specific phenolic compounds in blackberry responsible for the downregulation of Nox1 expression, inflammation and senescence; 2) to examine the contribution of the NF-κB/Nox1/COX-2 pathway to blackberry effects on ROS levels and senescence of VSMCs; and 3) determine the role of blackberry in reducing Nox1 expression, inflammation, vascular senescence and atherosclerosis in vivo. These studies will greatly advance our understanding of mechanisms regulating aging and will help to design nutritional therapies to delay inflammatory and age-related pathologies

Project Methods
Epidemiological studies demonstrate that consumption of fruits and vegetables is associated with decreased incidence of cardiovascular disease (CVD), which is mainly attributed to the polyphenol content of these foods. Polyphenols are bioactive compounds known for their strong antioxidant and anti-inflammatory capacity. However, a protective role of polyphenols from berries that prevent senescence induced by Ang II, a hormone involved in CVD, in VSMCs that could delay atherosclerosis has not been investigated. Our published and preliminary data fill this gap and provide evidence for the possible mechanism involved. We found that polyphenols from blackberries reduce oxidative stress and senescence induced by Ang II by preventing the upregulation of the NADPH oxidase (Nox) 1, an enzyme that produces superoxide. In VSMCs, Ang II promotes inflammation by upregulating the transcription factor nuclear factor kappa-B (NF-kB) and cyclooxygenase (COX)-2, which is also upregulated by the kinases ERK1/2, p38MAPK and the transcription factor NF-kB. Nox1 expression is also regulated by NF-kB. Supporting this notion, we recently reported that Nox1 is upregulated by NF-kB in response to zinc overload causing senescence of VSMCs. Based on these data, we hypothesize that blackberry polyphenols should prevent Ang II-induced Nox1 expression by inhibiting NF-kB.In this proposal, we will test the hypothesis that blackberry polyphenols prevent NF-kB activation by Ang II leading to reduced Nox1 expression and ROS levels, thus diminishing inflammation, senescence and atherosclerosis. We will investigate whether blackberry extract protect vascular cells from inflammation and oxidative stress during aging in vitro and in vivo and the molecular mechanism involved. The outcome of this study has the potential to provide further evidence of the benefit of functional foods to reduce the progression of CVDs. To achieve the goals of this proposal, we will use VSMCs that we isolated from rat aortas and Ang II, as a model of increased oxidative stress, inflammation and cellular senescence. For in vivo studies, we will use ApoE knockout mice, a mouse model of atherosclerosis, and high fat diet (HFD) to accelerate plaque deposition. We will test our hypothesis through the following aims:Specific Aims:Aim 1. To identify specific phenolic compounds in blackberry responsible for the downregulation of Nox1 expression, inflammation and senescence in VSMCs. In order to achieve this aim, we will incubate VSMCs with and without Ang II and blackberry polyphenol extract or purified phenolic compounds and determine Nox1 expression, ROS levels, inflammation and senescence. Based on our preliminary data, we hypothesize that the compounds gallic acid and/or 3-O-caffeoylquinic acid (3-CQA), abundant in blackberry extract, are responsible for the protective effects of this berry.Treatment of VSMCs. VSMCs isolated from rat aortas and cultured up to passage 12 will be growth-arrested for 24 h in DMEM containing 0.5% fetal bovine serum (FBS). VSMCs will be then incubated with and without phenolic compounds (gallic acid and 3-CQA), Ang II and blackberry polyphenol extract, as we previously reported.Cell Viability measurements. Viability will be evaluated by trypan-blue exclusion. Cells treated with and without phenolic compounds, blackberry extract and Ang II will be resuspended and incubated with trypan-blue for 5 min and then counted (trypan-blue negative cells) using a Neubauer chamber.Endpoint measurements. After treatment, VSMCs will be analyzed for Ang II signaling and expression of Nox1 and antioxidant enzymes by western blot. Conditions in which cell viability is diminished will not be used for further analysis. Quantification will be determined in triplicate in three independent experiments.Protein expression of target proteins will be assessed by western blot. We will measure the levels of Nox1 (Nox4, as a control) and the expression of antioxidant enzymes (SOD1, SOD2, catalase and GPx-1), inflammatory molecules (NF-kB, COX-2, COX-1, TNFa, IL-1a, and IL-6), senescence markers (p53 and p21) and cell signaling by measuring the phosphorylation of ERK1/2, p38MAPK and Akt.Senescence will be determined by measuring the levels of the senescence associated b-galactosidate (SA-b-gal). Cells will be washed with PBS, fixed with 0.2% glutaraldehyde and incubated in phosphate buffer containing FDG (Ex/Em: 488/515). Cells will be washed with PBS and fluorescence intensity will be measured in a plate reader and adjusted by cell number. Measurements will be done using 6 individual wells per condition in three independent experiments.Cellular ROS levels will be assessed by measuring hydrogen peroxide using the fluorescent probe H2DCFDA and superoxide using the fluorescent probe DHE.Statistical analyses. Analysis of variance (ANOVA) will be used to assess difference among treatments and Tukey-Kramer post-hoc test will be used for pairwise comparisons. Significant differences will be determined at p≤0.05. Values will be presented as mean ± standard deviation of mean (SD). Data analyses will be performed using SSPS version 20.0 for Windows (SPSS Inc., Chicago, IL).Aim 2. To examine the contribution of the NF-kB/Nox1/COX-2 pathway to blackberry effects on ROS levels and senescence of VSMCs. In this aim, we will determine the molecular mechanism of Nox1 downregulation by blackberry polyphenols and the contribution of the NF-kB/Nox1/COX-2 pathway to senescence mediated by Ang II. If we confirm that gallic acid and/or 3-CQA reduces senescence in a Nox1-dependent manner in aim 1, we will also determine the role of these compounds in NF-kB activity and COX-2 expression. To test this pathway, we will use NF-kB and COX-2 inhibitors and downregulation of Nox1 and COX-2 by siRNA.Changes in genes expression, senescence and ROS levels will be determined as described in aim 1. Downregulation by siRNA will be done using oligonucleotides previously reported by our lab. Downregulation of the target gene will be measured by western blotAim 3. Determine the role of blackberry in reducing Nox1 expression, inflammation, vascular senescence and atherosclerosis in vivo. To test the role of blackberry polyphenols in vivo, we will use ApoE-/- mice fed HFD, alone or supplemented with 2% blackberry freeze-dried powder for five weeks. If we confirm that gallic acid and/or 3-CQA reduces Nox1, we will also feed animals HFD and administer individual compounds by gavage. Postmortem, the aortas will be tested for senescence, plaque deposition, and protein expression of target gene.According to our published data and using a priori power analysis (G*Power3) with α=0.05, power=0.95 and d (effect size)= 1.8, we estimated that six animals per group will be necessary to obtain a significant difference (p<0.05). Animals will be used between four to six month of age.Four month-old male and female ApoE-/- mice will be fed HFD or HFD supplemented with 2% blackberry for five weeks. Aortas will be isolated, fixed in 0.2% glutaraldehyde, open longitudinally and photographed for measurements of plaque deposition. Quantification of plaque willbe performed by determining the area of plaque versus total area using Image J software. After measurements of plaque, aortas will be washed and analyzed for SA-b-gal activity using FDG. Fluorescence will be determined in a plate reader and will be adjusted by the weight of the aorta tissue.Aorta of animals fed in the same conditions will be used for measurements of protein expression of inflammatory genes, antioxidant enzymes, senescence markers and cell signaling.

Progress 03/01/18 to 02/28/22

Outputs
Target Audience:The target audience reached by this project includes graduate and undergraduate students that were trained because of NIFA funding, as well as the scientific community reached during the conferences in which the results of this research were presented Changes/Problems:This award has allowed us to explore different avenues by which polyphenols act to reduce disease burden. First, we uncovered a new role for gallic acid in regulating microbiome health. This new discovery will be the focus of a new grant proposal that will be submitted to the USDA. Second, we obtained an award from the Florida Department of Health to investigate the role of blackberry in reducing atherosclerosis caused by cigarette smoke. We are also interested in evaluating the effect of blackberry supplementation in adiponectin levels in vivo and in adiponectin signaling in vitro. This is a new direction that we are currently exploring. In terms of mechanisms, we are exploring possible sex-dependent differences in the phenotype of vascular smooth muscle cells. We isolated cells from the aortas of male and female mice and found major differences in terms of cell signaling, senescence and autophagy. This data is in the process of being published. We will explore the mechanism by which polyphenols act in male and female cells. In terms of problems, we encountered two major difficulties COVID19 in 2020 and the closing of Sandels building at FSU, which houses our laboratory, in January 2022. One major COVID19 restriction at FSU was that only a few studies using animals were allowed. We were fortunate to be able to complete all our in vivo studies. However, in vitro studies were delayed. The Sandels building is still closed for research and teaching due to biosafety concerns. What opportunities for training and professional development has the project provided?Abigail Cullen, a graduate student and Carolyn Deeb, an undergraduate student volunteered in my lab worked in this project and graduated in 2020. McKenzie Clark, an undergraduate student in honors thesis, worked on this project and graduated in Fall 2021. Ann Centner, a PhD student working on this projects will graduate Fall 2022. How have the results been disseminated to communities of interest?We have published several manuscripts and preseted the result of this work at local and national meetings as described in the "products" section of the report. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? This proposal aims to elucidate whether blackberry supplementation reduces the development of plaque in vivo in ApoE and whether this effect is mediated by downregulation of Nox1. Accomplishments under the 3 major goals are: 1) to identify specific phenolic compounds in blackberry responsible for the downregulation of Nox1 expression, inflammation and senescence. We identified gallic acid as a major polyphenol responsible for the effects of blackberry. In vitro, we tested 3-O-Caffeoylquinic acid (3C), 4-O-Caffeoylquinic acid (4C) and 5-O-Caffeoylquinic acid (5C) and found that gallic acid has the strongest effect on reducing cell signaling associated with oxidative stress and senescence in vascular smooth muscle cells (VSMCs). Gallic acid was selected for in vivo studies in aim 3. 2) to examine the contribution of the NF-κB/Nox1/COX-2 pathway to blackberry effects on ROS levels and senescence of VSMCs. This goal was the only one that was less developed mainly because the antibody against Nox1 stopped working. We use all the stock from a specific batch number and prioritize the in vivo studies. We tested several Nox1 antibodies from different companies, but no one worked. Instead, we developed a new direction by testing the effect of gallic acid in microbiome dysbiosis induced by high fat det. This new data is reported together with the effects of gallic acid in vivo. Additionally, progress for in vitro studies were delay due to the cessation of research activities due to COVID 19. Then, the Sandels building housing our research lab was closed due to biosafety concerns. The building was closed January 19, 2022 and remained closed until today (July 26, 2022). We recently were transferred to a new lab space in a different building (July 5, 2022). 3) determine the role of blackberry in reducing Nox1 expression, inflammation, vascular senescence and atherosclerosis in vivo. Under this goal we demonstrated that blackberry has sex-dependent effects on senescence and atherosclerosis in ApoE deficient mice. Blackberry reduced senescence and atherosclerosis in males showing no effect on females. Additionally, blackberry reduced Nox1 in the aortas of males, but no female mice. We also demonstrated that gallic acid mediates these effects. Both blackberry and gallic acid showed no improvement in the lipid profile. The effect of blackberry in atherosclerosis was published in the Journal of Nutritional Biochemistry in 2020. The manuscript reporting the effects of effects of gallic acid in vivo was recently accepted after minor revisions in the Journal of Nutritional Biochemistry.

Publications

• Type: Journal Articles Status: Published Year Published: 2021 Citation: Serino, A., Zhao, Y., Huang, J., Cullen, A. E., Deeb, C., Akhavan, N., Arjmandi, B. H., & Salazar, G. (2020). Gender differences in the effect of blackberry supplementation in vascular senescence and atherosclerosis in ApoE-/- mice. J Nutr Biochem, 80, 108375. doi:10.1016/j.jnutbio.2020.108375
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Salazar, G., Cullen, A., Huang, J., Zhao, Y., Serino, A., Hilenski, L., Patrushev, N., Forouzandeh, F., & Hwang, H. S. (2020). SQSTM1/p62 and PPARGC1A/PGC-1alpha at the Interface of Autophagy and Vascular Senescence. Autophagy, 16(6), 1092-1110. doi:10.1080/15548627.2019.1659612
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Cullen, A. E., Centner, A. M., Deitado, R., Fernandez, J., & Salazar, G. (2020). The Impact of Dietary Supplementation of Whole Foods and Polyphenols on Atherosclerosis. Nutrients, 12(7), 2069. doi:10.3390/nu12072069
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Centner, A. M., & Salazar, G. (2020). Nicotine in Senescence and Atherosclerosis. Cells, 9(4), 1035. doi:10.3390/cells9041035
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Serino, A., & Salazar, G. (2018). Protective Role of Polyphenols against Vascular Inflammation, Aging and Cardiovascular Disease. Nutrients, 11(1), 53. doi.org/10.3390/nu11010053
• Type: Journal Articles Status: Accepted Year Published: 2022 Citation: Clark, M., Centner, A. M., Ukhanov, V., Nagpal, R., & Salazar, G. Gallic acid reduces atherosclerosis and vascular senescence and remodels the microbiome in a sex-dependent manner in ApoE-/- mice. Under review J of Nutritional Biochemistry.
• Type: Journal Articles Status: Submitted Year Published: 2022 Citation: Centner, A. M., Cullen, A. E., Ukhanov, V., Hill, S., Deitado, R., Hwang, H. S., La Favor, J. D., Laitano, O., Parvatiyar, M. S., & Salazar, G. The Interaction of Sex and SQSTM1 in the Effects of Smoking and Nicotine in Cardiovascular Function, Atherosclerosis and Inflammation. Submitted to Circulation Research.
• Type: Journal Articles Status: Other Year Published: 2022 Citation: Cullen, A. E., Centner A. M., Deitado, R. and Salazar, G. The Duality of Adiponectin: The Role of Sex in Atherosclerosis. In preparation to submit to Circulation Research.
• Type: Journal Articles Status: Other Year Published: 2022 Citation: Centner, AM, Ukhanov, V, Cullen AE and Salazar G. Menthol and nicotine in E-cigarettes modulate atherosclerosis and gut microbiome diversity. In preparation to submit to the Journal Tobacco Control.
• Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Center, A., Clark, M., Ukhanov, V., Nagpal, R., & Salazar, G. Gallic acid reduces plaque burden in male mice. Presentation at Nutrition 2020, American Society for Nutrition. Current Developments in Nutrition, Volume 6, Issue Supplement_1, June 2022, Page 273
• Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Centner, A., Ukhanov, V., Laitano, O., & Salazar, G. Aerosolized E-liquids exacerbate atherosclerosis in mice. Presentation at Research Showcase College of health and Human Sciences, Florida State University, Tallahassee
• Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Centner, A., Cullen, A., Ukhanov, V., Hill, S., Laitano, O., & Salazar, G. Cigarette smoke and nicotine upregulate IL-17 and MMP3 levels promoting atherosclerosis in ApoE knockout mice. Presentation at Research Showcase, College of Health and Human Sciences, Florida State University.
• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Cullen, A., Centner, A. M., & Salazar, G. Regulation of the Phenotypic Switch of Vascular Smooth Muscle Cells by Adiponectin. Presentation at Research Showcase, College of Health and Human Sciences, Tallahassee, Florida.
• Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Centner, A., & Salazar, G. The Role of Sex in the Effects of Cigarette Smoke in Vascular Smooth Muscle Cells. Presentation at EB2022, Experimental Biology, Philadelphia.
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Cullen, A., Ismael, A., Koutakis, P., & Salazar, G. Adiponectin and the Regulation of Vascular Smooth Muscle Cell Phenotype. Presentation at American Heart Association Scientific Session, Philadelphia, Pennsylvania.
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Salazar, G., Zhao, Y., Cullen, A., & Deeb, C. Zinc and Polyphenols in Autophagy and Vascular Senescence. Presentation at ISZB Kyoto 2019, International Society for Zinc Biology, Kyoto, Japan.
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Salazar, G., Zhao, Y., & Huang, J. Blackberry supplementation reduces vascular aging and atherosclerosis induced by high fat diet in ApoE-/- mice. Presentation at Nutrition 2018, American Society for Nutrition, Boston MA.
• Type: Theses/Dissertations Status: Published Year Published: 2018 Citation: McKenzie Clark. The role of gallic acid in atherosclerosis in APOE-/- mice. Undergraduate Honors Thesis.
• Type: Theses/Dissertations Status: Published Year Published: 2018 Citation: Alexa Serino. Novel role of blackberries in vascular senescence and atherosclerosis in ApoE knockout mice. Undergraduate Honors Thesis.
• Type: Theses/Dissertations Status: Published Year Published: 2020 Citation: Abigail E. Cullen. The regulation of vascular smooth muscle cell phenotype by adiponectin. PhD dissertation.

Progress 03/01/20 to 02/28/21

Outputs
Target Audience:The target audience reached by this project includes graduate and undergraduate students that were trained because of NIFA funding, as well as the scientific community reached during the conferences in which the results of this research were presented. Changes/Problems:The major difficulty we faced was the slow recovery of research activities at Florida State University due to COVID-19 What opportunities for training and professional development has the project provided?Abigail Cullen, a graduate student and Carolyn Deeb, an undergraduate student volunteered in my lab worked in this project and graduated in 2020. Ann Centner, a PhD student, and McKenzie Clark an undergraduate student in honors thesis are currently working in this project. How have the results been disseminated to communities of interest?The results of this work were presented in the Experimental Biology annual meeting (EB2020) and the Research Showcase at Florida State University. What do you plan to do during the next reporting period to accomplish the goals?For this year we plan to finish all in vivo and in vitro data collection and analysis and prepare manuscript for publication.

Impacts
What was accomplished under these goals? This proposal aims to elucidate whether blackberry supplementation reduces the development of plaque in vivo in ApoE and whether this effect is mediated by downregulation of Nox1. Due to COVID19 we only were able to focus on aim 3 to define the role of polyphenols in the effects of blackberry in vivo. Aim 3) Determine the role of blackberry in reducing Nox1 expression, inflammation, vascular senescence and atherosclerosis in vivo. We have published a paper describing the role of blackberry supplementation (2% in the diet) in vascular senescence and atherosclerosis. We reported gender differences in the response to this fruit. Senescence, atherosclerosis and Nox1 expression were reduced in males, but not in female ApoE KOmice. We also reported that the protective effect of blackberry in males was independent of lipid profile, since cholesterol, LDL, and cholesterol were not affected by treatment. To determine whether the polyphenol content of blackberry and no other nutrients, such as fiber, mediates the protection against atherosclerosis, we treated animals with gallic acid. In our in vitro studies, we found that gallic acid had the strongest effect in reducing oxidative stress and senescence in VSMCs. Supplementation with 0.2% gallic acid in drinking water reduces plaque in male, but not female ApoE KOmice. Thus, gallic acid mimics the sex-dependent effect of blackberry. Similarly, gallic acid showed no effect on cholesterol levels in circulation.

Publications

• Type: Conference Papers and Presentations Status: Accepted Year Published: 2021 Citation: Sex Differences in Atherosclerosis in ApoE-/- Mice exposed to Nicotine and Cigarette Smoke
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2021 Citation: Cigarette smoke and nicotine upregulate IL-17 and MMP3 levels promoting atherosclerosis in ApoE knockout mice
• Type: Journal Articles Status: Submitted Year Published: 2021 Citation: The Interaction of Sex and SQSTM1 in the Effects of Smoking and Nicotine in Cardiovascular Function, Atherosclerosis and Inflammation

Progress 03/01/19 to 02/29/20

Outputs
Target Audience:The target audience reached by this project includes graduate and undergraduate students that were trained because of NIFA funding, as well as the scientific community reached during the conferences in which the results of this research were presented. Changes/Problems:The major difficulty we faced at the end of 2019 was an extended quarantine due to parvovirus infection that forced us to sacrificed 90% of our colony. At the beginning of this year, we were again in quarantine for a possible pinworm infection. Although tests came back negative, this prevented us form continuing our research for 2 months. Then in March Florida State University closed due to COVID-19. Although, we have faced these difficulties, we are confident we can finish this project if a non-cost extension (6 months) is granted. We plan to investigate new mechnaisms, as follows: a) Evaluate the effect of blackberry supplementation in adiponectin levels in vivo and in adiponectin signaling in vitro. b) Evaluate the mechanism by which polyphenol extracts regulate autophagy What opportunities for training and professional development has the project provided?Abigail Cullen, a graduate student and Carolyn Deeb, an undergraduate student volunteered in my lab worked in this project. Ann Centner, a new PhD student in my lab also contributed to this project. How have the results been disseminated to communities of interest?The results of this work were presented in the following conferences: Salazar, G., Zhao, Y., Cullen, A., & Deeb, C. Zinc and Polyphenols in Autophagy and Vascular Senescence. Presentation at ISZB Kyoto 2019, International Society for Zinc Biology, Kyoto, Japan. Akhavan, N., Parikh, K. R., Salazar, G., & Arjmandi, B. (accepted). The Antioxidative Effects of Borago Officinalis in Lipopolysaccharide and Hydrogen Peroxide-Activated RAW 264.7 Macrophages. Presentation to be given at Nutrition 2020, American Society for Nutrition, Seattle, WA. Cullen, A., Centner, A. M., & Salazar, G. (accepted). Regulation of the Phenotypic Switch of Vascular Smooth Muscle Cells by Adiponectin. Poster presentation to be given at Experimental Biology 2020, San Diego, California. What do you plan to do during the next reporting period to accomplish the goals?For this year (the final year of this proposal) we plan: a) Finish animal experiments and prepare manuscript. b) Finish the analysis of the in vitro data and manuscript preparation.

Impacts
What was accomplished under these goals? This proposal aims to elucidate whether blackberry supplementation reduces the development of plaque in vivo in ApoE and whether this effect is mediated by downregulation of Nox1. Below, we will describe our progress in the 3 aims that were proposed: Aim 1) Identify specific phenolic compounds in blackberry responsible for the downregulation of Nox1 expression, inflammation and senescence. We are preparing a manuscript in which we will report the effects of various polyphenols in vascular senescence, including 3-O-caffeoylquinic acid (3-CQA), the compound we hypothesized mediates the effect of blackberry, 4-O-caffeoylquinic acid (4-CQA) and 5-O-caffeoylquinic acid (5-CQA) found in different berries. Aim 2) Examine the contribution of the NF-κB/Nox1/COX-2 pathway to blackberry effects on ROS levels and senescence of VSMCs. We have found that in addition to reduce inflammation, berry polyphenols also regulate autophagy. We have expanded the focus of this aim to the characterization of this new phenotype and to finish the experiments related with the NF-κB/Nox1/COX-2 pathway. Aim 3) Determine the role of blackberry in reducing Nox1 expression, inflammation, vascular senescence and atherosclerosis in vivo. We have published a paper describing the role of blackberry supplementation (2% in the diet) in vascular senescence and atherosclerosis. We reported gender differences in the response to this fruit. Senescence, atherosclerosis and Nox1 expression were reduced in males, but not in female ApoE-/- mice. We also reported that the protective effect of blackberry in males was independent of lipid profile, since cholesterol, LDL, and cholesterol were not affected by treatment. We are currently evaluating whether 4% blackberry supplementation could be effective in females. We are also assessing whether pretreatment with 2 or 4% blackberry in the diet, before treatment with high fat diet is more effective in females. We have also collected animals (males and females) in which plaque regression was tested by treating animals with blackberry after high fat diet. We are currently analyzing the data. During last year, we also focused on additional mechanisms contributing to senescence of VSMCs, which is the main focus of this proposal. We published a manuscript in Autophagy (impact factor 11.1) describing the role of impaired autophagy in senescence. This work was supported by funds from this proposal. We also tested whether adiponectin deficiency promotes senescence of VSMCs that we intended to use as a new model to test polyphenol extracts. Surprisingly, VSMCs isolated form the adiponectin knockout mice showed reduced senescence and increased proliferation and migration. This data was presented at Florida State University and an abstract was accepted at EB 2020. Unfortunately, the conference was canceled due to COVID-19. The major difficulty we faced at the end of 2019 was an extended quarantine due to parvovirus infection that forced us to sacrificed 90% of our colony. At the beginning of this year, we were again in quarantine for a possible pinworm infection. Although tests came back negative, this prevented us form continuing our research for 2 months. Then in March Florida State University closed due to COVID-19. Although, we have faced these difficulties, we are confident we can finish this project if a non-cost extension (6 months) is granted.

Publications

• Type: Journal Articles Status: Published Year Published: 2020 Citation: Serino, A., Zhao, Y., Huang, J., Cullen, A., Deeb, C., Akhavan, N., Arjmandi, B., and Salazar, G. Gender differences in the effect of blackberry supplementation in vascular senescence and atherosclerosis in ApoE?/? mice. Journal of Nutritional Biochemistry, 2020, 80: 108375. PMID: 32248057
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Salazar, G., Cullen, A., Huang, J., Zhao, Y., Serino, A., Hilenski, L., Patrushev, N., Forouzandeh, F., and Hwang, H. SQSTM1/p62 and PPARGC1A/PGC-1alpha at the Interface of Autophagy and Vascular Senescence. Autophagy 2020, 16(6):1092-1110. doi:10.1080/15548627.2019.1659612
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Centner, AM and Salazar G. Nicotine in Senescence and Atherosclerosis. Cells 2020, 9(4): 1035.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2020 Citation: 1) Salazar, G., Zhao, Y., Cullen, A., & Deeb, C. Zinc and Polyphenols in Autophagy and Vascular Senescence. Presentation at ISZB Kyoto 2019, International Society for Zinc Biology, Kyoto, Japan
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2020 Citation: 2) Akhavan, N., Parikh, K. R., Salazar, G., & Arjmandi, B. (accepted). The Antioxidative Effects of Borago Officinalis in Lipopolysaccharide and Hydrogen Peroxide-Activated RAW 264.7 Macrophages. Presentation to be given at Nutrition 2020, American Society for Nutrition, Seattle, WA
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2020 Citation: 3) Cullen, A., Centner, A. M., & Salazar, G. (accepted). Regulation of the Phenotypic Switch of Vascular Smooth Muscle Cells by Adiponectin. Poster presentation to be given at Experimental Biology 2020, San Diego, California

Progress 03/01/18 to 02/28/19

Outputs
Target Audience:The target audience reached by this project includes graduate and undergraduate students that were trained because of NIFA funding, as well as the scientific community reached during the conferences in which the result of this research were presented. Changes/Problems:The only difficulty we faced this year was that a PhD student working on this project was unable to renew her Visa and re-enter the country in January 2019. Thus, no funds were used for the salary of graduate student during Spring 2019. A new PhD student will be assigned to this project starting Summer 2019. We will also adjust our plan related with female mice since no protective effects were seen in females supplemented with blackberry. We will increase blackberry from 2% to 4% in the diet. We will also pretreat females with blackberry in control diet before feeding them with the high fat diet. Although the lack of effect in females was unexpected, this result is relevant to human health and suggest gender differences in responses to nutrients. What opportunities for training and professional development has the project provided?During this past year one PhD student (Zahra Mostofinejad) and one undergraduate student (Alexa serino) in honors thesis worked with the PI on this project. As mentioned before, as a result of this funding, Alexa Serino graduated with honors in Fall 2019 after completing and defending her honors thesis. Additionally, our lab is also open to undergraduate students seeking experience in research. Carolyn Deeb, an undergraduate student volunteered in my lab by assisting with animal experiments. She worked with Alexa weighting animals in diet, genotyping and collecting tissues, as well as preparing samples for the analysis of protein expression by western blots. How have the results been disseminated to communities of interest?The results of this work were presented in the following conferences: 1) Nutrition 2018, American Society for Nutrition, Boston MA. Salazar G, Zhao Y, and Huang, J. (presented 2018, June). Blackberry supplementation reduces vascular aging and atherosclerosis induced by high fat diet in ApoE-/- mice. 2) Society for Redox Biology and Medicine (SfRMN) Annual Conference 2019, Chicago, IL. Salazar G, Huang J and Zhao Y. (presented 2018, November). SQSTM1/p62 deficiency induces oxidative stress and senescence in vivo and in vitro in VSMCs. What do you plan to do during the next reporting period to accomplish the goals?Based on these observations in female mice, we will adjust our original experimental plan as follows: a) Supplementing female mice with 4% blackberry instead of 2%. It is unknown whether absorption of polyphenols in the small intestine is reduced in males compared with females. It is also unknown whether the microbiome plays a role in the effect of blackberry and whether the microbiome respond differently to the HFD in females than males. b) Treating females with 2% blackberry (in control low fat diet) for 2 weeks before HFD treatment. Pretreatment with blackberry may improve the antioxidant capacity and/or other protective mechanisms that could prevent the formation of plaque during the HFD treatment. In the case of males, we will continue with the original plan by supplementing males with 3-O-caffeoylquinic acid (3-CQA) by gavage. A PhD student is being trained for these studies. Since blackberry was effective in reducing plaque in males, we will test whether blackberry induce the regression of plaque, as originally proposed. These experiment will also be performed in female mice. We will also continue with our in vitro studies testing the role of the NF-kB/COX2 signaling pathway in the regulation of Nox1 and senescence in VSMCs.

Impacts
What was accomplished under these goals? Cellular senescence influences organismal aging increasing predisposition to age-related and inflammatory diseases. This is because senescent cells have an active role in generating oxidative stress and pro-inflammatory molecules inducing tissue dysfunction. The increase in life expectancy, particularly in developed countries has considerably increased the population at risk of developing these diseases. It is anticipated that the population 85 and older will triple by 2050 (US Census Bureau 2008 national population projections). Thus, studies designed to understand the senescence or aging process leading to nutritional strategies, the focus of this proposal, to reduce inflammation and promote healthier aging, are urgently needed to cope with these imminent challenges for public health. We have previously demonstrated that blackberry polyphenol extract reduced senescence of vascular smooth muscle cells in culture. These cells are found in blood vessels and are critically involved in the formation of the atherosclerotic plaque. We also demonstrated that this extract prevented senescence by reducing the expression of the NADPH oxidase Nox1. Nox1 is an enzyme that produces superoxide inducing damage to proteins, lipids and the DNA. It is well known that Nox1 expression is upregulated in atherosclerotic plaque in human patients and that Nox1 mediates the formation of plaque in animal models of atherosclerosis, including the ApoE knockout (ApoE-/-) mice. This proposal aims to elucidate whether blackberry supplementation reduces the development of plaque in vivo in ApoE and whether this effect is mediated by downregulation of Nox1. Below, we will describe our progress in the 3 aims that were proposed: Aim 1) Identify specific phenolic compounds in blackberry responsible for the downregulation of Nox1 expression, inflammation and senescence. So far, we have tested the effect of blueberry, strawberry and cornus officionalis, a berry used in Chinese Traditional Medicine. The analysis of the polyphenol content of these berries showed that blueberry is low in 3-O-caffeoylquinic acid (3-CQA), the compound we hypothesized mediates the effect of blackberry. However, blueberry has a high content of 4-O-caffeoylquinic acid (4-CQA, 486.3) and also contains 5-O-caffeoylquinic acid (5-CQA, 118.6ppm). Cornus officionalis, on the other hand, has the highest levels of these compounds including gallic acid (1017.1 ppm), 3-CQA (775.9 ppm), 4-CQA (209.3 ppm) and 5-CQA (1371.9 ppm). Similar to blackberry polyphenols, we have found that blueberry and cornus officionalis reduced senescence of vascular smooth muscle cells (VSMCs) and that cornus officionalis also reduced Nox1 expression. We are in the process of testing the effect of blueberry polyphenols in Nox1 expression. We also found that the compounds 3-CQA, 4-CQA and 5-CQA act similarly by reducing senescence of VSMCs. The effect of these compounds in Nox1 expression is also being tested. Aim 2) Examine the contribution of the NF-κB/Nox1/COX-2 pathway to blackberry effects on ROS levels and senescence of VSMCs. We have mainly focused on aims 1 and 3 during this year. We plan to addressed this aim in this upcoming year. Aim 3) Determine the role of blackberry in reducing Nox1 expression, inflammation, vascular senescence and atherosclerosis in vivo. To address this aim, we fed the ApoE-/- male mice high fat diet (HFD) or a HFD supplemented with 2% freeze-dried powder for 5 weeks and measured plaque, senescence and the expression of Nox1. First, we confirmed the preliminary data that was presented in the original proposal showing that animals fed a HFD plus blackberry showed reduced plaque and senescence. We repeated this experiment using older male mice (6 months old males) to determine the effect of aging in the effect of blackberry. We observed a robust reduction in plaque and senescence, which correlated with a reduction in Nox1 expression in the aorta tissue. As we observed before (preliminary data), there was no differences in food consumption or body weight in response to blackberry supplementation. We measured the lipid profile in both groups and found no differences in total cholesterol, triglycerides or LDL levels. In order to identify possible blackberry metabolites in blood, we initiated a collaboration with Dr. Colin Kay at the Plants for Human Health Institute, North Carolina State University. Unfortunately, since these animals were fed a HFD, the high levels of lipids in the blood samples interfered with the analysis. We will repeat this analysis by feeding a low fat diet control with and without blackberry for the analysis of polyphenol metabolites. As proposed, we also used female mice to test the effect of blackberry in atherosclerosis. Surprisingly, blackberry showed no effect in plaque in female mice. Similar to males no change in lipid profile was observed in response to blackberry. In line with our hypothesis that downregulation of Nox1 mediates blackberry effects, no change in Nox1 was observed in females. A manuscript reporting these finding is in the last step of preparation and will be submitted to Nutrients at the end of June 2019. During this year, we also focused on additional mechanisms contributing to senescence of VSMCs, which is the main focus of this proposal. We submitted a manuscript that is under review in Autophagy (impact factor 11.1) describing the role of impaired autophagy in senescence. This work was supported by funds from this proposal. We are investigating whether Nox1 inhibits autophagy to promote senescence of VSMCs. Thus, during this year we have made significant progress in in vitro (aim 1) and in vivo (aim 3) experiments, as originally proposed in our timeline. Also, Alexa serino, an undergraduate student in honors thesis graduated in Fall 2018 with the dissertation entitle "Novel Role of Blackberries in Vascular Senescence and atherosclerosis in ApoE-/- Mice" in which she tested the role of blackberries in female mice. Alexa is also the first author of an invited review article published in January 2019 directly related with this proposal. In this manuscript, we reviewed published work on the effect of various fruits and vegetables rich in polyphenols in reducing cardiovascular disease in animal models.

Publications

• Type: Journal Articles Status: Published Year Published: 2018 Citation: Alexa Serino and Gloria Salazar. Protective Role of Polyphenols against Vascular Inflammation, Aging and Cardiovascular Disease. Nutrients 2019 Jan; 11(1):53.
• Type: Journal Articles Status: Submitted Year Published: 2019 Citation: Alexa Serino, Yitong Zhao, Jingwen Hwang, Abigail Cullen, Carolyn Deeb, Neda Akhavan, Bahram Arjmandi and Gloria Salazar. Gender Differences in the Effect of Blackberry Supplementation in Vascular Senescence and Atherosclerosis in ApoE-/- mice. Submitted to Nutrients.
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Salazar G, Zhao Y, and Huang, J. (presented 2018, June). Blackberry supplementation reduces vascular aging and atherosclerosis induced by high fat diet in ApoE-/- mice. Oral presentation in Nutrition 2018, annual meeting, Boston MA, June 2018
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Salazar G, Huang J and Zhao Y. SQSTM1/p62 deficiency induces oxidative stress and senescence in vivo and in vitro in VSMCs. Oral presentation in the Annual Conference of the Society for Redox Biology and Medicine (SfRMN), Chicago, November 2018.
• Type: Theses/Dissertations Status: Published Year Published: 2018 Citation: Novel Role of Blackberries in Vascular Senescence and atherosclerosis in ApoE-/- Mice. Dissertation of Alexa Serino, undergraduate student in Honors Thesis.
• Type: Journal Articles Status: Under Review Year Published: 2019 Citation: Gloria Salazar, Abigail Cullen, Jingwen Huang, Yitong Zhao, Alexa Serino, Lula Hilenski, Nikolay Patrushev, Farshad Forouzandeh and Hyun Seok Hwang. SQSTM1/p62 and PGC-1alpha at the Interface of Autophagy and Vascular Senescence. Under review in Autophagy.