Performing Department
(N/A)
Non Technical Summary
Ischemia with non-obstructive coronary artery disease (INOCA) afflicts mainly women and is responsible for an increased risk of cardiovascular deaths, myocardial infarction, and heart failure. The underlying pathological mechanism that drives INOCA is coronary microvascular dysfunction (CMD), an impairment of the cardiac microvessels of the epicardium. A myriad of untargeted medications are used to treat this condition; however, none of these drugs are fully efficacious, and no targeted therapy currently exists in treating INOCA. Thus, the long-term goal of this research is to develop an alternative safe, efficacious, and side-effect-free nutritional strategy to be used in the clinical setting to improve INOCA and advance our understanding of the interplay between diet, gut microbiota, and cardiovascular disease. Plant-based diets have been associated with reduced cardiovascular disease incidence, and pilot clinical studies indicate that a polyphenol-rich plant-based diet is protective of the macrovasculature both peripherally and in the heart. Additionally, the gut microbiota is critically involved in the metabolism of polyphenols which can also result in a microbial population shift. Thus, the central hypothesis of this proposed study is that the consumption of a polyphenol-rich plant-based diet can favorably alter gut microbial populations due to the polyphenol content of the diet and thereby improve INOCA by targeting CMD. The following specific aims have been proposed: 1) Investigate whether a plant-based diet via the microbiome can prevent or treat INOCA induced by hypertension in female spontaneously hypertensive rats (SHRs), and 2) Investigate whether a plant-based diet targets cardiac microvascular endothelial cells (ECs) and vascular smooth muscle cells (VSMCs) in CMD via the microbiome. SHR's will be utilized as they develop CMD and INOCA, and female animals will be utilized since INOCA afflicts mostly woman. Animals will be fed a diet supplemented with or without polyphenol-rich plant-foods in addition to with or without antibiotics to elucidate the role of the gut microbiome in mediating the efficacy of the intervention. Echocardiography and innovative magnetic resonance imaging techniques will be utilized to assess the microvascular function of the heart. Polyphenols will be quantified in both serum and within cardiac tissue via mass spectroscopy. Gut microbiota population shifts will be assessed with 16S RNA sequencing in feces. Additionally, ECs and VSMCs will be isolated from the heart and stimulated with their serum containing microbiota-derived polyphenol metabolites. Intracellular oxidative stress, inflammation, endothelial nitric oxide handling , and VSMC calcium homeostasis and elasticity will be assessed. This proposed project investigates the efficacy of a plant-based diet in the treatment of INOCA, an understudied disease that afflicts mostly females, a population for which research is lacking. This proposed study also will advance translational research, as substantial preliminary data will be generated and utilized for clinical trial funding in future grant applications.
Animal Health Component
0%
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Goals / Objectives
The goals of this award include both career and scientific goals. With respect to training, goals of this award include enhancement of research capabilities, improvement of scientific achievements, and development of career independency of the fellow. To achieve this goals, the following objectives will be met:Objective 1: Develop professional skills to facilitate career independenceObjective 2: Enhance scientific skills to develop a new and independent research programWith respect to the scientific goals, the long-term goal of this research is to develop an alternative safe, efficacious, and side-effect-free nutritional strategy to be used in the clinical setting to improve ischemia with non-obstructive coronary artery disease (INOCA) and advance our understanding of the interplay between diet, gut microbiota and cardiovascular disease.To achieve this long-term goal, the following specific aims have been proposed:Specific Aim 1. Investigate whether a plant-based diet via the microbiome can prevent or treat INOCA induced by hypertension in female spontaneously hypertensive rats (SHRs).Specific Aim 2. Investigate whether a PBD targets cardiac microvascular endothelial cells and vascular smooth muscle cells in coronary microvascular dysfunction via the microbiome.
Project Methods
To address Specific Aim 1, the following methods will be conducted:Experimental plan: Female, four-week-old spontaneously hypertensive rats (SHRs) will be divided into four dietary groups: 1) a control group will consume a control diet (AIN-93G), without any intervention, 2) a control group with antibiotics (ABX) in drinking water (0.2% sucrose to increase palatability) for the first 4 weeks (1 g/L of ampicillin, neomycin sulphate, metronidazole, and 0.5 g/L of vancomycin) followed by a ? concentration maintenance dose for the remainder of the study, 3) a plant-based diet (PBD), comprised of 28% (w/w; AIN-93G) polyphenol-rich plant-based foods (freeze-dried and powdered) that are commonly available and cultivated in the United States or 4) a PBD + ABX to determine the contribution of the gut microbiota to the PBDs hypothesized efficacy. Wistar Kyoto (WKY) rats will serve as normotensive controls due to their genetic similarities to SHRs, and will consume the control diet alone. The PBD used in this study will contain 4% each of the following seven fruits, vegetables, nuts and legumes: red bell pepper, Brussel sprouts, sweet potato, lemon, blueberry, black beans and walnuts. An innovative prevention and treatment model will be implemented to allow for the establishment of INOCA severity at 24 weeks in SHRs consuming the control diet. Changes in disease progression will be assessed 12 weeks after this 24-week time point utilizing the dietary and drug interventions, simulating a treatment model, typical of the clinical setting. BP will be moitored every two weeks. Hearts will be excised from animals from each group in both prevention and treatment arms during sacrifice (Fig. 6), and LVs will be utilized for protein and mRNA expression as well as polyphenol analysis (n=5) as well as histological evaluation (n=3). Additionally, serum and feces will be collected for downstream applications.To address Specific Aim 1, the following techniques will be utilized.Echocardiogram: Functional and morphological characteristics of the left ventricle (LV) of the heart will be assessed via echo in M-mode. In addition, to assess cardiac microvascular function diagnostically, coronary flow reserve (CFR) will be assessed by measuring coronary flow via color doppler. Briefly, following a basal measurement under resting conditions, an infusion pump will be utilized to deliver Dobutamine at a concentration of 20 µg/kg/min to facilitate cardiac stress and allow measurement of maximal flow velocity. Echocardiographic assessments will be conducted at weeks 12, 24 and 36.MRI: Animals will be injected with a novel collagen-specific molecular probe (ProCA32.collagen1) that allows for in vivo visualization of Type 1 collagen in the heart with T1 mapping via MRI. Additionally, T1 mapping will be used to assess CMD by injecting animals with L-NAME (100mg/kg; an eNOS inhibitor) and assessing changes in myocardial fluid flux as previously described. Changes in fluid balance indicate normal microvascular function, whereas no or little change indicates dysfunction. Lastly, to further evaluate the extent of ischemic damage, we will assess the myocardial area at risk via T1 and T2 mapping. MRI assessments will occur at weeks 24 and 36.Protein & mRNA expression: Protein will be extracted from the LVs of hearts and western blot will be used to detect the protein expression of NOX isoforms, inflammatory cytokines and signaling via mitogen-activated protein kinases (MAPKs) and nuclear factor kappa B (NF-κB) complex, as well as endogenous antioxidant defense enzymes and proteins involved in cardiac remodeling (matrix metalloproteinases) as previously described. mRNA will be isolated from LV of hearts. mRNA expression of hypertrophic genes ANP, BNP, β-MHC, GATA4, GATA6, SRF and Nkx2-5 will be assessed via qPCR as described previously.Histology: Hearts, once excised, will be stored in formalin, and subsequently paraffanized. Hypertrophy of cardiomyocytes will be assessed with H&E and WGA staining. Trichrome staining will be used to assess cardiac fibrosis and also corroborate fibrosis identification in MRI measures.Microbial analysis: Feces will be collected at baseline, week 4 (following acute antibiotic treatment), week 12, week 24 and week 36. Culture of fecal bacteria will occur via 16S rRNA gene sequencing to determine bacterial populations.To address Specific Aim 2, the following methods will be conducted:Experimental Plan: A subset of hearts (n=4/group) will be collected from all treated groups as described in Aim 1 and will be used for microvascular endothelial cell (EC) and vascular smooth muscle cell (VSMC) isolation with modified protocols. Hearts will be sliced, and a portion of these slices will be used for EC and VSMC isolation and purification. Purity will be assessed by staining cells with immunofluorescent antibodies specific to ECs and VSMCs. Isolated cells will be cultured in rat serum collected from the animals of their respective treatment group to simulate in vivo conditions. Polyphenols analysis in serum and tissue will be conducted. To address Specific Aim 2, the following techniques will be utilized.eNOS activity: Serum nitric oxide (NO_ metabolites will be quantified from a commercially available kit (Cayman Chemical, Ann Harbor, MI). Isolated microvascular ECs will be cultured in black 96-well plates, and incubated with 5 µM DAF 2-DA to fluorescently detect intracellular nitric oxide production as previously described. Separately, ECs will also be grown in 60 mm dishes. Following 24 h incubation in respective serum conditions, cells will be collected to detect the protein expression of eNOS and eNOS phosphorylation sites, Ser1177 and Thr495, activator and inhibitory sites, respectively, utilizing Western Blot.Intracellular oxidative stress and inflammation: Both isolated ECs and VSMCs will be treated with DHE and CM-H2DCFDA in 96-well black plates to detect intracellular O2- and H2O2 production fluorometrically, respectively. In ECs specifically, ONOO- will also be quantified fluorescently with a commercially available kit (Abcam, Waltham, MA). Additionally, proteins will be detected from both cells to quantify pro-oxidant NOX isoforms, xanthine oxidase, and antioxidant enzymes via Western Blot. Phosphorylation of inflammatory signaling via MAPKs and NF-κB will also be assessed.VSMC Ca2+ homeostasis and elasticity: Calcium flux in VSMCs will be monitored intracellularly via the fluorometric probe, Flou-8 (Abcam, Waltham, MA), by treating with phenylephrine, a Ca2+ stimulator. Protein will be isolated from VSMCs as described above, to detect the expression of redox-sensitive soluble guanylyl cyclase, an upstream effector of Ca2+ reduction, Ca2+-dependent and calmodulin-dependent protein kinase II (CaMKII), which can be increased by ROS or Ca2+ flux, via Western Blot. SR-specific release and sequestration proteins, ryanodine receptor 2 (RYR2) and SR-Ca2+ ATPase (SERCA2a), will also be quantified. VSMC stiffness will be measured at the single cell level and tissue level by using AFM ad 3D reconstituted tissue model as described previously.Evaluation planThe PD will engage in weekly meetings with Dr. Andrew Gewirtz (project mentor) to discuss progress, as well as weekly journal club meetings during the entirety of this project. Semi-annual presentation of data with Dr. Gewirtz's lab will ensure appropriate progress of this project in aspects of technical training, mentorship, career development and the overall progress of data collection.