Effects of Aging on Experimental Atherosclerosis in Nonhuman Primates

National Institute On Aging

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Abstract

Sixteen rhesus monkeys were assigned an atherogenic diet (high fat and high cholesterol diet: HCD) and ten monkeys a control diet (standard diet: SD) after baseline measurements were collected; blood pressure, pulse wave velocity (PWV) and lipid profile. Randomly chosen young (6-15 years-old, n=8) and old rhesus monkeys (16-28 years-old, n=8), were fed HCD for two years. Additional young and old monkeys (n=5 each) were maintained on a SD. Total blood cholesterol levels increased by 2-fold in both young and old HCD monkeys compared to SD from the sixth month forward. HCD-induced lipid deposition increased within the aortic intima of all subjects, and enhanced intimal thickness, atherosclerotic plaque burden and plaque necrotic core size were observed in a greater extent in old aortae. Aortic wall calcification increased with age regardless of diet. Thus, risk of atherosclerosis is attributable to age and is not due to longer exposure to dyslipidemia but rather stems from age-associated remodeling of the arterial wall, i.e., the soil in which atherosclerosis flourishes. A comprehensive quantitative proteomic study of the common carotid artery in monkeys fed with an HCD or SD was also performed and analyzed. We found 8 proteins that were less abundant and 12 proteins that are more abundant in old animals. Proteins that are less abundant in old animals are: Protein S100-A6, Isocitrate dehydrogenase NADP, Myelin P0 protein, Myosin-7, Aldo-keto reductase family 1-member B10, Protein S100-A4, Calmodulin and SPATS2-like protein. Proteins that are more abundant in old animals include Programmed cell death protein 6, Periostin, Apolipoprotein E, Erythrocyte band 7 integral membrane protein, Translation initiation factor IF-2, Complement component C9, Lactadherin (MFG-E8), Apolipoprotein C-I, Vitronectin, Annexin A7, Cysteine and glycine-rich protein 2, Serine protease HTRA1, Serum amyloid P-component , Complement C1q , and tumor necrosis factor-related protein. We are validating these molecules of interesting and investigation their bio-roles in the pathogenesis of age-associated atherogenesis. The above findings suggest that central arterial wall remodeling and stiffening, driven by a chronic inflammatory environment, accompanies arterial diseases, the leading cause of atherosclerosis burden and vulnerability in the older population. An increase in central arterial wall stiffening, measured as an increase in aortic PWV, is a major risk factor for adverse physiologic events. In rhesus monkeys, a 2-year diet high in fat and sucrose (HFS), increased not only body weight and cholesterol, but also induced prominent central arterial wall stiffening, increased PWV and arterial inflammation, which is an ideal experimental model for metabolic disorder syndrome. We found that the loss of endothelial cell integrity (destruction), lipid and macrophage infiltration, and calcification of the arterial wall are driven or robustly supported by genomic and proteomic signatures of oxidative stress and inflammation. Very importantly, resveratrol, a SIRT2 agonist, treatment prevented the HFS-induced arterial wall inflammation, destruction, and the accompanying increase in PWV. Dietary resveratrol has the potential of becoming a promising therapy to ameliorate increases in PWV and arterial health. The above studies demonstrate that collagen accumulation and remodeling in the vascular wall is a cardinal molecular event of vascular fibrosis that exacerbates stiffening, calcification, and the progression of atherosclerosis. Recent studies demonstrate that augmented collagen deposition and adverse matrix remodeling strongly correlate with an increased expression of the collagen-specific receptor tyrosine kinase, discoidin domain receptor 2 (DDR2), in the aorta of rhesus monkeys fed an HFS diet for 24 months. Together with findings demonstrating DDR2-dependence on collagen gene expression in vascular cells exposed to hyperglycemic conditions in vitro, our findings provide robust evidence that DDR2 is a novel determinant of vascular fibrosis associated with vascular pathologies such as calcification and atherosclerosis, and hence a therapeutic molecular target. Interestingly, we find that resveratrol also alleviates collagen deposition and remodeling in the vascular wall via the inhibition of DDR2 expression, which is associated with decreases in elastin degradation and calcification and atherosclerosis in nonhuman primates. In collaboration with Dr. Engler (Department of Bioengineering, University of California, San Diego), we found when studying the cardiac proteomes of both young and old rhesus monkeys, rats, and flies, certain age-associated remodeling events within the cardiomyocyte cytoskeleton are highly conserved and beneficial rather than deleterious. Targeted transcriptomic analysis in Drosophila confirmed conservation and implicated vinculin as a unique molecular regulator of cardiac function during aging. Cardiac-restricted vinculin overexpression reinforces the cortical cytoskeleton and enhances myofilament organization, leading to improved contractility and hemodynamic stress tolerance in healthy and myosin-deficient fly hearts. Moreover, cardiac-specific vinculin overexpression markedly increases the median life span in flies. In collaboration with Dr. Wang Wang (University of Washington, Seattle), we investigated the regulation and roles of the mitochondrial fission protein, the dynamin-related protein 1 (Drp1), in lipid overload-induced cardiomyocyte death and heart dysfunction in nonhuman primates. Monkeys fed a HCD (2 years) also exhibited myocardial damage and activated Drp1 levels in the heart. Interestingly, HCD decreased nicotinamide adenine dinucleotide (oxidized) (NAD+) levels and increased Drp1 acetylation. In adult monkey cardiomyocytes, palmitate increased Drp1 acetylation, phosphorylation, and protein levels, which were abolished by restoring the decreased NAD+ levels. Drp1 acetylation at lysine 642 (K642) is increased by HCD in monkey cardiomyocytes. The non-acetylated Drp1 mutation (K642R) attenuates palmitate-induced Drp1 activation, its interaction with voltage-dependent anion channel 1, mitochondrial fission, contractile dysfunction, and cardiomyocyte death. Excessive lipid supply created an intracellular environment to facilitate Drp1 acetylation, which increased its activity and mitochondrial translocation causing cardiomyocyte dysfunction and cell death. Thus, Drp1 could be a critical mediator for lipid overload-induced adverse myocardial remodeling as well as a potential target for therapy. In collaboration with Dr. Ji Li (University of South Florida, Tampa), we find that alterations in mitochondrial dynamics with age-related Sirtuin1/Sirtuin3 deficiency impair cardiomyocyte contractility in both mice and monkeys. Sirtuin1 (SIRT1) and Sirtuin3 (SIRT3) protects cardiac function with advancing age against ischemia/reperfusion (I/R) injury. The findings indicate that deficiency of SIRT1 and SIRT3 in aged mice hearts led to exacerbated cardiac dysfunction. Moreover, the deletion of SIRT1 or SIRT3 in young mice hearts impair cardiomyocyte contractility and shows aging-like cardiac dysfunction upon I/R stress, indicating the crucial role of SIRT1 and SIRT3 in protecting myocardial contractility from I/R injury. SIRT1/SIRT3 deficiency leads to the inactivation of Adenosine monophosphate-activated protein kinase (AMPK) and alterations in mitochondrial oxidative phosphorylation (OXPHOS) that causes impaired mitochondrial respiration in response to I/R stress. Furthermore, remodeling of the mitochondrial network goes accompanies hypoxic stress, and mitochondria. Cardiac SIRT1/SIRT3 deficiency in aging alters mitochondrial morphology characterized by the impairment of mitochondria fusion under I/R stress.