Effects of Aging on Experimental Atherosclerosis in Nonhuman Primates

National Institute On Aging

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Abstract

Aortic, carotid, and myocardial tissues harvested from sixteen rhesus monkeys fed an atherogenic diet (high fat and high cholesterol diet: HCD) and ten monkeys with a control diet (standard diet: SD) for two years have been analyzed. Blood pressure, pulse wave velocity (PWV), and lipid profile have been also analyzed. Young (6-15 years-old) and old rhesus monkeys (16-28 years-old) fed an HCD/ a SD have been randomly categorized into two age groups. Total blood cholesterol levels are increased by 2-fold in both young and old HCD monkeys compared to SD from after the sixth month of the experiment. HCD-induced increases in intimal thickening, lipid deposition, calcification, atherosclerotic plaque burden and necrotic core size have been observed in old versus young arterial walls. 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 has been 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 monkeys 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, driven by a chronic inflammatory environment, accompanies arterial diseases, the leading cause of an enhancement of arterial stiffening, atherosclerosis burden and vulnerability in the older population. An increase in central arterial wall stiffening, measured as an increase in aortic pulse wave velocity (PWV) an index of arterial stiffness, is a major risk factor for the development of atherosclerosis. We have found that in rhesus monkeys, a 2-year diet high in fat and sucrose (HFS), increases not only body weight and cholesterol, but also induces prominent central arterial wall stiffening, increases PWV and arterial inflammation, which is an ideal experimental model for metabolic disorder syndrome in humans. We have 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 effectively prevents 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 arterial stiffening and arterial health. The above studies demonstrate that inflammatory remodeling in the vascular wall is a cardinal molecular event of vascular fibrosis that exacerbates stiffening, calcification, and the progression of atherosclerosis. Recent studies have demonstrated 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. Our findings provide robust evidence that DDR2 is a novel determinant of vascular fibrosis associated with vascular pathologies such as calcification and atherosclerosis and a therapeutic molecular target. Interestingly, we have found that resveratrol 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 have found that 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. Targeted transcriptomic analysis in Drosophila has 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 have 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) exhibits myocardial damage and activates Drp1 levels in the heart. Interestingly, HCD decreases nicotinamide adenine dinucleotide (oxidized) (NAD+) levels and increased Drp1 acetylation. In adult monkey cardiomyocytes, palmitate increases Drp1 acetylation, phosphorylation, and protein levels, which is 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 creates an intracellular environment to facilitate Drp1 acetylation, which increases 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 have found 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 lead 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. Cardiac SIRT1/SIRT3 deficiency in aging alters mitochondrial morphology characterized by the impairment of mitochondria fusion under I/R stress.