Effects of Aging on Experimental Atherosclerosis in Nonhuman Primates

National Institute On Aging

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Abstract

Aortic, carotid, iliac, coronary arterial walls, peri-coronary fat tissue, 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) feed an HCD, or a SD, 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 and there is no significant difference between young and old HFD groups. HCD-induced increases in intimal thickening, inflammation, amyloid deposition, lipid deposition, calcification, atherosclerotic plaque burden, and necrotic core size have been observed in old versus young arterial walls. Thus, the risk of atherosclerosis is attributable to age and is not due to longer exposure to dyslipidemia but stems from age-associated vulnerability/susceptibility of the arterial wall, i.e., the fertile soil in which atherosclerosis origins and flourishes. A comprehensive 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, Milk Fat Globule EGF VIII (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. These increased proteins are closely associated with age-related increased amyloidosis and inflammation in carotid artery and arteriopathy. Central arterial wall remodeling is driven by a chronic metabolic disorder syndrome such as obesity and dyslipidemia in the older population. 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 thickening and stiffening, increases in aortic pulse wave velocity (PWV), and arterial inflammation. We have found that the loss of endothelial cell integrity (destruction), lipid and macrophage infiltration, elastolysis, fibrosis, 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, elastolysis, fibrosis, and the accompanying increase in PWV. Inflammatory remodeling in the vascular wall is a cardinal molecular event of vascular fibrosis that exacerbates stiffening, calcification, and the progression of atherosclerosis. 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 elastolysis, calcification and atherosclerosis and a therapeutic molecular target in non-human primates with aging. Interestingly, we have found that resveratrol alleviates collagen deposition, elastolysis, and other inflammatory 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. Importantly, we have found that the cardiac proteomes of both young and old rhesus monkeys, rats, and flies, that 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. Notably, we have found that cardiomyocytes exhibit progressive loss of Lamin C (mammalian Lamin A/C homologue) with advancing age, coincident with decreasing nuclear size and increasing nuclear stiffness in both flies and monkeys. We have found that premature genetic reduction of Lamin C phenocopies aging's effects on the nucleus and subsequently decreases heart contractility and sarcomere organization. Subsequently, we find a role for cardiac transcription factors in modulating adult heart contractility and show that maintenance of Lamin C, and cardiac transcription factor expression, slows age-dependent cardiac decline. We also 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 activated 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. We also have found that alterations in mitochondrial dynamics with age-related Sirtuin1/Sirtuin3 deficiency impair cardiomyocyte contractility in both mice and monkeys. 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. Cardiac SIRT1/SIRT3 deficiency in aging alters mitochondrial morphology characterized by the impairment of mitochondria fusion under I/R stress. In addition, Discoidin domain receptor 2 (DDR2), a collagen-activated receptor tyrosine kinase predominantly located in cardiac fibroblasts, has an obligatory role in cardiac fibroblast function and cardiovascular fibrosis. The detailed Investigation on the molecular basis of cardiovascular aging and dysregulated fibroblast function in the senescent heart would pave the way for effective strategies to mitigate cardiovascular diseases in a rapidly growing elderly population. Recently, we assessed age-related changes in the expression of Nuclear Factor (Erythroid derived 2)-Like 2 (NFE2L2 or NRF2) in the myocardium of humans, Rhesus monkeys, Fisher rats, and C57BL/6J mice. NRF2 is a central regulator of antioxidant and detoxification genes. Analyses of RNA-seq data from the Genotype-Tissue Expression (GTEx) project, which includes left ventricular samples from 294 male donors, showed an age-associated decline in NRF2 transcript and its downstream genes (SOD1, SOD2, CAT, GCLM, AKR1B). An age-dependent decrease in NRF2 mRNA or protein was observed in the myocardia of Rhesus monkeys and Fisher rats. To demonstrate that loss of NRF2 contributes to myocardial aging, the cardiac function of NRF2 knockout (KO) mice was evaluated at 19 and 24 months of age. At 19 months, NRF2KO mice showed diastolic dysfunction with increased end diastolic volume (EDV) and end systolic volume (ESV), while ejection fraction (EF) and fractional shortening (FS) were significantly reduced, indicating early onset heart failure. NRF2KO mice exhibited premature aging phenotypes and reduced lifespan. These findings support an age-related decline in NRF2 signaling across species and the early onset of maladaptive cardiac remodeling due to NRF2 loss. In collaboration with Drs. Myriam Gorospe/Ali Herman (National Institute on Aging, National Institutes of Health). We have reported that senescent vascular smooth muscle cells (VSMCs) accumulate in the vasculature with age and tissue damage and secrete factors such as dipeptidyl peptidase 4 (DPP4) that promote atherosclerotic plaque burden in murine, nonhuman primates, and human samples. Increased levels and activity of DPP4, a serine protease, in senescent VSMCs. Analysis of the conditioned media from senescent VSMCs revealed a unique senescence-associated secretory phenotype signature comprising many complement and coagulation factors; silencing or inhibiting DPP4 reduced these factors and increased cell death. Importantly, DPP4 inhibition reduced senescent cell burden and coagulation and improved plaque vulnerability through the endomorphic and senolytic effects of DPP4 inhibition in atherosclerosis. Thus, the inhibition of DPP4 is a potential molecular therapeutic approach to the prevention and treatment of age-associated arterial remodeling and disease.