Effects of Aging on Experimental Atherosclerosis in Nonhuman Primates

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Following collection of baseline data, 16 monkeys had been assigned to an atherogenic diet and 10 monkeys to a control diet. Baseline, cholesterol, triglycerides, and glucose were not different between the control and treated groups. At the second follow-up, cholesterol levels in the treated group were different from controls (p<0.0001). At the second follow-up, glucose was related with body weight in the controls but not in the treated group. After sacrifice, we harvested arterial tissue. Histochemical observation and morphological analysis indicated that age increases intimal thickness and medial thickness along with atherosclerotic lesions in these domestic monkeys fed normal diets. Interestingly, age affects fat deposition within arterial walls in these domestic monkeys with high cholesterol diets. Importantly, we found that a high cholesterol diet increases adverse histopathologic events and plaque burden, which are closely associated with changes in cholesterol, glucose, diastolic blood pressure, pulse wave velocity. Further studies indicate that high cholesterol diet reprograms the aged arterial wall via further damage of endothelial integrity and macrophage infiltration, foam cell formation, and fat or calcium deposits, contributing to a vulnerable thickened intima for the accelerated occurrence of adverse histopathologic events, including plaque burden. Comprehensive quantitative proteomic studies were designed to analyze proteomic changes of carotid arteries in the different conditions. We have found 8 proteins are less abundant and 12 proteins are more abundant in old animals. For example, the proteins which are less abundant 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. The proteins which are more abundant 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 and Complement C1q tumor necrosis factor-related protein. micro RNA arrays of carotid arteries in monkeys with or without high cholesterol diets indicate that there are totally 91 modified miRs, via a continuous age analysis. 15 miRs significantly change with aging, 67 miRs are significantly different in abundance associated with high cholesterol diet treatment. Part of PCR validation results showed that miR-21, miR-34a, miR-155, miR-210, miR-199a, miR-199a-3p, miR-423-5p, let-7i are indeed modulated with age and/or high cholesterol diet treatment. Furthermore, we found that carotid arteries of aged rhesus macaques exhibit significant oxidative stress (as indicated by the increased 8-iso-PGF2 and 4-HNE content and decreased glutathione and ascorbate levels) as compared with vessels of young macaques that is associated with activation of the redox-sensitive proinflammatory transcription factor, nuclear factor-kappaB. However, age-related oxidative stress does not activate Nrf2 and does not induce Nrf2 target genes (NQO1, GCLC, and HMOX1). In cultured vascular smooth muscle cells (VSMCs) derived from young monkeys, treatment with H(2)O(2) and high glucose significantly increases transcriptional activity of Nrf2 and upregulates the expression of Nrf2 target genes. In contrast, in cultured VSMCs derived from aged macaques, H(2)O(2)- and high glucose-induced Nrf2 activity and Nrf2-driven gene expression are blunted. High glucose-induced H(2)O(2) production was significantly increased in aged VSMCs compared with that in cells from young monkeys. Aged VSMCs cultured in the absence of systemic factors exhibited significantly increased secretion of interleukin-1, MCP-1, and tumor necrosis factor compared with young control cells. Secretion of interleukin-6 also tended to increase in aged VSMCs. This age-associated proinflammatory shift in the cellular secretory phenotype was associated with an increased mitochondrial O(2)(-) production and nuclear factor -light-chain-enhancer of activated B cells activation. Treatment of aged VSMCs with a physiologically relevant concentration of resveratrol exerted significant anti-inflammatory effects, reversing aging-induced alterations in the cellular cytokine secretion profile and inhibiting nuclear factor -light-chain-enhancer of activated B cells. Interestingly, resveratrol treatment, mimicking calorie restriction, attenuated mitochondrial O(2)(-) production and upregulated the transcriptional activity of Nrf2 in aged monkey VSMCs. Resveratrol treatment also prevents the proinflammatory properties of the aged monkey VSMC secretome, an effect that likely contributes to the demonstrated vasoprotective action of resveratrol in animal models of aging. Importantly, resveratrol treatment prevents the proinflammatory properties of the aortic wall, an effect that likely contributes to the demonstrated vasoprotective action of resveratrol in nonhuman primates with a high fat diet, an early arterial aging model. It is well known that central arterial wall stiffening, driven by a chronic inflammatory milieu, accompanies arterial diseases, the leading cause of cardiovascular (CV) morbidity and mortality in Western society. An increase in central arterial wall stiffening, measured as an increase in aortic pulse wave velocity (PWV), is a major risk factor for clinical CV disease events. 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 and increases PWV and inflammation. The observed loss of endothelial cell integrity, lipid and macrophage infiltration, and calcification of the arterial wall were driven by genomic and proteomic signatures of oxidative stress and inflammation. Very importantly, resveratrol prevented the HFS-induced arterial wall inflammation and the accompanying increase in PWV. Dietary resveratrol may hold promise as a therapy to ameliorate increases in PWV. Importantly, in collaboration with Dr. Engler (Department of Bioengineering, University of California, San Diego), we found the cardiac proteomes of young and old rhesus monkeys and rats, from which we show that 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 reinforced the cortical cytoskeleton and enhanced myofilament organization, leading to improved contractility and hemodynamic stress tolerance in healthy and myosin-deficient fly hearts. Moreover, cardiac-specific vinculin overexpression markedly increased median life span in flies. These findings suggest that the heart has molecular mechanisms to sustain performance and promote longevity in flies, rats, and monkeys.