Effects of Aging on Experimental Atherosclerosis in Nonhuman Primates

National Institute On Aging

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Abstract

Following collection of baseline data such as blood pressure, pulse wave velocity (PWV) and lipid profile, 16 rhesus monkeys were assigned an atherogenic diet (high cholesterol diet: HCD) and 10 monkeys a control diet (standard diet: SD). At baseline, cholesterol, triglycerides, and glucose levels showed no differences between the control and treated groups. At the second follow-up, cholesterol levels in the treated increased 2-fold compared to controls (p<0.0001). At the second follow-up, glucose levels were related with an increased body weight in control animals but not in the treated group. Arterial tissue was collected at the time of sacrifice. 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 the domestic monkeys fed a HCD. We also found that a HCD increases adverse histopathologic events and plaque burden and vulnerability, which are closely associated with changes in cholesterol and glucose levels, diastolic blood pressure, and PWV. Further our studies indicate that HCD 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 various conditions. 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. micro RNA (miR) arrays of carotid arteries in monkeys fed with or without a HCD indicate that there are 91 totally modified miRs, via a continuous age analysis. Fifteen miRs showed significant change with aging, 67 miRs were significantly different in abundance associated with HCD treatment. During PCR validation, we saw 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 HCD treatment. Furthermore, we found that carotid arteries of aged rhesus macaques exhibit significant oxidative stress (as indicated by the increased 8-iso-prostaglandin F2 (8-iso-PGF2) and 4-hydroxy-2-alkenals (4-HNE) content and decreased glutathione and ascorbate levels)when compared with vessels of young macaques that is associated with the 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 SD monkeys, treated with hydrogen peroxide (H2 O2) 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 SD macaques, treated with H2 O2- and high glucose, induced Nrf2 activity and Nrf2-driven gene expression were blunted. High glucose-induced H2 O(2) production was significantly increased in aged VSMCs compared with that of cells from young monkeys. Cultured VSMCs from old monkeys exhibited significantly increased secretion levels of interleukin-1, MCP-1, and tumor necrosis factor compared with young control cells. Increases in the secretion levels of interleukin-6 was also seen in aged VSMCs. This age-associated proinflammatory shift in the cellular secretory phenotype was associated with an increased mitochondrial oxygen 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 effects, attenuated mitochondrial O(2)(-) production and upregulated the transcriptional activity of Nrf2 in aged monkey VSMCs. Resveratrol treatment also prevented 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. 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 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, increases PWV and arterial inflammation. The observed loss of endothelial cell integrity, lipid and macrophage infiltration, and calcification of the arterial wall are driven by genomic and proteomic signatures of oxidative stress and inflammation. Very importantly, resveratrol treatment 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 showed 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. Abnormal matrix homeostasis is one of the hallmarks of vascular complications associated with diabetes. Vascular adventitial fibroblasts (AdF) as well as smooth muscle cells contribute significantly to matrix deposition in the vasculature. Identification of novel factors that regulate extracellular matrix gene expression in vascular cells and exploration of strategies to therapeutically target them to check excessive matrix production are important clinical goals. In the overall context of matrix biology, there has been a surge of interest in collagen receptors that mediate fibroblast responses during tissue remodelling. DDR2 is a fibroblast-specific collagen receptor tyrosine kinase, noted for its association with fibrotic diseases and regulation of a wide array of fundamental cellula