We had quarterly measured over a 2-year period: blood pressure, body weight, and morphometric measurements, blood pressure. Following collection of baseline data, 16 monkeys had been assigned to an atherogenic diet and 7 monkeys to a control diet. We have obtained measures of vascular stiffness: Doppler pulse wave velocity and applanation tonometry;morphologic characterization of atherosclerosis: CE-MRI;and blood chemistry measures: glucose, cholesterol, triglycerides, HDL, LDL, ox-LDL, CRP, MMPs, TIMPs, MTMMPs, MCP-1, TGF-beta1 and MFG-E8. Preliminary statistical analyses using a T test showed that at baseline, cholesterol (p=0.39), triglycerides (p=0.64), and glucose (p=0.18) were not different between the control and treated group. At the second follow-up, cholesterol levels in the treated group were different from controls (p<0.0001) but triglycerides and glucose significance were not altered. In a mixed models analyses, cholesterol, triglycerides and glucose were modeled separately using treatment group, first age and time as fixed effects along with their interaction terms. In addition to the blood chemistry, four imaging parameters, namely contrast enhancement, wall thickness, lumen diameter and T2thickest triad have been analyzed at the same time points as the other measures were done and correlate these parameters with the blood parameters to find associations at all-time points. After sacrifice, we harvested arterial tissue: ascending and descending thoracic, aorta arches;carotid arteries: right and left;coronary arteries: left descending coronary, right coronary. These tissues have been analyzed in the levels of transcription, translation, distribution, activation for the previously mentioned biomolecules. In addition, we have isolated arterial endothelial and smooth muscle cells from these monkeys for in vitro studies. Histochemical observation and morphological analysis indicate 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. Comprehensive quantitative proteomic studies were designed to analyze proteomic changes of carotid arteries in the different conditions. So far, we have finished one iTRAQ experiment, which gave protein abundance changes between young and old control monkeys. 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 include 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. Importantly, we have performed micro RNA array of carotid arteries in monkeys with or without high cholesterol diets. The microarray data normalized by Z score transformation were analyzed in the calculation of significant changes in gene expression changes between different samples and conditions. The statistical results show that there are totally 91 modified miRs, via a continuous age analysis (2-way ANOVA). 15 miRs significantly change with aging, 67 miRs are significantly different in abundance associated with high cholesterol diet treatment. microRNA array results were validated by real-time PCR. Expression of mature miRNAs was analyzed with the TaqMan MicroRNA Reverse Transcription Kit and TaqMan MicroRNA assays according to the instructions of the manufacturer (Applied Biosystems Inc). 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. Studies of bioroles of those miRs in aging and atherosclerosis are in progress. The in vivo and in vitro studies were designed to elucidate age-associated changes in the homeostatic role of Nrf2-driven free radical detoxification mechanisms in the vasculature of nonhuman primates. We found that carotid arteries of aged rhesus macaques (20 years) 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 (age:10 years) 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 M mulatta, 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 vascular smooth muscle cells cells 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 vascular smooth muscle cells compared with that in vascular smooth muscle cells from young M mulatta. Taken together, aging is associated with Nrf2 dysfunction in M mulatta arteries, which likely exacerbates age-related cellular oxidative stress, promoting nuclear factor-kappaB activation and vascular inflammation in aging. Furthermore, we tested the hypothesis that cell-autonomous mechanisms contribute to the proinflammatory changes in vascular phenotype that accompanies advancing age, we analyzed the cytokine secretion profile of primary vascular smooth muscle cells (VSMCs) derived from young (13y) and aged (21y) Macaca mulatta. 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 (1 M) 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. Resveratrol also attenuated mitochondrial O(2)(-) production and upregulated the transcriptional activity of Nrf2 in aged VSMCs. Thus, in nonhuman primates, cell-autonomous activation of nuclear factor -light-chain-enhancer of activated B cells and expression of an inflammatory secretome likely contribute to vascular inflammation in aging. Resveratrol treatment prevents the proinflammatory properties of the aged VSMC secretome, an effect that likely contributes to the demonstrated vasoprotective action of resveratrol in animal models of aging.

Agency
National Institute of Health (NIH)
Institute
National Institute on Aging (NIA)
Type
Investigator-Initiated Intramural Research Projects (ZIA)
Project #
1ZIAAG000238-05
Application #
8552339
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
5
Fiscal Year
2012
Total Cost
$372,211
Indirect Cost
Name
National Institute on Aging
Department
Type
DUNS #
City
State
Country
Zip Code
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Mattison, Julie A; Wang, Mingyi; Bernier, Michel et al. (2014) Resveratrol prevents high fat/sucrose diet-induced central arterial wall inflammation and stiffening in nonhuman primates. Cell Metab 20:183-90
Csiszar, Anna; Sosnowska, Danuta; Wang, Mingyi et al. (2012) Age-associated proinflammatory secretory phenotype in vascular smooth muscle cells from the non-human primate Macaca mulatta: reversal by resveratrol treatment. J Gerontol A Biol Sci Med Sci 67:811-20
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