Aging exacerbates obesity-induced oxidative stress and inflammation in perivascular adipose tissue in mice: a paracrine mechanism contributing to vascular redox dysregulation and inflammation

Abstract

Obesity in the elderly individuals is increasing at alarming rates and there is evidence suggesting that elderly individuals are more vulnerable to the deleterious cardiovascular effects of obesity than younger individuals. However, the specific mechanisms through which aging and obesity interact to promote the development of cardiovascular disease remain unclear. The present study was designed to test the hypothesis that aging exacerbates obesity-induced inflammation in perivascular adipose tissue, which contributes to increased vascular oxidative stress and inflammation in a paracrine manner. To test this hypothesis, we assessed changes in the secretome, reactive oxygen species production, and macrophage infiltration in periaortic adipose tissue of young (7 month old) and aged (24 month old) high-fat diet-fed obese C57BL/6 mice. High-fat diet-induced vascular reactive oxygen species generation significantly increased in aged mice, which was associated with exacerbation of endothelial dysfunction and vascular inflammation. In young animals, high-fat diet-induced obesity promoted oxidative stress in the perivascular adipose tissue, which was associated with a marked proinflammatory shift in the profile of secreted cytokines and chemokines. Aging exacerbated obesity-induced oxidative stress and inflammation and significantly increased macrophage infiltration in periaortic adipose tissue. Using cultured arteries isolated from young control mice, we found that inflammatory factors secreted from the perivascular fat tissue of obese aged mice promote significant prooxidative and proinflammatory phenotypic alterations in the vascular wall, mimicking the aging phenotype. Overall, our findings support an important role for localized perivascular adipose tissue inflammation in exacerbation of vascular oxidative stress and inflammation in aging, an effect that likely enhances the risk for development of cardiovascular diseases from obesity in the elderly individuals.

Keywords: Adiposity; Diabetes; Fat; Metabolic disease; Obesity.

Figure 1.

Aging exacerbates high-fat diet (HFD)–induced vascular impairment. (Panel A) Representative micrographs showing nuclear dihydroethidium (DHE) fluorescence (white), representing cellular production, in sections of aortas isolated from young and aged mice fed a HFD or standard diet (SD). For orientation purposes, overlay of the nuclear DHE signal (excitation: 490nm, emission: 525nm) and autofluorescence of elastic laminae (excitation: 490nm, emission: 525nm) is shown. Original magnification: 20×. (Panels B and C) Relaxation of aorta rings isolated from young and aged SD- or HFD-fed mice. Vasomotor responses were induced by the endothelium-dependent agent acetylcholine (B) and S-nitroso-N-acetylpenicillamine (SNAP, Panel C), an endothelium-independent vasodilator. Data are means ± SEM; *p < .05 vs respective SD-fed control (n = 5–7). (Panel D) Quantitative real-time RT-PCR data showing mRNA expression of Nox2 in the aortas of HFD-fed and SD-fed young and aged mice. Data are mean ± SEM (n = 5–7); *p < .05 vs SD. (Panel E) Hydroxyl Radical Antioxidant Capacity (HORAC) in the aorta of young and aged SD- and HFD-fed mice. Data are mean ± SEM (n = 5–7); *p < .05 vs SD. (Panel F) HFD-induced increases in caspase 3/7 activity in aortas of young and aged mice. Data are mean ± SEM (n = 5–7); *p < . 05. (Panels G and H) Protein expression of MCP-1 (G) and MIP-1α (H) in aortas of HFD-fed and SD-fed young and aged mice. Data are means ± SEM (n = 5–7); *p < . 05 vs SD, #p < .05 vs young. (Panel I) mRNA expression of the senescence marker p16^INK4a in aortas of HFD-fed and SD-fed young and aged mice. Data are mean ± SEM (n = 5–7); *p < . 05 vs SD, #p < .05 vs young.

Figure 2.

Aging exacerbates high-fat diet (HFD)–induced oxidative stress and inflammation in perivascular adipose tissue. (Panel A) Representative micrographs showing nuclear dihydroethidium (DHE) fluorescence (white), representing cellular production in sections of perivascular adipose tissue isolated from standard diet (SD)–fed and HFD-fed young and aged mice. Note that tissues derived from HFD-fed aged mice exhibit the most intense DHE fluorescence. Original magnification: 20×. (Panel B) Representative micrographs showing brown immunostaining for CD68 in sections of perivascular adipose tissues isolated from SD- and HFD-fed young and aged mice. Tissues derived from HFD-fed aged mice exhibit the greatest infiltration by CD68⁺ macrophages (arrows). In SD-fed young mice adipose tissue surrounding the distal portion of the thoracic aorta was primarily composed of multilocular brown adipocytes. In contrast, in aged mice surrounding the distal portion of the thoracic aorta adipose tissues consisting of both multilocular brown adipocytes and unilocular white adipocytes could be observed. Note that HFD feeding resulted in significant hypertrophy of the white adipocytes. (Panel C) Summary data for relative age- and HFD-induced changes in CD68⁺ macrophage content in perivascular adipose tissue. Data are mean ± SEM; *p < .05 vs control.

Figure 3.

(Panel A) Proteomic profiles of perivascular adipose tissue secretomes. Perivascular adipose tissues, derived from young (Y) and aged (A) mice fed a high-fat diet (HFD) or standard diet (SD), were kept in organoid culture and cytokine concentrations were assessed in the conditioned media (see Methods section). The heat map is a graphic representation of normalized cytokine concentration values in perivascular adipose tissue–conditioned media, depicted by color intensity, from highest (bright red) to lowest (bright green) expression. Values represent average secreted protein levels (log₂ [fold change, normalized to the respective control mean value]) in replicate samples (n = 6 in each group). Perivascular adipose tissues from aged mice on a HFD secrete the highest levels of inflammatory cytokines and chemokines. (Panel B) Correlation between cytokines secreted from the perivascular adipose tissue and cytokine levels in the sera from the same animals (p < .05, r² = .26). (Panel C) Relative levels of adiponectin secretion from perivascular adipose tissue derived from young and aged SD- or HFD-fed animals (see Methods section). Data are means ± SEM (n = 10 in each group); *p < .05 vs SD.

Figure 4.

(Panel A) Experimental design depicting the ex vivo organoid culture-based bioassay approach utilizing aortas isolated from control mice treated with perivascular adipose tissue–conditioned media. (Panel B) Production of H₂O₂ by detector vessels induced by proinflammatory factors secreted from the perivascular adipose tissue derived from young and aged standard diet (SD)–fed or high-fat diet (HFD)–fed mice. The Amplex Red/horseradish peroxidase assay was used to assess vascular reactive oxygen species (ROS) production. (Panels C and E) Quantitative real-time RT-PCR data showing mRNA expression of Nox2 (C), Nox1 (D), and Nos3 (E) in detector vessels cultured in the presence of perivascular adipose tissue–conditioned media. Data are means ± SEM (n = 10 in each group); *p < .05 vs young SD-fed, #p < .05 vs young HFD-fed, &p < .05 vs aged SD-fed.

Figure 5.

Quantitative real-time RT-PCR data showing mRNA expression of TNF-α (A), IL-6 (B), IL-1Β (C), and Nlrp3 (D) in detector vessels cultured in the presence of factors secreted from the perivascular adipose tissue derived from young and aged standard diet (SD)–fed or high-fat diet (HFD)–fed mice. Data are mean ± SEM (n = 10 in each group); *p < .05 vs young SD-fed, #p < .05 vs young HFD-fed, &p < .05 vs aged SD-fed.