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. 2009 Apr;296(4):H946-56.
doi: 10.1152/ajpheart.00693.2008. Epub 2009 Jan 30.

Longevity is associated with increased vascular resistance to high glucose-induced oxidative stress and inflammatory gene expression in Peromyscus leucopus

Nazar Labinskyy  1 , Partha Mukhopadhyay, Janos Toth, Gabor Szalai, Monika Veres, Gyorgy Losonczy, John T Pinto, Pal Pacher, Praveen Ballabh, Andrej Podlutsky, Steven N Austad, Anna Csiszar, Zoltan Ungvari
Affiliations

Longevity is associated with increased vascular resistance to high glucose-induced oxidative stress and inflammatory gene expression in Peromyscus leucopus

Nazar Labinskyy et al. Am J Physiol Heart Circ Physiol. 2009 Apr.

Abstract

Vascular aging is characterized by increased oxidative stress and proinflammatory phenotypic alterations. Metabolic stress, such as hyperglycemia in diabetes, is known to increase the production of ROS and promote inflammatory gene expression, accelerating vascular aging. The oxidative stress hypothesis of aging predicts that vascular cells of long-lived species exhibit lower steady-state production of ROS and/or superior resistance to the prooxidant effects of metabolic stress. We tested this hypothesis using two taxonomically related rodents, the white-footed mouse (Peromyscus leucopus) and the house mouse (Mus musculus), which show a more than twofold difference in maximum lifespan potential (8.2 and 3.5 yr, respectively). We compared interspecies differences in steady-state and high glucose (HG; 30 mmol/l)-induced production of O(2)(*-) and H(2)O(2), endothelial function, mitochondrial ROS generation, and inflammatory gene expression in cultured aortic segments. In P. leucopus aortas, steady-state endothelial O(2)(*-) and H(2)O(2) production and ROS generation by mitochondria were less than in M. musculus vessels. Furthermore, vessels of P. leucopus were more resistant to the prooxidant effects of HG. Primary fibroblasts from P. leucopus also exhibited less steady-state and HG-induced ROS production than M. musculus cells. In M. musculus arteries, HG elicited significant upregulation of inflammatory markers (TNF-alpha, IL-6, ICAM-1, VCAM, and monocyte chemoattractant protein-1). In contrast, the proinflammatory effects of HG were blunted in P. leucopus vessels. Thus, increased life span potential in P. leucopus is associated with decreased cellular ROS generation and increased resistance to prooxidant and proinflammatory effects of metabolic stress, which accord with predictions of the oxidative stress hypothesis of aging.

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Figures

Fig. 1.
Fig. 1.
Schematic representation of potential strategies by which long-lived, successfully aging animals can delay/limit oxidative stress-induced damage in the cardiovascular system and in other organs. A: lower initial ROS generation at young ages under steady-state conditions, so that it takes longer to reach the critical threshold even at the same rate of aging. B: lower ROS generation in response to metabolic stress (and other stressors). C: increased tolerance for increases in ROS production. D: slower rate of age-related increases in ROS generation (increasing the time to reach a threshold beyond which oxidative damage significantly impairs cellular function). Long-lived animals may use a combination of all of these strategies. Nonlinear/exponential characteristics of age-related ROS increases are based on O2•− production in rodent blood vessels (12) as well as on DNA oxidation in the liver, brain, kidney, heart, and skeletal muscle of the same strain (25).
Fig. 2.
Fig. 2.
Results from dihydroethidine (DHE) staining experiments in en face aortic preparations to measure vascular O2•− production [DHE reacts with O2•− to form the fluorescent product ethidium (EB)]. A–D: representative confocal images showing EB staining (red fluorescence) in endothelial cells of aortas from Mus musculus (A) and Peromyscus leucopus (C; original magnification: ×20). High glucose treatment (30 mM for 24 h) substantially increased EB staining in M. musculus endothelial cells (B), whereas P. leucopus cells were more resistant to high glucose-induced oxidative stress (D). E: time course of the build up of EB fluorescence in aortic segments of mice. Bar graphs are summary data for slope factors normalized to cell number (i.e., nuclear DNA content, as assessed by Hoechst fluorescence intensity), representing tissue O2•− production. Data are means ± SE [in arbitrary units (AU) of ΔEB fluorescence/dt/DNA content]; n = 5 animals/group. *P < 0.05 vs. untreated animals; #P < 0.05 vs. M. musculus. Inset, control experiments showing increases in EB fluorescence in the presence of a vascular sample (control) treated with or without polyethylene glycol (PEG)-SOD or the assay buffer (DHE dye) only.
Fig. 3.
Fig. 3.
A: relaxation to ACh was significantly impaired in high glucose (30 mM for 24 h)-treated aortic segments isolated from M. musculus compared with untreated controls. Preincubation with PEG-SOD restored ACh-induced relaxations in high glucose-treated aortic segments. Data are means ± SE; n = 5 vessels/group. *P < 0.05. B: relaxation to ACh was preserved in high glucose (30 mM for 24 h)-treated aortic segments isolated from P. leucopus. Data are means ± SE; n = 5 vessels/group. C: high glucose treatment did not affect relaxations to the nitric oxide (NO) donor S-nitroso-N-acetylpenicillamine (SNAP) in either group (n = 4 vessels/group) (not significant).
Fig. 4.
Fig. 4.
A–D: representative fluorescent images showing H2O2 production [measured by the 5 (and 6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate acetyl ester (C-H2DCFDA) fluorescence method] in endothelial cells of en face preparations of aortas of M. musculus (A and B) and P. leucopus (C and D). High glucose treatment (30 mM for 24 h) substantially increased dichlorofluorescein (DCF) staining in M. musculus vessels (B), whereas P. leucopus cells were more resistant to high glucose-induced oxidative stress (D). Green fluorescence shows DCF (original magnification: ×10). E: time course of the build up of DCF fluorescence in aortic segments of mice. Bar graphs are summary data for slope factors normalized to cell number (i.e., nuclear DNA content, as assessed by Hoechst fluorescence intensity), representing tissue H2O2 production. Bar graphs are summary data of DCF fluorescent intensities in endothelial cells of M. musculus and P. leucopus aortas. Data are means ± SE (in AU of ΔDCF fluorescence/dt/DNA content); n = 5 animals/group. *P < 0.05 vs. untreated animals; #P < 0.05 vs. M. musculus. F: results from Amplex red/horseradish peroxidas assays. Shown is the time course of the build up of resorufin (the fluorescence product formed from Amplex red) fluorescence by aortic segments of M. musculus and P. leucopus. M. musculus aortas showed a significantly steeper slope than P. leucopus vessels both under steady-state conditions and after high glucose treatment. Data are means ± SE; n = 5 animals/group. *P < 0.05 vs. untreated animals; #P < 0.05 vs. M. musculus. G, left: increases in resorufin fluorescence in response to exogenous H2O2. Also shown are the time-dependent increases in resorufin (middle) and DCF (right) fluorescence in the presence of a vascular sample (control) treated with or without PEG-catalase or the assay buffer (Amplex red or C-H2DCFDA, respectively) only.
Fig. 5.
Fig. 5.
Representative fluorescent images showing stronger cellular C-H2DCFDA staining (green fluorescence; an indicator of production of ROS, including H2O2) in primary M. musculus fibroblasts (A) than in P. leucopus fibroblasts (C). High glucose treatment elicited substantial increases in DCF fluorescence in M. musculus fibroblasts (B), whereas high glucose-induced increases in mitochondrial ROS generation were significantly lower in P. leucopus fibroblasts (D). Hoechst 33258 (blue fluorescence) was used for nuclear staining. E: bar graphs are summary data of DCF fluorescent intensities in control and high glucose-treated M. musculus and P. leucopus fibroblasts. Data are means ± SE. *P < 0.05. untreated animals; #P < 0.05 vs. M. musculus.
Fig. 6.
Fig. 6.
A: time course of the build up of MitoSox fluorescence in en face preparations of M. musculus and P. leucopus aortas. Bar graphs are summary data for the slope factor obtained from the time course experiments normalized to tissue mass, representing mitochondrial ROS production in the tissues. Shown are the effects of high glucose treatment (30 mM for 24 h) on mitochondrial ROS production in both species. Data are means ± SE; n = 5 animals/group. *P < 0.05 vs. untreated animals; #P < 0.05 vs. M. musculus. B and C: representative fluorescent images showing stronger perinuclear MitoSox staining (red fluorescence) in high glucose-treated primary M. musculus fibroblasts (B) than in P. leucopus fibroblasts (C). Hoechst 33258 (blue fluorescence) was used for nuclear staining. Scale bars = 50 μm. D: cellular MitoSox fluorescence intensities were assessed using flow cytometry as described in methods. Bar graphs are summary data for mean MitoSox fluorescent intensities. Data are means ± SE. Experiments were performed in quadruplicate with identical results. *P < 0.05 vs. untreated animals; #P < 0.05 vs. M. musculus.
Fig. 7.
Fig. 7.
High glucose-induced inflammatory gene expression in M. musculus and P. leucopus aortas. A: in cultured aortic segments isolated from M. musculus, high glucose treatment (30 mM for 24 h) elicited significant increases in the mRNA expression of TNF-α, IL-6, ICAM-1, VCAM, and monocyte chemoattractant protein (MCP)-1. B: in contrast, 24 h of high glucose treatment did not result in the significant induction of these inflammatory markers in aortas of P. leucopus. Analysis of mRNA expression was performed by real-time quantitative RT-PCR. β-Actin was used for normalization. Data are means ± SE; n = 5 animals/group. *P < 0.05. vs. untreated animals.
Fig. 8.
Fig. 8.
A: relaxation to ACh in aortic ring preparations from 3-, 18-, and 24-mo-old P. leucopus. Data are means ± SE; n = 5 animals/group. B: age-dependent decline in the maximal relaxation responses induced by ACh (open symbols) in aortas of M. musculus and P. leucopus as a function of chronological age. M. musculus data were replotted from previous publications (12, 43). Age-dependent increases in vascular O2•− production (as assessed by DHE staining) in arteries of M. musculus and P. leucopus (filled symbols) are superimposed.
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