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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online September 8, 2005 as doi:10.1096/fj.04-2864fje. |
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* Department of Clinical and Biological Sciences, University of Turin, Italy;
Interdepartmental Research Centre on Biology and Pathology of Aging, University of Pisa, Italy;
Italian National Research Council, Turin, Italy; and
Department of Biomedical Sciences and Human Oncology, University of Turin, Italy
1 Correspondence: Department of Clinical and Biological Sciences, University of Turin, Regione Gonzole 10, 10043 Orbassano, Turin, Italy. E-mail: elena.chiarpotto{at}unito.it
SPECIFIC AIMS
Calorie restriction (CR) has been proved to significantly retard age-related chronic diseases in all animal species tested so far, but the biological mechanisms underlying its beneficial effects are not clearly understood. Since CR has been shown effective against age-related oxidative damage and the latter may be responsible for tissue fibrosis through up-regulation of fibrogenic cytokines, we investigated whether CR might down-regulate fibrosis by preventing oxidant-dependent signaling and consequent fibrogenic stimulus.
PRINCIPAL FINDINGS
1. Calorie restriction protects rat aorta against the age-related increase of oxidative damage and fibrosis
Aortae from male Sprague-Dawley rats of different ages, subjected or not to CR, were examined for oxidative stress and fibrosis parameters. CR was achieved through starvation every other day (EOD) or through a 40% calorie restricted diet. Oxidative damage was evaluated in terms of adduct formation between lipid peroxidation-derived aldehydes [4-hydroxy-2,3-nonenal (HNE) and malonildialdehyde (MDA)] and tissue protein. To evaluate fibrosis, we determined the levels of transforming growth factor ß1 (TGFß1), the major profibrogenic cytokine, and collagen content. In the aortae from old (24 months of age) vs. young (3–6 months of age) rats we found a significant increase in oxidative stress parameters (HNE tissue protein adducts: 23% increase; MDA tissue protein adducts: 36% increase). CR exerted a protective effect against this process. The protective effect was exerted independent of the type of CR used [i.e., whether through starving on alternate days (every other day: EOD) or through feeding with a 40% reduced caloric intake (40% CR)].
The increase of the two parameters of fibrosis with age was even more evident: TGFß1 content was 23-fold higher in the aortae from 24- vs. 6-month-old rats (Fig. 1
) and CR again protected against this age-induced phenomenon (Fig. 1)
. The situation was similar with regard to collagen deposition in rat aortae, which markedly increased with age (1.7-fold at 24 months), and that CR in the form of either dietetic treatment prevented almost completely; the condition of the blood vessels of the aged group of animals returned to a situation very similar to that of their younger counterparts (6 months).
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Age-related increased fibrosis was associated with modifications of the cell composition of the artery wall, with a decrease in thickness and general derangement of the endothelial layer, a decrease and nonuniform distribution in 
-actin content, and an increase in vimentin-positive myofibroblast-like cells in older vs. younger tissues. CR strongly protected against all these artery wall alterations.
2. Calorie restriction protects against tissue sclerosis through down-regulation of mitogen-activated protein kinases (MAPKs) and of AP-1 binding activity
To elucidate the possible mechanism(s) responsible for the antifibrotic effect of CR, we then investigated the activity of MAPKs, known to be involved in TGFß1 expression and signaling. As Fig. 2
shows, Jun-N-terminal kinase (JNK) activity increased progressively with age, but CR, however achieved, fully protected against activation of this kinase. Results for p38 were similar: the increase detected at 12 and at 24 months was fully reversed by CR. In contrast, extracellular regulated kinases ERK1 and ERK2 did not show any age-related change, nor were they influenced by CR.
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DNA binding activity of AP-1 (i.e., the main redox-sensitive transcription factor involved in TGFß1 gene expression) increased with age (1.8-fold at 24 months) and was reduced by CR. Vimentin expression, another gene whose transcription depends on AP-1, also increased with age and CR fully reversed this effect.
CONCLUSIONS AND SIGNIFICANCE
Our study shows that CR protects tissues from increased both oxidative damage and fibrosis, further stressing the cause/effect correlation between the two phenomena. The protective effect of CR against peroxidative processes may be due in part to the maintenance of cell membrane order and fluidity achieved by quantitative and qualitative changes in phospholipid fatty acid membrane content and by lower mitochondrial production of oxygen free radicals. In regard to the CR-induced decrease of TGFß1, a similar decrement in the colon of genetically obese rats has been reported to be induced by mild energy restriction (20%), with a possible inhibitory effect on carcinogenesis; no relationship with animal age was found. On the other hand, CR has been demonstrated to reduce several age-dependent degenerative alterations in rat aorta, suggesting its possible anti-atherosclerotic action.
In an attempt to elucidate the mechanism(s) by which CR might protect against fibrosclerosis, we evaluated its effect on the MAPK pathway, involved in both TGFß1 expression and intracellular signal transduction. While with increasing age a progressive increase of JNK and p38 activity is evident, once again CR, achieved in either modality, significantly decreases the activity of these enzymes from 12 months of age. On the contrary, ERK was not modulated by aging or by CR. We demonstrated increased AP-1 DNA binding activity with age, which CR strongly protected against.
Thus, JNK hyperactivation might explain the increased fibrosis: JNK may up-regulate TGFß1 gene expression through AP-1 activation and may also enhance the response to TGFß1 by interacting with and activating the Smad pathway.
By down-regulating JNK and p38 activity, CR might act on gene transcription, since both kinases are involved in the activation of transcription factors such as AP-1 and ATF-2. Since JNK is fundamental for AP-1 activation, its inhibition or down-regulation resulted in a down-regulation of AP-1 and of AP-1-dependent gene transcription. This was shown to be true not only for TGFß1, but also for vimentin, another AP-1-dependent gene. By preventing the age-induced AP-1 hyper-activation, CR also reduced vimentin overexpression. Our data agree with another report where CR was found to prevent AP-1 activation in elderly rat kidney by suppressing nuclear levels of c-Jun and c-Fos and inhibiting phosphorylation of the c-Jun protein.
Our data further support the relationship between oxidative stress and fibrosis in different diseases and during aging. For the first time, we suggest that the protection exerted by CR against fibrosclerosis might be due to decreased oxidative stress, with consequent decreased mitogen-activated protein kinase (MAPK) activity and down-regulation of AP-1 nuclear binding and of TGFß1 expression and signaling (Fig. 3
). Down-regulation of oxidative stress-dependent cell signaling and fibrosis by CR may be considered in the treatment of age-associated diseases with sclerotic evolution.
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FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-2864fje;
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