Protective effect of calorie restriction on age-induced fibrosis

Editor: Jennifer V. Bentely and Mary Ann Keller © 2009 Nova Science Publishers, Inc.

Chapter 9

Protective Effect of Calorie Restriction

on Age-Induced Fibrosis

E. Chiarpotto

_{, L. Castello}

_{, E. Bergamini}

_{, and G. Poli}

Gonzole 10, 10043 Orbassano (TO), Italy.

2_{Research Center on the Biology and Pathology of Aging, Department of Experimental}

Pathology, University of Pisa, Via Roma 55, 56123 Pisa, Italy.

Abstract

Starting from research by McCay et al., several studies have demonstrated that controlled calorie restriction (CR) exerts an anti-ageing effect in different organisms, from invertebrates to mammals. Observational studies suggest that CR also has beneficial effects on human longevity. However, the anti-aging mechanisms of CR are still not clearly understood. One mechanism might be protection against the age-associated increase of oxidative stress and consequent cellular damage.

In parallel, a role of oxidative stress in fibrosis induction and progression has been demonstrated in many human diseases, such as pneumoconiosis, interstitial pulmonary fibrosis, cystic fibrosis, cirrhosis, neurodegenerative diseases and atherosclerosis.

In fibrosis, fibroblasts or fibroblast-like cells are activated by various cytokines, among which transforming growth factor 1 (TGF1) is prominent, to proliferate and produce high levels of extracellular matrix and collagen. High TGF1 levels have been found in human diseases of different organs all characterized by excessive ECM deposition and marked fibrosis (cirrhosis, chronic hepatitis, glomerulonephritis, diabetic nephropathy, atherosclerosis, sclerodermia, pulmonary fibrosis). Fibrosis is also a constant distinctive feature in tissue aging.

TGF1 exerts its multiple biological activities through interaction with type I and type II receptors. Signaling to the nucleus is principally through cytoplasmic proteins of the Smad family, but in various cell types TGF1 also activates mitogen activated protein kinase (MAPK) pathways, i.e. extracellular regulated kinase 1 and 2 (ERK1/2), c-Jun N-terminal kinase (JNK) and p38, leading to collagen type I synthesis through activation of the transcription factor activator protein 1 (AP-1).

In this context, it is of interest to study the possible protective effect of CR against the onset of fibrosis in the frame of tissue and soma aging.

In this connection, our group has shown that, with aging, there is an increase of oxidative stress in rat aorta, with a progressive hoarding of biologically-active end-products, in particular 4-hydroxy-2,3-nonenal (HNE). Moreover, the increase of oxidative stress with aging is accompanied by increased fibrosis in terms of TGF1 and collagen levels. CR protects against both phenomena.

With regard to possible protective mechanisms of CR against fibrosclerosis, we believe they may be closely connected to the reduction of oxidative stress: by decreasing HNE levels in older rat aortae, CR significantly decreases JNK and p38 activity. Since JNK is central for AP-1 activation, by negatively modulating JNK, CR also prevents AP-1 activation and consequently down-regulates transcription of AP-1-dependent genes, including TGF1, vimentin and collagen.

The possibility of controlling the fibrogenesis process by modifying dietary habits opens new nutritional horizons in the prevention and treatment of several pathological processes characterized by excessive fibrosis. However, since it seems difficult to transpose animal CR model as such to man, interest in natural and/or pharmacological CR mimetic molecules is increasing.

Introduction

Aging is a multifactorial process that affects nearly all organisms, and which is believed to be produced by intrinsic (developmental-genetic) (Atzmon et al., 2005; Novelli et al., 2008) and extrinsic (stochastic-environmental) causes (Burhans & Weinberger, 2007; Passos et al., 2007). These are not mutually exclusive, and both are important. Observations have been accumulating on both the genetic determinants of aging and the major cellular mechanisms involved in this process (e.g. free radicals, mitochondrial dysfunction and oxidative stress, autophagy, insulin-like growth factor/growth hormone signaling, cholesterol and glucose metabolism, telomere shortening, etc.).

Since we cannot act on our genetic background, any intervention on the environmental factors becomes extremely important as an anti-aging strategy.

One of the most enduring theories to explain aging is the free radical theory initially proposed by Harman in the 1950s (Harman, 1956) which implicates free radicals and other reactive molecules derived from oxygen (reactive oxygen species, ROS) as mediators of age-dependent cell decline. Oxidants are normal by-products of cellular metabolism, most being generated in the mitochondria along the electron transport chain during cellular respiration (for a review, see Poli et al., 2004). Mitochondria are not only the primary producers of ROS, they are also highly susceptible to oxidant-induced damage because of the proximity of ROS, and because mitochondrial DNA cannot be repaired (Grishko et al., 2005). This has led to the formulation of the mitochondrial theory of aging (Miquel et al., 1980; Trifunovic & Larsson, 2008) that suggests senescence is the result of damage caused by ROS to the mitochondrial genome in post mitotic cells. Once mitochondria become injured, they become less efficient at producing energy and produce larger amounts of ROS. In young cells, damaged mitochondria, as well as unwanted or redundant cell membranes and organelles, may be removed via autophagy (Bergamini et al., 2007; Kurz et al., 2007) and in this perspective autophagy may well be considered as an anti-aging mechanism (Donati, 2006).

Oxidative Stress, Lipid Peroxidation

and Fibrogenesis

The term fibrosis is used to define the excessive accumulation of connective tissue in parenchymal organs. Regardless of the underlying etiology, the process shows common features in terms of induction mechanisms, cells involved in the overproduction of extracellular matrix (ECM) components and the frequent progression to sclerosis. A central and essential role is played by macrophages and by ECM producing cells (hepatic stellate cells, smooth muscle cells, mesangial cells, myofibroblasts) which become the mediators of this process through the production of a complex network of growth factors, cytokines, chemoattractants and other reactive molecules (Wynn, 2008). Among these, transforming growth factor 1 (TGF1) is prominent (Branton & Kopp, 1999; Wynn, 2008). TGF1 is the most potent direct stimulator of collagen production, and it induces the transcription and synthesis of various other components of the ECM, such as fibronectin, glycosaminoglycans and proteoglycans (Verrecchia & Mauviel, 2002). In addition it stabilizes the newly formed ECM proteins by inhibiting their degradation (Hall et al., 2003). Thus, TGF-1is associated with fibrosis in a number of human diseases _{of different organs (including the lung, heart,} liver, kidney, skin, artery) (Blobe et al., 2000; Bartram & Speer, 2004; Sharma et al., 2006). Moreover, fibrosis is a constant distinctive feature of tissue aging (Abrass, 2000; de Souza, 2002).

In parallel, a role of ROS and of lipid peroxidation in fibrosis induction and progression is now generally acknowledged in many human diseases, such as pneumoconiosis, interstitial pulmonary fibrosis, cystic fibrosis, cirrhosis, neurodegenerative diseases and atherosclerosis (Poli & Parola, 1997; Poli, 2000; Chiarpotto et al., 2005). In this connection, at physiopathological doses 4-hydroxy-2,3-nonenal (HNE), the major aldehyde deriving from the peroxidative breakdown of membrane phospholipids, has been shown to stimulate TGF1 expression and synthesis (Leonarduzzi et al., 1997) and the binding to DNA of the nuclear transcription factor AP-1 (Leonarduzzi et al., 1997; Chiarpotto et al., 2005), responsible for the activation of several genes, among which TGF1 itself, procollagen type I and vimentin (Armendariz et al., 1994; Kim et al., 1989; Rittling et al., 1989).

In most cell types, intracellular signaling of TGF1 occurs via two receptor serine/threonine kinases. The active form of TGF1 binds to specific type II receptors, which trigger recruitment of type I receptors to form tetrameric complexes that become phosphorylated (Massaguè & Chen, 2000). This propagates the signal intracellularly downstream to the nucleus through the phosphorylation of cytoplasmic proteins of the Smad family, Smad2 and Smad3, which form complexes with Smad4. The complex finally translocates to the nucleus, where it may activate target genes (Massaguè & Chen, 2000). However, in recent years alternate modes of TGF1 signaling have been described, particularly the mitogen activated protein kinase (MAPK) pathways, i.e. extracellular regulated kinase 1 and 2 (ERK1/2), c-Jun N-terminal kinase (JNK) and p38 (Mulder, 2000). Subsequent to the activation of MAPKs by TGF1, transcriptional responses mediated by activator protein 1 (AP-1) can lead to type I collagen synthesis (Cheon et al., 2002; Sato et al., 2002).

Anti-Aging Effect of Calorie Restriction

Starting from research by McCay et al. in the 1930s (McCay et al., 1935), diet restriction has been shown to extend median and maximum life span in all animal species tested thus far, from invertebrates to larger mammals like dogs (Sohal & Weindruch, 1996). Studies on the effects of diet restriction have been extended to primates, and the results thus far obtained are positive (Ramsey et al., 2000; Mattison et al., 2007). Importantly, indices of risk for cardiovascular disease and diabetes, such as blood pressure, blood lipids, glucose tolerance, and insulin sensitivity, have been reported to improve in monkeys on calorie restriction (CR) and also in humans who practice CR (Fontana et al., 2004; Meyer et al., 2006; Holloszy & Fontana, 2007). Reduced body weight and adiposity are other common changes in body composition that are similar among different mammalian species subjected to CR. On the basis of the relationship in humans between body weight and adiposity on one hand, and general mortality and morbidity on the other (Shiner & Uehlinger, 2001; Samaras et al., 2002), these data indicate that CR should also enhance longevity in man. Additionally, other metabolic and hormonal indices, such as reduced body temperature, triiodothyronine (T3) and insulin, that are predictive of increased survival, have been reported in rodents and rhesus monkeys on CR (Mattison et al., 2003; Roth et al., 2004), and also in humans in the CALERIE study (Redman et al., 2008). Scientific evidence also supports the view that lower calorie diets in humans may decrease the incidence of many age-related diseases, including cardiovascular disease, diabetes, cancer and neurodegenerative disorders, such as Alzheimer’s and Parkinson’s diseases (Mattson, 2005; Roberts & Barnard, 2005) .

Despite the plethora of scientific studies on the anti-aging effect of CR, the underlying mechanism/s have not yet been fully clarified. A comprehensive review on the numerous hypotheses on this topics was attempted by Chiarpotto et al. (2006). One theory that still remains popular is that CR protects against the age-related increase of oxidative damage (Merry, 2004).

Even if CR regimens applied to man are not so restrictive (about 1700 Kcal/day) (Meyer et al., 2006), it is difficult for most individuals to practice CR. Increasing interest is thus now being shown for the search for organic or inorganic compounds that mime the biological effects of CR, often called “CR mimetics”.

In this context, it is of considerable importance to improve the caloric restriction approach, in view of its positive interference with most important mechanisms that lead to aging of the organism.

Molecular Mechanisms of Calorie Restriction’s

Protection Against Age-Induced Fibrosis

Aging is generally characterized by increased fibrotic tissue deposition in many organs; hence fibrosis/sclerosis is considered to be a significant index of tissue aging. It is thus of interest to study the possible protective effect of CR against the onset of fibrosis, in the framework of tissue and soma aging.

In this connection, we have shown that, with aging, oxidative stress increases in the rat aorta, with a progressive hoarding of biologically-active end-products, in particular 4-hydroxy-2,3-nonenal (HNE) (Table 1). Moreover, the increase with aging of oxidative stress

is accompanied by increased fibrosis in terms of collagen and TGF1 levels (Figure 1). It is conceivable that HNE is involved in the pathogenesis of sclerotic diseases through the up-regulation of pro-inflammatory and pro-fibrotic genes (Nitti et al., 2002; Leonarduzzi et al., 2005; Uchida, 2008).

Table 1. Effect of calorie restriction (CR) on oxidative stress markers in rat aorta homogenates during aging

Dietary Treatment HNE-protein adducts (Arbitrary Fluorescence Units/mg protein)

YR OR

AL 127.6 ± 14.9 156.6 ± 28.0a

EOD 133.0 ± 15.5 81.0 ± 22.0b

MDA-protein adducts (Arbitrary Fluorescence Units/mg protein)

YR OR

AL 89.9 ± 18.6 122.7 ± 19.8a

EOD 80.6 ± 9.1 77.2 ± 24.4b

Data are means ± SD of 5 animals per group.

AL: rats fed ad libitum; EOD: rats starved every other day from 2 months of age. YR: 6 months of age; OR: 24 months of age.

a _{Significantly different from AL YR, p < 0.01} b _{Significantly different from AL OR, p < 0.05}

Figure 1. Effect of calorie restriction on fibrosis markers in homogenates of aortas from rats of different ages. Collagen content is expressed as g/mg wet weight; TGF1 content is expressed as Arbitrary Densitometric Units. Data are means ± SD of 5 animals per group. * Significantly different from AL YR p < 0.001. ** Significantly different from AL OR p < 0.001. AL YR: rats aged 6 months fed ad libitum; AL OR: rats aged 24 months fed ad libitum; EOD OR: rats aged 24 months starved every other day from 2 months of age.

CR, achieved by feeding animals a standard diet on alternate days (EOD), protects against both oxidative stress and fibrosis (Table 1 and Figure 1). Possible protective mechanism/s of CR against fibrosclerosis might be closely connected to the reduction of oxidative stress: we observed increased JNK and p38 activation in rat aorta with age, both decreased by CR (Figure 2), while ERK1/2 are not influenced either by aging or by CR (data not shown). Moreover, AP-1 DNA binding is also increased in older rats and, as a consequence, the transcription of AP-1-dependent genes is also up-regulated, among which TGF1, collagen and vimentin (Figures 1, 3). CR is preventive against increased AP-1 activation and increased vimentin content (Figure 3); it may thus be assumed that, by decreasing HNE levels in older rat aortas, CR significantly decreases JNK and p38 activity. Since AP-1 activation is related to JNK activation (Karin & Gallagher, 2005), by down-regulating JNK, CR might prevent the age-related increase of AP-1 activation and consequently the transcription of AP-1-dependent genes, including TGF1, collagen and vimentin. Consistently, in the kidney, aging increases Fos and Jun levels and induces c-Jun phosphorylation, resulting in increased AP-1 activity, and CR prevents AP-1 activation

by suppressing nuclear levels of c-Fos and c-Jun (Kim et al., 2002). Moreover, Bhattacharya et al. (2006) reported significant inhibition of NF-kB (p65/p50) and AP-1 (c-Fos/c-Jun) activities in splenocytes from WT/CR mice compared to WT/AL mice, suggesting that CR inhibits the redox-sensitive NF-kB and AP-1-dependent inflammatory responses. In this connection, CR has also been found to modulate activity and expression of several pro-inflammatory cytokines including TNF-, IL-1 and IL-6, and to limit synthesis of other pro-inflammatory mediators (Chung et al., 2002; Chung et al., 2006). Moreover, inflammation markers are reduced in the plasma of animals and human volunteers subjected to CR (Ugochukwu & Figgers, 2007; Holloszy & Fontana, 2007; Johnson et al., 2007).

Figure 2. Effect of calorie restriction on MAPKs activation in homogenates of aortas from rats of different ages. Data are expressed as Arbitrary Densitometric Units and are means ± SD of 5 animals per group. * Significantly different from AL YR p < 0.001. § Significantly different from AL YR p < 0.05. ** Significantly different from AL OR p < 0.001. AL YR: rats aged 6 months fed ad libitum; AL OR: rats aged 24 months fed ad libitum; EOD OR: rats aged 24 months starved every other day from 2 months of age.

Figure 3. Effect of calorie restriction on AP-1 DNA binding activity (upper panel) and on vimentin levels (lower panel) in homogenates of aortas from rats of different ages. Data are expressed as Arbitrary Densitometric Units and are means ± SD of 5 animals per group. § Significantly different from AL YR p < 0.01. * Significantly different from AL OR p < 0.001. AL YR: rats aged 6 months fed ad libitum; AL OR: rats aged 24 months fed ad libitum; EOD OR: rats aged 24 months starved every other day from 2 months of age.

Conclusion

The free radical theory of aging proposed by Harman (1956) asserts that radicals endogenously produced during aerobic metabolism or induced by exogenous stimuli (irradiation, smoking, environmental pollution, dietary components) are major factors that contribute to aging. Oxidative damage to cells and tissues may be directly related to ROS and/or to final products of oxidative modifications of biological macromolecules, in particular lipids. Of these, lipid peroxidation-derived aldehydes (HNE) play a key role in the initiation and progression of fibrosis in various organs.

Among the several mechanisms proposed for the anti-aging action of CR, protection against oxidative damage is one of the most convincing.

In connection with fibrosclerosis, which is a constant and distinctive feature of tissue aging, CR decreases oxidative stress, with consequent reduction of the levels of HNE. CR may thereby also reduce fibrosis markers, TGF1 and collagen, most probably by reducing

JNK and AP-1 activation. Acting on ROS and oxidative stress, CR may also influence the inflammatory environment by down-modulating inflammatory mediators (Figure 4).

Figure 4. Possible model for the protective mechanism of calorie restriction against fibrosclerosis during aging.

The possibility of interfering in the fibrogenesis process by modifying dietary habits opens new nutritional horizons in the prevention and treatment of several pathological processes characterized by excessive fibrosis. The pharmacological or nutritional manipulation of endogenous cellular mechanisms is conceivably an innovative approach to therapeutic intervention in diseases causing tissue damage; this suggests potential novel therapeutic strategies relying upon the simultaneous activation of cytoprotective genes of the cell life program and down-regulation of pro-inflammatory and pro-oxidative genes. In light of the poor adherence of subjects to continuous low calorie restricted diets, studies are increasingly aimed at determining the feasibility and efficacy of natural and/or pharmacological CR mimetic molecules.

This work was supported by grants from the University of Turin, the Piedmont Regional Government, and the Italian Ministry for the Education, University and Research, PRIN 2007, Italy.

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