In short-lived organisms, the examination of survival curves is practical, and therefore useful in assaying aging rates. However, for most mammalian species, survival curves are less practical due to their relatively long lifespan and complexity. Given the long lifespan of mammals, it would be useful to establish organ-specific biomarkers of aging for evaluating the efficacy of interventions. Our comparison of gene expression patterns in heart and cerebellum of multiple strains of mice revealed tissue-specific biomarkers of aging that can be used to measure the aging process. Although thousands of genes were changed in expression with aging in each individual strain, there was a relatively small number of common genes changed in all strains tested: 20 genes in the heart and 99 genes in the cerebellum. One possible explanation for this finding is that many biological processes associated with aging are strain-specific. Aging resulted in up-regulation of several genes that are involved in immune and inflammatory responses related to innate immunity. The expression of C4 was induced with aging in both heart and cerebellum. C4 prevents early stage autoimmune disease (Paul et al., 2002
) and mice with a disrupted C4 locus showed an impaired immune response (Gadjeva et al., 2002
). In the heart, two more genes involved in the immune response were identified as biomarkers of aging. Cxcl14 is a potent chemoattractant that is ubiquitously expressed in normal tissues, but absent in many tumor cell lines (Frederick et al., 2000
) and Scap2 is required for the activation of immune system (Togni et al., 2005
In addition to C4, several genes involved in the immune and inflammatory response were increased in expression with aging in the cerebellum, including the lysosomal proteases cathepsin D, cathepsin S, cathepsin Z, and three components of C1q (alpha, beta, and gamma polypeptide) involved in innate imunity. The lysosomal protease cathepsin S is involved in degradation of protein antigens and controls intracellular trafficking of class II MHC molecules (Hsieh et al., 2002
). The activation of the classical complement system was reported in the nondemented aged human brain and also in early-stage Alzheimer’s disease (Zanjani et al., 2005
), and an increase of C1q beta polypeptide mRNA was found in aging rats (Pasinetti et al., 1999
). Taken together, our observations suggest that normal aging in the heart and brain is associated with a transcriptional pattern indicative of heightened immune and inflammatory responses. Interestingly, the expression of complement activation genes has been shown in skeletal muscle, kidney, and brain in humans (Zahn et al., 2006
). The expression of innate immunity genes may be due to the activation of an ancient NF-κB signaling pathway of host defense in multicellular organisms (Salminen et al., 2008
). This signaling system may connect genotoxic stress, inflammation, and apoptosis, and therefore play an important role in the origin of aging phenotypes and age-related diseases (Salminen et al., 2008
). Previous studies have shown that the expression of Gfap, the first validated brain aging transcriptional marker, increases progressively during aging in humans and rodent models (Nichols et al., 1993
). It is reassuring that our screen identified Gfap, and also that the expression of this gene is reduced by CR at all ages examined. Some of the inflammatory markers reported in this study were also identified in our original DNA microarray analysis of heart (Lee et al., 2002
) and brain (Lee et al., 2000
). Interestingly, gene sets related to the immune system, such as complement activation (GO:0006958) and regulation of the immune system (GO:0050776), were induced in both heart and cerebellum. Examination of these gene sets suggests that genes involved in innate immunity account for the majority of genes induced. Possibly, induction of these and other genes related to the immune system is a consequence of either increased levels of monocytes/macrophages in tissues, or increased levels of cytokines, as demonstrated in adipose (Wu et al., 2007
) and brain (Ye & Johnson, 1999
) tissues of aged mice. In contrast, GO categories related to mitochondria were induced in some strains, but suppressed in others. This observation suggests that a reduction in the expression of genes related to mitochondria and energy metabolism is not a universal feature of aging in mice.
Our data revealed two biomarkers of aging that are common in both heart and cerebellum: C4 and tissue inhibitor of metalloproteinase 2 (TIMP2). Aging is a major risk factor for the development of arterial stiffness and vascular disease such as hypertension and atherosclerosis (Lakatta, 2002
), and it is associated with the imbalance between matrix metalloproteinases and their endogenous inhibitors, tissue inhibitors of metalloproteinases (Dollery et al., 1995
; Zervoudaki et al., 2003
). In mice, new fibrovascular tissue from old animals expressed more TIMP2 than did corresponding tissue from young mice (Koike et al., 2003
). Comparison between young and old human microvascular endothelial cell lines revealed that TIMP2 is expressed at higher levels in cell lines from old humans (McNulty et al., 2005
). Interestingly, a transcriptional profiling of human tissues with aging identified TIMP1 as the gene displaying the highest change in gene expression in multiple tissues in humans (Zahn et al., 2006
). The transcriptional alterations of TIMP2 in multiple strains of aged mice are consistent with these previous observations, suggesting that elevated levels of TIMP2 may modulate impaired angiogenesis and fibrosis in aged tissues.
Previous studies reported the impact of antioxidants on age-related gene expression patterns in mice. Dietary supplementation with LA or CQ results in transcriptional changes associated with reduced oxidative stress in heart, but these antioxidants did not extend maximum life span and reduce tumor incidence (Lee et al., 2004
). Middle age-onset dietary supplementation of vitamin E also showed a partial inhibitory effect on age-related transcriptional alteration in heart and brain, but was not as effective as CR (Park et al., 2008
; Lee et al., 2002
). We have previously shown that RE can prevent age-related cardiac dysfunction and transcriptional alterations associated with cardiac aging (Barger et al., 2008
), and these findings are in agreement with the strong effect of RE in inhibiting transcriptional markers of aging reported in this study. In the heart, AC was the natural compound that displayed the largest inhibition in the expression of the transcriptional markers. In rats, short-term supplementation with AC reduced age-related alterations in lipid metabolism in multiple tissues including normalization of the cholesterol/phospholipid ratio (Tanaka et al., 2004
) and reduced DNA damage in the brain (Haripriya et al., 2005
). We have previously identified transcriptional evidence for alterations in lipid metabolism as a major feature of aging in the heart, and showed that CR, but not dietary antioxidants, can prevent these alterations (Park et al., 2008
; Lee et al., 2004
). Thus, we postulate that similar to RE, AC may be acting to mimic the metabolic effects of CR in the heart.
We note that a major finding of this study is the remarkable difference in efficacy of the tested compounds. In the heart, RE, LY, AC, and TP were at least as effective as CR, whereas LA and CQ were the most effective antioxidants tested in the cerebellum. Our studies provide support for an important role of oxidative stress in aging, but suggest that the effects of individual antioxidants are tissue-specific. Robust transcriptional biomarkers of aging will be useful for designing combinations of dietary antioxidants that will be effective in inhibiting the aging process in individual tissues in mammals.