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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online December 19, 2003 as doi:10.1096/fj.03-0849fje. |
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* College of Pharmacy, Pusan National University, Busan 609-735, Korea; and
Department of Physiology, The University of Texas Health Science Center at San Antonio, Texas, USA
2Correspondence: Department of Pharmacy, College of Pharmacy, Pusan National University, 30 Jangjeon-dong, Geumjeong-gu, Busan 609-735, Korea. E-mail: hyjung{at}pusan.ac.kr
SPECIFIC AIMS
To better understand vascular aging through the molecular events that result from increased inflammatory processes, it is pertinent to explore age-related serum levels of adhesion molecules (AMs), essential participants in the inflammation process. Knowing that calorie restriction (CR), the only established anti-aging paradigm, can reduce age-related oxidative stress and inflammatory reactions, we expected to show the effect of CR on age-related alterations of serum soluble AMs (sAMs) as oxidative stress was decreased.
PRINCIPAL FINDINGS
1. Expressions of sAM: E-selectin, P-selectin, VCAM-1, and ICAM-1 in rat serum increased during aging and these increases were blocked by CR
To determine whether protein levels of sAMs were changed by aging and CR, Western blot analysis was performed to measure levels of E-selectin, P-selectin, VCAM-1, and ICAM-1 in Fischer 344 rats at various ages (i.e., 6, 12, 18, and 24 months) in ad libitum (AL) and CR groups. Dramatically increased protein levels of all sAMs examined were found to be associated with the aging process in AL rats (Fig. 1
A). Among our findings when comparing old rats (24 months of age) to young rats (6 months of age): E-selectin level was 4.84-fold increased, P-selectin level was 2.45-fold higher, VCAM-1 level was 9.5-fold higher, and ICAM-1 level was 7.6-fold higher (Fig. 1B
). However, CR rats showed decreased sAM levels in comparison with their age-matched AL counterparts at all ages studied (Fig. 1A
).
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To correlate increased sAMs and tissue AMs expression, we ran experiments to detect AMs production in aorta homogenate. The results showed up-regulated expression of AMs during aging in AL rats and down-regulation by CR, i.e., the same tendency observed in alterations of sAMs. Results indicated that changes in levels of sAMs were reflective of alterations in AM levels in the vasculature.
2. Increased sAMs were correlated with accumulated oxidative stress during aging
Given that enhanced production of E-selectin, P-selectin, VCAM-1, and ICAM-1 is mainly a response by endothelial cells (ECs) in the inflammation process, we investigated whether age-related alterations of sAMs were due to oxidative stress during aging. Effects of aging and CR on total reactive oxygen species/reactive nitrogen species (ROS/RNS) and total thiol contents level in serum were examined as indicators of oxidative stress and reductive state, respectively. Total ROS/RNS were assayed by the fluorescent probe – 2', 7'-dichlorofluorescin diacetate. A gradual increase in ROS/RNS with age was found, more clearly in 24-month-old rats as evidenced by a significant 97% increase in fluorescence intensity, compared with 6-month-old AL rats (P<0.01). In contrast, the age-related increase in ROS/RNS was very efficiently blocked by CR – results showed CR rats in all age groups to have nearly the same levels of ROS/RNS as young rats. On the other hand, total thiol content levels in serum were suppressed in old AL rats (P<0.05 24-month vs. 6-month) whereas CR helped maintain total thiol content levels during aging.
To further identify the source of ROS/RNS generation, serum xanthine oxidase (XOD), a well-known source of superoxide generation and a marker of ECs damage, was evaluated and compared between AL and CR groups. Significantly elevated serum XOD was detected in old AL rats; in contrast, only a small change was observed in CR rats with age.
3. Lipopolysaccharide (LPS)-injected rats showed increased protein levels of sAMs
To duplicate our finding on the association between levels of sAMs and the aging process and to further understand the mechanism underlying these alterations with respect to inflammatory stress, young rats (13 months) and advanced-aged rats (31 months) were injected with a potent inflammatory stimulus, LPS. The levels of sAMs in serum from LPS-treated rats and their saline-injected control were measured by Western blotting. Results indicated that 1) old rats expressed higher levels of sAMs, duplicating previous findings; 2) the LPS-treated group showed increased production of sAMs, indicating a correlation between sAMs and inflammation; 3) LPS-treated old rats expressed the highest amount of sAMs among all groups, thereby, old rats exhibiting an enhanced sensitivity to inflammatory stimulation.
CONCLUSIONS AND SIGNIFICANCE
Currently, although the mechanisms underlying the aging process are poorly understood, most cellular and molecular elucidations of aging phenomena support the oxidative stress hypothesis, which proposes that an imbalance in the redox state due to increased oxidative stress coupled with a weakened anti-oxidative defense system may be the major underlying cause of aging, including vascular aging. This oxidative stress hypothesis has received its strongest support from the anti-aging action CR. CR, referring to a reduction in calorie intake without compromising essential nutrients to avoid malnutrition, is the only established anti-aging model. Although the cellular basis of CR’s life-prolonging effect has not been well elucidated, recent work on CR provides abundant evidence that CR likely extends diverse benefits by its anti-oxidative ability.
According to oxidative stress hypothesis of aging, among the more vulnerable tissues to stress, ECs that line the blood vessel, can be listed at the top. Among the major AMs are E-selectin, P-selectin, VCAM-1, and ICAM-1, and these are mainly produced by activated/dysfunctional ECs during the inflammation process. Up-regulation of AMs, including sAMs, is identified as the major factor underlying the EC-activation response to vascular inflammation and various diseases. Therefore, it is possible to propose a link between age and the expression of sAMs through EC activation by increased oxidative stress during aging.
However, only a few previous studies exist, with conflicting data. In the present study, we used various age groups covering the life span of rats—from young (6 months) to old (24 months), 24-month-old rats being the human equivalent of 70 to 75 years of age. We used CR as anti-aging positive control and proved our work using LPS-injection experiments. We reported that age-related increases of sAMs, which reflected changes in AM levels in tissues, were correlated with oxidative stress, contributed by increased ROS/RNS and decreased thiol contents. All alterations were blocked by CR. Increases in sAM levels were further duplicated in young and old rats injected with proinflammatory LPS. The findings are summarized in Fig. 2.
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Collectively, our study confirmed an association between sAMs and aging, which indicated EC activation/dysfunction during aging, further supporting oxidative stress hypothesis. Age-related sAM alterations may contribute to vascular aging as well as chronic age-related diseases. The confirmed association between sAMs and the aging process may be an important link between inflammation and vascular aging and/or vascular diseases.
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/1096/fj.03-0849fje; 
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