Alteration of soluble adhesion molecules during aging and their modulation by calorie restriction

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Abstract
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⤻ ). Figure 1. Effects of age and CR on expression of sAMs in rat serum. A) Western blot analysis was performed to detect E-selectin, P-selectin, VCAM-1, and ICAM-1 protein levels in serum from AL and CR rats, at ages 6, 12, 18, and 24 months. One representative blot of each protein is shown from three experiments that yielded similar results, respectively. B) The levels of protein quantified by densitometry as a percentage of the level of a 6-month-old AL rat. Statistical significance: *P < 0.05, **P < 0.01 vs. 6-month-old AL rats; #P < 0.05, ##P < 0.01 CR vs. age-matched AL rats. sAMs, soluble adhesion molecules; VCAM-1, vascular cell adhesion molecule-1; ICAM-1, intercellular adhesion molecule-1; AL, ad libitum; CR, calorie restriction. Download figure Download PowerPoint 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 (PP1) 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. ⤻ Figure 2. Schematic presentation of altered soluble adhesion molecules during aging and modulation by calorie restriction. During aging, accumulated reactive oxygen species/ reactive nitrogen species (ROS/RNS) are accompanied with the decrease in total thiol contents, which lead to a net increased oxidative stress during aging. Consequently vascular endothelial cells (ECs) are induced activation/dysfunction and, as the result, levels of soluble adhesion molecules (sE-selectin, sP-selectin, sVCAM-1 and sICAM-1) are elevated, contributing to increased incidence of vascular diseases during aging. In contrast, calorie restriction (CR) can suppress aging process by decreasing the level of ROS/RNS while maintaining the level of total thiol contents, thereby decreasing the age-related insult to ECs, then down-regulate level of sAMs. Accordingly, the incidence of age-related diseases is decreased by CR. Download figure Download PowerPoint 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. 1To read the full text of this article, go to http://www.fasebj.org/cgi/doi/1096/fj.03-0849fje;
Funding Information
  • Korean Foundation for Cancer Research (FS0004)
  • Korea Science and Engineering Foundation (R21‐2000‐000‐00021‐0)
  • National Institute on Aging (AG01188)