The FASEB Journal express article 10.1096/fj.03-0849fje. Published online December 19, 2003.
Alteration of soluble adhesion molecules during aging and their modulation by calorie restriction Yani Zou,* Kyung Jin Jung,* Jung Won Kim,* Byung Pal Yu,† and Hae Young Chung* *
College of Pharmacy, Pusan National University, Busan 609-735, Korea; †Department of Physiology, The University of Texas Health Science Center at San Antonio, Texas 78229-3900, USA Corresponding author: Hae Young Chung, Ph.D., Department of Pharmacy, College of Pharmacy, Pusan National University, 30 Jangjeon-dong, Geumjeong-gu Busan 609-735, Korea; E-mail:
[email protected] ABSTRACT To investigate the status of soluble adhesion molecules (sAMs) during aging, the present study determined protein levels of several major sAMs in serum samples obtained from rats at different ages. These sAMs include E-selectin, P-selectin, vascular cell adhesion molecule 1 (VCAM-1), and intercellular adhesion molecule 1 (ICAM-1). Fischer 344 rats, ages 6, 12, 18, and 24 months, fed ad libitum (AL) and calorie restricted (CR) diets were used in this study. Analysis by Western blotting showed that the levels of all sAMs studied increased during aging in AL rats, but were effectively blunted in the CR rats. Total reactive oxygen species/reactive nitrogen species (ROS/RNS) levels were measured by fluorescent probe 2’,7’-dichlorofluorescin diacetate. Increased ROS/RNS levels were found to coincide with increased levels of superoxidegenerating xanthine oxidase in serum during aging, but were found suppressed by CR. Increases in sAMs levels were duplicated in another experiment in which young (13-month-old) and old (31-month-old) rats were injected with proinflammatory lipopolysaccharide. These findings suggest that the altered expressions of sAMs may be due to increased oxidative stress with advanced age and that these increases were prevented by CR through its antioxidative action. Key words: vascular aging • inflammation • oxidative stress • endothelial cells • reactive oxygen/nitrogen species
A
dhesion molecules (AMs) are proinflammatory proteins that play a crucial role in cell−cell/cell−matrix interactions and are implicitly involved in the immune response and inflammation process. Among the major players, endothelial/leukocyte cell adhesion molecules, E-selectin, P-selectin, vascular cell adhesion molecule 1 (VCAM-1), and intercellular adhesion molecule 1 (ICAM-1) control the adhesion of leukocytes/monocytes to endothelium, consequently transmigrating to the vessel wall and mediating inflammation processes (1). These molecules maintain low levels in normal physiological conditions, but expressions can be enhanced when endothelial cells (ECs) are confronted by stimuli, including proinflammatory reactive oxygen species/reactive nitrogen species (ROS/RNS) (2), which are responsible for various pathological processes in vasculatures. A detailed mechanism by which soluble AMs (sAMs) are produced is still unclear, but sAMs seem to be shed off from the cell surface by
matrix metalloproteinases (3, 4). Recently, the levels of sAMs in plasma have been suggested as useful markers in stratifying disease severity or prognosis (5) and are used as markers, as well as vascular risk factors for many cardiovascular abnormalities such as peripheral arterial disease (5), coronary heart disease (6), stroke (7), and other conditions that are more prevalent and closely related to aging processes (8). Therefore, an investigation into the close association between aging and sAMs should produce experimental evidence on the underpinning mechanisms of vascular aging phenomena. 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 a disruption in the cellular redox balance (due to the net effect of increased oxidative stress and a decreased, counter-acting antioxidative force during aging) is responsible for abnormal cellular functions and the characteristic dysfunctions of aged organisms (9, 10). This oxidative stress hypothesis has received its strongest support from the antiaging action of calorie restriction (CR). CR, referring to a reduction in calorie intake without compromising essential nutrients to avoid malnutrition, is the only established antiaging, antioxidative experimental paradigm that consistently has shown to increase both median and maximum life spans in laboratory animals (9). CR is found to efficiently down-regulate all aforementioned agerelated proinflammatory proteins, thereby exhibiting powerful resistance against harmful stimuli (11−15). Although the molecular aspects of proinflammatory proteins have been investigated in relation to the oxidative stress hypothesis, the possible association of sAMs with vascular aging process has not attracted much attention from researchers in the field. Almost no information is available at this time on the age-related expression of sAMs, in particular on the effect CR has on the modulation of certain sAM expressions. For a better understanding of vascular aging through the molecular events that result from increased inflammatory processes, it seems pertinent to explore the age-related expressions of the following sAMs, soluble E-selectin (sE-selectin), soluble P-selectin (sP-selectin), soluble VCAM-1 (sVCAM-1), and soluble ICAM-1 (sICAM-1). Knowing that the CR paradigm can modulate age-related oxidative stress and inflammatory reactions, we hypothesized that the agerelated increases in the expression of sAMs are suppressed by CR as the level of oxidative stress was reduced. METHODS Animals and serum preparation Male, specific pathogen free, Fischer 344 rats were obtained from Chaels River Lab, MA. Rat maintenance procedures for specific-pathogen free status and dietary composition of chow have been previously reported (16, 17). Briefly, Fischer 344 rats were fed a diet of the following composition: 21% soybean protein, 15% sucrose, 43.65% dextrin, 10% corn oil, 0.15% αmethionine, 0.2% choline chloride, 5% salt mix, 2% vitamin mix and 3% Solka-Floc (cellulose). The semisynthetic diet was supplied by Purina Test Chow Co. (St. Louis, MO). The ad libitum (AL) fed group had free access to both food and water. The animals designated as CR were fed 60% of the food intake of their AL-fed littermates, beginning at 6 weeks of age. All protocols
used in the animal maintenance are approved by the Institutional Animal Care and Use Committee at University of Texas Health Science Center at San Antonio, TX, USA. Rats at 6, 12, 18, and 24 months of age were used in this study. Each group consisted of 6 animals. Body weights of rats in different groups were as follows: 6 months AL/CR rats 366.4 ± 15.9/212.3 ± 8.7 g; 12 months AL/CR rats 461.3 ± 46.9/246.3 ± 8.8 g; 18 months AL/CR rats 491.5 ± 17.8/280.8 ± 14.2 g; 24 months AL/CR rats 463.3 ± 32.6/257.7 ± 28.8 g. To obtain serum samples, rats were decapitated and blood was drawn and allowed to clot at room temperature (RT) for 30 min before being centrifuged at 3000 rpm at 4°C for 20 min. The supernatant was collected as serum, frozen and stored at −80°C until analyses were performed. This study complied with the Guide for the Care and Use of Laboratory Animals by the National Institutes of Health (Publication No. 85–23), USA. For the lipopolysccharide (LPS) challenge, LPS (5 mg/kg) was intraperitoneally injected into Fischer 344 rats, aged 13 and 31 months. Control group rats were treated with same volume of saline. Rats in all groups were killed 5 h after injection. Aorta sample preparation At 6 and 24 months of age, the rats were decapitated, the chest opened, and aortas quickly excised and immersed in ice-cold isotonic saline. The aortas were then rapidly frozen in liquid nitrogen, and stored in deep-freezer at −80°C until assayed. Aortas were homogenized with a polytron homogenizer in 7 volumes (v/w) of ice-cold homogenization solution of the following composition: 50 mM phosphate buffer (pH 7.4), 0.5 mM phenylmethylsulfonyl fluoride, 1 mM ethylenediaminetetraacetic acid disodium salt, 80 mg/L trypsin inhibitor and 1 µM leupeptin. The homogenate was centrifuged at 900×g at 4°C for 15 min, and supernatants (postnuclear fraction) were used for Western blotting analysis and biochemical assays in this study. Western blotting analysis Western blotting was carried out as described previously (18). Serum samples diluted 10 times or aorta homogenate (postnuclear fraction) were boiled for 5 min with a gel-loading buffer (pH 6.8, 0.125 M Tris [hydroxymethyl] aminomethane, 4% sodium dodecyl sulfate (SDS), 20% glycerol, 10% 2-mercaptoethanol, and 0.2% bromophenol blue) at a ratio of 1:1. Total protein-equivalents for each sample were separated by an SDS-polyacrylamide minigel, as described by Laemmli (19) at 100 V and transferred to a poly(vinylidene fluoride) membrane at 15 V for 45 min in a semidry transfer system. The membrane was immediately placed into a blocking solution (5% w/v skim milk powder in TBS-Tween buffer containing 10 mM Tris, 100 mM NaCl, and 0.1 mM Tween-20, pH 7.5) at RT for 1 h. The membrane was incubated with a primary antibody (0.5% w/v skim milk, diluted 1:200 in TBS-Tween buffer) at RT for 1.5 h, followed by incubation with a horseradish peroxidase-conjugated secondary antibody at RT for 1 h. Antibodies against E-selectin, P-selectin, VCAM-1, ICAM-1, and Rabbit anti-goat secondary antibody were obtained from Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA. Antixanthine oxidase (XOD) antibody was a gift from Dr. Takeshi Nishino (Nippon Medical School, Tokyo, Japan). Horseradish peroxidase-conjugated secondary antibody labeling was detected by ECL Western blotting detection reagents and analysis system (Amersham Biosciences, NJ) following the manufacturer’s instructions. Prestained blue protein marker was used for molecular
weight determination. The concentration of total protein in samples was measured with Sigma protein assay reagent kit containing bicinchoninic acid. Quantification of oxidative status in rat serum and aorta A previously published fluorescence assay method (20) was used to determine level of total ROS/RNS, including superoxide, hydroxyl radical, and hydrogen peroxide (20, 21). Briefly, 2’,7’-dichlorofluorescin diacetate (DCFH-DA, purchased from Molecular Probes, Inc., Eugene, OR) was dissolved in absolute ethanol at the concentration of 12.5 mM and kept at −80°C in the dark. It was freshly diluted with 50 mM phosphate buffer (pH 7.4) to 125 µM before experiment. DCFH-DA was added to serum (50 µL) or aorta homogenate in a 96-well plate to achieve a final concentration of 25 µM. The plates were incubated at 37°C for 30 min in the presence of exogenously added esterase (0.3 U at a final concentration) to ascertain a steady-level of 2’,7’dichlorofluorescein (DCF). The change in fluorescence intensity was monitored at two time points (0 and 30 min) using a microplate fluorescence reader (Bio-TEK Instruments, Inc., Winooski, VT) at excitation 485 nm/emission 530 nm. ROS/RNS status was calculated as fluorescence per milligram of protein and per min. Data were expressed by relative DCF concentrations; that is, all other groups were compared with the AL group at age 6 months. Quantification of reductive status in rat serum and aorta The total thiol content concentration assay was based on a previous report (22). Briefly, 200 mM Tris buffer (pH 8.2), 250 µL; 10 mM 5,5′- dithiobis-2-nitrobenzoic acid (DTNB), 25 µL; and methanol 1 ml were added to 25 µL of serum or aorta homogenate and stayed at 20°C for 15 min. After centrifugation at 4000 ×g for 20 min, optical density of the supernatant was determined at the wavelength of 412 nm (ε=13 mM−1cm−1) on a microplate reader (SpectraCount, Packard, Meriden, CT). Statistical analysis For Western blotting, one representative blot was shown from tripled independent experiments. For redox status determination, the results were expressed as mean ± standard error (n=6). The statistical significance of the difference between the treatments (age and diet) was determined by ANOVA method. One-factor ANOVA was conducted to analyze significant differences among all possible ages and diet pairs. Differences among the means of individual groups were assessed by the Fischer’s Protected LSD post hoc test. Values of P
