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The Journals of Gerontology Series A: Biological Sciences and Medical Sciences 59:B876-B885 (2004)
© 2004 The Gerontological Society of America

Vascular Aging: Molecular Modulation of the Prostanoid Cascade by Calorie Restriction

Jung Won Kim1, Yani Zou1, Sik Yoon2, Ji Hyeon Lee1, Yoon Kyung Kim1, Byung Pal Yu3 and Hae Young Chung1,

1 Department of Pharmacy
2 College of Medicine, Aging Tissue Bank, Pusan National University, Kumjung-Ku, Busan, Korea.
3 Department of Physiology, University of Texas Health Science Center at San Antonio.

Address correspondence to Hae Young Chung, PhD, Department of Pharmacy, College of Pharmacy, Pusan National University, Kumjung-Ku, Busan 609-735, Korea. E-mail: hyjung{at}pusan.ac.kr


   Abstract
Top
 Abstract
Methods
 Results
 Discussion
 References
 
The relevance of prostanoids to inflammation, thrombosis, and cardiovascular diseases is well known. The present study attempts to explore age effects on prostanoids and their biosynthesis cascade. Results from comparing prostanoid levels between young (6 months) and old (24 months) Fischer 344 rats showed rises of prostaglandin E2 (PGE2), PGI2, and thromboxane A2 (TXA2) levels in the old rats. Correlating evidence showed gene expression up-regulation of several prostanoid synthase enzymes in old rat aorta. Further, we found that expression of the antioxidant enzyme glutathione peroxidase was raised by age in the aorta, while superoxide dismutase and catalase expression showed no significant change during aging in the aorta. Moreover, calorie restriction (CR) was found to attenuate age-related prostanoid changes by suppressing inflammatory activities. In conclusion, the data from this study indicated that age-related increases in prostanoids and their biosynthesis might be closely associated with a weakened antioxidant capacity.

THE blood vessel is essential to an organism's end organs as a supplier of nutrients, oxygen, and hormones, and therefore, vascular aging is likely to play a crucial role in systemic aging. A major consideration in an organism's susceptibility to age-related vascular alterations is the role of redox-sensitive inflammatory mediators (1,2), including prostanoids.

In many parts of the body, prostanoids, consisting of prostaglandins (PGs), prostacyclins, and thromboxanes, mediate several key pathophysiological functions, from the host's inflammatory response to blood flow regulation. Prostanoid biosynthesis is a three-step sequence (3) of stimulus-initiated hydrolysis of arachidonate from membrane phospholipids. The first step involves secretory or cytosolic, or both types, phospholipase A2 (sPLA2 and cPLA2) (4,5); the second step is the oxygenation of arachidonate, which yields prostaglandin H2 (PGH2) by cyclooxygenases (COXs) (6); and the third is the conversion of PGH2 to the most important, biologically active end-products, PGD2, PGE2, PGF2{alpha}, PGI2, and thromboxane A2 (TXA2) via specific synthases (7).

The COXs play the important role as rate-limiting enzymes in PG biosynthesis, and they are reported to generate reactive species (RS) during the hydroperoxidation process (8). This RS generation is an important source of oxidative stress under both normal physiological and inflammatory conditions (9). In many cell types including endothelial cells and macrophages, COX-2, stimulated by some cytokines or lipopolysaccharides (LPS), is responsible for the large production of proinflammatory PGs (10–12).

Although the precise mechanism underlying vascular aging is unclear, enhanced oxidative stress, coupled with a weakened antioxidative defense system, has been reported as the major underlying cause of vascular aging (13–15). Damage to the vascular wall by RS, generated both within and outside the vessels, is strongly implicated in vascular aging and age-related vascular diseases, arteriosclerosis, hypertension, and diabetes (6). To defend against rampant attacks from RS, organisms have developed appropriate cellular antioxidative protective forces. A first-line antioxidant defense system includes typical enzymes such as superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and catalase. Any disruption in the balance between oxidative forces and counteracting antioxidative defenses could elicit oxidative damage to cellular structures and impair functions.

The most well-known physiological antioxidative action has been proved to be the life-prolonging action of calorie restriction (CR). CR has been investigated extensively in aging research and is the only established anti-aging experimental paradigm consistently shown to increase both median and maximum life spans in laboratory animals (16). Based on its suppressive action against RS, CR's anti-aging effects on vascular aging serves as a good example of its ability to maintain a proper redox balance by defending against age-related oxidative insults, thereby preserving vascular integrity during aging (13).

In this article, we delineate the molecular insights we gained from studying age-related alterations in the production of prostanoids, enzymatic gene expression during prostanoid biosynthesis, and CR's beneficial modulation of these processes.


   METHODS
Top
 Abstract
 Methods
Results
 Discussion
 References
 
Reagents and Materials
Enzyme immunoassay (EIA) kits for prostanoids were purchased from Amersham (Bucks, U.K.). RNAzol B was obtained from Tel-Test (Friendwood, TX). Primers for the reverse transcriptase-polymerase chain reaction (RT-PCR) were synthesized by Bioneer (Daejeon, Korea). Antibodies used for Western blotting were obtained from BioPur AG (Kanton, Bubendorf, Switzerland); antibodies against GSH-Px, SOD, and catalase were obtained from Rockland (Gilbertsville, PA). Polyvinylidene difluoride (PVDF) membranes were obtained from Millipore Corporation (Bedford, MA). Other chemicals were purchased from Sigma (St. Louis, MO).

Animal and Tissue Preparation
Male, specific pathogen-free Fischer 344 rats were raised in the barrier facilities at the University of Texas Health Science Center at San Antonio. Complete descriptions of animal housing, care, and feeding have been reported (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% {alpha}-methionine, 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). All protocols utilized in the animal maintenance are approved by the Institutional Animal Care and Use Committee of the University. All rats were fed ad libitum (AL) until 6 weeks of age, and then divided into AL and CR groups. Animals in the CR group were fed a whole food intake that was restricted by 40% of that for the AL group. Histopathological examination revealed no evidence of nephritic lesions in these soy protein-fed Fischer rats even at the advanced age of 24 months (18).

At 6 and 24 months of age, the rats were decapitated, and the chest was opened. To obtain serum samples, blood was drawn and allowed to clot at room temperature (RT) for 30 minutes before being centrifuged at 3000 rpm at 4°C for 20 minutes. The supernatant was collected as serum and frozen and stored at –80°C until analyses were performed.

Rat aorta was quickly excised and immersed in ice-cold isotonic saline, then rapidly frozen in liquid nitrogen and stored in deep-freeze at –80°C until assayed. The aorta was homogenized with a polytron homogenizer in 7 volumes (v/w) of ice-cold homogenization solution of the following composition: 50 mmol/L phosphate buffer (pH 7.4), 0.5 mmol/L phenylmethylsulfonyl fluoride (PMSF), 1 mmol/L ethylenediaminetetraacetic acid (EDTA), 80 mg/L trypsin inhibitor, and 1 µmol/L leupeptin. The homogenate was centrifuged at 900 x g for 15 minutes at 4°C, and the supernatants were used in this study. All studies complied with the Guide for the Care and Use of Laboratory Animals by the National Institutes of Health (Publication No. 85–23).

EIA for Prostanoids
One hundred microliters of serum or 400 µL of aorta homogenate samples were used in the EIA. Aorta homogenates were incubated with exogenous arachidonate (10 µmol/L) at 37°C for 30 minutes before detection. After acidification to pH 3 with 1 M citric acid, serum or aorta homogenate samples were applied to C-2 ethyl columns (Amersham Biosciences), prewashed with methanol and distilled water (DW), and subsequently washed with DW, 10% ethanol, and hexane. PGI2 and TXB2 were eluted with ethyl acetate, and elute was evaporated under vacuum to dryness. Because PGI2 and TXA2 are unstable and undergo spontaneous hydrolysis to more stable 6-keto-prostaglandin F1{alpha} and TXB2, respectively, they are determined by measuring the stable metabolites. All procedures followed the user's manual provided by the EIA kit.

Immunohistochemical Stain
Frozen sections (5 µm thick) were cut on a Reicheit cryostat and were placed on 3-aminopropyltriethoxysilane-coated slides. After being dried, the cryosections were fixed in acetone for 20 minutes at –20°C. Immunostaining was performed by using the streptavidin-biotin complex (ABC) method. In brief, the sections were incubated in a solution of phosphate-buffered saline (PBS) containing 3% H2O2 for 20 minutes to eliminate endogenous peroxidases. After washing in PBS, in order to block nonspecific binding, the sections were incubated with 5% normal donkey serum (Jackson Immuno Research Laboratories, West Grove, PA) for 30 minutes, and incubated with each of the primary antibodies, COX-1 (1:100) and COX-2 (1:100), at 4°C overnight. Following incubation with the primary antibodies, the sections were incubated for 60 minutes at room temperature with biotinylated donkey antimouse immunoglobulin (IgG) (1:100, Jackson Immuno Research Laboratories) and with the avidin-biotin complex reagent (Vectastain ABC Reagent Elite Kit, Vector Laboratories, Burlingame, CA) according to the manufacturer's instructions. The sections were developed with a 0.05% 3,3-diaminobenzidine and 0.003% H2O2- medium under microscopical control to visualize peroxidase activity. Afterwards, the sections were counterstained with Mayer's hematoxylin and mounted in a xylene-based mounting medium (Emtellan, Darmstadt, Germany).

Western Blotting
Aorta homogenates were boiled for 5 minutes with a gel-loading buffer consisting of 125 mmol/L Tris-Cl, 4% sodium dodecyl sulfate, 10% 2-mercaptoethanol, pH 6.8, and 0.2% bromphenol blue, at a 1:1 ratio. Total protein equivalents for each aorta homogenate (20 µg) were separated on an 8%–12% sodium dodecyl sulfate (SDS)-polyacrylamide minigel using a Laemmli buffer system and were transferred to a PVDF membrane at 100 V for 1.5 hours. The membrane was immediately placed in blocking buffer (5% nonfat milk) in 10 mmol/L Tris, pH 7.5, 100 mmol/L NaCl, and 0.1% Tween-20. The blot was allowed to block at RT for 1 hour. The membrane was incubated with specific primary antibody for 2 hours at 25°C followed by a secondary antibody for 1 hour at 25°C. The secondary antibodies used were a horseradish peroxidase-conjugated donkey antirabbit antibody (Amersham; 1:2,000) or a donkey antigoat antibody (Serotec, 1:3,000, Kidlington, Oxford, U.K.). Antibody labeling was detected using enhanced chemiluminescence (ECL) (Amersham) according to the manufacturer's instructions. Prestained blue protein markers were used for molecular weight determination. Protein concentrations were measured by the bicinchoninic acid method (19) using bovine serum albumin as a standard.

RT-PCR
Preparation of RNA.-- Tissue samples were homogenized in the presence of RNAzol B (2 mL/100 mg of tissue) with a Polytron homogenizer. Chloroform (0.2 mL) was added to 2 mL of homogenate. The samples were covered tightly, shaken vigorously for 15 seconds, and placed on ice for 5 minutes. The suspension was centrifuged at 12,000 x g at 4°C for 15 minutes. The aqueous phase was transferred to a fresh tube, followed by adding an equal volume of isopropanol, and then centrifuged again. The RNA pellet was washed once with 75% ethanol by vortexing. After a final centrifugation at 7500 x g at 4°C for 8 minutes, the pellet was dried for 10 minutes and dissolved in diethylpyrocarbonate (DEPC)-treated water.

RT.-- The first strand cDNA was synthesized from 2 µg total RNA. Reaction mixture contained DEPC-treated water, 250 ng of random primer, 2 µL of 100 mmol/L dithiothreitol (DTT), 4 µL of 5 x first strand buffer, 4 µL of 2.5 mmol/L dNTP, 100 U of RT (Gibco-Bethesda Research Laboratory, Gaithersburg, MD), and 16.5 U of RNase inhibitor. The RNA samples were added to the mixture and incubated at 37°C for 2 hours. The reaction was stopped by boiling at 100°C for 2 minutes, and the cDNA was stored at –20°C until use.

PCR.-- As a way to carry out the PCR, cDNA amplification was performed in a PCR master mix containing 1 x PCR buffer (Perkin Elmer, Gaithersburg, MD), 0.2 mmol/L deoxyribonucleotides (dNTP), 0.25 U of Taq DNA polymerase (Perkin Elmer), and 50 ng of sense and antisense primers. Reaction conditions were as follows: 94°C for 30 seconds denaturation, 54°C for 30 seconds annealing, and 72°C for 1 minute extension. Electrophoresis was performed in 1% agarose gel. After suitable separation was achieved, the gel was stained with ethidium bromide and photographed under ultraviolet transilluminator. Primer sequences, numbers of cycles, and product sizes are listed in Table 1.


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  		Table 1. Primer Sequences Used and Number of Cycles for Polymerase Chain Reaction Amplification.

 
Antioxidant Capacity Analysis
The reactive oxygen species' (ROS) scavenging capacity was measured by fluorometric method using 2',7'-dichlorofluorescin diacetate (DCFH-DA) (20). Aorta homogenate (30 µg) or the same volume of homogenate buffer (as control) was added to 0.2 mmol/L FeSO4/1 mmol/L H2O2, or 1 mmol/L menadione, or 1 mmol/L H2O2, followed by adding DCFH (2 µmol/L), which was prepared by esterase treatment on DCFH-DA. The changes in fluorescence intensity were measured by TECAN (GENios, GENios FL, and GENios Plus; TECAN Austria GmbH, Austria) with the excitation and emission wavelengths setting at 485 nm and 530 nm, respectively. After normalization by fluorescence intensity spontaneously generated from the sample, the difference between control and sample changes in fluorescence intensity within 30 minutes was regarded as antioxidant capacity and expressed as fluorescents/min.

Statistical Analysis
Results were analyzed statistically by the analysis of variance test via SPSS for Windows program (SPSS, Inc., Chicago, IL). Values of p <.05 were considered statistically significant.


   RESULTS
Top
 Abstract
 Methods
 Results
Discussion
 References
 
Effects of Age and CR on Prostanoids
Serum prostanoid contents were analyzed and compared for AL and CR rats at 6 and 24 months of age. The PGE2 content was strongly affected by age as shown in Figure 1. The serum content level of PGE2 for animals in the 24-month-old AL group was 2.2-fold higher than for animals in the 6-month-old group. Our results show that CR significantly attenuated this age-related increase in PGE2 (Figure 1). TXA2 content also increased with age. Serum from rats in the 24-month-old AL group showed an approximately 40% greater TXA2 content compared with rats in the 6-month-old group. In the case of PGI2, although analysis of serum PGI2 levels showed a substantial increase during aging, CR did not suppress this increase.



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  		Figure 1. Effects of age and CR on serum PGE2, TXA2 and PGI2 levels. After solid phase extraction, serum samples were assayed with prostanoid enzyme immunoassay kits as described in the "Methods" section. Because PGI2 and TXA2 are unstable and undergo a spontaneous hydrolysis to more stable 6-keto-prostaglandin F1{alpha} and TXB2, respectively, they were determined by measuring the stable metabolites. Each value is the mean ± SE (standard error) of 6 rats. Statistical significance: *p <.05 versus 6-month-old rats of the same group; **p <.01 versus 6-month-old rats of the same group; #p <.05 versus rats of the same age in the AL group and ##p <.01 versus rats of the same age in the AL group, respectively. PGE2 = prostaglandin E2; TXA2 = thromboxane A2; PGI2 = prostaglandin I2; AL = ad libitum group; CR = calorie restriction group

 
We also measured PGI2 and TXA2 levels in arachidonic acid-treated aorta homogenates. As shown in Figure 2, the age-related increase of PGI2 was estimated to be approximately 40% in the AL-fed animals, but was shown to be effectively attenuated in CR animals. When comparing young and old animals in the AL group, we found the level of TXA2 was enhanced about approximately 1.5-fold by the aging process. We also found that CR animals showed lower age-related increases in TXA2 levels than their AL-fed counterparts (Figure 2).



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  		Figure 2. Changes in PGI2 and TXA2 levels with age in rat aorta. Because PGI2 and TXA2 are unstable and undergo a spontaneous hydrolysis to more stable 6-keto-prostaglandin F1{alpha} and TXB2 respectively, they were determined by measuring their stable metabolites. Each value is the mean ± SE (standard error) of 6 rats. Statistical significance: *p <.05 versus 6-month-old rats of the same group; **p <.01 versus 6-month-old rats of the same group; #p <.05 versus rats of the same age in the AL group and ##p <.01 versus rats of the same age in the AL group, respectively. PGI2 = prostaglandin I2; AL = ad libitum group; CR = calorie restriction group

 
Effects of Age and CR on the Prostaglandin Cascade System
Production of prostanoids is catalyzed by an enzyme cascade that includes PLA2s, COXs, and terminal PG synthases. We evaluated the expression of prostanoids cascade enzymes by RT-PCR. Between two isoforms of PLA2, we found the mRNA levels of cPLA2 increased significantly during aging, while sPLA2 levels showed no change (Figure 3).




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  		Figure 3. Effects of age and CR on the mRNA levels of prostanoid-generating enzymes in aorta. A, One representative datum is shown. B, The levels of mRNA were quantified by densitometry as percent of 6-month-old AL rat. Each experiment was performed in triplicate. Statistical significance: *p <.05 versus 6-month-old rats of the same group; **p <.01 versus 6-month-old rats of the same group; #p <.05 versus rats of the same age in the AL group and ##p <.01 versus rats of the same age in the AL group, respectively. PLA2 = phospholipase A2; COX = cyclooxygenase; PGIS = prostaglandin I2 synthase; PGES = prostaglandin E2 synthase; GAPDH = glyceraldehyde-3-phosphate dehydrogenase; AL = ad libitum group; CR = calorie restriction group

 
COX also exists in two isoforms. COX-1 is expressed constitutively while COX-2 is induced in many cell types by the stimulation of inflammatory cytokines. COX-2 has been identified as a key enzyme in the regulation of PG synthesis and an important player in the inflammation process. We observed considerable amounts of COX-2 mRNA in the aorta of old rats, in contrast to the negligible amounts found in young rats. On the other hand, approximately equal amounts of COX-1 were found in young and old rats (Figure 3). Accordingly, in situ detection of COX-1 and COX-2 protein levels by immunohistochemical staining showed the same tendencies (Figure 4).



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  		Figure 4. Effects of age and CR on the COX-1 (1) and COX-2 (2) in the aorta. COX-1 and COX-2 expression in the aorta were performed by immunohistochemical stain. The strong staining in the tissue indicates the expression of interest. Scale bar = 100 µm. 6 = rat at 6 months of age; 24 = rat at 24 months of age; COX = cyclooxygenase; AL = ad libitum group; CR = calorie restriction group

 
Among the terminal PG synthases, we examined PGI2 synthase (PGIS) and PGE2 synthase (PGES) expression in the aorta of young and old rats fed AL and CR diets. Although no significant differences in PGES expression were shown between young and old rats, the PGIS mRNA level was significantly higher in old rats than in young rats (Figure 4). Taken together, these data suggested that up-regulated cPLA2, COX-2, and PGIS were likely responsible for increased PG production during aging.

The beneficial effects of CR on these enzymes were also detected. CR significantly suppressed age-associated increases in cPLA2 and COX-2.

Effects of Age and CR on Antioxidative Enzymes
SOD, catalase, and GSH-Px are three major antioxidant enzymes that play a central role in an organism's ability to protect against oxidative stress during ROS metabolism. The protein levels of these antioxidant enzymes during aging were determined by Western blot analysis. As shown in Figure 5, SOD and catalase levels were unchanged during the aging process, while GSH-Px levels were very high in old AL rats. CR rats were shown to have up-regulated catalase protein levels, but showed no change in SOD levels. On the other hand, we observed that CR blunted the age-related induction of GSH-Px.



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  		Figure 5. Effects of age and CR on antioxidant enzymes and antioxidant capacity in the rat aorta. A, One representative blot is shown. B, The levels of protein quantified by densitometry as a percent of the levels of a 6-month-old ad libitum rat. The protein levels were measured by Western blotting. Each experiment was performed in triplicate. C, Antioxidant capacity analysis for aorta homogenate of young (6 months) and old (24 months) from the AL and CR groups. Experiments were performed as described in the "Methods" section. Statistical significance: *p <.05 versus 6-month-old rats of the same group; **p <.01 versus 6-month-old rats of the same group; #p <.05 versus rats of the same age in the AL group and ##p <.01 versus rats of the same age in the AL group, respectively. AL = ad libitum group; CR = calorie restriction group; GSH-Px = glutathione peroxidase; SOD = superoxide dismutase

 
We also compared differences in antioxidant capacity between young and old aorta of CR and AL-fed rats. Data in Figure 5C indicate that, in the AL group, the aorta from old rats had a significantly weakened antioxidant capacity for H2O2 and the hydroxyl radical scavenging activity than that from young AL rats, while the impaired antioxidant capacity in old rats was attenuated by CR treatment.


   DISCUSSION
Top
 Abstract
 Methods
 Results
 Discussion
References
 
Many products of arachidonic metabolism, mainly prostanoids, play an important role in development of vascular diseases (21). Prostanoids are important mediators of both acute and chronic vascular inflammation and modulate a wide variety of cellular interactions in physiological and pathological processes; however, whether prostanoids and their biosynthesis systems are affected by the aging process, especially with respect to CR, is far from known. In the current study, we assessed age-related changes in prostanoid and related biosynthesis cascade in aorta and serum by comparing their levels in young and old CR and AL-fed rats.

In the present study, we found that the production of certain prostanoids was significantly changed during the aging process (Figure 1). PGE2 induces edema and vasodilation and contributes to the development of hyperalgesia. PGI2, a predominant PG produced by the vascular wall, causes vasodilation, inhibits platelet aggregation, and is generally thought to decrease during aging. In contrast, TXA2 has a strong vasoconstricting and platelet aggregating activity. The PGI2/TXA2 balance is generally accepted as an important marker for healthy vascular dynamics, and in the present study, we found no clear-cut change in the ratio of PGI2 and TXA2 between young and old AL rats in serum and in aorta homogenate (Figure 1 and Figure 2). Thereby, the increased PGI2 levels found in the present study could be related to a compensatory response to the elevated vasoconstricting factor TXA2.

Consistent with changes in the production of prostanoid metabolites, the expression of prostanoid-generating enzymes, including PGIS, PGES, PLA2, and COX2, were also found increased in aged AL aorta. As shown in the data (Figure 3), the age-related increased transcription of the PGIS gene may directly lead to enhanced production of PGI2 in the old AL rats. The liberation of arachidonic acid by cPLA2 upon cell activation is often the initial and rate-limiting step in PGs and leukotriene biosynthesis (22). Thus, an age-associated up-regulation of cPLA2 should affect production of prostanoids to a great extent.

In the present study, we also determined significant increased COX-2 expression during aging. We are particularly interested in COX-2 because it is the major source of RS generated during the aging process (13). Manev and colleagues and Dore and colleagues have reported that COX-2 expression appears to be up-regulated during aging (22,23). Dore and colleagues also suggested that increases in COX-2 enzymatic activity and prostaglandin production are associated with neuronal injury in age-related degenerative neurological diseases (23). Previously, we have reported an age-related increase in COX activity in rat brain (24) and enhanced COX-2 gene expression in rat heart (25). In the current study, we found enhanced COX-2 expression in both mRNA and protein levels of aged rat AL aorta.

Taken together, increased PGs synthase expressions during aging are responsible for enhanced level of prostaglandins, such as PGE2 and PGI2, which could be indicative of the proinflammatory status in aged tissue. An age-related increase in polyunsaturated fatty acids, including arachidonic acid-containing phospholipids, has already been reported previously (26), and in our present study, we detected higher prostanoid generating enzyme levels during aging. Therefore, we expect that the combination of these two events may lead to increased levels of prostanoids during aging.

The up-regulation of inflammatory enzymes likely occurs through the activation of redox-sensitive transcription factors, such as nuclear factor-kappa-B (NF-{kappa}B) (27). Kim and colleagues (28) reported that the nuclear binding activity of NF-{kappa}B in rat heart, liver, and kidney was significantly increased by the aging process and was effectively inhibited by CR. Here, COX-2, which has an NF-{kappa}B binding site on its promoter region (29), was found significantly up-regulated during aging. Although we did not determine aortic NF-{kappa}B binding activity in the present study, the age-related activation of NF-{kappa}B is most likely responsible for this induction.

Among three defensive antioxidative enzymes monitored during this study, GSH-Px expression was shown to increase with age, while no noticeable changes in SOD and catalase levels were observed during the aging process. Although the actual antioxidative enzyme activities were not measured in the present study, our past experiences and previous reports (30,31) indicate that these enzyme activities are always accompanied by their expressions. In addition, as shown in Figure 5C, antioxidative defenses capacity was decreased in the aorta during aging. Oltra and colleagues found that increased GSH-Px activity was accompanied by decreased SOD and catalase activities (32). Thus, our data showing increased aortic GSH-Px protein levels (Figure 5) can be viewed as compensatory responses that guard against cellular damage in the absence of increased SOD expression. The antioxidative action of CR was well exhibited in the significantly increased expression of catalase as we observed in both age groups.

In this context, it is most important to consider the effective action of CR in the suppression of age-related increases of the proinflammatory mediators we studied. 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 antioxidative ability in the suppression of age-related oxidative stress and, at the same time, by its ability to uphold the antioxidant defense system, including major RS scavengers during aging (18). The current data indicated that the suppression of age-related alterations in prostanoids and their generating enzymes by CR may be a major factor in the modulation of oxidative stress, which could preserve vascular function integrity during the aging process. Thus, this new information could provide further evidence to support the proposal that the inflammatory process is causally linked to vascular aging.

Conclusion
Through this study we documented data on prostanoid and prostanoid biosynthesis expression during aging. Our findings suggested that vascular oxidative stress and the inflammatory process could be closely related to vascular aging, and that CR inhibited these processes by its ability to maintain vascular integrity by fending off oxidative damage. Thus, the current investigation revealed molecular insights into age-related alterations of aorta.



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  		Figure 5. Continued

 

   Acknowledgments
 
This work was supported by a grant from the Korean Research Foundation (FS0004). The authors would like to thank the Brain Pool Program, sponsored by The Korean Federation for Science and Technology Societies, for its support of Dr. Gregory Y. Chang's visiting Professorship at Pusan National University. We thank Dr. Jeremiah T. Herlihy for his help. We also are grateful to the "Aging Tissue Bank" and Engineering Foundation and the National Institute on Aging (AG01188), for the supply of the aging tissue.


   Footnotes
 
Decision Editor: Steven N. Austad, PhD

Received February 5, 2004

Accepted May 28, 2004


   References
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 Methods
 Results
 Discussion
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