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The Journals of Gerontology Series A: Biological Sciences and Medical Sciences 59:B316-B323 (2004)
© 2004 The Gerontological Society of America

Food Restriction Attenuates Age-Related Increase in the Sensitivity of Endothelial Cells to Oxidized Lipids

Hong Yang1, MingJian Shi2, James Story2, Arlan Richardson4,5 and ZhongMao Guo2,3,

1 Department of Physiology
2 Department of Pathology, Anatomy & Cell Biology
3 Department of Internal Medicine, Meharry Medical College, Nashville, Tennessee.
4 Department of Physiology, University of Texas Health Science Center at San Antonio.
5 Geriatric Research, Education and Clinical Center, South Texas Veterans Health Care System, Audie L. Murphy Division, San Antonio.

Address correspondence to Dr. ZhongMao Guo, Department of Pathology, Anatomy & Cell Biology, Meharry Medical College, Nashville, TN 37208. E-mail: zguo{at}mmc.edu


   Abstract
Top
 Abstract
Methods
 Results
 Discussion
 References
 
Age-related endothelial dysfunction has been suggested to play a role in atherogenesis. Food restriction (FR) has been shown to retard the development of atherosclerosis. The goal of this report is to assess the effect of aging and FR on endothelial functions, including the release of endothelial nitric oxide (NO) and the adhesion of mononuclear cells (MNCs) to endothelial cells (ECs). ECs were obtained from the aorta of young mice fed ad libitum (Y-AL), old mice fed ad libitum (O-AL), or a food-restricted diet (O-FR). When compared with those obtained from Y-AL and O-FR mice, ECs obtained from O-AL mice decreased the basal level of NO release and increased the basal level of peroxynitrite, superoxide, and hydrogen peroxide. In addition, ECs obtained from O-AL elevated the response to CuSO4-oxidized low-density lipoprotein (oxLDL). For example, incubation with oxLDL reduced NO release approximately 52% in ECs obtained from O-AL mice. In contrast, the same dose of oxLDL reduced NO release only approximately 40% in ECs obtained from Y-AL and O-FR mice. Moreover, the level of oxLDL-induced adhesion of MNCs and oxLDL-induced expression of vascular cell adhesion molecule-1 and intercellular adhesion molecule-1 was markedly higher in ECs obtained from O-AL mice as compared with those obtained from Y-AL and O-FR mice. These results suggest that aging increases the sensitivity of ECs in response to oxLDL-reduced endothelial NO release and oxLDL-increased adhesion of MNCs to ECs. FR attenuates age-related increase in the sensitivity of ECs to oxLDL, which might be responsible, at least in part, for the antiatherogenic action of FR.

ATHEROSCLEROSIS is the underlying cause of coronary heart disease and cerebrovascular disease; the incidence of these cardiovascular diseases increases with age (1). This may, in part, be attributed to the steady progression of atherosclerotic lesions. However, there is evidence showing that aging is an independent risk factor for atherosclerosis. For example, when challenged with a similar degree of diet-induced hypercholesterolemia, older cynomolgus monkeys develop more severe atherosclerosis than their younger counterparts (2). More recently, a rapidly growing body of evidence suggests that aging causes changes in endothelial functions, which might predispose vasculature to atherosclerosis [for a review, see (3)]. For example, aging has been shown to reduce the bioavailability of endothelial nitric oxide (NO) (4) and, hence, to decrease the endothelium-dependent vasorelaxation in humans (5,6) and animals (7). In addition, aging has been reported to increase the production of endothelial adhesion molecules and elevate the adhesion and transmigration of leukocytes to the arterial wall. For example, the expression of vascular cell adhesion molecule-1 (VCAM-1) has been reported to increase with age in mouse aortas (8). In addition, the concentration of VCAM-1 has been reported to increase with age in human serum (9). Moreover, the aortic intima of aged rats has been found to accumulate a large number of leukocytes that adhered to the endothelium (10). Decrease in endothelial NO availability and increase in leukocyte adhesion to the arterial wall have been suggested to facilitate the development of atherosclerosis.

Food restriction (FR), which inhibits the progression of almost all age-related diseases, has been shown to retard the development of atherosclerosis. For example, Koletsky and Puterman (11,12) reported that FR reduced atherosclerosis in a genetically obese rat model. Subbiah and Siekert (13) reported that FR reduced the plasma and aortic cholesterol in the White Carneau pigeon. A previous study from our laboratory showed that FR inhibited the development of atherosclerosis without lowering plasma cholesterol in apolipoprotein E-deficient mice (14). At the present time, the mechanism underlying the antiatherogenic action of FR has not been defined. One of the possibilities is that FR reduces the age-related increase in the sensitivity of endothelial cells (ECs) to atherogenic factors. In the present article, we examined the effect of oxidized low-density lipoprotein (oxLDL), a major atherogenic factor, on leukocyte adhesion and NO release in ECs obtained from young mice fed ad libitum (Y-AL) and old mice fed ad libitum (O-AL) or a food-restricted diet (O-FR). Our results showed that oxLDL significantly reduced endothelial NO release and elevated the adherence of mononuclear cells (MNCs) to ECs. The magnitude of oxLDL-elevated MNC adhesion and oxLDL-reduced NO release in ECs obtained from O-AL mice was markedly greater than that in ECs obtained from Y-AL and O-FR mice. These results suggest that aging increases the sensitivity of ECs in response to oxLDL-reduced NO release and oxLDL-elevated MNC adhesion, and that FR attenuates the impaired endothelial functions caused by aging.


   METHODS
Top
 Abstract
 Methods
Results
 Discussion
 References
 
Animals and Cells
Male C57BL mice fed ad libitum or a food-restricted diet were obtained at age 6 months or 24 months from the animal colonies maintained by the National Institution on Aging (Bethesda, MD). The mice fed ad libitum had free access to the diet (NIH-31; Purina Mills, Inc., Richmond, IN). The food-restricted mice were fed a special NIH-31 fortified formula, which was enriched with vitamins, from 16 weeks of age. By feeding this diet, the food-restricted mice received the same amount of vitamins but 40% less calories as compared with the mice fed ad libitum (15). Under anesthesia with a rodent cocktail as previously described (16), aortas from the aortic arch to the branch point of renal arteries were collected from Y-AL, O-AL, and O-FR mice. Aortic ECs were isolated and characterized as previously described (16). Three lines of ECs for each age and diet group were used in this study. Each line of ECs was prepared with aortas obtained from 4 mice. ECs were cultured in Dulbecco's Modified Eagle's Medium (DMEM) containing 10% fetal bovine serum (FBS). ECs from passages 4, 5, or 6 were used for experiments. MNCs were isolated from Y-AL mice as previously described (16). For fluorochrome adhesion assay, MNCs were labeled with 2',7'-bis-(2-carboxyethyl)-5,6-carboxyfluorescein, acetoxymethyl ester (BCECF-AM) (16). All procedures for handling the animals were approved by the Institutional Animal Care and Use Committee of the Meharry Medical College, the University of Texas Health Science Center at San Antonio and the Subcommittee for Animal Studies at the Audie L. Murphy Memorial Veterans Hospital.

Measurement of NO Release, Nitric Oxide Synthase Activity, and Nitrotyrosine Concentration
The oxLDL used in this study was obtained by incubation of human low-density lipoprotein (LDL) with CuSO4 (5 µM) in phosphate-buffer saline (PBS) for 24 hours at 37°C (16). The level of LDL oxidation was approximately 56 to 62 nM of thiobarbituric acid-reacting substances (TBARS) per mg protein. The native LDL showed 0.5 to 1 nM of TBARS per mg protein. NO release from ECs was determined as previously described (16). Confluent ECs grown in a 96-well plate were incubated at 37°C for 2 hours in Krebs-HEPES medium (mM: NaCl 118, KCl 4.5, CaCl2 2.5, MgCL2 (??) 1.2, KH2PO4 1.2, Na-HEPES 25, NaHCO3 25, and glucose 5) containing oxLDL (5, 10, or 15 µg/ml) or equal concentrations of native LDL or lacking LDL as control. NO released from ECs was measured using a nitrate/nitrite colorimetric assay kit (Cayman Chemical Co., Ann Arbor, MI). At the end of the experiments, ECs were lysed and the protein level in the lysate was determined. The release of NO was expressed as nM/mg protein/hour.

The activity of nitric oxide synthases (NOSs) in ECs was detected as previously described (16). Confluent ECs grown in 6-well plates were incubated at 37°C for 2 hours in Krebs-HEPES medium containing oxLDL (10 µg/ml) or equal concentration of native LDL or lacking LDL as control. NOS activity was determined by measuring the conversion of [3H] L-arginine to [3H] L-citrulline. NOS activity was reported as the difference of the radioactivity between cells treated with and without L-NAME (nitro-L-arginine methyl ester).

Nitrotyrosine is an end product of the reaction between peroxynitrite and free or protein-bound tyrosine residues. Peroxynitrite is formed from the interaction of NO and . Nitrotyrosine in ECs was measured as described by Thom and colleagues (17) with slight modifications. Confluent ECs grown in 6-well plates were incubated at 37°C for 2 hours in Krebs-HEPES medium containing oxLDL (5, 10, or 15 µg/ml) or equal concentrations of native LDL or lacking LDL as control. The Krebs-HEPES medium was removed and 1 ml ice-cold PBS was then added to each well. Cells were scraped off the well and sonicated at ice temperature with a Sonicator (Model 100; Fisher Scientific, Hampton, NH). The lysate was centrifuged at 14,000 x g at 4°C for 10 minutes. The supernatant containing 10 µg protein was immobilized onto a polyvinylidene difluoride (PVDF) membrane using a 48-well BioDot SF microfiltration apparatus (Bio-Rad, Hercules, CA). After blocking with 5% nonfat milk, the PVDF membrane was reacted with a monoclonal antinitrotyrosine antibody (Cayman Chemical Co.). The membrane was then incubated with a horseradish peroxidase-conjugated secondary antibody and exposed to autoradiography film. The intensities of the immunoreactivity were determined by densitometric analysis (AlphaImager Analysis System; Alpha Innotech Co., San Leandro, CA).

Measurement of Reactive Oxygen Species
The amount of in ECs was measured as previously described (16). ECs grown in a 96-well plate at confluence were treated with oxLDL (10 µg/ml) or an equal concentration of native LDL or Krebs-HEPES medium in the absence of LDL for 2 hours at 37°C. Lucigenin (5 µM/ml) was added to the wells and luminescence was read using a luminometer (BL10000 Lumicount, Packard BioScience, Meriden, CT) for 5-minute intervals at room temperature. At the end of the experiments, ECs in each well of the 96-well plate were lysed and the protein level in the lysate was determined. The amount of in ECs was expressed as nM/mg protein/hour.

H2O2 released from ECs was measured using an Amplex Red Hydrogen Peroxide Assay kit (Molecular Probes, Eugene, OR) as previously described (16). ECs grown in a 96-well plate at confluence were incubated with native LDL, oxLDL (10 µg/ml), or Krebs-HEPES medium in the absence of LDL for 2 hours at 37°C. After incubation with Amplex red reagent for 30 minutes, fluorescence was read using a fluorometer (Fluoroskan Ascent FL, Thermo LabSystems, Inc., Beverly, MA). At the end of the experiments, ECs were lysed and the protein level in the lysate was determined. The release of H2O2 was expressed as nM/mg protein/hour.

Assay of MNC Adhesion to ECs
Adhesion of MNCs to ECs was measured as previously described (16). ECs grown in a 96-well plate at confluence were incubated with native LDL, oxLDL (5, 10, or 15 µg/ml), or Krebs-HEPES medium in the absence of LDL for 4 hours at 37°C. The medium was then changed to serum-free DMEM. The BCECF-AM-labeled MNCs (2 x 105) were added to each well. After 1-hour incubation, nonadherent MNCs were rinsed off and the firmly adherent MNCs were lysed with 0.5N NaOH. Fluorescence was read using a fluorometer (Fluoroskan Ascent FL, Thermo LabSystems). The number of adherent MNCs per well was determined based on a standard curve generated with known numbers of fluorescent-labeled MNCs. Data were expressed as adherent MNCs/mm2 (calculated with the area of a single well in the 96-well plate as 32 mm2).

ELISA Measurement of Endothelial Surface VCAM-1 and ICAM-1 Expression
The expression of VCAM-1 and intercellular adhesion molecule-1 (ICAM-1) on the EC surface was determined as previously described (16). Confluent ECs grown in 96-well plates were treated with oxLDL (10 µg/ml) or an equal concentration of native LDL or Krebs-HEPES medium in the absence of LDL for 4 hours at 37°C. VCAM-1 and ICAM-1 were determined with an enzyme-linked immunosorbent assay (ELISA) using antibodies against mouse VCAM-1 or ICAM-1.

Western Blotting for VCAM-, ICAM-1, and Endothelial NOS
The protein levels of VCAM-1 and ICAM-1 were determined with Western blots as described previously (16). ECs grown in 100 mm tissue culture dishes at confluence were incubated with native LDL, oxLDL (10 µg/ml), or culture medium in the absence of LDL for 4 hours. The ECs were lysed, the lysates were centrifuged at 14,000 rpm, and the supernatants containing 20 µg protein was separated by 10% SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Protein was transferred to PVDF membranes. The membrane was immunoblotted with VCAM-1 or ICAM-1 antibodies and then incubated with a horseradish peroxidase-conjugated secondary antibody (all antibodies from Santa Cruz Biotechnology, Inc., Santa Cruz, CA). The membrane was exposed to autoradiography film, and the relative intensity of the bands was quantified by an AlphaImager analysis system (Alpha Innotech Co.). To correct the differences in protein loading, the membrane was stripped in buffer containing 2% SDS, 63 mM Tris-HCl (pH 6.8), and 100 mM ß-mercaptoethanol, and reprobed with ß-actin antibody.

Statistical Analysis
For experiments using microplates and the microfiltration apparatus, the value of each experiment was averaged from 4 or 2 duplicates in the same plate. The number of experiments was indicated in figure and table legends. Data were reported as mean ± standard error of the mean. The differences among ECs treated with or without LDL as well as ECs obtained from young and old mice fed ad libitum or the food-restricted diet were analyzed by multiple-factor analysis of variance followed by the Shapiro-Wilk test. Differences were considered significant at a p value less than.05.


   RESULTS
Top
 Abstract
 Methods
 Results
Discussion
 References
 
A major aim of this investigation was to evaluate the influence of aging and FR on the release of endothelial NO. As shown in the top panel of Figure 1, the basal level of NO release was significantly lower in ECs obtained from O-AL mice than in those obtained from Y-AL mice. However, the difference is not significant between ECs obtained from Y-AL and O-FR mice. The addition of oxLDL to the culture medium reduced NO release at a concentration-dependent manner. The reduced magnitude differed in ECs obtained from Y-AL, O-AL, and O-FR mice. For example, 10 µg/ml of oxLDL reduced NO release about 38%, 52%, and 38% in ECs obtained from Y-AL, O-AL, and O-FR mice, respectively (Figure 1). Incubation of ECs with native LDL at the doses used in this study did not significantly alter the level of NO release (data not shown).



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  		Figure 1. The effects of age and food restriction (FR) on nitric oxide (NO) release, nitrotyrosine concentration, and nitric oxide synthase (NOS) activity in epithelial cells (ECs). ECs were obtained from young mice fed ad libitum (Y-AL) and old mice fed ad libitum (O-AL) or the food-restricted diet (O-FR). NO release and nitrotyrosine concentration were measured after ECs were treated with various concentrations of oxidized low-density lipoprotein (oxLDL) as indicated. NOS activity was measured after ECs were incubated with 10 µg/ml of oxLDL or equal concentration of native low-density liprotein (LDL) or in the medium lacking LDL (control). Values represent the mean ± SEM (standard error of mean) of 6 separate experiments. *p <.05 compared with age-matched and diet-matched controls. {dagger}p <.05 compared with Y-AL mice under the same culture condition. {ddagger}p <.05 compared with O-AL mice under the same culture condition

 
The age-related decline in endothelial NO release could result from a reduced NO synthesis and/or an enhanced NO breakdown. In this article, we measured NOS activity and nitrotyrosine concentration in EC treated with or without LDL. As Figure 1 illustrates, the basal level of NOS activity in ECs obtained from Y-AL, O-AL, and O-FR mice was comparable. In contrast, the basal level of nitrotyrosine was significantly higher in ECs obtained from O-AL mice than in those obtained from Y-AL and O-FR mice. Incubation of ECs with oxLDL increased nitrotyrosine at a concentration-dependent manner. The increase in nitrotyrosine differed in ECs obtained from Y-AL, O-AL, and O-FR mice. For example, 10 µg/ml of oxLDL increased nitrotyrosine by approximately twofold, threefold, and twofold in ECs obtained from Y-AL, O-AL, and O-FR mice, respectively (Figure 1). However, the same concentration of oxLDL did not alter NOS activity in ECs obtained from all 3 groups of mice (Figure 1).

This article examined the influence of age and FR on endothelial and H2O2. In the absence of LDL, and H2O2 in ECs obtained from O-AL mice were significantly higher than in those obtained from Y-AL mice (Table 1). In addition, the basal level of in ECs obtained from O-FR mice was significantly lower than in those obtained from O-AL mice. The basal level of H2O2 release in ECs obtained from O-FR mice was only slightly lower than in those obtained from O-AL mice; the difference was not statistically significant (Table 1). The addition of native LDL to the culture medium did not significantly alter the level of and H2O2 in ECs obtained from all 3 groups of mice (Table 1). However, the addition of oxLDL to the culture medium increased by 2.3-fold, 3.2-fold, and 2.8-fold, respectively, in ECs obtained from Y-AL, O-AL, and O-FR mice (Table 1). The addition of oxLDL to the culture medium increased H2O2 about 30%, 70%, and 50%, respectively, in ECs obtained from Y-AL, O-AL, and O-FR mice (Table 1). These results suggest that reactive oxygen species (ROS) in ECs under basal or oxLDL-stimulated conditions increase with age, and that FR attenuates the age-related increase in endothelial ROS.


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  		Table 1. The Effect of Age and Food Restriction on Reactive Oxygen Species Production in Mouse Aortic Endothelial Cells.

 
Figure 2 shows the effect of various concentrations of oxLDL on adherence of MNCs to ECs. Without pretreatment of ECs with oxLDL, an average of 35, 43, and 38 MNCs/mm2 firmly adhered to ECs obtained from Y-AL, O-AL, and O-FR, respectively (Figure 2). The number of MNCs adhering to the ECs increased with increasing concentrations of oxLDL, plateaued at concentration 10 µg/ml, and remained constant up to concentration 15 µg/ml. We have observed that oxLDL at concentrations higher than 20 µg/ml decreases the number of MNCs adhering to ECs because of EC death (data not shown). As data in Figure 2 illustrate, the number of MNCs adhering to ECs obtained from O-AL mice was greater than the number obtained from Y-AL and O-FR mice. For example, 10 µg/ml of oxLDL increased MNCs adherent to ECs obtained from Y-AL and O-FR mice by 2.8-fold and 3.3-fold, respectively (Figure 2). In contrast, the same dose of oxLDL increased the MNCs adhering to ECs obtained from O-AL mice by 4.2-fold. Pretreatment of ECs with the same concentration of native LDL did not significantly affect the adherence of MNCs to ECs obtained from Y-AL, O-AL, and O-FR mice (data not shown).



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  		Figure 2. The effects of age and food restriction (FR) on the adhesion of mononuclear cells (MNCs) to epithelial cells (ECs) and the expression of adhesion molecules on the EC surface. ECs were obtained from young mice fed ad libitum (Y-AL) and old mice fed ad libitum (O-AL) or the food-restricted diet (O-FR). The adhesion of MNCs to ECs was determined after ECs were treated with various concentrations of oxidized low-density lipoprotein (oxLDL) as indicated. The expression of vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) on the endothelial surface was determined after ECs were incubated with 10 µg/ml of oxLDL or equal concentration of native low-density lipoprotein (LDL) or in the medium lacking LDL (control). Values are mean ± SEM (standard error of mean) of 6 separate experiments. *p <.05 compared with age-matched and diet-matched controls. {dagger}p <.05 compared with Y-AL mice under the same culture condition. {ddagger}p <.05 compared with O-AL mice under the same culture condition

 
This study determined the effect of age and FR on the expression of VCAM-1 and ICAM-1 in mouse ECs. In the absence of LDL, the surface expression (Figure 2) and protein level (Figure 3) of VCAM-1 and ICAM-1 were comparable in ECs obtained from Y-AL, O-AL, and O-FR mice. Treatment of ECs with native LDL slightly increased the expression of VCAM-1 and ICAM-1, but this elevation was not statistically significant in ECs obtained from all 3 groups of mice (Figures 2 and 3). In contrast, the addition of oxLDL to the culture medium markedly elevated the expression of VCAM-1 and ICAM-1 in ECs. For example, 10 µg/ml of oxLDL increased the surface expression of VCAM-1 by 2.4-fold, 4.1-fold, and 2.6-fold in ECs obtained from Y-AL, O-AL, and O-FR mice, respectively (Figure 2). In addition, the surface expression of ICAM-1 was increased by 1.8-fold, 3.6-fold, and 1.9-fold in ECs obtained from Y-AL, O-AL, and O-FR mice, respectively (Figure 2). Correspondingly, the same concentration of oxLDL increased the protein level of VCAM-1 by 3.2-fold, 5.0-fold, and 3.8-fold in ECs obtained from Y-AL, O-AL, and O-FR mice, respectively (Figure 3). This concentration of oxLDL also induced approximately 1.8-fold, 2.9-fold, and 2.2-fold ICAM-1 protein in ECs obtained from Y-AL, O-AL, and O-FR mice, respectively (Figure 3).



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  		Figure 3. The effects of age and food restriction (FR) on the protein level of vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1). Epithelial cells (ECs) were obtained from young mice fed ad libitum (Y-AL) and old mice fed ad libitum (O-AL) or the food-restricted diet (O-FR). The confluent monolayer of ECs was incubated with 10 µg/ml of oxidized low-density lipoprotein (oxLDL) or equal concentration of native low-density lipoprotein (LDL) or culture medium alone (control). Western blotting was performed as described in Methods with antibodies against VCAM-1, ICAM-1, and ß-actin. The photographs show representative images of Western blots of VCAM and ICAM-1. The protein levels of VCAM-1 and ICAM-1 are expressed as the percentage of the intensity of these factors to the intensity of ß-actin in the autoradiography. Values are mean ± SEM (standard error of mean) of 6 separate experiments in which ECs are pooled from 4 mice for each experiment. *p <.05 compared with age-matched and diet-matched controls. {dagger}p <.05 compared with Y-AL mice under the same culture condition. {ddagger}p <.05 compared with O-AL mice under the same culture condition

 

   DISCUSSION
Top
 Abstract
 Methods
 Results
 Discussion
References
 
Data from in vivo and in vitro studies suggest that endothelial NO bioavailability declined with age. For example, the basal release of NO from rat arteries has been shown to decline with age in in vitro studies (18,19). In addition, it has been reported that NO-dependent vasodilation is reduced in older rats versus younger rats (20), as is the case in humans (5). In agreement with previous reports, we observed that cultured ECs obtained from aged mice had a reduced basal level of NO release and an increased magnitude of oxLDL-induced reduction in endothelial NO release. This study also demonstrated, for the first time, that FR attenuated both of these age-related deleterious effects: 1) the reduction in the basal release of endothelial NO, and 2) the increase in the sensitivity of ECs in response to oxLDL-reduced NO release.

Decline in endothelial NO release could result from an enhanced NO breakdown and/or a reduced NOS activity in ECs. Reports about the effect of aging on the NOS activity in ECs are controversial. For instance, several reports showed an age-related decrease in the activity (21) and expression of endothelial NOS (21,22) in ECs, while others showed that aging increased eNOS activity and expression in conjunction with an reduced free NO [for a review, see (3)]. In addition, the expression of inducible NOS (iNOS) in vascular cells has also been reported to either increase or maintain no change with age (21,23). In the present study, we did not observed an age-related or diet-related change in NOS activity in mouse ECs under basal and oxLDL-stimulated conditions. In addition, we measured the protein level of eNOS and iNOS with Western blots under the basal and oxLDL-stimulated conditions, and observed that the expression of these NOSs in ECs obtained from Y-AL, O-AL, and O-FR mice were comparable (data not shown). In contrast, we observed that the nitrotyrosine concentration was markedly higher in ECs obtained from O-AL mice than in those obtained from Y-AL and O-FR mice. These data suggest that aging increases the breakdown of endothelial NO through the peroxynitrite formation pathway, and that the increased NO breakdown, but not the reduced NOS activity, is responsible for the age-related decline in the basal level of endothelial NO release and the age-related increase in the sensitivity of ECs in response to oxLDL-reduced NO release. FR reduces endothelial peroxynitrite formation, and therefore attenuates the age-related decrease in NO release.

Peroxynitrite is formed from the nonenzymatic reaction of NO and with a diffusion-limiting rate. In this article, we observed that the basal level of endothelial and the magnitude of oxLDL-induced were significantly elevated in ECs obtained from O-AL mice compared with Y-AL mice. It has been suggested that the age-related increase in endothelial could result from an increased mitochondria dysfunction (19,24), an increased NOS uncoupling (uncoupling of NADPH [nicotinamide adenine dinucleotide phosphate] oxidation and NO synthesis) (22,25), or an increased NAD(P)H oxidase activity (25). In addition, an increase in cellular also could be derived from a reduced activity of superoxide dismutases (SODs), which convert to H2O2. However, a previous study from our laboratory showed that aging increased SOD activity in mouse aortas (15). In fact, the present article showed an increased H2O2 release in ECs obtained from O-AL mice compared with Y-AL mice. H2O2 has been reported to react with NO to produce hydroxyl radical and therefore reduce endothelial NO release (26). Thus, data from this article support the view that an increase in the generation of intracellular ROS (e.g., and H2O2) is one of the mechanisms responsible for the age-related increase in endothelial NO breakdown. In the present study, we also observed that the basal level of endothelial and the magnitude of oxLDL-induced were significantly reduced in ECs obtained from O-FR mice compared with O-AL mice, suggesting that FR attenuates the age-related increase in generation, and therefore reduces the production of peroxynitrite and H2O2 and reduces the breakdown of endothelial NO.

Modulation of leukocyte adhesion is one of the primary functions of ECs. In this study, we observed that the magnitude of oxLDL-increased MNC adhesion was significantly greater in ECs obtained from O-AL mice than in those obtained from Y-AL mice. Various explanations may be considered regarding the molecular mechanism by which aging increases leukocyte adhesion to ECs. A possibility is that the age-related increase in endothelial ROS activate the signal transduction cascades that modulate the activity of nuclear transcription factors and promote the expression of cell adhesion molecules [for a review, see (27)]. In this article, we did not examine the effect of aging on the activation of ROS-sensitive signaling pathways and nuclear transcription factors. However, we observed that aging increased endothelial and H2O2, and enhanced the expression of VCAM-1 and ICAM-1 induced by oxLDL. In addition, we observed that oxLDL-induced MNC adhesion and oxLDL-induced expression of VCAM-1 and ICAM-1 were significantly less in ECs obtained from O-FR mice than in those obtained from O-AL mice. These findings, together with data showing that FR reduces endothelial and H2O2, suggest that reduction in ROS production is, at least in part, a mechanism by which FR reduces the expression of cell adhesion molecules and reduces MNC adhesion.

In this study, we also noticed that the basal level of MNC adhesion and the basal expression of VCAM-1 and ICAM-1 in ECs obtained from O-AL mice were comparable with those in ECs obtained from Y-AL and O-FR mice. It is most likely that the age-related increase in endothelial and H2O2 under basal conditions was not sufficient to increase the expression of adhesion molecules. Thus, aging would not alter the basal level of leukocyte adhesion to ECs. Paradoxically, it has been reported that the expression of adhesion molecules increases with age in mouse aortas in the absence of hyperlipidemia (8). However, ECs are constantly exposed to various stimuli in the arterial wall and in plasma. Aging might increase the sensitivity of ECs to inflammatory stimuli. Moreover, the level of the inflammatory factors in the arterial wall and/or plasma might elevate with age (28). Thus, the age-related increase in expression of endothelial adhesion molecules under in vivo conditions might result from multiple factors.

In a previous study, we observed that combinational over-expression of Cu/Zn-SOD and catalase entirely blocked oxLDL-induced and H2O2 in ECs, but did not completely prevent oxLDL-induced increase in adherence of MNCs to ECs (16). These data implicate that mechanism(s) that are not associated with endothelial and H2O2 also play a role in oxLDL-induced leukocyte adhesion to ECs (16). It has been reported that NO is able to reduce leukocyte–EC interaction [for a review, see (29)], and that reduction in NO bioavailability elevates leukocyte–EC interaction (30–32). Thus, the decreased NO release in ECs obtained from aged mice might be another mechanism underlying the age-related increases in MNC adhesion. FR might attenuate age-related increase in MNC adhesion by increasing endothelial NO release.

Summary
This report confirmed that aging reduced the basal level of endothelial NO release, increased the magnitude of oxLDL-induced reduction in endothelial NO release, and increased oxLDL-induced leukocyte adhesion to ECs. Data in this article also demonstrated that aging increased endothelial and H2O2. Since both and H2O2 have been shown to neutralize NO and facilitate leukocyte adhesion, increase in intracellular ROS might be one of the mechanisms responsible for the age-related reduction in endothelial NO release and age-related increase in MNC adhesion to ECs. As importantly, we observed that FR attenuated age-related increase in ROS, and suppressed the response of ECs to oxLDL-reduced NO release and oxLDL-increased MNC adhesion. The oxLDL-reduced endothelial NO and oxLDL-increased adhesion of leukocyte to ECs have been suggested to play a role in atherogenesis. Thus, improvement of endothelial functions might be a mechanism by which FR inhibits atherogenesis.


   Acknowledgments
 
This study is supported by AFAR research grant A03109 (Hong Yang), and NIH grants PO3-AG13319 (Arlan Richardson), HL71525, and GM08037 (ZhongMao Guo).


   Footnotes
 
John Faulkner,, PhD, Decision Editor

Received September 16, 2003

Accepted January 12, 2004


   References
Top
 Abstract
 Methods
 Results
 Discussion
 References
 

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