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Am J Physiol Heart Circ Physiol 293: H2634-H2643, 2007. First published September 14, 2007; doi:10.1152/ajpheart.00397.2007
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Cardiovascular Aging

Development of progressive aortic vasculopathy in a rat model of aging

Steven J. Miller,1,2,3 William C. Watson,1 Kimberly A. Kerr,1 Carlos A. Labarrere,3,4 Neal X. Chen,5 Mark A. Deeg,3,6 and Joseph L. Unthank1,2,3

1Department of Surgery, Indiana University School of Medicine, 2Department of Cellular and Integrative Physiology, Indiana University School of Medicine, 3Indiana Center for Vascular Biology and Medicine, 4Division of Experimental Pathology, Methodist Research Institute/Clarian Health Partners, Inc., 5Department of Nephrology, Indiana University School of Medicine, and 6Departments of Medicine and Biochemistry and Molecular Biology, Indiana University School of Medicine, and Department of Veterans Affairs, Richard L. Roudebush Veterans Affairs Medical Center, Indianapolis, Indiana

Submitted 30 March 2007 ; accepted in final form 11 September 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Recent studies have established that age is the major risk factor for vascular disease. Numerous aberrant changes occur in vascular structure and function during aging, and animal models are the primary means to determine the underlying mechanisms of age-mediated vascular pathology. The Fischer 344/Brown Norway F1 hybrid (F344xBN) rat thoracic aorta has been shown to display age-related pathology similar to what occurs in humans. This study utilized the F344xBN rat aorta and both morphometric and global gene expression analyses to identify appropriate time points to study vascular aging and to identify molecules associated with the development and progression of vascular pathology. In contrast to some previous studies that indicated age-related abrupt changes, a progressive increase in intimal and medial thickness, as well as smooth muscle cell-containing intimal protrusions, was observed in thoracic aorta. This structural vascular pathology was associated with a progressive, but nonlinear, increase in global differential gene expression. Gene products with altered mRNA and protein expression included inflammation-related molecules: specifically, the adhesion molecules ICAM-1 and VCAM-1 and the bone morphogenic proteins osteopontin and bone sialoprotein-1. Intimal-associated macrophages were found to increase significantly in number with age. Both systemic and tissue markers of oxidant stress, serum 8-isoprostane and 3-nitrotyrosine, respectively, were also found to increase during aging. The results demonstrate that major structural abnormalities and altered gene expression develop after 6 mo and that the progressive pathological development is associated with increased inflammation and oxidant stress.

arterial remodeling; inflammation; microarray; oxidative stress

ADVANCING AGE IS THE MAJOR risk factor for vascular diseases (19, 31). This tenet is based both on epidemiological studies and on observations that functional and structural alterations occurring in the vasculature during aging have similarities to those observed in early stages of vascular disease development. With advancing age in humans, vascular stiffness is increased, and endothelial-dependent regulation of vascular tone is altered (19). Age-associated structural alterations in human large elastic arteries include luminal expansion and thickening of both the media and intima (19, 31). Intimal thickening is of special importance, as there are similarities with early plaque development, and it is correlated with the development of human vascular disease (32). Because aging is considered to provide an environment that promotes vascular disease, aging-related vascular alterations provide potential therapeutic targets for the treatment and even prevention of vascular disease.

Age-associated structural and functional arterial alterations are common to many species. Najjar et al. (31) have recently reviewed the similarities in age-associated changes that occur in large arteries of rats, primates, and humans. The occurrence of similar vascular changes with age in humans and rodents provides the opportunity to utilize these animals to investigate underlying mechanisms and to evaluate potential treatments. Although previous studies (1, 7, 12, 20, 21, 43) with rodent models have supplied significant insight into the development of vascular pathology, fundamental questions remain unanswered. One of the more basic questions concerns selection of the most appropriate ages to investigate mechanisms that regulate vascular aging events in specific rodent models. Results of studies with rats have suggested both progressive (45) and abrupt (6) vascular structural alterations during aging. In addition, many studies of altered gene expression (7, 8, 20, 34, 36, 43) have been done only at the extremes of age and thus were unable to correlate vascular structural alterations with altered gene expression during the aging process. Similar limitations apply to measures of age-related changes in inflammatory molecules and oxidative stress, and the role of macrophages in rat vascular aging is controversial (12, 17).

This study was designed using the Fischer 344/Brown Norway F1 hybrid (F344xBN) rat model of aging, the primary rat model promoted by the National Institute on Aging (NIA) (37), to address these limitations and controversies. Primary goals were to identify the most appropriate time points to study vascular aging in the F344xBN rat, based on both morphometric and gene expression data, and potentially important molecules associated with the development of vascular pathology. Ages spanning from young adult to near the mean survival age were used in determining when specific structural pathologies developed and in clarifying how changes in gene expression were temporally related to structural alterations. Results indicated that vascular structural pathology occurs in a progressive manner with aging in the F344xBN rat and is associated with increased expression of multiple inflammatory molecules, numbers of intimal macrophages, and markers of oxidative stress.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animal care and tissue isolation. The Indiana University School of Medicine Institutional Animal Care and Use Committee approved all procedures performed in this study. The F344xBN rat was adopted as an aging model, based in part on NIA recommendations (37). F344xBN rats were obtained from the NIA colony maintained by Harlan (Indianapolis, IN). Rats arrived at the stated ages, and studies were completed within 2 wk after arrival. Body masses of rats increased significantly (P < 0.003) from 3 to 12 mo (3 mo, 282 ± 2.6 g; 6 mo, 401 ± 5.3 g; 12 mo, 500 ± 14.9 g), but not from 12 to 24 mo (18 mo, 501 ± 13.8 g; 24 mo, 588 ± 47.5 g). To obtain rat thoracic aortas for morphometric and immunohistochemical analyses, rats at 3, 6, 12, 18, and 24 mo of age were anesthetized (pentobarbital sodium, 50 mg/kg ip), the caudal aorta cannulated with a double lumen cannula, and the vasculature perfused at the animal's approximate mean arterial pressure (~120 mmHg) with PBS containing dilator (10–4 M adenosine and 10–5 M sodium nitroprusside) followed by 4% paraformaldehyde. After perfusion, distal thoracic aortas (~1-cm segment superior to diaphragm) were isolated and processed, and separate segments were embedded in paraffin and plastic (JB-4, Polysciences), as described previously (41). Based on morphometric results and aged rat availability from the NIA rodent colony, an additional set of rats at 3, 6, 15, and 28 mo of age was used to obtain distal thoracic aorta total RNA for microarray analysis. Following anesthesia, the abdominal aorta was cannulated above the iliac bifurcation and perfused with 30 ml of cold phosphate-buffered saline followed by 10 ml of RNAlater (Ambion, Austin, TX). The descending thoracic aorta (1 cm) was isolated and excised from four rats of each age in randomized order, and the tissue was preserved in RNAlater at –20°C until use.

Morphometric analysis. Plastic embedded tissue sections were used for morphometric analysis, as previously described (41). Analysis was performed only on distal thoracic aortic sections where the media was of uniform thickness and vascular smooth muscle cells were oriented circumferentially. Digital images of arterial cross sections were analyzed using an image analysis system (Metamorph, Universal Imaging). Measurements of wall areas were completed on two sections from the same artery, at least 10 µm apart, and averaged.

Immunohistochemistry. Proteins and macrophages were detected in paraffin-embedded tissues using an immunoperoxidase technique. Aortic sections were deparaffinized in xylene and rehydrated through graded alcohols to water. Antigen retrieval was performed by immersing the slides in Target Retrieval Solution (DAKO) for 20 min at 90°C (in a water bath), cooling at room temperature for 10 min, washing in deionized water, and then proceeding with immunostaining. All subsequent staining incubation steps were done at room temperature, and Tris-buffered saline, pH 7.4 (Dako), plus 0.05% Tween 20 were used for all washes and diluents. Slides were thoroughly washed after each antibody incubation. Slides were blocked with protein blocking solution (Dako) for 25 min. After washing, 10 µg/ml of the primary antibody (von Willebrand factor, Dako), smooth muscle {alpha}-actin (clone 1A4, Sigma), intercellular adhesion molecule-1 (ICAM-1) (clone 1A29, Santa Cruz), vascular cell adhesion molecule-1 (VCAM-1) (H-276, Santa Cruz), macrophages (clone ED-1, Chemicon; clone ED-2, Serotec), nitrotyrosine (clone 1A6, Upstate), and bone sialoprotein (BSP) or osteopontin (OPN) (LF-100 or LF-124, 1:100 dilution; gifts of Dr. Larry Fisher, National Institutes of Health) were added to the slides and incubated for 60 min. A biotinylated link antibody plus streptavidin-horseradish peroxidase kit (Dako LSAB2) was utilized, along with a diaminobenzidine chromagen and peroxide substrate, to detect the bound antibody complexes. The slides were briefly counterstained with hematoxylin and dehydrated through graded alcohols to xylene. The slides were coverslipped with a permanent mounting media.

Gene expression analysis. Tissue was weighed and disrupted by using a bead homogenizer (FastPrep System; QBIOgene, Carlsbad, CA), and total RNA was purified by using an RNeasy Fibrous Tissue Mini Kit (Qiagen, Valencia, CA). To ensure high-quality RNA, sample concentration and integrity were determined by using the ratio of absorbance at 260 nm to 280 nm, agarose gels, and by analysis with an Agilent 2100 Bioanalyzer. Samples of total RNA were provided to the Indiana University Center for Medical Genomics (IUCMR) for microarray analysis. The microarray procedure was performed by following standard Affymetrix protocols (Affymetrix GeneChip Expression Analysis Technical Manual; Affymetrix, Santa Clara, CA). cRNA was hybridized to the Affymetrix GeneChip Rat Genome U34A Array for 17 h, followed by standard washing, staining, and scanning. Data were extracted using Affymetrix Microarray Suite 5.0 software (MAS5; Affymetrix MicroArray Suite 5.0 User's Guide, Santa Clara, CA). A complete data set is available at the NCBI GEO database (http://www.ncbi.nlm.nih.gov/projects/geo) under accession GSE7281. Microarray data analyses were carried out using the Microarray Data Portal, a proprietary analytic and informatics algorithm developed by the IUCMR. To eliminate noisy data from probe sets that reflected background signals, only those probe sets identified as "present" by MAS5 in at least one-half of the arrays were analyzed (26). False discovery rates were calculated as previously described (38, 39), and genes that had significant expression changes at P ≤ 0.05 using log-transformed signal values were analyzed for Gene Ontology categories.

Quantitative PCR. Relative differences in ICAM-1 and VCAM-1 mRNA expression were determined by using real-time quantitative RT-PCR with unused total RNA from the microarray studies. Aliquots of purified total RNA (0.5 µg) were treated to remove contaminating genomic DNA (DNA-free; Ambion) and then reverse transcribed using Ready-To-Go You Prime First Strand Beads (Amersham Biosciences, Piscataway, NJ) with random decamer priming. For PCR, an aliquot of the RT reaction (2.0–2.5 µl) was combined with the appropriate primers for the target in the presence of a PCR mastermix (QuantiTect Probe PCR Kit; Qiagen). Primer and probe sequences for rat ICAM-1 and VCAM-1 were obtained from Berti et al. (3). Reactions were run in an Applied Biosystems Prism 7700 Sequence Detection System using standard cycling conditions with dual-labeled (5'-FAM, 3'-TAMRA) probes as the product detection method. Standard curves were generated from serial dilutions of rat heart total RNA. Differences in RT-PCR target product yields for the age groups of aorta were determined from standard curves and expressed as fold differences in arbitrary units normalized to total RNA.

Serum analysis. Blood was harvested via aortic cannula and processed, and serum was stored at –70°C until assays were performed. All assays were performed by the Indiana University Endocrinology Analyte Core using commercial kits: cholesterol, glucose, and creatinine were determined using kits from Sigma-Aldrich (St. Louis, MO); oxidized LDL (OxLDL) was measured with a kit from Mercodia (Uppsala, Sweden); and 8-isoprostane was determined using an ELISA from Assay Designs (Ann Arbor, MI).

Data analysis. The Student t-test was used to establish statistical differences, unless indicated otherwise in the appropriate figure legends. All pairwise comparisons shown in Table 1 were performed using the Student-Newman-Keuls method. All measurements are reported as means ± SE, with the exception of the serum measurements, which are shown as SD.


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  		Table 1. Morphometric parameters for rat thoracic aorta

 

   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Morphometric analyses. Representative micrographs of plastic embedded thoracic aorta cross sections from male F344xBN 3-, 6-, 12-, 18-, and 24-mo-old rats are shown in Fig. 1. Age-related increases in medial thickness (MT) and the development of intimal protrusions were obvious changes that occurred in the aorta, and wall characteristics of these vessels are reported in Table 1. Linear regression analysis was performed to evaluate age-related changes in wall parameters. A positive correlation with age was found for aortic luminal radius (r), intimal thickness (IT) and MT, and the number of intimal cell nuclei per unit circumference length. A negative correlation was observed with medial cell nuclear density (number per cross-sectional area). The increase in intimal cell density required an increase in cell number as luminal circumference was increased. The decrease in medial cell density occurred without reduction in cross-sectional medial cell nuclear number (703 ± 120 vs. 800 ± 15 nuclei at 3 and 24 mo, respectively, P = 0.719). Ratios of IT and MT to luminal r were evaluated to determine whether age-related changes were simply the result of vessel enlargement. The MT/r relationship was unchanged during aging. However, the IT/r doubled from 3 to 24 mo. Consequently, the MT/IT ratio decreased with aging. It is apparent from the values presented in Table 1 that the changes progressed gradually rather than developing abruptly.


Figure 1
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  		Fig. 1. Effects of progressive aging in rat aorta. Representative micrographs show cross sections of 3-, 6-, 12-, 18-, and 24-mo-old Fischer 344/Brown Norway F1 hybrid (F344xBN) rat thoracic aorta. Sections from plastic-embedded tissues were stained with Lee's methylene blue. The internal elastic lamina was identified as the innermost continuous elastic lamina, which separated the intima from the media. The outer boundary of media is defined by dense staining of smooth muscle relative to adventitial connective tissue. Note the increasing thickness of the tunica intima and media and irregularity of the intimal surface with age. Arrows indicate typical intimal protrusions, which are first observed at 6 mo of age.

 
Matched paraffin tissue sections from the previously described vessels were subjected to an immunohistochemical analysis to determine potential changes in thoracic aorta structure with age. Antibody reactivity to von Willebrand factor indicated that the endothelium was continuous around the intima of the vessels at all ages, including the numerous intimal protrusions that were detected (Fig. 2A). These intimal protrusions significantly increased in size (area/unit length) with age from 6 to 24 mo. Reactivity to smooth muscle {alpha}-actin indicated that smooth muscle-like cells were present in all of the intimal protrusions (Fig. 2B).


Figure 2
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  		Fig. 2. Vascular structural changes. A: immunohistochemical detection of von Willebrand factor in rat thoracic aorta at 6 and 24 mo of age showing intact endothelial lining on atherosclerotic-like lesions (arrow). B: immunohistochemical detection of smooth muscle {alpha}-actin (clone 1A4) in atherosclerotic plaque-like lesions (arrow) in rat thoracic aorta at 6 and 24 mo of age. Linear regression analysis indicated that the intimal protrusion size (area/unit length) increased with age [Y = 154.7 + 72.5 * age (mo), P = 0.006, r2 = 0.59].

 
Differential gene expression. Changes in global gene expression with age for thoracic aorta were determined by oligonucleotide microarray analysis, and the results are summarized in Fig. 3. The number of differentially expressed genes increased progressively with age, although, unlike the morphometric alterations, which were gradual and linear, gene expression changes were greater at the later ages. The proportion of genes at a given age that showed increased expression was lower at 6 mo (29%) than at 15 mo (60%) or 28 mo (60%). Gene expression changes with age in thoracic aorta for classes of molecules were determined by an ontological analysis and are shown in Table 2. Selected molecules that had the most statistically significant (P < 0.05) and highest fold changes (>1.5x) in expression at 28 mo are listed in Table 3. The inflammation-related molecules ICAM-1 and VCAM-1 were among the genes with the greatest fold changes, and mRNA levels increased progressively from 6 to 28 mo of age (Fig. 4A), with relative expression at 28 mo being 2.1x and 4.7x, respectively, greater than 6 mo (P < 0.01). Real-time PCR was used to confirm the oligonucleotide microarray results for ICAM-1 and VCAM-1 mRNA expression (Fig. 4B).


Figure 3
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  		Fig. 3. Gene expression changes with age in thoracic aorta. Global gene expression was determined by oligonucleotide microarray analysis, and the diagram depicts the number of genes whose expression was altered (increased in parentheses) significantly (P < 0.05) at the indicated ages. Changes were determined relative to aortic gene expression at 3 mo of age. Overlap areas indicate the number of genes with altered expression common to both ages. The data analysis does not include filtering for fold change, low-expression level, or duplicate gene identities due to multiple probe sets, and expressed sequence tags are included as genes.

 

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  		Table 2. Gene ontology summary

 

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  		Table 3. Differential gene expression for selected genes between 3 and 28 mo of age

 

Figure 4
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  		Fig. 4. Adhesion molecule expression in aged rat thoracic aorta. A: vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) mRNA expression was determined by microarray analysis, and linear regression analysis demonstrated expression for both molecules increased progressively with age {VCAM-1 = 146 + [127 * age (mo)], P < 0.001, r2 = 0.82; ICAM-1 = 629 + [38 * age (mo)], P < 0.001, r2 = 0.76}. B: real-time PCR was used to verify microarray results for ICAM-1 and VCAM-1 mRNA expression at 6, 15, and 28 mo of age. *P < 0.05 vs. 6 mo; **P < 0.05 vs. 6 and 15 mo.

 
Inflammation-related protein expression and cells. Protein expression for ICAM-1 and VCAM-1 in tissue sections from aged thoracic aorta were analyzed immunohistochemically, and results showed that the aorta intimal reactivity increased progressively with age (Fig. 5). VCAM-1 appeared to be more abundant than ICAM-1 (not shown) in the aorta at all ages, based on a semiquantitative assessment of reactivity, and this correlated with mRNA expression levels.


Figure 5
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  		Fig. 5. VCAM-1 protein expression in thoracic aorta at 6, 12, 18, and 24 mo. VCAM-1 reactivity, as detected by immunohistochemistry, was essentially absent at 6 mo, showed discontinuous reactivity by 12 mo (arrows), and became continuous with the endothelium by 24 mo. These micrographs are representative of observations from aortas of 3 animals of each age.

 
Because microarray data suggested increased gene expression for bone matrix proteins, tissue sections of thoracic aortas from 6- and 24-mo-old rats were examined for protein expression of OPN and BSP by immunohistochemistry (Fig. 6). Staining for both BSP and OPN was detected primarily in the medial layer of aortas from 24-mo-old rats, but little or no staining was found in 6-mo-old rat aorta.


Figure 6
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  		Fig. 6. Expression of bone matrix proteins in aorta during aging. Bone sialoprotein (BSP) and osteopontin (OPN) were detected immunohistochemically in thoracic aortas of 6- and 24-mo-old F344xBN rats. Both proteins showed minimal medial reactivity at 6 mo and no apparent intimal staining. By 24 mo, BSP and OPN reactivity was prominent in the media, primarily near the medial/advential border, and was also detectable in the intima. Cross-sectional images represent typical reactivity obtained from 3 aortas at each age.

 
Reacting thoracic aorta tissue sections with an antibody specific to macrophages (clone ED2) showed that many ED2-positive cells present as nodes could be detected associated with the intima and appeared to be more concentrated at the areas where intimal protrusions occurred (Fig. 7A). The number of cell-containing nodes detected that were positive for macrophages also increased with advancing age (Fig. 7B). Identical results were obtained by using an alternative monocyte/macrophage-specific monoclonal antibody (ED-1; not shown).


Figure 7
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  		Fig. 7. Inflammatory cells increase during aging. A: ED-2-positive cells (macrophages) were rarely observed at 3 or 6 mo, but were commonly detected in thoracic aortic intima at 12 (not shown), 18, and 24 mo (arrows). Inset in 24-mo panel is a x2 enlargement to show additional detail. Micrographs are representative of results from 2 serial sections/aorta from 3 animals/age. B: quantitation of ED-2-positive nodes in aortic intima during aging. Nodes were counted in 2 serial sections/aorta from 3 animals/age, and results indicated an approximate sixfold increase from 3–6 to 12–24 mo. Mean cell numbers at 12, 18, and 24 mo and between 3 and 6 mo were not significantly different from each other. *P < 0.002.

 
Serum analyses and oxidative stress. An analysis of serum components showed that glucose and serum creatinine concentrations did not change with age (Table 4). Total cholesterol significantly increased between 6 and 18 mo and remained elevated at 24 mo. For indexes of systemic oxidative stress, we measured levels of serum OxLDL and total 8-isoprostane. Although changes in OxLDL with age were not statistically significant, mean values increased almost fourfold between 6 and 18 mo. Serum levels of 8-isoprostane increased threefold between 6 and 24 mo of age. These results are consistent with an age-related increase in oxidative stress. To assess whether oxidant stress increased in the vasculature during aging, immunohistochemistry was utilized to detect the presence of nitrotyrosine. Nitrotyrosine reactivity was present in the intima and selected cells in the media of aorta and increased with age, suggesting increased vascular oxidant stress with aging (Fig. 8).


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  		Table 4. Serum analyte results

 

Figure 8
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  		Fig. 8. Immunohistochemical detection of tissue nitrotyrosine. Representative micrographs (3 rats/age) of cross sections showed that discontinuous nitrotyrosine reactivity was present in the aortic intima of 6-mo-old F344xBN rats and became continuous at 24 mo, as well as becoming apparent in the media.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
There are several novel and important observations in this study that will be considered in the context of previous reports. These include 1) the progressive rather than abrupt development of age-related vascular structural pathology in the F344xBN rat; 2) the marked elevation in the number of genes with altered expression during aging; 3) the elevation in inflammation-associated molecule expression in the aging aorta; 4) the age-related increase in intimal macrophages; and 5) the increase in systemic and vascular oxidant stress with age.

Previous studies characterizing the nature of aging-associated vascular pathology have described both progressive and abrupt changes. Cantini et al. (6) evaluated thoracic aorta structure and composition in 3-, 10-, 15-, 20-, and 30-mo-old WAG/Rij rats. They observed that the inner diameter and medial cross-sectional area of thoracic aorta were increased relative to young (3 mo) rats at 20 but not 15 mo, indicating rather abrupt alterations after 15 mo. However, in studies with 2-, 8-, and 30-mo-old F344xBN rats, Wang and Lakatta (45) have reported a progressive, rather than abrupt, increase in MT and IT. While strain-dependent differences may exist, knowing the exact nature of the onset and development of age-related vascular pathology is fundamentally important in identifying the optimal ages to be used, both for determining molecular changes that may initiate pathological remodeling events, and for distinguishing from those that may occur as a result of pathology. Our data clearly demonstrate that age-related vascular pathology characterized by luminal expansion, and intimal and medial thickening, occurs in a progressive and linear manner in the F344xBN rat model. The observed intimal thickening and presence of intimal smooth muscle cells are consistent with previous findings (45) and are representative of the age-related pathology in human arteries that is thought to predispose them to future atherosclerotic events (17, 31).

Studies of global gene expression during aging have not previously been reported for the rat vasculature. Obtaining differential gene expression data for aging vasculature has been identified as an important goal for aging research (18). Results from the microarray analysis of thoracic aorta at four ages indicated an increase in the number of genes with altered expression, paralleling the increase in age-related pathology, except that gene expression changes were nonlinear, increasing more rapidly at the later ages. Genes showing early alterations that continued throughout aging included inflammation-related molecules, growth factors, and those involved in cellular energy production. It is interesting to note that expressions of multiple subunits of cytochrome-c oxidase were consistently downregulated. Other studies of aging in various tissues have indicated that decreased expression of mitochondrial enzymes occurs during aging (44, 46), and mitochondrial dysfunction is thought to be a key factor in the development of atherosclerosis (24, 25). Future studies are warranted to extend these observations to specific cell types or wall layers and to evaluate posttranscriptional modifications of proteins, such as phosphorylation, that regulate activity or function and have been shown to occur with aging (34).

The noted increase in pathology with age was associated with the increase in mRNA and protein expression of the adhesion molecules ICAM-1 and VCAM-1. These molecules are considered to be markers for vascular inflammation (30) and have been shown to increase with age in humans (28). In rodents, increased expression of inflammatory molecules during aging has been shown in coronary arteries (8) and aorta (15, 20, 36). This is consistent with the findings of Cybulsky et al. (9), whose results with a knockout mouse model suggest that VCAM-1 is involved in the initiation of atherosclerosis in coronary arteries. Their data did not show a role for ICAM-1, contrary to what has been described in humans (30). Our data also are similar to the findings of Merat et al. (27), who showed an increase in VCAM-1 expression with age in a mouse model of atherosclerosis, and with the results of Zou et al. (48) that recently demonstrated increased P-selectin and VCAM-1 expression in aged F344 rats. To the best of our knowledge, this is the first description of an age-associated increase in VCAM-1 expression in the F344xBN rat model of aging.

In addition to adhesion molecules, mRNA and protein expression increases were also detected with increasing age for OPN and BSP. Matrix cytokines, such as bone morphogenetic protein-4 and OPN, are thought to have an important role in vascular function (10, 11, 29). OPN has been shown to enhance advential myofibroblast and vascular smooth muscle cell migration and proliferation (14), as well as to promote matrix metalloproteinase-9 activation (5, 14, 16). OPN expression is upregulated during diabetes (4, 40) and may augment superoxide generation and oxylipid formation (16). Recent work by San Martin et al. (35) indicates that NAD(P)H oxidase activation, and the resultant superoxide, induced increased expression of bone morphogenetic protein-4 and OPN in a mouse model of diabetes. These results are consistent with our observation in the F344xBN rat of increased OPN expression and markers of oxidant stress and inflammation, along with pathological vascular remodeling. Thus, as suggested for diabetes, OPN may be a link between increased vascular oxidative stress and the inflammation that occurs during aging.

Although recent reviews (17, 31) have stated that inflammatory cells, i.e., macrophages, are not involved in age-associated vascular remodeling in the rat, results from several rat studies have suggested the presence of subendothelial macrophages in arteries during aging (1, 13, 22), and one study has reported over a sevenfold increase in subendothelial macrophages in aged vessels (12). Because we detected increases in inflammatory markers, and the presence of macrophages could provide important information regarding etiology, we investigated whether macrophages were associated with the aging thoracic aorta. Contrary to previous reports (17, 20, 31), we observed a significant increase in intimal-associated macrophages, consistent with results from earlier studies.

Progressive endothelial dysfunction occurs during aging (19), and accumulating evidence suggests that increases in age-related oxidative stress both activate inflammatory pathways and alter vascular cell phenotype (7, 8). To assess changes in systemic oxidant stress, serum 8-isoprostane levels were measured and found to be elevated at 18 mo and became statistically significant at 24 mo, suggesting progressive increases in oxidant stress with age. Cholesterol levels increased progressively with aging, but somewhat surprisingly OxLDL levels did not, although high variability in the OxLDL measurements may account for the lack of significance. The observation that tissue nitrotyrosine was increased with age suggests the presence of oxidative stress in the aorta: specifically, the presence of peroxynitrite due to scavenging of nitric oxide by superoxide (2, 42). It is interesting to note that nitrotyrosine could be detected in the intima of vessels even at 6 mo, suggesting that oxidative stress was present at the earlier stages of the rat's life. Nitration has been shown to have both positive (33) and negative (23, 47) effects on enzyme activity, and this modification could contribute significantly to vascular pathology during aging. Our results are consistent in part with the recent findings of Rice et al. (34), who showed that large increases in superoxide occurred with age in the thoracic aorta, and that this increase correlated with increased MT and total protein nitration.

The morphometric analyses (Table 1) indicate that most of the structural indexes are similar between young rats of 3–6 mo of age, but become statistically different by 12 mo and continue to change thereafter. The greatest change in the number of genes with differential expression occurs after 15 mo (Fig. 3). Similarly, while there is a progressive increase in the expression of inflammatory molecules, the largest increase also occurs after 15 mo. Together, these results suggest that changes between 6 and 12–15 mo are important for initiating the development of the age-related pathologies. This should be considered when selecting the most appropriate time points for investigating the mechanisms mediating vascular aging.

In summary, we have demonstrated that vascular aging in the F344xBN rat correlates with progressive changes in both differential gene expression and structural alterations. The results are consistent with the hypothesis that the development of age-related vascular pathology is associated with altered expression of inflammation-associated molecules and suggest that increased oxidative stress may contribute to this process. Therapies designed to modulate expression of these molecules and their related metabolic pathways may prove to be useful in diminishing the negative effects of vascular aging. The F344xBN rat model of aging displays many of the characteristic pathologies that progressively develop with aging in the human vasculature and thus appears to be an excellent model for studying the development of early stages of pathological vascular remodeling associated with aging.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported in part by National Heart, Lung, and Blood Institute Grant HL-42898 and a pilot grant from the Indiana Center for Vascular Biology and Medicine, funded by the Cryptic Mason Cardiovascular Research Fund. The Center for Medical Genomics is supported in part by grants from the Indiana 21st Century Research and Technology Fund and the Indiana Genomics Initiative (supported in part by the Lilly Endowment, Inc.).

Present address of M. A. Deeg: Eli Lilly & Co., Lilly Corporate Center, Indianapolis, IN 46285.


   ACKNOWLEDGMENTS
 
The microarray studies were carried out using the facilities of the Center for Medical Genomics at Indiana University School of Medicine. We thank Jennifer Stashevsky for expert technical assistance in embedding, sectioning, and staining the rat tissues, and Marcelo Sosa for excellent work in performing the immunohistochemistry.


   FOOTNOTES
 

Address for reprint requests and other correspondence: S. J. Miller, Dept. of Surgery, Indiana Univ. School of Medicine, 1001 West Tenth St., WD OPW 425, Indianapolis, IN 46202-2879 (e-mail: sjmiller{at}iupui.edu)

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   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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