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Am J Physiol Heart Circ Physiol 280: H2779-H2788, 2001;
0363-6135/01 $5.00
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Vol. 280, Issue 6, H2779-H2788, June 2001

Aging, oxidative responses, and proliferative capacity in cultured mouse aortic smooth muscle cells

Sung-Kwon Moon1, Larry J. Thompson3, Nageswara Madamanchi1, Scott Ballinger3, John Papaconstantinou4, Chris Horaist1, Marschall S. Runge1, and Cam Patterson1,2

1 Program in Molecular Cardiology and 2 Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, North Carolina 27599-7075; and 3 Division of Cardiology and Sealy Center for Molecular Cardiology and 4 Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch, Galveston, Texas 77555


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The cellular mechanisms that contribute to the acceleration of atherosclerosis in aging populations are poorly understood, although it is hypothesized that changes in the proliferative capacity of vascular smooth muscle cells is contributory. We addressed the relationship among aging, generation of reactive oxygen species (ROS), and proliferation in primary culture smooth muscle cells (SMC) derived from the aortas of young (4 mo old) and aged (16 mo old) mice to understand the phenotypic modulation of these cells as aging occurs. SMC from aged mice had decreased proliferative capacity in response to alpha -thrombin stimulation, yet generated higher levels of ROS and had constitutively increased mitogen-activated protein kinase activity, in comparison with cells from younger mice. These effects may be explained by dysregulation of cell cycle-associated proteins such as cyclin D1 and p27Kip1 in SMC from aged mice. Increased ROS generation was associated with decreased endogenous antioxidant activity, increased lipid peroxidation, and mitochondrial DNA damage. Accrual of oxidant-induced damage and decreased proliferative capacity in SMC may explain, in part, the age-associated transition to plaque instability in humans with atherosclerosis.

atherosclerosis; reactive oxygen species; cell cycle; DNA damage; lipid peroxidation


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

ATHEROSCLEROSIS, the most common cause of death in developed countries, is attributed to an excessive inflammatory and fibroproliferative response to arterial injury (33). The earliest stages of atherosclerotic lesions can be detected in coronary arteries from adolescents (26). However, the prevalence of coronary artery disease increases with age (26a), and age itself is an independent risk factor for atherogenesis (19), suggesting that the biological milieu in aging populations is conducive to atheromatous lesion formation. Consistent with the hypothesis that atherogenesis is an intrinsically age-related process, accelerated atherosclerosis is seen in diseases associated with premature senescence, such as Werner's syndrome and progeria (12).

Atherosclerotic lesions are heterogeneous at the cellular level, containing endothelial cells, smooth muscle cells (SMC), fibroblasts, macrophages, and other inflammatory cells, each of which participates in atheroma formation (33). This cellular heterogeneity, coupled with differences between animal models of atherosclerosis and the human disease, has made it difficult to ascertain the precise role that different cell types play in lesion formation. On the basis of the accelerated proliferative response of vascular SMC to mechanical injury in animal models (10, 11), it was initially presumed that SMC proliferation, and therefore increased numbers of SMC in atherosclerotic lesions, is a primary determinant of disease progression. However, it is now generally accepted that SMC protect the integrity of the otherwise unstable fibrous cap of atherosclerotic plaques and that a paucity of SMC in plaques, due in part to cellular senescence, is a detrimental feature of the disease (27). According to this hypothesis, SMC proliferation is deleterious in the early steps of atherosclerotic lesion formation, but SMC dysfunction due to cellular injury and loss of SMC proliferation in late stages of the disease is associated with an adverse outcome by participating in the process of plaque destabilization.

As we have come to better understand the multiple roles of inflammatory stimuli in atherogenesis, an important role has been assigned to reactive oxygen species (ROS) as effectors in inflammatory and noninflammatory cells. Recent data from our laboratories and others (15, 31, 41) demonstrated a critical role for ROS in vascular SMC proliferative responses. For example, alpha -thrombin, a potent SMC mitogen, activates an NADH oxidase that generates H2O2 (31). H2O2, in turn, is required for SMC proliferation in response to alpha -thrombin and other mitogens (31, 41), indicating that ROS elicit, and are required for, growth-regulating cell signaling. This relationship between ROS and vascular cell dysfunction is not surprising, given that ROS are linked to most risk factors for atherosclerosis (1, 29) and linked with aging-related diseases in general (40). At the same time, we do not know whether SMC alter their responsiveness to ROS during aging in a manner that might account for the increased frequency of coronary events in aging populations. Similarly, the effects of aging-associated increases in ROS may affect SMC independently of their effects on proliferation, for instance, by causing lipid peroxidation and DNA damage. These modifications may ultimately contribute to SMC dysfunction and disease progression (2, 17).

Previous studies aimed at determining the effects of aging on SMC proliferation have yielded confusing and, at times, contradictory results. The most commonly cultured primary SMC are derived from rat aortas. On the basis of careful reports from several different laboratories, it is generally considered that rat aortic SMC from aged animals have greater proliferative capacity than those derived from young animals (7, 24, 25), although it should be noted that growth advantages in SMC from older rats have not always been observed (44). In contrast with observations in rats, SMC derived from humans clearly demonstrate an age-related decline in proliferative capacity (37). The underlying mechanisms that account for these species-related differences have not been resolved.

We examined SMC derived from aortas of young and aged mice as a model to better understand the effects of aging on SMC proliferation and function. Mice are particularly advantageous to study because the genetic background can be carefully controlled and the kinetics of aging in mice are well characterized. We report that SMC from aged mice have a diminished proliferative capacity, although they produce higher basal and inducible amounts of H2O2 and have increased mitogen-activated protein (MAP) kinase activities. The discordance between ROS generation and MAP kinase activation, on the one hand, and proliferative capacity, on the other hand, can be attributed, at least in part, to dysregulation of cell cycle-regulating proteins cyclin D1 and p27. Cells from aged mice have decreased superoxide dismutase (SOD) activity and decreased pools of reduced glutathione (GSH). Constitutively increased levels of ROS are associated with increased lipid peroxidation products and mitochondrial DNA damage in cells from aged mice.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cell culture. Mouse aortic SMC were obtained from young (4 mo old) and aged (16 mo old) male mouse aortas. Briefly, the aortas were removed under sterile conditions. After the aortas were rinsed several times in Hanks' balanced salt solution, the adventitia was removed, and the aortas were minced and digested in 5 ml of digestion solution (0.125 mg/ml elastase, 0.25 mg/ml soybean trysin inhibitor, 10 mg/ml collagenase I, 2.0 mg/ml crystallized bovine albumin, and 15 mM HEPES) at 37°C for 45 min. The cellular digests were filtered through sterile 100-µM nylon mesh, centrifuged at 1,000 rpm for 10 min, and washed twice in DMEM containing 10% fetal calf serum before culture in the same medium. Before the experiments were performed, cells were growth arrested at 80% confluence for 48 h with medium containing 0.2% fetal bovine serum. Experiments shown are representative of results from three independent cultures from each group of mice.

Cell counts, [3H]thymidine incorporation, and apoptosis assays. For cell counts, SMC were plated overnight at equivalent densities (5 × 105 cells/plate) in 100-mm plates. At intervals after plating, cells were trypsinized, and cell numbers were determined using a Coulter Counter. For [3H]thymidine-uptake experiments, SMC grown to near confluence in 24-well tissue culture plates were made quiescent and treated with alpha -thrombin (1 U/ml), 4-hydroxynonenal (0.5 µM), diphenyliodonium (DPI, 10 µM), and/or vehicle, as indicated. Cells were incubated for an additional 24 h and labeled with [methyl-3H]thymidine (New England Nuclear; Boston, MA) at 1 µCi/ml during the last 3 h of this time period. After labeling, the cells were washed with phosphate-buffered saline and fixed in cold 10% trichloroacetic acid and then washed with 95% ethanol. Incorporated [3H]thymidine was extracted in 0.2 M NaOH and measured in a liquid scintillation counter as previously described (30). Values were expressed as means ± SE from six wells from two separate experiments. An ELISA-based assay based on the ability to detect free DNA-histone complexes was performed in triplicate according to the manufacturer's instructions (Roche; Indianapolis, IN).

Cellular release of H2O2. Cellular release of H2O2 was measured by a colorimetric assay that relies on the oxidation of o-dianisidine dihydrochloride (o-DD, Sigma; St. Louis, MO) in the presence of horseradish peroxidase (28). Medium from exponentially growing or growth-arrested SMC in six-well plates was aspirated and replaced by a 1-ml assay buffer (phenol red-free DMEM, 0.5 mM o-DD, and 5 U/ml horseradish peroxidase) with or without growth factors. After growth factor treatment, the assay buffer was removed and centrifuged for 5 min at 1,000 rpm. H2O2 concentration was determined by measuring specific absorbance at 470 nm using a standard curve generated with known concentrations of H2O2. Cells in each well were counted using a Coulter Counter, and H2O2 release was normalized to cells per well for each experiment. The specificity of this assay for measurement of H2O2 release was determined by pretreatment with catalase (100 U/ml).

Intracellular generation of H2O2. The fluorogenic substrate 2',7'-dichlorofluorescein diacetate (DCFDA, Molecular Probes; Eugene, OR) is a cell-permeable dye that is oxidized to 2',7'-dichlorofluorescein by H2O2 and can therefore be used to monitor intracellular generation of H2O2 (8). The medium of treated and untreated SMC grown in 96-well plates was replaced by Hanks' solution containing 10 µM DCFDA for 30 min. Generation of H2O2 was measured using a CytoFlor fluorescence plate reader (485-530 nm, PerSeptive Biosystems; Foster City, CA) (43). Cells in each well were counted using a Coulter Counter, and H2O2 release was normalized to cells per well for each experiment.

Western blot analysis. Quiescent mouse SMC were treated in the presence or absence of alpha -thrombin as indicated. Western blot analysis was performed as previously described (35). Polyclonal anti-cyclin D1 and anti-p27Kip1 antibodies were from Pharmingen (San Diego, CA), and polyclonal anti-copper/zinc-dependent SOD (SOD1) and anti-manganese SOD (SOD2) antibodies were from Upstate Biotechnology. Blots were stripped and rehybridized with beta -actin. Blots were quantitated by densitometry and normalized using the beta -actin signal to correct for differences in loading. For immunoblotting studies, experiments were repeated at least three times. Because the time at which cultured cells reentered the cell cycle varied between experiments, the results from different experiments were not pooled, and instead results from representative experiments are shown.

Early response kinase assay. Quiescent SMC were treated with alpha -thrombin for 10 min, and total cell lysates were prepared. Equal amounts of proteins were immunoprecipitated with an anti-early response kinase 2 antibody (Santa Cruz Biotechnology; Santa Cruz, CA). Immunoprecipitates were incubated with kinase buffer (150 µg/ml myelin basic protein, 20 µM ATP, and 30 µCi/ml [gamma -32P]ATP) for 20 min at 37°C. The samples were resolved on a polyacrylamide gel and analyzed by autoradiography.

SOD activity. Total and SOD2 activity were determined by inhibition of xanthine/xanthine oxidase-induced cytochrome c reduction. Appropriately treated cells were harvested by scraping in 10 volumes of phosphate-buffered saline and then homogenized. After centrifugation, protein concentration of the supernatant was determined. Supernatant (16.7 µl) was added to 967 µl of solution A (50 µM xanthine, 20 µM cytochrome c, 25 µM KH2PO4, 25 µM Na2HPO4, and 0.1 mM EDTA) in a cuvette. To this solution, 16.7 µl of solution B (0.2 U/ml xanthine oxidase in 0.1 mM EDTA) were added, and absorbance was read at 550 nm at 1-min intervals for 10 min. Results were compared with a standard curve, and SOD activity was expressed as units per microgram. To determine SOD2 activity, lysates were treated with 5 mM KCN to inactivate SOD1 before performing assays.

Reduced and oxidized glutathione concentrations. Glutathione levels were measured according to manufacturer's instructions (Calbiochem, San Diego, CA). GSH concentrations were determined from appropriately treated cell lysates based on their absorbance at 400 nm, in comparison with a GSH standard curve. For analysis of GSSG concentration, the supernatant was derivatized with 2-vinylpyridine before analysis.

Lipid peroxidation assay. Malondialdehyde (MDA) is an end product derived from peroxidation of polyunsaturated fatty acids and related esters, which is changed colorimetrically by N-methyl-2-phenylindole. This reaction generates a compound with increased absorbance at 586 nm. The experiments are performed using a lipid peroxidation assay kit (Calbiochem). Subconfluent, exponentially growing SMC in 150-mm plates were rinsed and lysed by repetitive freeze/thawing. N-methyl-2-phenylindole was added to the samples and incubated at 45°C for 60 min. The specific absorbance at 586 nm was measured in a spectrophotometer.

Quantitative PCR assay for mitochondrial DNA damage. Detection of DNA damage by quantitative PCR relies on the premise that any DNA template containing an oxidative lesion (such as strand breaks, base modifications, and/or apurinic sites) will arrest a thermostable polymerase (46). Therefore, only those templates that do not contain lesions will be amplified. DNA extraction and PCR conditions were performed as previously described (2) by using primers designed to amplify the entire mouse mitochondrial genome. Amplifications were corrected for mitochondrial copy number by simultaneously amplifying an 80-bp mitochondrial fragment, as well as by DNA slot-blot analysis. The reagent conditions for the mouse QPCR for the 16,059-bp mitochondrial DNA product (primers used: sense 5'-CCCAGCTACTACCATCATTCAAGTAG-3'; antisense 5'-GAGAGATTTTATGGGTGTAATGCGGTG-3') and the 80-bp fragment (primers used: sense 5'-GCAAATCCATATTCATCCTTCTCAAC-3', antisense 5'-GAGAGATTTTATGGGTGTAATGCGGTG-3') were 1× XL buffer II (Perkin-Elmer-Cetus), 1.1 mM Mg(OAc)2, 0.1 mg/ml BSA, 0.6 µM primers, and 2 µCi [32P]dATP. Each QPCR was initiated with a 75°C hot start addition of 1 unit of rTth polymerase (Perkin-Elmer-Cetus). The PCR consisted of 24 cycles of 94° for 15 s and 67° for 12 min for the large fragment and 18 cycles of 94° for 15 s and 65° for 1 min for the short fragment.

Statistical analysis. When appropriate, data were expressed as means ± SE. For multiple treatment groups, a factorial ANOVA followed by Fisher's least significant difference test was applied. Statistical significance was accepted at P < 0.05.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Impaired mitogenesis in SMC isolated from aged mice. We isolated SMC from the thoracic aortas of male C57BL/6 mice at ages 4 ("young") and 16 ("aged") mo by the collagenase method (16). Mice at 4 mo were chosen to represent mice that are in the early stages of the life cycle, yet are mature and fertile, whereas mice at 16 mo represent animals that are aged yet still within the median life span for this species. Isolated cells were rigorously characterized by morphological criteria, expression of smooth muscle alpha -actin, and absence of von Willebrand factor staining. Cells were used within five passages of primary culture in most experiments to prevent clonal selection of rapidly growing subsets of the proliferating cell population. Of note, these cells have been passaged up to 20 times without evidence of senescence or obvious changes in phenotype. Isolated SMC were obtained from young and aged aortas with equivalent efficiencies.

As a first step in characterizing these SMC, we measured their proliferative capacity at passage 2. Cells were trypsinized, plated at equivalent densities, and allowed to attach overnight. Cell counts were performed the following morning (day 0) to ensure that plating efficiencies for both SMC populations were equivalent. Cultures were maintained in complete SMC medium containing 10% fetal calf serum. By day 2, cell counts were significantly greater in SMC derived from young compared with aged mice (Fig. 1A). Over 6 days, SMC from young mice grew at a rate 2.8 times that of SMC from aged mice (P < 0.05).


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  		Fig. 1.   Impaired proliferative capacity in aged smooth muscle cells (SMC). A: growth curve analysis of cultured mouse aortic SMC. SMC from young (4 mo) and aged (16 mo) mice were plated at equal density overnight and cultured in standard growth medium for 6 days. Counts were performed in triplicate on alternating days. Results are presented as means ± SE for three independent experiments. *P < 0.05 compared with aged SMC. B: measurement of DNA replication by [3H]thymidine uptake as a marker for proliferation. Young and aged SMC were made quiescent for 3 days and then stimulated with alpha -thrombin (1 U/ml) or vehicle for 24 h, with or without diphenyliodonium (DPI) pretreatment (10 µM). Results are presented as means ± SE from three triplicate experiments. *P < 0.05 compared with SMC from young mice; **P < 0.05 compared with no thrombin treatment. C: detection of apoptosis in SMC treated with thrombin. Results are presented as means ± SE from three triplicate experiments. No significant differences were detected between young and aged SMC.

To characterize further the proliferative potential of these SMC populations, we measured the response of quiescent cultures to alpha -thrombin stimulation, by using [3H]thymidine uptake as a marker for DNA synthesis. We have previously shown that alpha -thrombin elicits mitogenesis of SMC through the protease-activated receptor-1 and that this effect requires generation of ROS via a DPI-inhibitable oxidase (9, 31); alpha -thrombin stimulation is therefore an attractive model for understanding the events that underlie SMC proliferation. We found that both basal and alpha -thrombin-stimulated thymidine uptake was greater in SMC from young compared with aged mice (2.3-fold and 3.2-fold increased, respectively, P < 0.05, Fig. 1B). Interestingly, alpha -thrombin-induced mitogenesis was inhibited by DPI to an equal degree in SMC from young and aged mice, implicating ROS generation in mitogenesis in both cell populations. Using a quantitative assay, no differences were found in rates of apoptosis in SMC from young and aged mice (Fig. 1C), indicating that cell counts and thymidine uptake represent differences in proliferation rather than in cell death. The differences in proliferative potential were not limited to responses to alpha -thrombin, because similar results were observed with the lipid peroxidation product 4-hydroxynonenal (Fig. 2), a known SMC mitogen (36).


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  		Fig. 2.   Decreased responsiveness to 4-hydroxynonenal (HNE) in aged SMC. Measurement of DNA replication was determined by [3H]thymidine uptake. Young and aged SMC were made quiescent for 3 days and then stimulated with HNE (0.5 µM) or vehicle for 24 h with or without DPI pretreatment (10 µM). Results are presented as means ± SE from three triplicate experiments. *P < 0.001 compared with SMC from aged mice; **P < 0.01 compared with no HNE treatment.

Increased ROS generation in SMC from aged mice. To explore ROS generation in these cells in more detail, we measured H2O2 production in unsynchronized SMC after the addition of fresh medium. H2O2 production was measured, because we have previously shown that it is a required intermediary in alpha -thrombin-mediated SMC proliferation (31). Extracellular H2O2 concentrations in SMC from young mice were 0.2-0.3 µM/105 cells, whereas SMC from aged mice secreted H2O2 at levels 10-fold higher (Fig. 3A). H2O2 release was characterized further in cells made quiescent by serum deprivation with or without alpha -thrombin stimulation. H2O2 concentrations in conditioned medium from quiescent cells were similar for SMC from young and aged mice (Fig. 3B). After alpha -thrombin treatment, secreted H2O2 levels were increased in both groups and were significantly higher in SMC from aged mice (P < 0.05). Similarly, we used DCFDA fluorescence to measure intracellular (and hence biologically available) H2O2 and found that alpha -thrombin-induced H2O2 generation was increased 3.1-fold in SMC from older animals (Fig. 3C).


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  		Fig. 3.   Increased H2O2 production in aged SMC. A: extracellular H2O2 production was measured in unsynchronized SMC in culture by measuring the oxidation of o-dianisidine dihydrochloride (o-DD). Results are expressed as means ± SE. H2O2 production was significantly greater (P < 0.05) in SMC from aged than those from young mice at all times points. *P < 0.05 compared with H2O2 production at 1 h. B: extracellular H2O2 production was measured in quiescent young and aged SMC after stimulation with alpha -thrombin (1 U/ml) or vehicle for 24 h. *P < 0.05 compared with young SMC. C: quantitative 2',7'-dichlorofluorescein (DCF) fluorescence was used as a measure of intracellular H2O2 production in SMC treated with alpha -thrombin or vehicle. Results are from three experiments in triplicate and are expressed as means ± SE. *P < 0.05, compared with SMC from young mice.

Increased MAP kinase activity in SMC from aged mice. These data argue that proliferative SMC in older animals exist at higher ambient ROS concentrations, an apparent paradox given the demonstrated role of ROS in SMC proliferation (32, 41) and the decreased proliferative capacity of SMC from aged mice. To determine how ROS generation and mitogenesis are dissociated in SMC from older mice, we examined cellular events downstream of ROS generation that are necessary for mitogenesis. As a first step, we measured activity of early response kinase 2, because this kinase is activated in SMC in response to ROS stimulation and is immediately upstream of transcriptional events occurring in response to growth stimuli such as alpha -thrombin and platelet-derived growth factor (14, 20, 39). Constitutive and thrombin-inducible MAP kinase activities were increased by 110% and 265%, respectively, in SMC from older mice compared with cells from young mice (Fig. 4). Therefore, despite their decreased proliferative capacity, ROS-inducible mitogenic pathways are intact in SMC and are in fact upregulated.


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  		Fig. 4.   Effects of aging on mitogen-activated protein (MAP) kinase activity in SMC. SMC from young and aged mice were treated in with alpha -thrombin (1 U/ml) or vehicle for 10 min. MAP kinase activity was measured by phosphorylation of myelin basic protein. The results are representative of three independent experiments. *P < 0.05 compared with no thrombin treatment; **P < 0.05 compared with young SMC. ERK, extracellular signal-regulated kinase.

Cyclin D1 and p27Kip1 are dysregulated in SMC from aged mice. The surprising dissociation between mitogenic signaling pathways and proliferative responses in SMC from aged mice led us to examine the activation of the cell cycle machinery in response to mitogenic stimulation. alpha -Thrombin-stimulated expression of the cyclin-dependent kinase inhibitor p21 was unchanged in SMC from aged mice compared with cells from young mice (not shown). However, expression of p27Kip1 and cyclin D1, two G1-associated factors, was dysregulated in SMC from older mice. In SMC from young mice, p27Kip1 protein, which negatively regulates proliferation, was downregulated by 48% within 6 h of alpha -thrombin treatment (Fig. 5A). In contrast, p27Kip1 downregulation was delayed in SMC from older mice after alpha -thrombin treatment. Similarly, cyclin D1 protein was upregulated (0.6- and 2.1-fold) at 12 and 24 h after alpha -thrombin treatment in SMC from young mice but was not induced in SMC from aged mice (Fig. 5B). These specific changes in the G1 cell cycle machinery in SMC from aged mice are consistent with, and likely to account for, their impaired proliferative capacity, and cannot be overcome by the persistently elevated levels of ROS observed in SMC from aged mice.


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  		Fig. 5.   Regulation of cell cycle-associated proteins in SMC from young and aged mice. SMC were treated with alpha -thrombin (1 U/ml) for the indicated times, and Western blot analysis was performed with antibodies specific for p27Kip1 (A) and cyclin D1 (B). Results from representative experiments were normalized to beta -actin expression by densitometry.

Impaired antioxidant defenses in SMC from aged mice. The age-associated differences in ROS generation in mouse SMC might result in the preferential accumulation of oxidative damage in cells from old mice, an effect that would be particularly amplified if antioxidant defenses were unable to compensate. We therefore measured the activity of ROS-scavenging systems in SMC. SOD2 protein levels were decreased approximately fivefold in comparison with SMC from younger mice, whereas expression of SOD1, the cytoplasmic and nuclear isoform of SOD, was not changed (Fig. 6A). Total SOD activity, as measured by inhibition of xanthine oxidase-induced ROS generation, was decreased by 25% in SMC from aged mice (Fig. 6B). The reduction in activity of SOD2 (manganese SOD), the mitochondrial isoform of SOD, was also decreased by 63% in these cells (Fig. 6C). In no case was antioxidant activity augmented by alpha -thrombin treatment, demonstrating that this system does not compensate in response to acute elevations in ROS generation.


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  		Fig. 6.   Superoxide dismutase (SOD) activity in SMC from young and aged mice. A: SMC were treated with alpha -thrombin (1 U/ml) for the indicated times, and Western blot analysis was performed with antibodies specific for coppper/zinc-dependent SOD (SOD1; top) and manganese SOD (SOD2; bottom). Results from a representative experiment examining SOD2 protein were normalized to beta -actin expression by densitometry and expressed in graph format. B: total SOD activity was measured by inhibition of xanthine/xanthine oxidase-induced cytochrome c reduction in SMC from young and old mice with or without alpha -thrombin treatment, as indicated. *P < 0.05 compared with young SMC. C: as in B except that MnSOD (i.e., SOD2) activity was measured by treating lysates with KCN to inactivate Cu/Zn SOD (SOD1). *P < 0.01 compared with SMC from young mice.

Glutathione serves as an additional means to buffer ROS that are generated intracellularly (45). GSH can accept electrons from peroxides and hydroxyl radicals derived from superoxide and in the process is converted to its oxidized disulfide form GSSG. We measured the concentration of GSH and GSSG as indexes of the activity of this protective pathway. Both GSH and GSSG concentrations were markedly decreased in SMC from aged mice (Fig. 7, A and B), indicating a global reduction in glutathione levels associated with aging. This reduction results in a 69% decrease in the concentration of reduced GSH that is available to quench ROS generated in cells from aged mice (Fig. 7A).


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  		Fig. 7.   Antioxidant pathways in SMC from young and aged mice. The concentrations of reduced glutathione (GSH, A) and also its oxidized form (GSSG, B) were measured in SMC from young and old mice with or without alpha -thrombin treatment, as indicated. *P < 0.05 compared with SMC from young mice.

Increased indexes of oxidative damage in SMC from aged mice. Our observation of age-associated changes in ROS production and antioxidant defenses in mouse SMC led us to consider whether SMC from aged mice might accumulate oxidative damage at a greater rate than SMC from young mice, which might in turn contribute to the atherogenic propensities of older blood vessels. To test this hypothesis, we measured different cellular indexes of oxidative damage. Intracellular MDA was measured as a marker of peroxidative damage to membrane lipids, based on the putative role for lipid peroxidation products in atherogenesis (17). MDA levels were increased 1.8-fold in SMC derived from aged mice compared with those from young mice (Fig. 8A), indicating that SMC from aged mice suffer increased peroxidative damage to membrane lipids.


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  		Fig. 8.   Oxidative injury in SMC from young and aged mice. A: intracellular malondialdehyde concentrations were measured in unsynchronized SMC from young and aged mice by N-methyl-2-phenylindole modification. Results are expressed as means ± SE of three experiments done in triplicate. *P < 0.05 compared with SMC from young mice. B: age-associated mitochondrial DNA damage in unsynchronized SMC of young and aged mice was measured by a quantitative PCR method. Results are expressed as means ± SEM of three experiments done in triplicate. *P < 0.05 compared with SMC from young mice.

Using a sensitive PCR-based assay, we previously demonstrated that ROS-induced mitochondrial DNA damage correlates with cellular dysfunction in SMC (2) and have shown that mitochondrial DNA damage precedes and probably accelerates atherogenesis in a mouse model of atherosclerosis (SW Ballinger, CA Knight, DL Burow, CA Conklin, Z Hu, J Reuf, C Horaist, R Lebovitz, C Patterson, and MS Runge, unpublished observations). We used this PCR-based assay, which relies on the inhibition of polymerase activity by oxidatively modified DNA, to measure the amount of mitochondrial DNA damage in SMC from aged and young mice. Mitochondrial DNA amplification was decreased by 36% in SMC from aged mice (Fig. 8B), demonstrating increased mitochondrial DNA damage incurred by SMC from aged mice. It is interesting to note that neither MDA generation nor mitochondrial DNA damage was significantly increased by alpha -thrombin treatment. This can be taken as an indication that intrinsic, long-term changes in ROS homeostasis, such as those observed with aging in SMC, are the major determinant of oxidative damage in this system.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Epidemiological data indicate that advanced age increases the risk of coronary artery disease independently of other well-established risk factors, such as diabetes, hypertension, and hypercholesterolemia (19). We investigated whether isolation of primary culture mouse aortic SMC, although a technically demanding task, may serve as a useful way to understand the mechanisms by which aging contributes to atherogenic risk. The phenotypic differences we observed in aged SMC indicate that this model may be particularly useful to define the genetic and molecular determinants that underlie the effects of aging on atherogenesis.

Although our observations in this mouse model differ from previous reports of SMC obtained from rats (5, 24, 25), the phenotype of SMC from aged mice bears a strong resemblance to the behavior of SMC from aged humans (37) and of intimal SMC isolated from advanced human atherosclerotic lesions, for which the proliferative capacity is clearly impaired (3, 34). There are several potential explanations for the differences between aged rat and mouse SMC as reported here. Adult rat arteries contain a population of SMC with an epithelioid morphology that retains expression of neonatal phenotypic markers (10, 38). This population may serve as the source for the rapidly proliferating cells derived from rats. However, this cellular phenotype has not been observed in SMC from other species, including mice and humans (27), and their presence or absence may explain the species-dependent differences in SMC phenotypes in older animals.

Our studies have been performed in SMC derived from mice in the same C57BL/6 background. Although this background is commonly used in studies of vascular function, it should be acknowledged that a body of evidence points to strain-related differences in end points of vascular function in mice (21). Further studies will be necessary to determine whether the cellular phenotypes observed in this report are altered when cells are derived from mice in different genetic backgrounds. Such studies may provide a means to identify genetic modifiers of the vascular aging process.

Our demonstration that both intracellular and extracellular H2O2 production are increased in SMC from older animals is not altogether surprising, given the previous association between ROS and aging in other circumstances (4, 18). Increased ROS production may be caused by impaired antioxidant defenses, by upregulation of specific cellular oxidases, or by impaired mitochondrial function (for example, by damage to the mitochondrial genome). We and others (15, 31) have previously shown that vascular SMC contain a DPI-inhibitable, NADH-consuming oxidase that generates H2O2 in response to stimuli such as alpha -thrombin and angiotensin II. Preliminary experiments from our laboratory indicate that SMC from mice deficient in the p47phox component of this oxidase have impaired oxidant-generating capacity, and the mice themselves in the ApoE (-/-) background are less prone to atherosclerotic lesion formation (P Barry-Lane, C Patterson, and MS Runge, unpublished observations), demonstrating the importance of this oxidase in SMC biology. Sustained increases in ROS have cellular effects that are likely to be important in the creation and perpetuation of the aged phenotype in SMC, including impaired gene regulation and oxidative damage to cellular compartments. In particular, damage to the lipid-rich cell membrane and the relatively unprotected mitochondrial genome may perpetuate cellular dysfunction and oxidant production in SMC (2, 36).

Although increased ROS production in aged SMC is not unexpected, the association between increased ambient ROS production and decreased proliferative potential in these cells is paradoxical. Previous studies have demonstrated that ROS themselves are mitogenic for SMC (32) and are necessary intermediates for SMC proliferation in response to potent mitogens such as platelet-derived growth factor and alpha -thrombin (31, 41). Why ROS and proliferation become uncoupled in SMC from aged mice is unclear; however, the regulation of cell cycle-related proteins in these cells provides some important clues. We did not see changes in regulation of the S phase-associated cyclin A or the cyclin-dependent kinase inhibitor p21 in aged SMC, but upregulation of the G1 phase-associated cyclin D1 was attenuated in these cells. This effect may be secondary to activation of upstream inhibitory pathways, to which cyclin D1 may be particularly responsive (23). We also observed that levels of the cyclin-dependent kinase inhibitor p27Kip1 failed to decrease in response to mitogenic stimulation in aged SMC. p27Kip1 is a critical check point protein that modulates progression of SMC through the cell cycle (22). Downregulation of cyclin-dependent kinase inhibitors such as p27Kip1 occurs through targeted degradation via the ubiquitin-proteasome pathway (42); therefore, it is attractive to speculate that aspects of this pathway may be impaired in aged SMC because they are in other tissues such as the aged liver (13).

Notably, although both aging and mitogenic treatment are independently associated with ROS generation in our studies (Fig. 3), the age of mice at the time of cell culture is the major variable that determines the accumulation of oxidative damage, as measured by mitochondrial DNA damage and MDA accumulation (Fig. 8). It is unclear why additional oxidative damage does not accrue in response to short-term ROS generation induced by mitogens such as alpha -thrombin; accumulation of oxidative damage may require sustained ROS generation or ROS generated in a particular cellular compartment. With regard to the latter possibility, it is interesting to note that mitochondrial DNA damage and impaired mitochondrial SOD expression and activity occur preferentially in aged SMC. In contrast, the oxidase that is activated by alpha -thrombin is cytosolic (31). We speculate that ROS generation within the mitochondria may be particularly detrimental to SMC function, and studies are underway in our laboratory to characterize SMC lacking either SOD1 or SOD2 to test this hypothesis.

We focused on the decreased proliferative potential of aged SMC as a potential contributing factor in atherosclerotic plaque instability, although this might not be the only aspect of the aged SMC phenotype that leads to disease progression. Aged SMC also have increased levels of the lipid peroxidation product MDA and increased amounts of mitochondrial DNA damage, both presumably secondary to chronically elevated ROS levels and/or impaired antioxidant defense pathways. We have shown previously in SMC that mitochondrial DNA damage, in particular, leads to further ROS generation and impaired cellular ATP production (2). Therefore, we postulate that this chronic cellular damage and dysfunction may also contribute to the proatherogenic phenotype of aged SMC, in synergy with their decreased proliferative capacity. Because they behave similarly to isolated human SMC with respect to aging and can be manipulated genetically, we believe that isolated aortic SMC from mice may be an ideal model for unraveling the molecular and genetic events that lead to age-associated increases in atherosclerotic plaque instability and cardiovascular morbidity and mortality.


    ACKNOWLEDGEMENTS

This work was supported by National Institutes of Health Grants AG-10514, HL-59652, and HL-57352 (to M. S. Runge) and HL-03658 and AG-10514 (to C. Patterson).


    FOOTNOTES

Address for reprint requests and other correspondence: C. Patterson, Univ. of North Carolina, Div. of Cardiology, 324 Burnett-Womack Bldg., Chapel Hill, NC 27599-7075

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 13 June 2000; accepted in final form 24 January 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Am J Physiol Heart Circ Physiol 280(6):H2779-H2788
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