(Hypertension. 1997;30:817-824.)
© 1997 American Heart Association, Inc.
Articles |
Anatomic Heterogeneity of Vascular Aging
Role of Nitric Oxide and Endothelin
From Cardiology and the Division of Hypertension (S.S.), University Hospital, Bern; Cardiology and Cardiovascular Research, Institute of Physiology (M.B., F.C., P.M., T.F.L.), University Zürich, Switzerland; and Division of Cardiology (R.P.B.), Hannover Medical School, Germany.
Correspondence to Thomas F. Lüscher, MD, FACC, FESC, Professor and Head of Cardiology, University Hospital, CH-8091 Zürich, Switzerland. E-mail 100771.1237{at}compuserve.com
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Abstract We investigated the effects of aging, a cardiovascular risk factor, on vascular function with regard to endothelial nitric oxide synthase (eNOS), superoxide dismutase (SOD), and endothelin (ET-1) in aorta and femoral artery of the rat. Concentration-response curves to acetylcholine, calcium ionophore A23187, norepinephrine, ET-1, big endothelin, sodium nitroprusside, and exogenous SOD were obtained. Expression of eNOS mRNA was analyzed by reverse-transcription polymerase chain reaction, SOD activity was assessed using a chemiluminescence-based cytochrome c assay, and ET-1 plasma concentrations were measured by radioimmunoassay. In aorta of old rats, relaxations to acetylcholine and calcium-ionophore A23187, basal NO release, and expression of eNOS mRNA in aortic endothelial cells were reduced (P<.05). In femoral arteries, relaxations to acetylcholine were preserved, whereas basal release of NO was attenuated (P<.05). Aging selectively increased contractions to norepinephrine and functional endothelin converting enzyme activity and attenuated contractions to ET-1 in aortas but not femoral arteries. Vascular SOD activity was higher in the femoral artery (P<.05) and unaffected by aging. Plasma ET-1 levels increased and plasma SOD activity decreased with age (P<.05). Aging was associated with an anatomic heterogeneity of endothelial dysfunction, functional endothelin converting enzyme activity, and vascular SOD activity. Vascular function was impaired in the aorta but not the femoral artery, which may be related to lower eNOS mRNA expression and SOD activity. These data suggest differential regulation of the vascular aging process that may contribute to the anatomic heterogeneity of atherosclerosis.
Key Words: aging • endothelium • endothelins • nitric oxide • superoxide dismutase
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Introduction |
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The endothelium serves as an important regulator of vascular tone by releasing several endothelium-derived relaxing factors.1 Cardiovascular risk factors such as hypercholesterolemia,2 hypertension,3 and diabetes4 impair endothelium-dependent relaxation as does aging, another risk factor for coronary artery disease.5 Endothelial dysfunction develops long before structural or atherosclerotic vascular changes occur. Although atherogenesis exhibits a marked heterogeneity—some arteries such as the aorta or the coronary circulation, which are particularly prone to atherosclerosis, whereas other vascular beds appear to be protected6 —it is still unknown whether vascular dysfunction observed with aging is also associated with anatomic heterogeneity.
Furthermore, the nature of age-related endothelial dysfunction is unclear and may involve mechanisms such as (1) increased breakdown of NO due to an augmented production of superoxide anions,7 (2) age-related alterations of antioxidant defense systems8 and/or increased oxidative injury,9 10 (3) decreased availability of L-arginine,11 or (4) changes in activity or expression of eNOS. Alterations in vascular smooth muscle responsiveness to catecholamines,12 13 ET,14 or endothelium-derived contracting factors15 have been reported. Finally, pulse pressure shows regional differences in human subjects16 and may locally affect vascular function with age.
We hypothesized that a regional heterogeneity of age-related changes in vascular function exists. Hence, we investigated the effects of aging on blood pressure, vascular function, and basal and stimulated NO release in aortas and femoral arteries and analyzed expression of eNOS mRNA in aortic endothelial cells. Furthermore, SOD activity in arterial tissues and plasma and plasma levels of ET-1 were determined.
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Methods |
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Animals and Blood Samples
Young and old Ro-Ro Wistar rats (female, 5 to 6 and 32 to 33 months of age, Biological Research Laboratories, Ltd, Füllinsdorf, Switzerland) were anesthetized with thiopental (50 mg/kg IP). A polyethylene catheter was inserted into the left femoral artery and connected to a pressure transducer (Letica PRI 256/2, Letica S/A Instruments, Hospitalet) for determination of blood pressure and heart rate. A recording of 15 minutes was used for calculation of mean and pulse pressures. Arterial blood samples for measurement of ET-1 levels and SOD activity were drawn. Rats were killed and arteries removed. All procedures and experimental protocols were performed in accordance with the local authorities for animal research (Kommission für Tierversuche des Kantons Bern, Switzerland) and the American Heart Association guidelines on research animal use.
Arterial Preparations
Thoracic aorta and the right common femoral artery were removed
and placed in cold Krebs-Ringer bicarbonate solution (in
mmol/L: NaCl 118.6, KCl 4.7, CaCl2 2.5,
KH2PO4 1.2, MgSO4 1.2,
NaHCO3 25.1, edetate calcium disodium 0.026, and glucose
11.1). Arteries were rinsed with a cannula to remove residual blood,
dissected in cold Krebs solution under a microscope (Wild-Heerbrugg),
cleaned from perivascular tissue, and cut into rings (aorta, 4 to
5 mm; femoral artery, 2 to 3 mm in length).
Organ Chamber Setup
Rings were suspended in organ chambers containing 25 mL of
Krebs-bicarbonate solution (37°C, pH 7.4, 95% O2 and 5%
CO2). To avoid endothelial damage of the
femoral artery, special tungsten stirrups (diameter, 100 µm)
were used for suspension to force transducers (model UTC 2, Gould
Statham). Arteries were allowed to equilibrate for 1 hour. Resting
tension was gradually increased, and rings were repeatedly exposed to
100 mmol/L KCl until the optimal tension for generating
force during isometric contraction was reached and were then
equilibrated for 30 minutes. Vessels were left at resting tension
throughout the study.
Experimental Protocols
Aortic and femoral artery rings were randomly assigned to
different protocols and exposed to cumulative concentrations of big ET
(10-7 to 10-11
mol/L), ET-1 (10-7 to
10-11 mol/L), or
norepinephrine (10-10 to
1x10-5 mol/L). Other rings were
preconstricted with norepinephrine (2 to
5x10-7 mol/L), and after a stable
plateau was reached (approximately 70% of KCl 100
mmol/L), relaxations to acetylcholine
(10-5 to 10-10
mol/L), calcium ionophore A23187 (10-10
to 3x10-6 mol/L), sodium nitroprusside
(10-5 to 10-10
mol/L), or SOD (0.075 to 75 U/mL) were obtained. In some
preparations, the endothelium was removed and its
absence verified by lack of response to acetylcholine
(3x10-6 mol/L). When indicated,
experiments were performed in the presence of L-NAME (0.1
mmol/L). All concentrations represent final
concentrations of drugs in the organ chamber. After the experiments,
surface areas of rings from aorta (n=44 from 7 animals in each group)
and femoral artery (n=34 from 7 animals in each group) were measured
planimetrically using a microscope containing a calibrated
eyepiece17 for calculation of index (surface
area/contraction).
RT-PCR
Expression of eNOS mRNA in aortic endothelial
cells was studied using a semiquantitative RT-PCR assay. Aortas
isolated from young and old (n=4 each) rats were cleaned of surrounding
connective tissue in cold Krebs-Ringer bicarbonate solution (pH 7.4),
they were opened longitudinally, and endothelial cells
were scraped off gently. Endothelial cells from two
rats of the same group were pooled. Total RNA was isolated by TRIzol
reagent. Concentrations of RNA were determined by measuring absorbance
at 260 nm (A260). The A260/A280
ratio of the samples ranged from 1.7 to 2.0. RNA (0.5 mg) was reverse
transcribed in 50 mL reaction mixture containing 200 U reverse
transcriptase (Superscript). To study eNOS expression, PCR was
performed using first-strand cDNA of young or old rat aortic
endothelial cells as a template. The sequence of PCR
primers for eNOS was kindly provided by Dr Thomas Michel (Brigham
and Women's Hospital, Boston). The sense primer was
5'-GGG-CCA-GGG-TGA-TGA-GCT-CTG-3', and the antisense primer was
5'-CCC-TCC-TGG-CTT-CCA-GTG-TCC-3'. For amplification of GAPDH the
primers used were 5'-CAG-GAA-TTC-GGT-GAA-GGT-CGG-AGT-CAA-CGG-3' and
5'-AGT-GGA-TCC-GGT-CAT-GAG-TCC-TTC-CAC-GAT-3'. The PCR primers were
purchased from MWG-Biotech. Each 50-mL reaction mixture contained 2 mL
of cDNA, 1 mL of each primer (20 mmol/L), 0.5 mL of Taq DNA
polymerase (5 U/mL), 5 mL of 10x buffer provided with Taq DNA
polymerase, and optimal concentrations of MgCl2. Samples
were placed onto a Biometra TRIO-Thermoblock and heated (94°C, 5
minutes) followed by 35 and 30 temperature cycles (exponential phase of
amplification for eNOS and GAPDH, respectively). Each cycle consisted
of three periods: denaturation for 30 seconds at 94°C, annealing for
30 seconds at 60°C for eNOS and 62°C for GAPDH, and extension for
90 seconds at 72°C. The PCR products were separated by 1%
agarose gel electrophoresis, visualized, and photographed using a
Visionary gel documentation system (Fotodyne, Bio Cell Consulting
Research). A comparison of ratios of densitometric measurements
(Macintosh II, NIH Image 1.4 software, public domain) of PCR
products for eNOS and GAPDH from endothelial cells
of young and old rats was made.
Measurement of Plasma ET-1 Levels
Blood samples were immediately transferred into a tube
containing EDTA and centrifuged at 4°C for 10 minutes at
5000g. Plasma was separated from blood cells at 4°C and
kept at -80°C until assayed. The investigator performing the
radioimmunoassay (S.S.) was unaware of the group to which plasma
samples belonged. Extraction of ET-1 from plasma was performed by
absorption on 500-mg SepPak Vac C18 cartridges
(Millipore).18 Columns were preactivated by
successive washes with 5 mL of 86% ethanol in 4% acetic acid, 5 mL of
methanol, 5 mL of sterile distilled water, and 5 mL of 4% acetic acid.
A 2 mL plasma sample acidified with 6 mL of 4% acetic acid was then
applied on the column at a flow rate of approximately 3 mL/min. The
columns were then washed with 18 mL of sterile distilled water and 18
mL of 24% ethanol in 4% acetic acid before ET-1 was eluted with 86%
ethanol in 4% acetic acid. The eluate was dried under nitrogen at
37°C and redissolved in 230 µL of assay buffer composed of 0.1%
phosphate buffer (pH 7.4), 0.05 mol/L NaCl, 0.1% Triton-X100,
0.02% sodium azide, and 0.1% bovine serum albumin. Synthetic
human/porcine ET-1 (Sigma), a rabbit antibody against synthetic ET-1
(Peninsula Laboratories), and 125I–ET-1 (Amersham) were
used. The antibody has 100% cross-reactivity with ET-1, 7% with ET-2
and ET-3, 17% with big ET, and no cross-reactivity with other
peptides. The anti-ET antibody was reconstituted according to the
manufacturer's instructions and then further diluted 1:3.5 with assay
buffer before adding 100 µL to the standards or reconstituted plasma
samples (100 µL) analyzed in duplicate. After 24 hours of
incubation, 100 µL of 125I–ET-1 (12 000 counts per
million [cpm]/tube) was added and incubation allowed to continue for
an additional 24 hours. Separation of bound and free antigen was
performed with a second antibody method, and pellets were counted by a
gamma counter (Canberra Packard). Using these procedures, the
sensitivity of the assay was increased compared with those previously
reported. Recovery averaged 78±4% (n=8). The effective range of the
standard curve was between 0.16 and 40 pg of ET-1 per tube, with a
lower limit of detection of 0.16 pg/tube and an IC50
value of 1.5 pg/tube. Intra- and interassay coefficients of
variation averaged 8.6% and 13.6%, respectively (n=10).
Measurement of SOD Activity
One milliliter of plasma was frozen at -80°C, and specimens
of aorta and femoral artery with endothelium from both
animal groups were frozen in liquid nitrogen and stored at -80°C
until assayed. On the day of the assay, artery specimens were minced
using an ultra-thurax and homogenated with a motor-driven
Teflon potter in 800 µL of 100 mmol/L Tris/HCl plus
1 mmol/L EDTA-buffer (pH 9.0). Samples were
centrifuged twice at 16 000g to remove insoluble
parts. The supernatant was stored on ice. SOD activity was determined
by the inhibition of O2- production
generated by xanthine–xanthine oxidase reaction.19
O2- production was determined using a
modified lucigenin-enhanced chemiluminescence
method.20 The reaction buffer contained 100
mmol/L Tris/HCL plus 1 mmol/L EDTA adjusted to pH
9.00 by HCl, xanthine (33 fmol/L), xanthine oxidase (0.334 U/L),
and lucigenin (250 fmol/L). These concentrations were chosen to
obtain a stable O2- production for at
least 5 minutes. By addition of defined amounts of exogenous SOD, a
standard curve was obtained that served as internal calibration
standard. After equilibration of the chemiluminescence signal, aliquots
of the cell homogenates were added, and inhibition of
superoxide production was recorded. SOD activity of tissue
homogenates was expressed in relation to protein content of
the buffer solution measured by the method of Lowry et
al.21
Materials
Acetylcholine chloride, calcium ionophore A23187 (dissolved in
DMSO, final concentration in organ chamber <0.01%), big ET-38
(human), EDTA, indomethacin (dissolved in sodium
carbonate 5 mmol/L), L-arginine, L-NAME,
norepinephrine bitartrate salt, potassium chloride, sodium
nitroprusside dihydrate, Cu,Zn-SOD (from bovine erythrocytes, 4300
U/mg), xanthine, and xanthine oxidase were purchased from Sigma
Chemical Co. ET was from Novabiochem AG, lucigenin from
Boehringer Mannheim, pentobarbital from Abbott Laboratories,
and TRIzol Reagent and reverse transcriptase (Superscript) from GIBCO
(BRL, Life Technologies, Inc). Substances were prepared daily and
dissolved in distilled water before use.
Calculations and Statistical Analysis
Data are given as mean±SEM and represent unpaired data;
n refers to the number of animals used. Relaxations of aorta or femoral
artery were calculated as percent relaxation of precontraction to
norepinephrine. Concentrations of the agonists causing
half-maximal (EC50) and maximal responses were calculated
for contractions and relaxations using non–linear regression
analysis. The EC50 value was expressed as negative
logarithm (pD2 value). Functional ECE activity was
expressed as a ratio of contractions (big ET/ET-1) of vessels from the
same animal. Because ECE activity cannot be solely assessed by
contractions to big ET, this ratio gives an approximation of the
functional enzyme activity by dividing the response to the substrate by
that of the enzyme product. Assuming that equimolar concentrations
of big ET are completely converted to ET-1, the ratio theoretically
should be 1. This ratio includes both conversion of big ET (enzyme
kinetics, enzyme activity) and receptor activation by ET-1. Differences
of pD2 values, maximal responses, and ratios were tested
for statistical significance. Data were analyzed using an
unpaired Student's t test when normally distributed or the
Wilcoxon test if not normally distributed. A value of
P<.05 was considered significant.
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Results |
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Body Weight, Heart Rate, Blood Pressure, and Pulse Pressure
Body weight was higher in old than in young rats (294±4 versus 222±5 g, respectively; P<.05). Heart rate (314±7 versus 302±11 bpm), mean blood pressure (85±5 versus 87±6 mm Hg), and systolic and diastolic blood pressures (data not shown) were not significantly different between both groups. However, pulse pressure increased with aging (25±1 versus 21±1 mm Hg, P<.05 versus young).
Endothelium-Dependent Relaxations
Acetylcholine and Calcium Ionophore A23187
Maximal responses and sensitivity (pD2 values) to
acetylcholine were markedly reduced with aging in the aorta (Fig 1
, left panel, n=6, P<.05)
but remained unaffected in the femoral artery (Fig 1, right panel;
Table 1, n=8, NS). In the aorta, aging
was also associated with a comparable reduction of relaxations to
calcium ionophore A23187 (Table 1; n=6, P<.05).
Precontraction to norepinephrine was not different between
arteries from old and young rats (data not shown).
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SOD
Endothelium-dependent relaxations to SOD were
inhibited by L-NAME (Fig 2, left panel,
n=6 to 9, P<.05) or denudation of the
endothelium (n=5 to 6, data not shown) to a similar
extent in vessels of young and old rats. Neither
indomethacin (10-5
mol/L) nor catalase (1200 U/mL), a hydrogen peroxide scavenger,
had any effect on these relaxations (data not shown). In old animals,
relaxations to SOD were markedly reduced in both aorta (Fig 2, left
panel, n=12, P<.05) and femoral artery (Fig 2, right panel,
n=12, P<.05). Threshold concentration of SOD was 0.15 U/mL.
In the femoral artery of young animals the presence of L-NAME not only
blocked relaxations to SOD but evoked
endothelium-dependent contractions after application of
SOD (Fig 2, right panel, n=5, P<.05). These contractions
were blunted in femoral arteries from old animals (Fig 2, right panel,
n=6, P<.05).
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) compared with young ones (
; P<.05). In
arteries from young rats (
) and old rats (
), relaxations
were blocked by the NOS inhibitor L-NAME
(10-4 mol/L). In femoral arteries,
contractions evoked by SOD after inhibition of NO synthesis were
blunted with aging (right). Data are mean±SEM.
Direct Relaxations to Sodium Nitroprusside
Aging had no effect on maximal responses (Table 1
) or
pD2 values of sodium nitroprusside (data not shown) in the
aorta or femoral artery. In both age groups,
endothelial denudation had no significant effect on
responses (data not shown).
Contractile Responses
Contractions to Potassium Chloride
With age, contractions to KCl (100 mmol/L) increased
in both the aorta and the femoral artery (P<.001, Table 2). Similarly, the size of the
arterial rings reflected by surface area increased with
aging in both the aorta (n=44 from 7 animals in each group,
P<.001) and the femoral artery (n=34 from 7 animals in each
group, P<.001). When contractions to KCl were corrected for
surface area (index SA/KCl), there was no difference between groups
(Table 2).
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Contractions to Norepinephrine
Aging differentially affected contractions to
norepinephrine. In the aorta, contractions to
norepinephrine were significantly increased at
concentrations from 10-8 to
3x10-7 mol/L as were pD2
values (Fig 3, left panel, n=7,
P<.05), although they remained unchanged in the femoral
artery (Fig 3, right panel, and Table 2; n=6, NS).
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Contractions to ETs
Maximal responses to ET-1, but not pD2 values,
were diminished in the aortas of old rats (Fig 4, left panel, n=10, P<.05).
However, neither maximal contractions nor pD2 values of
ET-1 were affected by aging in the femoral artery (Fig 4, right panel,
and Table 2; n=8 to 10, NS).
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Maximal contractions (10-7 mol/L)
induced by big ET were markedly higher in the femoral artery than in
the aorta in both young rats (87±14% versus 20±7%, n=6,
P=.002) and old rats (92±3% versus 41±11%, n=6,
P=.001). Contractions tended to increase with aging in both
the aorta and the femoral artery. This increase was significant only at
one concentration (3x10-8 mol/L) (Fig 5). However, functional ECE activity,
expressed as the ratio of contractions to big ET and ET-1
(10-7 mol/L), was increased in the
aorta but not in femoral arteries (Fig 6A, n=6, P<.05).
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Expression of eNOS in Aortic Endothelial Cells
Steady state levels of eNOS mRNA compared with steady state levels
of mRNA for GAPDH were determined by RT-PCR in aortic
endothelial cells. Densitometry measurements of PCR
products for eNOS and GAPDH from young and old rats showed a
reduction in expression of eNOS mRNA (0.15±0.01 versus 0.30±0.01
arbitrary densitometry units, n=4) in endothelial cells
from old animals.
Plasma and Vascular Tissue SOD Enzyme Activity
Plasma SOD activity decreased with age (0.10±0.01 versus
0.14±0.01 U/mg protein in young rats, n=6 to 7, P<.05).
Tissue SOD activity was higher in femoral arteries than in aortas
(about 1.5-fold; P<.05). Tissue SOD activity was similar in
aortas of old rats (6.6±0.3 U/mg protein, n=13) and young rats
(6.7±0.6 U/mg protein, n=10) and in femoral arteries (young versus
old: 10.15±0.8 versus 10.14±0.8 U/mg protein, n=7 to 13).
Plasma Levels of ET-1
Plasma levels of ET-1 increased with age, from 1.5±0.2
pg/mL in young rats to 2.1±0.2 pg/mL in old rats (Fig 6B, n=6 to 7, P<.05). Plasma ET-1 levels were correlated
with contractions to ET-1 (r=.72, P<.05, n=6)
and with functional ECE activity (r=.89, P<.05,
n=6) in aortas from young rats but not from old rats. However, an
inverse correlation between plasma SOD activity and plasma ET-1 levels
in young and old rats was found (n=6 to 7, r=-.502,
P<.05).
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Discussion |
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This study investigated functional consequences of vascular aging in aortas and femoral arteries from young and very old rats. Aging increased pulse pressure and selectively impaired vascular and endothelial function in the aorta. In this vessel, endothelium-dependent relaxations to acetylcholine and calcium ionophore A23187 were reduced with age as was the basal release of NO and expression of eNOS mRNA. Contractions to ET-1 were attenuated, whereas functional ECE activity and norepinephrine-induced contractions were enhanced. In plasma, aging increased ET-1 levels and reduced SOD activity.
Aging is a risk factor for atherosclerosis.5 Atherosclerosis is associated with endothelial dysfunction that precedes atherosclerotic changes of the vascular wall6 and is a common feature of several cardiovascular risk factors.22 23 Endothelial dysfunction due to aging has been studied in animal models24 25 and in human subjects.26 27 However, different vascular beds have never been compared in the same organism of very old animals as in this study, which in terms of maximal life span compare with very old humans.28 We found that endothelium-dependent relaxation to acetylcholine was markedly attenuated in aortas from very old rats, in line with our previous observation that release of NO is reduced in aortas of aging rats.24 These changes must be independent of receptor-mediated signal transduction mechanisms, since the response to a receptor-independent agonist of the L-arginine/NO pathway, calcium ionophore A23187, was similarly reduced. Also, an increase in contractility to norepinephrine in the aorta cannot account for this difference because the precontractions induced by the catecholamine were matched between young and old rats. Hence, a decrease in expression of eNOS mRNA in the aorta as demonstrated semiquantitatively by RT-PCR in this study more likely accounts for age-related changes in endothelium-dependent relaxation.
Relaxations to SOD were used to assess basal release of NO.29 30 Indeed, endothelium-dependent relaxations to SOD were blocked by the NOS inhibitor L-NAME or endothelial denudation. We found that relaxations were independent of cyclooxygenase or hydrogen peroxide, indicating that SOD prolongs the half-life of basal NO and in turn causes relaxation. Interestingly, relaxations to SOD were attenuated by aging in both the aorta and the femoral artery, whereas relaxations to exogenous NO as assessed by the response to sodium nitroprusside were unaffected. Thus, impaired basal NO production may result from decreased eNOS mRNA expression, whereas smooth muscle sensitivity to NO remains unaffected by aging. A decrease of basal release of NO could also explain why contractions to norepinephrine were enhanced in aortas of aging rats.
Unexpectedly, endothelium-dependent relaxations to acetylcholine in the femoral artery were preserved in old rats. Anatomic heterogeneity of endothelial dysfunction is a distinct feature of hypertension31 and has also been found in patients with early atherosclerosis and proven coronary artery disease.32 This study extends these findings to vascular aging. Endothelial function may also be modulated by pulse pressure, which was increased in old rats while other hemodynamic parameters were unchanged. Because the aorta is most exposed to pulsatility and because pulse pressure exhibits regional differences with aging,16 this could account, at least in part, for differences in vascular function observed in this study.
As with endothelium-dependent relaxations, aging attenuated contractions to ET-1 in the aorta but not the femoral artery. Indeed, in contrast to ET-1, aging increased contractions to norepinephrine and had no effect on the absolute tension evoked by potassium chloride. Since contractions to ET-1 in aortas of aging rats are independent of the endothelium,33 selective downregulation of ETA receptors and/or impaired signal transduction pathways may explain these findings.34 Interestingly, in rat coronary arteries aging is associated with increased contractions to ET-1,35 whereas in mesenteric resistance arteries the sensitivity but not the maximal response to ET-1 is reduced.14 Together with the data from the present study, it can be speculated that aging selectively alters ET-receptor activation in different parts of the arterial tree.
ECEs use big ET as a substrate to generate the biologically active product ET-1.36 37 38 In young rats, contractions to big ET were about fourfold higher in femoral arteries than in aortas, suggesting lower conversion of big ET in the latter vessel. Aging tended to increase contractions to big ET in both arteries investigated. However, this difference was significant only at a concentration of 0.03 µmol/L. Analysis of functional ECE activity (see "Methods") revealed that aging increased ECE activity in the aorta but not in femoral arteries. The reason for this difference is unclear. Preserved vascular ECE activity with aging in the femoral artery may, however, reflect maintained endothelial function (see above) that was markedly impaired in the aorta. Finally, increased ET-1 plasma levels and ECE activity in the aorta could explain a selective and possibly agonist-dependent downregulation of the response to ET-1 in this blood vessel.
Oxidative stress is an important factor contributing to vascular dysfunction7 and aging.10 39 The effect of aging on vascular SOD activity, an important antioxidant enzyme that determines the release of biologically active NO40 and that is abundant in the vascular wall,41 is unknown. Alterations in vascular SOD activity42 may be particularly important for endothelial function, since increased oxidative stress contributes to endothelial dysfunction.43 44 45 Hence, we investigated the effect of aging on SOD activity in arterial tissues and plasma. Vascular SOD activity was about 50% higher in femoral arteries, but no effect of aging on SOD activity in the aorta or the femoral artery was observed. Possibly, differential regulation of SOD activity in different vascular beds may predispose to age-related impairment of endothelial function as seen in the aorta. Indeed, several studies found that aging modulates SOD activity and expression in tissues such as heart and kidney,46 adrenal gland, liver, and brain.8 47 However, from our data it cannot be excluded that vascular antioxidant systems distinct from SOD are altered by the aging process.48 Circulating SOD activity concentrations in plasma are much lower than those found in tissue.49 The role of antioxidant and prooxidant enzymes in plasma and their effect on endothelial (dys)function is still unclear. Hypercholesterolemia causes endothelial dysfunction2 43 50 that is associated with a decrease in plasma SOD activity.51 There also is evidence that plasma-bound xanthine oxidase, a major cellular source of superoxide anion, impairs endothelial function.52 This raises the possibility of the existence of circulating redox system(s) in plasma. Tomoda and coworkers53 recently reported that plasma SOD activity is a predictor for myocardial reperfusion after myocardial infarction. Our results demonstrate that, at least in the rat, aging reduces plasma SOD activity and that this reduction inversely correlated with increases in ET-1 plasma levels. Since we previously demonstrated that oxidized low-density lipoprotein increases54 and that exogenous SOD inhibits ET-1 production in vitro,55 the data from the present study suggest a possible relationship between plasma SOD activity and ET-1 plasma levels in vivo.
In summary, we have demonstrated age-related impairment of vascular function in the aorta but not the femoral artery of the rat. Endothelial dysfunction in the aorta may be related to increased pulse pressure and/or reduced eNOS mRNA expression. Preserved vascular function in the femoral artery suggests that aging exhibits an anatomic heterogeneity that may protect certain blood vessels against the development of atherosclerosis.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This study was supported by grant No. 32-325 41.91/2 of the Swiss National Foundation. Dr Barton is supported by the Deutsche Forschungsgemeinschaft (Ba 1543/1-1). Dr Cosentino is the recipient of a research grant from Bristol Myers-Squibb. Dr Moreau holds a fellowship of the Medical Research Council of Canada. The help of Christian Binggeli, MD, with computer programming is much appreciated.
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Footnotes |
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Presented in part at the Scientific Sessions of the American College of Cardiology, Anaheim, Ca, March 17, 1997, and published in abstract form (J Am Coll Cardiol. 1997;29[suppl A]:304A.)
Received November 18, 1996; first decision January 7, 1997; accepted April 2, 1997.
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References |
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