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Aging is associated with endothelial dysfunction in healthy men years before the age-related decline in women
JACC Vol . 24, No. 2 August 1994: 47 1 -6
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DAVID S . CELERMAJER, PHD, FRAGP, :'- KELD E . SORENSEN, MD, DAVID J. SPIEGELHALTER, PHD,t DIMITRI GEORGAKOPOULOS, MD, JACQUI ROBINSON, RN, JOHN E . DEANFIELD, MB, BCHIR, FRCP London and Cambridge, England, United Kingdom
Objectives . This study assessed whether aging is associated with progressive endothelial dysfunction, whether the pattern of any age-related decline in vascular health is different in urea and women and whether any gentler difference is consistent with known changes in hormonal status. Background. Coronary and cerebrovascular disease are much less common in young and middle-aged women compared with men, although the gender difference in death from atherosclerosis is less marked after the menopause . Endothelial dysfunction is an early event in atberogenesis send is important in dynamic plaque stenosis in later life. Ile effect of aging on endothelial function in men and women, however, is not well known. Methods. We used high resolution ultrasound to study endothelium-dependent and endothelium-independent vascular respouses . Brachial artery physiology was investigated in 238 subjects (103 men, 135 women ; mean [tSD] age 38 ± 17 years, range 15 to 72) with no known risk factors for atherosclerosis . The responses to reactive hyperemia (slow-mediated dilation, which is
Life expectancy is greater for women than for men, in large part because of their lower incidence of cardiovascular death in middle age (1) . Male gender remains a cardiovascular risk factor, even after controlling for other influences, such as cigarette smoking and hypertension (2,3) . Many investigators have suggested that the basis for the protection of young and middle-aged women from atherosclerosis is their relative excess of estrogenic hormones (4-6) . Endothelial dysfunction is an important early event in experimental models of atherosclerosis (7) . It has recently been demonstrated in asymptomatic children and young
endulkelhim dependent) and to glyceryl trinitrate (an endothelimn independentt dilator) were assessed for all the subjects and then for men and women separately . Resalls . On multivariate analysis for the whole group, reduced flow-mediated dilation was related to older age (r = -6 .34, p < 0. 1). In urea, Pow-mediated dilation was preserved in subjects aged !!540 years but declined thereafter at 0 .21 %/year. In women, flow-mediated dllatl5ii was stable until the early 50s, after which it declined at 0 .49%lyear (p = 0 .002 compared with men) . In contrast, there was no significant change in the glyceryl trinitrate response with aging in either gender. Conclusions. Aging is associated with progressive endothelial dysfunction ha normal humans, and this appears to occur earlier in men than in women . In women, however, a steep decline commences at around the time of the menopause . This iscent tentwith a protective effect of estrogens on the arterial wall . (J Am Coll Cardiol 1994 ;24 :471®6)
adults with vascular risk factors (8) and may contribute to morbidity and mortality by disturbing arterial physiology in adults with established coronary stenoses (9,10) . Previous studies have shown that increasing age is associated with coronary endothelial dysfunction but have only examined small numbers of subjects, most of whom had multiple vascular risk factors and were undergoing catheterization for suspected coronary artery disease (11,12) . Using a noninvasive method, we examined endothelial function and its relation to age in a large number of healthy men and women who did not have these potentially confounding influences . Our observations suggest that there is a difference in the
From the Cardiothoracic Unit, Hospital for Sick Children, London and tMedical Research Council Biostatistics Unit, Cambridge, England, United Kingdom . Dr. Celetarajer is supported by the British Heart Foundation, London, Dr. Sorensen by the Danish Heart Foundation, Copenhagen, Denmark and Dr. Georgakopoulos by the European Society of Cardiology, Nice, France . This study was supported in part by a grant from Corda, London, England, United Kingdom. Manuscript received November 19, 1993 ; revised manuscript received March 8, 1994, accepted March 16, 1994 . *Present address and address h2r corresRgndente : Department of Cardiology, Royal Prince Alfred Hospital, Missenden Road, Camperdown 2050, Sydney, Australia.
01994 by the American College of Cardiology
pattern of age-related vascular injury in men and women, with a steep decline in endothelial function occurring in women after the menopause . This supports the concept of a hormonal influence on vascular disease and risk .
Methods Subjects . We studied 238 subjects (103 men, 135 women ; mean [±SD] age 38 ± 17 years, range 15 to 72) recruited from hospital staff, families, friends and community volun0735-10971941$7 .00
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CELERMAJER ET AL . VASCULAR AGING IN MEN AND WOMEN
teers over a 2-year period . Subjects were included only it they were asymptomatic, lifelong nonsmokers and had no family history of premature vascular disease . None were known to have hypertension, diabetes or hyperlipidemia . Because cholesterol levels increase with age in a normal population, we included all subjects with total cholesterol below the 90th centile for their age and gender (13). No subjects were taking regular medications . Thirty-six of the women had not had a menstrual period for >1 year ; their average age at menopause was 51 ± 1 years . None of these postmenopausal women was taking hormone replacement therapy . All studies were approved by the local ethics committee . Study design . Total plasma cholesterol and rest supine blood pressure were measured in every subject . All then had a noninvasive ultrasound study of the right brachial artery to test endothelial and smooth muscle function, as previously described (8) . Arterial diameter was measured at rest, during reactive hyperemia, again at rest and after administration of sublingual glyceryl trinitrate, using a standard 7 .0-MHz linear array transducer and 128 XP/10 system (Acuson) . Previous investigators have demonstrated that conduit artery dilation in response to flow increase is endothelium dependent (14,15), whereas the dilator response to glyceryl trinitrate is endothelium independent (9,16) . Hyperemic microvascular responses are also endothelium independent (17) . Each subject lay at rest for at least 10 min before the first rest scan was obtained . Increased flow was then induced by inflation of a pneumatic tourniquet placed around the forearm to a pressure of 300 mm Hg, followed by deflation after 4 .5 min . The second scan was taken for 30 s before and 90 s after cuff deflation . Fifteen minutes was then allowed for vessel' recovery, and a further rest scan was then recorded, Sublingual glyceryl trinitrate spray (400 µg) was then administered, and 3 to 4 min later the last scaai was done . Seven subjects declined sublingual glyceryl trinitrate . Dopplerderived flow measurements were obtained during the first rest scan and again during the 1st 15 s of reactive hyperemia . The electrocardiogram (ECG) was monitored continuously throughout the study . Data , tli scans were recorded on super VHS videotape for later analysis. Vessel diameter was measured by two independent observers unaware of the clinical details and stage of the experiment, as described previously (8). The mean vessel diameter and percent dilation for each patient were taken as the aver a of the measurements of the two observers . Statistics. The distributions of flow-mediated dilation, glyceryl trinitrate response, age, cholesterol, vessel size and hyperemia were summarized by sample means and standard deviations, and pairwise relations were expressed by Pearson correlation coefficients . Treating flow-mediated dilation and glyceryl trinitrate dilation as response variables and age, cholesterol and vessel size as independent variables, we investigated their simultaneous relation by multiple regres-
sion analyses, from which we obtained partial correlation coefficients. Men and women were then analyzed separately . For flow-mediated dilation, a multiple regression was carried out with a "vessel size" term plus two "age" terms, with coefficients depending on whether the subject's age was above or below a specified change point t,,,, x . Thus, for age below the change point t, the regression model is Flowmediated dilation = a + b(Size - Mean size) + c(Age -- t), where Mean size is the mean vessel size, whereas for age above the change point, the model is Flow-mediated dilation = a + b(Size - Mean size) + d(Age - t) . The "most likely" change point t m~x produced the minimal residual standard deviation rTto„~x over a range of possible change points from 30 to 65 years . To summarize the support for each possible change point I, the "profile likelihood" o,,,,,,,/o,1 was plotted, which has maximal I at t = as derived from a likelihood ratio test procedure (18) . To validate the twostraight-line model compared with a smooth curve, a quadratic function in age was also fitted to the data, the calculated residual standard deviation was smaller for the two-straight-line model . Men and women were compared with respect to the gradient of adjusted flow-mediated dilation with age, both before and after their estimated change point, using a Z test based on their estimated standard errors. For glyceryl trinitrate, the regression coefficient b arose from a simple linear regression on vessel size ; that is, Glyceryl trinitrate = a + b(Size - Mean size) . For each subject, flow-mediated and glyceryl trinitrateinduced dilation were adjusted for vessel size by subtracting b(Size - Mean size), and these adjusted values were then plotted against age .
Results The age distribution of the subjects and their vascular responses to increased flow and to glyceryl trinitrate are shown in Figures 1 and 2 . Scans from all 238 subjects were of sufficient quality for analysis, and no plaques were observed in any subject . Total plasma cholesterol was 191 ± 39 mg/dl (range 101 to 299) . and there was a positive correlation between age and cholesterol level (r = 0 .58, p < 0 .01). No subjects were hypertensive (blood pressure < 160/90 mm Hg in all) . In response to an increase in flow of 487 ± 172% after cuff deflation, there was an increase in brachial artery diameter of 7 .9 ± 3 .9% (range - 1% to 18%) . There was no correlation between age and degree of reactive hyperemia (r = -0 .06, p = 0 .49) or between flow-mediated dilation and hyperemia (r - 0.09, p = 0.16). In response to glyceryl trinitrate, arterial diameter increased by 19 .4 ± 5 .8% (range 4% to 38%) . On multivariate analysis of all subjects, flow-mediated dilation was independently related to age (r = -0 .34, p < 0.0001) and vessel size (r = -0 .35, p < 0 .0001) but not to the cholesterol level (r = -0 .09, p = 0.14) . In contrast, although the glyceryl trinitrate response was also related to vessel size
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CELERMAJER ET AL. VASCULAR AGING IN MEN AND WOMEN
FMD adjusted for vessel size
Figure 1 . Aging and flowmediated dilation (FMD) in 103 men (left) and 135 women (right) . Top, Individual data points for percent flow-mediated dilation adjusted for vessel size . Bottom, of a change .,a point existing at any given age ; this is isting highest for men at age 41 years and for women at 53 years. In women, there is a steeper decline in flow-mediated dilation after their change point .
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- 0.45, p < 0 .0001), there was no relation with age -0.10, p -- 0 .18) (Table 1) . Gender difference, In men, flow-mediated dilation (adjusted for vessel size) did not decline until the end of the fourth decade . Change-point analysis suggested that an age-related decline occurred subsequently, from -41 years of age, after which flow-mediated dilation decreased by
Figure 2 . Aging and glyceryl trinitrate (M)-induced dilation in 102 men (top) and 129 women (bottom) . The response to glyceryl trinitrate is preserved with increasing age in both genders . Seven of the 238 subjects had a decline in glyceryl trinitrate . GTN adjusted for vessel size
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0 .21%/year . In contrast, flow-mediated dilation was preserved in women until the early 50s (most likely change point at age 53 years), after which it declined significantly faster than in men (0 .49%)year, p = 0 .002) . There was a very narrow range in the age at menopause in this study, and separate analysis of years since menopause versus flowmediated dilation in women yielded a similar slope of decline . In contrast, there was no age-related decline in glyceryl trinitrate-induced dilation in either gender .
Discussion This study showed that aging is associated with progressive loss of flow-mediated dilation in the systemic arteries of healthy subjects with no risk factors for vascular disease . In contrast, responses to glyceryl trinitrate are preserved into old age, and this suggests that aging leads to endothelial dysfunction . Because there is no age-related change in the hyperemic flow response, it is unlikely that progressive proximal atherosclerosis could explain the arterial vasodilator abnormalities observed in this study . We also observed that the pattern of age-related changes in arterial physiology is different in men and women . In men, endothelial responses decline gradually after the fourth decade, whereas in women, vascular physiology remains healthy for a further 10 years . Subsequently, the rate of decline is faster in women, so that endothelial dysfunction is present in almost all subjects by 65 years of age . This pattern of vascular damage parallels the known difference between the genders in cardiovascular morbidity and mortality in middle and old age (1,19), where men are at much higher risk from age 40 to 50 years, but women "catch up" in later life . The process of atherosclerosis does not appear to differ
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Table 1 . Results of Multiple Regression Analysis for Determinants of Flow-Mediated Dilation (an endothelium-dependent response) and Glyceryl Trinitrate-Induced Dilation (an endothelium-independent response) FMD
Age (yr) Vessel size (mm) Cholesterol (10 mgfdl)
Coefficient (SD)
Partial Correlation Coefficient
. (0.02) -2 .13 (0.33) -0 .09 (0 .06)
-0.34 -0 .35 -0 .09
GIN
P Valve
Coefficient (SD)
Partial Correlation Coefficient
p Value
< 0.0001 < O.Ot0I 0.14
-0.03 (0.02) -4 .01(0.54) --0 .08 (0 .10)
-0.10 -0 .45 -0 .06
0 .18 < 0 .11 0 .41
FMD = flow-mediated dilation ; GIN ® glyceryl trinitrate-induced response,
between men and women, and the risk factors known to predispose to vascular disease affect both genders equally (20) . We have previously shown that this noninvasive method can detect flow-mediated dilation in healthy subjects and that risk factors such as hypercholesterolemia (21) and cigarette smoking (22) are associated with abnormal endothelial physiology in both male and female children and young adults . Aging is associated with an increasing prevalence of diabetes, hypertension and hypercholesterolemia, and in the previous studies of both Vita et al . (11) and Yasue et al . (12) . in which coronary endothelial dysfunction was found in most subjects >30 years of age, risk factors may have had an important confounding effect . Study furltations. In this study, by excluding all subjects who had ever smoked or who had known hyperlipidemia, hypertension, diabetes or a positive family history, we investigated the effects of aging per se on vasomotor responses and compared endothelial function in male and female subjects Previous studies enrolled too few patients to examine any gender differences in the effects of aging on endothelial function (11, 12) . The method we used to study endothelial and smooth muscle function is noninvasive and well tolerated, allowing investigation of a large number of subjects with neither risk factors nor symptoms . The accuracy of 7 .0-MHz ultrasound is sufficient to allow detection of small diameter differences (as little as 0 .1 mm), and the reproducibility of the technique has been established previously (8,22,23) . Although only superficial systemic arteries can be studied, atherosclerosis is a diffuse process, and there is a correlation between the presence of disease in different large arteries in the same subject (24) . Recently two groups have reported that brachial artery vasomotor responses are well correlated with coronary endothelial physiology and coronary atherosclerosis (25,26) . and the ovascular system . Aging is associated with a number of anatomic and hemodynamic changes in the vascular system of all subjects, including collagen degeneration, loss of elastin, increased intima-media thickness of the arteries and reduced vascular compliance (27,28) . Despite these age-related changes, the arterial response to glyceryl trinitrate was preserved in our study . This is consistent with other studies that have shown that pharmacologic doses of
exogenous nitrovasodilators remain effective in older adults (29,30) and that these drugs are still therapeutically effective in geriatric patients (16) . In contrast, we observed an agerelated decline in endothelial function in both men and women . Previous investigators have shown that the dilator capacity of both large and small coronary vessels in response to acetylcholine, an endothelium-dependent vasodilator, also decreases with increasing age in subjects with chest pain but angiographically smooth coronary arteries (11,12,31) . The mechanism whereby aging is associated with impaired endothelial function is not known . Possibilities include an age-related decrease in release of endotheliumderived relaxing factor(s), or their increased catabolism in the vessel wall, or increased release of constricting factors (31) . In vitro aging is associated with increased monocyteendothelial cell adherence, which may result from agerelated increases in peroxidative stress (32) . Our data suggest that women may be protected from these age-related changes for a decade longer than men . Gender differences . This gender difference in the pattern of age-related decline in endothelial function is best explained by the influence of hormonal factors . Estrogens may protect women from atherogenesis (33,34) . After the menopause, decreased estrogen levels are associated with a more atherogenic lipid profile (19,35) ; however, the effects of estrogen on plasma lipids can explain only a small part of the beneficial effects on the risk of vascular disease (36) . Estrogens are vasoactive hormones and have direct effects on the arterial wall (37,38). In experimental animals, surgically induced menopause is associated with a loss of endotheliumdependent coronary vasorelaxation (39) . In postmenopausal animals, estrogen replacement may restore normal endothelial function (6) . Intravenous estrogen acutely improves endothelium-dependent vasorelaxation in postmenopausal women with coronary artery disease (40), and sublingual estrogen has a beneficial effect on myocardial ischemia in similar patients (41) . In our study, the age at which endothelial dysfunction was seen in women was around the time of the menopause . These data therefore suggest, but do not prove, that estrogens may protect against endothelial injury . Injury of the endothelium is important both because it is a key early event in atherogenesis and because it may
JACC Val . 24, No . 2 August 1994:471-6
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increase risk in subjects with established atherosclerosis by predisposing to vasoconstriction and thrombosis (9) . The recent demonstration that L-arginme, the precursor of endothelium-derived relaxing factor, not only protects the endothelium but also retards atherosclerosis in the cholesterol-fed rabbit suggests that endothelial dysfunction may be intimately involved in the pathogenesis of atherotna (42) . Clinical i plications . Cardiovascular disease is the most common cause of death in the United States, and the known gender difference in life expectancy is largely related to the increased risk of coronary and carotid atherosclerotic complications in middle-aged men, Our data suggest that aging per se, irrespective of exposure to known risk factors, may be associated with earlier physiologic abnormalities in the systemic arteries of men compared with women . This is consistent with observations that both old age and mate gender arc independent risk factors for cardiovascular mortality and that estrogens may protect women from the age-related decline in vascular health . The association between menopause and the subsequent change in endothelial function that we have observed is provocative, but prospective studies will be required to see whether hormone replacement therapy prevents or retards the age-related changes in arterial physiology in postmenopausal women . Many such women are potentially candidates for hormone replacement therapy, not just for well-being and prevention for osteoporosis but also for protection of the arterial wall and reduction of vascular risk. The availability of a noninvasive method to assess ",ascular health will allow investigation of the appropriate type and timing of such hormonal therapy, both in individual subjects and in clinical trials .
References 1 . British Heart Foundation Statistics Database . Coronary Heart Disease Statistics, 1992, London : The Coronary Prevention Group, 1992 . 2. Castelli WP . Epidemiology of coronary heart disease . The Framingham Study . Am J Med 1984 ;76:4-12 . 3 . Lipid Research Clinics Program . The Lipid Research Clinics Primary Prevention Trial results . JAMA 1984;251 :351-64 . 4. Colditz GA, Wilett WC, Stampfer MI . Rosner B, Speizer FE . Hennekens CH . Menopause and the risk of coronary heart disease in women . N Engl J Med 1987 ;316 :1105-10. 5 . Witteman JCM, Grobbee DE, Kok FJ, Hofman A, Valkenburg HA . Increased risk of atherosclerosis in women after menopause . Br Med J 1989 ;298 :642-4 . 6 . Haarbo J, Leth-Espensen P, Stender S, Christiansen C . Estrogen monotherapy and combined estrogen/progestogen replacement therapy attenuate aortic accumulation of cholesterol in ovariectomized cholesterol-fed rabbits . J Clin Invest 1991 ;87:1274-9 . 7 . Ross R . The pathogenesis of atherosclerosis-an update . N Engl J Med 1986;8:488-5O0 . 8 . Celermajer DS, Sorensen KE, Gooch VM, et at . Non-invasive detection of endothelial dysfunction in children and adults at risk of atherosclerosis . Lancet 1992 ;340:1111-5 . 9. Ludmer PL, Selwyn AP, Shook TL, et al. Paradoxical vasoconstriction induced by acetyecholine in atherosclerotic coronary arteries . N Engl I Med 1986 ;315 :1046-51 . 10. Nobel EG, Selwyn AP, Ganz P. Large coronary arteries in humans are responsive to changing blood flow : an endothelitim-dependent mechanism
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that fails in patients with atherosclerosis . .1 Am Call Cardiol 1990 ;16: . j-56 . I1 . t'ita JA . Treasure CB, Nabel EG, et al . Coronary vasomotor response to acetylcholine relates to risk factors for coronary artery disease . Circulation 1990 ;81 :491-7. 12 . Yasue H . Matsuyama K, Matsuyama K, et al . Responses of angiographically normal human coronary arteries to intracoronary injection of acetylcholine by age and segment . Circulation 1990 ;81 :482-90. 13. Risk Factor Prevalence Study : Survey No . 3, 1989 . Sydney : National Heart Foundation of Australia . 14 . Pohl U, Holtz J, Busse R, Bassenge E . Crucial role of endothelium in the vasodilator response to increased flow in-vivo . Hypertension 1986 : 8 :37-44 . 15 . Smiesko V, Kozik J . Dolezel S . Role of endothelium in the control of arterial diameter by blood flow . Blood Vessels 1985 ;22 :247-51 . 16 . Mickelson IX, Simpson PJ, Lucchesi BR . lschemic Heart Disease : pathophysiology and pharmacologic management- In : Antonaccio M, editor, Cardiovascular Pharmacology . 3rd ed . New York : Raven Press, 1990 :293--340. 17 . Kuo L, Chilian WM, Davis MJ . Coronary arteriolar myogenic response is indepeudenl of endothelium . Circ Res 1990:66 :860-6 . 18 . Hinkley VV- Inference in two-phase regression . J Am Stat Assoc 1971 ; 66 :736-43 . 19 . Jacobs 115, Loefller FF . Post menopausal hormone replacement therapy . Br Med J 1992 ;305 :1403-8. 20. Farmer JA, Gotto AM . Risk factors for coronary artery disease . In : Braunwald E, editor. Heart Disease . 4th ed . Philadelphia: Saunders, 1992 :1125-60. 21 . Sorensen KE, Celermajer DS . Georgakopoulos D, Hatcher G, Betteridge J, DeanfieldJE . Impairment ofendothelium-dependent dilation is an early event in children with familial hypercholesterolemia, and is related to the Lp(a) level . J Clin Invest 1994;93 :50-5 . 22 . Celermajer DS, Sorensen ICE, Georgakopoulos 0 . et al . Cigarette smoking is associated with dose-related and potentially reversible impairment of endothelium-dependent dilation in healthy young adults, Circulation 1993;88 :2149-55 . 23 . Celermajer DS, Sorensen KE, Ryalls H, et al . Endothelial dysfunction occurs in the systemic arteries of children with homozygous homocystinutia but not in their heterozygous parents . J Am Coll Cardiol 1993 :22 ; 854-8 . 24 . Dlsson AG . Regression of femoral atherosclerosis . Circulation 1991 .83 : 698-700 . 25 . Uehata A, Gerhard MD, Meredith IT, et al . Close relationship of endothelial dysfunction in coronary and brachial artery [abstract] . Circulation 1993 ;88 : Suppi 1 :1-618 . 26. Vogel RA, Vaitkevicius PV, Plotnick GD . Ultrasound assessment of brachial artery endothelial-dependent vasoactivity as a means for diagnosing coronary artery disease [abstract] . J Am Coll Cardiol 1993 ;21 : 345A . 27 . Rosenthal J . Aging and the cardiovascular system . Gerontology 1987 ;33: Suppl 1 :1-3-8 . 28 . Smulyan H, Csermely TJ, Mookhetjee S, Warner RA . Effect of age on arterial distensibility in asymptomatic humans . Arteriosclerosis 1983 :2 : 199--205 . 29 . Feldman RL, Pepine CJ, Conti CR . Magnitude of dilation of large and small coronary arteries by nitroglycerin . Circulation 1981 ;64 :324-33, 30 . Parker JO, Vankoughnett KA, Farrell B . Nitroglycerin lingual spray : clinical efficacy and dose-response relation . Am J Cardiol 1986 ;57 :1-5 . . 31 Egashira K, Inou T, Hirooka Y, et al . Effects of age on endotheliumdependent vasodilation of resistance coronary artery by acetyicholine in humans . Circulation 1993 ;88 :77-81 . 32 . MolenaarR,VisserWI,VerkerkA .KosterIF,3ongkind .IF'.Peroxldative stress and in-vitro ageing of endothelial cells increases the tuonocyteendothelial cell adherence in a human in-vitro system . Atherosclerosis 1989 ;76:193-202 . 33 . Godsland IF, Wynn V, Crook D, Miller NE . Sex, plasma lipoproteins, and atherosclerosis - prevailing assumptions and outstanding questions . Am Heart J 1987;114 :1467-503. 34, Bush TL, Barrett-Connor E, Cowan LD, et al . Cardiovascular mortality and non-contraceptive use of estrogen in women : results from the lipid research clinics program follow-up study . Circulation 1987 ;75x102-9 .
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35. Gambrel RD Jr, Teran AZ . Changes in lipids and lipoproteins with long-term estrogen deficiency and hormone replacement therapy . Am J Obstet Gyoecol 1991 ;165 :3117-15 . 36. Williams 1K. Oestrogen therapy for myocardial ischaemia in women . Lancet 13 ;342:128. 37 . Mess RR, Rosenfeld CR . Local and systemic estradiol-17 beta: effects on uterine and systemic vasodilation, Am I Physiol 1989 ;256: E536-42 . 38 . Ganger KF, Vyas S, Whitehead M, Crook D, Heir H, Campbell S. Pulsatility index in internal carotid artery in relation to transdermal oestradial and time since menopause . Lancet 1991 ;338 :839-42.
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39 . Williams 3K . Adams MR. Klopfenstein HS . Estrogen modulates responses of atherosclerotic coronary arteries . Circulation 1990 ;81 :1640-7 . 40 . Williams 3K, Adams MR, Herrington DM, Clarkson TB. Short-term administration of estrogen and vascular responses of atherosclerotic coronary arteries. J Am Coll Cardiol 1992;29:452-7 . 41 . Rosano GMC, Sarrel PM . Poole-Wilson PA . Collins P. Beneficial efect of oestrogen on exercise-induced myocardial ischaemia in women with coronary artery disease. Lancet 1993;342:133-6 . 42. Cooke JP, Singer AH, Zera P, Rowan RA, Billingham ME . Antiatherogenic effects of t.-arginine in the hypercholesteroiemic rabbit . J Clin Invest 1992 ,,90:1163-72 .
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DAVID S . CELERMAJER, PHD, FRAGP, :'- KELD E . SORENSEN, MD, DAVID J. SPIEGELHALTER, PHD,t DIMITRI GEORGAKOPOULOS, MD, JACQUI ROBINSON, RN, JOHN E . DEANFIELD, MB, BCHIR, FRCP London and Cambridge, England, United Kingdom
Objectives . This study assessed whether aging is associated with progressive endothelial dysfunction, whether the pattern of any age-related decline in vascular health is different in urea and women and whether any gentler difference is consistent with known changes in hormonal status. Background. Coronary and cerebrovascular disease are much less common in young and middle-aged women compared with men, although the gender difference in death from atherosclerosis is less marked after the menopause . Endothelial dysfunction is an early event in atberogenesis send is important in dynamic plaque stenosis in later life. Ile effect of aging on endothelial function in men and women, however, is not well known. Methods. We used high resolution ultrasound to study endothelium-dependent and endothelium-independent vascular respouses . Brachial artery physiology was investigated in 238 subjects (103 men, 135 women ; mean [tSD] age 38 ± 17 years, range 15 to 72) with no known risk factors for atherosclerosis . The responses to reactive hyperemia (slow-mediated dilation, which is
Life expectancy is greater for women than for men, in large part because of their lower incidence of cardiovascular death in middle age (1) . Male gender remains a cardiovascular risk factor, even after controlling for other influences, such as cigarette smoking and hypertension (2,3) . Many investigators have suggested that the basis for the protection of young and middle-aged women from atherosclerosis is their relative excess of estrogenic hormones (4-6) . Endothelial dysfunction is an important early event in experimental models of atherosclerosis (7) . It has recently been demonstrated in asymptomatic children and young
endulkelhim dependent) and to glyceryl trinitrate (an endothelimn independentt dilator) were assessed for all the subjects and then for men and women separately . Resalls . On multivariate analysis for the whole group, reduced flow-mediated dilation was related to older age (r = -6 .34, p < 0. 1). In urea, Pow-mediated dilation was preserved in subjects aged !!540 years but declined thereafter at 0 .21 %/year. In women, flow-mediated dllatl5ii was stable until the early 50s, after which it declined at 0 .49%lyear (p = 0 .002 compared with men) . In contrast, there was no significant change in the glyceryl trinitrate response with aging in either gender. Conclusions. Aging is associated with progressive endothelial dysfunction ha normal humans, and this appears to occur earlier in men than in women . In women, however, a steep decline commences at around the time of the menopause . This iscent tentwith a protective effect of estrogens on the arterial wall . (J Am Coll Cardiol 1994 ;24 :471®6)
adults with vascular risk factors (8) and may contribute to morbidity and mortality by disturbing arterial physiology in adults with established coronary stenoses (9,10) . Previous studies have shown that increasing age is associated with coronary endothelial dysfunction but have only examined small numbers of subjects, most of whom had multiple vascular risk factors and were undergoing catheterization for suspected coronary artery disease (11,12) . Using a noninvasive method, we examined endothelial function and its relation to age in a large number of healthy men and women who did not have these potentially confounding influences . Our observations suggest that there is a difference in the
From the Cardiothoracic Unit, Hospital for Sick Children, London and tMedical Research Council Biostatistics Unit, Cambridge, England, United Kingdom . Dr. Celetarajer is supported by the British Heart Foundation, London, Dr. Sorensen by the Danish Heart Foundation, Copenhagen, Denmark and Dr. Georgakopoulos by the European Society of Cardiology, Nice, France . This study was supported in part by a grant from Corda, London, England, United Kingdom. Manuscript received November 19, 1993 ; revised manuscript received March 8, 1994, accepted March 16, 1994 . *Present address and address h2r corresRgndente : Department of Cardiology, Royal Prince Alfred Hospital, Missenden Road, Camperdown 2050, Sydney, Australia.
01994 by the American College of Cardiology
pattern of age-related vascular injury in men and women, with a steep decline in endothelial function occurring in women after the menopause . This supports the concept of a hormonal influence on vascular disease and risk .
Methods Subjects . We studied 238 subjects (103 men, 135 women ; mean [±SD] age 38 ± 17 years, range 15 to 72) recruited from hospital staff, families, friends and community volun0735-10971941$7 .00
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CELERMAJER ET AL . VASCULAR AGING IN MEN AND WOMEN
teers over a 2-year period . Subjects were included only it they were asymptomatic, lifelong nonsmokers and had no family history of premature vascular disease . None were known to have hypertension, diabetes or hyperlipidemia . Because cholesterol levels increase with age in a normal population, we included all subjects with total cholesterol below the 90th centile for their age and gender (13). No subjects were taking regular medications . Thirty-six of the women had not had a menstrual period for >1 year ; their average age at menopause was 51 ± 1 years . None of these postmenopausal women was taking hormone replacement therapy . All studies were approved by the local ethics committee . Study design . Total plasma cholesterol and rest supine blood pressure were measured in every subject . All then had a noninvasive ultrasound study of the right brachial artery to test endothelial and smooth muscle function, as previously described (8) . Arterial diameter was measured at rest, during reactive hyperemia, again at rest and after administration of sublingual glyceryl trinitrate, using a standard 7 .0-MHz linear array transducer and 128 XP/10 system (Acuson) . Previous investigators have demonstrated that conduit artery dilation in response to flow increase is endothelium dependent (14,15), whereas the dilator response to glyceryl trinitrate is endothelium independent (9,16) . Hyperemic microvascular responses are also endothelium independent (17) . Each subject lay at rest for at least 10 min before the first rest scan was obtained . Increased flow was then induced by inflation of a pneumatic tourniquet placed around the forearm to a pressure of 300 mm Hg, followed by deflation after 4 .5 min . The second scan was taken for 30 s before and 90 s after cuff deflation . Fifteen minutes was then allowed for vessel' recovery, and a further rest scan was then recorded, Sublingual glyceryl trinitrate spray (400 µg) was then administered, and 3 to 4 min later the last scaai was done . Seven subjects declined sublingual glyceryl trinitrate . Dopplerderived flow measurements were obtained during the first rest scan and again during the 1st 15 s of reactive hyperemia . The electrocardiogram (ECG) was monitored continuously throughout the study . Data , tli scans were recorded on super VHS videotape for later analysis. Vessel diameter was measured by two independent observers unaware of the clinical details and stage of the experiment, as described previously (8). The mean vessel diameter and percent dilation for each patient were taken as the aver a of the measurements of the two observers . Statistics. The distributions of flow-mediated dilation, glyceryl trinitrate response, age, cholesterol, vessel size and hyperemia were summarized by sample means and standard deviations, and pairwise relations were expressed by Pearson correlation coefficients . Treating flow-mediated dilation and glyceryl trinitrate dilation as response variables and age, cholesterol and vessel size as independent variables, we investigated their simultaneous relation by multiple regres-
sion analyses, from which we obtained partial correlation coefficients. Men and women were then analyzed separately . For flow-mediated dilation, a multiple regression was carried out with a "vessel size" term plus two "age" terms, with coefficients depending on whether the subject's age was above or below a specified change point t,,,, x . Thus, for age below the change point t, the regression model is Flowmediated dilation = a + b(Size - Mean size) + c(Age -- t), where Mean size is the mean vessel size, whereas for age above the change point, the model is Flow-mediated dilation = a + b(Size - Mean size) + d(Age - t) . The "most likely" change point t m~x produced the minimal residual standard deviation rTto„~x over a range of possible change points from 30 to 65 years . To summarize the support for each possible change point I, the "profile likelihood" o,,,,,,,/o,1 was plotted, which has maximal I at t = as derived from a likelihood ratio test procedure (18) . To validate the twostraight-line model compared with a smooth curve, a quadratic function in age was also fitted to the data, the calculated residual standard deviation was smaller for the two-straight-line model . Men and women were compared with respect to the gradient of adjusted flow-mediated dilation with age, both before and after their estimated change point, using a Z test based on their estimated standard errors. For glyceryl trinitrate, the regression coefficient b arose from a simple linear regression on vessel size ; that is, Glyceryl trinitrate = a + b(Size - Mean size) . For each subject, flow-mediated and glyceryl trinitrateinduced dilation were adjusted for vessel size by subtracting b(Size - Mean size), and these adjusted values were then plotted against age .
Results The age distribution of the subjects and their vascular responses to increased flow and to glyceryl trinitrate are shown in Figures 1 and 2 . Scans from all 238 subjects were of sufficient quality for analysis, and no plaques were observed in any subject . Total plasma cholesterol was 191 ± 39 mg/dl (range 101 to 299) . and there was a positive correlation between age and cholesterol level (r = 0 .58, p < 0 .01). No subjects were hypertensive (blood pressure < 160/90 mm Hg in all) . In response to an increase in flow of 487 ± 172% after cuff deflation, there was an increase in brachial artery diameter of 7 .9 ± 3 .9% (range - 1% to 18%) . There was no correlation between age and degree of reactive hyperemia (r = -0 .06, p = 0 .49) or between flow-mediated dilation and hyperemia (r - 0.09, p = 0.16). In response to glyceryl trinitrate, arterial diameter increased by 19 .4 ± 5 .8% (range 4% to 38%) . On multivariate analysis of all subjects, flow-mediated dilation was independently related to age (r = -0 .34, p < 0.0001) and vessel size (r = -0 .35, p < 0 .0001) but not to the cholesterol level (r = -0 .09, p = 0.14) . In contrast, although the glyceryl trinitrate response was also related to vessel size
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FMD adjusted for vessel size
Figure 1 . Aging and flowmediated dilation (FMD) in 103 men (left) and 135 women (right) . Top, Individual data points for percent flow-mediated dilation adjusted for vessel size . Bottom, of a change .,a point existing at any given age ; this is isting highest for men at age 41 years and for women at 53 years. In women, there is a steeper decline in flow-mediated dilation after their change point .
10
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so
40
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Age (years)
- 0.45, p < 0 .0001), there was no relation with age -0.10, p -- 0 .18) (Table 1) . Gender difference, In men, flow-mediated dilation (adjusted for vessel size) did not decline until the end of the fourth decade . Change-point analysis suggested that an age-related decline occurred subsequently, from -41 years of age, after which flow-mediated dilation decreased by
Figure 2 . Aging and glyceryl trinitrate (M)-induced dilation in 102 men (top) and 129 women (bottom) . The response to glyceryl trinitrate is preserved with increasing age in both genders . Seven of the 238 subjects had a decline in glyceryl trinitrate . GTN adjusted for vessel size
10
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40 Age yeas)
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GTN adjusted for vessel size
io
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FMD adjusted for vessel size
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0 .21%/year . In contrast, flow-mediated dilation was preserved in women until the early 50s (most likely change point at age 53 years), after which it declined significantly faster than in men (0 .49%)year, p = 0 .002) . There was a very narrow range in the age at menopause in this study, and separate analysis of years since menopause versus flowmediated dilation in women yielded a similar slope of decline . In contrast, there was no age-related decline in glyceryl trinitrate-induced dilation in either gender .
Discussion This study showed that aging is associated with progressive loss of flow-mediated dilation in the systemic arteries of healthy subjects with no risk factors for vascular disease . In contrast, responses to glyceryl trinitrate are preserved into old age, and this suggests that aging leads to endothelial dysfunction . Because there is no age-related change in the hyperemic flow response, it is unlikely that progressive proximal atherosclerosis could explain the arterial vasodilator abnormalities observed in this study . We also observed that the pattern of age-related changes in arterial physiology is different in men and women . In men, endothelial responses decline gradually after the fourth decade, whereas in women, vascular physiology remains healthy for a further 10 years . Subsequently, the rate of decline is faster in women, so that endothelial dysfunction is present in almost all subjects by 65 years of age . This pattern of vascular damage parallels the known difference between the genders in cardiovascular morbidity and mortality in middle and old age (1,19), where men are at much higher risk from age 40 to 50 years, but women "catch up" in later life . The process of atherosclerosis does not appear to differ
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Table 1 . Results of Multiple Regression Analysis for Determinants of Flow-Mediated Dilation (an endothelium-dependent response) and Glyceryl Trinitrate-Induced Dilation (an endothelium-independent response) FMD
Age (yr) Vessel size (mm) Cholesterol (10 mgfdl)
Coefficient (SD)
Partial Correlation Coefficient
. (0.02) -2 .13 (0.33) -0 .09 (0 .06)
-0.34 -0 .35 -0 .09
GIN
P Valve
Coefficient (SD)
Partial Correlation Coefficient
p Value
< 0.0001 < O.Ot0I 0.14
-0.03 (0.02) -4 .01(0.54) --0 .08 (0 .10)
-0.10 -0 .45 -0 .06
0 .18 < 0 .11 0 .41
FMD = flow-mediated dilation ; GIN ® glyceryl trinitrate-induced response,
between men and women, and the risk factors known to predispose to vascular disease affect both genders equally (20) . We have previously shown that this noninvasive method can detect flow-mediated dilation in healthy subjects and that risk factors such as hypercholesterolemia (21) and cigarette smoking (22) are associated with abnormal endothelial physiology in both male and female children and young adults . Aging is associated with an increasing prevalence of diabetes, hypertension and hypercholesterolemia, and in the previous studies of both Vita et al . (11) and Yasue et al . (12) . in which coronary endothelial dysfunction was found in most subjects >30 years of age, risk factors may have had an important confounding effect . Study furltations. In this study, by excluding all subjects who had ever smoked or who had known hyperlipidemia, hypertension, diabetes or a positive family history, we investigated the effects of aging per se on vasomotor responses and compared endothelial function in male and female subjects Previous studies enrolled too few patients to examine any gender differences in the effects of aging on endothelial function (11, 12) . The method we used to study endothelial and smooth muscle function is noninvasive and well tolerated, allowing investigation of a large number of subjects with neither risk factors nor symptoms . The accuracy of 7 .0-MHz ultrasound is sufficient to allow detection of small diameter differences (as little as 0 .1 mm), and the reproducibility of the technique has been established previously (8,22,23) . Although only superficial systemic arteries can be studied, atherosclerosis is a diffuse process, and there is a correlation between the presence of disease in different large arteries in the same subject (24) . Recently two groups have reported that brachial artery vasomotor responses are well correlated with coronary endothelial physiology and coronary atherosclerosis (25,26) . and the ovascular system . Aging is associated with a number of anatomic and hemodynamic changes in the vascular system of all subjects, including collagen degeneration, loss of elastin, increased intima-media thickness of the arteries and reduced vascular compliance (27,28) . Despite these age-related changes, the arterial response to glyceryl trinitrate was preserved in our study . This is consistent with other studies that have shown that pharmacologic doses of
exogenous nitrovasodilators remain effective in older adults (29,30) and that these drugs are still therapeutically effective in geriatric patients (16) . In contrast, we observed an agerelated decline in endothelial function in both men and women . Previous investigators have shown that the dilator capacity of both large and small coronary vessels in response to acetylcholine, an endothelium-dependent vasodilator, also decreases with increasing age in subjects with chest pain but angiographically smooth coronary arteries (11,12,31) . The mechanism whereby aging is associated with impaired endothelial function is not known . Possibilities include an age-related decrease in release of endotheliumderived relaxing factor(s), or their increased catabolism in the vessel wall, or increased release of constricting factors (31) . In vitro aging is associated with increased monocyteendothelial cell adherence, which may result from agerelated increases in peroxidative stress (32) . Our data suggest that women may be protected from these age-related changes for a decade longer than men . Gender differences . This gender difference in the pattern of age-related decline in endothelial function is best explained by the influence of hormonal factors . Estrogens may protect women from atherogenesis (33,34) . After the menopause, decreased estrogen levels are associated with a more atherogenic lipid profile (19,35) ; however, the effects of estrogen on plasma lipids can explain only a small part of the beneficial effects on the risk of vascular disease (36) . Estrogens are vasoactive hormones and have direct effects on the arterial wall (37,38). In experimental animals, surgically induced menopause is associated with a loss of endotheliumdependent coronary vasorelaxation (39) . In postmenopausal animals, estrogen replacement may restore normal endothelial function (6) . Intravenous estrogen acutely improves endothelium-dependent vasorelaxation in postmenopausal women with coronary artery disease (40), and sublingual estrogen has a beneficial effect on myocardial ischemia in similar patients (41) . In our study, the age at which endothelial dysfunction was seen in women was around the time of the menopause . These data therefore suggest, but do not prove, that estrogens may protect against endothelial injury . Injury of the endothelium is important both because it is a key early event in atherogenesis and because it may
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CELERMAJER ET AL . VASCULAR AGING IN MEN AND WOMEN
increase risk in subjects with established atherosclerosis by predisposing to vasoconstriction and thrombosis (9) . The recent demonstration that L-arginme, the precursor of endothelium-derived relaxing factor, not only protects the endothelium but also retards atherosclerosis in the cholesterol-fed rabbit suggests that endothelial dysfunction may be intimately involved in the pathogenesis of atherotna (42) . Clinical i plications . Cardiovascular disease is the most common cause of death in the United States, and the known gender difference in life expectancy is largely related to the increased risk of coronary and carotid atherosclerotic complications in middle-aged men, Our data suggest that aging per se, irrespective of exposure to known risk factors, may be associated with earlier physiologic abnormalities in the systemic arteries of men compared with women . This is consistent with observations that both old age and mate gender arc independent risk factors for cardiovascular mortality and that estrogens may protect women from the age-related decline in vascular health . The association between menopause and the subsequent change in endothelial function that we have observed is provocative, but prospective studies will be required to see whether hormone replacement therapy prevents or retards the age-related changes in arterial physiology in postmenopausal women . Many such women are potentially candidates for hormone replacement therapy, not just for well-being and prevention for osteoporosis but also for protection of the arterial wall and reduction of vascular risk. The availability of a noninvasive method to assess ",ascular health will allow investigation of the appropriate type and timing of such hormonal therapy, both in individual subjects and in clinical trials .
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