Am J Physiol Heart Circ Physiol 313: H890 –H895, 2017.

First published September 29, 2017; doi:10.1152/ajpheart.00416.2017.

RAPID REPORT Integrative Cardiovascular Physiology and Pathophysiology

Endothelial cell senescence with aging in healthy humans: prevention by

habitual exercise and relation to vascular endothelial function
Matthew J. Rossman,1 Rachelle E. Kaplon,1 Sierra D. Hill,1 Molly N. McNamara,1
Jessica R. Santos-Parker,1 Gary L. Pierce,2 Douglas R. Seals,1 and Anthony J. Donato3
1
Integrative Physiology, University of Colorado Boulder, Boulder, Colorado; 2Health and Human Physiology, University of
Iowa, Iowa City, Iowa; and 3Internal Medicine, University of Utah, Salt Lake City, Utah
Submitted 6 July 2017; accepted in final form 22 September 2017

Rossman MJ, Kaplon RE, Hill SD, McNamara MN, Santos- AGING is the primary risk factor for cardiovascular disease
Parker JR, Pierce GL, Seals DR, Donato AJ. Endothelial cell (CVD) (3, 14), which remains the leading cause of morbidity
senescence with aging in healthy humans: prevention by habitual and mortality in modern societies (3). Increased CVD risk with
exercise and relation to vascular endothelial function. Am J Physiol age is largely a consequence of the development of vascular
Heart Circ Physiol 313: H890 –H895, 2017. First published Septem-
endothelial dysfunction, as measured by a decline in endothe-
ber 29, 2017; doi:10.1152/ajpheart.00416.2017.—Cellular senescence
is emerging as a key mechanism of age-related vascular endothelial lium-dependent dilation (EDD) (26). Accordingly, the decline
dysfunction, but evidence in healthy humans is lacking. Moreover, the in EDD with sedentary aging is an independent predictor of
influence of lifestyle factors such as habitual exercise on endothelial future cardiovascular events (32). However, the molecular
cell (EC) senescence is unknown. We tested the hypothesis that EC events responsible for the adverse changes in vascular endo-
senescence increases with sedentary, but not physically active, aging thelial function with aging are not completely understood.
and is associated with vascular endothelial dysfunction. Protein ex- Cellular senescence, the essentially irreversible growth ar-
pression (quantitative immunofluorescence) of p53, a transcription rest of a cell (19), is emerging as a fundamental process
factor related to increased cellular senescence, and the cyclin-depen- contributing to physiological dysfunction with aging and as a
dent kinase inhibitors p21 and p16 were 116%, 119%, and 128% driver of numerous age-related pathologies, including CVD (1,
greater (all P ⬍ 0.05), respectively, in ECs obtained from antecubital
2, 9, 13, 16, 28). Cellular stressors such as DNA damage,
veins of older sedentary (60 ⫾ 1 yr, n ⫽ 12) versus young sedentary
(22 ⫾ 1 yr, n ⫽ 9) adults. These age-related differences were not dysfunctional telomeres, oxidative stress, or metabolic stimuli
present (all P ⬎ 0.05) in venous ECs from older exercising adults act via cellular signaling cascades to activate p53/p21 and p16
(57 ⫾ 1 yr, n ⫽ 13). Furthermore, venous EC protein levels of p53 tumor suppressor pathways to induce cell cycle arrest (19). The
(r ⫽ ⫺0.49, P ⫽ 0.003), p21 (r ⫽ ⫺0.38, P ⫽ 0.03), and p16 adverse consequences of increased cellular senescence are
(r ⫽ ⫺0.58, P ⫽ 0.002) were inversely associated with vascular mediated by the accumulation of senescent cells in tissues (28)
endothelial function (brachial artery flow-mediated dilation). Simi- as well as the proinflammatory and prooxidative senescence-
larly, protein expression of p53 and p21 was 26% and 23% higher associated secretory phenotype (SASP) of these cells, which
(both P ⬍ 0.05), respectively, in ECs sampled from brachial arteries can impair function of neighboring cells (1, 2, 11). As such,
of healthy older sedentary (63 ⫾ 1 yr, n ⫽ 18) versus young sedentary genetic clearance of senescent cells reverses multiple aspects
(25 ⫾ 1 yr, n ⫽ 9) adults; age-related changes in arterial EC p53 and of age- and disease-associated physiological dysfunction in
p21 expression were not observed (P ⬎ 0.05) in older habitually
exercising adults (59 ⫾ 1 yr, n ⫽ 14). These data indicate that EC
progeroid (1) and naturally aged (2, 13) mice and extends
senescence is associated with sedentary aging and is linked to endo- healthy lifespan (2).
thelial dysfunction. Moreover, these data suggest that prevention of Considerable in vitro and preclinical data support a delete-
EC senescence may be one mechanism by which aerobic exercise rious impact of senescence on vascular endothelial cell (EC)
protects against endothelial dysfunction with age. function. For example, ECs cultured to senescence exhibit
NEW & NOTEWORTHY Our study provides novel evidence in
reduced generation of the key vasodilatory and vasoprotective
humans of increased endothelial cell senescence with sedentary aging, molecule nitric oxide (NO) as well as increased production of
which is associated with impaired vascular endothelial function. reactive oxygen species (10, 15, 24, 31, 33). Expression of
Furthermore, our data suggest an absence of age-related increases in markers of senescence, including p21 and p16, are increased in
endothelial cell senescence in older exercising adults, which is linked arterial tissue from old mice, which is associated with oxida-
with preserved vascular endothelial function. tive stress-mediated suppression of NO-dependent vascular
Listen to this article’s corresponding podcast at http://ajpheart. endothelial function (4). Moreover, there is evidence that
podbean.com/e/aging-exercise-and-endothelial-cell-senescence/. treatment with “senolytics,” pharmaceuticals intended to selec-
cell cycle arrest; senescence-associated secretory phenotype; cardio- tively induce senescent cell death as well as genetic clearance
vascular disease of p16-expressing cells improves vascular function in old mice
(23, 34). Limited data from humans demonstrate that senescent
ECs and p16 expression are increased in atherosclerotic
plaques from patients with CVD (12, 16), and p53 and p21
Address for reprint requests and other correspondence: M. J. Rossman,
gene transcript levels are elevated in whole artery lysates,
Dept. of Integrative Physiology, Univ. of Colorado Boulder, 354 UCB, primarily composed of vascular smooth muscle cells, from
Boulder, CO 80309 (e-mail: [email protected]). healthy older adults (18). However, direct evidence for in-
H890 0363-6135/17 Copyright © 2017 the American Physiological Society http://www.ajpheart.org
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 AGING, EXERCISE, AND ENDOTHELIAL CELL SENESCENCE H891
creased senescence with aging in the vascular endothelium and enrolled in one of the following studies from our laboratory (6, 21, 22,
an impact on endothelial function in humans is lacking. 29) and, as such, subject characteristics data from these participants
Regular aerobic exercise preserves endothelial function with have been previously published. Twelve sedentary and 13 exercising
aging (8) and reduces CVD risk (5, 17). Preclinical data middle-aged and older adults as well as 9 young sedentary adults in
whom maximal O2 consumption (V̇O2max) and vascular endothelial
indicate that exercise decreases p53 and p16 protein expres-
function (FMD) had been assessed and who met the prespecified
sion in aortic lysates from young mice (30). Additionally, criteria were identified for venous EC analyses. To confirm that
exercise protects against high-fat diet-induced senescence in group-related changes observed in venous ECs were also present in
mice in adipose tissue, which is associated with improved arterial cells, arterial ECs were studied from a separate cohort from
health and physiological function (25). How aerobic exer- our database consisting of 18 sedentary and 14 exercising middle-
cise may modulate vascular EC senescence with aging is aged and older adults as well as 9 young sedentary adults. Because of
currently unknown. the more invasive nature of arterial EC collection, sample availability
In the present study, we tested the hypothesis that ECs was limited in participants in whom brachial artery FMD and V̇O2max
become senescent with sedentary but not physically active had been assessed. In addition, a sufficient arterial EC sample was
aging in healthy adults and that markers of EC senescence are only available for the assessment of the p53/p21 pathway.
Participants were nonobese (body mass index ⬍30 kg/m2), non-
associated with vascular endothelial dysfunction. To test this
smokers, nondiabetic, and free of other clinical diseases as determined
hypothesis, we measured protein expression of multiple senes- by medical history, physical examination, blood chemistry, and rest-
cence markers including p53, p21, and p16 in ECs obtained ing and exercise 12-lead ECG. Blood pressure, fasting circulating
from the antecubital veins and brachial arteries of young and lipids, and glucose were clinically normal in all participants. Seden-
older sedentary adults as well as older aerobic exercise-trained tary participants performed no regular aerobic exercise (i.e., ⬍30
adults. EDD was characterized by brachial artery flow-medi- min/day, ⱕ2 days/wk) for at least the preceding 2 yr. Exercise-trained
ated dilation (FMD), a clinically important measure of vascular participants performed regular vigorous aerobic exercise including
endothelial function. competitive cycling, running, and/or triathlons ⱖ5 days/wk for ⱖ45
min/session for at least the preceding 5 yr. All procedures were
MATERIALS AND METHODS approved by the Institutional Review Board of the University of
Colorado Boulder. The nature, benefits, and risks of the study were
Participants. A total of 75 healthy adults (Table 1) enrolled in explained to the volunteers, and their written informed consent was
studies during a 3-yr period were identified in our laboratory database obtained before participation.
according to prespecified age (young: 18 –30 yr and middle-aged and Measurements. All measurements were performed at the University
older: 50 –79 yr) and exercise status (sedentary or exercise trained) of Colorado Boulder Clinical Translational Research Center after a
criteria for this retrospective analysis. These subjects were previously 12-h overnight fast and 24-h abstention from alcohol and physical
exercise.
Participant characteristics. Body mass index and total body fat as
Table 1. Subject Characteristics
well as resting arterial systolic and diastolic blood pressures were
Young Older Older determined as previously described (7, 21). Leisure time physical
Sedentary Sedentary Trained activity was determined by the modifiable activity questionnaire (20).
V̇O2max was measured during incremental treadmill exercise testing
n 18 30 27
Men/women 13/5 21/9 20/7 performed to exhaustion (Balke protocol).
Age, yr 23 ⫾ 1 62 ⫾ 1* 59 ⫾ 1* EC protein expression. EC collection and protein expression mea-
(18–30) (51–75) (50–76) surements were performed as previously described (7, 21). Briefly,
Mass, kg 75 ⫾ 4 79 ⫾ 3 70 ⫾ 3† J-wires were advanced into an antecubital vein or brachial artery, and
(51–82) (56–98) (54–81) cells were recovered by washing and centrifugation. Collected cells
Body mass index, kg/m2 24 ⫾ 1 26 ⫾ 1 23 ⫾ 1† were fixed with 3.7% formaldehyde and plated on poly-L-lysine-
(20–28) (19–28) (20–25) coated slides (Sigma Chemical, St. Louis, MO) and then frozen at
Body fat, % 23 ⫾ 3 29 ⫾ 3* 23 ⫾ 2† ⫺80°C until analysis. At the time of analysis, cells were rehydrated
(14–30) (23–35) (15–29)
and incubated with primary antibodies against p53 (1:200, Cell
Leisure time physical activity, 39 ⫾ 11 32 ⫾ 6 83 ⫾ 9*†
MET h/wk (6–57) (10–55) (67–97) Signaling, Danvers, MA), p21 (1:500, Abcam, Cambridge, MA), and
Maximal O2 consumption, 44 ⫾ 2 34 ⫾ 2* 45 ⫾ 2† p16 (1:200, Abcam) along with Alexa Fluor 647 fluorescent second-
ml·kg⫺1·min⫺1‡ (29–54) (25–40) (32–55) ary antibody (Invitrogen, Carlsbad, CA). Cells were stained for
Total cholesterol, mg/dl 158 ⫾ 7 187 ⫾ 7* 187 ⫾ 9* vascular-endothelial (VE)-cadherin (1:500, Abcam) for positive iden-
(115–184) (145–209) (141–228) tification of the endothelial phenotype and DAPI for nuclear integrity.
HDL-cholesterol, mg/dl 45 ⫾ 4 52 ⫾ 3 56 ⫾ 5 Slides were viewed with a fluorescence microscope (Eclipse Ni-U,
(30–65) (36–70) (42–89) Nikon, Melville, NY), and whole cell fluorescence was assessed using
LDL-cholesterol, mg/dl 95 ⫾ 6 122 ⫾ 10* 112 ⫾ 6 Metamorph Software (Molecular Devices, Sunnyvale, CA). EC pro-
(54–117) (72–160) (73–140)
tein expression data are reported as ratios to human umbilical vein EC
Triglycerides, mg/dl 94 ⫾ 8 114 ⫾ 15 91 ⫾ 7
(50–131) (49–150) (52–119) protein expression. All EC sampling occurred during subjects’ initial
Systolic blood pressure, mmHg 114 ⫾ 5 121 ⫾ 3 119 ⫾ 3 visits to our laboratory; protein expression measurements were per-
(104–121) (98–141) (96–143) formed on EC slides that had been stored at that time for future use.
Diastolic blood pressure, mmHg 69 ⫾ 2 78 ⫾ 1* 75 ⫾ 2 Vascular endothelial function. Brachial artery FMD was deter-
(58–80) (63–93) (59–92) mined using duplex ultrasonography (PowerVision 6000, Toshiba,
Fasting blood glucose, mg/dl 78 ⫾ 4 87 ⫾ 2* 81 ⫾ 3† multifrequency linear-array transducer) as previously described (7, 8).
(75–99) (70–100) (60–95) FMD is expressed as the percent change in diameter from baseline.
Data are means ⫾ SE with ranges for relevant variables in parentheses; n, Data analysis. Statistical analyses were performed with SPSS
number of subjects. *P ⬍ 0.05 vs. the young sedentary group; †P ⬍ 0.05 vs. Statistics (IBM, Chicago, IL). Sample size estimates were based on
the older sedentary group. ‡Maximal O2 consumption was only assessed in preliminary analysis (n ⫽ 4 subjects/group) of venous and arterial EC
participants from whom venous endothelial cells were obtained. p21 expression levels in sedentary young and older adults. These

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H892 AGING, EXERCISE, AND ENDOTHELIAL CELL SENESCENCE

estimates indicated that 9 subjects/group would be adequate to detect

group differences with ⬎80% power; we increased the number of
older adults to be conservative and to account for additional variance
associated with EC protein expression in older adults. Main effects
were determined by one-way ANOVA, with least significant differ-
ences post hoc analyses used to determine prespecified differences
between specific groups. A general linear model with covariates and
least significant difference post hoc analysis was used to evaluate the
influence of participant characteristics that were significantly different
between groups within each cohort of participants (i.e., in the cohort
from whom venous ECs were collected and in the cohort from whom
arterial ECs were collected). Bivariate relations were assessed by
Pearson correlation analysis. Statistical significance was set at ␣ ⬍
0.05. All data are presented as means ⫾ SE.
RESULTS

Participant characteristics. There were no significant within

group differences between participants from whom venous or
arterial cells were collected, so participant characteristics data
were pooled (Table 1). Physical activity levels were similar
between older sedentary and young adults but were higher
(P ⬍ 0.05) in older exercising adults. V̇O2max, a measure of
maximal aerobic exercise capacity, was lower (P ⬍ 0.05) in
older sedentary adults but not different between young seden-
tary and older exercising adults.
Clinically normal values for body weight, body mass index,
blood pressure, and circulating factors were observed in all
groups (Table 1). However, modest differences were observed
with age in the participants from whom venous and arterial
ECs were collected, including increased body fat percentage as
well as total and LDL-cholesterol (all P ⬍ 0.05); diastolic
blood pressure and fasting blood glucose levels were also
increased with age, but only in the venous EC cohort (P ⬍
0.05). Additionally, in the participants from whom venous and
arterial ECs were collected, older exercising adults had lower
body mass, body mass index, and body fat percentage than
older sedentary adults (all P ⬍ 0.05); older exercising adults
from whom venous ECs were collected also had lower fasting
blood glucose levels than older sedentary adults (P ⬍ 0.05).
EC protein expression. Protein expression of p53, p21, and
p16 were 116%, 119%, and 128% greater (P ⬍ 0.05), respec-
tively, in venous ECs from older sedentary compared with
young adults (Fig. 1). Moreover, p53, p21, and p16 levels
were similar between young and older exercising adults.
These markers of senescence were 68%, 32%, and 71%
lower for p53, p21, and p16, respectively, in venous ECs
from older exercising adults compared with older sedentary
adults (Fig. 1).
Consistent with the observations in venous ECs, although
there were differences in the magnitude of group differences,
protein expression of p53 was 26% higher and p21 expression Fig. 1. Endothelial cell p53 (A), p21 (B), and p16 (C) protein expression in
was 23% greater (both P ⬍ 0.05) in arterial ECs from older endothelial cells sampled from antecubital veins of young sedentary (n ⫽ 9),
sedentary compared with young adults (Fig. 2). Arterial EC older sedentary (n ⫽ 12), and older exercising (n ⫽ 13) adults, with example
p53 and p21 protein expressions were 35% and 39% lower, immunofluorescent images below. Data are normalized to human umbilical
vein endothelial cell protein expression via immunofluorescence. Data are
respectively, in older exercising compared with sedentary older means ⫾ SE. *P ⬍ 0.05 vs. the young sedentary group; ‡P ⬍ 0.05 vs. the older
adults and similar to those of young adults (Fig. 2). sedentary group. AU, arbitrary units.
Group differences in p53, p21, and p16 EC protein expres-
sion remained significant (all P ⬍ 0.05) in venous ECs after
covarying for body mass, body mass index, body fat percent- the American Journal of Physiology-Heart and Circulatory
age, LDL-cholesterol, total cholesterol, diastolic blood pres- Physiology website). Group differences in p53 and p21 EC
sure, and fasting blood glucose (Table S1 in the Supplemental protein expression remained significant (all P ⬍ 0.05) in
Material; Supplemental Material for this article is available at arterial ECs after covarying for body mass, body mass index,

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 AGING, EXERCISE, AND ENDOTHELIAL CELL SENESCENCE H893
adults. Collectively, our findings may provide novel insights
into the molecular events underlying impaired endothelial
function and increased CVD risk with sedentary aging and the
protective effect of regular aerobic exercise.
EC senescence with age. In vitro and preclinical data impli-
cate increased cellular senescence as a driver of age-related
vascular endothelial dysfunction (4, 10, 15, 23, 24, 31, 33, 34).
To our knowledge, the present study is the first to evaluate
markers of cellular senescence in the vascular endothelium in
primary (i.e., noncultured) ECs obtained from human subjects.
These data extend preclinical observations and provide novel,
direct evidence of increased cellular senescence in ECs from

Fig. 2. Endothelial cell p53 (A) and p21 (B) protein expression in endothelial
cells sampled from brachial arteries of young sedentary (n ⫽ 9), older
sedentary (n ⫽ 18), and older exercising adults (n ⫽ 14), with example
immunofluorescent images below. Data are normalized to human umbilical
vein endothelial cell protein expression via immunofluorescence. Data are
means ⫾ SE. *P ⬍ 0.05 vs. the young sedentary group; ‡P ⬍ 0.05 vs. the older
sedentary group. AU, arbitrary units.

body fat percentage, LDL-cholesterol, and total cholesterol

(Table S1). Moreover, there were no within-group differences
between male and female subjects (P ⬎ 0.05).
Relation of EC expression of markers of senescence with
EDD. EDD, as assessed by brachial artery FMD, was reduced
in sedentary older adults compared with young adults (4.1 ⫾
0.7% vs. 8.9 ⫾ 0.9%, P ⬍ 0.05). Brachial artery FMD was
higher (P ⬍ 0.05) in older exercise-trained adults (9.0 ⫾ 0.6%)
compared with older sedentary adults and was not different
from young adults (P ⬎ 0.05). Protein expression of p53 (r ⫽
⫺0.49, P ⬍ 0.05), p21 (r ⫽ ⫺0.38, P ⬍ 0.05), and p16 (r ⫽
⫺0.58, P ⬍ 0.05) were all inversely associated with brachial
artery FMD (Fig. 3).
DISCUSSION

To our knowledge, our data provide the first direct evidence

that older sedentary, but not habitually exercising, adults ex-
hibit greater levels of EC senescence. Moreover, our data
indicate that vascular EC senescence is inversely associated Fig. 3. Inverse relation between endothelium-dependent dilation (brachial
artery flow-mediated dilation) and p53 (A), p21 (B), and p16 (C) protein
with a clinically relevant assessment of vascular endothelial expression in endothelial cells obtained from venous samples in a pooled group
function, suggesting that EC senescence may contribute to of young sedentary, older sedentary, and older exercising adults. AU, arbitrary
impaired vascular endothelial function with aging in healthy units.

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H894 AGING, EXERCISE, AND ENDOTHELIAL CELL SENESCENCE

healthy older adults. Specifically, our data show increased p53 present study are associational in nature and do not establish
and p21 expression in biopsied venous and arterial ECs and causation.
elevated p16 expression in venous ECs, indicative of increased Conclusions. The results of the present study provide novel
activation of the p53/p21 and p16 tumor suppressor pathways evidence in humans for increased EC senescence with healthy
(19). Collectively, our data indicate that EC senescence in- aging and a lack of increase in older adults who perform
creases with healthy aging, even in the absence of overt clinical habitual aerobic exercise. Additionally, our data suggest a link
vascular disease. between EC senescence and vascular endothelial dysfunction
EC senescence with age and exercise status. In contrast to and indicate that a reduction in endothelial senescence may be
their sedentary peers, older exercising adults did not exhibit one mechanism by which regular aerobic exercise affords
age-related increases in p53, p21, or p16 expression. These protection against increased CVD risk with aging.
data build on preclinical data indicating that exercise reduces
aortic protein expression of p53 and p16 in young mice (30) ACKNOWLEDGMENTS
and prevents the increase in adipose tissue senescence accom- The authors thank the staff of the University of Colorado Boulder Clinical
panying high-fat feeding (25). More broadly, these data sug- and Translational Research Center for technical assistance.
gest that habitual aerobic exercise preserves a healthy pheno- GRANTS
type in the vascular endothelium. This notion is in agreement
This work was supported by National Institute on Aging Awards AG-
with data from our laboratory showing reduced oxidative and 053009, AG-013038, AG-022241, AG-006537, AG-029337, AG-000279, AG-
inflammatory signaling in ECs obtained from older exercising 044031, AG-045339, AG-050238, and AG-053131.
adults compared with sedentary older adults (21, 29). Collec-
tively, these data may provide evidence for a novel molecular DISCLAIMERS
mechanism, reduced EC senescence, underlying the vascular Contents do not necessarily represent official National Institutes of Health
protective effects of aerobic exercise with aging, which may views.
contribute to reduced risk of CVD.
DISCLOSURES
Relation of EC senescence with vascular endothelial
function. The participants in the present study exhibited the No conflicts of interest, financial or otherwise, are declared by the author(s).
expected age-related reduction in EDD with sedentary aging, AUTHOR CONTRIBUTIONS
which was not present in older exercising adults (8, 26).
Importantly, the age- and exercise-related changes in EDD M.J.R., R.E.K., D.R.S., and A.J.D. conceived and designed research;
M.J.R., S.D.H., M.N.M., and A.J.D. performed experiments; M.J.R., S.D.H.,
were associated with EC senescence. These data suggest that M.N.M., and A.J.D. analyzed data; M.J.R., R.E.K., J.R.S.-P., and A.J.D.
senescence occurs in the vascular endothelium with sedentary interpreted results of experiments; M.J.R. prepared figures; M.J.R. drafted
aging and impairs vascular endothelial function. Moreover, in manuscript; M.J.R., R.E.K., S.D.H., J.R.S.-P., G.L.P., D.R.S., and A.J.D.
the absence of age-associated changes in EC senescence, edited and revised manuscript; M.J.R., R.E.K., S.D.H., M.N.M., J.R.S.-P.,
G.L.P., D.R.S., and A.J.D. approved final version of manuscript.
vascular endothelial function is preserved with healthy aging.
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