Am J Physiol Heart Circ Physiol 314: H1053–H1060, 2018.
First published December 22, 2017; doi:10.1152/ajpheart.00472.2017.
RESEARCH ARTICLE Advances in Cardiovascular Geroscience
Telomerase reverse transcriptase protects against angiotensin II-induced
microvascular endothelial dysfunction
Karima Ait-Aissa,1,2* X Andrew O. Kadlec,1,2* Joseph Hockenberry,1 X David D. Gutterman,1
and X Andreas M. Beyer1,2,3
1
Department of Medicine, Cardiovascular Center, Medical College of Wisconsin, Milwaukee, Wisconsin; 2Department of
Physiology, Medical College of Wisconsin, Milwaukee, Wisconsin; and 3Redox Biology Program, Medical College of
Wisconsin, Milwaukee, Wisconsin
Submitted 25 July 2017; accepted in final form 18 December 2017
Ait-Aissa K, Kadlec AO, Hockenberry J, Gutterman DD, Beyer
AM. Telomerase reverse transcriptase protects against angiotensin
II-induced microvascular endothelial dysfunction. Am J Physiol Heart
Circ Physiol 314: H1053–H1060, 2018. First published December 22,
2017; doi:10.1152/ajpheart.00472.2017.—A rise in reactive oxygen
species (ROS) may contribute to cardiovascular disease by reducing
nitric oxide (NO) levels, leading to loss of NO’s vasodilator and
anti-inflammatory effects. Although primarily studied in larger con-
duit arteries, excess ROS release and a corresponding loss of NO also
occur in smaller resistance arteries of the microcirculation, but the
underlying mechanisms and therapeutic targets have not been fully
characterized. We examined whether either of the two subunits of
telomerase, telomerase reverse transcriptase (TERT) or telomerase
RNA component (TERC), affect microvascular ROS production and
peak vasodilation at baseline and in response to in vivo administration
to angiotensin II (ANG II). We report that genetic loss of TERT
[maximal dilation: 52.0 ⫾ 6.1% with vehicle, 60.4 ⫾ 12.9% with
N␻-nitro-L-arginine methyl ester (L-NAME), and 32.2 ⫾ 12.2% with
polyethylene glycol-catalase (PEG-Cat) (P ⬍ 0.05), means ⫾ SD, n ⫽
9 –19] but not TERC [maximal dilation: 79 ⫾ 5% with vehicle,
10.7 ⫾ 9.8% with L-NAME (P ⬍ 0.05), and 86.4 ⫾ 8.4% with PEG-
Cat, n ⫽ 4 –7] promotes flow-induced ROS formation. Moreover,
TERT knockout exacerbates the microvascular dysfunction resulting
from in vivo ANG II treatment, whereas TERT overexpression is
protective [maximal dilation: 88.22 ⫾ 4.6% with vehicle vs.
74.0 ⫾ 7.3% with ANG II (1,000 ng·kg⫺1·min⫺1) (P ⫽ not signifi-
cant), n ⫽ 4]. Therefore, loss of TERT but not TERC may be a key
contributor to the elevated microvascular ROS levels and reduced
peak dilation observed in several cardiovascular disease pathologies.
NEW & NOTEWORTHY This study identifies telomerase reverse
transcriptase (TERT) but not telomerase RNA component as a key
factor regulating endothelium-dependent dilation in the microcircula-
tion. Loss of TERT activity leads to microvascular dysfunction but not
conduit vessel dysfunction in first-generation mice. In contrast, TERT
is protective in the microcirculation in the presence of prolonged
vascular stress. Understanding the mechanism of how TERT protects
against vascular stress represents a novel target for the treatment of
vascular disorders.
angiotensin II; flow-mediated dilation; microcirculation; telomerase;
telomerase reverse transcriptase
* K. Ait-Aissa and A. O. Kadlec contributed equally to this work.
Address for reprint requests and other correspondence: A. M. Beyer, Dept.
of Medicine, Medical College of Wisconsin, 8701 Watertown Plank Rd.,
Milwaukee, WI 53226 (e-mail: abeyer@mcw.edu).
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INTRODUCTION
Approximately one in every three deaths can be attributed to
cardiovascular disease (22). Previous research seeking to
lessen the cardiovascular disease burden has predominantly
explored disease-related pathological changes (e.g., oxidative
stress, inflammation, and remodeling) occurring in endothelial
and smooth muscle cell layers of large conduit arteries. Small
resistance arteries of the microcirculation have received less
attention (10), despite provocative reports illustrating their
pathophysiological (17, 18, 20) and prognostic (5, 27, 28)
importance in cardiovascular disease.
A major role of the microvascular endothelium is regulation
of vascular tone. Microvascular dilation is elicited through the
release of a variety of endothelium-derived factors in response
to chemical agonists or increases in blood flow. Nitric oxide
(NO) is the dominant flow-induced vasodilator in the micro-
circulation of subjects free of cardiovascular disease, whereas
H2O2 replaces NO in patients with coronary artery disease
(20). Although H2O2 maintains dilation as NO levels drop,
chronic and unopposed release of mitochondria-derived H2O2
(mtH2O2) from the microvascular endothelium, together with
loss of anti-inflammatory actions of NO, is believed to elicit
proinflammatory changes that contribute to disease develop-
ment (6). Clarification of the cellular factors responsible for
this microvascular transition from NO to H2O2 is needed to
identify potential therapeutic targets to combat excessive reac-
tive oxygen species (ROS) production and lessen microvascu-
lar dysfunction.
Existing evidence has established that loss of telomerase is
closely associated with elevations in mitochondrial ROS pro-
duction. Telomerase reverse transcriptase (TERT), the catalytic
subunit of the telomerase holoenzyme, and the telomerase
RNA component (TERC) are critical for canonical telomerase
function (25). While TERT is largely studied in models of
aging and cancer because of its telomere-lengthening effects in
the nucleus, TERT also acts to counteract oxidative stress in
the mitochondria of the microcirculation (1). Short-term ex
vivo pharmacological inhibition of TERT increases, and short-
term ex vivo pharmacological activation of TERT decreases,
release of H2O2 during flow-mediated dilation (FMD) in hu-
man microvessels (2). The impact of prolonged, genetic loss of
TERT and TERC on microvascular dilation and ROS release is
unknown, as is the potential protective effect of TERT over-
expression in vivo on the microvascular effects of established
H1053
H1054 TELOMERASE REVERSE TRANSCRIPTASE AND THE MICROCIRCULATION
pathological factors that augment ROS formation in the mi-
crocirculation, such as elevated angiotensin II (ANG II). To
explore the role of increased or decreased telomerase activity
under pathological conditions, we used the well-defined model
of ANG II infusion to induce ROS formation and endothelial
dysfunction (4, 11, 13, 29).
Understanding these in vivo relationships deepens our un-
derstanding of the potential pathophysiological effect of re-
duced TERT and/or TERC in the microcirculation and pro-
vides valuable information about the therapeutic role of TERT
in the microcirculation in an in vivo setting.
We hypothesized that genetic loss of TERT or TERC in-
creases flow-induced release of H2O2 in both coronary and
mesenteric microcirculations of TERT and TERC knockout
(KO) mice. We further hypothesized that TERT overexpres-
sion [TERT transgenic (Tg)] limits, whereas loss of TERT
exacerbates, the damaging effects of in vivo ANG II adminis-
tration on microvascular dilation.
MATERIALS AND METHODS
Overview. To address our hypotheses, we used established genetic
gain-of-function and loss-of-function models for TERT (TERT Tg
and TERT KO) and a loss-of-function model for TERC (TERC KO).
Later-generation TERT KO animals were also used to assess the
effect of progressive loss of TERT on microvascular dilation. Micro-
vascular phenotypes were examined using ex vivo video microscopy
(overall dilation and contribution of NO vs. H2O2 to dilation), fluo-
rescence detection (H2O2 release during dilation), and Western blot
quantification of NO synthase (NOS) levels. We also evaluated the
response to the commonly used vascular stressor ANG II to determine
whether TERT modulates microvascular tone under pathological
conditions.
General protocol in mice. All experiments were performed accord-
ing to the American Guidelines for the Ethical Care of Animals and
were approved by our Institutional Animal Care and Use Committee.
C57BL/6 TERT KO (TERT⫺/⫺) mice were obtained from Ronald
DePinho (MD Anderson Cancer Center, Houston, TX). TERC KO
(TERC⫺/⫺) mice were purchased from Jackson Laboratories (stock
004132 mTR⫺/⫺, Bar Harbor, ME). KO mice were bred as heterozy-
gotes to generate first-generation KO mice. To generate third-gener-
ation KO mice, homozygous KO mice of the first generation were
inbred for three consecutive generations. TERT Tg mice were pro-
vided by Dennis Bruemmer (University of Pittsburgh, Pittsburgh,
PA). Both male and female mice were used in the experiments
outlined in this study, and all animals were studied at 3– 4 mo of age
and compared with wild-type (WT) control mice of the same breeding
line. When possible, arteries from the same mouse were used for
multiple experiments (e.g., functional experiments and ROS evalua-
tion).
All mice were housed and maintained at a temperature of 23°C
with 12:12-h light-dark cycles and fed a solid standard diet (Na⫹
content: 0.4%) and water ad libitum. On the day of the experiment,
mice were euthanized between 8 AM and 12 PM, and tissues
(thoracic aorta, mesenteric resistance artery, and septal artery)
were harvested and immediately placed in cold 4°C HEPES (con-
taining 275 mM NaCl, 7.99 mM KCl, 4.9 mM MgSO4, 3.2 mM
CaCl2·2H2O, 2.35 mM KH2PO4, 0.07 mM EDTA, 12 mM glucose,
and 20 mM HEPES acid).
Cannulated arteriole preparation. Third- or fourth-order branch
arteries from the mesenteric artery (MA) and septal artery (SA)
(~200-␮m inner diameter) were cleaned of fat and connective tissue
and prepared for continuous measurements of internal diameter as
previously described (20, 21). In an organ chamber containing Krebs
buffer, both ends of the vessel were cannulated with glass micropi-
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pettes and pressurized (60 mmHg) before stepwise increases in flow
or administration of pharmacological agents, as we have previously
described (14, 19, 23).
Vascular response to flow and pharmacological interventions. The
contribution of NO or H2O2 to dilation was assessed in TERT KO,
TERC KO, and TERT Tg mice using FMD as previously described by
Kuo et al. (16). Adjustment of the height of each reservoir in equal
and opposite directions was used to generate flow via generation of a
pressure gradient without changes in intraluminal pressure (16). Di-
ameter at a given pressure gradient was recorded and expressed as
percent maximal diameter. Two flow-response curves were generated
for each vessel comparing untreated (vehicle) with effects of pharma-
cological inhibitors [NO inhibitor N␻-nitro-L-arginine methyl ester
(L-NAME; 100 mM) and H2O2 scavenger polyethylene glycol-cata-
lase (PEG-Cat; 500 U/ml)]. All pharmacological agents represent final
concentrations in the organ bath and were added at volumes of ⬍1%
of the total bath volume.
Arteries were constricted with norepinephrine (10 ␮M for MA) or
the thromboxane A2 analog U-46619 (100 nM for SA) to achieve a
20 –70% stable reduction in passive diameter. Dose-response curves
to acetylcholine (ACh; 1 nM⫺10 mM) and flow gradient (5–100
cmH2O) were performed to evaluate endothelium-dependent dilation.
At the end of each experiment, the endothelium-independent dilator
papaverine (Pap; 100 ␮M) was used to determine the maximal
(passive) diameter at 60 mmHg.
Measurement of mitochondrial ROS. Mito Peroxy Yellow 1
(mitoPY1) (7) was used as a secondary means to assess microves-
sel generation of mtH2O2 after loss of TERT. After cannulation in
a warmed chamber (37°C) containing HEPES buffer at pH 7.4,
MAs from TERT KO mice or from WT mice were perfused
intraluminally with mitoPY1 (5 ␮M, 1 h) at low levels of flow
below the threshold for dilation until the luminal surface was
bathed in mitoPY1-containing buffer. Next, the pressure gradient
was changed to 0 –100 cmH2O. Experiments were performed in the
presence or absence of the H2O2 scavenger PEG-Cat (500 U/ml).
Fluorescence was evaluated with a Nikon Eclipse TE200 micro-
scope using a krypton/argon laser at excitation wavelength of 488
nm and measured emission between 530 and 590 nm. For any
given experiment, baseline fluorescence was established without
flow. After 30 min of incubation with mitoPY1, each vessel was
compared with no-flow conditions as a measure of flow-induced
H2O2 production. In separate experiments, the specificity of the
probe was confirmed with PEG-Cat (H2O2 scavenger) present at
any time during the experiment. Levels of mtH2O2 are expressed as
relative average fluorescence intensity normalized to background
fluorescence and presented as percent change from baseline. All
comparisons were made using arteries studied at the same session
with constant microscope image display settings.
Microvascular response to ANG II. Nonpressor dose (400
ng·kg⫺1·min⫺1) or fast pressor dose (1,000 ng·kg⫺1·min⫺1) of ANG
II as previously described (15) was administered via osmotic mini-
pumps for 14 days to induce prolonged in vivo microvascular stress in
WT, TERT KO, and TERT Tg mice.
Western blot analysis. Protein expression was analyzed as previ-
ously described (9). Briefly, total protein (30 ␮g) from MA (6 – 8
pooled arteries/mouse) lysates was loaded and separated using SDS-
PAGE and then transferred into a polyvinylidene difluoride mem-
brane. The membrane was incubated with one of the following
primary antibodies: rabbit monoclonal anti-phosphorylated (Ser1177)
endothelial NOS (eNOS; dilution 1:1,000, catalog no. MA5-14957,
Invitrogen), rabbit polyclonal anti-total eNOS (dilution 1:1,000, cat-
alog no. PA5-16887, Invitrogen), or mouse monoclonal anti-GAPDH
(dilution 1:10,000, catalog no. ab8245, Abcam). Protein bands were
detected with X-ray film exposure and quantified by ImageJ software.
Materials. Norepinephrine and ANG II were obtained from Sigma-
Aldrich, and U-46619 was obtained from Cayman Chemical. PEG-
Cat was made by Quanta BioDesign (Plain City, OH), and L-NAME
 TELOMERASE REVERSE TRANSCRIPTASE AND THE MICROCIRCULATION H1055
was purchased from Sigma-Aldrich. mitoPY1 (Tocris) was prepared concentration were determined using two-way repeated-measures
in DMSO, and other agents were prepared in distilled water or Krebs ANOVA. A post hoc Tukey test was used for comparison of ⬎2
buffer. All concentrations represent the final concentrations in the means after ANOVA. P values of ⬍ 0.05 were deemed statistically
organ bath. significant. For fluorescence experiments and changes in gene/protein
Statistical methods. Data are presented as means ⫾ SD. For all expression, either a t-test or one-way ANOVA with a post hoc Tukey
concentration-response curves, differences between groups at each test was used as appropriate.

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H1056 TELOMERASE REVERSE TRANSCRIPTASE AND THE MICROCIRCULATION

RESULTS To examine whether TERC similarly influences H2O2 re-

Loss of TERT affects the magnitude of microvascular but not
conduit vessel dilation in first-generation mice. To examine
whether the dilation in isolated arteries is impaired by the
genetic loss of telomerase, we compared the overall peak
dilation to flow (FMD) and to ACh in both mesenteric (MA)
and septal (SA) microvessels and in the aorta. The overall
magnitude of FMD was similar in MAs and SAs of TERT KO
and WT mice [Fig. 1, A and E; maximal dilation: 77.4 ⫾ 7.1%
in the WT-MA group (n ⫽ 18), 53.6 ⫾ 7.1% in the WT-SA
group (n ⫽ 7), 52.0 ⫾ 6.1% in the TERT KO-MA group (n ⫽
18), and 64.2 ⫾ 14.6% in the TERT KO-SA group (n ⫽ 5)].
The magnitude of overall peak dilation was similar between
male and female mice [maximal dilation: 68 ⫾ 17% in female
WT mice (n ⫽ 7) and 42 ⫾ 17% in TERT KO mice (n ⫽ 4) vs.
76 ⫾ 6% in male WT mice (n ⫽ 7) and 54 ⫾ 9% in TERT KO
mice (n ⫽ 4), P ⫽ not significant; data not shown]. After
intercrossing TERT KO mice to obtain third-generation mice,
microvascular vasodilation to flow in the MA was eliminated
(Fig. 1I).
ACh-induced dilation was reduced in the MA of TERT KO
mice (first generation) compared with WT mice (P ⬍ 0.05; Fig.
1J). In contrast, we found no difference in the magnitude of
overall peak ACh-induced dilation in conduit arteries (aorta) of
first-generation TERT KO versus WT mice (Fig. 1K).
Loss of TERT decreases NO-mediated dilation and promotes
H2O2 as a compensatory vasodilator during FMD. We deter-
mined whether genetic loss of TERT or TERC alters the
mechanism of FMD in MAs and SAs using a NOS inhibitor
[L-NAME (100 ␮M)] or a H2O2 scavenger [PEG-Cat (500
U/ml)]. L-NAME impaired FMD in the MA and SA of WT
mice (Fig. 1, B and F), whereas PEG-Cat had no effect
[maximal dilation: 77.4 ⫾ 7.1% with vehicle (n ⫽ 18), 20.7 ⫾
8.6% with L-NAME (n ⫽ 7, P ⬍ 0.05), and 54.1 ⫾ 10.3% with
PEG-Cat (n ⫽ 7–18)]. Similarly, as with overall dilator capac-
ity, no differences in the mechanism of FMD were observed
between sexes. Endothelium-independent dilation to Pap (Fig.
1, D and H) was not altered compared with WT mice.
In contrast to WT mice, in the MA and SA of TERT KO
mice, PEG-Cat but not L-NAME reduced dilation to flow
[maximal dilation: 52.0 ⫾ 6.1% with vehicle (n ⫽ 18), 60.4 ⫾
12.9% with L-NAME (n ⫽ 7), and 32.2 ⫾ 12.2% with PEG-
Cat (n ⫽ 7, P ⬍ 0.05); Fig. 1, C and G]. Dilation to Pap was
unaltered (Fig. 1, D and H). Loss of NO was supported by a
reduced ratio of phosphorylated to total eNOS in lysates from
the MA of TERT KO mice (Fig. 2A). An elevation in mitoPY1
fluorescence, which detects mtH2O2, was observed in TERT
KO mouse microvessels but not in WT mouse microvessels
exposed to flow (Fig. 2, B and C).
lease during FMD, the contributions of NO and H2O2 to
dilation were also assessed in TERC KO mice. Interestingly, in
these mice, the magnitude and mediator of FMD were similar
to WT mice (NO dependent) [maximal dilation: 79 ⫾ 5% with
vehicle, 10.7 ⫾ 9.8% with L -NAME (P ⬍ 0.05), and
86.4 ⫾ 8.4% with PEG-Cat, n ⫽ 7; Fig. 3A]. Pap-induced
dilation was unchanged in TERC KO mice (Fig. 3B).
TERT overexpression protects against ANG II-induced mi-
crovascular dysfunction. We first performed experiments to
determine whether overexpression of TERT alters baseline
microvascular function (NO bioavailability or overall dilation)
using an established TERT Tg model (2). In the MA, endo-
thelium-dependent dilation to flow (Fig. 4, A and B) and ACh
(Fig. 4C) as well as endothelium-independent dilation to Pap
(Fig. 4E) were similar to that observed in WT mice. The
mechanism of FMD was unaltered relative to WT animals
(remained NO dependent) [maximal dilation: 64.1 5.8% in the
vehicle-WT group (n ⫽ 15) vs. 76.9 5.6 in the vehicle-TERT
Tg group (n ⫽ 12) and 16.1 7.8% in the L-NAME-WT group
(P ⬍ 0.05 vs. vehicle) vs. 24.4 12.5% in the L-NAME-TERT
Tg group (P ⬍ 0.05 vs. vehicle), n ⫽ 7–12; Fig. 4, A and B].
Next, we examined whether TERT modifies the in vivo
microvascular response to ANG II infusion via osmotic mini-
pumps (12). To evaluate the protective effects of TERT over-
expression on ANG II-induced microvascular endothelial dys-
function, we infused a pressor dose of ANG II (1,000
ng·kg⫺1·min⫺1) (3). In WT mice, prolonged high-dose ANG II
infusion caused a complete loss of endothelium-dependent
dilation to flow (Fig. 4D). However, TERT Tg animals showed
only a small reduction in overall dilator capacity [maximal
dilation: 88.22 ⫾ 4.58% with vehicle vs. 74.0 ⫾ 7.3% with
ANG II (1,000 ng·kg⫺1·min⫺1), n ⫽ 4, P ⫽ not significant;
Fig. 4D].
In TERT KO but not WT mice, the subpressor dose of ANG
II [400 ng·kg⫺1·min⫺1 for 14 days (26)] significantly decreased
FMD (Fig. 5A) without affecting endothelium-independent
dilation to Pap (Fig. 5B).
DISCUSSION
Novel findings. This study reports four major new findings.
First, loss of TERT impairs overall peak dilation to flow and
ACh in the microcirculation but not in larger conduit arteries in
first-generation mice and progressive loss of TERT in later
generations further decreases the magnitude of microvascular
FMD. Second, genetic loss of TERT shifts the primary medi-
ator of microvascular FMD from NO to H2O2. Third, absence
of TERT potentiates, whereas an increase in TERT prevents,
ANG II-induced microvascular dysfunction. Finally, loss of the

Fig. 1. Effect of telomerase deficiency on microvascular dilation to flow and acetylcholine (ACh) in first-generation mice. A: magnitude of flow-mediated dilation
(FMD) in wild-type (WT) and telomerase reverse transcriptase knockout (TERT KO; TERT⫺/⫺) mice (n ⫽ 18 mice). The mechanism of FMD in WT (B) and
TERT KO (C) mice was investigated after incubation with N␻-nitro-L-arginine methyl ester (L-NAME; nitric oxide synthase inhibitor) or polyethylene
glycol-catalase (PEG-Cat; H2O2 scavenger) in isolated mesenteric arteries (MAs) (n ⫽ 7 mice). *P ⬍ 0.05 vs. control at specific pressure gradients. D: dilation
to papaverine (Pap) in MAs treated with L-NAME and PEG-Cat. E⫺G: magnitude and mechanism of FMD in septal arteries (SAs) of WT (n ⫽ 7 mice) and
TERT KO mice (n ⫽ 5 mice). *P ⬍ 0.05 at specific pressure gradients. H: dilation to Pap in SAs treated with L-NAME and PEG-Cat. I: magnitude of FMD
in microvessels of first- vs. third-generation TERT KO mice. FMD was preserved in WT mice, reduced slightly in first-generation TERT KO (TERT⫺/⫺) mice
(n ⫽ 18 mice), and severely impaired in third-generation TERT⫺/⫺ mice (n ⫽ 10 mice). *P ⬍ 0.05 at specific pressure gradients. J and K: ACh-mediated dilation
in MAs and aortas of WT (n ⫽ 8 mice) and TERT KO mice (n ⫽ 6 mice) at specific pressure gradients. *P ⬍ 0.05 at specific pressure gradients. Values are
means ⫾ SD. *P ⬍ 0.05 via two-way repeated-measures ANOVA with a post hoc Tukey test. Max, maximal.

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 TELOMERASE REVERSE TRANSCRIPTASE AND THE MICROCIRCULATION H1057

Fig. 2. Absence of telomerase reverse tran-

scriptase (TERT) decreases endothelial ni-
tric oxide synthase (eNOS) protein levels
and increases mitochondrial reactive oxygen
species. A: protein levels of phosphorylated
eNOS (P-eNOS) and total eNOS (T-eNOS)
were evaluated via Western blot analysis. T-
eNOS and P-eNOS levels in mesenteric arter-
ies (MAs) from wild-type (WT), TERT knock-
out (KO), and TERT transgenic (Tg) mice are
shown. n ⫽ 6 mice/group. *P ⬍ 0.05. B and C:
change in Mito Peroxy Yellow 1 (mitoPY1)
fluorescence in WT and TERT KO mice in
response to elevations in flow and in the pres-
ence or absence of polyethylene glycol-cata-
lase (PEG-Cat). Values are means ⫾ SD; n ⫽
5 mice/group. *P ⬍ 0.05 vs. WT mice by t-test
and one-way ANOVA with a post hoc Tukey
test, respectively.

RNA component of telomerase, TERC, does not alter the
vascular phenotype.
Role of TERT in maintaining physiological vasodilation.
The present data support the idea that telomerase maintains
physiological NO-dependent dilation to flow. In particular, the
loss of the enzymatic TERT subunit contributes to the devel-
opment of microvascular endothelial dysfunction, character-
ized by a reduced overall dilator capacity and heightened
contribution of H2O2 to FMD. These findings suggest that loss
of TERT may be a key component of the pathophysiology of
cardiovascular diseases affected by overabundance of micro-
vascular ROS, which may precipitate proinflammatory
changes, or impaired overall dilator capacity, which may lead
to downstream ischemia. The data presented in this report are
consistent with our earlier findings using short-term pharma-
cological TERT inhibition (2, 9) and extend these initial
observations to chronic and in vivo effects of TERT in a
genetic model. Moreover, the observation that deletion of
TERC does not contribute the microvascular phenotypes iden-
tified in early generations underlines the physiological rele-
vance of TERT itself as an independent regulator of ROS
generation and FMD. This observation supports previous work
by Santos and colleagues (25) indicating that ablation of either
TERC or TERT alone does not result in equivalent functional
effects, likely because of TERT’s unique ability to localize to
the mitochondria. Additionally, our finding that progressive
loss of overall peak dilation occurs in later generations of
TERT KO mice suggests that telomere shortening may aug-
ment the effect of loss of TERT on the microcirculation.
Role of TERT in pathological vasodilation. Recently, it has
been shown that telomerase activity is negatively correlated
with the development of abdominal aortic aneurysm, which is
closely linked to elevated ANG II levels (8). Previous work has
shown that ANG II significantly diminishes telomerase activity

A TERC-/- B
TERC-/- +L-NAME
120 TERC-/- + PEG-CAT 120 Fig. 3. Effect of telomerase RNA component
%Max Dilation (MA)

(TERC) deletion on microvascular dilation to flow.

PAP %Max Dilation

100 100 A: mechanism of flow-mediated dilation in mesen-

80 teric arteries (MAs) of TERC knockout (KO) mice.
80
60 n ⫽ 7 mice/group. *P ⬍ 0.05 at specific pressure
40 60 gradients. B: dilation to papaverine (Pap) in MAs of
20 40
TERC KO mice. Values are means ⫾ SD. P ⫽ not
significant vs. vehicle via two-way repeated-mea-
0
20 sures ANOVA with a post hoc Tukey test. L-
-20
0
NAME, N␻-nitro-L-arginine methyl ester; max,
0 20 40 60 80 100
maximal; PEG-Cat, polyethylene glycol-catalase.
Pressure Gradient (cm H2O) Vehicle L-NAME PEG-CAT

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H1058 TELOMERASE REVERSE TRANSCRIPTASE AND THE MICROCIRCULATION

Fig. 4. Effect of telomerase reverse transcriptase overexpression (TERT Tg) on baseline flow-mediated dilation (FMD) and angiotensin II (ANG II)-induced
endothelial dysfunction. The mediator of FMD was determined by incubation with N␻-nitro-L-arginine methyl ester (L-NAME; nitric oxide synthase inhibitor)
or polyethylene glycol-catalase (PEG-Cat; H2O2 scavenger) in isolated mesenteric arteries (MAs) from wild-type (WT; A) mice (n ⫽ 15 mice) and TERT Tg
(B) mice (n ⫽ 12 mice). C: acetylcholine (ACh)-induced dilation in WT and TERT Tg mice (n ⫽ 5 mice/group). D and E: FMD was assessed in MAs from
WT and TERT Tg mice after treatment with 1,000 ng·kg⫺1·min⫺1 ANG II (osmotic minipump, n ⫽ 4 mice/group). E: dilation to papaverine (Pap) in all treatment
conditions. Dilation to ACh and Pap was unchanged in conduit arteries (aorta) in TERT Tg mice at specific pressure gradients. Values are means ⫾ SD. *P ⬍
0.05 via two-way repeated-measures ANOVA with a post hoc Tukey test. Max, maximal.

in endothelial progenitor cells (15), while we and others have
previously shown that ANG-(1–7), which opposes pathological
actions of ANG II, positively regulates telomerase activity (9).
This suggests that an intimate relationship exists between the
renin-angiotensin system and telomerase in the cardiovascular
system. We demonstrate that increased TERT itself can coun-
teract the negative effects of elevated ANG II on microvascular
vasodilator capacity. Further studies are needed to further
characterize TERT’s capacity to limit ANG II-induced patho-
logical changes in the microcirculation. As both loss of TERT
and elevations of ANG II are known to increase mitochondrial
ROS production, a mechanistic connection on this level should
be explored.
Study limitations. There are several limitations that should
be acknowledged. First, with the present experimental design,
we cannot differentiate endothelial-specific effects of TERT or
ANG II from smooth muscle or systemic effects. Second,
telomere shortening may have contributed to the observed
microvascular phenotype in first-generation TERT KO mice.
Importantly, it has been previously shown that early-generation

Fig. 5. Effect of telomerase reverse transcriptase

(TERT) deletion on the angiotensin II (ANG
II)-induced endothelial dysfunction. A: flow-medi-
ated dilation was assessed in isolated mesenteric
arteries (MAs) from wild-type (WT) and telomerase
reverse transcriptase knockout (TERT⫺/⫺) mice af-
ter treatment with 400 ng·kg⫺1·min⫺1 ANG II (os-
motic minipump). n ⫽ 6 mice/group. *P ⬍ 0.05 at
specific pressure gradients; #P ⬍ XXXXXX. B:
dilation to papaverine (Pap) in both treatment
conditions. Values are means ⫾ SD; n ⫽ 6. P ⫽
not significant via two-way repeated-measures
ANOVA with a post hoc Tukey test. Max, max-
imal.

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 TELOMERASE REVERSE TRANSCRIPTASE AND THE MICROCIRCULATION
TERT KO mice have relatively conserved telomeres (24),
rendering such a confounding effect unlikely.
In translating these findings, it will be important to consider
the effects of upregulating telomerase in noncardiac diseases.
For example, although not an oncogene, TERT is involved in
support of tumor growth and metastasis (1). The risk versus
benefit of TERT activation or inhibition needs to be carefully
considered in different cell types. Current evidence indicates
that the proliferative functions of TERT are driven by its
canonical telomere nuclear lengthening effects (1). Separating
mitochondrial and nuclear actions of telomerase would help
advance further discovery in this field.
Conclusions. This study demonstrates that loss of TERT but
not TERC increases ROS formation and precipitates microvas-
cular dysfunction. Decreased TERT augments, whereas over-
expression of TERT prevents, ANG II-induced microvascular
endothelial dysfunction. These data reveal a protective effect
of TERT on microvascular function in vivo with potential
implications for the treatment of cardiovascular diseases. Fu-
ture directions involve exploring potential mechanisms
whereby elevated TERT exerts protective effects on the mi-
crocirculation at baseline and in response to ANG II-induced
microvascular stress. Subcellular localization of TERT to the
mitochondria may reverse the negative long-term effects of
heightened microvascular H2O2 on remodeling, inflammation,
and other phenotypic changes associated with cardiovascular
disease pathogenesis.
ACKNOWLEDGMENTS
TERT Tg mice were obtained from Dr. Dennis C. Bruemmer. We thank
David Zhang for assistance with septal artery isolation.
GRANTS
This work was supported by National Heart, Lung, and Blood Institute
Grants R01-HL-133029 (to A. M. Beyer) and R01-HL-113612 (to D. D.
Gutterman). K. Ait-Aissa is the recipient of American Heart Association
Postdoctoral Fellowship 16POST26430075. A. O. Kadlec is a member of the
Medical Scientist Training Program at Medical College of Wisconsin (MCW),
which is partially supported by National Institute of General Medical Sciences
Training Grant T32-GM-080202. This work was partially funded by the
Advancing a Healthier Wisconsin Endowment through support of the MCW
Redox Biology Program (to A. M. Beyer).
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the authors.
AUTHOR CONTRIBUTIONS
K.A.-A., D.D.G., and A.M.B. conceived and designed research; K.A.-A.,
J.H., and A.M.B. performed experiments; K.A.-A. and A.M.B. analyzed data;
K.A.-A., A.O.K., D.D.G., and A.M.B. interpreted results of experiments;
K.A.-A., A.O.K., and A.M.B. prepared figures; K.A.-A. and A.O.K. drafted
manuscript; A.O.K., D.D.G., and A.M.B. edited and revised manuscript;
K.A.-A., A.O.K., J.H., D.D.G., and A.M.B. approved final version of manu-
script.
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