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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
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 INTRODUCTION

AM. Telomerase reverse transcriptase protects against angiotensin
II-induced microvascular endothelial dysfunction. Am J Physiol Heart Approximately one in every three deaths can be attributed to
Circ Physiol 314: H1053–H1060, 2018. First published December 22, cardiovascular disease (22). Previous research seeking to
2017; doi:10.1152/ajpheart.00472.2017.—A rise in reactive oxygen lessen the cardiovascular disease burden has predominantly
species (ROS) may contribute to cardiovascular disease by reducing explored disease-related pathological changes (e.g., oxidative
nitric oxide (NO) levels, leading to loss of NO’s vasodilator and stress, inflammation, and remodeling) occurring in endothelial
anti-inflammatory effects. Although primarily studied in larger con- and smooth muscle cell layers of large conduit arteries. Small
duit arteries, excess ROS release and a corresponding loss of NO also resistance arteries of the microcirculation have received less
occur in smaller resistance arteries of the microcirculation, but the attention (10), despite provocative reports illustrating their
underlying mechanisms and therapeutic targets have not been fully pathophysiological (17, 18, 20) and prognostic (5, 27, 28)
characterized. We examined whether either of the two subunits of
importance in cardiovascular disease.
telomerase, telomerase reverse transcriptase (TERT) or telomerase
RNA component (TERC), affect microvascular ROS production and
A major role of the microvascular endothelium is regulation
peak vasodilation at baseline and in response to in vivo administration of vascular tone. Microvascular dilation is elicited through the
to angiotensin II (ANG II). We report that genetic loss of TERT release of a variety of endothelium-derived factors in response
[maximal dilation: 52.0 ⫾ 6.1% with vehicle, 60.4 ⫾ 12.9% with to chemical agonists or increases in blood flow. Nitric oxide
N␻-nitro-L-arginine methyl ester (L-NAME), and 32.2 ⫾ 12.2% with (NO) is the dominant flow-induced vasodilator in the micro-
polyethylene glycol-catalase (PEG-Cat) (P ⬍ 0.05), means ⫾ SD, n ⫽ circulation of subjects free of cardiovascular disease, whereas
9 –19] but not TERC [maximal dilation: 79 ⫾ 5% with vehicle, H2O2 replaces NO in patients with coronary artery disease
10.7 ⫾ 9.8% with L-NAME (P ⬍ 0.05), and 86.4 ⫾ 8.4% with PEG- (20). Although H2O2 maintains dilation as NO levels drop,
Cat, n ⫽ 4 –7] promotes flow-induced ROS formation. Moreover, chronic and unopposed release of mitochondria-derived H2O2
TERT knockout exacerbates the microvascular dysfunction resulting (mtH2O2) from the microvascular endothelium, together with
from in vivo ANG II treatment, whereas TERT overexpression is loss of anti-inflammatory actions of NO, is believed to elicit
protective [maximal dilation: 88.22 ⫾ 4.6% with vehicle vs.
proinflammatory changes that contribute to disease develop-
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
ment (6). Clarification of the cellular factors responsible for
contributor to the elevated microvascular ROS levels and reduced this microvascular transition from NO to H2O2 is needed to
peak dilation observed in several cardiovascular disease pathologies. identify potential therapeutic targets to combat excessive reac-
tive oxygen species (ROS) production and lessen microvascu-
NEW & NOTEWORTHY This study identifies telomerase reverse lar dysfunction.
transcriptase (TERT) but not telomerase RNA component as a key
Existing evidence has established that loss of telomerase is
factor regulating endothelium-dependent dilation in the microcircula-
tion. Loss of TERT activity leads to microvascular dysfunction but not closely associated with elevations in mitochondrial ROS pro-
conduit vessel dysfunction in first-generation mice. In contrast, TERT duction. Telomerase reverse transcriptase (TERT), the catalytic
is protective in the microcirculation in the presence of prolonged subunit of the telomerase holoenzyme, and the telomerase
vascular stress. Understanding the mechanism of how TERT protects RNA component (TERC) are critical for canonical telomerase
against vascular stress represents a novel target for the treatment of function (25). While TERT is largely studied in models of
vascular disorders. aging and cancer because of its telomere-lengthening effects in
the nucleus, TERT also acts to counteract oxidative stress in
angiotensin II; flow-mediated dilation; microcirculation; telomerase;
telomerase reverse transcriptase 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
* K. Ait-Aissa and A. O. Kadlec contributed equally to this work.
Address for reprint requests and other correspondence: A. M. Beyer, Dept.
TERT and TERC on microvascular dilation and ROS release is
of Medicine, Medical College of Wisconsin, 8701 Watertown Plank Rd., unknown, as is the potential protective effect of TERT over-
Milwaukee, WI 53226 (e-mail: expression in vivo on the microvascular effects of established 0363-6135/18 Copyright © 2018 the American Physiological Society H1053
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pathological factors that augment ROS formation in the mi- pettes and pressurized (60 mmHg) before stepwise increases in flow
crocirculation, such as elevated angiotensin II (ANG II). To or administration of pharmacological agents, as we have previously
explore the role of increased or decreased telomerase activity described (14, 19, 23).
under pathological conditions, we used the well-defined model Vascular response to flow and pharmacological interventions. The
contribution of NO or H2O2 to dilation was assessed in TERT KO,
of ANG II infusion to induce ROS formation and endothelial
TERC KO, and TERT Tg mice using FMD as previously described by
dysfunction (4, 11, 13, 29). Kuo et al. (16). Adjustment of the height of each reservoir in equal
Understanding these in vivo relationships deepens our un- and opposite directions was used to generate flow via generation of a
derstanding of the potential pathophysiological effect of re- pressure gradient without changes in intraluminal pressure (16). Di-
duced TERT and/or TERC in the microcirculation and pro- ameter at a given pressure gradient was recorded and expressed as
vides valuable information about the therapeutic role of TERT percent maximal diameter. Two flow-response curves were generated
in the microcirculation in an in vivo setting. for each vessel comparing untreated (vehicle) with effects of pharma-
We hypothesized that genetic loss of TERT or TERC in- cological inhibitors [NO inhibitor N␻-nitro-L-arginine methyl ester
creases flow-induced release of H2O2 in both coronary and (L-NAME; 100 mM) and H2O2 scavenger polyethylene glycol-cata-
mesenteric microcirculations of TERT and TERC knockout lase (PEG-Cat; 500 U/ml)]. All pharmacological agents represent final
concentrations in the organ bath and were added at volumes of ⬍1%
(KO) mice. We further hypothesized that TERT overexpres-
of the total bath volume.
sion [TERT transgenic (Tg)] limits, whereas loss of TERT Arteries were constricted with norepinephrine (10 ␮M for MA) or
exacerbates, the damaging effects of in vivo ANG II adminis- the thromboxane A2 analog U-46619 (100 nM for SA) to achieve a
tration on microvascular dilation. 20 –70% stable reduction in passive diameter. Dose-response curves
to acetylcholine (ACh; 1 nM⫺10 mM) and flow gradient (5–100
MATERIALS AND METHODS cmH2O) were performed to evaluate endothelium-dependent dilation.
At the end of each experiment, the endothelium-independent dilator
Overview. To address our hypotheses, we used established genetic papaverine (Pap; 100 ␮M) was used to determine the maximal
gain-of-function and loss-of-function models for TERT (TERT Tg (passive) diameter at 60 mmHg.
and TERT KO) and a loss-of-function model for TERC (TERC KO). Measurement of mitochondrial ROS. Mito Peroxy Yellow 1
Later-generation TERT KO animals were also used to assess the (mitoPY1) (7) was used as a secondary means to assess microves-
effect of progressive loss of TERT on microvascular dilation. Micro- sel generation of mtH2O2 after loss of TERT. After cannulation in
vascular phenotypes were examined using ex vivo video microscopy a warmed chamber (37°C) containing HEPES buffer at pH 7.4,
(overall dilation and contribution of NO vs. H2O2 to dilation), fluo- MAs from TERT KO mice or from WT mice were perfused
rescence detection (H2O2 release during dilation), and Western blot intraluminally with mitoPY1 (5 ␮M, 1 h) at low levels of flow
quantification of NO synthase (NOS) levels. We also evaluated the below the threshold for dilation until the luminal surface was
response to the commonly used vascular stressor ANG II to determine bathed in mitoPY1-containing buffer. Next, the pressure gradient
whether TERT modulates microvascular tone under pathological was changed to 0 –100 cmH2O. Experiments were performed in the
conditions. presence or absence of the H2O2 scavenger PEG-Cat (500 U/ml).
General protocol in mice. All experiments were performed accord- Fluorescence was evaluated with a Nikon Eclipse TE200 micro-
ing to the American Guidelines for the Ethical Care of Animals and scope using a krypton/argon laser at excitation wavelength of 488
were approved by our Institutional Animal Care and Use Committee. nm and measured emission between 530 and 590 nm. For any
C57BL/6 TERT KO (TERT⫺/⫺) mice were obtained from Ronald given experiment, baseline fluorescence was established without
DePinho (MD Anderson Cancer Center, Houston, TX). TERC KO flow. After 30 min of incubation with mitoPY1, each vessel was
(TERC⫺/⫺) mice were purchased from Jackson Laboratories (stock compared with no-flow conditions as a measure of flow-induced
004132 mTR⫺/⫺, Bar Harbor, ME). KO mice were bred as heterozy- H2O2 production. In separate experiments, the specificity of the
gotes to generate first-generation KO mice. To generate third-gener- probe was confirmed with PEG-Cat (H2O2 scavenger) present at
ation KO mice, homozygous KO mice of the first generation were any time during the experiment. Levels of mtH2O2 are expressed as
inbred for three consecutive generations. TERT Tg mice were pro- relative average fluorescence intensity normalized to background
vided by Dennis Bruemmer (University of Pittsburgh, Pittsburgh, fluorescence and presented as percent change from baseline. All
PA). Both male and female mice were used in the experiments comparisons were made using arteries studied at the same session
outlined in this study, and all animals were studied at 3– 4 mo of age with constant microscope image display settings.
and compared with wild-type (WT) control mice of the same breeding Microvascular response to ANG II. Nonpressor dose (400
line. When possible, arteries from the same mouse were used for ng·kg⫺1·min⫺1) or fast pressor dose (1,000 ng·kg⫺1·min⫺1) of ANG
multiple experiments (e.g., functional experiments and ROS evalua- II as previously described (15) was administered via osmotic mini-
tion). pumps for 14 days to induce prolonged in vivo microvascular stress in
All mice were housed and maintained at a temperature of 23°C WT, TERT KO, and TERT Tg mice.
with 12:12-h light-dark cycles and fed a solid standard diet (Na⫹ Western blot analysis. Protein expression was analyzed as previ-
content: 0.4%) and water ad libitum. On the day of the experiment, ously described (9). Briefly, total protein (30 ␮g) from MA (6 – 8
mice were euthanized between 8 AM and 12 PM, and tissues pooled arteries/mouse) lysates was loaded and separated using SDS-
(thoracic aorta, mesenteric resistance artery, and septal artery) PAGE and then transferred into a polyvinylidene difluoride mem-
were harvested and immediately placed in cold 4°C HEPES (con- brane. The membrane was incubated with one of the following
taining 275 mM NaCl, 7.99 mM KCl, 4.9 mM MgSO4, 3.2 mM primary antibodies: rabbit monoclonal anti-phosphorylated (Ser1177)
CaCl2·2H2O, 2.35 mM KH2PO4, 0.07 mM EDTA, 12 mM glucose, endothelial NOS (eNOS; dilution 1:1,000, catalog no. MA5-14957,
and 20 mM HEPES acid). Invitrogen), rabbit polyclonal anti-total eNOS (dilution 1:1,000, cat-
Cannulated arteriole preparation. Third- or fourth-order branch alog no. PA5-16887, Invitrogen), or mouse monoclonal anti-GAPDH
arteries from the mesenteric artery (MA) and septal artery (SA) (dilution 1:10,000, catalog no. ab8245, Abcam). Protein bands were
(~200-␮m inner diameter) were cleaned of fat and connective tissue detected with X-ray film exposure and quantified by ImageJ software.
and prepared for continuous measurements of internal diameter as Materials. Norepinephrine and ANG II were obtained from Sigma-
previously described (20, 21). In an organ chamber containing Krebs Aldrich, and U-46619 was obtained from Cayman Chemical. PEG-
buffer, both ends of the vessel were cannulated with glass micropi- Cat was made by Quanta BioDesign (Plain City, OH), and L-NAME

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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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RESULTS To examine whether TERC similarly influences H2O2 re-

lease during FMD, the contributions of NO and H2O2 to
Loss of TERT affects the magnitude of microvascular but not dilation were also assessed in TERC KO mice. Interestingly, in
conduit vessel dilation in first-generation mice. To examine these mice, the magnitude and mediator of FMD were similar
whether the dilation in isolated arteries is impaired by the to WT mice (NO dependent) [maximal dilation: 79 ⫾ 5% with
genetic loss of telomerase, we compared the overall peak vehicle, 10.7 ⫾ 9.8% with L -NAME (P ⬍ 0.05), and
dilation to flow (FMD) and to ACh in both mesenteric (MA) 86.4 ⫾ 8.4% with PEG-Cat, n ⫽ 7; Fig. 3A]. Pap-induced
and septal (SA) microvessels and in the aorta. The overall dilation was unchanged in TERC KO mice (Fig. 3B).
magnitude of FMD was similar in MAs and SAs of TERT KO TERT overexpression protects against ANG II-induced mi-
and WT mice [Fig. 1, A and E; maximal dilation: 77.4 ⫾ 7.1% crovascular dysfunction. We first performed experiments to
in the WT-MA group (n ⫽ 18), 53.6 ⫾ 7.1% in the WT-SA determine whether overexpression of TERT alters baseline
group (n ⫽ 7), 52.0 ⫾ 6.1% in the TERT KO-MA group (n ⫽ microvascular function (NO bioavailability or overall dilation)
18), and 64.2 ⫾ 14.6% in the TERT KO-SA group (n ⫽ 5)]. using an established TERT Tg model (2). In the MA, endo-
The magnitude of overall peak dilation was similar between thelium-dependent dilation to flow (Fig. 4, A and B) and ACh
male and female mice [maximal dilation: 68 ⫾ 17% in female (Fig. 4C) as well as endothelium-independent dilation to Pap
WT mice (n ⫽ 7) and 42 ⫾ 17% in TERT KO mice (n ⫽ 4) vs. (Fig. 4E) were similar to that observed in WT mice. The
76 ⫾ 6% in male WT mice (n ⫽ 7) and 54 ⫾ 9% in TERT KO mechanism of FMD was unaltered relative to WT animals
mice (n ⫽ 4), P ⫽ not significant; data not shown]. After (remained NO dependent) [maximal dilation: 64.1 5.8% in the
intercrossing TERT KO mice to obtain third-generation mice, vehicle-WT group (n ⫽ 15) vs. 76.9 5.6 in the vehicle-TERT
microvascular vasodilation to flow in the MA was eliminated Tg group (n ⫽ 12) and 16.1 7.8% in the L-NAME-WT group
(Fig. 1I). (P ⬍ 0.05 vs. vehicle) vs. 24.4 12.5% in the L-NAME-TERT
ACh-induced dilation was reduced in the MA of TERT KO Tg group (P ⬍ 0.05 vs. vehicle), n ⫽ 7–12; Fig. 4, A and B].
mice (first generation) compared with WT mice (P ⬍ 0.05; Fig. Next, we examined whether TERT modifies the in vivo
1J). In contrast, we found no difference in the magnitude of microvascular response to ANG II infusion via osmotic mini-
overall peak ACh-induced dilation in conduit arteries (aorta) of pumps (12). To evaluate the protective effects of TERT over-
first-generation TERT KO versus WT mice (Fig. 1K). expression on ANG II-induced microvascular endothelial dys-
Loss of TERT decreases NO-mediated dilation and promotes function, we infused a pressor dose of ANG II (1,000
H2O2 as a compensatory vasodilator during FMD. We deter- ng·kg⫺1·min⫺1) (3). In WT mice, prolonged high-dose ANG II
mined whether genetic loss of TERT or TERC alters the infusion caused a complete loss of endothelium-dependent
mechanism of FMD in MAs and SAs using a NOS inhibitor dilation to flow (Fig. 4D). However, TERT Tg animals showed
[L-NAME (100 ␮M)] or a H2O2 scavenger [PEG-Cat (500 only a small reduction in overall dilator capacity [maximal
U/ml)]. L-NAME impaired FMD in the MA and SA of WT dilation: 88.22 ⫾ 4.58% with vehicle vs. 74.0 ⫾ 7.3% with
mice (Fig. 1, B and F), whereas PEG-Cat had no effect ANG II (1,000 ng·kg⫺1·min⫺1), n ⫽ 4, P ⫽ not significant;
[maximal dilation: 77.4 ⫾ 7.1% with vehicle (n ⫽ 18), 20.7 ⫾ Fig. 4D].
8.6% with L-NAME (n ⫽ 7, P ⬍ 0.05), and 54.1 ⫾ 10.3% with In TERT KO but not WT mice, the subpressor dose of ANG
PEG-Cat (n ⫽ 7–18)]. Similarly, as with overall dilator capac- II [400 ng·kg⫺1·min⫺1 for 14 days (26)] significantly decreased
ity, no differences in the mechanism of FMD were observed FMD (Fig. 5A) without affecting endothelium-independent
between sexes. Endothelium-independent dilation to Pap (Fig. dilation to Pap (Fig. 5B).
1, D and H) was not altered compared with WT mice.
In contrast to WT mice, in the MA and SA of TERT KO DISCUSSION
mice, PEG-Cat but not L-NAME reduced dilation to flow
[maximal dilation: 52.0 ⫾ 6.1% with vehicle (n ⫽ 18), 60.4 ⫾ Novel findings. This study reports four major new findings.
12.9% with L-NAME (n ⫽ 7), and 32.2 ⫾ 12.2% with PEG- First, loss of TERT impairs overall peak dilation to flow and
Cat (n ⫽ 7, P ⬍ 0.05); Fig. 1, C and G]. Dilation to Pap was ACh in the microcirculation but not in larger conduit arteries in
unaltered (Fig. 1, D and H). Loss of NO was supported by a first-generation mice and progressive loss of TERT in later
reduced ratio of phosphorylated to total eNOS in lysates from generations further decreases the magnitude of microvascular
the MA of TERT KO mice (Fig. 2A). An elevation in mitoPY1 FMD. Second, genetic loss of TERT shifts the primary medi-
fluorescence, which detects mtH2O2, was observed in TERT ator of microvascular FMD from NO to H2O2. Third, absence
KO mouse microvessels but not in WT mouse microvessels of TERT potentiates, whereas an increase in TERT prevents,
exposed to flow (Fig. 2, B and C). 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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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 genetic model. Moreover, the observation that deletion of
vascular phenotype. TERC does not contribute the microvascular phenotypes iden-
Role of TERT in maintaining physiological vasodilation. tified in early generations underlines the physiological rele-
The present data support the idea that telomerase maintains vance of TERT itself as an independent regulator of ROS
physiological NO-dependent dilation to flow. In particular, the generation and FMD. This observation supports previous work
loss of the enzymatic TERT subunit contributes to the devel- by Santos and colleagues (25) indicating that ablation of either
opment of microvascular endothelial dysfunction, character- TERC or TERT alone does not result in equivalent functional
ized by a reduced overall dilator capacity and heightened effects, likely because of TERT’s unique ability to localize to
contribution of H2O2 to FMD. These findings suggest that loss the mitochondria. Additionally, our finding that progressive
of TERT may be a key component of the pathophysiology of loss of overall peak dilation occurs in later generations of
cardiovascular diseases affected by overabundance of micro- TERT KO mice suggests that telomere shortening may aug-
vascular ROS, which may precipitate proinflammatory ment the effect of loss of TERT on the microcirculation.
changes, or impaired overall dilator capacity, which may lead Role of TERT in pathological vasodilation. Recently, it has
to downstream ischemia. The data presented in this report are been shown that telomerase activity is negatively correlated
consistent with our earlier findings using short-term pharma- with the development of abdominal aortic aneurysm, which is
cological TERT inhibition (2, 9) and extend these initial closely linked to elevated ANG II levels (8). Previous work has
observations to chronic and in vivo effects of TERT in a shown that ANG II significantly diminishes telomerase activity

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

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TERT KO mice have relatively conserved telomeres (24), 3. Beyer AM, Guo DF, Rahmouni K. Prolonged treatment with angiotensin
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of TERT on microvascular function in vivo with potential 9. Durand MJ, Zinkevich NS, Riedel M, Gutterman DD, Nasci VL,
implications for the treatment of cardiovascular diseases. Fu- Salato VK, Hijjawi JB, Reuben CF, North PE, Beyer AM. Vascular
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whereby elevated TERT exerts protective effects on the mi- telomerase. Arterioscler Thromb Vasc Biol 36: 1254 –1262, 2016. doi:10.
crocirculation at baseline and in response to ANG II-induced 10. Gutterman DD, Chabowski DS, Kadlec AO, Durand MJ, Freed JK,
microvascular stress. Subcellular localization of TERT to the Ait-Aissa K, Beyer AM. The human microcirculation: regulation of flow
mitochondria may reverse the negative long-term effects of and beyond. Circ Res 118: 157–172, 2016. doi:10.1161/CIRCRESAHA.
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ACKNOWLEDGMENTS 12. Hsueh WA, Wyne K. Renin-angiotensin-aldosterone system in diabetes
and hypertension. J Clin Hypertens (Greenwich) 13: 224 –237, 2011.
TERT Tg mice were obtained from Dr. Dennis C. Bruemmer. We thank doi:10.1111/j.1751-7176.2011.00449.x.
David Zhang for assistance with septal artery isolation. 13. Husain K, Hernandez W, Ansari RA, Ferder L. Inflammation, oxida-
tive stress and renin angiotensin system in atherosclerosis. World J Biol
GRANTS Chem 6: 209 –217, 2015. doi:10.4331/wjbc.v6.i3.209.
This work was supported by National Heart, Lung, and Blood Institute 14. Kadlec AO, Chabowski DS, Ait-Aissa K, Hockenberry JC, Otter-
Grants R01-HL-133029 (to A. M. Beyer) and R01-HL-113612 (to D. D. son MF, Durand MJ, Freed JK, Beyer AM, Gutterman DD.
Gutterman). K. Ait-Aissa is the recipient of American Heart Association PGC-1␣ (peroxisome proliferator-activated receptor ␥ coactivator 1-␣)
Postdoctoral Fellowship 16POST26430075. A. O. Kadlec is a member of the overexpression in coronary artery disease recruits NO and hydrogen
Medical Scientist Training Program at Medical College of Wisconsin (MCW), peroxide during flow-mediated dilation and protects against increased
which is partially supported by National Institute of General Medical Sciences intraluminal pressure. Hypertension 70: 166 –173, 2017. doi:10.1161/
Training Grant T32-GM-080202. This work was partially funded by the HYPERTENSIONAHA.117.09289.
Advancing a Healthier Wisconsin Endowment through support of the MCW 15. Kawada N, Imai E, Karber A, Welch WJ, Wilcox CS. A mouse
Redox Biology Program (to A. M. Beyer). model of angiotensin II slow pressor response: role of oxidative
stress. J Am Soc Nephrol 13: 2860 –2868, 2002. doi:10.1097/01.ASN.
DISCLOSURES 16. Kuo L, Chilian WM, Davis MJ. Interaction of pressure- and flow-
No conflicts of interest, financial or otherwise, are declared by the authors. induced responses in porcine coronary resistance vessels. Am J Physiol
Heart Circ Physiol 261: H1706 –H1715, 1991. doi:10.1152/ajpheart.1991.
17. Kuo L, Davis MJ, Cannon MS, Chilian WM. Pathophysiological
K.A.-A., D.D.G., and A.M.B. conceived and designed research; K.A.-A., consequences of atherosclerosis extend into the coronary microcirculation.
J.H., and A.M.B. performed experiments; K.A.-A. and A.M.B. analyzed data; Restoration of endothelium-dependent responses by L-arginine. Circ Res
K.A.-A., A.O.K., D.D.G., and A.M.B. interpreted results of experiments; 70: 465–476, 1992. doi:10.1161/01.RES.70.3.465.
K.A.-A., A.O.K., and A.M.B. prepared figures; K.A.-A. and A.O.K. drafted 18. Kuo L, Hein TW. Vasomotor regulation of coronary microcirculation by
manuscript; A.O.K., D.D.G., and A.M.B. edited and revised manuscript; oxidative stress: role of arginase. Front Immunol 4: 237, 2013. doi:10.
K.A.-A., A.O.K., J.H., D.D.G., and A.M.B. approved final version of manu- 3389/fimmu.2013.00237.
script. 19. Larsen BT, Bubolz AH, Mendoza SA, Pritchard KA Jr, Gutterman
DD. Bradykinin-induced dilation of human coronary arterioles requires
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