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Citation: Belakova, B.; Wedige, N.K.;
Awad, E.M.; Hess, S.; Oszwald, A.;
Fellner, M.; Khan, S.Y.; Resch, U.;
Lipovac, M.; Šmejkal, K.; et al.
Lipophilic Statins Eliminate
Senescent Endothelial Cells by
inducing Anoikis-Related Cell Death.
Cells 2023,12, 2836. https://doi.org/
10.3390/cells12242836
Academic Editors: Kay-Dietrich
Wagner and Ping Song
Received: 31 October 2023
Revised: 7 December 2023
Accepted: 11 December 2023
Published: 14 December 2023
Copyright: © 2023 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
cells
Article
Lipophilic Statins Eliminate Senescent Endothelial Cells by
inducing Anoikis-Related Cell Death
Barbora Belakova 1, Nicholas K. Wedige 1, Ezzat M. Awad 1,2, Simon Hess 1, AndréOszwald 1,3, Marlene Fellner 1,
Shafaat Y. Khan 1,4 , Ulrike Resch 1, Markus Lipovac 5, Karel Šmejkal 6, Pavel Uhrin 1,†,‡
and Johannes M. Breuss 1,*,†
1Institute of Vascular Biology and Thrombosis Research, Center for Physiology and Pharmacology,
Medical University of Vienna, 1090 Vienna, Austria; ezzat.awad@meduniwien.ac.at (E.M.A.);
andre.oszwald@meduniwien.ac.at (A.O.); shafaatyarkhan@hotmail.com (S.Y.K.);
ulrike.resch@meduniwien.ac.at (U.R.)
2Institute of Specific Prophylaxis and Tropical Medicine, Center for Pathophysiology,
Infectiology and Immunology, Medical University of Vienna, 1090 Vienna, Austria
3Department of Pathology, Medical University of Vienna, 1090 Vienna, Austria
4Department of Zoology, Government College University Lahore, Lahore 54000, Pakistan
5Karl Landsteiner Institute for Cell-Based Therapy in Gynecology, 2100 Korneuburg, Austria
6Department of Natural Drugs, Faculty of Pharmacy, Masaryk University, 612 00 Brno, Czech Republic
*Correspondence: johannes.breuss@meduniwien.ac.at
†These authors contributed equally to this work.
‡Deceased author This publication is dedicated to the memory of Pavel Uhrin, our dear and esteemed
colleague, who played a pivotal role in the conceptualization and development of this research.
Abstract:
Pre-clinical studies from the recent past have indicated that senescent cells can negatively
affect health and contribute to premature aging. Targeted eradication of these cells has been shown to
improve the health of aged experimental animals, leading to a clinical interest in finding compounds
that selectively eliminate senescent cells while sparing non-senescent ones. In our study, we identified
a senolytic capacity of statins, which are lipid-lowering drugs prescribed to patients at high risk
of cardiovascular events. Using two different models of senescence in human vascular endothelial
cells (HUVECs), we found that statins preferentially eliminated senescent cells, while leaving non-
senescent cells unharmed. We observed that the senolytic effect of statins could be negated with the
co-administration of mevalonic acid and that statins induced cell detachment leading to anoikis-like
apoptosis, as evidenced by real-time visualization of caspase-3/7 activation. Our findings suggest
that statins possess a senolytic property, possibly also contributing to their described beneficial
cardiovascular effects. Further studies are needed to explore the potential of short-term, high-dose
statin treatment as a candidate senolytic therapy.
Keywords: endothelial cells; senescence; senolytics; statins; anoikis; apoptosis
1. Introduction
Cellular senescence plays an important role in aging, and studies over the last two
decades have shown that accumulating senescent cells can affect many different organs
and contribute to numerous age-related pathologies including atherosclerosis, macular
degeneration, osteoporosis, neurodegeneration, and sarcopenia [1].
Senescent cells are defined by their permanent loss of the potential to proliferate.
Such loss can be induced by stresses, such as DNA damage and telomere dysfunction,
increased levels of reactive oxygen species (ROS), or mitochondrial dysfunction [
2
], and
is maintained by modulation of the p16/Rb and p21/p53 axis of cell cycle inhibitors.
Senescent cells furthermore develop a pro-inflammatory phenotype, termed the senescence-
associated secretory phenotype, SASP [
3
], in which they secrete pro-inflammatory and
Cells 2023,12, 2836. https://doi.org/10.3390/cells12242836 https://www.mdpi.com/journal/cells
Cells 2023,12, 2836 2 of 21
matrix-degrading molecules and typically undergo morphological changes such as cell
enlargement and flattening [1].
Endothelial senescence under physiological conditions is prompted by various factors
within the vascular microenvironment as they can be found in atherosclerotic plaques [
4
].
Disturbed hemodynamic flow, especially in areas like branching arteries and the aortic
arch, induces a high rate of cellular turnover and contributes to senescence [
5
–
7
]. Elevated
oxygen levels within arteries present an additional challenge to endothelial cells. Prolonged
exposure to factors associated with Western lifestyles, such as high glucose levels, insulin,
and reactive oxygen species (ROS), can promote senescence. Imbalances in vasodilators and
vasoconstrictors, along with impaired nitric oxide (NO) availability, create an environment
conducive to senescence. Chronic inflammation, often associated with increased ROS,
exacerbates the process. These factors collectively regulate endothelial cell senescence in
physiological contexts, as comprehensively reviewed in [2].
Pioneering pre-clinical experimental animal model studies over the past decade have
shown that the elimination of such malfunctioning pro-inflammatory senescent cells can
ameliorate age pathologies, improve the overall health status, and extend the life span
of experimental animals [
8
–
12
]. In particular, the removal of senescent endothelial cells
could promise broader health benefits [
2
]. Endothelial senescence increases monolayer
permeability [
13
] and induces expression of leukocyte-attractive (recruiting) adhesion
molecules [
14
]. Senescent endothelial cells also impede the regenerative capacity and
functionality of blood vessels, e.g., reduce the availability of the vasodilatory factors nitric
oxide (NO) and prostacyclin [
15
], further supporting the notion that the elimination of
senescent endothelial cells could be of clinical importance in vascular homeostasis and
tissue regeneration and remodeling and thus, fundamental in the fields of angiology
and cardiology.
Collectively, these studies support the idea that the administration of “senolytics”, com-
pounds that preferentially promote the elimination of senescent cells over non-senescent
ones could become a therapeutic approach to counteract age-related pathologies.
Statins represent widely used lipid-lowering drugs utilized for the state-of-the-art
treatment of patients with high risk of cardiovascular disease. The reported beneficial ef-
fects of these 3-hydroxyl-3-methyl-glutaryl-coenzyme A (HMG-CoA) reductase-inhibiting
drugs [
16
] are attributed to their lipid-lowering [
17
] and anti-inflammatory properties, in-
cluding the suppression of leukocyte-endothelial interactions [
18
,
19
]. Furthermore, statins
promote the synthesis of endothelial NO, an important regulator of blood vessel stiffness
that mediates vasorelaxation [
20
]. In addition, though, when applied at higher concentra-
tions, statins have shown a cytotoxic effect [21,22].
In our study, we investigated whether this cytotoxic effect of statins might show a
certain selectivity toward senescent versus non-senescent endothelial cells, which would
thereby demonstrate a senolytic capacity of this substance class. We addressed this question
by quantifying the cytotoxic effect of statins on two different types of senescent human
vascular endothelial cells (HUVECs) and comparing it with the effect on non-senescent
HUVECs. We also addressed the dependency of such cytotoxic capacity on the block-
ing of the mevalonate pathway and the kind of cell death that is induced by statins in
endothelial cells.
2. Materials and Methods
2.1. Isolation, Culturing, and Microscopic Characterization of HUVECs
HUVECs were isolated from human umbilical veins based on the granted permission
GS4-EK-4/562-2018 issued by the Ethic Commission of Lower Austria, Austria. Upon iso-
lation, using collagenase NB 4 (Serva, Heidelberg, Germany) as previously described [
23
],
the cells were cultured in M199-based medium supplemented with 20% fetal bovine serum
(FBS, Sigma-Aldrich, Saint Louis, MO, USA), 0.4% endothelial cell growth supplement with
heparin (ECGS/H, PromoCell, Heidelberg, Germany), and 2 mM L-glutamine, 0.1% peni-
cillin, 0.1% streptomycin, and 0.25
µ
g/mL fungizone (all from Lonza, Visp, Switzerland),
Cells 2023,12, 2836 3 of 21
designated as complete endothelial growth medium, on cell culture dishes pre-coated with
1% gelatin (Sigma-Aldrich). Primary cells obtained from nine single-donor cords were
pooled at passage 4 (P4). They were frozen or further propagated using serial passaging
until doubling times of approximately 10 days were reached. Associated limitations, such
as possible bias by donor cells reaching replicative senescence earlier than others, are
acknowledged and might in part be alleviated by the juxtaposition to the second model
of senescence.
For splitting, the complete endothelial growth medium was removed, and the cells
were washed once with calcium- and magnesium-free Hanks’ balanced salt solution
(CMFH). The cells were briefly exposed twice to a minimal volume of trypsin-EDTA (Lonza)
that was immediately removed, avoiding the need for subsequent centrifugation. Through-
out the serial passaging, morphological changes in the HUVECs were monitored with
imaging using a Nikon TMS inverted phase-contrast microscope (Nikon, Tokyo, Japan).
To prepare a stress-induced senescent population of HUVECs, cells at P9 (originating
from the same pool of donors), were grown to sub-confluency, trypsinized, and exposed
in suspension to 10 Gy of ionizing radiation provided by the cesium
137
-based irradiator
IBL-437 C (at the Department of Blood Group Serology and Transfusion Medicine, Medical
University of Vienna). The irradiated HUVECs were re-seeded to a growth area six times
larger than provided before to ensure enough space for their expected increase in size.
During the entire subsequent cultivation period lasting for 10 days, the irradiated HUVECs
were observed regularly, and morphological changes were recorded using phase-contrast
microscopy as described above. In some experiments, HUVECs were continuously moni-
tored (at a rate of 15 frames per hour) using an Olympus IX83 cellVivo live cell imaging
system (Olympus, Tokyo, Japan).
2.2. Immunocytochemical Characterization of the Proliferation Status of HUVEC Cultures
At passaging, cell numbers of trypsinized HUVECs were determined by counting with
the hemocytometer Luna-II (Logos Biosystems, Anyang-si, Republic of Korea), and their
proliferation status was characterized immunocytochemically, assessing the expression
levels of the proliferation marker Ki-67 one or two days after passaging, using a rabbit
monoclonal anti-Ki-67 antibody (RM-9106-S1, Thermo Fisher Scientific, Waltham, MA,
USA). In addition to the assessment of Ki-67, antibodies against several additional targets
were used for the characterization of the proliferation status/competence of HUVECs.
These included cyclin-dependent kinase inhibitor p16 (MA5-27905, rabbit monoclonal,
Thermo Fisher Scientific), a marker for double-strand breaks
γ
H2AX (613402, mouse
monoclonal, BioLegend, San Diego, CA), as well as the anti-apoptotic proteins XIAP (2045,
rabbit monoclonal, Cell Signaling Technology, Danvers, MA, USA) and Bcl-2 (AF6139,
rabbit polyclonal, Affinity Biosciences, Cincinnati, OH, USA).
For this, prior to staining, HUVECs were fixed for 10 min in 4% paraformaldehyde
(PFA, Sigma-Aldrich), washed with phosphate-buffered saline (PBS), permeabilized with
0.2% Triton-X 100 (Serva), and blocked with 3% goat serum (Abcam, Cambridge, UK).
The cells were then incubated with the primary antibodies (diluted in antibody-diluent,
Dako, Glostrup, Denmark) overnight at 4
◦
C. After washing with PBS, the cells were
exposed to secondary polyclonal antibodies, including goat anti-rabbit IgG Alexa 647 or
goat-anti-mouse IgG Alexa 488 (A21245 and A11029, both from Thermo Fisher Scientific)
for 2 h at room temperature. Nuclear counter-staining was performed using Hoechst 33258
(CAY-16756-50, Cayman Chemical, Ann Arbor, MI, USA). Immunofluorescent detection
was performed using the Olympus IX83 cellVivo live cell imaging system and 10
×
or
20×magnification.
Assessment of the senescence markers Ki-67 and
γ
H2AX from fluorescent images
was performed with manual counting. At least five different fields of view (comprising at
least 500 cells) were assessed and analyzed with an unpaired t-test using GraphPad Prism
version 5.04 for Windows (GraphPad Software, Boston, MA, USA). The significance level
**** indicates p< 0.0001.
Cells 2023,12, 2836 4 of 21
Differences in p16 expression were assessed as integrated cellular fluorescent signal
intensities per field of view divided by the number of cell nuclei present (given in arbitrary
units (a.u.)) by assessing at least five different fields of view using the Olympus software
package cellSens 2.1 and analyzed with an unpaired t-test using GraphPad Prism. The
significance level **** indicates p< 0.0001.
2.3. Monitoring the Senolytic Effects of Test Compounds on HUVECs with Microscopy-Based
Cell Enumeration
The senolytic effects of the test compounds were evaluated on HUVECs cultured
in a 384-well plate format. The following substances were examined for their senolytic
activity: simvastatin (MCE-HY-17502, MedChem Express, Monmouth Junction, NJ, USA),
atorvastatin (S5715, Selleck Chemicals, Houston, TX, USA) and lovastatin and pravastatin
(SC-200850A and SC-203218, both from Santa Cruz Biotechnology, Dallas, TX, USA) as well
as activated simvastatin.
Statins were applied to HUVECs either alone or in combination with mevalonic acid
(90469, Sigma-Aldrich). All non-activated statins, the positive control substances quercetin
(PHR1488, Sigma-Aldrich) and dasatinib (HY-10181, MedChem Express), and mevalonic
acid were prepared as 10 mM stocks by directly dissolving in DMSO and diluting to
working concentrations by mixing with complete endothelial growth culture medium.
Activated simvastatin was prepared using alkaline hydrolysis as previously described [
24
].
Briefly, 8 mg of simvastatin was dissolved in 200
µ
L of absolute ethanol, followed by the
addition of 300
µ
L 0.1 N NaOH and incubation for 2 h at 50
◦
C. Finally, 500
µ
L of sterile
water was added to obtain a 20 mM stock. For the negative control treatment, we used
complete endothelial growth medium only since the DMSO in the stocks became diluted at
least 1000-fold in complete endothelial growth medium during application.
To evaluate the senolytic effects of the tested compounds, the cell death-inducing
effects were examined on two types of senescent HUVECs and compared with the effect on
proliferation-competent HUVECs (“young”). The first type of HUVECs was exhaustively
propagated to senescence (“old”), and the second type was brought to senescence with
γ
-irradiation (“irradiated”). The irradiated cells were freshly prepared for each experiment,
starting approximately 14 days in advance, while “young” and “old” cells were used
directly after recovering from storage in liquid nitrogen. Three independent experiments
were conducted, each with duplicates.
For the analysis, HUVECs were trypsinized, and transferred into pre-gelatinized (0.2%
gelatin) Corning 384-well black plates (3985, Corning, Corning, NY, USA). The cells were
re-suspended in complete endothelial growth medium and seeded at a low seeding density
of 750 cells per well for young HUVECs, providing sufficient space for proliferation, and
1500 cells for old and irradiated HUVECs, which were larger and non-proliferative. After
24 h of cultivation, and again after 72 h, the culture medium was partially removed (leaving
20
µ
L to prevent drying out), and the test substances were added to achieve a 1
×
working
concentration. The HUVECs were then exposed to the test and control substances for a
period of four days in a CO2incubator.
Cell fate and the cell count of HUVECs were monitored daily using the Olympus IX83
cellVivo live cell imaging system. Each day, the 384-well plate was temporarily transferred
to a temperature- and CO
2
-controlled incubation chamber (PeCon, Erbach, Germany) for a
short visualization of each well. At the end of the experiments, the cells were fixed with 2%
PFA, washed three times with PBS, permeabilized with 0.2% Triton-X 100, and stained with
Hoechst 33258. The daily cell counts as well as end-point cell counts of stained cells, were
determined using cellSens software 2.1 (Olympus) and respective macros.
2.4. Western Blotting
Cultures of young, old, and irradiated HUVECs were kept in T25 cell culture flasks and
exposed for 72 h to either 0.33
µ
M, 0.6
µ
M, or 1
µ
M activated simvastatin or were left in full
growth medium as a negative control. Medium renewal was performed after an initial 48 h.
Cells 2023,12, 2836 5 of 21
As a positive control for apoptosis induction, young HUVECs were exposed for 3.5 h to
0.2
µ
M staurosporine (HY-15141, MedChem Express). After the 72-hour cultivation period
in the presence or absence of activated simvastatin or 3.5 h exposure to staurosporine,
respectively, the proteins from adhering cells were isolated. In addition, proteins were
isolated from the culture supernatants containing cellular debris of HUVECs subjected to
statin treatment (collected after 48 h during medium change).
Briefly, adhering cells were harvested using trypsinization and centrifuged at 365
×
g,
and the obtained pellet was washed twice with ice-cold PBS and then lysed using 100
µ
L
per 10
6
cells RIPA buffer (Sigma-Aldrich) supplemented with a protease inhibitor cocktail
(Bimake, Houston, TX, USA). The lysates were immediately vortexed, placed on ice for
30 min, and centrifuged at 14,000
×
gat 4
◦
C for 30 min. The pellet containing the cell debris
was discarded, and the supernatant was transferred to a new tube and immediately frozen.
In the case of culture supernatants, the collected fluids were ultracentrifuged for 45 min
at 4
◦
C and 100,000
×
gin an Optima TLX Ultracentrifuge using the TLA 100.3 rotor and
suited polycarbonate tubes (all Beckman Coulter, Brea, CA, USA). The obtained pellets
were resuspended in 40
µ
M protease inhibitor-supplemented RIPA buffer and processed
analogously to protein lysates from adhering cells.
A spectrophotometric assessment of protein concentration was carried out using the
NanoDrop 2000 instrument (Thermo Fisher Scientific). About 20–30
µ
g of total protein was
mixed with an equal volume of 2
×
reducing Laemmli solution and heated at 95
◦
C for
7 min on a thermomixer. Protein samples as well as a pre-stained protein marker (26619,
Thermo Fisher Scientific) were loaded and run on a 10% polyacrylamide gel. After separa-
tion, the proteins were transferred to polyvinylidene difluoride (PVDF) membranes, (Carl
Roth, Karlsruhe, Germany) pre-soaked in methanol, using wet blotting. The membranes
were subsequently blocked overnight in 5% skimmed milk (1% fat content; AppliChem,
Darmstadt, Germany) in PBS-T. Rabbit polyclonal
β
-actin antibody (1:1000; A2066, Sigma-
Aldrich), rabbit monoclonal lamin B1 antibody (1:500; ab133741), or Apoptosis Western Blot
Cocktail (1:250; ab136812) comprising antibodies against pro/p17-caspase-3, cleaved PARP-
1, and actin (both from Abcam), were used as primary antibodies. For
β
-actin and lamin B1
detection, donkey HRP (horseradish peroxidase)-conjugated secondary anti-rabbit (1:5000;
NA934, Amersham, Amersham, UK) was used. For Apoptosis Western Blot Cocktail
detection, HRP conjugated secondary antibodies (1:200; goat anti-mouse and anti-rabbit)
provided in the kit were applied. The membranes were exposed to HRP Western Bright
Sirius substrate (Advansta, San Jose, CA, USA), and the resulting chemiluminescence
was recorded using a CCD-based camera (Alpha Innotech, San Leandro, CA, USA). The
obtained images were analyzed using ImageJ software (version 1.53c) [
25
] and OlyVIA
software (version 2.9, Olympus).
2.5. Live Cell Imaging and Real-Time Detection of Apoptosis
Replicative senescent HUVECs were exposed to 0.33
µ
M activated simvastatin. On
day two of exposure, IncuCyte
®
Caspase-3/7 Green Apoptosis Assay Reagent 4440 (Essen
Bioscience, Ann Arbor, MI, USA), enabling real-time visualization of caspase-3/7 activation
in cells, was added to the complete endothelial growth medium. Time-lapse sequences were
recorded over 15 h (1 frame per 6 min) with the Olympus IX83 cellVivo live cell imaging
system using both the appropriate fluorescent channel as well as bright-field imaging.
2.6. Statistical Analysis
Daily recorded images of the 384-well test plates of three independent experiments
performed in duplicates were analyzed using the Olympus cellSens software’s Count and
Measure module. In preparation for parametric statistical testing, Shapiro–Wilk tests were
performed on all tested groups to confirm the assumption of a normal distribution, which
was confirmed for 77 of 78 conditions. A two-way analysis of variance (two-way ANOVA)
showed significant effects of both substance and concentration applied on all three tested
cell types. Finally, in order to ascertain which treatment groups differed significantly from
Cells 2023,12, 2836 6 of 21
their respective controls, a Dunnett’s Multiple Comparison test was performed post hoc.
All statistical tests were calculated in R using the packages: “tidyverse” for data processing
and plotting [26] and “DescTools” for statistical tests [27].
Concentration–response models were fit from the final-day responses of five sub-
stances: simvastatin (both, naïve and activated), lovastatin, atorvastatin, and pravastatin,
for all three types of HUVECs. All curves were modeled and plotted using R with the
additional packages “drc” [
28
] and “ggbreaks” [
29
]. All models were calculated using
log–logistic regression functions, with either one or two fixed parameters [
30
]. The general
4-parameter log–logistic function is described with the following equation:
f(x)=c+d−c
1+ex p(b(log(x)−log(e))
For most cases, both lower and upper asymptote limits were fixed (parameters c
and d, respectively), while only the parameters for the slope and the point of inflection
(parameters band e, where ecorresponds to the approximate relative ED50) were estimated
using functions provided in the “drc” package.
Furthermore, all mean functions of the concentration–response curves were assessed
for their fit to their respective data. Lack-of-fit tests showed that the mean structures of the
models accurately represent the given data.
Significance was assigned to pvalues less than 0.05. Results are given as means
±
SD.
3. Results
3.1. Generation of the Cells for the Two Types of Senescence Models, of Replicative Senescent
HUVECs with Extensive Passaging, and of Stress-Induced Senescent HUVECs with Exposure
to γ-Irradiation
We studied the senolytic aptitude of the tested compounds in two different senescence
models. On the one hand, we established replicative senescent cells by extensively prop-
agating a pool of proliferation-competent HUVECs, and on the other hand, we created
stress-induced senescent cells by exposing young HUVECs to 10 Gy of ionizing irradiation
(“irradiated” HUVECs).
For the induction of replicative senescence, we kept splitting HUVEC cultures and
monitored their proliferation capacity and morphology during the whole cultivation pe-
riod. With an increasing number of passages, the endothelial cultures became less pro-
liferative, and the cells gradually changed their morphological appearance. Specifically,
the cells progressively lost their typical cobblestone-like morphology (a hallmark of con-
fluent young HUVECs), became larger, more flat, and frequently multinucleated, and
formed long and branched protrusions (Supplementary Figure S1a). To generate cells that
became senescent prematurely, with stress exposure, we exposed HUVECs to ionizing
radiation. A single dose of 10 Gy induced cell death in approximately 30% of cells. The
surviving portion of irradiated cells exhibited features of cellular senescence similar to
the senescent phenotype observed in replicative senescent cells, namely: increased cell
size, multiple nuclei, flattened and granulated appearance, long protrusions, and irregular
shape (Supplementary Figure S1b).
We conducted an extensive analysis to validate the senescent status of both types of
generated HUVECs. The quantitative assessment focused on two key senescence markers:
Ki-67, for measuring the proportion of cells ceasing proliferation [
31
], and
γ
H2AX, for
evaluating the increase in lasting DNA damage [
32
] (Figure 1). In addition, we examined the
upregulation of the cell cycle inhibitor p16 [
33
], a well-established senescence marker, using
immunocytochemical staining (Figure 2). Moreover, given the importance of senescent cells’
ability to evade apoptosis [
34
], we used immunofluorescence to visualize the augmented
expression of apoptosis inhibitors Bcl-2 and XIAP [35,36] (Supplementary Figure S2). The
multitude of assessed senescence indicators/markers used allowed us to omit the execution
of the SA-ß-gal assay, an assay, which can lead to false positive results, e.g., in stressed cells,
as described [37,38] and as observed by us.
Cells 2023,12, 2836 7 of 21
Cells 2023, 12, x FOR PEER REVIEW 7 of 23
Ki-67, for measuring the proportion of cells ceasing proliferation [31], and γH2AX, for
evaluating the increase in lasting DNA damage [32] (Figure 1). In addition, we examined
the upregulation of the cell cycle inhibitor p16 [33], a well-established senescence marker,
using immunocytochemical staining (Figure 2). Moreover, given the importance of senes-
cent cells’ ability to evade apoptosis [34], we used immunouorescence to visualize the
augmented expression of apoptosis inhibitors Bcl-2 and XIAP [35,36] (Supplementary Fig-
ure S2). The multitude of assessed senescence indicators/markers used allowed us to omit
the execution of the SA-ß-gal assay, an assay, which can lead to false positive results, e.g.,
in stressed cells, as described [37,38] and as observed by us.
Figure 1. Assessment of Ki-67 and γH2AX expression in young and senescent HUVECs (old and
irradiated). (a) Immunouorescent co-detection of Ki-67 (red) and γH2AX (green); nuclear counter-
stain with DAPI scale bar 50 μm. (b) Quantication of the portion of Ki-67 positive cells and of cells
showing the senescence-associated doed paern of γH2AX. Statistical analysis was made using
unpaired t-tests. Signicance levels are indicated as follows: **** p < 0.0001.
Ki-67 γH2AX Merge DAPI
Irradiated
Young Old IrradiatedYoung Old Irradiated
Old Young
a
b
50µm
100100
80
60
40
20
0
80
60
40
20
0
% Ki-67 positive cells
% γH2AX positive cells
**** **** **** ****
Figure 1.
Assessment of Ki-67 and
γ
H2AX expression in young and senescent HUVECs (old and
irradiated). (
a
) Immunofluorescent co-detection of Ki-67 (red) and
γ
H2AX (green); nuclear counter-
stain with DAPI scale bar 50
µ
m. (
b
) Quantification of the portion of Ki-67 positive cells and of cells
showing the senescence-associated dotted pattern of
γ
H2AX. Statistical analysis was made using
unpaired t-tests. Significance levels are indicated as follows: **** p< 0.0001.
This analysis revealed, as expected, that the portion of cells expressing the prolifer-
ation marker Ki-67 was minimal in both types of senescent cultures (Figure 1). While in
cultures of young proliferating HUVECs, Ki-67 levels remained high, the percentage of
Ki-67-expressing
cells in senescent cultures was only about 9% (only one-third of these cells
had an intense and two-thirds rather faint remnant Ki-67 signal) in the case of replicative
senescence and about 3% in cultures prematurely senescent after irradiation. The expres-
sion of the double-strand break marker
γ
H2AX in a pattern of few bright spots per nucleus,
characteristic for senescent cells, was observed commonly in both types of senescent cell
cultures, while in cultures of young HUVECs, this pattern was rarely detected, and
γ
H2AX
expression was limited to mitotic cells, where
γ
H2AX is involved in DNA replication and
appears in a finely dotted pattern (Figure 1). In phase contrast, one can observe typical
alterations in size, shape, and granularity. As expected, the cell cycle-inhibitor p16 was
expressed in the majority of cells of both types of senescence models, while in young cells,
p16 was expressed to a much lower degree (Figure 2a,b). Further, we found that both types
of senescent HUVECs expressed higher levels of the anti-apoptotic proteins Bcl-2 and XIAP
than young HUVECs (Supplementary Figure S2), consistent with the higher resistance to
apoptosis reported for senescent cells [
34
], even though, in the case of endothelial cells, this
anti-apoptotic aspect might not be as pronounced as in fibroblasts [
39
]. In addition, we
also assessed the protein expression level of an integral part of the nuclear lamina, lamin
B1, which is commonly reduced in senescent cells [
40
,
41
]. Western blot analysis revealed
that compared with young HUVECs, lamin B1 expression was low in cells senescent with
propagation and virtually absent in cells senescent with irradiation (Figure 2c).
Cells 2023,12, 2836 8 of 21
Cells 2023, 12, x FOR PEER REVIEW 8 of 23
Figure 2. Assessment of p16 expression. (a) Immunouorescent staining for p16 showing the dier-
ent signal intensities of young versus senescent HUVECs (old and irradiated). P16 in red, nuclear
staining in blue, and corresponding phase contrast images; scale bar 150 μm. (b) Dierences in p16
expression were assessed as integrated cellular uorescent signal intensities per eld of view di-
vided by the number of cell nuclei present (given in arbitrary units (a.u.)) by assessing at least ve
dierent elds of view using the Olympus software package cellSens and analyzed with unpaired
t-tests using GraphPad Prism. Signicance levels are indicated as follows: ** p < 0.01, *** p < 0.005,
ns = not signicant. (c) Western blot analysis of lamin B1 expression in young, old, and irradiated
HUVECs. The detection of β-actin was used as an internal standard.
This analysis revealed, as expected, that the portion of cells expressing the prolifera-
tion marker Ki-67 was minimal in both types of senescent cultures (Figure 1). While in
cultures of young proliferating HUVECs, Ki-67 levels remained high, the percentage of
Ki-67-expressing cells in senescent cultures was only about 9% (only one-third of these
cells had an intense and two-thirds rather faint remnant Ki-67 signal) in the case of repli-
cative senescence and about 3% in cultures prematurely senescent after irradiation. The
expression of the double-strand break marker γH2AX in a paern of few bright spots per
nucleus, characteristic for senescent cells, was observed commonly in both types of senes-
cent cell cultures, while in cultures of young HUVECs, this paern was rarely detected,
and γH2AX expression was limited to mitotic cells, where γH2AX is involved in DNA
replication and appears in a nely doed paern (Figure 1). In phase contrast, one can
observe typical alterations in size, shape, and granularity. As expected, the cell cycle-in-
hibitor p16 was expressed in the majority of cells of both types of senescence models, while
in young cells, p16 was expressed to a much lower degree (Figure 2a,b). Further, we found
that both types of senescent HUVECs expressed higher levels of the anti-apoptotic pro-
teins Bcl-2 and XIAP than young HUVECs (Supplementary Figure S2), consistent with the
higher resistance to apoptosis reported for senescent cells [34], even though, in the case of
endothelial cells, this anti-apoptotic aspect might not be as pronounced as in broblasts
[39]. In addition, we also assessed the protein expression level of an integral part of the
nuclear lamina, lamin B1, which is commonly reduced in senescent cells [40,41]. Western
blot analysis revealed that compared with young HUVECs, lamin B1 expression was low
in cells senescent with propagation and virtually absent in cells senescent with irradiation
(Figure 2c).
3.2. Senescent-Rendered HUVECs Are More Susceptible to Simvastatin-Induced Cell Death
Figure 2.
Assessment of p16 expression. (
a
) Immunofluorescent staining for p16 showing the different
signal intensities of young versus senescent HUVECs (old and irradiated). P16 in red, nuclear staining
in blue, and corresponding phase contrast images; scale bar 150
µ
m. (
b
) Differences in p16 expression
were assessed as integrated cellular fluorescent signal intensities per field of view divided by the
number of cell nuclei present (given in arbitrary units (a.u.)) by assessing at least five different fields
of view using the Olympus software package cellSens and analyzed with unpaired t-tests using
GraphPad Prism. Significance levels are indicated as follows: ** p< 0.01, *** p< 0.005, ns = not
significant. (
c
) Western blot analysis of lamin B1 expression in young, old, and irradiated HUVECs.
The detection of β-actin was used as an internal standard.
3.2. Senescent-Rendered HUVECs Are More Susceptible to Simvastatin-Induced Cell Death
In our study, we aimed to identify compounds that can selectively eliminate senescent
endothelial cells while sparing young and healthy proliferating cells. We used two types
of senescent HUVECs in parallel: those that were rendered senescent with exhaustive
propagation and those that were rendered senescent with
γ
-irradiation, as well as young
HUVECs with intact proliferation competence. To measure the senolytic effect in conjunc-
tion with possible negative effects on the proliferation ability of young cells, we plated
different HUVEC types at their appropriate density in 384-well plates. For this, senescent
cells were seeded at a density more or less completely covering the provided culture surface.
Young cells were seeded at a sub-confluent density, allowing them to proliferate over the
full course of the four-day experiments. We then exposed the cells to the tested substances
at various concentrations for four days. We monitored the cells with live imaging and daily
cell counts.
Our preliminary experiments had identified simvastatin, a classic blood cholesterol-
lowering agent from the statin family of HMG-CoA reductase inhibitors [
42
], as a promising
substance with senolytic potential in HUVECs. To analyze the senolytic capacity of simvas-
tatin, we applied the substance at various concentrations ranging from 0.11
µ
M to 10
µ
M
every other day for a period of four days.
We found that treatment with simvastatin at concentrations greater than 0.11
µ
M in-
duced cell death in cultures of old HUVECs within four days in a concentration-dependent
manner (Figure 3a). HUVECs that had become senescent upon irradiation died to a compa-
rable or even greater extent (Figure 3b). Cultures of healthy young HUVECs treated with a
concentration of 1
µ
M only were inhibited in their proliferation, while at concentrations
of
2µM
and higher (Figure 3c), they also started to show signs of cell death (Figure 3c).
As a positive control for senolytic activity, we used a previously published senolytic treat-
ment [
10
] in which a combination of the kinase inhibitor dasatinib (at concentrations of
0.05 µM
, 0.15
µ
M, or 0.45
µ
M) with the antioxidant quercetin (at 10
µ
M) was applied,
as shown in Figure 3d–f. These concentration combinations of dasatinib and quercetin,
Cells 2023,12, 2836 9 of 21
as shown in our experiments, had an unexpectedly low eliminating effect on senescent
cells, yet they completely spared young cells from cell death induction and proliferation
hindrance at all tested concentrations.
Cells 2023, 12, x FOR PEER REVIEW 9 of 23
In our study, we aimed to identify compounds that can selectively eliminate senes-
cent endothelial cells while sparing young and healthy proliferating cells. We used two
types of senescent HUVECs in parallel: those that were rendered senescent with exhaus-
tive propagation and those that were rendered senescent with γ-irradiation, as well as
young HUVECs with intact proliferation competence. To measure the senolytic eect in
conjunction with possible negative eects on the proliferation ability of young cells, we
plated dierent HUVEC types at their appropriate density in 384-well plates. For this,
senescent cells were seeded at a density more or less completely covering the provided
culture surface. Young cells were seeded at a sub-conuent density, allowing them to pro-
liferate over the full course of the four-day experiments. We then exposed the cells to the
tested substances at various concentrations for four days. We monitored the cells with live
imaging and daily cell counts.
Our preliminary experiments had identied simvastatin, a classic blood cholesterol-
lowering agent from the statin family of HMG-CoA reductase inhibitors [42], as a prom-
ising substance with senolytic potential in HUVECs. To analyze the senolytic capacity of
simvastatin, we applied the substance at various concentrations ranging from 0.11 μM to
10 μM every other day for a period of four days.
We found that treatment with simvastatin at concentrations greater than 0.11 μM in-
duced cell death in cultures of old HUVECs within four days in a concentration-depend-
ent manner (Figure 3a). HUVECs that had become senescent upon irradiation died to a
comparable or even greater extent (Figure 3b). Cultures of healthy young HUVECs treated
with a concentration of 1 μM only were inhibited in their proliferation, while at concen-
trations of 2 μM and higher (Figure 3c), they also started to show signs of cell death (Fig-
ure 3c). As a positive control for senolytic activity, we used a previously published
senolytic treatment [10] in which a combination of the kinase inhibitor dasatinib (at con-
centrations of 0.05 μM, 0.15 μM, or 0.45 μM) with the antioxidant quercetin (at 10 μM)
was applied, as shown in Figure 3d–f. These concentration combinations of dasatinib and
quercetin, as shown in our experiments, had an unexpectedly low eliminating eect on
senescent cells, yet they completely spared young cells from cell death induction and pro-
liferation hindrance at all tested concentrations.
In all experiments, we accounted for spontaneous cell loss in the senescent cultures,
and therefore show the quantication of the treatment eect on the cell count in % of un-
treated control cells.
Figure 3.
Elimination of senescent HUVECs and inhibition of proliferation in young HUVECs
with 1
µ
M simvastatin. Time-response curves depict cell count changes for (
a
,
b
) senescent
(replication/irradiation-induced) and (
c
) young HUVECs when exposed to varying simvastatin
concentrations (ranging from 0.11 to 10
µ
M) over a 96-hour period. Additionally, time-response
curves for (
d
,
e
) senescent and (
f
) young HUVECs treated with combinations of quercetin (10
µ
M)
and dasatinib (0.05
µ
M, 0.15
µ
M or 0.45
µ
M) for 96 h are presented for comparison. Data in panels
(
a
,
b
,
d
,
e
) are expressed as percentages of the untreated control (depicted as a solid gray line as 100%),
while data in panels (
c
,
f
) are shown as percentages of the initial cell count (represented by the dotted
line at 100%; solid gray lines represent the values for the untreated control). These results are based
on three independent experiments conducted in duplicate. Statistical analysis was performed using
ANOVA followed by a post hoc Dunnett’s Multiple Comparison test. Significance levels are indicated
as follows: * p< 0.05, ** p< 0.01, *** p< 0.005, and ns = not significant.
In all experiments, we accounted for spontaneous cell loss in the senescent cultures,
and therefore show the quantification of the treatment effect on the cell count in % of
untreated control cells.
These
in vitro
results demonstrated the senolytic potential of simvastatin against both
replication-induced and radiation-induced senescent HUVECs at concentrations above
0.11 µM
and below 2
µ
M, prompting the question of the underlying mechanism and
whether this potential is specific to simvastatin or common to all statins.
3.3. All Investigated Lipophilic Statins Showed Senolytic Activity, Which Could Be Negated with
the Supplementation of Mevalonic Acid
We aimed to determine whether the selective removal of senescent endothelial cells is
a common property of statins, contingent on their blockage of the HMG-CoA reductase
enzyme. To address this, we tested the senolytic capacity of several other statins and we
investigated if such an effect would be negated with the concomitant provision of mevalonic
acid—a molecular entity whose synthesis is directly blocked by statins [
43
] and which
would therefore bypass the statin effect on the mevalonate/cholesterol
synthesis pathway
.
Cells 2023,12, 2836 10 of 21
To address the first point, we tested the effects of simvastatin, two other lipophilic
statins, i.e., lovastatin and atorvastatin, and the hydrophilic statin pravastatin [
42
]. As
above, we exposed both types of senescent HUVECs as well as young proliferation-
competent cells to these statins at concentrations ranging from 0.11
µ
M to 10
µ
M for
a period of four days. In addition, we examined if the pre-activation of simvastatin might
increase its senolytic activity, as some studies have reported that simvastatin would require
activation with alkaline hydrolysis for its optimal activity in vitro [24].
Our findings showed that lipophilic statins, including simvastatin, lovastatin, and
atorvastatin, were effective in eliminating senescent HUVECs generated with exhaus-
tive propagation while having little impact on young HUVECs (Figure 4a–c,e–g and
Supplementary Figure S3a–c,i–k
). Lovastatin required similar concentrations (starting at
0.33
µ
M, Figure 4b) for a comparable senolytic effect on old HUVECs, as previously ob-
served for simvastatin (Figure 4a), whereas the concentrations required for a comparable
senolytic effect were higher for atorvastatin, which started to eliminate senescent HU-
VECs only at concentrations above 1
µ
M (Figure 4a). Similar senolytic effects for all tested
lipophilic statins were observed using irradiation-induced senescent cells (Supplementary
Figure S3a–c). In contrast, in young HUVECs, the lipophilic statins became cytotoxic only
at high concentrations (2
µ
M for simvastatin and lovastatin and over 4
µ
M for atorvastatin,
Supplementary Figure S3i–k and Figure 4e–g) although a negative effect on the rate of
proliferation was noted at a lower concentration already (of about 1 µM).
The hydrophilic pravastatin, on the other hand, did not induce cell death in any type of
senescent cells analyzed at the tested concentrations (Figure 4d and
Supplementary Figure S3d
),
nor did it significantly affect young HUVECs (Supplementary Figure S3l and Figure 4h).
The latter findings are consistent with the fact that hydrophilic statins, such as pravas-
tatin [42], cannot penetrate the cell membrane on their own and thus require the presence
of a suitable membrane transporter for cellular uptake, which is primarily expressed on
liver and kidney cells [44].
As anticipated, the pre-activation of simvastatin resulted in the more effective removal
of senescent HUVECs (Supplementary Figure S4a,b and Figure 3a,b), whereas in young
HUVECs, activated simvastatin at the highest tested concentration of 1
µ
M did not cause a
reduction in the cell count until day 4 (Supplementary Figure S4c).
After demonstrating that all the tested lipophilic statins have senolytic potential, we
hypothesized that their ability to block the mevalonate pathway [
42
,
45
] is the molecular
basis for their senolytic capacity. To test this hypothesis, we co-administered mevalonic
acid (100
µ
M) during statin treatment, which provides the product of the step blocked
here. Indeed, circumventing the blocked step of the mevalonate pathway suppressed
the senolytic effect of all lipophilic statins studied (Figure 4i–k, presenting cell counts of
old HUVECs, Supplementary Figure S3e–g, showing cell counts of irradiated HUVECs,
and Supplementary Figure S3m–o, presenting cell counts of young HUVECs). Similar
results were also observed when pre-activated simvastatin was used in combination with
mevalonic acid (Supplementary Figure S4d–f). Expectedly, in the case of the hydrophilic
pravastatin, adding mevalonic acid to the treatment did not result in any significant changes
in senescent or young HUVECs (Figure 4l and Supplementary Figure S3h) nor in young
HUVECs (Supplementary Figure S3p).
These results demonstrate that the senolytic effects of lipophilic statins depend on
the inhibition of the mevalonate pathway, as evidenced by the selective removal of senes-
cent HUVECs by lipophilic statins, which can be blocked with the co-administration of
mevalonic acid.
Cells 2023,12, 2836 11 of 21
Cells 2023, 12, x FOR PEER REVIEW 12 of 23
Figure 4. Elimination of propagation-induced senescent HUVECs (old) by statins and prevention
thereof by mevalonic acid. (a–d) Time-response curves depict cell count changes for the eect of
selected lipophilic and hydrophilic statins on the cell count of senescent HUVECs over a 96-hour
period: (a) simvastatin, (b) lovastatin, (c) atorvastatin, and (d) the hydrophilic pravastatin. (e–h)
Concentration–response curves including EC50 values for old, irradiated, and young HUVECs
show the cell counts (in % of the initial count) 96 h after exposure to dierent concentrations of
statins. (i–l) Time–response curves for the cell count changes in old HUVECs exposed concomitantly
to dierent concentrations of (i) simvastatin, (j) lovastatin, (k) atorvastatin, and (l) pravastatin and
to 100 μM mevalonic acid. The used statin concentrations ranged from 0.11 to 10 μM and were ap-
plied for 96 h. Data in (a–d) are expressed as percentages of the untreated control (no statin, depicted
as a solid gray line as 100%), in the graph legend for (i–l), 0/0 labels the untreated control (no statin
and no mevalonate, black), while 0 labels the control only exposed to mevalonic acid but not to a
statin (gray). These results are based on three independent experiments conducted in duplicates.
Statistical analysis was performed using ANOVA followed by a post hoc Dunne’s Multiple Com-
parison test. * p < 0.05, *** p < 0.005, and ns = not signicant. All concentration–response curves,
which were ascertained using two dierent lack-of-t tests, are shown with 95% condence inter-
vals.
Figure 4.
Elimination of propagation-induced senescent HUVECs (old) by statins and preven-
tion thereof by mevalonic acid. (
a
–
d
) Time-response curves depict cell count changes for the ef-
fect of selected lipophilic and hydrophilic statins on the cell count of senescent HUVECs over a
96-h period: (
a
) simvastatin, (
b
) lovastatin, (
c
) atorvastatin, and (
d
) the hydrophilic pravastatin.
(e–h) Concentration–response
curves including EC50 values for old, irradiated, and young HUVECs
show the cell counts (in % of the initial count) 96 h after exposure to different concentrations of
statins. (
i
–
l
) Time–response curves for the cell count changes in old HUVECs exposed concomitantly
to different concentrations of (
i
) simvastatin, (
j
) lovastatin, (
k
) atorvastatin, and (
l
) pravastatin and to
100
µ
M mevalonic acid. The used statin concentrations ranged from 0.11 to 10
µ
M and were applied
for 96 h. Data in (
a
–
d
) are expressed as percentages of the untreated control (no statin, depicted as a
solid gray line as 100%), in the graph legend for (
i
–
l
), 0/0 labels the untreated control (no statin and
no mevalonate, black), while 0 labels the control only exposed to mevalonic acid but not to a statin
(gray). These results are based on three independent experiments conducted in duplicates. Statistical
analysis was performed using ANOVA followed by a post hoc Dunnett’s Multiple Comparison test.
*p< 0.05, *** p< 0.005, and ns = not significant. All concentration–response curves, which were
ascertained using two different lack-of-fit tests, are shown with 95% confidence intervals.
Cells 2023,12, 2836 12 of 21
3.4. Activated Simvastatin-Induced Apoptosis/Anoikis in Senescent HUVECs
To study the underlying mechanism of how statins might act senolytically on HUVECs
in more detail, we drew on the following two concepts. First, since senescent cells are
viewed as damaged cells that have successfully evolved resistance to apoptosis, and the
search for senolytic compounds often involves substances that would allow the induction
of apoptosis again [
9
,
46
,
47
]. Second, prior studies have reported that statins, although
applied at fairly high concentrations, can induce apoptosis in a variety of cancer cell lines
and primary cells, including endothelial cells [48–51].
Based on this, we set out to ascertain whether apoptosis, and which specific subtype
of apoptosis, plays a role in the elimination of senescent HUVECs when exposed to lower
statin concentrations. A hallmark of the initiation of apoptosis is the activation of caspases,
a family of cysteine proteases. Furthermore, the balance of pro-apoptotic (Bax) and anti-
apoptotic (Bcl-2) members of the Bcl-2 protein family at the mitochondrial level regulates
apoptosis [
36
]. Consequently, we explored whether statins differentially affect the pro- and
anti-apoptotic balance in senescent versus young HUVECs.
Initially, we examined the expression levels of Bax and Bcl-2 because these proteins
form mutually inhibiting heterodimers, and their quantitative ratio influences the decision
of whether cells undergo apoptosis or not [
36
]. However, after exposing senescent or young
cells to 0.33
µ
M activated simvastatin for 48 h, and we detected no significant change in the
mRNA-level ratio of pro-apoptotic Bax and anti-apoptotic Bcl-2.
To investigate the signs of apoptosis induction, we evaluated the proteolytic activation
of key apoptosis mediators, i.e., caspase-3 and its downstream target PARP-1, in response
to statins using Western blotting. Surprisingly, we did not observe evidence of cleaved,
activated caspase-3 or proteolytically activated PARP-1 in adherent HUVECs exposed
to 0.33
µ
M, 0.6
µ
M, or 1
µ
M activated simvastatin for 72 h (Figure 5a). In contrast, the
application of staurosporine as a positive control for apoptosis induction did result in the
proteolytic cleavage of caspase-3 and PARP1 in adherent HUVECs (Figure 5a), suggesting
that simvastatin induces cell death with distinct characteristics.
To gain deeper insights into the nature of cell death induced by simvastatin, we
used time-lapse microscopy. We closely tracked the morphological changes in and fate
of HUVECs exposed to activated simvastatin, capturing images at a rate of 10 frames
per hour. The resulting image sequences unveiled a distinct sequence of events: cells
initially contracted, lost their contact with the 2D matrix, and progressed into a phase of
pronounced membrane blebbing. Subsequently, observable cell movement came to an
abrupt halt, signifying cell death. Notably, the characteristic feature of apoptosis, membrane
blebbing, was exclusively observed in cells that had already detached from the matrix. This
observed sequence of events led us to consider that the mode of cell death induced by
statins might align with a specific subtype of apoptosis, known as anoikis [
52
,
53
]. Anoikis is
mediated by the loss of cell–matrix contact and associated cell survival signals. To provide
concrete evidence for our notion, we aimed to establish evidence for caspase activation in
response to statins occurring only after cell deadhesion.
To this end, we used a fluorescence-based sensor for detecting caspase-3/7 activation
to monitor the hypothesized induction of apoptosis in response to activated simvastatin
over time. Starting at 24 h after the addition of 0.33
µ
M activated simvastatin, cells were
recorded at the same frame rate as described previously, using both bright-field and
fluorescence channels, to correlate the timing of caspase activation with morphologically
discernible changes in the cells. Again, we observed that statin treatment resulted in cell
detachment, followed by the occurrence of blebbing. Remarkably, caspase activation was
only discernable in cells that had already severed their connection with the matrix (Figure 6,
Supplementary Figure S5, and Supplementary Movies S1 and S2). This observed sequence
of events, where caspase activation was only evident in cells that had already lost matrix
contact, strongly supports the conclusion that the mode of cell death induced by statins is
indeed anoikis.
Cells 2023,12, 2836 13 of 21
Cells 2023, 12, x FOR PEER REVIEW 14 of 23
Figure 5. Western blot analysis of caspase-3 and PARP-1 activation. (a) Young, old, and irradiated
HUVECs were exposed to 0.33 μM, 0.6 μM, and 1 μM activated simvastatin (aSV) for 72 h or alter-
natively, to 0.2 μM staurosporine (STS) for 3.5 h. (b) Caspase-3 and PARP-1 activation in superna-
tants collected after the initial 48 h of statin treatment. Samples in both cases, were analyzed with
an antibody cocktail consisting of antibodies against pro-caspase-3, activated caspase-3, cleaved
PARP-1, and actin as an internal standard.
To gain deeper insights into the nature of cell death induced by simvastatin, we used
time-lapse microscopy. We closely tracked the morphological changes in and fate of HU-
VECs exposed to activated simvastatin, capturing images at a rate of 10 frames per hour.
The resulting image sequences unveiled a distinct sequence of events: cells initially con-
tracted, lost their contact with the 2D matrix, and progressed into a phase of pronounced
membrane blebbing. Subsequently, observable cell movement came to an abrupt halt, sig-
nifying cell death. Notably, the characteristic feature of apoptosis, membrane blebbing,
was exclusively observed in cells that had already detached from the matrix. This ob-
served sequence of events led us to consider that the mode of cell death induced by statins
might align with a specic subtype of apoptosis, known as anoikis [52,53]. Anoikis is me-
diated by the loss of cell–matrix contact and associated cell survival signals. To provide
concrete evidence for our notion, we aimed to establish evidence for caspase activation in
response to statins occurring only after cell deadhesion.
To this end, we used a uorescence-based sensor for detecting caspase-3/7 activation
to monitor the hypothesized induction of apoptosis in response to activated simvastatin
over time. Starting at 24 h after the addition of 0.33 μM activated simvastatin, cells were
MW
cleaved PARP
a
b
-1
-
procaspase-3
cleaved caspase-3
~89 kDa
~42 kDa
~32 kDa
~17 kDa
aSV –0.33 0.6 1 –0.33 0.6 1 –0.33 0.6 1
young
old irrad
Figure 5.
Western blot analysis of caspase-3 and PARP-1 activation. (
a
) Young, old, and irradiated
HUVECs were exposed to 0.33
µ
M, 0.6
µ
M, and 1
µ
M activated simvastatin (aSV) for 72 h or
alternatively, to 0.2
µ
M staurosporine (STS) for 3.5 h. (
b
) Caspase-3 and PARP-1 activation in
supernatants collected after the initial 48 h of statin treatment. Samples in both cases, were analyzed
with an antibody cocktail consisting of antibodies against pro-caspase-3, activated caspase-3, cleaved
PARP-1, and actin as an internal standard.
In an effort to further validate these findings and demonstrate the activation of
caspase-3
in detached cells, we collected culture supernatants from senescent HUVECs
exposed to activated simvastatin for 48 h. These supernatants were subjected to ultracen-
trifugation, and the pelleted proteins were subsequently analyzed using Western blotting
with the same antibody cocktail used for adherent cells. The results revealed the presence of
activated caspase-3 fragments in the cell culture supernatants of both types of senescent HU-
VECs (Figure 5b). Notably, samples from irradiated HUVECs exhibited more pronounced
evidence of activated caspase-3 fragments compared with those from propagation-induced
senescent HUVECs. While the observed signals were relatively low overall, it is essential to
consider that the protein composition in the supernatants collected after the initial 48 h of
incubation likely represents a snapshot of the protein content at the time of harvest rather
than a cumulative picture, as a portion of the released proteins may have already degraded.
Thus, our results provide support for the hypothesis that statins induce anoikis, a
subtype of apoptosis induced by the loss of cell–matrix contact, in senescent HUVECs.
Cells 2023,12, 2836 14 of 21
Cells 2023, 12, x FOR PEER REVIEW 15 of 23
recorded at the same frame rate as described previously, using both bright-eld and u-
orescence channels, to correlate the timing of caspase activation with morphologically dis-
cernible changes in the cells. Again, we observed that statin treatment resulted in cell de-
tachment, followed by the occurrence of blebbing. Remarkably, caspase activation was
only discernable in cells that had already severed their connection with the matrix (Figure
6, Supplementary Figure S5, and Supplementary Movies S1 and S2). This observed se-
quence of events, where caspase activation was only evident in cells that had already lost
matrix contact, strongly supports the conclusion that the mode of cell death induced by
statins is indeed anoikis.
Figure 6. Examples of time-lapse sequences tracking morphological changes and caspase-3/7 acti-
vation in simvastatin-treated senescent HUVECs. (a) After exposing cells to a complete endothelial
growth medium containing 0.33 μM activated simvastatin for 24 hours, the Caspase-3/7 Green
Figure 6.
Examples of time-lapse sequences tracking morphological changes and caspase-3/7 acti-
vation in simvastatin-treated senescent HUVECs. (
a
) After exposing cells to a complete endothelial
growth medium containing 0.33
µ
M activated simvastatin for 24 hours, the Caspase-3/7 Green Apop-
tosis Assay Reagent 4440 was added, and the cells were recorded at a frequency of 10 images per hour
using both bright-field and fluorescence imaging (488/510 nm excitation/emission filter). The dashed
lines highlight the cell of interest. The full-length video of the presented sequence is available in the
Supplementary Material as Supplementary Movie S1. (
b
) For the control, vehicle-treated senescent
cells at the same time points.
Cells 2023,12, 2836 15 of 21
4. Discussion
Vascular dysfunction has been linked to many age-related diseases. Endothelial senes-
cence, characterized by an irreversible growth arrest and the secretion of pro-inflammatory
mediators that accompany senescence, is considered a culprit for vascular dysfunction.
In the last decade, research focused on identifying senolytic compounds that allow for
the selective elimination of such dysfunctional cells has led to the discovery of senolytic
compounds that have been successfully tested not only in pre-clinical experimental animal
models but also in a phase I pilot clinical study [54].
As senescent cells are known to be able to escape apoptosis, the search for senolytic
compounds has in many cases focused on finding substances that would enable the in-
duction of apoptosis in senescent cells [
34
,
46
,
47
]. Based on the knowledge that statins are
compounds that can induce apoptosis in primary and tumor cells when applied at very
high concentrations [
48
–
51
], we decided to explore the utility of statins for the selective
elimination of senescent endothelial cells.
Here, we report that statins, widely used cholesterol-lowering drugs [
17
], had a
senolytic effect on cultured vascular endothelial cells, as senescent HUVECs succumbed to
statin exposure at lower concentrations than their proliferation-competent counterparts.
Our decision to use actively proliferating young cells for control was driven by our intent
to evaluate the potential side effects of the tested senolytic candidate substances on cell
proliferation. Also, proliferating cells might exhibit higher susceptibility to toxicants
compared with quiescent cells. Concurrently, we recognize the limitation that cells in a
quiescent state may respond differently, potentially offering a more accurate reflection
of
in vivo
conditions. The senolytic capacity that we initially observed on simvastatin at
concentrations higher than 0.11
µ
M was not unique to simvastatin but was common to
the other lipophilic statins studied, i.e., lovastatin and atorvastatin, while the hydrophilic
pravastatin, unsurprisingly, and in accordance with its liver-specific uptake [
44
], did not
show senolytic activity toward the exposed endothelial cells.
In previous studies performed on non-senescent proliferation-competent endothe-
lial cells, statins have been noted to induce apoptosis when applied at concentrations
much higher than are clinically achieved during long-term therapy with these cholesterol-
lowering drugs. For example, 3 to 30
µ
M of lovastatin caused apoptotic cell death in a
majority of endothelial cells after 48 h [
50
], while in another study investigating apoptosis
as early as 24 h, 2.5
µ
M simvastatin in HUVECs induced apoptosis at only a low level of
about 10% [
55
]. The latter result is in accordance with our results, summed up in
Figure 3a
,
assessing the effect of simvastatin treatment over the time course of 96 h. In rat pulmonary
vein endothelial cells, the lipophilic statins simvastatin, lovastatin, atorvastatin, or flu-
vastatin applied at a very high concentration of up to 50
µ
M induced cell death within
24 h, which was associated with DNA fragmentation and activation of caspase-3 [
51
]. The
hydrophilic pravastatin, which is taken up selectively in the liver and kidneys via a specific
transporter [
44
], did not induce apoptosis in such cultured endothelial cells [
51
]. Our
results, where the onset of toxic effects of lipophilic statins on proliferative primary en-
dothelial cells occurred at concentrations greater than 1
µ
M for simvastatin and lovastatin
and greater than 4
µ
M for atorvastatin, are consistent with these data. Overall, these studies
demonstrated the ability of statins to induce cell death in proliferation-competent cells
when used at high concentrations.
In our experiments, examining the differential response of senescent and non-senescent
cells to statins, we demonstrated the senolytic potency of the lipophilic statins including
simvastatin, lovastatin, and atorvastatin. We found that senescent HUVECs were elim-
inated by simvastatin and lovastatin at concentrations higher than 0.11
µ
M. In young
cells, on the other hand, in accordance with the previous studies mentioned above, no
visible induction of cell death was observed at these concentrations or up to 1
µ
M within
the four-day treatment. Atorvastatin required higher concentrations for a comparable
senolytic effect, and the senolytic concentrations started at 1
µ
M for both types of senescent
HUVECs, whereas this compound was toxic only at concentrations of 4
µ
M and higher in
Cells 2023,12, 2836 16 of 21
proliferation-competent young cells. When comparing the senolytic effects of statins with
the combination of dasatinib and quercetin that we used in our study as a reference [
10
],
we found that dasatinib and quercetin eliminated senescent HUVECs at the used con-
centrations to a lower extent than statins. On the other hand, these compounds did not
significantly impact the proliferation of young HUVECs.
The fact that all three investigated lipophilic statins were senolytic suggested that
the underlying mechanism could be based on the inhibition of HMG-CoA reductase and
thus the synthesis of mevalonic acid and the synthetic cascades dependent on it. Our
results showing that the senolytic effect of statins could be blunted by the concomitant
administration of mevalonic acid are in agreement with the observations of other research
groups who showed that the apoptotic effect of statins on various tumor cells and en-
dothelial cells was also prevented with the concomitant administration of mevalonic acid.
Mevalonate, the synthesis of which can be directly inhibited by statins, is not only required
for cholesterol production but is also important for the synthesis of sterol products such as
geranylgeranyl pyrophosphate and farnesyl pyrophosphate; which deficiency is associated
with statin-mediated apoptosis induction in endothelial cells [
51
,
56
]. It has been shown
repeatedly that the prenylation of small G proteins such as RhoA, which are important for
cell adhesion, is negatively influenced by statin administration. Exposure of cells to statins
caused the retention of RhoA in the cytosol, reduced its activity at the cell membrane and
impeded the formation of focal adhesion complexes, and compromised the integrity of the
cytoskeleton, subsequently leading to apoptosis in endothelial cells [
50
,
55
] and other cell
types [
57
]. Taken together, these findings suggest that statin-induced anoikis/apoptosis of
senescent HUVECs too, is mediated by blocking mevalonate signaling.
The question of the actual mode of statin-induced cell death of senescent HUVECs
was addressed here. Using bright-field time-lapse video microscopy, we first showed that
cells exposed to senolytic concentrations of simvastatin initially contract, then detach from
the matrix, and subsequently show membrane blebbing, followed by apparent cell death.
Such cell detachment is consistent with a previous report describing the detachment of
healthy proliferative HUVECs in response to 1 or 5
µ
M simvastatin treatment [
56
]. Yet,
although the authors of this study suggested that simvastatin fosters cell detachment by
inhibiting prenylation and presumably caspase-8 activation, they acknowledged an open
question: whether caspase activation precedes detachment or is a consequence thereof [
56
].
This question was addressed by our approach, where we visualized caspase activation
in real-time using a fluorescent caspase-3/7 sensor. In these simvastatin-treated HUVEC
cultures, we observed caspase activation only after cell detachment, supporting the notion
that induction of cell death by statins might be primarily caused by anoikis, a form of
apoptosis induced by the loss of cell–matrix contact [
52
,
53
]. This finding of caspase-3/7
activation in detached cells is in line with published Western blot data [
56
], where the active
caspase fragment was found only in cell lysates derived from detached cells, not in those
from adherent cells. In addition, this finding also fits the outcome of our Western blots
where, similarly, we did not find activation of caspase-3 or its downstream target
PARP-1
(Figure 5a) when analyzing lysates stemming from the adherent cells remaining after
simvastatin treatment, whereas we did see hints on caspase-3 activation when analyzing
protein extracts obtained from cell culture supernatants (Figure 5b). Overall, the presented
literature data and our current results led us to conclude that lipophilic statins induce
anoikis in senescent HUVECs.
Although the senolytic property of statins in endothelial cells has not been reported
previously, several studies performed in pre-clinical models could indirectly support our
results. For example, symptoms of experimentally induced pulmonary hypertension in
rats were alleviated with simvastatin treatment [
58
]. In this case, the beneficial effects
of statin treatment were coupled with the activation of caspase-3 in endothelial cells, as
demonstrated in histological sections of pulmonary aortas [
58
]. Furthermore, the number
of
β
-galactosidase positive, i.e., probably senescent cells, detected in the aortas of old rats
exposed to oxidized low-density lipoprotein (OX-LDL), could be reduced with concomitant
Cells 2023,12, 2836 17 of 21
simvastatin treatment [
59
]. Nevertheless, it also has to be mentioned that long-term
treatment of aged mice with simvastatin—in contrast with rapamycin treatment–did not
increase their life span [60].
Our current study raises the question of whether short-term treatment with appro-
priate statin concentrations could provide a therapeutic benefit for “blood vessel reju-
venation” that surpasses the reported benefits of statins including lipid-lowering and
anti-inflammatory properties as well as their ability to enhance NO synthesis [
17
,
19
,
20
].
Clinically, statins are often used as long-term therapy for patients at high risk of cardio-
vascular events, administered in daily doses of about 20 to 40 mg, resulting in maximum
plasma concentrations between 0.01 and 0.1
µ
M [
61
,
62
]. The feasibility of transient therapy
with short-term or intermittent high-dose statins is suggested by studies on oncological
patients. For example, a daily dose of lovastatin of 25 mg/kg for 7 consecutive days was
well-tolerated, with peak plasma levels reaching 2.32
±
1.27
µ
M and mean plasma levels
of 0.28
±
0.09
µ
M [
63
]. More than one week of simvastatin treatment at a daily dose of
15 mg/kg
has been described as being tolerated by oncological patients without severe
side effects [64].
While the elimination of senescence endothelial cells is in general considered to
beneficial [
65
], such an approach might also result in adverse effects. The elimination of
p16
High
-expressing cells was detrimental to health and lifespan due the disruption of the
blood–tissue barrier and resulted in liver fibrosis in experimental p16 knock-in mice [
66
].
The pitfalls of the elimination of senescence cells have been reviewed in more detail [
3
].
Thus, further thoroughly designed (pre)-clinical studies would have to be tackled in order
to evaluate the possible benefits of “hit and run” treatment with high-dose lipophilic statins.
The results reported here may invite the exploration of other naturally occurring HMG-
CoA reductase inhibitors in addition to lovastatin [
67
] as lead compounds for senolytic
therapeutics. For instance, policosanols, which are long-chain aliphatic alcohols found
in sugar cane extracts, have shown lipid-lowering effects, possibly through increased
AMP-kinase phosphorylation [
68
,
69
] or decreased HMG-CoA reductase expression [
70
].
Similarly, compounds like 4, 17(20)-pregnadiene-3, 16-dione (E- and Z-guggulsterone)
from the resin of Commiphora wightii have demonstrated hypolipidemic activity [
71
] and
appeared to directly inhibit HMG-CoA reductase in studies using HepG2 cells [
72
]. Such
natural compounds might serve as candidates for developing novel senolytic therapies.
5. Conclusions
In summary, our study demonstrates the senolytic capacity of lipophilic
statins—specifically
simvastatin, lovastatin, and atorvastatin—on cultured human endothelial cells. Beyond a
concentration threshold of 0.11
µ
M, these cholesterol-lowering drugs exhibit a distinct yet
limited range of effectiveness in selectively eliminating senescent cells, offering promise in
addressing age-related vascular dysfunction. The mechanism underlying statin-induced
senolysis involves anoikis, triggered by the disruption of the mevalonate pathway.
Our findings encourage the exploration of short-term, high-dose statin regimens as a
means for vascular rejuvenation, introducing a novel dimension to the well-established
benefits of these drugs. Additionally, our study suggests investigating alternative naturally
occurring HGM-CoA reductase inhibitors for potential senolytic therapies. Thoroughly
designed (pre)-clinical studies will be essential to evaluate the feasibility and risk associated
with such interventive strategies.
Thus, this research not only advances our comprehension of senolytic interventions
but also hints at a broader role for lipophilic statins in mitigating the impact of senescent
endothelial cells. The translation of these findings into further investigations holds the
potential to unveil new therapeutic avenues for these widely prescribed drugs.
Cells 2023,12, 2836 18 of 21
Supplementary Materials:
The following supporting information can be downloaded at: https://www.
mdpi.com/article/10.3390/cells12242836/s1, Supplementary Figure S1: Phase-contrast images docu-
menting the development of senescence in HUVECs; Supplementary Figure S2: Immunofluorescent
comparison of Bcl-2 and XIAP expression in young versus senescent HUVECs; Supplementary Figure
S3: Effects of different statins on the cell count of irradiated and young HUVECs; Supplementary
Figure S4: Effects of pre-activated simvastatin on the cell counts of old, irradiated, and young
HUVECs; Supplementary Figure S5: Additional example of a time-lapse sequence tracking morpho-
logical changes and caspase-3/7 activation in simvastatin-treated senescent HUVECs; Supplementary
Movies S1 and S2: Video sequences of senescent HUVECs exposed to activated simvastatin.
Author Contributions:
Conceptualization, J.M.B. and P.U.; methodology, S.H., A.O., M.F., S.Y.K.,
U.R. and M.L.; validation, B.B., E.M.A. and N.K.W.; formal analysis, N.K.W., B.B., S.H., J.M.B. and
P.U.; investigation, B.B., E.M.A., J.M.B. and P.U.; resources, J.M.B., P.U. and M.L.; data curation, J.M.B.,
N.K.W., B.B. and P.U.; writing—original draft preparation, J.M.B., B.B., K.Š. and P.U.;
writing—review
and editing, J.M.B. and P.U.; visualization, J.M.B., N.K.W. and B.B.; supervision, J.M.B. and P.U.;
project administration, P.U.; funding acquisition, J.M.B. and P.U. All authors have read and agreed to
the published version of the manuscript.
Funding:
This work was supported by the FWF (Austrian Science Fund) project Nr. P31743-B30
granted to PU and by GA ˇ
CR (Czech Sciences Foundation) project Nr. AT-CZ 21-38204L. In addition,
the contribution of SYK was supported by the OeAD GmbH (Austria’s Agency for Education and
Internationalisation) with an Ernst Mach Follow-up Grant.
Institutional Review Board Statement:
HUVECs were isolated from human umbilical veins based on
the granted permission GS4-EK-4/562-2018 issued by the Ethic Commission of Lower
Austria, Austria
.
Informed Consent Statement: Not applicable.
Data Availability Statement:
The data presented in this study are available upon request from the
corresponding author.
Acknowledgments:
We would like to thank Judit Mihaly-Bison for her excellent technical support,
and Reinhold Breuss and Johannes Schmid for support during manuscript finalization. Open Access
Funding by the Austrian Science Fund (FWF).
Conflicts of Interest: The authors declare no conflict of interest.
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