Effects of some anti-diabetic and cardioprotective agents on proliferation and apoptosis of human coronary artery endothelial cells.

Abstract

The leading cause of death for patients suffering from diabetes is macrovascular disease. Endothelial dysfunction is often observed in type 2 diabetic patients and it is considered to be an important early event in the pathogenesis of atherogenesis and cardiovascular disease. Many drugs are clinically applied to treat diabetic patients. However, little is known whether these agents directly interfere with endothelial cell proliferation and apoptosis. This study therefore aimed to investigate how anti-diabetic and cardioprotective agents affect human coronary artery endothelial cells (HCAECs).
The effect of anti-diabetic and cardioprotective agents on HCAEC viability, proliferation and apoptosis was studied. Viability was assessed using Trypan blue exclusion; proliferation in 5 mM and 11 mM of glucose was analyzed using [3H]thymidine incorporation. Lipoapoptosis of the cells was investigated by determining caspase-3 activity and the subsequent DNA fragmentation after incubation with the free fatty acid palmitate, mimicking diabetic lipotoxicity.
Our data show that insulin, metformin, BLX-1002, and rosuvastatin improved HCAEC viability and they could also significantly increase cell proliferation in low glucose. The proliferative effect of insulin and BLX-1002 was also evident at 11 mM of glucose. In addition, insulin, metformin, BLX-1002, pioglitazone, and candesartan significantly decreased the caspase-3 activity and the subsequent DNA fragmentation evoked by palmitate, suggesting a protective effect of the drugs against lipoapoptosis.
Our results suggest that the anti-diabetic and cardioprotective agents mentioned above have direct and beneficial effects on endothelial cell viability, regeneration and apoptosis. This may add yet another valuable property to their therapeutic effect, increasing their clinical utility in type 2 diabetic patients in whom endothelial dysfunction is a prominent feature that adversely affect their survival.

Full text

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ORIGINAL INVESTIGATIONOpen Access
Effects of some anti-diabetic and cardioprotective
agents on proliferation and apoptosis of human
coronary artery endothelial cells
Linnéa Eriksson, Özlem Erdogdu, Thomas Nyström, Qimin Zhang*and Åke Sjöholm*
Abstract
Background: The leading cause of death for patients suffering from diabetes is macrovascular disease. Endothelial
dysfunction is often observed in type 2 diabetic patients and it is considered to be an important early event in the
pathogenesis of atherogenesis and cardiovascular disease. Many drugs are clinically applied to treat diabetic
patients. However, little is known whether these agents directly interfere with endothelial cell proliferation and
apoptosis. This study therefore aimed to investigate how anti-diabetic and cardioprotective agents affect human
coronary artery endothelial cells (HCAECs).
Methods: The effect of anti-diabetic and cardioprotective agents on HCAEC viability, proliferation and apoptosis
was studied. Viability was assessed using Trypan blue exclusion; proliferation in 5 mM and 11 mM of glucose was
analyzed using [3H]thymidine incorporation. Lipoapoptosis of the cells was investigated by determining caspase-3
activity and the subsequent DNA fragmentation after incubation with the free fatty acid palmitate, mimicking
diabetic lipotoxicity.
Results: Our data show that insulin, metformin, BLX-1002, and rosuvastatin improved HCAEC viability and they
could also significantly increase cell proliferation in low glucose. The proliferative effect of insulin and BLX-1002
was also evident at 11 mM of glucose. In addition, insulin, metformin, BLX-1002, pioglitazone, and candesartan
significantly decreased the caspase-3 activity and the subsequent DNA fragmentation evoked by palmitate,
suggesting a protective effect of the drugs against lipoapoptosis.
Conclusion: Our results suggest that the anti-diabetic and cardioprotective agents mentioned above have direct
and beneficial effects on endothelial cell viability, regeneration and apoptosis. This may add yet another valuable
property to their therapeutic effect, increasing their clinical utility in type 2 diabetic patients in whom endothelial
dysfunction is a prominent feature that adversely affect their survival.
Keywords: Diabetes, Endothelium, Coronary artery disease, Insulin, Statin, Metformin, Lipids, AT1-receptor antago-
nist, Peroxisome proliferator-activated receptor gamma agonist
Background
The prevalence of diabetes among adults worldwide was
estimated in 2010 to 6.4%, thus affecting 285 million
adults. This figure is predicted to rise to 7.7%, in num-
bers 439 million, by 2030 [1]. In patients with diabetes,
the major cause of death is macrovascular disease [2,3],
and in individuals with type 2 diabetes, the main etiology
for up to 75% of the mortality is atherosclerotic
cardiovascular disease [4]. In contrast to microangiopa-
thies (e.g. nephropathy and retinopathy), where the causal
relation to hyperglycemia is well supported, the link
between hyperglycemia and macroangiopathy is uncer-
tain, at least in terms of the possibility of reducing
macrovascular morbidity solely by reducing hyperglyce-
mia. Most patients with diabetes are consequently being
treated with one or more antidiabetic drugs, a lipid-
lowering statin and an ACE inhibitor or angiotensin
receptor antagonist for hypertension and/or albuminuria.
* Correspondence: qmnzhang@yahoo.com; ake.sjoholm@sodersjukhuset.se
Department of Clinical Science and Education, Södersjukhuset, Karolinska
Institutet, Stockholm SE-11883, Sweden
Eriksson et al. Cardiovascular Diabetology 2012, 11:27
http://www.cardiab.com/content/11/1/27
CARDIO
VASCULAR
DIABETOLOGY
© 2012 Eriksson et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited.

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The endothelium is composed of a monolayer of cells
that line the lumen of blood vessels and form a physical
barrier between circulating blood and the vascular smooth
muscle cells. The endothelium plays a very important role
in maintenance of vascular integrity by protecting the
vessels from activation of clotting and proinflammatory
factors. It also participates in the regulation of blood flow
and blood pressure [5]. Loss of physiological features of
the endothelium, such as its preference to support vasodi-
latation, fibrinolysis and antiaggregation, is referred to as
endothelial dysfunction [6], which has been observed in
diabetes (type 1 and type 2), in obesity and in patients
with insulin resistance. In fact, the extent of endothelium-
dependent vasodilatation correlates in obese and insulin
resistant subjects with their individual insulin sensitivity
[7,8]. Endothelial dysfunction has thus emerged as an
important early target for preventing atherosclerosis and
cardiovascular disease [9].
Hyperglycemia and hyperlipidemia are important factors
in the development of endothelial dysfunction. Free fatty
acids (FFAs), formed during lipolysis from triglycerides,
and hyperglycemia are known to impair the endothelial-
dependent vasodilatation [10,11]. Increased plasma levels
of FFAs and glucose are characteristic features in patients
with type-2 diabetes. Both factors are known induce apop-
tosis of endothelial cells and endothelial cell death is
believed to be involved in, and contribute to, endothelial
dysfunction and atherosclerosis [12-14].
Current anti-diabetes drugs are mainly aimed to correct
hyperglycemia by promoting pancreatic b-cell insulin
secretion, increasing insulin sensitivity, or reducing intest-
inal glucose uptake and hepatic gluconeogenesis. Apart
from insulin, glimepiride, a third generation sulfonylurea
[15], pioglitazone, a peroxisome proliferator-activated
receptor gamma (PPAR-g) agonist, and a member of the
thiazolidinedione family (TZD) [16], and metformin, a
biguanide, are used for treatment of type 2 diabetes [9].
Candesartan, an angiotensin II receptor antagonist, and
rosuvastatin, a competitive inhibitor of the enzyme HMG-
CoA reductase, are used for management of hypertension
and hyperlipidemia, respectively [17,18]. In addition, we
have also studied BLX-1002, a novel thiazolidinedione
with no structural resemblance to other TZDs [19], which
does not appear to affect PPARs. There is evidence that
BLX-1002 can improve hyperglycemia in diabetic animal
models without the body weight gain typically associated
with PPARg-mediated adipocyte differentiation [19,20].
Not much is known about BLX-1002, but it has been
shown to potentiate insulin secretion from islets in a phos-
phatidylinositol 3-kinase (PI3K)-dependent manner. BLX-
1002 also activates AMP-activated protein kinase (AMPK)
[20] perhaps through its ability to inhibit the mitochon-
drial complex 1 [19,21].
Compared to studies on the actions of the above drugs
on glycemia, little has been done regarding their direct
actions on proliferation and apoptosis of human coronary
artery endothelial cells (HCAECs). Understanding the
effects of these agents is important as endothelial growth
and apoptosis are involved in endothelial repair and func-
tion. Disruption of the intimal layer subjects the arterial
wall to greater risk for macrovascular disease [14], the
most common etiology for morbidity and mortality in dia-
betic patients [15]. Therefore, the aim of our study was to
investigate the effects of drugs used in treatment of dia-
betic patients on proliferation and lipotoxicity-induced
apoptosis in HCAECs.
Methods
Materials
Clonetics™ HCAECs, culture medium EGM-2 MV and
cell culture supplements were purchased from Lonza
(Basel, Switzerland). Insulin lispro was from Eli Lilly and
Company (Indianapolis, IN), C-peptide was generously
provided by Prof. John Wahren (Creative Peptides, Inc.,
Stockholm, Sweden), and pioglitazone was graciously
donated by Takeda Pharmaceuticals North America, Inc.
(Lincolnshire, IL). Metformin, glimepiride, sodium palmi-
tate, and bovine serum albumin (BSA) (fatty acid free)
were purchased from Sigma-Aldrich (St. Louis, MO).
Rosuvastatin and candesartan were kindly given by Astra-
Zeneca (London, United Kingdom). BLX-1002 was
graciously donated by Bexel Pharmaceuticals, Inc. (Union
City, CA). [3H]thymidine was bought from Amersham
Biosciences (Piscataway, NJ), EnzChek®caspase-3 activity
assay kits from Molecular Probes®, Life Technologies
(Carlsbad, CA), DNA fragmentation ELISA kits from
Roche Diagnostics (Mannheim, Germany), and DC™
Protein Assay from BioRad Laboratories (Hercules, CA).
Cell culture
HCAECs, isolated from normal human coronary arteries
[22], were grown in EGM-2 MV medium supplemented
with hydrocortisone, human epidermal growth factor
(hEGF), 5% fetal bovine serum (FBS), vascular endothelial
growth factor (VEGF), human fibroblast growth factor
(hFGF)-B, R3-insulin-like growth factor (IGF)-1, ascorbic
acid and gentamicin/amphotericin-B at 37°C in a humidi-
fied (5% CO2, 95% air) atmosphere as recommended by
the supplier. Passage 5-12 were used in the study for eva-
luation of cell viability, [3H]thymidine incorporation
rates, DNA fragmentation ELISA and caspase-3 activity
assays. Confluent cultures were detached by trypsination
and seeded onto tissue culture dishes and grown until
80-90% confluence. Cells were incubated overnight in
serum-deficient EGM medium containing 0.5% FBS and
2 mM L-glutamine prior to 24 or 48 h incubation in the
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presence or absence of the desired agents. The use of the
cells was approved by the Research Ethics Committee,
Stockholm South, Dnr: 232/03.
Cell viability
Cells were incubated with serum deficient medium, in
the presence or absence of the drugs for 48 h. Cell
number was manually counted in a hemocytometer and
cell viability assessed by Trypan blue exclusion.
[3H]thymidine incorporation
Rates of [3H]thymidine incorporation into DNA were ana-
lyzed as previously described [22,23] and used as a measure
of DNA synthesis. In brief, cells were cultured until 80%
confluence. After serum starvation over night, cells were
incubated in the presence of the agents or vehicle for 24 h
at 5 mM or 11 mM glucose to simulate a normoglycemic
and hyperglycemic milieu, respectively. Cells were pulsed
with [3H]thymidine (1 μCi/ml) 8 h prior to the end of the
incubation. Cells were then collected and homogenized
through ultrasonication. The labeled cells were precipitated
in ice-cold 10% trichloroacetic acid (TCA). The precipitate
was washed with 10% TCA and [3H]thymidine incorpora-
tion into DNA was measured using a microplate scintilla-
tion and luminescence counter (Wallac MicroBeta®Trilux,
PerkinElmer) [24]. The protein concentration of the sam-
ples was measured using DC™ Protein Assay (Bio-Rad
Laboratories).
Caspase-3 activity
Cells were cultured to 90% confluence. After incubation
in serum deficient medium overnight, cells were pre-
treated for 1 hour with the drugs or solvents, after which
the incubation was continued for 24 h in the presence of
0.125 mM palmitate/0.25% BSA or vehicle [13]. Caspase-
3 activity, a measure of apoptosis, was evaluated using
the EnzChek®Caspase-3 Assay Kit (Molecular Probes®,
Life Technologies) according the manufacturer’s instruc-
tions. The assay is based on the 7-amino-4-methylcou-
marin-derived substrate Z-DEVD-AMC, which yields a
fluorescent product (excitation/emission ~342/441 nm)
upon proteolytic cleavage by active caspase-3. All results
were normalized to the protein concentration of the cor-
responding sample using DC™ Protein Assay (Bio-Rad
Laboratories).
DNA fragmentation
Cells were cultured to 90% confluence and treated in the
same way as described for the caspase-3 activity assay. Cell
apoptosis was analyzed using the Cell Death Detection Kit
plus (Roche Diagnostics) according the manufacturer’s
instructions. The Cell Death Detection Kit measures cyto-
plasmic DNA-histone nucleosome complexes generated
during apoptotic DNA fragmentation. Samples were
measured at 405 nm and corrected for background signals
caused by irregular microtiter plates or light scattering due
to solid particles in the solution at the reference wavelength
of 492 nm. Separate wells were seeded for protein concen-
tration measurements using DC™ Protein Assay (Bio-Rad
Laboratories). Absorbance data was normalized to the pro-
tein concentration of the corresponding treatment.
Statistical analysis
Results are expressed as mean ± SEM. Statistical analysis
was performed using Student’s t-test or ANOVA, as appro-
priate. P < 0.05 was considered statistically significant.
Results
Agents that were used in this study were the following:
insulin lispro (100 pM, 1 nM), C-peptide (1 nM), pioglita-
zone (2.5 μM), metformin (500 μM), glimepiride (1 μM),
rosuvastatin (10 nM), candesartan (100 nM) and BLX-
1002 (1 μM). Of the compounds tested, we were unable to
detect any effect of C-peptide on either proliferation or
apoptosis of the cells. All agents have been tested in all
conditions, but only compounds that were able to exert
significant effects are displayed in the graphs.
Cell viability of HCAECs is increased by the agents
As shown in Figure 1, among the agents tested, BLX-1002
and insulin (the latter at both 100 pM and 1 nM)
increased cell viability. Exposure of the cells to metformin
also resulted in a slight, but statistically significant,
increase in cell viability. Under the same conditions, cell
viability was also augmented by rosuvastatin, pioglitazone,
candesartan and glimepiride.
Insulin, BLX-1002, metformin and rosuvastatin stimulate
proliferation of HCAECs at normal glucose concentration
Since the increased viability noted above (Figure 1) could
be explained by increased proliferation, decreased apopto-
sis, or a combination thereof, we investigated if the drugs
exert any effect on HCAECs’ DNA synthesis. Rates of [3H]
thymidine incorporation were analyzed after a 24 hour
exposure to the agents at 5 mM glucose. The mitogenic
effect of the compounds was also verified by measuring
protein concentrations of the samples to reflect an
increase in cell number. As shown in Figure 2, insulin,
BLX-1002, metformin and rosuvastatin increased both
DNA synthesis and protein concentration significantly.
Insulin and BLX-1002 stimulate proliferation of HCAECs at
high glucose
Since insulin, BLX-1002, metformin and rosuvastatin
were able to increase the proliferation of HCAECs in 5
mM glucose (Figure 2), we further investigated whether
they had any effect on cell proliferation at high glucose.
HCAECs were therefore exposed to 11 mM of glucose,
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to simulate a diabetic setting, in the presence or absence
of the agents. Similar to the effect observed at 5 mM glu-
cose, insulin and BLX-1002 were both able to increase
DNA synthesis and protein concentration (Figure 3). In
contrast to their effects at 5 mM glucose, metformin and
rosuvastatin did not exert any effect on cell proliferation
under these conditions (data not shown).
Insulin, BLX-1002, metformin, pioglitazone and
candesartan suppress lipoapoptosis in HCAECs
Plasma FFA levels are increased in type-2 diabetic
patients and the FFA palmitate is known to induce
apoptosis of endothelial cells [13]. We therefore evalu-
ated how the agents affected the activity of cleaved cas-
pase-3, a crucial mediator of apoptosis [25], in HCAECs
after 24 hour exposure to 0.125 mM palmitate. As
shown in Figure 4, palmitate significantly increased cas-
pase-3 activity, thus confirming its pro-apoptotic effect,
in HCAECs. Insulin, BLX-1002, metformin, pioglitazone
and candesartan significantly decreased the palmitate-
induced caspase-3 activation, suggesting a protective
effect of the drugs against lipotoxicity in HCAECs. Gli-
mepiride significantly decreased basal caspase-3 activity
with ~20% (data not shown), which might explain why
an increased viability of the cells in the presence of gli-
mepiride is noted (Figure 1).
To corroborate the anti-apoptotic effect of the agents
above, we investigated their influence on palmitate-
induced DNA fragmentation by analyzing cytoplasmic
DNA-histone nucleosome complexes generated during
apoptosis. Palmitate alone gave rise to a markedly
increased apoptosis of the HCAECs (Figure 5). Co-incuba-
tion with insulin, BLX-1002, metformin, pioglitazone or
candesartan significantly countered the palmitate-induced
apoptosis, thus confirming the protective effect of these
agents against lipoapoptosis in HCAECs.
Discussion
Our major findings in this study are that insulin, BLX-
1002, metformin and rosuvastatin exert positive effects on
regeneration and viability of HCAECs. Insulin, BLX-1002,
metformin, pioglitazone and candesartan also conferred
protection of HCAECs against lipotoxicity.
Proliferation of HCAECs is induced in normal and high
glucose levels
By studying how the agents affect DNA synthesis, through
analysis of the incorporation of radiolabeled thymidine
into DNA, we found that insulin, BLX-1002, metformin
and rosuvastatin increased the HCAECs’ DNA synthesis
and subsequently the protein concentration in 5 mM glu-
cose. Taken together, this suggests that the above agents
stimulate HCAEC proliferation at normal glucose concen-
trations. The mitogenic effect seen with the compounds
probably explain, at least in part, the increased viability.
Kageyama et al. recently showed that when HCAECs
were exposed to glucose concentrations ranging from
5.6-16.7 mM apoptosis was dose dependently increased.
This was also coupled to an increased expression of the
death receptors TNF-R1 and Fas [26]. High glucose is
also known to cause oxidative stress through generation
of reactive oxygen species (ROS) in HCAECs as recently
shown by Serizawa et al. [27]. This might be due to on
uncoupling of endothelial nitric oxide synthase (eNOS)
and increased activity of the NADPH oxidase [27].
Increased levels of glucose might also cause proliferative
Figure 1 Insulin, metformin, BLX-1002, rosuvastatin, pioglitazone,
candesartan and glimepiride increase HCAEC viability. Cells were
incubated for 48 h in medium supplemented with 0.5% FBS in the
presence or absence of insulin lispro (100 pM and 1 nM), metformin
(500 μM), BLX-1002 (1 μM), rosuvastatin (10 nM), pioglitazone (2.5 μM),
candesartan (100 nM) and glimepiride (1 μM). Viability was assessed
with Trypan blue exclusion. * denotes P < 0.05, ** denotes P < 0.01,
*** denotes P < 0.001 for chance differences compared to controls by
Student’s t-test.
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dysfunction of endothelial cells [28,29], which is believed
to contribute to premature development of atherosclero-
sis [30]. We therefore wanted to test if the above agents
also stimulate HCAEC proliferation at 11 mM glucose,
simulating a diabetic milieu. We found that insulin and
BLX-1002 were the only two agents that could promote
cell proliferation in high glucose. The mitogenic effect
of the agents in HCAECs may prove beneficial in
dampening or delaying coronary atherosclerosis, by
rapidly covering a vascular wall lesion with endothelium
and thus protecting it from further atherothrombotic
events. It may also be favorable for diabetic patients with
coronary artery disease undergoing percutaneous coron-
ary revascularization and treated with drug-eluting stents.
It is believed that delayed re-endothelization is a major
underlying problem for late-stent thrombosis [31] and
Figure 2 Insulin, BLX-1002, metformin and rosuvastatin stimulate HCAEC proliferation at normal glucose concentration. In order to
study the effects of agents on proliferation of HCAECs, cells were incubated at 5 mM of glucose in medium supplemented with 0.5% FBS for 24
h in the presence or absence of insulin lispro (100 pM), metformin (500 μM), BLX-1002 (1 μM), rosuvastatin (10 nM). Eight hours prior to the end
of the incubation, cells were pulsed with [3H]thymidine. Cells were then harvested and [3H]thymidine incorporation into DNA was measured
using a microplate scintillation & luminescence counter, and the protein concentration of the samples was measured using DC™ Protein Assay.
* denotes P < 0.05, ** denotes P < 0.01, *** denotes P < 0.001 for chance differences vs. controls by Student’s t-test.
Figure 3 Insulin and BLX-1002 stimulate HCAEC proliferation in high glucose. HCAECs were incubated at 11 mM of glucose in medium
supplemented with 0.5% FBS for 24 h in the presence or absence of insulin lispro (100 pM) or BLX-1002 (1 μM). Eight hours prior to the end of
the incubation, cells were pulsed with [3H]thymidine. Cells were then harvested and [3H]thymidine incorporation into DNA was measured using
a microplate scintillation & luminescence counter and the protein concentration of the samples was measured using DC™ Protein Assay. *
denotes P < 0.05, ** denotes P < 0.01, *** denotes P < 0.001 for a chance difference vs. control by Student’s t-test.
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that rapid re-endothelization is essential for preventing
restenosis, which is the major limitation with this treat-
ment [32].
The underlying mechanisms involved in the agents’
proliferative effects were not investigated in this scanning
study, but will be subject to future work. However, the
effects are consistent with previous findings insofar that
insulin at supraphysiological concentrations increases
[3H]thymidine incorporation in endothelial cells [33].
Insulin and rosuvastatin have also been shown to activate
the PI3K/Akt pathway, a pathway that is well known to
beinvolvedincellsurvival
[18,22,34-36]. BLX-1002 is a novel compound of the
TZD family, but with no apparent PPAR affinity. We
andproliferation
have previously demonstrated that it can signal through
PI3K and is able to activate AMPK in insulin-secreting
cells [20]. Also, metformin is a known activator of
AMPK, and AMPK activates eNOS [37], an important
factor for proliferation of HCAECs [22].
HCAECs are protected from lipoapotosis
Plasma levels of FFAs are typically elevated in diabetic
patients and FFAs are known to impair endothelial-
dependent vasodilatation [11,38]. FFAs are also known to
cause apoptosis of endothelial cells [13,38]. Apoptosis of
HCAECs might disrupt the endothelial monolayer of the
coronary artery wall and might contribute to a proinflam-
matory environment that could lead to premature
Figure 4 Insulin, metformin, BLX-1002, pioglitazone and candesartan protect HCAECs against palmitate-induced caspase-3 activation.
Palmitate-induced caspase-3 activity, a measure of apoptosis, was evaluated using the EnzChek®Caspase-3 Assay Kit. HCAECs were incubated in
medium containing 5 mM glucose, supplemented with 0.5% FBS, with or without insulin lispro (1 nM), BLX-1002 (1 μM), metformin (500 μM),
pioglitazone (2.5 μM), candesartan (100 nM) in the presence of 0.125 mM palmitate or vehicle (ethanol and BSA) for 24 h. * denotes P < 0.05 for
a chance difference vs. controls by ANOVA. # denotes P < 0.05 for a chance difference vs. palmitate by ANOVA.
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endothelial dysfunction and unfolding of coronary ather-
osclerosis [13,38]. We therefore addressed whether the
above agents protect HCAECs against apoptotic cell
death caused by the fatty acid palmitate, simulating dia-
betic hyperlipidemia. Our results are commensurate with
other reports in the literature. Insulin has previously
been shown to protect human umbilical vein endothelial
cells (HUVECs) from palmitate-induced apoptosis
through activation of the PI3K/Akt pathway [38]. BLX-
1002, whose functions are essentially unknown, has been
shown to promote insulin secretion in pancreatic b-cells,
an effect that involves PI3K and AMPK activation [20].
Since both PI3K and AMPK convey anti-apoptotic sig-
nals in other cell types [38-41], these pathways may also
be operative in HCAECs. This also holds true for
metformin, which can activate AMPK and indeed, speci-
fic activation of AMPK has been shown to protect bovine
aortic endothelial cells from palmitate-induced apoptosis
[41]. The anti-apoptotic effect of pioglitazone observed in
the present study is consistent with findings in both
HUVECs and endothelial progenitor cells (EPCs) [42,43].
Although the mechanisms behind such effect remain
unclear and will be addressed in forthcoming studies, pal-
mitate induces ROS in endothelial cells [41] and pioglita-
zone has been shown to decrease ROS formation in such
cells [44]. This might contribute to its anti-apoptotic effect
since oxidative stress is known to induce cell death
[14,44]. Pioglitazone has also been shown to protect
HCAECs from tumor necrosis factor alpha induced cas-
pase-3 activity and apoptosis [45]. To the best of our
Figure 5 Insulin, metformin, BLX-1002, pioglitazone and candesartan protect HCAECs against palmitate-induced DNA fragmentation.
To further corroborate the protective effects of the agents against lipotoxicity-induced apoptosis, DNA fragmentation was evaluated using the
Cell Death Detection kit ELISAplus. Cells were exposed for 24 h to 0.125 mM palmitate or vehicle at 5 mM glucose with or without insulin lispro
(1 nM), BLX-1002 (1 μM), metformin (500 μM), pioglitazone (2.5 μM), candesartan (100 nM). * denotes P < 0.05 for a chance difference vs controls
by ANOVA, # denotes P < 0.05 for a chance difference vs palmitate by ANOVA.
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knowledge, there is no literature regarding putative effects
of candesartan on lipoapoptosis of endothelial cells. But,
in support to our findings, candesartan has been shown to
protect from hypoxia-induced endothelial cell apoptosis
through pathways involving increased eNOS expression
and decreased caspase-3 activity [17]. Candesartan’s ability
to modulate the caspase-3 activity might also contribute to
its positive effects on cell viability.
Conclusions
Abnormal proliferation and apoptosis of endothelial cells
are involved in endothelial dysfunction, damage and
repair. It thus contributes to the premature development
of atherosclerosis and vascular complications in diabetes,
making it important to understand the influence of cur-
rently applied anti-diabetic and cardioprotective agents on
endothelial function. Our results suggest that drugs such
as insulin, metformin, rosuvastatin, pioglitazone and can-
desartan that are oftentimes used in the clinical manage-
ment of diabetic patients have direct and beneficial effects
human coronary artery endothelial cells by promoting
their proliferation and conferring protection against lipo-
toxicity. These findings may add yet another valuable
property to their therapeutic effects, increasing their clini-
cal utility in type 2 diabetic patients in whom endothelial
dysfunction is a prominent feature that adversely affect
their survival.
Abbreviations
AMPK: AMP-activated protein kinase; BSA: Bovine serum albumin; eNOS:
Endothelial NOS; EPC: Endothelial progenitor cells; FBS: Fetal bovine serum;
FFA: Free fatty acid; HCAEC: Human coronary artery endothelial cells; HUVEC:
Human umbilical vein endothelial cells; hEGF: Human epidermal growth
factor; hFGF-B: Human fibroblast growth factor-B; IGF-1: R3-insulin-like
growth factor-1; NO: Nitric oxide; PI3K: Phosphatidylinositol 3-kinase; PPAR-γ:
Peroxisome proliferator-activated receptor gamma; ROS: Reactive oxygen
species; TCA: Trichloroacetic acid; TZD: Thiazolidinedione; VEGF: Vascular
endothelial growth factor.
Acknowledgements
Financial support was provided through the regional agreement on medical
training and clinical research (ALF) between Stockholm County Council and
the Karolinska Institute, and by Diabetes Research and Wellness Foundation,
the Janne Elgqvist family foundation, the Swedish Society for Medical
Research, the Swedish Society of Medicine, Stiftelsen Serafimerlasarettet, the
Swedish Heart and Lung foundation, Eli Lilly & Company, the European
Foundation for the Study of Diabetes, Karolinska Institutet Foundations, and
Stiftelsen Olle Engkvist Byggmästare.
Authors’ contributions
LE and ÖE performed the experimental procedures, performed the statistical
calculations, and contributed to results interpretation and discussion. TN, QZ
and ÅS provided expertise in diabetes and endothelial dysfunction, the in
vitro model, and conceived, designed and co-ordinated the research plan,
respectively. LE, QZ and ÅS wrote the manuscript. LE, TN, QZ and ÅS
contributed to discussion and edited the paper prior to submission. All
authors read and approved the final manuscript.
Competing interests
Dr. Sjöholm has received research grants, and provided lectures,
consultancies and expert testimony to several pharmaceutical companies
involved in diabetes care, such as GlaxoSmithKline, Takeda Pharmaceuticals
North America, Schering-Plough, Novo-Nordisk, Eli Lilly, Novartis, Aventis,
Sanofi-Synthelabo, Bristol-Myers, Squibb, Merck Sharp & Dohme, Pfizer,
Boehringer-Ingelheim, Selena-Fournier, Roche Diagnostics, Astra-Zeneca,
Bayer, Pharmacia, and Hässle Pharmaceuticals.
Dr. Sjöholm is also on the National and/or European Advisory Boards in
Diabetes Care for Eli Lilly, Takeda Pharmaceuticals, Novartis, Sanofi-Aventis,
Merck Sharp & Dohme, Boehringer-Ingelheim and Astra-Zeneca.
Received: 8 March 2012 Accepted: 21 March 2012
Published: 21 March 2012
References
1.Shaw JE, Sicree RA, Zimmet PZ: Global estimates of the prevalence of
diabetes for 2010 and 2030. Diabetes Res Clin Pract 2010, 87(1):4-14.
2.Sjoholm A, Nystrom T: Inflammation and the etiology of type 2 diabetes.
Diabetes Metab Res Rev 2006, 22(1):4-10.
3. Sjoholm A, Nystrom T: Endothelial inflammation in insulin resistance.
Lancet 2005, 365(9459):610-612.
4.Dandona P, Ghanim H, Chaudhuri A, Mohanty P: Thiazolidinediones-
improving endothelial function and potential long-term benefits on
cardiovascular disease in subjects with type 2 diabetes. J Diabetes
Complications 2008, 22(1):62-75.
5.Calles-Escandon J, Cipolla M: Diabetes and endothelial dysfunction: a
clinical perspective. Endocr Rev 2001, 22(1):36-52.
6.Avogaro A, de Kreutzenberg SV, Fadini G: Endothelial dysfunction: causes
and consequences in patients with diabetes mellitus. Diabetes Res Clin
Pract 2008, 82(Suppl 2):S94-S101.
7.Rask-Madsen C, King GL: Mechanisms of Disease: endothelial dysfunction
in insulin resistance and diabetes. Nat Clin Pract Endocrinol Metab 2007,
3(1):46-56.
8.Steinberg HO, Chaker H, Leaming R, Johnson A, Brechtel G, Baron AD:
Obesity/insulin resistance is associated with endothelial dysfunction.
Implications for the syndrome of insulin resistance. J Clin Invest 1996,
97(11):2601-2610.
9.Nathanson D, Nystrom T: Hypoglycemic pharmacological treatment of
type 2 diabetes: targeting the endothelium. Mol Cell Endocrinol 2009,
297(1-2):112-126.
10.Beckman JA, Goldfine AB, Gordon MB, Garrett LA, Creager MA: Inhibition of
protein kinase Cbeta prevents impaired endothelium-dependent
vasodilation caused by hyperglycemia in humans. Circ Res 2002,
90(1):107-111.
11.Lundman P, Tornvall P, Nilsson L, Pernow J: A triglyceride-rich fat
emulsion and free fatty acids but not very low density lipoproteins
impair endothelium-dependent vasorelaxation. Atherosclerosis 2001,
159(1):35-41.
12. Ho FM, Liu SH, Liau CS, Huang PJ, Lin-Shiau SY: High glucose-induced
apoptosis in human endothelial cells is mediated by sequential
activations of c-Jun NH(2)-terminal kinase and caspase-3. Circulation
2000, 101(22):2618-2624.
13. Chai W, Liu Z: p38 mitogen-activated protein kinase mediates palmitate-
induced apoptosis but not inhibitor of nuclear factor-kappaB
degradation in human coronary artery endothelial cells. Endocrinology
2007, 148(4):1622-1628.
14.Cifarelli V, Geng X, Styche A, Lakomy R, Trucco M, Luppi P: C-peptide
reduces high-glucose-induced apoptosis of endothelial cells and
decreases NAD(P)H-oxidase reactive oxygen species generation in
human aortic endothelial cells. Diabetologia 2011, 54(10):2702-2712.
15.Hamaguchi T, Hirose T, Asakawa H, Itoh Y, Kamado K, Tokunaga K,
Tomita K, Masuda H, Watanabe N, Namba M: Efficacy of glimepiride in
type 2 diabetic patients treated with glibenclamide. Diabetes Res Clin
Pract 2004, 66(Suppl 1):S129-S132.
16.Smith U: Pioglitazone: mechanism of action. Int J Clin Pract Suppl 2001,
121:13-18.
17.Matsumoto N, Manabe H, Ochiai J, Fujita N, Takagi T, Uemura M, Naito Y,
Yoshida N, Oka S, Yoshikawa T: An AT1-receptor antagonist and an
angiotensin-converting enzyme inhibitor protect against hypoxia-
induced apoptosis in human aortic endothelial cells through
upregulation of endothelial cell nitric oxide synthase activity. Shock 2003,
19(6):547-552.
Eriksson et al. Cardiovascular Diabetology 2012, 11:27
http://www.cardiab.com/content/11/1/27
Page 8 of 9

Page 9

18.Li X, Yang G, Zhao G, Wu B, Edin ML, Zeldin DC, Wang DW: Rosuvastatin
attenuates the elevation in blood pressure induced by overexpression
of human C-reactive protein. Hypertens Res 2011, 34(7):869-875.
Brunmair B, Staniek K, Lehner Z, Dey D, Bolten CW, Stadlbauer K, Luger A,
Furnsinn C: Lipophilicity as a determinant of thiazolidinedione action in
vitro: findings from BLX-1002, a novel compound without affinity to
PPARs. Am J Physiol Cell Physiol 2011, 300(6):C1386-C1392.
Zhang F, Dey D, Branstrom R, Forsberg L, Lu M, Zhang Q, Sjoholm A: BLX-
1002, a novel thiazolidinedione with no PPAR affinity, stimulates AMP-
activated protein kinase activity, raises cytosolic Ca2+, and enhances
glucose-stimulated insulin secretion in a PI3K-dependent manner. Am J
Physiol Cell Physiol 2009, 296(2):C346-C354.
Brunmair B, Staniek K, Gras F, Scharf N, Althaym A, Clara R, Roden M,
Gnaiger E, Nohl H, Waldhausl W, et al: Thiazolidinediones, like metformin,
inhibit respiratory complex I: a common mechanism contributing to
their antidiabetic actions? Diabetes 2004, 53(4):1052-1059.
Erdogdu O, Nathanson D, Sjoholm A, Nystrom T, Zhang Q: Exendin-4
stimulates proliferation of human coronary artery endothelial cells
through eNOS-, PKA- and PI3K/Akt-dependent pathways and requires
GLP-1 receptor. Mol Cell Endocrinol 2009, 325(1-2):26-35.
Edwards SJ, Reader KL, Lun S, Western A, Lawrence S, McNatty KP,
Juengel JL: The cooperative effect of growth and differentiation factor-9
and bone morphogenetic protein (BMP)-15 on granulosa cell function is
modulated primarily through BMP receptor II. Endocrinology 2008,
149(3):1026-1030.
Sjoholm A, Zhang Q, Welsh N, Hansson A, Larsson O, Tally M, Berggren PO:
Rapid Ca2+ influx and diacylglycerol synthesis in growth hormone-
mediated islet beta -cell mitogenesis. J Biol Chem 2000,
275(28):21033-21040.
Nicholson DW, Ali A, Thornberry NA, Vaillancourt JP, Ding CK, Gallant M,
Gareau Y, Griffin PR, Labelle M, Lazebnik YA, et al: Identification and
inhibition of the ICE/CED-3 protease necessary for mammalian
apoptosis. Nature 1995, 376(6535):37-43.
Kageyama S, Yokoo H, Tomita K, Kageyama-Yahara N, Uchimido R,
Matsuda N, Yamamoto S, Hattori Y: High glucose-induced apoptosis in
human coronary artery endothelial cells involves up-regulation of death
receptors. Cardiovasc Diabetol 2011, 10:73.
Serizawa K, Yogo K, Aizawa K, Tashiro Y, Ishizuka N: Nicorandil prevents
endothelial dysfunction due to antioxidative effects via normalisation of
NADPH oxidase and nitric oxide synthase in streptozotocin diabetic rats.
Cardiovasc Diabetol 2011, 10:105.
Varma S, Lal BK, Zheng R, Breslin JW, Saito S, Pappas PJ, Hobson RW,
Duran WN: Hyperglycemia alters PI3k and Akt signaling and leads to
endothelial cell proliferative dysfunction. Am J Physiol Heart Circ Physiol
2005, 289(4):H1744-H1751.
McGinn S, Saad S, Poronnik P, Pollock CA: High glucose-mediated effects
on endothelial cell proliferation occur via p38 MAP kinase. Am J Physiol
Endocrinol Metab 2003, 285(4):E708-E717.
Berk BC, Abe JI, Min W, Surapisitchat J, Yan C: Endothelial atheroprotective
and anti-inflammatory mechanisms. Ann N Y Acad Sci 2001, 947:93-109,
discussion 109-111.
Finn AV, Joner M, Nakazawa G, Kolodgie F, Newell J, John MC, Gold HK,
Virmani R: Pathological correlates of late drug-eluting stent thrombosis:
strut coverage as a marker of endothelialization. Circulation 2007,
115(18):2435-2441.
Nickson CM, Doherty PJ, Williams RL: Novel polymeric coatings with the
potential to control in-stent restenosis-an in vitro study. J Biomater Appl
2010, 24(5):437-452.
Gousseva N, Kugathasan K, Chesterman CN, Khachigian LM: Early growth
response factor-1 mediates insulin-inducible vascular endothelial cell
proliferation and regrowth after injury. J Cell Biochem 2001, 81(3):523-534.
Enomoto S, Sata M, Fukuda D, Nakamura K, Nagai R: Rosuvastatin prevents
endothelial cell death and reduces atherosclerotic lesion formation in
ApoE-deficient mice. Biomed Pharmacother 2009, 63(1):19-26.
Engelman JA, Luo J, Cantley LC: The evolution of phosphatidylinositol 3-
kinases as regulators of growth and metabolism. Nat Rev Genet 2006,
7(8):606-619.
Zeng G, Nystrom FH, Ravichandran LV, Cong LN, Kirby M, Mostowski H,
Quon MJ: Roles for insulin receptor, PI3-kinase, and Akt in insulin-
signaling pathways related to production of nitric oxide in human
vascular endothelial cells. Circulation 2000, 101(13):1539-1545.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
34.
35.
36.
37.Davis BJ, Xie Z, Viollet B, Zou MH: Activation of the AMP-activated kinase
by antidiabetes drug metformin stimulates nitric oxide synthesis in vivo
by promoting the association of heat shock protein 90 and endothelial
nitric oxide synthase. Diabetes 2006, 55(2):496-505.
Piro S, Spampinato D, Spadaro L, Oliveri CE, Purrello F, Rabuazzo AM: Direct
apoptotic effects of free fatty acids on human endothelial cells. Nutr
Metab Cardiovasc Dis 2008, 18(2):96-104.
Hermann C, Assmus B, Urbich C, Zeiher AM, Dimmeler S: Insulin-mediated
stimulation of protein kinase Akt: a potent survival signaling cascade for
endothelial cells. Arterioscler Thromb Vasc Biol 2000, 20(2):402-409.
Franke TF, Kaplan DR, Cantley LC: PI3K: downstream AKTion blocks
apoptosis. Cell 1997, 88(4):435-437.
Kim JE, Kim YW, Lee IK, Kim JY, Kang YJ, Park SY: AMP-activated protein
kinase activation by 5-aminoimidazole-4-carboxamide-1-beta-D-
ribofuranoside (AICAR) inhibits palmitate-induced endothelial cell
apoptosis through reactive oxygen species suppression. J Pharmacol Sci
2008, 106(3):394-403.
Artwohl M, Holzenbein T, Furnsinn C, Freudenthaler A, Huttary N,
Waldhausl WK, Baumgartner-Parzer SM: Thiazolidinediones inhibit
apoptosis and heat shock protein 60 expression in human vascular
endothelial cells. Thromb Haemost 2005, 93(5):810-815.
Gensch C, Clever YP, Werner C, Hanhoun M, Bohm M, Laufs U: The PPAR-
gamma agonist pioglitazone increases neoangiogenesis and prevents
apoptosis of endothelial progenitor cells. Atherosclerosis 2007,
192(1):67-74.
Fujisawa K, Nishikawa T, Kukidome D, Imoto K, Yamashiro T, Motoshima H,
Matsumura T, Araki E: TZDs reduce mitochondrial ROS production and
enhance mitochondrial biogenesis. Biochem Biophys Res Commun 2009,
379(1):43-48.
Chen J, Li D, Zhang X, Mehta JL: Tumor necrosis factor-alpha-induced
apoptosis of human coronary artery endothelial cells: modulation by the
peroxisome proliferator-activated receptor-gamma ligand pioglitazone. J
Cardiovasc Pharmacol Ther 2004, 9(1):35-41.
38.
39.
40.
41.
42.
43.
44.
45.
doi:10.1186/1475-2840-11-27
Cite this article as: Eriksson et al.: Effects of some anti-diabetic and
cardioprotective agents on proliferation and apoptosis of human
coronary artery endothelial cells. Cardiovascular Diabetology 2012 11:27.
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