Journal of Diabetes and Its Complications 18 (2004) 289–299

Aspirin rectifies calcium homeostasis, decreases reactive oxygen species,

and increases NO production in high glucose-exposed

human endothelial cells

Elena Dragomir, Ileana Manduteanu, Manuela Voinea, Gabi Costache,Adrian Manea, Maya Simionescu*

Institute of Cellular Biology and Pathology ‘‘Nicolae Simionescu’’, 8, BP Hasdeu Street, PO Box 35-14, 79691 Bucharest, Romania

Received 1 August 2003; received in revised form 13 February 2004; accepted 5 March 2004

Abstract

Aspirin’s pharmacological action is mainly related to its property to inhibit prostaglandin synthesis; apart from this, aspirin has some

beneficial side effects that are not completely understood, yet. Since aspirin possesses antioxidant properties and antioxidants prevent high D-

glucose enhanced endothelial [Ca2+]i, we questioned whether aspirin also has an effect on this process as well as on high-glucose-impaired

nitric oxide (NO) production. For these purposes, human endothelial cells (HECs) were cultured in normal concentration (5 mM) glucose

(NG) or high concentration (33 mM) glucose (HG) and after confluence, exposed for 48 h to HG in the absence or presence of 1 mM aspirin.

Then, the [Ca2+]i was measured fluorimetrically using fura-2, NO production was determined by Griess reaction, superoxide anions (O2) was

evaluated by ferricytochrome c reduction, the intracellular reactive oxygen species (ROS) were evaluated by fluorimetry, and the levels of

protein kinase C (PKC) by Western blot. The results showed that HECs exposed to HG displayed: (i) increased [Ca2+]i; (ii) enhanced O2

release; (iii) augmented level of intracellular ROS; and (iv) PKC translocation to the membrane fraction. By comparison, exposure to cells

grown in HG to 1 mM aspirin resulted in: (i) a reduction of histamine stimulated [Ca2+]i release to control level and of [Ca2+]i entry by 30% ;

(ii) a twofold increase in NO production; (iii) a decrease of O2� accumulation in both culture medium and cell homogenate (by 60.4% and

70%, respectively); (iv) a decline of ROS to the control levels; and (v) a reduction of PKC translocation to the control levels. These data

indicate that aspirin corrects the high-glucose-induced changes in cellular Ca2+ homeostasis and NO production, via a mechanism involving

the reduction of the O2� levels possible by acting on PKC-induced NADPH activity.

D 2004 Elsevier Inc. All rights reserved.

Keywords: High glucose; Aspirin; Nitric oxide; Superoxide anions; PKC

1. Introduction

Aspirin is a nonsteroidal anti-inflammatory drug

(NSAID) widely used in the treatment of nonsevere

human inflammatory disorders. In the last decade, various

studies have described prostaglandin-independent actions

of aspirin such as blockade of transcription factor NF-kB

(Kopp & Ghosh, 1994), prevention of colon cancer

development conferring a 40% risk decrease (Kune, Kuns,

& Watson, 1988; Thun, Namboodiri, Calle, Flanders, &

Health, 1993; Thun, Namboodiri, & Health, 1991), pro-

1056-8727/04/$ – see front matter D 2004 Elsevier Inc. All rights reserved.

doi:10.1016/j.jdiacomp.2004.03.003

* Corresponding author. Tel.: +40-21-411-0860; fax: +40-21-411-1143.

E-mail address: simionescum@simionescu.instcellbiopath.ro (M. Sim-

ionescu).

tection of endothelial cells (ECs) from oxidative stress (El

Midaoui, Wu, & De Champlain, 2002; Podhaisky, Abate,

Polte, Oberle, & Schroder, 1997) and beneficial effects in

diabetes mellitus (Hundal et al., 2002). Recently, it has

been demonstrated that chronic in vivo treatment with

aspirin prevents the development of hypertension and

significantly reduces the insulin resistance in chronically

glucose-fed rats (El Midaoui et al., 2002). However, the

mechanism of action of aspirin on type 2 diabetic patients

is less clear.

Endothelial dysfunction in diabetes mellitus has been

demonstrated (Cohen, 1993; Manduteanu, Voinea, Serban,

& Simionescu, 1999; Simionescu et al., 1996). Although the

mechanism of high glucose (HG) concentrations-induced

endothelial dysfunction is not yet known, it has been found

that the production of different mediators by EC (including

E. Dragomir et al. / Journal of Diabetes and Its Complications 18 (2004) 289–299290

nitric oxide [NO]) is impaired in HG conditions (Mon-

cada, Palmer, & Higgs, 1988). Thus, several investigators

have shown that in various blood vessels of experimental

diabetic rats and hamsters, the endothelium-dependent

relaxation is impaired (Costache et al., 2000; Diederich,

Skopec, Diederich, & Dai, 1994; Mayhan, Simmons, &

Sharpe, 1994).

The biosynthesis of endothelium-derived autacoids criti-

cally depends on the increase of intracellular Ca2+ concen-

tration, which also plays an important role in the signaling

pathways that regulate endothelial secretion of vasorelaxants.

It has been reported that HG exposure of human umbilical

ECs or porcine aortic ECs (for 24 h) increases resting [Ca2+]iby twofold, enhances agonist-stimulated Ca2+ elevation

(Sobrevia, Nadal, Yudilevich, & Mann, 1996; Wascher

et al., 1994) and endothelium-derived relaxing factor (EDRF)

production (Graier, Simecek, Kukovetz, & Kostner, 1996;

Wascher et al., 1994). In hyperglycemic conditions, an

enhanced Ca2+/EDRF signaling has been reported (Graier

et al., 1996); an increase in the formation of superoxide

anions (O2�) was suggested to be involved in the process.

The increase of plasma glucose concentration (found in

diabetes) induces the oxidative stress as a result of an

imbalance between the production of reactive oxygen species

(ROS) and the antioxidant defense mechanism (Bonnefont-

Rousselot, Bastard, Jaudon, & Delattre, 2000; Catherwood

et al., 2002). The production of ROS has been reported to be

increased in patients with diabetes (Baynes, 1991;Mullarkey,

Edelstein, & Brownlee, 1990; Oberley, 1988; Sano, Umeda,

Hashimoto, Nawata, & Utsumi, 1998; Wolf, Jiang, & Hunt,

1991) and recent papers indicate that glucose-related ROS

production has a central role in diabetic pathology (Lander,

1997; Sundaresan, Yu, Ferrans, Irani, & Fienkel, 1995). HG

concentration stimulates ROS production through the PKC-

dependent activation of NADPH oxidase in cultured vascular

cells (Inoguchi et al., 2000). NADPH oxidase is one of the

important sources of ROS in EC and smooth muscle cells.

Numerous stimuli that activate the vascular NADPH oxidase

generate ROS and increase intracellular Ca2+ (Allen, Khan,

Al-Mohana, Batten, & Yacoub, 1998; Honda et al., 1999;

Zaho, Ehringer, Dierichs, & Miller, 1997). Recent data

indicate that NADPH oxidase-generated ROS affect [Ca2+]ioscillations during histamine stimulation in human endothe-

lial cells (HECs) (Hu et al., 2002). Also, it was demonstrated

that the activity of the NADPH oxidase was increased in

diabetic vessels (Hink et al., 2001) and that HG concentra-

tion stimulated ROS production through a PKC-dependent

activation of NADPH oxidase in cultured vascular cells

(Inoguchi et al., 2000).

Since it has been shown that antioxidants such as vitamin

E, probucol, GSH, and vitamin C prevent high D-glucose

enhanced endothelial Ca2+/cGMP response by scavenging

the overshoot O2� (Graier, Simecek, Hoebel, Wascher, &

Kostner, 1997) and aspirin has antioxidant properties (El

Midaoui et al., 2002; Ghiselli, Laurenti, De Maiani, Maiani,

& Ferro-Luzzi, 1992; Podhaisky et al., 1997), we investi-

gated the effect of aspirin on the high D-glucose-induced

changes in intracellular Ca2+ concentration and NO release.

To elucidate the mechanism of action of aspirin, the effect of

the drug on HG-induced increased levels of ROS was

investigated. Also, because PKC membrane translocation

has been widely used as an indication of PKC activation

(Murphy, McGinty, & Godson, 1998), we further performed

Western blot analysis to test the effect of aspirin on PKC

activation. We provide evidence that aspirin has a favorable

effect on HECs grown in HG, reducing the increased level

of [Ca2+]i, decreasing the intracellular level of ROS and

superoxide anions by acting on PKC-induced activation of

NADPH oxidase, and increasing the NO production.

2. Materials and methods

2.1. Cells

HECs, an immortalized human EC line derived from

HUVEC-EA hy926 cell (kindly donated by Dr. Cora-Jean S.

Edgell, Department of Pathology University of North Car-

olina, Chapel Hill), were grown in Dulbecco’s modified

Eagle medium (DMEM) containing normal glucose (NG) or

high glucose concentration (HG), supplemented with 10%

fetal bovine serum (FBS), 100 U/ml penicillin, 100 Ag/ml

streptomycin in a humidified incubator with 5% CO2 and

95% air, at 37 jC. The cells were seeded at a concentration

of 5�104 cell/ml in 6- and 24-well tissue culture plates.

2.2. Reagents

The dye 2V,7V-dichlorofluoroscein diacetate (DCFH-DA)

was purchased from Molecular Probes (Eugene, OR, USA)

and the culture medium, amino acids, vitamins, and peni-

cillin/streptomycin were from Gibco. Monoclonal antibody

anti-PKC, fura-2 AM, aspirin and all other chemicals were

procured from Sigma-Aldrich Chemie (Germany) and the

FBS from Sebak (Suben, Austria).

2.3. Experimental protocol

HECs were subjected to three experimental conditions:

(i) the cells grown in DMEM containing normal, 5 mM D-

glucose (NG) for 7 days; (ii) cells grown in DMEM

containing 33 mM D-glucose (HG) in the culture medium

for 7 days; and (iii) cells grown in HG concentration (as in

ii) except that in the last 2 days, 1 mM aspirin was added.

The concentration of aspirin used was at therapeutic plasma

concentration as in Shimpo et al. (2000).

2.4. Measurement of intracellular Ca2+ concentration

Intracellular free Ca2+ concentration ([Ca2+]i) was deter-

mined using the fura-2 technique, as previously described

(Graier, Simecek, & Sturek, 1995). Briefly, HECs subjected

E. Dragomir et al. / Journal of Diabetes and Its Complications 18 (2004) 289–299 291

to the above experimental conditions were harvested (after

2-min treatment with 0.06% trypsin in PBS), centrifuged,

washed once, and resuspended in Hepes buffered saline

solution (HBSS) in the absence of Ca2+ but containing 3 AMfura-2/AM. After 1 h at 37 jC in the dark (under shaking),

the cells were washed twice and resuspended in HBSS. To

determine the cell fluorescence, a thermostatically controlled

(37 jC) dual-wavelength spectrofluorimeter (Shimadzu RF-

5001PC) was employed. The excitation wavelengths were

set at 340 and 380 nm and emission was monitored at 510

nm with a standard bandpass filter. Intracellular Ca2+ release

was established by stimulation with 25 AM histamine, in the

absence of free extracellular Ca2+. To assess the Ca2+ entry

into the cells, the increase in [Ca2+]i upon addition of 2.5

mM extracellular Ca2+ to histamine prestimulated cells was

measured. Intracellular calcium concentration was calculat-

ed according to the following equation, as described in

(Hallam, Pearson, Needham, 1988):

½Ca2þ�i ðnMÞ ¼ Kd

R� Rmin

Rmax � R

where R is the ratio of fluorescence of the sample at 340 and

380 nm. Rmax is the fluorescence ratio obtained by adding

10% Triton X-100 and Rmin is the fluorescence ratio subse-

quently obtained by 5 mM EGTA. Kd is the dissociation

constant of calcium-bound fura-2, taken to be 224 nM.

2.5. Determination of ROS

HECs were assayed for intracellular ROS using DCFH-

DA. Upon incubation, the latter is taken up by cells, where

intracellular esterase cleaves the molecule to DCFH, which,

in the presence of H2O2, is oxidized to DCF. The total

fluorescence was measured using a spectrofluorimeter (Shi-

madzu RF-5001PC) at an emission wavelength of 527 nm

and an excitation wavelength of 516 nm (Eremeeva, Dasch,

& Silverman, 2001). After measuring the fluorescence

intensity, the cells were lysed and the protein concentration

was determined using the bicinchoninic acid protein assay

reagent (Sigma). The ROS level was expressed as fluores-

cence unit per milligram of protein.

2.6. Measurement of O2� production

Superoxide anions were determined by the reduction of

the ferricytochrome c by O2� as described (Heinecke, Rosen,

Suzuki, & Chait, 1987). Briefly, HEC cultured in DMEM

(without phenol red) supplemented with 10% FBS, contain-

ing either 5 mM glucose, 33 mM glucose, and 1 mM aspirin

were incubated with 16 AM ferricytochrome c in the absence

or presence of 400 U/ml superoxide dismutase (SOD).

Reduction of ferricytochrome c was monitored at 550 nm

for 30 min. The difference in absorption between samples,

in the absence or presence of SOD, indicated directly the

extinction due to O2� reduction of ferricytochrome c. Reac-

tion that occurs has a rate constant i1.5�105 at pH 8.5 at

room temperature.

Fe3þ þ cyt cþ O�2 ! Fe3þcyt cþ O�

2

The concentration of O2� was calculated using the molar

extinction coefficient of reduced form of ferricytochrome c

that is: e=21,000 (Tarpey & Fridovich, 2001). Rate of O2�

release is given in micromoles per 106 cells. Cell counts

were determined for calculating results as micromoles O2�

per 106 cells per minute.

2.7. Assessment of NADPH-dependent O2� production

The NADPH-dependent O2� production by EC was

investigated using SOD-inhibitable cytochrome c reduction

assay as previously described (Li et al., 2002). The HECs

resuspended in lysis buffer (1 mM EGTA, 20 mM mono-

basic potassium phosphate pH 7.0) containing protease

inhibitors (0.5 mM phenylmethylsulfonyl, 0.5 Ag/ml leu-

peptin, 10 Ag/ml aprotinin) were disrupted using a Dounce

homogenizer. The homogenate was disrupted in 96-well

flat-bottom culture plates, and cytochrome c (200 AM) and

NADPH (100 AM) were added in the presence or absence of

SOD (200 U/ml) and incubated at room temperature for

30 min. Reduction of cytochrome c was determined reading

the absorbance at 550 nm on a microplate reader. O2�

generation was expressed as nanomoles O2� per minute

per milligram of protein.

2.8. Detection of NO

NO production was evaluated by measuring the level of

nitrite (NO2�), the oxidized product of NO, using the Griess

reaction as previously described (Hishikawa, Nakaki,

Suzuki, Saruta, & Kato, 1992). NO is an electrochemically

reactive species that can be oxidized. To measure the total

NO oxidation products, NO3� is first reduced to NO2

�, by

NADH-dependent nitrate reductase. Briefly, 50 Al cell su-pernatant (exposed to the three experimental conditions

described above) was mixed with 25.5 Al of 0.1 M potas-

sium phosphate buffer (pH 7.4), 11 Al of 2 mM NADPH,

1.5 Al of 1 mM FAD and 12 Al (1.25 U/ml) of nitrate

reductase. All samples were incubated for 2 h at room

temperature in the dark and then 75 Al of 1% sulphanilamide

and 75 Al 1% N-(1-napthyl)-ethylenediamine was added.

Using a microplate reader, the nitrite production was deter-

mined by reading the absorbance at 550 nm. The amount of

NO produced was normalized against the number of cells.

2.9. Detection of PKC level by Western blot analysis

To evaluate the PKC levels, Western blot analysis was

carried out as previously described (Haller, Lindschau,

Quass, Distler, & Luft, ND). The cells exposed to different

experimental conditions (described above) were washed

three times with ice-cold isotonic buffer (phosphate-buffered

E. Dragomir et al. / Journal of Diabetes and Its Complications 18 (2004) 289–299292

saline, containing 136 mM, NaCl, 2.6 mM KCl, 1.4 mM

KH2PO4, and 4.2 mM Na2HPO4, pH 7.2). After washing,

the cells were collected into 2 ml of homogenization buffer

(20 mM Tris–HCl [pH 7.5], 5 mM NaCl, 1 mM EDT,

5 mM MgCl2, 2 mM dithiothreitol, 200 AM phenylmethyl-

sulfonyl fluoride, 1 Ag/ml aprotinin, 1 Ag/ml leupeptin).

Cells were disrupted with 20 strokes in a Dounce homog-

enizer and the lysate was centrifuged at 100,000�g for 1 h.

The supernatant was collected (the cytosolic fraction) and

the membrane pellet was suspended in the same volume of

homogenization buffer containing 1% Triton X-100 for 30

min on ice. The same concentration of protein from

cytosolic and membrane fractions was subjected to SDS-

polyacrylamide gel electrophoresis using 10% acrylamide

separating gel. This was followed by transfer to nitrocellu-

lose membrane that was successively incubated, first with

blocking buffer containing 137 mM NaCl, 20 mM Tris–

HCl, pH 7.5, 5% nonfat dry milk powder and 0.05% Tween

20 for 2 h at room temperature and then with monoclonal

antibodies anti-PKC (Sigma, Clone MC5) in Tris-buffered

saline with 0.05% Tween 20 (overnight). Peroxidase-con-

jugated anti-mouse IgG was used for detection, using ECL

system (Pierce).

2.10. Statistical analysis of the data

Statistical processing was performed using the ANOVA

method of Origin and Student’s t test for a single comparison.

Statistical significance was considered as P value < .05.

3. Results

3.1. Effect of high D-glucose on intracellular Ca2+ in HECs

As determined by measurement of calcium-bound fura-2,

exposure of EC for 7 days to elevated glucose (33 mM)

resulted in an increase in cytosolic free calcium above the

control values (cells exposed to 5 mM glucose). Thus, the

nominal absence of extracellular elevated glucose (33 mM)

in the culture medium resulted in an increase in cytosolic

free calcium above Ca2+; the basal cytosolic free calcium in

HG-treated cells had a mean value off106.75 nM, whereas

in control cells wasf66.88 nM; these figures were stable in

time in both experimental conditions (Fig. 1A).

Application of histamine induced a transient elevation

of [Ca2+]i in both normal (5 mM) and HG (33 mM)

concentrations, which in time, declined to a new plateau

level without reaching the baseline. The peak increase of

[Ca2+]i in response to histamine was higher in HECs

exposed to HG than in control cells (158.4 vs. 105 nM).

As shown in Fig. 1A, subsequent addition of 2.5 mM Ca2+

generated a great enhancement in the fluorescence of

HECs; consistently, the Ca2+ entry was higher for the cells

grown in 33 mM glucose (f266.45 nM) than in control

cells (f148.5 nM).

The data obtained upon addition of histamine and the

basal values were used to calculate the differential increase

in [Ca2+]i release in HECs exposed to 5 and 33 mM

glucose (Fig. 1B). The results indicated a significant

[Ca2+]i release in response to histamine in the presence

of HG (P= .007). The difference in [Ca2+]i entry in HECs

grown in 5 and 33 mM glucose were calculated as the

variation between the values obtained upon calcium addi-

tion and the basal values (Fig. 1C) and the results were

significant [P=1.07�10�7].

3.2. Effect of aspirin on high D-glucose mediated cellular

calcium homeostasis

Having detected that HG (33 mM) induced accumula-

tion of [Ca2+]i in HECs exposed to histamine and an

increase in Ca2+ entry within the cells, we questioned

whether aspirin has an effect on these processes. The

experiments showed that incubation of HG-treated EC with

1 mM aspirin for 48 h prior to exposure to histamine

reduced the effect of elevated glucose on intracellular

calcium concentration. As shown in Fig. 2, in response

to histamine, the [Ca2+]i in ECs grown in 33 mM glucose

and exposed to aspirin was decreased when compared to

the values obtained for HECs subjected to HG in the

absence of aspirin treatment (from 158.4 to 111.75 nM.)

Moreover, aspirin had an effect on Ca2+ entry into the

cells by decreasing the accumulation of [Ca2+]i from a

medium value of f266 nM in HECs grown in HG

(33 mM) to a medium value of f197 nM for cells grown

in HG and exposed to aspirin (Fig. 2A). To find out if the

difference was significant, we calculated the changes in the

calcium release and entry, measured (as above) in HECs

grown in HG in the absence or presence of aspirin; as

shown in Fig. 2B and C, aspirin decreased significantly the

[Ca2+]i release (P= .002) and the [Ca2+]i entry (P= .02) in

these cells.

In the experimental conditions tested, the cell viability

was over 90% as determined by the cell viability assay using

0.1% crystal violet as described (Podhaisky et al., 1997).

3.3. Influence of aspirin on HG-induced ROS in HECs

Since it was reported that HG induces accumulation of

ROS in ECs, we inquired whether aspirin, due to its

antioxidant properties, may affect this process. To test this,

experiments were performed on cultured ECs grown in HG,

in the presence or absence of 1 mM aspirin. We have found

that high concentrations of glucose (33 mM) in the culture

medium generated a significant accumulation of intracellu-

lar ROS in ECs. As compared to control cells (grown in

5 mM glucose), the ROS levels increased by f100% in the

cells exposed to 33 mM glucose (Fig. 3). Exposure of ECs

grown in high D-glucose to aspirin for 48 h decreased the

production of intracellular ROS to a level close to the

control values (Fig. 3).

Fig. 1. (A) Intracellular Ca2+ concentration in HECs cultured in DMEM containing 5 mM (n) or 33 mM (.) D-glucose. Confluent cells grown in low and high

glucose concentration were harvested, washed, and exposed for 1 h to Ca2+ free Hepes buffer in the presence of fura-2 AM. Cells were stimulated with 25 AMhistamine and after 5 min of recording the changes in intracellular Ca2+, 2.5 mMCa2+ was added and the effect of high D-glucose on Ca2+ entry was recorded. By

comparison with ECs grown in NG, HG induces a cellular increase in basal calcium ( P= 2.6�10�6), an enhance response to histamine stimulation

( P= 1.2�10�5) and a significant augmentation of [Ca2+]i upon Ca2+ addition in the culture medium ( P= 6.6�10�8). (B) The differential increase in [Ca2+]irelease in HECs exposed to 5 and 33mMglucose, calculated from the data of (A) as the difference (D) between the values obtained upon addition of histamine and

the basal value ( P= .007). (C) The differential increase in [Ca2+]i entry in HECs (grown in 5 and 33 mM glucose) calculated from data of (A), as the difference

between the values obtained upon calcium addition and the basal values ( P= 1.07�10�7). The graphs represent the media of six independent experiments.

E. Dragomir et al. / Journal of Diabetes and Its Complications 18 (2004) 289–299 293

3.4. Role of aspirin on the generation of superoxide

anion in HG-treated ECs

The amount of O2� released from HECs exposed to

various experimental conditions was determined by mea-

suring the O2� reduction of ferricytochrome c (see Materials

and methods). As shown in Fig. 4, compared with HECs

maintained under normal glucose concentration (5 mM), the

amount of O2� generated in the cells grown in 33 mmol D-

glucose raised f3-fold above the normal values, giving

Fig. 2. Effect of aspirin on high D-glucose-induced changes in endothelial calcium mobilization. (A) ECs were grown in 5 mM D-glucose (n), 33 mM D-

glucose (.), or 33 mM D-glucose followed by 48 h exposure to 1 mM aspirin (E). Note that by comparison, in response to histamine, the [Ca2+]i accumulation

in ECs grown in HG and exposed to aspirin decreases ( P= .007), attaining a value close to that of the control cells (grown in 5 mM glucose). Also, when Ca2+

was added in the culture medium, the significant increase in calcium accumulation within the cells is significantly reduced by prior exposure of cells to aspirin

( P= .02). The calculated changes in the calcium release (B) and calcium entry (C), measured as the difference (D) between the [Ca2+]i in response to histamine

(B) or calcium addition (C) and the basal value in HECs grown in HG versus cells kept in HG and aspirin. Note that aspirin decreases significantly the [Ca2+]irelease ( P= .002) and the [Ca2+]i entry ( P= .02). The results represent the statistic media of six independent experiments.

E. Dragomir et al. / Journal of Diabetes and Its Complications 18 (2004) 289–299294

Fig. 5. Effect of aspirin on NADPH-dependent O2� production (measured as

NADPH oxidase activity) in HECs grown in HG concentration. NADPH

oxidase activity was determined in homogenates of endothelial cells grown

in NG (5 mM) or HG (33 mM) concentration and in HG conditions

followed by 48 h exposure to 1 mM aspirin. The results show that HG

induces a great increase in NADPH oxidase activity (*P= .001), a process

that is significantly reduced by incubation of endothelial cells with aspirin

(**P= .02, n= 4).

Fig. 3. Effect of aspirin on the production of ROS in HECs grown in high

D-glucose. The ROS were determined by measuring the fluorescence of

DCF-AM by spectrofluorometry (see Materials and methods) in HECs

grown in 5 mM glucose, 33 mM glucose, and in 33 mM glucose and ex-

posed to aspirin for 48 h. Averaged data indicate that there is an increase in

intracellular ROS in HG condition as compared to control (*P= 5.8�10�4);

they also reveal that exposure to aspirin of HECs prevents the glucose

induced-augmented ROS (**P= 7.4�10�4, n= 4) to a level close to that of

cells grown in standard glucose concentration.

E. Dragomir et al. / Journal of Diabetes and Its Complications 18 (2004) 289–299 295

values of 0.232 and 0.642 nmol O2�/min/106 cells, respec-

tively. Exposure to aspirin for 48 h of HECs grown in HG

concentration inhibited the production of superoxide anions

that appeared in the culture medium at values close to those

obtained for HECs grown in normal glucose concentration

(0.254 nmol O2�/min/106 cells).

In other control experiments, we found that in the

absence of the ECs, O2� was absent in the culture medium

containing normal or high D-glucose concentration.

Fig. 4. Effect of aspirin on the generation of O2� in HECs exposed to high

(33 mM) D-glucose. Superoxide anion production in HECs was determined

by measuring the SOD-inhibitable reduction of cytochrome c to the ferrous

form. Culture of HECs for 7 days in high D-glucose concentration in the

culture medium increases the generation of O2� about 3-fold above the value

obtained from cells grown in NG concentration (*P= .004). Aspirin (1 mM)

significantly inhibited the HG-induced O2� release (**P= .008, n= 4). The

rate of O2� released is given in nmol O2

�/min/106 cells.

3.5. Assay of NADPH oxidase activity

To investigate a possible mechanism involved in the

inhibitory effect of aspirin on O2� production by HG-

exposed HEC, we further searched whether aspirin

inhibited NADPH oxidase activity. To this purpose, the

aspirin effect on SOD-inhibitable reduction of cytochrome

c by an EC homogenate preparation was measured. The

results showed that high D-glucose (33 mM) triggered in

Fig. 6. Effect of aspirin (1 mM) on the NO production by ECs grown in

high D-glucose concentration. The NO production was determined in ECs

cultured in NG (5 mM) or HG (33 mM) concentration and in 33 mM

glucose with 1 mM aspirin by nitrite (NOx) evaluation using the Griess

reagents. Exposure of ECs to HG does not affect the production of NO

( P= .165 vs. control cells). ECs grown in HG in the presence of aspirin

increase significantly the NOx concentration in the culture medium as

compared to the cells grown in the absence of aspirin (*P= .074�10�5).

The data are expressed as micromolar nitrite per 1�106 cells and they

represent the statistic media for seven independent experiments.

E. Dragomir et al. / Journal of Diabetes and Its Complications 18 (2004) 289–299296

HECs a significant increase in NADPH oxidase activity,

which attained value high above those obtained on cells

grown in normal (5 mM) glucose (Fig. 5). Exposure of

HECs to aspirin generated a significant decrease of

NADPH oxidase activity, which attained values close to

that of control cells grown in the presence of 5 mM

glucose in the culture medium (Fig. 5).

3.6. Effect of aspirin on NO production by ECs exposed

to high D-glucose

The possible relationship between the effect of aspirin

on calcium mobilization and NO production in HG-treated

HECs was investigated. To this purpose, before the cul-

tured HECs were assessed for calcium measurements, the

cell supernatant was collected and analyzed for nitrite

concentrations. As shown in Fig. 6, in the ECs grown

for 7 days in 33 mM glucose, the NO production by the

cells was slightly increased but not significantly different

when compared with the cells cultured in NG concentra-

tion (5 mM) in the culture medium. Compared with HECs

grown in 33 mM glucose, exposure of HECs (maintained

in HG) to aspirin induced a significant increase (by 108%)

of nitrite in the culture medium, suggesting an increased

production of NO in ECs.

3.7. Effect of aspirin on HG-induced PKC

translocation/activation

PKC has been shown to be activated in diabetes, and

PKC activation has been found to increase vascular

superoxide production by activating NADPH oxidase.

To demonstrate whether the aspirin may decrease NADPH

oxidase activity by acting on PKC activation, we exam-

Fig. 7. Effect of aspirin on HG-induced intracellular translocation of

PKC. Cytosolic and membrane fractions obtained from homogenates of

HECs exposed to 5 mM, 33 mM glucose, or 33 mM glucose and aspirin

were subjected to SDS-PAGE, transferred to nitrocellulose, and immuno-

blotted with a monoclonal antibody specific for PKC. Note that aspirin

(1 mM) significantly ( P= .046) inhibited the HG-induced PKC translo-

cation from the cytosolic to the membrane fraction (representative for

three experiments).

ined the effect of aspirin on HG-induced PKC by Western

blot analysis. The effect of aspirin on PKC translocation

is shown in Fig. 7. The results showed that under basal

conditions (5 mM glucose), PKC was mostly located in

the cytosolic fraction. HG concentration (33 mM) induced

a significant translocation of PKC in the membrane

fraction, with a concomitant decrease in the cytosolic

fraction. Incubation of cells grown in HG condition with

aspirin reduced PKC translocation in the control levels.

4. Discussion

Intracellular Ca2+ is a key regulator of cell function

(Berridge, 1993) and changes in Ca2+ homeostasis affect

vascular functions such as permeability (Hempel et al.,

1997), the secretion of inflammatory cytokines (Conboy,

Manoli, Mhaiskar, & Jones, 1999), gene expression

(Ronald, Muldoon, Lenormand, & Magun, 1990), and

protein synthesis (Brostron & Brostron, 1990). The effect

of high D-glucose on the Ca2+ response to different

agonists is dependent of the period of glucose overload,

cellular species, and agonist (Pieper & Dondlinger, 1996).

Several earlier studies have revealed that both short- and

long-term exposure to high D-glucose impairs endothelial

Ca2+ signaling. Thus, incubation of the bovine cerebral EC

for 2 h in solutions containing 23 mM glucose did not

change the resting levels of [Ca2+]i but histamine applica-

tion failed to mobilize Ca2+ from both intracellular store

sites and extracellular space (Kimura, Oike, & Ito, 1997).

Incubation of porcine aortic EC for 24 h with HG solution

(44 mmol/l) increased resting [Ca2+]i and enhanced ago-

nist-stimulated Ca2+/EDRF signaling via the generation of

O2� by an unknown mechanism (Graier et al., 1996;

Wascher et al., 1994).

It has been shown that antioxidants (vitamin E, probucol,

GSH, and vitamin C) diminish the high D-glucose-enhanced

endothelial Ca2+ response by scavenging the overshoot O2�

(Graier et al., 1997). Also there is evidence that aspirin has

antioxidant properties (El Midaoui et al., 2002; Ghiselli

et al., 1992; Podhaisky et al., 1997) and has favorable

effects in diabetes mellitus (Hundal et al., 2002), but its

mechanism of action is not fully elucidated. To our knowl-

edge, there are no data about the effect of aspirin on calcium

mobilization in ECs exposed to HG concentrations. We

designed experiments to study the effect of aspirin on the

impaired Ca2+ response in HECs grown concentrations

(condition simulating the diabetes mellitus) and to uncover

its mechanisms of action.

Our results corroborate well and extend other reports

(Graier et al., 1996; Wascher et al., 1994), demonstrating

that both intracellular calcium release and extracellular

calcium entry are altered following HEC exposure to HG

concentration. The data revealed that the resting level of

[Ca2+]i is increased in HG concentration for 7 days. Both

peak and plateau levels of [Ca2+]i in response to histamine

E. Dragomir et al. / Journal of Diabetes and Its Complications 18 (2004) 289–299 297

were increased in HG-cultured cells in the absence of Ca2+

and subsequent addition of Ca2+ in the medium, resulted in

a sustained increase in [Ca2+]i. These data are in agreement

with the results obtained by Graier et al. (1996), who

reported an increase of [Ca2+]i in bradykinin-stimulated

ECs after 24 h exposure to HG.

Aspirin exposure of HECs grown in HG induces a

decrease to the control level of [Ca2+]i in response to

histamine stimulation of the cells, and a decline in calcium

accumulation upon extracellular calcium addition. To reveal

the mechanisms underlying the favorable effects of aspirin,

the effect of the drug on intracellular ROS levels was tested.

In our study, chronic exposure (7 days) of ECs to HG

concentration induced a significant increase in ROS, as

determined using DCFH-DA. The superoxide anion (O2�)

release by HECs in the culture medium was greater in HG,

as compared to NG-grown cells. Aspirin restored to control

values the increase in free radical production induced by

HG. These results corroborate well and complete previous

studies that showed the beneficial effect of aspirin in

preventing the development of hypertension in chronically

glucose-fed rats through its antioxidative properties, by

reducing the aortic O2� production (El Midaoui et al.,

2002). Accumulation of (O2�) is one of the reported candi-

dates for the pathogenesis of vascular and/or endothelial

damage in a diabetic environment. It has been recently

shown that NADPH oxidase-derived ROS are critical in

the generation of Ca2+ oscillation during histamine stimu-

lation (Hu et al., 2002). The vascular NADH/NADPH

oxidase is a major source of ROS in the vasculature

(Griendling, Minieri, Ollerenshaw, & Alexander, 1994).

Recent studies have revealed that smooth muscle cells and

ECs can produce ROS through activation of NADPH

oxidase (Bayraktutan, Draper, Lang, & Shah, 1998; Mohaz-

zab, Kaminski, & Wolin, 1999; Rajagopalan et al., 1996;

Warnholtz et al., ND). It is well known that the activation of

NADPH oxidase is at least in part mediated by PKC

(Hempel et al., 1997) and that the PKC activation is

considered a key event in the generation of ROS (Inoguchi

et al., 2000).

The above data lead us to further evaluate the antioxidant

potential of aspirin, and to test its effects on NADPH

activity in HECs grown in HG and treated with aspirin.

The results showed that aspirin inhibited NADPH oxidase

activity in HECs grown in HG. It is interesting to observe

that the effect of the drug was similar to that obtained on the

O2� release in culture medium of HEC grown in HG

conditions. The association of HG levels increased ROS

production, and activation of PKC is well established

(Inoguchi et al., 2000). The role of PKC in mediating

endothelial dysfunction has also been postulated by Tesfa-

mariam, Brown, and Cohen (1991). In agreement with these

observations, it was found (Hink et al., 2001) that in vitro

incubation of aortic tissue from diabetic animals with the

PKC inhibitor chelerytrine had a marked inhibitory effect on

superoxide production.

Taken together, we questioned whether aspirin has any

effects on PKC activation. Our results show that aspirin

inhibited the HG-induced PKC activation by reducing

translocation of PKC from cytosol to the membrane. Thus,

we may safely assume that in HECs grown in HG

concentration, aspirin restores the Ca2+ homeostasis by

inhibiting PKC-mediated activation of NADPH oxidase

activity and consequently reducing the production of O2.

Since it is well known that endothelial NO synthesis

(i.e., constitutive NO synthase) depends on intracellular

calcium (Moncada, Palmer, & Higgs, 1991), we investi-

gated whether aspirin has an effect on NO production by

EC grown in HG concentration. The results showed that

the NO production was not significantly different in high

D-glucose exposed HECs compared to the normal medi-

um-growing cells. However, our data cannot evaluate the

functionality of NO that may well be changed especially

due to the overproduction of O2�. This assumption is

backed by the experiments that showed that in HECs

exposed to HG conditions, aspirin lead to a significantly

increase in NO production as compared to that detected in

untreated HG-grown HEC. These data are in good agree-

ment with reports that indicate that aspirin increases NO

release in ECs (Bolz & Pohl, 1997). It was stated that

HG increases the O2� production which rapidly react with

NO (to produce peroxynitrite, a potent oxidant) leading to

NO destruction (Sqardito & Pryor, 1995). Thus, our

results are in line with the findings that in HG, the NO

biodisponibility is affected by reaction with overexpressed

O2� (Langenstroer & Pieper, 1992). Therefore, we can

conclude that aspirin may improve production and/or

bioavailability of NO by scavenging O2� radicals pro-

duced under HG conditions.

This study confirms and extends previous reports

showing that exposure of ECs to HG concentration

impairs intracellular Ca2+ mobilization and NO production

and/or biodisponibility by a mechanism involving the

accumulation of O2�. In addition, our results provide

evidence that aspirin improves the calcium response to

agonist and NO production and/or biodisponibility in HG

condition. We assume that the antioxidant properties of

aspirin are accountable for these effects, since our data

show that aspirin may scavenge O2� by reducing NADPH

activity in HG conditions. It is likely that through the

combination of its effects (anti-inflammatory, antiplatelet

aggregation, antioxidant agent) aspirin may represent a

valuable drug for protection of ECs from the deleterious

effects of HG (hyperglycemia) associated with diabetes.

Acknowledgments

The authors are indebted to G. Mesca, D. Rogoz, and I.

Manolescu. We are grateful to Dr. Cora-Jean S. Edgell

(Department of Pathology, University of North Carolina,

Chapel Hill) for kindly providing the Ea hy926 cells. The

E. Dragomir et al. / Journal of Diabetes and Its Complications 18 (2004) 289–299298

work was supported by grants from the Romanian Academy

and from the Ministry of Education and Research.

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