www.elsevier.com/locate/vph

Vascular Pharmacology 4

Coordinated induction of iNOS–VEGF–KDR–eNOS

after resveratrol consumption

A potential mechanism for resveratrol preconditioning of the heart

Samarjit Dasa, Vijay K.T. Alagappanb, Debasis Bagchic, Hari S. Sharmab,

Nilanjana Maulika, Dipak K. Dasa,*

aCardiovascular Research Center, University of Connecticut, School of Medicine, Farmington, CT 06030-1110, United StatesbInstitute of Pharmacology, Erasmus University Medical Center, Rotterdam, The Netherlands

cCreighton University, Omaha, NE, USA

Abstract

Existing evidence indicates that resveratrol, a red wine and grape-derived polyphenolic antioxidant, can pharmacologically precondition

the heart in a nitric oxide (NO)-dependent manner. To further explore the role of NO in resveratrol-mediated cardioprotection, the induction

for the expression of the potential molecular targets of NO including VEGF and KDR as well as iNOS and eNOS were examined by Western

blot analysis and immunohistochemistry. Two groups of rats were studied, one group of animals was fed resveratrol for 7 days while the other

group was given water only. After 1, 3, 5 and 7 days, the rats were sacrificed and the expression of the proteins was examined by Western blot

analysis. Western blot detected an overexpression of iNOS and VEGF within 24 h of resveratrol treatment while the induction of KDR was

not increased until after 3 days and eNOS expression after 5 days of resveratrol treatment. These expressions were further increased after 7

days of resveratrol treatment, when the rats were sacrificed for the isolated working heart preparation. Resveratrol provided cardioprotection

as evidenced by superior post-ischemic ventricular recovery, reduced myocardial infarct size and decreased number of apoptotic

cardiomyocytes. Immunohistochemistry was performed in the hearts at baseline, and at the end of 30-min ischemia/2-h reperfusion. The

hearts obtained from resveratrol-treated rats revealed enhanced expression for iNOS, eNOS and VEGF and KDR compared to control hearts

at the end of reperfusion. The results of this study demonstrate that resveratrol leads to a coordinated upregulation of iNOS–VEGF–KDR–

eNOS, which is likely to play a role in resveratrol-mediated cardioprotection.

D 2005 Elsevier Inc. All rights reserved.

Keywords: Resveratrol; iNOS; eNOS; NO; VEGF; KDR; Cardioprotection; Apoptosis

1. Introduction

Resveratrol (trans-3,5,4¶-trihydroxystilbene) is a phe-

nolic phytoalexin present in grape skins and wines,

especially red wines (Creasy and Coffee, 1988; Paul et al.,

1999). It exerts a wide variety of biological effects including

an estrogenic property (Gehm et al., 1997), an anti-platelet

activity (Bertelli et al., 1996) and anti-inflammatory

function (Das et al., in press; Das et al., 2005). Recently,

resveratrol has been found to protect kidney, brain and heart

cells from ischemia/reperfusion injury (Giovannini et al.,

1537-1891/$ - see front matter D 2005 Elsevier Inc. All rights reserved.

doi:10.1016/j.vph.2005.02.013

* Corresponding author. Tel.: +1 860 679 3687; fax: +1 860 679 4606.

E-mail address: DDAS@NEURON.UCHC.EDU (D.K. Das).

2001; Huang et al., 2001; Ray et al., 1999). Resveratrol has

also been found to pharmacologically precondition hearts by

a nitric oxide (NO)-dependent manner (Hattori et al., 2002;

Imamura et al., 2002).

NO is a pleiotropic molecule that affects diverse

biochemical and physiological function including regulation

of vascular tone and vascular remodeling (Garthwaite and

Boulton, 1995). A potential therapeutic target for NO is

angiogenesis (Ziche and Morbidelli, 2000). Incubation of

human vascular smooth muscle cells with NO donors

enhances vascular endothelial-derived growth factor

(VEGF) synthesis and inhibition of NO synthase abrogates

VEGF production (Jozkowicz et al., 2001). Inhibitors of

eNOS block VEGF-induced endothelial cell migration,

2 (2005) 281 – 289

S. Das et al. / Vascular Pharmacology 42 (2005) 281–289282

proliferation, and tube formation in vitro and VEGF-

induced angiogenesis in vivo. In the absence of eNOS

inhibition, VEGF stimulates phosphoinositide 3-kinase

(PI3K) and Akt-dependent phosphorylation of eNOS,

resulting in an activation of eNOS and increased NO

production (Dimmeler et al., 1999). In endothelial cells, the

KDR/Flk-1 receptor of VEGF is predominantly involved in

eNOS phosphorylation.

Although both tyrosine kinase receptors VEGFR-1/Flt-1

and VEGFR-2/Flk-1(KDR) are necessary for VEGF signal-

ing, there is a basic difference between the two receptors

(Kranenburg et al., 2005). While stimulation of Flt-1 is

linked to cell migration, Flk-1/KDR receptor activation is

associated with both cell migration and proliferation, and

most importantly by the mitogen-activated protein (MAP)

kinase cascade (Kroll and Waltenberger, 1997). Interest-

ingly, while induction of the expression of VEGF and Flt-1

occurs within a very short time, induction of the KDR

receptor does not occur until days later (Li et al., 1996). The

KDR receptor is believed to be involved in eNOS

expression, because a KDR-receptor-selective mutant, and

not an Flt-1 receptor-selective mutant, can increase eNOS

expression.

To further explore the role of NO in resveratrol

preconditioning, we examined the molecular targets of

NO. The results of our study demonstrate a coordinated

upregulation of iNOS–VEGF–KDR–eNOS after resvera-

trol consumption, suggesting that this pathway may be

involved in resveratrol-induced preconditioning.

2. Materials and methods

2.1. Animals and resveratrol treatment

Six Sprague–Dawley rats weighing 275–300 g were

gavaged with resveratrol (Sigma Chemical Co.) 2.5 mg/kg

in 50% ethanol using a stomach needle (1.2 mm diameter)

every day for 7 days. Six Control rats were similarly

gavaged with 0.5 ml of 50% ethanol every day forcefully

into the stomach. After 7 days, the rats were anesthetized

with pentobarbital sodium (80 mg/kg i.p. injection, Abbott;

North Chicago, IL). After intravenous administration of

heparin (500 IU/kg, Elkins-Sinn; Cherry Hill, NJ), the

chests were opened, and the heart from each rat was

rapidly excised and mounted on a nonrecirculating Lan-

gendorff perfusion apparatus. The perfusion buffer used in

this study consisted of a modified Krebs–Henseleit

bicarbonate buffer (KHB) (in mM: 118 NaCl, 4.7 KCl,

1.2 MgSO4, 1.2 KH2PO4, 25 NaHCO3, 10 glucose, and 1.7

CaCl2), pH 7.4, gassed with 95% O2–5% CO2, and filtered

through a 5-Am filter to remove any particulate contami-

nants. The buffer was maintained at a constant temperature

of 37 -C and was gassed continuously for the entire

duration of the experiment. Left atrial cannulation was then

carried out, and after allowing for stabilization of 10 min in

the retrograde perfusion mode, the circuit was switched to

the antegrade working mode, which allowed for the

measurement of myocardial contractility as well as aortic

and coronary flows. Essentially, this is a left heart

preparation in which the heart is perfused with a constant

preload of 17 cm H2O (being maintained by means of a

Masterflex variable speed modular pump, Cole Parmer

Instrument; Vernon Hills, IL) and pumps against an

afterload of 100 cm H2O. At the end of 10 min, after the

attainment of steady-state cardiac function, baseline func-

tional parameters were recorded. The circuit was then

switched back to the retrograde mode. The hearts were then

subjected to 30 min of global ischemia followed by 120

min of reperfusion with the same KHB buffer. The first 10

min of reperfusion was in the retrograde mode to allow for

post-ischemic stabilization and thereafter in the antegrade-

working mode to allow for assessment of functional

parameters, which were recorded at 10 min, 30 min, 60

min and 120 min into reperfusion.

2.2. Cardiac function assessment

Aortic pressure was measured using a Gould P23XL

pressure transducer (Gould Instrument Systems Inc., Valley

View, OH, USA) connected to a side arm of the aortic

cannula, the signal was amplified using a Gould 6600 series

signal conditioner and monitored on a CORDAT II real-time

data acquisition and analysis system (Triton Technologies,

San Diego, CA, USA) (Garthwaite and Boulton, 1995).

Heart rate (HR), left ventricular developed pressure (LVDP)

(defined as the difference of the maximum systolic and

diastolic aortic pressures), and the first derivative of

developed pressure (dp/dt) were all derived or calculated

from the continuously obtained pressure signal. Aortic flow

(AF) was measured using a calibrated flow meter (Gilmont

Instrument Inc., Barrington, IL, USA) and coronary flow

(CF) was measured by timed collection of the coronary

effluent dripping from the heart.

2.3. Infarct size estimation

At the end of reperfusion, a 10% (w/v) solution of

triphenyl tetrazolium in phosphate buffer was infused into

aortic cannula for 20 min at 37 -C (Hattori et al., 2001). The

hearts were excised and stored at� 70 -C. Sections (0.8 mm)

of frozen heart were fixed in 2% para-formaldehyde, placed

between two cover slips and digitally imaged using a

Microtek Scan Maker at 600z. To quantitate the areas of

interest in pixels, a NIH image 5.1 (a public-domain software

package) was used. The infarct size was quantified and

expressed in pixels.

2.4. TUNEL assay for assessment of apoptotic cell death

Immunohistochemical detection of apoptotic cells was

carried out using TUNEL (Maulik et al., 1999). The sections

S. Das et al. / Vascular Pharmacology 42 (2005) 281–289 283

were incubated again with a mouse monoclonal antibody

recognizing cardiac myosin heavy chain to specifically

recognize apoptotic cardiomyocytes. The fluorescence

staining was viewed with a confocal laser microscope.

The number of apoptotic cells were counted and expressed

as a percent of the total myocyte population.

2.5. Western blot method

Total protein (50 Ag) in the Clontech Extraction buffer

was added to an equal volume of sodium dodecyl sulphate

(SDS) buffer and boiled for 10 min before being separated

on 7–15% SDS polyacrylamide gels in running buffer (25

mM Tris, 192 mM glycine, 0.1% (w/v) SDS, pH 8.3) at

200 V. The Precision plus Protein Kaleidoscope standards

(10 Al) (Bio-Rad Laboratories, CA, USA) were used as

molecular weight standards. The gel was transferred onto a

nitrocellulose membrane (Bio-Rad Laboratories, CA, USA)

at 100 V for 1 h in transfer buffer (25 mM Tris base, 192

mM glycine, 20% (v/v) methanol, pH 8.3). After blocking

the membranes for 1 h in Tris-buffered saline (TBS-T) (50

mM Tris, pH 7.5, 150 mM NaCl) containing 0.1% (v/v)

Tween-20 and 5% (w/v) non-fat dry milk, blots were

incubated overnight at 4 -C with the primary antibody. All

the antibodies were purchased from BD Transduction

Laboratories and were used at manufacturer’s recommen-

ded dilutions. Membranes were washed three times in

TBS-T prior to incubation for 1 h with horseradish

peroxide (HRP)-conjugated secondary antibody diluted

1:2000 in TBS-T and 5% (w/v) non-fat dry milk. Western

blots were developed with the ECL Detection Reagents 1

and 2 (Amersham Biosciences) and exposed to Kodak X-

OMAT film.

2.6. Immunohistochemistry

Immunohistochemical detection of iNOS/eNOS was

performed on 6 Am thick paraffin-embedded myocardial

tissue sections, and mounted on 3-amino-propyl-trioxysi-

lane (Sigma, St Louis, MO, USA) coated glass slides.

Immunostaining for iNOS and eNOS was performed using

the peroxidase–antiperoxidase (PAP) technique. Slides

were incubated in 0.3% H2O2 to quench endogenous

peroxidase, then boiled for 15 min in citrate buffer, rinsed

with PBS and processed for staining. After pre-incubation

with normal rabbit serum, slides were incubated overnight at

4 -C with mouse monoclonal antibody for iNOS (Trans-

duction). After PBS wash, the sections were incubated with

rabbit-anti-mouse serum (Dako Corp, Glostrup, Denmark),

washed and further incubated with mouse PAP-complex

(Sigma). The chromogen reaction was allowed to take place

in the dark, using 0.025% 3,3-diaminobenzidine (Sigma).

Subsequently, slides were counterstained with Mayer’s

hematoxylin and visualized and photographed under a light

microscope. Negative controls consisted of omission of

primary antibody.

Immunohistochemical detection of VEGF was performed

on 5 Am thick paraffin-embedded myocardial tissue

sections, using a multiple step avidin–biotin complex

(ABC) method (Biogenex, San Ramon, CA, USA). Affinity

purified rabbit anti-human-iNOS, eNOS or VEGF anti-

bodies were applied in a dilution of 1:200 (v/v) as primary

antibody followed by biotinylated anti-immunoglobulin and

tertiary streptavidin conjugated alkaline phosphatase. The

VEGF antibody used was raised against a 20 amino acid

synthetic peptide corresponding to residues 1–20 of the

amino terminus of human VEGF (Santa Cruz Biotechnol-

ogy Inc., Santa Cruz, CA, USA). Color was developed

using Naphtol AS-MX phosphate and new fuchsine.

In case of flk-1/KDR staining, after deparaffinization in

xylene and rehydration through graded alcohol, slides were

rinsed with phosphate buffered saline (PBS). Endogenous

peroxidase was blocked with 0.3% hydrogen peroxide. For

flk/KDR staining, slides were pre-treated by boiling in

citrate buffer (10 mM citrate buffer, pH=6.0) for 10 min in a

microwave oven. Subsequently, sections were preincubated

with 10% normal goat serum diluted in 5% bovine serum

albumin in phosphate buffered saline (5% BSA/PBS,

pH=7.4), and afterwards incubated for 30 min at room

temperature with a rabbit polyclonal antibody against mouse

KDR/Flk-1 (Neomarkers, RB-1527, Fremont, CA, USA) in

a dilution of 1:200 v/v. Consecutive tissue sections were

also stained with a monoclonal mouse anti-human alpha-

smooth muscle actin (a-SMA) antibody (clone 1A4:

Biogenex, San Ramon, USA) in a dilution of 1:1000 v/v.

The optimal dilution of the first antibody was identified by

examining the intensity of staining obtained with a series of

dilutions of the antibody from 1:50 to 1:1000. Negative

controls were prepared by omission of the primary antibody.

After washing with Tris-base buffered saline (TBS,

pH=7.4), the test and control slides were incubated for 15

min with Powervision+i Post-antibody Blocking solution

(Immunovision Technologies, Daly City, CA, USA). Next,

slides were washed and incubated with Powervision+ipolymerised horseradish peroxidase conjugates (Immunovi-

sion Technologies, Daly City, CA, USA). Finally, the

sections were stained with 3,3¶-diaminobenzidine tetrahy-

drochloride (Sigma, Zwijndrecht, NL) as a chromogen,

counterstained with Mayer’s heamatoxylin and visualized

with light microscopy.

2.7. Statistical analysis

The values for myocardial functional parameters, myo-

cardial infarct size and cardiomyocyte apoptosis are all

expressed as the meanTstandard error of the mean (S.E.M.).

Analysis of variance test was first carried out to test for any

differences between the mean values of all groups. If

differences between groups were established, the values of

the treated groups were compared with those of the control

group by a modified t-test. The results were considered

significant if p<0.05.

40

30

20

10

0Control Resveratrol

Control ResveratrolIn

farc

t S

ize/

Are

a o

f R

isk

(%)

30

25

20

15

10

5

0

*

*

Ap

op

toti

c M

yocy

tes

(%)

Fig. 1. Effects of resveratrol on myocardial infarct size and cardiomyocyte

apoptosis. The isolated hearts from control (n =6) and resveratrol-fed (n =6)

rats were subjected to 30 min of global ischemia followed by 2 h of

reperfusion in a working mode. Infarct size was measured by the TTC dye

method while cardiomyocyte apoptosis was evaluated by TUNEL method

in conjunction with antibody against a-myosin heavy chain. Results are

expressed as meanTS.E.M. *p <0.05 vs. control.

S. Das et al. / Vascular Pharmacology 42 (2005) 281–289284

3. Results

3.1. Effects of resveratrol on cardioprotection

There were no differences in baseline cardiac function

between the two groups. In general, there were no significant

differences between resveratrol vs. control on heart rates and

coronary flow (Table 1). As was expected, on reperfusion, the

absolute values of all functional parameters were decreased in

all the groups as compared with the respective baseline

values. Resveratrol-treated hearts displayed a significantly

better recovery of post-ischemic myocardial function. The

cardioprotective effects of resveratrol were evidenced by

significant differences in LVDP from R-30 onward (Table 1),

the difference is especially apparent at R-60 (112.66T1.17mm Hg vs. 88.01T9.57 mm Hg) and at R-120 (95.26T2.14mm Hg vs. 42.5T7.62 mm Hg) and also in the LVdp/dt at R-

30 onwards; R-30 (3302.4 T122.21 mm Hg vs.

2471.67T235.48 mm Hg), R-60 (2999.8T61.93 mm Hg vs.

1880.5T403.3 mm Hg) and R-120 (2096.2T125.4 mm Hg/s

vs. 899.83T86.75 mm Hg/s). Aortic flow was markedly

higher in the resveratrol group from R-30 onwards at all rest

three points. R-30 (62.6T2.43 ml/min vs. 36.03T12.7 ml/

min), R-60 (47.24T3.79 ml/min vs. 19.24T6.48 ml/min) and

R-120 (10.5T1.74 ml/min vs. 4.29T1.43 ml/min) (Table 1).

Infarct size (percent of infarct vs. total area at risk) was

noticeably reduced in the resveratrol group as compared to

the control (20.58T3.19% vs. 33.79T2.74%) (Fig. 1). The

percent of apoptotic cardiomyocytes was significantly

reduced in the resveratrol group as compared to the control

(3.5T0.9 vs. 22.7T1.5%).

3.2. Effects of resveratrol on the expression of iNOS, eNOS,

VEGF and KDR

Western blot analysis revealed an increased induction of

iNOS, eNOS, VEGF and KDR proteins in the resveratrol-

Table 1

Effects of resveratrol on the recovery of post-ischemic ventricular function

HR

(beats/min)

LVDP

(pr/mm

Control Baseline 311.98T31.29 126.7

10 R 315.6T25.9 107.4

30 R 365.07T16.99 103.5

60 R 344.43T34.57 88.0

120 R 414.28T24.38 42.

Resveratrol (2.5 mg/kg) 7 days Baseline 434.78T15.9 123.2

10 R 387.68T8.09 116.

30 R 412.36T19.81 119.9

60 R 402.6T13.78 112.6

120 R 424.38T14.3 95.2

Rats were fed resveratrol (2.5 mg/kg for 7 days) by gavaging. The control rats wer

At the end of 7 days, the rats were sacrificed, the hearts excised for the isolated wo

followed by 2-h reperfusion.

HR: heart rate; LVDP: left ventricular developed pressure; LVdp/dt: maximum firs

of six animals as group.

* p <0.05, resveratrol vs. control.

fed rat hearts in a coordinated fashion (Fig. 2). An increased

induction of iNOS and VEGF was found within 24 h of

resveratrol treatment. Significant amount of KDR expres-

sion became apparent only after 3 days of resveratrol

Hg)

LVdp/dt

(mm Hg/s)

Aortic flow

(ml/min)

Coronary flow

(ml/min)

6T3.16 3318.66T115.19 71.55T5.15 29.75T0.89

3T5.4 2411.5T250.43 42.64T12.84 26.6T1.61

6T7.1 2471.67T235.48 36.03T12.7 26.6T2.3

1T9.57 1880.5T403.3 19.24T6.48 25.2T1.615T7.62 899.83T86.75 4.29T1.43 21.51T2.4

6T1.95 3450.2T79.72 73.04T1.62 21.36T2.11

9T1.6 3153.2T83.73 62.6T2.43 21.12T2.15

4T1.06 3302.4T122.21* 62.6T2.43* 22.08T2.116T1.17* 2999.8T61.93* 47.24T3.79* 21.84T1.68

6T2.14* 2096.2T125.4* 10.5T1.74* 22.08T1.1

e given 50% ethyl alcohol by gavaging, and kept under identical conditions.

rking heart preparation. The hearts were made globally ischemic for 30 min

t derivatives of developed pressure. Results are expressed as meanTS.E.M.

Fig. 2. Western blot analysis of iNOS, eNOS, VEGF and KDR receptor proteins. The blots were scanned, normalized, and the average (meansTS.E.M.) of

three experiments are shown on the right side. Representative blots are shown on the left side.

S. Das et al. / Vascular Pharmacology 42 (2005) 281–289 285

treatment, while eNOS upregulation became apparent only

after 5 days of resveratrol treatment. Thus, the induction of

these proteins appeared to be expressed in the following

order: iNOS/VEGFYKDRYeNOS.

3.3. Immunohistochemical localization of iNOS, eNOS,

VEGF and KDR

Visualization of the proteins after immunoperoxidase

color reaction revealed increased activity of these proteins in

the resveratrol fed rat hearts that were subjected to 30 min of

ischemia followed by 2 h of reperfusion. Immunoreactive

iNOS, eNOS and VEGF were localized mainly in the

cytoplasm of cardiomyocytes and vascular smooth muscle

cells and not in fibrotic areas (Figs. 3–5). KDR was

predominantly localized in the endothelial cells of blood

vessels in sham and IR rats (Fig. 6).

4. Discussion

Several salient features were noticed from our study. First,

the results of our study demonstrated that short-term

resveratrol consumption for only 7 days could render the

myocardium resistant to ischemia/reperfusion injury. Resver-

atrol-fed hearts revealed improved post-ischemic ventricular

recovery, reduced myocardial infarct size, and decreased

number of apoptotic cardiomyocytes compared to non-fed

animal hearts. Western blot analysis showed the induction of

the expression of iNOS, eNOS, VEGF and KDR in a

coordinated fashion in the order of iNOS/VEGFYeNOSYKDRYeNOS. Immunohistochemistry detected increased

expression of iNOS/eNOS/VEGF/KDR in the resveratrol-

fed hearts subjected to 30 min of ischemia and 2 h of

reperfusion as compared to those in non-fed hearts.

A growing body of evidence indicates that resveratrol

can pharmacologically precondition a heart through in a

NO-dependent manner (Hattori et al., 2002; Imamura et al.,

2002). A number of other studies also demonstrated a direct

role of NO in resveratrol-mediated cardioprotection (Chen

and Pace-Asciak, 1996; Hung et al., 2004; Hung et al.,

2001; Kiziltepe et al., 2004; Zou et al., 2003; Bradamante et

al., 2003; El-Mowafy, 2002; Orallo et al., 2002; Bruder et

al., 2001; Giovannini et al., 2001; Hung et al., 2000;

Fitzpatrick et al., 1993). Several reports exist in the literature

to show that resveratrol can induce eNOS and iNOS

expression. For example, resveratrol induced an expression

of eNOS in the human umbilical vein endothelial cells

(HUVEC) (Wallerath et al., 2002). In addition to its long-

term effects on eNOS expression, resveratrol also enhanced

the production of bioactive NO in the short term (within 2

min), suggesting a role of iNOS. Our results support these

previous observations as we also observed iNOS expression

within 24 h, while eNOS expression did not become

apparent until after 3 days. In another study, resveratrol

induced the expression of iNOS in cultured bovine

pulmonary artery endothelial cells (Hsieh et al., 1999). In

a recent study, resveratrol-mediated cardioprotection was

abrogated by pretreating the heart with aminoguanidine, an

iNOS inhibitor Hattori et al., 2002). In another study,

resveratrol preconditioned a normal mouse heart; however,

it was unable to precondition a heart from an iNOS

knockout mouse (Imamura et al., 2002).

Resveratrol shares many common physiological func-

tions with NO. For example, both resveratrol and NO

possess anti-inflammatory and anti-platelet activities

(Engelman et al., 1995a,b; Ferrero et al., 1998) and can

exert vasodilatory effects on blood vessels (Chen and Pace-

Asciak, 1996). Similar to NO, resveratrol is a potent

scavenger for peroxyl radicals (Sato et al., 2000; Kotamraju

et al., 2001). NO exists as a free radical, and resveratrol is a

weak free radical scavenger in vitro, but both possess potent

antioxidant capacity in vivo, and can attenuate lipid

peroxidation. The fact that resveratrol augments NO

availability and both of them share a common physiological

function strongly suggests that resveratrol exerts its car-

dioprotective effects through NO. Both resveratrol and NO

are implicated in myocardial preconditioning.

While NO has been implicated in resveratrol-mediated

cardioprotection, the exact mechanism remains unknown.

S. Das et al. / Vascular Pharmacology 42 (2005) 281–289286

There are several downstream molecular targets for NO. A

potential therapeutic target for NO is angiogenesis (Francis

et al., 2001). Treatment of cells with NO donors increases

Fig. 3. Immunohistochemical localization of iNOS. Paraffin sections of

myocardial tissue obtained from sham, I/R and Resveratrol+I/R rats were

incubated with antibodies against iNOS. Micrograph showing the

immunohistochemical localization of iNOS in the vascular endothelium

(arrow heads) of the rats employing the peroxidase–antiperoxidase (PAP)

method (brown color). (A) Negative control where primary antibody was

omitted showing blue nuclear staining without immunohistochemical

localization of iNOS. (B) Endothelial expression of iNOS in coronary

vessels in sham operated rats. (C) Intense iNOS expression in the

endothelium of coronary vessels and cytoplasmic localization in cardio-

myocytes in rats subjected to ischemia and reperfusion. (D) Enhanced

endothelial expression of iNOS in coronary vessels as well as in

cardiomyocytes in rats treated with resveratrol and subjected to ischemia

and reperfusion. [Bar for all=100 Am.]

Fig. 4. Immunohistochemical localization of eNOS. Paraffin sections of

myocardial tissue obtained from sham, I/R and Resveratrol+ I/R rats were

incubated with antibodies against eNOS. Micrograph shows the immuno-

histochemical localization of eNOS in the vascular endothelium of the rat

hearts employing the peroxidase–antiperoxidase (PAP) method (brown

color). (A) Endothelial expression of eNOS in coronary small and large

vessels in sham operated rats. (B) Intense eNOS expression in the

endothelium of all size coronary vessels in rats subjected to ischemia and

reperfusion. (C) Very intense endothelial expression of eNOS in all size

coronary vessels in rats treated with resveratrol and subjected to ischemia

and reperfusion. [Bar for all=100 Am.]

the angiogenic factor, VEGF, and inhibitors of NO synthase

such as l-NAME can block VEGF generation (Murohara et

al., 1998). The limbs from eNOS knockout mice exhibited

significant impairment in angiogenenic response, suggesting

that NO can induce angiogenesis through VEGF (Sen et al.,

2002). A recent study demonstrated upregulation of

S. Das et al. / Vascular Pharmacology 42 (2005) 281–289 287

inducible VEGF expression at dermal wound site with a

combination of resveratrol and grape seed proanthocyani-

dins (Ziche et al., 1997). Consistent with this previous

report, our results also demonstrate the induction of the

expression of iNOS/VEGF by resveratrol. While VEGF

mediates angiogenesis, NO and VEGF together may interact

to promote angiogenesis (Shen et al., 1999).

Angiogenesis is tightly regulated by two families of

growth factors, the VEGF and the VEGF receptor such as

Flk-1/KDR (El-Gendi et al., 2002). VEGF can activate

Fig. 5. Immunohistochemical localization of VEGF. Paraffin sections of

myocardial tissue obtained from sham, I/R and Resveratrol+ I/R rats were

incubated with antibodies against VEGF. Color was developed using

Naphtol AS-MX phosphate and new fuchsine and visualized under light

microscope. Immunoreactive VEGF is localized in the smooth muscle cells

and in the cardiomyocytes.

Fig. 6. Immunohistochemical localization of KDR. Paraffin sections of

myocardial tissue obtained from sham, I/R and Resveratrol+I/R rats were

incubated with antibodies against KDR. Color was developed using

Naphtol AS-MX phosphate and new fuchsine and visualized under light

microscope. Immunoreactive KDR is localized in the endothelial cells of

blood vessels.

eNOS through KDR and eNOS inhibitors block VEGF-

induced endothelial cell migration, proliferation, and tube

formation in vitro and VEGF-induced angiogenesis in vivo

(Benndorf et al., 2003). Another study showed that VEGF

increased eNOS expression via activation of the KDR

receptor tyrosine kinase in porcine aorta endothelial cells

(Shen et al., 1999). Inactivation of eNOS expression

significantly impaired VEGF-induced angiogenesis in an

eNOS knockout mouse model (Sen et al., 2002). Our results

support this notion, and further confirm that initial induction

S. Das et al. / Vascular Pharmacology 42 (2005) 281–289288

of iNOS–VEGF by resveratrol potentiates the expression of

KDR, which in turn upregulates eNOS expression.

In summary, the results of the present study demonstrate

a sequential and coordinated activation of iNOS/VEGF–

KDR–eNOS, indicating the existence of a positive feedback

loop for NO production. Resveratrol mediate early activa-

tion of iNOS and late activation for eNOS have been

recognized previously. This study shows for the first time

that VEGF and its tyrosine kinase receptor KDR regulates

this feed back loop for NO production. An increase in NO

can in turn activate VEGF. As mentioned earlier, NO is

known to play a crucial role in myocardial preconditioning.

It appears that VEGF is a potential target for NO for

cardioprotection achieved by resveratrol.

Acknowledgements

This study was supported by NIH HL 34360, HL22559,

HL56322 and HL75665.

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