The role of eNOS, iNOS and NFκB in upregulation and activation of ...

Page 1 of Articles 35 in PresS. Am J Physiol Heart Circ Physiol (May 9, 2008). doi:10.1152/ajpheart.01350.2007 

The role of eNOS, iNOS and NF B in upregulation and activation of 

cyclooxygenase-2 and infarct size reduction by atorvastatin 

Yumei Ye, MD a,b ; Juan D. Martinez, MD a ; Regino J. Perez-Polo, PhD c ; Yu Lin, BSc a,b 

; Barry F. Uretsky, MD a,b , Yochai Birnbaum, MD a,b,c . 

a- The Department of Internal Medicine, b- The Division of Cardiology, c- The 

Department of Biochemistry and Molecular Biology, University of Texas Medical 

Branch, Galveston, TX 

Running Title: Ye Y, et al. Mechanisms of COX2 upregulation by atorvastatin 

Corresponding author: 

Yochai Birnbaum, MD 

The Division of Cardiology 

University of Texas Medical Branch 

5,106 John Sealy Annex 

301 University Blvd. 

Galveston, Texas 77555-0553 

E-mail: yobirnba@utmb.edu 

Office: 409-772-2794 

Fax: 409-772-4982 

Copyright Information 

Copyright © 2008 by the American Physiological Society.

Abstract: 

Ye Y, et al. Mechanisms of COX2 upregulation by atorvastatin 2 

Objectives: Pretreatment with atorvastatin (ATV) reduces infarct size (IS) and increases 

myocardial expression of phosphorylated endothelial nitric oxide synthase (P-eNOS), 

inducible NOS (iNOS), and cycloxygenase-2 (COX2) in the rat. Inhibiting COX2 

abolished the ATV-induced IS limitation without affecting P-eNOS and iNOS expression. 

We investigated: 1) whether 3-day ATV pretreatment limits IS in eNOS -/- and iNOS -/- 

mouse; 2) whether COX2 expression and/or activation by ATV is eNOS-, iNOS and/or 

NF B-dependent. Methods: Male C57BL/6 wild-type (WT), University of North 

Carolina eNOS -/- , and iNOS -/- mice received ATV 10 mg/kg/d (ATV+) or water alone 

(ATV-) for 3 days. Mice underwent 30min coronary artery occlusion and 4h of 

reperfusion, or hearts were harvested and subjected to ELISA, immunoblotting, biotin 

switch and electrophoretic mobility shift assay (EMSA). Results: ATV reduced IS only 

in the WT mice. ATV increased eNOS, P-eNOS, iNOS and COX2 levels, and activated 

NF B in WT mice. It also increased myocardial COX2 activity. In eNOS -/- mice, ATV 

increased COX2 expression, but not COX2 activity or iNOS expression. NF B was not 

activated by ATV in the eNOS -/- mice. In the iNOS -/- mice eNOS and P-eNOS levels were 

increased, but not iNOS and COX2 levels; however, NF B was activated. Conclusions: 

Both eNOS and iNOS are essential for the IS-limiting effect of ATV. Expression of 

COX2 by ATV is iNOS- but not eNOS- or NF B dependent. Activation of COX2 is 

dependent on iNOS. 

Key words: eNOS, iNOS, COX2, NF B, atorvastatin, infarct size. 

Introduction 


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The 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) 

protect against ischemia-reperfusion injury and when administered before ischemia [2; 5; 

7; 25; 26; 34-36; 43; 44; 46-48; 52; 53] or immediately upon reperfusion [4; 17; 47] 

limits myocardial infarct size (IS) in various animal models. Several investigators have 

shown that activation of endothelial nitric oxide synthase (eNOS) is essential for this 

protective effect, as non-specific nitric oxide synthase (NOS) inhibitors blunt the IS- 

limiting effect of statins [5; 48] and statins do not reduce IS in eNOS -/- mice [1; 4; 18; 25; 

52]. However, most of these studies used a particular eNOS -/- line (Harvard). As reported 

by Sharp et al, there are two distinct lines of eNOS -/- mice, the Harvard line lacks 

compensatory increases in inducible nitric oxide synthase (iNOS) and has IS bigger than 

the corresponding wild-type mice, and the University of North Carolina line, which has 

compensatory increases in iNOS expression and IS smaller than the corresponding wild- 

type mice [37]. It has been suggested that iNOS can be protective and compensate for the 

lack in eNOS and eNOS may compensate for a lack of iNOS in adenosine-triggered 

preconditioning [3]. Others have suggested that neuronal NOS (nNOS) is upregulated in 

the University of North Carolina eNOS -/- mice and can restore NOS-dependent 

vasodilation [13; 30]. It has not been demonstrated whether statins limit IS in the 

University of North Carolina eNOS -/- mouse. Moreover, while there is abundant literature 

on the pathways of activation of eNOS by statins [21; 28], less is known about the 

signaling pathways of protection downstream from eNOS. It has been suggested that 

statins activates the Reperfusion Injury Salvage Kinase (RISK)-pathway [4; 17], which 

appears to be independent of NOS activation [22; 23]. On the other hand, we have shown 

that statins also activate iNOS [2; 7; 34; 53], cytosolic phospholipase A2 (cPLA2) [7; 53], 

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cyclooxygenase 2 (COX2) [2; 7; 53] and the specific prostaglandin synthases, PGI2 

synthase [7], PGE2 synthase [7], and PGD2 synthase [54]. Blocking either iNOS [7; 34] 

or COX2 [2; 7] abrogates the IS-limiting effect of atorvastatin (ATV). In the rat, iNOS 

activates COX2 by S-nitrosylation [2]. However, it is unclear whether iNOS is needed to 

upregulate COX2 expression, as pioglitazone, a peroxisome proliferator-activated 

receptor- (PPAR- ) agonist, increases COX2 expression and activity without 

upregulating iNOS [53]. It has been shown that NF B mediates the upregulation of iNOS 

and/or COX2 in delayed ischemic preconditioning [10; 40]. However, several studies 

have suggested that statins suppress NF B activation [14; 15; 32; 33; 41; 49]. Therefore, 

we asked whether 3-day ATV pretreatment limits IS in the University of North Carolina 

line of eNOS -/- mouse and iNOS -/- mouse. We also asked whether eNOS, iNOS and NF B 

are needed for the upregulation and/or activation of COX2 by ATV. 

Methods: 

Male C57BL/6 wild-type (WT), University of North Carolina eNOS -/- , and iNOS -/- mice 

were purchased from the Jackson Laboratory (Maine, USA) and received humane care in 

compliance with ‘The Guide for the Care and Use of Laboratory Animals’ published by 

the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). The 

protocol was approved by UTMB IACUC. 

Treatment 

Mice received 3-day pretreatment with ATV (10 mg/kg per day) dissolved in water or 

water alone, administered by oral gavage once daily. On the fourth day mice underwent 

coronary artery ligation for 30 min followed by 4 hours of reperfusion (IS protocol)(n=10 





in each group), or the mice were euthanized under anesthesia, hearts were explanted 

without being subjected to ischemia, rinsed in cold PBS (pH 7.4), containing 0.16 mg/ml 

heparin to remove red blood cells and clots, frozen in liquid nitrogen and stored at -80°C 

for further analyses (immunoblotting, calcium dependent and independent NOS activity, 

6-keto-PGF1 levels, COX2 activity, EMSA for NF B, and immunofluorescence for 

NF B). There were 4 mice in each group. 

Infarct size 

On the fourth day mice were anesthetized with intraperitoneal injection of ketamine (60 

mg/kg) and xylazine (6 mg/kg), intubated and ventilated (FIO2=30%). The rectal 

temperature was monitored and body temperature was maintained between 36.7 and 

37.3 0 C throughout the experiment. The chest was opened and the left coronary artery 

was encircled with a suture and ligated for 30 minutes. Ischemia was verified by regional 

dysfunction and discoloration of the ischemic zone. Isofluorane (1-2.5% titrated to effect) 

was added after the beginning of ischemia to maintain anesthesia. At 30 minutes of 

ischemia, the snare was released and myocardial reperfusion was verified by change in 

the color of the myocardium. Subcutaneous 0.1 mg/kg buprenorphine was administered, 

the chest was closed and the mice were recovered from anesthesia. Four hours after 

reperfusion the mice were re-anesthetized, the coronary artery was reoccluded, Evan's 

blue dye 3% was injected into the right ventricle and the mice euthanized under deep 

anesthesia [2; 7; 43; 53]. 

The pre-specified exclusion criteria were lack of signs of ischemia during coronary artery 

ligation, lack of signs of reperfusion after release of the snare, prolonged ventricular 

arrhythmia with hypotension, and area at risk 10% of the LV weight. 


Determination of area at risk (AR) and infarct size (IS) 


Hearts were excised and the left ventricle was sliced transversely into 6 sections. Slices 

were incubated for 10 minutes at 37°C in 1% buffered (pH=7.4) 2,3,5-triphenyl- 

tetrazolium-chloride (TTC), fixed in a 10% formaldehyde and photographed in order to 

identify the AR (uncolored by the blue dye), the IS (unstained by TTC), and the non- 

ischemic zones (colored by blue dye). The area of AR and IS in each slice were 

determined by planimetry, converted into percentages of the whole for each slice, and 

multiplied by the weight of the slice and the results summed to obtain the weight of the 

myocardial AR and IS [2; 7; 43; 53]. 

The effect of NF B and JAK inhibitors on the induction of iNOS and COX2 by ATV 

WT mice were treated with intraperitoneal ATV (5 mg/kg); vehicle alone (DMSO 5%); 

ATV + AG-490 (JAK inhibitor, 40 µg/kg); or ATV + SN50 (NF B inhibitor, 400 µg/kg) 

(n=4 in each group). Hearts were harvested 8h later and assessed for iNOS, COX2, I B, 

and -actin expression in the whole cell lysate and phosphorylated STAT-1 and lamin B 

in the nuclear fraction. 

Immunoblotting 

Myocardial samples from the left ventricular wall were homogenized in RIPA lysis 

buffer (Santa Cruz Biotechnology) and centrifuged at 14,000 rpm for 15min at 4 0 C. The 

supernatant was collected and the total protein concentration was determined using the 

Lowry protein assay. The protein samples (50 µg) with loading buffer were run in 4-20% 

Tris-HCl Ready Gel at a 100V for 2h until the desired molecular weight bands were 

separated. After electrophoresis, the gel was equilibrated in transfer buffer (25mM Tris, 

193mM glycine, 0.1% SDS and 10% methanol) and the proteins were transferred to 





nitrocellulose membrane. The protein signals were quantified by an image-scanning- 

densitometer and the strength of each protein signal was normalized to the corresponding 

-actin stain signal. Signals for p-STAT-1 in the nuclear fraction were normalized to the 

corresponding lamin B signal. Data are expressed as a ratio between the protein and the 

corresponding -actin or p-STAT-1 signal density. 

NOS activity 

Myocardial samples were homogenized in a buffer (25mM Tris-HCL (PH 7.4); 1 mM 

EDTA; and 1 mM EGTA), centrifuged at 10,000 X g for 15 min. The supernatant, 

containing the soluble enzyme iNOS, and the pellet, containing the membrane-bound 

eNOS and neuronal NOS (nNOS) [calcium dependent NOS (cNOS)] were separated. The 

pellet was resuspended in homogenization buffer. NOS activity was determined by 

measuring the conversion of L-[14C]-arginine to L-[14C]-citrulline using a commercial 

kit (Cayman Chemicals, Ann Arbor, MI). For assessing calcium dependent NOS (cNOS) 

activity CaCl2 was added to the samples. For assessing calcium independent (ciNOS) 

activity, CaCl2 was omitted from the solution. NOS activity was defined as counts per 

minute (cpm) [34]. 

6-keto-PGF1 and Phospholipase A2 (PLA2) activity 

Myocardial samples of the anterior wall of the left ventricle were perfused with a PBS 

solution (pH 7.4) containing 0.16mg/ml heparin to remove red blood cells and clots, 

homogenized in cold PBS (pH 7.4), and centrifuged. The supernatants were collected and 

stored on ice. Measurement of 6-Keto-PGF1 , the stable metabolite of prostacyclin, and 

PLA2 activity were made using immunoassay assay kits. 

COX activity 



Myocardial samples of the anterior wall of the left ventricle were perfused and rinsed 

with 0.05M Tris buffer, PH 7.4, containing 0.16 mg/ml heparin to remove any red blood 

cells and clots. Samples were homogenized in 5-10 ml of cold buffer (0.1 M Tris-HCL, 

pH 7.8 containing 1 mM EDTA) per gram tissue, centrifuged at 10,000Xg for 15 minutes 

at 4 0 C, and the supernatant was collected and stored on ice. The COX activity assay kit 

measures the peroxidase activity of COX, assayed colorimetrically by monitoring the 

appearance of oxidized N,N,N’,N’-tetramethyl-p-phenylenediamaine (TMPD) at 590 nm. 

Each myocardial sample was tested in triplicate (the first without an inhibitor; the second 

with DuP-697 (0.286 µM), a specific COX2 inhibitor; and the third with Sc560 (0.314 

µM), a specific COX1 inhibitor. COX1 activity was calculated as the difference between 

total COX activity in the sample without an inhibitor and the sample with Sc560, and 

COX2 activity as the difference between total COX activity in the sample without an 

inhibitor and the sample with DuP-697. 

Nuclear extraction 

Myocardial samples (0.25 g) were homogenized, mixed with Buffer A Mix [Hepes (pH 

7.9) 10 mM, KCl 10 mM, EDTA 10 mM, DTT 100 mM, protease inhibitor cocktail, and 

IGEPAL 10%, (Sigma, St Lois, MO)], homogenized again and incubated for 15 min on 

ice, and centrifuged at 850 x g for 10 min at 4 0 C. The supernatants were discharged, 

Buffer A Mix was added again and the samples incubated for an additional 15 min on ice, 

and centrifuged at 15,000 x g for 3 min at 4 0 C. The supernatant were discharged and the 

pellets resuspended in 150 µl of Buffer B Mix [Hepes (pH 7.9) 20mM, NaCl 0.4M, 

EDTA 1 mM, glycerol 10%, protease inhibitor cocktail, and IGEPAL 10%], the tubes 

were shake on ice at 200 rpm for 2h, centrifuged at 15,000 x g for 5 min at 4 0 C, and the 





supernatants collected as the nuclear fraction and stored at -80 0 C until used (Modified 

from Dignam et al [16]. 

Electrophoretic mobility shift assay (EMSA) 

EMSA were carried out as described elsewhere with some modifications [19]. Briefly, 

the EMSA gel is made with 30% polyacrylamide, 10 x TBE, 10% AP, H2O, and TEMED 

to yield 7.5% gel. NF B probes: Oligonucleotides encompassing the IgG- B enhancer 

sequence (GGGACTTTCC) were 5' labeled with a- 32 P-ATP and T4 polynucleotide 

kinase. Binding reactions with 40 µg of nuclear extracts were performed in a 20 µl 

volume containing 20,000 cpm of probe, 2 µg of poly dI-dC, 10 µl of TK100 buffer (25 

mM HEPES, pH 7.9, 20% glycerol, 1 mM EDTA, 100 mM KCl, 2 mM MgCl2, 2 mM 

dithiothreitol, and 2 mM PMSF) and competitor. Nuclear extracts were incubated with 

the poly dI-dC on ice for 10 min, and then the buffer and probe was added. Incubation 

was continued for 20 min at room temperature, after which reaction mixtures was loaded 

on the gel in 0.25× TBE buffer (pH 7.2). Gels were dried and exposed to x-ray film. 

When antibodies were used in EMSA for immunodepletion/supershift study, nuclear 

extracts were incubated with the different antibodies for 30 min at 4°C before the 

addition of the poly dI-dC. 

Immunofluorescence 

Myocardial sections were incubated in a blocking solution (1-3% normal serum/5% 

BSA/0.1% Triton X-100) for 30 min at room temperature, and rinsed in PBS. The 

primary anti-NF B p65 antibodies were centrifuged at 12,000 rpm at 4 0 C for 2 min and 

diluted in PBS. 50-100 µl of the primary antibodies were added to each section, and the 

samples were incubated for 1-24 hr at 4 0 C in a humidified box, washed three times in 



PBS. The Alexa-secondary antibodies [Goat anti-mouse IgG with Alexa 488 (488-GAM) 

for monoclonal primary antibodies (Molecular Probe, A-11029), and goat anti-rabbit IgG 

with Alexa 594 (594-GAR) for rabbit polyclonal primary antibodies (Molecular Probe, 

A-11037)] were centrifuged at 12,000 rpm at 4 0 C for 2 min, diluted 1:200-1:400 in PBS. 

The sections were incubated with the Alexa-secondary antibodies in dark at room 

temperature for 1h, washed in PBS for 3 times, incubated in 1µg/ml DAPI (1:1000 

dilution) at room temperature for 5 min in the dark, and rinsed with PBS. The sections 

were dried, and Fluoromount-G was added onto the slides, and the slides were covered 

with a cover-slip. Slices were stored at 4 0 C, until examined and photographed under 

microscope. 

Biotin switch assay 

S-nitrosylation of COX2 was determined with the biotin switch method, as has been 

previously described [2]. Myocardial samples were homogenized with HEN buffer [25 

mM HEPES (pH 7.7)-0.1 mM EDTA-0.01 mM necuproine]. The supernatant containing 

membrane fragments and the cytosolic protein was recovered. The samples were 

incubated for 30 min at 4°C with blocking solution containing HEN buffer, 0.1% SDS, 

and 20 mM N-ethylmaleimide [NEM] to block free thiols. Lysates were centrifuged at 

16,000 Xg for 10 min at 4°C. Cold acetone was added to precipitate the proteins. The 

pellets were resuspended in HEN buffer with 1% SDS, with 20 mM sodium ascorbate 

added to decompose the SNO bonds. The resulting free thiols in the sample were reacted 

with 0.05 mM biotinylating agent, biocytin [MPB] for 30 min at room temperature. The 

excess MPB was removed by additional protein precipitation in cold acetone. COX2 was 

immunoprecipitated with anti-COX2 polyclonal antibody. Immunoprecipitates were 





washed three times with HEN buffer and resuspended in 50 µl of HEN containing 

Laemmli sample buffer, boiled at 95°C for 5 min, loaded on 10% acrylamide gels, and 

transferred to nitrocellulose. The biotinylated COX2 protein was detected with 

horseradish peroxidase-linked streptavidin. All procedures up to biotinylation were 

performed in the dark. The membranes were stripped with a stripping buffer and blotted 

again with anti-COX2 antibodies. The signal densities of the biotinylated COX2 and total 

COX2 were quantified by an image-scanning densitometer and the ratio of the densities 

was calculated for each animal. 

Materials 

ATV was purchased from Pfizer Pharmaceuticals (New York, NY). NOS-activity kit, and 

ELISA kit for 6-keto-PGF1 , and COX2 activity were purchased from Cayman Chemicals 

(Ann Arbor, MI). Polyclonal anti-iNOS antibodies were purchased from Cayman 

Chemical; polyclonal anti ser-1177 phosphorylated-eNOS (P-eNOS) antibodies from Cell 

Signaling (Beverly, MA); monoclonal anti-eNOS antibodies and monoclonal anti-COX2 

antibodies from BD Bioscience (San Jose, CA); and monoclonal anti- -Actin antibody 

from Sigma (St.Louis, MO). Anti- NF B antibodies (NF B p65, NF B p50) were 

purchased from Millipore Corporation, Billerica, MA. Anti lamin B, anti I B, and anti p- 

STAT-1 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). 

[N-(3-malemidylpropionyl) biocytin was purchased from Molecular Probes (Eugene, 

OR). ImmunoPure Streptavidin, Horseradish Peroxidase Conjugated was purchased from 

PIERCE Biotechnology, Inc. (Rockford IL). AG-490 was purchased from Sigma (St. 

Louis, MO) and SN50 from Calbiochem (San Diego, CA). 

Statistical analysis 



Data are presented as mean ±SEM. The significance level is 0.05. Body weight, left 

ventricular weight and the size of the AR were compared using analysis of variance 

(ANOVA). Data on IS (as a percentage of the AR) and enzyme expression and activity 

were compared between the ATV treated and not treated groups using t-test or Mann- 

Whitney Rank Sum test when appropriate. Values of P



the eNOS -/- or iNOS -/- mice (Figure 2c). On the other hand, COX2 expression was 

upregulated by ATV only in the WT and eNOS -/- , but not the iNOS -/- mice (Figure 2d). 

NOS activity 

All enzyme activity analyses were done in hearts that were explanted on the fourth day of 

the experiment without being subjected to regional ischemia. In agreement with the P- 

eNOS immunoblotting results, ATV augmented cNOS activity in the WT and iNOS -/- 

mice, but not in the eNOS -/- mice (Figure 3a). ciNOS activity was augmented by ATV 

only in the WT mice (Figure 3b). 

Myocardial 6-keto-PGF1 levels and COX activity 

ATV significantly increased myocardial 6-keto-PGF1 levels only in the WT mice 

(Figure 3c). ATV did not affect COX1 activity in all three strains (data not shown). 

COX2 activity was increased by ATV only in the WT mice, but not in the eNOS -/- and 

iNOS -/- mice (Figure 3d). Thus, the upregulated COX2 protein in the eNOS -/- mice treated 

with ATV was inactive. 

NF B activation 

EMSA was done in hearts that were explanted on the fourth day of the experiment 

without being subjected to regional ischemia. EMSA showed that NF B was activated in 

the WT and iNOS -/- mice, but not in the eNOS -/- mice (Figure 4a). Immunoblotting of I B 

in the cytosol confirmed the EMSA results, showing decreased I B levels in the ATV 

treated WT and iNOS -/- mice (Figure 4b). Immunofluorescent staining of myocardial 

tissue of WT mice showed increased staining of NF B in the cell nuclei of mice treated 

with ATV (Figure 5). 

The effect of NF B and JAK inhibitors on the induction of iNOS and COX2 by ATV 



SN50, an NF B inhibitor, blocked the ATV induced decrease in I B levels, indicating 

blocking NF B activation. AG49 blocked ATV induced increase in p-STAT-1 levels in 

the nuclear fraction, indicating blockade of the JAK-STAT activation. SN50 completely 

blocked the induction of iNOS, whereas AG490, a JAK inhibitor, had no effect (Figure 6). 

In contrast, AG490 and SN50 alone did not block the induction of COX2 by ATV. 

S-nitrosylation of COX2 

ATV induced S-nitrosylation of COX2 only in the WT mice. The COX2, induced by 

ATV in the eNOS -/- mice was not S-nitrosylated, in agreement with its being inactive 

(Figure 7). 

Summary of the findings is presented in table 2. 

Discussion 

Our main finding is that ATV did not limit IS in the University of North Carolina eNOS -/- 

and iNOS -/- mice, as reported for the Harvard eNOS -/- mice [1; 18; 25; 52]. The induction 

of COX2 by ATV was eNOS and NF B independent, as ATV increased COX2 expression 

in eNOS -/- mice and in these mice NF B was not activated by ATV. On the other hand, in 

the iNOS -/- mice, NF B was activated by ATV; however, COX2 expression was not 

changed. Moreover, SN50, an NF B inhibitor, did not block COX2 expression, although 

it blocked iNOS expression. These data are in agreement with the delayed ischemic 

preconditioning literature that NF B activation is downstream to eNOS [10]. On the other 

hand, COX2 activity was increased by ATV only in the WT mice. In the eNOS -/- mice, 

ATV increased COX2 expression, but the COX2 was inactive. Of note, the upregulated 

COX2 in the eNOS -/- mice was not S-nitrosylated. Previously we have shown that 





myocardial COX2, upregulated by ATV is inactivated by an iNOS inhibitor that 

decreased its Sinitrosylation [2] (Table 2). 

We have previously shown that 3-day ATV treatment does not upregulate nNOS levels in 

the rat myocardium [7]. As the activities of both eNOS and nNOS are calcium dependent, 

cNOS activity reflects the activity of both enzymes. In the present study we did not 

observe an increase in cNOS activity in the eNOS -/- mice, suggesting that ATV did not 

induce a significant upregulation of nNOS in these mice. Moreover, ATV did not 

decrease IS in the University of North Carolina eNOS -/- mice, suggesting that in contrast 

to the effect on vascular relaxation [13; 30], nNOS does not compensate for the lack of 

eNOS in our model. 

iNOS 

Our findings are in disagreement with those of Sharp et al that iNOS expression is 

upregulated in the University of North Carolina eNOS -/- mice [37]. Myocardial levels of 

iNOS were very low in the eNOS -/- mice and were not upregulated by ATV in these mice. 

In accordance, ciNOS activity was not increased in the eNOS -/- mice compared to the WT 

mice. The present study confirms our previous results that iNOS upregulation is essential 

for the IS-limiting effect of ATV. Scalia has also shown that simvastatin does not limit IS 

in iNOS -/- mice [36]. We have previously shown that 1400W, a selective iNOS inhibitor, 

abrogates the IS-limiting effect of 3-day pretreatment with ATV, by inhibiting the 

activation of COX2 via S-nitrosylation [2]. In that study, 1400W did not affect total and 

Ser-1177 phosphorylated eNOS levels and cNOS activity, although it blocked the ATV 

induction of ciNOS activity. In the present study iNOS expression and ciNOS activity 

were not upregulated in the eNOS -/- mice [2], suggesting that iNOS is downstream of 



eNOS, as has been described for the delayed form of ischemic preconditioning [10] [23]. 

It has been suggested that eNOS activation leads to activation of soluble guanylate 

cyclase, protein kinase C (PKC ), NF B, and Janus kinase (JAK)-signal transducers and 

activators of transcription (STAT) pathways, leading to activation of iNOS and/or COX2 

[3; 9-11] [23]. In contrast, in other models eNOS has been reported to inhibit NF B 

activation [45]. This may reflect that NF B activation involves both inflammatory 

signaling (p65/p50) and anti-inflammatory signaling (cRel/p52) as well as other non- 

canonical pathways. It is probable that the upregulation of iNOS by ATV is NF B- 

dependent (Figure 6). Indeed, both NF B and iNOS expression and activity were not 

increased by ATV in the eNOS -/- mice. 

Our data suggest that the expression of COX2 is dependent on the presence of intact 

iNOS, as COX2 expression was not increased in the iNOS -/- mice despite activation of 

NF B. Previously it has been shown that 24 hours after ischemic preconditioning 

stimulus, COX2 expression was increased in both WT and iNOS -/- mice, suggesting that 

with a more robust stimulus than statin pretreatment, such as preconditioning, iNOS is not 

essential for COX2 upregulation [50]. Although iNOS expression and ciNOS activity 

were not increased by ATV in the eNOS -/- mice, the presence of intact iNOS gene may 

enable COX2 upregulation. Alternatively, a gene adjacent to iNOS on chromosome 10 

that is responsible for upregulating COX2 expression could have been erroneously 

deleted in the iNOS -/- mice. However, as pioglitazone, a PPAR- agonist augments COX2 

expression in the same iNOS -/- mice strain [8], this is probably not the explanation. 

The over-expressed COX2 in the ATV-treated eNOS -/- mice was not activated and not S- 

nitrosylated. Although we cannot exclude the possibility that eNOS S-nitrosylates COX2 





in the present study, previously we have shown that 1400W, a selective iNOS inhibitor, 

prevents COX2 S-nitrosylation by ATV in the heart without affecting cNOS activity [2]. 

Kim et al have shown that S-nitrosylation by iNOS is essential for COX2 activation in 

inflammatory cells [27]. Moreover, Xuan et al have shown the although ischemic 

preconditioning increased COX expression in iNOS -/- mice, the COX2 was inactive [50]. 

Thus, iNOS is needed for both the expression and activation of myocardial COX2 by 

ATV. eNOS is a membrane bound enzyme, whereas iNOS is a soluble enzyme, thus, the 

localization of these two enzymes in the cells is different and although COX2 is a 

membrane bound enzyme [42], it seems that iNOS, but not eNOS is responsible for COX2 

S-nitrosylation. 

We [2] and others [27] have used the biotin switch assay to assess S-nitrosylation. 

Recently, it has been suggested that artifacts may interfere with the interpretation of the 

test [24]. However, currently there are no reliable alternative assays to assess for S- 

nitrosylation. It is plausible that other mediators may activate COX2 independent of S- 

nitrosylation, especially in inflammatory models. However, it seems that when COX2 is 

upregulated by ATV without an inflammatory stimulus, iNOS-induced S-nitrosylation is 

essential for COX2 activation in the rat and mouse myocardium. It may be that 

nitrosylation of COX2 is not a general prerequisite, but rather is restricted to the 

myocardium. 

Of note, ciNOS activity in the iNOS -/- mice was comparable to that of the WT control 

group and not nil. This represents background noise of the method and not residual 

activity, as has also been shown by Guo et al [20]. 

COX2 



Previously we have shown that ATV upregulates COX2 expression and activity [2; 7; 

53]. Inhibiting COX2, but not COX1 abrogated the IS-limiting effect of ATV [2; 7]. 

COX2 is essential for mediating the protective effect of delayed ischemic 

preconditioning, as COX2 inhibition abrogates IS limitation by preconditioning [10; 12; 

38; 39]. It is well established that NF B affects COX2 expression [10; 29; 40; 45]. 

However, other pathways such as the JAK-STAT signaling pathway [10; 45; 50; 51], 

PI3K through C/EBP or ERK via CREB can also upregulate COX2 expression 

independent of NF B [45]. Our data suggest that the upregulation of COX2 expression by 

oral ATV in the heart is independent of NF B, as COX2 expression was increased by 

ATV in eNOS -/- mice, despite the fact that NF B was not activated. Moreover, in the 

iNOS -/- mice, COX2 expression was not increased by ATV despite activation of NF B. 

Furthermore, SN50 did not block the ATV-induction of COX2 expression, although it 

blocked iNOS induction, supporting the fact that the induction of iNOS, but not COX2 is 

NF B-dependent (Figure 6). In addition, AG490 also did not affect COX2 induction by 

intraperitoneal ATV, suggesting that COX2 induction may be independent of JAK-STAT, 

in contrast to the findings in ischemic preconditioning [10]. Further studies are needed to 

clarify the role of JAK-STAT in mediating statin-induced myocardial protection. 

The dose of ATV used in the present study (10 mg/kg/d) may be considered high. Indeed, 

several investigators have shown reduction of IS with lower doses of statin; however, in 

all these studies statins were administered intraperitoneally, subcutaneously or 

intravenously. In a dose ranging study we have shown that 3-day oral pretreatment with 

ATV at 10 mg/kg/d and 75 mg/kg/d reduces IS in the rat [5], whereas at a dose of 1-2 

mg/kg/d oral ATV has no effect [5; 34]. Moreover, we have shown that in the rat, blood 





levels of ATV 16 hours after a third dose of 10 mg/kg/d are comparable to those seen in 

humans treated with ATV 80 mg/d [6]. Other investigators have also used equivalent or 

even higher doses of oral statins to show myocardial protection [31; 48]. 

In conclusion, we have shown that both iNOS and eNOS are essential for mediating the 

IS-limiting effect of ATV. Induction of eNOS leads to NF B-dependent iNOS 

upregulation. On the other hand, the induction of COX2 expression by ATV is eNOS- and 

probably NF B- independent. However, iNOS is needed for both the increased expression 

and activation of COX2. 


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Table 1: Protocol 1: body weight, left ventricular weight, and AR. 

ATV- 

n=10 

Wild type eNOS -/- 

ATV+ 

n=8 

ATV- 

n=10 

ATV+ 

n=10 


ATV- 

n=10 

iNOS -/- 

ATV+ 

Body weight (g) 23.0±1.1 24.7±0.5 24.3±0.6 25.1±0.3 24.0±0.7 25.2±0.4 

LV weight (mg) 125±1 118±1 119±1 119±1 117±2 118±2 

AR (% of LV) 44.7±1.0 46.3±1.7 44.8±0.9 45.2±1.8 46.0±1.2 47.0±1.3 

AR- ischemic area at risk; IS- infarct size; LV- left ventricle. 


n=10


Table 2: Summary of the effects of ATV in wild-type, eNOS -/- and iNOS -/- mice. 

Wild-type eNOS -/- iNOS -/- 

Infarct size - - 

eNOS expression - 

P-eNOS expression - 

cNOS activity - 

iNOS expression - - 

ciNOS activity - - 

COX2 expression - 

COX2 activity - - 

COX2 S-nitrosylation - - 

NF B activation - 





Figure 1: The effect of ATV on infarct size (IS) in the wild-type (WT), eNOS -/- and 

iNOS -/- mice. ATV limited IS in the WT, but not in the eNOS -/- and iNOS -/- mice. There 

were 8 mice in the WT ATV+ group and 10 mice in each of the other groups. 

Figure 2: Samples of immunoblots and densitometric analyses of myocardial expression 

of total eNOS (a), Ser-1177 P-eNOS (b), iNOS (c), and COX2 (d) in WT, eNOS -/- and 

iNOS -/- mice with or without ATV pretreatment. Total eNOS and Ser-1177 P-eNOS levels 

were increased by ATV in the WT and iNOS -/- mice. There was no expression of eNOS in 

the eNOS -/- mice. ATV augmented iNOS levels only in the WT mice. On the other hand, 

ATV increased COX2 levels in the WT and eNOS -/- mice, but not in the iNOS -/- mice. 

There were 4 mice in each group. 

Figure 3: Myocardial cNOS activity (a), ciNOS activity (b), 6-keto-PGF1 levels (c) and 

COX2 activity (d) in WT, eNOS -/- and iNOS -/- mice with or without ATV pretreatment. 

ATV augmented cNOS activity in the WT and iNOS -/- mice. In contrast, ciNOS activity 

was increased by ATV only in the WT mice. Similarly, 6-keto-PGF1 levels and COX2 

activity were augmented by ATV only in the WT mice. There were 4 mice in each group. 

Figure 4: a: EMSA showing activation of NF B (p50 and p65) in the WT and iNOS -/- 

mice, but not in the eNOS -/- mice. The NF B and the supershift bands are marked with 

arrows. b: A sample of immunoblot and densitometric analysis of cytosolic I B showing 

a decrease in I B levels in the WT and iNOS -/- mice, but not in the eNOS -/- mice. There 

were 4 mice in each group. 



Figure 5: Immunofluorescent staining of NF B in heart tissue of WT mice not receiving 

(ATV-) or receiving (ATV+) ATV. The nuclei are stained in blue (DAPI) and NF B p65 

in red. Translocation of NF B into the nuclei is seen in the ATV treated mice. 

Figure 6: Myocardial COX2, iNOS, I B and -actin levels and nuclear p-STAT-1 and 

Lamin B levels in mice treated with intraperitoneal ATV alone or with AG490 (a JAK 

inhibitor) or SN50 (an NF B inhibitor). * p


% of the Area at risk 

60 

50 

40 

30 

20 

10 

0 

p

a. b. 

c. 

d. 




a. b. 

c. d. 


a. 

b. 




ATV- 

ATV+ 

DAPI NF B DAPI + NF B 


X100




a. 

b. 

S- Nitrosylation COX-2 

WT eNOS -/- iNOS -/- 

ATV - + - + - +

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