| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Regular Articles |
From the Division of Cardiovascular and Respiratory Medicine,Department of Internal Medicine, Kobe University Graduate School ofMedicine, Kobe, Japan
 |
Abstract |
|---|
 Top Abstract Materials and MethodsResultsDiscussionReferences |
|---|
Nitric oxide (NO) is a key regulator of vascular functions that play important roles in physiology as well as in pathophysiology of various cardiovascular diseases. There has been a controversy about the generation of NO during I/R. NO production is shown to be increased during ischemia and in the very early periods of reperfusion in rat cerebral or isolated heart I/R model.9,10 In contrast, other studies suggested that NO generation decreases due to reduced expression and inactivation of endothelial NO synthase (eNOS) in the affected vessels.11,12 The roles of NO in I/R injury have not been established because of its biphasic aspects, cytoprotective or cytotoxic, in different organs and a variety of experimental conditions.13-16 L-arginine, a substrate for NOS enzymes, and NO donors have been demonstrated to exert protective effects against I/R injury in rat skeletal muscle and rat mesenteric artery in vivo.17,18 In contrast, studies using NOS inhibitors have shown either protective or detrimental effects of NO in I/R tissue injury.16,19 Thus, the roles of endogenous NO, particularly that derived from eNOS, in the pathogenesis of I/R have not been clearly elucidated.
We have previously established transgenic mice in which eNOS is overexpressed mainly in the endothelium,20 and have presented protective effects of eNOS-derived NO in lung inflammation induced by lipopolysaccharide21 or various types of vascular remodeling.22,23 In the present study, we investigated the roles of NO in I/R injury in skeletal muscle of mice hindlimb by the use of eNOS-transgenic (eNOS-Tg) and their littermates wild-type (WT) mice. Particularly, we focused on the vascular integrity such as assembly of endothelial junctions and in vivo vascular permeability of plasma proteins and leukocytes. Also, we examined the effects of eNOS overexpression on oxidants production in the skeletal muscle tissues after I/R.
|
Materials and Methods |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
All chemicals were obtained from Sigma Chemical Co. (St. Louis, MO, USA) unless specified. Pentobarbital sodium was purchased from Abbot Laboratories (North Chicago, IL, USA). Evans Blue dye (EBD) was purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). The following antibodies were used: a rabbit polyclonal anti-eNOS antibody, and a mouse monoclonal anti-ß-catenin antibody (Transduction Laboratories, Lexington, KY, USA); a rat monoclonal anti-VCAM-1 antibody (CHEMICON International, Temecula, CA, USA); a goat polyclonal anti-VE-cadherin antibody (Santa Cruz, Santa Cruz, CA, USA); and a mouse monoclonal anti-ICAM-1 antibody (Genzyme/Techne, Cambridge, MA, USA).
I/R Injury Model in Mice
Male eNOS-Tg (n = 166) mice and their
littermates WT (n = 100) were derived from the
same genetic background (C57BL/6) as previously
described.20
In eNOS-Tg mice, to evaluate the precise
effects of eNOS overexpression in the pathological process of I/R, 73
animals were treated with a non-selective NOS inhibitor,
N
-nitro-L-arginine methyl ester (L-NAME), in
drinking water (1 mg/ml) for one week before the experiment. Complete
inhibition of the endogenous NOS activity was confirmed as described
previously.21,22
Mice were anesthetized with pentobarbital
sodium (80 µg/kg) intraperitoneally and placed on a heated table, and
the body temperature was maintained at 37°C throughout the
experiments. Ischemia was induced by occluding right femoral arteries
for 30 minutes with the use of vascular clips (AESCULAP), and the
arteries were subsequently reperfused for 60 or 240 minutes by
releasing the clips. In the following experimental protocol, all tissue
samples were obtained from gastrocnemius muscles. All animal
experiments were conducted according to the Guidelines for Animal
Experimentation at Kobe University Graduate School of Medicine.
Protein Analysis for eNOS
The expression of eNOS in hindlimb skeletal muscle was analyzed by immunoblotting, and NOS enzymatic activity was determined by conversion of [3H]-L-arginine to [3H]-L-citrulline as described previously.20 Enzyme activity was expressed as citrulline production in picomole per milligram of protein per minute.
Measurements of NO Release from Skeletal Muscles
The skeletal muscles in hindlimbs were excised, and incubated in O2-saturated Krebs’ solution at 37°C for 60 minutes. After the supernatants were collected, the muscles were again incubated in O2-saturated Krebs’ solution containing 30 µmol/L acetylcholine at 37°C for 60 minutes. Then, the supernatants collected under these two conditions were subjected to nitrite measurement by using a NOx analyzer (ozone chemiluminescence), as reported previously.24,25 The difference in NO release, calculated by subtracting NO release in the non-stimulated condition from that in the acetylcholine-stimulated condition, was expressed as picomole per milligram of protein per hour.
Imuunohistochemistry and Immunofluorescence
Immunohistochemical stainings for eNOS, ICAM-1, and VCAM-1 of the vasculatures in gastrocnemius muscles were performed by the labeled streptavidin biotin method as previously described.20 VE-cadherin and ß-catenin were visualized by immunofluorescence microscopy. Briefly, frozen sections were fixed in acetone for 10 minutes, blocked with carrier protein for 60 minutes, and incubated with a primary antibody for 3 hours. In the case of mouse primary antibodies, fixed sections were blocked with blocking solution from the HistoMouse plus kit (Zymed, South San Francisco, CA, USA) following the manufacturer’s instructions. Each section was then incubated with the biotinylated secondary antibody for 30 minutes and subsequently with horseradish peroxidase-labeled streptavidin or FITC-labeled streptavidin for 20 minutes. To detect horseradish peroxidase activity, endogenous peroxides were quenched with 0.03% hydrogen peroxide. Fluorescence was examined by the laser scanning confocal imaging system (MRC-1024, Bio-Rad Laboratories).
Measurements and in Situ Detection of Superoxide
Superoxide levels in muscles were measured by the lucigenin-enhanced chemiluminescence method with the use of 10 µmol/L lucigenin. In situ dihydroethidium fluorescence was performed according to the method described previously.26 Briefly, the unfixed frozen tissues were cut into 10-µm-thick sections, and incubated with 2 x 10-6 mol/L dihydroethidium at 37°C for 30 minutes in a light-protected humidified chamber. The images were obtained by the laser scanning confocal imaging system (MRC-1024, Bio-Rad).
Evaluation of Vascular Permeability
EBD, which binds to plasma proteins, was used as a tracer for extravascular protein leakage.27 At 60 or 240 minutes after reperfusion, EBD (30 mg/kg) was injected through the tail vein under anesthesia. Five minutes after EBD administration, the peripheral vascular bed was perfused with 0.9% NaCl containing heparin (10 units/ml) followed by 4% paraformaldehyde in 0.05 mol/ml citrate buffer (pH 3.5). The gastrocnemius muscle was removed, rinsed in ice cold phosphate-buffered solution, gently blotted, and weighed. Half of the tissue was dried by incubation at 60°C for 48 hours, and reweighed. The remaining tissue was incubated in 1 ml of formamide at room temperature for 48 hours to extract EBD. EBD contents in tissues were quantified by measuring the optical density of the formamide extract at the wavelength of 620 nanometer and adjusted with tissue weight. Vascular permeability was expressed as a ratio of EBD extravasation in the ischemic muscle to that in the non-ischemic contralateral muscle.
Myeloperoxidase Assay
Myeloperoxidase (MPO) activity in skeletal muscle was examined on the basis of the method described previously.21,28 In brief, the excised skeletal muscle was rinsed with phosphate-buffered saline, blotted, and weighed. The muscle was homogenized in 50 mmol/L potassium-phosphate-buffered solution (pH 6.0) containing 0.5% hexadecyltrimethylammonium bromide (HTAB). After centrifuging at 25,000 x g for 15 minutes at 4°C, the supernatant was collected. The pellet was resuspended in 1 ml of potassium-phosphate buffered solution containing HTAB, and sonicated for 30 seconds. The suspension was centrifuged again at 25,000 x g for 15 minutes at 4°C, and the supernatant was collected. This extraction procedure was repeated three times. The supernatant collected in the above procedure was mixed 1:30 (v/v) with 50 mmol/L potassium-phosphate-buffered solution containing 0.167 mg/ml o-dianisidine dihydrochloride and 0.0005% hydrogen peroxide. Then, the absorbance was measured at 460 nm for 5 minutes. MPO activity, calculated by a change in the absorbance for 5 minutes, was adjusted with the protein content, and shown as delta absorbance (ABS) per minutes per milligram of protein.
Quantitative Analysis of Tissue Viability (WST-1 Assay)
To quantify tissue viability, mitochondrial dehydrogenase activity was evaluated by assaying its cleaving activity of the tetrazolium salt WST-1 (Roche Molecular Biochemicals, Basel, Switzerland), with modifying the method described by Belkin.29 Briefly, the ischemia-reperfused gastrocnemius muscle and the contralateral non-injured muscle as a control were homogenized in 500 µl of 0.25 mol/L sucrose. After centrifugation at 3,000 x g for 5 minutes, 100 µl of supernatant was mixed with 10 µl of WST-1 reagent in microtiter plates. After shaking thoroughly for 1 minute, the reaction mixture was incubated under a humidified atmosphere (37°C, 5% CO2). The absorbance of the red colored formazan dye cleaved from WST-1 was measured at 450 nm by the spectrophotometer U2000 (Hitachi). Reactions were performed in triplicate. The absorbance was adjusted with the protein content, and the result was expressed as a ratio of the reduced WST-1 activity in the ischemic muscle to that in the contralateral muscle.
Statistical Analysis
Data were expressed as mean ± SEM. Unpaired Student’s t-test was used to compare the protein levels of eNOS, NOS activity, and NO release in the skeletal muscles between WT and eNOS-Tg mice. For other data, two-way analysis of variance was used to compare the differences between two groups with Bonferroni’s test for post hoc analysis. Values of P <0.05 were considered statistically significant.
|
Results |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Expression of eNOS in hindlimb skeletal muscle was analyzed by
both immunoblotting and immunohistochemistry. As shown in Figure 1A and B
, the protein expression of eNOS
in skeletal muscles was significantly increased in eNOS-Tg mice
compared to WT mice. The Ca2+-dependent NOS
activity in skeletal muscle of eNOS-Tg mice was also 1.5-fold higher
than that of WT mice (Figure 1C)
. In addition, the
acetylcholine-stimulated NO release in skeletal muscle of eNOS-Tg mice
was approximately twofold higher than that of WT mice (24.7 ± 1.5
vs. 12.8 ± 5.7 pmol/mg protein/hour respectively; P
<0.05; Figure 1D
). Immunohistochemistry showed that eNOS expression in
endothelial cells of arterioles, capillary microvessels, and
postcapillary venules was higher in eNOS-Tg mice than in WT mice
(Figure 2)
.
|
|
Reoxygenation by I/R is known to enhance oxidant production and
exaggerate reperfusion tissue injury.2
As shown in Figure 3
a and c, I/R caused a significant
increase in in situ detection of superoxide with
dihydroethidium fluorescence in the ischemic limb of WT mice after 60
minutes of reperfusion. This increase was markedly suppressed in the
affected limbs of eNOS-Tg mice (Figure 3, b and e)
. In contrast, L-NAME
treatment increased the intensity of dihydroethidium fluorescence in
eNOS-Tg mice after reperfusion, showing the reversed effect of NOS
inhibitor on superoxide production (Figure 3, c and f)
. At 240 minutes
after reperfusion, similar results were obtained (data not shown). As
revealed also by lucigenin-enhanced chemiluminescence method,
superoxide levels in the ischemic muscles of WT mice were increased by
twofold of the non-injured control muscles during reperfusion (Figure 4)
. In eNOS-Tg mice, however, the
superoxide levels were significantly lower than in WT mice at both
reperfusion time points. On the other hand, in L-NAME-treated eNOS-Tg
mice, superoxide production in the reperfused muscles were comparable
to the levels in WT mice at 60 minutes, and increased at 240 minutes
after reperfusion.
|
|
It is established that endothelial cells form cell-cell junctions
composed of VE-cadherin-based adherens junction and tight junction to
maintain vascular integrity, including the regulation of vascular
permeability.6,30
As adherens junction assembly is
considered to be essential for the formation of tight
junction,6
we studied the effects of I/R on endothelial
adherens junction assembly by immunofluorescence of key components such
as VE-cadherin and ß-catenin which interacts with cytoplasmic tail of
VE-cadherin.6
After reperfusion for 60 minutes following
ischemia for 30 minutes, VE-cadherin and ß-catenin were hardly
detectable at cell-cell contact sites of endothelial cells in the
reperfused vessels in WT mice (Figure 5
c
and i). In contrast, this disappearance of adherens junction components
did not occur in eNOS-Tg mice at the same time point (Figure 5, d and j)
. After reperfusion for 240 minutes, VE-cadherin and ß-catenin were
seen at cell-cell contacts of endothelial cells in the reperfused
vessels in both genotypes (Figure 5, e, f, k, and l)
. In eNOS-Tg mice
treated with L-NAME, the similar findings seen in WT mice were observed
(data not shown). Thus, it seems that the reorganization of endothelial
cell-cell junctions occurs at the earlier phase of reperfusion in WT
mice, which is prevented by overexpression of eNOS. In the later phase
of reperfusion, the assembly of endothelial junctions appears to be
maintained in both genotypes.
|
. In contrast, the
ischemia-reperfused muscle in eNOS-Tg mice showed no significant
increase in the EBD extravasation at 60 minutes after reperfusion. In
L-NAME-treated eNOS-Tg mice, the inhibitory effect of eNOS
overexpression on the EBD extravasation was completely cancelled. In
the later phase of reperfusion (at 240 minutes after reperfusion), the
EBD extravasation in the ischemia-reperfused muscle in the three groups
was not significantly different from that of non-ischemic muscle
(Figure 6)
. Thus, in accordance with the reorganization of endothelial
cell-cell junctions (Figure 5)
, vascular permeability in
vivo appears to increase in the earlier phase of reperfusion, and
this increase was attenuated by eNOS overexpression.
|
There is accumulating evidence that exaggerated trafficking of
leukocytes in postischemic venules is one of important pathogenic
factors in I/R tissue injury.1,2
Leukocyte extravasation
is mediated by leukocyte-endothelial interactions via adhesion
molecules expressed on endothelial cells such as ICAM-1 and
VCAM-1.2
Therefore, the expression of ICAM-1 and VCAM-1 in
postischemic microvessels was examined by immunohistochemistry. In
non-ischemic control muscle of both genotypes, ICAM-1 and VCAM-1 were
hardly detectable in microvessels (Figure 7
b, d, f, and h). After 240 minutes, but
not 60 minutes, of reperfusion, the immunoreactivities of both ICAM-1
and VCAM-1 were increased in the reperfused microvessels of WT mice
(Figure 7, a and e)
, whereas this increase of ICAM-1 or VCAM-1
expression did not occur in eNOS-Tg mice (Figure 7, c and g)
. On the
other hand, the up-regulated expression of these adhesion molecules was
observed in L-NAME-treated eNOS-Tg mice (data not shown).
|
. However, at 240 minutes after
reperfusion, MPO activity in the affected limb of WT mice was
remarkably increased, when compared to that in the non-ischemic
contralateral muscle (Figure 8)
. In contrast, the increase in MPO
activity was significantly repressed in eNOS-Tg mice. L-NAME treatment
for eNOS-Tg mice increased MPO activity to the level comparable to that
in WT mice. These results indicate that eNOS overexpression suppresses
expression of ICAM-1 and VCAM-1 leading to leukocyte infiltration in
the later phase of reperfusion.
|
Tissue injury triggers disruption of mitochondria and subsequent
depletion of essential reduction-oxidation coenzymes like mitochondrial
dehydrogenase.31
To evaluate the effects of I/R on tissue
viability, mitochondrial dehydrogenase activity was quantified by
spectrophotometrically measuring its cleaving activity of tetrazolium
salt WST-1 to formazan. After 30 minutes of ischemia, mitochondrial
dehydrogenase activity of limb muscle was declined to about 80% of
non-ischemic control limb muscle in all groups (Figure 9)
. This observation indicates that
ischemia for 30 minutes produced tissue injury to similar extents among
three groups and that eNOS overexpression did not affect the
ischemia-induced tissue injury.
|
. In eNOS-Tg mice, however, the
decrease in mitochondrial dehydrogenase activity in the affected limb
muscle was significantly attenuated and hardly detectable (Figure 9)
.
In L-NAME-treated eNOS-Tg mice, as in WT mice, the mitochondrial
dehydrogenase activity progressively declined after reperfusion. These
results suggest that eNOS overexpression is protective against
I/R-induced tissue injury in mice skeletal muscle. |
Discussion |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
. In eNOS-Tg mice, but not L-NAME-treated eNOS-Tg mice,
however, superoxide levels in the postischemic muscles were not
enhanced in the reperfusion phase. In situ detection of
superoxide supports these observations (Figure 3)
. The superoxide
levels detected by the method used in this study may not provide the
accurate quantity of endogenous superoxide generation because of the
methodological limitation as previously pointed out.32,33
However, the decreased levels of superoxide detected in the affected
tissues of eNOS-Tg mice were completely reversed by L-NAME treatment
(Figure 4)
. Therefore, our present findings reveal that eNOS
overexpression decreases superoxide levels in the reperfused skeletal
muscles.
Vascular permeability, a key component of vascular integrity, is
regulated mainly by endothelial cell-cell adhesion maintained by
cell-cell junctions.4-6
We found transient and reversible
disassembly of endothelial junctions in the earlier phase of
reperfusion in our I/R model, which showed a good correlation with an
increase in vascular plasma leakage observed in a similar time course
(Figures 5 and 6)
. eNOS overexpression seems to prevent the
reorganization of endothelial junctions leading to an increase in
vascular plasma leakage. Our findings demonstrate a novel function of
NO in the maintenance of endothelial cell-cell adhesion in
vivo. On the other hand, NO is shown to mediate increases in
vascular permeability, particularly in vascular endothelial growth
factor-induced vascular permeability.34,35
In some animal
models of I/R, NO is also shown to promote vascular leakage after
I/R.36,37
The discrepant results may be related to the
differences in disease models, including duration and severity of
ischemia, targeted organs examined, or animal species. However, most of
those studies were performed by using NO donors or NOS inhibitors,
which did not allow the direct assessment of NO derived from eNOS on
vascular permeability. In the present study, we examined, for the first
time, the role of eNOS-derived NO on vascular leakage elicited by I/R
in skeletal muscle with the use of eNOS gene-engineered mice. Our study
may serve to elucidate the discrepancy on the role of NO on I/R-induced
vascular leakage.
I/R-induced increases in vascular permeability have been linked to
pathological events such as oxidant stress38
and
interaction between leukocytes and endothelial cells.7,8
Activated leukocytes and injured endothelial cells generate enormous
amounts of reactive oxygen species, and adhesion of leukocyte to the
vessel wall is proposed to trigger the intracellular signals that
disorganize VE-cadherin/ß-catenin complex in endothelial cells and
increase leukocyte extravasation.7
In eNOS-Tg mice,
superoxide levels in the postischemic tissues were consistently reduced
throughout the reperfusion periods, and eNOS overexpression inhibited
both the expression of adhesion molecules and the concomitant leukocyte
infiltration in the reperfused tissues (Figures 7 and 8)
. These results
suggest that the overproduced NO derived from eNOS maintains the
microvascular integrity by decomposing superoxide as well as abrogating
leukocyte adhesion to the endothelial cells through inhibition of
adhesion molecule expression.
A possibility that the reduced perfusion pressure in postcapillary vessels, due to systemic hypotension in eNOS-Tg mice,20 contributes to the attenuation of vascular protein leakage is to be argued. EBD extravasation levels in non-ischemic muscles were, however, similar between WT and eNOS-Tg mice. Also, in L-NAME-treated eNOS-Tg mice, where systemic blood pressure is increased by 10 mmHg compared to WT mice,20 EBD extravasation levels in the reperfused tissues were similar to WT mice. Therefore, the reduced perfusion pressure is unlikely to be responsible for the attenuated EBD extravasation.
Subsequently, we analyzed viability of ischemia-reperfused muscle to
evaluate the effects of NO on I/R injury. The extent of tissue injury
induced by short-time ischemia was similar between WT and eNOS-Tg mice,
however the reperfusion-induced aggravation of tissue injury was
prevented by eNOS overexpression (Figure 9)
. Because of the lack of
definitive evidence on roles of endogenous NO from eNOS in I/R injury,
genetically engineered eNOS-Tg mice are useful to study in
vivo pathophysiological roles of NO. In this study, the relative
short duration of ischemia was chosen because mice are shown to be more
susceptible to I/R injury compared with other species such as rats,
rabbits, and humans.16
The differences in susceptibility
to tissue injury among species are an important issue for data
interpretation. To apply the data obtained from our I/R model to
clinical situations, further studies are required.
In contrast to our study, there are reports implicating NO as a mediator of reperfusion injury in various tissues. In a variety of I/R model, NO derived from inducible NOS (iNOS) is shown to react with superoxide and generates peroxynitrite, resulting in the reperfusion injury.15,16 Considerable amounts of NO derived from neuronal NOS (nNOS) are also suggested to contribute to I/R injury in brain via peroxynitrite formation or induction of intracellular Ca2+ overload in neuronal cells.39 In these studies, reperfusion injury was protected by NOS inhibitors via inactivation of iNOS or nNOS, which decreases NO production and inhibits peroxynitrite generation. On the other hand, the effects of NOS inhibitors on I/R injury are suggested to diversify depending on the doses and timing of drug administration.40 We found that L-NAME treatment cancelled the beneficial effects of eNOS overexpression and rather worsened the reperfusion injury. In the present experimental model, the detrimental effects of L-NAME support the idea that eNOS overexpression serves to prevent I/R injury.
Our study demonstrates that overexpression of eNOS in the endothelium could attenuate reperfusion injury after short-time ischemia in mice skeletal muscle by decreasing superoxide production, preventing protein leakage, and abrogating leukocyte-endothelial interactions. Up-regulation of eNOS or augmentation of eNOS-derived NO production is indicated to be beneficial in the treatment of I/R injury. The present findings that eNOS overexpression maintains vascular integrity in I/R could contribute to identify a novel therapeutic approach for I/R injury.
|
Acknowledgements |
|---|
|
Footnotes |
|---|
Supported by the Ministry of Education, Science, Sports and Culture of Japan (10470165) and the Japan Heart Foundation/Pfizer Grant for Research on Hypertension and Vascular Metabolism.
Accepted for publication January 4, 2002.
|
References |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
-nitro-L-arginine methyl ester and dexamethasone. Eur J Pharmacol 1997, 332:273-278[Medline]
Related articles in Am J Pathol:
This article has been cited by other articles:
 |
|
 |
|
  H. T. Hua, H. Albadawi, F. Entabi, M. Conrad, M. C. Stoner, B. T. Meriam, R. Sroufe, S. Houser, G. M. LaMuraglia, and M. T. Watkins Polyadenosine Diphosphate-Ribose Polymerase Inhibition Modulates Skeletal Muscle Injury Following Ischemia Reperfusion Arch Surg, April 1, 2005; 140(4): 344 - 351. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. K. Walsh, H. Huang, Z. Wang, J. Williams, R. de Crom, R. van Haperen, C. I. Thompson, D. J. Lefer, and T. H. Hintze Control of myocardial oxygen consumption in transgenic mice overexpressing vascular eNOS Am J Physiol Heart Circ Physiol, November 1, 2004; 287(5): H2115 - H2121. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. R. Tejedo, G. M. Cahuana, R. Ramirez, M. Esbert, J. Jimenez, F. Sobrino, and F. J. Bedoya Nitric Oxide Triggers the Phosphatidylinositol 3-Kinase/Akt Survival Pathway in Insulin-Producing RINm5F Cells by Arousing Src to Activate Insulin Receptor Substrate-1 Endocrinology, May 1, 2004; 145(5): 2319 - 2327. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. P. Jones, J. J. M. Greer, A. K. Kakkar, P. D. Ware, R. H. Turnage, M. Hicks, R. van Haperen, R. de Crom, S. Kawashima, M. Yokoyama, et al. Endothelial nitric oxide synthase overexpression attenuates myocardial reperfusion injury Am J Physiol Heart Circ Physiol, January 1, 2004; 286(1): H276 - H282. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. P. Chung, M. M. Monick, N. Y. Hamzeh, N. S. Butler, L. S. Powers, and G. W. Hunninghake Role of Repeated Lung Injury and Genetic Background in Bleomycin-Induced Fibrosis Am. J. Respir. Cell Mol. Biol., September 1, 2003; 29(3): 375 - 380. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
P <0.05 vs. the value
for WT mice and L-NAME-treated eNOS-Tg mice at 60 or 240 minutes after
reperfusion, respectively.
,
P <0.05 vs. the
value for WT mice or L-NAME-treated eNOS-Tg mice at 240 minutes after
reperfusion, respectively.