(Circulation. 1997;95:2441-2447.)
© 1997 American Heart Association, Inc.
Articles |
In Vivo Gene Transfection of Human Endothelial Cell Nitric Oxide Synthase in Cardiomyocytes Causes Apoptosis-Like Cell Death
Identification Using Sendai Virus–Coated Liposomes
From the Second Department of Internal Medicine, Health Service Center, University of Tokyo, Tokyo, and the Institute for Molecular and Cellular Biology (Y.K.), Osaka University, Osaka, Japan.
Correspondence and reprint requests to Professor T. Toyo-oka, The Second Department of Internal Medicine, Tokyo University Hospital, University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113, Japan.
 |
Abstract |
|---|
 Top Abstract IntroductionMethodsResultsDiscussionReferences |
|---|
Background Nitric oxide (NO) has various actions on the cardiovascular system, although its pathophysiological significance in myocardial cells remains obscure. The aim of the present study was to identify direct NO actions on cardiomyocytes by gene transfection in vivo using a newly developed vector under physiological conditions.
Methods and Results Liposomes containing the
ß-galactosidase (ß-gal) gene alone or with the human
endothelial cell nitric oxide synthase (ecNOS) gene
were coated with UV-inactivated Sendai virus and injected
into the left ventricular wall of rat heart in vivo.
Histological examination confirmed that the
transfection efficiency was comparable to adenovirus-mediated
transfection and that the new vector per se caused no inflammation.
ß-Gal expression was confined to cardiomyocytes between
two intercalated discs, suggesting that the transfected gene did not
permeate the discs. An immunohistochemical study showed that
cotransfection of the ecNOS gene induced massive myocardial cell
shrinkage in both transfected cells and the adjacent myocytes in a
time- and dose-dependent manner. Histochemical findings in shrunk cells
coincided with apoptosis as identified by terminal
deoxynucleotidyl transferase–mediated dUTP-biotin
nick-end labeling. Electron microscopy of the lesion revealed
myofibrillar degradation and accumulation of mitochondria but no
apoptotic bodies. Pretreatment with the NOS
inhibitor N
-nitro-L-arginine
methyl ester abolished these morphological alterations.
Conclusions The efficient expression of the human ecNOS gene in vivo suggests that NO or its toxic metabolite caused myocardial degradation, a part of which was compatible with apoptosis of the transfected cardiomyocytes themselves and the adjacent cells as a paracrine effect. These morphological features mimicked acute myocarditis or ischemic injury.
Key Words: genes • endothelium-derived factors • immunohistochemistry • myocardium • molecular biology
|
Introduction |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Nitric oxide has a variety of actions on the cardiovascular system: vessel relaxation, inhibition of the proliferation of smooth muscle cells and endothelial cells, and suppression of platelet adhesion.1 2 The recent report of Balligand et al3 that ecNOS protein is constitutively expressed not only in coronary endothelial cells but in myocardial cells suggests autocrine and paracrine effects on cardiomyocytes, as is the case in vascular cells.4 5 Compared with NO released from activated macrophages, which might contribute to the pathogenesis of viral or bacterial myocarditis or septic shock,1 2 the pathophysiological significance of NO produced from myocardial cells is still obscure.
In general, overexpression or knockout of a specific gene in myocardial tissue is of great use for the accurate elucidation of the actual function of its corresponding product. In a physiological setting, transmural pressure, passive stretching during diastole, and local coronary flow in the presence of several cytokines, which might modify gene expression, are preserved. Accordingly, local modulation of gene expression would be very significant for evaluation of the gene product.
In this study, the human ecNOS gene was successfully transfected by using Sendai virus–coated liposomes. This vector caused less inflammation than the adenovirus-mediated methods previously reported.6 7 Massive necrosis of myocardial cells, a part of which satisfied the criteria of apoptosis, is also described.
|
Methods |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Plasmid Construction and Design of Vectors
The ß-gal gene with a CMV promoter at an Xba I site and SV40 poly A signal at a BamHI site was inserted into a pBluescript II KS+ plasmid (Stratagene). A 4.1-kb Xba I fragment of human ecNOS cDNA8 was inserted into a plasmid containing the CMV promoter with SV40 poly A signal (pcDNA 3, InVitrogen) at an EcoRI site.
Sendai virus (hemagglutinating virus of Japan)–coated liposomes were prepared.9 10 Nuclear protein (high-mobility group 1, 65 µg) and histochemical marker (ß-gal) gene alone (200 µg) or with the ecNOS gene (200 µg) were mixed and incubated for 1 hour at room temperature. The liposomes were then vortexed and sonicated with a lipid mixture (phosphatidylcholine, cholesterol, and phosphatidylserine) and incubated with UV-inactivated Sendai virus for 1 hour at 37°C.
Gene Transfer In Vivo
After anesthetizing 300-g male Wistar rats (n=28) with
pentobarbital sodium (25 mg/kg IP), the Sendai virus–coated liposomes
were percutaneously injected into the left
ventricular wall under the guidance of two-dimensional
echocardiography. To examine the dose dependency of
ecNOS gene transfection on histology, a high (50 µg) or low (5 µg)
dose of ecNOS gene was administered.
NOS Inhibition by L-NAME and Its Verification
L-NAME was administered to rats in drinking water (12 mg·kg
body wt-1·d-1,
100 mg/L) for 7 days before the transfection11 to clarify
the effects of L-NAME on NOS inhibition. After transfection, L-NAME
treatment was continued in both the control (ß-gal transfection
alone) and NOS gene (NOS plus ß-gal transfection) groups for an
additional 7 days. NOS inhibition was
physiologically verified by measuring systemic
pressure in the tail artery by sphygmomanometry and
histologically examined by inspecting the medial
thickening of coronary arteries.12
Light and Electron Microscopy
Rats were killed on days 3, 7, 10, and 14 (n=4 in each group)
after the in vivo transfection. The hearts were excised, fixed in 2%
paraformaldehyde in PBS for 4 hours at 4°C and sliced
(2-mm thickness). After X-gal staining,13 the extent of
ß-gal expression was measured by obtaining the ratio of the
X-gal–stained area to the cross-sectional area of the left
ventricular wall. Paraffin-embedded 4-µm-thick sections
were then prepared for light microscopic assessment.
Other tissues fixed in PBS containing 2.5% glutaraldehyde and 2% paraformaldehyde for 12 hours at 4°C were postfixed in 2% osmium tetroxide for 2 hours at 4°C, embedded in epoxy resin 812, sectioned to 60-nm thickness, and stained with uranyl acetate for 15 minutes and lead citrate for 20 seconds at room temperature. Electron microscopy was performed with a Hitachi H 7000 electron microscope.
Immunohistochemical Assessment of Gene Expression and
Macrophage Infiltration
Cryostat sections (6 µm thick) of the hearts removed on
day 7 after the transfection were fixed with 2%
paraformaldehyde in PBS for 10 minutes.
Endogenous peroxidase activity was quenched with 2%
hydrogen peroxide in 60% methyl alcohol for 30 minutes at room
temperature. The specimen was then permeabilized with
0.1% Triton X-100 in PBS for 20 minutes and incubated with either
monoclonal antibody specific to ecNOS protein (5 µg/mL, Transduction
Laboratories) or monoclonal antibody specific to rat macrophage
(2 µg/mL, Serotec) overnight at 4°C. These sections were incubated
with biotinylated rabbit anti-mouse IgG for 30 minutes at room
temperature, and immunoproducts were visualized by using the
avidin-biotin complex method (ABC kit, Vectastain). After rinsing, the
slides were counterstained with Mayer's hematoxylin solution and
mounted for light microscopy.
Assessment of Apoptosis
DNA was extracted from the transfected heart, and
electrophoresis was performed in agarose gel to detect the
ladder.14 For the in situ detection of apoptosis,
cryostat sections fixed in 10% paraformaldehyde
solution for 10 minutes at room temperature were treated according to
the instructions for the apoptosis detection kit (ApopTag Plus,
Oncor), which is a modification of the TUNEL method.15
Briefly, after endogenous peroxidase was quenched with 2% hydrogen peroxide in PBS for 5 minutes at room temperature, specimens were incubated with terminal deoxynucleotidyl transferase enzyme in a humidified chamber for 1 hour at 37°C and then anti–digoxigenin peroxidase for 30 minutes at room temperature. They were then stained with diaminobenzidine substrate for 3 minutes at room temperature and counterstained in Mayer's hematoxylin solution for 1 minute at room temperature.
Statistical Analysis
For morphometry, pictures of five or six vision fields were
taken at x400 magnification; observation fields were restricted to the
area where the ß-gal expression was evident in X-gal staining. Values
are expressed as mean±SE. The results were considered significant if
P<.05. Statistical significance was estimated among the
various groups by using one-way ANOVA. Group-to-group comparisons were
conducted by using Student's t test.
|
Results |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Efficiency of Gene Transfection by Sendai Virus–Coated Liposomes
The expression of ß-gal was clearly detected in the cross section of the transfected hearts (Fig 1
). Enzyme
activity was visualized as blue color in the left
ventricular wall in all hearts transfected with the ß-gal
gene alone or cotransfected with ecNOS genes. ß-Gal expression was
detected 3 through 14 days after transfection (Fig 2).
It peaked on day 7, when the extent of ß-gal expression reached
11.6±1.1% (n=4) in cross section (Fig 2).
|
|
In contrast, expression after the administration of plasmid DNA alone
without Sendai virus–coated liposomes attained only 1.1±0.1% (n=4,
P=.0003 versus virus-coated liposome vector; Fig 2
).
Cotransfection of the ecNOS gene did not affect the extent of ß-gal
expression (11.6±1.7%, n=4; Figs 1 and 2). However, when animals were
treated with L-NAME to examine the effect of NOS inhibition, ß-gal
expression decreased even when Sendai virus–coated liposome vectors
were used in the cotransfection with ecNOS (3.4±0.4%, n=4,
P=.0022 versus without treatment; Fig 2) or ß-gal
transfection alone (3.6±0.2%, n=4). It is unclear why ß-gal
expression decreased with L-NAME treatment. Changes in systemic or
coronary pressure and circulation with L-NAME treatment may
influence ß-gal gene expression.
Histological and Physiological
Findings of ß-Gal Expression and ecNOS Transfection With or Without
L-NAME Treatment
Microscopic examination revealed characteristic features of gene
expression after transfection (Fig 3). ß-Gal activity
was detected throughout the entire myoplasm of the transfected cells
and was clearly distinguished by an intercalated disc from adjacent
cells where ß-gal was not expressed. Infiltration of white blood
cells in the transfected portion was slight, and the inflammatory
process due to vector administration was much less than for the
adenovirus-mediated method (Fig 3A).6 7
|
Furthermore, ecNOS gene transfection together with the ß-gal gene
demonstrated markedly different findings from the marker gene
transfection alone (Fig 3B and 3C). At the low dose (5 µg) of the
ecNOS gene no or minimal lesions were documented (Fig 3B), but at the
high dose (50 µg) a clear border was delineated between the intact
cardiac tissue and the injured area (Fig 3C). The lesion corresponded
to the transfected area of the ecNOS gene because ß-gal staining was
restricted to that lesion (Fig 3B and 3C). The sizes of most cells
in the transfected area were greatly reduced, and the myoplasm of
the degenerated cells was much decreased (Fig 3C).
Small cells in the lesion could be classified into four types according
to the shape (elongated or round) and stain (blue or not stained).
Elongated cells stained blue were scattered throughout the necrotic
lesion (arrows in Fig 3), while elongated, unstained cells were
detected in the periphery (arrowheads in Fig 3B and 3C). These
elongated cells may have originated from denatured myocardial cells,
while the round cells might be infiltrating macrophages, as
identified later. For ecNOS gene transfection alone without the marker
(ß-gal) gene, the injured area had the same changes as observed for
cotransfection (Fig 3D).
The pathological alterations in the NOS gene group were markedly
reduced with L-NAME treatment (Fig 3E) because the number of shrunk
cells decreased to the same level as the control. The
cardiomyocytes transfected with ß-gal preserved their
original histological structure. These findings suggest
that the pathological degeneration after ecNOS gene transfection was
due to NO or its metabolites. The medial thickening of coronary
arteries was not histologically obvious in the
L-NAME–treated group, probably because the dose and period were
smaller than in the previous report.12 Accordingly, these
drastic alterations in the transfected lesion would be due to the ecNOS
gene and not the ß-gal gene.
L-NAME treatment confirmed the NOS inhibitory effect since the systolic pressures of rats increased from 117±3 to 158±3 mm Hg (n=4, P<.01) in the control group (ß-gal transfection alone) and from 120±2 to 160±2 mm Hg (n=4, P<.01) in the NOS gene group (NOS plus ß-gal transfection).
Characterization of the Transfected Lesion
Immunohistochemical staining with antibody specific to ecNOS
demonstrated endogenous NOS protein on myocardial
cells3 as well as endothelial cells in
coronary arteries (Fig 4A). In overtransfected
myocardial cells, additional staining was visualized as thick staining.
Cotransfection of the ecNOS and ß-gal genes resulted in the mixed
color of brown (immune complex with ecNOS protein) and blue (ß-gal
activity; arrows in Fig 4A). Small elongated cells without blue
staining (arrowheads in Fig 4A) were also documented at the border
between the degraded lesion and normal myocardial cells. Occasionally,
either ß-gal activity or ecNOS protein was clearly detected on
normal-sized myocardial cells, and sarcomeres were clearly visible in
these cells. This might be due to the selective transfection of one of
the two genes in each myocardial cell.
|
The infiltration of a large number of macrophages was detected
with specific antibody in the injured area, indicating that a portion
of the round small cells found in the transfected lesion (Fig 4B)
represent invading leukocytes and macrophages.
Recent studies have indicated that apoptosis occurs with cell
necrosis.16 End labeling of fragmented DNA with the TUNEL
method demonstrated that 0.5% of the nuclei in small cells in the
degenerated lesion were positive for the apoptotic reaction in
situ, whereas none of the control myocardial cells were positive (Fig 4C). All cells that were positively stained shrank in volume.
Azan staining revealed deposition of collagen fibers in the necrotic
lesion (Fig 4D), suggesting that the degenerative process after the
ecNOS gene transfection was accompanied by fibrosis. The pathological
findings of leukocyte invasion and fibrosis were similar to those of
the inflammatory lesion in myocardial infarction at the subacute
stage or myocarditis of viral or bacterial origin.
Electron Microscopic Assessment
Electron microscopy of the untransfected area showed no
pathological deterioration, and myofibrillar array and mitochondrial
structure were both well preserved (Fig 5A). However, in
the transfected area, mitochondrial accumulation and swelling were
found between thin myofibrils (Fig 5B). The mean numbers of these
mitochondria in the transfected and untransfected areas, respectively,
were 95±7 and 82±5 per 100 µm2
(P<.05). The mean diameter of these mitochondria in the
transfected area was 1.16±0.04 µm, compared with
0.91±0.04 µm (P<.01) in the untransfected area. The
mitochondria were round or elliptical in the untransfected area; some
of the mitochondria between the sparse myofibrils in the transfected
area were deformed and shaped like confetti.
|
In addition to these mitochondrial abnormalities, various changes
within the myocytes were observed, including intracellular edema,
depletion of glycogen granules, and sparse myofilament arrays (Fig 5C).
However, the integrity of the cytoplasmic membrane was preserved, with
both apoptotic bodies and chromatin condensation being absent
(Fig 5C). As described in the light microscopic study (Fig 3A), a clear
contrast bordered by an intercalated disc was observed in two myocytes
(Fig 5D). In other words, most of the mitochondria were destroyed on
one side of the intercalated disc.
|
Discussion |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
We have made five major observations. First, in contrast to adenovirus vector,6 7 the newly developed Sendai virus–coated liposomes were very useful for the efficient transfection of gene in vivo with little associated inflammation. Second, both the reporter gene ß-gal and the physiologically significant gene ecNOS were transfected to cardiomyocytes in living animals. Third, expression of the ß-gal gene was limited to the cardiomyocytes between two intercalated discs, providing a clear contrast with adjacent cells. Fourth, overexpression of the ecNOS gene caused unique cell degeneration, including a reduction in myoplasm volume, mitochondrial accumulation, collagen deposition, and macrophage infiltration. Finally, these morphological alterations were ameliorated with L-NAME pretreatment.
Biological Characteristics of Sendai Virus–Coated Liposome
Vector
To elucidate the specific function of an expressed gene, gene
transfer is superior to transgenic animals for the following reasons.
When the gene product is systemically expressed, it may obscure the
local function. This is especially true when the gene product is
essential for maintaining cell viability or the overexpressed
product is lethal to the animal. Local expression or knockout of a
specific gene is of great significance for identifying the
physiological function of the gene product in
situ. In this setting, the vector should not cause a secondary
effect.
Adenovirus vector is not appropriate for this purpose because the
vector itself causes local inflammation.6 7 Sendai
virus–coated liposomes were vastly superior to adenovirus-mediated
transfection because Sendai virus has no pathogenicity in either humans
or rodents. UV-irradiation of Sendai virus is beneficial for preventing
infection in other species, and the process does not weaken the
transfection efficiency of the liposomes. Local administration of the
newly developed vector in vivo induced no harmful action other than the
needle puncture (Fig 3A). Furthermore, expression of the ß-gal gene
was confined to myocytes between two intercalated discs, suggesting
that the transfected gene did not permeate the discs. Comparison of
transfected cardiomyocytes and nontransfected adjacent
cells with electron microscopy (Fig 5D) was particularly useful.
Morphological Alterations After ecNOS Gene Transfection and
Relation to Apoptosis
The classic criteria for apoptosis17 18 19
include a reduction of cytoplasm volume, which was shown in most cells
in the degraded lesion (Fig 3B and 3C), detection of apoptotic
reaction as identified by end labeling of fragmented DNA (Fig 4C), and
no disruption of the cytoplasmic or nuclear membranes of the degraded
cells as assessed by electron microscopy (Fig 5C). Other
characteristics of apoptosis not observed in the present
study include the presence of apoptotic bodies, chromatin
condensation, and a DNA ladder after gel electrophoresis. Thus, the
present data satisfied some but not all of the features of typical
apoptosis.
Pathological examination also demonstrated an inflammatory process in
the transfected myocardium. Double staining of ß-gal
activity and ecNOS protein (Fig 4A) or macrophages (Fig 4B)
using specific antibodies, TUNEL analysis of apoptosis
(Fig 4C), and azan staining (Fig 4D) revealed coexisting myocardial
cell necrosis, apoptosis, and lymphoplasmacytic infiltration in
the transfected myocardium. Pinsky et al20
have indicated that NO production in vitro from
activated macrophages causes cytotoxic action on
myocardial cells in tissue culture. Their results, together with the
present findings, suggest that an inflammatory process does not
exclude the possibility of apoptosis.
Pathological Significance of Injured Cells
Occasional myocytes located at the periphery of the transfected
lesion showed degradation without expression of ecNOS protein (Fig 4A).
This finding may indicate that the cells transfected by the ecNOS gene
and adjacent nontransfected cells were injured by NO or its secondary
metabolites, such as peroxynitrite (ONOO-), in an
autocrine or paracrine manner, respectively. This scenario is likely
since NO is a diffusible gas and is actually excreted from NO-producing
cells.5 20
A very small number of atrophic myocytes stained positively for the
apoptotic reaction in cryostat sections in situ, whereas none
of the control myocardial cells were positive (Fig 4C). All cells that
stained positively by the TUNEL method had decreased cell volumes. It
should be emphasized that nucleus fragmentation, which is
characteristic of apoptosis, was not detected at all. The
heterogeneity of the apoptotic reaction among
the atrophic cells suggests that a stage in the degradation process is
required for apoptotic reaction; hence, the other denatured
cells could not react to TUNEL staining.
The electron microscopy results revealed mitochondrial accumulation and
swelling of the injured cells (Fig 5B) compared with control cells in
the untransfected portion of the same myocardial tissue (Fig 5A). This
indicates that mitochondria in a limited area were affected by ecNOS
gene transfection and that the overexpression of ecNOS did not cause
any morphological changes in myocytes distant from the transfected
lesion. Impairment of oxidative phosphorylation and
energy metabolism might lead to failure of the
mitochondrial cation pump, and subsequently progressive swelling before
mitochondrial rupture.21 The accumulation and swelling of
mitochondria in degenerated lesions may be a defense mechanism against
free radicals, including ONOO-, because mitochondria are
one of the main sources of toxic free radicals.22 Hibbs et
al23 report that activated macrophages
cause inhibition of mitochondrial respiration.
In addition to the mitochondrial abnormalities, other changes within
the myocytes were observed, including intracellular vacuole formation,
depletion of glycogen granules, and sparse myofilament arrays. These
findings suggest deterioration of the myofibrils and their subsequent
absorption (Fig 5C). Most damaged cells with mitochondrial
abnormalities did not exhibit typical apoptotic bodies or dense
chromatin, which are characteristic of
apoptosis.19 Although these changes satisfied some
of the oncosis criteria proposed by Majno and Joris,24 the
present findings lacked blebbing and increased permeability of the
cytoplasm membrane.
Conclusions
The findings of the present study suggest that overexpression
of ecNOS by in vivo gene transfection probably produced NO or its toxic
metabolites, which caused unique myocardial cell death. These cell
injuries fulfilled some but not all of the criteria for
apoptosis in the transfected cardiomyocytes
themselves and in the adjacent cells as a paracrine effect. It would be
attractive to assume that similar cell death may be involved in the
progression of acute myocardial infarction, viral myocarditis, the
development of cardiomyopathy, or the regression of
cardiac hypertrophy.
|
Selected Abbreviations and Acronyms |
|---|
|
|
Acknowledgments |
|---|
A portion of the present study was financially supported by the Ministry of Education, Science and Culture and the Ministry of Health and Welfare, Japan, the Research Foundation for Clinical Pharmacology and Molecular Cardiology, the Fugaku Trust Foundation for Medical Research, and the Mochida Research Foundation. The authors thank Dr James K. Liao, Division of Vascular Medicine, Brigham and Women's Hospital, Boston, Mass, for kindly donating human ecNOS gene. The assistance of Dr Satoru Fukuda, Chief Director, Laboratory of Electron Microscopy, University of Tokyo, Mari Kataoka, and Yoshitaka Nakagawa is gratefully acknowledged.
|
Footnotes |
|---|
Drs Kawaguchi and Shin contributed equally to the present study.
Received September 25, 1996; revision received December 11, 1996; accepted December 11, 1996.
|
References |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
-
Furchgott RF, Vanhoutte PM.
Endothelium-derived relaxing and contracting
factors. FASEB J.. 1989;3:2007-2018.[Abstract]
-
Vane JR, Anggard EE, Botting RM. Regulatory
functions of the vascular endothelium.
N Engl J Med. 1990;323:27-36.[Medline]
[Order article via Infotrieve]
-
Balligand JL, Kobzik L, Hant X, Kaye DM, Belhassent L,
O'Hara DS, Kelly RA, Smith TW, Michel T. Nitric oxide-dependent
parasympathetic signaling is due to activation of constitutive
endothelial (type III) nitric oxide synthase in cardiac
myocytes. J Biol Chem. 1995;270:14582-14586.
[Abstract/Free Full Text]
-
Shin WS, Sasaki T, Kato M, Hara K, Seko A, Yang WD,
Shimamoto N, Sugimoto T, Toyo-oka T. Autocrine and paracrine
effects of endothelium-derived relaxing factor on
intracellular Ca2+ of endothelial cells and
smooth muscle cells. J Biol Chem. 1992;267:20377-20382.
[Abstract/Free Full Text]
-
Wang YP, Shin WS, Kawaguchi H, Inukai M, Kato M,
Sakamoto A, Uehara Y, Miyamoto M, Shimamoto N, Korenaga R, Ando J,
Toyo-oka T. Contribution of sustained Ca2+ elevation
for nitric oxide production in endothelial
cells and subsequent modulation of Ca2+ transient in
vascular smooth muscle cells in coculture. J Biol
Chem. 1996;271:5647-5655.
[Abstract/Free Full Text]
-
Guzman RJ, Lemarchand P, Crystal RG, Epstein SE,
Finkel T. Efficient gene transfer into myocardium by
direct injection of adenovirus vectors. Circ Res. 1993;73:1202-1207.
[Abstract/Free Full Text]
-
French BA, Mazur W, Geske RS, Bolli R. Direct
in vivo gene transfer into porcine myocardium using
replication-deficient adenoviral vectors.
Circulation. 1994;90:2414-2424.
[Abstract/Free Full Text]
-
Liao JK, Shin WS, Lee WY, Clark SL. Oxidized
low-density lipoprotein decreases the expression of
endothelial nitric oxide synthase. J
Biol Chem. 1995;270:319-324.
[Abstract/Free Full Text]
-
Kaneda Y, Iwai K, Uchida T. Increased
expression of DNA cointroduced with nuclear protein in adult rat
liver. Science. 1989;243:375-378.
[Abstract/Free Full Text]
-
Kaneda Y. Virus (Sendai virus
envelopes)-mediated gene transfer. In: Celis JE, ed. Cell
Biology: A Laboratory Handbook. San Diego, Calif: Academic Press,
Inc; 1994:50-57.
-
Ujiie K, Yuen J, Hogarth L, Danziger R, Star RA.
Localization and regulation of endothelial NO synthase
mRNA expression in rat kidney. Am J Physiol. 1994;267:F296-F302.
[Abstract/Free Full Text]
-
Ito A, Egashira K, Kadokami T, Fukumoto Y, Takayanagi
Y, Nakaike R, Kuga T, Sueishi K, Shimokawa H, Takeshita A.
Chronic inhibition of endothelium-derived nitric oxide
synthesis causes coronary microvascular structural changes and
hyperreactivity to serotonin in pigs.
Circulation. 1995;92:2636-2644.
[Abstract/Free Full Text]
-
Bonnerot C, Rocancourt D, Briand P, Grimber G, Nicolas
JF. A ß-galactosidase hybrid protein targeted to nuclei as a
marker for developmental studies. Proc Natl Acad Sci
U S A. 1987;84:6795-6799.
[Abstract/Free Full Text]
-
Zekeri ZF, Quaglino D, Latham T, Lockshin RA.
Delayed internucleosomal DNA fragmentation in programmed cell
death. FASEB J.. 1993;7:470-478.[Abstract]
-
Gavrieli Y, Sherman Y, Ben-Sasson SA.
Identification of programmed cell death in situ via
specific labeling of nuclear DNA fragmentation. J Cell Biol. 1992;119:493-501.
[Abstract/Free Full Text]
-
Carson DA, Ribeiro JM. Apoptosis and
disease. Lancet. 1993;341:1251-1254.[Medline]
[Order article via Infotrieve]
-
Kerr JFR. Shrinkage necrosis: a distinct mode of
cellular death. J Pathol. 1971;105:13-20.[Medline]
[Order article via Infotrieve]
-
Kerr JFR, Wyllie AH, Currie AR.
Apoptosis: a basic biological phenomenon with wide-ranging
implications in tissue kinetics. Br J Cancer. 1972;26:239-257.[Medline]
[Order article via Infotrieve]
-
Wyllie AH, Duvall E. Cell death. In: McGee JOD,
Isaacson PG, Wright NA, eds. Oxford Textbook of
Pathology, I: Principles of Pathology. Oxford, England: Oxford
University Press; 1992:141-157.
-
Pinsky DJ, Cai B, Yang X, Rodriguez C, Sciacca RR,
Cannon PJ. The lethal effects of cytokine-induced
nitric oxide on cardiac myocytes are blocked by nitric oxide synthase
antagonism or transforming growth factor ß. J Clin
Invest. 1995;95:677-685.
-
Toner PG, Kamel HMH, Cameron S. Organelle
pathology in cell injury. In: McGee JOD, Isaacson PG, Wright NA,
eds. Oxford Textbook of Pathology, I: Principles of
Pathology. Oxford, England: Oxford University Press; 1992:157-181.
-
Turrens JF, Alexandre A, Lehninger AL.
Ubisemiquinone is the electron donor for superoxide formation by
complex III of heart mitochondria. Arch Biochem
Biophys. 1985;237:408-414.[Medline]
[Order article via Infotrieve]
-
Hibbs JB Jr, Vavrin Z, Taintor RR. L-arginine is
required for expression of the activated macrophage
effector mechanism causing selective metabolic inhibition
in target cells. J Immunol. 1987;138:550-565.[Abstract]
-
Majno G, Joris I. Apoptosis, oncosis,
and necrosis: an overview of cell death. Am J
Pathol. 1995;146:3-15.[Abstract]
This article has been cited by other articles:
 |
|
 |
|
  H. Ly, Y. Kawase, R. Yoneyama, and R. J. Hajjar Gene Therapy in the Treatment of Heart Failure Physiology, April 1, 2007; 22(2): 81 - 96. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E Belcher, J Mitchell, and T Evans Myocardial dysfunction in sepsis: no role for NO? Heart, June 1, 2002; 87(6): 507 - 509. [Full Text] [PDF]
|
|
|
|
 |
|
 T. Kawada, M. Nakazawa, S. Nakauchi, K. Yamazaki, R. Shimamoto, M. Urabe, J. Nakata, C. Hemmi, F. Masui, T. Nakajima, et al. Rescue of hereditary form of dilated cardiomyopathy by rAAV-mediated somatic gene therapy: Amelioration of morphological findings, sarcolemmal permeability, cardiac performances, and the prognosis of TO-2 hamsters PNAS, January 22, 2002; 99(2): 901 - 906. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Iwata, S. Sai, Y. Nitta, M. Chen, R. de Fries-Hallstrand, J. Dalesandro, R. Thomas, and M. D. Allen Liposome-Mediated Gene Transfection of Endothelial Nitric Oxide Synthase Reduces Endothelial Activation and Leukocyte Infiltration in Transplanted Hearts Circulation, June 5, 2001; 103(22): 2753 - 2759. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. J. Szabolcs, N. Ma, E. Athan, J. Zhong, M. Ming, R. R. Sciacca, J. Husemann, A. Albala, and P. J. Cannon Acute Cardiac Allograft Rejection in Nitric Oxide Synthase-2-/- and Nitric Oxide Synthase-2+/+ Mice : Effects of Cellular Chimeras on Myocardial Inflammation and Cardiomyocyte Damage and Apoptosis Circulation, May 22, 2001; 103(20): 2514 - 2520. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. E. McDonald, M. N. Grinman, C. M. Carthy, and K. R. Walley Endotoxin infusion in rats induces apoptotic and survival pathways in hearts Am J Physiol Heart Circ Physiol, November 1, 2000; 279(5): H2053 - H2061. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Hikiji, W. S. Shin, T. Koizumi, T. Takato, T. Susami, Y. Koizumi, Y. Okai-Matsuo, and T. Toyo-Oka Peroxynitrite production by TNF-alpha and IL-1beta : implication for suppression of osteoblastic differentiation Am J Physiol Endocrinol Metab, June 1, 2000; 278(6): E1031 - E1037. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 U. Weiland, J. Haendeler, C. Ihling, U. Albus, W. Scholz, H. Ruetten, A. M. Zeiher, and S. Dimmeler Inhibition of endogenous nitric oxide synthase potentiates ischemia-reperfusion-induced myocardial apoptosis via a caspase-3 dependent pathway Cardiovasc Res, February 1, 2000; 45(3): 671 - 678. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Mortensen, J. Skouv, D. M. Hougaard, and L.-I. Larsson Endogenous Endothelial Cell Nitric-oxide Synthase Modulates Apoptosis in Cultured Breast Cancer Cells and Is Transcriptionally Regulated by p53 J. Biol. Chem., December 31, 1999; 274(53): 37679 - 37684. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Veit, J.-P. Boissel, M. Buerke, T. Grosser, J. Meyer, and H. Darius Highly efficient liposome-mediated gene transfer of inducible nitric oxide synthase in vivo and in vitro in vascular smooth muscle cells Cardiovasc Res, August 15, 1999; 43(3): 808 - 822. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Koglin, D. J. Granville, T. Glysing-Jensen, J. S. Mudgett, C. M. Carthy, B. M. McManus, and M. E. Russell Attenuated Acute Cardiac Rejection in NOS2 -/- Recipients Correlates With Reduced Apoptosis Circulation, February 16, 1999; 99(6): 836 - 842. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. J. Ing, J. Zang, V. J. Dzau, K. A. Webster, and N. H. Bishopric Modulation of Cytokine-Induced Cardiac Myocyte Apoptosis by Nitric Oxide, Bak, and Bcl-x Circ. Res., January 22, 1999; 84(1): 21 - 33. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. Stein, T. Eschenhagen, J. Rudiger, H. Scholz, U. Forstermann, and I. Gath Increased expression of constitutive nitric oxide synthase III, but not inducible nitric oxide synthase II, in human heart failure J. Am. Coll. Cardiol., November 1, 1998; 32(5): 1179 - 1186. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Koglin, T. Glysing-Jensen, J. S. Mudgett, and M. E. Russell NOS2 Mediates Opposing Effects in Models of Acute and Chronic Cardiac Rejection : Insights from NOS2-Knockout Mice Am. J. Pathol., November 1, 1998; 153(5): 1371 - 1376. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Gawaz, F.-J. Neumann, T. Dickfeld, W. Koch, K.-L. Laugwitz, H. Adelsberger, K. Langenbrink, S. Page, D. Neumeier, A. Schomig, et al. Activated Platelets Induce Monocyte Chemotactic Protein-1 Secretion and Surface Expression of Intercellular Adhesion Molecule-1 on Endothelial Cells Circulation, September 22, 1998; 98(12): 1164 - 1171. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Jeppsson, C. Pellegrini, T. O'Brien, V. M. Miller, H. D. Tazelaar, and C. G.A. McGregor Transbronchial gene transfer of endothelial nitric oxide synthase to transplanted lungs Ann. Thorac. Surg., August 1, 1998; 66(2): 318 - 324. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. A Doggrell and L. Brown Rat models of hypertension, cardiac hypertrophy and failure Cardiovasc Res, July 1, 1998; 39(1): 89 - 105. [Full Text] [PDF]
|
|
|
|
|
|
J. Chen, Y. Wang, Y. Wang, T. Nakajima, K. Iwasawa, H. Hikiji, M. Sunamoto, D.-K. Choi, Y. Yoshida, Y. Sakaki, et al. Autocrine Action and Its Underlying Mechanism of Nitric Oxide on Intracellular Ca2+ Homeostasis in Vascular Endothelial Cells J. Biol. Chem., September 8, 2000; 275(37): 28739 - 28749. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||