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INTRODUCTION |
Fas ligand (FasL)1 is a type II membrane protein
belonging to the tumor necrosis factor
(TNF) family. It induces apoptosis in various cell types that bear the
receptor Fas (also called APO-1 or CD95) (1). FasL and Fas-deficient
mice, gld and lpr mice, respectively, develop
similar phenotypes including splenomegaly, lymphadenopathy, and the
accumulation of autoantibodies (2). These phenotypes suggest that this
receptor/ligand pair functions to regulate immune cell homeostasis.
Cells differ markedly in their sensitivity to Fas-mediated apoptosis
due in part to differences in the expression of FLIP, an inhibitor of
Fas-mediated death signaling (3). Endothelial cells express high levels
of FLIP and are relatively resistant to FasL-induced apoptosis (4-6). In contrast, cardiac myocytes will undergo apoptotic cell death when
presented with membrane-bound FasL (7). Furthermore, FLIP levels are
down-regulated in ischemic regions of the heart following reperfusion
(8), suggesting that Fas-mediated apoptosis may contribute to tissue
damage under these conditions.
The role of FasL in inflammation has received considerable attention.
FasL expression is detected at some immune privileged sites, for
example, eye and testis (9, 10), where it is believed to protect those
tissues by inducing apoptosis in the intruding Fas-bearing immune cells
(9). Although some studies have shown that FasL expression can promote
allograft survival in transplantation models, other studies have
reported that ectopic FasL promotes neutrophil infiltration and
accelerates rejection (reviewed in Ref. 11). FasL-induced inflammatory
responses can result from the release of interleukin-1
(12)
and other cytokines that recruit neutrophils (13). In this regard, it
has been shown that ectopic FasL expression in cardiac myocytes
promotes proinflammatory cytokine expression, leukocyte infiltration,
and hypertrophy (14). Heart grafts overexpressing FasL in cardiac
myocytes also undergo accelerated rejection, accompanied by massive
neutrophil infiltration (15).
Inflammatory reactions involving leukocytes have been shown to
contribute to the pathogenesis of myocardial ischemia-reperfusion injury (MI/R) (16). Necrotic cardiomyocytes release cytokines causing
leukocytes to transmigrate through the endothelium and accumulate at
the ischemic site. Once in the myocardium, leukocytes release
proteolytic enzymes and reactive oxygen species, causing further damage
to the heart. Neutrophils play a critical role in reperfusion injury,
and it has been shown that neutrophil depletion results in reduced
myocardial infarct size (17, 18).
Besides its expression on immune cells, FasL is also expressed at low
levels on the vascular endothelium (4). Administration of the
pro-inflammatory cytokine TNF-
down-regulates FasL expression in
endothelial cells, and overexpression of FasL on the endothelium attenuates leukocyte extravasation induced by TNF-
(19). It has also
been shown that FasL overexpression on the vascular endothelium of
carotid arteries inhibits transplant-associated intimal hyperplasia (20) and that adenovirus-mediated delivery of FasL to injured vessels
inhibits intimal hyperplasia in animals with preexisting immunity to
adenovirus (21, 22). These data suggest that when expressed in
endothelial cells, FasL may have anti-inflammatory actions.
To elucidate further the function of FasL in endothelium, we
constructed transgenic mouse lines that stably express FasL in vascular
endothelium under the control of the vascular endothelial cadherin
(VEcadherin) promoter. When subjected to ischemia-reperfusion, the
hearts of VEcadFasL transgenic mice displayed significantly smaller
infarct size and a reduction in functional impairment when compared
with nontransgenic mice. Consistent with these data, a decrease in
neutrophil activity was observed at an early time point in transgenic
hearts compared with nontransgenic hearts. No differences in infarct
size were seen between transgenic and nontransgenic mice that were
treated with cytotoxic anti-neutrophil serum prior to injury. These
data support the hypothesis that endothelial FasL acts to minimize
neutrophil extravasation and organ damage following
ischemia-reperfusion injury.
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EXPERIMENTAL PROCEDURES |
Generation of Transgenic Mice--
Human FasL cDNA was
ligated to mouse VEcadherin promoter (Fig. 1A). The 2.5-kbp
promoter fragment has been reported previously (23) to express the
chloramphenicol acetyltransferase reporter gene exclusively and
constitutively in endothelial cells. Human FasL cDNA with mouse
VEcadherin promoter was originally subcloned into pBluescript vector
(pBSmVEhFasL). The transgene construct was liberated from the vector by
ClaI and SalI digestion and purified by agarose
gel electrophoresis and Geneclean kit (Qiagen, Valencia, CA). Donor
eggs were prepared from B6C3 mice (a hybrid of C57/B6 and C3H) and were
microinjected with transgene construct. After microinjection, eggs were
transferred into oviducts of pseudopregnant ICR foster mothers. Founder
mice were identified by PCR with genomic DNA isolated from tail snips
(forward primer VEFas1, 5'-CCT CCG ATG AGA CCT AGA ATT GAG-3'; reverse
primer VEFas2, 5'-ACA GAG GTT GGA CAG GGA AGA AC-3'). All mice
in this study were maintained in a facility accredited by the
Association for the Assessment and Accreditation of Laboratory Animal
Care, International. Animals were maintained at a constant temperature
of 70-72 °F, 40-70% humidity, with 10-15 air changes per hour.
All manipulations performed on the mice were approved by the
Institutional Animal Care and Use Committee.
Determination of mRNA Transcript Expression--
Total RNA
was isolated from hearts or lungs of transgenic mice or wild-type
littermates. FasL transgene and cytokine mRNA expression were
examined by RiboQuantTM MultiProbe RNase Protection Assay
System according to the manufacturer's instructions (Pharmingen).
Total RNA blots were also hybridized to transcript-specific probes for
atrial natriuretic factor (ANF),
- and
-myosin heavy chain (
-
or
-MyHC), Serca2a (SERCA), and phospholamban (PLB). To control for
loading, all steady-state transcript levels were normalized to the
signal intensity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH).
Hybridization signals were quantified by PhosphorImager analysis
(Amersham Biosciences).
RT-PCR Analysis--
Total RNA was isolated from hearts of
2-month-old transgenic mice with ULTRASPECTM RNA isolation
system (Biotecx Laboratories, Houston, TX) according to the
manufacturer's instructions. Total RNA was reverse-transcribed to
cDNA and amplified by PCR according to the directions of the manufacturer (Applied Biosystems, Foster City, CA), generating a 528-bp
fragment of FasL (forward primer RTVE5, 5'-GGC CCT GGA CAG ACT CGA GTC
TAG TAA C-3'; reverse primer RTVE3.1, 5'-GAG GCA TGG ACC TTG AGT TGG
ACT TGC C-3') or a fragment of glyceraldehyde-3-phosphate dehydrogenase
(forward primer, 5'-CGA TGC TGG CGC TGA GTA C-3'; reverse primer,
5'-CGT TCA GCT CAG GGA TGA CC-3'). The following PCR conditions were
used: 1 cycle at 94 °C for 1 min, 35 cycles at 94 °C for 30 s and 68 °C for 3 min. The PCR products were electrophoresed using
1.5% agarose gel.
Immunohistochemistry--
Thoracic arteries were collected from
mice and fixed with 4% paraformaldehyde. Paraffin-embedded
specimens were cut into 6-µm sections and then deparaffinized and
blocked with 5% goat serum and 0.01% Triton-X in phosphate-buffered
saline (PBS) for 1 h. Sections were immuno-stained using anti-FasL
antibody (N-20) and anti-von Willebrand factor antibody (Santa Cruz
Biotechnology, Santa Cruz, CA), respectively, followed by New Fuchsin
substrate system (Dako, Carpinteria, CA).
Isolation of Endothelial Cells from Mice--
Mouse endothelial
cells were grown from mouse thoracic aorta using an explant technique
(24). Isolated thoracic aorta was opened longitudinally and cut into
small pieces (2-3 mm). Pieces of vessels were placed with intima side
down on Matrigel-coated 6-well plates in the presence of minimum volume
of endothelial growth medium (Clonetics, San Diego, CA). After 4 days,
the pieces of vessels were removed, and migrated endothelial cells were
allowed to grow to confluence. Migrated cells were identified as
endothelial cells on the basis of typical morphology and CD31
immunostaining. Endothelial cells on Matrigel-coated dishes were
harvested and transferred to gelatin-coated 10-cm dishes or 6-well
plates. They were grown and maintained with complete media for
experiments. The human monocyte (myeloid leukemia) cell line, U937, was
obtained from American Type Culture Collection (Manassas, VA) and
maintained in RPMI 1640 medium with 10% fetal bovine serum.
Protein Detection by Flow Cytometry--
Endothelial cells
derived from transgenic mice or wild-type littermates were harvested
with PBS containing 0.5% EDTA. Cells were incubated with PBS
containing 5% fetal calf serum and 10 µg/ml hamster antibody against
human FasL (4H9) (MBL, Nagoya, Japan) or hamster IgG for 1 h at
4 °C. After three washes in phosphate-buffered saline, cells were
incubated with 10 µg/ml fluorescein isothiocyanate-conjugated goat
anti-hamster IgG (Kirkegaard & Perry Laboratories, Inc., Gaithersburg,
MD) for 30 min at 4 °C. Immunofluorescence staining on the cell
surface was analyzed using flow cytometer (FACS, BD Biosciences).
Co-culture Toxicity Assay--
Endothelial cells cultured with
lipopolysaccharide in 6-well plates were co-cultured with monocyte
(myeloid leukemia) cell line, U937 (106 cells), in direct
contact under rotating conditions (60 rpm) overnight. Nonadherent
U937 cells were collected and washed with cold PBS twice and then fixed
with 3.7% formaldehyde in PBS for 20 min. After fixation cells were
washed with PBS twice and stained with Hoechst 33342. Cells were
analyzed for the appearance of pyknotic nuclei using a Nikon Diaphot
microscope using a ×40 objective.
Morphometric and Histopathologic Analysis--
Under general
anesthesia, the heart was perfused with heparinized relaxation buffer
(25 mM KCl and 5% dextrose in PBS). After removal, the
heart was weighed and normalized to total body weight. For
histopathologic assessment, hearts were fixed in 10% buffered formalin
and embedded in paraffin. Paraffin-embedded myocardial sections (5 µm) from each heart were stained with hematoxylin and eosin or
Gomori's one-step trichrome.
Myocardial Ischemia-Reperfusion--
The surgical protocol and
infarct size determination were performed similar to methods described
previously (25). Mice were anesthetized with intraperitoneal injections
of pentobarbital sodium (50 mg/kg) and ketamine hydrochloride (50 mg/kg). The mice were then orally intubated with polyethylene-90
(PE-90) tubing, connected to a rodent ventilator (model 683, Harvard
Apparatus) via loose junction, and supplemented with oxygen. The
ventilator was set to a tidal volume of 2.5 ml and a rate of 120 strokes per min. Body temperature was maintained at 37 °C using a
rectal thermometer and infrared heating lamp. After performing a median sternotomy, the left anterior descending (LAD) coronary artery was
visualized and ligated with 7-0 silk suture mounted on a tapered needle. Ischemia was confirmed by the appearance of myocardial hypokinesis and pallor distal to the occlusion. Following 30 min of LAD
occlusion, the ligature was removed, and reperfusion was visually
confirmed. The chest wall was closed, and the mice were given
subcutaneous butorphanol tartrate (0.1 mg/kg) for analgesia. The mice
were then allowed to recover in a temperature-controlled area
supplemented with oxygen. At 6 or 72 h of reperfusion, the mice
were anesthetized with pentobarbital and ketamine and ventilated, and a
thoracotomy was performed. The LAD was re-ligated and Evans blue (1.5 ml of 1.0% solution) was retrogradely infused into the carotid artery
to delineate the nonischemic from the ischemic zones. The hearts were
sliced in five 1-mm thick sections along the long axis. Ex
vivo incubation of each heart slice in 1.0% 2,3,5-triphenyltetrazolium chloride for 5 min at 37 °C allowed differentiation between the viable and necrotic areas of the previously ischemic myocardium. The left ventricular area (LV), area at risk (AAR), and area of infarction (INF) for each slice were determined by
computer planimetry using NIH Image software (version 1.57).
Evaluation of Arterial and Ventricular Hemodynamics--
To
assess the closed-chest hemodynamic status of both groups of mice after
3 days of reperfusion, a 1.4Fr Millar (SPR-671) ultraminiature pressure
catheter was inserted into the left ventricle as described previously
(26). Mice were anesthetized with ketamine (50 mg/kg intraperitoneally)
and pentobarbital (50 mg/kg intraperitoneally) and supplemented with
oxygen via a nasal cone. The right common carotid artery was isolated;
the catheter was inserted into the artery and advanced to the aorta.
Data were recorded for 10 s using MacLab acquisition hardware and
software. Offline assessment of these data yielded heart rate, systolic
blood pressure, diastolic blood pressure, and mean arterial blood
pressure. The catheter was then advanced through the aortic valve into
the LV. If more than three attempts were needed to advance the catheter
into the LV, ventricular assessment was aborted to avoid damage to the catheter. Data were recorded for 10 s. Subsequent offline
evaluation provided LV systolic pressure, LV end diastolic pressure, LV
developed pressure, and the first derivative of the LV pressure curve
(minimum and maximum dp/dt). Data were calculated
from the arithmetic mean of 40-50 cardiac cycles per parameter per animal.
Measurement of Tissue Myeloperoxidase Activity--
The assay
procedures are similar to methods described previously (27). Hearts
were divided into ischemic and non-ischemic parts after
ischemia-reperfusion surgery and were snap-frozen in liquid nitrogen.
The frozen tissues were weighed and homogenized in 500 µl of 50 mM potassium phosphate buffer (pH 6.0) with 0.5% hexadecyltrimethylammonium bromide (Sigma). The tissues were then sonicated for 10 s, freeze-thawed three times, and sonicated for another 10 s. The samples were ultracentrifuged at 30,000 × g at 4 °C for 30 min. Supernatant was taken out, and the
volume was measured. To measure the myeloperoxidase activity, 290 µl of 50 mM potassium phosphate buffer (pH 6.0) with 0.167 mg/ml o-dianisidine hydrochloride (Sigma) and 0.0005%
hydrogen peroxide were added to 10 µl of supernatant. The absorbance
was measured at 450 nm every 30 s for 4 min. One unit of activity
produces one milli-optical density unit increase per min.
Neutrophil (PMN) Depletion--
Neutrophil depletion was
performed using adsorbed rabbit anti-mouse PMN serum (Accurate
Chemicals, Westbury, NY) as described previously (28). The anti-PMN
serum was diluted 1:10 in sterile 0.9% saline, and mice were injected
(intraperitoneally) with 300 µl per day for 3 consecutive days prior
to myocardial ischemia-reperfusion injury. The extent of PMN depletion
was confirmed in each mouse prior to the induction of myocardial
ischemia and reperfusion.
Statistical Analysis--
All findings were analyzed with a
Student's unpaired t test using StatView 4.5 (Abacus
Concepts). Values are reported as means ± S.E. with significance
set at p < 0.05.
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RESULTS |
FasL Expression in VEcadFasL Transgenic Mice--
Several lines of
transgenic (Tg) mice were generated using the vascular endothelial
cadherin (VEcadherin) promoter upstream of human FasL cDNA (Fig.
1A). Human FasL cDNA was
chosen because it is distinguishable from endogenous mouse FasL.
Transcripts of FasL transgene were analyzed by RNase protection assay
(Fig. 1B) and RT-PCR (Fig. 1C). Heart and lung
tissues were examined because these organs are highly vascularized.
FasL transgene mRNA levels were highest in Line 2 and lowest in
Line 12, with Line 17 expressing an intermediate level of transcript.
The probes used in RNase protection assay and the primers used in
RT-PCR were all specific for the human FasL transgene, and no signals were detected in nontransgenic (NTg) littermates.

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Fig. 1.
RNA analysis of VEcadFasL transgenic
mice. A, structure of FasL transgene with mouse
VEcadherin promoter. Primers VEFas1 and VEFas2 were used for DNA PCR
analysis, and primers RTVE5 and RTVE3.1 were used for RT-PCR.
B, RNase protection assay with RNA extracted from lungs of
NTg mice and different lines (L) of Tg mice. The probe is
specific for human FasL. C, RT-PCR analysis with RNA
extracted from hearts of NTg mice and different lines of Tg mice.
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To determine whether FasL transgene is specifically expressed in
endothelium at the protein level, immunohistochemical analyses were
performed on artery sections from Tg mice or NTg mice. Overexpression of FasL protein was detected on the luminal surface of Tg vessels compared with NTg vessels, and the staining was absent in the underlying smooth muscle cell layer (Fig.
2A). A similar staining pattern was obtained with anti-von Willebrand factor antibody, indicating that the FasL transgene expression was localized to endothelial cells.

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Fig. 2.
Transgenic FasL protein is
specifically expressed in vascular endothelial cells.
A, immunohistochemistry of aortic sections from Tg mice and
NTg littermates. Left panels are stainings for FasL.
Right panels are stainings for von Willebrand factor
(vWF). B, FACs analysis of FasL transgene
expression on surface of endothelial cells derived from Tg mice Lines
2, 17, and 12 (L2, L17, and L12) and NTg
littermates. Surface expression of FasL transgene was detected on all
transgenic lines (shaded curve) with hamster anti-human FasL
antibody. Hamster IgG was used as negative control (open
curve).
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Transgenic Endothelial Cells Express Functional FasL on the Cell
Surface--
It is reported that endothelial FasL expression is
largely cytoplasmic with little or no cell surface expression (29). To examine the cell surface expression of FasL transgene, endothelial cells were cultured from Tg and NTg aortae. FACS analysis with anti-human FasL antibody revealed the presence of human FasL on the
surface of transgenic endothelial cells (Fig. 2B).
Endothelial cells from Line 2 displayed highest FasL expression,
followed by Line 17 and Line 12, which is consistent with the RNA
analysis data. Human FasL was not detectable on endothelial cells
derived from NTg littermates.
The monocytic cell line U937 is readily susceptible to Fas-mediated
apoptosis (30). To assess the functionality of FasL transgene,
endothelial cells derived from Tg or NTg aortae were co-cultured with
U937 cells. Non-adherent U937 cells were collected and stained with
Hoechst 33342 (Fig. 3A).
Microscopic analysis revealed a significantly larger portion of
non-adherent U937 cells with pyknotic nuclei following co-culture with
L2- and L17-derived endothelial cells, compared with NTg endothelial
cells (Fig. 3B). Endothelial cells from Line 2 showed higher
cytotoxicity compared with those from Line 17. Similarly, a larger
portion of non-adherent cells appeared to have pyknotic nuclei
following incubation with L12-derived cells, but this was not
statistically significant relative to NTg cells. Again, the levels of
cytotoxicity from the endothelial cells of different lines correlated
with the different expression levels of the FasL transgene detected in
mRNA assays and FACS analysis.

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Fig. 3.
Cytotoxicity of endothelial cells derived
from transgenic mice toward co-cultured U937 cells. A,
Hoechst staining of U937 cells co-cultured with endothelial cells
derived from Tg mice (L2, L17, and L12) and NTg
mice. B, the number of apoptotic U937 cells co-cultured with
endothelial cells from Tg mice and NTg littermates were quantified by
counting the number of cells with pyknotic nuclei. Results are from two
separate experiments (n = 6, *, p < 0.05).
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Characterization of VEcadFasL Transgenic Mice--
VEcadFasL Tg
mice did not display detectable developmental abnormalities or
detectable pathological abnormalities. Autopsy analysis did not reveal
differences in organ appearance between Tg and NTg littermates.
Analyses of peripheral blood leukocytes did not reveal differences in
the levels of CD19+ B cells, CD3+ T cells, or granulocytes between any
Tg line and NTg littermates (data not shown). Tg Lines 12 and 17 bred
with normal Mendelian frequency. However, Line 2 did not breed as often
as the other transgenic lines, produced smaller litter sizes, and gave
rise to transgene-positive pups at a reduced frequency (data not shown).
Transgenic mice that overexpress murine FasL from the cardiac
myocyte-specific
-myosin heavy-chain promoter (
-MyHC-FasL Tg)
develop cardiac hypertrophy and display mild leukocyte infiltration, accompanied with corresponding changes in gene expression (14). To
examine whether the consequences of endothelial FasL overexpression differ from those of cardiomyocyte FasL overexpression, age-matched (18 week-old) VEcadFasL Tg mice with high level endothelial FasL expression
(Line 2) were compared with
-MyHC-FasL Tg mice with high level
cardiomyocyte-specific FasL expression (Line 61). The heart-to-body
weight ratio of VEcadFasL Tg mice is normal, compared with the 123%
increase of the
-MyHC-FasL Tg mice. VEcadFasL Tg hearts demonstrate
no histologic or gross morphologic evidence of inflammation including
cardiomyocyte hypertrophy, leukocytic infiltration, and
interstitial fibrosis that is characteristic of
-MyHC-FasL Tg hearts
(data not shown).
Total RNA isolated from Tg and NTg hearts was probed for ANF,
- and
-myosin heavy chain (MyHC), Serca2a (SERCA) and PLB. Consistent with
histologic and morphometric data, VEcadFasL Tg hearts do not
demonstrate molecular changes characteristic of pathological
hypertrophy, i.e. the changes in ANF or MyHC isoform switches characteristic of
-MyHC-FasL Tg mice (Fig.
4A). In contrast, expression
of SERCA and PLB, components of cardiomyocyte calcium handling, is
slightly reduced in the VEcadFasL Tg hearts but not to the extent seen
in the
-MyHC-FasL Tg mice (Fig. 4A).

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Fig. 4.
VEcadFasL Tg mice show neither hypertrophic
nor inflammatory changes in the hearts. Total RNA isolated from
VEcadFasL Tg hearts was analyzed for characteristic gene expression
changes seen with -MyHC-FasL Tg mice. A, VEcadFasL Tg
hearts do not demonstrate molecular changes characteristic of
hypertrophy which is seen in -MyHC-FasL Tg hearts, i.e.
induction of ANF and MyHC isoform switches and changes in the
expression level of SERCA and PLB. B, VEcadFasL Tg hearts
show only minimal alterations in cardiac pro- and anti-inflammatory
cytokine transcripts expression, in contrast to marked changes of
cytokine expression in -MyHC-FasL Tg hearts. B6C3 NTg and VEcadFasL
Tg, n = 3; FVB/N NTg and -MyHC-FasL Tg,
n = 5. *, p < 0.05. IL,
interleukin; TGF, transforming growth factor.
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Proinflammatory consequences of enforced cardiomyocyte FasL expression
are also associated with altered expression of pro- and
anti-inflammatory cytokines in the heart. In contrast, VEcadFasL Tg
hearts show only minimal alterations in cardiac cytokine transcript expression (Fig. 4B). Therefore, with the exception of some
minor changes in cardiac gene expression, pro-inflammatory and
hypertrophic consequences of cardiomyocyte FasL overexpression are
undetectable in hearts with VEcadherin-directed endothelial FasL expression.
Reduced Infarct Size and Improved Myocardial Function in VEcadFasL
Tg Mice Following Ischemia-Reperfusion Injury--
To determine the
consequences of endothelial FasL overexpression on inflammatory injury,
VEcadFasL Tg mice were subjected to myocardial ischemia and reperfusion
(MI/R). Line 17 Tg mice were used for this analysis because a
sufficient number of Line 2 Tg mice could not be bred for these
analyses. The left anterior descending coronary artery (LAD) of both Tg
and NTg groups of mice were subjected to 30 min of occlusion followed
by 3 days of reperfusion. As shown in Fig.
5, the NTg and Tg mice experienced similarly sized areas-at-risk (AAR) as assessed by retrograde perfusion
with Evans blue dye. However, myocardial infarct size with respect to
the area-at-risk (INF/AAR) was 42% less in Tg compared with NTg hearts
(p < 0.05).

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Fig. 5.
The infarction area in VEcadFasL hearts is
significantly reduced following ischemia-reperfusion injury.
A, AAR/LV and INF/AAR in Tg mice and NTg mice were
determined following 30 min of myocardial ischemia and 72 h of
reperfusion. The AAR/LV was not significantly different between the two
groups. However, INF/AAR was significantly (*, p < 0.05) reduced in the Tg group compared with NTg group. NTg,
n = 10; Tg, n = 15. B,
representative photomicrograph of heart taken at mid-ventricular level
following 30 min of myocardial ischemia and 72 h of
reperfusion.
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To examine heart performance, peripheral and ventricular hemodynamics
were assessed after ischemia-reperfusion injury. As shown in Fig.
6A, LV hemodynamics were
augmented in Tg mice compared with NTg mice. Although LV systolic
pressure was somewhat elevated in the Tg group, it did not reach
statistical significance. However, LV end diastolic pressure was
significantly lower in the Tg group (p < 0.05).
Similarly, LV developed pressure was significantly greater in the Tg
group compared with the NTg group (p < 0.05). The
indices of contractility, maximum and minimum
dp/dt, were significantly greater in the Tg
hearts compared with the NTg hearts (p < 0.05) (Fig.
6B). Mean arterial blood pressure, systolic blood pressure,
and diastolic blood pressure were all significantly higher in Tg
compared with NTg mice (Fig. 6C). Taken together, these
hemodynamics data indicate that VEcadFasL Tg mice showed better heart
performance compared with NTg mice after MI/R injury, and these data
are consistent with a smaller infarct size in Tg mice.

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Fig. 6.
VEcadFasL Tg hearts show better performance
after ischemia-reperfusion injury. A, ventricular
hemodynamics in Tg mice and NTg mice following 30 min of myocardial
ischemia and 72 h of reperfusion. Although left ventricular
systolic pressure (LVSP) was not significantly higher in Tg
group, LV end diastolic pressure (LVEDP) and LV developed
pressure (LVDevP) were significantly (*, p < 0.05) improved in Tg mice compared with NTg mice. NTg,
n = 7; Tg, n = 7. B, maximum
and minimum dp/dt in Tg mice and NTg mice
following 3 days of reperfusion. Transgenic expression of FasL
significantly (*, p < 0.05) improved LV
dp/dt in mice subjected to myocardial ischemia
and reperfusion. NTg, n = 7; Tg, n = 7. C, peripheral hemodynamics in Tg and NTg mice following 30 min of myocardial ischemia and 72 h of reperfusion. Mean arterial
blood pressure (MABP), systolic blood pressure
(SBP), and diastolic blood pressure (DBP) were
all significantly (*, p < 0.05) higher in Tg mice
compared with NTg mice. NTg, n = 9; Tg,
n = 11.
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Decreased Myocardial Neutrophil Activity Following MI/R in
VEcadFasL Tg Mice--
Early neutrophil accumulation has been shown to
be critical in MI/R injury (17, 18). To investigate whether endothelial FasL abates inflammatory injury by reducing neutrophil entry into the
ischemic area, neutrophil myeloperoxidase activity was assayed in Tg
and NTg mice following 30 min of ischemia and 6 h of reperfusion. As shown in Fig. 7, myeloperoxidase
activity was reduced by 54% in ischemic segments of VEcadFasL Tg
hearts compared with NTg hearts (p < 0.01). This
corresponds to the 42% decrease of infarction size compared with NTg
hearts (Fig. 5). Sham-operated hearts from Tg and NTg groups did not
show any significant difference in myeloperoxidase activity.

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Fig. 7.
Neutrophil activity is reduced in Tg mice
during ischemia-reperfusion injury. Myeloperoxidase
(MPO) activity within the ischemic region was significantly
(*, p < 0.01) decreased in Tg mice compared with NTg
mice following 30 min of myocardial ischemia and 6 h of
reperfusion. NTg, n = 9; Tg, n = 7.
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Abrogation of the Protective Action of Endothelial FasL
Overexpression by Neutrophil Depletion--
To test whether the
protective action of endothelial FasL is mediated by a reduction in
neutrophil activity in the ischemic tissue, the effects of neutrophil
depletion prior to ischemia-reperfusion injury were assessed in Tg and
NTg mice. Treatment with anti-PMN serum for 3 consecutive days prior to
injury led to an 88% reduction in the average level of circulating
neutrophils (p < 0.001). Neutrophil depletion led to
reductions in infarct size in both strains of mice (Fig.
8). Although expression of the transgene
led to a 42% decrease in infarct size in the untreated mice (Fig. 5),
no protection was evident in the Tg group following neutrophil
depletion (Fig. 8).

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Fig. 8.
Neutrophil depletion eliminates the
protective action of the FasL transgene in myocardial
ischemia-reperfusion injury. NTg and Tg mice underwent 3 consecutive days of neutrophil depletion by anti-PMN serum injection.
Mice then underwent 30 min of myocardial ischemia followed by 72 h
of reperfusion. AAR/LV and INF/AAR were not significantly different
between groups. NTg, n = 6; Tg, n = 6.
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DISCUSSION |
In this study we described the effects of endothelial FasL
overexpression on myocardial damage following ischemia-reperfusion injury. We established a new mouse model that specifically expresses FasL in vascular endothelial cells. These mice developed normally and
had normal hearts but exhibited a 42% reduction in infarct size
following myocardial ischemia-reperfusion injury. Consistent with the
smaller infarct size, VEcadFasL mouse hearts displayed less functional
impairment than nontransgenic mice following the injury. The
cardioprotective effect in the transgenic mice may be attributable to a
54% reduction in neutrophil accumulation in the reperfused tissue.
Thus, overexpression of FasL on vascular endothelium may serve to
minimize neutrophil recruitment, thereby reducing myocardial damage.
A number of studies have analyzed the role of the Fas/FasL system in
the heart. The
-MyHC-FasL transgenic mice that ectopically express
FasL in cardiac myocytes develop mild cardiac hypertrophy associated
with interstitial fibrosis and leukocyte inflammation (14). Although
apoptosis was not observed in the
-MyHC-FasL hearts, other
experiments have provided direct evidence that Fas pathway can induce
apoptosis in cardiomyocytes. In the study of Lee et al. (7),
it was shown that FasL expression promotes cardiomyocyte apoptosis both
in vivo and in vitro and that Fas-deficient lpr mice displayed less cardiomyocyte apoptosis and
correspondingly smaller infarct size after ischemia-reperfusion injury.
Hearts from lpr mice also display fewer apoptotic cells
ex vivo after ischemia-reperfusion (31), and intravenous
administration of FasL-blocking antibody attenuates inflammation in the
heart and reduces myocardial infarct size following
ischemia-reperfusion injury (32). It is possible that the inflammation
and hypertrophy in
-MyHC-FasL mouse hearts result from chronic, but
undetectably low, levels of apoptosis in cardiac myocytes or
tissue-resident inflammatory cells (13). Alternatively, the cardiac
hypertrophy observed in
-MyHC-FasL mice may result from an
activation of intrinsic growth regulatory signaling cascades that
promote the phosphorylation of glycogen synthase kinase 3
within
cardiac myocytes (33). In this regard, Fas is reported to positively affect the growth of a number of cell types (reviewed in Ref. 34);
however, cell cycle entry in some of these cases may result from the
execution of the apoptotic process (for example see Ref. 35).
In contrast to cardiomyocytes, vascular endothelial cells express FasL,
and overexpression of FasL in these cells normally does not affect
viability or cell cycle status (4). Thus, the overexpression of FasL by
the endothelial cells of transgenic mice would be expected to alter
immune responses in the absence of cytotoxic or proliferative effects
on the endothelium. The marked differences between the heart phenotypes
of VEcadFasL and
-MyHC-FasL mice clearly illustrate that FasL can
exert either pro-inflammatory or anti-inflammatory actions within an
organ depending upon differences in cell type-specific expression or microenvironment. Ectopic expression of FasL in cardiac myocytes leads
to inflammation and tissue damage, whereas overexpression of FasL on
the vascular endothelium has an anti-inflammatory effect and protects
the heart from injury. Similar to our findings in heart, both pro- and
anti-inflammatory effects of FasL expression have also been observed in
other transgenic models. Transgenic mice expressing FasL in pancreatic
beta cells display neutrophil infiltration and develop diabetes at a
young age (36). In this setting, ectopic expression of FasL will lead
to the destruction of Fas-positive islet cells (37). In contrast, FasL
is expressed on follicular cells of the thyroid (38), and transgenic
expression of FasL on thyroid follicular cells prevents thyroid
allograft rejection (39). These data support the hypothesis that
FasL-induced inflammation involves FasL that is expressed ectopically,
whereas anti-inflammatory effects are associated with overexpression in cell types that normally express endogenous FasL (11).
Neutrophils accumulate at the site of ischemia within hours of
reperfusion and damage the tissue by releasing proteases and oxidants
(40). It is well established that neutrophil depletion will reduce
ischemia-reperfusion injury in the heart (17, 18). Neutrophils express
both Fas and FasL and are susceptible to Fas-mediated cell death (41).
Thus, it is reasonable to hypothesize that FasL expressed on the
endothelial surface may induce apoptosis in Fas-bearing neutrophils,
thereby preventing their transmigration to myocardial tissue. In
support of this hypothesis, we have shown that neutrophil depletion
will abrogate the protective action of the FasL transgene on myocardial
ischemia-reperfusion injury. In contrast, expression of membrane-bound
FasL is reported to promote neutrophil chemotaxis (42-44), although
FasL has no direct chemoattractant activity (45). In these situations,
neutrophil chemotaxis may be the indirect result of pro-inflammatory
cytokine release that is caused by the cytotoxic activity of FasL (12, 13). These effects may be minimized in the VEcadFasL mice due to the
resistance of endothelial cells to Fas-mediated apoptosis (4, 5, 46)
and to the co-expression of immunosuppressive cytokines by vascular
cells (47, 48), potentially creating a microenvironment that promotes
immunological tolerance to FasL.
In summary, we constructed strains of mice that overexpress FasL on the
vascular endothelium. These mice were protected from ischemia-reperfusion injury in the heart, displaying smaller infarct size, improved performance, and diminished neutrophil infiltration. These data show that endothelial cells can be engineered to express high levels of FasL, leading to reductions in injury-induced
inflammation and organ damage.