Endothelial Cell Overexpression of Fas Ligand Attenuates Ischemia-Reperfusion Injury in the Heart*

Jiang YangDagger §, Steven P. Jones||, Toshimitsu Suhara**, James J. M. Greer, Paul D. Ware, Nhan P. Nguyen, Harris PerlmanDagger Dagger , David P. Nelson§§, David J. Lefer, and Kenneth WalshDagger §¶¶

From the Dagger  Molecular Cardiology, Whitaker Cardiovascular Institute, Boston University School of Medicine, Boston, Massachusetts 02118, § Program in Cellular, Molecular and Developmental Biology, Tufts University School of Medicine, Boston, Massachusetts 02111, the  Department of Molecular and Cellular Physiology, Louisiana State University Health Sciences Center, Shreveport, Louisiana 71130, || Division of Cardiology, The Johns Hopkins University, Baltimore, Maryland 21205, the ** Department of Geriatric Medicine, Osaka University Medical School, Suita City, Osaka, Japan, the Dagger Dagger  Department of Molecular Microbiology and Immunology, St. Louis University, St. Louis, Missouri 63104, and §§ Divisions of Cardiology and Molecular Cardiovascular Biology, Children's Hospital Medical Center, Cincinnati, Ohio 45229

Received for publication, November 18, 2002, and in revised form, February 3, 2003

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Fas ligand (FasL) is a member of tumor necrosis factor family that induces apoptosis in target cells that express Fas. The function of FasL during inflammation remains controversial. In this study, we examined the role of vascular endothelial FasL during acute myocardial ischemia-reperfusion that is closely associated with inflammation. Transgenic mouse lines were established that overexpress human FasL on endothelium under the control of the vascular endothelial cadherin promoter. Expression of FasL transgene was detected at both mRNA and protein levels, and functional transgene-encoded FasL protein was specifically expressed on the surface of vascular endothelial cells. Transgenic mice developed normally and had normal hearts. When subjected to 30 min of myocardial ischemia and 72 h of reperfusion, myocardial infarct size was reduced by 42% in the transgenic mice compared with nontransgenic littermates (p < 0.05). Moreover, hemodynamic data demonstrated that transgenic hearts performed better following ischemia and reperfusion compared with nontransgenic hearts. Myocardial neutrophil infiltration was reduced by 54% after 6 h of reperfusion in transgenic hearts (p < 0.01). Neutrophil depletion prior to ischemia-reperfusion injury led to smaller infarcts that were not different between transgenic and nontransgenic mice, suggesting that endothelial FasL may attenuate ischemia-reperfusion injury by abating the inflammatory response. These results indicate that vascular endothelial FasL may exert potent anti-inflammatory actions in the setting of myocardial ischemia-reperfusion injury.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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-1beta (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-alpha down-regulates FasL expression in endothelial cells, and overexpression of FasL on the endothelium attenuates leukocyte extravasation induced by TNF-alpha (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.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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), alpha - and beta -myosin heavy chain (alpha - or beta -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.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.

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).

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).

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 alpha -myosin heavy-chain promoter (alpha -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 alpha -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 alpha -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 alpha -MyHC-FasL Tg hearts (data not shown).

Total RNA isolated from Tg and NTg hearts was probed for ANF, alpha - and beta -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 alpha -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 alpha -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 alpha -MyHC-FasL Tg mice. A, VEcadFasL Tg hearts do not demonstrate molecular changes characteristic of hypertrophy which is seen in alpha -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 alpha -MyHC-FasL Tg hearts. B6C3 NTg and VEcadFasL Tg, n = 3; FVB/N NTg and alpha -MyHC-FasL Tg, n = 5. *, p < 0.05. IL, interleukin; TGF, transforming growth factor.

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.

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.

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.

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.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 alpha -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 alpha -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 alpha -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 alpha -MyHC-FasL mice may result from an activation of intrinsic growth regulatory signaling cascades that promote the phosphorylation of glycogen synthase kinase 3beta 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 alpha -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.

    ACKNOWLEDGEMENT

We thank A. Hofmann for providing the pBSmVEhFasL plasmid.

    FOOTNOTES

* This work was supported by National Institutes of Health Grants AG-15052, AG-17241, AR40197, HD-23681, HL-66957 (to K. W.), HL60849, and DK43785 (to D. J. L.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¶¶ To whom correspondence should be addressed: Molecular Cardiology/CVI, Boston University School of Medicine, 715 Albany St., W611, Boston, MA 02118-2526. Tel.: 617-414-2392; Fax: 617-414-2391; E-mail: kxwalsh@bu.edu.

Published, JBC Papers in Press, February 7, 2003, DOI 10.1074/jbc.M211707200

    ABBREVIATIONS

The abbreviations used are: FasL, Fas ligand; TNF, tumor necrosis factor; RT, reverse transcriptase; Tg, transgenic; NTg, nontransgenic; FACS, fluorescence-activated cell sorter; ANF, atrial natriuretic factor; PLB, phospholamban; LV, left ventricle; PBS, phosphate-buffered saline; MI/R, myocardial ischemia-reperfusion injury; INF, infarction; AAR, area at risk; MyHC, myosin heavy chain; VEcadherin, vascular endothelial cadherin; LAD, left anterior descending; PMN, polymorphonuclear leukocytes; SERCA, Serca2a.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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