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1 Division of Cardiothoracic Surgery, Department of Surgery, and 2 Division of Cardiology, Department of Medicine, University of California Medical Center and University of California School of Medicine, Los Angeles, California 90095
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Virus- and nonvirus-mediated immunosuppressive cytokine gene therapy prolongs cardiac allograft survival in various nonfunctional heart transplant animal models, but its cardiac adverse effects have not been addressed. Recently, we developed a functional heterotopic heart transplant model in rabbits. For the first time, we were able to systematically compare the efficiency, efficacy, and adverse effects of optimized adenovirus- and liposome-mediated ex vivo interleukin (IL)-10 gene transfer in functional donor hearts. The efficiency of liposome-mediated gene transfer was greatly improved in physiologically functioning donor hearts and was only three- to fourfold lower than adenovirus-mediated gene transfer. The efficacy of liposome-mediated IL-10 gene transfer was much higher than that mediated by adenovirus. Significant negative inotropic and arrhythmogenic adverse effects on transplanted hearts were observed due to viral cytotoxicity and immunogenesis, which greatly abated the therapeutic efficacy of this first generation adenovirus-mediated gene therapy.
heart transplantation; acute allograft rejection; adenovirus-mediated gene transfer; liposome-mediated gene transfer; interleukin-10
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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APPLYING A GENE
THERAPY APPROACH to facilitate transplantation would augment the
production of immunosuppressive agents within the allograft, markedly
impair effective antigen presentation, reduce or eliminate
immunogenecity, prevent rejection, and prolong allograft survival
(27). Gene transfer ex vivo has the potential to introduce
immunosuppressive molecules into only the graft, which would limit
systemic side effects (2). Several studies (8,
19) have shown that both adenovirus and liposome could successfully transfer reporter or therapeutic genes, such as two multifunctional cytokine genes, transforming growth factor (TGF)-
and interleukin (IL)-10, to the cardiac allografts. In both mouse and
rabbit heterotopic heart transplant models, both cytokines have shown
potent inhibitory functions in critical pathways of alloreactivity,
which holds promise for immunosuppressive gene therapy ex vivo
(5, 19). However, the gene transfer efficiency and
efficacy have not been systematically compared between the two gene
transfer systems. Most importantly, the adverse effect of viral and
nonviral gene transfer strategies on cardiac function has not been
examined, because all the previous studies were performed on a
nonfunctional heart transplant model. Recently, we developed a new
functional cervical heterotopic heart transplant model in rabbits. The
purpose of the present study is to systematically compare the
efficiency, efficacy, and the cardiac adverse effects of adenovirus-
vs. liposome-mediated ex vivo IL-10 gene transfer in functional cardiac allografts.
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METHODS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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New heterotopic functional heart transplant model and ex vivo gene delivery. New Zealand White donor rabbits weighing 3.5 kg (Charles River Laboratories; St. Constant, Quebec, Canada) and recipient rabbits weighing 4 kg (Irish Farms; Norco, CA) were purchased from geographically unrelated vendors. After a sternotomy was performed, a cannula was inserted through the right carotid artery for cardioplegic and gene infusion. The systemic venous return to the donor heart was occluded. The pulmonary artery was transsectioned, and the left atrium was opened for ventricular decompression. University of Wisconsin (UW) solution (20 ml) was injected (induced arrest, 50 mmHg; after arrest, 40 mmHg) into the cannula, and topical cooling was applied. The pulmonary veins were ligated, and the heart was excised and then placed in cold (4°C) UW solution. Adenovirus vector- or liposome-IL-10 gene complexes suspended in 3 ml normal saline were administrated by continuous ex vivo intracoronary infusion at 3 ml/20 min to the donor heart. Donor hearts were then transplanted to the recipient rabbits at the heterotopic cervical position. The donor ascending aorta was anastomosed to the recipient's proximal right carotid artery. The donor left atrium was anastomosed to the recipient's distal right carotid artery. The donor pulmonary artery was anastomosed to the recipient's proximal right common jugular vein. The donor right atrium was anastomosed to the recipient's distal common jugular vein. The recipient's common jugular vein between the donor right atrium and pulmonary artery anastomosis sites was ligated and cut.
Liposome-DNA complex and adenovirus vector preparations. The plasmid pSVIL-10 containing human recombinant IL-10 cDNA coupled to the simian virus 40 (SV40) early promoter was used as DeBruyne et al. (8) have previously described. The new generation of the cationic liposome GAP:DLRIE (kindly provide by GIBCO-BRL) has, typical of the 2,3-dioxy-propaniminium class of cationic lipids, a basic skeleton that also includes (+)-N-(2-hydroxyethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propaniminium bromide (DLRIE), 1,2-bis(oleoyloxy)-3-(trimethyl-ammonio) propane, N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride, and 2,3-dioleyloxy-N-[2(sperminecarboxiamido)ethyl]-N,N-dimethyl-1-propanaminium trifluoroacetate (23). Recombinant IL-10 cDNA (50 µg) was complexed with liposome (75 µg) at a ratio of 1:1.5 (wt/wt). The liposome and gene mixture was gently vortexed over 15 min just before use. Recombinant replication-deficient "first generation" adenoviruses are constructed by replacing the E1 and E3 region from the adenoviral genome with foreign DNA sequences of interest using homologous recombination as previously described (5). An expression cassette encoding the human recombinant IL-10 gene and an SV40 early promoter gene was inserted to replace the deleted region. The control "empty" virus Ad5d1434 has deletions of the entire E1A region and most of the E1B region. All recombinant viruses were plaque purified on 293 cells and grown to a high-titer stock of 109 plaque-forming units (pfu)/ml.
Semiquantitative RT-PCR and quantitative Northern blot analysis
for cytokine gene expression.
Total RNA was isolated from frozen specimens collected after the
transplantation at the desired time course (7, 8). cDNA
was obtained by random primer RT of 1 µg total RNA using 200 units
SuperScript II Reverse Transcriptase. cDNA was amplified with 1 unit
Taq DNA polymerase (GIBCO-BRL) and 0.2 µM primer to a
final volume of 100 µl using the primers for human IL-10 and -actin. After an initial denaturing step at 95°C for 2 min, the amplification was carried out for 30 cycles (94°C for 1 min, 55°C for 1 min, and 72°C for 2 min) on a thermocycler (GeneAmp PCR System
9600, Perkin-Elmer). -Actin was coamplified and used as an internal
control. RT-PCR products were analyzed by electrophoresis on 1.2%
agarose gels stained with ethidium bromide, illuminated with
ultraviolet light, measured by quantitative scanning densitometry of a
digitally acquired photograph, and analyzed by an imaging analysis
program (NIH Image). The logarithmic amplification phase of
RT-PCR was determined by conducting amplification on undiluted and
serial dilutions from 2- to 128-fold of 20 µg RNA. The linear regression analysis of the template concentration and end-product signal yielded a correlation coefficient of 0.99. Each reaction was
within the linear phase. The optimal number of PCR cycles was
determined by preliminary experiments in which individual amplification
reactions were prepared by using 1 µg total RNA from the same sample.
The resulting cDNAs were amplified for 20-38 cycles with -actin
and IL-10 primer pairs. The efficiency was determined by plotting log
optical density vs. cycle number. Equivalent efficiency in the PCR
amplification of the -actin and IL-10 was indicated by the
equivalent slopes obtained over the range of cycle numbers from 20 to
38. The slope of the curve is directly proportional to the efficiency.
The optical number of PCR was 30, because it has the highest
amplification efficiency in the linear range of logarithmic
amplification phase of the reaction. The repeated PCR analysis on the
same sample showed a measurement variability of <12%.
-actin cDNA probes were uniformly labeled with
random primers using Klenow and [
-32P]dCTP. The total
cellular RNA was size fractionated on formaldehyde-agarose gels run at
30 mA overnight to provide resolution of high-molecular-weight mRNAs.
RNA transferred onto nitrocellulose filters was conducted overnight
through use of 10× SSC (1× SSC = 0.15 mol NaCl-0.015 mol sodium
citrate; pH 7.0) transfer buffer. Gels were photographed before and
after the transfer, and nitrocellulose filters were photographed after
the transfer to confirm transfer efficiency of the 28S and 18S rRNAs.
With the use of these transfer conditions, no residual rRNAs were
detected in the gels after transfer. The filters were then baked at
80°C for 2 h in a vacuum oven and prehybridized at 42°C
overnight in buffer with 1× Denhardt's solution (0.02% polyvinylpryrolidone-0.02% Ficoll-0.02% BSA), 5× SSC, 0.025 mol sodium phosphate (pH 7.4), 50 mg/ml sonicated calf thymus DNA, 0.1%
SDS, and 50% (vol/vol) formamide. The filters were hybridized to cDNA
probes overnight at 42°C in the same buffer mixture. Filters were
washed simultaneously to a final stringency of 0.2× SSC-0.1% SDS at
55°C for 15 min. Ten individual blots containing all the samples for
the study were hybridized. RNA samples for control and gene-transferred
allografts were loaded onto each gel to avoid potential bias caused by
variability in the efficiency of transfer of a particular gel or region
of a gel. Each filter was exposed for three different exposure times to
obtain signals in the linear range for densitometric analysis of each
mRNA species. Filters were exposed on Storage Phosphor Screen and on
X-ray film (XOMAT AR, Eastman Kodak; Rochester, NY) at 
80°C with a
single intensifying screen. Relative amounts of mRNA were determined
with the Phosphorimager using Imagequant software. Each densitometric
score for IL-10 mRNA was then normalized by dividing its optical
density with the corresponding -actin optical density as an internal
control. The mean value for this ratio for the normal group was set at 1.0. For internal control and normalization of mRNA Phosphorimager scores, a synthetic 28S cDNA oligonucleotide based on the human 28S
rRNA sequence was used after exchange labeling with polynucleotide kinase and [-32P]ATP.
ELISA analysis and immunofluorescent staining. The standard PharMingen protocol for sandwich ELISA was used to quantify the amount of human IL-10 in the myocardium (22). Standard 96-well flat-bottomed plates were coated overnight at 4°C with 50 µl monoclonal mouse anti-human IL-10 antibody (PharMingen; San Diego, CA), washed two times with phosphate-buffered saline (PBS)-Tween solution, and blocked with PBS solution-10% fetal calf serum for 1 h. Samples were added for 2 h at room temperature. After the six washes, 100 µl secondary biotinylated monoclonal mouse anti-human IL-10 antibody was added at room temperature for 45 min. Avidin peroxidase was added for 30 min, and 0.1% hydrogen peroxide in 2,2'-azino-bis(3-ethylbenz-thiazoline)-6-sulfonic acid substrate was then used to visualize detection. Quantification was performed on an automated ELISA plate reader (Dynatech Laboratories).
Intracellular IL-10 expression was examined by immunofluorescent staining (26). Samples were sectioned on a cryostat to 8-µm-thick slices and removed by flash condensation. Sections were washed with a bath of PBS (0.01 M; pH = 7.2) and then incubated for 15 min with 25 µl of primary monoclonal antibody to human IL-10 (mouse IgG) from Biosource International (Camarillo, CA). After the sections were rinsed in PBS, they were incubated with 25 µl FITC-labeled secondary antibody [anti-mouse IgG, goat F(ab')2, Protos Immuno Research; San Francisco, CA] for 15 min. Control staining was done using an isotype antibody, goat anti-human IgG (H + L, Jackson ImmunoResearch Laboratories; West Grove, PA). The distribution of IL-10 expression was quantified by the mean peak intensity of the fluorescence in five high-power microscopic fields (×40 magnification) per section of the left and right ventricles, septum, and left and right atria.Adenovirus ELISA. ELISA plates were coated with adenovirus-antigen, incubated overnight, and blocked by the addition of 200 µl of blocking buffer. After the plates were washed three times, serial dilutions of the serum samples were added to the wells and incubated for 1 h. Plates were then incubated with 100 µl/well of sheep anti-rat IgOPD Fab fragments and incubated with 100 µl/well OPD solution (20). The reaction was stopped with 50 µl/well of 0.1 M H2SO4. Plates were then read at 492 nm on an ELISA plate reader.
Histological evaluation. The clinical severity of allograft rejection was evaluated by histological examination. Standard hematoxylin-eosin staining was also performed. Rejection scores were determined based on the standard grading nomenclature established by the International Society for Heart and Lung Transplantation. Paraffin sections of formalin-fixed heart specimens were stained with hematoxylin-eosin to evaluate the general morphology and grade of myocardial rejection (13).
Measurement of infiltrate lymphocyte subpopulation by flow cytometry. Transplanted hearts were perfused with Hanks' balanced salt solution (pH 7.3) at 4°C until the perfusate was clear. The myocardium was then diced in Hanks' solution and expressed through a cell strainer (70 µm, Falcon) for the flow cytometry test. After centrifugation, the red blood cells were lysed, the cell suspension was passed through a cell strainer, and the pellet was then washed three times with 1× PBS. The surface differentiation of antigens was analyzed by flow cytometry after staining with FITC-conjugated monoclonal antibodies directed against CD3, CD4, and CD8 (Spring Valley Lab; Woodbine, MD) as T cell subset markers (11). Cells were stained by 1 µl of each antibody incubated in 50 µl PBS solution supplemented with 0.5% BSA with each antibody for 45 min at 4°C in round-bottomed 96-well plates. After cells were washed three times, they were analyzed by fluorescence-activated cell scan (FACScan) flow cytometry.
Assessment of cardiac function. To assess cardiac function, the grafts from both control and treatment groups underwent transatrial catheterization at 4-day intervals beginning on the day of transplantation to quantify atrial and ventricular pressures. An Ag-AgCl bipolar electrode catheter was used to measure the amplitude of the mean arterial pressure and action potential duration at 90% depolarization (APD90) (21). A His bundle catheter was used to determine the atrioventricular conduction time.
Data analysis. All data are expressed as mean ± SD. Allograft survival curves were produced with the Kaplan-Meier product limit method. Paired or unpaired t-tests and two-way ANOVA were performed on quantitative data to test for statistical differences between groups.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Functional heterotopic heart transplant model.
As shown in Fig. 1A, the
advantages of this newly developed functional heart transplant model
compared with the vascularized nonworking model are 1) both
atria are at normal working condition; 2) the left ventricle
is filled with only oxygenated blood; and 3) both ventricles
have sufficient preload and have normal physiological function. One
hundred twenty-eight heart transplantations were performed (98 allografts and 30 isografts; heart transplant was performed on third-
generation sibling-inbred New Zealand White rabbits). The
average ischemic time was 75 ± 20 min. The average time
of harvesting was 20 min. All hearts resumed beating spontaneously within 2 min. In both allografts and isografts, normal cardiac function
was achieved within 2 h after reimplantation. Isograft mean
survival was more than 5 wk, and histological rejection was not
observed. In allografts, the histological grade of rejection was zero
at postoperative day 2, gradually increased during
postoperative days 3-6, and then became exacerbated
between postoperative days 6 and 8. The graft
contractile activity ceased at 9 ± 2 days. The hemodynamic and
electrophysiological parameters recorded from the allografts were not
significantly different from those of the isografts at 3 h after
transplantation. However, at that time, the left ventricular pressure
in isografts and allografts (85 ± 7 mmHg) was slightly lower than
that in the control hearts (95 ± 9 mmHg; Fig. 1B). At
24 h, left ventricular pressure recovered to 92 ± 8 mmHg,
which was comparable with that in the control heart. However, both left
and right ventricular pressures were significantly decreased in
the allografts (60 and 46%, respectively) compared with those in the
isografts at postoperative day 4 (P < 0.01)
and further decreased (84 and 78%, respectively) at postoperative day 6. In the allografts, the endomyocardium
APD90 in both right and left ventricles was slightly
prolonged at postoperative day 2 (P < 0.05)
and greatly prolonged at postoperative days 4 and 6 (P < 0.01) compared with those in the
isografts and control hearts. However, the significant atrioventricular
conduction prolongation assessed by the His bundle electrogram was not
observed until postoperative day 6.
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Efficiency of gene transfer mediated by adenovirus vs. liposome.
Adenovirus-mediated ex vivo intracoronary human IL-10 gene transfer
induced a transient transgene expression in the cardiac allografts. As
shown in Fig. 2A, the
significant increase in human IL-10 gene overexpression could be
observed in the donor hearts as early as postoperative day
2, reached a peak at postoperative day 4, and then
sharply declined. In contrast, the liposome-mediated human IL-10 gene
transfer-induced transgene expression had a relatively late onset, long
steady state, and much slower decline. At postoperative day
8, the IL-10 mRNA level was 8.1 ± 2.2-fold higher in the
liposome group than in the adenovirus group, as determined by RT-PCR.
An 8.7 ± 2.9-fold increase in the mRNA level, as determined by
Northern blot analysis, further confirmed this finding (Fig.
2B). The time course of the change in the mRNA level
quantified by Northern blot analysis was consistent with that
determined by RT-PCR analysis. Liposome-mediated transgene expression
is initiated and reaches peak levels earlier in the functional model
than in the nonfunctional model. However, the time course of transgene
expression induced by adenovirus-mediated gene transfer was not changed
in the functional model compared with that in the nonfunctional model.
The distribution of human IL-10 was not different in the left and right
ventricles or septum in both groups (Fig. 2C). In the
nonfunctional heart transplant model, the efficiency of gene transfer
was much lower in the atrium than the ventricle. In the functional
model, the gene transfer efficiency was greatly improved, and the human
IL-10 protein level in both atria was almost same as that in the
ventricles (Fig. 2C). Most importantly, the efficiency of
liposome-mediated gene transfer in the functional heart transplant
model was improved 2.87-fold in the left ventricle (Fig.
3A). However, the efficiency of the adenovirus-mediated gene transfer was only increased 1.22-fold. Thus the efficiency of the adenovirus-mediated gene transfer was sixfold higher than liposome-mediated gene transfer in the
nonfunctional heart transplant model but only threefold higher in the
functional model. Dynamically, the maximal human IL-10 protein
expression in the left ventricle induced by 50 µg IL-10-DNA-liposome
complex was lower than that from 109 pfu/ml AdSvIL10 at
postoperative day 4. At postoperative day 10,
however, the IL-10 protein level was higher in the liposome-IL-10 group
than that in the adenovirus-IL-10 gene-treated group (Fig. 3B). At postoperative day 14, the human IL-10
level was still fourfold higher in the liposome-IL-10 gene-treated
group compared with that in the control group, but it was no longer
detectable in the adenovirus-IL-10 gene-treated group.
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Efficacy of gene transfer mediated by adenovirus vs. liposome.
The longevity of the allograft increased from 9 ± 2 days in the
control group to 28 ± 6 days in the liposome-IL-10 gene-treated group and 15 ± 3 days in the adenovirus-IL-10 gene-treated group. Kaplan-Meier survival curves for Ad5d1434, liposome only, AdSvIL10, and
liposome-IL-10-transfected allografts are shown in Fig.
4A. The survival curve of the
adenovirus-treated group was slightly but statistically significantly
shifted to the left compared with that of the control group or
"empty" liposome-treated group (P < 0.05).
However, the rightward shift of the survival curve was much greater in
the liposome-IL-10 gene-treated group (P < 0.01). The
rejection process in the allografts was significantly decelerated by
localized human IL-10 gene transfection. In Fig. 4B, the
rejection scores were plotted against the allograft survival period for individual grafts, and average slopes were then compared among the
study groups. The average rejection slopes were significantly lower in
allografts that had human IL-10 gene transfer mediated by either
liposome or adenovirus (0.39 ± 0.06 and 0.21 ± 0.04, respectively) compared with controls (0.52 ± 0.15, P < 0.005 and P < 0.001, respectively). However, rejection was significantly less severe in the
liposome-IL-10 gene therapy group than in the AdSvIL10 gene therapy
group (P < 0.05). The rejection score was significantly lower in functional cardiac allografts than in
nonfunctional cardiac allografts in both adenovirus and liposome
groups, but this improvement was more pronounced in the liposome group.
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Effects of gene transfer mediated by adenovirus vs. liposome on
cardiac function.
With the use of the new functional heterotopic heart transplant model,
for the first time, two major sets of cardiac function parameters
measuring hemodynamics and electrophysiology can be assessed in cardiac
allografts for the evaluation of gene therapy efficacy. As shown in
Fig. 5A, empty liposome and
liposome-antisense IL-10 gene transfer did not alter the dynamic change
in allograft function caused by acute rejection, which was the same as
that in the control group. In the liposome-mediated IL-10 gene-treated group, all hemodynamic parameters remained in the normal physiological range until postoperative days 12-20. At postoperative
day 24, both right and left ventricular systolic pressures
in donor hearts were decreased ~35% (from 23 ± 4 and 92 ± 8 mmHg at postoperative day 1 to 15 ± 3 and 63 ± 8 mmHg, respectively). Both right and left ventricular end-diastolic
pressures were increased from 4 ± 1 and 6 ± 2 mmHg at
postoperative day 2 to 12 ± 3 and 18 ± 5 mmHg at
postoperative day 24, respectively. The change of left ventricular systolic pressure was significantly correlated with the
changes of the IL-10 transgene expression, IL-10 protein level in the
allografts, and the rejection score of the allografts (Fig. 5B). In contrast, Ad5d1434 and adenovirus-IL-10 gene
transfer caused a slight but statistically significant decrease in the left ventricular pressure (22 ± 9%) and a 2.5-fold increase in the left ventricular end-diastolic pressure in allografts at
postoperative day 2. This negative inotropic effect was also
observed in isografts. At postoperative day 4, acute
rejection was decelerated in the adenovirus-mediated IL-10 gene therapy
group; however, the improvement in ventricular function was not as
pronounced as that in the liposome-IL-10-treated allografts. In
parallel, both left and right atrial contraction were also diminished,
which resulted in a significant increase in atrial pressure. The
correlation between the rejection score and left ventricular function
in the adenovirus-IL-10 gene-treated group was not as significant as
that in the liposome-IL-10-treated group. At postoperative day
14, 80% of the allografts stopped beating in this group.
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Effects of adenovirus- vs. liposome-mediated IL-10 gene transfer on
the electrophysiology of cardiac allograft.
At the same time, changes in electrophysiological parameters,
which included the right and left ventricular monophasic action potentials and His bundle electrogram, were also recorded in the allografts. There was no significant change in His bundle electrograms and in the monophasic action potential recorded in allografts in 2 days
after transplantation in the control group and 6 days in the
liposome-mediated IL-10 gene transfer group. In the adenovirus-treated group with or without the IL-10 gene, the monophasic APD90
in both the atrium and ventricle was significantly prolonged in the allografts, associated with mild lymphocytic infiltration at
postoperative day 2 compared with the control hearts (Fig.
6A). Prolongation of
APD90 was much greater in the atrium than in the ventricle. At postoperative day 6, a significant prolongation of
atrioventricular conduction time was observed in 3 of 10 Ad5d1434-transfected allografts and also in 5 of 15 adenovirus-IL-10-treated allografts (data not shown). Considerable
episodes of ventricular tachycardia and ventricular fibrillation
occurred in adenovirus-treated allografts during postoperative
days 1-4 (Fig. 6B). Supraventriuclar
tachycardia and atrial fibrillation not only occurred in
adenovirus-IL-10 gene-treated allografts at postoperative day
2 but also occurred in a sizable amount of Ad5d1434-treated
allografts and even isografts (data not shown). However, in the
liposome-only and liposome-IL-10 gene-treated groups, arrhythmias
were rarely observed before rejection occurred, and the incidence was
less than that in the control group (Fig. 6C). Premature
ventricular beat was also more frequently seen in the
adenovirus-treated group than in the liposome-treated group
(P < 0.01).
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Cellular and humoral immunogenesis of transfected adenovirus
vector.
A focal and diffuse lymphocytic infiltrate was present in 6 of
10 adenovirus-treated isografts at postoperative day 2 but absent in liposome-treated isografts (0 of 10 isografts). At
postoperative day 2, significant CD3+, CD4+, and CD8+
lymphocytic infiltrates were also observed in the adenovirus only and
adenovirus-IL-10 gene-transfected allografts but not in control,
liposome-only, or liposome-IL-10 gene-treated allografts (Fig.
7). At postoperative day 6,
both CD4+ and CD8+ infiltrates were significantly decreased in the
adenovirus-IL-10 gene-treated and liposome-IL-10 gene-treated allografts compared with those in the control, adenovirus-treated, or
liposome-treated allografts. A greater reduction in CD8+ than CD4+
cells was observed in the liposome-IL-10-treated allografts than in the
adenovirus-IL-10 gene-treated allografts (P < 0.01) (Fig. 7).
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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The findings of the present study are threefold: First, the new heterotopic functional heart transplant model is suitable for evaluating the gene transfer efficiency and efficacy because it closely resembles the clinical setting and redresses the misinterpretations caused by the nonfunctional model per se. Second, the efficacy of liposome-mediated ex vivo IL-10 gene transfer was higher, although the gene transfer efficiency was lower, than that in adenovirus-mediated gene therapy. Third, adenovirus transfection may induce significant adverse effects on cardiac allograft function and various arrhythmias.
Previously, various animal models have been developed for systematically studying acute and chronic rejection. The nonvascularized heart transplantation model was used for some early gene therapy studies, and its limitations in providing clinically relevant information about the alloreactive immune response were soon recognized (1). Thereafter, a heterotopic vascularized nonfunctional transplanted heart model became the most widely used model for recent gene therapy studies, because it allowed the study of allograft rejection on a beating heart without systemic complications (17). Although several groups have developed working left heart models of heterotopic heart transplantation, they were not popularized. In these models, however, left ventricle preload was still insufficient, coronary arteries were perfused with nonoxygenated or low oxygenated blood, and cardiac output was still only half that of the normal physiological condition (28). In the functional heterotopic heart transplant model that we have developed, which closely resembles orthotopic heart transplantation in humans, the physiological function in the whole heart is completely preserved. Additionally, this model enables us to measure the hemodynamic and electrophysiological parameters for monitoring the cardiac adverse effects of gene transfection in the transplanted heart. Normal atrial filling not only preserves atrial function, it also provides sufficient ventricular preload and improves ventricular contractile function. Although the ventricular function was impaired during the first 24 h postoperation without inotropic agents, as in human orthotopic heart transplantation, right and left ventricular function fully recovered by 24-48 h postoperation, and the peak systolic pressures in both chambers were almost the same as in the normal heart. On the other hand, in the functional model, normal sinus rhythm was preserved, and atrial and ventricular arrhythmias were greatly reduced compared with that in the nonfunctional model. Premature termination of function in isografts and allografts often occurred in the nonfunctional model due to allograft rejection and surgical complications, including hemodynamic complications, arrhythmias, and massive thrombus formed in the atrial and ventricular cavities. These surgical complications were in turn interfering with transgene expression, further promoting early allograft termination. This vicious circle is terminated in the functional model. Gene transfer efficiency is significantly improved in both adenovirus- and liposome-mediated gene transfer using this model.
The improvement of gene transfer efficiency by model amelioration was much greater in liposome-mediated gene transfer than in adenovirus-mediated gene transfer. A better coronary perfusion and oxygen supply that keeps cardiac myocytes under normal metabolism improved myocardial contraction, facilitating both viral- and nonviral vector-mediated gene transfer. However, in the liposome-mediated gene transfer, a healthy myocyte membrane and vigorous myocardium contraction may facilitate lipid fusion and promote gene transfection extensively (24). Prolonged allograft survival may extend the action of liposome on the membrane, because liposome-mediated gene transfer has a relatively slower onset but longer steady state (9). In contrast, the efficiency of adenovirus-mediated gene transfer may already have reached a maximum level by the active and relatively fast virus invasion, and thus derives little or no benefit from the functional heart. Instead, better coronary perfusion may facilitate the antiviral immune response. Most importantly, our results demonstrate that liposome-mediated gene transfer has a 20% peak efficiency in the working heart, which was only three to four times lower than that with adenovirus-mediated gene transfer.
Concomitantly, the efficacy of ex vivo liposome-mediated IL-10 gene therapy in acute allograft rejection in the functional heart transplant model was also much greater than previously reported (2). A physiologically functional heart reduces hemodynamic complications, increases coronary artery perfusion, and maintains normal cardiac rhythm, and these factors complement each other to significantly prolong allograft survival and also improve IL-10 gene expression. Long and steady IL-10 expression, in turn, reduces allograft rejection, improves cardiac function, and prolongs allograft survival. In this functional model, increase of IL-10 gene expression is initiated earlier and reaches a peak level earlier than that in the nonfunctional model and that may also be beneficial for allograft survival. Although complete tolerance was not achieved, allograft survival in the liposome-mediated IL-10 gene therapy group was longer than that in the adenovirus-mediated IL-10 gene therapy group. Overall, the observation of higher therapeutic efficacy carried by a still relatively lower gene transfer efficiency further suggests a great therapeutic potential for ex vivo liposome-mediated IL-10 gene transfer. Future studies focused on improving the lipid-base DNA formulation to induce earlier gene transfection with higher efficiency, repeated gene infusion, or incorporating multi-immunosuppressive genes into the liposome may lead to achieving the goal of complete tolerance (4). On the other hand, these findings challenge the accuracy of previous evaluations in gene transfer efficiency and efficacy, suggest possible misinterpretations due to the fundamental pathophysiology of the nonfunctional heart transplant model, and underscore the importance of the reevaluation of gene transfer efficiency in the functional heart transplantation model.
A striking finding of the present study is the adverse effects of adenovirus-mediated gene transfer on donor hearts. The immunogenicity and toxicity of the attenuated adenovirus vector has long been recognized (18). The toxicity was predominantly found in the liver and brain either as a primary target organ or as a secondary affected organ in adenovirus-mediated in vivo and ex vivo gene transfer in other organs (10, 25). A relatively high humoral immune response was also found in patients that had a single intramyocardial viral vector administration (12). In the present study, the findings of focal and diffuse lymphocytic infiltration in isografts and allografts transfected with adenovirus-IL-10 gene or adenovirus alone, but not in any of liposome-IL-10-, empty liposome-, or saline-treated isografts, indicate a viral-induced cellular immunoresponse (16). In parallel, adenovirus transfection induced a negative inotropic effect and life-threatening arrhythmias in the functional cardiac isografts and allografts with or without significant lymphocytic infiltration, suggesting a host antiviral humoral response and/or cardiac toxicity of the viral vector (15). Arrhythmias in acute adenovirus myocarditis have been reported previously, although the mechanism is still unclear. The arrhythmogenic effect of attenuated adenovirus in the transplanted heart was never evaluated due to the lack of a functional heart transplant model. The observation of cardiac toxicity suggests the attenuated virus might still share certain features of the wild-type virus. The observation of the relatively high intensity of the antiviral cellular immune response manifested by the earlier and severe infiltration in allografts than isografts suggests the interaction between the antiviral and allogenic immune responses (3). Early-occurring functional alterations followed by structural alteration in the integrity of the cardiac myocytes may be also attributable to the transient transgene expression pattern of the viral vector (14).
There are three important conclusions from these results that have implications for future gene therapy studies. First, the functional heterotopic heart transplant model we developed has paved the way for us to systematically evaluate the applicability, efficacy, and adverse effects of gene therapy in the heart and may be useful to redress previous misinterpretations based on observations from the nonfunctional heart transplant model. Second, liposome-mediated IL-10 gene transfer has a higher efficacy than adenovirus-mediated gene transfer due to the lack of immunogenicity and toxicity and holds greater potential for long-term transgene therapy. Third, the observations regarding the significant negative inotropic and arrhythmogenic effects of this first generation adenovirus suggest viral cytotoxicity in the transplanted heart, and we should pay particular attention to this in clinical applications.
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FOOTNOTES |
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Address for reprint requests and other correspondence: L. Sen, UCLA Medical Center and UCLA School of Medicine, 47-123 Center for the Health Sciences, 10833 Le Conte Ave., Los Angeles, CA 90095-1679 (E-mail: lsen{at}mednet.ucla.edu).
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.
Received 5 December 2000; accepted in final form 3 May 2001.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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