Transfection of lung cells in vitro and in vivo: effect of
antioxidants and intraliposomal bFGF
Xiaoping
Luo1,
Rosetta
Belcastro2,3,
Judy
Cabacungan2,3,
Vicky
Hannam3,
Anna
Negus3,
Yanxia
Wen3,
Jonathan
Plumb3,
Jim
Hu2,3,
Brent
Steer3,
David R.
Koehler2,
Gregory P.
Downey3,4,5, and
A. Keith
Tanswell2,3,5,6
1 Department of Pediatrics, Tongji Hospital, Tongji
Medical College, Huazhong University of Science and Technology,
Wuhan 430074, People's Republic of China; 2 The
Canadian Institutes for Health Research Group in Lung Development
and 3 Lung Biology Programme, Hospital for Sick
Children Research Institute, and the Departments of
4 Medicine, 5 Physiology, and
6 Paediatrics, University of Toronto, Toronto, Ontario
M5G 1X8, Canada
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ABSTRACT |
We hypothesized that constitutive
formation of reactive oxygen species by respiratory cells is a barrier
to gene transfer when liposome-DNA complexes are used, by contributing
to rapid degradation of plasmid DNA. When plasmid DNA is complexed to
liposomes it is protected against H2O2-mediated
degradation but not that mediated by the hydroxyl radical. Treatment of
distal rat fetal lung epithelial cells (RFL19Ep) with the
vitamin E analog Trolox (50 µM) reduced intracellular plasmid
degradation. Both Trolox (50 µM) and an iron chelator,
phenanthroline (0.1 µM), significantly increased transgene
expression in RFL19Ep approximately twofold, consistent
with a hydroxyl radical-mediated inhibition of transgene expression.
When basic fibroblast growth factor (bFGF; 20 ng/ml), a growth factor
with antioxidant properties, was included within liposomes, we observed
a significantly greater enhancement of RFL19Ep transgene
expression (~2-fold) over that seen with Trolox or phenanthroline.
Inclusion of bFGF within liposomes also significantly enhanced
(~4-fold) transgene expression in mice following intratracheal instillation.
gene transfer; reactive oxygen species; growth factors; basic
fibroblast growth factor
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INTRODUCTION |
THE INTRACELLULAR
FORMATION of reactive oxygen species (ROS) under pathological
conditions has been implicated in a wide variety of degenerative
processes, diseases, and syndromes (12). However, it has
more recently been appreciated that constitutively formed intracellular
ROS are also important stimulators of signal transduction during a
variety of cell processes (31), including DNA synthesis (9, 31). The hydrogen peroxide
(H2O2) content of normal human breath
condensate is ~0.26 µM (1, 18), with a range from 0.01 to 0.4 µM (21), presumably reflecting constitutive
formation of ROS by lung cells. Consistent with this observation are
the findings that both type II pneumocytes from adult animals
(2) and distal rat fetal lung epithelial cells
(RFL19Ep) (24) generate and release into their
culture medium significant quantities of H2O2.
In conditions in which there is pulmonary inflammation, the
H2O2 concentration of breath condensate may
increase considerably to >1 µM (15).
We have previously reported that liposome-mediated transfection of
primary cultures of distal RFL19Ep cells is, at least in part, limited by rapid intracellular degradation of the transfected DNA
(34). That these cells generate and release into their
culture medium significant quantities of H2O2,
even when maintained in a fetal oxygen tension of 20 mmHg
(24), suggested to us that constitutive formation of ROS
by these cells may contribute to degradation of transfected DNA.
H2O2 in the culture medium of distal
RFL19Ep cells is derived from the reduction of cell-derived superoxide through spontaneous or enzymatic dismutation
(14). In the studies reported here, we have examined
whether constitutive ROS production by these cells is an impediment to
transfection using liposome-DNA complexes, as assessed by antioxidant
interventions. In addition, we have evaluated the use of intraliposomal
basic fibroblast growth factor (bFGF), a growth factor with antioxidant properties (36) that has the potential to enhance
transgene expression not only by its antioxidant properties
(36) but also through enhanced cellular uptake of DNA
(29) or, because bFGF produced within a cell is largely
directed toward receptors on the nuclear membrane (5, 22),
through facilitating delivery of DNA in the liposome-DNA complex to the
nuclear membrane.
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MATERIALS AND METHODS |
Materials.
1,[2-14C]Dioleoyl-sn-glycero-3-phosphoethanolamine
([14C]DOPE), [methyl-3H]thymidine, and
triethylammonium deoxycytidine 5'[
-33P]triphosphate
were from Amersham Canada (Oakville, Ontario, Canada). [8-14C]Adenine,
[5,6,8,9,11,12,14,15-3H(N)]prostaglandin
F2
, and 125I-bFGF were from New England
Nuclear (Boston, MA). Proteinase K was from Promega (Madison, WI).
Recombinant human bFGF, recombinant human FGF receptor 1
(IIIc)/Fc
chimera, and recombinant human nerve growth factor (NGF) receptor/Fc
chimera were from R&D Systems (Minneapolis, MN).
1,10-Phenanthroline was from ICN Biomedicals (Costa Mesa, CA).
Diphenyl-p-phenyldiamine was from Eastman Kodak (Rochester,
NY). 6-Hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid
(Trolox) was from Sigma-Aldrich (Oakville, Ontario, Canada). All other
chemicals were from Sigma (St. Louis, MO). Vetbond tissue adhesive was
from 3M (St. Paul, MN). Human erythrocyte catalase was from
Calbiochem-Novabiochem (San Diego, CA). DEAE Sepharose FF and CM
Sepharose FF were from Amersham (Baie d'Urfé, Québec, Canada). Porcine trypsin, heat-inactivated fetal bovine serum (FBS),
and Dulbecco's modified Eagle's medium (DMEM) were from GIBCO Canada
(Burlington, Ontario, Canada). Collagenase and type I DNase (2,367 U/mg) were from Worthington (Freehold, NJ). Restriction enzymes were
from Pharmacia (Baie d'Urfé, Québec, Canada).
1,2-Dioleoyldimethylammonium chloride (DODAC) was from Inex
Pharmaceuticals (Vancouver, British Columbia, Canada).
1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) was
from Avanti Polar Lipids (Alabaster, AL).
GL67/dimyristylphosphatidyl-ethanolamine-polyethylene glycol 5000 (DOPE/DMPE-PEG5000) was from Genzyme (Framingham, MA). A 10-ml
thermobarrel liposome extruder was from Lipex Biomembranes (Vancouver,
British Columbia), and extrusion filters were from Nucleopore
(Pleasanton, CA). The pCMV4-chloramphenicol acetyl transferase (CAT)
construct was a generous gift from Drs. K. Brigham and J. Conary
(Vanderbilt University School of Medicine, Nashville, TN). The secreted
human placental alkaline phosphatase plasmid (pSEAP) construct was
purchased from Tropix (Bedford, MA). An 8-isoprostane enzyme
immunoassay kit was from Cayman Chemical (Ann Arbor, MI). A rhodamine
nucleic acid labeling kit was from PanVera (Madison, WI), and the
labeling procedure was according to the manufacturer's instructions.
Fluorescent labeling of cell nuclei with 4',6-diamidino-2-phenylindole
(DAPI) was performed when cells were mounted with a DAPI-containing
mounting medium from Vector Laboratories (Burlingame, CA).
Cell culture.
Primary cultures of 19-day-gestation distal RFL19Ep were
prepared as previously described (20, 33, 34). Briefly,
the lungs of 19-day-gestation fetal rats were isolated and separated from major vessels and airways. The lungs were minced and gently vortexed to remove erythrocytes. This step was repeated until the
supernatant was clear. The minced lung tissue was then subjected to
proteolytic digestion with trypsin and DNase, a process that was
arrested by the addition of FBS. The resultant cell suspension was
eluted through 100-µm-mesh nylon bolting cloth. The eluted cells were
next subjected to a collagenase digestion, which was also arrested by
the addition of FBS, and fibroblasts were removed by differential
adherence. Epithelial cells of >95% purity, as assessed by staining
for cell cytoskeleton components, were obtained using this technique
(20). Cells were seeded in DMEM with 10% (vol/vol) FBS at
a sufficient cell density to allow near-confluence 24-48 h after
seeding, at which time the cells were changed to serum-free medium for
the individual experiments. Cells were maintained in an humidified gas
mixture of 3% O2, 5% CO2, and 92%
N2 to maintain the cells at a normal fetal arterial oxygen
tension of ~20 mmHg. Concentrations of catalase-inhibitable
H2O2 released into serum-free culture medium
collected over 24 h were confirmed (data not shown) to be similar
to those previously reported (24), when measured using an
alternate methodology (16).
Cationic liposome preparation.
An equimolar solution of DODAC/DOPE in chloroform was dried under
vacuum in a rotary evaporator, then maintained under vacuum for
24-48 h. The resulting lipid film was rehydrated at 1 µg total lipid/µl phosphate-buffered saline (PBS) and sonicated for 2× 30 s. For calculations of lipid recovery [14C]DOPE
was included in the initial lipid mixture. Liposomes were initially
extruded through sequentially smaller gauge syringe needles from 18- to
27-Fr and were then subjected to five freeze-thaw cycles with liquid
N2 and extrusion through a 400-nm filter (25). Liposomes were recovered by centrifugation at 165,000 g for
1 h. For DODAC/DOPE liposomes, plasmid DNA was added at a 1:10
(wt/wt) DNA-to-lipid ratio. The charge ratio that allows optimal uptake of DODAC/DOPE liposome/DNA complexes by RFL19Ep occurs in a
narrow band between DNA/lipid molar ratios of 1:10-1:15
(34). Preparations were diluted with culture medium as
necessary. GL67/DOPE/DMPE-PEG5000 was provided as a lyophilized powder,
which was vortexed in ultrapure water ± 20 ng/ml bFGF. For in
vivo studies with GL67/DOPE/DMPE-PEG5000 liposomes, DNA was added at a
3.6:0.6 (mol/mol) DNA-to-lipid ratio. The mixture was allowed to stand
at 30°C for 15 min, without mixing, to allow the formation of
lipid-DNA complexes.
When bFGF was included within liposomes, it was added to the
rehydration buffer at a concentration of 20 ng/ml, which we have previously defined to be the optimal concentration for stimulation of
RFL19Ep DNA synthesis when added to the culture medium
(32). In preliminary experiments (data not shown), it was
observed that bFGF added to preformed liposomes modestly reduced
transfection efficiency, presumably by altering the charge ratio of the
liposome-DNA complex. Liposomes were, therefore, washed in 0.05%
(wt/vol) trypsin and 0.02% (wt/vol) EDTA to degrade extraliposomal
bFGF. In preliminary experiments, liposomes were incubated with
125I-bFGF to allow 125I-bFGF/liposome complexes
to form. These were washed free of unassociated 125I-bFGF,
then washed in 0.05% (wt/vol) trypsin and 0.02% (wt/vol) EDTA for
1 h, which resulted in a 71 ± 2% (n = 3;
means ± SE) removal of 125I-bFGF from
125I-bFGF/liposome complexes.
For in vitro transfection experiments, liposome-DNA complexes were
added to cell cultures for 24 h at a lipid concentration of 2.5 nmol/cm2, as previously described (34). Uptake
of DODAC/DOPE liposome-DNA complexes by RFL19Ep is linear
up to 24 h, and exposure to a lipid concentration of 2.5 nmol/cm2 is not associated with cytotoxicity
(34).
Cytotoxicity index.
Cytotoxicity was assessed by the release of
[8-14C]adenine from cells preincubated with 0.2 µCi/ml
[8-14C]adenine (27). As reported elsewhere,
this assay has an ability to detect cell injury equivalent to
measurements of lactate dehydrogenase release (33) or
trypan blue exclusion (11). The medium containing [8-14C]adenine was added for 2 h before the
monolayer was washed 2× with fresh medium. The percentage of
preincorporated [8-14C]adenine released into the culture
medium was assessed after 48 h and expressed relative to the basal
release by cells not exposed to additives, which was given an arbitrary
index value of 1.
DNA synthesis.
This was assessed by incorporation of [3H]thymidine (1 µCi/ml) into cell DNA (17) under serum-free conditions.
The isotope was added 48 h after plating, and the duration of the
incubation was 24 h, as previously described (18).
Oxidants and antioxidants.
For studies of intracellular plasmid DNA stability, Trolox (50 µM)
was added to the culture medium at the time of liposome addition. After
24 h, when the medium was changed, fresh medium containing 50 µM
Trolox was added for a further 24 h. For studies of transgene
expression, Trolox (50 µM), diphenyl-phenyldiamine (0.2 µM), and
phenanthroline (100 nM) were introduced into the culture medium at the
time of liposome addition and maintained in fresh culture medium for
48 h after the 24-h transfection period. Cell counts were
performed on an automated cell counter (Coulter Electronics, Hialeah,
FL). Accuracy of cell counts was regularly validated against
hemocytometer counts. Measurement of 8-isoprostane by immunoassay
was used as an index of lipid peroxidation (26). We have
previously validated this measurement against aldehyde production by
RFL19Ep as assessed by gas chromatography-mass spectrometry (34). Results have been presented as picograms per
milligrams of protein, with protein measurements made according to the
method of Bradford (6).
DNA degradation.
Plasmid DNA alone or an equivalent amount of DNA in a liposome-DNA
complex was exposed to 0.5, 1, 2, or 5 µM
H2O2 for 24 h or to 20 mM
H2O2 ± 0.1 or 1 µM cupric
(Cu2+) sulphate for 10 min, to generate the hydroxyl
radical (3). In preliminary studies,
H2O2-mediated degradation of plasmid DNA was
shown to be inhibitable by preincubation of the
H2O2 solution with either erythrocyte catalase
(100 U/ml) or Trolox (2 mM). Catalase was removed from the
solution, prior to further treatment of the plasmid, by stirring with
sequential additions of DEAE Sepharose FF and CM Sepharose FF, followed
by centrifugation. The plasmid was linearized using 100 units of the
restriction enzyme SmaI with 100 µg of pCMV4-CAT in 100 µl of buffer (containing in mM: 100 Tris acetate, 100 Mg acetate, and
500 K acetate, pH 7.5) at 30°C overnight. After a 24-h exposure to
DNA with cationic liposomes, cells were washed twice with culture
medium to remove any free liposome-DNA complexes. Cells were harvested
immediately after washing or, after a further 24 h in culture, by
treatment with 0.1% (wt/vol) trypsin and 0.001% (wt/vol) DNase and
centrifugation, to remove cell surface-associated liposomes and DNA
(8). After resuspension in PBS, a DNA extract was prepared
from the cells with proteinase K (13). DNA extracts were
subjected to electrophoresis in 1% (wt/vol) agarose gels. Blots were
probed with probes specific to the CAT nucleotide sequence.
DNA uptake.
For studies of DNA uptake, pCMV4-CAT plasmids were nick translated with
triethylammonium deoxycytidine 5'[
-33P]triphosphate.
We removed unincorporated nucleotides by passing the reaction mixture
through a small column of Sephadex G-50. For uptake experiments,
[33P]plasmid DNA was mixed with unlabeled plasmid DNA to
give a final specific activity of ~1.6 × 108
dpm/µg DNA. Integrity of the labeled DNA was confirmed by
electrophoresis on a 1% (wt/vol) agarose gel with transfer onto a
nylon membrane and autoradiography. For studies of liposome-associated
plasmid uptake, the cells were harvested at the end of the incubation period by treatment with 0.1% (wt/vol) trypsin and 0.001% (wt/vol) DNase and centrifugation. Trypsin removes cell surface-associated liposomes (8), and DNase was included to degrade any
surface-associated DNA not associated with liposomes. Total DNA uptake
was calculated from the 33P-content of the cell pellet.
Reporter gene activity.
The bacterial CAT gene, which is not present in eukaryotic cells, was
used as one reporter gene to measure transgene expression. The gene
product is an enzyme that catalyses the transfer of acetyl groups to
the substrate chloramphenicol from acetyl 1-CoA. Catalytic activity was
determined by a radiometric assay, as described by Stribling et
al. (30). In other studies, a mammalian
expression vector containing SEAP was used as a reporter gene
(4) with chemiluminescent detection. Reporter gene
activities were measured 48 h after the end of the exposure of
cultured cells to liposome-DNA complexes or 48 h after
intratracheal instillation of liposome-DNA complexes.
Fluorescent DNA localization.
Digital images of fluorescent DNA and cell nuclei were superimposed for
calculations of the number of nuclear membranes in direct contact with
fluorescent DNA. For these studies, the cells were cultured on
chambered glass slides from Nalge Nunc International (Naperville, IL).
Two to three random fields were used from each of four separate
chambers, and mean values from each chamber used for calculations of
means ± SE.
Instillation of liposomes into mouse lungs.
Female CD1 mice at 10-12 wk of age were used. Inhalational
anesthesia was established with 2% ethrane in 60% nitrous oxide with
40% oxygen in a chamber. Anesthesia was maintained with a nose cone. A
small skin incision was made over the trachea for insertion of a 30-Fr
needle to inject the liposome-DNA suspension into the trachea. The skin
incision was closed with veterinary tissue adhesive. Liposome-DNA
suspensions were in a final volume of 100 µl of ultrapure water. This
contained 33.9 µg of GL67/DOPE/DMPE-PEG5000 with 118.8 µg of
plasmid DNA. For comparison, some animals received plasmid DNA alone.
Animals were killed 48 h after instillation of the DNA or
liposome-DNA suspensions to allow removal of lung tissue for
assay of reporter gene activity.
Statistical analysis.
All values are shown as means ± SE of each group. Where error
bars are not evident in the figures they fall within the plot point.
Where figures show data from representative experiments, these
experiments have been replicated on two to four occasions. Statistical
significance (P < 0.05) was generally determined by analysis of variance followed by assessment of differences using Duncan's multiple range test (28).
 |
RESULTS |
To determine whether plasmid DNA was susceptible to
H2O2-mediated injury, we exposed plasmid DNA to
0.5-5 µM H2O2 for 24 h before and
after being complexed with liposomes. As shown in Fig. 1, plasmid DNA exposed to 0.5-5 µM
H2O2 showed a concentration-dependent loss of
supercoiled DNA, with an increase in the relaxed form, consistent with
oxidant injury. However, when the plasmid DNA was complexed to cationic
liposomes, the DNA was protected against injury. In contrast,
complexing DNA with liposomes did not protect it against a 10-min
exposure to hydroxyl radicals (Fig. 2).
When hydroxyl radical generation was initiated with low concentrations of Cu2+ sulfate (0.1 µM), an increase in relaxed DNA was
evident with or without conjugation with liposomes. At an increased
concentration of Cu2+ sulfate (1 µM), DNA was totally
degraded with a complete loss of DNA bands, and no protection was seen
when DNA was complexed with liposomes. In control experiments (data not
shown), 0.1 and 1 µM Cu2+ sulfate alone had no effect on
the DNA bands.

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Fig. 1.
Plasmid DNA was separated on 1% agarose gels before (Pl) and after
(L) linearization as standards. Compared with control plasmid DNA (C),
pCMV4-chloramphenicol acetyl transferase (CAT) DNA that had been
exposed to 0.5, 1, 2, or 5 µM H2O2 (H0.5, H1,
H2, H5) for 24 h showed a concentration-dependent loss of the
supercoiled form with a reciprocal increase in the damaged relaxed
form. However, when plasmid DNA was complexed with liposomes, the DNA
was protected against injury at these concentrations of
H2O2.
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Fig. 2.
pCMV4-CAT DNA was separated on 1% agarose gels before
(Pl) and after (L) linearization as standards. The hydroxyl radical was
generated by the addition of cupric (Cu2+) sulfate to 20 mM
H2O2 (H) for 10 min and compared with unexposed
control samples (C). In the presence of 0.1 µM Cu2+
sulfate, a small increase in the relaxed form of plasmid DNA was
evident. In the presence of 1 µM Cu2+ sulfate, all DNA
forms were completely degraded, and no protection was evident from
complexing DNA with liposomes.
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That cell-derived generation of ROS contributes to degradation of
transfected DNA intracellularly was suggested by an experiment in which
plasmid DNA was extracted from RFL19Ep after transfection in the presence or absence of the antioxidant Trolox, which we have
previously shown to scavenge H2O2, arrest lipid
peroxidation, and attenuate hydroxyl radical formation in
oxidant-stressed RFL19Ep (24). The loss of
supercoiled DNA with time in culture was slowed by the presence of
Trolox, with an ~2-2.5-fold difference in supercoiled DNA
content (Fig. 3). In contrast, cells
transfected and maintained in the presence of Trolox had clearly
detectable bands of supercoiled DNA, consistent with reduced
oxidant-mediated DNA injury. That antioxidants can not only preserve
cell content of transfected DNA with time in culture but also enhance
transgene expression is shown in Fig.
4A. The presence of Trolox (50 µM), diphenyl-phenyldiamine (0.2 µM), or phenanthroline (0.1 µM)
in the culture medium resulted in a 2-2.5-fold increase
(P < 0.05) in transgene expression. When liposomes
containing bFGF (20 ng/ml) were used for transfection, transgene
expression was significantly enhanced (P < 0.05) over and above that seen with either Trolox or phenanthroline in the culture
medium (Fig. 4B). No additional transgene expression was observed by the combined addition of Trolox or phenanthroline with bFGF
(data not shown), suggesting that the effect of bFGF was not simply due
to an antioxidant effect alone. An increased intracellular content of
plasmid DNA following transfection with liposomes containing bFGF was
confirmed, as shown in Fig. 5.

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Fig. 3.
pCMV4-CAT DNA was separated on 1% agarose gels before
(Pl) and after (L) linearization as standards. Separated simultaneously
was 20 µg of DNA that had been extracted from rat fetal lung
epithelial cells (RFL19Ep) immediately after transfection
(C24, T24) or 24 h later (C48, T48). Cells were cultured in the
presence (T24, T48) or absence (C24, C48) of 50 µM Trolox. An
increased abundance of the supercoiled form of plasmid DNA was evident
at 24 h (2.56-fold) and 48 h (2.05-fold) in cells that had
been cultured in the presence of Trolox.
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Fig. 4.
A: the addition of 50 µM Trolox (T) , 0.2 µM
diphenyl-phenyldiamine (D), and 0.1 µM phenanthroline (P) as
antioxidant interventions before and during transfection of
RFL19Ep significantly enhanced pCMV4-CAT reporter gene
expression compared with additive-free control (C) cells. B:
RFL19Ep transfected with liposome-DNA complexes in which
the liposomes contained 20 ng/ml of basic fibroblast growth factor
(bFGF, B) had a significantly increased expression of the pCMV4-CAT
reporter gene relative to cells transfected with the liposome-DNA
complex in the absence of intraliposomal bFGF (C). This effect was
significantly greater than the enhanced reporter gene expression
observed with cells transfected in the presence of 50 µM Trolox (T)
or 0.1 µM phenanthroline (P) in the culture medium (n = 4; means ± SE; *P < 0.05 vs. control cells;
#P < 0.05 vs. all other groups).
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Fig. 5.
pCMV4-CAT DNA was separated on 1% agarose gels before
(Pl) and after (L) linearization as standards. Separated simultaneously
was 20 µg of DNA that had been extracted from replicate
RFL19Ep immediately after a 24-h transfection with the
liposome-DNA complex in the presence (B) or absence (C) of
intraliposomal bFGF. An increased abundance of intracellular plasmid
DNA was evident at 24 h in cells that had been transfected with
the liposome-DNA complex containing intraliposomal bFGF.
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That bFGF did have antioxidant activity when RFL19Ep were
treated with liposomes (2.5 nmol/cm2) containing 20 ng/ml
bFGF for 24 h is shown in Fig. 6.
Control cells or cells treated with liposomes containing buffer alone exposed to 50% O2 had a significant (P < 0.05) increase in 8-isoprostane release, as previously described
(24). Cells treated with liposomes containing bFGF had
both a significantly (P < 0.05) reduced basal level of
8-isoprostane release in 3% O2 and a significant
(P < 0.05) inhibition of the 50%
O2-mediated increase in 8-isoprostane production. There was
no liposome- or bFGF-mediated effect on protein recovered from cells
exposed to 50% O2. Another potential contributor to the
enhanced transgene expression observed with intraliposomal bFGF was
enhanced binding to the cell surface through an FGF receptor. Should
some bFGF escape the liposome but remain liposome-DNA complex
associated, bFGF that had not been completely removed by washing
liposomes in a trypsin and EDTA solution after preparation could act as
a ligand for this receptor. To test this, accessible bFGF was exposed
to a truncated soluble receptor to reduce its availability for binding
to cell surface receptors. That the selected concentration of truncated
receptor (5 ng/ml) could block bFGF activity was confirmed with DNA
synthesis as an index of bFGF activity secondary to binding to FGF
receptors at the cell surface. As shown in Fig.
7A, the truncated soluble receptor attenuated the significant (P < 0.05)
bFGF-mediated increase in cell DNA synthesis and also significantly
(P < 0.05) reduced basal levels of DNA synthesis. The
specificity of this effect was studied in separate experiments using a
similarly constructed truncated soluble receptor to NGF. The truncated
soluble receptor to NGF had no effect on cell DNA synthesis, and
neither soluble receptor was cytotoxic at a concentration of 5 ng/ml
(data not shown). Having confirmed that the truncated soluble FGF
receptor was active at a concentration of 5 ng/ml, we added this
concentration to the culture medium during transfections in the
presence or absence of intraliposomal bFGF. The soluble truncated FGF
receptor had no significant (P > 0.05) inhibitory
effect on the enhanced (P < 0.05) transgene expression
brought about by the presence of intraliposomal bFGF, and there was no
independent effect (P > 0.05) of extraliposomal bFGF
(Fig. 7B).

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Fig. 6.
RFL19Ep were exposed to 3 or 50%
O2, and lipid peroxidation was assessed by measurement of
8-isoprostane. Exposure to 50% O2 increased 8-isoprostane
formation. Cells treated with liposomes containing 20 ng/ml of bFGF
(solid bars) had a significantly reduced 8-isoprostane formation in
both 3 and 50% O2-exposed cells, relative to untreated
cells (open bars). Cells treated with liposomes containing buffer only
(hatched bars) had no significant change in 8-isoprostane formation
(n = 4; means ± SE; *P < 0.05 vs. additive-free control cells at the same O2
concentration; #P < 0.05 vs. additive-free control
cells at 3% O2).
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Fig. 7.
A: exposure of RFL19Ep to 20 ng/ml
of bFGF (B) in the culture medium significantly increased cell DNA
synthesis over that observed under additive-free control conditions
(C). Addition of 5 ng/ml of chimeric truncated soluble FGF receptor to
the culture medium had no significant effect on DNA synthesis under
control conditions (SR) but did attenuate the increase in DNA synthesis
observed in response to bFGF (B/SR). B: expression of the
secreted human placental alkaline phosphatase (SEAP) transgene was
enhanced by the presence of intraliposomal bFGF (lB) compared with
transfection in the absence of intraliposomal bFGF (C). Neither the
addition of 5 ng/ml of truncated soluble FGF receptor (IB + SR and C + SR) nor the presence of 20 ng/ml of bFGF in the medium (mB) had
significant effects on transgene expression (n = 4;
means ± SE; *P < 0.05 vs. control cells).
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Recognizing that liposome-DNA complexes prepared with intraliposomal
bFGF might also affect DNA uptake by a variety of other mechanisms, we
examined radiolabeled DNA uptake in the presence or absence of
intraliposomal bFGF (Fig. 8).
Liposome-DNA complexes prepared with intraliposomal bFGF had a
significantly (P < 0.05) reduced uptake after 1, 6, and 12 h of incubation but not after 24 or 48 h.

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Fig. 8.
Uptake of radiolabeled liposome-DNA complexes by
RFLE19Ep in the presence (closed circles) or absence (open
circles) of intraliposomal bFGF. The presence of intraliposomal bFGF
significantly inhibited uptake of liposome-DNA complexes after 1, 6, and 12 h of incubation, but not after 24 or 48 h
(n = 4; means ± SE; *P < 0.05 vs. control cells at the same time point).
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Next, we sought to determine whether intraliposomal bFGF enhanced
perinuclear localization of transfected DNA, using plasmid DNA that had
been tagged with a fluorescent marker. Cells transfected with
bFGF-containing fluorescent DNA-liposome complexes for 24 h had
very different patterns of fluorescence when compared with cells
transfected with fluorescent DNA-liposome complexes not containing
bFGF. Fluorescent DNA appeared randomly distributed over the slide when
bFGF-free liposomes were used (Fig.
9D), whereas cells that had
been transfected with bFGF-containing liposomes had a proportion of
cells with a distinctive perinuclear localization of fluorescent
plasmid DNA (Fig. 9B). At 48 h, more fluorescent DNA
was evident in cells that had been transfected with liposomes containing bFGF (Fig. 10B)
than cells treated with control liposomes (Fig. 10D).
Fluorescent DNA was observed in contact with the nuclear membrane of
9.7 ± 1.1% of cells transfected with control liposomes, but this
was significantly increased (P < 0.05;
n = 4; means ± SE) to 37.3 ± 3.4% for
cells transfected with liposomes containing bFGF.

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Fig. 9.
Photomicrographs to show localization of
4',6-diamidino-2-phenylindole (DAPI)-stained nuclei (A,
C, E) or rhodamine-labeled DNA (B,
D). RFL19Ep were transfected with fluorescent
(A-D) or control (E, F)
liposome-DNA complexes for 24 h. Fluorescent DNA (arrow,
B) encircling nuclei (arrow, A) was observed in
some cells treated with the liposome-DNA complex containing bFGF
(B), but not in cells treated with the bFGF-free
liposome-DNA complex (D).
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|

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Fig. 10.
Photomicrographs to show localization of DAPI-stained nuclei
(A, C, E) or rhodamine-labeled DNA
(B, D). RFL19Ep were transfected with
fluorescent (A-D) or control (E,
F) liposome-DNA complexes for 24 h, followed by a
further 24 h in culture. Loss of fluorescent DNA during time in
culture was significantly greater in cells treated with the bFGF-free
liposome-DNA complex (D) than in cells treated with the
liposome-DNA complex containing bFGF (B).
|
|
Last, we used mice to determine whether our observations of enhanced
reporter gene expression with bFGF in vitro would also be evident in
vivo. As shown in Fig. 11, when
GL67/DOPE/DMPE-PEG5000 liposomes, prepared containing either control
buffer or 20 ng/ml of bFGF, were instilled via the trachea into mice, a
significant increase in pulmonary transgene expression was observed
with the inclusion of bFGF in the liposomes (n = 5).
There were no grossly obvious differences in lung histology following
instillation of the two types of liposomes.

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Fig. 11.
GL67/1,2-dioleoyl-sn-glycero-3-phosphoethanolamine/dimyristylphosphatidyl-ethanolamine/polyethylene
glycol 5000 (DOPE/DMPE-PEG5000) liposomes were prepared containing
either control buffer (hatched bar) or 20 ng/ml of bFGF (solid bar) for
in vivo instillation in mice. Naked DNA alone was also instilled for
comparison (open bar). A significant increase in pulmonary transgene
(pCMV4-CAT) expression was observed with the inclusion of bFGF in the
liposomes (n = 5; means ± SE; *P < 0.05 vs. control liposome expression or naked DNA alone).
|
|
 |
DISCUSSION |
The prototypic lung disease for which gene therapy has been
proposed is cystic fibrosis. Adenovirus-mediated transfer of the human
cystic fibrosis transmembrane conductance regulator gene has been less
successful than originally anticipated, in that it is associated with
dose-limiting toxicity, low efficiency, and brief efficacy, in part due
to induction of an adenovirus-specific cell-mediated immunity
(38). Liposome-mediated gene transfer has certain
advantages over viral gene transfer, in that this approach is both
nonimmunogenic and nononcogenic, but it suffers from being less
efficient than the viral approach (37). Several barriers
to efficient gene transfer have been identified, including inactivation
by cystic fibrosis sputum (23) and the presence of
preexisting inflammation (35). We hypothesized that
intracellular and extracellular ROS may contribute to these barrier
effects not only in cystic fibrosis but also in the various acute lung injuries that may be amenable to a gene therapy approach
(7).
The feasibility of cationic lipid-mediated gene transfer to the
developing human and rodent lung has been established
(19). Our interest has been in using this approach for
studies of neonatal lung injury, for which reason we conducted our
experiments with distal lung epithelial cells from preterm rat fetuses.
We assume that the majority of H2O2 found in
the aqueous hypophase at the surface of the lung epithelium will be
derived from lung epithelial cells and macrophages. Consistent with
this, fetal lung epithelial cells in culture have a medium
concentration of H2O2 similar to that reported
for normal breath condensate (24). Under normoxic conditions, 1-2% of mitochondrial oxygen consumption can be
accounted for in the production of superoxide and, upon dismutation,
H2O2 (10). It has been recently
recognized that ROS are important signal transduction molecules for
various cell processes, including growth, under physiological
conditions (31). In the absence of phagocyte
myeloperoxidase, to form hypochlorous acid, the amount of
H2O2 present in normal breath condensate
(1, 18) and cell culture medium from RFL19Ep
(24) would not be sufficient to significantly damage
plasmid DNA. However, moderate damage would be evident at
concentrations obtained from inflamed lungs (15). Plasmid
DNA was protected from supraphysiological concentrations of
H2O2 when complexed with liposomes, although
not from hydroxyl radical-mediated injury.
Trolox, at a concentration that attenuates hydroxyl radical
formation, arrests lipid peroxidation and scavenges
H2O2 (24), and limits
intracellular degradation of transfected supercoiled DNA, consistent
with this degradation being mediated by ROS. Transfection in the
presence of either of two antioxidants, Trolox and
diphenyl-phenyldiamine, increased transgene expression
~2-2.5-fold. That these agents act through limiting hydroxyl
radical-mediated DNA injury is suggested by an equivalent effect on
transgene expression of phenanthroline, a cell-permeant iron chelator,
at a concentration that we have previously shown to prevent increased
hydroxyl radical production when RFL19Ep were exposed to
hyperoxic conditions (24).
Of particular interest are the findings using intraliposomal
bFGF, which resulted in an approximately fourfold increase in transgene
expression. This growth factor has been reported to have antioxidant
properties (36), which we have been able to confirm. It
is, therefore, likely that an antioxidant effect contributes to its
capacity to enhance transgene expression. The mechanism by which the
remainder of this effect is mediated is not clear. Treatments of
bFGF-containing liposomes with soluble FGF receptor and trypsin suggest
that enhanced attachment of the liposome-DNA complex to the cell
through binding of monovalent bFGF to FGF receptors on the cell is not
the mechanism. Such treatments do not exclude such an effect through
multimeric multivalent bFGF complexes masked in the liposome-DNA
complex, which become unmasked on attachment of the liposome-DNA
complex to the cell. However, because inclusion of bFGF in liposomes
did not enhance their uptake, the bFGF-mediated enhancement of
transgene expression cannot be attributed to an effect of enhanced
binding to an FGF receptor, a conformational change, or a modification
of surface charge on uptake of the liposome-DNA complex. Our finding of
enhanced localization of fluorescent DNA to the nuclear membrane should
be interpreted with caution. It is possible that enhanced nuclear
targeting, through a chaperone effect of bFGF directed at FGF receptors
on the nuclear membrane (5, 22), may contribute to this
finding. However, this observation may simply reflect a reduction in
DNA degradation with enhanced retention of DNA either at the nuclear membrane or within endosomes.
In summary, intraliposomal bFGF significantly enhances transgene
expression both in vitro and in vivo. This effect is in part due to the
antioxidant properties of bFGF, but other mechanisms also appear to be
involved. Intraliposomal bFGF may be suitable for use as an adjuvant
for studies in humans, in that a recombinant human protein, which would
be nonimmunogenic, is available.
 |
ACKNOWLEDGEMENTS |
This work was supported by a Research and Development Programme
Grant from the Canadian Cystic Fibrosis Foundation, a Group Grant from
the Canadian Institutes for Health Research, and Chinese National
Science Fund for Distinguished Young Scholars Grant 30125019. A. K. Tanswell holds the Hospital for Sick Children Women's Auxiliary Chair in Neonatology.
 |
FOOTNOTES |
Address for reprint requests and other correspondence:
A. K. Tanswell, Div. of Neonatology, Hospital for Sick
Children, 555 Univ. Ave., Toronto, Ontario, Canada, M5G 1X8
(E-mail: keith.tanswell{at}sickkids.ca).
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.
First published January 3, 2003;10.1152/ajplung.00479.2001
Received 14 December 2001; accepted in final form 18 December 2002.
 |
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