Enhanced Collateral Growth by Double Transplantation of Gene-Nucleofected Fibroblasts in Ischemic Hindlimb of Rats
et al. (2011) Enhanced Collateral Growth by Double Transplantation of Gene-Nucleofected
Fibroblasts in Ischemic Hindlimb of Rats. PLoS ONE 6(4): e19192. doi:10.1371/journal.pone.0019192
Enhanced Collateral Growth by Double Transplantation of Gene-Nucleofected Fibroblasts in Ischemic Hindlimb of Rats
Ziyang Zhang 0
Alex Slobodianski 0
Wulf D. Ito 0
Astrid Arnold 0
Jessica Nehlsen 0
Natalie Lund 0
Jihong Liu 0
Jose -Toma s Egan a 0
Jo rn A. Lohmeyer 0
Daniel F. Mu ller 0
Hans-Gu nther Machens 0
Carlo Gaetano, Istituto Dermopatico dell'Immacolata, Italy
0 1 Department of Plastic Surgery and Hand Surgery, Faculty of Medicine, University Hospital Rechts der Isar, Technische Universita t M u nchen , Munich, Germany , 2 Department of Plastic Surgery and Hand Surgery, University of Lu beck , Lu beck, Germany , 3 Cardiovascular Center Oberallgaeu, Academic Teaching Hospital, University of Ulm , Immenstadt, Germany , 4 Department of Cardiovascular Diseases, School of Medicine, Sir Run Run Shaw Hospital, Zhejiang University , Hangzhou, China, 5 Experimental Angiology , Medical Department II, University Hospital Lu beck , Lu beck, Germany , 6 Department of Urology, Tongji Hospital, Huazhong University of Science and Technology , Wuhan , China , 7 Facultad de Ciencias, Center for Genome Regulation, Universidad de Chile , Santiago , Chile
Background: Induction of neovascularization by releasing therapeutic growth factors is a promising application of cellbased gene therapy to treat ischemia-related problems. In the present study, we have developed a new strategy based on nucleofection with alternative solution and cuvette to promote collateral growth and re-establishment of circulation in ischemic limbs using double transplantation of gene nucleofected primary cultures of fibroblasts, which were isolated from rat receiving such therapy. Methods and Results: Rat dermal fibroblasts were nucleofected ex vivo to release bFGF or VEGF165 in a hindlimb ischemia model in vivo. After femoral artery ligation, gene-modified cells were injected intramuscularly. One week post injection, local confined plasmid expression and transient distributions of the plasmids in other organs were detected by quantitative PCR. Quantitative micro-CT analyses showed improvements of vascularization in the ischemic zone (No. of collateral vessels via micro CT: 6.862.3 vs. 10.162.6; p,0.05). Moreover, improved collateral proliferation (BrdU incorporation: 0.4860.05 vs. 0.5760.05; p,0.05) and increase in blood perfusion (microspheres ratio: gastrocnemius: 0.4160.10 vs. 0.5060.11; p,0.05; soleus ratio: soleus: 0.4260.08 vs. 0.6060.08; p,0.01) in the lower hindlimb were also observed. Conclusions: These results demonstrate the feasibility and effectiveness of double transplantation of gene nucleofected primary fibroblasts in producing growth factors and promoting the formation of collateral circulation in ischemic hindlimb, suggesting that isolation and preparation of gene nucleofected cells from individual accepting gene therapy may be an alternative strategy for treating limb ischemia related diseases.
Funding: This work was supported by grants from Innovations fund Schleswig-Holstein and University Hospital rechts der Isar, Technische Universitat M unchen
to HGM; a clinic research grant from Technische Universitat M unchen to ZZ (KKF. No. 8744556); German Research Council (DFG) IT-13/1, IT- 13/2, IT-13/3 to WDI
and FONDAP (Nr. 15090007) to JTE. ZZ was supported by a scholarship from the China Scholarship Council. The funders had no role in study design, data
collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
. These authors contributed equally to this work.
Lower extremity ischemia causes many clinic disorders. Patients
suffer from a slight muscle pain or walking problems to lower leg
ulceration and gangrene or even amputation. Although surgical
procedures can help some patients with arterial occlusions, new
treatment approaches are still needed for the patients who are not
suitable for surgery . Therapeutic neovascularization based on
proangiogenic growth factors has been suggested as a possible
clinical approach [2,3,4,5]. In this regard, the use of recombinant
proangiogenic growth factors has been tested in both pre-clinic
and clinical trials in previous studies. For example, Baffour R et al.
proved that recombinant bFGF could enhance angiogenesis and
growth of collaterals by in vivo administration in a rabbit hindlimb
ischemic model . In addition, VEGF was found to improve
neovascularization in a hindlimb ischemia model . However,
although some of the results were encouraging from the past
researches and randomized placebo-controlled double-blind
clinical trials with recombinant proteins, there are still some
suboptimal results which are needed to be addressed. In particular,
Lederman et al. reported that an improvement occurred in peak
walking time 90 days after treatment in TRAFFIC trial with
bFGF. However, this therapeutic effect was not observed at other
time points . In a randomized VEGF clinical trial (RAVE trial)
with AdVEGF-121, there was no clinical therapeutic effects
observed . One of the possible explanations for failure of growth
factor delivery in those studies could be owing to the direct
administration of single growth factor, which could not induce
enough therapeutic effects.
The combined administration of both bFGF and VEGF has
been tested recently and found to have synergistic effects. In 1995,
Asahara et al. proved for the first time that such synergistic effects
of bFGF and VEGF enhanced collateral growth in a rabbit
hindlimb ischemia model when they were administrated with a
ratio of around 1:50 (bFGF:VEGF) . However, the usual
limitations of protein therapies are the low half life of the
recombinant proteins (VEGF: 3 to 6 minutes in vivo and bFGF
1.5 minutes to 3 minutes in vivo [11,12,13]) and the possible side
effects associated with high doses of exogenous proteins (apart
from the possible tumorigenic effects because of inducement of
angiogenesis, high dose VEGF could cause edema due to an
increased microvascular permeability[14,15], high dose bFGF
could cause hypotension related to a dose-dependent vasodilating
Currently, gene therapy has emerged as a rational approach to
constantly produce and release proangiogenic molecules in the
ischemic area [17,18,19]. In this regard, Lee et al. demonstrated
that injection of both bFGF and VEGF plasmids together
intramuscularly improved the therapeutic effects in ischemic
damage. However, the short biological half life of the both factors
owing to the immune clean effects was found to lead to failure of
long-term therapeutic efficacy. To improve the efficiency of gene
transfection, many previous studies used viral vector to enhance
transfection efficiency by direct delivery of gene construct or via
injection of cells transfect with viral vectors. Kondoh et al.
reported that virus-based ex vivo gene transfer method 
increased transfection efficiency; in addition, high efficiency of
adenovirus-based ex vivo fibroblast gene transfer was also
established for bFGF and VEGF genes. However, owing to the
biohazard associated with the use of viral vectors, viral gene
therapy is not yet widely accepted for clinical use.
In the present study, we developed a new strategy using ex vivo
gene transfer of autologous cells via non-viral nucleofection
technique to maximize the bFGF and VEGF expression in in vitro
and in vivo experiments. This strategy was proven to have the
highest transfection efficiency when compared to other classic
nonviral methods, including the original nucleofection method. In the
animal model of hindlimb ischemia, the delivered primary
fibroblasts remarkably increased the formation of collateral vessels
and improved blood supply to ischemic tissue area, as measured by
micro-CT 3D reconstructions of microvascular networks and
quantification of arteriogenesis and angiogenesis.
Materials and Methods
Cell isolation and characterization
Fibroblasts were isolated from skin obtained from the back of
inbred rats. Briefly, the samples were cut into 4 cm61 mm strips
and then incubated with Dispase-2 (Roche, Penzberg, Germany).
Subsequently, the epidermal layer was carefully removed and the
dermis was minced and then incubated for 3 hours under
magnetic rotation with 0.1% collagenase (Roche, Penzberg,
Germany). After that, the mixture was filtered through a
100mm Cell Strainer (BD biosciences, Hamburg, Germany) to obtain
the primary cells. For characterization, cytospinned cells and
normal cells seeded for 48 hours were fixed in ice-cooled ethanol
for 30 minutes. Subsequently, the cells were stained with anti
fibroblast antibody (Prolyl 4-hydroxylase subunit beta: P4H beta,
Acris, Heidelberg, Germany) and tetramethyl rhodamine
isothiocyanate conjugated phalloidin (Sigma-Aldrich, MO, USA)
according to the manufacturers instructions. Then, the cells were
mounted in Prolong-containing DAPI (Invitrogen, Oregon, USA).
Afterwards, cell morphology was analyzed by phase-contrast/
fluorescence microscopy (Nikon, eclipse te2000-s).
High-efficient ex vivo nucleofection
Nucleofection was performed under different conditions, and
16106 primary rat skin fibroblasts (passage 2, 8090% confluent)
were trypsinized and harvested by centrifugation at 1006g for
10 min. The original nucleofection protocol was conducted
following the suggested instructions (Basic Primary Mammalian
Fibroblast NucleofectorH Kit, Lonza, Cologne, Germany). The
supernatant was removed and the cell pellets were resuspended in
100 ml of nucleofection solution or 100 ml of Dulbeccos Modified
Eagle Medium(DMEM) supplemented with 10% Fetal Calf Serum
(FCS) for the purpose of comparing different nucleofection
solutions. Afterwards, 4 mg of pmaxGFPH plasmids (Lonza) were
mixed with the cells and subsequently transferred into
nucleofection cuvettes. We also tested different transfection cuvettes and
found that Eppendorf electroporation cuvettes (4-mm gap,
Eppendorf, Hamburg, Germany) were as efficient as Amaxa
cuvettes (2-mm gap, Amaxa, Cologne, Germany). Subsequently,
the cells were nucleofected by using the U30 program from the
nucleofection device and 500 ml of pre-warmed culture medium
was immediately added to the cells. The cells were trypsinized and
analyzed with a CASYH system (Innovatis AG, Reutlingen,
Germany) for cell number and cell viability 48 hours after
transfection. Transfection efficiency and apoptosis were quantified
by fluorescent activated cell sorting (FACS; Cytomation MoFloH
Flow Cytometer, Dako, Denmark). DMEM+10% FCS,
Eppendorf cuvettes (4-mm gap, Eppendorf, Hamburg, Germany) and
U30 program were chosen for the in vitro and in vivo study.
Plasmids encoding for VEGF165 and bFGF were constructed
based on pmaxGFPH backbone (Lonza). The modified VEGF165
nucleofection protocol was as following: 1) Fibroblasts (36106,
passage 2) were mixed with 12 mg of VEGF165 plasmid and
100 ml of nucleofection solution (DMEM+10% FCS); 2) the
mixture was then transferred into Eppendorf electroporation
cuvettes (4-mm gap) and nucleofected with single electropulse
program U30. After that, cells were transferred into a T25 culture
flask and 4 ml DMEM+10% FCS were added; 3) the above two
steps were repeated for 5 times with a total of 156106 cells. 4)
After 24 hours, VEGF165 nucleofected cells were collected and
counted, only 56106 cells were obtained for in vivo administration.
The bFGF nucleofection protocol was conducted at the same time
as VEGF165 nucleofection with another 156106 cells to obtain
56106 bFGF nucleofected cells. Cells (56106 with bFGF and
56106 with VEGF165) were then mixed before in vivo cell
Transfected cells (1.46105) were seeded in 12-well plates and
cultured in 1.5 ml of DMEM + 10% FCS. Subsequently, medium was
changed and collected daily. Concentrations of VEGF165 and bFGF
were quantified by Bio-Plex Suspension Array System, following
manufacturers instructions (Bio-Rad, Hamburg, Germany).
Hindlimb ischemia model
Experiments were performed on male Lewis inbred rats (weight
200 grams, Charles River Laboratories, Sulzfeld, Germany).
Femoral artery ligation was performed as described previously
. All in vivo procedures were approved by animal committee of
Luebeck University (No.1/1m/09).
Animals were randomly divided into 2 groups of treated and
control animals. Directly after ligation of the right femoral artery,
the cells (16107) transfected with VEGF165 or bFGF (56106
each) were intramuscularly injected into the gracilis muscle and
adductor muscles in the middle part of the thigh because it has
been demonstrated that this is the site of major collateral growth in
the rat model employed but also in other animal models [21,22].
This is also the site of major collateral growth in humans when the
superficial femoral artery is occluded and was consequently chosen
as main injection site in the TAMARIS and WALK trials .
The same number of cells without transfection was injected into
the same site in control group.
Quantitative micro-CT system
High-resolution desktop X-ray micro-CT system (SkyScan1072,
SkyScan, Belgium) was used to visualize and quantify the vascular
networks. Seven days after femoral artery ligation, micro-CT
angiographies were performed as described before with modifications
, After perfusion with the contrast medium, the ischemic zone of
limbs was reconstructed from Z-axis cross section slices. After
visualization and reconstruction of the vascular networks in the
ischemic zone, collateral vessels were quantified and compared
between the control and treated animals as described earlier .
Typical corkscrew-like collateral vessels were counted in 3D view.
Compared to X-ray view, collaterals were easier to identify. After 3D
reconstructions, the bone structures were subtracted and the vessel
volume was quantified. Voxel number information from
reconstruction slices of the ischemia zone (600 cross-sections below the proximal
end of the ligation point) was collected for quantification of blood
volume (see Materials and Methods S1 for detailed protocol).
The distribution of the plasmid was assessed by real-time PCR
detecting the pmaxGFPH plasmid backbone sequences. At day 3,
7, 14, and 28 after injections, animals were sacrificed and organs
were carefully collected and frozen for plasmid detection.
Afterwards, the DNA was isolated with NucleoSpinH Tissue kit
(MACHEREY-NAGEL, Dueren, Germany) and detected with
specific primer for the plasmid backbone (see Materials and
Methods S1 for detailed protocol).
Local gene expression
Seven days post injection, animals were sacrificed and samples
were obtained from the muscles of the treated and control animals.
Total RNA was then isolated with NucleoSpinH RNA II kit
(MACHEREY-NAGEL, Dueren, Germany) according to the
manufacturers instructions. VEGF165 and bFGF mRNA
expression was detected with specific primer for both the genes (see
Materials and Methods S1 for detailed protocol).
Collateral proliferation and blood perfusion ratio
Animals were sacrificed, the middle zone of the collaterals on day
7 including the surrounding tissues were taken, and proliferation
assays were performed as described previously [21,24]. Proliferation
of the collateral artery was detected by 5-Bromo-29-Deoxyuridine
(BrdU) labeling and detection Kit 2 (Roche Diagnostics, Penzberg,
Germany). The proliferative index was calculated as the number of
BrdU-positive nuclei to the total number of nuclei inside the vessel
wall. Blood flow in the lower hindlimb was detected on day 7 after
ligation by FluoSpheres polystyrene microspheres (15 mm,
redfluorescent, Invitrogen, USA). The microspheres were injected via
catheters through the carotid artery of rats running on a treadmill.
After injection, rats were sacrificed and gastrocnemius and soleus
from both hindlimbs were taken. Blood flow was expressed as ratios
of occluded and non-occluded hindlimb perfusion (microspheres
numbers) of the soleus and gastrocnemius (see Materials and
Methods S1 for detailed protocol).
All data were evaluated by at least 3 independent experiments.
The data were shown as Mean 6 SEM. Statistical comparisons
between 2 groups were performed with two-tail Students t-test.
One-way ANOVA followed by post-hoc analyses was used for
comparison of differences within multiple groups. The differences
between the groups were considered significant when p,0.05.
Isolation and characterization of primary dermal
Isolated rat dermal fibroblasts were spindle-shaped and adhered
rapidly to the culture flasks (Figure 1A). The characteristics of
Figure 2. Optimization of nucleofection in primary cultures of fibroblasts. (A) Improved method (improved) and original method (control)
were compared under fluorescence microscope to show optimization of nucleofection. Representative pictures of each method are shown in this
panel. Scale bar represents 100 mm. (B) Cell number and transfection rate were significantly enhanced by the improved method (experiment was
repeated for more than 5 times; * p,0.05; ** p,0.01).
these primary fibroblasts were detected by cytospin for the
expression of prolyl 4-hydroxylase subunit beta (P4H beta). A
fluorescent microscopic imaging for the expression of P4H beta
is presented (Figure 1B). In addition, the actin cytoskeleton
morphology of the fixed and permeabilized cells was also
examined by FITC-label phalloidin under sub-confluent and
confluent conditions (Figure 1 C and D), which indicated the
nature of smooth muscle cells. The cell growth curve was obtained
with normal culture medium (DMEM+10%FCS), showing an
active cell replication with a doubling time of 2448 hours. Cell
growth was linear during the first 7 days and then reached a
plateau phase on day 8 (Figure 1E).
Optimization of nucleofection with improved reagents
We first examined transfection efficiency with six nucleofector
programs (including A24, T16, U12, U23, U30, and V13). The cells
were transfected with commercial protocol provided by the
manufacturer. The highest transfection efficiency (around 60%)
and cell viability (.95%) were obtained with program U30.
Therefore, program U30 was chosen for further in vitro and in vivo
To achieve the maximal therapeutic effects, nucleofection
reagents were modified to improve gene transfection efficiency
in primary cell cultures of fibroblasts (details described in Materials
and Methods). Two days after nucleofection, the total number of
cells significantly decreased in the control group, in which
commercial kit reagents were used (Figure 2A, upper panels).
When transfected reagents were modified, transfection efficiency,
as shown by more GFP-positive cells, was enhanced (Figure 2A,
lower panels). With such improved reagents, 2 days after
nucleofection, more transfected cells were obtained in contrast to
the control standard protocol (Figure 2B, left panel: 0.4260.35
(Improved) vs. 1.0760.29 (Control); *p,0.05). By FACS, we
found that the use of modified solution did not affect cell growth,
but induced higher transfection rate (Figure 2B, right panel: 80.
565.0 (Improved) vs. 51.567.9 (Control); **p,0.01).
Figure 3. Cell growth and gene expression in primary cultures of fibroblasts transfected with bFGF and VEGF plasmids. After
Nucleofection, 1.46105 cells were seeded in a 12 wells plate, 1.5 ml of the culture medium was changed every day. (A) 7 days after Nucleofection,
more cells were observed in transfected groups. Quantification showed that significant differences were found between bFGF nucleofected group
and control group (* P,0.05). (B) Recombinant growth factors were detected for up to 21 days in the culture media. Scale bar represents 100 mm.
Dermal fibroblast nucleofection with VEGF and bFGF
After optimization with pmaxGFP vector, VEGF165 and bFGF
sequences were inserted into the pmaxGFPH backbone as coding
sequences. Seven days later, significantly more cells were observed
in the group transfected with plasmids expressing bFGF when
compared with the control (Figure 3A). Next, the growth-factor
release was evaluated daily for 21 days (Figure 3B). Long-term
expressions of both VEGF165 and bFGF proteins were found after
nucleofection, and the dynamics of their release were similar. Both
growth factors reached the expression or release peak at the first
week. However, the amount of secreted growth factors was
different between both the groups, with about 50-fold higher
release of VEGF165 than bFGF.
In vivo gene-delivery efficiency by nucleofected primary
After administration of genetically modified fibroblasts,
distribution of the plasmids in the peripheral tissues or organs was detected
by real-time PCR. No plasmid could be detected in any organs of
the control rats injected with nonmodified cells. The plasmid was
detected for more than 28 days at the site of injection (Figure 4A).
The peak of expression at the injection site was on day 3, and then
remained at a fairly high level. From day 3 to day 7, the plasmid
concentration dropped rapidly. After day 7, the local concentration
was maintained at the level of 105 copies/100 ng DNA, and then
deceased to the order of 103 copies/100 ng DNA on day 28.
Expression of bFGF and VEGF165 was also detected by PCR at the
site of injection. On day 7 post injection, the expression of both
angiogenic growth factors in the muscles was more than 100-folds
higher than that in the control group (Figure 4B). In all peripheral
tissues and organs from the experimental rats, no plasmid was
detected after day 14 (Figure 4C).
Administration of nucleofected fibroblasts enhanced
angiogenesis and arteriogenesis in the ischemic zone
After cell administration, the ischemic zone was reconstructed
(Figure 5A) and the collateral was counted in 3D reconstruction
views (Figure 5B). A significant increase in the number of vessels
was triggered by the injection of nucleofected cells (Figure 6A and
B; *p,0.05). Quantification was conducted after reconstruction of
the ischemic zone (Figure 7A). Both angiogenesis (Figure 7B, right
upper panel; 3.6160.56 vs. 5.1361.08 *p,0.05) and arteriogenesis
(Figure 7B, right lower panel; 1.3360.09 vs. 1.6360.06, *p,0.05)
increased, indicating an improvement in the medium-size vessel
and related small and thin vascular networks.
Increased collateral growth and blood flow in ischemic
hindlimbs treated with nucleofected fibroblasts
To further examine the effects of nucleofected cells on collateral
growth, collateral proliferation index was analyzed after local
administration of nucleofected cells. The results showed an
enhanced collateral proliferation in the same model (Figure 8A,
BrdU incorporation: 0.4860.05 vs. 0.5760.05; *p,0.05).
Reestablishment of a collateral circulation in ischemic lower
hindlimbs was detected by fluorescent microspheres. These
microspheres were trapped inside the lumen of the small arteries
and the number of the microspheres inside each muscle is related
to the blood flow (Figure 8B, left panel). Microspheres from both
soleus and gastrocnemius (ligated and nonligated hindlimbs) were
Figure 4. Gene delivery efficiency and expression in vivo by modified primary dermal fibroblasts. (A) High levels of plasmids were
detected in the injected area for more than 28 days. (B) 7 days after cell injection, gene expressions of bFGF and VEGF165 from injected area were
measured. Results showed that the expressions of both bFGF and VEGF165 were 100 times increased related to the control group. (C) After cell
administration, organs or tissues were collected at 4 different time points (experimental group: n = 3; control group: n = 3) and the sequence of the
plasmid backbone was used for detection of its distribution. Results are shown as the average value obtained from 100 ng DNA of each organ or
tissue. N represents that no plasmids were detected.
Figure 5. Comparison of micro-CT 3 dimensional collateral detection with planar method. (A) Ischemic hindlimb was reconstructed
through 1024 Z-axis slices (left panel). (B) Vessels in 3 dimensional (3D) reconstruction views were analyzed. Red arrows show the same corkscrew
collateral vessel in both X-ray review and 3D reconstruction view.
counted under fluorescent microscope to obtain the perfusion ratio
from each animal. Blood flow from soleus and gastrocnemius
significantly increased after administration of nucleofected cells
(Figure 8B, right; microspheres ratio: gastrocnemius: 0.4160.10
vs. 0.5060.11; *p,0.05; soleus ratio: soleus: 0.4260.08 vs.
Although several technologies of gene therapy have been
extensively applied in animal models of ischemia, their use in
clinical settings is still limited . Among the main reasons
behind the poor clinical translation of animal studies into humans
Figure 6. Increased 3D collateral vessels in ischemic hindlimbs treated with nucleofected fibroblasts. (A) One week after cell
administration, micro-CT angiography was performed and the ischemic zone from both treated and control samples were reconstructed. (B)
Summarized data showed that injection of transfected cells increased the number of visible collateral vessels (treated group: n = 7; control group:
n = 6 *p,0.05).
Figure 7. Increased vascular volume in ischemic hindlimbs treated with nucleofected fibroblasts. (A) The ischemic zone was defined as
600 slices below the proximal ligation point (slices from the left panel to right panel), including the distal ligation point in the middle panel. (B)
Quantification was done with 2 different thresholds: T20 and T40. With threshold 20, all vessels (angiogenesis and arteriogenesis related) were
counted. Threshold 40 was used to quantify only vessels with higher caliber (arteriogenesis related). (C and D) Vessels related to angiogenesis and
arteriogenesis were significantly enhanced after nucleofected fibroblasts injection (treated group: n = 7; control group: n = 6; Angiogenesis: * p,0.05,
Figure 8. Increased collateral growth and blood flow in ischemic hindlimbs treated with nucleofected fibroblasts. (A) One week after
cell administration, collateral growth was detected and quantified by BrdU and Hoechst staining. The proliferation index is expressed as BrdU (green)
positive nuclei vs. total (Hoechst/blue). Quantification showed that proliferation index was significantly enhanced by the injection of transfected cells
(Experimental group: n = 8; control group: n = 7,* p,0.05). Scale bar represents 50 mm. (B) Blood flow was analyzed by FluoSpheresH blood flow
detection method. Cryosections were analyzed by fluorescent microscopy and the numbers of the microspheres per muscle sample were counted
(left panel). The white arrows show the fluorescent microspheres trapped in the small capillaries. Blood flow ratio was calculated as numbers of
microspheres trapped in the ligated muscle vs. the number trapped in the same part of the non-ligated leg in the same animal. Results are shown as
gastrocnemius perfusion ratio between control and experimental group; soleus perfusion ratio between control group and experimental group (right
panel). In both muscles, the blood flow was significantly improved (Experimental group: n = 8; control group: n = 7 soleus: **p,0.01 gastrocnemius
*p,0.05). Scale bar represents 50 mm.
is the lack of both a highly efficient gene delivery system and
relatively easy procedures for the clinical translation. Here, we
described a modified nucleofection technology based on
alternative reagents which owns a high transfection efficiency (.80%)
and also a relatively easy operation procedures.
Nucleofection technology is a newly developed nonviral
transfection method based on electroporation technology. It could
be useful for applications in gene therapy with autologous cells
[26,27,28]. Different approaches have been used for nucleofection
based gene transfer into primary culture cells [29,30]. Several
previous studies have demonstrated the possibility of using this
technology to promote neovascularization in ischemic tissues.
Aluigi et al. have reported that nucleofection of human
mesenchymal stem cells in vitro could produce gene expression
for more than 20 days . Although stem/progenitor cells were
considered to be better candidates for ex vivo transfection,
uncontrolled differentiation that could lead to a serious side effect
was always a concern. Thus, in our study, the physiological stable
autologous fibroblasts were used to deliver targeted genes into the
ischemic tissue to demonstrate the efficiency of gene therapy.
Compared with a recent study by Mueller et al., who
demonstrated that a high-efficiency ex vivo rat dermal fibroblast
transfection with VEGF nucleofection technology could induce
local neovascularization effects in a rat skin flap model , our
findings at least overcame two major drawbacks, namely the
relative high cost (Our method cost only 10 folder less than the
original method) and the low transfection efficiency (550 pg/ml
peak expression of VEGF in the previous study and 10 ng/ml
peak expression of VEGF in our present study). Thus, improved
reagents for nucleofection in the present study will largely reduce
the cost for high efficiency of nucleofection. This modified
nucleofection may direct toward the development of similar
protocols for human autologous cell gene therapy. However, it
should be noted that much work may be needed to translate the
findings from the present study to a clinical setting. Most
importantly, there is a need to establish efficient protocols for
preparation of primary cell cultures from human tissues and to
establish the nucleofection efficiency of related genes into such cell
cultures. In addition, the applicability for different ischemic
diseases in human needs more careful evaluation because the
ischemic changes or stages observed in the animal model we used
in the present study may be not necessarily seen under human
conditions. Certainly there are a number of promising candidates
and promising ways of therapeutic application [32,33,34,35]. In
the light of previously published studies, the complex mechanism
of collateral growth and last not least the results of the present
study we think a gene therapy with combination of different
growth factors may be likely to have a sustained effect in the
treatment of lower extremity ischemia. However, as pointed out
above, it is still a very long way to clinical application.
The delivery of our cells directly into the middle part of the
thigh is also another important aspect which may favor the
therapeutic effects while lower the possible systematic side effects.
Our results also confirmed a possible localized expression pattern.
It was found that the plasmid can be detected for more than 28
days in the injected area, but it was undetectable in other organs
after 14 days. The peak of expression of the plasmid in vivo was
determined to occur on day 3 (Figure 4A), which is consistent with
the in vitro data (Figure 3B). Although most of the previous studies
showed that less than 10% of the plasmid can reach the general
circulation after direct plasmid intramuscular injection [36,37], it
was reported that plasmid can be detected by PCR up to 1 year in
mice . Here, we showed that the use of ex vivo transfection has
substantially decreased this time. When compared with the local
plasmid concentration, the plasmids in the other organs were fairly
low. On day 7, plasmid accumulation in the spleen could be
explained by cleaning effects of the circulating plasmid by the
immune system. All these findings indicate that autologous cell
gene delivery can make sustained gene expression in the injected
After injections of gene-modified fibroblasts into an ischemia
model, we also analyzed the neovascularization effects using a 3D
micro-CT system. This technology was used here because it
presents several advantages when compared to the planar area
measurement methods. In our previous studies, angiographies had
to be taken from different angles to obtain sufficient information
about the collateral vessels [21,24]. Even under the best
circumstances, reliable collateral vessel counting is difficult to
perform. In the present study, we were able to easily reconstruct
the 3D structures of the ligated hindlimbs and quantify the
collateral vessels in 3D views (Figure 5A and B). With the use of
this method, we were also able to quantify the blood volume by
voxel rendering in the reconstruction sections. Vessels related to
arteriogenesis and angiogenesis were analyzed separately
(Figure 7), and both were found to be improved after cell
administration. This result was confirmed by measuring the blood
flow in lower hindlimb (Figure 8). Using this technique, we
analyzed the efficiency of this autologous cell therapy with gene
delivery in the hindlimb ischemia model. Our results strongly
suggest that autologous cell therapy with mixture of VEGF165 and
bFGF nucleofected cells is very efficient to promote the formation
and growth of collateral vessels and re-establish circulation in
ischemic area of hindlimb.
In summary, we established a gene delivery method based on
highly efficient modified nucleofection approach using double
transplantation of nucleofected primary fibroblasts. This
technology owns 2 major advantages over other local application: 1) No
exogenous vectors are needed; 2) High transfection efficiency with
a relatively low cost. This technology might have the potential to
be used in the future clinically.
The authors gratefully acknowledge the support of the TUMs Thematic
Graduate Center/Faculty Graduate Center of Medical Life Science and
Technology at Technische Universitat M unchen. The authors would like
to thank Dr. Zou for his helpful advices and critical revision of the
Conceived and designed the experiments: ZZ AS WDI HGM. Performed
the experiments: ZZ AA JN SW NL. Analyzed the data: ZZ AA JN.
Contributed reagents/materials/analysis tools: ZZ JAL DFM JL. Wrote
the paper: ZZ JTE JAL DFM JL HGM.
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