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Cardiovascular Research 1999 43(3):808-822; doi:10.1016/S0008-6363(99)00172-8
© 1999 by European Society of Cardiology
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Copyright © 1999, European Society of Cardiology

Highly efficient liposome-mediated gene transfer of inducible nitric oxide synthase in vivo and in vitro in vascular smooth muscle cells

Kerstin Veit, Jean-Paul Boissel, Michael Buerke, Tilo Grosser, Jürgen Meyer and Harald Darius*

Department of Medicine II, Johannes Gutenberg-University, 55101 Mainz, Germany

* Corresponding author. Tel.: +49-6131-173-628; fax: +49-6131-176-613 darius{at}2-med.klinik.uni-mainz.de

Received 16 November 1998; accepted 4 May 1999


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Objective: The efficient introduction of regulatory genes into vascular smooth muscle cells (SMCs) is one of the most promising options for gene therapy of cardiovascular diseases. Cationic liposome-mediated gene transfer may become a favorable transfection technique with regard to patient’s safety for in vivo administration. However, this method until now has its limitation in a low transfection efficiency. Therefore, the present study was designed to improve cationic liposome-mediated transfection of rabbit vascular SMCs in vitro and in vivo, in order to enhance transfection efficiency and present an optimized system which may offer a potential therapeutic benefit for in vivo application. Methods and results: Optimized lipofection of rabbit SMCs with the mammalian expression vector pE-N1 and the reporter gene green fluorescent protein resulted in a mean transfection efficiency of about 50%. The unique transfection of rabbit SMCs in vitro and in vivo with the inducible isoform of human nitric oxide synthase (NOSII), using the same vector, resulted in a successful transient transcription and translation of a functionally active human NOSII in rabbit SMC, persisting 5–6 days. We could further demonstrate that the transfection procedure and the transgene product did neither induce necrosis nor apoptosis under the conditions chosen and did not result in the induction of endogenous NOSII of transfected SMCs. Conclusion(s): These findings indicate potential therapeutic relevance for this nonviral gene transfer system for in vivo gene therapy for cardiovascular diseases.

KEYWORDS Angioplasty; Gene therapy; Nitric oxide; Restenosis; Smooth muscle


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
The field of cardiovascular gene therapy has developed rapidly during the last decade investigating a variety of gene delivery techniques in vitro and in vivo [1–3]. However, the methods currently used, e.g., retroviral vectors [4,5] adenoviral vectors [6,7] adenovirus-augmented receptor-mediated gene delivery [8], liposomes [9,10], HVJ liposome conjugates [11,12], and others suffer from one or more problems due to either cell toxicity, low transfection efficiency, induction of an inflammatory response or mutagenesis. Because of the safety issues and the ease of handling [13–15] we decided to optimize the nonviral cationic liposome-mediated transfection of rabbit vascular smooth muscle cells in vitro. It is well known that the efficacy of lipofection varies among different cell types and most studies reported investigated immortalized animal cell lines [9,16].

Vascular smooth muscle cells are a suitable target for a gene transfer approach because of their proximity to the lumen surface and abundance in the vessel wall [17]. Furthermore, abnormal proliferation of smooth muscle cells is a critical event in the feature of atherosclerosis and of certain accelerated forms of vascular disease, for example, restenosis following balloon angioplasty [18,19].

Nitric oxide synthases (NOS) generate nitric oxide (NO), an autacoid with important regulatory functions in the cardiovascular system including inhibition of platelet aggregation [20,21], leukocyte adhesion and smooth muscle cell proliferation [22–25]. Therefore, the NOS represent attractive targets for vascular gene transfer. The inducible isoform of NOS (NOSII) has furthermore the advantage, if compared to the other isoforms, of exerting high enzyme activity with a continuous release of NO without requiring agonist stimulation [26,27].

For the demonstrated reasons we decided (i) to develop an efficient, nontoxic, highly reproducible, cationic liposome-mediated gene transfer system for the inducible NOS using the reporter gene green fluorescent protein for optimization, (ii) to investigate the expression and functional activity of the transfected inducible isoform of human NOS in rabbit vascular smooth muscle cells in vitro, and (iii) to confirm the potential of the vector developed to transfect vascular smooth muscle cells within the vessel wall in vivo.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
2.1 Materials
Buffers and other chemicals were obtained in p.a. quality from Sigma–Aldrich (Deisenhoven, Germany), Pharmacia Biotech (Freiburg, Germany) and Boehringer-Mannheim (Mannheim, Germany).

2.2 Tissue culture
Smooth muscle cells were isolated from the thoracic aorta of white New Zealand rabbits by the explant technique and smooth muscle cells purity was characterized by positive staining with smooth muscle specific {alpha}-actin monoclonal antibodies (Sigma–Aldrich) [28,29]. Smooth muscle cells were maintained in RPMI-1640, (Gibco BRL, Eggenstein, Germany) supplemented with 10% fetal calf serum (FCS) (PAA Laboratories, Cölbe, Germany), penicillin (100 IU/ml) and streptomycin (50 µg/ml) (Gibco BRL) and passaged by trypsinization (0.25% trypsin–EDTA solution) (Gibco BRL) and incubated at 37°C in a humified atmosphere of 95% air and 5% CO2 with exchange of media every 3 days. Only cells of passages 3–5 were involved in the in vitro experiments.

2.3 Plasmid vector constructs
A 885 base pair (bp) transfection time fragment corresponding to the coding cDNA sequence of human NOSII was isolated by reverse transcriptase polymerase chain reaction (RT-PCR) from a cytokine-stimulated human colon carcinoma cell line DLD-1 (ATCC: CCL221), controlled by sequencing and inserted into the XHO1/NOT1 cloning site of the commercially available cytomegalovirus controlled mammalian expression vector pE-N1 (Clontech, Heidelberg, Germany) pE-NOSII-N1. Optimization of transfection efficiency was performed using the same expression vector containing the reporter gene green fluorescent protein pE-GFP-N1 (Clontech) [30–32].

2.4 Transfection procedure
Plasmid DNA was isolated and purified from DH5-{alpha} Escherichia coli (Clontech) using the Qiagen Plasmid Maxi Kit (Qiagen, Hilden, Germany). In case of transfecting rabbit smooth muscle cells in 24-well plates, cells were seeded the day before transfection at a density of 5·104 per well and maintained in RPMI-1640 with 10% FCS.

DNA was ethanol-precipitated over night and dissolved in Hanks’ balanced salt solution (HBSS; Sigma) (250 µl/24-well plate) or in a specific Transfection-Kit-containing buffer, according to the manufacturers instructions.

Positively charged liposomes (DOTAP, Fugene, Boehringer-Mannheim; Effectene, Superfect, Qiagen; Lipofectin, Lipofectamin, Cellfectin, DMRIE-C, Gibco) were used for the transfection experiments and investigated in the experiments following manufacturers’ instructions. Amount of DNA, liposomes, DNA/liposome-ratio, dissolving-buffer and transfection time were varied in order to improve transfection conditions for smooth muscle cells.

In case of optimized DOTAP transfection, 5 µg DNA per well were dissolved in HBSS and gently mixed with 15 µl DOTAP, incubated at RT for 30 min and then added to the cells. Transfection was then performed for 6 h in medium containing 10% FCS. Control cells were not stimulated and nontransfected, control-transfected cells were transfected using the reporter-gene under identical conditions as used for NOSII transfection, and human NOSII transfection was performed using the pE-NOSII-N1 plasmid.

2.5 Flow cytometry
Optimization of transfection efficiency of rabbit smooth muscle cells was performed using the reporter gene GFP. The percentage of GFP positive transfected cells was determined by flow cytometric analysis (FACScan, Becton Dickinson, Heidelberg, Germany). Cells were harvested 24 h after transfection by trypsinization, washed twice with phosphate buffered saline (PBS) and measured without fixing. The lower limit for positive fluorescence was determined with β-galactosidase transfected smooth muscle cells.

2.6 RT-PCR
Total cellular RNA was extracted 18 h after transfection from rabbit smooth muscle cells (in vitro) and rabbit femoral artery (in vivo) using the method of Chomczynski and Sacchi [33]. To exclude contamination of RNA samples with transfected plasmid DNA, RNA was first digested with DNase1 (200 U/ml; 60 min; 37°C) and phenol chloroform extracted. RT was then performed with the SuperScript RNaseH– first strand cDNA synthesis Kit (Gibco BRL) with 2 µg total RNA or water as negative control, 200 U/µg RT and 15-mer oligo dt primer (Gibco BRL) according to the manufacturer’s instructions (70° C, 10min; 4°C, 5 min, 37°C, 30 min; 45°C, 30 min). One µl cDNA template was used per 100 µl PCR reaction [primers 2.5 pM; dNTP 2 µM; 1 U Tag-DNA-polymerase (Boehringer -Mannheim)]. The primers, which amplify a specific 707 bp fragment corresponding to a highly conserved region of NOSII from position 475 form the ATG start codon up to position 1181, were designed as follows: sense strand: 5'-ATAGAGGAACATCT-GGCCAG-3', and antisense strand: 5'-TCCTCCAGGATG-TTGTAGCG-3' (Gibco BRL) and used in PCR at a concentration of 0.05 pM. The PCR reaction was performed in the order of 95°C for 5 min; 55°C for 2 min; 72°C for 3 min for 40 cycles. The PCR products were controlled by electrophoresis on a 1.5% agarose gel and stained with ethidium bromide.

2.7 Western blot analysis
Total cellular protein from rabbit smooth muscle cells (in vitro) and rabbit femoral artery (in vivo) was isolated after unique transfection with ice-cold lysis buffer (50 mM Tris–HCL, 0.5 mM EDTA, 0.5 mM EGTA, 20 mM 3-[(cholamidopropyl)dimethylamino]-1-propanesolphonate, 10% glycerol and the protease inhibitors leupeptin [1 µg/ml], phenylmethylsulfonyl fluoride [1 mM], pepstatin A [1 µg/ml], aprotinin [0.2 µg/ml]). Protein concentration was determined by the DC protein assay method (Bio-Rad, München, Germany). Thirty µg of each sample were separated on 8% sodium dodecyl sulphate polyacrylamide gel electrophoresis and transblotted onto nitrocellulose membranes (Minitransblot, Tank-method, Bio-Rad). Additional protein binding sites on the membrane were blocked with 5% defatted milk in TTBS (0.01 M Tris base, 150 mM NaCl pH 7.4 with 0.05% Tween 20). Blots were incubated with the monoclonal anti-NOSII antibody (Transduction Laboratories, Dianova, Hamburg, Germany) (1:2000 dilution in 1% defatted milk in TTBS) as primary antibody for 1 h at room temperature. The primary antibody was detected using a goat antimouse IgG1 alkaline phosphatase conjugated antibody (1:25 000 dilution in 1% defatted milk in TTBS) (Sigma–Aldrich), incubated for 1 h at room temperature and visualized with BCIP/NBT staining as indicated by the manufacturer (Sigma–Aldrich).

2.8 Immunostaining
Rabbit smooth muscle cells were fixed in ice-cold methanol/acetone (1:1 v/v; 30 s; –20°C), washed twice with PBS. Nonspecific binding of antibodies was blocked with 1% bovine serum albumin (BSA)/PBS (1 h, room temperature). Primary antibodies were incubated overnight at 4°C (anti-NOSII, mouse IgG, 1:100 dilution) (Transduction Laboratories). After washing, the secondary antibody was incubated for 1 h at room temperature (antimouse IgG FITC conjugate, 1:200 dilution; Sigma–Aldrich) and immunofluorescence was viewed at 490 nm.

Vessel sections of rabbit femoral artery were fixed in ice-cold methanol/acetone (1:1 v/v; 30 s; –20°C) and washed twice with 0.1% BSA/PBS. Nonspecific binding of antibodies was blocked with 1% BSA/PBS (1 h room temperature). Primary antibodies were incubated overnight at 4°C (anti-NOSII, mouse IgG, 1:100 dilution) (Transduction Laboratories). After washing the secondary antibody was incubated for 1 h at room temperature (goat antimouse IgG1 alkaline phosphatase conjugated antibody, 1:200 dilution) (Sigma–Aldrich), and visualized with BCIP/NBT staining as indicated by the manufacturer (Sigma–Aldrich). Nuclei staining was performed using hematoxilin (Merck, Darmstadt, Germany).

2.9 Arginine–citrulline conversion
Total cellular protein from rabbit smooth muscle cells was isolated 2 days after transfection with ice-cold lysis buffer (50 mM Tris–HCL pH 7.4, 2.5 mM EDTA, 2.5 mM EGTA, 1% NP-40) and the protease inhibitors dithiothreitol [1 mM], leupeptin [10 µg/ml], phenylmethylsulfonyl fluoride [100 µM], pepstatin A [10 µg/ml], aprotinin [2 µg/ml]. Protein concentration was determined by the DC protein assay method (Bio-Rad). Then, 5 µg of each protein were incubated with the NOS cofactors FAD (5 µM), FMN (5 µM), NADPH (1 mM), BH4 (3 µM) and 14C-L-arginine (50 µCi/ml) (Amersham, Braunschweig, Germany), ±L-NAME (NG-nitro-L-arginine methylester hydrochloride, 200 µM) (Clinalfa, Läufelingen, Switzerland) gently shaking for 30 min at 37°C. The reaction was stopped with ice-cold methanol, protein was removed by centrifugation, and the supernatant was lyophilized. After dissolving the samples in water, 14C-L-arginine was separated from 14C-L-citrulline using thin layer chromatography (Polygram SIL N-HR, Macherey–Nagel, Düren, Germany) with the solvent chloroform/methanol/ammonium hydroxide/water (volume ratio 0.5:4.5:2.0:1.0). The densitometric analyses were performed with the Molecular Imager system (Bio-Rad).

2.10 c-GMP enzyme immunoassay
c-GMP determination was performed using the BIOTRAK c-GMP EIA system (Amersham) according to the manufacturer’s instructions. Briefly, rabbit smooth muscle cells were washed twice with HBSS and then incubated with HBSS containing 30 U/ml superoxide dismutase, 10 µM dipyridamole, 2.5 mM EDTA, 2.5 mM EGTA and ±200 µM L-NAME for 15 min at 37°C and 30 rpm. After removing the supernatant, the reaction was stopped by homogenizing the cells with ice-cold 5% trichloroacetic acid. Protein determination was performed with the pellet (DC protein assay method, Bio-Rad), and the supernatant was washed four times with water saturated diethyl ether. The extract was lyophilized, dissolved in assay buffer (0.5 M sodium acetate, 0.02% BSA, pH 5.8) and the nonacetylation EIA for c-GMP determination was performed.

2.11 Cell proliferation
Cell proliferation was determined using the colorimetric immunoassay (Boehringer-Mannheim) for the quantification of cell proliferation, based on the measurement of 5-bromo-2'-deoxyuridine (BrdU) incorporation during DNA synthesis. Briefly, rabbit smooth muscle cells were seeded in 96-well plates at a density of 1·104 cells per well, the next day transfected, then synchronized for 48 h with RPMI-1640 containing 0.1% FCS. Cell proliferation was then stimulated for 24 h with RPMI-1640 medium containing 10% FCS. BrdU incorporation was allowed for 4 h (±L-NAME 200 µM), before performing the enzyme-linked immunosorbent assay (ELISA) following manufacturer’s instructions.

2.12 PMN adhesion
Neutrophil granulocytes were isolated from anticoagulated whole blood (2 U/ml heparin) from healthy male volunteers by Histopaque (Sigma–Aldrich) density gradient centrifugation. Isolated PMNs were radioactive labeled with 111indium (1 µl/ml Krebs Henseleit buffer, pH 7.4; 30 min, 37°C) (Amersham), washed twice to remove nonbound activity and cell number was adjusted at 5·106/ml. Smooth muscle cells were transfected in 24-well plates as described and the adhesion assay was performed 24 h later as follows: smooth muscle cells were either incubated in RPMI-1640/10% FCS or in RPMI-1640/10% FCS with TNF{alpha} 300 U/ml for 6 h at 37°C. Four h later, smooth muscle cells were washed twice and incubated with HBSS+2% BSA, ±TNF{alpha} (300 U/ml) and ±L-NAME (200 µM). After washing three times, PMNs (1.25·106 per well) were added to the smooth muscle cells and incubated in a shaker bath (30 rpm) for 30 min at 37°C. Nonadhering PMNs were then removed by washing five times and the adhering cells were solubilized overnight with 1 N NaOH and measured with 10 ml scintillation liquid (Rotiscint, Roth, Karlsruhe, Germany) in a β-counter (Wallac). One hundred percent adhesion was determined by counting 1.25·106 PMNs.

2.13 Apoptosis
The programmed cell death was investigated with the photometric enzyme-immunoassay (Boehringer-Mannheim) for the qualitative and quantitative determination of cytoplasmatic histone-associated-DNA-fragments. Rabbit smooth muscle cells were seeded in 96-well plates, the next day transfected or treated with actinomycin D (100 ng/ml) as a positive control. DNA fragmentation was measured from lysates of cytoplasmatic fractions after 24 h.

2.14 Balloon angioplasty of rabbit femoral artery (in vivo transfection)
The investigations conform with the Guide for the Care and Use of Laboratory Animals, published by the National Institutes of Health.

New Zealand White male rabbits (2.8–3.8 kg) were housed individually in a controlled-temperature, standard light/dark environment and allowed to stabilize before any intervention.

Rabbits were anaesthetized with xylazinhydrochloride (4 mg/kg i.m., Bayer, Leverkusen, Germany) and pentobarbital (bolus: 10 mg/kg, supplementary: 4 mg/kg i.v., Sanofi, Nürtingen, Germany). Under sterile conditions the right femoral artery was isolated, a 2 cm incision was made, and a femoral arteriotomy was performed to advance a 6F Transport coronary dilatation–infusion catheter (Transport®, Boston Scientific, Ratingen, Germany) retrogradly to an area within the femoral artery. The position of the balloon catheter was ascertained by anatomic landmarks and controlled angiographically. Thereafter the inner balloon was inflated with saline to a pressure of 6 atm and the outer balloon was infused with DNA (pE-NOSII-N1 or pE-GFP-N1, 100 µg) liposome (300 µl) complexes in 1 ml HBSS or HBSS alone. After 2 min the balloon catheter was deflated and removed. The femoral artery was sutured and the patency of the vessel was controlled by Doppler distal to the insertion site. The surgical incision was closed, the animals received fragmin (30 IU/kg s.c., Grünenthal, Aachen, Germany), aspirin (30 mg/kg i.v., Bayer), perioperative and postoperative gentamicin (6 mg/kg i.v. and i.m., Ratiopharm, Ulm, Germany). Two days following angioplasty the rabbits were anesthetized as described and the femoral arteries were excised after application of heparin (600 IU/kg i.v.). The experiment was terminated by an injection of sodium pentobarbital (40 mg/kg i.v.).

Vessel segments were washed with PBS, embedded in tissue freezing medium (Tissuetec, Jung, München, Germany), frozen in liquid nitrogen and cut into 8 µm cryosections.

2.15 Statistics
Results were expressed as mean±standard deviation (SD). Statistically significant differences of mean values were evaluated using a paired Student’s t-test following Bonferroni’s adaptation of the level of significance if indicated. Values of * P<0.05 and ** P<0.01 were accepted to denote statistical significance.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
3.1 Optimization of lipofection for rabbit smooth muscle cells in vitro (Fig. 1)
The nonviral transfection conditions for rabbit smooth muscle cells were optimized with regards to the DNA concentration, expression vector, type of nonviral transfection reagent, DNA/liposome ratio, dissolving buffer, transfection time and time interval until protein expression. Nonviral-mediated transfection of rabbit smooth muscle cells was performed using cationic liposomes (DOTAP, Fugene, Boehringer-Mannheim; Effectene, Superfect, Qiagen; Lipofectin, Lipofectamin, Cellfectin, DMRIE-C, Gibco) and the reporter gene GFP. The percentage of GFP positive transfected rabbit smooth muscle cells was determined by flow cytometry versus β-galactosidase control transfected smooth muscle cells (0.34±0.1%) as lower limit for positive fluorescence.


Figure 1
Figure 1
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  		Fig. 1 Optimization of transfection efficiency of rabbit smooth muscle cells using various cationic liposomes and the reporter gene GFP. The percentage of GFP positive transfected smooth muscle cells was determined by flow cytometry versus β-galactosidase control transfected smooth muscle cells (0.34%) as lower limit for positive fluorescence.

 
Fig. 1A and Table 1 demonstrate transfection optimization for rabbit smooth muscle cells varying the type of cationic liposome and the transfection time. Optimized transfection conditions for rabbit smooth muscle cells were obtained with the cationic liposome DOTAP after 6–8 h transfection time and 24 h protein expression (51.3%), and the Effectene transfection system after 8 h transfection and 48 h protein expression (52.4%). Other investigated liposomes were much less effective in transfecting rabbit smooth muscle cells; the respective values of the efficiencies are listed in Table 1.


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  		Table 1 Optimization of transfection efficiency of rabbit smooth muscle cells using various cationic liposomes and the reporter gene GFPa

 
Fig. 1B shows transfection optimization for rabbit smooth muscle cells using the liposome DOTAP, varying the DNA amount (µg) and DNA/DOTAP (µg/µl) ratio. Transfection efficiency for rabbit smooth muscle cells was optimal using 5 µg GFP–DNA and 15 µl DOTAP. Other concentrations were either less effective or toxic.

Fig. 1C exerts photo documentation of fluorescence microscopy of GFP positive rabbit smooth muscle cells (excitation wavelength: 490 nm; 200-fold magnification) with the GFP typical bright green-yellow fluorescence. Optimized transfection conditions for rabbit smooth muscle cells (Fig. 1C, graph) were obtained with the cationic liposome DOTAP and the Effectene transfection system. Other investigated liposomes were much less effective in transfecting rabbit smooth muscle cells.

3.2 RNA of human NOSII in rabbit cells following in vitro and in vivo transfection (Fig. 2)
RNA of transfected NOSII cDNA (pE-NOSII-N1) in rabbit smooth muscle cells in vitro and in vivo in rabbit femoral artery after PTA was determined by RT-PCR (n=6; n=5). Contamination of plasmid DNA could be excluded by digesting total RNA with DNase1 before performing RT.


Figure 2
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  		Fig. 2 RNA of transfected NOSII in rabbit smooth muscle cells in vitro and in rabbit femoral artery after PTA was determined by RT-PCR. There was no detectable human NOSII cDNA in RNA harvested 18 h following transfection in nonstimulated (lane 2, in both gels) and control transfected (lane 3, in both gels) rabbit smooth muscle cells and vessels. In contrast, the transfection of rabbit smooth muscle cells and rabbit femoral artery with NOSII lead to the amplification of the specific 707 bp NOSII cDNA fragment as expected with the primers used (lane 4, in both gels).

 
There was no detectable human NOSII cDNA in the RNA harvested 18 h following transfection in nonstimulated (lane 2, in both gels) and control transfected (pE-GFP-N1, lane 3, in both gels) rabbit smooth muscle cells and vessels.

In contrast, the transfection of rabbit smooth muscle cells and rabbit femoral artery with NOSII lead to the amplification of the specific 707 bp NOSII cDNA fragment corresponding to the primer involved (lane 4, in both gels).

Furthermore, specific human NOSII mRNA was detectable in NOSII transfected smooth muscle cells, however not in control cells if measured by RNase protection assay (data not shown).

3.3 Protein of human NOSII in rabbit cells after in vitro and in vivo transfection (Figs. 3 and 4Go)
Transfected human NOSII protein in rabbit smooth muscle cells (in vitro transfection) and balloon dilated rabbit femoral artery (in vivo transfection) was assessed by Western blot (n=4; n=5) (Fig. 3) and positive immunostaining (Fig. 4, in vitro transfection; Fig. 5, in vivo transfection).


Figure 3
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  		Fig. 3 Transfected human NOSII protein in rabbit smooth muscle cells and balloon dilated rabbit femoral artery was assessed by Western blot. Human NOSII protein was detectable with the typical protein mass of 130 kDa in the NOSII positive control (lane 1, in both blots) and in NOSII transfected rabbit smooth muscle cells and vessels. After single transfection of NOSII in vitro in smooth muscle cells, NOSII protein was detectable until day 5 (lanes 3, 4 and 5, in vitro transfection). There was no NOSII protein detectable in controls (lane 2 and 6). After single in vivo transfection, human NOSII protein was detectable until day 3 postPTA (lanes 4 and 5, in vivo transfection) but not in controls respectively (lanes 2 and 3, in vivo transfection).

 

Figure 4
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  		Fig. 4 Fluorescence microscopy of transfected human NOSII in rabbit smooth muscle cells. NOSII immunofluorescence was positive in NOSII transfected rabbit smooth muscle cells after staining with specific antihuman NOSII and FITC-conjugate (left picture, NOSII transfected). Transfection resulted in positive staining of 10.3±4.2% of smooth muscle cells. In contrast, there was no positive NOSII staining in unstimulated, control transfected (β-galactosidase) rabbit smooth muscle cells (right picture).

 

Figure 5
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  		Fig. 5 Protein of transfected human NOSII after in vivo administration in rabbit femoral artery was assessed by immunostaining. Human NOSII immunoreactivity, indicated by marked brown coloration of cells, was observed in the luminal part of the medial layer of balloon dilated, NOSII transfected rabbit femoral artery sections (left picture). In contrast, there was no staining for human NOSII in control transfected, balloon dilated rabbit femoral artery (right picture).

 
In Western blot, NOSII protein was detectable using a specific anti-NOSII antibody (Fig. 3) with the typical protein mass of 130 kiloDaltons (kDa) in the NOSII positive control (lane 1, in both blots) and in pE-NOSII-N1 transfected rabbit smooth muscle cells and vessels.

After single administration of pE-NOSII-N1 in vitro in smooth muscle cells, human NOSII protein was detectable until day 5 (lanes 3, 4 and 5, in vitro transfection). There was neither NOSII protein in nontransfected unstimulated smooth muscle cells (lane 2), nor in control-transfected smooth muscle cells (lane 6).

After single in vivo transfection following balloon dilatation of rabbit femoral artery, human NOSII protein was detectable until day 3 postPTA (lanes 4 and 5, in vivo transfection) but not in controls respectively (lanes 2 and 3, in vivo transfection).

NOSII immunofluorescence (n=5) was positive in pENOSIIN1 transfected rabbit smooth muscle cells in vitro (Fig. 4) exerting a protein expression sufficient for detection by fluorescence microscopy of 10.3±4.2%. In contrast, there was no positive NOSII staining in nonstimulated control transfected rabbit smooth muscle cells.

In vivo transfected human NOSII protein was assessed by positive immunostaining of frozen sections of rabbit femoral artery using the alkaline phosphatase/BCIP/NBT system (n=3) (Fig. 5). Marked human NOSII immunoreactivity, indicated by brownish coloration of cells, was observed in the luminal part of the medial layer of balloon dilated, NOSII transfected rabbit femoral artery sections (pE-NOSII-N1 transfected). In contrast, there was no staining for human NOSII in control transfected, balloon dilated rabbit femoral artery (negative control).

3.4 Enzyme activity of transfected human NOSII in vitro in smooth muscle cells
3.4.1 Arginine to citrulline conversion (Fig. 6A) and c-GMP (Fig. 6B)
Activity of transfected human NOSII protein in rabbit smooth muscle cells in vitro was assessed by measuring arginine to citrulline conversion (Fig. 6A) (n=6) and NOs’ second messenger c-GMP (Fig. 6B) (n=7).


Figure 6
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  		Fig. 6 Functional active transfected human NOSII protein in rabbit smooth muscle cells in vitro was demonstrated by arginine to citrulline conversion (A) and c-GMP, NOs’ second messenger (B). (A) This shows on the left side the typical separation of arginine from citrulline in thin layer chromatography. The densitometric analysis of the radioactive spots gives rise to the arginine to citrulline conversion rate (graph). Nontransfected, unstimulated (control), and control transfected protein of smooth muscle cells exerts basal levels of arginine to citrulline conversion. In contrast, the NOSII transfection of rabbit smooth muscle cells resulted in a significant 13-fold increase in the arginine to citrulline conversion rate. The coincubation of protein of pE-NOSII-N1 transfected smooth muscle cells with the L-arginine analogue L-NAME caused a decrease of the arginine to citrulline conversion on basal levels. c-GMP of nontransfected, unstimulated (control) and control transfected rabbit smooth muscle cells were at basal values below 200 fmol/mg protein. Single transfection of smooth muscle cells with NOSII resulted in a significant increase in c-GMP levels (** P<0.01 at day 1–5 and * P<0.05 at day 6), persisting 5–6 days, up to a maximal 16-fold increase in c-GMP. The parallel coincubation with L-NAME prevented the rise in c-GMP levels at any point of time investigated.

 
Fig. 6A shows on the left side the characteristic separation of arginine from citrulline by thin layer chromatography. The densitometric analyses of the radioactive spots allowed for the calculation of the arginine to citrulline conversion rate (Fig. 6; graph). Nontransfected, nonstimulated (control) and control transfected (pE-GFP-N1) rabbit smooth muscle cells showed a basal arginine to citrulline conversion rate of 6.8±3.8% and 5.0±3.2%, respectively. In contrast, the transfection of rabbit smooth muscle cells with pE-NOSII-N1 resulted in a highly significant 13-fold increase of the arginine to citrulline conversion rate to 70.6±16.6%. The incubation of pE-NOSII-N1 transfected rabbit smooth muscle cells with the L-arginine analog L-NAME (200 µM) lead to a decrease in the arginine to citrulline conversion rate to basal levels (2.1±1.8%).

c-GMP levels (Fig. 6B) of nontransfected, nonstimulated (control) and control transfected smooth muscle cells were 182.3±49.9 and 160.6±37.8 fmol/mg protein, respectively. There was no difference in c-GMP levels of control smooth muscle cells between day 1 and day 6 (data not shown). In contrast, the transfection of rabbit smooth muscle cells with pE-NOSII-N1 resulted in a highly significant maximal 16-fold increase in c-GMP levels to 2891±1568 (day 1 posttransfection), 2634±1376 (day 3), 2289±1583 (day 5), and 907±345 fmol/mg protein (day 6). The simultaneous incubation of pE-NOSII-N1 transfected rabbit smooth muscle cells with L-NAME completely prevented the increase in c-GMP levels at any point of time investigated [day 1: 186±63; day 3: 189±96; day 5: 159±75 and day 6 posttransfection: 178±65 (fmol/mg protein)].

3.5 Physiologic effects of NOSII transfection in vitro in smooth muscle cells
3.5.1 Smooth muscle cell proliferation (Fig. 7A) and PMN adhesion (Fig. 7B)
To assess some of the physiological effects of NOSII transfection on rabbit smooth muscle cells in vitro, proliferative activity was studied by BrdU incorporation (Fig. 7A), and with regards to cell–cell interactions by measuring PMNs adhesion to smooth muscle cells (Fig. 7B).


Figure 7
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  		Fig. 7 To determine physiologic consequences of NOSII transfection, rabbit smooth muscle cell proliferation (A) and PMN adhesion (B) were assessed in vitro. One hundred percent smooth muscle cell proliferation (A) was determined by stimulating nontransfected cells (control) with 10% FCS and was unaffected by coincubation with L-NAME as well as by control transfection (GFP; ±L-NAME). In contrast, transfection of rabbit smooth muscle cells with NOSII lead to a significant (* P<0.05 at 0.3 and 0.6 µg DNA, and ** P<0.01 at 1.25 and 2.5 µg DNA) and dose-dependent decrease in smooth muscle cell proliferation from 100% to 50% (0.3 µg DNA), 40% (0.6 µg DNA), 25% (1.25 µg DNA) and 16% (2.5 µg DNA). Coincubation of NOSII transfected smooth muscle cells with L-NAME resulted in a complete abolition of proliferation-inhibition at all concentrations studied. Adhesion of 111indium labeled PMNs on unstimulated smooth muscle cells (B) did not differ significantly between untransfected, control transfected and NOSII transfected smooth muscle cells. Stimulation of the smooth muscle cells with TNF{alpha} resulted in an increase in PMN adhesion on untransfected and on control transfected rabbit smooth muscle cells in comparison with the TNF{alpha}-stimulated controls. PMN adhesion to NOSII transfected smooth muscle cells was reduced to about a third of control (** P<0.01). The reduced adhesion of PMNs to NOSII transfected smooth muscle cells was almost completely abolished following coincubation with L-NAME.

 
The proliferative activity was assessed (Fig. 7A) following maximal stimulation of untreated, nontransfected cells (control) using 10% FCS [100% proliferation{cong}1.06±0.11 (optical density [OD] at 450nm)] and was not affected by coincubation with L-NAME (1.02±0.12). Comparable results were obtained by stimulating smooth muscle cell proliferation with ADP or thrombin (data not shown). Additionally, the proliferation was unaltered by control transfection with pE-GFP-N1 with and without L-NAME: 0.96±0.05 and 1.04±0.13 (OD at 450 nm). In contrast, transfection of rabbit smooth muscle cells with pE-NOSII-N1 lead to a significant and dose-dependent (0.3–2.5 µg DNA) decrease in BrdU incorporation indicating a reduction in cell proliferation rate with OD values of 0.61±0.13 (0.3 µg DNA), 0.45±0.12 (0.6 µg DNA), 0.34±0.14 (1.25 µg DNA) and 0.25±0.15 (2.5 µg DNA). The simultaneous incubation of pE-NOSII-N1 transfected rabbit smooth muscle cells with L-NAME (200 µM) resulted in the complete abolition of the proliferation inhibitory effects caused by the NOSII transfection at all concentrations investigated with OD values (OD 450 nm) of 1.06±0.20 (0.3 µg DNA),1.04±0.15 (0.6 µg DNA), 1.01±0.15 (1.25 µg DNA), and 1.04±0.20 (2.5 µg DNA).

In order to exclude the induction of necrosis as a possible mechanism of action resulting in a diminished BrdU incorporation, the release of LDH was determined. LDH release in the supernatant was unaltered by the NOSII or control transfection confirming that the proliferation inhibitory effect of the NOSII transfection was not due to a toxic effect, e.g., induction of necrosis (data not shown).

The percentage of 111-indium labeled PMNs adhering to unstimulated smooth muscle cells was unaffected by transfection with values of: 9.5±1.5% (nontransfected), 9.2±1.2% (pE-GFP-N1), and 7.2±2.4% (pE-NOSII-N1). In contrast, stimulation of smooth muscle cells with TNF{alpha} resulted in a significant augmentation of PMN adhesion to 17.1±2.7% on nontransfected smooth muscle cells, and to 16.6±2.2% on pE-GFP-N1 transfected smooth muscle cells. However, PMN adhesion on pE-NOSII-N1 transfected smooth muscle cells amounted to 6.4±3.2%, being significantly diminished if compared to TNF{alpha}-stimulated controls. The reduction in PMN adhesion on NOSII transfected smooth muscle cells was almost completely abolished by coincubation with L-NAME with values of 16.6±2.7 for nontransfected, 15.9±3.2% for pE-GFP-N1 transfected, and 14.8±2.8% for pE-NOSII-N1 transfected smooth muscle cells.

3.6 Programmed cell death (Fig. 8)
In order to investigate, whether the transfection procedure itself or the transgene product(s) of pE-NOSII-N1 transfected rabbit smooth muscle cells may induce programmed cell death DNA fragmentation was measured using a commercially available ELISA system (n=4). The results demonstrate no significant difference in DNA fragmentation between nonstimulated, nontransfected (OD at 405 nm: 0.26±0.18), control transfected (OD: 0.18±0.12), and pE-NOSII-N1 transfected (OD: 0.22±0.17) rabbit smooth muscle cells. As a positive control apoptosis was induced in smooth muscle cells by treatment with actinomycin D for 24 h (OD: 1.48±0.28).


Figure 8
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  		Fig. 8 Programmed cell death measured by DNA fragmentation in transfected rabbit smooth muscle cells. Maximal induction of apoptosis was seen in smooth muscle cells treated for 24 h with actinomycin D as a positive control. In contrast, neither the transfection procedure itself (GFP; 2.5 µg) nor the transgene product of NOSII transfected rabbit smooth muscle cells (2.5 µg) induced programmed cell death.

 

   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
During the last two decades balloon dilatation of vessels has become the treatment of choice if atherosclerosis has resulted in a flow limiting vascular stenosis. Although the initial success rates of balloon angioplasty of stenosed peripheral and coronary arteries are about 90–95%, following an observation period of 3 to 6 months 30 to 50% of the patients develop a restenosis. This process of restenosis formation is primarily due to the migration and proliferation of the vascular smooth muscle cells, resulting in the formation of a neointima. However, injury of the normal protective barrier of the endothelial cells during PTCA plays a key role. The resulting local loss of endothelial cells, associated with platelet adhesion on exposed subendothelial layers may be one of the important vascular responses to injury. Therefore, a paracrine mechanism in which smooth muscle cell proliferation is regulated by endothelium-derived factors has been proposed [34,35].

Endothelial-derived NO may be one important endogenous mediator maintaining smooth muscle cells in their normal quiescent state. The NOSIII isoform (ec-NOS) is constitutively expressed by vascular endothelial cells. Synthesized NO diffuses to underlying smooth muscle cells, stimulating soluble guanylate cyclase. The c-GMP generated may contribute to a variety of cellular responses, including inhibition of smooth muscle cell migration and proliferation [22–25], regulation of adhesion molecule expression and thrombo-resistance [20,21]. Systemic delivery of organic NO donors or the physiologic NO precursor L-arginine, the substrate for NOS, has been shown to reduce neointima formation following balloon injury significantly [36].

Therefore, gene transfer of NOS cDNA in order to cause a locally enhanced production of NO might be beneficial in reducing vascular response to injury. Previous in vitro and in vivo studies have already suggested the feasibility and efficacy of NOS gene transfer for gene therapy of neointimal hyperplasia [37–39]. However, one of the main limitations of the methods involved, is the use of adenovirus or even Sendai virus–liposome complex systems in order to achieve a high yield of NOS transfer in the vascular cells. The safety or potential toxicity of such transfection systems for human therapy are still unresolved, and several studies have demonstrated cytotoxicity and induction of immune response in cell lines and animal models. Furthermore, recently evidence has been presented that viral gene transfer into somatic cells may ultimately result in cDNA transfer to germ line cells [11,13,14].

In this paper, we present the results of our investigations on a nonviral cationic liposome-mediated transfection system for vascular smooth muscle cells. Cationic liposomes are regarded as safe vectors for direct gene transfer and transfection is limited primarily to the site of delivery [13,14,40,41]. Although they can accept large amounts of DNA, a rather poor transfection efficiency has been reported previously [42,43]. However, using the cationic liposome DOTAP under optimized conditions a 50% efficiency of transfer of the reporter gene GFP was obtained in primary cultures of rabbit smooth muscle cells. For comparison, De Martin et al. have previously obtained a 70% transfection efficiency of GFP gene in human smooth muscle cells but using an adenovirus-based system [44]. Other cationic liposomes studied e.g. Lipofectamin, Lipofectin, DMRIE-C, Superfect resulted in a poor transfection efficiency in rabbit vascular smooth muscle cells. The specificity of the transfection procedure, depending on the cell type investigated, the transfection reagent, DNA/transfection reagent ratio, incubation time and various other parameters was also reported by other authors in vitro as well as in vivo [12,45].

Previous gene transfer experiments involving NOS have been conducted using the constitutive NOS isoforms, NOSI or NOSIII. However, the activities of these two enzymes depend on availability of calcium and the generation of NO follows a sharp peak and low steady state release pattern following cellular stimulation. In order to obtain a more advantageous pattern of NO release we used NOSII as a transgene in this study. The inducible isoform NOSII has the advantage of releasing NO continuously without requiring agonist stimulation [26,27]. The transgene used in this study corresponds to the coding sequence of the human NOSII. Five' and 3' were excised to remove any potential NOS specific regulatory sequences which could lead to a transcriptional regulation by factors induced during vascular injury.

We postulated that the transfer efficiency observed with the reporter gene GFP was sufficient to induce biological effects. Using the identical experimental conditions, human NOSII transgene was introduced into rabbit smooth muscle cells in vitro and in vivo. A very high level of expression of NOSII transcript and protein was achieved and using immunohistochemistry between 10–15% of NOSII immunoreactive cells were detected. A direct comparison to the percentage of positive cells counted following GFP transfection (about 50%) is difficult, since GFP was detected due to its spontaneous fluorescence. Comparable results of a successful transfection have been shown by Iwashina et al. [54] by transfecting inducible NOS into vascular smooth muscle cells of rat and human using Lipofectin but β-Gal as reporter gene, by Channon et al. [46] transfecting human and rabbit smooth muscle cells with the neuronal NOS isoform using adenovirus-based vectors or Porteous et al. [47] transfecting pCMV–CTFR–DOTAP to nasal epithelial cells.

The functional activity of the transfected human NOSII was convincingly demonstrated in vitro by the substantial increase in arginine to citrulline conversion rate and in c-GMP levels, both detectable up to 6 days posttransfection. These effects were completely abolished in the presence of the L-arginine analog L-NAME. Comparable results of functional active transfected NOS (ec-NOS) were shown by Chen et al. [48] using canine basilar arteries and an adenoviral transfection system. Six days after transfection a rapid reduction of NOSII expression was observed. If the reduced NOS expression after day 6 is due to RNA and protein degradation or cellular turnover is not known yet. However, previously it had been shown that smooth muscle cell migration and proliferation following balloon dilatation already occur at day 4 after injury, and that the inhibition of this process during the initial phase finally prevents neointima formation [49,50]. Thus, a transgene being actively transcribed and translated into NOSII protein only during this critical period may be of advantage, since a long-lasting elevation of NO generation can eventually have deleterious long term effects on cell viability. Furthermore, during this transient expression of NOSII, we were unable to detect any signs of apoptosis or necrosis in rabbit vascular smooth muscle cells studied in our model. In contrast Iwashina et al. [54] demonstrated induction of apoptosis by inducible NO lipofection into rat and human vascular smooth muscle cells. These contradictory findings are possibly due to the other cell-species used, as it was shown, for example, by some of our coauthors that apoptosis by NO donors is hardly induced in rabbit cells requiring mM doses, or other tissue culture conditions (e.g. passage number of cells, FCS content of medium).

The biological effectiveness of the transgene product was tested in vitro in cell proliferation and cell adhesion assays. Rabbit smooth muscle cells transfected with increasing doses of human NOSII cDNA exhibited a dose dependent reduction in cell proliferation. Comparable results were obtained with NO donors, SIN-1 and SNP (data not shown). Moreover, a decrease in PMN adhesion by about 70% was observed in TNF-stimulated smooth muscle cells carrying the NOSII transgene if compared to nontransfected control cells. Both effects were completely abolished by the NOS inhibitor L-NAME.

Scott Burdon et al. [51] reported that the transfection itself triggered the expression of endogenous inducible NOS. However, under our experimental conditions the culture of primary rabbit smooth muscle cells in the presence of liposomes or transfection using liposome/reporter gene complex did not lead to the induction of the inducible isoform of rabbit NOS. Therefore, the observed biological effects are clearly due to the overexpression of the human NOSII transgene. The functional capacity of the endogenous NOSII in primary cultures of rabbit smooth muscle cells was demonstrated by induction upon treatment with LPS or IL1β (data not shown), as previously reported [52,53].

In summary, this study presents a nontoxic and highly efficient, cationic liposome-mediated gene transfer system for the inducible isoform of human NOS into rabbit vascular smooth muscle cells in vitro and in vivo, which results in a transient transcription, resulting in the translation of a biologically active NOSII enzyme. Our data demonstrated the feasibility of this highly efficient nonviral approach for the cellular transfer of NOS cDNA. The observed transient overproduction of NO resulted in both a decrease in cell proliferation and a diminished leukocyte adhesion.

Further in vitro and in vivo studies using this nonviral gene transfer methodology are presently planned in order to demonstrate the potential usefulness of this approach for gene therapy to reduce or even prevent neointima formation following percutaneous coronary angioplasty in humans.

Time for primary review 28 days.


   Acknowledgements
 
This study was supported by Robert Müller Foundation, Mainz (Germany). The authors wish to thank Professor Dr. U. Förstermann, Department of Pharmacology, Johannes Gutenberg-University, Mainz for permitting us to perform some of the experiments in his laboratories and Ms. Anne-Kristin Gröning for her excellent technical assistance.


   References
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 

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