(Circulation. 2000;102:2005.)
© 2000 American Heart Association, Inc.
Basic Science Reports |
Gene Transfer of Human Prostacyclin Synthase Ameliorates Monocrotaline-Induced Pulmonary Hypertension in Rats
From the Division of Cardiology, Department of Medicine, National Cardiovascular Center (N. Nagaya, S.K., M.U., T.N., N. Nakanishi, M.Y., K.M.); Department of Pharmacology, National Cardiovascular Center Research Institute (C.Y., M.S., T.T.); and Departments of Gene Therapy Science (R.M., Y.K.) and Geriatric Medicine (T.O.), Osaka University Medical School, Osaka, Japan.
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Abstract |
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Background—Prostacyclin is a potent vasodilator that also inhibits platelet adhesion and cell growth. We investigated whether in vivo gene transfer of human prostacyclin synthase (PGIS) ameliorates monocrotaline (MCT)-induced pulmonary hypertension in rats.
Methods and Results—The cDNA encoding PGIS was intratracheally
transfected into the lungs of rats by the hemagglutinating virus of
Japan–liposome method. Rats transfected with control vector lacking
the PGIS gene served as controls. Three weeks after MCT injection, mean
pulmonary arterial pressure and total
pulmonary resistance had increased significantly; the increases
were significantly attenuated in PGIS gene–transfected rats compared
with controls [mean pulmonary arterial pressure,
31±1 versus 35±1 mm Hg (-12%); total pulmonary
resistance, 0.087±0.01 versus 0.113±0.01 mm Hg · mL
· min-1 ·
kg-1 (-23%), both P<0.05].
Systemic arterial pressure and heart rate were unaffected.
Histologically, PGIS gene transfer inhibited the
increase in medial wall thickness of peripheral
pulmonary arteries that resulted from MCT injection. PGIS
immunoreactivity was intense predominantly in the bronchial epithelium
and alveolar cells. Lung tissue levels of 6-keto-PGF1
, a
stable metabolite of prostacyclin, were significantly increased for 
1
week after transfer of PGIS gene. The Kaplan-Meier survival curves
demonstrated that repeated transfer of PGIS gene every 2 weeks
increased survival rate in MCT rats (log-rank test,
P<0.01).
Conclusions—Intratracheal transfer of the human PGIS gene augmented pulmonary prostacyclin synthesis, ameliorated MCT-induced pulmonary hypertension, and thereby improved survival in MCT rats.
Key Words: prostaglandins • gene therapy • hypertension, pulmonary • viruses
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Introduction |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Prostacyclin, a metabolite of arachidonic acid, has vasoprotective effects, including vasodilation, antiplatelet aggregation, and inhibition of smooth muscle cell proliferation.1 2 Prostacyclin synthase (PGIS) catalyzes the conversion of prostaglandin H2 to prostacyclin. In 1994, we determined the amino acid sequence of human endothelial PGIS by cDNA cloning.3 Recently, we showed that introduction of the PGIS cDNA into rat carotid artery after balloon-induced injury results in increased prostacyclin synthesis and inhibition of smooth muscle cell proliferation.4 These results raise the possibility that in vivo gene transfer of PGIS could have beneficial effects in patients with vascular disease.
Primary pulmonary hypertension (PPH) is a rare but life-threatening disease characterized by progressive pulmonary hypertension, ultimately producing right ventricular (RV) failure and death.5 Interestingly, PGIS deficiency in the lungs and impaired prostacyclin production have been linked to the development of pulmonary hypertension in this disease.6 7 As a result, continuous intravenous infusion of prostacyclin has become recognized as a therapeutic breakthrough that can improve hemodynamics and survival in patients with PPH.8 9 10 11 Recently, transgenic mice with lung PGIS overproduction have been shown to be protected from the development of pulmonary hypertension after exposure to hypoxia.12 However, whether in vivo gene transfer of human PGIS can ameliorate pulmonary hypertension remains unknown.
The hemagglutinating virus of Japan (HVJ)–liposome method is a liposome-based gene delivery system that enables introduction of the contents of liposomes directly into living cells by means of the virus-cell fusion machinery.13 14 15 16 Because HVJ-liposomes are prepared after ultraviolet irradiation of the virus, this gene transfer system is less toxic and less immunogenic than adenovirus and herpesvirus vectors.
Thus, the purpose of this study was to investigate whether intratracheal transfer of the human PGIS gene by use of HVJ-liposomes augments pulmonary prostacyclin synthesis, ameliorates monocrotaline (MCT)-induced pulmonary hypertension, and improves survival in MCT rats.
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Methods |
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Animals
Male Wistar rats weighing 100 to 120 g were used in this study. Rats were randomly assigned to receive intratracheal injection of either the HVJ-liposome complex with PGIS expression plasmid or control vector, followed by a subcutaneous injection of either 60 mg/kg MCT or 0.9% saline. This protocol resulted in the creation of 4 groups: MCT rats transfected with PGIS expression plasmid (MCT-PGIS, n=8); MCT rats transfected with control vector (MCT-CON, n=8); normal rats transfected with PGIS expression plasmid (NL-PGIS, n=8); and normal rats transfected with control vector (NL-CON, n=8). Another 40 rats injected with MCT were studied to assess the time course of prostacyclin synthesis after transfer of either PGIS expression plasmid or control vector. An additional 24 rats were studied to evaluate the effects of PGIS gene therapy on survival in MCT rats.
Construction of Plasmid DNA
The expression vector for human PGIS was constructed as
described previously.3 In brief, the blunted
HindIII/BamHI fragment of the full-length human
PGIS cDNA was ligated into the blunted XhoI site of the
pUC-CAGGS expression plasmid. To verify that the pUC/PGIS construct
encoded a biologically active PGIS protein, pUC/PGIS was transfected
into 293 cells, and PGIS activity in the transfected cells was measured
as conversion of
[14C]PGH2 to
6-keto-[14C]PGF1. The
pUC-CAGGS vector lacking the insert encoding human PGIS served as the
control vector.
Preparation of HVJ-Liposomes
Preparation of HVJ-anionic liposomes with a lipid composition
similar to that of the viral envelope (AVE) has been described
elsewhere.15 In brief, lipid mixtures were prepared by
mixing lipid solutions (phosphatidylcholine/dioleoylphosphatidyl
ethanolamine/sphingomyelin/phosphatidylserine/cholesterol,
4:4:4:3:15 in molar ratio). Dried lipid mixture was propagated with
plasmid DNA (200 µg) in 200 µL balanced salt solution (mmol/L: NaCl
137, KCl 5.4, Tris-HCl 10 [pH 7.6]). HVJ-liposomes were prepared by
mixing the lipid with HVJ virus inactivated by ultraviolet
irradiation. After incubation and sucrose gradient
centrifugation, the HVJ-liposome complex was collected
with a Pasteur pipette.
Experimental Protocol
After the rats were anesthetized by
intraperitoneal injection of pentobarbital (30
mg/kg), tracheostomy was performed to introduce a polyethylene catheter
(PE-50) into the left and right main bronchi. Through the catheter, 400
µL HVJ-liposome complex including 100 µg plasmid DNA was
administered into the bronchi. Next, rats were intubated with a
polyethylene tube (PE-240) and artificially ventilated with a
volume-regulated respirator for 30 minutes. Then, the polyethylene tube
was removed and the tracheotomy was closed. Finally, rats were given a
single subcutaneous injection of either 60 mg/kg MCT or 0.9% saline 24
hours after gene transfer. Animals were maintained on standard rat
chow.
Hemodynamic studies were performed 22 days after gene transfer. Rats were anesthetized with intraperitoneal pentobarbital (30 mg/kg) and placed on a heating pad to maintain body temperature at 37°C to 38°C throughout the study. A polyethylene catheter (PE-10) was inserted into the right femoral artery to measure heart rate and mean arterial pressure. A 3.5F umbilical vessel catheter was inserted through the right jugular vein into the pulmonary artery for measurement of RV pressure and pulmonary arterial pressure. These hemodynamic variables were measured with a pressure transducer (model P 23 ID, Gould) connected to a polygraph and recorded with a thermal recorder (7758 B System, Hewlett-Packard). A thermomicroprobe was advanced into the ascending aorta via the right carotid artery and connected to a cardiac output computer (Cardiotherm-500, Columbus Instruments). Cardiac output was measured in triplicate by the thermodilution method. Total pulmonary resistance was calculated by dividing mean pulmonary arterial pressure by cardiac output. After completion of the above measurements, cardiac arrest was induced by injection of 2 mmol KCl through the catheter. The ventricles and lungs were excised, dissected free, and weighed. The measurement of the RV weight excluded the intraventricular septum. The ratio of RV weight to body weight (RV/BW) and the ratio of left ventricular weight to body weight (LV/BW) were calculated as indexes of ventricular hypertrophy.
Morphometric Analysis of Pulmonary Arteries
Paraffin sections 4 µm thick were obtained from the
middle region of the right lung and stained with hematoxylin and eosin
for examination by light microscopy. Statistical analysis of
the medial wall thickness of the pulmonary arteries was
performed as described previously.17 In brief, the
external diameter and the medial wall thickness were measured in 30
muscular arteries (ranging in size from 25 to 50 and from 51 to
100 µm in external diameter) per lung section. For each artery,
the medial wall thickness was expressed as follows: % wall
thickness=[(medial thicknessx2)/external diameter]x100. A lung
section was obtained from individual rats for comparison among 4 groups
(MCT-PGIS, MCT-CON, NL-PGIS, and NL-CON groups, n=5 each).
Immunohistochemical Analysis
Seven days after transfer of PGIS expression plasmid or control
vector, lungs of MCT rats were stained immunohistochemically with a
rabbit polyclonal antibody raised against a partial sequence of the
human PGIS sequence. Sections 4 µm thick were incubated in 3%
hydrogen peroxide for 30 minutes to block endogenous
peroxidase activity and to permeabilize the cells.
Nonspecific binding of rabbit serum was prevented by preincubating the
sections with 0.2% normal goat serum. The sections were sequentially
incubated at 4°C overnight with rabbit antibody against human PGIS at
a concentration of 1:1000 and then with biotinylated goat anti-rabbit
IgG (Dako Japan Co) for 30 minutes, followed by peroxidase labeling
with streptavidin (LSAB kit, Dako Japan Co) for an additional 20
minutes at room temperature. Each incubation was followed by washing in
Tris-buffered saline. Staining was visualized with a chromogen, 0.06%
3,3'-diaminobenzidine/0.03% hydrogen peroxide in 8 mmol/L
Tris-HCl (pH 6.85). Hematoxylin was used as a counterstain. Control
sections were incubated with nonimmune rabbit IgG at a concentration of
1:1000.
Measurement of 6-keto-PGF1
Lung tissue levels of 6-keto-PGF1, a
stable metabolite of prostacyclin, were analyzed to assess
prostacyclin production. To determine the time course of
prostacyclin synthesis in the lung, a total of 80 lungs in MCT rats
were evaluated 2, 7, 14, and 21 days after transfer of PGIS expression
plasmid (n=10 each) or control vector (n=10 each).
6-keto-[3H]PGF1
(10 000 dpm, 6.55 TBq/mmol, Amersham Pharmacia Biotech) was added as a
tracer for calculation of the recovery factor. After extraction in
ice-cold ethanol and purification with a C-18 reverse-phase cartridge
(Sep-Pak Plus, Waters), 6-keto-PGF1 was
quantified with an enzyme immunoassay kit (Cayman Chemical
Co).18 The protein content of the precipitate after
ethanol extraction of the lung was determined by the Lowry
method.19 The results were expressed as picograms of
6-keto-PGF1 per milligram of protein.
Survival Analysis
To evaluate the effects of PGIS gene therapy on survival in
MCT-injected rats, 24 rats received repeated administration of
HVJ-liposome complex with PGIS expression plasmid (n=12) or control
vector (n=12) every 2 weeks. After the rats were anesthetized
by intraperitoneal injection of pentobarbital (30
mg/kg), the repeated administration of 200 µL HVJ-liposome complex
including 50 µg plasmid DNA was done via tracheal tube (PE-240). Rats
were then ventilated with a volume-regulated respirator for 30 minutes.
Survival was estimated from the date of MCT injection to the death of
the rat or 10 weeks after the injection.
Statistical Analysis
All data were expressed as mean±SEM unless otherwise
indicated. Comparisons of parameters between 2 groups were
made by unpaired Student’s t test. Comparisons of
parameters among 4 groups were made by 1-way ANOVA,
followed by Newman-Keuls test. Survival curves according to the
presence or absence of PGIS gene transfer were derived by the
Kaplan-Meier method and compared by log-rank tests. A value of
P<0.05 was considered statistically significant.
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Results |
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Physiological Profiles
The physiological profiles of the 4 experimental groups are summarized in the Table
. Body weight and mean
arterial pressure were significantly lower in MCT rats
(MCT-CON and MCT-PGIS groups) than in normal rats (NL-CON and NL-PGIS
groups). There was no significant difference in heart rate among the 4
groups. Both RV/BW and RV systolic pressure were significantly
increased in rats injected with MCT.
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Effect of PGIS Gene Transfer on MCT-Induced Pulmonary
Hypertension
Three weeks after MCT injection, pulmonary hypertension
developed in both MCT groups, but the rise in mean pulmonary
arterial pressure was significantly smaller (by 12%) in
the MCT-PGIS group than in the MCT-CON group (31±1 versus 35±1
mm Hg, P<0.05, Figure 1).
Cardiac output was significantly higher (by 13%) in the MCT-PGIS than
in the MCT-CON group (360±11 versus 320±16 mL ·
min-1 ·
kg-1, P<0.05).
Cardiac output tended to be decreased in the MCT-CON compared with the
NL-CON group, but no reduction in cardiac output was observed in the
MCT-PGIS group. Therefore, total pulmonary resistance was
significantly lower (by 23%) in the MCT-PGIS than in the MCT-CON group
(0.087±0.01 versus 0.113±0.01 mm Hg · mL ·
min-1 ·
kg-1, P<0.05).
Similarly, increases in RV systolic pressure and RV/BW were
significantly attenuated in the MCT-PGIS compared with the MCT-CON
group (Table
). In contrast, neither systemic
arterial pressure nor heart rate differed between the 2
groups. There were no significant differences in
hemodynamic parameters between the NL-CON
and NL-PGIS groups.
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P<0.05 vs MCT-CON.
Morphometric Analysis of Pulmonary Arteries
Representative photomicrographs showed that the
hypertrophy of the pulmonary vessel wall was
attenuated in MCT-PGIS rats compared with MCT-CON rats (Figure 2). Quantitative analysis of
peripheral pulmonary arteries demonstrated
significant increases in percent wall thickness in both MCT groups, but
these changes were significantly attenuated in the MCT-PGIS compared
with the MCT-CON group (25±2% versus 31±2% in the vasculature with
external diameters of 25 to 50 µm; 26±2% versus 33±3% in the
51- to 100-µm vasculature, both P<0.05, Figure 3). There was no significant difference
in either parameter between the NL-CON and NL-PGIS
groups.
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Immunohistochemical Analysis
PGIS immunoreactivity was intense in distal bronchial
epithelium in MCT rats transfected with PGIS gene (Figure 4). Diffuse
immunostaining for PGIS was detected in alveolar cells
after PGIS gene transfer. In addition, intense immunoreactivity for
PGIS was observed in the bronchial epithelium near the
pulmonary vasculature. No major adverse effects of gene
expression were observed in the transfected lung segments.
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Lung Tissue Level of 6-keto-PGF1
Lung tissue content of 6-keto-PGF1 in MCT
rats was significantly increased 2 days (999±173 versus 534±103 pg/mg
protein, P<0.05) and 7 days (1045±133 versus 716±68 pg/mg
protein, P<0.05) after a single transfer of PGIS expression
plasmid (Figure 5). Lung
6-keto-PGF1 levels remained elevated 14 days
after PGIS gene transfer, although this was statistically not
significant.
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Survival Analysis
Kaplan-Meier survival curves demonstrated that MCT rats
transfected with PGIS gene had a significantly higher survival rate
than those transfected with control vector alone (33% versus 0% in
10-week survival, log-rank test, P<0.01, Figure 6). No definite adverse effects were
detected after repeated transfer of PGIS gene.
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Discussion |
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In the present study, we demonstrated that (1) intratracheal transfer of the human PGIS gene by use of HVJ-liposomes induced overexpression of PGIS protein in bronchial epithelium and alveolar cells in MCT rats, resulting in augmenting prostacyclin synthesis in the lungs; and (2) transfer of the PGIS gene ameliorated MCT-induced pulmonary hypertension and pulmonary vascular remodeling. Finally, we demonstrated that (3) repeated transfer of the PGIS gene improved survival in MCT rats without definite adverse effects.
Patients with PPH have significantly decreased production of prostacyclin relative to that of thromboxane.6 Recently, PGIS expression has been shown to be particularly decreased in remodeled pulmonary arteries containing plexiform lesions in patients with PPH.7 These findings raise the possibility that impaired prostacyclin synthesis resulting from decreased PGIS may be implicated in the pathogenesis of PPH. In fact, continuous intravenous infusion of prostacyclin markedly lowers pulmonary vascular resistance in patients with PPH and improves survival beyond that attained with conventional therapy alone.8 9 10 11 However, prostacyclin is metabolized rapidly, requiring continuous intravenous administration. Quality of life still remains an important issue to be resolved. Therefore, it would be more desirable to enhance endogenous prostacyclin production.
In the present study, transfer of human PGIS gene to the lung could be achieved by intratracheal injection of HVJ-liposome complex. Intense immunoreactivity for PGIS was observed predominantly in bronchial epithelium and alveolar cells. Earlier studies have shown that HVJ-liposomes have strong affinity for bronchial epithelium and alveolar cells.20 21 Recently, Saeki et al15 developed a novel anionic liposome with a lipid composition similar to the AVE, which showed up to a 10-fold improvement in transduction efficiency in vivo over conventional HVJ-liposomes. Thus, in the present study, this HVJ-AVE liposome was used for successful intratracheal gene transfer. The human PGIS gene discovered in our institute was used in the present study because the cDNA for human PGIS has high identity with its rat counterpart.3 22
Recently, Champion et al23 showed that intratracheal gene
transfer of calcitonin gene–related peptide to bronchial epithelial
cells and alveolar cells attenuates chronic hypoxia-induced
pulmonary hypertension in the mouse, suggesting that lung cell
transduction with a vasodilator peptide may be sufficient to alter
vascular function. Geraci et al12 showed that
pulmonary prostacyclin synthase overexpression in transgenic
mice protects against development of hypoxic pulmonary
hypertension. Because they chose to use the epithelial cell–specific
promoter instead of a vascular system–specific promotor, PGIS gene was
expressed in bronchial epithelial cells and alveolar cells but not in
pulmonary vessels. Nevertheless, PGIS-expressing mice did
inhibit a pulmonary hypertensive response to acute
hypoxia. In clinical settings, inhaled aerosolized prostacyclin
acts transepithelially with pulmonary selectivity and improves
pulmonary hypertension and
oxygenation.24 25 These findings suggest
that transepithelial delivery of prostacyclin to the vasculature may
have beneficial effects on pulmonary
hemodynamics. In the present study, lung tissue
content of 6-keto-PGF1 was significantly
increased for at least 1 week after transfer of PGIS gene, suggesting
increased prostacyclin synthesis in the lung. The consequence of this
synthesis in rats injected with MCT was a significant decrease in mean
pulmonary arterial pressure and total
pulmonary resistance. Importantly, the improvement in
pulmonary hemodynamics was not accompanied by
systemic hypotension, paralleling earlier results with gene transfer of
endothelial nitric oxide synthase to the
lung.26 27 Considering increased PGIS in the bronchial
epithelium near the pulmonary vasculature, it is possible that
endogenous prostacyclin produced by epithelial PGIS acts
transepithelially with pulmonary vascular selectivity and
ameliorates MCT-induced pulmonary hypertension.
Histological examination of MCT rats revealed that PGIS gene transfer inhibited an increase in the medial wall thickness of peripheral pulmonary arteries. In MCT-injected rats, endothelial cell injury caused by MCT activates platelets and vasoconstrictive factors, resulting in pulmonary hypertension and pulmonary vascular remodeling.28 Given the known potent vasoprotective effects of prostacyclin, such as vasodilation, antiplatelet aggregation, and inhibition of smooth muscle cell proliferation,1 2 it is interesting to speculate that endogenous prostacyclin produced by epithelial PGIS overexpression may act as a paracrine factor in the regulation of progressive pulmonary vascular remodeling in MCT rats.
Single gene transfer with HVJ-liposomes induced overexpression of PGIS
and increased production of
6-keto-PGF1 for at least 1 week. The
HVJ-liposome method has been shown to be less toxic and less
immunogenic than viral vectors such as adenovirus and herpesvirus
because HVJ-liposomes are prepared after ultraviolet irradiation of the
virus. Thus, MCT rats received repeated administration of the
HVJ-liposome complex with PGIS gene every 2 weeks. Interestingly,
repetitive transfer of PGIS gene significantly improved survival in MCT
rats without definite inflammation in the lungs. Thus, PGIS gene
therapy may be a promising treatment for severe pulmonary
hypertension. However, the initial success of PGIS gene therapy
reported here should be confirmed by long-term experiments, and
extensive toxicity studies in animals are needed before clinical
trials.
Conclusions
Intratracheal transfer of the human PGIS gene with
HVJ-liposomes augmented pulmonary prostacyclin synthesis,
inhibited MCT-induced pulmonary hypertension, and thereby
improved survival in MCT rats. PGIS gene therapy may be a new
therapeutic strategy for the treatment of pulmonary
hypertension.
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Acknowledgments |
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This work was supported in part by the Japan Intractable Diseases Research Foundation. We thank Michiko Tamakoshi, Toru Satoh, Fumio Sakamaki, and Fumiki Yoshihara for their technical assistance.
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Footnotes |
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Reprint requests to Noritoshi Nagaya, MD, Division of Cardiology, Department of Medicine, National Cardiovascular Center, 5-7-1 Fujishirodai, Suita, Osaka 565-8565, Japan.
Received February 8, 2000; revision received May 23, 2000; accepted May 24, 2000.
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 K. Kuwano, A. Hashino, T. Asaki, T. Hamamoto, T. Yamada, K. Okubo, and K. Kuwabara 2-{4-[(5,6-Diphenylpyrazin-2-yl)(isopropyl)amino]butoxy}-N-(methylsulfonyl)acetamide (NS-304), an Orally Available and Long-Acting Prostacyclin Receptor Agonist Prodrug J. Pharmacol. Exp. Ther., September 1, 2007; 322(3): 1181 - 1188. [Abstract] [Full Text] [PDF]
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A. Hara, K.-i. Yuhki, T. Fujino, T. Yamada, K. Takayama, S. Kuriyama, O. Takahata, H. Karibe, Y. Okada, C.-Y. Xiao, et al. Augmented Cardiac Hypertrophy in Response to Pressure Overload in Mice Lacking the Prostaglandin I2 Receptor Circulation, July 5, 2005; 112(1): 84 - 92. [Abstract] [Full Text] [PDF]
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T. Itoh, N. Nagaya, S. Murakami, T. Fujii, T. Iwase, H. Ishibashi-Ueda, C. Yutani, M. Yamagishi, H. Kimura, and K. Kangawa C-type Natriuretic Peptide Ameliorates Monocrotaline-induced Pulmonary Hypertension in Rats Am. J. Respir. Crit. Care Med., December 1, 2004; 170(11): 1204 - 1211. [Abstract] [Full Text] [PDF]
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C. Yokoyama, T. Yabuki, M. Shimonishi, M. Wada, T. Hatae, S. Ohkawara, J. Takeda, T. Kinoshita, M. Okabe, and T. Tanabe Prostacyclin-Deficient Mice Develop Ischemic Renal Disorders, Including Nephrosclerosis and Renal Infarction Circulation, October 29, 2002; 106(18): 2397 - 2403. [Abstract] [Full Text] [PDF]
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M. Ono, Y. Sawa, K. Matsumoto, T. Nakamura, Y. Kaneda, and H. Matsuda In Vivo Gene Transfection With Hepatocyte Growth Factor via the Pulmonary Artery Induces Angiogenesis in the Rat Lung Circulation, September 24, 2002; 106(12_suppl_1): I-264 - I-269. [Abstract] [Full Text] [PDF]
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H. Suhara, Y. Sawa, N. Fukushima, K. Kagisaki, C. Yokoyama, T. Tanabe, S. Ohtake, and H. Matsuda Gene transfer of human prostacyclin synthase into the liver is effective for the treatment of pulmonary hypertension in rats J. Thorac. Cardiovasc. Surg., May 1, 2002; 123(5): 855 - 861. [Abstract] [Full Text] [PDF]
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