(Circulation. 2001;103:2090.)
© 2001 American Heart Association, Inc.
Basic Science Reports |
Selective Augmentation of Prostacyclin Production by Combined Prostacyclin Synthase and Cyclooxygenase-1 Gene Transfer
From the Vascular Biology Program, Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan (S-K.S., M-J.T., J-Y.L., K.K.W.), and Vascular Biology Research Center and Department of Internal Medicine, University of Texas–Houston Medical School, Houston, Tex (J.T.W., K.K.W.).
Correspondence to Dr Kenneth K. Wu, Division of Hematology, University of Texas–Houston Medical School, 6431 Fannin, MSB 5.016, Houston, TX 77030. E-mail Kenneth.K.Wu{at}uth.tmc.edu
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Abstract |
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Background—We tested the hypothesis that combined cyclooxygenase-1 (COX-1) and prostacyclin synthase (PGIS) gene transfer selectively augments prostacyclin production without a concurrent overproduction of other prostanoids.
Methods and
Results—ECV304 cells were transfected with
bicistronic pCOX-1/PGIS versus pCOX-1 or pPGIS, and prostanoids were
analyzed. Contrary to the high prostaglandin E2
synthesis in pCOX-1 transfected cells, selective prostacyclin formation
was noted with bicistronic plasmid transfection. Next, we determined
the optimal ratio of Ad-COX-1 to Ad-PGIS by transfecting human
umbilical vein endothelial cells with various titers of these 2
adenoviral constructs and determined the level of protein expression
and prostanoid synthesis. Our results show that optimal ratios of
adenoviral titers to achieve a large prostacyclin augmentation without
overproduction of prostaglandin E2 or
F2
were 50 to 100 plaque forming units (pfu)
of Ad-COX-1 to 50 pfu of Ad-PGIS per cell. A higher Ad-PGIS to Ad-COX-1
ratio caused a paradoxical decline in prostacyclin
synthesis.
Conclusions—Prostacyclin synthesis can be selectively augmented by cotransfecting endothelial cells with an optimal ratio of COX-1 to PGIS. Combined COX-1 and PGIS gene transfer has the potential for therapeutic augmentation of prostacyclin.
Key Words: gene therapy • prostaglandins • hypertension, pulmonary • heart diseases
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Introduction |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Prostacyclin (PGI2) is a potent inhibitor of platelet aggregation and a vasodilator, and it acts in concert with nitric oxide, ectonucleotidase, and other endothelial molecules to maintain vascular homeostasis and vasoprotection.1 It is synthesized primarily in vascular endothelial and smooth muscle cells after appropriate stimulation by specific agents. Its biosynthesis is catalyzed by a series of enzymes: cytosolic phospholipase A2 cleaves arachidonic acid (AA) from the sn-2 position of phospholipids, cyclooxygenase (COX) converts AA to prostaglandin (PG) H2, and PGI2 synthase (PGIS) converts PGH2 to PGI2.2 PGH2 is a precursor of several biologically active prostanoids, including PGE2, PGD2, PGF2
, and
thromboxane A2. Two COX isoforms (COX-1 and COX-2) have been identified in endothelial cells (EC): COX-1 is expressed constitutively, whereas COX-2, which is undetectable in resting cells, is induced by proinflammatory and mitogenic factors.3 4 COX expression is considered a key step in determining the capacity for the synthesis of PGI2 and other PGs.4 We previously showed by retrovirus-mediated COX-1 gene transfer that the overexpression of COX-1 in ECs is accompanied by a marked increase in prostanoid synthesis, notably PGI2 and PGE2 in response to stimulation by AA, ionophore A23187, or thrombin.5 Similarly, adenovirus-mediated COX-1 gene transfer in ECs enhanced the production of PGI2, and the direct administration of Ad-COX-1 into injured porcine carotid arteries abrogated thrombus formation, as determined by histological examinations and flow measurements, as a result of increased PGI2 production by the injured artery.6 The antithrombotic effect depended on the titer of Ad-COX-1.6
Overexpression of PGIS by gene transfer reportedly increased PGI2 production and inhibited smooth muscle cell proliferation in a rat carotid artery injury model.7 These experimental results suggest that overexpression of PGI2 synthetic enzymes may potentially be useful in treating pulmonary hypertension, peripheral vascular disease, and other vascular disorders. However, there are drawbacks to overexpressing a single synthetic enzyme such as COX-1 or PGIS for enhancing PGI2 synthesis. In COX-1 gene-transferred cells, besides augmented PGI2 synthesis, a large quantity of PGE2 is also produced. PGE2 is a proinflammatory mediator, and its overproduction may have undesirable effects.8 In PGIS-overexpressed cells, augmentation of PGI2 synthesis is limited because of low cellular levels of COX-1, which is further compromised by autoinactivation during catalysis.9 10 11 12 We postulate that the cotransfection of ECs with COX-1 and PGIS genes at appropriate ratios will shunt PGH2 through the PGIS pathway, with a selective augmentation of PGI2 synthesis.
In this report, we tested this hypothesis by cotransfecting ECs with bicistronic COX-1/PGIS plasmids and by co-transfecting ECs with different titers of replicating defective adenoviruses containing COX-1 (Ad-COX-1) or PGIS (Ad-PGIS) cDNA insert and analyzing the metabolite profile in the transfected cells. Our results indicate that cotransfection of human umbilical vein endothelial cells (HUVECs) with 50 to 100 plaque-forming units (pfu) of Ad-COX-1 and 50 pfu of Ad-PGIS per cell yielded a large increase in PGI2 without a concurrent overproduction of PGE2 or other eicosanoids.
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Methods |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Cell Culture and Materials
ECV304 cells were initially used in our transfection experiments before the report that they exhibit endothelial, epithelial, and bladder cancer cell characteristics.13 We noted that the ECV304 cells used in our experiments stained positively for von Willebrand factor, were capable of expressing adenovirus-transferred COX-1 and PGIS transgenes that were colocalized to the endoplasmic reticulum (as were the natively expressed enzymes), and had a large PGI2 synthetic capacity.14 Thus, ECV304 cells retain EC properties, and they were suitable for the initial feasibility test. ECV304 and human embryonic kidney (HEK)-293 cells were obtained from the American Type Culture Collection (Manassas, Va). They were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 µg/mL streptomycin at 37°C in a humidified 5% CO2 atmosphere.
HUVECs were prepared from freshly obtained umbilical veins and cultured as previously described.5 We routinely used HUVECs in passages 3 through 6. Although COX-1 expressions decline with increasing HUVEC passages, we found that HUVECs from passages 3 through 6 are capable of synthesizing prostanoids that are quantitatively reduced but qualitatively similar to cells at earlier passages. Furthermore, HUVECs from passages 3 through 6 expressed adenovirus-mediated COX-1 and PGIS transgenes competently, as demonstrated by confocal immunofluorescent microscopy.14 There was no significant difference in transfection results among cells at passages 3 to 6. Cell culture media and antibiotics were obtained from BRL Life Technologies. [1-14C]-AA (55 mCi/mmol) was obtained from Amersham.
Recombinant Plasmid Construction
pCOX-1 and pPGIS were constructed in pSG5, as
previously
described.6 15 The
bicistronic pCOX-1/PGIS was constructed in pSG5 by SalI digestion to
remove the COX-1 expression cassette, and it was then further subcloned
into the NdeI site of pPGIS. The final construct contained 2 expression
cassettes that were driven by independent SV40 promoters
(Figure 1A
).
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Recombinant Adenovirus Production
Replication-defective adenoviruses were produced as
described
previously.6 16 17
The adenovirus shuttle plasmid vector pAd-cytomegalovirus (CMV), which
was kindly supplied by Dr S.-H. Chen at Mount Sinai School of Medicine
(New York, NY), contains a CMV promoter and a polyadenylation
signal of bovine growth hormone. The recombinant adenovirus (rAd) was
prepared by cotransfecting HEK-293 cells with pAd-CMV containing the
candidate cDNAs in expression cassettes and pJM17, which was kindly
provided by Dr. L. Chan at Baylor College of Medicine (Houston, Tex),
using an Effectene (Qiagen) transfection system. Two to 3 weeks after
transfection, rAd plaques were picked, propagated, and screened for
specific cDNA sequence using polymerase chain reaction and protein
expression by Western blot analysis. A large-scale production of high
titer rAd was performed as described
previosly,6 with minor
modifications.
HEK-293 cells grown to 
95% confluence were infected with
rAd for 36 to 44 hours, harvested by centrifugation, and resuspended in
fresh culture medium. After freezing and thawing 5 times, cells were
centrifuged to remove cell debris. The supernatant was collected, and
rAd was harvested by CsCl gradient ultracentrifugation. The opalescent
band containing viral particles was collected, loaded onto the top of
1.33 g/mL CsCl, and centrifuged again at the same condition for 18
hours. The opalescent band recovered was dialyzed 3 times against 1
liter of buffer containing 10 mmol/L Tris (pH 7.4), 1 mmol/L
MgCl2, and 10% (v/v) glycerol at 4°C for 18
hours. Virus stocks were separated into aliquots and stored at
-80°C. Viral titers were determined by a plaque-assay method. The
HEK-293 cells were infected with serially diluted viral preparations
and then overlaid with low-melting-point agarose after infection.
Numbers of plaques formed were counted within 2 weeks. Plaque-forming
units per cell (pfu/cell) are referred to as multiplicity of infection
(moi).
Extraction and Analysis of AA
Metabolites
ECV304 cells were transfected with recombinant
plasmids by lipofectamine (Gibco). Forty-eight hours after plasmid
transfection and 24 hours after rAd infection, cells were washed and
incubated in serum-free Dulbecco’s modified Eagle’s medium containing
10 µmol/L [1-14C] AA at 37°C for 10
minutes. The media were collected, and eicosanoids in the media were
extracted by Sep-Pak Cartridge (Waters Associates), as previously
described.18 The extracted
eicosanoids were analyzed by reverse-phase high-pressure liquid
chromatography (HPLC), as previously
described.19 The eicosanoid
peaks were identified by the retention time of the authentic
radiolabeled standards. The quantity of each eicosanoid peak was
determined by relating the integrated area (mV · s) of the peak to
the standard obtained from authentic radiolabeled eicosanoids and AA. A
1000-mV · s integrated area was equivalent to 6.18 ng of AA, 7.44 ng
of 6-keto-PGF1, 7.12 ng of
PGE2, 7.16 ng of PGF2,
and 5.66 ng of 12-hydroxy-5,8,10-heptadecatrienoic acid (HHT),
respectively.
Western Blot Analysis
A total of 15 µg of cell lysate proteins were
applied to each lane and analyzed by Western blots, as previously
described.20 PGIS
antibodies21 and COX-1
antibodies (Santa Cruz) were each diluted to 1:2000.
Peroxidase-conjugated anti-rabbit or anti-mouse IgG (1:2000 dilution)
was used as the second antibody to detect PGIS and COX-1 bands,
respectively, by enhanced chemiluminescence
(Amersham).
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Results |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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To evaluate the feasibility of our proposal, we initially transfected ECV304 cells with pCOX-1, pPGIS, or the bicistronic pCOX-1/PGIS and determined the protein expression and the arachidonate metabolites in the transfected cells. Basal COX-1 and PGIS protein levels were low in untransfected or pUC18-transfected ECV304 (Figure 1B
). There was a concentration-dependent increase in
COX-1 and PGIS when cells were transfected with 1, 2, or 4 µg of
bicistronic pCOX-1/PGIS
(Figure 1B). The COX-1 protein level expressed in cells
transfected with 4 µg of pCOX-1/PGIS was comparable to that with 4
µg of pCOX-1, whereas the PGIS protein level in
pCOX-1/PGIS–transfected cells was only 50% of that in
pPGIS-transfected cells
(Figure 1B).
There was a marked difference in the HPLC profile of cells
transfected with pCOX-1/PGIS versus pCOX-1 or pPGIS. Transfection of
cells with pCOX-1 (4 µg) increased PGE2
synthesis by 100-fold over control levels
(Figure 2 and
Table 1). In contrast, pCOX-1/PGIS transfection increased
6-keto-PGF1 levels in a
concentration-dependent manner, without a concurrent
PGE2 increase
(Figure 2 and
Table 1). The 6-keto-PGF1 level
produced by cells transfected with 4 µg of pCOX-1/PGIS was 12-fold
higher than that produced by cells transfected with 4 µg of pCOX-1
(Figure 2 and
Table 1). HHT production was increased in cells transfected
with pCOX-1 and pCOX-1/PGIS. These transient transfection results
support the notion that co-overexpression of COX-1 and PGIS redirects
PGH2 through the PGIS pathway.
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Because ECV304 cells possess epithelial cell and bladder
cancer cell characteristics, we performed subsequent experiments in
cultured HUVECs. We transfected HUVECs with a mixture of Ad-COX-1 and
Ad-PGIS at different pfu ratios to determine whether the relative
quantities of prostanoids produced are influenced by different ratios
of COX-1 to PGIS overexpression. HUVECs were transfected with 50 moi
(pfu/cell) of Ad-COX-1 or Ad-PGIS alone, a fixed 50 moi of Ad-COX-1
with 10 to 100 moi of Ad-PGIS, or a fixed 50 moi of Ad-PGIS with 10 to
100 moi of Ad-COX-1. The transfected cells were lysed, and COX-1 and
PGIS protein levels were determined by Western blot analysis. The
transgenic COX-1 and PGIS protein levels were markedly elevated by
individual Ad-COX-1 and Ad-PGIS transfection, respectively, compared
with untransfected or Ad-CMV controls
(Figure 3). Combined Ad-COX-1 and Ad-PGIS transfections
produced COX-1 and PGIS protein levels comparable to those of
individual Ad-COX-1 or Ad-PGIS transfections
(Figure 3).
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Eicosanoids generated by transfected cells treated with
[1-14C]AA (10 µmol/L) are shown in
Figure 4. The left panel shows the eicosanoid profile of
cells transfected with a fixed 50 moi of Ad-COX-1 combined with 0 to
100 moi of Ad-PGIS, and the right panel shows the control vector
profile and the profile of cells transfected with a fixed 50 moi of
Ad-PGIS and 0 to 100 moi of Ad-COX-1. Quantitative data on the major
prostanoid peaks shown in this figure are displayed in
Table 2. PGE2 was the major product
(Figure 4); it constituted 78% of the total prostanoids
produced, whereas PGF2 and
6-keto-PGF1 made up 13% and <1%,
respectively, of the total prostanoids produced by HUVECs transfected
with Ad-COX-1 (50 moi) alone
(Table 2). By contrast,
6-keto-PGF1 was the predominant peak in cells
transfected with combined Ad-COX-1/Ad-PGIS
(Figure 4).
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Cotransfection of HUVECs with 100 moi of Ad-COX-1 and 50 moi
of Ad-PGIS yielded the highest 6-keto-PGF1
level, which constituted 85% of total prostanoids
(Table 2). When Ad-PGIS moi was in excess of Ad-COX-1 moi,
the 6-keto-PGF1 and the total prostanoids
produced were markedly reduced
(Figure 4 and
Table 2). For example, the
6-keto-PGF1 and the total prostanoid levels
produced by cells transfected with 50 moi of Ad-COX-1 plus 100 moi of
Ad-PGIS were only 44% and 40%, respectively, of those produced by
cells transfected with 50 moi each of Ad-COX-1 and Ad-PGIS
(Table 2). HHT levels were increased by Ad-COX-1
transfection and by Ad-COX-1/Ad-PGIS cotransfection but not by Ad-PGIS
transfection
(Table 2). The highest value of HHT was produced by cells
transfected with Ad-COX-1/Ad-PGIS in a 50/10 to 50/20 ratio
(Table 2). Its level was reduced when the Ad-PGIS titer was
in excess of the Ad-COX-1 titer. Only trace amounts of
hydroxyeicosatetraenoic acid (HETE)-like eicosanoids were detected, and
their values did not vary by combined transfections. Therefore, we did
not characterize these HETE-like peaks further.
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Results from this study demonstrate that PGI2 synthesis can be augmented without overproduction of other prostanoids by cotransfecting ECs with Ad-COX-1/Ad-PGIS. The extent of PGI2 augmentation depends on the ratio of Ad-COX-1 to Ad-PGIS titers. Our data with HUVECs indicate that transfection with 100 moi of Ad-COX-1 and 50 moi of Ad-PGIS produced the highest PGI2 augmentation when PGE2 and PGF2
levels remained very low (only slightly
above the basal level). This is in contrast to the large increase in
PGE2 synthesis seen in HUVECs transfected with
Ad-COX-1 alone. Because the total prostanoids produced by Ad-COX-1
versus Ad-COX-1/Ad-PGIS–transfected cells are equivalent, the data are
consistent with the concept that an equivalent amount of
PGH2 is produced by the overexpressed COX-1, but
a majority of PGH2 is metabolized via the PGIS
pathway when there is an abundant PGIS. Our results provide direct
evidence to support the hypothesis that the formation of diverse
prostanoids from PGH2 in cells is governed by
the level of the final synthetic enzymes. Further, they suggest that
combined gene transfer has a general application for engineering
selective augmentation of prostanoid synthesis. It is intriguing that PGI2 synthesis declines significantly when the Ad-PGIS titer is in excess of the Ad-COX-1 titer. At Ad-PGIS excess, the level of HHT, a breakdown product of PGH2, was proportionally reduced, which is consistent with reduced PGH2 synthesis. Because no major peaks at the HETE fractions were detected in individual or combined transfections, it is unlikely that the reduced PGH2 is due to a shunt to the lipoxygenase pathways. The mechanism by which an excess of PGIS expression reduces PGH2 generation is unclear. It may be speculated that PGI2 synthesis requires a certain spatial and quantitative relationship between COX-1 and PGIS located on the endoplasmic reticulum membrane and an excessive PGIS expression may perturb this relationship, thereby hindering PGI2 synthesis. We recently showed that overexpressed COX-1 and PGIS,14 like constitutively expressed enzymes,22 are colocalized to the endoplasmic reticulum. COX-1 binds to the luminal endoplasmic reticulum membrane by hydrophobic interactions, whereas PGIS anchors to the cytosolic side of the endoplasmic reticulum membrane through a single transmembrane domain.21 It has been presumed that COX-1 and PGIS are located close to each other to facilitate metabolite transfer, but the quantitative and spatial relationship of these 2 enzymes are unclear and require further investigation.
The quantity of HHT produced by cotransfected cells was
higher than that of control cells and proportionally higher than that
of PGE2 or PGF2
(Table 2). HHT is generated from PGH2
by several potential pathways. Thromboxane synthase catalyzes the
conversion of PGH2 to HHT, malondialdehyde, and
thromboxane A2 in a 1:1:1
ratio.23 Cytochrome P450 was
recently reported to convert PGH2 to HHT and
malondialdehyde.24 HHT can
also be converted from PGH2 by a nonenzymic
pathway. Cultured HUVECs possess very low levels, if any, of
thromboxane synthase activity. It is unlikely that HHT is derived from
this pathway. However, we cannot exclude the possibility that HHT was
derived from cytochrome P450 or from nonenzymic conversion.
Several clinical trials have shown that PGI2 and its stable analog iloprost are efficacious in treating primary pulmonary hypertension.25 26 27 A recent randomized, multicenter, controlled trial provides objective evidence for the beneficial effect of beraprost, a stable analog of PGI2, on peripheral vascular disease.28 PGI2 is likely to have broad effects on diverse vascular disorders. However, systemic administration of these drugs is associated with undesirable side effects. PGI2 acts locally in an autocrine and paracrine manner. It will be desirable to administer PGI2 locally at a targeted site. Local administration of PGI2 and its more stable analogs to the targeted vascular region remains a challenge because of the relatively short half-life of these drugs. Gene transfer to overexpress PGI2 synthetic enzymes, thereby augmenting PGI2 productions at a targeted site of the vascular system, has been shown to be feasible in several animal models.6 7 In addition to local PGI2 productions, gene transfer approaches have the following other advantages: (1) PGI2 is produced only when stimuli are present, and (2) PGI2 synthetic enzyme(s) are expressed for a longer period of time than drugs.
Previous experimental work used a single gene approach to augment PGI2 production. Results from the present study indicate that overexpression of COX-1 alone is accompanied by an overproduction of PGE2, which is a key proinflammatory mediator and may potentially contribute to vascular inflammation, whereas overexpression of PGIS alone has a minimal effect on increasing PGI2 synthesis. In fact, PGIS overexpression may suppress PGI2 synthesis. In contrast, combined COX-1/PGIS gene transfer at appropriate ratios selectively augments PGI2 productions and should be a better approach than the single gene transfer approach. Work is in progress in our laboratory to evaluate the effects of combined Ad-COX-1/Ad-PGIS transfer on vascular lesions in appropriate experimental animal models.
In summary, it has become possible for the first time to engineer selective augmentation of PGI2 synthesis by combined transfer of Ad-COX-1 and Ad-PGIS. By selecting an optimal ratio of COX-1 to PGIS overexpression, it is possible to shunt a majority of PGH2 through the PGIS pathway. Combined COX-1/PGIS gene transfer is potentially useful in gene therapy for diverse vascular diseases.
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Acknowledgments |
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This work was supported by grants from the Department of Health and the National Science Council of Taiwan under "The Frontier Program of Medical Genetics Research" (DOH89-TD-1132) and grants from the National Institutes of Health (P50-NS 23327 and RO1-HL 50675). We thank Angela Wang and Susan Mitterling for secretarial assistance.
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Footnotes |
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The first 2 authors contributed equally to this work.
Guest Editor for this article was Joseph Loscalzo, MD, PhD, Boston University School of Medicine, Boston, Mass.
Received June 16, 2000; revision received November 8, 2000; accepted November 20, 2000.
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H. Lin, T.-N. Lin, W.-M. Cheung, G.-M. Nian, P.-H. Tseng, S.-F. Chen, J.-J. Chen, S.-K. Shyue, J.-Y. Liou, C.-W. Wu, et al. Cyclooxygenase-1 and Bicistronic Cyclooxygenase-1/Prostacyclin Synthase Gene Transfer Protect Against Ischemic Cerebral Infarction Circulation, April 23, 2002; 105(16): 1962 - 1969. [Abstract] [Full Text] [PDF]
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