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【摘要】
Objectives- The major immediate-early cytomegalovirus enhancer/promoter (MIECMV), widely used in cardiovascular gene therapy, contains several positively regulatory cAMP response elements (CRE). Catecholamine signaling via ß-adrenoceptors might increase transgene expression from MIECMV, and if so, ß-blockers may have a detrimental effect on the efficacy of clinical cardiovascular gene therapy strategies.
Methods and Results- Cultured smooth muscle cells were exposed to isoprenaline, atenolol, or propranolol, alone and in combination before infection with adenoviruses expressing ß-galactosidase. ß-galactosidase expression was assayed 72 hours later. Isoprenaline increased transgene expression from MIECMV up to 8-fold ( P <0.001), but had no effect on a promoter containing no CRE. The effect of isoprenaline was inhibited by ß-blockade and by specific CRE-decoy oligonucleotides. ß-blockers did not reduce transgene expression below basal levels. After adenovirus-mediated porcine intracoronary gene transfer, however, ß-blockade reduced ß-galactosidase expression by up to 250-fold compared with non-ß-blocked animals ( P <0.01).
Conclusions- Enhancement of promoter activity by endogenous catecholamines is essential for high-level transgene expression from MIECMV within the vasculature. ß-blocker-mediated suppression of transgene expression from MIECMV in vascular tissues has a significant bearing on clinical studies of cardiovascular gene transfer. This is the first described interaction to our knowledge between widely prescribed pharmaceuticals and a commonly used promoter of clinical transgene expression.
The major immediate-early cytomegalovirus enhancer/promoter (MIECMV) is used extensively in studies of clinical cardiovascular gene transfer. Transgene expression from MIECMV within the vasculature is suppressed by ß-blockade. This interaction is highly pertinent to the interpretation of current studies and to the planning of future studies of therapeutic cardiovascular gene transfer.
【关键词】 ßadrenergic receptor blockers catecholamines gene therapy
Introduction
Intracellular signaling after ligand-mediated activation of ß-adrenoceptors is mediated by intracellular adenosine-3', 5'-cyclic monophosphate (cAMP). Ligand-binding leads to the activation of adenylyl cyclase by G-proteins coupled to the cytosolic domain of the ß-adrenoceptor. Activation of adenylyl cyclase increases cAMP synthesis, which leads to activation by protein kinase A of the transcriptional factor cAMP-response element-binding protein (CREB). The cAMP-response element (CRE) is an ubiquitous 8-base-pair-long palindromic DNA sequence that mediates the effects of cAMP on transcriptional activity. 2 Once activated, CREB binds to CRE and stimulates assembly of the RNA polymerase complex which promotes transcription. 2
The major immediate-early enhancer/promoter from human cytomegalovirus (MIEhCMV) is a commonly used promoter of transgene expression, particularly in vascular gene therapy. In a recent review of experimental vascular gene transfer, of the 69 studies cited 80%) used MIEhCMV to promote transgene expression. 3 MIEhCMV contains several copies of CRE. Clearly there is potential for enhancement by ß-adrenergic agonists of transgene expression under the transcriptional regulation of MIEhCMV and for inhibition of any such effect by antagonists of ß-adrenoceptors (ß-blockers). Because ß-adrenoceptors are widely distributed throughout the vasculature and ß-blockers are frequently prescribed in a range of cardiovascular diseases, such an interaction might prove very pertinent with respect to the clinical application of gene therapy to those pathologies for which ß-blockers may be coprescribed.
To establish whether ß-adrenoceptor-mediated signaling enhances transgene expression from MIECMV by activation of CREB, we studied the effect of the nonsubtype-specific ß-adrenoceptor agonist isoprenaline on transgene expression in cultured smooth muscle cells (SMCs) infected with 1 of 3 recombinant adenoviruses. Ad5- lacZ contains Escherichia coli lacZ under transcriptional regulation of MIEhCMV, 4 RAd36 contains lacZ regulated by the more potent homologue from murine cytomegalovirus (MIEmCMV), 5 and RAd122 contains lacZ under regulation of the Rous sarcoma virus long terminal repeat promoter (RSV-LTR), which contains no CRE. 6 We have studied the effects of the nonselective ß-blocker propranolol, the ß 1 -selective ß-blocker atenolol and the ß 2 -selective agent ICI118,551 on transgene expression. ß-blockers were studied alone and in combination with isoprenaline or forskolin, the latter of which directly activates adenylyl cyclase. 7 We have also investigated the effect of a specific CREB-binding decoy oligonucleotide on transgene expression. 8 To determine to what extent endogenous catecholamines affect transgene expression in vivo, we subsequently studied the effects of atenolol and propranolol on ß-galactosidase expression after intracoronary delivery of RAd36.
Methods
Recombinant Adenoviruses and Oligonucleotides
Viruses were generated by homologous recombination in 293 cells. Stocks were purified by cesium chloride gradient centrifugation and titered by serial-dilution end-point assay.
Two phosphorothioate-oligonucleotides were purchased (Sigma-Genosys). The CRE-decoy sequence comprises 3 copies of the palindromic CRE sequence 8 : 5'-TGACGTCA-TGACGTCA-TGACGTCA-3'. The control oligonucleotide was a 24-bp nonsense-sequence palindrome: 5'-CTAGCTAG-CTAGCTAG-CTAGCTAG-3'.
SMC Culture
Primary-cultured porcine coronary SMCs were established as previously described. 9 Porcine and human (TCS Cellworks) coronary SMCs were seeded in 6-well plates and grown to 90% confluence, then rendered quiescent by exposure to 2%-serum medium for 72 hours. To establish the maximum concentrations of isoprenaline, atenolol, propranolol, ICI118,551, and forskolin, to which SMC could be exposed without toxicity, medium supplemented with one of each agent at progressive 2-fold increases in concentration, starting at 12.5 µmol/L, were added to SMCs in different wells. The medium was replaced with fresh supplemented medium daily for 72 hours. In subsequent experiments, quiescent SMCs were exposed to atenolol, propranolol, ICI118,551, isoprenaline, forskolin, isoprenaline+atenolol, isoprenaline+propranolol, isoprenaline+ICI118,551, forskolin+ atenolol, forskolin+propranolol, or unsupplemented medium only for 24 hours before infection with Ad5- lacZ, RAd36, or RAd122 at multiplicities of infection of 50, 10, and 500, respectively. For each virus, 4 wells of SMCs were exposed to each agent or combination of agents. Freshly supplemented medium was added daily to infected SMCs until 72 hours postinfection, after which ß-galactosidase activity was quantified by chromogenic assay in lysates of the cell monolayers, as described previously. 5
SMC Transfection
To establish the maximum concentration of phosphorothioate-oligonucleotide to which cells could be exposed without inducing toxicity, SMCs were transfected with escalating doses of each oligonucleotide. Single doses of CRE-decoy or nonsense oligonucleotide were complexed with 20 µL of Lipofectamine 2000 transfection reagent (Invitrogen) to achieve a final concentration in the SMC medium of 125, 250, 500, or 1000 nM. Those SMC used for oligonucleotide-transfection studies were subsequently transfected, at the same time as adenovirus infection, by addition of a single dose of CRE-decoy or nonsense oligonucleotide complexed with 20 µL of Lipofectamine 2000 sufficient to achieve the maximum concentration in the SMC medium that elicited no toxicity.
Intracoronary Gene Transfer
Fifteen large white pigs (21 to 26 kg; Biological Sciences Facility, University of Manchester) underwent intracoronary delivery of RAd36. Procedures conformed to the UK Animals (Scientific Procedures) Act 1986 and were authorized by the Home Office. Intracoronary gene transfer was performed using a stent-based gene transfer device. Collagen-bound CoverStents (Medtronic) were loaded with 2.5 x 10 9 IU of RAd36 immediately before procedure. Five pigs received 100 mg atenolol orally once daily for 96 hours before and 72 hours after procedure, 5 pigs received oral propranolol (80 mg twice daily) for the same period and 5 pigs received no ß-blocker. All animals received 150 mg aspirin daily, commencing 24 hour before procedure. Anesthesia was induced by inhalation of 4% isoflurane and maintained, after endotracheal intubation, with 2.5% isoflurane. The heart rate of each animal was recorded electrocardiographically immediately postintubation. The left carotid artery was exposed and an 8-F guide catheter inserted; 5000 IU of heparin were injected before coronary angiography was performed. Vessel segments in the LAD (diameter 2.2 to 2.7 mm) were selected for stenting. CoverStents (3.0 mm x 15 mm) were deployed by balloon inflation to 8 atmospheres for 30 seconds. The carotid artery was ligated, the neck incision closed, and the animals allowed to recover. Animals were euthanized 72 hours after stent deployment by intravenous bolus of pentobarbitone (100 mg/kg). Stented arterial segments were harvested and cut open longitudinally. The stents were carefully removed. Sections of artery lying outwith the stented segment were trimmed off and the adventitia carefully stripped off using ophthalmological tweezers. The central 5 mm of each segment were embedded in OCT medium and snap-frozen in liquid nitrogen (LN 2 ) for X-gal staining. The remaining lengths of each infected vessel segment were frozen in LN 2 before ß-galactosidase assay.
Transgene Expression In Vivo
OCT-embedded segments were cut, using a cryostat, into as many 7-µm-thick sections as could be obtained. X-gal cytochemistry was performed on each section as described previously. 5 The area of X-gal staining in each section was measured using a Leica Quantimet 600s digital analysis system. Frozen vessel segments were homogenized in LN 2 using a mortar and pestle, then resuspended in lysis solution. 5 ß-galactosidase activity was assessed by chromogenic assay in vessel homogenates as described previously. 5
Statistical Analysis
Multiple groups were analyzed by 1-way analysis of variance (ANOVA). Comparisons were made between groups by Newman-Keuls post-hoc test.
Results
Isoprenaline Increases ß-galactosidase Expression Under Transcriptional Regulation of the Major Immediate-Early Cytomegalovirus Enhancer/Promoter in Cultured SMCs
The maximum concentration of isoprenaline to which cultured coronary SMC could be exposed for 72 hours was 50 µmol/L. The maximum concentrations of propranolol, atenolol, ICI118,551 and forskolin that coronary SMCs could survive for the same period were 50 µmol/L, 400 µmol/L, 25 µmol/L, and 50 µmol/L, respectively. Reagents were used at these concentrations in subsequent in vitro studies.
Cells exposed to isoprenaline revealed significantly greater transgene expression 72 hours after infection with Ad5- lacZ and RAd36. By comparison with cells exposed to unsupplemented medium, increases in ß-galactosidase activity of 4.8- and 7.6-fold (both P <0.001) were observed in Ad5- lacZ -infected porcine and human coronary SMCs, respectively, after exposure to isoprenaline ( Figure 1 ). Exposure to isoprenaline increased ß-galactosidase expression 3.4-fold in porcine coronary SMCs and 5.6-fold in human coronary SMCs infected with RAd36 (both P <0.001; Figure 2 ). Subsequent exposure of Ad5- lacZ -infected HCSMC to lower concentrations revealed significant enhancement of ß-galactosidase expression with 6.25 µmol/L isoprenaline, although no effect was observed at 3.125 µmol/L ( Figure 3 a).
Figure 1. ß-galactosidase activity in lysates of cultured coronary SMCs 72 hours after infection with Ad5- lacZ. In (a) porcine and (b) human SMCs, isoprenaline and forskolin increase transgene expression from MIEhCMV. The effect of isoprenaline is antagonized by both atenolol and propranolol, although the effect of propranolol is more marked in both species.
Figure 2. ß-galactosidase activity in lysates of cultured coronary SMCs 72 hours after infection with RAd36. In both (a) porcine and (b) human SMCs, isoprenaline and forskolin increase transgene expression from MIEmCMV. As with MIEhCMV, the effect of isoprenaline is antagonized by atenolol and propranolol, although propranolol is more efficacious in both species.
Figure 3. ß-galactosidase activity in Ad5- lacZ -infected human coronary SMCs. a, 3.125 µmol/L isoprenaline does not enhance transgene expression, but higher concentrations elicit progressively greater ß-galactosidase expression. b, The specific ß 2 -antagonist ICI118,551 (25 µmol/L) abolishes isoprenaline-induced enhancement of transgene expression.
Cosupplementation of culture medium with atenolol or propranolol in addition to isoprenaline significantly inhibited isoprenaline-induced enhancement of transgene expression. Coexposure with propranolol reduced ß-galactosidase activity in Ad5- lacZ - and RAd36-infected SMC to levels that did not differ from those observed in SMC exposed to unsupplemented medium ( Figures 1 and 2 ). Exposure to atenolol in addition to isoprenaline decreased isoprenaline-induced enhancement by 50% in porcine and human SMC infected with Ad5- lacZ ( Figure 1 ). ß-galactosidase expression was nonetheless significantly greater than that observed in SMC exposed to unsupplemented medium (both P <0.05). In cells infected with RAd36, atenolol 20% ( Figure 2 ). Again, ß-galactosidase expression was significantly greater in SMCs exposed to isoprenaline+atenolol than in SMC exposed to unsupplemented medium (both P <0.001). SMC exposed to atenolol or propranolol in the absence of isoprenaline expressed levels of transgene that did not differ significantly from cells exposed to unsupplemented medium ( Figures 1 and 2 ). Subsequent exposure of Ad5- lacZ -infected HCSMC to ICI118,551 in addition to isoprenaline reduced ß-galactosidase activity to the level observed in SMCs exposed to unsupplemented medium ( Figure 3 b).
Exposure to forskolin increased transgene expression by 1.6- to 4.6-fold in porcine and human coronary SMCs infected with either virus (all P <0.05; Figures 1 and 2 ). Transgene expression in SMCs exposed to atenolol or propranolol in addition to forskolin did not differ significantly from levels observed in SMCs from the same species infected with the same virus and exposed to forskolin alone ( Figures 1 and 2 ). All cells exposed to forskolin, in the absence or presence of ß-blocker, expressed more ß-galactosidase than cells exposed to unsupplemented medium (all P <0.05).
The Effect of Isoprenaline on Transgene Expression From MIECMV Is Mediated by CREB
Isoprenaline did not increase transgene expression in SMCs infected with a recombinant adenovirus in which expression of ß-galactosidase was under transcriptional regulation of RSV-LTR ( Figure 4 a).
Figure 4. ß-galactosidase activity in coronary SMCs 72 hours after infection with (a) RAd122 and (b) Ad5- lacZ. Isoprenaline (a) has no effect on transgene expression from RSV-LTR. Transfection of RAd122-infected SMC with CRE-decoy oligonucleotide (500 nM) does not affect transgene expression. Transfection (b) of Ad5- lacZ -infected human SMC with CRE-decoy (500 nM) abolishes isoprenaline-induced enhancement of transgene expression.
The maximum concentration of CRE-decoy oligonucleotide to which SMC could be exposed without toxicity was 500 nM. Both oligonucleotides were used at this concentration in vitro. Transfection with CRE-decoy inhibited isoprenaline-induced enhancement of ß-galactosidase expression in Ad5- lacZ -infected SMC ( Figure 4 b), but did not affect levels of transgene expression in RAd122-infected SMC ( Figure 4 a). In contrast, transfection with nonsense oligonucleotide did not affect ß-galactosidase expression in Ad5- lacZ -infected SMC ( Figure 4 b). CRE-decoy had no effect on levels of transgene expression in Ad5- lacZ -infected SMCs in the absence of isoprenaline.
ß-Blockers Significantly Decrease Transgene Expression Regulated by the Major Immediate-Early Murine Cytomegalovirus Enhancer/Promoter In Vivo
Mean heart rates in pigs given oral ß-blockers for 96 hours before gene transfer were significantly lower immediately postendotracheal intubation than in animals receiving no ß-blocker (atenolol=121±17beats/min; propranolol=117± 6beats/min; no ß-blocker=174±9beats/min: P =0.02).
Expression of ß-galactosidase in coronary arteries 72 hours after CoverStent-mediated delivery of RAd36 was substantially greater in animals receiving no ß-blocker than in animals administered atenolol or propranolol. ß-galactosidase activity in lysates of snap-frozen artery segments was 20-fold greater ( P <0.01) in non-ß-blocked animals than in atenolol-treated animals and 500-fold greater ( P <0.01) than in those receiving propranolol ( Figure 5 a). The mean area of staining with X-gal in cryostat-sections of infected arteries was 80-fold greater ( P <0.001) in animals that received no ß-blocker than in those that received atenolol and 260-fold greater ( P <0.001) than in those that received propranolol ( Figures 5b and 6 ). At least one X-gal-positive cell was observed in 60% of sections from non-ß-blocked animals. In contrast only 2.9% of sections from atenolol-treated animals and 0.9% of sections from propranolol-treated animals contained one or more X-gal-positive cells ( P <0.001 by 2 ).
Figure 5. ß-galactosidase expression in porcine coronary arteries. ß-galactosidase (a) activity in atenolol- and propranolol-exposed vessels is 20- and 500-fold less, respectively, than in vessels exposed to no ß-blocker. Mean area (b) of X-gal staining in vessels exposed to no ß-blocker is 80-fold and 260-fold greater than in atenolol- and propranolol-exposed vessels.
Figure 6. ß-galactosidase expression in coronary arteries 72 hour after RAd36 delivery. Sections demonstrate the greatest area of X-gal staining seen in any section from each group. Transgene expression, present on the luminal surface and in the peri-luminal media, is many-fold greater in the non-ß-blocked vessel. (bar=100 µm)
Discussion
We have investigated the effect of the ß-adrenoceptor agonist isoprenaline on the activity of the major immediate-early enhancer/promoter from human and murine cytomegalovirus as promoters of transgene expression in coronary SMCs. Exposure to isoprenaline at concentrations in excess of 3.125 µmol/L increased transgene expression in cultured SMCs infected with Ad5- lacZ or RAd36. Transgene expression from a promoter lacking CRE was not enhanced by isoprenaline, confirming that the enhancer effect of isoprenaline in Ad5- lacZ - and RAd36-infected SMCs is not the consequence of a nonspecific increase in cellular transcription. Concomitant ß-blockade reduced the effect of isoprenaline in Ad5- lacZ -infected and RAd36-infected SMCs. Propranolol was a more potent antagonist of this effect than atenolol. The mode of action of both ß-blockers (inhibition of ß-adrenoreceptor-mediated upregulation of adenylyl cyclase) is verified by the lack of effect of either on forskolin-mediated enhancement of transgene expression.
Greatest isoprenaline-induced enhancement of transgene expression was observed in Ad5- lacZ -infected human SMCs. We therefore studied the effects in such cells of the ß 2 -adrenoceptor-specific antagonist ICI118,551 and of a well-characterized CRE-decoy oligonucleotide that specifically interferes with CREB-dependent transcription. 8 In keeping with the ß 2 -adrenoceptor being the predominant subtype in vascular tissues, 10 ICI118,551 completely abolished isoprenaline-induced enhancement of transgene expression.
Transfection with CRE-decoy at the time of Ad5- lacZ infection abolished isoprenaline-mediated enhancement of transgene expression. Control oligonucleotide did not affect isoprenaline-mediated enhancement, confirming that the effect of isoprenaline on MIECMV is dependent on the integrity of the CREB pathway.
These results suggest that endogenous catecholamines may have a significant effect in vivo on transgene expression regulated by MIECMV. Our observations after intracoronary gene transfer demonstrated that transgene expression from MIECMV within the vasculature is heavily dependent on endogenous catecholamine-mediated enhancement of promoter activity. This has a significant bearing on attempts to translate preclinical data into viable clinical gene therapies, particularly within the vasculature, as ß-blockers are prescribed widely for myocardial ischemia, hypertension, and heart failure, all of which are targets for the clinical application of therapeutic gene transfer. We did not assay plasma ß-blocker levels in our in vivo studies, as our interest lay in ß-blockers as they would be used clinically. The significantly reduced heart rate immediately after intubation indicates clinically adequate ß-blockade. Because the collagen membrane that invests the CoverStent (please see http://atvb.ahajournals.org) prevents direct contact between the blood and the luminal surface of the stented segment, the reduction in transgene expression is unlikely to be a consequence of changes in shear stresses resulting from reduced heart rate in ß-blocked animals.
MIEmCMV has been used infrequently for gene transfer in vivo. In contrast, MIEhCMV is used widely in experimental studies of "therapeutic" gene transfer and has been used in many clinical studies of cardiovascular gene therapy. MIEhCMV was used to promote expression of vascular endothelial growth factor in the first human trial of arterial gene transfer. 11 It has been used subsequently in studies of gene transfer to peripheral arteries, 12,13 coronary arteries, 14 and ischemic myocardium. 15-18 Several of these studies reported the number of patients concomitantly receiving ß-blockers. Rajagopalan et al reported that 6% of patients received ß-blockers (although 45% had an excellent clinical indication in the form of previous myocardial infarction). 12 In the study by Losordo et al, 80% of patients took a ß-blocker. 16 Grines et al reported that ß-blockers were among the most frequent concomitant medications 17 and 90% of recipients of gene therapy in the study by Hedman et al received a ß-blocker. 14 Although some improvements in clinical end points were reported 14,17,18 the effects of gene transfer were not striking. It is impossible to state that concomitant ß-blockade in these studies is the reason for their limited clinical success. However, the potential for ß-blockade to have influenced the outcome of such studies cannot be ignored.
Studies in vitro have shown that MIECMV is the most potent promoter of transgene expression in vascular tissues. It elicits greater transgene expression in SMCs than SMC-specific promoters. 19,20 When compared with other viral promoters in SMCs (RSV-LTR, simian virus 40 [SV40] early promoter) MIEhCMV elicited most expression. 19,21 Because the RSV-LTR and SV40 promoters are not cAMP-responsive, 6 it is likely that an even greater advantage would be conferred on MIEhCMV in the presence of catecholamines. Our observations also show that ß-blockers do not reduce transgene expression to sub-basal levels in SMC, in keeping with reports that CRE does not govern basal levels of activity of MIEhCMV in CMV-infected cells. 22 The use of cAMP-insensitive promoters would therefore be unlikely to confer any advantage over MIEhCMV within the vasculature, as those that have been compared with MIEhCMV do not achieve greater basal levels of transgene expression in SMCs; unlike MIEhCMV, they would not permit increased transgene expression in response to endogenous catecholamines.
Our observations have implications that spread beyond the field of vascular gene therapy. MIEhCMV is a potent promoter in cells from nonvascular tissues. 23,24 Clinical studies of gene therapy for cystic fibrosis, 25 liver metastases, 26 1-antitrypsin deficiency, 27 factor IX deficiency, 28 and esophageal carcinoma 29 have used MIEhCMV to promote therapeutic transgene expression. Furthermore, activation of MIEhCMV is the rate-limiting step in cytomegalovirus replication. 22 Our observations suggest that ß-blockade may restrict cytomegalovirus replication in humans, which is pertinent not only to the setting of clinical infection with cytomegalovirus but also might account for some of the cardioprotective effects of ß-blockers in the setting of atherosclerosis, as cytomegalovirus is implicated in atherogenesis and is associated with increased mortality from coronary artery disease. 30
The requirement of the CRE for maximal transcriptional activity has also been demonstrated in herpes simplex virus-1, 31 the human T-cell leukemia virus type 1, 32 the Epstein-Barr virus Bam HI W promoter, 33 and the varicella-zoster virus immediate-early gene 62 promoter. 34 In these viruses, too, ß-blockade may act as adjunctive therapy to limit viral replication.
We have investigated the effects of ß-blockers on transgene expression elicited by MIECMV in vivo. The finding that ß-blockers severely inhibited transgene expression after intracoronary gene transfer has implications for all studies of clinical gene therapy. It is likely that the effect of ß-blockade on transgene expression from MIECMV in vivo is just the first clinically significant interaction between pharmacological agents and promoters of transgene expression. The 1 -adrenoceptor agonist phenylephrine, for example, acting through a cAMP-independent pathway, enhances MIEhCMV-regulated transgene expression in rat cardiomyocytes, 35 raising the possibility that -adrenoceptor antagonists might also have significant effects on transgene expression in vivo. Our findings stress the importance of the potential for interactions between gene therapy and other therapeutic modalities. To date, these have largely been neglected in the undertaking of clinical trials to assess the therapeutic potential of gene transfer.
Acknowledgments
RAd122 was provided by Professor Andrew Baker (University of Glasgow, UK).
Sources of Funding
This work was funded by Heart Research UK grant RG2483. Mr Salem is a Medical Research Council PhD student. Dr Appleby was a British Heart Foundation Junior Research Fellow. Dr Kingston is a British Heart Foundation Intermediate Research Fellow.
Disclosures
None.
【参考文献】
Edelstein ML, Abedi MR, Wixon J, Edelstein RM. Gene therapy clinical trials worldwide 1989-2004-an overview. J Gene Med. 2004; 6: 597-602.
Montminy M. Transcriptional regulation by cyclic AMP. Annu Rev Biochem. 1997; 66: 807-822.
Appleby CE, Kingston PA. Gene therapy for restenosis-what now, what next? Curr Gene Ther. 2004; 4: 153-182.
Kingston PA, Sinha S, David A, Castro MG, Lowenstein PR, Heagerty AM. Adenovirus-mediated gene transfer of a secreted transforming growth factor-beta type II receptor inhibits luminal loss and constrictive remodeling after coronary angioplasty and enhances adventitial collagen deposition. Circulation. 2001; 104: 2595-2601.
Appleby CE, Kingston PA, David A, Gerdes CA, Umana P, Castro MG, Lowenstein PR, Heagerty AM. A novel combination of promoter and enhancers increases transgene expression in vascular smooth muscle cells in vitro and coronary arteries in vivo after adenovirus-mediated gene transfer. Gene Ther. 2003; 10: 1616-1622.
Mellon PL, Clegg CH, Correll LA, McKnight GS. Regulation of transcription by cyclic AMP-dependent protein kinase. Proc Natl Acad Sci U S A. 1989; 86: 4887-4891.
Seamon KB, Padgett W, Daly JW. Forskolin: unique diterpene activator of adenylate cyclase in membranes and in intact cells. Proc Natl Acad Sci U S A. 1981; 78: 3363-3367.
Park YG, Nesterova M, Agrawal S, Cho-Chung YS. Dual blockade of cyclic AMP response element- (CRE) and AP-1-directed transcription by CRE-transcription factor decoy oligonucleotide. gene-specific inhibition of tumor growth. J Biol Chem. 1999; 274: 1573-1580.
Christen T, Bochaton-Piallat ML, Neuville P, Rensen S, Redard M, van Eys G, Gabbiani G. Cultured porcine coronary artery smooth muscle cells. A new model with advanced differentiation. Circ Res. 1999; 85: 99-107.
Chruscinski A, Brede ME, Meinel L, Lohse MJ, Kobilka BK, Hein L. Differential distribution of beta-adrenergic receptor subtypes in blood vessels of knockout mice lacking beta(1)- or beta(2)-adrenergic receptors. Mol Pharmacol. 2001; 60: 955-962.
Isner JM, Pieczek A, Schainfeld R, Blair R, Haley L, Asahara T, Rosenfield K, Razvi S, Walsh K, Symes JF. Clinical evidence of angiogenesis after arterial gene transfer of phVEGF165 in patient with ischaemic limb. Lancet. 1996; 348: 370-374.
Rajagopalan S, Mohler ER, 3rd, Lederman RJ, Mendelsohn FO, Saucedo JF, Goldman CK, Blebea J, Macko J, Kessler PD, Rasmussen HS, Annex BH. Regional angiogenesis with vascular endothelial growth factor in peripheral arterial disease: a phase II randomized, double-blind, controlled study of adenoviral delivery of vascular endothelial growth factor 121 in patients with disabling intermittent claudication. Circulation. 2003; 108: 1933-1938.
Kim HJ, Jang SY, Park JI, Byun J, Kim DI, Do YS, Kim JM, Kim S, Kim BM, Kim WB, Kim DK. Vascular endothelial growth factor-induced angiogenic gene therapy in patients with peripheral artery disease. Exp Mol Med. 2004; 36: 336-344.
Hedman M, Hartikainen J, Syvanne M, Stjernvall J, Hedman A, Kivela A, Vanninen E, Mussalo H, Kauppila E, Simula S, Narvanen O, Rantala A, Peuhkurinen K, Nieminen MS, Laakso M, Yla-Herttuala S. Safety and feasibility of catheter-based local intracoronary vascular endothelial growth factor gene transfer in the prevention of postangioplasty and in-stent restenosis and in the treatment of chronic myocardial ischemia: phase II results of the Kuopio Angiogenesis Trial (KAT). Circulation. 2003; 107: 2677-2683.
Fortuin FD, Vale P, Losordo DW, Symes J, DeLaria GA, Tyner JJ, Schaer GL, March R, Snell RJ, Henry TD, Van Camp J, Lopez JJ, Richenbacher W, Isner JM, Schatz RA. One-year follow-up of direct myocardial gene transfer of vascular endothelial growth factor-2 using naked plasmid deoxyribonucleic acid by way of thoracotomy in no-option patients. Am J Cardiol. 2003; 92: 436-439.
Losordo DW, Vale PR, Symes JF, Dunnington CH, Esakof DD, Maysky M, Ashare AB, Lathi K, Isner JM. Gene therapy for myocardial angiogenesis: initial clinical results with direct myocardial injection of phVEGF165 as sole therapy for myocardial ischemia. Circulation. 1998; 98: 2800-2804.
Grines CL, Watkins MW, Mahmarian JJ, Iskandrian AE, Rade JJ, Marrott P, Pratt C, Kleiman N. A randomized, double-blind, placebo-controlled trial of Ad5FGF-4 gene therapy and its effect on myocardial perfusion in patients with stable angina. J Am Coll Cardiol. 2003; 42: 1339-1347.
Kastrup J, Jorgensen E, Ruck A, Tagil K, Glogar D, Ruzyllo W, Botker HE, Dudek D, Drvota V, Hesse B. Direct intramyocardial plasmid vascular endothelial growth factor-A165 gene therapy in patients with stable severe angina pectoris: A randomized double-blind placebo-controlled study: The Euroinject One trial. J Am Coll Cardiol. 2005; 45: 982-988.
Ribault S, Neuville P, Mechine-Neuville A, Auge F, Parlakian A, Gabbiani G, Paulin D, Calenda V. Chimeric smooth muscle-specific enhancer/promoters: valuable tools for adenovirus-mediated cardiovascular gene therapy. Circ Res. 2001; 88: 468-475.
Kim S, Lin H, Barr E, Chu L, Leiden JM, Parmacek MS. Transcriptional targeting of replication-defective adenovirus transgene expression to smooth muscle cells in vivo. J Clin Invest. 1997; 100: 1006-1014.
DeYoung MB, Zamarron C, Lin AP, Qiu C, Driscoll RM, Dichek DA. Optimizing vascular gene transfer of plasminogen activator inhibitor 1. Hum Gene Ther. 1999; 10: 1469-1478.
Keller MJ, Wheeler DG, Cooper E, Meier JL. Role of the human cytomegalovirus major immediate-early promoter?s 19-base-pair-repeat cyclic AMP-response element in acutely infected cells. J Virol. 2003; 77: 6666-6675.
Cara A, Lucarelli E, Cornaglia-Ferraris P. Engineering viral promoters for gene transfer to human neuroblasts. Cell Mol Neurobiol. 2000; 20: 409-415.
Zarrin AA, Malkin L, Fong I, Luk KD, Ghose A, Berinstein NL. Comparison of CMV, RSV, SV40 viral and Vlambda1 cellular promoters in B and T lymphoid and non-lymphoid cell lines. Biochim Biophys Acta. 1999; 1446: 135-139.
Boucher RC, Knowles MR, Johnson LG, Olsen JC, Pickles R, Wilson JM, Engelhardt J, Yang Y, Grossman M. Gene therapy for cystic fibrosis using E1-deleted adenovirus: a phase I trial in the nasal cavity. The University of North Carolina at Chapel Hill. Hum Gene Ther. 1994; 5: 615-639.
Habib NA, Hodgson HJ, Lemoine N, Pignatelli M. A phase I/II study of hepatic artery infusion with wtp53-CMV-Ad in metastatic malignant liver tumours. Hum Gene Ther. 1999; 10: 2019-2034.
Flotte TR, Brantly ML, Spencer LT, Byrne BJ, Spencer CT, Baker DJ, Humphries M. Phase I trial of intramuscular injection of a recombinant adeno-associated virus alpha 1-antitrypsin (rAAV2-CB-hAAT) gene vector to AAT-deficient adults. Hum Gene Ther. 2004; 15: 93-128.
Fabb SA, Dickson JG. Technology evaluation: AAV factor IX gene therapy, Avigen Inc. Curr Opin Mol Ther. 2000; 2: 601-606.
Shimada H, Matsubara H, Ochiai T. p53 gene therapy for esophageal cancer. J Gastroenterol. 2002; 37 Suppl 14: 87-91.
Muhlestein JB, Horne BD, Carlquist JF, Madsen TE, Bair TL, Pearson RR, Anderson JL. Cytomegalovirus seropositivity and C-reactive protein have independent and combined predictive value for mortality in patients with angiographically demonstrated coronary artery disease. Circulation. 2000; 102: 1917-1923.
Marquart ME, Zheng X, Tran RK, Thompson HW, Bloom DC, Hill JM. A cAMP response element within the latency-associated transcript promoter of HSV-1 facilitates induced ocular reactivation in a mouse hyperthermia model. Virology. 2001; 284: 62-69.
Langlois M, Audet B, Legault E, Pare ME, Ouellet M, Roy J, Dumais N, Mesnard JM, Rothstein DM, Marriott SJ, Tremblay MJ, Barbeau B. Activation of HTLV-I gene transcription by protein tyrosine phosphatase inhibitors. Virology. 2004; 329: 395-411.
Bell A, Skinner J, Kirby H, Rickinson A. Characterisation of regulatory sequences at the Epstein-Barr virus BamHI W promoter. Virology. 1998; 252: 149-161.
Moriuchi H, Moriuchi M, Cohen JI. Proteins and cis-acting elements associated with transactivation of the varicella-zoster virus (VZV) immediate-early gene 62 promoter by VZV open reading frame 10 protein. J Virol. 1995; 69: 4693-4701.
Maass A, Langer SJ, Oberdorf-Maass S, Bauer S, Neyses L, Leinwand LA. Rational promoter selection for gene transfer into cardiac cells. J Mol Cell Cardiol. 2003; 35: 823-831.
作者单位：Vascular Gene Therapy Unit (H.K.S., P.R., C.E.A., P.A.K.), University of Manchester, Manchester, UK; Medtronic Bakken Research Centre B.V. (A.D.), Maastricht, Netherlands; Department of Medicine (A.M.H.), University of Manchester, Manchester, UK.