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Cardiovascular Research 1997 35(3):567-574; doi:10.1016/S0008-6363(97)00158-2
© 1997 by European Society of Cardiology
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Copyright © 1997, European Society of Cardiology

Regulated expression of a foreign gene targeted to the ischaemic myocardium

Howard Prenticed, Nanette H Bishoprica,b, Martin N Hicksc, Daryl J Dischera,b, Xiaosu Wua, Andrew A Wylied and Keith A Webstera,b,*

aPharmaceutical Discovery Division, SRI International, Menlo Park, CA, USA
bDepartment of Molecular and Cellular Pharmacology, University of Miami, Miami, FL, USA
cDepartment of Medical Cardiology, Glasgow Royal Infirmary, University of Glasgow, Glasgow, UK
dDivision of Molecular Genetics, Institute of Biomedical and Life Sciences and Department of Medicine and Therapeutics, University of Glasgow, Glasgow, UK

* Corresponding author. Department of Molecular and Cellular Pharmacology, Rosenstiel Medical Science Building, Rm. 6038, University of Miami, 1600 NW Tenth Avenue, Miami, FL 33136, USA. Tel.: +1 (305) 243-6779; fax: +1 (305) 243-4555; e-mail: kwebster@chroma.med.miami.edu

Received 18 February 1997; accepted 28 May 1997


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Objectives: Regulated expression of transferred foreign genes may be an important feature of gene therapy. Because coronary artery disease often involves intermittent myocardial ischaemia followed by periods of normal cardiac function it will probably be necessary to regulate the expression of putative therapeutic/cardioprotective genes directly in response to ischaemia-associated signals. The objectives of the current study were to develop a combination of gene regulatory components that can be used to target a product to the myocardium and limit the expression of the gene to periods of ischaemic activity. Methods: Expression plasmids were constructed containing muscle-specific promoters and hypoxia-responsive enhancer elements linked to a reporter gene. The regulation of these constructs by hypoxia or experimental ischaemia was measured following transient expression in cultured cells or after direct injection of DNA into the rabbit myocardium. Results: A single set of hypoxia response elements placed immediately upstream of the minimal muscle-specific {alpha}-myosin heavy chain promoter conferred potent positive regulation of this promoter by hypoxia in vitro and by ischaemia in vivo. Induction by ischaemia persisted for at least 4 h and returned to the baseline level within 8 h. Conclusions: Hypoxia responsive regulatory elements, in combination with weak tissue-restricted promoters incorporated into an appropriate vector system may allow controlled expression of a therapeutic gene in ischaemic myocardium.

KEYWORDS Gene therapy; Hypoxia; Cardioprotection; Redox


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
A number of procedures have been described for the efficient delivery and expression of foreign genes in the heart and coronary vasculature. Replication-defective adenoviral vectors [1–7], adeno-associated viral vectors [8], and disabled herpes virus vectors [9]can result in highly efficient transfer to rat, dog, and pig cardiac myocytes in vivo. Naked DNA and liposome complexes have also provided substantial, although perhaps more localized, delivery to cardiac myocytes in vivo and in vitro [10–14]. In both cases either direct intramuscular injection or infusion into the coronary artery with or without the assistance of a catheter appears to be quite effective in transferring significant amounts of transcriptionally active genetic material into both the vascular cells and the cardiac myocytes. Although most of the studies to date have analyzed the expression of transferred reporter genes, biologically relevant genes have also been transferred to both the vasculature and myocardial tissue [15–20].

Existing vector systems can provide adequate delivery of a gene, and it seems likely that immunogenic responses will eventually be reduced or even eliminated [8, 21]. However, appropriate regulation of the therapeutic gene after delivery remains a problem. Localized injections, or the use of tissue-specific cell surface recognition molecules can afford one level of vector targeting, reviewed in [10, 11], and the use of tissue-specific promoters (e.g. von Willebrand factor [22], {alpha}-myosin heavy chain [3, 23], myosin light chain-2 [24]) can provide another degree of localized expression. Unfortunately, these elements generally direct constitutive expression in the target cell or tissue that is unrelated to the (pathophysiological) cellular environment. A third possible control manoeuvre involves the inclusion of an externally regulatable promoter element, such as a hormone-, antibiotic-, or metal-responsive enhancer, in the therapeutic gene promoter [25–27]. In this case the expression of the targeted gene can be modulated by treatment or withdrawal of a specific effector (e.g. hormone, antibiotic, metal). While this clearly may be advantageous in some circumstances, such an approach will require additional monitoring and intervention. It would be preferable for expression of the targeted gene to be directly responsive to a component of the disease phenotype.

Our understanding of the pathophysiology of coronary artery disease and myocardial ischaemia is incomplete, but there is substantial evidence that myocardial cell damage occurs during both the ischaemic episode and the subsequent reperfusion [28–35]. Ischaemic hearts experience regional cycles of hypoxic and oxidative stress that punctuate normal cardiac function, resulting in progressive cell loss and tissue damage. Animal studies have identified a number of interventions that can reduce the damage caused by ischaemia and reperfusion, including overexpression of heat shock proteins [36, 37], infusion of anti-inflammatory agents [35, 38], treatments with tissue plasminogen activator, vasodilators, and antioxidants (reviewed in [39, 40]), and overexpression of anti-apoptotic genes such as Bcl2 [41, 42]. These agents are all candidates for gene therapy that could afford significant cardioprotection during ischaemic episodes if their delivery and activity were appropriately regulated. Appropriate regulation in this case should involve elevated activity of the agent during heightened periods of ischaemic activity, and quenched levels when the ischaemia subsides. This may be possible if the transferred gene is under the control of elements that respond directly to a component of ischaemic or oxidative stress.

In the present study we have examined the possibility that hypoxia response elements (HREs) incorporated into a minimal muscle specific promoter can be used to regulate the expression of a test gene (firefly luciferase) by hypoxia and ischaemia using cultured cardiac myocytes in hypoxic culture and a rabbit model of experimental myocardial ischaemia. We show that a cartridge containing multiple copies of a hypoxia response element from the human erythropoietin gene placed upstream of a minimal {alpha}-myosin heavy chain (MHC) promoter, is tightly regulated by hypoxia in vitro and by ischaemia in vivo. The results indicate that this combination of regulatory elements may be useful in the design of vectors for the targeting and regulated expression of a therapeutic gene in the ischaemic myocardium.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
2.1 Plasmid constructions
pGEM4-HRE, containing four copies of the erythropoietin HRE cloned into the BamHI site of pGEM4 (Promega Biotech, Madison, WI), was a gift from Greg Semenza (Johns Hopkins University, Baltimore, MD) [43]. The {alpha}-MHC promoter constructs truncated to –1.2 kb and –86 bp were generous gifts from Tom Gustafson [44]and Bruce Markham [45]respectively. pGL (Gene Light) vectors were obtained from Promega. pSVHRE was made by inserting a 240 bp HindIII PCR fragment from pGEM4-HRE into the multiple cloning site of pGLBP which contains the proximal SV40 promoter linked to the luciferase gene (Promega). To insert the {alpha}-MHC promoter fragments the SV40 promoter was removed from pSVHRE, and replaced with the 1.2 kb or 86 bp fragments of the {alpha}-MHC promoter to create p{alpha}-MHC1.2HRE, and p{alpha}-MHC86HRE. Internal controls pRSV-luciferase and pCAT3PV (SV40 promoter linked to the Escherichia coli chloramphenicol acetyltransferase (CAT) gene), were from Promega Biotech.

2.2 Cell culture
The isolation of cardiac myocytes from neonatal rat hearts has been described in detail elsewhere [46, 47]. Briefly, hearts from 30–40 pups were minced and subjected to serial trypsin digestion to release single cells. After the final digestion the cells were washed and pre-plated for 0.5 h in MEM with 5% fetal calf serum. Non-attached cells were replated in 60-mm Falcon dishes at 4x106 cells per plate in the same medium with 0.1 mM bromodeoxyuridine (BrdU) for 3 days after which time the BrDU was removed. Cultures were transfected on day 1 after isolation and used for experiments after a further 3 or 4 days. The preplating and BrdU steps reduce the background of non-myocardial cells. These procedures reproducibly generate cultures that contract synchronously at >250 beats per minute consistent with the normal beating rate of the intact rat heart [48]. Cultures were maintained in humidified air with 5% CO2 at 37°C [46, 47]except where indicated. Culture of HeLa cells and C2C12 skeletal myoblasts have been described previously [49–51]. Calcium phosphate transfections, luciferase and CAT quantitation, and protein determinations in cell and tissue extracts were performed as described previously [22, 49, 50, 52, 53].

2.3 Hypoxia
Transfected cultures were maintained in MEM with 5% fetal calf serum supplemented with 3 g/l glucose until exposure to hypoxia (pO2=8–12 mmHg). Details of our methods for exposing cells to hypoxia have been described previously [47, 54]. Oxygen was continuously monitored with an oxygen electrode (Controls Katharobic, Philadelphia, PA) inside the chamber and contractility was monitored by edge detection as described previously [47, 54]. After 16 h of hypoxia, plates were harvested and lysed for protein and luciferase assays. Under these conditions, cardiac myocytes retained contractility for the duration of all experiments, and intracellular ATP and medium glucose levels were maintained throughout as previously described (data not shown, see references [47, 54, 55]).

2.4 Gene transfer and ischaemia/reperfusion in rabbit heart
Male New Zealand White rabbits (2.5–3.0 kg) were premedicated with intramuscular Hypnorm, Jansen Pharmaceuticals [0.3 ml/kg; fluanisone (10 mg/ml), fentanyl citrate (0.315 mg/ml)] and treated with intravenous midazolam (0.25–0.5 mg/kg) to permit endotracheal intubation (size 3–4). Animals were ventilated (0.3–0.4 l/min/kg) on a small animal ventilator with 1–2 cm H2O of positive end-expiratory pressure. Animals were maintained under anaesthesia with an inhaled mixture of equal concentrations of nitrous oxide and oxygen plus 1% halothane at a flow rate of 2 l/min. The chest was opened by a left thoracotomy, and the pericardium removed for DNA injection. DNA was introduced by injection (100 µl per injection in a Hamilton syringe; 50 µg SV40-CAT plasmid and 50 µg of {alpha}-MHC86±HRE plasmid suspended in phosphate-buffered saline), firstly into the cardiac apex (three injections) and secondly into the proximal left ventricular wall (three injections). Postoperative analgesia was administered (0.2 mg/kg buprenorphine i.m.). Seven days after DNA injection rabbits were subjected to a second thoracotomy for implementation of transient myocardial ischaemia as described previously [56]. Premedication, ventilation and anaesthesia were performed as described above. After thoracotomy, the 1st obtuse marginal artery was ligated by insertion of a suture at the midpoint between the atrioventricular groove and the cardiac apex. The presence of an area of ischaemia was confirmed by visualisation of tissue discoloration and by ECG changes as described previously [56]. After 15 min of ischaemia the suture was removed to allow the heart to be reperfused. Intravenous quinidine was administered 5 min before ligation to decrease the likelihood of ischaemia or reperfusion-induced arrhythmia. Ventricular arrhythmias during surgery were treated with an 8 joule epicardial DC shock. Post-operative analgesia was administered as described above. Sham-operated animals were subjected to the same procedures but without coronary ligation. One to eight hours after surgery animals were sacrificed with a lethal dose of pentobarbital sodium and the heart excised for assays of reporter gene expression [22]. All procedures were performed under license in accordance with NIH guidelines or in accordance with the United Kingdom Animals (Scientific Procedures) Act, 1986.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
To characterise the expression and hypoxia response of the promoter elements, the different plasmids were transfected into muscle and non-muscle cells and assayed for the transient expression of luciferase under aerobic and hypoxic conditions as described in Section 2. Luciferase activities are expressed relative to the aerobic activity of the RSV plasmid (Fig. 1a) or the SVHRE plasmid (Fig. 1b), arbitrarily designated as 1 for each cell type. The results from control plasmids, without HREs, are shown in Fig. 1a. The RSV promoter construct was expressed at high levels in all cell types and was induced slightly during exposure to hypoxia. The other control plasmids, pGLSV2 and {alpha}-MHC86, expressed at a fraction of the RSV levels, as expected for truncated promoters [44, 45]and were also induced slightly (<2-fold) by hypoxia. The molecular mechanism(s) for the small inductions of these diverse promoters by hypoxia is not clear although it may be related to the presence of hypoxia responsive promoter elements. We have previously described activation of transcription factor AP1 by hypoxia, and the transcription factor Sp1 may also be sensitive to hypoxia ([52, 54], and Webster et al. unpublished observations). The {alpha}-MHC86 construct expressed preferentially in muscle cells, in agreement with previous reports [45], and the expression increased by 1.4±0.3-fold (n = 3) under hypoxia.


Figure 1
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  		Fig. 1 Tissue specificity and hypoxic response of HRE vectors. Subconfluent cultures of HeLa cells, C2C12 myoblasts, or day 1 isolated cardiac myocytes were transfected using the calcium phosphate procedure as described in Section 2. After 2–3 days the cultures were transferred to a hypoxic incubator (also described in Section 2), or remained aerobic. After an additional 16 h the cells were harvested and lysed for luciferase activity assays as described previously [22, 49, 52]. All results are means of at least 3 separate experiments±S.E.M., absent error bars means that the S.E.M. was <10%. The expression of constructs containing the HRE elements in aerobic cells (b) was slightly increased compared with the parental plasmid without HREs (data not shown). In (a) RSV=RSV-luciferase; SV=pGL2PV; {alpha}1=p{alpha}-MHC86 (GL). In (b), V=pGL2PV-HRE; {alpha}1=p{alpha}-MHC86-HRE (GL); {alpha}2=p{alpha}-MHC1.2-HRE (GL). These constructs are described in Section 2. Please note the different scale gradations in (a) and (b).

 
The hypoxic induction of the HRE-containing promoters was enhanced compared with the parent plasmid (minus HRE) in all cases (Fig. 1b). The non-tissue selective SV40-HRE promoter was expressed at high levels in all cells tested and was induced 4- to 6-fold by hypoxic incubation in all cell types (V columns in Fig. 1b). In agreement with previous reports, the level of expression of the {alpha}-MHC promoters was muscle specific [23, 44, 45]. The –1.2 kb {alpha}-MHC promoter (labeled {alpha}2 in Fig. 1b) directed expression poorly in HeLa and C2C12 cells but was stronger in cardiac myocytes. This result is consistent with repressor activities within the –1.2 kb region that may reduce expression in non-cardiac cells including skeletal muscle [57], and is also consistent with the thyroid hormone requirements for high level expression of this promoter [23, 45]. The 1.2 kb {alpha}-MHC construct was expressed at relatively higher levels in aerobic cardiac myocytes and was induced by about 4-fold under hypoxia, similar to the SV40-HRE promoter. The smaller {alpha}-MHC-HRE construct (labeled {alpha}1 in the figure), truncated to –86, contains the minimal elements that retain muscle specificity [45]. This construct was active in both C2C12 myocytes and cardiac myocytes but the expression was significantly lower than the other two test plasmids in aerobic cardiac myocytes. This is also consistent with previous reports that have described multiple elements upstream of –86 that are required for high level cardiac specific expression of the {alpha}-MHC promoter [23, 45]. Both of the {alpha}-MHC promoter-HRE constructs were induced by hypoxia, but fold induction was higher (9.2±1.2) for the –86 bp promoter. This effect may be related to the low aerobic expression of this promoter in the cardiac cells or to the closer proximity of the HRE elements to the transcription initiation site compared with the 1.2 kb promoter.

These in vitro results confirm the tissue specificity, hypoxia inducibility, and relative expression of the {alpha}-MHC-HRE promoter constructs in skeletal and cardiac myocytes in vitro. The –86 {alpha}-MHC-HRE construct was selected for analyses in vivo because it had lower basal expression in aerobic cardiac myocytes and showed the highest relative induction by hypoxia. In vivo expression studies were carried out after intramuscular injection of control (RSV-CAT, SV2CAT, or GL2PV) and test plasmids as described in Section 2. Repeat thoracotomies for ischaemia or sham procedures were performed one week after the initial injections to allow recovery and stabilization of the tissue. An ischaemic interval was imposed for 15 min as described in Section 2, after which time the hearts were reperfused for varying times before sacrificing the animals and removing tissue for reporter assays as described above. The results of these assays are shown in Fig. 2.


Figure 2
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  		Fig. 2 Induction of p{alpha}-MHC86-HRE in ischaemic rabbit hearts. The surgical procedures, injections, and assays are described in Section 2. One week after the first thoracotomy and injection of DNA, a second thoracotomy was performed and the animals were subjected to ischaemia by left marginal artery ligation as described or to sham operation only. Fifteen minutes after the ligation the suture was removed and the hearts were reperfused for varying times as indicated on the figure. At each time point rabbits were sacrificed and the hearts were removed, the ventricles and ischaemic zones were dissected, and the appropriate regions of tissue were lysed and analysed for reporter gene expression as described. The results represent data from a minimum of 3 separate experiments in each case with S.E.M. as indicated. The 1 h time point for p{alpha}-MHC86-HRE includes results from 5 experiments, and the 4 h time point includes data from 10 experiments. Induction of luciferase gene expression by ischaemia at the 1 and 4 h time points was significantly different from the shams with p<0.001 at both time points by t-test, comparing related samples.

 
For all data points the expression of the test promoter ({alpha}-MHC86) was normalised by reference to the internal control (SV40) and each time point represents the relative expression of luciferase in the ischaemic hearts compared to the sham treatments after normalization. One hour after the ischaemia, expression of the luciferase reporter gene in p{alpha}-MHC86HRE injected hearts was increased by 4.05±1.0-fold (n = 5) compared with the sham. Four hours post ischaemia luciferase expression was 4.6±0.74-fold (n = 10) higher than the sham. After 8 h the luciferase expression of p{alpha}-MHC86HRE in the ischaemic hearts was equivalent to the sham-operated level (n = 3). There was no difference in the expression of an {alpha}-MHC86 construct lacking HREs or of the RSV-CAT construct between sham and ischaemic hearts at 4 h, indicating that the induction by ischaemia required HREs (Fig. 2). These results demonstrate that hypoxia-activated transcription and translation following experimental ischaemic episodes in the rabbit heart is initiated rapidly (within 1 h) and persists for at least 4 h. Therefore it may be possible to limit the expression of a therapeutic gene to periods of myocardial ischaemia.


   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
Our results demonstrate that four copies of the human erythropoietin HRE linked to the basal promoter of the muscle selective {alpha}-MHC gene conferred inducible expression of luciferase to the promoter by both hypoxia in vitro and by a short interval of ischaemia in vivo. The response to ischaemia was rapid, with almost maximal induction within 1 h, and enhanced expression remained for at least 4 h after the ischaemic episode. The promoter activation is consistent with a direct response to hypoxia because the effect was dependent on the presence of the HRE enhancer elements. Expression returned to basal (sham) levels after 8 h. All control plasmids including p{alpha}-MHC86 were induced slightly by subjecting cells to hypoxia in vitro (Fig. 1a), but not by ischaemia (Fig. 2). The absence of any induction of the controls during ischaemia suggests that different elements with different response times may be responsible for these two effects. Induction of HIF-1 by hypoxia is known to occur rapidly [43, 61], and probably determines the induction of p{alpha}-MHC-HRE following ischaemia. However, in our in vitro experiments, the cells were exposed to prolonged hypoxia (16 h) therefore additional factors may be activated during this period that mediate the small inductions of the control plasmids. Although we did not directly assay the cellular distribution of the injected plasmids in the rabbit heart, numerous other studies using similar protocols to ours have shown that cardiac myocytes are the major recipients of DNA transferred by this method [22, 23, 53, 58, 59]. Also the {alpha}-MHC86 promoter retains muscle specificity ([45], and see Fig. 1), therefore it will be preferentially expressed in myocytes.

There are a number of interesting features associated with the response of the transferred promoter-HRE to ischaemia. Firstly the induction was rapid, occurring within the first hour of reperfusion (Fig. 2). The timing of the response is determined by the rate of accumulation and activity of the HRE-binding transcription factor HIF-1 (reviewed in [60]) during ischaemia and reperfusion. Previous studies indicate that this response is quite rapid [61], and our own analyses of HIF-1 accumulation in C2C12 cells exposed to cycles of hypoxia and reoxygenation indicate that the binding activity appears within 20–30 min after exposure to hypoxia (data not shown). We are currently examining earlier time points following ischaemia in rabbit and rat hearts for gene expression and accumulation of HIF-1 binding activity to characterize the response time. Secondly, 15 min of ischaemia caused a level of induction of {alpha}-MHC-HRE (~5-fold) that was equivalent to the induced levels following 16 h of hypoxia in vitro (~9-fold), and the induction during reperfusion was sustained for at least 4 h. These results suggest that the HRE-mediated regulation is extremely sensitive to ischaemia in the heart; they also indicate that the heart may experience ischaemia-related alterations in gene expression for several hours after reperfusion. The duration of expression of the transferred gene after reperfusion will be determined by the stability of the DNA constructs, mRNA transcripts and encoded protein, as well as by the persistence of HIF-1 activity after reoxygenation.

These results indicate that it is possible to regulate directly the expression of a transferred gene in response to ischaemia in the rabbit heart. It may therefore be possible to regulate a therapeutic gene so that it is expressed only during episodes of ischaemia and reperfusion, so that tissue levels of the gene product will fluctuate in accordance with the duration, frequency, and severity of the ischaemia. In this study we have tested a minimally regulated promoter, but it will be possible to further manipulate and optimize the regulatory units for gene therapy purposes. In our studies a single unit of four tandemly repeated copies of the HRE was placed immediately upstream of the {alpha}-MHC promoter at bp –86. This conferred an approximately 9-fold induction of expression by hypoxia in vitro. Other studies indicate that this level of induction could be increased possibly up to 40-fold by placing HRE-like elements in both 5' and 3' positions of the target gene [60]. Elements contained in the promoter and 3' regions of the erythropoietin gene confer up to 1000-fold induction by hypoxia or anaemia in vivo [62].

Techniques and procedures are being developed for the successful delivery and expression of genes in the heart, and there are a number of potential therapeutic genes that may protect against ischaemic damage [35, 37–40, 63, 64]. The probability that any of these will be used for gene therapy treatment of myocardial ischaemia depends on numerous factors including reliable delivery, sustained retention, positive therapeutic efficacy, and long-term safety. The latter two issues may be related to levels of expression, regulation, and side effects. In many instances it may be undesirable to transfer a ‘therapeutic’ gene that will be expressed at high constitutive levels, that are not related to the (patho)physiological state of the target tissue. For myocardial ischaemia, the use of a weak, preferably silent promoter that is strongly inducible by ischaemia/hypoxia may allow therapeutic levels of cardioprotective agents to accumulate when symptoms are severe or frequent, and to disappear when symptoms subside. This approach provides an internal sensor to trigger an endogenous source of medication, potentially reducing the need to leave the tennis court for nitroglycerine, and averting the deleterious consequences of recurrent myocardial hypoxia and oxidative stress.

Time for primary review 36 days.


   Acknowledgements
 
This work was supported by grants HL44578 (K.A.W.) and HL49891 (N.H.B.), from the National Institutes of Health, by the Cigarette and Tobacco Surtax of the State of California through the Tobacco-Related Disease Research Program of the University of California, Grant 1RT-402 (K.A.W.), by grants from the British Heart Foundation, Wellcome Trust, Medical Research Council, Royal Society, and Scottish Home and Health Department (H.P.), and by a Wellcome Trust Foundation Collaborative Travel Grant (K.A.W., N.H.B., H.P.). A.A.W. is the recipient of a studentship from the BBSRC. The authors are grateful to Ms Karen Jess for excellent technical assistance.

A preliminary report of this work has been published in abstract form (Webster et al., Hypoxia regulated vectors for targeting genes to ischaemic myocardium. Circulation 1995;92:I-756).


   References
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 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 

  1. Muhlhauser J., Jones M., Yamada I., et al. Safety and efficacy of in vivo gene transfer into the porcine heart with replication-deficient, recombinant adenovirus vectors. Gene Ther (1996) 3:145–153.[Web of Science][Medline]
  2. Kirshenbaum L.A., MacLennan W.R., Mazur W., French B.A., Schneider M.D. Highly efficient gene transfer into adult ventricular myocytes by recombinant adenovirus. J Clin Invest (1993) 92:381–387.[Web of Science][Medline]
  3. Subramaniam A., Gulick J., Neumann J., Knotts S., Robbins J. Transgenic analysis of the thyroid-responsive elements in the {alpha}-cardiac myosin heavy chain gene promoter. J Biol Chem (1993) 268:4331–4336.[Abstract/Free Full Text]
  4. Magovern C.J., Mack C.A., Zhang J., et al. Direct in vivo gene transfer to canine myocardium using a replication-deficient adenovirus vector. Ann Thorac Surg (1996) 62:425–433. Discussion.[Abstract/Free Full Text]
  5. Barr E., Carroll J., Kalynych A.M., et al. Efficient catheter-mediated gene transfer into the heart using replication-defective adenovirus. Gene Ther (1994) 1:51–58.[Web of Science][Medline]
  6. Li J.J., Ueno H., Pan Y., et al. Percutaneous transluminal gene transfer into canine myocardium in vivo by replication-defective adenovirus. Cardiovasc Res (1995) 30:97–105.[Abstract/Free Full Text]
  7. French B.A., Mazur W., Geske R.S., Bolli R. Direct in vivo gene transfer into porcine myocardium using replication-deficient adenoviral vectors. Circulation (1994) 90:2414–2424.[Abstract/Free Full Text]
  8. Kaplitt M.G., Xiao X., Samulski R.J., et al. Long-term gene transfer in porcine myocardium after coronary infusion of an adeno-associated virus vector. Ann Thorac Surg (1996) 62:1669–1676.[Abstract/Free Full Text]
  9. Coffin R.S., Howard M.K., Cummings D.V.E., et al. Gene delivery to the heart in vivo and to cardiac myocytes and vascular smooth muscle cells in vitro using herpes virus vectors. Gene Ther (1996) 3:560–566.[Web of Science][Medline]
  10. Miller N., Vile R. Targeted vectors for gene therapy. FASEB J (1995) 9:190–199.[Abstract]
  11. Nabel E.G. Gene therapy for cardiovascular disease. Circulation (1995) 91:541–548.[Free Full Text]
  12. Sawa Y., Suzuki K., Bai H.Z., et al. Efficiency of in vivo gene transfection into transplanted rat heart by coronary infusion of HVJ liposome. Circulation (1995) 92:II479–II482.[Medline]
  13. Ellison K.E., Bishopric N.H., Webster K.A., et al. Fusigenic liposome-mediated DNA transfer into cardiac myocytes. J Mol Cell Cardiol (1996) 28:1385–1399.[CrossRef][Web of Science][Medline]
  14. Allen T.M. Long-circulating (sterically stabilized) liposomes for targeted drug delivery. Trends Physiol Sci (1994) 15:215–220.[CrossRef]
  15. Metzger J.M., Parmacek M.S., Barr E., et al. Skeletal troponin C reduces contractile sensitivity to acidosis in cardiac myocytes from transgenic mice. Proc Natl Acad Sci USA (1993) 90:9036–9040.[Abstract/Free Full Text]
  16. Milano C.A., Allen L.F., Rockman H.A., et al. Enhanced myocardial function in transgenic mice overexpressing the β-2-adrenergic receptor. Science (1994) 264:582–586.[Abstract/Free Full Text]
  17. Giordano F.J., Ping P., McKirnan M.D., et al. Intracoronary gene transfer of fibroblast growth factor-5 increases blood flow and contractile function in an ischaemic region of the heart. Nat Med (1996) 2:534–539.[CrossRef][Web of Science][Medline]
  18. Ping P., Yang Q., Hammond H.K. Altered beta-adrenergic receptor signaling in heart failure, in vivo gene transfer via adeno and adeno-associated virus. Microcirculation (1996) 3:225–228.[Medline]
  19. Acsadi G., Lochmuller H., Jani A., et al. Dystrophin expression in muscles of mdx mice after adenovirus-mediated in vivo gene transfer. Hum Gene Ther (1996) 7:129–140.[Web of Science][Medline]
  20. Marian A.J., Yu Q.T., Mann D.L., Graham F.L., Roberts R. Expression of a mutation causing hypertrophic cardiomyopathy disrupts sarcomere assembly in adult feline cardiac myocytes. Circ Res (1995) 77:98–106.[Abstract/Free Full Text]
  21. Quinones M.J., Leor J., Kloner R.A., et al. Avoidance of immune response prolongs expression of genes delivered to the adult rat myocardium by replication-defective adenovirus. Circulation (1996) 94:1394–1401.[Abstract/Free Full Text]
  22. Prentice H., Kloner R., Prigozy T., et al. Tissue restricted gene expression assayed by direct DNA injection into cardiac and skeletal muscle. J Mol Cell Cardiol (1994) 26:1393–1401.[CrossRef][Web of Science][Medline]
  23. Molkentin J.D., Kalvakolanu D.V., Markham B.E. Transcription factor GATA-4 regulates cardiac muscle-specific expression of the alpha-myosin heavy-chain gene. Mol Cell Biol (1994) 14:5056–5065.[Abstract/Free Full Text]
  24. Franz W.-M., Breves D., Klingel K., Brem G., Hofschneider P.H., Kandolf R. Heart-specific targeting of firefly luciferase by the myosin light chain-2 promoter and developmental regulation in transgenic mice. Circ Res (1993) 73:629–638.[Abstract/Free Full Text]
  25. Kitsis R.N., Buttrick P.M., McNally E.M., Kaplan M.L., Leinwand L.A. Hormonal modulation of a gene injected into the rat heart in vivo. Proc Natl Acad Sci USA (1991) 88:4138–4142.[Abstract/Free Full Text]
  26. Fishman G.I., Kaplan M.L., Buttrick P.M. Tetracycline-regulated cardiac gene expression in vivo. J Clin Invest (1994) 93:1864–1868.[Web of Science][Medline]
  27. Koh G.Y., Kim S.J., Klug M.G., et al. Targeted expression of transforming growth factor-beta 1 in intracardiac grafts promotes vascular endothelial cell DNA synthesis. J Clin Invest (1995) 95:114–121.[Web of Science][Medline]
  28. Entman M.L., Michael L., Rossen R.D., et al. Inflammation in the course of early myocardial ischaemia. FASEB J (1991) 5:2529–2537.[Abstract]
  29. Williams F.M., Kus M., Tanda K., Williams T.J. Effect of duration of ischaemia on reduction of myocardial infarct size by inhibition of neutrophil accumulation using an anti-CD18 monoclonal antibody. Br J Pharmacol (1994) 111:1123–1128.[Web of Science][Medline]
  30. Maclean D., Fishbein M.C., Braunwald E., Maroko P.R. Long term preservation of ischaemic myocardium after experimental coronary artery occlusion. J Clin Invest (1978) 61:541–551.[Web of Science][Medline]
  31. Satriano J.A., Shuldiner M., Hora K., et al. Oxygen radicals as second messengers for expression of the monocyte chemoattractant protein, JE/MCP-1, and the monocyte colony-stimulating factor, CSF-1, in response to tumor necrosis factor-{alpha} and immunoglobin G. J Clin Invest (1993) 92:1564–1571.[Web of Science][Medline]
  32. Ivey C.L., Williams F.M., Collins P.D., Jose P.J., Williams T.J. Neutrophil chemoattractants generated in two phases during reperfusion of ischaemic myocardium in the rabbit. J Clin Invest (1995) 95:2720–2728.[Web of Science][Medline]
  33. Galinanes M., Lawson C.S., Ferrari R., et al. Early and late effects of leukopenic reperfusion on the recovery of cardiac contractile function. Circulation (1993) 88:673–683.[Abstract/Free Full Text]
  34. Gottlieb R.A., Burleson K.O., Kloner R.A., Babior B.M., Engler R.L. Reperfusion injury induces apoptosis in rabbit cardiomyocytes. J Clin Invest (1994) 94:1612–1628.
  35. Ma X., Weyrich A.S., Lefer D.J., et al. Monoclonal antibody to L-selectin attenuates neutrophil accumulation and protects ischaemic reperfused cat myocardium. Circulation (1993) 88:649–658.[Abstract/Free Full Text]
  36. Marber M.S., Latchman D.S., Walker J.M., Yellon D.M. Cardiac stress protein elevation 24 hours after brief ischaemia or heat stress is associated with resistance to myocardial infarction. Circulation (1993) 88:1264–1272.[Abstract/Free Full Text]
  37. Mestril R., Chi S.H., Sayen M.R., O'Reilly K., Dillmann W.H. Expression of inducible stress protein 70 in rat heart myogenic cells confers protection against simulated ischaemia-induced injury. J Clin Invest (1994) 93:759–767.[Web of Science][Medline]
  38. Yang B.C., Virmani R., Nichols W.W., Mehta J.L. Platelets protect against myocardial dysfunction and injury induced by ischaemia and reperfusion in isolated rat hearts. Circ Res (1993) 72:1181–1190.[Abstract/Free Full Text]
  39. Taylor S.H. Congestive heart failure: towards a comprehensive treatment. Eur Heart J (1996) 17(Suppl_B):43–56.[Free Full Text]
  40. Steare S.E., Yellon D.M. The potential for endogenous myocardial antioxidants to protect the myocardium against ischaemia-reperfusion injury. J Mol Cell Cardiol (1995) 27:65–74.[Web of Science][Medline]
  41. Stenman C.L., Shutter J.R., Hockenbery D., Kanagawa O., Korsemeyer S.J. Bcl-2 inhibits multiple forms of apoptosis but not negative selection in thymocytes. Cell (1991) 67:879–888.[CrossRef][Web of Science][Medline]
  42. Kiefer M.C., Brauer M.J., Powers V.C., et al. Modulation of apoptosis by the widely distributed Bcl-2 homologue Bak. Nature (1995) 374:736–739.[CrossRef][Medline]
  43. Semenza G.L., Nejfelt M.K., Chi S.M., Antonarakis S.E. Hypoxia-inducible nuclear factors bind to an enhancer element located 3' to the human erythropoietin gene. Proc Natl Acad Sci USA (1991) 88:5680–5684.[Abstract/Free Full Text]
  44. Gustafson T.A., Markham B.E., Bahl J.J., Morkin E. Thyroid hormone regulates expression of a transfected alpha-myosin heavy chain fusion gene in fetal heart cells. Proc Natl Acad Sci USA (1987) 84:3122–3126.[Abstract/Free Full Text]
  45. Molkentin J.D., Jobe S.M., Markham B.E. Alpha-myosin heavy chain gene regulation: delineation and characterization of the cardiac muscle-specific enhancer and muscle-specific promoter. J Mol Cell Cardiol (1996) 28:1211–1225.[CrossRef][Web of Science][Medline]
  46. Bishopric N.H., Simpson P.C., Ordahl C.P. Induction of the skeletal actin gene in {alpha}1-adrenoceptor mediated hypertrophy of rat cardiac myocytes. J Clin Invest (1987) 80:1194–1199.[Web of Science][Medline]
  47. Webster K.A., Bishopric N.H. Molecular regulation of cardiac myocyte adaptations to chronic hypoxia. J Mol Cell Cardiol (1992) 24:741–751.[CrossRef][Web of Science][Medline]
  48. Penney DG. Carbon dioxide induced cardiac hypertrophy. In: Zak R. editor. Growth of the heart in health and disease. New York: Raven Press, 1984:337–62.
  49. Webster K.A., Muscat G.E.O., Kedes L. Adenovirus E1A products suppress myogenic differentiation and inhibit transcription from muscle-specific promoters. Nature (1988) 332:553–558.[CrossRef][Medline]
  50. Webster K.A., Kedes L. The c-fos cyclic AMP-responsive element conveys constitutive expression to a tissue-specific promoter. Mol Cell Biol (1990) 10:2402–2406.[Abstract/Free Full Text]
  51. Bodi I., Bishopric N.H., Discher D.J., Wu X., Webster K.A. Cell-specificity and signaling pathway of endothelin-1 gene regulation by hypoxia. Cardiovasc Res (1995) 30:975–984.[Abstract/Free Full Text]
  52. Wu X., Bishopric N.H., Discher D.J., Murphy B.J., Webster K.A. Physical and functional sensitivity of zinc finger transcription factors to redox change. Mol Cell Biol (1996) 16:1035–1046.[Abstract]
  53. Prentice H., Kloner R.A., Li Y., Newman L., Kedes L. Ischaemic/reperfused myocardium can express recombinant protein following direct DNA or retroviral injection. J Mol Cell Cardiol (1996) 28:133–140.[CrossRef][Web of Science][Medline]
  54. Webster K.A., Discher D., Bishopric N.H. Induction and nuclear accumulation of fos and jun proto-oncogenes in hypoxia cardiac myocytes. J Biol Chem (1993) 268:16852–16859.[Abstract/Free Full Text]
  55. Webster K.A., Discher D.J., Bishopric N.H. Regulation of fos and jun immediate-early genes by redox or metabolic stress in cardiac myocytes. Circ Res (1994) 75:361–371.
  56. Denver M.A., MacFarlane N.G., Miller D.J., Cobbe S.M. Enhance SR function in saponin-treated ventricular trabeculae from rabbits with heart failure. Am J Physiol (1996) 271:H850–H859.[Medline]
  57. Mahdavi V, Koren G, Michaud S, Pinset C, Izumo S. Identification of the sequences responsible for the tissue-specific and hormonal regulation of the cardiac myosin heavy chain genes. In: Kedes LH, Stockdale FE, editors. Cellular and molecular biology of muscle development. New York: Alan R. Liss, 1989:369–79.
  58. Buttrick P.M., Kass A., Kitsis R.N., Kaplan M.L., Leinwand L.A. Behavior of genes directly injected into the rat heart in vivo. Circ Res (1992) 70:193–198.[Abstract/Free Full Text]
  59. Buttrick P.M., Kaplan M.L., Kitsis R.N., Leinwand L.A. Distinct behavior of cardiac myosin heavy chain gene constructs in vivo. Circ Res (1993) 72:1211–1217.[Abstract/Free Full Text]
  60. Bunn H.F., Poyton R.O. Oxygen sensing and molecular adaptation to hypoxia. Physiol Rev (1996) 76:839–885.[Abstract/Free Full Text]
  61. Wang G.L., Jiang B.-H., Rue E.A., Semenza G.L. Hypoxia-inducible factor 1 is a basic helix-loop-helix-PAS heterodimer regulated by cellular O2 tension. Proc Natl Acad Sci USA (1995) 92:5510–5514.[Abstract/Free Full Text]
  62. Jelkmann W. Erythropoietin: structure, control of production, and function. Physiol Rev (1992) 72:449–489.[Free Full Text]
  63. Hockenbery D.M., Oltval Z.L., Yin X.-M., Milliman C.L., Korsmeyer S.J. Bcl-2 functions in an antioxidant pathway to prevent apoptosis. Cell (1993) 75:241–251.[CrossRef][Web of Science][Medline]
  64. Gibbons G.H., Dzau V.J. Molecular therapies for vascular diseases. Science (1996) 272:689–693.[Abstract]

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