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Ann Thorac Surg 1999;68:1929-1933
© 1999 The Society of Thoracic Surgeons
a Department of Surgery, University of Connecticut School of Medicine, Farmington, Connecticut, USA
b Department of Surgery, Baystate Medical Center, Springfield, Massachusetts, USA
Address reprint requests to Dr Das, Department of Surgery, University of Connecticut School of Medicine, Farmington, CT 06030-1110
e-mail: ddas{at}neuron.uchc.edu
Presented at the International Symposium on Myocardial Protection From Surgical Ischemic-Reperfusion Injury, Asheville, NC, Sep 21–24, 1997.
Abstract
Ischemic reperfused heart represents a potential target for gene therapy because gene transfer can represent an alternate pharmacological approach to protect the heart from cellular injury. Gene therapy may be particularly useful to deal with previously unapproachable problems. For myocardial preservation, gene therapy could replace those pharmacological interventions when drugs are delivered locally by sustained release with the help of mechanical device, eg, implants. In this review, attempts are made to define the molecular targets for gene therapy primarily applicable to myocardial preservation associated with ischemia and reperfusion. It has been emphasized that for successful gene transfer, not only characterization of proper targets and elimination of undesirable side effects are necessary, but also the therapy must be proven superior compared to other pharmacological interventions including surgery.
Gene therapy is a technique in which a normal allele of a gene is administered into a cell in order to modify the genetic repertoire of the target cell which either cannot express its own copy or produces a defective copy. It is usually a two-step process which involves identification of appropriate DNA sequences and cell types and successful delivery to the target tissue. To date, gene therapy is limited to somatic cells only.
Usually cells that can be grown in vitro are attractive targets for gene transfer. A variety of cells have been used for this purpose which include fibroblasts, keratinocytes, hepatocytes, endothelial cells, and myocytes. However, a great deal of care must be attributed to select a target cell because after the genes are expressed in the cell they often require posttranslational modification. Since the heart is the site of expression of many genetic disorders, it comprises an attractive target tissue for gene transfer, and the use of gene therapy to cure cardiovascular diseases appears to be a rational approach in clinical medicine.
The scope of gene therapy has been broadened in recent years and, especially for the cardiovascular system, gene therapy has been redefined to include a variety of systemic diseases which are not of genetic origin. For example, based on the observation that angiogenic peptide growth factors can enhance collateral blood flow to the ischemic heart, bypassing coronary artery stenoses [1], a clinical trial of percutaneous catheter-based delivery of the gene encoding endothelial growth factor in peripheral vascular disease has recently been approved by the Food and Drug Administration. Intracoronary delivery of an adenoviral vector coding for a secreted tfibroblast growth factor was found to increase blood flow and contractile function in the ischemic porcine heart [2]. Gene therapy, thus, may represent an alternate mode of pharmacological intervention to combat cardiovascular diseases.
In this review, attempts will be made to define the molecular targets for gene therapy primarily applicable to myocardial preservation associated with ischemia and reperfusion. Proper identification of the targets for gene transfer in the setting of ischemia-reperfusion obviously depends on the clear understanding of the pathophysiology of the disease. A great deal of attention has been given to the problem of intimal hyperplasia and restenosis after coronary angioplasty [3]. A similar approach for gene transfer may be proven highly beneficial for myocardial preservation in the setting of ischemia reperfusion.
Transduction and expression of genes
There are five important components which must be considered in the development of gene therapy: (i) the isolation and cloning of a target gene; (ii) the development of a proper vector for gene transfer; (iii) the identification of a target cell; (iv) in vivo gene delivery, and (v) the identification of potential therapeutic targets. After successful gene delivery, the foreign gene must be expressed in the target cell and improvement in the pathologic phenotype must be apparent. Finally, for successful gene therapy, there should not be any deleterious effects after the transfection with the foreign gene.
Both viral and nonviral vectors have been used for the purpose of gene therapy [4]. It is important to characterize target cells before the optimization of gene delivery because cells may differ significantly in proliferation features. For example, unlike vascular cells, cardiac myocytes are terminally differentiated cells and, hence, they require a vector which is independent on cell replication for their expression. Among many techniques of gene transfer, viral vectors have been proven to be the most effective [5]. These vectors can be directly administered to intact animals in vivo or to cells in vitro for subsequent grafting into recipient organs. Two types of vectors have been used for the purpose of gene delivery: retrovirus and adenovirus. Retrovirus contains RNA genomes that are reverse transcribed after infection yielding a double-stranded cDNA copy of the genome flanked by identical elements (long terminal repeats) which contains the regulatory sequences necessary for the expression of intervening genes. Retroviral vectors are produced by deleting viral genes from the provirus and replacing with the target gene of therapeutic potential. Although retroviral vectors were successfully employed for direct gene transfer, their use has been proven difficult because of certain limitations. One of the major problems is that they are very unstable in vivo and often tend to shut off gene expression. Furthermore, their capacity for foreign DNA is limited to less than 10 kb, and they can be produced only in low to moderate amounts.
In contrast to retroviruses, adenoviruses contain relatively larger double stranded DNA of 36 kb and 150 kb with broad host range. These viruses can be produced in very high titers and usually do not integrate into the host cell genome. They enter cells through specific surface receptors and travel to the nucleus [6]. Adenoviruses do not require host cell proliferation for gene transfer and subsequent expression. Adenoviral vectors have been successfully used for gene therapy in cases of atherosclerosis, hereditary myopathies, coronary artery restenosis, and many other cardiovascular diseases. Target cells include respiratory airway epithelial cells, hepatocytes, endothelial cells, skeletal muscle cells, and cardiomyocytes [7]. However, cytotoxicity and immunological reactions following infection with adenovirus remain a significant issue both in vivo and in vitro [8]. During recent years, the second and third generation of adenoviral vectors are being produced which claim to possess no adverse effects on the host cells. Table 1 lists both viral and non-viral vectors which can be used for transduction and expression of genes.
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Delivery of genes to the desired cell requires a targeting mechanism. Direct gene transfer can be achieved using balloon catheters. To deliver genes directly to rabbit aortas, Flugelman and coworkers used perfusion balloons and retroviral vectors encoding neomycin phosphotransferase gene as well as several other genes [9]. Double balloon catheters were used with adenoviral vectors expressing a nuclear-targeted beta-galactosidase gene in the carotid and femoral arteries of sheep [10]. Catheter-based direct gene transfer was also performed by French and associates [11]. Murry and colleagues performed myogenic determination (MyoD-based) gene therapy for myocardial infarction [12].
A variety of catheters have been used in the past for arterial gene transfer including double balloon catheters [10], hydrogel-coated balloon catheters [13], porous balloon catheters [14], and channeled balloon catheters [15]. For adenoviral vectors, the transduction efficiency falls with decreasing exposure times [16]. In order to achieve proper transduction, at least 30 min is necessary to deliver adenoviral vectors [17]. However, in the coronary vasculature, such protracted balloon inflations is simply impossible. To overcome this problem, a dispatch catheter (Scimed Life Systems, Inc, Maple Grove, MN) which is a specially designed autoperfusion balloon catheter, was recently used for arterial gene transfer [18]. Distal perfusion allowed for prolonged infusion without ischemia.
The pattern of genes expressed may vary qualitatively depending on the mode of gene transfer. Selective genetic modification of vascular endothelium is achieved when vessels are infected with dwell or double-balloon techniques, whereas genes are primarily expressed in medial smooth muscle cells when they are introduced through a high-pressure balloon system [19]. Catheter-based delivery remains the most attractive and meaningful method for gene transfer and warrants clinical application.
Finally, adenovirus seems to be a useful vector which can be introduced into the heart with the help of a catheter. This process was found to express recombinant genes in the myocardium. Adenovirus is a highly efficient vector of gene transfer into cardiomyocytes in vitro [20]. Intravenous administration of a recombinant E coli beta-galactosidase adenovirus resulted in highly efficient gene transfer throughout mouse cardiac muscle [21]. Percutaneous coronary gene transfer was used to transfect the coronary sinus and coronary arteries of mice and rabbits using adenoviral vectors [22].
Therapeutic targets for myocardial preservation
As described in Table 2, a wide variety of diseased hearts may represent potential targets for gene therapy. We will restrict our discussion only to ischemic reperfused myocardium (the infarcted heart). Is ischemic reperfused heart a target for gene therapy? The answer is yes. A recent study has demonstrated that direct injection of reporter genes into hearts subjected to coronary artery occlusion followed by reperfusion could result in gene expression comparable to the levels observed in non-occluded normal hearts [23]. The fact that ischemic reperfused myocardium remains a target for gene therapy receives further support from the observation that intracoronary administration of an adenoviral vector encoding fibroblast growth factor could ameliorate ischemic reperfusion injury [24].
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Nitric oxide (NO) represents another potential candidate for gene therapy for myocardial preservation. Numerous reports are available in the literature indicating the cardioprotective role of NO [40, 41]. Providing hearts with an excess amount of NO in the face of ischemia results in the amelioration of ischemic reperfusion injury [42]. In fact, direct gene transfer of nitric oxide synthase was reported very recently [43]. Many other compounds that have been found to be cardioprotective may become targets for gene therapy. For example, bradykinin has recently been found to reduce myocardial injury associated with ischemia–reperfusion [44]. Enhancement of the calcium extrusion mechanism by upregulating Ca2+-ATPase by direct Ca2+-ATPase gene transfer have been proven to be cardioprotective [45]. Fatty acid binding proteins (FABP) and vasoactive intestinal peptide (VIP) were found by our laboratory to play a crucial role in myocardial ischemia reperfusion injury. FABP binds with the free fatty acids and lysophosphoglycerides that are generated in the ischemic reperfused myocardium thereby ameliorating the detrimental effects of these amphipathic compounds [46], while VIP functions both as a signal transduction agent and free radical scavenger in the reperfused heart [47]. Both FABP and VIP are lost from the postischemic heart during reperfusion [47, 48]. It has been demonstrated by us that the intracoronary delivery of FABP or VIP could protect the heart from ischemic reperfusion injury [49]. FABP was encapsulated inside liposome and directly delivered into the isolated perfused rat heart.
A growing body of evidence suggests that reperfusion of ischemic myocardium is associated with apoptotic cell death and DNA fragmentation [50]. It appears that part of reperfusion injury may be mediated by apoptosis. Very recently, reperfusion of ischemic myocardium was found to result in the downregulation of Bcl-2, an anti-death gene for apoptotic cell death [51]. A recent study from this laboratory demonstrated that myocardial adaptation to ischemia by repeated short durations of ischemia and reperfusion resulted in the upregulation of Bcl-2 gene (Fig 1), suggesting that this gene is likely to be part of the heart’s defense system. Incidentally, Bcl-2 gene is believed to function as an antioxidant [52]. These results imply that in the near future, Bcl-2 may become a potential target for gene therapy for myocardial preservation.
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It should be clear from this presentation that the ischemic reperfused heart represents a potential target for gene therapy. As mentioned earlier, the original concept of gene therapy aimed at replacing defective or missing genes is not applicable for the ischemic heart. Here gene transfer is merely an alternate pharmacological approach to protect the heart from ischemic reperfusion injury. The obvious question that comes to mind is why gene therapy when pharmacological modalities can serve the purpose? The answer is simple. Gene therapy may be used in this case to deal with previously unapproachable problems. For example, NO has been proven to play a crucial role in myocardial preservation. However, while it is beneficial for the protection of ischemic myocardium, excess NO may be detrimental to the heart and delivery of NO itself is not feasible. Upregulation of inducible nitric oxide synthase (iNOS) by direct gene transfer to supply sustained and local release of NO when it is needed could be extremely beneficial to the heart. Similarly, controlled release of agents to dissolve thrombus is desirable, and antithrombotic drugs must function locally within a narrow therapeutic range to avoid risk of hemorrhage [53]. Gene therapy will also find its place in myocardial preservation because drugs are often necessary to act over an extremely short time frame. Again NO is a good example of this.
In summary, for myocardial preservation, gene therapy could replace those pharmacological interventions when drugs are delivered locally by sustained release with the help of a mechanical device, eg, implants. However, for successful gene transfer, not only characterization of proper targets and elimination of undesirable side effects are necessary, but also the therapy must be proven superior compared to other pharmacological interventions including surgery. Clear definition of the target cell (eg, endothelial or myocytes) is obviously necessary.
Acknowledgments
This study was supported in part by National Institutes of Health grants HL 22559, HL 34360, HL 33889, and a Grant-in-Aid from the American Heart Association.
References
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