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Gene- and cell-based therapies for cardiovascular diseases: current status and future directions

L.G Meloa,b, M Gnecchia, A.S Pachoria, K Wangb and V.J Dzaua,*,1

a Brigham and Women's Hospital and Harvard Medical School, Boston, MA, USA
b Queen's University, Ontario, Canada

* Professor V.J. Dzau, Chairman, Department of Medicine, Brigham and Women's Hospital, Tower 1, Room 210, 75 Francis Street, Boston, MA 02115-6110, USA. Tel.: +1-617-732-6340; fax: +1-617-732-6439
vdzau{at}partners.org

Abstract

Recent advances in understanding the molecular and cellular basis of cardiovascular diseases, together with the availability of tools for genetic manipulation of the cardiovascular system, offer possibilities for new treatments. Gene therapies have demonstrated potential usefulness in treating complex diseases such as hypertension, atherosclerosis and myocardial ischaemia based on studies of various animal models. Some of these experimental therapies have transitioned into clinical trials to assess their safety, feasibility and therapeutic potential in humans with cardiovascular disease. The recent isolation of adult progenitor cells with the capacity to differentiate into endothelial and cardiomyocyte phenotypes has opened an exciting era of cell therapies for vascularization and repair of ischaemic tissues and injured blood vessels. Despite these significant developments, we believe that the successful translation of these experimental therapies into clinical practice will require safer and more effective vectors and delivery tools, a deeper knowledge of progenitor cell biology and, finally, the documentation of efficacy and safety through multicentre randomized trials.

Key Words: Bone marrow • Cardiac progenitors • Cellular cardiomyoplasty • Coronary artery disease • Endothelial progenitor cells • Heme oxygenase • Protective gene therapy

Introduction

The serious socio-economic implications of cardiovascular disease, in terms of burden and economic loss to the patient and the financial strain that it imposes on health delivery systems, calls for new therapeutic strategies for the management of these diseases. Recent advances in the understanding of the molecular and cellular mechanisms of cardiovascular disease, together with the availability of tools for genetic manipulation of the cardiovascular system, offer new possibilities for treatment of cardiovascular diseases.1–5 Gene transfer may be used as a gain of function strategy to replace or augment the function of defective or under-compensating genes that are involved in disease progression (Fig. 1) (for review, see6). The therapeutic potential of gene transfer has been demonstrated in several animal models of cardiovascular disease, using a wide range of therapeutic targets.6–9 Strategies for gene silencing have also been successfully used to inhibit the activity of pathogenic genes (Fig. 1).6,10–12 The recent identification of adult progenitor cells with the capacity to differentiate into endothelial and myogenic phenotypes has opened a new and exciting era in cell therapy with the potential for vascularization and repair of ischaemic and infarcted myocardium and injured vessels.13–15 Some of these strategies have progressed to early phase clinical trial testing and others are currently in advanced clinical evaluation.16,17



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  		Fig. 1 Strategies for genetic manipulation in the cardiovascular system. (a) Gene transfer involves the delivery of exogenous genes (transgenes) by a vector capable of expressing the therapeutic protein in the host cells in order to increase the activity of the gene(s) (gain-of-function) whose endogenous function may be deficient and cause disease. (b) Gene blockade involves inhibition of genes involved in the pathogenesis of disease. Four strategies are commonly used to inhibit gene activity at the transcriptional or translational level. The first strategy employs double stranded deoxyoligonucleotides containing the consensus binding sequences (decoy oligonucleotides) for transcriptional factors involved in the activation of pathogenic genes. Alternatively short single-stranded deoxyoligonucleotides complementary to the target gene mRNA (antisense oligonucleotides) are delivered to the target cells or tissue by transfection or with the aid of a vector. The antisense deoxyoligonucleotide binds to the target mRNA transcript and prevents it from being translated. The second, transfection of a molar excess of the decoy oligonucleotide prevents the binding and transactivation of the genes regulated by the target transcriptional factor. The third strategy is novel and uses small interfering RNA's (siRNA) to knockdown the endogenous activity of pathogenic genes. Less commonly, short segments of RNA with enzymatic activity (ribozymes) are used to degrade target mRNA transcripts. Reproduced with permission.

 
In this article, we provide an overview of the current state of gene- and cell-based therapies for cardiovascular disease, and discuss the opportunities and challenges lying ahead in this burgeoning field. We use select examples to illustrate the potential of these novel therapies in the treatment of common cardiovascular diseases, such as coronary artery disease, restenosis and vein graft failure.

Tools for genetic manipulation of the cardiovascular system
Most vectors used in cardiovascular gene transfer lack tissue specificity, incurring the risk of systemic biodistribution and ectopic transgene expression.2–4,18 Furthermore, transgene expression by most vectors is transient, rendering them unsuitable for use in chronic vascular diseases.2–4

Vectors
A number of vector systems have evolved over the years. Nonviral vectors usually yield low and transient gene transfer efficiency due to lack of genomic integration and rapid degradation of the vector.4 A promising new delivery strategy uses synthetic peptide carriers containing a nuclear localization signal to facilitate nuclear uptake of the target cDNA.19 These peptide–DNA heteroplexes are recognized by intracellular receptor proteins and imported into the nucleus, where the target cDNA is transcribed.

Recombinant viruses have become the preferred vectors for cardiovascular gene transfer. These are replication-deficient viral particles that retain their ability to penetrate target cells and deliver genetic material with significantly higher efficiency than non-viral vectors.2,3,18 Some viral vectors, such as adeno-associated virus (AAV) and lentivirus are capable of sustained expression of the therapeutic gene,2,3,18,20 rendering them suitable for use in chronic myocardial and vascular diseases. Some viral proteins may trigger a robust immune reaction which may reduce the duration of transgene expression;21 however, some recent developments have led to the production of vectors with attenuated immunogenicity.22 Furthermore, there is a risk, albeit remote, that these vectors may revert to replication proficiency, thus, raising safety concerns about biological hazards such as oncogenesis and insertional mutagenesis.19,20 The current impetus is for the development of physiologically regulated vectors that will be capable of mediating tissue specific expression of the transgene in response to pathophysiological cues such as hypoxia, oxidative stress or inflammation.23

Targets of gene and cell therapy for cardiovascular diseases
Several genes have emerged as potential targets for cardiovascular diseases. Over-expression of vasodilator genes such as nitric oxide synthase (NOS) and atrial natriuretic peptide24,25 or the inhibition of vasoconstrictor peptides such as angiotensin26,27 have been effective as antihypertensive strategies in animal models, whereas inhibition of pro-proliferative genes has shown promise in the treatment of vascular proliferative diseases.28–30 Over-expression of antithrombotic and/or the inhibition of pro-inflammatory and cell adhesion molecules may have therapeutic potential in the treatment of thrombosis and inflammation of the vessel wall in atherosclerosis,31–34 and the recent isolation of endothelial progenitor cells (EPC) may allow the design of cell based therapies for repair of damaged vessels and bioengineering of vein grafts and vascular prostheses.13,14,35,36

In the setting of coronary artery disease, the over-expression of cytoprotective genes such as antioxidant enzymes8,37–39 and survival proteins40–42 has emerged as a potential strategy for myocardial protection from ischaemic injury. Inhibition of endothelial cell activation may be useful as treatment for acute myocardial infarction or for immunosuppression during cardiac transplantation,43 and strategies for plaque stabilization and inhibition of platelet adhesion may be beneficial in preventing the occurrence of acute coronary events and myocardial infarction.44 Gene therapy for rescuing the failing ischaemic myocardium may also be attainable in certain conditions. Targeted delivery of pro-angiogenic factors such as vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF) may be useful in the treatment of myocardial and peripheral ischaemia.45–50 Cell-based therapies for repair and vascularization of infarcted myocardium are also currently being evaluated using a variety of substrates, such as skeletal muscle myoblasts,51,52 fetal cardiomyocytes53,54 and bone marrow-derived progenitor cells.55,56 The use of adult autologous cardiac progenitors may be favoured for cellular cardiomyoplasty because this circumvents many of the ethical, moral and technical issues associated with other sources of cell.

Gene- and cell-based therapies for vascular diseases
Elevated vascular tone, hypercholesterolaemia, inflammation and vascular smooth muscle cell (VSMC) proliferation and migration are prominent features of hypertension and atherosclerosis that coexist under a common denominator of endothelial dysfunction.57,58 VSMC proliferation into the intimal space is a major cause of post-angioplasty and in stent restenosis, and vein graft bypass failure.59,60 Several experimental gene therapy strategies aimed at reducing vascular tone, plasma cholesterol levels and vessel wall proliferation have been assessed as potential treatments for these diseases in various animal models.24–34,61,62 Inhibition of cell cycle progression has emerged as an important therapeutic target for prevention of restenosis and graft atherosclerosis, and transplantation of autologous EPC has shown promise as a strategy for bioengineering of vein grafts and vascular prostheses and in repair of injured blood vessels.35,36 More recently, exogenous mobilization of circulating EPC with cytokines has shown potential as a non-invasive prophylactic therapy for prevention of restenosis following balloon angioplasty.63,64

Vascular protection
Atherosclerosis and thrombosis
Gene therapy aimed at reducing cholesterol level and/or at increasing thromboresistance and tensile strength within the plaque could potentially offer the possibility to achieve long-term plaque stabilization and prevent the occurrence of acute coronary events.44 For example, the over-expression of apoprotein ApoA-1 in mice by intravenous adenoviral gene delivery increases serum HDL levels.65 Blockade of monocyte infiltration and activation in the arterial wall by inhibition of monocyte chemoattractant protein-1 (MCP-1) receptor activation was shown to retard the onset of atheroma and to limit progression and destabilization of established atherosclerotic lesions in ApoE deficient mice,66 whereas over-expression of antithrombotic genes at sites in the vessel wall at risk of thrombosis may be a feasible protective strategy for vulnerable plaque and prevention of acute coronary events.31–34

Another important target for vascular protection is NOS.67 Endothelium-derived nitric oxide exerts a plethora of vasculoprotective actions, including vasorelaxation, inhibition of VSMC proliferation and migration, and inhibition of platelet activation and adhesion.68 NOS gene transfer provides a mechanism to increase nitric oxide bioactivity and enhance the anti-atherogenic properties of the vessel wall. For example, delivery of inducible nitric oxide synthase (iNOS) by adenovirus has been reported to abrogate aortic allograft atherosclerosis in rats,69 and neuronal (nNOS) and endothelial (eNOS) nitric oxide synthase gene transfer reduces inflammatory cell infiltration and lipid deposition in carotid arteries of cholesterol-fed rabbits.70 Gene transfer of cytoprotective genes such as heme oxygenase-1 (HO-1) and superoxide dismutase (SOD) has also been shown to exert vasculoprotective effects. Adenovirus-mediated delivery of HO-1 attenuated the development of aortic lesions in ApoE-deficient mice, in parallel with a decrease in iron deposition,71 whereas delivery of manganese superoxide dismutase gene improved vascular function in pre-atherosclerotic carotid arteries from hypercholesterolaemic rabbits.72

Lipid-lowering gene therapy may be useful in the treatment of inherited disorders of lipid metabolism, such as familial homozygotic hypercholesterolaemia (FHH) and apoE deficiency, because of their refractoriness to medical treatment. For these patients, gene therapy may offer hope for a cure because of the monogenic nature of these diseases. The use of lipid-lowering gene therapy in humans has to date only been evaluated in a small Phase I feasibility clinical trial with patients diagnosed with FHH. Three out of five patients treated with a retroviral vector expressing the wild-type LDL receptor showed a reduction of 6–23% in plasma LDL levels,73 but the duration of transgene expression was relatively short, possibly due to retroviral gene silencing. The development of clinically effective therapies for FHH and apoE deficiency will be conditioned by the availability of vectors capable of providing stable, long-term expression of the therapeutic gene. This will require a vector that is capable of directing chromosomal integration of the transgene without triggering insertional mutagenesis. Furthermore, the need for chronic therapeutic gene expression in these diseases will require regulated and tissue-specific expression of the transgene. One potential strategy is to use a physiologically sensitive AAV vector for skeletal muscle- or liver-specific expression of apoE or the LDL receptor.

Neointima proliferation
Percutaneous coronary intervention (PCI) and coronary artery bypass grafting (CABG) are common treatment options for patients with coronary artery disease requiring revascularization. However, the failure rate of these procedures due to restenosis and atherosclerosis remains relatively high. The ability to inhibit proliferation of the medial smooth muscle using gene therapy techniques provides the opportunity to genetically engineer native vessels and grafts to render them resistant to atherosclerosis and neointimal hyperplasia (for review, see74).

Delivery of antiproliferative genes, such as those coding for the nitric oxide synthases, offers an approach to achieve inhibition of neointima hyperplasia. All three isoforms of nitric oxide have been shown to exert vasculoprotective and antiproliferative effects after gene transfer (for review, see67). Endothelial and iNOS gene transfer were both efficacious in reducing neointimal thickening in balloon injured vessels.75,76 This has led to at least one Phase I clinical trial (Restenosis Gene Therapy Trial, REGENT-I), currently in progress, to evaluate the efficacy of catheter-based iNOS gene delivery to prevent restenosis of coronary arteries treated by percutaneous transluminal coronary angioplasty (PTCA). Local delivery of antioxidant enzymes such as HO-177 and ecSOD78 by adenovirus has also been shown to inhibit neointima hyperplasia in various animal models of restenosis, possibly due to reduction in inflammation and oxidative stress during the early phase of vascular injury, and the subsequent inhibition of vascular smooth muscle proliferation.

Another approach is the direct inhibition of cell cycle. Cytostatic strategies involve the inhibition of key proteins regulating cell cycle progression in order to arrest neointimal cell proliferation.28–30 We used this strategy to treat jugular veins in vivo with HVJ–liposome complexes containing antisense oligonucleotide against cell cycle regulators PCNA and cdc2 kinase in atherosclerotic New Zealand rabbits prior to carotid artery interpositional grafting.79 Subsequent histological and functional analysis of the vein graft showed that the intra-operative gene therapy led to inhibition of graft atherosclerosis in parallel with improved endothelial function and reduced inflammation.80 More significantly, the gene therapy led to adaptive remodeling of the graft, successfully inducing medial hypertrophy while inhibiting neointimal hyperplasia, to yield conduits that resemble normal arteries.79 Thus, these studies showed the therapeutic potential of cell-cycle inhibition for the genetic engineering of vein grafts that are resistant to failure. Similarly, we showed that treatment of vein grafts prior to implantation with a decoy deoxyoligonucleotide bearing the consensus binding sequence of E2F-1, a transcriptional factor involved in cell-cycle progression, resulted in prolonged resistance to neointimal hyperplasia and improved patency of the graft after transplantation.81

These findings led us to initiate a Phase I prospective, randomized, double-blind trial of human saphenous vein graft treatment with E2F decoy (Project In Ex-Vivo Vein Graft Engineering Via Transfection, PREVENT-1) in high risk patients suffering form peripheral arterial occlusive disease.82 Using non-distending pressure to deliver the E2F decoy oligonucleotide ex vivo prior to arterial interpositional grafting, we demonstrated that E2F decoy treatment was safe and feasible. Although the results were preliminary, the study provided evidence that antiproliferation gene therapy is feasible for clinical application. More recently, the PREVENT II has largely confirmed the finding of the PREVENT I trial. The PREVENT II is a randomized, double-blinded, placebo-controlled Phase II trial designed to evaluate the effect of E2F decoy treatment on CABG failure (unpublished findings, Grube et al., American Heart Association meeting, November 2001. For commentary, see83). The interim results confirmed the feasibility and safety of using E2F-1 decoy. Analysis of the secondary endpoints using quantitative coronary angiography and three-dimensional intravascular ultrasound demonstrated increased patency and adaptive vessel remodeling characterized by reduction in neointimal size and volume in the treated group 1 year after treatment, leading to 40% reduction in critical stenosis. These encouraging results now need adequately sampled and powered Phase III studies in patients with coronary and peripheral vessel disease in order to further validate the therapeutic value of this approach. The ease with which the treatment can be incorporated during routine vein grafting procedures offers a potentially effective and cost-effective approach for the prevention of coronary and peripheral vein graft atherosclerosis and failure.

Despite these promising preclinical data, the use of gene therapy in treatment of vasculoproliferative diseases still has to overcome various feasibility and efficacy issues. The complexity of the pathological processes leading to restenosis implies that genetic manipulation of multiple targets may be necessary for effective and sustained therapeutic benefit.

Vascular repair
Endothelial progenitor cell transplantation
An emerging area with therapeutic potential is the use of autologous EPC transplantation for repair of damaged blood vessels and bioengineering of bioprosthetic grafts.35,36,84–88 EPC are thought to originate from a common hemangioblast precursor in the bone marrow89 and express endothelial lineage markers such as CD34, Flk-1, VE-cadherin, PECAM-1, von Willebrand factor, eNOS and E-selectin. The cells can be isolated from peripheral blood, expanded and genetically modified to yield sufficient numbers for therapeutic application13,14 (for review, see15). In vivo, the cells are mobilized to sites of injury, such as ischaemic myocardium and injured vessels, where they may participate in local vasculogenesis and tissue repair90–92 (for review, see93).

Autologous endothelial cell transplantation may be useful as adjunctive therapy for rapid re-endothelialization and restoration of homeostasis in blood vessels injured during revascularization procedures,35,86,88,94 or for seeding prosthetic grafts, stents or engineering of artificial vessels.35,36,84–88,95,96 Furthermore, the cells can be genetically engineered ex vivo to express therapeutic genes that could impart desirable qualities to the grafts, such as an enhanced antithrombotic surface97–99 or the capability to synthesize vasodilatory and cytoprotective factors for maintenance and survival of the grafts. We35 and several other groups36,84,85,94–96,100 have already demonstrated the feasibility of transplanting autologous endothelial cells to create a biosurface in ePTFE grafts and in denuded native blood vessels. We also showed that transplantation of autologous EPC from peripheral blood leads to rapid endothelialization of ePTFE graft segments and balloon-denuded carotid arteries. Furthermore, the injured vessels transplanted with autologous EPC showed significant inhibition of neointima deposition compared to the saline-treated control vessels.35 Using a similar approach, Kaushal et al.36 showed that implantation of EPC into decellularized porcine iliac vessels implanted as coronary interposition grafts reconstituted a functional endothelial layer that conferred improved vasodilatory function and prolonged patency of the grafts. More recently, we reported that the therapeutic benefit of transplanted EPC can be enhanced by genetically modifying the cells ex vivo with a retrovirus to over-express eNOS.99 Our results showed that the denuded carotid vessels treated with eNOS-expressing EPC had a significantly greater reduction in neointima deposition than vessels treated with cells over-expressing the reporter gene green fluorescence protein (GFP). Others, using endothelial cells transduced with a retrovirus over-expressing -PA to seed stainless steel intravascular stents, reported sustained retention of the transplanted cells to the stent surface after implantation,85 suggesting that this may be an option for delivery of therapeutic genes for prevention of in-stent restenosis and thrombosis. Thus, EPC may have a dual therapeutic role as a cell substrate for tissue repair and as a vector for gene delivery.

Endothelial progenitor cell mobilization
A less cumbersome and potentially more effective strategy to enhance re-endothelialization of damaged vessels involves the mobilization and recruitment of endogenous EPC from the bone marrow and other sources to the injured sites. Bhattacharya et al.101 and Shi et al.102showed that mobilization of bone marrow by exogenous granulocyte colony stimulating factor (G-CSF) enhanced endothelialization and patency of small calibre prosthetic grafts. Using a similar strategy, we have recently demonstrated that pre-treatment with G-CSF led to accelerated re-endothelialization and concomitant reduction in neointima hyperplasia in balloon-injured carotid artery, in association with an increase in the abundance of circulating EPC.63,103 Others have shown that statin therapy104,105 and oestrogen106 increases the number of circulating EPC and reduces neointima hyperplasia in animal models of arterial injury. Thus, the therapeutic potential of EPC could be harnessed by non-invasive pharmacological manipulation and used to accelerate the endogenous repair mechanisms for inhibition of neointimal hyperplasia and prevention of restenosis following revascularization procedures.

Gene- and cell-based therapies for myocardial protection and rescue
Gene-based strategies for protection of the myocardium at risk
Reperfusion after prolonged ischaemia can lead to myocardial injury. This process, known as ischaemia/reperfusion (I/R) damage, is characterized by increased reactive oxygen species (ROS) formation, endothelial dysfunction and inflammatory cell activation and infiltration.107 The increased ROS production during reperfusion of the ischaemic myocardium may eventually deplete the buffering capacity of endogenous antioxidant systems108 leading to increased myocardial oxidative stress. The development of gene therapies for acute myocardial infarction is untenable at present, because the time required for transcription and translation of therapeutic genes with the current generation of vectors exceeds the time window for successful intervention. For this reason, gene transfer of anticoagulant genes is not feasible as primary thrombolytic therapy for acute myocardial infarction. An alternative gene therapy approach for myocardial protection is to devise strategies that could "prevent" I/R injury by using a method that could confer long-term expression of cytoprotective genes in the myocardium. This novel concept of "pre-emptive" gene therapy would protect the heart from future I/R injury, thereby minimizing the need for acute intervention.8

Gene therapy for protection against oxidative stress-induced injury
Gene therapy aimed at increasing endogenous anti-oxidant reserves should be a useful protective strategy for the ischaemic myocardium, given the prominent role of oxidative stress in CAD and I/R injury.107 Such strategy could be used to increase the native protective response of the myocardium, and render it resistant to future I/R insults. We have evaluated the feasibility of anti-oxidant enzyme gene transfer as a long-term first line of defense against I/R-induced oxidative injury using a rAAV vector for intramyocardial delivery of heme oxygenase-1 (HO-1).8 HO-1 is an enzyme that catalyses the breakdown of heme into equimolar amounts of bilirubin, iron and carbon monoxide. The byproducts of heme catabolism exert wide ranging cytoprotective effects, indicating that HO-1 plays an essential role in the adaptation to cellular stress. Our findings show that HO-1 gene delivery to the left ventricular risk area several weeks in advance of myocardial infarction in a rat model of myocardial I/R injury results in approximately 80% reduction in infarct size. The reduction in myocardial injury in the treated animals is accompanied by decreases in oxidative stress, inflammation and interstitial fibrosis, leading to post-infarction functional recovery and normalization of left ventricular dimensions.8,109 We found comparable findings with extra cellular superoxide dismutase (ec-SOD) gene transfer.110 This secreted metalloenzyme plays an essential role in maintenance of redox homeostasis by dismutating the oxygen free radical superoxide. Our findings showed improved long-term survival after myocardial infarction in the ec-SOD-treated animals compared with the animals treated with the control vector, in parallel with smaller infarcts and decreased myocardial inflammation.110 Significant protection from I/R injury has also been achieved by over-expression of other major antioxidant enzyme systems, such as Cu/Zn SOD38 catalase,39 glutathione peroxidase111 and stress-induced heat shock proteins.112

Gene therapy for myocardial protection against apoptosis and inflammation
Marked myocardial protection from I/R injury has also been reported with over-expression of survival genes such as Bcl-2 and Akt,40–42as well as immunosuppressive cytokines,113 adenosine A1 and A3 receptors114 and hepatocyte growth factor.115 A potential strategy for acute protection from I/R injury may be the inhibition of pro-inflammatory genes activated by I/R. Morishita et al.10 showed that pretreatment with a decoy oligonucleotide capable of inhibiting the trans-activating activity of NF-{kappa}B reduces myocardial infarct size after coronary artery ligation in rats. Although the rapid in vivo degradation of oligonucleotides precludes their use in long-term myocardial protection, this strategy may be useful in treatment of acute myocardial ischaemia during cardiac transplantation. For example, treatment with antisense oligonucleotide directed against intercellular adhesion molecule-1 (ICAM-1) prolongs cardiac allograft tolerance and long-term survival when administered ex vivo prior to transplantation into the host.43 Therefore, such an approach could be useful in the preparation of donor hearts for transplantation (for review, see116).

Despite the compelling pre-clinical evidence of the therapeutic potential of long-term myocardial over-expression of antioxidant enzymes and cytoprotective genes as a strategy for myocardial protection, the suitability and efficacy of these experimental therapies in patients with coronary artery disease has not been assessed. Our findings suggest that pre-event delivery of cytoprotective genes with AAV vector may be useful as a "pre-emptive" strategy for sustained myocardial protection from future episodes of I/R injury in patients with or at high risk of developing coronary artery disease. However, a number of issues need to be resolved. First, the optimal method and timing for administering these genes in the clinical setting needs to be established. Second, there are concerns about the potential biodistribution and germ cell contamination by the vector, amid recent findings from our laboratory of ectopic expression of vector and transgene sequences in the testis, long after intramyocardial gene transfer.117 We anticipate that these unresolved issues will likely be areas of intense investigation in the next few years.

Rescue strategies for ischaemic myocardium
Gene therapies for rescuing the ischaemic myocardium may be attainable in certain cases. Therapeutic angiogenesis by delivery of genes coding for pro-angiogenic factors such as VEGF, FGF and hepatocyte growth factor (HGF) has been shown to promote neovascularization and functional recovery of ischaemic myocardium in several animal models and in humans with coronary artery disease9,45–50,118–122 (for review, see6,17). Recently, circulating endothelial progenitor cells and bone-marrow derived mononuclear cells have been reported to induce new vessel formation in the myocardium after direct injection123–129 or systemic infusion.92 The ability to expand and genetically modify these cells ex vivo may offer unprecedented opportunities to develop novel cell-based gene therapy strategies for efficient neovascularization of ischaemic myocardium in patients with inoperable coronary artery disease and peripheral artery disease.

Gene transfer to induce therapeutic angiogenesis
Therapeutic angiogenesis by exogenous supplementation of pro-angiogenic factors is emerging as a potential treatment option for patients for whom PCI or surgical revascularization has been excluded. Evidence of enhanced neovascularization and functional recovery has been demonstrated in several animal models of hind limb and myocardial ischaemia by gene transfer of pro-angiogenic growth factors28,47–50,92 (for review, 7). In all cases tissue perfusion was reported to be improved in association with histological and angiographic evidence of new vessel formation.

These pre-clinical studies led to several small scale non-randomized Phase I and II clinical trials with patients suffering from myocardial ischaemia45,46,118–121 (for review, see130). These trials, consisting of small non-randomized patient samples, have demonstrated the feasibility and safety of angiogenic gene therapy for treatment of ischaemic heart disease, but questions remain regarding the long-term sustainability of the therapeutic effect of angiogenic gene transfer. In a Phase I study in five male patients aged 53–71 years of age with coronary artery disease that did not respond to conventional anti-angina therapy, intramyocardial delivery of naked plasmid encoding VEGF165 into the ischaemic myocardium led to reduction of anginal symptoms and improvement, albeit modest, in left ventricular function concomitant with reduced ischaemia.118 Rosengart et al.45 reported significant improvement in regional ventricular function and wall motion in the region of vector administration after intramyocardial delivery of VEGF121 in patients undergoing conventional CABG compared with patients receiving placebo, whereas Vale et al.119 reported significant reductions in weekly anginal attacks for as long as 1 year after catheter-based delivery of naked VEGF-2 (VEGF-C) assisted by electromechanical NOGA mapping of the left ventricle in patients with chronic myocardial ischaemia. The recently published results of the Angiogenic GENe Therapy (AGENT) double-blinded, randomized, placebo-controlled trial using dose-escalating adenovirus-mediated intracoronary delivery of FGF-4 in 79 patients (mean age 65 years) with angina showed a general trend towards an increase in exercise tolerance and improved stress echocardiograms at 4 and 12 weeks after gene transfer in the patients treated with FGF-4 gene therapy compared with the patients receiving placebo, in association with angiographic evidence of neovascularization.131 However, the trial was not sufficiently powered to detect statistically significant differences between the treated and placebo groups in the treadmill exercise time to fatigue. The outcome beyond 12 weeks has not been reported for these patients. More recently the AGENT 2 trial has been completed.132 The trial assessed the effect of intracoronary Ad5FGF-4 gene transfer on myocardial perfusion in 52 patients (mean age 58 years) with stable angina 8 weeks after gene transfer, using stress-related reversible perfusion defect size (RPDS) as the primary endpoint. The results of this trial showed a decrease in RPDS and improved perfusion in the treated group compared with the placebo group, but the difference did not reach statistical significance, presumably due to the confounding effect of an outlier in the placebo group. In addition, no significant differences between the two groups were found in angina parameters (Canadian Cardiovascular Society angina class). However, a greater number of patients in the Ad5FGF-4 treated group reported complete resolution of anginal symptoms and no nitroglycerin use.

Despite the evidence from these small-scale trials supporting the feasibility and safety of angiogenic gene transfer, we believe that there are several outstanding issues relating to the safety of the approach. It was reported that constitutive over-expression of VEGF in mouse heart led to intramural angiomas followed by heart failure and death133 and that VEGF may accelerate plaque progression in atherosclerotic vessels.134 Furthermore, constitutive over-expression of VEGF may result in sustained increase in plasma level of this cytokine that may induce angiogenesis of occult neoplasms. These concerns underscore the need for regulated expression of pro-angiogenic factors as a measure to enhance the safety of this therapeutic strategy. Another approach to achieve regulated therapeutic angiogenesis is to employ engineered transcription factors capable of activating endogenous VEGF expression in response to pathophysiological cues.135 These novel strategies may allow endogenous regulation of angiogenesis so that the extent of neovascularization matches the severity of the ischaemic insult. It is also necessary to determine the safest and most efficacious route and method for therapeutic gene delivery in order to avert potentially hazardous late-onset side effects, such as neovascularization of occult neoplasms or peripheral vascular effects that may result in oedema and hypotension. This may require incorporation of cell-specific promoters for targeted expression of the transgene. Regarding the therapeutic sustainability of angiogenic gene transfer, it is necessary to establish whether the desired long-term therapeutic effect can be achieved with a single administration of the therapeutic gene, or whether multiple treatments may be required. This is an important issue because VEGF-induced neovessels tend to regress soon after termination of transgene expression in the absence of adequate blood supply.7

Cell-based therapy to induce angiogenesis
An alternative strategy for neovascularization of the ischaemic heart involves the use of EPC as angiogenic substrate. The cells are isolated from the mononuclear cell fraction of peripheral blood or bone marrow by density centrifugation. The mononuclear fraction is then injected whole, submitted to further selection, or cultured and expanded ex vivo under selective growth conditions to obtain sufficient number of cells for the therapeutic application. The cells are then used without any further manipulation, or they may be genetically modified with vectors expressing therapeutic genes and then delivered to the target area, where they may engraft and promote new vessel growth.35,136

Myocardial transplantation of autologous CD31+ cells isolated from peripheral blood induced new vessel formation and improved left ventricular perfusion and performance in pig hearts rendered ischaemic by placement of an ameroid constrictor in the circumflex coronary artery.32 Similarly, whole or CD34+-selected human mononuclear cells from peripheral blood induced revascularization and repair of infarcted myocardium when injected immediately after acute myocardial infarction in nude rats,123 leading to reduction in interstitial fibrosis and improvement in ventricular function. Evidence of neovascularization has also been seen with bone marrow-derived mononuclear cells (BM-MNC). Kocher et al.92 reported that intravenous delivery of human CD34+ BM-MNC to nude rats with myocardial infarction led to significant neovascularization of the infarcted myocardium, resulting in reduced apoptosis of myocytes in the peri-infarct area, decreased fibrosis and sustained recovery of left ventricular function. Others have reported that implantation of bone marrow-derived Lin–ckit+55 in the infarct border resulted in improved left ventricular function in association with new vessel formation.

EPC may also be employed as vectors for delivery of pro-angiogenic factors. Presumably, the transplanted genetically modified EPC could contribute to new vessel growth by proliferating and differentiating at the site of implantation, and by secreting pro-angiogenic growth factors. This concept was recently validated by Iwaguro and colleagues.136 Using athymic mice with hind limb ischaemia, this group showed that the transplantation of murine EPC transduced ex vivo with an adenoviral vector expressing VEGF resulted in more efficient neovascularization and blood flow recovery than treatment with untransduced EPC.

Another potential strategy for angiogenesis of ischaemic myocardium in coronary artery disease involves the mobilization of EPC to the ischaemic region using cytokines or conventional pharmacological therapeutic agents used in treatment of coronary artery disease, such as statins. Orlic et al.137 showed that mobilization of bone-marrow by G-CSF and stem cell factor-1 (SCF-1) led to decreased post-infarction mortality and functional recovery in mice with myocardial infarction in association with significant regeneration and angiogenesis of the infarcted myocardium. In athymic nude mice with hind limb ischaemia, local injection of stromal cell-drived factor-1 (SDF-1) stimulated homing of systemically delivered human peripheral blood-derived mononuclear cells (PB-MNC) to the ischaemic muscle and induced vasculogenesis.138 Other groups showed that statin therapy increases the number of circulating EPC both in experimental models and in patients with stable coronary artery disease,139–141 suggesting that the beneficial therapeutic effect of these drugs may be mediated, at least in part, via mobilization of EPC and subsequent neovascularization of ischaemic myocardium.

These pre-clinical studies has led to several recent small-scale feasibility and safety studies to evaluate the use of bone marrow cell transplantation in treatment of ischaemic heart disease and myocardial infarction.142–146 In a recent small scale Phase I clinical trial, Stamm and colleagues142 injected autologous AC133+ bone marrow cells into the infarct border during CABG in six patients that had suffered earlier acute transmural myocardial infarction. The authors reported improved perfusion of the infarcted area and significant enhancement of global left ventricular function 3–9 months after surgery. The transplantation protocol appeared to be safe and did not cause adverse cardiac effects. Strauer et al.143 reported that intracoronary delivery of unfractionated autologous mononuclear bone marrow cells 6 days after infarction led to a reduction in infarct size and improvement in ventricular function and chamber geometry 10 weeks after transplantation. Intracoronary infusion of either BM-MNC or PB-MNC 4 days after infarction to a randomized group of 20 patients with reperfused acute myocardial infarction, led equally to significant improvements in global left ventricle ejection fraction and wall motion in the infarct zone and reduced end systolic dimensions at 4 months follow-up, in association with increases in coronary flow reserve in the infarct artery and in myocardial viability.144 No adverse effects were reported to be associated with the transplantation protocol, and the authors suggested that autologous progenitor cell may be a feasible strategy for preventing post-infarction ventricular remodeling and failure. Two other groups have reported recently that transendocardial delivery of autologous BM-MNC using NOGA mapping led to significant improvements in left ventricular perfusion and performance, and reduced incidence of ischaemic episodes in patients with end-stage ischaemic heart disease145or stable angina,146 suggesting that BM-MNC transplantation may be useful as a strategy to improve myocardial function in patients with severe ischaemic heart disease.

Cell-based therapies for myocardial repair and regeneration
The vast majority of adult cardiac myocytes are terminally differentiated and unable to divide.147 When the infarct size is too big it may lead to maladaptive remodeling of the left ventricle and heart failure.148 Cell transplantation (cellular cardiomyoplasty) may offer a potential alternative for repair of infarcted myocardium (for review, see15,149). In addition to EPC, several other cell sources, such as skeletal myoblasts, embryonic and fetal cardiomyoctes and bone-marrow derived mesenchymal (stromal) cells have been used to repair infarcted myocardium in experimental models (for review, see150). However, the efficacy of this strategy has been inconsistent and more experiments need to be done in order to better understand the real potentiality of cardiomyoplasty.

Myocardial repair by bone marrow-derived progenitors
Autologous mesenchymal cells from the bone marrow stroma of long bones (MSC) may be ideally suited for repair of infarcted myocardium. These cells exhibit a high degree of plasticity.151 It has been reported that MSC could differentiate into synchronously beating cardiomyocytes in vitro after treatment of primary cultures of mouse bone marrow with the cytosine analog 5-azacytidine.56,152 Toma and colleagues153 showed that human MSC transplanted into the left ventricular wall of immunodeficient mice differentiate into cardiac myocytes without the need for myogenic differentiation prior to transplantation. Tomita et al.56 reported that transplantation of 5-azacytidine-treated bone marrow cells repopulate the scar and significantly improve left ventricular function in cryoinjured rat hearts. Wang et al.154 detected several cell types, including cardiomyocytes, endothelial cells, and fibroblasts within and on the border of the scar 1 month after intracoronary delivery of retrovirally transduced (reporter gene LacZ) bone marrow cells to infarcted rat hearts, suggesting that the microenvironment present around the injured myocardium may induce transdifferentiation of bone marrow progenitors into the various cell types required for regeneration and maintenance of the myocardium.

Myocardial repair by bona fide cardiac progenitor cells
In addition to the marrow-derived precursor cells, clusters of highly proliferating primitive cells have been detected in the infarcted myocardium by two independent groups.155,156 In a recently published study, Beltrami and colleagues155 reported the identification and isolation of a population of undifferentiated lineage negative (Lin–) cells that express stem cell markers such as the stem cell factor receptor (c-kit+), and the stem cell antigen 1 (Sca-1+). These cells were found to be clonogenic and self-renewing and capable of differentiating into all myocardial cell types, including cardiomyocytes, endothelial cells and vascular smooth muscle cells. Within the clusters, the progenitor cells were found to be at different stages of cardiomyogenic differentiation, reflecting their cardiogenic potential. This was further supported by the ability of early passage cells to induce significant myocardial regeneration and improve ventricular performance after transplantation into infarcted hearts from syngeneic rats. Recently, the same group reported a dramatic 13-fold increase in the abundance of these resident stem cells in the myocardium of patients suffering hypertrophic cardiomyopathy secondary to aortic stenosis.157 This observation raises the intriguing hypothesis that the compensatory cardiac wall thickening in these patients may be due not only to the well-described hypertrophy of the cardiomyocytes but also to hyperplastic growth of these proliferating cells.

Outstanding issues with cell-based therapy for myocardial and repair
Although the recent pre-clinical and clinical studies using cell transplantation for myocardial repair and regeneration have yielded promising results, we consider these findings preliminary until the time when the therapeutic values of these strategies are more comprehensively assessed. The nature of the mobilizing, migration and homing signals for bone marrow progenitor cells and the mechanism of differentiation and incorporation into the target tissues need to be more fully investigated. Controlled trials are necessary in order to define and standardize the optimal time and method of delivery, and the subpopulation and number of cells required to achieve a sustained therapeutic effect. Strategies to improve survival and engraftment of the transplanted cells may be necessary to optimize the positive effects of cardiomyoplasty. Finally, the morphological and functional complexity of the myocardium could raise the possibility that the optimal protocol for myocardial repair and regeneration may require more than one cell type.

Perspectives and future directions
The last decade has witnessed the development of several gene- and cell-based strategies with potential therapeutic value for treatment of cardiovascular disease. Some of these strategies have made the transition from the pre-clinical phase into early clinical trials and their feasibility and safety has been established in some cases. In the gene therapy arena, there is urgent need for further developments in vector and delivery technologies with improved safety and efficacy profiles. The future will likely see increased demand for the use of regulatable vector systems with the capability to confer tissue-specific expression of therapeutic transgenes.

The outlook for cell-based regenerative therapies for vascular and myocardial disease is promising. However, the issues of the timing of administration, the appropriate clinical condition (acute myocardial infarction versus heart failure), the optimal cell number/composition and, importantly, the safety of stem/progenitor cell transplantation must be determined. There is a pressing need to define and standardize the protocols for isolation and therapeutic application of cells to use in cell-based therapies. Large scale randomized trials are necessary in order to evaluate the long-term safety and therapeutic efficacy of cell-based therapies amid potential concerns about delayed onset complications such as visualization of occult tumours, development of age or diabetic-induced retinopathies, or the appearance of ectopic foci in the myocardium that could lead to life-threatening arrhythmias.

Acknowledgments

Dr. Melo is Canada Research Chair in Molecular Cardiology and a New Investigator of the Heart and Stroke Foundation of Canada, and is supported by grants from the Canadian Institutes of Health Research, Heart and Stroke Foundation of Saskatchewan, Canadian Foundation of Innovation and the Ontario Innovation Fund. Dr. Dzau is supported by grants from the National Institutes of Health #HL 35610, HL 058316, HL 072010 and HL 073219. Dr. Pachori is the recipient of a NRSA postdoctoral scholarship from the National Institutes of Health. Dr. Gnecchi is the recipient of a research award from the University of Pavia and IRCCS Policlinico San Matteo, Pavia, Italy.

Footnotes

1 Dr. Dzau has stock ownership in Corgentech Inc. Back

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