Gene and Cell Therapy for Heart Disease - Wiley Online Library

Life, 54: 59–66, 2002 c 2002 IUBMB Copyright ° 1521-6543/02 $12.00 + .00 DOI: 10.1080/15216540290114234

IUBMB

Review Article Gene and Cell Therapy for Heart Disease Regina M. Graham, Nanette H. Bishopric, and Keith A. Webster Department of Molecular and Cellular Pharmacology, University of Miami Medical Center, Miami, Florida, 33136, USA

Summary Heart disease is the most common cause of morbidity and mortality in Western society and the incidence is projected to increase significantly over the next few decades as our population ages. Heart failure occurs when the heart is unable to pump blood at a rate to commensurate with tissue metabolic requirements and represents the end stage of a variety of pathological conditions. Causes of heart failure include ischemia, hypertension, coronary artery disease, and idiopathic dilated cardiomyopathy. Hypertension and ischemia both cause infarction with loss of function and a consequent contractile deficit that promotes ventricular remodeling. Remodeling results in dramatic alterations in the size, shape, and composition of the walls and chambers of the heart and can have both positive and negative effects on function. In 30–40% of patients with heart failure, left ventricular systolic function is relatively unaffected while diastolic dysfunction predominates. Recent progress in our understanding of the molecular and cellular bases of heart disease has provided new therapeutic targets and led to novel approaches including the delivery of proteins, genes, and cells to replace defective or deficient components and restore function to the diseased heart. This review focuses on three such strategies that are currently under development: (a) gene transfer to modulate contractility, (b) therapeutic angiogenesis for the treatment of ischemia, and (c) embryonic and adult stem cell transfer to replace damaged myocardium. IUBMB Life, 54: 59–66, 2002 Keywords

Angiogenesis; gene therapy; hypertrophy; ischemia; SERCA2; stem cells; VEGF.

FAILURE OF CONTRACTION AND β-ADRENERGIC RESPONSES Abnormal myocardial function affecting both relaxation (diastole) and contraction (systole) is central to the pathogenesis

Received 12 December 2001; accepted 2 July 2002. Address correspondence to Keith A. Webster, Department of Molecular and Cellular Pharmacology, University of Miami Medical Center, 1600 NW 10th Avenue, RMSB 6038, Miami, Florida 33136, USA. Fax: 305-243-6082. E-mail: [email protected]

of all forms of heart disease. Impairment of left ventricular diastolic function often occurs before systolic dysfunction particularly in dilated cardiomyopathy and ischemic heart disease (1) and there is debate over which elements of the contractile system should be targeted. At the molecular level the cause of defective muscle contractility can be broadly attributed to abnormal sarcoplasmic reticulum (SR) calcium handling and alterations in myofilament calcium sensitivity, both of which are under β-adrenergic control. In this section of this review we focus on the evidence that implicates abnormal intracellular calcium handling and altered β-adrenergic receptor signaling in heart failure and the molecular approaches that are being pursued to correct them.

Molecular Regulation of Muscle Contraction Muscle contraction and relaxation occurs through a series of coordinated steps. Electrical excitation across the sarcolemma leads to the opening of voltage-gated (L-type) Ca2+ channels. Entry of a small amount of Ca2+ into the cell triggers the release of larger stores of Ca2+ from the sarcoplasmic reticulum (SR) through the Ca2+ release channels known as ryanodine receptors (RyR). The rise in the intracellular Ca2+ concentration ([Ca2+ ]i) initiates contraction by binding to the myofilament protein troponin C. Conformational changes within the troponin complex disrupt the high-affinity binding sites on tropomyosin, resulting in acto-myosin cross-bridge formation and muscle contraction. Maximum force-generating capacity of the sarcomere is proportional to the number of crossbridges formed, which in turn is determined by the [Ca2+ ]i and the affinity of troponin C for Ca2+ . Once maximum tension is attained, diastole begins; Ca2+ is released from the myofilaments and cross-bridges are detached. Ca2+ is removed from the cytosol primarily through two active processes, the SR Ca2+ ATPase pump (the SERCA2a isoform in cardiac muscle) and the Na+ -Ca2+ exchanger (NCX). The force of contraction and the efficiency of relaxation are regulated by the phosphorylation state of proteins such as phospholamban (PLB), which modulate the activity of the calcium handling proteins (SERCA) (Fig. 1). Alterations in the activity and/or 59

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the β-adrenergic signaling pathway may be prime targets for therapeutic intervention.

Figure 1. Excitation-contraction and β-AR regulation. Ca2+ entry through the voltage-activated Ca2+ channel (L-type Ca2+ ) causes a local increase in [Ca2+ ]i, inducing an even greater release of Ca2+ from the SR via RyRs. As the [Ca2+ ]i increases, Ca2+ to binds myofilaments, and contraction results. Relaxation occurs when the [Ca2+ ]i is removed from the cytosol primarily by the SR Ca2+ ATPase (SERCA2). β-ARs are Gprotein-coupled receptors and agonist stimulation (A) activates the classic Gαs -AC-cAMP-PKA signaling cascade. The phosphorylation (P) of PLB by PKA enhances myocardial relaxation by relieving PLB’s inhibitory effect on SERCA, thus promoting Ca2+ sequestration by the sarcoplasmic reticulum (SR). βARK phosphorylation of β-AR results in receptor desensitization and reduced β-AR signaling responses. relative quantity of these proteins have been implicated in the pathogenesis of heart failure and are thus potential gene therapy targets.

Targeting PLB and SERCA2a The prominent roles of SERCA2a and PLB in the regulation of myocardial contractility have been confirmed in genetically engineered mice as well as in gene transfer studies. Although PLB gene knockout mice demonstrate enhanced contractile parameters, augmenting PLB levels (which reduces the SERCA2a:PLB ratio) in intact hearts produces contractile changes similar to those seen in heart failure (2, 3). SERCA2a levels have been reported to be decreased or unchanged in endstage heart failure (4); however, depressed activity of the SR Ca2+ ATPase is widely acknowledged to be an integral feature of heart failure. SERCA2a transgenic mice demonstrate increased contractility and faster relaxation rates (5). The activity of SERCA is negatively regulated by PLB (3). Phosphorylation of PLB relieves this inhibition thereby stimulating ATP-dependent sequestration of Ca2+ by the SR during relaxation. PLB is under tight regulation by the β-adrenergic pathway. The β-adrenergic receptor (β-AR) stimulation activates the stimulatory G protein alpha (Gαs )-adenylyl cyclase (AC)cAMP-protein kinase A (PKA) cascade, leading to the phosphorylation of PLB and other proteins involved in cardiac excitationcontraction coupling (Fig. 1). As a consequence, components of

Gene Transfer Approaches to Augment β -Adrenergic Responsiveness Depressed responsiveness and downregulation of the β-adrenergic pathway is an established feature of heart failure. Possible causes include increased receptor degradation, decreased receptor synthesis, decreased activity of adenylyl cyclase, enhanced turnover of Gs , and increased expression of inhibitory G-protein alpha (Gi ) (reviewed in (6, 7). There is evidence that each of these pathways contributes to loss of β-adrenergic responsiveness in failing hearts (4, 6). An important question relating to treatment of heart disease is whether this downregulation is adaptive, or whether it is part of the etiology of heart failure and contributes to deterioration. Gene transfer experiments examining the effects of constitutively overexpressing individual components of the β-adrenergic signaling pathway in rodent and rabbit models have suggested a number of novel therapeutic targets. Overexpression of β2 -Adrenergic Receptor. The effects of β2 -adrenergic receptor (β2 -AR) overexpression were analyzed in transgenic mice (8) and by adenoviral-mediated gene transfer into rabbit cardiac myocytes in vitro (9) and in vivo (10). Transgenic mice had increased basal myocardial adenylyl cyclase activity, enhanced atrial contractility, and increased left ventricular function in vivo. No change in heart physiology in terms of hypertrophy or dilation was apparent at 2 months, and it was concluded from these studies that gene therapy aimed at enhancing the level of β2 -AR might be a viable approach to improve myocardial function in the failing heart. Additionally, adenovirus-mediated delivery of β2 -AR was shown to restore agonist-stimulated PKA activity and increase the inotropic responses of cardiac myocytes isolated from failing rabbit hearts (9). In vivo delivery of β2 -AR DNA to rabbit hearts produced 5to 10-fold increases in the levels of β-AR expression, increasing basal and isoproterenol-stimulated cAMP production and contractile performance (8). These results indicate that at least short-term benefits may be achieved by β2 -AR gene delivery to diseased hearts. Overexpression of βARK and βARK Inhibitor. The effects of overexpressing βARK1 or the βARK1 inhibitor (βARKct) in the heart were analyzed in transgenic mice (11) and after adenoviral-mediated gene delivery in vitro (12). βARK1 is a member of the G-protein coupled receptor kinase (GRK) family that is selective for β1 - and β2 -adrenergic receptors; phosphorylation by βARK1 desensitizes the receptors and targets them for degradation. The phenotype of the βARK1 overexpressing mouse included blunted inotropic and chronotropic responses to β-adrenergic agonists, consistent with desensitization of both receptors. These results demonstrated that excessive levels of βARK could contribute to the diminished β-adrenergic responsiveness of failing hearts. The opposite was observed with the βARK inhibitor-overexpressing mice. These mice displayed

GENE AND CELL THERAPY FOR HEART DISEASE

enhanced systolic and diastolic function, quantitatively similar to that reported for β2 -adrenoreceptor-overexpressing mice. No abnormal cardiac physiology was reported for these transgenic mice at 2 to 4 months of age and the authors of the studies suggested that the prevention of β-adrenoreceptor desensitization through βARK inhibition could improve function of the failing heart. Similarly, adenoviral-mediated gene transfer of the βARK1 inhibitor potentiated β-adrenergic signaling in rabbit ventricular myocytes and significantly inhibited agonist induced desensitization (12). Overexpression of Gs . Excitement about the possibility of targeting β-adrenergic receptors or βARKs to enhance β-adrenergic responsiveness in the myocardium has recently been tempered by evidence that such interventions may actually accelerate the progression of heart failure. This would be consistent with previous clinical studies showing that chronic treatment with β-adrenergic agonists or phosphodiesterase inhibitors results in higher mortality rates (13). Transgenic mice overexpressing Gs α develop features of classical hypertrophic and dilated cardiomyopathy and show a significantly increased age-related mortality (14). Heart failure occurred in these mice and was attributed to the absence of β-adrenergic desensitization in the face of chronically enhanced catecholamine stimulation, a classical feature of late stage heart failure. Similar results were reported for transgenic mice overexpressing the β1 -adrenergic receptor (β1 -AR) (15). These results suggest the transfer of these genes to augment β-AR responsiveness may in fact promote the progression of heart failure especially at later stages and is not an appropriate therapeutic target. Overexpression of Adenylyl Cyclase. In an approach aimed at augmenting β-adrenergic responsiveness “safely,” transgenic mice overexpressing type IV adenylyl cyclase (AC) were analyzed (16). The rationale for these experiments was that overexpression of AC, unlike that of β-adrenergic receptors or Gs , will mediate an increased inotropic response only in the stimulated state and will not cause continuous activation. These mice had improved contraction parameters and increased isoproterenol stimulated chronotropy. No abnormalities in myocardial physiology were reported at 19 months. The authors concluded that AC is a potential target for safely increasing cardiac responsiveness to β-adrenergic stimulation. However the authors did not address the potential adverse effects of overactivity in response to catecholamine surges.

Summary A controversy still exists over whether desensitization of βadrenergic responses in the failing heart is adaptive or maladaptive. It seems probable that either can be true depending on the circumstances, and that there will be some benefit from conditionally and/or partially resensitizing the system. The demonstrated clinical benefits of β-adrenergic blockers that act by partially protecting against receptor desensitization (17) as well as the results of transgenic mice overexpressing βARK inhibitor (11) support this possibility. β1 -AR and β2 -AR transgenic mice

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demonstrate distinct phenotypes, indicating that these receptor subtypes mediate, at least in part, different signal transduction pathways and point to β2 -AR as a therapeutic target. However, as with pharmacologic intervention, determining the correct “dosage” is important. Transgenic mice with high levels of β2 -AR expression (100–350-fold) developed varying degrees of fibrotic cardiomyopathy in a dose-dependent manner, yet mice with 60-fold β2 -AR expression continued to demonstrate enhanced contractility and normal pathology for ≥1 year (longest time point examined) (18). Direct constitutive enhancement of specific components of β-adrenergic signaling, including adenylyl cyclase seems problematic. Abnormal calcium handling is an overriding feature of heart failure and any stimulus that activates force development aspects of the β-adrenergic pathway is likely to worsen this problem. Because of such complications inherent in the enhancing β-adrenergic system in failing hearts, diastole may be the preferred therapeutic target. Targeting PLB, or SERCA2a, may improve ventricular function by removing calcium more efficiently from around the contractile elements, significantly improving relaxation and filling. In support of this approach, adenoviral gene transfer of SRCA2a or antisense of PLB was shown to improve contractility in human failing myocytes (19). Several reviews have been written discussing gene delivery techniques (20) and it is not the focus of this survey. However, it should be noted that although adenoviral vectors can infect both dividing and non-dividing cells with high efficiency, gene expression is transient and an inflammatory response is possible.

THERAPEUTIC ANGIOGENESIS FOR THE TREATMENT OF ISCHEMIA Coronary artery disease (CAD) is the most common cause of heart disease, leading to more than 0.5 million deaths in the US annually (21). Patients with severe CAD may experience more than 10 ischemic episodes per day, each with ECG ST-segment depression more than 2 mm and is often nonsymptomatic (22). The condition is progressive as plaque continues to build up in the arteries, reducing blood flow and oxygen delivery, and increasing the risk of a major coronary event. Cardiac tissue is progressively lost through necrosis and apoptosis (23, 24 ), hypertrophy increases, and congestive heart failure is the most probable outcome. Although there have been significant advances in the prevention and treatment of CAD in recent years, a large number of patients suffer from disabling symptoms and the prognosis for many is poor.

Therapeutic Angiogenesis Therapeutic angiogenesis involves the transfer of growth factors to ischemic tissues to promote development of collateral blood vessels and resupply the circulation (for reviews see (25, 26). It is an obvious approach for the treatment of ischemic diseases and has been gaining momentum not only to treat the previously untreatable conditions such as peripheral arterial

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Table 1 Gene therapy trials∗ Investigator

Disease

Gene(s)

Number of trials

Phase

Annex, B. H. Chronos, N. A. F. Comerota, A. J. Crystal, R. G. Crystal, R. G. Dreiling, R. J. Grines, C. L. Hinohora, T. Iskandrian, A. E. Isner, J. M. Isner, J. M. Isner, J. M. Isner, J. M. Kornowski, R. Kuntz, R. Lee, J. S. McCarthy, P. Rajagopalan, S. Riddell, J. Rosengart, T. K. Rosengart, T. K. Sanborn, T. A. Steed, D. Stewart, D. J. Yla-Herttuala, S. Yla-Herttuala, S.

PAOD PAOD PAOD CAD PAOD CAD CAD PAOD CAD CAD CAD PAOD PAOD CAD CAD CAD CAD PAOD PAOD CAD CAD CAD PAOD CAD CAD PAOD

VEGF + VEGF2-CAD-CL-009 (plasmid) FGF (plasmid) FGF (plasmid) VEGF (Ad) VEGF (Ad) Del-1 (cDNA-lipofection) FGF (Ad) Del-1 (cDNA-lipofection) FGF (Ad) VEGF-2 (plasmid) VEGF + VEGF2-CAD-CL-005 (plasmid) HIF-1alpha/Vp16 (Ad) VEGF-2 (plasmid) VEGF (Ad) iNOS (cDNA-lipofection) FGF-4 (Ad) VEGF (plasmid) VEGF (Ad) FGF-4 (Ad) HIF-1alpha/Vp16 (Ad) VEGF (Ad) VEGF (Ad) PDGF (Ad) VEGF (Ad) VEGF (Ad) LacZ + VEGF (Ad)

1 1 1 5 2 1 1 1 1 5 1 3 6 1 1 1 1 1 2 1 1 1 1 1 1 1

I/II I I I/II I I II/III I II I/II II I I/II II I I/II I/II II I I I I I II I/II I/II

∗

Adapted partly from Gene Therapy Clinical Trials/Journal of Gene Therapy hhttp://213.80.3.170:80/trials/index.htmli.

occlusive disease (PAOD), but also as a general strategy for multiple ischemic conditions including CAD. Therapeutic angiogenesis combined with pharmacotherapy may eventually eliminate the need for angioplasty and bypass surgery for treating CAD and PAOD (25). Results from studies in animal models of PAOD and CAD, initiated in the early 1990s, have been very encouraging, to the extent that numerous clinical trials are currently in progress testing vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF) proteins and genes (Table 1). In this section we will discuss the stages in the development of angiogenic gene therapy, its current status, and future prospects.

Animal Studies The first experiments on therapeutic angiogenesis involved delivery of recombinant vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF) proteins to ischemic limbs and myocardium of rabbits, dogs, and rats (15, 27–29). Ischemia was induced in canine hearts by gradually occluding the left circumflex coronary artery (LCx) using an ameroid constrictor, and dogs were instrumented with catheters for drug

delivery to the ischemic zone. VEGF treatments (45 µg protein per day) were initiated 10 days after LCx constriction and blood flow measurements were performed weekly. At the end of the experiment animals were sacrificed and capillaries and vessels in the ischemic tissues were quantitated. The results of these experiments were encouraging; chronic treatment with VEGF caused a 40% increase in collateral blood flow and an 89% increase in the numerical density of intramyocardial distribution vessels at 4 weeks. Similar results were reported for FGF administration to heart and skeletal muscles of mice, rats, and dogs (30–33). Neoangiogenesis in response to FGF-1 was reported to occur in both ischemic and non-ischemic tissues. These experiments established the “therapeutic angiogenic” benefit of VEGF and FGF-1 delivered as pure proteins in animal models. The next stage in development of the technology was to transfer genes that encode angiogenic growth factors rather than recombinant proteins, which are costly and unstable. In the first of such studies, ischemic hindlimb models were used to test the effects of plasmids encoding VEGF injected directly into the muscle (34 ). The results of these experiments were consistent with the results of VEGF protein delivery although the effects


were less dramatic because of the relatively low efficiency of plasmid transfer to tissues. Further studies confirmed that angiogenesis and neovascularization could be induced in ischemic heart and skeletal muscles of rabbits, rats, dogs, and pigs by the direct injection of VEGF- or FGF-encoding DNA in plasmid or adenoviral (Ad) vectors. Direct injection of Ad-FGF-5 into the ischemic canine myocardium mediated 300% improvement in wall thickening of the ischemic region and improved blood flow at 2 weeks and 12 weeks after treatment (33). Similar results were reported using plasmid encoded VEGF in pigs (35). In further studies the genes for angiopoietin and hepatocyte growth factor (HGF) were shown to be as effective as VEGF and FGF in promoting angiogenesis in both PAOD and CAD animal models (36, 37).

Clinical Studies Results from the animal models provided a unified and compelling case for therapeutic angiogenesis whether the angiogenic factor is delivered as a recombinant protein or by means of a plasmid or adenoviral vector. This promise seems to be translating into similar positive results in clinical trials including some phase III studies (Table 1). Preliminary results from patient studies using VEGF and FGF-1 to treat myocardial ischemia have been encouraging. The first of these was a randomized controlled trial in which recombinant FGF-1 protein was injected into the myocardium of 20 patients undergoing two or three venous bypass grafts (38). Transfemoral intra-arterial digital subtraction angiography 12 weeks after surgery showed pronounced accumulation of contrast medium at the site of injection extending peripherally around the LAD. A capillary network could also be visualized sprouting out from the coronary artery into the myocardium. Similar positive results were obtained when VEGF plasmid was injected directly into the ischemic left ventricles of 5 male patients with angina and CAD (39). At 60 days follow-up, left ventricle ejection fractions and angiograms were improved and all patients reported significant reduction in angina. Multiple trials testing VEGF and FGF in CAD patients are currently ongoing (Table 1); the available results from gene delivery protocols are mixed but generally positive. A possible limitation to the clinical trials currently in progress is the use of plasmid and adenoviral vectors that may be less than optimal (20). Transfer of plasmid DNA generally results in low levels of gene expression although ischemic muscle is more permissive to transduction. Adenoviral vectors as discussed previously can elicit an immune reaction and limit further gene transfer into the area of interest due to the presence of neutralizing antibodies. On the other hand, gene expression with AAVs tends to be protracted and widely distributed, with little or no immune response (20). Recent animal studies have demonstrated positive results of VEGF delivered to the heart using the more stable AAV vector (40). Combined with techniques to regulate the expression of the angiogenic gene (41), it seems possible that this method of gene transfer will become the method of choice.

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Summary Animal data, as well as clinical results, support the feasibility of therapeutic angiogenesis. We propose that this will become a major procedure for the treatment of CAD and PAOD when the gene delivery techniques are optimized, and when safety issues have been addressed (26). These latter issues may be resolved by using a permanent gene delivery strategy such as AAV in which the pro-angiogenic transgene is directly responsive to the disease. Such a system involving conditional silencing and hypoxia-regulated gene expression has been described by the authors and is currently in the final stage of preclinical testing (42). Successful implementation of therapeutic angiogenesis will set the stage for the introduction of a variety of additional genes that may protect the heart and vasculature from disease. Candidate genes include nitric oxide synthetase to augment vasodilation, caspase inhibitors to prevent apoptosis, and insulin-like growth factor to protect against ischemic damage (43, 44). STEM CELL REPLACEMENT TECHNIQUES Loss of cardiac tissue and contractile function is a central feature of hypertension and ischemic heart disease. The irreversible loss of cardiac myocytes during myocardial infarction (MI) promotes hypertrophy, ventricular remodeling, and ultimately heart failure. The potential for repair following MI is limited by the low regeneration capacity of cardiac muscle (45 ) and by inadequate vascularization of the remaining viable tissue. The only effective therapeutic intervention for end-stage heart disease is transplantation. Given the paucity of donor hearts and the increasing incidence of heart disease, alternate methods of treatment are highly desirable. One such strategy may be to replace lost myocytes in the diseased myocardium with stem cells. The final section of this review focuses on recent advances in this area.

Cell-Mediated Therapy Recent demonstrations that skeletal myoblasts and neonatal cardiac myocytes can be introduced into the myocardium to potentially form viable grafts have lent support to the feasibility of cell-based therapeutic approaches (reviewed in 46, 47). Although skeletal muscle cell engraftment attenuated progressive heart dysfunction in MI models, the lack of electromechanical coupling to cardiac myocytes makes this cell type problematic. Engrafted fetal cardiomyocytes, on the other hand, are able to form intercalated discs and gap junctions with host cells, and have been shown to improve hemodynamics and left ventricle function. Given that fetal cardiomyocytes may not be available in sufficient numbers if at all for cell replacement therapy, an alternate source of muscle cells is highly desirable. Embryonic Stem Cells Embryonic stem (ES) and embryonic germ (EG) cells are pluripotential and may provide essentially limitless sources of compatible cardiac tissue (48). ES cells are derived from the

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inner cell mass of the developing blastocyst that differentiates into all three embryonic germ layers, the mesoderm, endoderm, and ectoderm. EG cells are derived from primordial germ cells formed from the migration of epiblast cells following implantation and gastrulation of the embryo. When injected back into a recipient blastocyst, both ES and EG cells contribute to all cell lineages of the chimeric embryo including the germ line. Under the appropriate culture conditions in vitro these cells can proliferate indefinitely while retaining their pleuripotential character (48). Pure populations of cardiac myocytes were derived from mouse ES cells by transfecting the aminoglycoside phosphotransferase cDNA driven by the cardiac specific α-myosin heavy chain promoter and selecting cells with G418. These ES-derived cardiomyocytes were injected into the hearts of adult dystrophic mice and formed stable grafts for up to 7 weeks; however, functional improvements were not demonstrated in this model (49). Cardiac myocytes have also been derived from human embryonic stem cells (50), confirming ES cells as a feasible source of human tissue. Limitations of ES cells include their potential to produce teratomas and immune rejection of grafted cells by the host. It may be possible however to genetically engineer ES cells with host MHC antigens or derive compatible cells by nuclear transfer to generate ES cells that are identical to the transplant recipient.

Adult Stem Cells Like ES cells, adult stem cells are self-renewing and pluripotential. These cells have been identified in most tissues including bone marrow, brain, skin, liver, and pancreas, but not yet in the heart (48). Recent studies have demonstrated the plasticity of adult stem cells including the capacity to contribute to all 3 germ layers in chimeric chick and mouse embryos (51). Exciting results obtained with bone marrow-derived stem cells in animal models support their potential to regenerate multiple cell types in the myocardium and provide therapeutic benefit for ischemic heart disease. Injection of bone marrow mononuclear cells (BM-MNC), a mixed population of cell lineages, including hematopoietic, fibroblasts, osteoblasts, myogenic cells, and endothelial precursors, following left anterior descending coronary artery ligation (LAD) resulted in decreased infarct size, and increased regional blood flow in a porcine model of MI (52). These effects were primarily a result of increased angiogenesis in the infarcted tissue. The potential for neovascularization and myocardial repair was examined in a rat model of MI using a subpopulation of human BM-MNC enriched for hematopoietic and endothelial progenitors (53). Mobilized mononuclear cells were obtained from single donor leukopheresis products of humans treated with recombinant granulocyte colony-stimulating factor (G-CSF) and isolated based on expression of the hematopoietic lineage marker, CD34. Isolated cells were injected into the tail vein of athymic rats subjected to LAD ligation. Injection of the CD34+ fraction resulted in an increase in microvascularity and a reduction in the number of apoptotic cardiac myocytes

compared to controls. The increase in the number of capillaries within the infarct zone was exclusively of human origin while the increase in capillaries in the peri-infarct zone was of rat origin. As a result systolic function was augmented by 30–40% and persisted for at least 15 weeks. The potential of bone marrow stromal cells (MSC), a population of BM cells considered to be mesenchymal stem cells, to develop into cardiomyocytes in vivo was also examined in rats (54). Isolated MSCs, were expanded and labeled with the LacZ reporter gene ex vivo and infused into the aorta 2 weeks after coronary artery ligation. Histological analysis 4 weeks later indicated that the donor cells differentiated into cardiomyocyte-like cells, fibroblast, and endothelial cells in 8 out of 12 recipient rats. The effect of MSC transplant on heart function was not reported. Orlic et al. recently confirmed the potential of BM-derived stem cells to produce functional cardiac cells as well as blood vessels (55 ). Lineage negative (lin− ), c-kit+ bone marrow cells, a population of hematopoietic cells enriched for hematopoietic stem and progenitor cells, were isolated from male transgenic mice expressing enhanced green fluorescent protein (EGFP). MI was induced by coronary artery ligation and 3 to 5 hours later donor lin− , c-kit+ hematopoietic cells were injected into the area adjacent to the infarct. Examination 9 days later indicated that myocardial regeneration was obtained in 12 out of 30 recipient female mice. The regenerating myocardium contained donorderived cardiac myocytes as well as smooth muscle and endothelial cells. Donor derived cardiac cells occupied 68% of the infarcted left ventricle in some mice, and hemodynamics were significantly improved compared with controls. In a second strategy, endogenous bone marrow cells were mobilized by infusion of G-CSF and stem cell factor (SCF). This treatment increased the in vivo population of stem cells able to contribute to myocardial repair (56). Cytokine treatment significantly improved survival following MI and resulted in the formation of vascular structures and myocytes occupying an average of 76 ± 11% of the infarcted myocardium. Hemodynamics and left ventricle function were again significantly improved and scar formation was reduced. These studies indicate that stem cells can promote myocardial regeneration after infarction in animal models and support their potential use in the treatment of heart disease. Bone marrow-derived stem cells have the advantage of being more readily available and histocompatible. There are several hurdles that need to be surmounted before these become useful clinical strategies. Survival of stem cells may be an issue because viable grafts were demonstrated in only a fraction of the recipient animals (55 ). While the mobilization of a patient’s own bone marrow by cytokine treatment is an attractive concept, the therapeutic window for such treatment is unclear. Orlic et al. treated the mice 5 days prior and 3 days post-MI (56). It is not known whether a sole treatment post-MI will be effective. A possible adjunct to all of these techniques may be to engineer the stem cells prior to implantation. The introduction of appropriately regulated prosurvival and antiapoptosis genes such as Akt (44) or factors that may promote angiogenesis within the


infarcted tissue (26) are attractive possibilities. The stem cell as a vehicle for gene therapy is a novel concept combining two exciting new approaches for the treatment of heart disease.

Summary Demonstrations that adult stem cells can home into sites of tissue injury, engraft, differentiate, and improve organ function make them an exciting prospect for the treatment of heart disease. It could be argued that the quest for therapeutic applications is premature because the fundamental biology of stem cells, including the signals that determine migration and differentiation, are not well understood. Despite these concerns, the remarkable results of the animal studies point to an enormous therapeutic potential and highlight the need for further research and developmental trials. CONCLUSIONS Our increased understanding of heart disease at the cellular and molecular level combined with major advances in our ability to manipulate genes and cells will lead to revolutionary changes in the way that heart failure is managed. The strategy that is the closest to the clinic is therapeutic angiogenesis. With more than 25 clinical trials completed or underway and more in the pipeline, this technique has the potential to reverse ischemia in both heart and peripheral vascular disease. When the associated safety issues have been addressed and the protocols optimized, therapeutic angiogenesis is likely to become a major clinical procedure. The delivery of genes such as SERCA2a that augment contractility of the failing heart may ultimately attain a significant clinical presence as well. Finally adult stem cells combined with genetic engineering, although more futuristic, may ultimately provide a revolutionary approach to disease treatment. When these therapies are jointly implemented we can expect to see a profound impact on the prognosis for heart and limb disease patients with significant improvements in survival and quality of life, as well as reduced treatment costs. ACKNOWLEDGEMENTS Supported by National Institutes of Health grants HL44578, AI60198, and grant BM034 from the Chiles Endowment Biomedical Research Program of the Florida Department of Health (to K.A.W.). The opinions, findings, and conclusions or recommendations expressed in this publication are those of the authors and do not necessarily reflect the views of the Biomedical Research Program or the Florida Department of Health. REFERENCES 1. Brutsaert, D. L., and Sys, S. U. (1997) Diastolic dysfunction in heart failure. J. Card. Fail. 3, 225–242. 2. Arai, M., Alpert, N. R., MacLennan, D. H., Barton, P., and Periasamy, M. (1993) Alterations in sarcoplasmic reticulum gene expression in human heart failure. A possible mechanism for alterations in systolic and diastolic properties of the failing myocardium. Circ. Res. 72, 463–469.

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