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Ruel M, Kelly RA, Sellke FW. Therapeutic Angiogenesis, Transmyocardial Laser Revascularization, and Cell Therapy.
In: Cohn LH, Edmunds LH Jr, eds. Cardiac Surgery in the Adult. New York: McGraw-Hill, 2003:715750.

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Chapter 27

Therapeutic Angiogenesis, Transmyocardial Laser Revascularization, and Cell Therapy

Marc Ruel/ Ralph A. Kelly/ Frank W. Sellke

????Vasculogenesis, Arteriogenesis, and Angiogenesis
????Mechanisms of Angiogenesis
????Spontaneous Angiogenesis in Adult Tissues
????Growth Factors and Delivery Strategies
????Gene Delivery Vectors
????Preclinical Studies
????Indications and Surgical Technique
????Clinical Studies
????Efficacy Issues
????Types of Lasers
????Mechanisms of TMR
????Preclinical Studies
????Indications and Surgical Technique
????Clinical Studies
????????CO2 LASER
????Skeletal Myoblast Implants for Heart Failure
????Enhancing the Survival of Transplanted Cells
????Inducing Proliferation of Adult Cardiac Myocytes
????Stem Cell Approaches to Cardiac Repair and Regeneration

Despite increased awareness and better management of cardiovascular risk factors, coronary artery disease (CAD) may involve the epicardial vasculature of some patients so diffusely that repeated attempts at catheter-based interventions and coronary artery bypass grafting (CABG) can prove unsuccessful at alleviating ischemic symptoms and preventing complications. Although this situation may be encountered in any given subgroup of patients (particularly those who have previously undergone CABG), it is more common in diabetics,1,2 heart transplant recipients with cardiac allograft vasculopathy,3,4 and patients of Indian descent or with a family history of early-onset CAD.5,6 Overall, it is estimated that patients with ungraftable coronary artery disease account for approximately 5% of patients who undergo coronary angiography at large referral centers.7 The failure to revascularize even a single ischemic myocardial territory due to poor graftability is associated with a decrease in both survival and freedom from angina in these patients, regardless of the presence of a patent left internal thoracic artery bypass to the left anterior descending artery.8 Consequently, patients with ungraftable coronary disease may potentially benefit from alternative therapies such as therapeutic angiogenesis and transmyocardial laser revascularization, either of which could serve as the sole therapy or as an adjunct towards complete myocardial revascularization.

There are also a large number of patients with poor left ventricular function and congestive symptoms due to one or several myocardial infarctions for whom medical therapy or conventional revascularization procedures have been unsuccessful or are likely to fail in the context of an unfavorable myocardial viability profile. Although many of these patients may be eligible for orthotopic heart transplantation, current waiting times for donor hearts and limitations in organ availability render this option unlikely to occur before the patient has become severely ill and reached status I priority level. Cell-based modalities for heart failure, collectively referred to as cell therapy, may eventually constitute a therapeutic option for these patients by enabling repopulation of their infarcted myocardial territories with functional cardiomyocytes.

Therapeutic angiogenesis, transmyocardial laser revascularization, and cell therapy share a common goal of directly restoring perfusion and function to chronically ischemic myocardial territories without intervening on the epicardial coronary arteries. In spite of these approaches having received considerable scientific attention over the last several years, they have not yet been proven to provide clinical benefit and are consequently reserved for patients who have failed conventional therapies. Nevertheless, as the understanding of the physiologic mechanisms that constitute their scientific foundations improves over time, their more widespread applications could potentially revolutionize the practice of cardiovascular medicine. For these reasons, all members of the multidisciplinary cardiac team may be called to play a role in their development and implementation, and will benefit from an understanding of the mechanistic basis, current status of research, clinical data, and therapeutic potential behind these approaches. This chapter provides a synopsis of these key points as they pertain to each of these three modalities.

Vasculogenesis, Arteriogenesis, and Angiogenesis

At least three different processes may result in the growth of new blood vessels: vasculogenesis, arteriogenesis, and true angiogenesis (Table 27-1).9,10 Vasculogenesis occurs early in fetal development, within new avascular tissue, and consists of the differentiation of endothelial cells from angioblasts and endothelial progenitor cells, followed by their proliferation, coalescence, and recruitment of other cell types to complete the process of vascular formation in situ.11 Although long considered to play little or no role in the response to chronic ischemia in adult tissues, vasculogenesis has been shown to occur in adult mice from the recruitment of circulating bone marrow endothelial progenitor cells after cutaneous wounding and hindlimb ischemia.12

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TABLE 27-1 Biological processes leading to the formation of new blood vessels

Arteriogenesis refers both to the process by which an individual's postnatal vascular network is remodeled later in life by the maturation of preexisting collaterals in response to supply-demand imbalances ("angiogenic remodeling"), and to the de novo formation (by sprouting) of mature blood vessels that contain pericytes and smooth muscle cells, which in fact constitutes the true goal of "therapeutic angiogenesis."

Angiogenesis, in its strictest definitional meaning, refers to the sprouting into surrounding tissues of newly formed capillaries derived from preexisting vessels, which spontaneously occurs at the border zone of myocardial infarction or in granulation tissue during wound healing. However, these newly formed capillaries lack a fully developed medial layer, have abnormal permeability, and do not undergo vasomotor regulation. Thus the term "therapeutic angiogenesis" may constitute a misnomer, and "therapeutic arteriogenesis" better decribes a process that is likely to result in the alleviation of ischemia; nevertheless, the former designation is widely accepted and the distinction will not be carried further in this text.

Mechanisms of Angiogenesis

Vasodilation and increased capillary permeability from the sequestration of intercellular adhesion molecules such as vascular endothelial (VE)-cadherin and platelet endothelial cell adhesion molecule (PECAM)-1 are believed to constitute initial steps in the physiologic angiogenic process (Fig. 27-1).13 These events are mediated by the combined actions of nitric oxide (NO) and vascular endothelial growth factor (VEGF) on endothelial and smooth muscle cells and result in the extravasation of plasma proteins, although limited by the negative feedback actions of angiopoietin-1 (Ang1), an agonist for the Tie2 receptor of endothelial cells.14 The detachment of smooth muscle cells and the degradation of the surrounding perivascular matrix constitute a next step that involves the Tie2 antagonist angiopoietin-2 (Ang2) and several metalloproteinases. This allows for the migration of endothelial cells and the liberation of endogenous growth factors such as fibroblast growth factor (FGF)-2 and VEGF from the matrix.13 The subsequent proliferation of endothelial cells and their migration to distant sites are induced by several growth factors including VEGFs, FGFs (which recruit endothelial, mesenchymal, and inflammatory cells),15 Ang1 (chemotactic for endothelial cells and an inducer of sprouting),16 and platelet-derived growth factors (which recruit pericytes and smooth muscle cells around nascent vessel sprouts).17 Endothelial cells can then assemble into cords, form a lumen, and reexpress adhesion molecules such as VE-cadherin, three processes believed to make endothelial cells resistant to apoptosis and mediated by VEGF, FGF-2, and Ang1.18,19 These rudimentary cords can remain dormant or develop, branch, recruit periendothelial cells, and mature as functional vessels in order to meet local demands. The signals and mechanisms responsible for these maturation processes are incompletely understood, but may involve the combined actions of PDGF-BB (smooth muscle chemotaxis),17 Ang1 and Tie2 (smooth muscle to endothelial cell interactions),16 and FGF-2 (smooth muscle growth and vessel enlargement).13 Table 27-2 summarizes the role of select substances involved in the angiogenic process.

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FIGURE 27-1 Mechanisms implicated in the angiogenic process. SMC = smooth muscle cell; EC = endothelial cell.


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TABLE 27-2 Substances involved in the angiogenic process (partial listing)

Spontaneous Angiogenesis in Adult Tissues

While vasculogenesis may not significantly contribute to increasing the vascularity of adult tissues under ischemic or inflammatory conditions, the spontaneous occurrence of both arteriogenesis and angiogenesis has been demonstrated in animals models and humans under a variety of stresses that include wound healing and inflammation,20 peripheral vascular disease, chronic coronary insufficiency,21,22 and acute myocardial ischemia.2328

Spontaneous angiogenesis may be enhanced by a number of commonly encountered substances. Nicotine, for instance, is proangiogenic and may worsen atherosclerotic plaques by promoting intimal proliferation.29,30 Moderate ethanol concentrations and low-dose statins also have proangiogenic properties, and the use of statins has been associated with increased tissue perfusion in a hindlimb ischemia model.31,32 Adenosine and heparin appear to stimulate angiogenesis in the presence of ischemia. In a trial of IV adenosine and heparin administered daily for 10 days in patients with chronic stable angina, a 9% reduction in the extent and a 14% improvement in the severity of perfusion defects was noted on exercise thallium imaging in patients who received adenosine and heparin versus those who received placebo.33

A number of commonly used medications have been shown to potentially interfere with the angiogenic process. These include captopril,34 isosorbide dinitrate,35 furose-mide,36 spironolactone,37 as well as ASA and other anti-inflammatory drugs whose antiangiogenic effects relate to the inhibition of COX-2, the inducible isoform of cyclo-oxygenase.38,39

Growth Factors and Delivery Strategies

Despite the complexity of the angiogenic process, therapeutic angiogenesis regimens have mainly focused on the administration of a single growth factor, with select isoforms of VEGF-A (VEGF121, VEGF165) and FGF (FGF-1, FGF-2) having been most extensively studied.40 Delivery strategies may involve the actual angiogenic protein or the gene encoding for it; while proteins are administered directly, gene-based approaches usually employ naked plasmid DNA or a viral vector that encodes the gene to be incorporated by the host endothelial cells. Several routes of administration have been developed to deliver angiogenic substances to the ischemic myocardium in a single or repeated fashion, and include intravenous, intracoronary, left atrial, surgical perivascular, intrapericardial (via a catheter placed under echo guidance), and catheter-based intramyocardial approaches. Table 27-3 outlines the relative advantages and disadvantages of protein- versus gene-based approaches.

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TABLE 27-3 Protein vs. gene therapy for therapeutic angiogenesis

Gene Delivery Vectors

Gene-based approaches require vectors in order to incorporate an angiogenic gene into a target host cell and induce production of the encoded protein. Although naked plasmid DNA can be used for these purposes, its efficiency is limited by the small fraction of plasmid DNA that actually enters the cell nucleus.41 The use of adenoviruses as vectors is associated with much higher transfection efficiency, and these viruses can be readily produced as replication-deficient mutants for gene transfer applications. However, elevated titers of circulating antibodies to adenoviruses are common in the general population and can elicit an inflammatory response that may compromise the incorporation and expression of the gene.42 Alternatives have therefore been developed that include adeno-associated viruses, which are unique in their ability to transduce nondividing cells (thereby allowing for a more prolonged transgene expression), and retroviral vectors.41 Retroviruses differ from plasmid and other viral vectors in that their RNA is reverse transcribed to DNA and integrated into the host cell genome. While this induces long-lasting expression of the incorporated gene, it also raises legitimate safety concerns linked to potentially deleterious overexpression.10 In an attempt to increase the efficacy and safety profile of gene delivery vectors, several laboratories are now focusing on the development of viral vectors that would allow for up- or downregulation of the gene of interest.

Preclinical Studies


A large animal model of chronic myocardial ischemia constitutes one of the requirements for the preclinical evaluation of direct revascularization modalities such as therapeutic angiogenesis. Since laboratory animals do not spontaneously develop CAD, some intervention that leads to coronary insufficiency must be performed. Vessel embolization, surgical ligation, or thrombogenic copper coil implantation results in acute coronary occlusion and myocardial infarction; although a chronic ischemic area exists at the border zone of this infarct, it does not adequately model the ischemic myocardial territory of patients with severe angina.

A collateral-dependent, chronically ischemic myocardial territory with minimal infarction can be created with the surgical implantation and intermittent inflation of an external pneumatic coronary occluder,43,44 or by inserting an ameroid constrictor around a major coronary artery (usually the proximal left circumflex).45 The ameroid method results in progressive stenosis and occlusion of the encircled vessel over a period of 2 to 4 weeks (Fig. 27-2A). The constrictor consists of hygroscopic compressed casein made into a cylindrical shape and enclosed within a stainless steel collar; when in contact with fluid, the casein expands in an inward radial direction as a result of the fixed metal ring and occludes the artery (Fig. 27-2B). Ameroid constrictors have been used clinically to produce gradual occlusion of the portal vein in conjunction with hepatic artery ligation for the palliation of unresectable hepatic tumors in children.46

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FIGURE 27-2 Ameroid constrictor model of chronic myocardial ischemia. (A) The ameroid is surgically inserted around the proximal left circumplex artery (LCx). (B) Expansion of the inner casein core is limited by the outer metal ring, resulting in progressive occlusion of the vessel.

Pigs are an ideal model for the study of chronic myocardial ischemia due to their relatively few native collateral vessels (unlike dogs, which have an extensively interconnected coronary network) and their lack of propensity to develop normal perfusion to the collateral-dependent myocardium induced by the placement of an ameroid constrictor. Even so, swine mount an endogenous angiogenic response to chronic myocardial ischemia that results in increased intercoronary collateral flow, and which mandates for the use of ischemic controls in preclinical studies.47,48 Compared to normal vessels, these collaterals have less medial smooth muscle, impaired endothelial-mediated vasodilatation, and altered endothelium-independent relaxation properties.48,49


Preclinical experience with VEGF has mainly involved its VEGF165 and VEGF121 isoforms derived from alternative splicing of the VEGF-A gene. Both protein- and gene-based approaches have been employed, and success has been observed using either modality in limb ischemia models as well as in the setting of myocardial ischemia.5057 For instance, 2 ?g of VEGF were administered perivascularly over 4 weeks to swine whose lateral myocardial territory had been rendered ischemic by a circumflex ameroid occluder.52 Treated animals developed higher coronary flow and a 4-fold increase in capillary density of the collateral-dependent myocardium when compared with controls; furthermore, a decrease in the size of the ischemic zone was demonstrated on magnetic resonance imaging.55

The use of intracoronary or intrapericardial routes for the administration of VEGF has not consistently led to similar preclinical success. In dog studies, a 28-day course of intracoronary VEGF injections was effective in increasing flow to the collateral-dependent territory above that of controls,57 but a 7-day course did not produce any effect and actually exacerbated neointimal accumulation following endothelial injury.58 In another study, the same authors reported that while injection of an adenoviral vector encoding for VEGF165 through an indwelling pericardial catheter resulted in sustained pericardial transgene expression, no increase in perfusion of the collateral-dependent territory could be demonstrated.59


Like VEGF, several isoforms of fibroblast growth factor exist, of which FGF-1 and FGF-2 have been the most studied. Both FGFs are believed to induce angiogenesis as well as arteriogenesis by stimulating the growth of a variety of cell types, including vascular smooth muscle cells and endothelial cells.13

FGF-1 is strongly expressed in ischemic myocardium and may play a key role in the spontaneous formation of collaterals.60 Its initial use to stimulate angiogenesis in large animals produced disappointing results that were perhaps due to the instability of wild-type FGF-1, which has a biologic half-life of 15 min at 37?C.6163 Following recognition that heparin binding and the replacement of a single cysteine residue with a serine increased the half-life of FGF-1 1000-fold,63 perivascular administration of its S117 mutant form was studied in a porcine ameroid model and resulted in improvements of collateral-dependent myocardial blood flow and left ventricular function.64

FGF-2 has been extensively studied in canine and porcine models of myocardial ischemia using a variety of delivery strategies. Unger et al gave daily intracoronary bolus injections of 110 ?g of FGF-2 in the distal circumflex artery of dogs starting 10 days after placement of an ameroid constrictor and continuing for 28 days.65 The transmural collateral flow in FGF-2-treated dogs significantly exceeded that of controls by the second week of treatment and was associated with an increase in the density of distribution (>20 ?m) vessels. These investigators also conducted chronic studies with daily left atrial injections of FGF-2 for up to 13 weeks, in which the maximum effect attributable to the growth factor was temporally related to the presence of myocardial ischemia.66 In these prolonged studies, chronic FGF-2 therapy was not associated with the occurrence of any structural or vasoproliferative adverse effect for up to 6 months after treatment initiation.66

Local perivascular administration of FGF-2 using 10 sustained-release heparin-alginate capsules that each contained 1 or 10 ?g of FGF-2 was studied in swine, and was associated with increased perfusion of the collateral-dependent territory and a dose-dependent improvement in left ventricular ejection fraction both at rest and during pacing.6769 Furthermore, perivascular FGF-2 administration resulted in normalization of ischemia-induced impairments of endothelial-dependent vasodilatation in the collateral-dependent territory.70 Single-dose intrapericardial and intracoronary delivery of FGF-2 were also studied and led to comparable perfusion and contractility improvements; however, single-dose intravenous infusion was not effective.71,72

The myocardial and tissue distribution of I125 labeled FGF-2 after intracoronary and intravenous administration in swine were studied with organ autoradiography.73 The liver accounted for the majority of I125-labeled FGF-2 activity at 1 hour after injection with either route; total cardiac-specific activity at 1 hour was 0.88% for intracoronary and 0.26% for intravenous administration, and further decreased to 0.05% and 0.04% at 24 hours, respectively (Fig. 27-3A). In another study, the distribution of intravenous injections of FGF-2 was compared with that of perivascular sustained-release delivery.74 The amount of FGF-2 deposited in arteries adjacent to sustained-release devices was 40 times that deposited in animals who received a single intravenous bolus of FGF-2 (Fig. 27-3B). FGF-2 was also 5- to 30-fold more abundant in the kidney, liver, and spleen after intravenous injection than following perivascular release, supporting perivascular delivery as being far more efficient and specific than intravenous delivery at achieving local deposition of FGF-2.

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FIGURE 27-3 Tissue deposition after intravenous (IV), intracoronary (IC), and perivascular (using heparin-alginate sustained released capsules) administration of FGF-2. (A) Total cardiac specific activity after IC and IV injection of 25-?g Ci of I125-FGF-2 in swine. Total specific cardiac activity with either method was less than 1% at 1 hour and 0.1% at 24 hours, with the liver accounting for most of the I125-FGF-2 activity. (Reproduced with permission from Laham RJ, Rezaee M, Post M, et al: Intracoronary and intravenous administration of basic fibroblast growth factor: myocardial and tissue distribution. Drug Metab Dispos 1999; 27:821.) (B) Tissue deposition after a single intravenous injection versus perivascular administration of FGF-2 in intact carotid arteries ("native"), in arteries whose endothelium was denuded, and in arteries allowed to develop intimal hyperplasia 2 weeks after endothelial denudation. Deposition of FGF-2 was substantially lower with IV injection than with perivascular release for native arteries (factor of 40.0 less), denuded arteries (factor of 18.9 less), and hyperplastic arteries (factor of 67.1 less). bFGF = FGF-2. (Reproduced with permission from Edelman ER, Nugent MA, Karnovsky MJ: Perivascular and intravenous administration of basic fibroblast growth factor: vascular and solid organ deposition. Proc Natl Acad Sci U S A 1993; 90:1513.)


Although overexpression of VEGF in mice has been associated with the formation of angiomas and vascular tumors,75,76 the occurrence of these adverse events or of proliferative retinopathy has not been reported in any study of growth factor therapy that has involved large animals. Most of these studies were of short-term duration, however, and may not have involved a sufficient number of animals to detect a rare occurrence of these events.

VEGF and FGF-2 are known to be associated with systemic hypotension that occurs in a dose-dependent fashion; in this regard, the doses of FGF-2 leading to hypotension are substantially higher than for VEGF.77,78 FGF-2 has also been associated with proliferative membranous nephropathy leading to proteinuria in mice, but this has not been observed in preclinical or clinical studies.79,80

Indications and Surgical Technique


Table 27-4 outlines the current potential indications and contraindications to angiogenic therapy. As of this writing, the clinical effectiveness of this modality has not yet been proven. Therapeutic angiogenesis should therefore be considered experimental and reserved only for patients who have failed or are not amenable to conventional revascularization procedures.

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TABLE 27-4 Potential indications and contraindications to angiogenic therapy

Patients considered for therapeutic angiogenesis should have persistent, severe chronic stable angina imputed to a myocardial territory that can not adequately or safely be revascularized by conventional methods.81 Dobutamine-stress echocardiography or nuclear imaging to confirm ischemia and viability in the target territory is recommended. Since therapeutic angiogenesis implies a slow course of new vessel development, it should not be employed to attempt emergency revascularization or salvage of threatened proximal coronary occlusion.10

Absolute contraindications to the administration of angiogenic growth factors include a history of malignancy within the last 5 years, with the exception of basal or early-stage squamous skin cancers that are considered cured. Therapeutic angiogenesis should not be carried out in patients with proliferative retinopathy, vascular malformations, and chronically low blood pressure. In addition, FGF-2 is contraindicated in patients with decreased creatinine clearance or proteinuria. Severely compromised left ventricular ejection fraction constitutes a relative contraindication to angiogenic therapy, since most delivery techniques involve procedural stress that could precipitate cardiac decompensation.


The surgical perivascular implantation of angiogenic growth factors can be performed in conjunction with CABG or as sole therapy.82,83 Both strategies are amenable to the use of minimally invasive approaches in combination with multivessel off-pump CABG, ipsi- or contralateral minimally invasive direct coronary artery bypass (MIDCAB), or as sole therapy (through a subxiphoid or small thoracotomy incision). In the future, the safe implantation of angiogenic growth factors via a closed-chest, videoscopic approach may constitute an ideal method of delivery.

Although a surgical approach can be used for the administration of virtually any type of angiogenic protein or gene vector, most of the experience has involved the perivascular delivery of FGF-2 protein using sustained-release beads implanted at the time of CABG (Fig. 27-4).81,82,84 Controlled release of the FGF-2 is derived from its avidity for heparin, which is bound to sepharose beads and hardened into a capsule using a calcium chloridealginate solution, without causing any substantial reduction in the biological activity of the growth factor.67,74,85 Once implanted, release of FGF-2 from the polymer occurs via first-order kinetics over a 4- to 5-week period, without any inflammatory reaction resulting from polymer placement.

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FIGURE 27-4 Implantation of sustained-release FGF-2 beads in the myocardial distribution of an ungraftable right coronary artery, in conjunction with CABG. PDA = posterior descending artery; LVB = left ventricular branches of the right coronary artery. (Adapted with permission from Sellke FW, Laham RJ, Edelman ER, et al: Therapeutic angiogenesis with basic fibroblast growth factor: technique and early results. Ann Thorac Surg 1998; 65:15.)

The perivascular implantation of FGF-2 using this delivery system in conjunction with CABG has been carried out through a median sternotomy.82,84 After the institution of total cardiopulmonary bypass and cardioplegic arrest, distal coronary anastomoses were performed to graftable coronaries and the nongraftability of the target territory was confirmed by direct inspection of the vessel. Multiple linear incisions were made in the epicardial fat surrounding the ungraftable vessel and in the transition zone between the target territory and that supplied by a grafted or patent coronary artery in order to enable the development of subepicardial collaterals between ischemic and normally perfused myocardium. Ten heparin-alginate beads, each containing 1 or 10 ?g of human recombinant FGF-2, were inserted in the subepicardium and secured in place with a 6-0 polypropylene suture, with up to 3 beads being placed in a single incision. Proximal anastomoses were then constructed, the patient separated from cardiopulmonary bypass, and routine closure performed. As a quality control measure, 2 to 6 heparin-alginate beads from each batch were cultured aerobically and anaerobically to ensure sterility.

Clinical Studies


Perivascular delivery The safety of surgical FGF-1 administration was demonstrated in a series of 20 patients conducted by Schumacher et al, who injected 0.01 mg/kg of FGF-1 directly into the myocardium along a diffusely diseased left anterior descending (LAD) coronary artery to which the left internal thoracic artery (LITA) was also grafted.86,87 Patients were followed up 12 weeks and 3 years later, when the LITA was selectively injected and the degree of anterior myocardial collateralization quantitatively evaluated by digital subtraction angiography. Although a local increase in collateral blush was observed along the LAD, the investigators did not report nuclear imaging assessments of ischemia or functional parameters such as exercise capacity, CCS angina class, or freedom from angina recurrence.

The safety and efficacy of perivascular FGF-2 administration was evaluated in a phase I, double-blind, randomized controlled trial that involved 24 patients concomitantly undergoing CABG.84 In this study, patients in whom high-dose FGF-2 sustained-release capsules had been implanted in an ungraftable territory at the time of CABG had complete relief from angina and showed significant improvements in stress nuclear perfusion defect size at 3-month follow-up. These patients were subsequently followed up to a mean of 32 months postoperatively with clinical assessment and nuclear imaging.81 At this late follow-up, patients treated with either dose of FGF-2 had experienced significantly greater freedom from angina recurrence than controls (Fig. 27-5). Double-blinded late nuclear imaging studies revealed that all but one patient in the control group had either persistence of a reversible perfusion defect or evidence of a new fixed defect in the ungraftable myocardial territory; in contrast, this was observed in only 1 of 9 patients treated with FGF-2 (Fig. 27-6). The remaining FGF-treated patients had disappearance of their ungraftable territory reversible perfusion defect and stability or decrease in the size of their fixed defect. FGF-treated patients also showed better late global left ventricular perfusion scores during pharmacologic stress.81 Although this study only involved a small number of patients and may have been confounded by concomitant CABG, it nevertheless suggested that perivascular FGF-2 therapy may be associated with persistent freedom from angina recurrence and sustained improvements in left ventricular perfusion.

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FIGURE 27-5 Baseline and 3 years postoperative SPECT images of patients who received FGF-2 versus placebo beads implanted in an ungraftable inferoapical myocardial territory at the time of CABG.81 Horizontal long-axis views show complete resolution of the large inferoapical perfusion defect at rest and stress in the patient who received FGF-2, and no detectable change from baseline in the patient who received placebo.


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FIGURE 27-6 Freedom from angina in patients who underwent implantation of FGF-2 versus placebo in a major, nongraftable myocardial territory at the time of CABG. (Reproduced with permission from Ruel M, Laham RJ, Parker JA, et al: Long-term effects of surgical angiogenic therapy with FGF-2 protein. J Thorac Cardiovasc Surg 2002; 124:28.)

Intravascular delivery Laham et al conducted a phase I, open-label dose-escalation trial that evaluated the efficacy and safety of a single-bolus intracoronary administration of FGF-2 in 52 patients. Their results suggested that this therapy may improve symptoms and myocardial perfusion.88 These investigators proceeded with a multicenter, randomized, double-blind, placebo-controlled trial of a single intracoronary infusion of FGF-2 at 0, 0.3, 3, or 30 ?g/kg in 337 patients (FGF-2 Initiating Revascularization Support [FIRST]trial).89 Efficacy was evaluated at 90 and 180 days by exercise tolerance test, nuclear perfusion imaging, and angina questionnaire. Exercise tolerance was increased at 90 days in all groups but was not significantly different between placebo and FGF-treated groups. FGF-2 reduced angina symptoms, and these differences were more pronounced in highly symptomatic patients with baseline CCS angina class scores of III or IV. However, this benefit did not persist at 180 days because of continued improvement in the placebo group, and the trial was considered negative with respect to all main end points. Adverse events were similar across all groups, except for hypotension, which occurred more frequently in the 30-?g/kg FGF-2 group.

Intravenous and intracoronary administration of VEGF was studied in a randomized, double-blind, phase II trial (VEGF in Ischemia for Vascular Angiogenesis [VIVA]trial), which was also completely negative with respect to symptom improvement, exercise time, and nuclear imaging end points.90 It is possible that the effects of FGF-2 and VEGF in the FIRST and VIVA trials may have been compromised by the choice of intravascular delivery routes, which not only are nonspecific in their tissue distribution, but also carry the potential to worsen atherosclerosis.30


Perivascular delivery Rosengart et al have examined the effects of direct administration of an adenoviral vector encoding for VEGF121, either as an adjunct to conventional CABG (in 15 patients) or as sole therapy (in 6 patients).83 There was no control group and the principal end points of this phase I trial were related to safety. In this regard, no systemic or cardiac-related adverse events related to vector administration were observed. All patients reported improvements in angina class, and postoperative nuclear imaging suggested increased contractility during stress conditions in the area of vector administration, but did not reveal an increase in myocardial perfusion.

Losordo et al initiated a small phase 1 trial to determine the safety and bioactivity of direct myocardial gene transfer (using naked plasmid DNA) of VEGF165 as the sole therapy in 5 patients with inoperable CAD and symptomatic myocardial ischemia.91 The vector was administered by four 2.0-mL needle injections into the anterolateral left ventricular free wall through a small left anterolateral thoracotomy. The injections were not associated with any side effect other than isolated premature ventricular complexes at the time of needle penetration. All patients had a significant reduction in angina as measured by nitroglycerin use, improved collateral scores on angiography, and reduced size of the ischemic defect on dobutamine nuclear imaging. These data were reproduced in another open-label, uncontrolled study in which 20 patients received either 125 or 250 ?g of naked plasmid VEGF121 injected directly into the myocardium via a minimally invasive thoracotomy.92 Plasma VEGF concentrations increased at 14 days to a level 2-fold over pretreatment values and returned to baseline by 3 months. As in the previous study, patients reported decreased angina and reduced nitroglycerine use, and improvement was seen on radionuclide perfusion imaging.92

Catheter-based delivery A pilot study of catheter-based myocardial gene transfer involved 6 patients with chronic myocardial ischemia who were randomized to receive 200 ?g of VEGF-2 or placebo.93 A steerable, deflectable 8F catheter incorporating a 27-gauge needle was advanced percutaneously and guided into the left ventricular myocardium by left ventricular electromechanical mapping. Despite the small number of patients, end points of angina frequency, nitroglycerin consumption, and stress myocardial perfusion on nuclear imaging showed a trend in favor of the group of subjects transfected with VEGF-2 versus controls. These trends were confirmed by the same group of investigators in a subsequent phase 1/2 trial involving 19 patients.93a

Efficacy Issues


From the evidence currently available, perivascular administration of angiogenic proteins may arguably constitute the route of choice for angiogenic therapy, since it presents a more specific tissue distribution profile than intravascular techniques, does not result in rapid washout, is not limited by the endothelial barrier, and does not carry the potential of exacerbating intimal plaques. Protein administration also offers safety advantages over gene-based approaches, and may be delivered perivascularly using a sustained-delivery system without provoking an inflammatory response.10,82,84 Although a surgical approach is required, it allows for the precise placement of beads within the ischemic territory as well as at the transition between normally perfused and collateral-dependent myocardium, thereby promoting the formation of subepicardial collaterals that may provide arterial inflow to the ischemic zone. Overall, these properties may account for the greater efficacy of perivascular over intravascular approaches in preclinical and clinical studies,81,84 which in our opinion makes it the currently preferred route for angiogenic therapy (in patients with end-stage CAD who have no alternative treatment option).

No tangible evidence currently exists that clearly supports the clinical efficacy of myocardial gene transfer modalities. Furthermore, these approaches have fallen into public and scientific disfavor following the highly publicized deaths of an 18-year-old patient and other subjects enrolled in gene transfer angiogenesis studies.94 At the present time, gene-based approaches should only be used in an experimental and closely regulated setting until further safety and efficacy data are available.


There is an interaction between the local availability of NO and the regulation of blood vessel growth mediated by the actions of VEGF and to a lesser extent FGF-2.9598 Diminished NO availability has been implicated in the inhibition of spontaneous and exogenous angiogenic responses in hypercholesterolemic rodents,99,100 and recently in preclinical, large-animal models of hypercholesterolemia-induced endothelial dysfunction (Ruel M, Sellke FW: submitted data). Given these data and the fact that therapeutic angiogenesis is not nearly as effective in patients with inoperable CAD as it has been in laboratory animals, the failure of effect observed in clinical trials may relate to a deficiency in the stimulated release of NO, whose production as well as that of other endothelium-derived substances is altered in advanced CAD. The current clinical indications for angiogenic therapy may therefore paradoxically target a subgroup of patients for whom the modality therapy is least likely to work, and it is plausible that the clinical efficacy of therapeutic angiogenesis may depend on the concomitant modulation of the coronary microvascular endothelium in patients with end-stage CAD. Research aimed at elucidating these questions is ongoing and may help clarify the missing link between successful animal models and disappointing clinical trials of angiogenic therapy.


Although the physiological events that result in angiogenesis are incompletely understood and likely of formidable complexity, therapeutic approaches so far have mostly concentrated on the administration of a single growth factor. While this strategy was made necessary by safety considerations and limited knowledge of potential interactions between growth factors, it is debatable whether it will ever result in clinically reproducible de novo formation of functionally competent vessels.40 Alternatively, the safety and efficacy of therapeutic approaches could be increased by stimulating endogenous angiogenesis in response to ischemia or by using combinations of exogenous growth factors. For instance, the experimental stimulation of endogenous angiogenesis without administration of exogenous growth factors was recently achieved by modulating the proangiogenic properties of the gastric submucosa in order to accomplish myocardial revascularization in a swine model of chronic ischemia (Fig. 27-7).101

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FIGURE 27-7 Gastroepiploic (GEA) arteriogram of a swine with chronic myocardial ischemia 7 weeks after apposition of a pedicled gastric submucosal patch. Patch contour (P), circumflex artery reconstitution (LCx) and occlusion at the ameroid constrictor (A) level, and drainage of contrast via the coronary sinus (CS) are seen.101 The rationale for this model was to evaluate whether endogenous angiogenesis could be stimulated without the need for growth factors. (Reproduced with permission from Ruel M, Sellke FW, Bianchi C, et al: Endogenous myocardial angiogenesis and revascularization using a gastric submucosal patch. Ann Thorac Surg (in press).)

A developing alternative is the use of angiogenesis master-switch genes, which are capable of inducing whole cascades of angiogenesis-related genes.40 These master-switch genes are physiologically induced by ischemia and mediate the endogenous angiogenic responses of animals and humans.28,102 One example is hypoxia-induced factor (HIF)-1{alpha}, a gene expressed in tissues subjected to ischemia, which initiates the cascade of VEGF-dependent angiogenesis.40 PR39 and relaxin are two other master-switch genes that have the propensity to induce both VEGF and FGF systems.40,103105 However, the therapeutic use of exogenous master-switch genes may potentially lead to excessive, uncontrolled angiogenesis by upregulating hundreds of proangiogenic genes. Their use should therefore be restricted until they are better understood and their tissue distribution reliably confined to a predetermined myocardial territory.


The notion of achieving myocardial revascularization by directly creating channels into the myocardium dates back more than half a century. Arthur Vineberg had long suggested that some application of this concept could result in the alleviation of angina106 when Sen et al, after studying the reptilian heart, reported in 1965 on the experimental use of "transmyocardial acupuncture" for myocardial revascularization.107,108 Hershey and White demonstrated that multiple myocardial punctures with an 18-gauge needle could allow for chronic survival in dogs after total excision of the LAD, as well as following ameroid occlusion of all 3 coronary arteries.109112 Nevertheless, the interpretation of these experimental data was already controversial and the concept termed "a physiologic impossibility" as early as 1969.113

The limited success of these procedures was overshadowed by the invention and widespread popularity of CABG until interest in them was revived in 1981 by Mirhoseini,114 who used a 450-W carbon dioxide laser to revascularize the hypokinetic anterior wall of a patient with a nongraftable LAD in conjunction with the construction of bypass grafts to other territories.115,116 Still, interest in transmyocardial laser revascularization (TMR) did not rise until 1995, largely catalyzed by the work of Frazier and Cooley.117 These authors reported promising preliminary results on 21 patients in whom TMR had been used as sole therapy, which suggested that this modality may improve anginal status, endocardial perfusion as measured by photon emission tomography (PET), and regional contractile function during stress. Following subsequent phase I studies, the FDA approved TMR as a therapy for angina in the presence of ungraftable coronary disease in 1998.118

Types of Lasers

Although historically involving mechanical needle punctures,107,108 TMR has since used lasers to create a series of 1-mm channels placed approximately 1 cm apart in the myocardial territory to be lased. Three main types of lasers exist: carbon dioxide (CO2), holmium:yttrium-aluminum garnet (YAG) and xenon-chloride (excimer).119122 The key characteristics of these lasers are outlined in Table 27-5. Regardless of the type of laser, the end result is generally the creation of a channel approximately 1 mm in diameter, surrounded by a 1- to 2-mm rim of necrosis and a 1- to 3-mm zone of myofibrillary degeneration in the periphery of this rim.123 The CO2 laser has the advantage of producing high-energy pulses that create a transmural channel with a single pulse, low peak power, and high photonic absorption that may, in comparison to other laser types, minimize structural tissue trauma. The pulse can be synchronized with the R wave of the ECG and delivered during end diastole to transect the heart within 10 to 60 milliseconds, thereby minimizing the interference with ventricular conduction. The holmium:YAG and excimer lasers are low-energy, require multiple firing for the creation of a single channel, and cannot be synchronized with the ECG; however, they have the capacity of being coupled to a fiberoptic catheter for transluminal endocardial delivery.124 To avoid uncontrolled pericardial hemorrhage, channels created by a percutaneous approach are not transmural; it has been hypothesized that this may lead to less stimulation of subepicardial collateralization and arteriogenesis between normally perfused and collateral-dependent myocardial territories after TMR.

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TABLE 27-5 Main types of lasers used for TMR

Mechanisms of TMR


Since the initial experimental evaluation of this modality, a major matter of debate has been whether the channels created with TMR remained patent or not. Cooley reported in 1994 a patient who died of an extracardiac cause 3 months after undergoing TMR, and in whom histologic analysis revealed multiple patent channels running perpendicular to and interconnecting with the native vasculature.125 Although reactive fibrous scar tissue had caused narrowing of the original laser tract, the channels had endothelialized and contained red blood cells, suggesting that they were functional. Amid support from other investigators126,127 and the documentation of patent channels 2 hours after TMR,123 this mechanism was called into question by autopsy results obtained 4 1/2 weeks after TMR, which did not show patency.128 Canine studies with CO2 and holmium:YAG lasers also showed that, regardless of the laser source, channels were occluded by thrombus within 6 to 24 hours, after which organization and neovascularization of the channel region occurred.129,130 In swine studies by another group, all channels became occluded by scar within 6 weeks.131 Furthermore, in an excised canine heart model, channels created with a CO2 laser did not provide acute myocardial perfusion or preserve myocardial viability in the presence of acute ischemia secondary to LAD ligation.129,130

Subsequent histologic studies of patients who died after TMR validated these experimental findings and showed that laser-induced channels were filled with abundant granulocytes and thrombocytes, fibrinous network, clots, and detritus as early as 3 days after operation.132 This was also documented in a report of three patients who had died 1 to 11 days following laser revascularization and in whom the internal lining surface of laser channels was not endothelialized, was composed of vacuolated and condensed myocardial debris, and was surrounded by zones of necrotic cardiomyocytes, without any obvious connections between laser channels and the ventricular cavity.133 It is now widely recognized that the postulated long-term patency of TMR channels is a myth; in this regard, it is possible that early reports may have confused thebesian veins with the so-called ventriculo-myocardial connections that would have resulted from TMR.134


TMR could stimulate angiogenesis by a specific mechanism or by a wound-healing response to injury.135 In a study of TMR in swine, Hughes et al found a highly disorganized pattern of neovascularization consistent with angiogenesis located predominantly at the periphery of the channels.136 Immunohistochemistry confirmed the presence of endothelial cells within these neovessels, and a higher vascular density was demonstrated in lased ischemic myocardium than in nonlased ischemic myocardium. Horvath et al found a 2-fold increase in VEGF mRNA expression in the ischemic territory of swine treated with TMR using a CO2 laser versus control animals subjected to ameroid constriction of the left circumflex alone.137 There was a 3-fold increase in the number of new blood vessels in the ischemic zone of the TMR group compared with the control group. TMR also resulted in a higher number of von Willebrand factorpositive microvessels with increased expression of matrix metalloproteinases and platelet-derived endothelial cell growth factor in dogs at 2 weeks,138 and TMR led to a significantly greater vascular density and increased expression of FGF-2 and transforming growth factor-beta in rats.139

Whether this angiogenic response is specific to TMR, enhanced by the use of one specific type of laser versus another, or equivalently brought on by the mechanical (versus laser) creation of channels remains controversial. It has been found that both mechanical and laser transmyocardial revascularization lead to an increase in the expression of VEGF and FGF-2.140,141 This was, however, contradicted by Horvath et al, who showed that only laser TMR resulted in a significant increase in new blood vessels in the ischemic zone at 6 weeks, in addition to causing less fibrosis and better preserving the contractility of the collateral-dependent zone than mechanical TMR using a hot needle, a normothermic needle, or an ultrasonic needle.142


The denervation of cardiac sympathetic afferent fibers is another mechanism that has been proposed to explain the relief of anginal symptoms imputed to TMR. TMR with a holmium:YAG laser has been shown in dogs to result in a decreased response to the epicardial application of bradykinin and a 66% decrease in the protein expression of tyrosine hydroxylase in lased regions, consistent with a destruction of cardiac nerve fibers.143,144 However, these findings were refuted by subsequent studies that suggested that TMR does not acutely change sympathetic or parasympathetic efferent neuronal activation in the affected ventricle either after electrical or chemical, combined with isoproterenol, stimulation.145 Furthermore, TMR had no acute effect on reflexes mediated by left ventricular receptors with sympathetic afferent fibers after epicardial and intracoronary administration in anesthetized dogs.146

The chronic neural response was studied by Aurora et al, who showed that TMR made the intrinsic cardiac nervous system less responsive to chemical stimulation with nicotine, although it did not alter responses to electrical stimulation.147 A human correlate of this finding was suggested by the report of decreased myocardial PET hydroxyephedrine uptake (a marker of sympathetic innervation) in patients treated with TMR in excess of perfusion defects.148 Critics of the denervation theory maintain that the myocardial area treated by TMR represents less than 1% of the left ventricular mass and that denervation is unlikely to account for the alleviation of ischemia.


TMR produces fibrosis in the lased segments,135,149 and it has been hypothesized that the resultant diastolic tethering of the left ventricle may improve wall stress characteristics, prevent further ischemia and dilatation of the ischemic segment, and promote favorable remodeling.150 No experimental model has been used to test this hypothesis, however.

Preclinical Studies


The use of a CO2 laser has been championed in preclinical and clinical studies by Horvath et al. These investigators have shown in a porcine ameroid model of chronic ischemia that TMR improved myocardial perfusion and wall motion at rest.151 These findings were supported by Hughes et al, who used a hibernating myocardium model and reported significantly improved regional stress function in the lased segments (using both CO2 and holmium:YAG lasers) at 6 months postoperatively, consistent with a reduction in ischemia. Global left ventricular wall motion at peak stress improved as well.152 TMR was reported to prevent necrosis and scarring and to improve myocardial function in a sheep model of acute myocardial infarction,153 but these findings were not reproduced in other studies.154156 Horvath also developed a closed thoracoscopic technique for TMR that proved feasible in swine.157


Like the CO2 laser, the preclinical feasibility and efficacy of these two types of lasers were demonstrated in several studies.152,158161 However, one report involving the holmium:YAG laser failed to show that TMR increased perfusion assessed by thermal imaging and postmortem MRI, both acutely after occlusion of the first diagonal branch in sheep, as well as 28 days later.162 Rosengart et al reported that TMR with an excimer laser resulted in increased channel derivatives and neovascularization compared with nonlased channels while preserving normal ventricular function, and that there was a dose-response relationship with the number of channels created.158 The feasibility of percutaneous TMR using a holmium:YAG laser in dogs was reported in 1997.163


A comparison of the three main types of lasers in swine showed that the CO2 laser synchronized with the R wave was significantly less arrhythmogenic than the Ho:YAG and excimer lasers.164 In addition, the interaction of the CO2 laser with porcine cardiac tissue appeared significantly less traumatic than that of the Ho:YAG and excimer lasers. Ho:YAG channels were highly irregular and were surrounded by a relatively wide ring of coagulation necrosis, which was also observed after use of the excimer laser. In contrast, the CO2 channels were straight, well demarcated, and the zone of structural and thermal damage was smaller than with the other two types of laser. These lasers were also compared in another study, which found that only TMR performed with CO2 and holmium:YAG lasers resulted in a significant improvement in myocardial blood flow to the lased left circumflex regions, with no increase observed in sham- or excimer-lased animals.165 Furthermore, there was significant improvement in regional stress function on dobutamine stress echocardiography of the lased segments 6 months postoperatively in animals undergoing holmium:YAG and CO2 laser TMR, but no change in wall motion in sham- or excimer-lased animals.


Experience in preclinical models of TMR combined with angiogenic therapy has been mixed, with some investigators reporting an increased inflammatory response from the addition of VEGF therapy to TMR in lieu of the enhanced angiogenic response that was anticipated.166 Yamamoto et al combined TMR and intramyocardial FGF-2 treatment (directly into the channels) in chronically ischemic dogs and found that this combination resulted in an increase in the density of large vessels (>50 ?m), in keeping with the postulated arteriogenic effects of FGF-2 in ischemic tissues, but no augmentation of myocardial blood flow.167 The combination of gene transfer vectors with TMR also produced mixed results, with one group showing that TMR enhanced the transfection efficiency of an expression plasmid encoding for VEGF at 6 weeks,168 but another reporting impaired transgene expression of an adenoviral vector gene and increased myocardial inflammation from combination of the two therapies.169

Indications and Surgical Technique


The potential indications and contraindications to TMR are outlined in Table 27-6. As in the case of therapeutic angiogenesis, data confirming the effectiveness of TMR in double-blind, randomized controlled trials have not been produced. Although the procedure has been approved by the FDA since 1998, TMR is best considered semiexperimental and reserved for patients who have failed or are not amenable to conventional revascularization procedures.

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TABLE 27-6 Potential indications and contraindications to TMR

Patients eligible for TMR should have persistent, severe chronic stable angina imputed to a myocardial territory that cannot be adequately or safely revascularized by conventional methods. Most patients have previously had CABG.170 The procedure should not be employed to attempt emergency revascularization or salvage of threatened proximal coronary occlusion. TMR has been used for the relief of unstable angina when attempts at weaning intravenous antianginal medications have failed; intermediate results for this indication have showed a two-class improvement in angina severity in 82% of patients and a mortality of 13% to 22.4% at 1 year, but were associated with an increased in perioperative mortality compared to when the procedure is used for chronic stable angina.171,172

TMR has been proposed as a therapeutic adjunct for the heart transplant patient population, either as a bridge to transplant for patients with a low ejection fraction and ungraftable coronary disease,173,174 or for the treatment of allograft vasculopathy.175178 Hetzer's group, in a report of 30 patients with ischemia and reduced left ventricular ejection fraction (treated with TMR as a sole therapy, showed that myocardial perfusion and ejection fraction did not significantly improve.179 Survival was only 50% at 1 year, and the authors did not recommend the use of TMR for this indication. Similarly, the use of TMR for the treatment of cardiac allograft arteriosclerosis appeared feasible in initial case series,175178 but did not lead to any benefit 24 months after treatment.180

It is unknown whether redo TMR should be considered for select patients; one case performed 4 years following initially successful TMR resulted in angina class and functional improvements.181


Surgical TMR may be performed via a small anterolateral thoracotomy,182 in conjunction with CABG through a sternotomy, or using a videoscopic approach. Attentive anesthetic management is crucial in order to minimize intraoperative cardiac ischemia. In this regard, particular attention should be given to the constant optimization of coronary perfusion pressure and ventricular afterload, oxygenation (in anticipation of one-lung ventilation), and potassium and magnesium levels (to prevent ventricular irritability). The monitoring of mixed venous oxygen saturation with a Swan-Ganz catheter and the assessment of regional contractility and mitral valve function with transesophageal echocardiography (TEE) are recommended; TEE is also mandated to confirm endocardial penetration by the laser beam. If intraoperative ischemia arises despite these measures and leads to hemodynamic instability, an intra-aortic balloon pump (IABP) is inserted. Postoperative pain should be managed attentively, and the use of a thoracic epidural catheter is advisable unless contraindicated by coagulopathy or heparin use.

The patient is positioned in a 45-degree right lateral decubitus position with the left arm suspended at a right angle to retract the latissimus dorsi laterally. External defibrillator pads are placed over the right chest and left shoulder. The left groin is prepared and available for the establishment of femoro-femoral cardiopulmonary bypass or the placement of an IABP. A cell-saver suction and a carbon canister smoke evacuator are installed, and the operating room personnel are provided with protective eyewear.

A 6- to 10-cm anterolateral thoracotomy is performed in the fifth intercostal space at a level over the point of maximum impulse, which brings the inferior border of the incision to the level of the diaphragm and left ventricular apex. The ribs are progressively opened with two small rib spreaders placed at right angles to each other (Fig. 27-8). Since many of these patients have previously undergone CABG, care is taken to avoid lung or internal thoracic artery pedicle injury that could result from the shearing of adhesions, as well as to avoid the manipulation of diseased saphenous vein grafts. The left lung is selectively decompressed or reduced tidal volume ventilation is used, and the pericardium is exposed and incised vertically anterior to the left phrenic nerve. The pericardium is freed and retracted laterally, and the diaphragm retracted caudally, thereby allowing for exposure of the entire free wall of the left ventricle (Fig. 27-9). Complete dissection of all epicardial adhesions is essential to prevent dimpling of the left ventricle wall, which can bring mitral valve chordae closer to the endocardial surface and make them susceptible to injury during activation of the laser (Fig. 27-10).

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FIGURE 27-8 Exposure of the left ventricle for TMR using a CO2 laser is obtained by progressively opening the thoracotomy incision with two small rib spreaders placed at right angle to each other and by suspending the pericardium. (Reproduced with permission from March RJ: Laser revascularization of ischemic myocardium, in Naunheim KS (ed): Minimal Access Cardiothoracic Surgery. Philadelphia, WB Saunders, 2000.)


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FIGURE 27-9 Surgical exposure and performance of TMR using a holmium:YAG laser. (Reproduced with permission from March RJ: Laser revascularization of ischemic myocardium, in Naunheim KS (ed): Minimal Access Cardiothoracic Surgery. Philadelphia, WB Saunders, 2000.)


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FIGURE 27-10 Complete dissection of all epicardial adhesions is essential to prevent dimpling of the left ventricle wall during TMR, which can bring mitral valve chordae close to the endocardial surface and make them susceptible to injury during activation of the laser. (Reproduced with permission from March RJ: Laser revascularization of ischemic myocardium, in Naunheim KS (ed): Minimal Access Cardiothoracic Surgery. Philadelphia, WB Saunders, 2000.)

The laser handpiece is put against the myocardial area to be lased with care taken to avoid epicardial vessels, the anticipated location of papillary muscles (Fig. 27-11), and the indentation of the left ventricular wall. One channel is created per every cm2 of myocardial surface to be treated. With the CO2 laser, each 30-J pulse is gated on the R wave of the ECG (when the ventricle is distended with blood and electrically quiescent), and creates a transmural scar without causing arrhythmias. Transmural penetration is confirmed with TEE; a higher energy level may be used depending on the amount of epicardial fat and wall thickness (which is thicker towards the apex), or a lower energy employed if the procedure is performed on CPB in order to minimize bleeding. With the holmium:YAG, 3 to 8 pulses are usually needed to result in a transmural penetration at a rate of 5 pulses per second. Arrhythmias are minimized if these channels are created slowly. Once approximately 5 channels are made in an area, direct pressure with a sponge is used to control the bleeding and the laser procedure restarted once hemostasis is achieved. If necessary, a 6-0 polypropylene horizontal mattress suture may be used to close the epicardium. A typical TMR procedure involves the placement of 30 to 50 channels. The pericardial cavity is then irrigated with warm saline, and each channel is checked for bleeding. The pericardium is loosely reapproximated, and a single chest tube is left in the pleural space in communication with the pericardial space.

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FIGURE 27-11 Anticipated location of the anterolateral (AL) and posteromedial (PM) papillary muscles, which should be excluded from the area of myocardium to be lased during TMR. (Reproduced with permission from March RJ: Laser revascularization of ischemic myocardium, in Naunheim KS (ed): Minimal Access Cardiothoracic Surgery. Philadelphia, WB Saunders, 2000.)

Surgical TMR has also been performed though a sternotomy in conjunction with CABG183185 and thoracoscopically with minimal morbidity.186189 Percutaneous direct myocardial revascularization involves the insertion of a 9F deflectable guiding catheter in the common right femoral artery under local anesthesia. An optical fiber connected to a holmium:YAG laser is passed over a guide wire into the left ventricle and electromechanical mapping is used to create non-transmural channels in the desired area, each channel being approximately 1 mm in diameter and 2 to 5 mm in depth.190


TMR is associated with a considerable risk (up to 47%) of perioperative cardiac morbidity, which consists mostly of myocardial infarction, low cardiac output syndrome, and ventricular arrhythmias (Table 27-7). 170,191 Pericardial tamponade, delayed chordal rupture from laser injury, and micro air emboli from the creation of channels on the beating left ventricle have also been reported.192 TMR is associated with universal rises in CPK-MB and a 50% incidence of ischemic electrocardiographic changes in the first 48 hours.193 A TEE should be performed and compared to the last operative view whenever the suspicion of a mechanical cardiac complication is raised. Low cardiac output and ischemia are indications for the insertion of an IABP. If chordal rupture has occurred, IABP insertion and aggressive afterload reduction are carried out, and the necessity of surgical repair is reevaluated under these conditions; the prognosis with this complication is poor regardless of the approach elected. The use of TMR for unstable angina and the presence of poor LVEF (as independent predictors of perioperative morbidity170,191; conversely, good blood flow to at least one myocardial territory, female gender, and prior CABG have been associated with a decreased risk of mortality after TMR.194,195

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TABLE 27-7 Perioperative complications of TMR

Clinical Studies


Case series The feasibility and safety of TMR with a CO2 laser have been evaluated in a number of case series published over the last 7 years.196198 Overall, these have shown that TMR is associated with a perioperative mortality of approximately 9% to 15%197,199201 (reduced to 3% to 5% in subsequent randomized trials),202204 significant decreases in angina class at 3, 6, and 12 months compared to pretreatment,197,199201 improvements in exercise tolerance,200,201 a decrease in the number of perfusion defects in the lased areas of the left ventricle,197199 and a significant decrease in the number of admissions for angina in the year following the procedure when compared with the year before treatment.197,205 However, a conflicting report noted that technetium perfusion scans in 94 TMR patients were significantly worse than those of matched controls both at rest and during stress at 3-, 6-, and 12-month follow-up.206 This was also supported by Landolfo et al, who reported that despite improvements in mean anginal class at 3, 6, and 12 months, no improvement in perfusion imaging parameters could be demonstrated at any time point after TMR.205 Furthermore, TMR has been associated in other series with no improvement200 or an actual decrease in left ventricular ejection fraction.207

The long-term effects of TMR were evaluated by Horvath et al in a series of 78 patients treated with a CO2 laser and followed up to a mean of 5 years.208 Benefits similar to those observed at 1-year follow-up were noted, including a persistent improvement in angina class and Seattle Angina Questionnaire scores. Eighty-one percent of patients were of CCS class II or better, and 17% of the patients had no angina 5 years after TMR. Late nuclear imaging studies were not conducted.

Clinical trials Randomized controlled trials of TMR using a CO2 laser have been less numerous than case series, and were all open-label. A multicenter randomized controlled trial that involved 91 patients who underwent transmyocardial revascularization and 101 patients who received continued medical treatment showed impressive results: angina had improved by at least two CCS classes at 12 months in 72% of the patients assigned to TMR versus 13% of the patients who received medical treatment, and was associated with a significantly improved quality of life in the TMR group.202 In the first year of follow-up, only 2% of patients who underwent TMR were hospitalized because of unstable angina, compared with 69% of patients assigned to medical treatment. Myocardial perfusion improved by 20% in the TMR group and worsened by 27% in the medical treatment group. These differences were all statistically significant.

Almost simultaneously, a randomized controlled trial that involved a total of 188 patients showed that TMR was associated with only modest improvements in angina class in TMR-treated patients at 12 months, and with no significant difference in treadmill exercise time.203 Another open-label randomized controlled trial demonstrated that TMR was associated with a decrease in angina symptoms, and decreased time to chest pain during exercise, but no difference in total exercise time or Mvo2 during exercise.204 These investigators subsequently reported a small but significant reduction in left ventricular ejection fraction and an increase in left-ventricular end-diastolic volume in the TMR group on nuclear imaging at 12-month follow-up, without any myocardial perfusion benefit attributable to this modality.207


Case series The feasibility of TMR using a holmium:YAG laser either as sole therapy or in conjunction with CABG has been reported compared to that of TMR with a CO2 laser. Perioperative mortality ranged from zero to 12%,170,209212 improvements in angina class170,172,209211 and exercise tolerance were noted,210,211 but lack of improvement210,212 or worsening170 in perfusion of the lased areas and in left ventricular ejection fraction was also documented.210,212 Furthermore, Mohr et al did not find any sustained benefits in angina relief 36 months after the performance of TMR with a holmium:YAG laser as sole therapy.212

Percutaneous direct myocardial revascularization has been evaluated in a number of case series and was associated with a periprocedural mortality ranging from zero to 2.9%213,214 and improved time to onset of angina during exercise with improved exercise duration,190,213,214 but again with some conflicting nuclear imaging results that showed either no improvement at 1 and 6 months213,214 or improvement at stress only.215 However, decreased size of the ischemic zone on cardiac MRI was reported by another group,216 whose authors also documented the feasibility of combining percutaneous direct myocardial revascularization with coronary angioplasty and stenting.217,218

Clinical trials Open-label, randomized controlled trials of surgical TMR using a holmium:YAG laser have shown benefit in reducing angina and improving exercise tolerance.219,220 A large, nonblinded trial that involved 275 patients demonstrated that surgical TMR with a holmium:YAG laser was also associated with a significantly higher survival without major cardiac events, and freedom from rehospitalization at one year.220 These surprising results were not supported by any myocardial perfusion differences between TMR and control patients, however. These investigators subsequently evaluated surgical TMR in combination with CABG versus CABG alone (i.e., where an ungraftable myocardial territory was not addressed) and found that the TMR-CABG combination was associated with a trend towards greater survival and freedom from major adverse cardiac events at 1 year, but no difference in angina relief and treadmill improvement.221

The Angina TreatmentsLasers and Normal Therapies in Comparison (ATLANTIC) trial recruited 182 patients who were randomized either to surgical TMR or best medical therapy.222 All patients had an ejection fraction of over 30%, no severe congestive symptoms, and at least one myocardial territory supplied by a normal or mildly diseased graft or coronary artery. Angina class and exercise tolerance were significantly improved in the TMR versus the medical treatment group at all study time points, but on nuclear imaging the changes in the percentage of myocardium with fixed and reversible defects from baseline to the 3-month, 6-month, and 12-month visits did not differ significantly between the two treatment groups. In the TMR group, a small but significant decrease in ejection fraction was noted from baseline to 3 months, and the rate of heart failure or new onset left ventricular dysfunction was higher in the TMR group. As a sequel to the ATLANTIC study, the Potential Class Improvement from Intramyocardial Channels (PACIFIC) trial was conducted to evaluate the efficacy of percutaneous TMR in a total of 221 patients randomized to undergo this modality or receive medical treatment.223 The percutaneous procedure was associated with low morbidity, increased exercise tolerance time, lower angina scores, and improved quality of life.

The only double-blind, randomized controlled trial of any type of direct myocardial revascularization modality is the Direct Myocardial Revascularization in Regeneration of Endomyocardial Channels Trial (DIRECT), whose final results were presented at the 2001 meeting of the American College of Cardiology.224 This multicenter trial conducted by Leon et al examined whether percutaneous myocardial revascularization using a holmium:YAG laser improved exercise duration or angina frequency when compared to medical management. It also evaluated the incidence of major adverse cardiac events; nuclear perfusion imaging was conducted at 6 months. The trial involved 298 predominantly male patients with a mean age of 63 years and preserved LV function who had coronary disease and severe symptoms despite best medical therapy, were no longer candidates for either PTCA or CABG, and had reproducible positive exercise tests associated with reversible ischemia. Patients were randomized into three arms: placebo (sham procedure), low-dose PDMR (1015 channels/zone), or high-dose PDMR (2025 channels/zone). No crossovers were permitted for the full year of follow-up. The trial was essentially negative with respect to all end points, with no differences in angina class and exercise duration among the three groups at baseline, 6 months, or 12 months, although there were statistically significant improvements in each group in terms of 6-month and 12-month to baseline differences for these two end points. The magnitude of ischemia on nuclear perfusion imaging at 6 months also showed no consistent differences that would suggest a therapeutic effect related to percutaneous direct myocardial revascularization. Although it is possible that the results of the DIRECT trial may not be entirely generalizable to surgical TMR, especially if involving a CO2 laser, they are highly suggestive that a strong placebo effect may have plagued most if not all case series and open-label clinical trials in this field, whose results should therefore be viewed with caution and skepticism until efficacy is reevaluated in a blinded and randomized fashion.

At the beginning of the 21st century, while large gains have been made in treating or preventing the most common underlying causes of many forms of heart failure, particularly ischemic vascular disease, the treatment of established heart failure of any cause remains unsatisfactory. Drug treatment with vasodilators and "neurohormonal" antagonists resulted in important but largely marginal gains in mortality and the functional status of many patients with heart failure. In contrast, drugs that markedly improved symptoms and functional status were associated with increased mortality.225 The development of mechanical devices to assist or replace heart function has also had an impact, and indeed has saved lives in patients with end-stage disease.226228 However, much additional development will be necessary to lower patient morbidity and achieve economies of scale before widespread adoption of these devices is possible. Finally, while gene therapy approaches to either the prevention of heart failure (e.g., by inducing angiogenesis in ischemic but viable muscle) or the improvement of heart function directly (e.g., by inhibiting myocyte apoptosis or manipulation of adrenergic signaling) remain promising, none has yet proven safe and effective in humans.229

An alternative approach is to examine the possibility of replacing damaged myocardial cells with either newly formed cardiac myocytes (e.g., either by inducing cardiac myocytes to divide or by de novo generation of myocytes from a stem cell population), or by adding other cell types to damaged heart tissue that may prevent further ventricular dilation and deleterious "remodeling." In this section, we review the current literature on "cell therapy" approaches to the treatment of heart failure, some of which have now entered clinical trials. While a detailed description of earlier literature on this topic is beyond the scope of this chapter, the reader is referred to a comprehensive review of the data up to 2000 by P.D. Kessler and B.J. Byrne.230

Skeletal Myoblast Implants for Heart Failure

Cardiac myocytes in adult hearts may have a limited ability to undergo DNA synthesis and cytokinesis.231,232 Moreover, recent data document that a reservoir of pluripotent stem cells, whether derived from the bone marrow or resident in tissues of adult humans, may be able to repopulate damaged cardiac muscle (vide infra). Regardless, none of these mechanisms is normally sufficient to replace the function of more than a small number of damaged myocytes. In contrast, fetal or neonatal myocytes, which retain a substantial proliferative capacity, can "replace" damaged cardiac tissue, by integrating into areas of myocyte necrosis and forming gap junctions with adjacent viable cells.233236 For obvious reasons, use of fetal or neonatal human tissue for this purpose is impractical, while transplanting fetal cells from other species remains problematic due to issues of tissue rejection and the possibility of infectious agents crossing a species barrier.

The cell type that has been most extensively examined to date for "repair" of injured cardiac muscle is immature skeletal muscle cells known as skeletal myoblasts, a lineage-restricted form of stem cell that serves as a reservoir of cells for injured striated muscle or muscle placed under increased mechanical stress. Chiu et al were among the first to systematically examine the effects of skeletal myoblast transplantation, documenting in both canine and rat models that transplanted skeletal myoblasts survived in scar tissue created by cryoinjury.237,238 Field et al documented the engraftment of an immortalized skeletal myocyte cell line (C2C12 cells) into cardiac muscle, including the formation of intercalated discs.233,234,239242 Murry et al also demonstrated that myoblasts derived from fetal skeletal muscle would integrate into cardiac muscle damaged by cryoinjury. The transplanted myoblasts altered their typical fast-twitch muscle phenotype to include some slow-twitch, ?-myosin heavy chain (?-MHC)containing fibers as well.

Since slow-twitch fibers are much less resistant to fatigue, this held the promise that autologous skeletal myoblasts from juvenile or adult humans might also exhibit the ability to "adapt" to the microenvironment within the human heart. However, although undifferentiated skeletal myoblasts in vitro express connexin 43, which facilitates formation of electromechanical ("gap") junctions with cardiac myocytes in tissue culture, connexin 43 was rapidly downregulated when myoblasts were implanted in vivo, making it unlikely that skeletal myoblasts would spontaneously integrate with cardiac myocytes in situ in the heart.235,242,243 Indeed, when injected into normal cardiac muscle in a syngeneic rat model, skeletal myoblasts initially engrafted and adopted a slow-twitch phenotype, but clearly did not adopt a characteristic cardiac myocyte phenotype. Furthermore, again, when injected into normal cardiac muscle, the skeletal muscle grafts never expressed intercalated disc proteins such as N-cadherin and connexin 43, and typically atrophied over time.244,245

Taylor et al have reported the most detailed hemodynamic assessments of the consequences of implantation of autologous skeletal myoblasts into the injured heart.246249 Autologous skeletal myoblast grafts were examined in rabbit hearts scarred by cryoinjury, a model that replicates with some fidelity the hemodynamics observed in infarcted human hearts. Successful engraftment of skeletal myoblasts into infarcted muscle resulted in improved ventricular compliance with limited diastolic "creep" or progressive remodeling and dilatation of infarcted ventricular muscle, and appeared to be superior to a nonmyocyte cell type (i.e., fibroblasts).250 These authors also performed a comprehensive analysis of myocardial function in the in situ rabbit heart. In addition to the improvement in diastolic strain and stiffness, Taylor et al noted a modest improvement in systolic function in some animals, although this remains a controversial finding.249,251

Apstein et al252 also examined the effects of transplantation of skeletal myoblasts from syngeneic donor animals into adult rat hearts following a myocardial infarction in an ischemia-reperfusion model. Thus, this model differs from that of Taylor by using a more clinically relevant mode of injury, and by using myoblasts of fetal origin, which may have had a higher replicative potential, and also may integrate more readily into adult myocardium. Using echocardiographic analysis of the rat heart in situ, and ex vivo Langendorff retrograde (blood) perfused heart function analysis, the data indicated both less global ventricular dilatation and improved ex vivo systolic pressure generation in hearts that had received myoblast implants (Figs. 27-12 and 27-13). Interestingly, exercise tolerance (i.e., running time on a treadmill in animals conditioned to perform the test) was also enhanced in animals that had been treated with myoblast injections. Although the use of fetal skeletal myoblasts might have confounded the analysis of these data, this laboratory has repeated these studies using skeletal myoblasts from adult syngeneic donor animals, with similar results (Apstein C, Liao R, et al: manuscript in preparation).

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FIGURE 27-12 Systolic pressurevolume relationship at 6 weeks following cell transplantation. Data are shown for three groups of animals in the study by Jain et al, which compared the ex vivo systolic pressure volume relationship among hearts from control, noninfarcted hearts (dashed line), control infarcted hearts (dark boxes), and infarcted hearts that received injections of fetal skeletal myoblasts (light boxes).252*, p p Reproduced with permission from Jain M, DerSimonian H, Brenner DA, et al: Cell therapy attenuates deleterious ventricular remodeling and improves cardiac performance after myocardial infarction. Circulation 2001; 103:1920.)


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FIGURE 27-13 Skeletal myoblast survival following transplantation. Immunohistochemical analysis of skeletal myoblast survival in the infarcted hearts of rats 9 days following cell injections. Panels A-C show trichrome staining at progressively increasing magnification in which fibrotic tissue stains blue, and viable muscle stains red (bars represent 1 mm, 200 ?m, and 100 ?m, respectively). Panels D-F show staining for myogenin, a skeletal musclespecific transcription factor.

Another group that has contributed extensively to this literature is the laboratory of Weisel et al, beginning with a report in 1996 on the hemodynamic effects of fetal rat cardiac myocytes implanted into cryoinjured muscle and scar tissue of adult rats.253 Additional studies from this laboratory have compared the effects on cardiac function and angiogenesis of a variety of cell types, including fetal rat enteric smooth muscle cells, fetal and adult skeletal myoblasts, and skin fibroblasts, among other cell types.254,255 As expected, not all cell types contributed equally to the maintenance of systolic and diastolic function, and animals that received injections of enteric smooth muscle cells or skin fibroblasts showed no significant improvement over controls.255 Importantly for the design of clinical trials of cell therapies, in a study in an adult rat heart cryoinjury model using syngeneic fetal cardiac myocytes, in which cells were given either immediately, or at 2 weeks or 4 weeks following injury, Li et al determined that neonatal myocytes transplanted immediately (i.e., at the time of injury) could not be detected at 8 weeks, nor did these "0" time point transplanted hearts perform better that control animals that had received injections of culture medium alone. Animals transplanted at 2 weeks after cryoinjury exhibited the greatest functional improvement.256 The presumed reason for the absence of benefit of myocytes transplanted immediately following injury was the presence of a robust inflammatory response in the recently infarcted muscle, a process that takes a week or more to completely resolve in rodents (this observation contrasts with results obtained with immediate injection of some stem cell populations, as discussed below).

Finally, although it is often assumed that skeletal myoblasts in these preparations are the "active" cell type responsible for the diastolic and possibly systolic functional improvements observed in hearts that had received transplants, this remains a hypothesis. In all the experiments described above that used skeletal "myoblast" preparations, other cell types (mostly fibroblasts) account for the large minority or even a majority of the cells injected, depending on the preparation. Apstein et al have directly compared relatively pure populations of fibroblasts with skeletal myoblasts, both prepared from adult rat tissues, in their rat infarct/reperfusion model of cardiac injury. When examined at 8 weeks following the infarct, as determined by both echocardiography and Langedorff retrograde (blood) perfusion hemodynamics, both cell types yielded nearly identical improvements in cardiac function, when compared to infarcted control hearts that received culture medium alone (Apstein C, Wentworth BM, Liao R: manuscript in preparation).

Perhaps the most extensive experience to date with skeletal myoblast transfer for the treatment of heart failure has been accrued by the laboratory of Menasche et al. In a series of reports, they have documented that syngeneic skeletal myoblasts from neonatal rats were equivalent to fetal cardiac myocytes in their ability to delay maladaptive ventricular remodeling.257,258 Although neither group of cells is directly relevant to the currently proposed clinical trials in humans, these results did address whether fetal cardiac myocytes (which retain a significant proliferative capacity compared to adult cardiac myocytes, and can integrate with surviving myocytes in the injured myocardium) were clearly superior to skeletal myoblasts. The answer is: apparently not, at least based on these data.

In one of the first rodent experiments to use autologous skeletal myoblasts (analogous to currently ongoing trials in humans), Menasche et al also documented an improvement in cardiac function in animals that received myoblast injections compared to animals that received injections of cell culture medium alone.258 In these experiments, on average 3.5 x 106 cells were injected, a "dose" that when adjusted for heart size approximates that being used in their clinical trials. Note however that, once again, only approximately 50% of the cells were desmin positive (a characteristic of muscle, as opposed to fibroblasts), again raising the issue of the relative contribution of skeletal myoblasts versus other cell types in these primary cultures.

While encouraging, these data begged the question of whether the appropriate comparisons were being done in animal studies in which skeletal myoblast implantation was compared to untreated control animals. Since virtually all heart failure patients now receive at a minimum an angiotensin-converting enzyme (ACE) inhibitor, Menasche et al next asked whether skeletal myoblast therapy added anything in addition to current medical therapy for heart failure.259 The rat infarct model was a relevant animal model in which to test this hypothesis, since proof-of-concept animal studies validating the survival benefit of ACE inhibitors (ACEI) in heart failure had first been done in the rat by Pfeffer et al (reviewed in Bristow et al225). Menasche et al259 studied 4 groups of animals (n = 99 animals): MI controls, MI with ACEI, MI with myoblast injections, and MI with myoblast injections and ACEI. Although mortality was not a primary end point at 8 weeks of follow-up, there was a clear separation of groups based on 2D echocardiography. Control animals that received injections of culture medium alone exhibited a continuing decline in ejection fraction (to an average of 19%) compared to either ACE inhibitortreated or myoblast-treated animals (ejection fractions of 32% vs. 37%, respectively). The animals that received both ACEI and myoblast injections had an average ejection fraction of 44%, clearly indicating a biological andif transferable to humans clinically meaningful improvement (Fig. 27-14).259

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FIGURE 27-14 Heart failure, ACE inhibitors, and skeletal myoblast injections. The data shown are the left ventricular ejection fractions (LVEF) for four groups of animals at three time points: baseline (BL), 1, and 2 months: (1) control infarcted animals; (2) infarcted animals treated with myoblast injections alone; (3) infarcted animals treated with ACE inhibitors alone; and (4) infarcted animals treated with both skeletal myoblast injections and ACE inhibitors. *, p = .0048 vs. ACEI; #, p , p = .0084 vs. skeletal myoblasts; ** p p Reproduced with permission from Masferrer JL, Koki A, Seibert K: COX-2 inhibitors: a new class of antiangiogenic agents. Ann NY Acad Sci 1999; 889:84.)

To validate whether the observations made in rodents were applicable to larger animals, Menasche et al have also examined the effect of autologous skeletal myoblasts in an ovine circumflex artery embolization infarct model. Cells were injected through a lateral thoracotomy 2 weeks following the infarct, with injections both within and surrounding the infarct zone. Although the number of animals was relatively small, and the primary end point for most animals was just 4 months, there was a statistically significant improvement in ejection fraction and end-diastolic volume in the animals that received myoblasts, when compared to control sheep.260

In separate studies in rodents, two groups have independently shown that transplantation of skeletal myoblasts also can improve cardiac function in animals with cardiomyopathies not of ischemic origin. Scorsin et al261 demonstrated that direct intramyocardial injection of fetal murine cardiomyocytes improved LV function compared to medium-injected control mice, as measured by echocardiography, in animals with heart failure induced by doxorubicin, an anthracycline antibiotic widely used in cancer chemotherapy.261 Data similar to these were subsequently published by Weisel et al.262 Neonatal ventricular myocytes were injected directly into the hearts of hamsters with a cardiomyopathy due to an abnormal delta-sarcoglycan gene. Although both sets of experiments were performed on animals with distinctly different global cardiomyopathies, ventricular function, as measured in situ by fractional shortening on echo261 or on ex vivo Langendorff isolated perfused heart functional analysis,262 was improved in both experimental models following injections of neonatal cardiac myocytes when compared to controls, at least during the time course of these experiments (1 month for both sets of experiments). Once again, however, it is not clear that cardiac myocytes are essential for modifying the progressive decline in cardiac function in the cardiomyopathy model, since Weisel et al noted that smooth muscle cells harvested from the vas deferens of cardiomyopathic hamsters and implanted into the hearts of recipient animals of the same hamster strain maintained smaller ventricular dimensions and better contractile function than controls.262

Similar data have been obtained by Suzuki et al263 in a rat model of doxorubicin toxicity, in which syngeneic neonatal rat skeletal myoblasts were injected in a heterotopic cardiac transplant model, in which the transplanted heart was obtained from rats treated for 2 weeks with high doses of the anthracycline. Once again, function of the transplanted heart was improved compared to controls, as measured by end-of-life Langendorff perfusion studies. One important difference in the experimental protocol employed by Suzuki et al was that the transplanted cells (approximately 106 cells) were injected into the coronary arteries using a Langendorff retrograde perfusion technique. Presumably a proportion of the injected cells were trapped in the microcirculation of the heart, and subsequently migrated into the surrounding muscle, a technique that this group had previously demonstrated resulted in successful cell transplantation.264

Enhancing the Survival of Transplanted Cells

Suzuki et al have also investigated several methods that might enhance survival of cells transplanted into ischemic muscle. Reasoning that exposing cells ex vivo to increased temperature (i.e., to a "heat shock" stimulus, induced by incubation at 42?C for 60 minutes) might enhance graft viability, they demonstrated greater survival of heat-shocked L6 rat myoblasts after 28 days, as judged by staining for ? galactosidase (used to track the injected cells), compared to untreated myoblasts.265 Carrying this line of experimentation to its logical next step, this group then demonstrated that ex vivo transfection of intact rat hearts with a protein known to play an important role in the heat shock response, HSP70, encoded within a viral/liposome complex, resulted in an improvement in myocardial function following ischemic stress compared to control hearts.266 This group has also employed skeletal myoblasts that had been transfected with a gene encoding a protein that could have enhanced the function or survival of implanted myoblasts. Rat skeletal myoblasts transfected with connexin 43, a gap junction protein, enhanced myotube development in vitro (i.e., the cells adopted a differentiated phenotype more readily).267

With regard to survival of transplanted cells, particularly in animal models of coronary ischemia, one primary determinant will be the blood supply to cells transplanted into an ischemic environment. Weisel et al have shown that implantation of (allogeneic) aortic endothelial cells in cryoinjured rat hearts resulted in increased regional blood flow as measured by microsphere analysis,138 suggesting that cell transplantation alone induces at least a moderate angiogenic response. Suzuki et al have transfected skeletal myoblasts with a vascular endothelial growth factor (VEGF) type-A isoform (VEGF-A165) ex vivo, which were then injected into the peri-infarct border zone of rats hour following LAD occlusion.268 This VEGF-A isoform retains the heparin binding motif (unlike VEGF-A121) and has been demonstrated to induce angiogenesis in a number of animal models (clinical trials with this reagent are currently ongoing 269). Infarcted animals treated with myoblasts that had been transfected ex vivo with VEGF-A165 had a reduced mortality rate at 28 days, and a significant reduction in infarct size, possibly due in part to an increased angiogenic response, as judged by counting capillary density (a relatively insensitive technique for detecting angiogenesis) (Fig. 27-15).268 Similar data were reported by Li et al following ex vivo transfection of cardiac myoyctes and fibroblasts with plasmids encoding VEGF-A165.270

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FIGURE 27-15 Ex vivo transfection of skeletal myoblasts with a VEGF-A isoform. Suzuki et al tested the hyopothesis that ex vivo transfection of primary cultures of skeletal myoblasts isolated from juvenile rats with an angiogenic genein this case, the VEGF-A165 isoformwould induce a greater angiogenic response than myoblast therapy alone, and thereby improve myocardial contractile function. Panels A and B illustrate sections from hearts treated with (A) myoblasts alone or (B) myoblasts treated ex vivo with VEGF-A165. Scale bar = 50 ?m. (C) Capillary density is illustrated for animals treated with injections of culture medium alone (i.e., no cells), skeletal myoblasts alone (ControlCell), and skeletal myoblasts transfected with VEGF-A165 (VEGFCell).*, p p Reproduced with permission from Suzuki K, Murtuza B, Smolenski RT, et al: Cell transplantation for the treatment of myocardial infarction using vascular endothelial growth factor-expressing skeletal myoblasts. Circulation 2001; 104:1207.)

One caveat to this approach has been raised by the work of Blau et al. They examined the effects of high levels of VEGF-A expression observed in mice whose hearts had been implanted with primary cultures of murine skeletal myoblasts that had been transfected ex vivo with the murine VEGF-A gene driven by a retroviral promoter.271,272 Animals that received the VEGF-A gene-transfected myoblasts all exhibited highly vascularized intramural tumors. It is presumed that the continuous high levels of all VEGF-A isoforms within the hearts of these mice, possibly the consequence of using a retroviral promoter, were the reason for the observed increase in angioma formation. There have been no reports of similar tumors in humans treated with recombinant proteins, or either plasmid or viral vectors encoding a VEGF-A isoform, although most of the studies reported to date are relatively small Phase I and Phase II clinical trials. Despite this caveat, the current safety profile of this technology is encouraging. Moreover, it is reasonable to assume that with the development of tissue-specific and regulatable promoter technologies, it will be possible in the future to calibrate the amount of transgene expressionand therefore target protein levelswithin a given cell type and tissue. This should enhance the safety of adjunctive gene therapy, whether given in vivo or ex vivo, when applied to cell transplantation protocols.

Finally, an alternative approach to the induction of angiogenesis by cell therapy is to inject relatively undifferentiated autologous bone marrow cells into the myocardium. Chiu et al documented increased neovascularization in infarcted rat hearts following injection of a population of "mesenchymal" bone marrowderived cells from syngeneic rat donors.238 Fuchs et al also demonstrated that endomyocardial injections of autologous bone marrow resulted in enhanced myocardial perfusion in a porcine ameroid constrictor model, as judged by microsphere analysis.273 The presumptive mechanisms include the likelihood that a number of injected cells secrete cytokines such as GM-CSF that are known to facilitate angiogenesis, as well as the presence of a population of "endothelial progenitor cells" or "EPCs" present in relatively undifferentiated bone marrow preparations, which may undergo amplification and differentiation in the ischemic intracellular milieu of the healing, postinfarct scar tissue, and contribute to angiogenesis and arteriogenesis.274,275

Inducing Proliferation of Adult Cardiac Myocytes

Although recent data, noted above, support the hypothesis that cardiac myocytes may possess a limited capacity for cell division (cytokinesis), the inability of the large majority of myocytes within the heartand the limited recruitment of pluripotent stem cells from within the heart itself or other tissue sourcesto contribute to repair of the injured myocardium is an important cause of morbidity and mortality. A number of approaches have been attempted to induce "terminally differentiated" cardiomyocytes to divide, but most myocytes typically undergo programmed cell death (apoptosis) when "forced" into the cell cycle. One approach to addressing this problem that has provided important insights into the signaling pathways that regulate cardiomyocyte division has been developed by Field.276,277 This laboratory has reported on the actions of several "tumor suppressor" cell cycle regulatory genes,278 and had identified a 193-kDa protein, in addition to other known tumor suppressor proteins within cardiac myocytes (such as p53 and p107), which participated in the induction of apoptosis following exposure to agents such as simian virus 40 (SV40) large T antigen, or the adenoviral E1A oncoprotein. In a screening assay to determine which portions of the p193 protein were important for the bioactivity of this tumor suppressor protein, Field et al generated a truncated mutant termed 1152stp that, instead of inducing apoptosis, enhanced myocyte proliferation, particularly in combination with a known prosurvival mutant of p53 (termed CB7). Cotransfection of both mutant proteins into embryonic stem (ES) cell-derived cultures of beating cardiomyocytes prevented apoptosis in cells exposed to a potent growth stimulus, such as adenoviral E1A. Thus, despite the recent demonstration that stem cells derived from bone marrowand possibly a reservoir of cells within the myocardium itself, reviewed belowmay contribute to cardiac repair following injury, the controlled induction of DNA synthesis, karyokinesis, and cytokinesis of adult cardiac myocytes in situ in the adult heart remains a promising if daunting therapeutic challenge.

Stem Cell Approaches to Cardiac Repair and Regeneration

The ability of the human heart to repair damaged muscle after injury is limited after the first year or two of life. This had been attributed to an irreversible exit from the cell cycle in "terminally differentiated" cardiac myocytes, although the underlying mechanism(s) limiting further myocyte replication remained unclear. However, recent evidence from a number of sources has cast doubt on this long-held tenet of cardiac cell biology. As will be discussed briefly below, cardiac "regeneration" following injury may be possible using several approaches.

Since myocardial ischemia and infarction due to advanced coronary atherosclerosis are among the commonest causes of death and disability in adult humans, the ability to induce new blood vessel formationangiogenesisparticularly when accompanied by progressive vascular remodeling that contributes to the growth of larger arterioles and conduit vessels, a process termed arteriogenesis,279281 would be an important advance on current therapeutic alternatives. "Angiogenesis" induced by cytokines such as isoforms of the VEGF-A gene or members of the fibroblast growth factor (FGF) family, whether given as gene therapy or as recombinant proteins, or transcription factors such as hypoxia-inducible factor-1 (HIF-1), all have potential to induce new blood vessel growth, and clinical trials employing each of these approaches are now underway.269,274 There is now good evidence that further remodeling of new blood vessels into large-caliber vessels is dependent upon recruitment of inflammatory cells279281 and a discrete lineage-restricted bone marrow stem cell population termed "angioblasts,"282 or "endothelial progenitor cells" (EPCs) by Asahara et al.275,283 There also is increasing evidence that recruitment of EPCs by cytokines, or ex vivo expansion of EPCs, followed by direct injection into ischemic tissue or by systemic administration, accelerates new blood vessel formation and minimizes cardiac damage.282,283

Following large amounts of tissue damage due to ischemia or infarction, even restoration of adequate blood flow will not restore normal cardiac function and is often insufficient to prevent subsequent ventricular remodeling. However, as noted above, the received wisdom that the heartlike the brainis a "terminally differentiated" tissue, incapable of substantive repair with restoration of function, must be reevaluated based on recent data (for both organs). It is well known, of course, that cardiac myocytes in juvenile and adult mammals, including humans, can undergo a limited degree of DNA synthesis and even karyokinesis (nuclear division), but the capacity for cytokinesisactual cell divisionwas believed to be quite limited or nil. This assumption has been challenged by a wealth of recent evidence, reviewed below.

Perhaps the most striking example from the recent literature is in a specific strain of mouse known as the "MRL" strain. This strain has been well known for years to have a remarkable ability to heal wounds, a capacity that has been likened to that of amphibians. To determine whether MRL mice also have the capacity to regenerate injured cardiac muscle, Haber-Katz et al284 induced full-thickness right ventricular infarctions using a cryoprobe, and compared the response in this mouse strain to a control mouse strain (C57Bl/6). Remarkably, within 60 to 90 days following the injury, the MRL mice had essentially completely regrown replacement, vascularized muscle with restoration of normal right ventricular function, while control animals went on to develop a thinned, scarred, and dysfunctional right ventricular remnant. Note that myocardial infarcts in the mouse, whether due to cryoinjury or ischemia, undergo a repair process and induce progressive ventricular remodeling elsewhere in the ventricle that is similar to that observed in humans, although the time course is "telescoped" into days to weeks, as opposed to months or years in patients. This strain is now being intensively examined for clues to its remarkable ability for repairing damaged tissues.

The data reviewed above in the MRL strain do not mean that human hearts do not exhibit any capacity for cardiac myoycte replacment following injury. Anversa et al have demonstrated that, following a myocardial infarction, cardiac myocytes in adult human hearts (harvested at autopsy) express the nuclear antigen Ki-67, a marker limited to cells undergoing replication. Moreover, myocytes caught in the act of cell division, with clearly identifiable mitotic spindles and division into daughter cells, could also be identified. Ki-67positive cells were most readily identifiable in the peri-infarct region (i.e., the "border zone"), but could be identified, at lower frequencies, in areas of the heart remote from the infarct. In contrast, hearts harvested from subjects who had died from noncardiac causes exhibited little or no Ki-67.

While these observations suggested that myocytes in situ within the heart were being induced to replicate, albeit at a rate inadequate to replace the damaged tissue, there was another, not mutually exclusive, possibilitythat myocytes were being formed by a reservoir of pluripotent stem cells recruited from within the heart itself and/or other tissues, such as bone marrow. In male heart transplant recipients, for example, who had received a heart from a female donor and who later came to autopsy, the transplanted hearts were found to contain a number of cell types, including fully differentiated cardiac myocytes, that contained a "Y" chromosome, indicating that that particular cell must have been of host origin.285 While it is possible that at least some of the Y-chromosomepositive cardiac cells had migrated from the atrial remnant of the recipienta hypothesis for which Anversa et al provide intriguing evidencesome proportion of the cells had likely been recruited from other tissues, with the bone marrow the most obvious candidate.

In support of this hypothesis, recent data in experimental animals and humans document that bone marrowderived cells in recipient animals can populate organ grafts. Krause et al286 demonstrated that female mice, following doses of radiation sufficient to ablate the bone marrow and subsequently infused with bone marrow constituents from syngeneic male donors, had cells that exhibited Y-chromosome positivity in virtually all tissue beds, when examined several months later. Similar data have been generated by Goodell et al,287 who performed a myocardial infarction in female mice that had previously been lethally irradiated and subsequently reconstituted with bone marrow, once again from male donor mice. Although the percentage of male donorderived myocytes in the hearts of the female bone marrow recipient mice was low (about 0.02%), they were easily detectable. That this process clearly occurs in humans has been demonstrated by Grimm et al,288 who examined male recipients of kidney allografts from a female donor, who also documented the presence of Y-chromosomepositive cells in all cellular constituents of the kidney, including blood vessels, mesangial cells, endothelial cells, and tubular epi-thelium.

This phenomenon of bone marrowderived pluripotent progenitor cells continuously repopulating tissues may have important implications for other disease processes of relevance to cardiac and vascular surgeons, such as transplant arteriopathy. Long-term survival of, for example, cardiac allografts is limited by the development of an arteriopathy that involves neointimal thickening and gradual occlusion of coronary arteries and arterioles. The source of the neointimal cells has been presumed to be inflammatory cells of host origin as well as vascular smooth muscle cells derived from the media of vessels within the allograft itself. However, Mitchell et al,289 in a murine model of aortic transplant arteriopathy, in which aortas from a standard strain of laboratory mice were transplanted into HLA mismatched mice that also constitutively express the marker gene ?-galactosidase ("?Gal"), demonstrated that virtually all the smooth muscle cells populating the neointima of the aortic allograft were of recipient origin; i.e., they were all ?Gal-positive, indicating that the neointimal cells must have been of host originpresumably from bone marrowand not due to increased replication of smooth muscle cells of donor origin within the graft (Fig. 27-16). 289

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FIGURE 27-16 Recipient (presumably bone marrow origin) of intimal hyperplasia in aortic allografts. To determine whether cells contributing to the intimal hyperplasia characteristic of solid organ transplants were of graft or host origin, Mitchell et al transplanted portions of the thoracic aortic from one strain of mice, BALB/c ("B/c"), into another MHC mismatched strain, C57Bl/6, and vice versa. The C57Bl/6 mice were also ROSA 26 ?-galactosidase transgenic animals ("B6 ROSA26"); the cells from those animals can be tracked because they stain blue with the appropriate reagents (X-gal). Sections of the native aortas of B6 ROSA26 mice and B/c mice are shown in (A) and (B), respectively. Note the absence of any neointimal proliferation in either panel. Panels (C) and (D) show a B6 ROSA 26 aorta transplanted into a B/c recipient, and a B/c aorta transplanted into a B6 ROSA 26 recipient. Note that in both cases the robust neointimal proliferation present in panels (C) and (D) exhibited the phenotype of the recipient animal; i.e., the cells comprising the neointimal proliferation did not originate from cells migrating into the neointima from the media of the graft, but were of host originpresumably recruited from the bone marrow of the recipient animal. (Reproduced with permission from Shimizu K, Sugiyama S, Aikawa M, et al: Host bone-marrow cells are a source of donor intimal smooth-muscle-like cells in murine aortic transplant arteriopathy. Nat Med 2001; 7:738.)

What then is the potential of pluripotent stem cells, whether of marrow origin or recruited from peripheral tissues, for inducing repair of damaged cardiac tissue? Once again, recent data provide reason for optimism. Anversa et al,290 in a murine myocardial infarction model, examined whether a specific subset of bone marrow derived cells that were negative for common hematopoietic lineage markers (i.e., "lin-"), but positive for stem cell factor (i.e., "c-kit+"), when injected within hours of a myocardial infarction in mice, would induce the formation of new cardiac muscle replete with regenerated cardiac myocytes, blood vessels, and stromal cells (Figs. 27-17 and 27-18). A similar bone marrowderived cell population that also contained "lin-" cells, but was, however, negative for c-kit (i.e., "c-kit-"), did not result in substantive cardiac repair. The injected cells were tracked over time using bone marrow cells from syngeneic donor mice that were sex mismatched (i.e., male donors into female recipients) and that constitutively expressed enhanced green fluorescent protein (EGFP), providing an additional marker with which to track the injected cells. Interestingly, injections of lin-, c-kit+ cells were ineffective if given after the reparative inflammatory response had begun in the infarcted tissue, typically after 12 hours or longer following the infarction.290

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FIGURE 27-17 Bone marrow-derived cells reconstitute infarcted cardiac muscle. (A) In a murine model of myocardial infarction, Orlic et al injected a specific subset of bone marrowderived cells (lin-, c-kit+) into the newly infarcted muscle shortly after the infarct was created directly. Compared to control animals that received a different subset of bone marrowderived cells (lin-, c-kit-) whose infarcts exhibited only normal healing (E), animals that received injections of lin-, c-kit+ cells exhibited evidence of reconstitution of the infarct area with newly formed cardiac myocytes, vasculature, and stromal cells (AD). The asterisk denotes necrotic myocytes. Arrowheads note the placement of lin-, c-kit+ cells. VM = viable muscle; MI = myocardial infarction. Magnification is x 12 (A), x 25 (C), x 50 (B, D, and E); red, cardiac myosin; green: propidium iodide labeling of nuclei. (Reproduced with permission from Orlic D, Kajstura J, Chimenti S, et al: Bone marrow cells regenerate infarcted myocardium. Nature 2001; 410:701.)


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FIGURE 27-18 Ventricular function was analyzed in sham-operated mice (SO; n = 11), control mice that had a myocardial infarction but received control injections of (lin-, c-kit-) cells (MI; n = 6), and in mice that had had a myocardial infarction and received injections of (lin-, c-kit+) bone marrowderived cells (MI+BM; n = 9), Hemodynamic testing was performed prior to sacrifice on anaesthetized animals with a Millar microtip pressure transducer in the ventricular cavity (*, p p Reproduced with permission from Orlic D, Kajstura J, Chimenti S, et al: Bone marrow cells regenerate infarcted myocardium. Nature 2001; 410:701.)

An extensive discussion of the cell biology of mammalian stem cell populations is beyond the scope of this chapter. However, the advances in our understanding of the phenotypic plasticity of many "committed" stem cell lineages are also requiring the construction of new paradigms.291,292 The phenotypic "barriers" that are assumed to exist among, for example, the mesenchymal, hematopoietic, and neurologic lineages have also yielded to new data, as highlighted by Moore's evocative 1999 editorial entitled "Turning brain into blood."293 In addition to bone marrow, Verfaille et al have recently shown that pluripotent stem cells also reside in bone marrow and in other tissues in adult humans, cells that exhibit a remarkable ability to assume any of the three principal germline lineages,291 which she has termed "multipotential adult progenitor cells" or "MAPCs."

Whether MAPCs are indeed a unique reservoir of pluripotent stem cells in the adult animal, or represent just a more thorough understanding of the phenotypic plasticity of the conventional stem cell lineages, will require additional experimentation. For example, Condorelli et al294 have demonstrated that even apparently differentiated cells of one "traditional" lineageendothelial cells, of hematopoietic lineage origincan be induced to form a cell type of mesenchymal lineagecardiac myocytesunder specific culture conditions in vitro or when injected into injured, ischemic myocardium. Importantly, MAPCs isolated from both juvenile and adult human bone marrow could be propogated through at least 80 population doublings in vitro, without any sign of senescence, indicating that this reservoir of stem cells in the adult animal could provide a renewable resource for facilitating tissue repair.292

Indeed, our understanding of stem cell biology is now expanding so rapidly that it is unclear whether a cell phenotype such as that described for MAPCs is importantly different than bone marrow or other tissue-derived adult stem cells that can undergo differentiation into a number of phenotypes, such as "mesenchymal" stem cells, a stem cell phenotype that had been reported earlier.295 Toma et al296 have documented that human bone marrowderived mesenchymal stem cells will engraft in infarcted hearts of immunocompromised (CB17 SCID/beige) adult mice when injected directly into ventricular muscle. A portion of the injected human cells became immunohistochemically indistinguishable from the resident murine cardiac myocytes, expressing a number of (human) cardiac myocyte-specific markers.296

Alternatively, Ogawa et al have demonstrated that "mesenchymal" stem cells, isolated from murine bone marrow, can undergo differentiation into a cardiac myocyte-like phenotype after exposure to a demethylating agent, 5-azacytidine.297,298 Although these cells expressed a number of characteristics of cardiac myoyctes, including the expected complement of adrenergic and muscarinic receptors, among other markers, the safety of this approach, which uses a demethylating agent to induce expression of cardiac-specific genes, may limit its application in studies in humans.

Although the data reviewed above suggest that it may be possible to repair damaged myocardium, as noted by Anversa et al in the infarcted mouse heart, the timing of therapeutic interventions may be critical to the repair process. Since collection and ex vivo expansion of autologous stem cell populations would of necessity take time, certainly days and perhaps weeks, this approach may have practical limitations for limiting, for example, the extent of tissue necrosis following a myocardial infarction accompanied by pharmacologic, percutaneous, or surgical revascularization. Interestingly, cells of the "mesenchymal" lineage appear to induce a remarkable degree of immunologic tolerance; i.e., they appear not to elicit rejection as allografts or even as xenografts.299,300 While the mechanism(s) by which selected subsets of adult stem cells induce tolerance remains unknown, if this were true, it would permit the development of "off-the-shelf" stem cell products, thereby eliminating the 2- to 4-week period required to harvest, culture, and expand lineage-restricted autologous stem cells (such as skeletal myoblasts). Regardless, these new data suggest that repair of diseased or damaged tissues by cellular therapeutics holds substantial promise for the future.

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