Chapter 27. Transmyocardial Laser Revascularization and Extravascular Angiogenetic Techniques to Increase Myocardial Blood Flow

Despite the success of medical therapy, percutaneous coronary interventions (PCIs), and coronary artery bypass grafting (CABG) in the treatment of coronary artery disease (CAD), there are a significant number of patients with refractory angina owing to diffuse CAD that is not amenable to PCI or CABG. This severe CAD can lead to incomplete revascularization and is noted to occur in up to 25% of CABG surgery.1 This incomplete revascularization is a powerful independent predictor of operative mortality and perioperative adverse events.1,2 Additionally, the presence of diseased but nongrafted arteries carries a poor prognosis and poses a significant negative influence leading to an increased incidence of death, recurrent angina, myocardial infarction (MI), and the need for repeat CABG.3,4

Strategies to treat patients with end-stage coronary disease have sought to create or enhance myocardial angiogenesis. Such techniques include TMR as well as protein-, gene-, and cell-based therapies. TMR was founded, in part, on previous methods of providing direct perfusion to the myocardium. Prior attempts at direct perfusion were based on Wearn's description of sinusoids that allowed blood to flow directly from the ventricle into the myocardium.5 These arterioluminal connections provide perfusion in more primitive vertebrate hearts and occur clinically in children with pulmonary atresia, an intact ventricular septum, and proximal obstruction of the coronary arteries. Sen and colleagues6 used myocardial acupuncture to establish direct perfusion and theoretically to recreate a coronary microcirculation similar to that of the reptilian heart. Additional methods of attempting to improve myocardial blood flow include Beck's creation of a form of superficial angiogenesis as a response to epicardial and pericardial inflammation.7 Combining the acupuncture, implantation, and inflammation techniques, Boffi8 and Borst9 used hollow tubes implanted in the myocardium to establish direct perfusion. Results from all these procedures yielded limited success. The angina relief obtained was not long lasting, was difficult to replicate, and most important, eventually was overshadowed by the ability to perform CABG. The mechanical trauma that resulted in poor long-term patency of myocardial acupuncture was overcome in theory by using a laser to create the channels. Although Mirhoseini and colleagues10 and Okada and colleagues11 pioneered the use of a laser to perform this type of revascularization in conjunction with CABG in the early 1980s, the use of a laser as sole therapy to establish its efficacy required advancements in the technology. The carbon dioxide (CO2) laser used by Mirhoseini had a peak output of 80 W and, therefore, required a significant amount of time to complete a transmural channel. As a result, to perform TMR optimally, the heart had to be chilled and still. Increasing the output of the laser to 800 W allowed TMR to be performed on a beating heart. This breakthrough led to the widespread clinical application of TMR. Since then, more than 25,000 patients have been treated with TMR around the world, and results from individual institutions, multicenter studies, and prospective, randomized, controlled trials have been reported.12–20

The early nonrandomized trials demonstrated that sole-therapy TMR could be performed safely on patients with severe CAD who previously had no options. The significant angina relief seen in such patients led to prospective, randomized, controlled studies to further demonstrate the efficacy of TMR. In these pivotal trials, more than 1100 patients were enrolled and randomized to receive either TMR or medical management as treatment for their severe angina.16–20 The trials employed a 1:1 randomization in which half the patients were treated with laser and those in the control group continued on maximal medical therapy. All patients were followed for 12 months.

Patients

The entry criteria for these studies and for sole-therapy TMR in general are as follows. Patients had refractory angina that was not amenable to standard methods of revascularization, as verified by a recent angiogram; they had evidence of reversible ischemia based on myocardial perfusion scanning; and their left ventricular ejection fractions were greater than 25%.

The typical patient profiles of TMR patients in the randomized, controlled trials are listed in Table 27-1. Because the patients were equally randomized to the medical management group, there were no significant demographic differences between the TMR and control groups for any of these trials. Two different wavelengths of laser light were used. Two studies16,17 employed a holmium:yttrium-aluminum-Garnett (Ho:YAG) laser, and three18–20 used a CO2 laser. The average patient age was 62 years, and most were male (86%). Although there were significant differences in the baseline distribution of patients according to Canadian Cardiovascular Society (CCS) angina class, the majority of the patients were in angina class IV (61%). The ejection fractions for all the patients were mildly diminished at 48 ± 10%. Many of the patients had suffered at least one previous MI, and most had some prior revascularization, CABG, and/or PCI. Two of the trials16,18 permitted a crossover from the medical management group to laser treatment for the presence of unstable angina that necessitated intravenous antianginal therapy for which they were unweanable over a period of at least 48 hours. By definition, these crossover patients were less stable than and significantly different from those who had been randomized initially to TMR or medical management alone.

Operative Technique

For sole-therapy TMR, the patient is placed in a supine position with his or her left side slightly elevated. General anesthesia is established using a double-lumen endotracheal tube or a bronchial blocker to isolate the left lung. Although not mandatory, this facilitates the operation, particularly because most of the patients have pleural and mediastinal adhesions from previous bypass surgery. Additionally, a thoracic epidural catheter can be employed to provide postoperative pain control.

A left anterior thoracotomy in the fifth intercostal space is the usual incision site. Once the ribs are spread by a retractor, the pericardium is opened to expose the epicardial surface of the heart (Fig. 27-1). Care must be taken to avoid previous bypass grafts. The left anterior descending (LAD) artery is identified and used as a landmark for the location of the septum. The inferior and posterolateral portions of the heart can be reached through this incision with a combination of manual traction, placement of packing behind the heart, and as illustrated, with the use of a right-angled laser handpiece. Channels are created starting near the base of the heart and then serially in a line approximately 1-cm apart toward the apex, starting inferiorly and working superiorly to the anterior surface of the heart. Because there is some bleeding from the channels, commencement of the TMR inferiorly keeps the anterior area clear and expedites the procedure. The number of channels created depends on the size of the heart and the size of the ischemic area. Myocardium that is thinned by scar, particularly when the scar is transmural, should be avoided because TMR will be of no benefit in these regions, and bleeding from channels in these areas may be problematic. The thoracotomy then is closed after placement of a chest tube, and in the majority of the cases the patient is extubated in the operating room.

The handpiece in Fig. 27-2 is from a CO2 laser and illustrates one of the differences between the two lasers employed for TMR. The CO2 laser energy is delivered via hollow tubes and is reflected by mirrors to reach the epicardial surface. One-millimeter channels are made with a 15- to 20-J pulse. Firing of the laser is synchronized to occur on the R wave of the electrocardiogram (ECG) to avoid arrhythmias. The transmural channel is created by a signal pulse in 40 milliseconds and can be confirmed by transesophageal echocardiography (TEE). The vaporization of blood by the laser energy as the laser beam enters the ventricle creates an obvious and characteristic acoustic effect on TEE. The Ho:YAG laser achieves a 1-mm channel by manually advancing a fiber bundle through the myocardium while the laser fires. Typical pulse energies are 2 J for this laser at a rate of five pulses per second; 10 to 20 pulses are required to traverse the myocardium. Detection of transmural penetration is done primarily by tactile and auditory feedback.

End Points

The principal subjective end point for all the trials was a change in angina symptoms. This was assessed by the investigator and/or a blinded independent observer. In addition to assigning an angina class, standardized questionnaires such as the Seattle Angina Questionnaire, the Short Form Questionnaire 36 (SF-36), and the Duke Activity Status Index were employed. These tests were used to detect changes in symptoms and quality of life. Objective measurements consisted of repeat exercise tolerance testing as well as repeat myocardial perfusion scans. Patients were reassessed at 3, 6, and 12 months after randomization.

Mortality

Before the randomized studies, mortality rates in the 10 to 20% range12–15 were reported for TMR patients. In the randomized trials, lower perioperative mortality rates were reported ranging from 1 to 5%16–20 One of the important lessons learned from these controlled trials that differs from the earlier studies was a decrease in the mortality when patients taken to the operating room were stable, specifically not on intravenous (IV) heparin or nitroglycerin. When patients were allowed to recover from their most recent episode of unstable angina and were able to be weaned from intravenous medications such that their operation could be performed 2 weeks later, the mortality dropped to 1%.18 The 1-year survival for TMR patients was 84 to 95% and for medical management patients was 79 to 96%. Meta-analysis of the 1-year survival demonstrated no statistically significant difference between the patients treated with a laser and those who continued with their medical therapy. Long-term survival of the randomized patients from two of these studies has been reported. Four-year follow-up from Aaberge and colleagues, in which both the TMR and medical management groups were kept intact, demonstrated a 78% survival for TMR versus 76% for medical management (p = ns).21 In a 5-year follow-up using an intent-to-treat analysis, Allen reported a survival for TMR patients at 65% versus 52% for medical management patients (p = .05).22

Morbidity

Unlike mortality, the exact definition of various complications varied from one study protocol to the next, and therefore, morbidity data are difficult to pool. Nevertheless, the typical TMR patient's postoperative course had a lower incidence of MI, heart failure, and arrhythmias than what has been documented in a similar cohort of patients, those who have reoperative CABG.16–20

Angina Class

The principal reason for performing TMR is to reduce the patient's anginal symptoms. This can be quantified by assessing the angina class before and after the procedure. Angina class assessment was performed by a blinded independent observer in all studies. This was done as the only angina assessment or as comparison with the investigators' assessment. Significant symptomatic improvement was seen in all studies for patients treated with the laser. Using a definition of success as a decrease of two or more angina classes, all the studies demonstrated a significant success rate after TMR, with success rates ranging from 25 to 76% (Fig. 27-3). A meta-analysis of this angina reduction yielded a summary odds ratio of 9.3 (95% CI 4.6–18.5; p < .000001). Significantly fewer patients in the medical management group experienced symptomatic improvement, and the success rate for these patients ranged from 0 to 32%. The seemingly broad range of success is caused by differences among the baseline characteristics of the studies. It is more difficult to achieve a two-angina–class improvement if the baseline angina class is III. Studies that started with most of their patients in angina class III, not surprisingly, showed the lowest success rate. In contrast, the largest success rate for TMR was seen in the trial in which all the patients were in CCS class IV at enrollment. Of note, the medical management group in this study also showed the largest success rate.16 This underscores some of the baseline differences among the studies.

Quality of Life and Myocardial Function

Quality-of-life indices as assessed by the Seattle Angina Questionnaire, the SF-36, and the Duke Activity Status Index demonstrated significant improvement for TMR-treated patients versus medical management in every study. Global assessment of myocardial function by ejection fraction using echocardiography or radionuclide multigated acquisition scans showed no significant change in overall ejection fraction for any of the patients regardless of group assignment or study.

Hospital Admission

Another indicator of the efficacy of TMR was demonstrated in a reduction in hospital admissions for unstable angina or cardiac-related events postprocedure. A meta-analysis of the data provided indicates that the 1-year hospitalization rate of patients in the laser-treated group was statistically significantly less than for those treated medically. Medical management patients were admitted four times more frequently than TMR patients over the year of follow-up.

Exercise Tolerance

Additional functional test assessment using exercise tolerance also was performed in three of the trials.17,19,20 Although the method of treadmill testing differed among the trials, the results demonstrate an improvement in exercise tolerance for TMR-treated patients. Two studies showed an average of 65- to 70-second improvement in the TMR group at 12 months compared with their baseline, whereas the medical management group had either an average of 5-second improvement or a 46-second decrease in exercise time over the same interval.19,20 One additional trial demonstrated that the time to chest pain during exercise increased significantly, and fewer patients were limited by chest pain in the TMR group, whereas the medical management group showed no improvement.20

Medical Treatment

All the studies employed protocols that continued all the patients on maximal medical therapy. For each study, the frequencies and dosages of antianginal and cardiovascular drugs were similar between the two groups at baseline. TMR patients, as a result of their symptomatic improvement, had a reduction in their medication use over the year of follow-up. Because many of these patients used a combination of short- and long-acting nitrates preoperatively, the trials demonstrated a significant decrease in the use of nitrates in TMR-treated patients, whereas the medical management patients showed a slight increase in their nitrate usage. At 1 year, the overall medication use decreased or remained unchanged in 83% of the TMR patients, and conversely, the use of medications increased or remained unchanged in 86% of the medical management patients.18 The significant angina relief seen following TMR was not owing to medication changes or increases for the TMR-treated patients.

Myocardial Perfusion

As stated, myocardial perfusion scans were obtained preoperatively to verify the extent and severity of reversible ischemia. The four largest randomized trials included follow-up scans as part of the study.16–19 These results reflect more than 800 of the patients randomized. The methodology of recording and analyzing these results differed in each study, so it is difficult to pool the data. Nevertheless, review of the results demonstrated an improvement in perfusion for CO2 TMR-treated patients. Fixed (scar) and reversible (ischemic) defects were tallied for both the TMR-treated patients and the medical management groups. One study demonstrated a significant decrease in the number of reversible defects for both the TMR and the medical management patients.19 This improvement in the reversible defects in the TMR group was seen without a significant increase in the fixed defects at the end of the study. However, the number of fixed defects in the medical management group had nearly doubled over the same 12-month interval. Similarly, there was a 20% improvement in the perfusion of previously ischemic areas in the CO2 TMR group of another trial, and in that same trial there was a 27% worsening of the perfusion of the ischemic areas in the medical management group at 12 months.18 There was no difference in the number of fixed defects among the groups at 12 months, nor was there a significant change in the number of fixed defects for each patient compared with his or her baseline scan. The remaining two Ho:YAG studies that obtained follow-up scans showed no significant difference between the TMR and medical management groups at 12 months and no significant improvement in perfusion in the TMR-treated patients over the same interval.16,17

Nonrandomized data previously had demonstrated an improvement in perfusion using dual isotope scanning at 1 and 2 years after CO2 TMR.12 Additionally, using N-13 ammonia position-emission tomographic (PET) assessment, subendocardial perfusion improved significantly compared with subepicardial perfusion after CO2 TMR treatment.23

Long-Term Results

Two reports of long-term follow-up of prospectively randomized patients are available. Similar to the 1-year results, intention-to-treat analyses determined that significantly more TMR than medical management patients continued to experience at least two-class angina improvement from baseline (88% versus 44%; p < .001) or were free from angina symptoms altogether (33% versus 11%; p = .02) at a mean of 5 years.22 In long-term follow-up of the randomized trial that kept both the TMR and medical management groups intact (ie, no crossover), it was shown that angina symptoms still were improved significantly (24% versus 3%, TMR versus medical management; p = .001), and unstable angina hospitalizations were reduced significantly (p < .05) at a mean follow-up of 43 months.21 Follow-up of a series of nonrandomized patients who received TMR and survived long term support these findings.24 At a mean of 5 years and up to 7 years postprocedure, 81% of these patients improved to class II or better, 68% were found to have improved at least two angina classes from baseline, 17% were angina-free, and quality of life remained significantly improved. These last two reports reflect the sustained angina relief seen with CO2 TMR because these patients had no additional procedures to account for their symptom improvement over the long term.

Based on an assessment of the cumulative results from these multiple randomized trials, the recently updated American College of Cardiology/American Heart Association (ACC/AHA) practice guidelines25 and the Society of Thoracic Surgeons (STS) practice guidelines26 have determined that the weight of the evidence favors the use of TMR in the treatment of stable, medically refractory angina patients.

Clinical Trials

Owing to its success as sole therapy, TMR has been evaluated in conjunction with CABG in patients with diffuse CAD who would be incompletely revascularized by CABG alone. The safety and effectiveness of adjunctive TMR have been somewhat difficult to assess owing to the influence of coronary bypass grafts and the lack of randomized control arms in some studies.27–29

Two prospective, randomized, controlled multicentered trials have been performed using TMR adjunctively with CABG in patients. In these studies, patients with one or more viable myocardial target areas served by coronary vessels that were not amenable to bypass grafting received either CABG plus TMR or CABG alone.30,31 Baseline and operative characteristics were similar between groups, including the location and number of bypass grafts placed (3.1 ± 1.2, CABG + TMR; 3.4 ± 1.2, CABG alone; p = .07). Patients were blinded to their treatment group through 1-year follow-up.

Mortality

Improved outcomes following TMR + CABG versus CABG alone in terms of a reduced operative mortality rate (1.5% versus 7.6%; p = .02), reduced postoperative inotropic support requirements (30% versus 55%; p = .001), increased 30-day freedom from major adverse cardiac events (97% versus 91%; p = .04), and improved 1-year Kaplan-Meier survival (95% versus 89%; p = .05) have been reported.30 Multivariable predictors of operative mortality were CABG alone (OR 5.3; p = 0.04) and increased age (OR 1.1; p = .03).30 A similar trend in operative mortality following TMR + CABG versus CABG alone (9% versus 33%; p = .09) was reported in a study of high-risk patients.31

Efficacy

The use of TMR adjunctively with CABG has been shown to decrease intensive care unit (ICU) times and length of hospitalization stay.29 In a long-term follow-up of the randomized, controlled trial, the effectiveness of TMR + CABG versus CABG alone has been reported.32 At a mean of 5 years, both groups experienced significant angina improvement from baseline; however, the TMR + CABG group had a lower mean number of angina-free patients (78% versus 63%; p = .08) compared with the CABG-alone group. Long-term survival was similar between randomized groups.

Observational data on the practice of TMR + CABG have been collected in the STS National Cardiac Database.33 From 1998 to 2003, 5618 patients underwent TMR + CABG. These were compared with 932,715 patients who underwent CABG-only operations. The TMR + CABG patients therefore account for 0.6% of the surgical revascularization practice in the database. Table 27-2 outlines the significant baseline differences between the CABG-only patients and the TMR + CABG patients. The TMR + CABG patients have an increased incidence of every surrogate marker of diffuse arterial disease, and therefore, it is not surprising that their observed mortality was higher at 3.8% (versus 2.7% for CABG-only patients; p < .001). When unstable angina patients were removed, the observed mortality for TMR + CABG was decreased to 2.7%, and the observed:expected (O/E) ratio was 0.87. Comparison of use and outcomes from sites that have a TMR laser versus those that showed no evidence of overuse of TMR or difference in outcomes.33

Laser–Tissue Interactions

Understanding the mechanism of TMR starts with understanding the laser-tissue interaction. While numerous devices,34 including ultrasound,35 cryogenic ablation,36 radiofrequency revascularization,37,38 heated needles,39,40 and the aforementioned hollow and solid needles have been used; none has engendered the same response that is seen with a laser. Additionally, numerous wavelengths of laser light also have been employed. These include xenon chloride (XCl),41 neodymium:YAG (Nd:YAG),42 erbium:YAG (Er:YAG),43 and thulium-holmium-chromium:YAG lasers (THC:YAG).44 All these devices have been explored experimentally but have not been pursued on a significant scale clinically. Only CO2 and Ho:YAG lasers are used for TMR. The result of any laser–tissue interaction depends on both laser and tissue variables.43–45 A CO2 laser has a wavelength of 10,600 nanometers, whereas a Ho:YAG laser has a wavelength of 2120 nanometers. These infrared wavelengths are absorbed primarily in water and therefore rely on thermal energy to ablate tissue. One significant difference, however, is that the Ho:YAG laser is pulsed, and the arrival of two successive pulses must be separated by time to allow for thermal dissipation. Otherwise, the accumulated heat will cause the tissue to explode under pressure. Such explosions create acoustic waves that travel along the planes of lower resistance between muscle fibers and cause structural trauma as well as thermocoagulation.45 The standard operating parameters for the Ho:YAG laser are pulse energies of 1 to 2 J and 6 to 8 W/pulse. The energy is delivered at a rate of five pulses per second through a flexible 1 mm optical fiber bundle. Despite the low energy level and short pulse duration, very high levels of peak power are delivered to the tissue so that with each pulse there is an explosion (Fig. 27-4). Additionally, the fiber is advanced manually through the myocardium, and it is therefore impossible to know whether the channel is being created by the kinetic energy delivered via the mechanical effects of the fiber or there has been enough time for thermal dissipation before the next pulse.

In contrast, the CO2 laser was used at an energy level of 15 to 20 J/pulse with a pulse duration of 25 to 40 milliseconds. At this level, the laser photons do not cause explosive ablation, and the extent of structural damage is limited. Additionally, a transmural channel can be created with a single pulse (see Fig. 27-4). Confirmation of this transmurality is obtained by observing the vaporization of blood within the ventricle using TEE.

Finally, the CO2 laser is synchronized to fire on the R wave, and with its short pulse duration, arrhythmic complications are minimized. The Ho:YAG device is unsynchronized and, owing to the motion of the fiber through the myocardium over several cardiac cycles, is more prone to produce ventricular arrhythmias.

Patent Channels

As noted, the original concept of TMR was to create perfusion via channels connecting the ventricle with the myocardium. Clinical work has demonstrated some evidence of long-term patency.46 Additional experimental work showed some evidence of patency as well.47–49 There are also significant reports from autopsy series and laboratories that indicate that the channels do not remain patent.50–52 The consensus is that while channels occasionally may remain patent, this is not the principal mechanism of TMR.

Denervation

In contrast to the open-channel mechanism, damage to the sympathetic nerve fibers may explain the angina relief noted in clinical trials. The nervous system of the heart can function independent of inputs from extracardiac neurons to regulate regional cardiac function by reflex action. This intrinsic system contains afferent neurons, sympathetic efferent postganglionic neurons, and parasympathetic efferent postganglionic neurons. Because of this complex system, it is difficult to demonstrate true denervation. However, several experimental studies have demonstrated that denervation indeed may play a role in Ho:YAG TMR.53,54 Experimental evidence to the contrary was performed in a nonischemic animal model.55 Although the studies were carried out carefully, it is difficult to isolate the sympathetic afferent nerve fibers, and the experiments were in the acute setting and only address short-term effects. Regardless of the methodology employed in the laboratory, there is significant evidence of sympathetic denervation following PET scanning of Ho:YAG TMR–treated patients.56

Angiogenesis

The likely underlying mechanism for the clinical efficacy of TMR is the stimulation of angiogenesis. This mechanism fits the clinical picture of significant improvement in symptoms over time, as well as a concomitant improvement in perfusion, as seen with the CO2 laser. Numerous reports have demonstrated a histologic increase in neovascularization as a result of TMR channels.51,57–61 More molecular evidence of this angiogenic phenomenon was derived from work that demonstrated an upregulation of vascular endothelial growth factor (VEGF) messenger RNA, expression of fibroblast growth factor 2 (FGF2), and matrix metalloproteinases following TMR.62–64 Histologically, similar degrees of neovascularization have been noted after mechanical injury of various types. Needle injury has been demonstrated by immunohistochemistry to also stimulate growth factor expression and angiogenesis. The conclusion is that TMR-induced angiogenesis is a nonspecific response to injury.65,66 Investigation of this using hot and cold needles, radiofrequency energy, and laser energy to perform TMR clearly demonstrates a spectrum of tissue response to the injury.39 The results in a model of chronic myocardial ischemia to mimic the clinical scenario indicate that indeed neovascularization can occur after mechanical TMR, but if these new blood vessels grow in the midst of a scar, there will be little functional contribution from blood flow through these new vessels. The recovery of function with laser TMR was to the result of a minimization of scar formation and maximization of angiogenesis.

This then becomes a critical question: If TMR induces angiogenesis, is there an ensuing improvement in function? Clinically, this has been demonstrated subjectively with quality-of-life assessments, but more important, it has been demonstrated objectively with multiple techniques, including dobutamine stress echocardiography, 67 PET scanning,23 and cardiac magnetic resonance imaging (MRI).68,69 As further evidence of the angiogenic response, experimental data have mirrored the clinical perfusion results noted, with improvements in perfusion in porcine models of chronic ischemia where the ischemic zone was treated with CO2 TMR.70–73 This improved perfusion did lead to an improvement in myocardial function as well.

Myocardial laser revascularization has been performed percutaneously, 74 thoracoscopically,75 via thoracotomy,16–20 and via sternotomy.27–30 Aside from the PMR approach, any of the other surgical approaches have yielded similar symptomatic improvement. Several percutaneous trials have attempted to demonstrate a symptomatic improvement with the creation of 2- to 3-mm deep subendocardial divots achieved with a laser fiber fed via a peripheral artery into the left ventricle.74 Even with the use of electromechanical mapping to verify the position of the fiber and creation of the channel, the results from PMR have been less favorable than those seen with TMR. A double-blinded, randomized, controlled trial showed no benefit to the laser-treated patients compared with the untreated control group.74 Because the patients were blinded to their treatment, the possibility of a significant placebo effect for PMR has been raised. Of note, the morbidity and mortality of PMR reportedly are similar to those seen with TMR. As a result, the U.S. Food and Drug Administration (FDA) rendered PMR unapprovable.

The failure of PMR to achieve the same clinical results that have been seen with TMR may result from several significant limitations, the first of which is the partial-thickness treatment of the left ventricle. Even at the maximal estimated depth of 6 mm that has been reported with PMR, this is significantly less than the full-thickness treatment of the myocardium that is achieved with an open TMR approach. Furthermore, typically fewer of these partial-thickness channels are created with PMR. The exact location of the channel and the establishment of a wide distribution of the channels from inside a moving ventricle are also problematic. Finally, the limitations of Ho:YAG TMR are also applicable to PMR because that is the wavelength of light that has been employed.

Other potential applications include the use of TMR in the treatment of cardiac transplant graft atherosclerosis. Although the procedure has been performed on a small number of patients, the results have indicated a benefit.76 Finally, the combination of TMR plus other methods of angiogenesis may provide an even more robust response. Experimental work investigating these combinations has verified a synergistic effect with regard to histologic evidence of significant angiogenesis and, perhaps more important, an improvement in myocardial function with a combination of TMR and gene therapy versus either therapy alone.77–79

Cardiothoracic surgeons increasingly are faced with a more complex patient who has developed a pattern of diffuse CAD and has exhausted nonsurgical options. Results replicated in multiple randomized, controlled trials augmented by recently available long-term results have validated the safety, effectiveness, and substantially improved health outcomes achieved through application of TMR for the treatment of selected patients with severe angina owing to diffuse disease when used alone and as adjunctive therapy to achieve a more complete revascularization.

Before TMR, investigators have been trying different mechanical strategies to increase blood flow in ischemic hearts since the 1930s by obliterating the pericardial sac with mechanical abrasion and the addition of asbestos powder, tacking omentum to ischemic hearts, removing the epicardium, or combining several of these with the addition of implanting the internal mammary artery into the myocardium. Subsequently, pharmacologic strategies were employed in both experimental and clinical studies to ischemic hearts by using heparin and growth factors such as VEGF and fibroblast growth factors (FGF1 and FGF2). The angiogenic growth factors were first applied via direct delivery of specific proteins, and then gene delivery techniques, in both experimental and clinical studies. In the late 1990s, cell-based therapy was introduced in therapeutic angiogenesis for ischemic heart diseases and has been the most rapidly progressing and hot research field over the past 12 years. This section discusses the topic of protein-, gene-, and cell-based therapeutic angiogenesis for the treatment of ischemic cardiovascular diseases.

Proteins

Experimental Studies

In early in vitro studies, VEGF, FGF1, and FGF2 proteins showed angiogenic potential.80,81 These studies were followed by in vivo work showing that these factors actually do stimulate the growth of new vessels.82 The more recent genetic studies of vasculogenesis in mouse embryogenesis documented the critical importance of synergetic effects of multiple molecules, such as VEGF and angiopoietin-1, to the development of mature, branching blood vessels.83

Basic FGF and VEGF proteins stimulate the development of collaterals to tissues supplied by an obstructed artery and augment tissue blood flow were first demonstrated in the early 1990s. In experiments on myocardial ischemia, a portion of the left ventricle of dogs was made ischemic by gradual occlusion of the circumflex coronary artery. The intracoronary or left atrial administration of bFGF or intracoronary administration of VEGF proteins daily for 28 days significantly increased collateral flow.84 Likewise, studies in the rabbit ischemic hind limb model demonstrated that intramuscular administration of bFGF protein daily for 2 weeks improved limb perfusion significantly.85

However, when the effect of VEGF protein studied in the canine myocardial ischemia model using two different methods, controversial results were observed: While 28 days of administering boluses of VEGF into the left atrium improved collateral flow, 7 days of administration did not,86 and although 7 and as few as 2 days of intracoronary administration of bFGF improved collateral flow, a single bolus injection did not.87 These results demonstrated, at least in this model of myocardial ischemia, that the duration of exposure of the vessels supplying the ischemic tissue to angiogenesis factors is critical for a therapeutically relevant effect.

Additional studies employing 125I-labeled bFGF demonstrated that route of administration is another critical factor in determining local tissue uptake and, potentially, therapeutic response.88 The results showed that whereas 3 to 5% of an intracoronary dose of bFGF was recovered in the myocardium, only 0.5% of an intravenous dose was. The most plausible explanation of these findings derives from the fact that myocardial uptake depends on peak serum concentration; because bFGF has a heparin-binding domain, considerable first-pass uptake in the lungs will occur after intravenous administration (the lungs contain large amounts of heparin sulfates), resulting in a blunted peak serum concentration presented to the myocardium when compared with the very high concentrations presented to the myocardium with bolus injection directly into the coronary artery.

The biologic consequences of these differences were demonstrated in angiogenesis studies of the same canine ischemia model. Collateral flow improved with intracoronary administration of bFGF but did not increase when the drug was given intravenously, despite its being given for 1 week.87 Although similar uptake studies have not been performed with VEGF, its 165 isoform (VEGF165) also has a heparin-binding domain (whereas VEGF121 does not), suggesting that similar results would be seen.

Animal studies that appear to be at variance with these results also have been reported. Thus, Lopez and colleagues89 delivered VEGF165 to a porcine model of myocardial ischemia (ameroid occlusion of the circumflex coronary artery) by three different local intracoronary delivery systems (via an InfusaSleeve catheter, intracoronary bolus infusion, and epicardial implantation of an osmotic delivery system). VEGF was administered 3 weeks after ameroid placement, and indices of collateral function were assessed at that time (baseline) and 3 weeks later. Whereas there was no significant improvement in circumflex territory perfusion in control pigs, improved circumflex perfusion was demonstrable within each VEGF-treated group using paired t-tests to compare pretreatment and posttreatment perfusion values. Although these data are suggestive of a VEGF treatment effect, they are not convincing. First, ongoing collateral development has been observed in pigs throughout the 6-week period after circumflex ameroid placement.90 In the Lopez study, however, the control group did not exhibit the expected increase in circumflex territory perfusion during that interval. Second, direct comparisons between individual VEGF treatment groups and the control group were not statistically significant. Only when all three VEGF treatment groups were combined in a post hoc analysis was a statistically significant difference demonstrable between VEGF groups and the control group. Third, there were three deaths in VEGF-treated animals during the investigation. Elimination of three animals in a small study such as this could have an important effect on the results through selection bias. Thus, while suggestive, the data from this experiment do not demonstrate unequivocally that a single bolus intracoronary injection of VEGF is capable of increasing collateral flow to a greater extent than that which occurs in the absence of therapy.

Hariawala and colleagues91 also reported improved flow in a similar model. However, this study is flawed by the fact that intracoronary bolus administration of VEGF (2 mg) caused severe hypotension that led to the acute death of four of eight animals in the treated group; hence the surviving animals, which were found to have greater collateral flow than the untreated controls, may have survived only because they had greater intrinsic collateral flow. These investigators also demonstrated in the rabbit hind limb model of ischemia that a single dose of intrafemoral bFGF or VEGF165 improves collateral flow and, surprisingly, that a single intravenous dose of VEGF165 also improves flow.92 The conflicting results are reported in the literature relating to whether a single intra-arterial bolus injection of VEGF or bFGF protein improves collateral flow and whether improvement occurs following intravenous administration, at least for heparin-binding agents.

Clinical Trials

The clinical trials of protein-based angiogenesis have been reported by using growth factors like FGF and VEGF families. The effect of FGF-1 on the ischemic myocardium was first performed by Schumacher and colleagues93 in a series of 20 patients. In the study, 0.01 mg/kg of FGF-1 protein was injected directly into the ischemic myocardium along LAD while patients were undergoing bypass surgery. Three months later, neoangiogenesis, together with the development of a normal vascular appearance, was demonstrated angiographically. The first randomized, double-blind, placebo-controlled clinical trial for basic FGF was reported by the Simons' group.94 Twenty-four patients undergoing CABG were randomized to three groups, receiving either 10 μg or 100 μg of bFGF, or placebo through delivery of microcapsules capable of sustained-release which were implanted in ischemic myocardium. During 16 months of follow-up, all patients in the 100 μg bFGF remained angina-free and a stress nuclear perfusion imaging test at baseline and 3 months showed significant improvement. The efficacy of single intracoronary infusion of FGF-2 (0, 0.3, 3, or 30 μg/kg; n = 337 patients) was tested by a multicenter FIRST trial.95 At 90 days, in all FGF-2 treated groups angina symptoms were significantly reduced compared with placebo, but not in 180 days because of the continued improvement in the placebo. The effect of intracoronary injection of recombinant human VEGF on myocardial perfusion was first performed on 14 severe CAD patients,96 and followed by a multicenter VIVA trial on a total of 178 patients.97 In the small patients group study, seven patients who received high-dose VEGF (0.05–0.167 μg/kg) showed improvement in resting myocardial perfusion and collateral count density at 60 days follow-up.96 In the VIVA trial, patients were randomized to receive a 20-minute intracoronary infusion of placebo, low-dose (17 ng/kg/min) or high-dose (50 ng/kg/min) rhVEGF and followed by 4-hour intravenous infusion on days 3, 6, and 9. However, the study showed no significant differences in primary end point of the trial and ETT time compared with placebo at 60 days follow-up. At day 120, high-dose patients showed improvement in angina class and favorable trends in exercise treadmill test time and quality of life.97

Genes

Experimental Studies

Gene therapy presents one of the solutions to the possible dosing conundrum because gene therapy can be considered a sophisticated form of a sustained delivery system. Once transfected, the target cell expresses gene product for days, weeks, or longer depending on the specific tissue transfected and the specific vector used.

Proof of concept that gene therapy can improve collateral function was demonstrated by Giordano and colleagues.98 They found in a porcine model of myocardial ischemia (ameroid occlusion of the circumflex coronary artery) that a single-dose intracoronary administration of an adenoviral vector carrying the FGF5 transgene into the nonoccluded right coronary artery increased myocardial flow and function. Surprisingly, they found that about 95% first-pass myocardial uptake was achieved with intracoronary administration. Hammond and colleagues have since demonstrated that FGF4 produces similar effects in restoring myocardial flow and function.99 Improvement of myocardial contractility in a porcine model of chronic ischemia has been reported recently by using a combined TMR and FGF-2 gene therapy approach.100 In this study, adenoviral vector encoded FGF-2 was formulated in a collagen-based matrix and directly injected into ischemic myocardium. Other investigators also have performed studies employing the rabbit hind limb model of ischemia and have reported that injection into the femoral artery of the VEGF165 transgene carried in a plasmid vector improves collateral flow.101

Direct Intramyocardial Injection

No matter how efficient first-pass uptake is, a considerable proportion of an angiogenesis factor injected into an artery supplying the target tissue will enter the systemic circulation and thereby expose nontarget tissues to its biologic effects.102 Although there is no definitive evidence yet that such systemic spillover will produce serious side effects, there is always that possibility (see the following). Therefore, it appears that if direct intramuscular injection of the angiogenesis factor, either by the transepicardial or transendocardial route, does result in enhanced collateral flow, such an approach might be preferable.

A protein injected once intramuscularly would be unlikely to persist in the tissue long enough to exert an important biologic effect.102 Although multiple injections of protein might well improve collateral flow,85 such a strategy has practical limitations. Therefore, once it was demonstrated that an adenoviral vector carrying a reporter transgene efficiently expresses its gene product after intramyocardial injection,103 this approach to gene delivery was explored as an approach for gene therapy.

Proof of concept that intramyocardial injection could enhance collateral flow and improve impaired myocardial function was demonstrated in a porcine model of myocardial ischemia. This was achieved by the transepicardial injection of an adenoviral vector carrying the VEGF121 transgene performed after thoracotomy.104 The feasibility of catheter-based transendocardial delivery of angiogenesis genes has been shown recently,105 demonstrating that the direct injection of angiogenesis factors into the myocardium can be accomplished without the need for thoracotomy.

However, because VEGF induces marked increases in vascular permeability and tissue edema, excessive VEGF administration could develop deleterious effects. Recent reports showed that in a chronic ischemic rabbit ear model, both adenoviral encoded VEGF and Angiopoietin-1 (Ang-1) increased flow at one week after injection. However, Ang-1-induced flow was localized to larger vessels, with no visible inflammatory response, but VEGF produced a diffuse increase in flow that was associated with pronounced swelling, vessel leakage, and inflammatory cell infiltration. At 4 weeks, the flow in the VEGF treated group decreased from pretreatment values. In contrast, the Ang-1–induced improvement was maintained.106 Similar deleterious effects of VEGF were also reported by Masaki et al in their mouse hind limb ischemia model.107

Clinical Trials

The first clinical trial on gene-based therapy using adenoviral vector was reported by Rosengart and colleagues.108 In this phase I trial, 15 patients received adenoVEGF121 by direct intramyocardial injection as an adjunct to conventional CABG and six patients received gene-therapy only. Thirty days after the treatment, all patients showed improvement of wall motion in the area of vector administration by coronary angiography and stress 99mTc-sestamibi perfusion scan, as well as improvement in angina class after therapy. There was no evidence of systemic or cardiac-related adverse events related to vector administration. The effect of plasmid encoded VEGF2 on chronic myocardial ischemia was studied in 19 patients using catheter-based delivery.109 In this small phase 1/2 study, Losordo and colleagues used an injecting catheter device guided by NOGA mapping technique and administered 200 to 800 μg of phVEGF2 plasmid directly into the endomyocardium. The end point analysis at 3 months disclosed improvement in angina class and strong trends favoring efficacy of phVEGF2 versus placebo in exercise duration, functional improvement, and Seattle Angina Questionnaire data. The adenoviral encoded FGF4 was studied by AGENT trial by Grines et al. in a group of 79 patients with chronic stable angina pectoris.110 In this trial, patients received one time intracoronary injection of five different doses of adenoviral encoded FGF4 (from 3.3 × 108 to 1011 viral particles in half-log increments). In this first report of a randomized, double-blind and placebo-controlled trial, ad5-FGF4 showed a trend to have greater improvements in exercise time versus placebo at 4 weeks' follow-up. The same group also reported an AGENT-2 study of 52 patients with chronic stable angina using a single dose of 1010 viral particle of ad5FGF4 intracoronary infusion. At 8 weeks after treatment, ischemic defect size was significantly decreased in ad5-FGF4–injected patients compared with placebo-treated patients and the viral vector was well tolerated and did not result in any permanent adverse sequelae.111 Hedman et al. conducted a phase II Kuopio Angiogenesis Trial (KAT) to study the effects of VEGF on restenosis and chronic myocardial ischemia by intracoronary injection of either adenoviral or plasmid encoded VEGF165 on 103 patients who underwent PTCA and stenting.112 At 6 months' follow-up, no difference was found in restenosis rate or minimal lumen diameter between all study groups. However, myocardial perfusion showed a significant improvement in the adenoVEGF treated group. Recently, the Euroinject One Trial was reported by Kastrup and colleagues113 on direct intramyocardial plasmid VEGF-A165 gene therapy in patients with stable severe angina pectoris. In this study, 80 "no option" patients with severe stable ischemic heart disease were randomly assigned to receive either 0.5 mg of phVEGFA165 or placebo plasmid in the myocardial region showing stress-induced perfusion defects under the guidance of the NOGA-MyoStar system. At 3 months' follow-up, the VEGF gene transfer did not significantly improve stress-induced myocardial perfusion defect compared with placebo. However, improved regional wall motion indicated a favorable anti-ischemic effect.

In summary, single protein or gene-based therapy so far has not achieved significant convincing beneficial results in improving myocardial perfusion both experimentally and clinically. More clinical studies are needed to determine how to achieve optimal myocardial angiogenesis. Many aspects of gene transfer, including the appropriate vector dose, formulation, and administration route, are still to be tested.

Cell-Based Therapy

Two classical concepts have been challenged in recent years by cell-based therapy using either embryonic or adult stem cells. First, vasculogenesis, which previously referred to a process that occurred only in the embryo, in which the vascular system develops from mesodermal precursor cells called angioblasts that invade the different embryonic organs and assemble in situ to form the primary capillary plexus. However, many investigators now believe that in adults, bone marrow–derived stem cells or endothelial progenitor cells can be recruited to and incorporated into tissues undergoing neovascularization. Second, cardiac myocytes originally were considered to be terminally differentiated cells that cannot be regenerated in adulthood. However, recent studies have shown that a limited number of cardiomyocytes may be regenerated by locally sited or recruited circulating stem cells. Therefore, stem cells, which include hematopoietic stem cells (HSCs), endothelial progenitor cells (EPCs), mesenchymal stem cells/stromal stem cells (MSCs), myoblasts, and undifferentiated side population cells, have been used as an alternative therapeutic strategy for ischemic cardiovascular diseases that cannot be treated by routine interventional approaches. Theoretically, embryonic stem cells have more potential to differentiate into cardiomyocytes; however, clinical trials are now only limited to adult stem cells owing to the fact that they are relatively easier to handle, and autologous transplantation can be performed in clinical patients. Several centers around the world, including the United States, had reported an improved functional status in experimental animals and clinical patients after such therapy; however, it is still unclear how bone marrow–derived stem/progenitor cells are mobilized and recruited into ischemic tissues, if the injected cells in clinical trial patients differentiated into functional cardiomyocytes, and what particular cell type is better to use compared with others. This section focuses on the progress status of clinical trials, mechanisms of involving functional improvements by stem cell therapy, limitations for applications, and potential risks.

Clinical Trials

Intracoronary Infusion of Autologous Bone Marrow–Derived Cells

TOPCARE-AMI

Assmus and colleagues first reported cell-based therapy for 20 acute myocardial infarction (AMI) patients in 2002.114 In this study, the authors performed intracoronary infusion of autologous bone marrow–derived mononuclear cells (n = 9) or circulating blood-derived progenitor cells (n = 11) 4 to 5 days after AMI. The circulating blood-derived progenitor cells were expanded ex vivo for 3 days before injection. The bone marrow–derived cells were extracted on the same day as injection without expansion. At 4 months after cell injection, patients' cardiac function was improved compared with 11 matched controls. The authors also reported postinfarction remodeling outcome using serial contrast-enhanced MRI.115 A total of 28 patients with reperfused AMI who received bone marrow–derived cells or circulating blood progenitor cells were analyzed. They found that intracoronary infusion of adult progenitor cells in patients with AMI beneficially affects postinfarction remodeling processes. The migratory capacity of the infused cells is a major determinant of infarct remodeling, suggesting a causal effect of progenitor cell therapy on regeneration enhancement.115 In 2004, the same group reported final 1-year results of 59 patients that showed similar functional improvements.116

TOPCARE-CHD

Assmus and colleagues also performed a clinical study of using functional competent BMCs in chronic postinfarction heart failure patients. In this study, 121 patients treated with BMCs showed statistically significant improvement in the serum level of natriuretic peptide and favorable clinical outcomes.117

Strauer and colleagues conducted a study to test the effect of autologous bone marrow–derived mononuclear cells (BMCs) on myocardial repair or regeneration. The authors first reported the data with 10 AMI patients who received BMCs by intracoronary injection and compared with 10 compatible patients treated with standard therapy alone. At 3 months after cell therapy, they found that the infarct region had decreased significantly and wall motion also significantly improved.118 More recently, the same group reported another study using same cell therapy technique on 18 patients with chronic MI (5 months to 8.5 years old) for their effects on myocardial regeneration.119 At 3 months, the patients with cell therapy showed that the infarct size was reduced by 30% and global left ventricular ejection fraction and infarction wall movement velocity increased significantly, whereas in the control group no significant changes were observed. The authors also found that following BMC transplantation there were improvements in maximum oxygen uptake and regional 18F-fluor-desoxyglucose uptake into infarct tissue suggesting a regeneration of myocardium after infarction.

BOOST Trial

The initial BOne marrOw transfer to enhance ST-elevation infarct regeneration (BOOST) trial was reported by Meyer GP and colleagues,120 which showed significant improvements in global and regional left ventricular systolic function. However, the 5-year follow-up from this trial proved that a single intracoronary infusion of BMCs did not promote a sustained improvement of LVEF in ST-elevation myocardial infarction patients.121

Catheter-Based Transendocardial Cell Injection

Perin and colleagues122 reported their results using a NOGA catheter technique to transendocardialy inject autologous bone marrow stem cells for severe chronic ischemic heart failure patients. Fourteen patients who received cell injection showed significant functional improvement compared with seven controls. Similar methods also were reported by Fuchs and colleagues in Washington Hospital Center in 10 no-option patients with advanced CAD. The authors first initiated a porcine ischemic model to test the effect of freshly extracted autologous bone marrow on myocardial blood perfusion. Improved collateral flow and contractility in a treated group of animals was found.123 Subsequently, in a pilot clinical study, the 10 no-option patients with advanced CAD autologous bone marrow direct myocardial injection induced a significant improved Canadian Cardiovascular Society angina score and stress-induced ischemia occurring within the injected territories.124

Intracoronary Injection of Extra Vivo Expanded BMCs

Chen and colleagues125 were the first group to use autologous ex vivo expanded bone marrow–derived mesenchymal stem cells in patients with AMI. In their study, a total of 69 patients who received PCI 12 hours after AMI were chosen randomly for either cell injection (n = 34) or control (n = 35). The bone marrow–derived mononuclear cells were cultured in vitro for 10 days, and then patients underwent intracoronary injection of the fibroblast-like mesenchymal stem cells. Patients who received mononuclear stem cell injection showed significant improvement in left ventricular (LV) function at 3 to 6 months of follow-up.

Mobilized Peripheral Blood Mononuclear Cell Studies

Kang and colleagues126 reported a mobilized peripheral blood mononuclear cell study for AMI patients after coronary stenting. A total of 27 patients with AMI who underwent PCI 48 hours later were studied. Ten patients received intracoronary injection of mobilized (granulocyte colony-stimulation factor [G-CSF] 10 μg/kg for 4 days) PBMC, and 10 patients received injection of G-CSF alone, seven as control. At 6 months, the cell infusion group showed improvement in LV function compared with the other two groups.

However, from later randomized placebo-controlled trials (REVIVAL-2) reported by Zohlnhofer and colleagues127 showed that stem cells mobilized by G-CSF therapy did not improve the infarct size, left ventricular function, or coronary restenosis in patients with acute myocardial infarction who had successful mechanical reperfusion.

Autologous Myoclast Studies

Taylor and colleagues128 first reported to use autologously transplanted skeletal myoblasts to improve ventricular function in animal models of heart failure. This has been a hot topic in the field of cell-based therapy for the last 10 years. However, recently reported first randomized placebo-controlled clinical study of autologous myoblast transplantation showed no evidence of functional improvement of the left ventricle, but did show an increased number of early postoperative arrhythmic events.129

Direct Epimyocardial Cell Transplantation

Patel and colleagues130 recently reported direct myocardial injection of autologous bone marrow–derived stem cells in 10 patients who underwent bypass surgery. Six months later, the cell injection patients showed improvement in LV function compared with 10 patients who received bypass surgery alone. No side effects were found with this direct stem cell injection.

Allogenic Human Mesenchymal Stem Cell Therapy

Most recently, Osiris Therapeutics Inc. completed its first allogenic hMSCs clinical trial reported by Hare and colleagues.131 In this study, hMSCs, developed by Osiris Therapeutics Inc. more than 10 years ago, were administered intravenously to patients with acute myocardial infarction at a dose range of 0.5, 1.6, and 5 million cells/kg. They found that the allogenic stem cell therapy was safe, and the arrhythmia events were significantly reduced, global symptom score and LV ejection fraction in a subset of anterior MI patients was significantly better in hMSCs treated compared with placebo patients. The study suggested the potential usage of allogenic cell therapy in the future.

Safety and Efficacy

Potential for Deleterious Effects

For most potent therapeutic interventions, therapeutic efficacy is rarely free of the potential for harmful effects to occur. The biologic activities of most of the angiogenesis agents currently being tested clinically are very potent, and it is likely that the same activities that lead to a therapeutic effect also could cause unwanted side effects. It is therefore probable that some side effects consequent to the cellular effects of these agents inevitably will occur. If this concept is true, then the critical question we will have to address in large clinical trials is whether the incidence of these risks is sufficiently low that they will be outweighed by the therapeutic benefits.

Among the side effects that might occur as a result of the biologic effects of these agents is the development of new blood vessels in nontargeted tissues, a complication that would be particularly devastating if it were to occur, for example, in the retina. It is possible that this particular complication may not develop unless a tissue is "primed" to respond with an angiogenesis response. That is, quiescent cells have low constitutive expression of receptors for the VEGF and FGF family of agents—thus, unless the tissue is exposed to very high doses of the ligands for prolonged periods, it is possible that normal tissue is resistant to the neovascularization effects of angiogenesis factors, a result suggested by the study by Banai and colleagues.132

Other VEGF-specific complications could develop as a result of the potent activity of VEGF as an inducer of vascular permeability.133 Although angiogenesis and vascular permeability might be considered two separate biologic activities, it is also possible that the vascular permeability properties of VEGF are essential for angiogenesis to occur.

Whatever the interrelation between these two actions, if vascular permeability increases in tissues other than the tissue targeted for angiogenesis, serious consequences could accrue. That this could occur was demonstrated in a recent study in which the effects of overexpression of VEGF (achieved by injecting an adenovirus carrying the VEGF transgene) in adult mice was investigated.133 The mice, as expected, developed elevated circulating levels of VEGF following injection of the adenoviral vector. However, a high percentage died within days, developing increased vascular permeability and severe multiple-organ edema.

Other potential complications based on biologic activities are the expansion and induction of instability of atherogenic plaque and the growth of tumors. For example, Flugelman and colleagues demonstrated an association between unstable angina and the intraplaque presence of aFGF and bFGF.134 They suggested that these agents might play a role in plaque instability. In addition, the broad range of cells on which the FGF family of agents exerts mitogenic effects could result in the growth of cells resident within plaques or of malignant cells.

Although the direct mitogenic effects of VEGF are limited largely to endothelial cells, it is of note that VEGF and its receptors, VEGFR1 and VEGFR2 (flt-1 and Flk-1), are overexpressed in atherosclerotic lesions.135 Moreover, a number of nonendothelial tumor cells have been found to possess low levels of functional VEGFR1 and VEGFR2.136 Also of possible relevance is the fact that the uterus possesses functional VEGF receptor tyrosine kinases137 and VEGF is mitogenic for uterine smooth muscle. These observations raise the possibility that the atherosclerotic lesion, certain tumors, and the common leiomyoma (fibroid) could at least theoretically respond to direct exogenous stimulation by VEGF.

There is also increasing evidence suggesting that growth of microvessels into plaque or tumors, through angiogenesis processes, is critical to growth of both tumor and plaque.138,139 Thus, microvascular angiogenesis per se, an activity inherent in most angiogenesis factors, could predispose to plaque or tumor growth. In addition, the potent vascular permeability effect of VEGF could result in exposing a plaque or tumor to many cytokines and growth factors that normally are confined to the plasma and through this indirect mechanism stimulate their growth.

It must be emphasized that there have been no conclusive reports in clinical studies demonstrating that angiogenesis agents actually induce new tumor development, increase growth of in situ tumors, or increase plaque size. However, several experimental studies have demonstrated that prolonged exposure of skeletal muscle or myocardium to high local levels of VEGF or FGF family peptides can cause hemangioma-like tumors and vascular malformations140 and can increase neointimal development.141

A phase I randomized, dose-escalation trial also demonstrated that high doses of bFGF can lead to the development of thrombocytopenia and renal toxicity.142 In addition, the immune surveillance system is not normally exposed to large amounts of these proteins. Therefore, it is possible that antibodies can develop to these cytokines and these could either impair the efficacy of repeated administration of the agents or even possibly lead to immunopathogenic processes. It also should be noted that one of the clinical trials in progress employs FGF2 of porcine origin94,95; although the high homology between the FGFs in different species makes it unlikely that recognition of nonself protein will occur, this certainly is not beyond the realm of possibility.

FGF and VEGF proteins, administered acutely, can produce hypotension through, at least in part, a nitric oxide–mediated pathway143 and, in the case of FGF2, a potassium channel–mediated mechanism.144 The hypotensive effect has resulted in the death of pigs that had chronic myocardial ischemia and were treated with the intracoronary injection of VEGF165 protein91 and in a prolonged hypotensive episode of a patient entered into a phase I study testing the safety of intracoronary administration of bFGF.142 This complication appears to occur only when high systemic levels of bFGF and VEGF develop rapidly. Thus, it would appear to be of little or no concern if bFGF and VEGF proteins are not administered rapidly and of no concern when the factors are given as genes—which express the proteins they encode slowly.

We also need to consider, in the case of gene therapy employing viral vectors, the potential for the vectors themselves to cause deleterious effects. The administration of large amounts of virus can lead to massive immune responses that could cause serious, even fatal immunopathology. Such responses are unlikely given the amount of adenovirus administered in current clinical cardiovascular protocols. However, the foreign proteins presented by the virus, even when administered in relatively small amounts, probably will induce immune responses that conceivably could decrease subsequent sensitivity to the beneficial effects of the transgene delivered by the virus if administered repeatedly or could possibly lead to immune-mediated tissue damage.

In cell-based therapy, most of the clinical trials so far are using non ex vivo expanded or short term expanded (4 to 5 days) cells. In animal studies, stem cells potentially can transform via in vitro expansion. These transformed cells can create tumors in nude mice.145 Similar incidents have occurred occasionally in adult human bone marrow–derived mesenchymal stem cells as well. It is still not clear which cell type is best for myocardial ischemia patients; however, if it is decided to use ex vivo expanded cells, one has to be sure that they are not tumorigenic. It is absolutely necessary to test these cells for tumorigenic potential in nude mice and to perform a karyotyping test before injecting the cells into patients.