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.
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.
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
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
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.
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.
Intracoronary Infusion of Autologous Bone Marrow–Derived Cells
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
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.
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.
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.