Angiogenesis

# 1. Overview

# 1.1 The Magnitude of the Problem
Atherosclerotic disease remains the leading cause of morbidity and mortality in the Western Hemisphere despite advancements in the preventive health care, medical management, interventional cardiology, and cardiovascular surgery. In 2001, cardiovascular disease accounted for 38.5 percent of all deaths in the United States. This is equivalent to 1 out of every 2.6 deaths in the United States. In this year, cardiovascular disease mortality was about 60 percent of total mortality. It was listed as a primary or contributing cause of the death of about 1,408,000 death certificates out of 2,400,000 deaths from all causes. Since 1900, cardiovascular disease has been the number one killer in the United States every year except for 1918.  Risk factor modification, anti-platelet agents, medical therapy designed to decrease myocardial oxygen demand, coronary vasodilators, and coronary revascularization procedures such as percutaneous coronary interventions (PCI) or coronary artery bypass grafting (CABG) all play a role in treatment of coronary artery disease. The number of cardiac interventions continues to rise. In the United States alone, a total of 1.3 million inpatient cardiac catheterizations, 561,000 PTCA procedures, and 519,000 coronary artery bypass procedures were performed in 2000. The vast amount of procedures is due to the progressive nature of the disease and the lack of a permanent fix by performing a procedure. Lastly, the cost of caring for cardiovascular disease and stroke is astronomical. In the United States in 2004, this figure was estimated to be $368.4 billion. This includes both health expenditure and lost productivity secondary to morbidity and mortality.
Additionally, ischemic heart disease remains the leading cause of congestive heart failure (CHF). The incidence of this disease has reached epidemic proportions. According to the 44 year follow-up of the NHLBIs Framingham heart study, CHF incidence approaches 10 per 1000 population after age 65 with 22 percent of male and 46 percent of female patients with myocardial infarction becoming disabled with heart failure. Hospital discharges for CHF was 377,000 in 1979. In 2001, it had risen to 995,000. 
Unfortunately, a large number of patients (5-21%) with ischemic heart disease wither can not undergo revascularization or receive incomplete revascularization by these procedures.  Despite maximal medical therapy, many are crippled by residual angina and congestive heart failure symptoms. Consequently, novel treatment strategies are needed. Therapeutic angiogenesis may play a role by providing increased blood supply to ischemic myocardium. Additionally, about five percent of patients with peripheral vascular disease have residual symptoms despite maximum medical and surgical therapy. Consequently, they may benefit from treatment with angiogenesis factors. 
Congestive heart failure is a progressive disease resulting from myocyte loss. Myogenesis, the process of myocyte regeneration, may play a pivotal role in its treatment. While implantable cardioverters-defibrillators improve survival, it has no positive effect on quality of life. The lack of organs for transplantation and the slow development of mechanical assist devices make myogenesis the major therapeutic option for these patients.
First, it is important to define the target patient population for such therapy. These patients are more commonly referred to as no-option patients. One angiogenesis application study of 500 patients yielded 59 patients (12%) who were considered poor candidates for PCI/CABG.  However, wide variability in treatment patterns exist. The different treatment styles cause different estimates of the magnitude of the no-option problem. It has been estimated that about 5 to 21% of CAD patients have no option for revascularization. The most common reasons for residual unrevascularized but still ischemic myocardium include: recurrent restenosis (less frequent with drug eluting stents), prohibitive expected failure, chronic total occlusion, poor targets for CABG/PCI, saphenous graft total occlusion with patent left internal mammary artery graft, degenerated saphenous vein grafts (less frequent with introduction of distal protection), no conduits, calcified aorta, and co-morbidities such as renal failure, cancer, or cerebrovascular disease. The management strategies for these patients are severely limited. A cocktail of medications, including antiplatelet agents, nitrates, beta-blockers, angiotensin converting enzyme inhibitors, angiotensin II receptor blockers, and calcium channel blockers, is often prescribed to help minimize symptoms. However, despite maximal medical therapy, many of these patients continue to be poorly controlled. The target population for angiogenesis therapy is constantly in flux. Advances in interventional and surgical techniques have helped improve quality of life and reduce the number of no-option patients. Development of drug (sirolimus and paclitaxel) eluting stents has all but solved the problem of recurrent restenosis. The target population for myogenesis application is easier to define. It essentially includes patients with Class III and IV New York Heart Association (NYHA) symptoms refractory to currently therapy.
Extensive preclinical testing of various agents, vectors, and cells in an attempt to achieve therapeutic angiogenesis and myogenesis have been performed with promising results. Clinical investigations have rapidly followed. Scientific basis and careful controls have been lacking in these clinical trials, which have led to poor results and ultimately disappointing phase II trials. Unfortunately, it seems that the same mistakes that were made with the development of angiogenesis are now being repeated in myogenesis investigations.

# 1.2 Therapeutic Angiogenesis
Angiogenesis is a complex process that ultimately leads to the formation of new blood vessels. It involves multiple steps, including endothelial cell proliferation and migration, extracellular matrix breakdown, attraction of pericytes and macrophages, followed by smooth muscle cell proliferation and migration, formation and sealing of new vascular structures, and finally deposition of new matrix. A coordinated action of several mitogens, cascades, and inhibitors is need for the process to work effectively. In patients with atherosclerosis, gradual occlusion of coronary arteries is frequently associated with development of collateral circulation (Figure 1). While collateral circulation is associated with improved clinical outcome, this process is often inadequate in many patients. The number of revascularization procedures done attests to this inadequacy. It appears that myocardial ischemia is a potent stimulus for angiogenesis. The concentrations of growth factors and chemo-attractants have been shown to increase during ischemia. This suggests that ischemia-induced angiogenesis may be mediated by these molecules. Specific growth factors include fibroblast growth factors  and vascular endothelial growth factor (VEGF) which are the most widely known and studied.
The definition of angiogenesis is an important concept to understand. While angiogenesis is often used as a general term to describe the process of neovascularization, it is actually one of three separate processes: vasculogenesis, angiogenesis, and arteriogenesis. The formation of new vascular structures from stem cells during embryogenesis is termed vasculogenesis. This may contribute to adult neovascularization as well.  Angiogenesis, by strict definition, refers to the formation of thin walled endothelium lined structures from preexisting vessels but lack a smooth muscle layer. These structures generally sprout from post-capillary venules. This is the manner by which capillaries form along the edge of an infarction or within healing wounds. The proliferation of vessels with a complete smooth muscle wall is known as arteriogenesis and can be visualized angiographically in patients with collaterals in the setting of advanced coronary artery disease. Although not well established, the process is believed to result from remodeling of existing collateral vessels as well as the formation of new vessels. Patients display significant variability in their angiogenic response to ischemia. While some patients develop complex collateral circulation, some have little to no response at all.

# 1.3 Molecular Mechanisms
The underlying mechanisms behind angiogenesis are beginning to be discovered. The process may begin with vasodilatation mediated by nitric oxide followed by an increase in permeability mediated by VEGF. This increased permeability allows for plasma protein extravasation and scaffold formation. Endothelial cell migration is supported by adhesion molecules such as PECAM-1 and cadherins. The vascular smooth muscle cells detaching and loosening signaled by Ang2 enables the migration and sprouting of endothelial cells. The process of angiogenesis is initiated by VEGF by Ang1 and is required to stabilize the endothelial networks and to increase periendothelial cell interactions. Platelet derived growth factor (PDGF) stimulates inflammatory cells and promotes cell-cell interactions by molecules such as v3 integrin. VEGF has morphogenic effects which allow endothelial cells cords to acquire and enlarge their lumen. Unfortunately, muscularization of the network is poorly understood. The process appears to be tissue specific. In the coronary arteries, the epicardial layer appears to be the source of smooth muscle cells which migrate under PDGF-BB and VEGF stimulation. TGF-  and downstream transcription factors Smads promote extracellular matrix production and solidify cell-cell interactions.  FGF can help further this process resulting in arteriogenesis. In pathologic conditions such as ischemic myocardium, arteriogenesis can allow for as much as a 20-fold enlargement of collateral network vessels. Chemokines and cytokines are upregulated by increased collateral flow which results in monocyte recruitment. Monocytes produce proteinases which cause medial destruction and further remodeling. Hypoxia-inducible transcription factors (HIF) and their stabilization by peptide regulator 39 help induce and potentiate the neovascularization process. Newer imaging techniques utilize knowledge of molecular mechanisms to help enhance image resolution and improve sensitivity and specificity.

# 2. Growth Factor Therapy

# 2.1 Growth Factors for Myocardial Angiogenesis
As described earlier, a complex molecular signaling cascade is responsible for angiogenesis. Many cytokines involved in the process have been identified, including the fibroblast growth factor (FGF) family, vascular endothelial growth factor (VEGF) family, platelet derived growth factor (PDGF) family, and angiopoietins.  The most studied and widely used in clinical studies are VEGFs and FGFs.

## 2.1.1 VEGF
Vascular endothelial growth factors are a family of heparin-biding glycoproteins. They have been shown to act as mitogens for vascular endothelial cells as well as stimulants for the endothelial progenitor cell mobilization from the bone marrow.  VEGF  as well as placental growth factor (PIGF) are all part of the family of VEGF. These factors interact with several different tyrosine kinase receptors (flt-1, flk-1, and flt-4). VEGFs expression is increased in the setting of vascular injury, acute and chronic ischemia as well as hypoxia of the cardiac myocytes, vascular smooth muscles and endothelial cells. Their actions are mediated through downstream activation of Akt and eventual release of nitric oxide (NO) leading to increased vascular permeability,  endothelial cell growth, and formation of tubular structures.
Evidence for VEGF as a pro-angiogenic agent has been provided in pre-clinical studies with animal models of chronic myocardial ischemia (Figure 2) with improvement in myocardial perfusion after treatment with VEGF. In a porcine ameroid model of chronic ischemia (Figure 3), perivascular and intracoronary administration of VEGF has been shown to improve myocardial blood flow and ventricular function. Since the actions of VEGF are mediated through NO release, disease entities that cause decreased bioavailable NO and endothelial dysfunction are associated with impairment in growth factor induced angiogenesis. An example of this is hypercholesterolemia.
The release of nitric oxide and arteriolar vasodilation leads to hypotension. Thus, hypotension is associated with intravenous and intracoronary VEGF administration and has been proven to be dose limiting in phase I trials. A theoretical risk associated with growth factor administration is the development of plaque angiogenesis. This may precipitate the growth and destabilization of atherosclerotic plaques. Another theoretical concern of growth factor therapy is the accelerated growth of primary tumors and the stimulation of metastasis. This is based on the well documented role of angiogenesis in tumor biology. Proliferative retinopathy in the diabetic population is another disease which may be worsened by growth factor therapy by pathologic angiogenesis. These potential complications may be avoided by local, rather than regional or systemic, delivery strategies. It is important to note that none of these matters have become apparent clinically. Instead, studies have shown the lack of efficacy of VEGF in phase II clinical studies via intracoronary and intravenous administration.

## 2.1.2 FGF
Twenty three different proteins collectively make up the FGF family. They are classified based on their expression pattern, receptor binding preference, and protein sequence. Normal myocardium contains FGF and its expression is stimulated by hypoxia and hemodynamic stress. FGF-2 modulates various cellular functions in multiple cell types. Its functions in the context of angiogenesis include inducing endothelial cell proliferation, survival, and differentiation. Additionally, it is involved in cell migration of endothelial cells, smooth muscle cells, macrophages, and fibroblasts. These effects are mediated through its interaction with the tyrosine kinase receptor FGFR1 which also leads to the downstream release of NO. Additionally, endothelial cells  are stimulated by FGF-2 to produce several proteases, including plasminogen activator and matrix metalloproteinases that promote chemotaxis.
Researchers have shown that FGF can induce angiogenesis in mature tissue. Animal studies have demonstrated increased vascularity after intracoronary injections of FGF in acute coronary thrombosis models. FGF-2 treatment with perivascular and intrapericardial delivery improved coronary blood flow and regional left ventricular function in studies using the ameroid constrictor model of chronic myocardial ischemia. Improvements in myocardial perfusion and function were also noted in studies using intracoronary infusions as the delivery method. Similar to VEGF, FGF-2 can also cause acute vasodilation and hypotension. Long term use of high dose FGF can also cause renal failure which may manifest as membranous nephropathy accompanied by proteinuria.

# 2.2 Growth Factor Delivery
Two approaches, protein therapy and gene transfer, have generally been used to achieve therapeutic angiogenesis.

## 2.2.1 Protein Therapy
Protein therapy has several advantages, including controlled delivery, established safety, predictable pharmacokinetics and tissue levels, and absence of long-term unexpected side effects. The main disadvantages of this strategy include short tissue half-life of many proteins and expensive recombinant molecules. These shortcomings can be potentially overcome with sustained delivery systems, such as the use of heparin alginate capsules for perivascular FGF-2 delivery in surgical angiogenesis trials. It is important to note that some angiogenic agents can not be delivered as proteins and thus may necessitate gene transfer. Examples include HIG-1 and PR39  which are transcription factors involved in the angiogenic cascade. However, for FGFs and VEGFs, protein therapy may supersede gene transfer, especially given the limitations of current vectors.

## 2.2.2 Gene Transfer
In this approach, the target cells become factories for the desired angiogenic cytokines. It relies on the ability of injected genetic material to provide sustained and effective expression of the desired protein in the appropriate tissues. Advantages of this method include sustained expression of the factor in the tissues, ability to express transcriptional factors, potential for regulated expression, and the ability to express multiple genes simultaneously. However, there has been little experience to date with this method in the clinical setting. Consequently, the toxicities and side effects of various vectors are poorly understood. Additionally, gene-based therapy can theoretically cause detrimental sustained expression potentially leading to pathologic angiogenesis. Other complications include inflammatory reactions to delivery vectors and mutated pathologic vectors (for viral therapy). Several phase I and phase II studies involving gene therapy have recently been published. However, the protein based (growth factor) therapy has been more extensively studied than gene transfer techniques. 
Various vectors and vehicles exist for the transfer of target DNA to the cells and tissues of interest.

### 2.2.2.1 Non-Viral Vectors
Naked DNA is a poor medium to achieve gene transfer. When naked DNA comes into contact with the cell membranes, very little will enter the cell. Consequently, a carrier or a virus vector is generally used to increase transfection efficiency and provide adequate expression of the therapeutic agent. Plasmid or liposomal  complexes are often used carrier molecules. A small amount of plasmid will enter the nucleus, where it remains in an episomal location (not integrated into the genome), resulting in limited duration of transgene expression. This occurs in both proliferating and non-proliferating cells. While it is simple to produce large amounts of plasmid and liposomal complexes, low transfection efficiency, short duration and low levels of transgene expression limit this approach.  Transfection efficiency can be improved by phospholipid formulas, such as 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-dodecanoyl/ 1, 2-dioleoyl-sn-glycero-3-phosphocholine and cationic polymers such as polyL-ornithine with galactose and the fusigenic peptide mHA2 (Galactose-polyLornithine-mHA2).

### 2.2.2.2 Viral Vectors
Specific receptors bind with adenoviruses allowing them to enter the cell. Subsequent lysosomal degradation occurs and viral DNA is released into the cytoplasm. This DNA eventually finds its way to the nucleus and remains in an extra chromosomal location. Replication-deficient adenoviruses are produced in-vitro in specific packaging cells that complement gene products deleted from the viral genome to prevent in-vivo replication. Advantages of adenoviruses include easy production in high titirs, high transduction efficiency, and the ability to express in both proliferating and non-proliferating cells. Unfortunately, both the first and second generation adenoviral vectors are associated with a significant local inflammatory reaction. Eventually, transgene expression is extinguished. Circulating anti-adenoviral antibody, common for some adenoviral subtypes, can greatly reduce the duration and magnitude of expression. Less inflammatory reactions may be produced by newer encapsulated (gutted) adenoviruses. These modifications also allow the adenovirus to carry full length genes such as the dystrophan gene.
With their ability to transduce non-dividing cells and permit lasting transgene expression in a wide variety of tissues, recombinant adeno-associated viruses (AAV) are promising candidates as gene vectors. However, AAV remain difficult to produce and have a small expression cassette. Newer procedures for screening, characterization and high throughput production of AAV vectors may circumvent this scalability problem. The size limitation may be overcome by using a dual vector approach.
Retroviruses are another class of viruses that can be used as vectors. After retroviruses enter cells via specific receptors, the viral RNA is reverse transcribed to DNA. This DNA is then integrated into the cellular chromosomal architecture, leading to stable, prolonged, and high expression of the therapeutic transgene.  Retroviruses that are replication deficient are produced in-vitro in specific packaging cell lines containing retroviral genes (G, P, E) that have been deleted from the retroviral genome. Consequently, their target cell population is limited to actively dividing cells. Additionally, they can not be produced in high titer. The efficient insertion of genes by retroviruses is often complicated by transcriptional inactivation of the retroviral long terminal repeats (LTRs) and the production of replication competent retroviruses is feared. Fortunately, advancements to overcome these limitations have been made, including the development of lentivirus vectors allowing efficient gene transfer to non-dividing cells. Moreover, viral titers have improved with the use of pseudotyping.

## 2.2.3 Cell-Based Therapy
This promising new strategy utilizes autologous cells or cell lines transfected with a transgene of interest to express that transgene in vivo. Because autologous cells are used, the inflammatory response is not invoked. Ultimately, the hope is that prolonged expression can be achieved by stable transfection using various measures including electroporation, in vitro retroviral, or lentiviral transfection. The use of autologous cells can also provide functional benefits independent of actual transgene expression. However, this has yet to be studied. Cell-based therapy allows for complex constructs to be built. This would allow for stable regulated expression and multiple transgene expression. Myocardial regeneration and angiogenesis is currently being studied with cell-based therapies by numerous investigations using hematopoetic cells, skeletal myoblasts, and endothelial progenitor cells.

# 2.3 Delivery Strategies
Delivery strategies can be classified into four categories. These include systemic (intravenous), local/regional infusion (intracoronary for myocardial angiogenesis and intra-arterial for limb angiogenesis), local periadventitial delivery (catheter based or surgical implantation), and intramyocardial (catheter-based or surgical for myocardial therapy, intramuscular for peripheral vascular disease). While vector development and angiogenic potential of carious cytokines have been extensively studied, the optimal delivery strategy for these agents has not been investigated. In many studies, the delivery of these agents has been arbitrarily chosen and tested for efficacy. Delivery parameters, such the mode of delivery, the volume to be delivered, the rate of infusion or injection, and the biocompatibility with materials used for each particular agent has not been adequately examined to date. The different delivery methods that have been used by investigators for gene transfer to the heart and peripheral vasculature have included intracoronary, epicardial , and endocardial  injections. Several investigators have found that gene expression after intravenous administration of an adenoviral construct was highest in the kidney, followed by the lung, liver, brain, and then heart. Consequently, intravenous administration has not been advocated. Intracoronary administration of adenoviral vectors have been studied by several investigators. However, most studies have been performed with intramuscular and intramyocardial delivery.

# 2.4 Clinical Studies of Protein Therapy

## 2.4.1 Phase I Trials
The first phase I clinical trial of coronary angiogenesis demonstrated the safety of intramyocardial injection of 0.01 mg/kg of FGF-1. The study enrolled forty patients undergoing CABG of the internal mammary artery (IMA) to the left anterior descending (LAD) coronary artery. These patients were randomized to receive intramyocardial injections of either 0.01 mg FGF-1 or placebo. All patients had further stenoses of the LAD distal to the anastomosis. Twelve weeks after treatment, coronary angiography showed increased capillary refill in patients that received FGF-1 compared to placebo patients.  The safety and efficacy of FGF-1 was confirmed at 3 years follow-up. Mortality was similar in both groups. The capillary network seen at 12 weeks post treatment persisted on angiography. Lastly, echocardiography suggested improved left ventricular ejection fraction (LVEF).
A preliminary study of surgically-delivered, intramyocardial FGF-2 that demonstrated the safety of this technique was followed by a phase II randomized, double-blinded, placebo-controlled trial. Twenty-four patients undergoing CABG with ungraftable areas of myocardium were randomized to 10g FGF-2, 100 g FGF-2, or placebo.  The patients that received the FGF-2 were implanted with slow release heparin-alginate microcapsules into ischemic but viable ungraftable myocardial tissue. Average follow up was 16 months with clinical assessment and nuclear perfusion imaging. There were no reports of recurrent angina or repeat revascularizations for the 100 g FGF-2 groups versus three reports of recurrent angina and two repeat revascularizations in the control group.  Nuclear defect size was significantly reduced in the 100 g FGF-2 group (Figure 4). After a 32 month average follow-up, patients treated with either dose of FGF-2 had significant benefits in both myocardial perfusion and angina free period compared to the placebo group.  Nuclear perfusion scans revealed a persistent reversible or a new fixed defect in 4 of 5 patients who received placebo. However, only 1 of 9 patients treated with FGF-2 showed these defects (p=0.03). Additionally, a trend toward improved LVEF was observed in patients who received FGF-2. 
Phase I trials have also examined the safety of less invasive methods of delivery of FGF-2, including intracoronary, and intravenous administration. These were open label dose escalating studies. Fifty-two patients with CAD and inducible ischemia who were suboptimal candidates for either PTCA or CABG received intracoronary FGF-2. The dose varied from 0.33 g/kg to 48 g/kg. Thirty six g/kg being the maximally tolerated dose secondary to treatment induced hypotension. At six month follow-up, patients reported improvement in quality of life assessments. Additionally, reduced angina frequency and improved exertional capacity scores were noted. Significant improvements were seen in exercise treadmill time, LVEF, target wall thickening and myocardial perfusion as measured by MRI.  However, no correlation was noted between the dose used and the improvement in parameters studied. Efficacy of the treatment could not be examined secondary to the lack of a control group and the open label design of the study. Next, the intracoronary delivery of FGF-2 was evaluated in a randomized, placebo-controlled, dose escalation phase I trial. Twenty five patients were randomized at a 2:1 ratio to a single intracoronary dose of FGF-2 or placebo. Hypotension occurred in two patients and bradycardia in three patients in the FGF-2 therapy group. While the FGF-2 group showed significantly increased epicardial coronary artery diameter compared to the placebo group, no improvement in treadmill exercise tolerance was observed.
The safety and tolerability of VEGF has also been studied in several phase I trials. Intravenous and intracoronary delivery of recombinant VEGF using dose escalation regimens were studied in two trials. Follow-up period was 60 days. Results showed VEGF delivered by intracoronary and intravenous routes was well tolerated and suggested dose dependent improvements in myocardial blood flow by nuclear perfusion studies.
Overall, the results of phase I trials show evidence for the safety of protein-based angiogenic therapy with VEGF and FGF and also suggest their efficacy.

## 2.4.2 Phase II Trials
Unfortunately, randomized, double blind, controlled phase II trials have shown modest, if any, benefit of protein based angiogenic therapy.  The VEGF in Ischemia for Vascular Angiogenesis (VIVA) trial was a multi-center, randomized, double-blind, placebo-controlled study of an intracoronary and intravenous regimen of recombinant VEGF  that randomized a total of 178 patients to low dose, high dose, or placebo groups. VEGF was delivered by an intracoronary infusion, followed by intravenous infusions at three-day intervals.  Treadmill exercise time was the measured primary end point. The study showed no significant improvement in treadmill time or angina class in the treatment groups compared to placebo at two months follow-up. One year follow-up showed a trend to sustained improvement in angina that was not significant. However, the VEGF infusions were well tolerated and their was no increased risk of cancer or myocardial infarction. All three groups in the trial had significant improvements in exercise treadmill time, angina class, and quality of life measures which showed the marked placebo effect in end-stage CAD patients. The persistence of the placebo effect has been demonstrated at up to 30 months.
The FGF-2 Initiating Revascularization Support Trial (FIRST) was another multi-center, randomized, double-blind, placebo-controlled phase II study. It was designed to examine the safety, pharmacokinetics, and efficacy of FGF-2.  Three hundred thirty seven patients who were poor candidates for percutaneous or surgical revascularization were randomized to treatment with intracoronary FGF-2 at 0, 0.3, 3, or 30 g/kg doses. Again, exercise tolerance test time was the measured primary endpoint. The mean change in exercise tolerance test time was not significantly different between treatment and control groups at 90 or 180 days after treatment.  However, patients older than 63 years of age showed a significant benefit in treadmill time. The Seattle Angina Questionnaire at 90 days showed significantly decreased angina frequency at 90 days, but the difference became insignificant at 180 days because of continued improvement of the control group. Stress nuclear imaging showed no significant difference.
The long term follow-up of the randomized double blind controlled trial of surgical intramyocardial delivery of FGF-2 (described above) showed persistent improvement in time free of angina. Additionally, nuclear perfusion at 32 months was significantly improved in treated patients versus control patients. However, the sample population was small in this study and a larger study is necessary to confirm these results.
Several factors can help explain the disappointing results of the phase II trials. Recombinant proteins have a relatively short plasma half life. Additionally, less than one percent of 125I-FGF-2 administered using the intracoronary route is deposited in the myocardium at 1 hour based on animal studies. Even less remains at 24 hours. Lastly, endothelial dysfunction, a common finding in patients with coronary disease, can diminish the effect of growth factors.

# 2.5 Clinical Studies of Gene Therapy

## 2.5.1 Phase I Trials
The first clinical trial included five end stage coronary disease patients with intractable angina and inoperable coronary disease that failed medical management. VEGF165 plasmid was injected into each patient through a small left anterior thoracotomy. No complications were noted related to the insertion of the plasmid. Decreased nitroglycerin use between 10 and 60 days after treatment was noted in all patients. SPECT-sestamibi showed improved blood flow. Angiography showed increased collateral flow to previously ischemic areas. LVEF remained unchanged. A larger nonrandomized, uncontrolled, dose escalating trial followed to assess the safety and bioactivity of intramyocardial delivery of the VEGF165 plasmid. Twenty patients received the VEGF165 plasmid and were followed for 180 days. Although there were no intraoperative complications, one patient suffered a cardiac arrest on the second postoperative day. The patient died four months later from aspiration pneumonia. Plasma VEGF levels were measured and peaked at day 14. They returned to baseline by 90 days. SPECT-sestamibi perfusion scans showed improvement in 13 of 17 patients at 60 days. Angiography also demonstrated collateral filling.
A phase I clinical trial of 21 patients using direct intramyocardial injection of adenovirus encoding VEGF121 was conducted by Rosengart and colleagues. Fifteen patients received the therapy along with CABG. Six received only the intramyocardial injection. There were no complications related to vector administration. Results showed trends toward improvement in angina classification and treadmill exercise testing at six months. At thirty days, the 99m Tc-sestamibi images showed improvement in wall motion abnormalities in the region of vector administration in the majority of patients. Angiography showed increased collaterals.
Fortuin et al conducted a phase I, open-label, dose escalation study of VEGF-2 naked DNA delivered by direct myocardial injection through a thoracotomy. Thirty patients with end-stage coronary disease were recruited and received 200, 800, or 2000 g of naked plasmid DNA as sole therapy.  No adverse effects were noted and most patients had a reduction in angina frequency, CCS angina class, and nitroglycerin use. However, a correlation between dose and clinical benefit could not be established. Angiography failed to provide evidence of angiogenesis.
Next, a percutaneous method of gene delivery was attempted. The NOGA system, a catheter that electromechanically maps the myocardium to distinguish between infracted and normal myocardium, was studied in a single blinded pilot study. VEGF-2 plasmid DNA was delivered through intramyocardial injections in 3 patients and compared to 3 patients who received placebo injections. Nitroglycerin use and reduction in angina frequency was significantly reduced in the patients who received the VEGF-2 plasmid versus placebo at one year follow-up. At 90 days after treatment, the area of ischemia and perfusion scores had improved.

## 2.5.2 Phase II Trials
Larger phase II, double-blinded, placebo controlled trials followed the early success of phase I trials. In one study, nineteen patients were randomized in a 2:1 ratio to either receive VEGF-2 plasmid injection or placebo by the NOGA system. Patients were followed until 12 weeks.  Nitroglycerin use was decreased in both treatment and placebo groups. However, angina classification significantly improved in the treatment group compared to no improvement in the placebo group. Additionally, the treatment group had a significant increase in the mean duration of exercise compared to placebo patients. Electromechanical mapping demonstrated a reduced area of ischemic myocardium in the patients that received VEGF-2 plasmid injections while patients in the control group demonstrated no change.
The Angiogenic Gene Therapy trial (AGENT) was a double blinded, phase I/II trial. It used intracoronary infusion of increasing doses of adenovirus encoding for FGF-4.  Seventy-nine patients were followed for twelve weeks after being randomized to receive either placebo or one of five doses of Ad5-FGF-4. While exercise tolerance was not significantly increased in treatment groups over placebo, a subgroup analysis of patients with initial ETTs of 10 minutes or less did have a significant improvement in treated patients versus controls. No differences were noted in stress-induced wall motion scores by echocardiography. Because of the lack of effectiveness or likelihood of a positive outcome, the trial was terminated early.
The Kuopio Angiogenesis Trial (KAT) was a randomized, double-blinded trial of intracoronary delivery of VEGF165 gene transfer. This occurred during PTCA. Out of 109 patients that were included in the study, 37 patients received VEGF adenovirus, 28 patients received VEGF plasmid liposome, and 38 control patients received Ringers lactate solution.  While several patients had complications during the procedure or soon after, none were attributed to the gene therapy. Overall, the therapy was feasible and well tolerated. At 6 months, the clinical restenosis rate was 6%. In quantitative coronary angiography analysis, the study groups did not differ significantly between the minimal lumen diameter and the percent diameter stenosis. However, VEGF-Adv treated patients showed a significant improvement in myocardial perfusion at 6 months. While no increases in the incidences of serious adverse effects were noted in any of the study groups, some inflammatory responses were transiently present in the VEGF-Adv group. These included transient fever and increases in CRP and LDH levels.
The double-blinded Euroinject One study  randomized 80 no-option (end-stage CAD) patients to intramyocardial plasmid gene transfer of VEGF165 or placebo. The trial delivered the treatment percutaneously via the NOGA catheter system and only included patients with stress-induced myocardial perfusion defects. An improvement in wall motion abnormalities was noted. However, no improvement in myocardial perfusion by 99mTc sestamibi SPECT imaging was shown. While CCS angina class improved in both groups, no significant difference between the two groups was noted.

# 2.6 Future Prospects
Despite some disappointing initial results in clinical trials, therapeutic angiogenesis can potentially provide new treatment strategies for end-stage coronary disease. Initial concerns about the safety of FGF and VEGF have not been realized. The trials discussed have repeatedly shown the safety of protein and gene transfer therapy with no evidence of angioma, neoplasms, plaque angiogenesis, or retinopathy at the doses used in human trials.
Several reasons explain why some trials have failed to show improvement with treatment.  Patients selected for trials are potentially the ones mostly likely to fail, having had multiple percutaneous and surgical revascularization attempts. They often have multiple comorbidities such as diabetes mellitus, hypercholesterolemia, as well as endothelial dysfunction.  Growth factor therapy has a diminished response in the setting of endothelial dysfunction. Oral L-arginine (a NO donor) can reverse endothelial dysfunction and can improve the angiogenic response. Consequently, modulation of endothelial dysfunction may represent a novel strategy to enhance myocardial angiogenesis.
Outcomes measured in many of the studies include changes in angina class, frequency, quality of life, and exercise tolerance. They are all very subjective measures and are thus susceptible to a large placebo effect. End points of morbidity and mortality, including myocardial infarction and death may provide objective measures of outcome.  Because these events occur in low frequency, extremely large study populations may be required to show significant improvements in outcome.  A non-invasive, objective, and sensitive device to assess the efficacy of angiogenic therapy may help solve these problems. Potential candidates include single photon emission computer tomography (SPECT), positron emission tomography (PET), and magnetic resonance imaging (MRI) (Figure 5). This is discussed later in the article.
The majority of the phase II trials reported to date have primarily focused on intravascular delivery. However, phase I trials of intramyocardial protein and gene delivery have shown promising results. Adequately powered, randomized, double-blind, placebo controlled phase II trials of intramyocardial delivery techniques are needed. Intravenous and intracoronary delivery results in systemic release and side effects such as nitric-oxide mediated hypotension. Consequently, the dose delivered is limited. Intramyocardial delivery by a percutaneous catheter or a minimally-invasive surgical procedure may help provide adequate doses and prevent systemic side effects. Furthermore, it has become clear that the timing and duration of growth factor therapy may be critical in inducing angiogenesis. Consequently, the use of sustained release systems of gene transfer may be necessary to provide prolonged exposure to growth factors.
Lastly, it is important to remember that the process of angiogenesis is extremely complex and involves a large number of chemical messengers. A multi-agent therapy may be necessary to obtain a therapeutic benefit. Research supports a synergistic mechanism of action between growth factors in angiogenesis. An alternate strategy may be using gene transfer methods that use transcription factors, such as HIF-1, that regulate the expression of multiple angiogenic genes. As we further our knowledge of the basic mechanisms of angiogenesis and the techniques of angiogenic therapy, the strategies used in the design of technologies and clinical trials will be based on a more sound scientific foundation.  Hopefully, this will allow therapeutic angiogenesis to become a reality in the treatment of coronary artery disease.

# 3. Cellular Therapy

# 3.1 Overview

## 3.1.1 Regenerative Potential of the Heart
Recently, there has been challenge to the dogma that the heart is a terminally differentiated organ without the ability to regenerate. Consequently, a new exciting possibility of cellular transplant therapy to regenerate infracted myocardium and prevent the sequela of ACS.  During an ST elevation myocardial infarction, cytokines are produced that allow recruitment of both resident and circulating stem cells for cardiomyocyte regeneration. However, the processes of apoptosis, fibrosis and compensatory hypertrophy usually overwhelm these mechanisms. Consequently, disadvantageous ventricular remodeling results. Additionally, advanced age seems to impair the regenerative potential of the progenitors.

## 3.1.2 Definition of Cellular Therapy
Cellular cardiomyoplasty is a technique by which progenitor cells are delivered to ischemic or infracted myocardium to help restore blood flow and contractility. Fetal cardiomyocytes, autologous skeletal myoblasts, adult cardiac myocytes, embryonic stem cells and both peripheral and autologous bone marrow stem cells have all been tested in various animal models. Clinical trials have been conducted mostly with autologous bone marrow derived cells in the acute setting of reperfusion therapy. Skeletal myoblasts have been used in clinical trials in the setting of chronic scar and myocardial dysfunction.

# 3.2 Cell Types

## 3.2.1 Bone Marrow Stem Cells
Bone marrow stem cells can be subdivided into CD34 positive hematopoetic progenitors that can give rise to endothelial cells and blood cells , and CD34 negative mesenchymal stem cells that can give rise to multiple lineages including cardiomyocytes in vitro . These progenitor cells are mobilized during acute myocardial infarction.
It has been shown in mouse models of myocardial infarction that both Lin negative c-kit+ cells and sca-1+ cells were capable of incorporating themselves as both endothelial cells and smooth muscle cells (support cells of neovessels), as well as cardiomyocytes.
It has also been found that new capillaries and vessels could be formed by injecting bone marrow cells into matrigels within skeletal muscle (Figure 6).
However, the specific identity of the pleuripotent stem cell and its lineage markers still remain undiscovered. Additionally, the viability of the cells after intramyocardial injection into murine infarcted myocardium has been doubted. As few as one percent of injected cells are viable four days after implantation.

## 3.2.2 Skeletal Myoblasts
Because of the so-called satellite cells that can be readily mobilized at the time of injury, skeletal muscle cells are capable of regeneration and repair more readily than cardiac myocytes. They can be harvested easily from autologous hosts and can be easily expanded in culture. Additionally, they are fairly resistant to hypoxia. A major disadvantage is that skeletal myocytes lose their gap junctions as they mature and become electrically isolated, predisposing to reentry arrhythmias. Also, the viability of dissociated myoblasts in suspension injected into myocardial scar without appropriate trophic environment has been doubted  despite some encouraging data. In one study, myoblasts injected into canine myocardium could not be identified four weeks after implantation. Additionally, the needle tract caused fibrosis and scarring (Figure 7).

# 3.3 Delivery Methods and Procedures
Under local anesthesia, bone marrow can be aspirated from the iliac crest. Very little processing is required if bone marrow cells without enrichment are used for a particular population. Some investigators enrich the monocyte population in endothelial progenitors by culturing for three days in endothelium-specific medium. Cells can be injected intramyocardially either by surgical techniques or by catheter-based approaches, including intracoronary, intravenous and intramyocardial from the left ventricular cavity. The thigh muscle is the usual site for skeletal myoblast harvesting. The cells undergo 2 to 3 weeks of expansion in culture before implantation.  Because of the grave risk of embolization with an intracoronary technique, injection approaches are limited to the intramyocardial route.

# 3.4 Clinical Trials

## 3.4.1 Autologous Bone Marrow Stem Cell (ABMSC) Intracoronary Injections in Acute Myocardial Infarction
Maximal restoration of blood flow to the ischemic/hibernating myocardium is the goal in acute myocardial injury. In this situation, bone marrow cells are optimal because of their angiogenic potential and their ability to either fuse with  or transdifferentiate  into cardiomyocytes.  In small animals, improved collateral perfusion and regional myocardial function has been demonstrated. It has also been demonstrated in large animals models. To date, a total of five hundred patients were studied who received either selected or unselected bone marrow stem cells. The majority of these studies were small, non-randomized pilot studies.

### 3.4.1.1 Repair of infarcted myocardium by autologous intracoronary mononuclear bone marrow cell transplantation in humans
Stauer et al (2002) enrolled twenty patients with transmural acute myocardial infarction in one of the earliest studies. The mean duration of anginal pain was 12 hours.  Left coronary angiography and revascularization was performed in all patients. Ten of the patients were assigned to treatment with 1.5-4 million unselected ABMSCs delivered by an over-the-wire intracoronary balloon catheter with 6-7 repeated injections at 5 to 9 days after their initial infarction.  Left ventricular ejection fraction on left ventriculography and dobutamine echocardiography was the primary outcome measured. Hemodynamic assessment was also made at 3 months after treatment. Patients in the treatment group had significant decrease in wall motion abnormalities and infarct size based on ventriculography at initial hospitalization and at 3 months (30 vs. 12%; p=0.005). In the non-treatment group, infarct size remained unchanged (20%). Ejection fraction increased from 57 to 62% in the treatment group and 60 to 64% in the control group (SD= 7-10%) However, these changes were statistically insignificant. While the end systolic volumes decreased significantly in the treated patients, the end-diastolic volumes did not. Stress echocardiography revealed an increase in the ratio of the systolic pressure to end-diastolic volume. Additionally, the treatment group experienced a 26% decrease in perfusion defect on thallium imaging (p=0.016).  In summary, there was a decrease in infarct size, an increase in perfusion, and an improvement in hemodynamics that could be attributed to the ABMSC transplant. While not randomized, the control group was well matched to the treatment group. Because of longer time to treatment from time of symptom onset, the effect of PTCA on the outcomes was likely attenuated. The delivery and retention of cells in the myocardium was enhanced by the novel intracardiac injection technique used in the study. The angiogenesis effects of the ABMSCs likely benefited from the timing of injection within 5-9 days of the acute infarction, a time when there is still active chemokine and cytokine expression. The concomitant revascularization itself probably also ameliorated the effect. The study raised issues of standardization and optimization of the number of bone marrow cells transplanted. It also addressed the careful choice of timing of the intracoronary injection, given the kinetics of the inflammatory response in myocardial infarction. 

### 3.4.1.2 Transplantation Of Progenitor Cells And Regeneration Enhancement in Acute Myocardial Infarction (TOPCARE-AMI)
Initially a randomized study of 20 patients comparing bone marrow and peripheral blood-derived stem cell injection, the Transplantation Of Progenitor Cells And Regeneration Enhancement in Acute Myocardial Infarction (TOPCARE-AMI) study was later expanded to include 60 patients. All patients enrolled in the study had an acute ST elevation MI treated with stenting and 2b3a inhibitors. Ten matched patients treated with similar revascularization technique but received no stem cell treatment served as a control group. The twenty patients in the treatment group were randomized to receive either of the two cell types at 24 hours after the initial myocardial infarction (time of cell harvest).
Four days after the MI, intracoronary cell injection was performed. Blood derived cells were expanded in the media containing VEGF and human plasma. The resulting culture had 90% endothelial cell population. Density gradient centrifugation was used to isolate bone marrow cells which contained a heterogeneous progenitor cell population that was CD34 and CD45 positive. LV ejection fraction on ventriculography and stress echo, wall motion abnormalities, coronary flow reserve (CFR) on intracoronary Doppler, myocardial infarct size on FDG-PET scan at initial hospitalization and at 4 months post-MI all served as primary outcomes. Serum inflammatory marker elevation or troponin elevation secondary to stem cell intracoronary injections with balloon occlusion was not observed. Malignant arrhythmias were not observed either.
Five of the 19 patients had evidence of in-stent restenosis at the time of the intracoronary injection. The patients in both the born marrow and peripheral blood endothelial progenitor groups showed a nine percent improvement (from 51 to 60%) in ejection fraction (p=0.003). Additionally, a decrease in end-systolic but not end-diastolic volume as well as a decrease in the degree of wall motion abnormalities in both the infarct and its border-zone was noted (p<0.0001). The effect was more pronounced in the border zone. This change was not observed in the untreated controls. The control group experienced a mild 2.5-3% increase in ejection fraction, within the range expected with revascularization alone. The effect between the two cell types was not significantly different. 
At four months, wall motion indexes improved even in the patients who had in-stent restenosis. Coronary flow reserve (CFR) measurements showed normalization of perfusion in most patients, and some improvement in those patients with re-stenosis. This suggests angiogenic effects at the tissue perfusion level. FDG-PET viability study showed significant decrease in infarct territory, with an increase in uptake from 54 to 63% (p<0.0001) in the affected coronary territory. Some of the important safety issues of the cardiomyoplasty and cell delivery technique, such as arrhythmogenesis and impact of vessel occlusion, were addressed by the study. Restenosis in the treatment group did not occur at markedly higher rates than the control group. The magnitude of improvement in the ejection fraction was greater than that demonstrated by Stauer et al. possibly because of earlier infusion time (4 days after 
infarction).
Neither of the studies addressed the contribution of ischemic conditioning from repeated balloon inflations as potential contributors to myocardial perfusion improvement. This could have also played a role considering that the control group did not undergo sham ballooning.
In the follow up study, these patients were evaluated by cardiac MRI at 4 months to further delineate the mechanism of improvement in the LV function associated with cellular therapy. Late enhancement volume, i.e. infarct volume, decreased by 20% over 4 months (p<0.05).  There was a proportional increase in the regional ejection fraction within the infracted areas as well as global ejection fraction (r=0.8; p < 0.001). A correlation between decrease in infarct volume (late enhancement) and improvement in wall thickening and wall motion score could be established. Importantly, improvement in function from cellular therapy was not dependent on initial size or transmural extent of infarction. Because full thickness infarcts improved their function as much as non-transmural infarct areas, this technology could be potentially beneficial in acute MI patients presenting with congestive heart failure and shock. While these patients were excluded from all the current studies, they are a group that may possibly derive the greatest benefit. The cell number or type did not impact the degree of improvement in function either. However, the in vitro migratory response to angiogenic factors such as VEGF and SDF-1 appeared to be a significant predictor.  Functional cardiac MRI, with its excellent ability to examine myocardial regional contractility  may be preferable in the future to other modalities in assessment of the efficacy of cell therapy. Because migratory capacity is important in mediating the therapeutic effect, it is possible that combining angiogenic factor gene or protein transfer with cellular therapy may enhance cardiomyoplasty. However, this assertion must first be examined in an in vivo setting. Lastly, it was noted that contractility and not just perfusion improve within the infracted area itself. The effect is not limited solely to the peri-infarct zone. However, the assumption that this is a direct result of the ability of progenitors to transdifferentiate into cardiomyocytes is yet to be proven.
An additional 40 patients (total of 60) underwent long-term follow up in the TOPCARE-AMI trial  and provided further evidence of good safety profile as well as sustained improvement in ejection fraction and LV contractility by MRI. The LV mass total decreased overall suggesting that deleterious compensatory hypertrophy and remodeling of adjacent myocardium did not occur. No lethal arrhythmias were reported and the restenosis rate remained at 21%.

### 3.4.1.3 Experimental and clinical regenerative capability of human bone marrow cells after myocardial infarction
Fernandez-Aviles et al. enrolled twenty patients with extensive ST elevation MI (> 6 mm) and treated them with primary or rescue angioplasty and stenting. Additionally, bone marrow stem cells were also administered at median time of 13 days after infarction. Stress echocardiography, functional MRI, and coronary angiography with LV gram and CFR were done at 30 days and 6 months. Patients were followed up to 21 months. The 13 patients who refused treatment were used as matched controls. While greater than ten times the number of ABMSCs were used in this study compared to other studies (70 million cells on average), a markedly higher increase in ejection fraction was not noted. CD34 positive cells made up only one percent of the treatment population.   Despite a later time of injection (13 days when infarct scar is considered to have formed), the beneficial effects on the ejection fraction and contractility (wall thickening by MRI) were comparable to prior studies. Other in vitro experiments showed that ABMSCs grown in the presence of murine cardiomyocytes begin to display some of the cardiomyocyte surface markers. However, whether these were contractile elements or whether these were a result of transdifferentiation or fusion with murine cardiomyocytes was not proven.

### 3.4.1.4 Bone marrow transfer to enhance ST-elevation infarct regeneration (BOOST)
The first prospective randomized trial of ABMSCs versus placebo was entitled BOne marrOw transfer to enhance ST-elevation infarct regeneration (BOOST). The study enrolled 60 patients with ST elevation MI who were treated with PCI.  Inclusion criteria included wall motion abnormalities in 2/3 of the anterior, inferior, or lateral wall without pulmonary edema or cardiogenic shock. Baseline EF was 51-53% in both the treatment and placebo group. Five to six days after MI, 250 million ABMSCs were administered via intracoronary route to 30 randomly selected patients. Ejection fraction change at 6 months post-treatment as determined by MRI was the primary end point. Ejection fraction increased by 6.7% in the treatment group (50 to 56.7%) and was unchanged in the control group (51.3 to 51%) treated with PCI alone (p=0.0026). This was similar to results reported in other studies. However, this trial was able to control for the baseline benefit of PCI. Global EF improvement was observed in both older and younger and patients, men and women, and regardless of whether the right or left coronary territory was involved. Those with greater symptom to PCI times appeared to benefit more. Similar to the TOPCARE-AMI trial, both patients with large and small late-enhancement areas on MRI benefited from the stem cell treatment. However, this trial showed a greater benefit in those patients with larger baseline defects. The functional MRI demonstrated improvement in wall motion and wall thickening only in the peri-infract border zone, but not in the actual infarct zone. This differs from the TOPECARE-AMI study.  Most recent communications report that the improvement in global ejection fraction is maintained at 18 months post-infarction. A good safety profile was confirmed by the trial. The rates of in-stent restenosis between the two groups were comparable (30%) and there was no increase in the incidence of inducible VT on electrophysiological studies.

### 3.4.1.5 Lack of regeneration of myocardium by autologous intracoronary mononuclear bone marrow cell transplantation in humans with large anterior myocardial infarctions.
To date, only one trial has failed to demonstrate any benefit of ABMSCs infusion. This was a small, non-randomized trial of 5 patients  with ST elevation MI  who underwent primary PCI or failed thrombolysis and underwent rescue PCI. A single injection was given containing thirty million cells. Compared to baseline, there was no improvement in ejection fraction at 3 months based ventriculography or stress echocardiography (42 to 44%; p=NS). Neither wall motion nor contractility index improved either. While CFR improved from1.7 to 2.5, it was not statistically significant because of the small sample size (n=5). At twelve months, ejection fraction increased by 4% to 46%. Again, this was not statistically significant. In general, the ejection fractions in these 5 patients were 10% lower than in other studies indicating larger myocardial infarction. Consequently, this may undermine the TOPCARE-AMIs claim that the response to therapy is not based on baseline infarct size. The measured benefit of cell transplant may have been difficult to demonstrate in this study because the time to PCI was shorter that in other studies. The small sample size may not have provided adequate power to measure the additional benefit.

### 3.4.1.6 Large Randomized Controlled Trials
The preliminary phase I trials of ABMSCs in AMI showed a great safety profile and provided promise for improved heart function in the long term. However, large randomized trials of this therapy are needed to make any definitive claims. Two such studies were presented at the American Heart Associating meeting (November, 2005).  Reinfusion of Enriched Progenitor cells And Infarct Remodeling in  Acute Myocardial Infarction (REPAIR-AMI) randomized 204 patients to 236 million marrow cell infusion or supernatant injections four days after MI. While both groups had an improvement in ejection fraction, the cellular therapy group had a somewhat greater effect. The ejection fraction increased from 48 to 54% in the treated group and from 47 to 50% in the control group (p=0.02). The clinical significance of these results is not certain. Long term follow-up will be needed to explore the sustainability of these results. The Autologous Stem Cell Transplantation in Acute Myocardial Infarction (ASTAMI)  arrived at results that contradicted REPAIR-AMI. The ASTAMI used sensitive imaging end-points at 6 months after treatment with bone marrow stem cells. One hundred patients were randomized to stem cell injection versus placebo 5 to 8 days after acute myocardial infarction. At 6 months post-procedure, no difference in ejection fraction could be detected by echocardiography, nuclear imaging, or by the most sensitive cardiac MRI. The placebo group appeared to have a greater improvement in function. Table 1 summarizes the results of all of the trials reviewed here.

## 3.4.2 Autologous Bone Marrow Stem Cells for Chronic Stable Angina and Ischemic Cardiomyopathy without option of revascularization
In patients with refractory chronic angina who were not candidates for traditional revascularization due to diffuse coronary artery disease, initial studies with transmyocardial revascularization gave promising results. However, the studies failed to account for a strong placebo effect observed. Additionally, follow up studies failed to show efficacy. The idea of using ABMSCs as a source of angiogenic factors has been tested in several trials. Given that the patients are not candidates for revascularization, a transendocardial route with electromechanical mapping (EMM) is necessary to deliver the cell injection. This can be accomplished with a NOGA catheter or other intramyocardial injection catheters.
Eight patients with angina refractory to medical therapy were enrolled  into the first feasibility study to assess this new methodology. At three months, functional cardiac MRI was used for assessment. Ejection fraction did not increase significantly, but remained close to baseline at 57%. Wall thickening improved (11%; p=0.004) as well as wall motion (5.5%; p=0.008) of the target wall. There was as a decrease in hypoperfused myocardium (3.9%; p=0.004). EMM times approached 200 minutes.
Fuchs et al. study (2003)  was another second feasibility pilot trial of ten patients with chronic angina secondary to non-intervenable coronary disease but with ejection fraction above 30% (mean of 47%). Via transendocardial approach, patients received 12 injections of ABMSCs using EMM into the ischemic territory as determined by SPECT. Ejection fraction change by echocardiography at 3 months after the procedure and change in the size of reversible ischemia on SPECT served as outcomes. While the ejection fractions did not change, there was a decrease in semi-quantitative stress scores on SPECT imaging within the injected segments (p<0.0001). Eight out of the ten patients showed an improvement in angina score. No arrhythmic or procedural complications occurred. EMM time was 30 minutes.
The largest study to date was an open-label study with 14 patients who received treatment and 7 who served as controls. Unlike the pilot studies, inclusion criteria included an EF of less than 40%. SPECT imaging demonstrated a reversible defect in all patients. Consequently, the group represented a population who was at high risk for morbidity and mortality. A mean of 25 million cells were injected (split into 15 aliquots) in the area of viability and reversible defect. Viability was determined by unipolar voltage with NOGA catheter while reversible defect was determined by prior Single Photon Emission Computer Tomography (SPECT) imaging. Control group patients did not undergo a sham procedure. The change in cardiopulmonary exercise tolerance, echocardiographic ejection fraction, and viability on SPECT were all evaluated at two months after treatment. Angiographic LV function and EMM were evaluated at 4 months. However, the control group did not undergo the EMM assessment at 4 months. At two months, a significant decrease in BNP levels and an improvement in creatnine was noted in the treatment group (BNP 282 vs 565; p=0.06; cr 1.1 vs 1.62; p=0.03). The patients in the treatment group also had increased exercise capacity and fewer anginal symptoms. METs increased from 5 to 6.7 in the treatment group and did not change in the control group (p=0.0085). A six percent increase in EF was noted on echocardiography (p=0.027). A reduction in reversible defect by SPECT from 15 to 4.5% (p=0.022) without a change in fixed defect percentage. This means that the reduction in reversible defect was not secondary to scar formation from the injection site. Although there was an increase in the ischemic area in the control group, the difference was not statistically significant. LV function improvement was sustained at 4 months. EMM showed a change in linear shortening 5.7 to 10.8 in the injected regions which indicated an increase in contractility (p<0.0005).  The investigators believed that the effect of ABMSCs injections was secondary to angiogenic properties of cells and hence improved contractility of hibernating myocardium. EMM was particularly useful to determine the viability of the tissue treated and guide the procedure. Neither arrhythmia nor myocardial injury was associated with the procedure. The lack of a sham procedure control was the major limitation of the study. The magnitude of the placebo effect can not be determined, which can affect the subjective NYHA class and exercise tolerance data. However, the improvement in SPECT imaging does provide objective evidence of improvement.
The same investigators decided to extend their patient follow up to 6 and 12 months and tried to stratify the magnitude of improvement by progenitor cell characteristics. This would help elucidate the potential mechanism of improvement . Although BNP levels increased in both groups, the treatment group experienced less of a rise (BNP 507 vs 740 at 12 months; p=0.08). A beneficial difference was noted in the NYHA class (2.7 vs 1.4; p=0.01) and exercise tolerance (METs 7.2 vs 5.1; p=0.02). The size of the reversible defect on repeat SPECT scans at 12 months was smaller in the treated group (11 vs 34%; p=0.01).  However, there was no longer a difference in the ejection fraction between the two groups. Early hematopoeitic cell and monocyte phenotype correlated with better perfusion at 6 months by SPECT, particularly the monocyte lineage, suggesting their possible role in angiogenic factor secretion.
Cellular therapy use in chronic angina patients also needs validation in larger randomized trials that are controlled with sham procedures.  Additionally, pre-clinical and clinical studies are needed to establish the best approach to administer cells for maximal cell survival and retention. The NOGA catheter may not be the best method. Thompson recently noted  that endoventricular catheters can be subject to motion due to the cardiac cycle, interference from the subvalvular apparatus, or inadequate tract formation within the myocardium by the needle. Perhaps  injections via the coronary sinus and the great cardiac vein with the guidance of intravascular ultrasound (IVUS) and an extendable nitinol needle may help overcome some these limitations.  The procedure may also be shortened by this approach (80-90 minutes as reported by Perin for EMM mapping).

## 3.4.3 Skeletal Myoblast Intramyocardial Injection for Ischemic Myocardial Dysfunction
In acute or chronic ischemia, the angiogenic potential of transplanted cells needs to be evaluated. The patients who are most likely 
to benefit from cells are those with chronic non-viable scar and myocardial dysfunction. The injected cells, either directly or indirectly (via paracrine effect) , improve the contractility of the treated myocardium . Autologous skeletal myoblasts have also been successfully expanded in vitro and implanted in myocardium of animals. They have been shown to improve contractility although they do no contract synchronously with the rest of the myocardium and do not integrate into it  . The need for manipulation in culture, the need for epicardial implantation during an open surgical procedure, and the arrhythmogenic potential of these cell islands all limit the use of skeletal myoblast. 
The first clinical study enrolled 10 patients with LVEF <35% and non-viable scar due to past myocardial infarction on FDG-PET and indication for coronary artery bypass grafting (CABG).  Two to three weeks before CABG, myocytes were cultured from the patients autologous vastus lateralis biopsy. With a 27-gauge needle, 800 million myoblasts were injected epicardially during CABG into a scar supplied by a non-graftable diseased vessel. Prednisone was given post-operatively in all patients. Patient safety and ability to obtain myocytes after culture served as primary outcomes. LV ejection fraction at one, three, and six months post-procedure was the secondary outcome. Ninety percent of the expanded cells were viable and sixty percent were myogenic. Although there were no major complications, four of them developed inducible ventricular tachycardia (VT) within 11-22 days and required Automatic Internal CardioverterDefibrillator (AICD). Ejection fraction increased from 23 to 32% (p=0.002). However, revascularization alone may account for this increase. Both non-revascularized segments that were transplanted with cells and those that were revascularized improved their contractility, suggesting that myoblasts indeed improved contractility locally.
Another small study enrolled 12 patients undergoing CABG. These patients had ejection fractions between 25 and 45% and non viable scar. After implantation, there was no need for AICD placement and ejection fractions improved from a mean of 35 to 53% at 3 months (p=0.002).  One explanation for the lower incidence of inducible VT in this study could be the use of autologous patient plasma for muscle culture instead of fetal bovine serum. This may have decrease the degree of inflammation around skeletal myoblasts and caused less VT. The suggestion that VT can be reduced by autologous plasma has been also shown in another more recent study by Chachques et al (2004) of 20 patients. Although a smaller amount of myoblasts were injected (200 million) that in the Menasche study, it was equally effective. However, the scar area in this study was also revascularized. Consequently, it is difficult to establish the contribution of myoblast transplant to heart function. However, myoblast treated areas had larger semi-quantitative improvement in wall motion that revascularized areas (wall motion score index 2.6 down to 1.6; p=0.0001). FDG-PET showed increase in uptake in transplanted scar areas (from 0.126 to 0.231; p=0.01). In revascularized areas, similar changes were observed (0.170 to 0.284; p=0.014). This may imply presence of viable myoblasts in the scar area. However, definitive proof is lacking. This improvement may solely represent the effect of revascularization on hibernating myocardium that was previously undetected by FDG-PET and thus originally thought to be non-viable.
Another small, five patient study , used a catheter approach to inject  skeletal myoblasts transendocardially with EMM guidance. No revascularization was done. A trend towards improvement in ejection fraction by echocardiography and LV angiography was noted. However, this could not be determined by MRI. Wall thickening in the injected areas showed significant improvement over untreated segments 0.9 to 1.8 mm; (p=0.008). One of the patients developed a long NSVT which forced the placement of an AICD.
Twelve months is the longest follow up to date of patients who underwent skeletal myoblast implantation . Ten patients undergoing CABG with low ejection fractions were treated with skeletal myoblasts. While all patients were placed on prophylactic amiodarone, two patients developed NSVT after the operation necessitating the amiodarone infusion. Ejection fraction improvement was similar to other studies mentioned above and was maintained at twelve months.
Skeletal myoblast implantations require better investigation into the efficacy of implantation and viability of injected myocytes. This data could potentially be obtained through biopsy of treated patients. The treatment continues to be associated with the risk of arrhythmia, but its etiology is still unclear. It may be necessary to have patients enrolled in further studies to receive prophylactic AICDs not only for treatment, but also for better recording and monitoring of abnormal heart rhythms.  EMM guided catheters for epicardial implantation may be less invasive for implantation than open heart surgery. CellFix, an example of such a catheter, allows for possible repeat administration of cells  as well as co-administration with angiogenic factors to improve survival. The effect of myoblast transplant from that of revascularization also needs to be separated by larger safety and efficacy randomized trials. Additionally, the use of adult cardiac myocytes  or fetal cardiomyocytes  for transplantation is another option that has not yet been investigated in human trials. These cells have a better potential to integrate with the rest of the myocardium, possibly making them less susceptible to arrhythmia and improving contractility even greater.

## 3.4.4 Angiogenesis and Cytokine Clinical Trials
Losordo et al.  recently reviewed the results of preclinical and clinical trials of angiogenic factor protein and gene therapy. This is also reviewed earlier in this article. However, the synergistic co-administration of angiogenic factors and cytokine with cellular therapy is an important consideration. FGF Initiating RevaScularization Trial (FIRST) of 300 patients with CAD who underwent intracoronary FGF-2 protein administration did not show any advantage over placebo but demonstrated a substantial placebo effect.  Additionally, VEGF-2 gene therapy studied in a phase I trail with direct myocardial injection showed no evidence of angiogenic effect by angiography.  Perhaps extracellular matrix or cellular vehicle is needed for sustained and effective administration of angiogenic factors.  It is also possible that the angiogenic network elaborated after a myocardial infarction is so elaborate that administration of a single cytokine cannot replicate it. Additionally, the endogenous endothelium may be too diseased to respond to angiogenic proteins. New endothelial progenitor cells may be needed for angiogenesis and vasculogenesis to take place. On the other hand, transplanted cells may also survive and retain better when co-administered with growth factors that mimic their natural humoral and structural environment. Evidence for this is provided by a study that showed that skeletal myoblast survival improves when fibrin biodegradable scaffold is used.   Nugent and Edelman recently reviewed other tissue engineering approaches to myocardial regeneration.  Pre clinical trials of cardiomyoplasty for acute and chronic myocardial infarction are currently being conducted with myotissue transplantation, a technology that would provide the ultimate preservation of structure and angiogenic milieu of the transplanted cardiomyocytes (Figure 8).
The MAGIC trial  studied the combination of cytokine treatment and cell therapy in a prospective randomized trial of 27 patients undergoing stenting for acute myocardial infarction. The additional treatment was intracoronary infusion of unselected peripheral blood stem cells with administration of intravenous Granulocyte-colony stimulating factor (G-CSF). The investigators hoped that the G-CSF would increase endothelial progenitor/stem cell mobilization from the bone marrow that usually occurs in the acute setting of MI  and that peripheral blood could be used instead of bone marrow for infusion. Additionally, recent evidence suggested that G-CSF is capable of preventing unfavorable ventricular remodeling.  However, the trial was stopped prematurely because of an increased incidence of in-stent restenosis in G-CSF treated patients. While benefits of left ventricular ejection fraction and exercise tolerance were noted, the safety concern of restenosis was more prominent.

# 3.5 Conclusions and Future Directions
Cellular therapy holds great promise, especially for patients with end-stage coronary artery disease or refractory angina. It can potentially reduce incidence of left ventricular dysfunction and heart failure. Small phase I trials conducted to date show encouraging results. However, the therapy needs to be perfected before it can be widely applied. We run the danger of disappointment with cellular therapy in larger randomized Phase II and III trials unless the mechanistic foundation of this potent therapy is elucidated first to help refine the technology (Lee, Wykrzykowsha, and Laham, 2006 in press). The viability of injected cells remains rather poor, which calls into question the direct contribution of cellular therapy to the improvement in contractility. It is possible that the paracrine effect of apoptosis of these cells may be the underlying mechanism leading to the improvement in function. Poor viability has been shown in many preclinical studies (Figure 7). Cell delivery catheters need to be developed better. Matrix scaffolds to allow for better cell survival and imaging techniques to track cell viability need to be developed.  Ideally, MRI technology would allow for gene and protein expression imaging. MRI is already the most sensitive and specific technique for assessing left ventricular performance (wall motion, wall thickening, ejection fraction) and perfusion as well as viability (late enhancement).

# 4. Imaging Technology in Angiogenesis and Myogenesis

# 4.1 Clinical Experience To-Date and Limitations of Imaging Technology
As reviewed elsewhere, pre-clinical and phase I clinical gene therapy trials testing angiogenic factors have been very promising.  However, placebo controlled phase II clinical studies have been disappointing.  Limitations of imaging and non-invasive detection techniques as well as a very strong placebo effect in this patient population may help explain these contradictory findings. Angiography can not image vessels less than 180 m in diameter. Nuclear imaging has a spatial resolution of 10 mm, which is the thickness of the myocardium. This makes it unsuitable to detect small capillary networks that are often intramyocardial. While Positron Emission Tomography (PET) and Single Photon Emission Computed Tomography (SPECT) have slightly better resolution, it is still not precise enough to image new vascular growth. The best technology to date is MRI, which has a spatial resolution of 25-100 m. 
Myogenesis imaging faces similar challenges. It is difficult to determine the effects of myogenesis on myocardial perfusion and function. The following reviews currently available technologies for imaging of angiogenesis and myogenesis. While these therapeutic modalities were previously viewed as an attempt to influence poorly understood processes with poorly developed tools and outcome measures, tremendous advances have been made on all of these fronts.

# 4.2 Specific Imaging Methods
Current technologies to image angiogenesis include immunohistochemistry, electron microscopy, optical imaging with fluorescence , ultrasound, laser doppler , radionuclide imaging including PET, x-ray angiography, multislice computed tomography and MRI.
Imaging used in pre-clinical studies that allowed for histologic evaluation include vascular casts, microscopy, and microscopic CT. Intravascular injection of gelatin or other casting material is used to make vascular casts. They are then imaged with transmission microscopy or electron microscopy (Figure 9). Immunohistochemical staining for vascular markers such as CD31, von Willebrand factor and basement membrane proteins (integrins) improved histologic analysis. Counting vessels per unit area is allowed by staining in thicker sections (Figure 10 and 11). Endothelial cell labeling is also feasible in animals transgenic for Green Fluorescent Protein reporter under the regulation of endothelial cell-specific protein. However, these histologic methods fail to distinguish functional vessel from those without a functional lumen. Intravital labeling with intravascular tracers such as fluorescent lectin is necessary to make such a  determination. Angiography, the clinical equivalent of this, does not have adequate resolution to visualize the microvessels of angiogenesis (25 m). It is limited to larger coronary arteries and veins (Figure 12). CT and MR angiography has marginally improved resolution (200 m) in humans (Figure 13 and 14). While the micro-MR and micro-CT  technologies used in mice have a resolution of 10 m, they have poor temporal resolution  and require a high radiation dose. However, the use of contrast agents such as supramagnetic iron oxide particles and gadolinium labeled albumin that can allow for blood volume measurements. Additionally, MRI and PET scanning can be enhanced by molecular tracers. Microvascular perfusion can be observed with color doppler combined with microbubble contrast. However, resolution is limited. Unfortunately, these methods are unfeasible for angiogenesis or cell tracking imaging in a clinical setting. Correlations can be established between these benchmark methods and imaging technology in pre-clinical models that is applicable to clinical settings.

## 4.2.1 Vascular Corrosion Casts and Microspheres for Myocardial Perfusion
In contrast to immunohistochemistry and basic histology, vascular corrosion casts allow for imaging vessels with patent lumina. A gelatin-dye mixture is injected into the vessel of interest and allows visualization of all collaterals originating from that vessel (Figure 9). It may be perfusion fixated first. The vessels are then excised, embedded in plastic, sectioned, and then imaged for vessel size and structure by electron microscopy. Imaging also shows collaterals and neovascular network architecture. The structural analysis is usually confirmed with colored, radioactive, or neutron-activated microspheres. With a diameter of 15 m and labeled with tracers such as samarium, gold, rhenium, or lutetium, the microspheres are injected into the left atrium. Two samples are drawn from the arterial system for reference concentration measurements. Generally, one type of microsphere is injected at rest while another is injected after pacing or administration of adenosine or other stress agent. After excision of the heart, the tissue is fixed in paraformaldehyde and radioactivity is measured in sections by a Germanium detection system after neutron activation (BioPal). The radioactivity of microspheres reflects perfusion to the sectioned area of myocardium. In contrast to optical imaging technologies, tissue penetrance does not limit this technique.

## 4.2.2 Fluorescein Video Angiography
This  minimally invasive and low-cost method to quantify angiogenesis and solute exchange has been validated in different human tumors. Fluorescein is a hydrophilic solute capable of diffusion and brightly highlighting all the plasma perfused microvessels. Videoprints can detect vessels filled with fluorescein and can quantify the time course of fluorescein concentration and the diffusion from vessels. In basal cell carcinoma, fluorescein video angiography reveals a 2-fold increase in microvessel image area fraction and a 3-fold increase in the slope of accumulation. This represents increased angiogenesis compared to normal controls. Although video-microscopy use is limited by tissue perfusion, this technique could be used for imaging myocardial angiogenesis. Advances in optical imaging make this technology usable for clinical application. Its limitations are similar to that of many bioluminescence reporters such as green fluorescence protein or luciferase.

## 4.2.3 Two Photon Excitation (TPE) Fluorescence Imaging
TPE microscopy, much more advanced than single conventional single photon confocal microscopy, allows for three-dimensional resolution of cellular fluorescence signals within strongly scattering thick samples such as myocardium. TPE of calcium-sensitive fluorophore rhod-2 can image calcium flux and differentiate between transplanted and endogenous myocardial cells. While single photon microscopy causes photon excitation above and below the plane of focus, requiring an adjustable pinhole to increase spatial resolution, a two photon excitation provides a quantitative measure of the probability of a molecule absorbing two photons simultaneously (cross section area) multiplied by the fluorescence emission quantum efficiency. Consequently, a three-dimensional image can be produced. TPE also has a much better penetration depth than conventional single photon confocal microscopy. Fluorophore excitation by the second light intensity confines photon absorption to one narrow plane and reduces linear absorption. Consequently, the energy is allowed to reach deeper tissues. Because the wavelength of the photon is longer, scatter is reduced. Subcellular resolution can be achieved even several millimeters in depth. The fluorophores are often caged compounds that are biologically inert unless irradiated.  Using Ti-sapphire laser with near infrared light, calcium fluxes could be quantified down to femtoliter amounts and 1 m resolution using caged fluorophores as read out. This technology could be used to study gap junctions and other electrical communications between transplanted cardiomyocytes and fibroblasts. Transplanted fetal cardiomyocytes have been demonstrated to communicate with resident myocytes. However, skeletal myoblasts and resident myocytes show no communication.  Temporal resolution of erythrocyte flow within newly formed capillaries may also be detected with TPE.

## 4.2.4 Nuclear Imaging
The distribution of an injected radioactive isotope in heart muscle can be shown by nuclear imaging. A relative reduction in isotope uptake is shown in ischemic areas with impaired blood delivery. Thallium-201 scintigraphy was the earliest clinical imaging of inducible ischemia and infarction. However, it suffers from poor spatial resolution (about 10 mm). Small but significant changes in perfusion are sometimes missed, as would be seen after angiogenesis therapy and branch vessel disease. Next, SPECT imaging was developed. Using a collimated array of detectors, SPECT enabled resolution of the isotope distribution to select planes and 3D summaries rather than just a volume projection. Additionally, technetium 99m sestamibi allows high dosage and better image quality than thallium-201.
Besides poor image spatial resolution, nuclear imaging is not sensitive to partial thickness progressive changes. Instead, only all-or-none determinations of inducible ischemia can be made at maximally tolerated stress. While this is very useful clinically, it is not suitable for angiogenesis or cardiomyoplasty studies. This is because successful angiogenesis and myogenesis can produce new vascular and cardiomyocyte elements that protect tissue and even improve exercise tolerance without necessarily abolishing inducible ischemia. Consequently, it may be more useful to test at a fixed stress level attempting to match baseline exercise peak. However, stress is not accurately reproducible and can be affected by conditioning, medications, hormonal effects, fluid loading, and other variables.  Additionally, the diagnostic value of thallium and sestamibi are based on maximal stress. Current radionuclide methods have a resolution that is at a scale two orders of magnitude larger than the process of angiogenesis. Consequently, clinical trials of therapeutic angiogenesis and laser revascularization using SPECT imaging have cast doubt on the ability of nuclear imaging to quantify the progressive changes secondary to therapeutic angiogenesis.
Resolution may be improved by combining non-invasive nuclear imaging with molecular targeted approaches. As endothelial cells develop cell to cell and cell to extracellular matrix adhesion during angiogenesis, integrin v3 is expressed which can be bound to radiolabelled quinolone In-RP748.  This can be tracked by a single photon emission computed tomographic imaging (SPECT) and was successfully used to image ischemia induced angiogenesis in rat and dog models of chronic myocardial infarction.  Molecular imaging will become more easily applicable to the clinical setting with the development of hybrid x-ray CT and SPECT imaging systems. In the future, it may be possible to track cardiac stem cells in myogenesis and cardiomyoplasty in a similar manner by tagging cardiac stem cell markers such as sca-1 or c-kit with radiolabelled compounds.
Perhaps loading stem cells with another tracer that emits photons of different energy such as indium 111In and perform simultaneous imaging for both viability with 201T1 and cell engraftment with 111In is an easier method. The 111In signal persists for as long as 96 hours after injection. However, because the 111In signal remains after cell death, the signal can not be correlated with graft cell viability. However, it may be useful in guiding or verifying initial delivery success. Histologic confirmation of cell implantation may be accomplished by simultaneous labeling of a reported gene such as luciferase, which is under transcriptional control of a sarcomeric structural protein, by fluorescence microscopy and iron particle labeling for electron microscopy.

## 4.2.5 Myocardial Contrast Echocardiography
By detecting frequency shifts from moving blood, color-flow doppler ultrasound can assign color based on a scale proportional to blood velocity. However, 200 m is its highest resolution. Microbubbles in myocardium allow blood volume assessment indirectly because of changes in small capillary bed resistance. Myocardial contrast echocardiography combines microbubbles with ultrasound. The 1-10 mm hydrophobic vesicles are filled with perfluorohydrocarbon gas. The high intensity ultrasound burst these microbubbles and the back scatter echo reflects the myocardial perfusion.  Capillary blood volume is reflected by the myocardial signal which is related to the microbubble concentration. During hyperemic vasodilation, the microbubble velocity increases in proportion to the coronary blood flow. In stenotic epicardial vessels, changes in the capillary bed resistance results in large changes in coronary blood flow. Because the capillary resistances are in parallel, the change in resistance translates into a change in volume. The greater the signal detected by myocardial contrast echocardiography, the greater the myocardial blood volume and the more capillaries that are in parallel. Consequently, this method can be used to indirectly detect angiogenesis and new capillary growth. It also permits assessment of myocardial perfusion under stress and hyperemia conditions induced by adenosine infusion. In animal models with chronic myocardial ischemia treated with VEGF-121, Myocardial Contrast Echocardiography (MCE) could detect improved endocardial and epicardial perfusion.  The detection was similar to that achieved with radiolabelled microsphere injection. Additionally, MCE could distinguish between the VEGF effect on endocardial versus epicardial perfusion and showed a greater effect on the endocardium. While this method appears promising, it is limited in the clinical setting. It is difficult to obtain identical images at rest and under stress to allow for the ultrasound backscatter to be similar. Similar blood microbubble concentration is non-uniform between the two conditions. Although it is less invasive that catheterization methods of determining coronary blood flow, MCE remains technically difficult and time consuming.  Future advancements may eventually make it a useful technique in the clinical setting.

## 4.2.6 Coronary Angiography
Even with the high quality 7 inch magnification mode, 30 frames per second cine, and tailored background and contrast, assessing collateral circulation and angiogenesis is challenging by x-ray angiography secondary to its limited resolution. Catheter engagement and rate of contrast injection, both of which are variable, can affect the outcomes of longitudinal studies. On angiography, collaterals typically are very faint and are usually between 0.3 to 0.7 mm. They are identified based on their size and anastomosis with the same vessel or another collateral. They may also extend more than half of the distance between two epicardial vessels. They can also appear on follow up angiography where they were previously absent. Generally, they branch at angles less than 135 degrees and are quite tortuous. Collateral flow grade is expressed on scale from zero to four, where zero is no flow and four is a well opacified vessel that is greater than 0.7 mm in diameter that fills antegrade. Other parameters that are assessed include collateral frame count (number of cine frames required for contrast media to reach the recipient vessel) and myocardial blush grade (opacification of myocardial microvasculature i.e. vessels that are not angiographically apparent). The myocardial blush grade is very significant in indirect neovascularization assessment. It is assessed on a scale from zero to four, where zero is no blush and four is blush persistent until the next contrast injection long after contrast washout from the epicardial artery. Careful evaluation of the recipient vessel filling grade, where zero is no collaterals and four is complete filling of a totally occluded epicardial vessel by a collateral, should also be performed. The complexity of the collateral network, which is based on the number of collateral branches, can also be noted. The collateral length is planimetered using a catheter as a scaling device. It is graded based on the number of eighths of the parent vessel length. Quantitative and semiquantitative methods for collateral network assessment have been proposed by several investigators. In conclusion, angiogenesis and the effects of transplanted cells on myocardial perfusion can only be indirectly and semi-quantitatively measured by X-ray angiography because the technology is limited by its current resolution (Figure 12).

## 4.2.7 Coronary and Myocardial Fractional Flow Reserve (FFRcor and FFRmyo)
X-ray angiography can help quantify improved myocardial perfusion by measuring myocardial fraction flow reserve. FFRcor is the maximum blood flow in the presence of a stenosis divided by the normal maximum flow of the artery. FFRmyo is the maximum myocardial blood flow distal to an epicardial stenosis divided by its value if no epicardial stenosis existed. Essentially, FFR reflects the percentage of normal maximum flow that remains despite the presence of a lesion. The FFR of a coronary artery and its dependent myocardium can be ascertained by the following equations: FFRcor = (Pd-Pw)/(Pa-Pw) and FFRmyo = (Pd-Pv)/(Pa-Pv), where Pa, Pd, and Pv are taken at maximum vasodilation. Pw is taken at coronary occlusion. FFRcor can be calculated only during PTCA because Pw must be measured.  However, FFRmyo can be calculated during a diagnostic catheterization. The difference between FFRcor and FFRmyo reflects the collateral flow to the vessel distal to the stenosis and is termed fractional collateral flow.  FFRmyo is the most useful clinically and reflects both antegrade and collateral contributions to myocardial perfusion in the presence of an epicardial coronary stenosis.  The formula for FFRmyo can be simplified further to FFR = Pd/Pa. A significant FFR is less than 0.75 based on prior studies in patients with ischemia on exercise testing.  The technique is safe and has been well validated (Figure 15). However, the results may be confounded by a steal phenomenon induced by systemic adenosine administration. The hyperemic response to adenosine may be blunted by endothelial dysfunction secondary to hyperlipidemia and diabetes. In angiogenesis or cellular therapy, one would expect no FFRcor improvement but improvement in FFRmyo. Additionally, the vasodilator response of immature vessels may be less pronounced thus underestimating the degree of neovascularization.

## 4.2.8 Multislice Computer Tomography imaging of angiogenesis and new applications in stem cell transfers
Transport of intravenous iodinated contrast to the tissue via the blood flow and diffusion of contrast between the intravascular and extravascular space forms the basis for CT angiogenesis imaging. Tissue contrast distribution can be determined by multislice scanners that measure tissue and vascular enhancement over time (Figure 14). This allows for estimating tissue blood flow, blood volume and capillary surface permeability. X-ray contrast used by CT is a hydrophilic, inert derivative iodobenzoic acid with a substantial molecular size (700 Da). It only distributes in the blood and interstitial space and is excluded from the cells. Extraction efficiency (E) can be measured as contrast moves from arterioles to the extravascular space. X-ray attenuation increases in the presence of contrast and causes enhancement on CT. The enhancement is not dependent on the tissue microenvironment unlike MRI.  In a typical study, the contrast agent is administered and the signal to noise ratio is proportional to administered concentration, rate, and volume. Scans are done in two phases: a vascular phase and interstitial wash-in and wash-out phase.  By calculating tissue attenuation curves from the contrast arrival time in the arterial, interstitial, and venule phases, perfusion assessment can be performed. Eventually, the contrast-enhanced image is subtracted from a baseline CT image. Microsphere distribution measurements have helped validate CT angiogenesis in multiple studies in tumor angiogenesis models and cerebral ischemia. Disadvantages to perfusion CT include sensitivity to motion artifact and a limited field of view (20 mm). Two volumes of contrast distribution are compared by elastic match imaging and the difference between the two identifies the myocardium subtended by collaterals and angiogenesis. Saline is used to pressurize the territory beyond the coronary stenosis and contrast is injected proximally to form the first volume. The second volume is obtained without saline backpressure. This technique is limited by higher x-ray doses and its sole ex-vivo use.
Lima and Lardo (personal communications) recently used multidetector CT with contrast to establish the viability of injected stem cells. The borders of the injected transplanted cell islands are outlined as the CT contrast diffuses into the interstitium. The study is limited by its signal to noise ratio, which is not quite as good as that for contrast enhanced magnetic resonance imaging. Additionally, the study requires a threshold number of injected cells.

## 4.2.9 MR Imaging of Angiogenesis
MRI offers some unique characteristics that are unlike other techniques. It helps define the mechanism and impact of angiogenic and cell therapy. Furthermore, it is cost-effective because its potentially high sensitivity to treatment effects allows for smaller trials. The use of the MRI has several specific techniques that are well suited for assessment of angiogenesis and cellular tracking.

### 4.2.9.1 Perfusion-sensitive MRI
By adjusting imaging parameters, the myocardium is represented as dark (Figure 13). With every heart beat, an image is obtained for forty seconds after the injection of contrast. The contrast-labeled blood first arrives at the right ventricle and appears as a bright signal. The contrast then moves to the lungs and then fills the left ventricle, the aorta, and coronary arteries to arrive finally in the myocardium.
Generally, a target level is designated by its distance from base to apex and is studied in detail. Cine images track the out-of-plane target motion due to left ventricular contraction. With the SMART technique, cardiac twisting from diastole to systole is tracked both prospectively and retrospectively.  Blood arrival to the myocardium that is impaired can by measured by tracking signal change at each pixel using a space-time map. It can also be tracked by measuring other variables such as time of arrival, slope of signal intensity, or time to 50% peak signal intensity.
The measurements can be based on one of two parameters. Either they are based on a time-intensity relation for pixels in impaired vs. normal supply zones or on derived time-concentration curves that take into account MRI relaxivity and hematocrit. While one category of measurements is purely descriptive and is based on the upslope or time to peak, another measurement category is based on mathematical modeling of contrast arrival based on dye dilution principles of convolution. A more comprehensive model of blood arrival based on differential equations, reflecting multiple pathways, multiple compartments, conservation of mass, and stable exchange rates between compartments comprises a third category. Perfusion-sensitive MRI can follow two approaches, with the ultimate goal of establishing a distinction between signal intensity at baseline from the arrival of contrast-labeled blood. This is accomplished by adjusting the imaging conditions so that the myocardium is dark and becomes bright with contrast-labeled blood.

### 4.2.9.2 Functional Assessment
Regardless of stress conditions, improved blood supply leads to an enhancement of tissue function. Radial wall motion or wall thickening can represent regional cardiac function. Both of these variables are measured as a change from diastole to systole divided by the systolic dimension. Additionally, wall thickness can be expressed as thickness release, the change from diastole to systole divided by the systolic dimension. This measurement reflects the metabolically active component of the cycle and is less subject to interference because the denominator is larger.
With heart contraction, the base approaches the apex, the axis tilts, and the heart rotates. Both the TOPCARE-AMI and the BOOST trial employed MRI technology to measure myocardial contractility and showed a benefit with the use of autologous peripheral and bone-marrow derived progenitors injected into patients with acute myocardial infarction. One limitation of this technology is incompatibility with internal cardioverter-defibrillators and pacemakers, which are very common in patients with ischemic cardiomyopathy. Add out MRI data from FGF studies and laser studies.

### 4.2.9.3 Cell Tracking by MRI
The use of stem cells to help repair injured tissue has been studied to help regain lost functionality. The hope has been that the undifferentiated cells can help improve heart contractility and accelerate formation of vascular elements. Cells can be labeled to produce a distinctive signal when the heart is imaged by MRI to help track their location and verify their delivery. MRI labeling compounds include T1 agents, T2- (susceptibility) agents, and chemical shift agents. T1 agents facilitate magnetization change and increase signal recovery, which is termed positive contrast. T2- or magnetic susceptibility agents disturb the uniformity of the magnetic field thus accelerating signal loss. This is termed negative contrast. Chemical shift agents work by changing resonance frequency. To date, susceptibility agents have been preferred because the effect blooms much larger than the target.
Investigators have successfully imaged iron-labeled bone marrow and skeletal muscle cells after injection into the myocardium with catheter-based delivery.  This was accomplished using intracellular magnetic susceptibility, the proportionality between the applied magnetic field strength and the magnetization established in atoms with an unpaired nucleon. The superparamagnetic iron oxide particles produce a strong augmentation of the local magnetic field causing a regional increase in T2 and T2*. This is represented as a loss of signal intensity on T2- sensitive MRI.
The most accurate stem cell delivery to a scar region involves combining delayed enhancement MRI (to outline the scar) and iron-labeled stem cells delivered by a MRI-safe active nitinol catheter. This is a catheter whose tip is highlighted. Hyperenhanced regions were identified and a green-highlighted needle was guided towards these regions. The injected cells could be immediately visualized as a reduced signal secondary to iron particle effect on relaxivity. There are two major limitations to this method. The first is that the iron itself may have a functional and survival effect on injected cells. Secondly, the decrease in signal may not be necessarily proportional to the number of cells injected or retained. Hence, a quantitative measurement can not be established. However, a threshold number of cells must be present for the MRI to detect them. Other guidance methods for cell injections, such as ultrasound and electromechanical maps, are far less precise.

## 4.2.10 Molecular Imaging
The desire to locate and measure specific molecules has increased as we discover more about signaling pathways and specific regulators involved in angiogenesis. The distribution of a specific molecule can be determined by PET, MRI, and optical methods with specific labels. PET studies substitute a positron emitting isotope for an ordinary atom in the target molecule. MRI relies on larger nanoparticle attachment to an antibody fragment or ligand that associates with the target molecule. Together, these techniques can tract agent distribution (PET) and measure effect (MRI). The larger the molecular tag, the worse its ability to enter extravascular tissue. At 120 kDaltons and above, minimal extravasation occurs. Cross-linked iron oxide (CLIO), a strong MRI contrast agent, bound to antibody fragments targeted to human E-Selectin has been shown in studies to achieve a 100 to 200 times high binding to cells stimulated to over express E-Selectin than control cells. The cells in the study were human endothelial umbilical vein cells stimulated with IL-1 and studied in vitro. In vivo, alpha(nu)beta(3)-integrin, a biomarker  that has been highly expressed on activated neovascular endothelial cells and is absent on mature quiescent cells, has been tracked in rabbits. This was accomplished by using paramagnetic nanoparticles to mark the location in MRI, and non-paramagnetic agent to displace the marker. Even though the nanoparticles have a relatively large size, they still penetrated deep into leaky tumor neovasculature, and produced signal changes of 56-126%.

# 4.3 Conclusions and Future Directions
A wide array of technology can be employed to assess angiogenesis and cellular therapy effects in the ischemic myocardium. Through a better understanding of the molecular mechanisms of angiogenesis, clinically applicable imaging technologies may be further developed such as optical, ultrasound, nuclear, computer tomography, or magnetic resonance imaging. Clinical imaging can be improved by designating specific molecular probes that mark different stages of angiogenesis. Two photon emission microscopy is an example of combining activated probes to follow subcellular processes in the myocardium in vivo.  By conjugating molecular markers to iron and gadolinium, MRI can image angiogenesis and cardiomyoplasty. Indirect methods of imaging functional effects of angiogenesis therapy and cardiomyoplasty have progressed tremendously and have now been validated.  MRI and other sensitive assessment methods evaluate functional and delayed enhancement and provide more effective clinical outcomes measures. This translates into proof of concept studies with smaller sample sizes and limits the powerful placebo effect shown in previous studies. Using novel imaging technologies with better sensitivity and specificity, future investigations should not be plagued with inadequate outcomes measures and insensitivity to detect treatment effect.

# 5. Conclusion

# 5.1. Explanation of Failures and Disappointments
Angiogenesis has followed the usual path for emerging technologies: incredible results  unrealistic expectations  sobering disappointments  cautious optimism. Development strategies generally strive to follow the righteous path (Figure 16): understanding the biology  developing therapeutic agents, vectors, and animal models  site specific delivery  adequate outcome measures. Failures and disappointments have followed initial promising results in angiogenesis for several reasons.
First, angiogenesis is a complex process that requires the action of multiple growth factors, angiogenesis inhibitors, and modulators in specific cascades. The delivery of a single growth factor for a short duration can not be expected to result in a long term therapeutic response. Additionally, these growth factors are already expressed in high levels in the setting of ischemia. The administration of additional exogenous growth factors may provide little benefit. Moreover, evidence suggests that endothelial dysfunction resulting from advanced age, diabetes, or elevated cholesterol can impair angiogenic response. In a study with a porcine model of chronic myocardial ischemia, animals were fed either a high cholesterol or a normal diet. An ameroid constrictor was placed on the left coronary circumflex artery. After four weeks, FGF-2 loaded in heparin alginate beads for slow release was implanted in the circumflex territory. The high cholesterol diet group showed significant endothelial dysfunction and impaired angiogenesis as compared to the normal diet group. This resulted in decreased circumflex perfusion compared to the control. 
Animal model choice may also contribute to the poor results. For angiogenesis, the primary animal models have been the porcine, rabbit, and murine models. For cardiac angiogenesis research, the porcine ameroid constrictor model, particularly the Juvenile Yorkshire pig, is the most commonly used. Most of these animals have been young and healthy, with normal cholesterol and endothelial function. This differs strikingly from the older, diabetic population studied in phase II trials with hypercholesterolemia and endothelial dysfunction. This may explain why promising pre-clinical studies have been followed by disappointing phase II trials. Additionally, effective delivery to the myocardium with adequate distribution and retention has been an obstacle. Intravenous and intracoronary has been proven to be especially poor. The liver metabolizes most of the growth factor delivered intravenously. Intrapericardial delivery has been shown to improve myocardial distribution and retention but with poor penetration into the endocardium. While intramyocardial delivery resulted in the best myocardial deposition and retention, still less than 20% of the therapy administered is retained with injections localized to administration site. It is very important to first determine the best route of administration to optimize the delivery strategy before subjecting a specific agent, cell, or vector to clinical study.
Another major problem with clinical studies is outcome measures. The means used to assess angiogenesis in patients have been adopted from cardiology and cardiac surgery studies. They may not be sensitive enough to detect the small changes seen with therapeutic neovascularization. Animal studies have shown an improvement in blood flow in ischemic territory with angiogenic therapy to be about 20-40 percent. This is far less than the revascularization seen with angioplasty or bypass surgery. Consequently, outcome measure must be altered to expect this relatively smaller improvement.  This improvement may be all what is needed to improve the quality of life of no-option patients. However, small tissue-level increases in perfusion with angiogenic therapy can not be expected to be perceived by a nuclear perfusion scan, which has a spatial resolution of about 8-10 mm. The development of newer outcome measures more sensitive to angiogenic therapy is as important as developing newer angiogenic agents themselves. Generally, outcome measures consist of hard endpoints and soft endpoints. Hard endpoints include death, myocardial, infarction, stroke, and recurrent ischemia  MACE while soft endpoints include parameters such as angina class and quality of life measures. While hard endpoints are preferable for clinical study, the rarity of these events even in high-risk no-option patients requires prohibitively large studies in order to measure a significant effect. Softer endpoints can be made more objective with the use of independent assessments and validated questionnaires. Surrogate endpoints play an important part in reducing numbers of patients needed for preliminary efficacy and in providing insight into the mechanism of treatment. Such end points include exercise assessment, nuclear perfusion scan (SPECT and PET), magnetic resonance functional and perfusion imaging, multidetector computer tomography, and echocardiography. As discussed above, magnetic resonance imaging is particularly very promising. 
Appropriate study design is also essential. Efficacy data should not be claimed by small open label studies. These studies should solely be used to assess safety and tolerability. Patients with end stage heart disease experience an extremely powerful placebo effect that is associated with an improvement in symptoms, exercise time, and even perfusion scans.  However, this placebo effect has been shown to be sustained for only up to two years of follow-up. Before efficacy claims can be made, adequately powered, randomized, double-blinded, placebo-controlled studies must be successfully completed.

# 5.2 Pathway Towards Therapeutic Angiogenesis
Therapeutic angiogenesis can and will be achieved. However, a robust translational model and several general principles must be established:
- 1.Because angiogenesis is such an intricate process, sustained delivery of multiple growth factors, master switch molecules (HIF-1, RTEF-1) , cell based therapy , and microtissue transplantations are more likely to be beneficial. Short term delivery of single growth factors is unlikely to provide an improvement.
- 2.Optimized organ specific and agent specific delivery strategy should be determined before preclinical and clinical studies in order to avoid ineffective delivery and agent inactivation. An example of this is adenoviral vector inactivation with catheter delivery.  After this analysis is performed, further development can take place.
- 3.Adequate survival and treatment specific trans-differentiation should be confirmed before cell and tissue transplants should be performed.
- 4.Animal models should reflect the disease states of the human population most likely to receive treatment. Adult, aging, hypercholesterolemic animals should be used to confirm results found in juvenile normal animals. Adequate power and multiple outcome measures are necessary to account for the marked variability in many animal models and to avoid a chance finding.
- 5.Well designed studies and outcome measures in clinical studies:
- a.Adequately powered to prevent possibility of a chance finding.
- b.The powerful placebo effect seen in this patient population should be accounted for with a randomized, double blinded, placebo controlled design. Patient and investigator blinding are the best way to reduce crossover (which dilutes treatment effect) and minimize differences with other parameters.
- c.Multiple outcome measures with endpoints that help clarify mechanism, including quality of life assessment and imaging studies.
Only by following a rigorous development plan can angiogenesis as a treatment for coronary artery disease become a reality. Specific experimental objectives that will help lead to therapeutic angiogenesis include:
- 1.Discovering the mechanism of action and role in physiologic and pathologic angiogenesis of a particular agent.
- 2.Testing in vitro with endothelial cell proliferation (cell lines followed by primary human endothelial cells), migration (wounding assay), and tube formation (matrigel plates)
- 3.Testing in vivo in matrigel mouse plug model
- 4.Testing in vivo in murine myocardial infraction or hind limb ischemia model assessing infarct or limb salvage, perfusion, and endothelial cell density (CD31 staining)
- 5.Large animal model delivery optimization
- 6.Preclinical study in juvenile normal animals for proof of concept
- 7.Preclinical study in adult or aging disease population
- 8.Phase I clinical study for safety and tolerability only.
- 9.Phase II, randomized, double blind, placebo- controlled study with endpoints that reveal mechanism and adequate outcome measures.

# Figure Legend
- Figure 1. (=Figure 1 from Position Statement)
Cine angiography in the right anterior oblique cranial project of the left coronary system.  Extensive collaterals (black arrows) filling a totally occluded right coronary artery (white arrows) can be appreciated
- Figure 2. (=Figure 2 from Growth Factor)
The porcine ameroid constrictor model remains the most frequently used preclinical model for therapeutic angiogenesis.  Two animals with an ameroid constrictor (black arrows) placed on the left circumflex artery are shown here. Total occlusion of the artery 2-3 weeks after placement is noted. On the left is an angiogram from a control animal with no reconstitution of the left circumflex artery (white arrows). On the right is an angiogram from an animal that received perivascular VEGF (via a pump). Prompt filling of the left circumflex artery (whiter arrows) by collaterals (both left to left and right to left) is noted. Experiments are performed mostly on juvenile pigs.
- Figure 3. (=Figure 3 from Growth Factor)
Histological analysis in the ameroid constrictor model showing increased neovascularization after VEGF administration (B) compared to control animal (A). Batson Casting (C) showing Left Circumflex artery in Blue, Left Anterior Descending in Red and Right Coronary Artery in White. Left Circumflex distribution is being supplied by collaterals from other territories. Corresponding Angiography (D) of ameroid contrictor model of Left Circumflex Artery occlusion and patent Left Anterior Descending with bridging collaterals from the Left Anterior Descending to the Left Circumflex Artery territory.
- Figure 4. (=Figure 4 from Growth Factor)
Rest Thalium (bottom row) and stress-SestaMibi (upper row) scans at baseline (left) and 3 months (right) after CABG in a patient who received 100 g of perivascular (intramyocardial) basic FGF showing improvement in inferolateral wall perfusion. 
- Figure 5. (=Figure 5 from Growth Factor)
Myocardial perfusion/contrast arrival as assessed using MR Imaging after bolus administration of gadodiamide. Time sequence display of selected short axis diastolic images shows contrast arrival to the right ventricle, the left ventricle, followed by left ventricular myocardium. The mean size of the delayed contrast arrival zone (underperfused area of myocardium) was reduced significantly after Basic FGF administration (at 180 days). 
- Figure 6. (=Figure 1 from Cell Therapy)
Bone marrow cell induced capillary and neovessel formation in the matrigel placed in the skeletal muscle of a dog.
- Figure 7. (=Figure 2 from Cell Therapy)
Fibrosis and lack of viability of skeletal myoblasts injected into myocardium of dogs.
- Figure 8. (=Figure 3 from Cell Therapy)
- A. Viability of myotissue (autologous myocardial septal biopsy tissue) implanted into anterior wall scar at 4 weeks post-implantation in a porcine model of myocardial infarction. 12 Yorkshire pigs underwent an anterior myocardial infarction by balloon occlusion of the LAD and were randomized to implantation of 6-9 septal intact myocardial biopsy tissues into the anterior infarct area versus sham operation. Animals underwent cardiac MRI for anterior wall perfusion and delayed enhancement imaging for infarct volume at 4 weeks post implant and were subsequently sacrificed. Tissues were harvested for histology.
- B. Bar graph showing results of cardiac MRI showing increased perfusion in the anterior wall of treated animals as compared to the septal (non-implanted) wall perfusion, and decreased infarction volume after myotissue implantation as measured by delayed enhancement cardiac MRI in the same porcine model of myocardial infarction.
- Figure 9. (=Figure 1 from Imaging)
Batson casts or microfill technology enables visualization of collaterals after digesting the myocardium.  Here RCA territory was filled with white, LAD territory with red, and LCX (occluded) with blue.  Note the collateral vessels from LAD and RCA to LCX territory (arrows).
- Figure 10. (=Figure 2 from Imaging)
BrdU staining in control animals (left) and animals treated with growth factors (VEGF) showing BrdU positive cells (arrows) in treated animals corresponding to neovascular growth.
- Figure 11. (=Figure 3 from Imaging)
CD-31 (PECAM-1) staining showing CD31+ cells corresponding to endothelial cells. No single marker is able to identify all endothelial cells (including vwF, eNOS, CD-31, flt-1, flk-1).
- Figure 12. (=Figure 4 from Imaging)
Coronary Angiography can identify collateral vessels (black arrows) from Left coronary artery to the occluded right coronary artery in this patient.  Filling of collateral dependent vessel (white arrows) depends on resolution, catheter engagement, strength and duration of contrast injection and cannot be used for longitudinal assessment.
- Figure 13. (=Figure 5 from Imaging)
MRI functional (top) and perfusion (bottom) imaging. Functional imaging is performed using an ECG-triggered Steady-State Free Procession sequence at a field strength of 1.5 T during a breathhold. Regional systolic function is determined for each of the 16 segments taking into consideration wall thickening during systole and also inward endocardial motion. Perfusion imaging was done by making the heart dark and obtaining images for 40 s following the injection of a contrast agent. The sequence of pictures (from left to right) depicts the arrival of contrast in the right ventricle, then left ventricle, aorta, coronary arteries to arrive in the myocardium.
- Figure 14. (=Figure 6 from Imaging)
Multidetector CT assessment of function (top) during diastole (left) and systole (right).  Anatomic information about the coronary anatomy (RCA on the right and LAD on the left bottom panel) can be obtained and with faster imaging, may be able to detect collaterals.
- Figure 15. (=Figure 7 from Imaging)
Fractional flow reserve in the myocardium (FFRmyo) as measured by intracoronary Doppler (left) and pressure wire (right) after administration of adenosine which maximally dilates the normal segments and the microvessels. FFRmyo reflects both antegrade flow and myocardial perfusion. FFRmyo would be expected to improve with angiogenesis studies while FFRcor (which reflects antegrade perfusion alone) should stay the same.
- Figure 16. (=Figure 2 from Position Statement)
Schematic of the steps necessary for the development of any therapeutic intervention.  Successful angiogenesis hinges on adhering to these steps.