Gene Therapy to Stimulate Angiogenesis to Treat Diffuse Coronary Artery Disease

Abstract

Cardiac gene therapy offers a strategy to treat diffuse coronary artery disease (CAD), a disorder with no therapeutic options. The use of genes to revascularize the ischemic myocardium has been the focus of two decades of preclinical research with a variety of angiogenic mediators, including vascular endothelial growth factor, fibroblast growth factor, hepatocyte growth factor, and others encoded by DNA plasmids or adenovirus vectors. The multifaceted challenge for developing efficient induction of collateral vessels in the ischemic heart requires a choice for route of delivery, dosing level, a relevant animal model, duration of treatment, and assessment of phenotype for efficacy. Overall, studies of gene therapy for ischemia in experimental models are very encouraging, with clear evidence of safety and efficacy, strongly supporting the concept that gene therapy to induce angiogenesis is a viable therapeutic approach for CAD. Clinical studies of cardiac gene therapy with angiogenic factors have added substantially to the evidence for efficacy, but definitive studies have not yet led to commercial approval. This review provides the general concepts for angiogenesis-based therapeutic approaches for diffuse CAD and summarizes the results from key studies in the field with recommendations for refinement to a successful product design and evaluation.

Introduction

C oronary artery disease (CAD) is characterized by plaques of lipids, calcium, and inflammatory cells along the inner wall of the coronary arteries, resulting in narrowing of the arterial lumen, with consequent reduction in oxygen-rich blood flow to the myocardium (Klein and Greenland, 2007; Nabel and Braunwald, 2012). The consequences of CAD are profound, with angina, decreased exercise tolerance, myocardial infarction, cardiac failure, and significantly increased mortality (Jolicoeur et al., 2012). CAD is the most common cause of death worldwide, and is responsible for significant morbidity, hospital admissions, and economic costs (Heidenreich et al., 2011; WHO, 2013). The disease is initially treated medically with pharmacologic agents, diet, weight control, smoking cessation, and exercise. With advancing disease and/or with acute coronary syndrome, obstruction in the large coronary arteries is alleviated with percutaneous coronary intervention and/or with coronary artery bypass grafting (CABG) (Hlatky et al., 2009; Stiermaier et al., 2013).

Medical, angioplasty/stents, and CABG therapies have markedly reduced morbidity and mortality for CAD (Hlatky et al., 2009). However, many patients, particularly those with diabetes, also have diffuse disease affecting small coronary vessels that cannot be stented or bypassed (Jolicoeur et al., 2012). In addition, up to 50% of individuals undergoing CABG or percutaneous coronary intervention are incompletely revascularized, an outcome associated with morbidity and mortality rates as great as twice that of completely revascularized patients. These individuals are also potential candidates for angiogenic interventions. The focus of this review is the use of in vivo gene transfer to treat this population of patients with CAD.

The Biology of Angiogenesis

The mechanism by which a tissue is revascularized through the establishment of collateral vessels involves a cascade of steps with numerous growth factors and their associated receptors functioning in a concerted fashion. This process is initiated by the hypoxia-induced transcription factors, commonly in response to ischemic conditions (Carmeliet, 2003; Hayakawa and Shibasaki, 2013).

The basic strategy to treat diffuse small vessel CAD is to induce the growth of new branches of coronary arteries from preexisting patent vessels. This process, referred to as “angiogenesis,” is distinct from “vasculogenesis,” which is the de novo formation of endothelial cells and functional vessels from mesoderm progenitor cells (Flamme et al., 1997). As the heart develops in embryogenesis, the initial process of coronary artery formation is by vasculogenesis after which most blood vessels are generated by angiogenesis (Flamme et al., 1997). Downstream arteriogenesis remodels and matures the existing capillary beds in response to shear stress, resulting in larger mature vessels (Helisch and Schaper, 2003).

Angiogenesis begins with increased permeability of the existing vasculature for exomigration of endothelial cells in concert with the release of matrix metalloproteinases that degrade the basement membrane (Markkanen et al., 2005). An extracellular scaffold is established to support proliferating endothelial cells derived from the existing vessel. This initiates the formation of neovascular buds and ultimately the formation of capillary beds (Emanueli and Madeddu, 2001).

The biology of these processes provides an opportunity for therapeutic intervention with one or more of the angiogenic growth factors as a mechanism to revascularize the ischemic myocardium. The most important angiogenic-specific signal is vascular endothelial growth factor A (VEGF-A), a member of a gene family that is responsible for growth and differentiation of vascular-related tissues (Ferrara et al., 2003). The gene for VEGF-A has eight exons that generate the three major isoforms, VEGF-A121, VEGF-A165, and VEGF-A189, each indicative of the number of amino acids in the processed protein (Park et al., 1993; Patil et al., 2012). The smallest, 121 isoform, is freely soluble and the 165 isoform has intermediate solubility, while the 189 isoform binds to heparin in the extracellular matrix (Ferrara et al., 2003). Although these isoform forms have overlapping functions for angiogenesis, each has a distinct role in the process with apparent synergy (Whitlock et al., 2004; Amano et al., 2005). Upregulated or recruited factors that respond to VEGF-A include platelet-derived growth factor, fibroblast growth factor (FGF), angiopoietin 1 (ANGPT1), and transforming growth factor β1 (TGFB1). Each of these mediators contributes to the formation of a functional vascular bed (Carmeliet and Jain, 2011).

In addition to VEGF-A, the VEGF gene family includes five other growth factors, VEGF-B, C, D, E, and F, as well as placental growth factor (Ferrara, 2004; Patil et al., 2012). These other VEGF family genes share sequence homology and have overlapping function, but the gene regulation, target tissue, and specific effector functions vary and have likely evolved to provide fine control to the timing, location, and extent of angiogenesis. For example, VEGF-A expression is induced by hypoxia, whereas VEGF-B is not (Roy et al., 2006).

The FGFs are members of a large gene family grouped into subfamilies by homology and phylogeny and are associated with a diverse array of functions such as inner ear development or mood disorders (Beenken and Mohammadi, 2009). Evidence that FGFs have angiogenic-related functions derives from observations that FGF-1 is found in the pericardial fluid of ischemic hearts and that endothelial cells treated with FGF-1 form capillary networks in vitro (Iwakura et al., 2000; Uriel et al., 2006). Some FGF family members have angiogenic potential and have been used in gene therapy applications.

Angiopoietins are a family of growth factors that bind Tie, the endothelial cell-specific tyrosine-protein kinase receptor that contributes to blood vessel development (Brindle et al., 2006). Overexpression of angiopoietin in the skin of mice results in larger vessels and more complex vasculature, whereas mice with angiopoietin knocked out have poorly developed vasculature, are missing larger vessels, and lack normal vascular branch structures (Brindle et al., 2006). Gene-targeting studies suggest that angiopoietin functions downstream in vascular development from VEGF, but there are data suggesting that these growth factors can act upstream as well (Suri et al., 1998; Iwakura et al., 2000).

Hepatocytic growth factor (HGF) is a mesenchyme-derived pleiotropic growth factor with potent angiogenic function (Yuan et al., 2008). HGF is a mitogen for endothelial cells and, when delivered to the myocardium in experimental animals, can induce angiogenesis (Sala and Crepaldi, 2011).

The chemokine stromal-derived factor-1α (SDF-1α) recruits endothelial cells to ischemic tissue, inducing neovascularization (Salcedo et al., 1999). The proangiogenic mechanism of SDF-1α is based on its chemoattractant function, which differs from the proliferative mechanism of VEGF. SDF-1α also stimulates the release of VEGF and thus may have therapeutic effects that complement and amplify VEGF (Salcedo et al., 1999).

In addition to the specific angiogenic growth factors, there are additional angiogenesis-related strategies that leverage other physiological mechanisms for the establishment of collateral vessels in the ischemic myocardium. For example, the anti-apoptotic regulator BCL-2 stabilizes VEGF mRNA in ischemic tissue, leading to increased angiogenesis (Iervolino et al., 2002). The hypoxia-induced transcription factor HIF-1α induces the expression of VEGF and other angiogenic mediators (Heinl-Green et al., 2005). The peptide PR39 is an inhibitor of proteasome degradation of HIF-1α and enhances the expression of HIF-1α-regulated VEGF (Post et al., 2006).

Gene Therapy to Stimulate Angiogenesis

Although coronary artery stenting and CABG are effective therapies for the treatment of large vessel CAD, neither is useful in treating diffuse CAD. The basic concept of gene therapy for the treatment of CAD is to deliver to the myocardium a gene that will mimic the normal process of angiogenesis during cardiac development of the coronary vasculature. There are several challenges to accomplishing this: the choice of angiogenic gene; the choice of the vector to deliver the gene; and the choice of route of administration of the gene transfer vector/gene drug.

Choice of Angiogenic Genes

The stimulation of collateral vessel creation in the ischemic myocardium has been the focus of preclinical and clinical studies for two decades and the topic of numerous reviews (Carmeliet, 2003; Lavu et al., 2011; Zachary and Morgan, 2011; Giacca and Zacchigna, 2012; Henry and Satran, 2012; Rosengart et al., 2012). With the knowledge that many growth factors that are involved in the cascade of events that lead to collateral vessel growth, a critical issue is whether a single gene or multiple genes are necessary. The goal for almost all gene therapy strategies has been to nucleate the angiogenesis process in the ischemic region with the initiation of neovascular growth. Because there is a limitation in the gene transfer vector of gene cargo space for the coding sequences of the gene(s) to be expressed, most studies have focused on using a single gene proximal in the angiogenesis cascade, with the context that expression of this gene will initiate a cascade of biologic processes in the correct amount and timing for the formation of collateral vessels. The angiogenic genes that have been assessed in humans are the VEGF-A121 and VEGF-A165 isoforms, the VEGF family genes VEGF-C and VEGF-D, FGF-2, FGF-4, HIF1α, and HGF. Inherent in the use of a single gene is the assumption that downstream arteriogenesis will be evoked by the natural processes that come from intravessel shear stress secondary to blood flow (Helisch and Schaper, 2003).

Choice of Vector

Angiogenesis occurs during cardiac development and postdevelopmentally in response to ischemia, stress, and hypertrophy. Over a short period, the process is initiated, the blood vessels are constructed, and the stimulus ends (Flamme et al., 1997). In the context that this process is often inadequate, gene therapy is a good approach to stimulate the natural process of cardiac angiogenesis; that is, only a short pulse of the expression of a gene(s) is necessary. To achieve this, the experimental models and clinical studies relevant to cardiac angiogenesis have used either plasmids or adenovirus vectors to deliver the angiogenic gene. Both of these gene transfer vectors have short (1–2 weeks) expression times, ideal to initiate the angiogenesis process (Huang and Viroonchatapan, 1999). Most of the studies with plasmids have used naked plasmids without liposome carriers. Except for the HIF-1α studies, all of the adenovirus studies have used the human adenovirus serotype 5 (Hinkel et al., 2013).

Plasmids, almost always used as naked DNA, are the safest and simplest way of transferring exogenous nucleic acid into a target cell. However, transfection efficiency is very low and therapeutic levels of the protein might not be reached (Huang and Viroonchatapan, 1999). Viral vectors, with high transduction efficiency and easy-to-manipulate expression cassettes, offer a great advantage. The disadvantages of viral delivery are the development of an immune and inflammatory response to the vector, primarily the vector capsid (Thomas et al., 2003). However, for angiogenesis applications, the immune response is a built in mechanism to limit expression of these potent factors to a time frame that minimizes risks associated with persistent expression (Lee et al., 2000). Adeno-associated virus and lentivirus vectors have not been used to induce angiogenesis, as they mediate persistent expression that would be a safety risk, although theoretically these could be mitigated with a regulatable promoter (Hofmann et al., 1996).

Choice of Route of Administration

The challenge for delivery of an angiogenic gene therapy drug is to genetically modify cardiac cells to generate angiogenic signals in the myocardium. Intravenous (systemic) delivery is the least invasive method, but inefficient delivery and distribution to other organs are major limitations (Gyongyosi et al., 2005; Stewart et al., 2009). Delivery by percutaneous coronary artery catheterization (intracoronary delivery) is clinically routine, and can be performed safely to deliver genes into the coronary circulation. However, this delivery may be limited because of the inherent disease (coronary artery occlusion) preventing delivery of the gene to the ischemic region, and by rapid movement of the vector in the circulation and thus limited exposure of the coronary artery endothelial cells to the vector, resulting in inefficient gene transfer (Gyongyosi et al., 2005). Intraventricular delivery via left ventricular catheterization uses an electromagnetic mapping catheter system to identify sites of delivery. The method presents difficulties in localizing the target area, and it cannot guarantee that the entire dose is actually administered to the myocardium, leading to loss of efficacy and the risk of systemic vector delivery to other organs. In contrast, epicardial delivery via intramyocardial injection insures a direct, targeted, high local concentration and avoids endothelial cell barriers and accidental delivery to the circulatory system. Although epicardial administration requires surgical intervention, the minimally invasive procedures for this delivery have excellent safety records (Crystal et al., 2002; Harvey et al., 2002). Another advantage of epicardial administration is direct visual identification of the myocardial target area during vector administration. Compared with other delivery techniques, epicardial administration provides the highest levels of localized transgene expression that have been achieved (Guzman et al., 1993; Muhlhauser et al., 1996; Vassalli et al., 2003; Yuan et al., 2008); vectors can be delivered with a high degree of accuracy (French et al., 1994; Vassalli et al., 2003; Yuan et al., 2008); a number of targeted injections can be performed (French et al., 1994; Vassalli et al., 2003); and there is limited systemic spread of the vector (Macgovern et al., 1996).

Experimental Animal Models

Preclinical animal studies for angiogenic therapies provide the ischemic tissue for screening the potency of genes, the efficiency of gene transfer for vectors, and an in vivo proof-of-concept system to evaluate phenotypes for efficacy and toxicology. Large numbers of gene therapy-based studies for cardiac angiogenesis have been carried out in mice, rats, rabbits, dogs, and pigs (Tables 1, 2, and 3). Since it is difficult to model cardiac ischemia in small animals, most murine studies utilize a surrogate tissue, such as the ischemic hind limb established by constriction of blood flow through the iliac artery. Alternatively, myocardial infarction has been used as an ischemic model for testing an angiogenic gene therapy in rodents (Aoki et al., 2000; Miyagawa et al., 2002; Retuerto et al., 2004; Jiang et al., 2006; Ruixing et al., 2006; Li et al., 2007; Kaminsky et al., 2013). In larger animals, such as the pig, candidate gene therapy vectors have been delivered directly to the myocardium of an ischemic heart induced with an ameroid constrictor on a coronary vessel. Although the use of these animal models could provide the basis for a large-scale systematic screening of the therapeutic genes, their respective isoforms, and splicing variants, this has not been done. Instead, all of the studies have used one, or at most two, angiogenesis gene(s), animal model, vector type, route of delivery, and measure of efficacy. Thus, there have been no published, large sets of comparative data that would inform a clear path forward for the “best” route of clinical therapeutic development. Despite this, the positive results for many of these studies strongly support specific efficacious options.

Table 1.

Preclinical Studies of Plasmid-Mediated Gene Transfer to Induce Angiogenesis

Gene	Model	Outcome	Reference
VEGF-A165	NZ white rabbits, hind limb ischemia	Increased development of vessels and capillary density	Takeshita et al. (1996)
	Dogs, intermittent left coronary artery occlusions	Improved collateral blood flow	Hattan et al. (2004)
	Male Sprague-Dawley rats, left coronary artery ligated	Increased microvessel count, decrease in infarct size	Ruixing et al. (2006)
	Young female Wistar rats, left coronary artery ligated	VEGF plasmid transfected human skeletal myoblasts, increased vessel density	Ye et al. (2007b)
VEGF-C	NZ white rabbits, hind limb ischemia	Increased capillary density	Witzenbichler et al. (1998)
ANGPT1/ANGPT2	NZ white rabbits, hind limb ischemia	ANGPT1 increased capillary density, ANGPT2 did not	Shyu et al. (1998)
ANGPT1/VEGF-A165	NZ white rabbits, hind limb ischemia	Increased number of capillaries with each gene separately, larger effect using both genes	Chae et al. (2000)
	C57Bl/6 mice, with and without left coronary artery ligated	VEGF increased capillary angiogenesis; the addition of ANGPT1 increased arteriogenesis but had no effect on angiogenesis	Siddiqui et al. (2003)
BCL-2	Myocardial infarcted rat	Ex vivo BCL-2-transfected MSCs improved capillary density in the border zone of the infarct compared with control and reduced the infarct zone	Li et al. (2007)
HGF	Sprague-Dawley rats, with and without myocardial infarct	Number of vessels increased in both cases leading to increased blood flow	Aoki et al. (2000)
	Sprague-Dawley rats and NZ white rabbits, hind limb ischemia	Angiogenesis in both rats and rabbits	Taniyama et al. (2001)
	Lewis rats, myocardial infarcted	Plasmid in liposomes combined with rat cardiomyocytes, increased cardiac performance and perfusion	Miyagawa et al. (2002)
	Canine (beagles), left coronary artery ligated	Number of capillaries in the ischemic area increased, blood flow improved	Funatsu et al. (2002)
	Sprague-Dawley rats, left coronary artery ligated	Smaller scar and increased capillary density	Kondo et al. (2004)
SDF-1α	Female Fischer rats, left coronary artery ligated	Skeletal muscle myoblasts overexpressing SDF-1α, increased stem cell migration to the heart and enhanced angiomyogenesis	Elmadbouh et al. (2007)
Shh	Female Fischer-344 rats, left coronary artery ligated	MSCs transfected with Shh had increased blood vessel density	Ahmed et al. (2010)

ANGPT1, angiopoietin 1; Bcl-2, B-cell lymphoma 2; HGF, hepatocyte growth factor; hHGF, human HGF; HSM, human skeletal myoblasts; MSCs, mesenchymal stem cells; NZ, New Zealand; SDF-1α, stromal cell-derived factor; Shh, sonic hedgehog; VEGF-A165, 165 amino acid variant of isoform A of vascular endothelial growth factor; VEGF-C, isoform C of vascular endothelial growth factor.

Table 2.

Preclinical Studies of Adenovirus-Mediated Transfer of VEGF to Induce Angiogenesis

Gene	Model	Outcome	Reference
VEGF-A121	Yorkshire swine, ameroid constrictor on coronary artery	Reduction of the ischemic area, improved wall thickening, and increased collateral vessel development	Rosengart et al. (1998)
	C57/Bl6 mice; Yorkshire swine with a constrictor on coronary artery	In both groups, the vector was demonstrated safe with no major toxicity	Patel et al. (1999)
	C3H/He mice and mice treated with dexamethasone	Wound healing model; treatment increased vascularity and bursting strength	Ailawadi et al. (2002)
	Yorkshire swine with heart failure induced by pacing	Improvement in myocardial function	Leotta et al. (2002)
	NZ white rabbits and Sprague-Dawley rats	Enhanced regional perfusion in rabbits and prolonged exercise tolerance in rats	Schalch et al. (2004b)
	Fischer 344 rats with left coronary artery ligated	Increased capillary density in the infarct region and improved survival of transplanted cardiomyocytes	Retuerto et al. (2004)
VEGF-A165	Sprague-Dawley rats with hindlimb ischemia	Increased vascularity and blood flow	Mack et al. (1998)
	Female Yorkshire pigs, left coronary artery ligated	Increased capillary density and blood flow	Haider et al. (2004)
	Male Lewis rats, left coronary artery ligated	Increased capillary density	Matsumoto et al. (2005)
	Female Fischer rats, left coronary artery ligated^a	Increased angiogenesis	Hao et al. (2007)
VEGF-All	C57/Bl6 mice, hindlimb ischemia	100-fold more potent in restoring blood flow compared with the administration of an individual isoform	Whitlock et al. (2004)
VEGF-All6A+	C57/Bl6 mice, hindlimb ischemia	100-fold more potent than individual splice forms and improved safety because of lower dose requirement, reduced VEGF-mediated pulmonary edema	Amano et al. (2005)
VEGF-B	Domestic pigs, occlusion of the left coronary artery, NZ white rabbits with hindlimb ischemia	Improvement in vessel density and blood perfusion	Lahteenvuo et al. (2009)
VEGF-C	Landrace piglets, occlusion of the left artery	Prevention of progression of ischemia	Patila et al. (2006)
VEGF-A121 and Egr1	Wild-type mice and Egr-1 knockout mice, femoral artery ligation	AdVEGF-A121 increased in perfusion, Ad-Egr1 showed partial recovery	Schalch et al. (2004a)
VEGF-A165 and VEGF-D	Domestic pigs	VEGF-D similar to VEGF-A165 for angiogenesis	Rutanen et al. (2004)

Egr1, early growth response protein 1; VEGF-A121, 121 amino acid variant of isoform A of vascular endothelial growth factor; VEGF-All, genomic and cDNA hybrid of isoform A of vascular endothelial growth factor that produces all three variants, 121, 165, 189; VEGF-All6A+, variant of VEGF-All, expressing all 3 variants but where the splicing site of exon 6A was changed to promote expression of variant 189; VEGF-B, isoform B of vascular endothelial growth factor.

Tested both plasmid and adenovirus vectors; the adenovirus vector resulted in higher expression of VEGF, but both methods resulted in similar levels of improvement.

Table 3.

Preclinical Studies of Adenovirus-Mediated Gene Transfer to Induce Angiogenesis Using Genes Other than VEGF

Active factor	Model	Outcome	Reference
ANGPT-1	Yorkshire pig, coronary artery ligation	Ad-ANGPT-1- and Ad-ANGPT-1-transduced skeletal muscles improved angiogenesis and heart function	Ye et al. (2007a)
FGF-1	LWD pig, ameroid constrictor on coronary artery	AdFGF-1-transduced fibroblasts increased collateral vessels density and improved contraction in the ischemic area	Ninomiya et al. (2003)
FGF-5	Yorkshire pig, ameroid constrictor placed around the coronary artery	Increased blood flow and heart function	Giordano et al. (1996)
HIF-1α	Wistar rats, left coronary artery ligated	Skeletal myoblasts coadministered with Ad2-HIF-1α treatment improved cell survival and increased angiogenesis above either constituent alone	Azarnoush et al. (2005)
HIF-1α	Pig, ameroid constrictor on coronary artery	Increased myocardial perfusion during stress, improved left ventricular function; no evidence of measurable change in vessel density	Azarnoush et al. (2005)
HIF-1α	Pig, reduction stent into the circumflex artery^a	Increase of small and large vessels, improved perfusion of the ischemic myocardium, and return of myocardial function	Hinkel et al. (2013)
PR39	Yorkshire pig, ameroid constrictor on coronary artery	Increase blood flow and angiogram-detectable collateral vessels, increased expression of VEGF and FGF-1	Post et al. (2006)
ANGPT-1 and AKT	Infarcted Fischer rat, ligated left coronary artery	Increased blood vessel density and heart function	Jiang et al. (2006)

AKT1, Rac protein kinase alpha; FGF-1, fibroblast growth factor-1; FGF-5, fibroblast growth factor-5; HIF-1α, hypoxia-inducible factor 1α; PR39, proline/arginine rich peptide.

Ad 2 serotype used.

Studies with Plasmid Vectors

Survival studies have demonstrated that despite the low transduction efficiency, plasmid vectors with angiogenic genes induce angiogenesis and are effective in treating ischemia in experimental animal models. Included in these studies are plasmid-mediated transfer of VEGF-A165, VEGF-C, ANGPT1 and 2, the combination of VEGF-A165 and ANGPT1, BCL-2, HGF, SDF-1α, and Shh (Table 1).

Studies with Adenovirus Vectors

Adenovirus vectors mediating expression of angiogenic factors provide robust, transient expression in a wide variety of tissue types. The most commonly assessed genes delivered by adenovirus vectors have the VEGF family (Table 2), but ANGPT1, FGF1, FGF5, HIF-1α, PR39, and the combination of ANGPT1 and AKT have also been assessed (Table 3).

Adenovirus vector delivery of numerous VEGF isoforms have been studied, the most common has been VEGF-A with delivery to the ischemic heart and hind limb of small and large animal models. The two most commonly used VEGF-A splice variants have been VEGF-A121 and VEGF-A165. All animal studies with these growth factors demonstrated measures of efficacy by either increases in blood flow, capillary density, or exercise tolerance. A new paradigm in gene therapy for angiogenesis is the use of an adenovirus vector that coexpresses three splice variants of VEGF-A: VEGF-A121, VEGF-A165, and VEGF-A189. This vector is 10- to 100-fold higher in potency over each of the individual VEGF-A splice forms (Whitlock et al., 2004). Further modification of the transgene to bias the expression to the 189 higher-molecular-weight splice form of VEGF-A resulted in improved survival, reduced pulmonary edema, and reduced capacity to support tumor growth (both VEGF-related safety issues).

Although VEGF is likely the controlling gene switch for angiogenesis, other mediators delivered by adenovirus vectors also induced angiogenesis (Table 3). For example, AdFGF-1-treated fibroblasts transplanted to heart mediated an increase in collateral vessels and improved contraction in the ischemic heart (Ninomiya et al., 2003). Direct administration to the ischemic heart of adenovirus serotype 5 expressing FGF-5 resulted in increased blood flow in a porcine model (Giordano et al., 1996). Similarly, adenovirus vector delivery of ANGPT-1, PR39, and HIF-1α all induced angiogenesis (Hinkel et al., 2013). Collectively, these studies suggest that angiogenesis in ischemic tissues can be triggered directly by numerous growth factors as well as by gene products normally attributed to other pathways.

Clinical Trials

The extensive preclinical studies of angiogenesis in animal models of ischemia provide a large dataset supporting the use of many candidate genes for clinical development of biological treatments for diffuse CAD. As a result, multiple candidate vector/gene combinations have been tested in human clinical trials (Tables 4 and 5). The challenge for developing a successful gene therapy for diffuse CAD is the choice of the angiogenic gene, the vector for delivery, the dose, the target disease state, and the phenotype to evaluate. Since 1997, dozens of clinical trials have been conducted to evaluate the potential of angiogenic therapy for treating patients with diffuse CAD. Despite the many successful studies to establish safety, and encouraging results regarding efficacy, as yet there is no approved angiogenic gene therapy for commercial licensure. Here we present the data from representative clinical studies. Both plasmid and adenovirus vectors have been used. The most common angiogenic genes used in clinical studies have been from the VEGF family.

Table 4.

Clinical Studies of Plasmid-Mediated Gene Transfer to Induce Angiogenesis

Gene	Trial name (ID number)	Phase	CCS angina class (patient status)	Delivery method	Outcome summary	Reference
VEGF-A165		I	III–IV, inoperable CAD	Epicardial intramyocardial, minithoractomy	Safety studies, improved collaterals for at least one occluded vessel seen in all patients by day 60, second trial showed that VEGF-A165 augments perfusion of ischemic myocardium	Symes et al. (1999); Vale et al. (2000)
	EUROINJECT-ONE (not listed)	II/III	III–IV, CAD with no therapeutic options	Epicardial percutaneous intramyocardial, NOGA-guided Cordis catheter injection	Gene therapy did not significantly improve stress-induced perfusion abnormalities, but improved regional wall motion was noted	Gyongyosi et al. (2005); Kastrup et al. (2005)
	NORTHERN (NCT00143585)	II/III	III–IV, CAD, patients who reached maximal medical therapy	Percutaneous intramyocardial, NOGA-guided Cordis catheter injection	No benefit from VEGF-A165 therapy in primary endpoints, no difference between treated and placebo groups in myocardial perfusion as assessed by SPECT despite observed revascularization in the ischemic regions	Stewart et al. (2009)
	GENESIS-1 (not listed)	I	II–III, severe CAD, not amendable by revascularization	Endocardial intramyocardial with Cordis catheter injection	No adverse events occurred over the 2-year study; angina class and myocardial perfusion increased significantly; stress ejection fraction did not change	Favaloro et al. (2012)
VEGF-A165/FGF-2	VIF-CAD (NCT00620217)	II	III–IV, CAD	Percutaneous intramyocardial, NOGA-guided Johnson and Johnson catheter injection	1st trial to study the use of a bicistronic VEGF-A165/FGF-2 plasmid, no improvement in perfusion by SPECT; patients in the treated group improved their ETT time	Kukula et al. (2011)
VEGF-A165 (with G-CSF co-treatment)	(NCT00135850)	I/II	III–IV, severe chronic CAD, with no option for revascularization	Intramyocardial, NOGA VEGF-A165 followed 1 week later with subcutaneous G-CSF	No improvement in myocardial stress perfusion	Ripa et al. (2006)
VEGF-C ^a	VEGF-2 (not listed)	I/II	III–IV, refractory to maximum medical therapy, multivessel occlusive CAD not amendable to CABG or PCI	Percutaneous intramyocardial, NOGA-guided Biosense-Webster catheter injection	Significant improvement for angina and ETT, treatment vs. placebo groups 2 years posttreatment	Losordo et al. (2002); Reilly et al. (2005)
		I	III–IV, “no option” patients, refractory to maximum medical therapy or multivessel occlusive CAD	Epicardial intramyocardial via a minithoractomy	At 1 year, clinical improvement observed but no evidence of angiogenesis	Fortuin et al. (2003)
	GENASIS (NCT00090714)	IIb	III–IV, CAD, not suitable for conventional revascularization procedures	Endocardial injection by Stiletto Boston Scientific catheter	No significant difference between treated and placebo groups for ETT at 3 months	Medical News Today (2006)

CABG, coronary artery bypass grafting; CAD, coronary artery disease; CCS, Canadian Cardiovascular Society class of angina; ETT, exercise tolerance test; HGT, hepatocyte growth factor; NOGA, endoventricular electromechanical mapping system; NORTHERN, NOGA angiogenesis revascularization therapy: evaluation by radionuclide imaging; PCI, percutaneous coronary intervention; PCTA, percutaneous transluminal coronary angioplasty; SPECT, single-photon emission computed tomography.

VEGF-C is the current nomenclature for VEGF-2.

Table 5.

Clinical Studies of Adenovirus-Mediated Gene Transfer to Induce Angiogenesis

Gene ^a	Trial name (ID number)	Phase	CCS angina class (patient status)	Delivery method	Outcome summary	Reference
VEGF-A121	(NCT01174095)	I/II	III–IV, severe CAD, not suitable for CABG	Epicardial; minithoractomy	Improvement in exercise tolerance and angina at 1 year, improvement in patient survival and decreased angina scores from long-term follow-up (median>11 years), shows safety of AdVEGF-A121 therapy for CAD	Rosengart et al. (1999, 2012)
	REVASC trial (not listed)	II/III	II–IV, CAD, not amendable to revascularization	Epicardial intramyocardial via minithoractomy	Significant improvement in primary end point, exercise time, at 26 weeks; improved anginal symptoms were seen at both time points	Stewart et al. (2006)
	NOVA trial (not listed)	II/III	II–IV, CAD, not amendable to revascularization or CABG	Percutaneous intramyocardial, with NOGA guidance	Midterm analysis at 12, 26, and 52 weeks showed no difference between treated and placebo groups for ETT or perfusion	Kastrup et al. (2011)
VEGF-A165	KAT trial (not listed)	II/III	II–III, CAD with stable angina but not diffuse CAD	Intracoronary following PTCA	Improvement in coronary perfusion at 6 months; long-term follow-up (>7 years) showed safety of Ad-VEGF-A165 therapy for CAD	Hedman et al. (2003, 2009)
VEGF-All6A+ ^b	(NCT01757223)	I/II	III–IV, CAD with angina refractory to medical therapy	Intramyocardial	Recruiting soon	ClinicalTrials.gov ^c
VEGF-D ^d	KAT301 trial (NCT01002430)	I	II–III, “no option” patients with CAD, not amendable to revascularization or CABG	Endocardium injections by NOGATM catheter	Safety trial with 3 viral doses; trial ongoing; results pending	ClinicalTrials.gov ^c
FGF-4	AGENT trial (not listed)	I/II	II–III, stable angina	Intracoronary	Safety trial, no adverse events with therapy; no significant improvement observed	Grines et al. (2002)
	AGENT-2 trial (not listed)	II/III	II–IV, maximal antianginal therapy who were not candidates for PCI or CABG	Intracoronary	Improved stress-induced myocardial perfusion defect, positive results with SPECT at 8 weeks	Grines et al. (2003); Kapur and Rade (2008)
	AGENT-3 trial (NCT00346437)	II/III	II–IV, patient on antianginal therapy who did not require immediate PCI or CABG	Intracoronary	Negative results overall, but positive for patients age >55 years who had CCS III–IV stage of CAD at trial start; female patients showed significant improvement in exercise time and anginal class	Henry et al. (2007)
	AGENT-4 trial (NCT00185263)	II/III	II–IV, patients on antianginal therapy who were not candidates for PCI or CABG	Intracoronary	Negative results overall, but female patients showed significant improvement in exercise time and anginal class; interim analysis indicated that trial would not reach statistical significance; trial was terminated	Henry et al. (2007)
	AWARE trial (NCT00438867)	III	III–IV, CAD, women only trial	Intracoronary	Trial is ongoing, no results published	Kapur and Rade (2008)
	ASPIRE trial (NCT01550614)	III	Angina class not listed, patients with stable angina	Intracoronary	Trial is ongoing with no preliminary results published	Kaski and Consuegra-Sanchez (2013); ClinicalTrials.gov ^c
HGF		I	Angina class not listed, CAD	Intramyocardial	Safe and well tolerated with dose-dependent myocardial perfusion improvements	Yuan et al. (2008); Yang et al. (2009)
	(NCT01925352)	I/II	II–III, severe and diffuse CAD	Intracoronary	Trial is ongoing, no results published	ClinicalTrials.gov ^c
HIF-1α ^e		I	I–III, multivessel CAD	Intramyocardial administered during CABG	Safe with no adverse reactions during 1 year postoperation, with trend toward ventricular function improvements in treated group	Kilian et al. (2010)

AGENT, angiogenic gene therapy.

Serotype of adenovirus is Ad5, unless noted.

VEGF-All6A+, Ad5 vector that produces all 3 VEGF-A splice variants: VEGF-A121, VEGF-A165, and VEGF-A189 (Amano et al., 2005).

For trials with unreleased interim or final study reports, refer to ClinicalTrials.gov for trial specifics.

Serotype of adenovirus not specified.

Ad 2 serotype used.

Evaluating Efficacy

One of the critical aspects of the design of the clinical studies has been the endpoints used to evaluate efficacy. Most of the clinical studies have used measures of exercise capacity or myocardial perfusion and collateral coronary vessels. Exercise stress testing is the most commonly used endpoint, using the time a patient can exercise on a treadmill with progressive increases in the speed and elevation (Bruce et al., 1973; Gibbons et al., 2002). Alternatively, time to electrocardiogram ST-segment depression has been used (Okin and Kligfield, 1995; Gibbons et al., 2002). Echocardiography permits the dynamic evaluation of cardiac structure and function at rest and during stress provoked by exercise or a pharmacologic agent, but is relatively insensitive because of poor anatomical resolution. Two-dimensional echocardiographic imaging performed during or immediately after stress has been used to assess the extent of ischemia and to estimate ventricular systolic function (Pellikka et al., 2007). Nuclear stress testing (myocardial perfusion imaging) has been used for assessment of ischemia after exercise stress or pharmacologic stress by adenosine, dipyridamole, or dobutamine. Two scanning techniques are frequently used: cardiac positron emission tomography (Tillisch et al., 1986; Tio et al., 2004) and single-photon emission computed tomography (SPECT) (Grines et al., 2003; Kastrup et al., 2005; Ripa et al., 2006). Positron emission tomography imaging by fluorine-18-tagged deoxyglucose provides a means of detecting glucose metabolism in the myocardium. SPECT imaging analyzes the relative blood flow in different regions of the myocardium (Marcassa et al., 2008; Rischpler et al., 2013). The most commonly used tracers for SPECT are thallium-201, a potassium analog that uses the cell membrane Na/K pumps, and Tc-99m sestamibi that accumulates in the mitochondria as a marker of viability. Magnetic resonance imaging can be used to evaluate heart wall motion abnormality and myocardium perfusion during pharmacologic stress (Hundley et al., 1999; Hendel et al., 2009). Ischemic myocardium typically shows abnormal thickening or contractility when stressed. Coronary angiography involves the selective injection of a radiopaque contrast agent into the coronary arteries allowing the evaluation of both flow and anatomy of the arteries. Although angiography provides an assessment of the anatomy, it does not assess function, and this modality has not generally been used for gene therapy trials.

Clinical Studies Using Plasmid Vectors

As in preclinical studies VEGF family members have been the most common angiogenic factors used in the clinical plasmid trials for CAD (Table 4). Among the VEGF-A isoforms, both VEGF-A121 and VEGF-A165 splice variants are the most studied; these variants are potent in heart tissue (Lavu et al., 2011). All of the plasmid VEGF-A trials have used VEGF-A165 or a combination of VEGF-A165 with another angiogenic-related genes (Table 4). Early phase I studies showed that the VEGF treatments were safe and effective with improved collateral blood filling of occluded vessels and improved perfusion of the ischemic myocardium, but none of these were placebo controlled (Symes et al., 1999; Vale et al., 2000; Sarkar et al., 2001; Favaloro et al., 2012). Several completed large-scale phase II/III trials with plasmid VEGF-A165 included a randomized placebo control such as the EUROINJECT-One and NORTHERN trials (Kastrup et al., 2005; Stewart et al., 2009). In the EUROINJECT-One trial, plasmid-delivered VEGF-165 did not significantly improve the stress-induced perfusion abnormalities, although a subset had improved regional wall motion indicating positive anti-ischemia effect (Gyongyosi et al., 2005; Kastrup et al., 2005). The Canadian NORTHERN trial found no efficacious benefit from VEGF-165 plasmid therapy with no statistical difference between treated and placebo groups of SPECT-assayed myocardial perfusion, despite improved revascularization in the ischemic regions (Stewart et al., 2009).

The VIF-CAD trial used concomitant plasmid delivery of VEGF-A165 and FGF-2 genes via a bicistronic plasmid. The data showed an improvement in exercise testing, but the primary goal of increased cardiac perfusion as assessed by SPECT was not significant (Kukula et al., 2011). No improvement in myocardial perfusion or angina class was observed in a phase I trial of intramyocardial injections of plasmids coding for VEGF-165 followed by the cytokine G-CSF 1 week later; the addition of G-CSF was designed to stimulate bone marrow stem cells to repopulate the myocardial ischemia (Ripa et al., 2006). Finally, there have been clinical studies with plasmid delivery of the alternate VEGF family member VEGF-C. The initial safety of intramyocardial injections of plasmid VEGF-C study showed clinical improvement at 1 year posttreatment. However, because there was no evidence of angiogenesis per se and because the study lacked controls, the authors indicated concern for the placebo effect (Fortuin et al., 2003). Two clinical trials were carried out with plasmid-directed VEGF-C, one by direct myocardial administration and the other by catheter to the myocardium. The catheter-based trial included a placebo control. Both plasmid VEGF-C trials yielded statistically significant clinical improvement in angina class (Losordo et al., 2002; Reilly et al., 2005). A third study (GENASIS) with catheter-delivered plasmid-directed VEGF-C showed no significant improvement in exercise testing at 3 months over placebo difference, and the trial was terminated (Medical News Today, 2006).

Clinical Studies Using Adenovirus Vectors

On the basis of the more efficient cardiac transduction by adenovirus-mediated gene transfer vectors, several trials were carried out to deliver one of several VEGFs (VEGF-A121, VEGF-A165, VEGF-D) to the ischemic heart (Table 5). Long-term follow-up (median>11 years) to one of the earliest trials, a phase I/II study with adenovirus-mediated expression of VEGF-A121 to the ischemic myocardium of patients with diffuse CAD, provided anecdotal evidence for improvement in both patient survival and decreased angina class scores (Rosengart et al., 1999, 2012). Two larger phase II/III, randomized, controlled trials using adenovirus expressing VEGF-A121 have been completed (REVASC and NOVA). In the REVASC trial, direct intramyocardial injections of AdVEGF-A121 resulted in statistically significant improvement in primary end point and exercise time, at 26 weeks, along with improved angina and pain class, although improvement in perfusion was not observed (Stewart et al., 2006). In contrast, the NOVA trial with adenovirus expressing VEGF-A121 had disappointing results. Mid-term analysis at 12, 26, and 52 weeks showed no statistical difference between the treated and placebo groups for exercise tolerance time or perfusion. The trial was subsequently terminated early (Kastrup et al., 2011). The Kuopio Angiogenesis Trial (KAT) compared plasmid and adenovirus vectors with VEGF-A165 delivered by catheter to coronary artery. The study concluded that, although there were no safety-related outcomes, the treatment had no significant effect on restenosis. Interestingly, however, the adenoviral vector-based therapy did improve myocardial perfusion (Hedman et al., 2003). Long-term follow-up (>7 years) suggested that adenovirus VEGF-A165 gene therapy was safe and efficacious for CAD (Hedman et al., 2009). A single clinical trial evaluating adenovirus-vectored VEGF-D via endocardial administration has begun, but no results of this phase I trial have been published.

A phase I, randomized, placebo-controlled, double-blind study of gene therapy for cardiac ischemia with adenovirus-directed delivery of FGF-4 has demonstrated a good safety profile and improved exercise testing (Grines et al., 2002). The large, randomized, placebo-controlled trials with adenovirus-mediated FGF-4 delivery noted a gender difference in response to the treatment. While the AGENT-2 trial showed improvements at 8 weeks in the primary end point of stress-induced myocardial perfusion and SPECT analysis (Grines et al., 2003; Kapur and Rade, 2008), the subsequent larger trials (AGENT-3 and AGENT-4) did not support this conclusion; no significant improvement was observed between the treated patients and placebo controls. Interestingly, however, a subset of patients (>55 years who had severe angina at the start of the trial) did show positive improvements, and female patients overall showed significant improvement in exercise time and anginal class (Henry et al., 2007). The gender issue is currently being explored by the adenovirus-based FGF-4 gene therapy study with women only (AWARE), but there are no published results to date. The ongoing ASPIRE trial is based on adenovirus-directed FGF-4 delivered by catheter with a primary endpoint of myocardial perfusion (Kaski and Consuegra-Sanchez, 2013), but there are no published results. A clinical trial with HGF by adenovirus vector delivered by catheter was well tolerated, but the sample size was too small to evaluate efficacy (Yang et al., 2009). The delivery of HIF-1α with an adenovirus and saline control group was assessed by epicardial administration to the ischemic myocardium of patients undergoing CAGB; there were no significant safety concerns, but the study group was too small to evaluate efficacy.

Future Directions

There is substantial preclinical evidence that angiogenic gene therapy improves ischemia, and the positive results of the REVASC, VEGF-2, GENESIS, and VIF-CAD trials suggest that clinical efficacy is a reachable goal. One of the challenges for the clinical studies has been to conduct blinded-placebo-controlled studies that would convincingly establish efficacy of the gene drug. In that context, indirect delivery methods such as intracoronary and endocardial administration facilitate the use of a placebo control because they encompass relatively noninvasive and, therefore, presumably safe interventions. On the other hand, preclinical data and the outcomes observed in clinical trials suggest that these approaches are often associated with inadequate vector delivery, and ineffectual therapeutic angiogenic interventions as a result. While expectations of acceptable risk have been standard in the traditional drug development arena, gene therapy trials are subject to higher levels of scrutiny, and there have been very few gene therapy trials using a placebo-controlled, randomized design. For cardiac angiogenic gene therapy, the regulatory and ethics hurdles may be a challenge, but we believe that it is necessary to move angiogenic gene therapy to approval (Crystal et al., 2012). From the extensive preclinical and clinical studies with angiogenic gene therapy that have been carried out to date, there is strong evidence that it will be successful. With appropriate clinical design, we predict that regulatory approval of angiogenic gene therapy will be achieved.

Footnotes

Acknowledgments

We thank Megumi Matheson for helpful suggestions and N. Mohamed and D.N. McCarthy for help in preparing this article. These studies were supported, in part, by the Qatar Foundation and the Weill Cornell Medical College in Qatar.

Author Disclosure Statement

No competing financial interests exist.

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