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Cardiovascular Research 1997 35(3):480-489; doi:10.1016/S0008-6363(97)00167-3
© 1997 by European Society of Cardiology
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Copyright © 1997, European Society of Cardiology

Gene therapy for collateral vessel development

Guido Melilloa, Marco Scocciantia, Imre Kovesdib, Jorge Safi, Jrc, Teresa Riccionia,c and Maurizio C Capogrossia,c,*

aLaboratorio di Patologia Vascolare, Istituto Dermopatico dell'Immacolata, Via dei Monti di Creta 104, 00167 Rome, Italy
bGenVec Inc., Rockville, MD, USA
cGene Therapy Unit, Laboratory of Cardiovascular Science, National Institute on Aging, National Institutes of Health, Baltimore, MD, USA

* Corresponding author. Tel.: +39 6 66467106; Fax: +39 6 66462430; e-mail: capogrossi@idi.it

Received 25 April 1997; accepted 10 June 1997


   Abstract
Top
 Abstract
1 Introduction
 2 Angiogenesis and growth...
 3 Methods for gene...
 4 Therapeutic angiogenesis
 5 Discussion: future directions...
 References
 
Transfer of the cDNA coding for angiogenic factors represents a novel and promising approach to induce therapeutic angiogenesis and enhance blood flow to ischemic tissues which cannot be revascularized otherwise. This review will focus on therapeutic angiogenesis based on gene transfer techniques for the treatment of myocardial and limb ischemia. The experimental studies demonstrating the angiogenic effect of recombinant growth factors in animal models and in humans, as well as the most promising methods for gene transfer, will be described. Further, gene transfer studies to induce therapeutic angiogenesis will be reviewed to identify critical questions that still need to be answered before gene therapy with angiogenic factors may be considered for routine clinical application.

KEYWORDS Gene therapy; Angiogenesis; Ischemia; FGF; VEGF; Heparin; Adenovirus


   1 Introduction
Top
 Abstract
 1 Introduction
2 Angiogenesis and growth...
 3 Methods for gene...
 4 Therapeutic angiogenesis
 5 Discussion: future directions...
 References
 
Vasculogenesis represents the de novo formation of new blood vessels and it occurs in the embryo by association of angioblasts. In contrast, angiogenesis is the biologic process by which new blood vessels develop from the preexisting vasculature and results from the interplay of cell–cell, cell–matrix and cell–cytokine interaction. The formation of a novel vascular network is a rare event in the adult organism and occurs in a tightly controlled and transient manner in the female reproductive organs. Neovascularization occurs also in a variety of pathologic conditions including ischemia, inflammation, wound healing, tumor growth, diabetic retinopathy, rheumatoid arthritis, psoriasis and chronic wounds. Induction of angiogenesis has been proposed as a therapeutic strategy to treat myocardial and limb ischemia due to atherosclerosis of coronary and peripheral arteries. Encouraging preclinical studies in animal models of myocardial infarction, chronic myocardial ischemia and hindlimb ischemia suggest that in the future, therapeutic angiogenesis may play a role in the management of patients affected by these conditions.


   2 Angiogenesis and growth factors
Top
 Abstract
 1 Introduction
 2 Angiogenesis and growth...
3 Methods for gene...
 4 Therapeutic angiogenesis
 5 Discussion: future directions...
 References
 
Angiogenesis is due to the sprouting of new small vessels from the preexisting vasculature (Table 1) and includes a series of events occurring in precise sequence [1–3]and involving endothelial cells and pericytes [4, 5]. Since the growth of the capillary network contributes minimally to the regulation of blood flow, increase in the tissue density of resistance vessels, which are the major determinants of blood supply, must be achieved in order to improve perfusion of ischemic tissues such as myocardium and skeletal muscle [6, 7]. The development of an arteriole from a capillary involves apposition and differentiation of pericytes and smooth muscle cells and matrix remodeling [8]. Although relatively little is known about capillary and arteriole growth in adult tissue, the stimulus for development appears largely due to the metabolic requirements of the tissue [9]. The angiogenic process is characterized and modulated by the interactions between stimulators and inhibitors of angiogenesis [10, 11](Table 2) as well as by blood flow through the newly formed vascular network [9]: an increase in endothelial mitogens and their membrane receptors has been found in response to a variety of angiogenic stimuli, such as hypoxia [31–34], ischemia [35–38], mechanical stretch [39, 40], and inflammation [41, 42].


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  		Table 1 Overview of main events in angiogenesis

 

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  		Table 2 Receptor/ligand systems involved in angiogenesis (modified from Ref. [10])

 
Among the growth factors identified and used to induce angiogenesis experimentally, the fibroblast growth factor (FGF) family and vascular endothelial growth factor (VEGF) have been already used to induce therapeutic angiogenesis in clinically relevant animal models.

Acidic FGF (aFGF or FGF-1) and basic FGF (bFGF or FGF-2) are the most extensively characterized members of the large FGF family, comprising at least nine polypeptides. The FGFs enhance proliferation of a variety of cells including endothelial and smooth muscle cells [13, 43–47], and share some important features: they bind heparin with high affinity, exhibit a high degree of protein homology and bind either high-affinity receptors with tyrosine kinase activity or low-affinity/high-capacity receptors in association with heparan sulphate proteoglycans. The binding of FGFs to the high-affinity receptors is modulated by attachment to the low-affinity receptors [48–50]. To date, at least four high-affinity FGF receptors (FGFR) have been identified and their cDNAs have been cloned [13–16].

Vascular endothelial growth factor is a heparin-binding angiogenic glycoprotein which also enhances vascular permeability. It is noteworthy that VEGF and bFGF have a synergistic effect on endothelial cell proliferation, differentiation and angiogenesis [51, 52]. VEGF is produced by different cell types, including vascular smooth muscle cells [53]. Interestingly, endothelial cells produce VEGF only under anoxic conditions but possess VEGF binding sites and the mitogenic action of VEGF, unlike other growth factors, is selective for endothelial cells [54, 55]. There are four forms of VEGF produced by alternative splicing of mRNA [56]which differ for their ability to bind heparan sulphate proteoglycan in the extracellular matrix. Endogenous VEGF may have a role in blood vessel development in the embryo [57]and in the adult in response to ischemia: in the latter situation, hypoxia may upregulate VEGF by hypoxia inducible factor-1 (HIF-1)-mediated transcription, increased mRNA stability [31–34, 58]or by increased VEGF receptor expression [59]. As with the FGF family described above, VEGF binds heparan sulphate proteoglycan with low-affinity. Additionally, in human cells two tyrosine kinases have been identified as high-affinity VEGF receptors; fms-like tyrosine kinase (Flt1) [60]and kinase domain region (KDR, or Flk1) [61]. Knockout mice models have been created for either receptor and in both cases the embryos die in utero at day 8.5–9.5. Flk-1 null embryos lack vascular structures almost completely [62]whereas Flt-1 null embryos have a grossly abnormal and disorganized vascular system [63]. These data suggest that Flk-1 is crucial for vessel formation at an earlier stage than Flt-1. The ligand of another member of the same family of endothelial cell-specific tyrosine-kinase receptors, Flt4, has been recently identified and named VEGF-C. This novel factor shares a 32% amino acid identity to VEGF, does not bind Flt1 or Flk1, and stimulates the migration of bovine capillary endothelial cells in collagen gel [64, 65].


   3 Methods for gene transfer
Top
 Abstract
 1 Introduction
 2 Angiogenesis and growth...
 3 Methods for gene...
4 Therapeutic angiogenesis
 5 Discussion: future directions...
 References
 
There are a variety of gene transfer systems available, each with its own positive characteristics as well as its own shortcomings. These systems can be roughly divided into two major categories, viral and nonviral. Adenovirus (AV), retrovirus (RV), adeno-associated virus (AAV) and herpes simplex virus (HSV) are the most commonly used viral vectors for gene transfer. The nonviral methods include the use of liposomes as well as the introduction of naked DNA into the target area.

A detailed description and evaluation of these systems is beyond the scope of this review and has been extensively published [66–68].

The application of in vivo gene therapy techniques to standard clinical practice will require gene delivery systems which are both safe and efficient. In addition, ideal gene therapy vectors would be engineered to transfect only the desired target cells. Among the variety of different approaches for gene transfer into the cardiovascular system, the most commonly used are: direct incubation with unmodified (naked) DNA, coupling of DNA with lipophilic/hydrophobic agents, and replication deficient adenoviral vectors.

The use of unmodified or ‘naked’ DNA is simple and well tolerated by the recipient organism due to the low toxicity and low immune response. However, transfection efficacy is significantly lower than with other methods; also, cell penetration by the hydrophilic DNA decreases significantly as DNA length increases. Plasmids of DNA have been incorporated in hydrogel coated angioplasty balloons and utilized in the first approved phase I gene therapy clinical trial to induce therapeutic angiogenesis [69–72].

To enhance cell uptake, a variety of lipophilic/hydrophobic compounds, e.g. cationic phospholipids (liposomes), have been coupled to DNA. Under these conditions, DNA is taken up by the cell via endosomes and, although a large amount is degraded by lysosomal action, some DNA reaches the nucleus for expression. Cell targeting may be achieved by conjugating specific target proteins to the DNA/liposome complex. After conjugation, the liposome particle will preferentially enter those cells with appropriate receptors on their surface [73–81].

In the last few years, adenovirus has frequently been the vector of choice for gene transfer because it can infect a variety of cells. Adenovirus, being itself a relatively large virus, is also capable of taking on a reasonably large payload of up to approximately 8 kilobases (kb). Since it has been in laboratory use for approximately forty years, it has also been shown to be a relatively safe virus, inducing little to no adverse reaction for example when administered as a vaccine agent.

The major limitations of adenoviral vectors are: (a) lack of sustained expression, as the viral DNA does not integrate into the host genome; (b) antigenicity against viral proteins by both humoral and cytotoxic T-lymphocytes (CTL); and (c) possible toxicity at high doses.

The lack of sustained expression is not likely to present a problem for acute applications such as cancer, restenosis after angioplasty or therapeutic angiogenesis. In fact, sustained expression of a growth factor(s) in vivo may have detrimental effects such as the development of angiomas and tumorigenesis due either to cell transformation or induction of new blood vessel formation in occult tumors. Therefore, when the other two shortcomings are addressed, adenovirus based vector systems may be the best suited for these applications. Vectors presently in the clinic and in research laboratories are providing the knowledge base for the development of improved systems for gene transfer and eventually for gene therapy [82].

The first generation adenovirus vectors have been rendered replication deficient by deletion of their E1A and E1B genes [83]. Although these vectors are considered safe for clinical use, antigenicity against viral proteins due to both humoral and cytotoxic T-lymphocytes has been documented. Ongoing work in the field aims at overcoming this drawback by deleting additional essential regions of the adenovirus genome to attenuate the host inflammatory response [84–89]. Furthermore, vectors are being designed to include antiinflammation and antiimmune genes to reduce MHC Class I molecule presentation on the surface of infected cells [90, 91]. Longer gene expression and more efficacious repeat administration is expected from these new vectors.

The promise of adenovirus vectors for gene delivery has been tempered by their lack of tissue specificity [92]. Without alteration, adenoviruses cannot efficiently infect cells such as endothelial and smooth muscle cells. In order for adenoviruses to work effectively in these cells, their tropism must be expanded. Recently, several new approaches have been taken to redirect adenovirus tropism from its native receptors to nonnative receptors [93–98].

One of the most promising new adenoviruses is the universal targeting vector (UTV) [95]. The transduction efficiency of cells not easily infected by adenovirus can be increased by as much as two logs in many cases using this vector. UTV was designed to genetically modify one of the virus capsid proteins, the fiber. The virus fiber was extended to incorporate a targeting sequence on the end: this modification essentially alters the tropism of the virus. The incorporation of lysine residues as the targeting sequence allows the vector to become capable of infecting most cells which express heparin-containing cell surface moieties.

It is hoped that the new generation of adenovirus vectors with tissue-specific targeting capability, especially in combination with cell-specific promoters, will result in the production of more effective reagents for vascular therapy, with either elimination or minimization of toxic side effects.

Different methods have been suggested to deliver the vector to the target tissue. The ideal delivery method should be capable of transfecting the target tissue with no systemic exposure to the vector. With the presently available vectors for gene transfer, local delivery is thus preferred to systemic administration of solutions containing plasmids or recombinant virus. Local delivery has been mainly accomplished by direct injection (in the myocardium or in skeletal muscle segments) [99, 100]or by selective infusion of the vector solution in the artery perfusing the target organ [101]. Alternatively, direct gene transfer to the endothelium of an arterial segment has been obtained by catheters inserted percutaneously and advanced to the target artery. A study comparing three different catheter-based strategies and a surgical technique has shown that catheters permit relatively efficient adenovirus-mediated gene transfer to vascular endothelium [102]. In the first clinical protocol approved for gene therapy of hindlimb ischemia, naked cDNA plasmids were suspended in a hydrogel polymer coating the balloon of an angioplasty catheter to obtain contact between plasmids and endothelial cells by balloon inflation, as previously done in animal models [70, 103].


   4 Therapeutic angiogenesis
Top
 Abstract
 1 Introduction
 2 Angiogenesis and growth...
 3 Methods for gene...
 4 Therapeutic angiogenesis
5 Discussion: future directions...
 References
 
The term therapeutic angiogenesis was first proposed in 1993 by Höckel [104], although the concept that angiogenic processes might be useful in the treatment of vascular diseases had been already proposed for a variety of ischemic conditions, not only affecting the heart [105–108]or the lower limb [109, 110], but also skin flaps [111], peripheral nerves [112], bone [113]and tracheal grafts [114], and as a strategy to accelerate wound healing. This review will focus on therapeutic angiogenesis based on gene transfer techniques for the treatment of myocardial and limb ischemia. The experimental background for the use of gene transfer techniques is derived from studies describing the effects of recombinant angiogenic factors to induce angiogenesis in animal models and in humans.

Heparin has been the first vasoactive factor administered to induce angiogenesis in animal models [115, 116], and in patients with coronary [105, 106, 108]and peripheral artery disease [117]: its role in potentiating angiogenesis initiated by other mechanisms and augmenting collateral circulation has been clearly established. Subsequently, recombinant members of the FGF family were tested to induce therapeutic angiogenesis in ischemic limbs and heart. The majority of the studies on angiogenesis in peripheral ischemia have utilized the rabbit model of hindlimb ischemia following femoral artery removal: in this model, partial compensation by spontaneous angiogenesis and recruitment of collaterals is known to occur naturally after a variable amount of time from the surgical procedure. Animal models of myocardial ischemia have also been used. Both models are characterized by the presence of an ischemic environment, which is itself a potential confounding factor, since it can trigger angiogenic stimuli able per se to enhance collateral blood flow. Different strategies for in vivo growth factor delivery have been employed. In a rat model of hindlimb ischemia it was shown that exogenous bFGF, slowly released from pellets applied in the thigh, enhanced blood flow in the ischemic limb [109]. This study also demonstrated that heparin enhanced and protamine inhibited the rate of formation of the collateral circulation. However, differences in blood flow between treated and control groups were present in the first 3 weeks but did not persist at the 4-week time point. The canine model of acute myocardial infarction was also utilized to examine the therapeutic potential of exogenous recombinant bFGF infused into the circumflex coronary artery within 6 h after occlusion of the left descending coronary artery [107]. Although the infarct had already developed completely at the time of treatment, 1 week later the group treated with bFGF demonstrated improved left ventricular ejection fraction and angiographic evidence of neovascularization. Histology demonstrated a reduced infarct size and neovascularization in the treated group, although the methodology used for infarct size measurement did not follow established protocols. Two additional studies have shown, in a dog model of chronic myocardial ischemia, the angiogenic effect of exogenous bFGF infused either intracoronary [118]or into the systemic circulation [119]. In the first study, myocardial blood flow to the ischemic territory was increased in the bFGF-treated group at 2 and 4 weeks after the beginning of treatment. In the other study from the same group, using a similar animal model of chronic myocardial ischemia, bFGF was administered daily into the left atrium and an improvement in blood flow to the ischemic myocardium in the bFGF-treated group was observed 1 and 2 weeks after beginning treatment. However, blood flow to the ischemic area in the control animals improved toward the end of the 4-week study period and approached that of bFGF-treated group, so that the difference between groups were not statistically significant beyond 4 weeks.

Both studies showed that bFGF had acute effects as arterial vasodilator and also exhibits a profile consistent with tissue binding and chronic hemodynamic effects. This study [119]was also the first to demonstrate that 4 weeks after treatment withdrawal, no regression of collateral development was detectable. A very recent study from the same group utilized the same animal model to evaluate whether the beneficial effect on collateralization shown in the short term by administration of bFGF would be maintained at 6 months. The results indicated that in dogs with chronic single vessel coronary occlusion and mature collateral vessels, repeat administration of bFGF by left atrial bolus did not induce further collateralization [120]. This study supports the concept that ischemia may be required to induce effective angiogenesis by systemic arterial administration of recombinant growth factors.

The potential of bFGF to induce angiogenesis and to improve the performance of skeletal muscle used for cardiomyoplasty has been studied in the latissimus dorsi muscle transferred into the pericardial cavity of goats. Treatment with bFGF resulted in better muscle preservation and higher capillary density compared to controls [121].

The first study on the therapeutic potential of aFGF in the cardiovascular system used a dog model of chronic ischemia [122]. In this study, local delivery of aFGF induced smooth muscle cell hyperplasia in arterioles and small arteries in the area of subendocardial infarction, but no development of collaterals bridging the ischemic myocardium to an arterial pedicle positioned over the epicardium was observed.

In a rabbit model of hindlimb ischemia, aFGF was given daily by intramuscular injection in the ischemic limb for 10 days, beginning 11 days after femoral artery excision [123]. The study lasted 40 days and it documented better perfusion in the treated animals with neovascularization and reconstitution of the distal arterial tree.

Because of the specificity of VEGF on endothelial cell stimulation, studies have been performed with this growth factor, both in the rabbit model of hindlimb ischemia and in a dog model of chronic myocardial ischemia. In the first study [124], 10 days after the femoral artery removal, rabbits received injections of VEGF or vehicle alone into the iliac artery supplying the ischemic territory. In the treated group both hindlimb blood pressure and capillary density were significantly enhanced when compared to control at day 30 after treatment. In this study peripheral pressures were measured only indirectly by blood pressure cuff. Further, the histological evaluation was limited to capillaries rather than resistance vessels and did not determined red and white muscles separately. A comparable effect could be achieved in the same animal model, using either systemic intravenous [125]or local intramuscular injection of VEGF [126].

In a canine model of chronic myocardial ischemia, progressive constriction of the left circumflex coronary artery was induced and, 10 days after surgery, VEGF was infused daily into the artery distally to the constricting device [127]. VEGF increased collateral blood flow and density of intramyocardial vessels greater than 20 µm in diameter up to 28 days after beginning treatment. In another similar study, VEGF was compared to bFGF. Both factors were infused into the left atrium [128]: basic FGF enhanced collateral blood flow to the ischemic myocardium, while VEGF-treated animals did not show any improvement in blood flow when compared to control. By contrast, as expected from in vitro studies [51, 52], simultaneous intraarterial administration of both bFGF and VEGF has been shown to have synergistic effects in enhancing collateral development in the rabbit ischemic hindlimb [129].

Additional beneficial effects of both bFGF and VEGF have been demonstrated in swine models of chronic myocardial ischemia. Increased vasomotor relaxation responses to these factors in the ischemic, collateral perfused myocardium has been observed [130, 131], related to increased gene expression of the respective tyrosine kinase receptors [38].

In summary, therapeutic angiogenesis can be successfully obtained by treatment with angiogenic factors either infused in the systemic circulation or delivered locally. However, the effect of angiogenic growth factors has been shown only in short-term studies, no longer than 4–6 weeks, and, except for heparin, none of these factors has been tested in clinical trials.

The first studies on gene therapy for ischemic diseases have used the cDNA for VEGF, FGF-5 and aFGF. Either intraarterial and intramuscular administration of a plasmid encoding the cDNA for VEGF165 enhances collateral blood flow in the ischemic rabbit hindlimb [100, 103]. In the same animal model, human cDNA plasmids encoding for VEGF121, VEGF165 and VEGF189 have similar biological activity in inducing angiogenesis [132].

A replication-deficient herpes simplex virus type 1 coding for the human VEGF165 was used to infect NIH 3T3 fibroblasts, injected subcutaneously into mice after resuspension in Matrigel plugs. One week later, histologic evidence of neovascularization and higher hemoglobin content was shown in the plugs [133]. The cDNA for the same growth factor, encoded in a replication-deficient adenovirus vector, (AdCMV.VEGF165) induced both human endothelial cell proliferation and differentiation into capillary-like structures in vitro, and, resuspended in Matrigel and injected in mice subcutaneous tissues, infected the surrounding tissues determining neovascularization [134].

Phase 1 clinical studies on therapeutic angiogenesis induced by VEGF165 gene transfer techniques are in progress. Men and women with critical chronic lower limb ischemia who are not considered good candidates for conventional revascularization are eligible for enrollment in either of these protocols, which differ in the method of delivery of cDNA plasmids coding for human VEGF165. The purpose of these studies is to document the safety of plasmid gene transfer as well as the anatomic and physiological extent of collateral artery development after intraarterial [70, 72]or intramuscular delivery (Isner JM, personal communication). All patients are closely monitored for tumor development and eye neovascularization. The therapeutic effects are assessed by clinical examination, conventional and magnetic resonance angiography, duplex color/flow Doppler and quality of life questionnaires. In a case report recently published [71], evidence was provided for angiogenesis in the ischemic limb, namely blood flow enhancement assessed by intravascular Doppler and spider angiomas development following intraarterial gene transfer. However, the magnitude of neovascularization was not enough to prevent leg amputation, 5 months after gene transfer.

In addition to VEGF, other growth factors are being evaluated in animal studies on therapeutic angiogenesis. In a swine model of latent myocardial ischemia mimicking angina pectoris, the intracoronary injection of adenovirus expressing human FGF-5 resulted in improved myocardial performance secondary to increased regional blood flow. Such effects persisted for 12 weeks after FGF-5 gene transfer; further, histology revealed that the number of capillaries surrounding each myocardial fiber was greater in the endocardium of the treated animals. This was the first report in a clinically relevant animal model to demonstrate a benefit of adenovirus-mediated gene transfer of an angiogenic growth factor [101].

In a rabbit model of coronary occlusion, a small area of myocardium next to a major epicardial artery was infected with an adenovirus vector coding for recombinant, secreted acidic FGF [135]. After 2 weeks there was histologic evidence of neovascularization and, upon coronary artery ligation, the area at risk for myocardial infarction was reduced by approximately 50%. This study demonstrated that adenovirus-mediated gene transfer of angiogenic growth factors can induce therapeutic angiogenesis in the absence of chronic and acute ischemia [99]. Finally, as a general caveat, most gene transfer studies have used animals whose vessels are free of atherosclerotic lesions: it has been shown that atherosclerosis can reduce the transfection efficiency typically found with adenovirus vectors in normal arteries [136]. The importance of this potential limitation to adenovirus-based arterial gene therapy will be better defined as results from human studies will be available.


   5 Discussion: future directions and role of gene therapy for therapeutic angiogenesis
Top
 Abstract
 1 Introduction
 2 Angiogenesis and growth...
 3 Methods for gene...
 4 Therapeutic angiogenesis
 5 Discussion: future directions...
References
 
In the last years, an increasing amount of experimental work has been committed at providing enough background evidence to initiate clinical trials on gene therapy of ischemic vascular diseases. Although the available data have been deemed sufficient to approve the first phase 1 clinical studies on chronical peripheral obstructive arterial disease, a number of questions still need to be addressed.

It is not yet determined whether the new vascular network obtained by angiogenic interventions is stable. With the gene transfer methods more likely to be used in clinical studies, transgene expression in vivo is frequently limited to a few weeks: although this period of time is adequate to induce the formation of new blood vessels, it is still unknown whether new vessels will persist after the production of the angiogenic factor has ceased. In this context, issues such as vessel remodeling, vessel regression and competition of flow need to be evaluated. In case the newly formed vessels have a limited life span, experimental work is needed to investigate if additional exposure to growth factors may be helpful in the preservation of the novel vascular network.

Another relevant question addressed only recently is whether ischemia is required for the angiogenic factor to exert its effect. This question has major clinical relevance because most patients with severe coronary artery disease have normal resting myocardial blood flow and a diminished coronary reserve, therefore they are not constantly ischemic; rather they develop ischemia only when the metabolic demand exceeds the blood supply through the stenotic artery. The available data are controversial: studies employing recombinant proteins such as bFGF in dog models of chronic ischemia suggest that ischemia is required for angiogenesis [120]. However, preliminary results in a rabbit model of coronary artery ligation suggest that adenovirus-mediated gene transfer of recombinant, secreted aFGF induces neovascularization in the nonischemic heart [99]. More studies will be necessary to address this issue and to establish if angiogenesis can be induced as a preventive measure to avoid tissue damage at the time ischemia develops.

The presently available experimental work is not sufficient to define the best strategy to induce angiogenesis, since very few studies compared different methods of delivery and/or constructs expressing different angiogenic factors in the same model [128]. The studies performed so far show that the VEGF, FGF-5 and recombinant, secreted aFGF1–154 genes can induce therapeutic angiogenesis in animal models. Other growth factors have not yet been tested, and so far only one study has been designed to evaluate the synergistic effect of two different growth factors in vivo [129].

Similarly, the choice of the delivery system best suited to induce therapeutic angiogenesis is not yet based on a direct comparison of different approaches.

Specific attention is mandated by the issue of safety of clinical gene therapy interventions. Significant side effects must be ruled out by studies specifically designed to ensure that, in response to gene therapy protocols, neovascularization will not develop in areas not supposed to be targeted by the angiogenic intervention and that a tumorigenic effect is not observed. Potential adverse effects linked to the delivery method must also be considered in the context of the effect expected from tissue exposure to angiogenic growth factors. For example, catheter-based techniques may result in damages to the endothelial lining and to atherosclerotic plaques that, if eventually exposed to growth factors, could increase neointimal proliferation or plaque growth and neovascularization.

Answers to these and other questions can be reasonably expected in the near future. Carefully designed studies must include multiple experimental groups to control for nonspecific effects related to the vector itself, or to the inflammatory process ensuing vector delivery. Further, as recently pointed out [137], the choice of the appropriate experimental endpoints is critical, since a gold-standard to document angiogenesis in humans is not currently available. Thus, studies should demonstrate the association between anatomical evidence of angiogenesis, physiological indices of enhanced perfusion and functional improvement.

Hopefully, the ongoing clinical trials in patients with severe peripheral artery disease and, eventually, forthcoming trials aimed at treating ischemic heart disease will provide important information to substantiate the potential that gene transfer techniques have demonstrated so far. The development of this form of biotechnology may play a significant role in the treatment of ischemic diseases, and may provide a new therapeutic option for patients who do not respond to medical therapy and are not candidates for either surgical revascularization or angioplasty.

Time for primary review 15 days.


   References
Top
 Abstract
 1 Introduction
 2 Angiogenesis and growth...
 3 Methods for gene...
 4 Therapeutic angiogenesis
 5 Discussion: future directions...
 References
 

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