Recent advances in gene transfer technologies and better understanding of molecular and genetic bases of cardiovascular disease have made gene therapy an emerging alternative treatment strategy. Promising results have been obtained in animal models of restenosis and vein graft thickening, and limb and cardiac ischemia. Gene therapy for the induction of angiogenesis is based on the concept that myocardial and peripheral ischemia can be improved by stimulating neovessel formation and collateral development from the existing vasculature. Therapeutic vascular growth includes stimulation of angiogenesis, arteriogenesis, and lymphangiogenesis (Yla-Herttuala and Martin, 2000). Prevention of restenosis can be achieved by inhibiting smooth muscle cell proliferation, migration, matrix synthesis, remodeling, and thrombosis.

Gene therapy has the potential advantage of enabling gene expression for a sufficiently long period, at an adequate concentration to stimulate effective therapeutic response from a single administration. However, full potential of this therapy can be achieved only after further development of gene transfer technology and selection of effective treatment genes.

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2. Choice of angiogenic factors

Angiogenic signals are mediated by a number of growth factors and cytokines. The endothelial specific growth factors include members of the vascular endothelial growth factor (VEGF) family of proteins and the angiopoietin (Ang) family. Different members of the VEGF family act as key regulators of endothelial cell function, controlling vasculogenesis, angiogenesis, vascular permeability, and endothelial cell survival (Ferrara and Davis-Smyth, 1997). Other factors that promote angiogenesis are fibroblast growth factors (FGFs), hepatocyte growth factor (HGF), epherins, platelet derived growth factors (PDGFs), and hypoxia-inducible factor-1 (HIF-1). Vessel survival is dependent on VEGF and other exogenous survival factors. Angs act during remodeling of vascular plexus and a combination therapy with VEGF and Ang-1 may produce more stable vessels. Another important aspect is induction of angiogenesis by angiogenic master switch genes, such as HIF-1 α and HGF, which stimulate multiple neovascularization cascades.

VEGF family comprises of six members, VEGF-A, -B, -C, -D, -E, and placental growth factor (PIGF), which differ in their molecular mass and biological properties. The mechanism of action is through tyrosine kinase receptors, VEGFR-1, VEGFR-2, and VEGFR-3. VEGF-A is known to play a crucial role in angiogenesis and vasculogenesis and is a ligand for VEGFR-1 and VEGFR-2 (Ferrara, 2001). VEGF-A promotes increased microvascular permeability and fibrin deposition that may be responsible for enhanced migration of endothelial cells in extracellular matrix. It supports the survival of endothelial cells by expressing antiapoptotic proteins in these cells. In phase I and II clinical trials, VEGF-A plasmid/liposome or adenovirus vector has been used for coronary artery disease (CAD), in-stent restenosis, and peripheral artery occlusive disease. Vascular endothelial growth factor B (VEGF-B) is structurally related to VEGF-A and binds only to VEGFR-1 (Olofsson et al., 1998). VEGF-B is a very weak mitogen when tested in mammalian cells. The receptors for VEGF-B are located on endothelial cells; thereby, it is more likely to act in a paracrine manner.

Expression of VEGF-C occurs during early embryonic development before the emergence of lymphatics, which is suggestive of its role in vasculogenesis and angiogenesis. VEGF-D has angiogenic and lymphangiogenic potentials (Bhardwaj et al., 2003; Rissanen et al., 2003). Both VEGF-C and VEGF-D act through VEGFR-2 and VEGFR-3 (Hamada et al., 2000; Achen, 1998). Biological activity of VEGF-C and -D depends on proteolytic cleavage. VEGF-E was discovered in the genome of the Orf virus, and signaling through VEGFR-2 and neuropilin receptor (NRP-1) causes endothelial cell mitogenesis. PIGF-1 binds specifically to VEGFR-1 and is a nonheparin binding protein. PIGF-2 is a heparin binding protein that binds to VEGFR-1 and NRP-1. VEGF and PIGF form heterodimers have been found to bind to VEGFR-2. High concentration of PIGF saturates VEGFR-1 binding sites and augments the action of VEGF, which then acts through VEGFR-2. PIGF is chemotactic to endothelial cells and monocytes.

Angs are a group of growth factors that affect the growth of endothelial cells. They bind to the receptor Tie2 (tyrosine kinase with immunoglobulin and epidermal growth factor homology domain) (Sato et al., 1995). Ang1/Tie-2 along with VEGF/VEGFR-2 is critical for mobilization and recruitment of hemopoietic stem cells and the circulation of endothelial cell precursors. Property of Ang-1 to reduce vascular leakage and inflammation might prove beneficial in vascular gene therapy. Ang-2 is an antagonist for Ang-1 and is probably needed for vascular remodeling.

HIF-1 is a transcription factor that acts as a regulator of oxygen homeostasis. It acts as a transcriptional activator of VEGF gene. A cellular enzyme HIF-1 α prolyl hydrolase (HIF-PH) probably serves as a cellular oxygen sensor. HIF-1 α administered via gene transfer induces expression of VEGF, which leads to therapeutic neovascularization of ischemic tissues. This property of HIF-1 has been used for promoting therapeutic angiogenesis.

The family of PDGFs currently compromises of four members, PDGF-A, -B, -C, and -D, which bind to receptors PDGFR- α and PDGFR- β. They are major mitogens for fibroblasts, smooth muscle cells, and several other cell types. PDGF-A and PDGF-B form homo and heterodimers with their tyrosine kinase receptors, whereas PDGF-C and PDGF-D form apparently only homodimers. Increased PDGF activity has been implicated in several pathological conditions in adults, including atherosclerosis, restenosis, fibrosis, and tumorigenesis. PDGF receptor (PDGFR) inhibition is known to reduce restenosis in experimental animals.

FGFs are known to stimulate cell migration and cell mitosis and affect cellular senescence. FGF signaling contributes to multiple, distinct steps in vessel formation. These steps include proliferation and differentiation of Flk1-positive hemangioblastic precursor cells from mesoderm, assembly of endothelial cells during vasculogenesis, and sprouting angiogenesis. FGFs can regulate vascular morphogenesis by acting either directly through FGFRs or indirectly by inducing other angiogenic factors like VEGFs. FGF is produced by angiogenic tissue, and it can be released to stimulate endothelial cells, smooth muscle cells, and pericytes. Thus, FGFs might be responsible for the maturation of blood vessels. Adenoviral mediated FGF-4 gene delivery has been used in Phase II clinical trials for peripheral artery occlusive disease and coronary artery disease.

Hepatocyte growth factor stimulates proliferation and migration of endothelial cells through c-Met (a transmembrane tyrosine kinase) receptor present on endothelial cells and some other cell types including smooth muscle cells and pericytes. Overexpression of HGF in the skin increases granulation tissue formation, angiogenesis, and VEGF levels. It has been used to promote therapeutic angiogenesis in animal models and in a clinical trial.

The Eph family of receptor tyrosine kinases is the largest known family of receptor tyrosine kinases (RTKs) identified so far. Expression of ephrin ligands may be induced by growth factors and cytokines in various cell types. Ligands of EphB family induce capillary sprouting in vitro. Expression of ephrin-B2 and its cognate EphB receptors in mesenchymal cells adjacent to vascular endothelial cells suggests an EphB/ephrin-B2 interaction at endothelial-mesenchymal contact zones. Ephrin-A1 is expressed at sites of vascular development. The Eph receptor family represents a new class of receptor tyrosine kinases, and their role in angiogenesis is yet to be defined.

Certain risks are associated with therapeutic angiogenesis and include formation of hemangiomas or vascularization of tumors, neovascularization in atherosclerotic lesions leading to plaque rupture, development of nonfunctional vessels, and edema. Increasing the tissue specificity of the gene constructs and promoters and regulating the transgene expression should minimize these risks.

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3. Targeted gene delivery systems

The efficacy and safety of gene therapy also depends on targeting genes to particular cells and effectively controlling their expression. Developing vectors with defined cell-type trophism or using cell-specific promoters and regulatory elements can produce better targeting (Harris and Lemoine, 1996).

Receptor-mediated targeting is based on receptor ligand interaction. Modified vectors have been prepared that target binding to alternative attachment receptors, improving vector specificity. Adenoviruses are widely used vectors for gene transfer to dividing and nondividing cells (see Adenovirus vectors). However, they have broad cell tropism and transgene expression is often detected in various ectopic organs. Novel adenoviruses targeted to vascular wall have been developed. They include matrix metalloproteinase-2 and -9 (MMP-2 and -9) targeted TIMP-1 encoding adenoviruses, αν integrin targeted human interleukin-2 encoding adenoviruses, and endothelial cell targeted adenoviruses. Additionally, blocking of (Coxsackie and adenovirus) CAR receptors may lead to targeted expression by adenoviral vectors (Kibbe et al., 2000).

In transcriptional targeting, tissue or cell type–specific promoters and regulatory elements are used to restrict expression in nontarget tissues. Endothelial specific promoters include fms-like tyrosine kinase-1 (FLT-1), intercellular adhesion molecule-2 (ICAM-2), von Willebrand factor, and Tie promoters. The SM22alfa promoter restricts transgene expression exclusively to smooth muscle cells after adenovirus-mediated gene transfer to arterial wall. Along with the use of viral promoters in cardiovascular gene transfer, vectors containing inducible promoters are now being used to regulate gene expression and to optimize therapeutic effect. An example of inducible promoters is Escherichia coli tetracycline responsive element tet, which activates transcription of the transgene only in the presence of tetracycline. Another strategy is the use of endogenous stimuli to regulate transgene expression. Examples of this approach are vectors containing transcription regulatory elements sensitive to hypoxia, which can be effectively used for the regulation of transgene expression in ischemic tissues. The hypoxia response element (HRE) is introduced into an expression cassette and gene expression is activated by HIF-1 under ischemic conditions (Dachs et al., 1997).

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4. Potential therapeutic targets

Atherosclerosis is characterized by deposition of atheromas or plaques in the inner layers of arteries. These plaques can ultimately occlude an artery, or an unstable plaque can result in thromboembolic episodes. Complex etiology of atherosclerosis makes the use of a single or local gene transfer for its prevention or treatment a controversial issue. But several genetic disorders with a single gene defect, which predispose to the development of atherosclerosis, can be treated with gene therapy. In cases of low-density lipoprotein (LDL) receptor deficiency, LDL receptor and very low density lipoprotein (VLDL) receptor gene transfers to liver may prove beneficial. Lecithin cholesterol acyl transferase (LCAT) or lipid transfer protein gene transfer can be used to treat certain dyslipoproteinemias. It is possible to inhibit the elevated levels of atherogenic apolipoprotein (apo) B100 by apobec-1 gene transfer, which is a catalytic subunit of apoB editing enzyme. ApoA1 gene transfer that promotes reverse cholesterol transport might be used to treat apoA1 deficient patients. ApoE gene transfer might be useful for decreasing lipoprotein levels in the treatment of Type III Hyperlipoproteinemia. Lipoprotein lipase and hepatic lipase gene transfers could benefit patients having deficiency of these enzymes. Class A soluble scavenger receptor gene transfer could decrease lipid accumulation in macrophages and class B soluble scavenger receptors can alter high-density lipoprotein (HDL) levels. Decreased nitric oxide (NO) bioavailability probably results in endothelial dysfunction, occurring in early atherosclerosis. It could be corrected by using endothelial nitric oxide synthase (eNOS) and VEGF genes. In advanced cases of atherosclerosis, however, an increased NO production may not be useful. Rho family GTPases participates in the regulation of actin cytoskeleton and cell adhesion. Inhibiting Rho kinase (RhoK) by dominant negative RhoK gene transfer decreases atherosclerosis. Overexpression of antioxidant enzymes like superoxide dysmutase (SOD) also helps in decreasing atherosclerosis. It has been seen that Interleukin-10 (IL10) and platelet activating factor acetyl hydrolase (PAF-AH) gene transfers decrease atherosclerosis probably via their antiinflammatory effects. Rupture of an unstable plaque and subsequent thrombosis in an atherosclerotic artery might precipitate an acute ischemic episode. TIMP gene transfer may prove useful to stabilize unstable plaques. Other gene transfers used to decrease thrombotic episodes in animal models include cyclooxygenase, hirudin, thrombomodulin, tissue plasminogen activator, and tissue factor pathway inhibitor.

Coronary artery occlusion due to atherosclerosis can result in myocardial ischemia. Angiogenic gene therapy is aimed at promoting new blood vessel formation in ischemic myocardium, thereby improving cardiac perfusion, exercise tolerance, and quality of life. Therapeutic angiogenesis using VEGF, FGF, HGF, and HIF-1α has proved beneficial in many animal models. Improvement in exercise tolerance was reported after adenovirus-mediated VEGF gene transfer to ischemic myocardium. Targeted delivery of angiogenic growth factors using sophisticated delivery systems like NOGA catheters might further improve chances of rescuing ischemic myocardium.

In PAD, there is decreased blood supply to the limbs because of arterial obstruction and vasoconstriction. Many of these patients suffer from disabling symptoms like severe ischemic rest pain, and amputation is often required to alleviate suffering. Therapeutic angiogenesis using angiogenic growth factors has been recently used to treat critical limb ischemia. VEGF, FGF, and HGF gene transfers have been used to promote development of collateral blood vessels in animal models and clinical trials. Angiopoietins can also possibly enhance the maturity of new vessels formed after VEGF gene therapy. Other cytokines like Monocyte chemotactic protein-1 (MCP-1) and PDGFs can also promote angiogenesis indirectly.

Maladaptive response to injury can result in occlusion of an artery as seen after balloon angioplasty, stenting, or in bypass vein graft. Restenosis is defined as a diameter stenosis of 50% at follow up. Restenosis occurs in 10–30% of patients after balloon angioplasty and stenting. Multiple factors including smooth muscle cell proliferation, matrix accumulation, remodeling, thrombosis, and platelets and leukocyte adhesion are involved in the development of arterial restenosis after angioplasty, stenting, and in vein graft disease. Various gene therapy strategies have been employed to decrease cellular proliferation. These include antisense oligonucleotides and ribozymes against c-myb, c-myc, cdc-2, cdk-2, ras, bcl-x, or decoy constructs against transcription factors such as E2F and NFkB. Cell cycle inhibitors like nonphosphorylated retinoblastoma gene, p21, p27, p53, and gax can decrease cellular proliferation and neointima formation. Similarly, (Herpes Simplex Virus-Thymidine Kinase) HSV-TK, cytosine deaminase, preprocecropine A, and fas ligand gene transfers have been shown to decrease cellular proliferation and smooth muscle cell migration in the blood vessels. Transfer of VEGF and HGF genes to vessel wall has been shown to decrease neointima formation, possibly by enhancing endothelial repair. It has been hypothesized that rapid regeneration of endothelial cells results in secretion of antiproliferative substances like nitric oxide, C-type natriuretic peptide, and prostacyclin I₂. Gene transfer of TIMP-1, nitric oxide synthtase, and dominant negative Rho kinase has resulted in decreased neointima formation in animal models. Inhibition of thrombosis by recombinant hirudin (inhibitor of thrombin) gene transfer resulted in decreased neointima formation in animal models. Only a limited number of gene therapy clinical trials have been conducted for restenosis and vein graft disease. Ex vivo gene transfer of E2F decoy in vein grafts has been successful in decreasing graft failure rate in human trials. Other clinical trials for gene therapy in restenosis and vein graft disease have so far been inconclusive.

Essential hypertension is a progressive disease characterized by chronically elevated blood pressure of unknown etiology (see Hypertension). Multifactor and intricate etiology of systemic hypertension has led to the question of feasibility of gene therapy in hypertension. But it has been shown that altering certain mediators by gene therapy can result in effective lowering of systemic blood pressure. Argument in favor of gene therapy for hypertension has been that a single gene transfer might be able to control systemic hypertension for a long term, thereby improving patient compliance. One approach has been to transfer genes, which increase vasodilator proteins like tissue kallikrein, atrial natriuretic peptide (ANP), adrenomedullin, and eNOS. Another approach has been to decrease the vasoconstrictor proteins. Antisense oligonucleotides and DNA have been used against -adrenoreceptors, angiotensin converting enzyme (ACE), angiotensin type-1 receptors, angiotensin gene activating element, thyrotropin releasing hormone (TRH) and TRH receptor, carboxypeptidase y, c-fos, and CYP4A1. Although promising results have been obtained in animal models for hypertension, no clinical trial for gene therapy in systemic hypertension has yet taken place.

Pulmonary hypertension is characterized by progressively increasing pulmonary artery pressure. Primary pulmonary hypertension (PPH) is a disease of unknown etiology, while secondary pulmonary hypertension results from diseases like collagen vascular disease, congenital heart disease, chronic thrombotic/ and or embolic disease, chronic obstructive pulmonary disease, chronic hypoxia, and certain drugs. Mutations in BMPR-II (encoding bone morphogenetic protein receptor II) have been reported in many cases of sporadic cases of PPH. It is usually a progressive and fatal disease. Gene therapy to decrease cellular proliferation and vasospasm in pulmonary vessels has so far been limited to animal studies.

MCP-1, Prepro-calcitonin gene related peptide, atrial natriuretic peptide, eNOS,

prostacyclin synthtase and VEGF gene transfers have been used with varying degree of success in animal models for pulmonary hypertension.

Alterations in myocardial β-adrenergic receptor system and intracellular calcium signaling play a crucial role in the pathophysiology of heart failure. Ability to genetically manipulate beta-adrenergic receptor system and calcium signaling might prove beneficial in the management of chronic congestive cardiac failure of primary myocardial origin, where conventional drug therapy is often inadequate. HGF gene transfer that has an antifibrosis and antiapoptosis action in the myocardium was beneficial in an animal model of cardiomyopathy with heart failure. Recent genetic studies have revealed that mutations in genes for cardiac sarcomere components lead to dilated cardiomyopathy. Mutations in the Z-line region of titin were found along with decreased binding affinities of titin to Z-line proteins. Gene therapy directed at correcting defective sarcomeric proteins may prove beneficial in cases of familial cardiomyopathy.

Various therapeutic genes and their disease targets are listed in Table 1.

 

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5. Delivery systems for cardiovascular gene transfer

Selection of an appropriate system results in efficient expression of the therapeutic gene. Gene delivery to the cardiovascular system could be local, regional, and systemic. Local gene transfer is required in cases of atherosclerotic lesions, vein graft, ischemic conditions of myocardium or skeletal muscle, regional as in the case of pulmonary hypertension or systemic as in atherosclerosis and hypertension. Perivascular collars or sheaths, needle injection catheters, and biodegradable gels can be used for delivering the vectors to adventitia (Laitinen et al., 1997; Laitinen and Yla-Herttuala, 1998). When specific physical or biological targeting methods are available, they usually improve transgene expression (Yla-Herttuala and Alitalo, 2003). Ultrasound, microbubbles and electronic pulses can be used to enhance gene transfer efficiency. Local delivery to small arterioles and capillaries can also be achieved by using coated biodegradable microspheres.

Successful transfection of smooth muscle cells in media could be achieved by high-pressure intra luminal gene delivery. Disruption of intima and internal elastic lamina by balloon angioplasty and subsequent delivery of the vector by infusion catheter results in transfection of medial SMC. Coated stents and hydrogel coated balloon catheters are also useful for delivering vectors to endothelial cells or medial smooth muscle cells (Riessen et al., 1993). Different types of catheters have been developed for the delivery of vectors to blood vessels, such as double, gel coated, porous channel balloon and dispatch catheters.

Vector delivery to ischemic myocardium for CAD has been achieved using intramyocardial injections via thoracotomy and intracoronary injections (Hedman et al., 2003). A recent introduction to myocardial gene transfer is nonfluoroscopic catheter-based electromechanical mapping system (NOGA, Biosense Webster, Inc). NOGA system assesses the reduction in electrical voltage and mechanical activity in ischemic myocardium, thereby differentiating it from healthy tissue. Pericardial delivery of the vectors can also be useful for transferring genes to myocardium and coronary arteries. Gene delivery for the treatment of peripheral arterial disease (PAD) can be done by the use of direct intramuscular injections (Isner et al., 1998), infusion–perfusion catheters, and hydrogel coated balloon catheters.

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6. Issues in clinical trial design

The field of therapeutic angiogenesis for CAD is fast growing from basic and preclinical investigations to clinical trials although many new issues need to be addressed. These include a deep understanding of the biology of angiogenesis, selection of appropriate patient populations for clinical trials, choice of therapeutic end points for curative or palliative purposes, choice of therapeutic strategy, route of administration, and the side effect profile (Isner et al., 2001).

The induction of arteriogenesis is clinically more relevant in maintaining the cardiac function and patient survival than angiogenesis as demonstrated by the fact that myocardial infarction patients are less likely to develop ventricular aneurysms and show improved survival, if they have collateral arteries. Patient selection is another important feature, both with respect to age and genetic background. There is age dependent impairment of VEGF expression at least in part caused by impaired induction of HIF-1 activity in response to ischemia or hypoxia. Combination therapies using different growth factors according to their role in the initiation of growth and maintenance of blood vessels are needed to ensure long-term viability of these vessels. An alternative to this is the use of “angiogenic master switch” genes like HIF-1α that is capable of initiating multiple neovascularization cascades.

Presently, most gene transfer studies in cardiovascular gene therapy are preclinical, although a number of high profile vascular gene therapy clinical trials are in progress. The most important targets for cardiovascular gene therapy are CAD and PAD. The clinical trials for cardiovascular gene therapy are summarized in Table 2A and 2B.