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its angiogenic effects. Increases in blood flow, induced by a vasodilator, may stimulate the proliferation of endothelial cells. Increased shear stress in skeletal muscle microcirculation is associated with increased uptake of bromodeoxyuridine by capillary endothelial cells.71 Increased shear stress also plays a role in the remodeling of existing capillary structures, leading to the development of arterialized collateral vessels.72
3. NOS Gene Transfer
The lack of effect of systemic NO donor treatment on post-ischemic revascularization in NOS-III-KO mice58 may be due (at least in part) to insufficient local NO concentration in the ischemic hindlimb. In order to overcome this limitation, the effect of local delivery of NOS-III gene was tested in several animal models of limb ischemia.
3.1. Gene delivery vectors
Local and systemic delivery of NOS-III gene has been achieved using both viral and non-viral gene delivery vectors.73−77 Viral vectors currently used for cardiovascular gene transfer include adenovirus, adenoassociated virus, and most recently, lentivirus.78−81 These recombinant viruses are genetically modified to be replication-incompetent, and they contain an inserted cDNA sequence of interest (“transgene”) and an appropriate promoter (i.e. CMV). Non-viral vectors that have been studied in vascular gene transfer include naked plasmids and liposomes. Viral vectors generally have higher transfection efficiency but are also more immunogenic than non-viral vectors. A detailed comparison of these vectors with major advantages and disadvantages for cardiovascular gene transfer and the various ways to deliver them can be found in a recent review article.82
Naked plasmid and recombinant adenovirus have become the most frequently used vectors for cardiovascular gene transfer studies.83 The human adenovirus is a non-enveloped linear double-stranded DNA virus with a 36 kb viral genome.84 The advantage of adenoviral vectors for cardiovascular gene transfer includes their ability to transduce both dividing and non-dividing cells with high efficiency and the ability to generate high titer stock vector (i.e. up to 1012 pfu/ml). The viruses enter
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the host cells via an endocytosis process through the interaction between viral fiber and penton proteins and their cell surface receptors (CAR and integrins, respectively).85,86 Upon entry into the cell, the virus is taken up by endosomes, which are disrupted by the virus, resulting in viral DNA release into the cytoplasm. The viral DNA then enters the nucleus, where it is not incorporated into the host chromosome but remains episomal. The major drawbacks of first generation adenoviral vectors include the capsid protein-induced, cell-mediated (i.e. CD8+ T-cell) immune response which may limit the duration of transgene expression and may prevent repeated administration of the vector.
3.2. NOS-III gene transfer
Potential advantages of targeted local delivery of the NOS enzyme gene include sufficient (local) increase in NO production in target tissues without systemic hypotensive side effects, as observed after systemic treatment with small molecule NO donors. Attempts so far included plasmid/liposome gene transfer of NOS-III gene resulting in restored NO production and concomitant inhibition of intimal hyperplasia in balloon-injured rat carotid arteries.76 Ex vivo, adventitial delivery of NOS-III gene using adenoviral vectors demonstrated improved NOdependent vasorelaxation.77,87 NOS-III gene transfer was also shown to reverse hyperlipidemia-induced endothelial dysfunction.73
Although these studies demonstrated favorable outcome of NOSIII gene transfer under well-defined experimental conditions, utility of NOS overexpression will depend on the disease and the mechanism of endothelial dysfunction. In conditions when substrate or co-factor availability may be limited or NOS-III activation is prohibited by risk factors (such as ox LDL or hyperglycemia), wild-type NOS-III overexpression may not result in efficacious restoration of NO-mediated functions.
3.3. NOS-II gene transfer
The inducible isoform of NOS (NOS-II) is associated with disease pathophysiology such as systemic vasodilation and hypotension in sepsis88 as well as in the pathogenesis of autoimmune diseases.89−92
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Nonetheless, there may be advantages of using NOS-II over NOS-III for certain cardiovascular disease indications. An example is the prevention of re-stenosis after balloon angioplasty. With even the most efficient delivery system available (adenoviral vectors), gene transfer efficiency may be low during clinical applications. The potential advantage of NOS-II gene transfer is that NOS-II synthesizes much larger quantities of NO93 which can diffuse to a large number of neighboring cells.94 In comparison, many more cells would have to express NOSIII to synthesize a similar amount of NO. In addition, NOS-II enzymatic activity will be maximally activated in the absence of agonist stimulation.
4.Therapeutic Angiogenesis with NOS-III Gene Transfer for Critical Limb Ischemia
4.1.Impaired angiogenesis and arteriogenesis in patients with critical limb ischemia
In healthy young individuals, myocardial ischemia induces collateral vessel development, which provides certain protection from subsequent coronary events.95 Ischemia-induced arteriogenesis and subsequent angiogenesis are compensatory mechanisms to restore adequate blood supply to ischemic tissues. In the coronary circulation and peripheral vasculature, ischemia-initiated opening of pre-existent collaterals and arterialization of these immature vascular channels preserves blood flow and contributes to the extent of ischemic reserve capacity in the heart and leg.72 However, in older patients with chronic diseases and multiple risk factors, arterioand angiogenesis in response to ischemia is severely limited or absent. Indeed, it has been shown that common risk factors, such as diabetes, hypercholesterolemia and advanced age, impair angiogenesis and arteriogenesis96−100 which may contribute to the increased severity of cardiovascular diseases in this patient population.
Peripheral arterial occlusive disease (PAOD) is a highly prevalent disease lacking adequate treatment especially for its most advanced stage, critical limb ischemia (CLI).101 Local delivery of genes encoding angiogenic growth factors such as vascular endothelial growth factors
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(VEGF), fibroblast growth factors (FGF) or hepatocyte growth factor (HGF) emerged as a promising new therapeutic approach (termed “therapeutic angiogenesis”) for ischemic cardiovascular disease.102 However, clinical results so far lack unequivocal proof for efficacy by therapeutic angiogenesis. It may be due to inefficient gene delivery or because some patients are refractory to exogenously administered growth factors.102
4.2.Animal models of impaired ischemia-induced angiogenesis and arteriogenesis
Impaired ischemic limb arteriogenesis and angiogenesis was described in mice deficient in placental-derived growth factor,103 interleukin-1,104 the angiotensin II type-1 receptor,105 matrix metalloproteinase-9,106 adiponectin,107 and caveolin-1.108 In addition, studies in experimental mouse models of hindlimb ischemia demonstrated impaired revascularization and CLI-like symptoms (i.e. tissue necrosis) in old animals98,109 and in animals with compromised immune system.110 Diminished angiogenesis in some of these animal models corresponded with decreased expression of VEGF.98,99,111 Interestingly, substituting endogenous VEGF by exogenously delivered protein or gene has not shown therapeutic benefit in either aged mice109 or in the immunecompromised balb/c mice,110 unlike in other mouse models of hindlimb ischemia.99
4.2.1. NOS-III-KO mice
Mice deficient in NOS-III gene (NOS-III-KO) also show a severe form of critical limb ischemia after femoral artery ligation.58,112−114 Moreover, the ability of statin-based drugs, angiotensin II, neuropeptide Y, and stromal cell-derived factor-1α (SDF-1α) to improve limb angiogenesis are absent in mice deficient in NOS-III.115−118 The molecular mechanisms for how NOS-III regulates ischemia-triggered arteriogenesis and angiogenesis are related, in part, to the inability of NOS-III-KO mice to respond to vascular endothelial growth factor (VEGF). Indeed, VEGF-mediated increased permeability, angiogenesis, and endothelial cell precursor mobilization is markedly impaired in mice lacking
NOS-III.58,112,119,120