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enzyme substitutes for Akt-phosphorylated wild-type NOS-III enzyme, which results from KDR-mediated VEGF activation. Therefore the mutant gene will not require intact agonist pathway for its full activity. Furthermore, common risk factors for cardiovascular disease (oxLDL or hyperglycemia)129−131 and angiogenesis inhibitors, such as endostatin,132 interrupt Akt-mediated Serine 1177 phosphorylation in wildtype NOS-III which can all be avoided with the mutant enzyme.129

4.5.NOS-III-S1177D gene transfer in animal models of hindlimb ischemia

Two recent publications showed that transfer of this constitutively active mutant form of NOS-III gene using either adenovirus113 or naked plasmid (combined with electroporation)114 effectively rescued the structural and functional defects in angiogenesis and arteriogenesis in the ischemic hindlimbs of NOS-III-KO mice. In addition, improvement in blood flow recovery and angiogenesis in response to NOS-III gene transfer have been also demonstrated in several animal models of hindlimb ischemia without genetic deficiency in NOS-III (Akt-KO miceand non-obese diabetic (NOD) mice).

4.5.1. Plasmid delivery of the NOS1177D gene

Skeletal muscle expression of NOS1177D (the human form of mutant NOS-III) was tested in NOS-III-KO mice using intramuscular plasmid injection in combination with electroporation.114 There was no detectable NOS-III protein (measured by specific ELISA) in hindlimb musculature of NOS-III-KO mice (Fig. 6). In wild-type mice, the average level of NOS-III protein was approximately 30 pg/mg skeletal muscle wet weight. Quantitation of NOS-III protein expression from skeletal muscle homogenates by NOS-III-specific ELISA showed that the transgene expression after NOS1177D gene delivery can reach or exceed levels seen in wild-type mice (Fig. 6).

To evaluate the therapeutic potential of local mutant human NOS-III (NOS1177D) gene delivery, two groups of six-month-old NOS-III-KO mice were injected intramuscularly either with NOS1177D plasmid (pNOS1177D) or an empty vector (pNull).114 Treatment with the

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Fig. 7. Intramuscular injection of plasmid NOS1177D augments post-ischemic flow recovery (A) and prevents limb loss (necrosis and auto-amputation) (B) in NOS-III-KO mice. NOS-III-KO mice underwent unilateral femoral artery resection and three days later were injected with an empty plasmid (“pNull”) (n = 8) or a plasmid carrying the mutant NOS-III-S1177D gene (pNOS1177D) (n = 8) followed by electroporation. (A) Limb blood flow was measured by laser Doppler perfusion imaging (LDPI) at various time points after surgery. Data are shown as mean ± SEM and expressed as the ratio of perfusion in the ischemic versus non-ischemic hindlimb. NOS-III S1177D gene transfer (filled columns) significantly improved LDPI flow on days 7 and 28 (D7, D28) compared to pNull treatment (open columns). p < 0.05; BI = before ischemia). (B) Ischemic tissue damage of the hindlimb was evaluated by taking photographs of the limbs on the same days when LDPI measurements were made. By day 28, the evidence of limb loss was significantly greater in the pNull–treated (four out of eight animals lost their limb) than in the pNOS1177D–treated group (none of the animals lost their limb). (Reproduced with modifications by permission from the Nature Publishing Group.)

406 G. M. Rubanyi

Fig. 8. Intramuscular injection of Ad5NOS1179D improves ischemia–induced angiogenesis (A) and arteriogenesis (B) in NOS-III-KO mice. NOS-III-KO mice were injected with Ad5GFP (n = 5) or Ad5NOS1179D (n = 5) in the adductor muscle of the ischemic left hindlimb. Four weeks after surgery and gene injection, mice were sacrificed and the gastrocnemius muscle harvested and tested for PECAM-1 (angiogenesis; A) and PECAM-1 + SMA (arteriogenesis; B) immuno-staining (as described in detail in legend to Fig. 2). Data are shown as mean ± SEM. p < 0.05: statistically significant differences between Ad5GFP and Ad5NOS1179D treatment. In contrast to Ad5GFP, injection of Ad5NOS1179D significantly increased post-ischemic angiogenesis and arteriogenesis. (Reproduced with modifications by permission from the National Academy of Sciences, USA.)

4.5.3.Effect of NOS1177D gene transfer in mouse CLI models without genetic deficiency of NOS-III

In a rat model of hindlimb ischemia, intramuscular injection of adenovirus carrying the wild-type human NOS-III gene increased postischemic flow recovery and angiogenesis.122 Similar to NOS-III-KO mice, balb/c mice also respond to unilateral surgical femoral artery occlusion with severe, VEGF refractory hindlimb ischemia and autoamputation.110,111 In this mouse strain, which does not have a genetic deficiency of NOS-III expression, delivery of pNOS1177D resulted in significantly improved post-ischemic blood flow recovery (K. Kauser and G. M. Rubanyi — unpublished observation). Non-obese diabetic (NOD) mice develop severe impairment in hindlimb blood flow recovery after femoral artery ligation, potentially due to diabetes-induced endothelial dysfunction resulting from lost nitric oxide activity.133 This

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In coronary post-capillary (venular) endothelial cells, nitric oxide mediates the angiogenic effect of VEGF57 by activation of ERK 1/2.134 In a canine model of repetitive myocardial ischemia, collateral blood flow (measured by microspheres) progressively increased during the 21-day experimental period, which was prevented by treatment of dogs with the NOS inhibitor L-NAME.135 Similarly, in a rat model of chronic myocardial ischemia, treatment with L-NAME significantly reduced basal and maximum left ventricular blood flow (measured by MRI) and angiogenesis.136 Ischemia-induced upregulation of VEGF production in the myocardium was not prevented by L-NAME, indicating, that similar to the peripheral vasculature, NO mediates VEGF-induced angiogenesis and collateral growth in the coronary circulation as well. An additional mechanism of NO-induced facilitation of collateral growth in the ischemic heart is suppression of anti-angiogenic molecules such as angiostatin, via downregulation of tissue matrix metalloproteinases (MMPs), MMP-2 and MMP-9, which generate angiostatin.135,137

5.2.NOS-III-derived nitric oxide facilitate myocardial gene transfer by adenoviral vectors

In pre-immunized pigs (by intravenous injection of control adenovirus causing elevation of anti-adenoviral neutralizing antibody titer similar to that found in the majority of coronary artery bypass graft patients), co-injection of Ad5Luc and Ad5NOS-III via the great cardiac vein (retrograde) resulted in > 200-fold higher luciferase expression than after retrograde injection of Ad5Luc alone.138 Ad5NOS-III co-injection also reduced Ad5Luc injection-induced T-cell-mediated inflammation and cardiac myocyte apoptosis.138 These results suggested that intracardiac NOS-III gene transfer may reduce some of the known barriers to adenovirus-mediated myocardial gene transfer. Another limitation of effective myocardial gene transfer by adenoviral vectors is poor penetration of the microvascular (endothelial) barrier. It has been reported that increasing vascular permeability by VEGF pre-administration significantly augments adenoviral transfection efficiency in the isolated perfused rabbit heart, which can be inhibited by L-NAME and mimicked by the NO donor nitroglycerin.139