In vivo electroporation of constitutively expressed HIF-1α plasmid DNA improves neovascularization in a mouse model of limb ischemia

Objective Hypoxia-inducible factor-1 alpha (HIF-1α) is a transcription factor that stimulates angiogenesis during tissue ischemia. In vivo electroporation (EP) enhances tissue DNA transfection. We hypothesized that in vivo EP of plasmid DNA encoding a constitutively expressed HIF-1α gene enhances neovascularization compared with intramuscular (IM) injection alone. Methods Left femoral artery ligation was performed in mice assigned to three groups: (1) HIF-EP (n = 13); (2) HIF-IM (n = 14); and (3) empty plasmid (pVAX)-EP (n = 12). A single dose of HIF-1α or pVAX DNA (20 μL of 5 μg/μL each) was injected into the ischemic adductor muscle followed by EP (groups one and three). Mice in group two received IM injection of HIF-1α plasmid DNA alone. From preligation to days 0, 3, 7, 14, and 21 postligation, limb perfusion recovery quantified by laser Doppler perfusion imager, limb function, and limb necrosis were measured. On day 21, the surviving mice (4-5 per group) were sacrificed and adductor muscle tissues stained for necrosis using hematoxylin and eosin, capillary density (anti-CD31 antibodies), and collateral vessels via anti-α-smooth muscle actin antibodies. Results In vivo EP of HIF-1α DNA significantly improved limb perfusion (HIF-EP: 1.03 ± 0.15 vs HIF-IM: 0.78 ± 0.064; P < .05, vs pVAX-EP: 0.41 ± 0.019; P < .001), limb functional recovery (HIF-EP: 3.5 ± 0.58 vs HIF-IM, 2.4 ± 1.14; P < .05, vs pVAX-EP: 2.4 ± 1.14; P < .001), and limb autoamputation on day 21 (HIF-EP: 77% ± 12% vs HIF-IM: 43% ± 14%; P < .05 vs pVAX-EP: 17% ± 11%; P < .01). Adductor muscle tissue necrosis decreased (HIF-EP: 20.7% ± 1.75% vs HIF-IM: 44% ± 3.73; P < .001, vs pVAX-EP: 60.05% ± 2.17%; P < .0001), capillary density increased (HIF-EP: 96.83 ± 5.72 vessels/high-powered field [hpf] vs HIF-IM: 62.87 ± 2.0 vessels/hpf; P < .001, vs pVAX-EP: 39.37 ± 2.76 vessels/hpf; P < .0001), collateral vessel formation increased (HI-EP: 76.33 ± 1.94 vessels/hpf vs HIF-IM: 37.5 ± 1.56 vessels/hpf; P < .0001, vs pVAX-EP: 18.5 ± 1.34 vessels/hpf; P < .00001), and the vessels were larger (HIF-EP: 15,521.67 ± 1298.16 μm2 vs HIF-IM: 7788.87 ± 392.04 μm2 ; P < .001 vs pVAX-EP: 4640.25 ± 614.01 μm2 ; P < .0001). Conclusions In vivo EP-mediated delivery of HIF-1α plasmid DNA improves neovascularization in a mouse model of limb ischemia and is a potentially suitable nonviral, noninvasive intervention to facilitate therapeutic angiogenesis in critical limb ischemia.