title: In vivo electroporation of constitutively expressed HIF-1α plasmid DNA improves neovascularization in a mouse model of limb ischemia.
abstract: 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 μm(2) vs HIF-IM: 7788.87 ± 392.04 μm(2); P < .001 vs pVAX-EP: 4640.25 ± 614.01 μm(2); 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.
authors.affiliation: Department of Medicine, Cardiovascular Division, Vascular Medicine Section, Perelman School of Medicine at the University of Pennsylvania and University of Pennsylvania Health System, Philadelphia, Pa. Electronic address: geoffrey.ouma@uphs.upenn.edu.
authors.forename: Geoffrey O
authors.initials: GO
authors.lastname: Ouma
authors.affiliation: Department of Surgery, Perelman School of Medicine at the University of Pennsylvania and University of Pennsylvania Health System, Philadelphia, Pa.
authors.forename: Eduardo
authors.initials: E
authors.lastname: Rodriguez
authors.affiliation: Department of Pathology and Laboratory Medicine, Perelman School of Medicine at the University of Pennsylvania and University of Pennsylvania Health System, Philadelphia, Pa.
authors.forename: Karuppiah
authors.initials: K
authors.lastname: Muthumani
authors.affiliation: Department of Pathology and Laboratory Medicine, Perelman School of Medicine at the University of Pennsylvania and University of Pennsylvania Health System, Philadelphia, Pa.
authors.forename: David B
authors.initials: DB
authors.lastname: Weiner
authors.affiliation: Cardiovascular Division, Hospitial of the University of Pennsylvania and Cardiovascular Institute, Perelman School of Medicine at the University of Pennsylvania and University of Pennsylvania Health System, Philadelphia, Pa.
authors.forename: Robert L
authors.initials: RL
authors.lastname: Wilensky
authors.affiliation: Department of Medicine, Cardiovascular Division, Vascular Medicine Section, Perelman School of Medicine at the University of Pennsylvania and University of Pennsylvania Health System, Philadelphia, Pa.
authors.forename: Emile R
authors.initials: ER
authors.lastname: Mohler
mesh_terms: D000818:Animals; D001783:Blood Flow Velocity; D003097:Collateral Circulation; D004195:Disease Models, Animal; D018274:Electroporation; D018014:Gene Transfer Techniques; D015316:Genetic Therapy / Q000379:methods*; D006614:Hindlimb; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000096:biosynthesis* / Q000235:genetics; D007273:Injections, Intramuscular; D007511:Ischemia / Q000235:genetics / Q000378:metabolism / Q000473:pathology / Q000503:physiopathology / Q000628:therapy*; D017078:Laser-Doppler Flowmetry; D051379:Mice; D008807:Mice, Inbred BALB C; D018482:Muscle, Skeletal / Q000098:blood supply* / Q000378:metabolism / Q000473:pathology; D009336:Necrosis; D018919:Neovascularization, Physiologic; D020127:Recovery of Function; D012039:Regional Blood Flow; D013997:Time Factors
chemical_list: C497443:Hif1a protein, mouse; D051795:Hypoxia-Inducible Factor 1, alpha Subunit