title: Overexpression of endothelial nitric oxide synthase increases skeletal muscle blood flow and oxygenation in severe rat hind limb ischemia.
abstract: OBJECTIVE
  Although nitric oxide (NO) has a critical role in angiogenesis, the therapeutic potential of NO synthase overexpression in severe ischemia remains undefined. We tested the hypothesis that overexpression of endothelial NO synthase (eNOS) would improve tissue perfusion in severe hind limb ischemia.
  
  
  METHODS
  Severe hind limb ischemia was induced in 122 adult male Sprague-Dawley rats. Ten days after the induction of hind limb ischemia, vascular isolation and intraarterial delivery of an adenoviral vector encoding eNOS (AdeNOS), a control adenoviral vector (AdE1), or phosphate-buffered saline solution (PBS) was performed. Skeletal muscle blood flow, muscle oxygen tension, angiography, and immunohistochemistry for capillary counts were measured.
  
  
  RESULTS
  Gene transfer of AdeNOS increased eNOS protein expression and enzyme activity. Two weeks after gene transfer, skeletal muscle blood flow was fourfold higher in eNOS-transduced than in AdE1-transduced or PBS treated rats and was similar to exercise-induced maximal flow in nonischemic muscle. eNOS overexpression increased muscle oxygen tension in a titer-dependent fashion. This increase persisted 1 month after transduction, even though eNOS enzyme activity had declined to normal levels. Angiography and capillary counts showed that eNOS overexpression increased the size and number of collateral arteries, but did not significantly increase the capillary-muscle fiber ratio.
  
  
  CONCLUSIONS
  eNOS overexpression in an ischemic rat hind limb significantly increased skeletal muscle blood flow, muscle oxygen tension, and collateral arteries (arteriogenesis). Furthermore, eNOS overexpression did not result in capillary angiogenesis above control levels. These studies demonstrate the potential for eNOS overexpression as treatment for severe limb ischemia in human beings.
authors.affiliation: Division of Vascular Surgery, Pacific Vascular Research Laboratory, University of California-San Francisco, 505 Parnassus Avenue, San Francisco, CA 94143, USA.
authors.forename: Lucy S
authors.initials: LS
authors.lastname: Brevetti
authors.forename: David S
authors.initials: DS
authors.lastname: Chang
authors.forename: Gale L
authors.initials: GL
authors.lastname: Tang
authors.forename: Rajabrata
authors.initials: R
authors.lastname: Sarkar
authors.forename: Louis M
authors.initials: LM
authors.lastname: Messina
mesh_terms: D000256:Adenoviridae; D000792:Angiography; D000818:Animals; D001783:Blood Flow Velocity; D002196:Capillaries / Q000473:pathology; D002455:Cell Division; D005822:Genetic Vectors; D006614:Hindlimb / Q000098:blood supply / Q000000981:diagnostic imaging; D007511:Ischemia / Q000000981:diagnostic imaging / Q000378:metabolism / Q000503:physiopathology*; D008297:Male; D018482:Muscle, Skeletal / Q000098:blood supply* / Q000378:metabolism / Q000473:pathology; D009389:Neovascularization, Pathologic / Q000503:physiopathology; D019001:Nitric Oxide Synthase / Q000235:genetics / Q000502:physiology*; D052250:Nitric Oxide Synthase Type III; D010100:Oxygen / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D012039:Regional Blood Flow; D014161:Transduction, Genetic
chemical_list: D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III; C496316:Nos3 protein, rat; D010100:Oxygen