title: Deletion of prolyl hydroxylase domain proteins (PHD1, PHD3) stabilizes hypoxia inducible factor-1 alpha, promotes neovascularization, and improves perfusion in a murine model of hind-limb ischemia.
abstract: BACKGROUND
  There is an emerging focus on investigating innovative therapeutic molecules that can potentially augment neovascularization in order to treat peripheral arterial disease (PAD). Although prolyl hydroxylase domain proteins 1 and 3 (PHD1 and PHD3) may modulate angiogenesis via regulation of hypoxia inducible factor-1α (HIF-1α), there has been no study directly addressing their roles in ischemia-induced vascular growth. We hypothesize that PHD1(-/-) or PHD3(-/-) deficiency might promote angiogenesis in the murine hind-limb ischemia (HLI) model.
  
  
  METHODS
  Wild type (WT), PHD1(-/-) and PHD3(-/-) male mice aged 8-12weeks underwent right femoral artery ligation. Post-procedurally, motor function assessment and laser Doppler imaging were periodically performed. The mice were euthanized after 28days and muscles were harvested. Immunohistochemical analysis was performed to determine the extent of angiogenesis by measuring capillary and arteriolar density. VEGF expression was quantified by enzyme-linked immunosorbent assay (ELISA). Bcl-2 and HIF-1α were analyzed by immunofluorescence. Fibrosis was measured by picrosirius red staining.
  
  
  RESULTS
  PHD1(-/-) and PHD3(-/-) mice showed significantly improved recovery of perfusion and motor function score when compared to WT after femoral artery ligation. These mice also exhibited increased capillary and arteriolar density, capillary/myocyte ratio along with decreased fibrosis compared to WT. VEGF, Bcl-2 and HIF-1α expression increased in PHD1(-/-) and PHD3(-/-) mice compared to WT.
  
  
  CONCLUSIONS
  Taken together these results suggest that PHD1 and PHD3 deletions promote angiogenesis in ischemia-injured tissue, and may present a promising therapeutic strategy in treating PAD.
authors.affiliation: Molecular Cardiology and Angiogenesis Laboratory, Department of Surgery, University of Connecticut Health Center, Farmington, CT, USA; Stanley J. Dudrick Department of Surgery, Saint Mary's Hospital, Waterbury, CT, USA.
authors.forename: Muhammad T
authors.initials: MT
authors.lastname: Rishi
authors.affiliation: Molecular Cardiology and Angiogenesis Laboratory, Department of Surgery, University of Connecticut Health Center, Farmington, CT, USA.
authors.forename: Vaithinathan
authors.initials: V
authors.lastname: Selvaraju
authors.affiliation: Molecular Cardiology and Angiogenesis Laboratory, Department of Surgery, University of Connecticut Health Center, Farmington, CT, USA.
authors.forename: Mahesh
authors.initials: M
authors.lastname: Thirunavukkarasu
authors.affiliation: Molecular Cardiology and Angiogenesis Laboratory, Department of Surgery, University of Connecticut Health Center, Farmington, CT, USA; Stanley J. Dudrick Department of Surgery, Saint Mary's Hospital, Waterbury, CT, USA.
authors.forename: Inam A
authors.initials: IA
authors.lastname: Shaikh
authors.affiliation: Department of Cell Biology, University of Connecticut Health Center, Farmington, CT, USA.
authors.forename: Kotaro
authors.initials: K
authors.lastname: Takeda
authors.affiliation: Department of Cell Biology, University of Connecticut Health Center, Farmington, CT, USA.
authors.forename: Guo-Hua
authors.initials: GH
authors.lastname: Fong
authors.affiliation: Stanley J. Dudrick Department of Surgery, Saint Mary's Hospital, Waterbury, CT, USA.
authors.forename: J Alexander
authors.initials: JA
authors.lastname: Palesty
authors.affiliation: Molecular Cardiology and Angiogenesis Laboratory, Department of Surgery, University of Connecticut Health Center, Farmington, CT, USA.
authors.forename: Juan A
authors.initials: JA
authors.lastname: Sanchez
authors.affiliation: Molecular Cardiology and Angiogenesis Laboratory, Department of Surgery, University of Connecticut Health Center, Farmington, CT, USA. Electronic address: nmaulik@uchc.edu.
authors.forename: Nilanjana
authors.initials: N
authors.lastname: Maulik
mesh_terms: D000818:Animals; D004195:Disease Models, Animal; D005355:Fibrosis; D017353:Gene Deletion; D006614:Hindlimb; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000378:metabolism*; D007511:Ischemia / Q000235:genetics / Q000378:metabolism* / Q000473:pathology / Q000503:physiopathology; D008297:Male; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D009043:Motor Activity; D018482:Muscle, Skeletal / Q000098:blood supply* / Q000378:metabolism* / Q000473:pathology; D018919:Neovascularization, Physiologic; D011393:Procollagen-Proline Dioxygenase / Q000172:deficiency* / Q000235:genetics; D055550:Protein Stability; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D020127:Recovery of Function; D012039:Regional Blood Flow; D013997:Time Factors; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism
chemical_list: C497443:Hif1a protein, mouse; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D019253:Proto-Oncogene Proteins c-bcl-2; D042461:Vascular Endothelial Growth Factor A; C467480:vascular endothelial growth factor A, mouse; C052617:Bcl2 protein, mouse; C522721:PHD1 protein, mouse; C522722:PHD3 protein, mouse; D011393:Procollagen-Proline Dioxygenase
keywords: Bcl-2; HIF; Hindlimb ischemia; Prolyl hydroxylase; VEGF