title: Double knockdown of prolyl hydroxylase and factor-inhibiting hypoxia-inducible factor with nonviral minicircle gene therapy enhances stem cell mobilization and angiogenesis after myocardial infarction.
abstract: BACKGROUND
  Under normoxic conditions, hypoxia-inducible factor (HIF)-1α is rapidly degraded by 2 hydroxylases: prolyl hydroxylase (PHD) and factor-inhibiting HIF-1 (FIH). Because HIF-1α mediates the cardioprotective response to ischemic injury, its upregulation may be an effective therapeutic option for ischemic heart failure.
  
  
  RESULTS
  PHD and FIH were cloned from mouse embryonic stem cells. The best candidate short hairpin (sh) sequences for inhibiting PHD isoenzyme 2 and FIH were inserted into novel, nonviral, minicircle vectors. In vitro studies after cell transfection of mouse C2C12 myoblasts, HL-1 atrial myocytes, and c-kit(+) cardiac progenitor cells demonstrated higher expression of angiogenesis factors in the double-knockdown group compared with the single-knockdown and short hairpin scramble control groups. To confirm in vitro data, shRNA minicircle vectors were injected intramyocardially after left anterior descending coronary artery ligation in adult FVB mice (n=60). Functional studies using MRI, echocardiography, and pressure-volume loops showed greater improvement in cardiac function in the double-knockdown group. To assess mechanisms of this functional recovery, we performed a cell trafficking experiment, which demonstrated significantly greater recruitment of bone marrow cells to the ischemic myocardium in the double-knockdown group. Fluorescence-activated cell sorting showed significantly higher activation of endogenous c-kit(+) cardiac progenitor cells. Immunostaining showed increased neovascularization and decreased apoptosis in areas of injured myocardium. Finally, western blots and laser-capture microdissection analysis confirmed upregulation of HIF-1α protein and angiogenesis genes, respectively.
  
  
  CONCLUSIONS
  We demonstrated that HIF-1α upregulation by double knockdown of PHD and FIH synergistically increases stem cell mobilization and myocardial angiogenesis, leading to improved cardiac function.
authors.affiliation: Department of Medicine, Division of Cardiovascular Medicine, Stanford University School of Medicine, Stanford, CA 94305-5454, USA.
authors.forename: Mei
authors.initials: M
authors.lastname: Huang
authors.forename: Patricia
authors.initials: P
authors.lastname: Nguyen
authors.forename: Fangjun
authors.initials: F
authors.lastname: Jia
authors.forename: Shijun
authors.initials: S
authors.lastname: Hu
authors.forename: Yongquan
authors.initials: Y
authors.lastname: Gong
authors.forename: Patricia E
authors.initials: PE
authors.lastname: de Almeida
authors.forename: Li
authors.initials: L
authors.lastname: Wang
authors.forename: Divya
authors.initials: D
authors.lastname: Nag
authors.forename: Mark A
authors.initials: MA
authors.lastname: Kay
authors.forename: Amato J
authors.initials: AJ
authors.lastname: Giaccia
authors.forename: Robert C
authors.initials: RC
authors.lastname: Robbins
authors.forename: Joseph C
authors.initials: JC
authors.lastname: Wu
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000502:physiology; D053595:Embryonic Stem Cells / Q000166:cytology / Q000502:physiology / Q000637:transplantation*; D005260:Female; D055785:Gene Knockdown Techniques; D015316:Genetic Therapy / Q000379:methods*; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000378:metabolism; D051379:Mice; D008815:Mice, Inbred Strains; D006899:Mixed Function Oxygenases / Q000235:genetics* / Q000378:metabolism; D023421:Models, Animal; D032386:Myoblasts, Cardiac / Q000378:metabolism / Q000473:pathology; D009203:Myocardial Infarction / Q000378:metabolism / Q000473:pathology / Q000628:therapy*; D009206:Myocardium / Q000378:metabolism / Q000473:pathology; D032383:Myocytes, Cardiac / Q000378:metabolism / Q000473:pathology; D018919:Neovascularization, Physiologic / Q000502:physiology*; D011393:Procollagen-Proline Dioxygenase / Q000235:genetics* / Q000378:metabolism; D033581:Stem Cell Transplantation; D016896:Treatment Outcome
chemical_list: C497443:Hif1a protein, mouse; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D006899:Mixed Function Oxygenases; C551240:factor inhibiting hypoxia-inducible factor 1, mouse; D011393:Procollagen-Proline Dioxygenase