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

Circulation. 2011 Sep 13;124(11 Suppl):S46-54. doi: 10.1161/CIRCULATIONAHA.110.014019.

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.

Methods and 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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Apoptosis / physiology
  • Embryonic Stem Cells / cytology
  • Embryonic Stem Cells / physiology
  • Embryonic Stem Cells / transplantation*
  • Female
  • Gene Knockdown Techniques*
  • Genetic Therapy / methods*
  • Hypoxia-Inducible Factor 1, alpha Subunit / metabolism
  • Mice
  • Mice, Inbred Strains
  • Mixed Function Oxygenases / genetics*
  • Mixed Function Oxygenases / metabolism
  • Models, Animal
  • Myoblasts, Cardiac / metabolism
  • Myoblasts, Cardiac / pathology
  • Myocardial Infarction / metabolism
  • Myocardial Infarction / pathology
  • Myocardial Infarction / therapy*
  • Myocardium / metabolism
  • Myocardium / pathology
  • Myocytes, Cardiac / metabolism
  • Myocytes, Cardiac / pathology
  • Neovascularization, Physiologic / physiology*
  • Procollagen-Proline Dioxygenase / genetics*
  • Procollagen-Proline Dioxygenase / metabolism
  • Stem Cell Transplantation
  • Treatment Outcome

Substances

  • Hif1a protein, mouse
  • Hypoxia-Inducible Factor 1, alpha Subunit
  • Mixed Function Oxygenases
  • factor inhibiting hypoxia-inducible factor 1, mouse
  • Procollagen-Proline Dioxygenase