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

Mei Huang, Patricia Nguyen, Fangjun Jia, Shijun Hu, Yongquan Gong, Patricia E. De Almeida, Li Wang, Divya Nag, Mark A. Kay, Amato J. Giaccia, Robert C. Robbins, Joseph C. Wu

Research output: Contribution to journalArticlepeer-review

61 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)S46-S54
JournalCirculation
Volume124
Issue number11 SUPPL. 1
DOIs
StatePublished - Sep 13 2011
Externally publishedYes

Keywords

  • DNA minicircles
  • hypoxia-inducible factor 1
  • laser capture microdissection
  • myocardial ischemia
  • prolyl hydroxylase
  • RNA interference
  • stem cells

ASJC Scopus subject areas

  • Cardiology and Cardiovascular Medicine
  • Physiology (medical)

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