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
; 124(11 Suppl): S46-54, 2011 Sep 13.
Article
em En
| MEDLINE
| ID: mdl-21911818
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 ANDRESULTS:
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.
Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
Terapia Genética
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Pró-Colágeno-Prolina Dioxigenase
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Neovascularização Fisiológica
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Células-Tronco Embrionárias
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Técnicas de Silenciamento de Genes
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Oxigenases de Função Mista
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Infarto do Miocárdio
Tipo de estudo:
Prognostic_studies
Limite:
Animals
Idioma:
En
Ano de publicação:
2011
Tipo de documento:
Article