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

被引:70
作者
Huang, Mei [1 ,2 ]
Nguyen, Patricia [1 ]
Jia, Fangjun [2 ]
Hu, Shijun [2 ]
Gong, Yongquan [1 ,3 ]
de Almeida, Patricia E. [1 ,2 ]
Wang, Li [1 ,2 ]
Nag, Divya [1 ]
Kay, Mark A. [4 ]
Giaccia, Amato J. [5 ]
Robbins, Robert C. [3 ,6 ]
Wu, Joseph C. [1 ,2 ,6 ]
机构
[1] Stanford Univ, Sch Med, Dept Med, Div Cardiovasc Med, Stanford, CA 94305 USA
[2] Stanford Univ, Sch Med, Dept Radiol, Mol Imaging Program, Stanford, CA 94305 USA
[3] Stanford Univ, Sch Med, Dept Cardiothorac Surg, Stanford, CA 94305 USA
[4] Stanford Univ, Sch Med, Dept Pediat, Stanford, CA 94305 USA
[5] Stanford Univ, Sch Med, Dept Radiat Oncol, Stanford, CA 94305 USA
[6] Stanford Univ, Sch Med, Cardiovasc Inst, Stanford, CA 94305 USA
关键词
prolyl hydroxylase; hypoxia-inducible factor 1; RNA interference; DNA minicircles; myocardial ischemia; stem cells; laser capture microdissection; ENDOTHELIAL PROGENITOR CELLS; HIF-1-ALPHA/VP16; HYBRID; TRANSCRIPTION FACTOR; ISCHEMIC-HEART; IN-VIVO; EXPRESSION; GAMMA; HYPOXIA-INDUCIBLE-FACTOR-1-ALPHA; NEOVASCULARIZATION; FACTOR-1-ALPHA;
D O I
10.1161/CIRCULATIONAHA.110.014019
中图分类号
R5 [内科学];
学科分类号
1002 ; 100201 ;
摘要
Background-Under normoxic conditions, hypoxia-inducible factor (HIF)-1 alpha is rapidly degraded by 2 hydroxylases: prolyl hydroxylase (PHD) and factor-inhibiting HIF-1 (FIH). Because HIF-1 alpha 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-1 alpha protein and angiogenesis genes, respectively. Conclusions-We demonstrated that HIF-1 alpha upregulation by double knockdown of PHD and FIH synergistically increases stem cell mobilization and myocardial angiogenesis, leading to improved cardiac function. (Circulation. 2011; 124[suppl 1]:S46-S54.)
引用
收藏
页码:S46 / S54
页数:9
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