Short hairpin RNA interference therapy for ischemic heart disease.
BACKGROUND
During hypoxia, upregulation of hypoxia inducible factor-1 alpha transcriptional factor can activate several downstream angiogenic genes. However, hypoxia inducible factor-1 alpha is naturally degraded by prolyl hydroxylase-2 (PHD2) protein. Here we hypothesize that short hairpin RNA (shRNA) interference therapy targeting PHD2 can be used for treatment of myocardial ischemia and this process can be followed noninvasively by molecular imaging.


RESULTS
PHD2 was cloned from mouse embryonic stem cells by comparing the homolog gene in human and rat. The best candidate shRNA sequence for inhibiting PHD2 was inserted into the pSuper vector driven by the H1 promoter followed by a separate hypoxia response element-incorporated promoter driving a firefly luciferase reporter gene. This construct was used to transfect mouse C2C12 myoblast cell line for in vitro confirmation. Compared with the control short hairpin scramble (shScramble) as control, inhibition of PHD2 increased levels of hypoxia inducible factor-1 alpha protein and several downstream angiogenic genes by >30% (P<0.01). Afterward, shRNA targeting PHD2 (shPHD2) plasmid was injected intramyocardially following ligation of left anterior descending artery in mice. Animals were randomized into shPHD2 experimental group (n=25) versus shScramble control group (n=20). Bioluminescence imaging detected plasmid-mediated transgene expression for 4 to 5 weeks. Echocardiography showed the shPHD2 group had improved fractional shortening compared with the shScramble group at Week 4 (33.7%+/-1.9% versus 28.4%+/-2.8%; P<0.05). Postmortem analysis showed increased presence of small capillaries and venules in the infarcted zones by CD31 staining. Finally, Western blot analysis of explanted hearts also confirmed that animals treated with shPHD2 had significantly higher levels of hypoxia inducible factor-1 alpha protein.


CONCLUSIONS
This is the first study to image the biological role of shRNA therapy for improving cardiac function. Inhibition of PHD2 by shRNA led to significant improvement in angiogenesis and contractility by in vitro and in vivo experiments. With further validation, the combination of shRNA therapy and molecular imaging can be used to track novel cardiovascular gene therapy applications in the future.
Huang
Mei
M
Stanford University School of Medicine, Edwards Building R354, Stanford, CA 94305-5344, USA.
Chan
Denise A
DA
Jia
Fangjun
F
Xie
Xiaoyan
X
Li
Zongjin
Z
Hoyt
Grant
G
Robbins
Robert C
RC
Chen
Xiaoyuan
X
Giaccia
Amato J
AJ
Wu
Joseph C
JC
D000818:Animals; D015687:Cell Hypoxia; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000235:genetics*; D004452:Echocardiography; D005260:Female; D051793:Hypoxia-Inducible Factor 1 / Q000378:metabolism; D064799:Hypoxia-Inducible Factor-Proline Dioxygenases; D017874:Immediate-Early Proteins / Q000235:genetics*; D008163:Luminescent Measurements; D051379:Mice; D008815:Mice, Inbred Strains; D032446:Myoblasts / Q000166:cytology / Q000378:metabolism; D017202:Myocardial Ischemia / Q000000981:diagnostic imaging / Q000235:genetics / Q000503:physiopathology*; D009206:Myocardium / Q000378:metabolism; D018919:Neovascularization, Physiologic / Q000187:drug effects; D010957:Plasmids / Q000494:pharmacology; D011393:Procollagen-Proline Dioxygenase; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000494:pharmacology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013318:Stroke Volume / Q000187:drug effects; D014162:Transfection; D015854:Up-Regulation; D016277:Ventricular Function, Left
D004268:DNA-Binding Proteins; D051793:Hypoxia-Inducible Factor 1; D017874:Immediate-Early Proteins; D034741:RNA, Small Interfering; D011393:Procollagen-Proline Dioxygenase; C490189:Egln1 protein, mouse; D064799:Hypoxia-Inducible Factor-Proline Dioxygenases