[Inhibitory effect of siRNA targeting HIF-1alpha on differentiation of peripheral blood endothelial progenitor cells].
OBJECTIVE
Transcription factor hypoxia inducible factor-1 alpha (HIF-1alpha) is a key determinant of oxygen-dependent gene regulation in angiogenesis. Suppression of HIF-1alpha is important for exploring HIF-1-dependent processes and hypoxia-induced pathophysiologic events. This study applied RNA interference targeting HIF-1alpha to human peripheral blood endothelial progenitor cells (EPCs) for therapeutic antineovascularization in vitro.


METHODS
Small interference RNA (siRNA) targeting HIF-1alpha was constructed and transfected into human EPCs. Transfection efficiency of siRNA-HIF-1alpha was measured by reverse transcription-polymerase chain reaction (RT-PCR), immunocytochemistry, and flow cytometry. The content of endothelial nitric oxide synthase (eNOS) was measured by ELISA. The morphology of EPCs/endothelial cells (ECs) cultured in normoxia or hypoxia was observed after siRNA-HIF-1alpha transfection.


RESULTS
SiRNA-HIF-1alpha was successfully constructed with the transfection efficiency of about 20%. After transfected for 6 h, HIF-1alpha mRNA was detected in EPCs cultured in normoxia or hypoxia with suppression; the expression of vascular endothelial growth factor (VEGF) was significantly attenuated (P < 0.05); the expression of HIF-1beta had no obvious change (P > 0.05). Stable expression of HIF-1alpha protein was induced by hypoxia, and was inhibited by siRNA-HIF-1alpha (P < 0.05). When cultured in 1% or 21% oxygen pressure for 3 days, CD31+ EPCs were inhibited more efficiently by siRNA-HIF-1alpha than by pEGFP (P < 0.05). The content of eNOS was significantly attenuated by siRNA-HIF-1alpha in time- and VEGF concentration-dependent manners (P < 0.01). EPCs differentiation and proliferation were induced by hypoxic culture, and suppressed by siRNA-HIF-1alpha.


CONCLUSIONS
The constitutive or hypoxia-induced expression of HIF-1alpha in EPCs is sufficient to affect the expression of downstream target genes. SiRNA targeting HIF-1alpha could inhibit the expression of the genes which promote neovascularization, and suppress the differentiation of EPC to EC.
Cardiology Department, Affiliated Renji Hospital, Medical College, Shanghai Jiaotong University, Shanghai, 200001, PR China.
Meng
M
Jiang
Chang-Qian
CQ
Wang
Bin-Yao
BY
Wang
Ding-Jiu
DJ
Huang
D051784:Aryl Hydrocarbon Receptor Nuclear Translocator / Q000378:metabolism; D002454:Cell Differentiation; D015687:Cell Hypoxia; D049109:Cell Proliferation; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000166:cytology*; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000096:biosynthesis / Q000235:genetics*; D009389:Neovascularization, Pathologic / Q000209:etiology; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism; D010957:Plasmids; D034622:RNA Interference; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D034741:RNA, Small Interfering; D013234:Stem Cells / Q000166:cytology* / Q000378:metabolism; D014162:Transfection; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism
D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D042461:Vascular Endothelial Growth Factor A; D051784:Aryl Hydrocarbon Receptor Nuclear Translocator; D052250:Nitric Oxide Synthase Type III