[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.
Jiang
Meng
M
Cardiology Department, Affiliated Renji Hospital, Medical College, Shanghai Jiaotong University, Shanghai, 200001, PR China.
Wang
Chang-Qian
CQ
Wang
Bin-Yao
BY
Huang
Ding-Jiu
DJ
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