[Overexpression of hypoxia inducible factor-1alpha (HIF-1alpha) promotes the differentiation of endothelial progenitor cell ex vivo].

To investigate the influence of HIF-1alpha overexpression on the differentiation of endothelial progenitor cells (EPCs) ex vivo, EPCs were isolated from human peripheral blood by density gradient centrifugation, overexpressed HIF-1alpha was transfected to EPCs by electroporation; HIF1alpha, HIF1beta, vascular endothelial growth factor (VEGF) mRNA level were measured with RT-PCR; HIF-1alpha protein was detected with immunohistochemistry in a time course. CD31(+) cells were measured with flow cytometry. Cell morphology was observed after transfection. The results showed that the transfection efficiency of HIF-1alpha to EPCs was about 20%. HIF-1alpha and its controlled target gene VEGF were markedly induced by HIF-1alpha vector (P < 0.05). HIF1beta had its same level as it before interference (P > 0.05). HIF-1alpha protein was induced by HIF-1alpha transfection after 12 hours but was undetectable at 24 hours. After 7 - 14 days cultured in 21% oxygen pressure, fluorescence-trace experiments revealed that CD31 + EPCs/EC could be generated more efficiently from overexpressed HIF-1alpha than that from pEGFP transfected group (P > 0.05). EPC morphology was observed by light microscopy. HIF-1alpha-transfected cells under normoxia sprouted more rapidly from the EPC colonies than the untransfected cells or cells transfected with an GFP vector, which essentially maintained the original colony formation. HIF-1alpha transfected cells took on an array-like arrangement rather than random dispersal, suggesting that they were in an advanced state of differentiation. It is concluded that the utility of overexpression of HIF-1alpha can induce target genes which have influence on cell differentiation. HIF-1alpha transfection was found to give a prospected way to do the insight research on ischemic treatment in vivo.

[Effects of TIMP-2 gene transfer on atherosclerotic plaque in rabbits].

OBJECTIVE: To evaluate the effects of TIMP-2 local gene transfer on atherosclerotic plaque. METHODS: Atherosclerosis models were induced by denuding femoral artery endothelium plus high lipid diet in rabbits. TIMP-2 gene was transferred locally by balloons eluted with pcDNA3-TIMP-2. RT-PCR and Western blot were performed to verify exogenous genes transfer. MMPs activity in atherosclerotic plaque was evaluated by zymography. HE and VG staining and automatic image analysis system were used for pathological analysis of atherosclerotic femoral arteries. The lumen area of the vessel and the collagen contents in the atherosclerotic plaque were measured. RESULTS: The expression of TIMP-2 gene in pcDNA3-TIMP-2 transferred group was significantly higher than control-vector transferred group at the end of week 2 after operation and reached the peak at the end of week 4. Comparing with the control group, the expression of TIMP-2 protein in treated group was also higher at the end of week 2, 4, and 8 after operation. Correspondingly, the MMP-2 and MMP-9 activities were lower in treated group. The thickness of fibrous cap of atherosclerotic plaque and the amount of collagen of the lesion were increased significantly in treated group compared with the control group, but there were no significant differences in vessel lumen area. CONCLUSION: TIMP-2 gene transfer locally in atherosclerotic plaque could inhibit the activities of MMP-2 and MMP-9 in the lesion, increase the thickness of fibrous cap and the amount of collagen of the lesion, but may have no effect on the degree of the stenosis.

[Inhibitory effect of siRNA targeting HIF-1alpha on differentiation of peripheral blood endothelial progenitor cells].

BACKGROUND & 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.