The targeting expression of the vascular endothelial growth factor gene in endothelial cells regulated by HRE.ppET-1

Abstract

The success of gene therapy depends largely on the efficacy of gene delivery vector systems that can deliver genes to target organs or cells selectively and efficiently with minimal toxicity. Here, we show that by using the HRE.ppET-1 regulatory element, we were able to restrict expression of the transgene of vascular endothelial growth factor (VEGF) to endothelial cells exclusively in hypoxic conditions. Eukaryotic expression vectors such as pEGFP-HRE.ppET-1, pcDNA3.1-VEGF+Pa, pcDNA3.1-ppET-1+ EGF+Pa, and pcDNA3.1-HRE.ppET-1+VEGF+Pa were constructed by using a series of nuclear molecule handling methods like PCR, enzyme digestion. The recombinant vectors were transfected into HUVEC cells and HL7702 cells by the lipofectin method. GFP expression was observed with a fluorescence microscope to validate the specificity of expression in endothelial cells under the regulation of HRE.ppET-1 element. Cobalt chloride (final concentration 100 mumol/L) was added to the medium to mimic hypoxia in vitro. After transfection of vectors, the expression of VEGF mRNA was detected by RT-PCR, and the expression of VEGF was detected by Western blotting and ELISA methods under normoxia and hypoxia, respectively. The cell proliferation rate was detected by the MTT test. The expression of GFP revealed that the exterior gene was transcripted effectively in endothelial cells regulated by the HRE.ppET-1 element, while the expression of GFP was very weak in nonendothelial cells. The results of RT-PCR, Western blotting and ELISA showed that VEGF gene expression in the pcDNA3.1-HRE.ppET-1+VEGF+Pa group and in the pcDNA3.1-ppET-1+VEGF+Pa group was higher in hypoxia than it was in normoxia (P<0.05). The MTT test showed that the proliferation rate of HUVEC transfected with HPVA under hypoxia exceeded that of the control group. We conclude that the HRE.ppET-1 element was expressed specifically in endothelial cells, and can increase the expression of VEGF in hypoxia and stimulate proliferation of endothelial cells. Taking advantage of these facts could greatly improve the efficiency of gene therapy. The vector would be valuable for various gene transfer studies targeting endothelial cells.

Hypoxia effects on proangiogenic factors in human umbilical vein endothelial cells: functional role of the peptide somatostatin

“…1a, the predicted length of each qPCR product was confirmed by agarose gel electrophoresis (148, 106, 92 and 60 bp which correspond to the predicted length of VEGF, VEGFR-1, VEGFR-2 and GAPDH mRNA, respectively). In agreement with previous results demonstrating that hypoxia is a potent stimulus for VEGF upregulation in HUVEC (Avouac et al 2008;Cho et al 2008;Liu et al 2009;Nilsson et al 2004;Takata et al 2008;Zheng et al 2008), we found that hypoxia caused a drastic increase in the level of the VEGF mRNA (∼163%, **P < 0.01; Fig. 1b) while we also determined a significant decrease in the expression of both VEGFR-1 (∼58%, **P<0.01; Fig.…”

“…Our study shows that hypoxia is a potent stimulus for VEGF expression and production, confirming previous findings in HUVEC (Avouac et al 2008;Cho et al 2008;Liu et al 2009;Nilsson et al 2004;Takata et al 2008;Zheng et al 2008). Hypoxia has been proposed to play an important role in the regulation of VEGF receptor expression although previous studies have yielded conflicting results.…”

“…VEGF is one of the most potent proangiogenic factors which promotes the proliferation and migration of endothelial cells and increases vascular permeability. In human umbilical vein endothelial cells (HUVEC), which are widely used as an endothelial cell model in vitro, hypoxia increases VEGF expression and secretion (Avouac et al 2008;Cho et al 2008;Liu et al 2009;Nilsson et al 2004;Takata et al 2008;Zheng et al 2008).…”