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Novel in vitro gene transfer method for study of local modulators in vascular smooth muscle cells
R Morishita1, G H Gibbons, Y Kaneda
1Division of Cardiovascular Medicine, Falk Cardiovascular Research Center, Stanford University School of Medicine, Calif. 94305-5246.
Abstract:
Although many in vitro gene transfer methods already exist, such as calcium phosphate precipitation, electroporation, or cationic liposomes, these methods cause significant cell injury and cell death. The study of the biology of endogenous autocrine-paracrine vasoactive systems such as the renin-angiotensin system in vascular cells is limited by the lack of a suitable gene transfer method with high efficiency of transfection and expression that will permit cell biology studies. Recently, the Sendai virus (hemagglutinating virus of Japan, HVJ)-liposome-mediated gene transfer method has been shown to be an efficient and nontoxic method of gene transfer. In this study, we characterized the efficiency and suitability of the HVJ method for vascular biology research. Using SV40 T-antigen complementary DNA (cDNA), we initially compared the efficiency of the HVJ method and lipofection for transfection of cultured vascular smooth muscle cells (VSMCs). We observed that after 35 minutes of incubation, the HVJ method exhibited a 10-fold higher efficiency of transfection than lipofection. We used this method to study vascular angiotensin converting enzyme (ACE) expression in cultured VSMCs and cultured rat carotid arteries in vitro. The HVJ method of transfection of human ACE cDNA into VSMCs and COS cells was significantly more efficient than lipofection. Using this method, we demonstrated that transfection of ACE cDNA resulted in increased DNA synthesis, which was inhibited by the specific angiotensin II receptor antagonist DuP 753 (10(-6) M).(ABSTRACT TRUNCATED AT 250 WORDS)
Insights
Sendai virus (HVJ)-liposome gene transfer is a highly efficient and non-toxic method for vascular biology research, significantly outperforming lipofection in vascular smooth muscle cells.
Area of Science:
- Vascular Biology
- Gene Transfer Technology
- Cell Biology
Background:
- Existing in vitro gene transfer methods often cause significant cell damage.
- Studying endogenous vasoactive systems in vascular cells requires efficient and non-toxic gene transfer.
Purpose of the Study:
- To characterize the efficiency and suitability of Sendai virus (HVJ)-liposome-mediated gene transfer for vascular biology research.
- To compare HVJ-liposome method with lipofection for gene transfer in vascular cells.
Main Methods:
- Comparison of HVJ-liposome and lipofection for transfection of vascular smooth muscle cells (VSMCs) using SV40 T-antigen cDNA.
- In vitro study of vascular angiotensin converting enzyme (ACE) expression in VSMCs and rat carotid arteries using HVJ-liposome mediated gene transfer.
- Assessment of DNA synthesis following ACE cDNA transfection and inhibition by angiotensin II receptor antagonist DuP 753.
Main Results:
- HVJ-liposome method showed 10-fold higher transfection efficiency than lipofection in VSMCs after 35 minutes.
- HVJ-mediated transfection of human ACE cDNA was significantly more efficient than lipofection in VSMCs and COS cells.
- Transfection of ACE cDNA led to increased DNA synthesis, which was inhibited by DuP 753.
Conclusions:
- The HVJ-liposome method is a highly efficient and non-toxic gene transfer technique suitable for vascular biology studies.
- This method enables detailed investigation of gene expression and cellular responses in vascular cells.
- HVJ-liposome mediated gene transfer facilitates the study of pathways like the renin-angiotensin system in vascular cells.