Novel in vitro gene transfer method for study of local modulators in vascular smooth muscle cells.
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)
Morishita
R
R
Division of Cardiovascular Medicine, Falk Cardiovascular Research Center, Stanford University School of Medicine, Calif. 94305-5246.
Gibbons
G H
GH
Kaneda
Y
Y
Ogihara
T
T
Dzau
V J
VJ
D000818:Animals; D002339:Carotid Arteries / Q000276:immunology; D020212:Carotid Artery Injuries; D002404:Catheterization; D002478:Cells, Cultured; D004247:DNA / Q000096:biosynthesis / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods; D008081:Liposomes; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000378:metabolism*; D009924:Organ Culture Techniques; D010222:Parainfluenza Virus 1, Human / Q000235:genetics; D007703:Peptidyl-Dipeptidase A / Q000235:genetics / Q000378:metabolism; D051381:Rats; D013194:Staining and Labeling; D014162:Transfection
D008081:Liposomes; D004247:DNA; D007703:Peptidyl-Dipeptidase A