Novel in vitro gene transfer method for study of local modulators in vascular smooth muscle cells

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 (hemagglu- tinating 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 ‘M). Transfected human ACE cDNA into the rat carotid artery in organ culture yielded ACE expression in the tunica media as detected by immunohistochemistry at 3 days after transfection, whereas there was no positive staining In the control vector-transfected and untreated vessels. The present study demonstrates that the HVJ-mediated method is a very effective and efficient gene transfer method for VSMCs. Furthermore, the successful gene transfer into intact blood vessels in organ culture suggests that the HVJ-mediated gene transfer technique may provide a new and useful tool for in vivo study of endogenous autocrine-paracrine vascular modulators (such as the vascular renin-angiotensin system) and may be a useful delivery method for gene therapy for vascular diseases.(Hypertension 1993;21:894-899) © 1993 American Heart Association, Inc.