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(Hypertension. 1999;33:354-359.)
© 1999 American Heart Association, Inc.

Scientific Contributions

Antisense Inhibition of AT₁ Receptor in Vascular Smooth Muscle Cells Using Adeno-Associated Virus-Based Vector

Dagmara Mohuczy; Craig H. Gelband; M. Ian Phillips

From the Department of Physiology, College of Medicine, University of Florida, Gainesville, Fla.

Correspondence to Dr M. Ian Philips, Department of Physiology, College of Medicine, University of Florida, Box 100274, Gainesville, FL 32610. E-mail IP{at}phys.med.ufl.edu

Abstract—Vascular smooth muscle cells (VSMCs) are the main peripheral target for vasoconstriction and growth-promoting activity of angiotensin II (Ang II), acting through angiotensin type 1 receptors (AT₁-R). Current antihypertension treatments include daily reductions in the effects of Ang II. To decrease an effect of Ang II in a prolonged fashion, we have developed an adeno-associated virus (AAV) vector with antisense DNA for AT₁-R. AAV has many advantages over other viral vectors. AAV is nonpathogenic, does not stimulate inflammation or immune reaction and enters nondividing cells, and provides stable long-term gene expression. To test AAV in VSMCs, we constructed and tested plasmid AAV (pAAV) and recombinant AAV (rAAV) with AT₁-R antisense DNA. rAAV was constructed with a cassette containing a cytomegalovirus promoter and the cDNA for the AT₁-R inserted in the antisense direction. The cassette was packaged into the virion. Transfection of VSMCs with the pAAV antisense to AT₁-R produced a significant reduction in the amount of AT₁-R (P<0.01). Transduction of VSMCs with the rAAV-AT₁-R-AS at MOI of 5 also showed significant reduction of AT₁-R and long-lasting expression of the transgene for at least 8 weeks. The reduction of AT₁-R number in VSMCs was concomitant with a decrease in the Ang II–stimulated increase of intracellular calcium. The results show that AAV vector delivers AT₁-R antisense to inhibit AT₁-R in VSMCs. For the purpose of gene therapy for hypertension, it is necessary to demonstrate the effectiveness of a vector system in VSMCs. This study provides support for the potential use of AAV AT₁-R antisense in VSMCs.

Key Words: recombinant adeno-associated virus vector • angiotensin • receptors, angiotensin • antisense • muscle, smooth, vascular • protein, green fluorescent • calcium

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