Inhibition of the α1D-adrenergic receptor gene by RNA interference (RNAi) in rat vascular smooth muscle cells and its effects on other adrenergic receptors

Abstract

Sympathetic-induced vasoconstriction is mediated by various adrenergic receptor (AR) subtypes located on membranes of vascular smooth muscle cells (VSMC) located on the arterial wall, but is mostly attributed to activation of the α 1D -AR. In order to study interaction and crosstalk among AR genes, we induced post-transcriptional silencing of the α 1D -AR gene in cultured VSMC using the RNAi technique. A pSEC neo expression plasmid vector containing a small interfering RNA (siRNA) sequence selected to bind to the targeted mRNA of the α 1D -AR gene was transfected into cultured VSMC from rat aorta. The RNA expression of all AR-subtype genes was assessed by Q-RT-PCR and the α 1D and α 2A -AR proteins quantified by Western blot. In siRNA transfected cells, the α 1D -AR protein levels decreased by 55%, 69% and 75% at 24h, 48h and 72h, respectively (p < 0.03-0.01) with progressive increases in its gene expression by 50%-61% and concurrent increase in α 2A -AR protein peaking at 48 h. Decreases were noted in expression of the α 1A, α 2A , and β 3 AR genes. We conclude that post-transcriptional silencing of the α 1D -AR gene leads to significant decrease in receptor protein despite reactive increase in gene expression. However, suppression of one AR leads to reactive changes in other subtypes, indicating that cross-talk among related genes, whose products have overlapping functions, may partly offset anticipated effects in vivo.

New Aspects of Gene-Silencing for the Treatment of Cardiovascular Diseases

“…examined the silencing effect of siRNA targeting the mRNA of α1 D -ARs in rats. The time-dependent decrease of the α1 D -AR protein level raises high interest for further examinations [ 165 ]. Blocking receptors of the renin-angiotensin-aldosterone system (RAAS) could be another method for influencing the blood pressure (BP).…”

Gene-Silencing for Treatment of Cardiovascular Diseases

“…The reduced blood pressure was also not greater if the 1-adrenoreceptor were blocked by -blockers. Sun et al also used RNAi to inhibit 1D-adrenerig receptor gene in rat VSMCs and the therapeutic implications of this treatment are of great interest, for the reason that pharmacologic blockage of 1-ARs is a commonly used treatment for hypertension [64]. A other area of gene targeting in hypertension are the blockage of receptors that are affected by potent vasoconstrictors like Angiotensin II (Ang II).…”