Abstract 5461 In vivo Knockdown of Aortic Nicotinic Acetylcholine Receptor Alpha1 (nAChRα1) Diminishes Aortic Atherosclerosis and Valve Calcification
Urokinase plasminogen activator (uPA) contributes to atherosclerosis, restenosis and vascular remodeling. We have recently identified nAChRα1 as a functional cell receptor for uPA in addition to its classic receptor, uPAR. Here, we test the hypothesis that nAChRα1 plays a role in the pathogenesis of atherosclerosis. C57BL/6J ApoE−/− mice (male) were initially fed a Western diet for 8 wks. Plasmid DNA encoding scramble RNA (pScr) or siRNA (pSir2) for nAChRα1 was then injected into the mice (n=15) using an aortic hydrodynamic gene transfer protocol. Three mice from each group were sacrificed 7 days after DNA injection to confirm the nAChRα1 gene silencing. The rest of the mice continued on the Western diet for an additional 16 wks. Aortas were harvested for paraffin-embedding (aorta root), protein (ascending aorta and aortic arch) and RNA (descending aorta) (n=8). Whole aortas were isolated for oil red staining in 4 mice of each group. The nAChRα1 was highly up-regulated in aortic atherosclerotic lesions. Knockdown of the nAChRα1 gene was achieved as early as day 7 (76%↓) after DNA injection, and aortic nAChRα1 in pSir2-treated mice remained lower (78%↓) at 16 wks compared to the pScr (n=8, P<0.05). The nAChRα1 reduction resulted in less severe aortic atheroslcerosis at 16 wks. The ratio of the intimal/medial thickness (I/M ratio) was smaller in the nAChRα1-silenced aortas (3.1±1.8 versus 8.4±1.4, pSir2 versus pScr, n=8, P<0.05). There were significantly fewer F4/80+ macrophages (60%↓) and αSMA+ myofibroblasts (80%↓) in the neointima of the pSir2-treated mice compared to the pScr (both P<0.05). The TGF-β1 and αSMA mRNA levels were down-regulated by 80% and 72% respectively in the nAChRα1-silenced aortas (pSir2 vs pScr; n=5, both P<0.05). Since nAChRα1 is a ligand-gated Ca2+ channel that may be associated with osteopathic responses in aortic intimal myofibroblasts, aortic calcification was evaluated with the Von Kossa staining. The calcification was most prominent in the aortic valves; a 41% reduction in area stained was observed in the pSir2-treated mice (pSir2 vs pScr; n=8 P<0.05). These data suggest that the nAChRα1 gene plays a significant role at the artery wall and reducing its expression serves to reduce aortic plaque development and valve calcification.
This research has received full or partial funding support from the American Heart Association, National Center.