Aortic stenosis (AS) is an age-related disease characterized by fibrotic aortic valve narrowing, resulting in high shear stress (SS). We demonstrated that SS can activate TGF-β1 released from platelets, and that N-acetylcysteine (NAC) partially inhibits SS-dependent platelet TGF-β1 activation in vitro. Here, we show that NAC blocks AS progression by inhibiting platelet TGF-β1 activation in an improved Ldlr-/-Apob100/100 (LDLR) spontaneous AS mouse model.
90%, as opposed to ~30% in the previously published model, of our LDLR mice (n=30) on a modified high-fat diet (HF) developed AS within 6 mo and showed >30% decreased cusp-separation (0.59 ± 0.02 vs. 0.86 ± 0.01; p<0.0001) and increased SS (328 ± 7 dyn/cm2 vs. 724 ± 50 dyn/cm2, p<0.0001) compared to basal levels. Valve thickness increased ~30% and collagen levels were >40% higher than basal levels. Total plasma TGF-β1 increased significantly and strongly correlated with SS (r=0.4, p=0.019). Physical association of activated platelets with valvular cells was seen, and the latter were also positive for p-Smad2 staining in nuclei, indicating that TGF-β1 is released from platelets and activated by SS.
To test whether blocking platelet TGF-β1 activation prevents AS progression, LDLR mice received pharmacological doses of NAC (2% in drinking water) at different stages of AS (mild to moderate at 3 mo and moderate to severe at 5 mo after HF). Significantly lower active TGF-β1 was generated after 2 h of SS and NAC treatments inhibited AS progression as seen by improved cusp separation (0.59 ± 0.02 in HF, 0.66 ± 0.02 at 3 mo NAC; 0.70 ± 0.02 at 5 mo NAC; p<0.05 for both NAC groups compared to HF) and SS (724 ± 50 dyn/cm2 in HF, 560 ± 38 dyn/cm2 at 3 mo NAC; 507 ± 28 dyn/cm2 at 5 mo NAC; p<0.05 for both NAC groups compared to HF), lower p-Smad2 (p=0.001) and valve thickness (p=0.02) compared to mice receiving HF alone.
Thus, results using our robust AS model suggest that shear-activated platelet TGF-β1 plays a critical role in the pathogenesis of AS progression. This model can be used for preclinical evaluation of preventative therapies, and assessing the benefits of NAC treatment in clinical trials.