Abstract 16217: Oxidative Stress Induced Calpain and Caspase-3 is Increased in Aortic Smooth Muscle Cells Derived From Bicuspid Aortic Valve Patients.

Introduction: Bicuspid aortic valve (BAV) associated aortopathy portends an increased risk of ascending aortic aneurysm formation and dissection/rupture. We previously showed increased oxidative stress-induced cell death for aortic smooth muscle cells (SMCs) isolated from BAV patients when compared to SMCs isolated from patients with the morphologically normal tricuspid aortic valve (TAV). Furthermore, there is evidence of increased activity of calpain, a calcium dependent protease that can regulate oxidative stress in vasculature, in BAV aortic tissue. Hypothesis: We hypothesize that oxidative stress-induced cell death in BAV-associated aortopathy is mediated by calpain-induced caspase-3. Methods: Calpain and caspase-3 activity were quantified in serum-deprived primary human SMC populations isolated from BAV (n=9) and TAV (n=12) aortic specimens treated with 1mM of tert-butyl hydroperoxide (tBHP) for 3 hours. We measured tBHP-induced cell death in the presence and absence of the calpain inhibitor calpeptin (30μM) in BAV (n=9) and TAV (n=8) using a MTS-based assay. Results: tBHP-induced calpain activity was higher for BAV SMCs when compared with TAV SMCs (65 ± 16.9% vs. 45.9 ± 5.9%, p=0.02). tBHP-induced caspase 3 activity was higher for BAV SMCs compared with TAV SMCs (222 ± 69% vs. 140 ± 8%, p<0.01, respectively). Calpain inhibition alleviated tBHP-induced cell death in TAV SMCs (66.4 ± 6.7% vs. 93.2 ± 9.3%, p=0.05), and did not affect BAV SMCs (53.6 ± 6.8% vs. 66.3 ± 5.9%, p=0.34). Conclusions: These data highlights the clear differences in cellular biology between TAV and BAV SMCs. It demonstrates that the increased vulnerability of BAV SMCs to oxidative stress compared to TAV SMCs may be due to caspase-3, which has been previously shown to be induced by calpain. Despite increased induction of calpain by tBHP in BAV SMCs, calpain inhibition did not affect oxidative stress induced cell death for BAV SMCs as it did for TAV SMCs. Therefore, calpain is unlikely to be an important effector in BAV SMCs as it is for TAV SMCs in regulating oxidative stress-induced cellular death. [ABSTRACT FROM AUTHOR]