ROS Mechanisms in BAV Aortopathy

描述（由申请人提供）：本项目将验证主动脉平滑肌细胞（SMCs）中活性氧（ROS）积累介导双尖瓣主动脉瓣（BAV）患者主动脉病变的假设。跨学科研究小组拥有独特的专业知识组成，并设计了一种新方法来证明在bav升主动脉瘤（TAA）标本中ROS积累导致SMCs去分化，细胞外基质（ECM）组成和结构异常以及主动脉壁生物力学强度的改变。该假设得到了该团队强有力的初步数据的支持，这些数据表明，与三尖瓣主动脉瓣(TAV)- taa和非动脉瘤患者相比，BAV患者的升主动脉中ROS积累、ROS存在下细胞活力降低、氧化应激反应减弱、SMCs去分化、基质结构破坏以及生物力学拉伸和分层强度的改变。该创新策略有两个目的：1)确定SMC表型、ECM组成和结构以及生物力学拉伸和分层强度的变化与BAV主动脉病变中的ROS积累有关；2)证明主动脉SMCs中的ROS积累介导了BAV主动脉病变。本研究将利用1)PI广泛的人升主动脉标本组织库和从以下患者队列中分离的原发SMCs: a) BAV- taa与b)非动脉瘤性BAV， c) TAV- taa, d)非动脉瘤性TAV“正常”；2)该团队建立的基于支架的三维组织培养模型。利用研究者在电子顺磁共振自旋捕获和荧光基ROS探针共聚焦显微镜方面的专业知识，对ROS进行评估。利用二次谐波产生的多光子显微镜来确定胶原蛋白和弹性蛋白纤维的排列，胶原蛋白和弹性蛋白的组织学检测，以及MMP活性的量化，将实现对ECM成分和结构的创新评估。

DESCRIPTION (provided by applicant): This project will test the hypothesis that reactive oxygen species (ROS) accumulation in aortic smooth muscle cells (SMCs) mediates the aortopathy in patients with bicuspid aortic valve (BAV). The interdisciplinary investigative team possesses a unique composition of expertise and has devised a novel approach to prove that ROS accumulation in BAV-ascending aortic aneurysm (TAA) specimens leads to de-differentiation of SMCs, abnormal extracellular matrix (ECM) composition and architecture and altered biomechanical strength of the aortic wall. The hypothesis is supported by the team's strong preliminary data demonstrating ROS accumulation, reduced cell viability in the presence of ROS, diminished oxidative stress responses, de- differentiation of SMCs, disrupted matrix architecture and altered biomechanical tensile and delamination strengths in the ascending aorta of BAV patients compared with tricuspid aortic valve (TAV)-TAA and non- aneurysmal patients. The innovative strategy is accomplished in a two-aim approach: 1) Define what changes in SMC phenotype, ECM composition and architecture, and biomechanical tensile and delamination strengths are associated with ROS accumulation in BAV aortopathy and 2) Prove that ROS accumulation in aortic SMCs mediates the BAV aortopathy. This study will exploit 1) the PI's extensive tissue bank of human ascending aortic specimens and primary SMCs isolated from the following patient cohorts: a) BAV-TAA compared with b) non-aneurysmal BAV, c) TAV-TAA, and d) non-aneurysmal TAV "normal"; and 2) the team's established scaffold-based 3-D tissue culture model. ROS will be evaluated using the investigators' expertise in electron paramagnetic resonance spin trapping and confocal microscopy of fluorescence-based ROS probes. Innovative assessment of ECM composition and architecture will be achieved using multi-photon microscopy with second harmonic generation to define alignment of collagen and elastin fibers, histological detection of collagen and elastin, and quantification of MMP activity. The team's pioneering techniques for studying blood vessel biomechanics will be utilized throughout the research design. This study will, for the first time, define and prove that ROS mechanisms mediate the BAV aortopathy. The results will potentiate significant long-term clinical benefits including the development of improved diagnostic tools for earlier detection and better surveillance of the BAV aortopathy and the discovery of pharmacologic therapies directed at modulation of ROS in the aortic wall to prevent aneurysm formation in BAV patients.