1 Project Summary 2 3 The overall goal of this project is to develop accurate and reliable prediction tools and pharmacological targets 4 for the prevention of rupture of intracranial aneurysms (IAs). Abnormal hemodynamic stress such as 5 impingement flow with high wall shear and oscillating flow with low wall shear, is intimately linked with the growth 6 and rupture of IAs. However, detailed mechanisms underlying weak IA walls are not yet defined due to (1) the 7 absence of technologies for profiling the spatial distribution of gene expression of endothelial cells (ECs) induced 8 by the complex hemodynamic flow stressors created in IAs, (2) difficulties in collecting sequential clinical images 9 of growing IAs and acquiring human IA tissue samples to validate biologic mechanisms, and (3) the absence of 10 technologies allowing integration of the data from 3D multimodal techniques. To overcome these obstacles, we 11 have built a strong, multidisciplinary team and created a new experimental system that bridges human samples, 12 imaging, and dynamic modeling platforms. In this project, we challenge two fundamental questions regarding 13 hemodynamic stress and induced responses within the IAs. First, does complex abnormal hemodynamic stress 14 within human IAs induce abnormal regulation of EC signaling pathways? Second, what signaling pathways in 15 EC link unstable wall remodeling during IA growth and rupture? To address these questions, we have pioneered 16 a 3D Live EC Aneurysmal Flow Simulator (3D LEAFS) for profiling the spatial distribution of EC responses to 17 complex hemodynamic flow stress created in patient-specific IAs. Preliminary studies demonstrate that abnormal 18 flow in IAs induces abnormal EC morphology, cellular dysfunction and inflammation, and increased permeability. 19 We have developed an extensive database of clinical images of growing IAs and also tissue samples, exploiting 20 integrated flow analysis and 3D histological imaging of human IA tissue scanned with micro-CT and multiphoton 21 microscopy. With this database, we have linked abnormal flow with IAs to growth, wall thinning and weak wall 22 remodeling leading to rupture. By combining these state-of-the-art technologies, we propose to examine 23 fundamental impact of abnormal flow stress on ECs, and identify relationships between EC pathophysiological 24 responses and wall changes leading to fragile walls, growth and rupture. The proposed research is innovative 25 because this will be the first research to answer the above questions by utilizing multimodalities including 26 longitudinal follow-up images, surgical video, micro-CT, multiphoton microscopy, in vitro 3D endothelialized flow 27 simulator, and flow analysis for development of a pipeline for linking flow-induced EC responses to pathologic 28 changes in human IA tissue. The specific aims of this project are: 1) determine the EC signaling pathways 29 associated with unstable wall remodeling, 2) correlate...