SUMMARY Arterial stiffness is a key risk factor for cardiovascular disease (CVD) events. A change in arterial stiffness is a significant pathology in vascular injury, atherosclerosis, and coronary disease. Stiffening of the vessel wall promotes anomalous migration and proliferation of vascular smooth muscle cells (VSMCs), leading to neointima formation on the vessel wall. It is not clear, however, how the extracellular matrix (ECM) influences these pathological processes. This research proposal will address this by exploring how changes in arterial stiffness elicit VSMC behaviors that contribute to cardiovascular disease. Specifically, this work draws upon preliminary data revealing that the protein survivin is a key regulator of stiffness-mediated VSMC proliferation and migration and an effector of arterial stiffening and remodeling. Using mouse and human VSMCs, we will first explore how vascular ECM stiffness impacts VSMC migration, proliferation, and chromatin organization at the single-cell level (early stage of disease progression; Aim 1) and, second, determine how pathological ECM stiffness drives neointima formation, altering the local mechanical environment of VSMCs in vitro (advanced stage of disease progression; Aim 2). Lastly, we will confirm survivin’s role in regulating both ECM production and arterial stiffness (in vivo animal model; Aim 2). These aims will be achieved using 3D cell culture with a novel in vitro porcine decellularized aorta ECM-based fibrous scaffold system and mouse injury models. Briefly, VSMCs isolated from mouse and human aortas will be cultured on nanofibrous scaffolds of different stiffnesses and structures that mimic normal and pathological conditions in the body. The VSMC responses to pathological ECM stiffness will be analyzed using advanced microscopy to observe changes in cellular/nuclear structure and biomechanical properties in vitro, and the RNA and protein expression will be assessed at the single-cell level. Finally, arterial stiffness and VSMC function will be studied in intact arteries of injured mice; histology and biochemical analyses of dissected tissues will be conducted after arterial stiffness has been manipulated by arterial injury, drug treatment, or genetic mutations. This project will, for the first time, study the molecular and biophysical mechanisms by which survivin (i) mediates stiffness-sensitive VSMC functions and (ii) contributes to neointima formation and stiffening, revealing a completely new aspect of survivin biology in VSMCs and in the pathology of arterial stiffness. Overall, this proposal is unique in its ability to identify potential new therapeutic targets for the treatment of CVDs.