Arterial stiffening is a risk factor for cardiovascular disease, but how arteries stay supple and how arterial stiffness contributes to disease are unknown. Our preliminary studies show that arterial elasticity is maintained by Apo lipoprotein E (apoE) and apoE-containing HDL through a suppressive effect on the expression of extracellular matrix genes. ApoE interrupts a mechanically driven feed-forward loop that increases the expression of collagen-I, fibronectin, and lysyl oxidase in response to substratum stiffening. These effects are independent of the apoE lipid-binding domain. Arterial stiffness is increased in apoE-null mice, this stiffening can be reduced by administration of the lysyl oxidase inhibitor, BAPN, and BAPN treatment attenuates atherosclerosis despite highly elevated cholesterol. Macrophage abundance in lesions is reduced by BAPN in vivo, and monocyte/macrophage adhesion is reduced by substratum softening in vitro. Mechanistically, we show that apoE and apoE-containing HDL inhibit Rho-GTP activity and reduce intracellular force in VSMCs. These changes in VSMC mechanics then affect ECM gene expression. Finally, we show that in addition to regulating (fibrillar) collagen-I, apoE and apoE-HDL inhibit the expression of collagen-VIII, a non-fibrillar collagen that has profound effects on VSMC function and atherosclerosis, yet is largely unexplored in terms of its mechanical properties and mechanistic effects. Overall, our data describe a completely new role for apoE and apoE-HDL that is independent of plasma cholesterol levels, intimately connected to cell and tissue mechanobiology, and causally linked to protection from atherosclerosis. We now propose three specific aims to characterize the relationships between apoE, intracellular force, matrix remodeling, and protection from atherosclerosis. In Aim 1, we will use a new micro fabrication platform of VSMC micro-tissues to study the effect of collagen-VIII on Rho-activity, contractility, ECM gene expression, and tissues stiffness in 3D. We will also use this system to determine how collagen-VIII controls the mechanical response to apoE. These in vitro studies will be complemented with an ex vivo analysis of arterial stiffness in apoE+/+ and apoE-/- arteries isolated from WT and collagen-VIII deficient mice. In Aim 2, we examine the mechanism by which stiffness controls atherosclerotic lesion development, with the particular goal of identifyin mechano-sensitive adhesion receptors that account for stiffness-dependent attachment of monocytes and macrophages to sub endothelial ECM protein. As in Aim 1, complementary in vivo experiments with existing mouse models will test the effect of arterial stiffness on monocyte abundance in vivo. Aim 3 will link the results in the first two aims by developing a new mouse model that can delete RhoA from VSMCs and establish the effect of reduced intracellular force on ECM gene expression, arterial stiffness, and atherosclerosis in vivo. This work brings together a team of three PI's (Assoian, Chen and Bendeck) with complementary expertise and an established track record of co- publication who, jointly, will establish how this novel regulatin of the ECM and VSMC mechanics by apoE provides cholesterol-independent protection against cardiovascular disease.