In every form of heart disease, the secretion of extracellular matrix (ECM) by activated fibroblasts, or myofibroblasts, results in cardiac fibrosis. Fibrosis impedes compliance and pumping function, ultimately leading to heart failure due to left ventricular dilation and loss of mechanical function. Little is known about endothelial cell and vessel adaptations to the environment or how local mechanics and chemistry impact vessel structure and flow in vivo. Combinatorial fibroblast and ECM mechanical and chemical crosstalk with endothelial cells are unknown. Moreover, in vitro models aiming to assess vascular adaptations to an extracellular environment lack physiologically relevant ECMs and instead provide exogenous ECM components to optimize control of variables. A system for in vivo, cell-specific phenotypic manipulation will allow for controlled perturbations at an organ level while maintaining relevant, native ECM remodeling over time. Thus, I propose to examine transgenic mice with cardiac fibroblast-specific overexpression of a constitutively active mitogen-activated protein kinase kinase 6 (MKK6) to study vascular remodeling with respect to the fibroblasts and the ECM they secrete. These mice were previously shown to develop interstitial and perivascular fibrosis after 16-20 weeks of the MKK6 gene activation without an injury stimulus, serving as an effective model of the interstitial fibrosis preserved across the results of aging, hypertension, aortic stenosis, and other diseases of the heart. Importantly, the remodeling seen in these diseases does not involve a massive loss of cardiomyocytes, as in a myocardial infarction, but rather a conserved fibroblast phenotypic change, an altered extracellular space, and/or restricted vascular flow over time. Similarly, manipulation of the MKK6 pathway allows for overexpression or knockdown of cardiac fibroblast activation, corresponding to increased ECM or the inability to secrete ECM as a response to a stimulus, respectively. First, I propose to study the biochemical, structural, and mechanical properties of the ECM as well as the macro- and microvascular responses to activated, quiescent, and control cardiac fibroblast phenotypes in vivo. Second, three-dimensional vessel-like structures with controlled fibroblasts and ECMs will be engineered as in vitro platforms to define the molecular regulators of vascular remodeling induced by microenvironmental cues. The effects of combined signaling will be resolved by global characterization along with a reductionist method. The goal of this work is to inform heart therapies by providing targets for steering cardiac vascular remodeling.