Cardiovascular pathologies are a leading cause of death in the Western world. Atherosclerosis is the development of lipid-rich plaques within the arterial wall, and is considered the major underlying pathology that contributes to cardiovascular disease development. Intriguingly, age is a primary risk factor for atherosclerosis, and increased artery stiffness increases with advanced age and atherosclerosis. It is now established that endothelial cell feedback to mechanical stimuli on the cellular level, including age-related arterial stiffness can promote a pro-atherogenic endothelium. In response to increased matrix rigidity, endothelial cells exert increased RhoA-mediated contractile forces that disrupt monolayer barrier function and increase endothelium permeability. Identifying therapeutic approaches to overcome the cellular response to extracellular matrix stiffness cues and understanding how the altered mechanical properties of the arteries associated with aging contribute to endothelium disruption is important to the prevention of atherosclerosis. Using hydrogel scaffolds with tunable crosslinking, I fabricated physiologically relevant models to study the effects of age-related matrix mechanical cues on endothelial cell behaviors in the translational context of atherosclerosis. I identified that therapeutically targeting the Rho GTPase family members RhoA and Rac1 with the available statin, simvastatin, attenuated the endothelial cell response to increased matrix stiffness. Importantly, the altered GTPase activity was associated with endothelial cell-cell junction reorganization and decreased intercellular junction tension to promote an atheroprotective endothelium. Motivated by the increased spatial stiffness heterogeneity observed in the aged arterial intima, I then developed and characterized a second hydrogel platform that employed photocrosslinking to vary the presentation of matrix stiffness. My data demonstrated that increased complexity of a heterogeneously stiff substrate in addition to a mean increase in the elastic modulus was deleterious to monolayer integrity and disrupted cell-cell junctions. Collectively, these two studies identified a pharmacological approach to overcome the cellular response to extracellular matrix stiffening and identified a novel matrix mechanical factor that has implications for the development of age-related atherosclerosis. Finally, because pathological matrix stiffening drives disease progression, I investigated the emerging field of mechano-medicine and potential therapeutic approaches to overcome stiffness-mediated pathologies.