Recent studies on endothelial dysfunction in relation to vascular diseases including atherosclerosis have highlighted the key contribution of the microenvironment of endothelial cells (ECs). By mimicking the microenvironment of early atherosclerotic lesions, here, we replicate the pathophysiological phenotype and function of ECs within microchannels. Considering the elevated deposition of fibronectin (FN) in early atherosclerotic plaques and the close correlation between the vascular stiffness and the progression of atherosclerosis, we utilized FN coated hydrogels with increased stiffness for endothelial substrates within the microchannels. As a result, we demonstrated that endothelial integrity on FN coated microchannels is likely to be undermined exhibiting a random orientation in response to the applied fluid flow, notable disruption of vascular endothelial cadherins (VE-cadherins), and higher endothelial permeability as opposed to that on microchannels coated with collagen (CL), the atheroresistant vascular model.