Project Summary/Abstract Mechanisms of capillary plexus formation have been well studied in avian, zebrafish, and mammalian embryos, with the precise timing of these processes being found to have profound importance in the formation of an efficient and robust vasculature. However, due to the limitations of in vivo developmental models, no results of modifying initial conditions or temporally modifying the initiation of flow and the importance of increased viscosity when blood cells enter circulation are unknown. Recent reports have leveraged the precise control available within microfluidic in vitro devices to elucidate novel angiogenic mechanisms. The objective of this proposed research is to determine how temporally modulating the shear stress, media viscosity, and increasing cortical actin assembly through a non-canonical Notch pathway affect the transition of a nascent capillary bed into an aligned quiescent vascular network, and determine the molecular cues that cause a transition from a stable quiescent network to a highly dynamic phenotype. Experiments will be carried out in an in vitro dual-channel microfluidic device to precisely control experimental conditions that are unavailable in in vivo models. The overall hypothesis of this proposal is that changes in shear force and fluid viscosity initiated immediately after formation of the primitive plexus, enable rapid and efficient vascular remodeling, which is stabilized by a cortical reinforcing non-canonical Notch pathway. We will address this hypothesis and achieve the proposed goals by first determining how precisely timed shear force, dynamic viscosity changes, and addition of exogenous protein expression on nascent vasculature affects cortical reinforcement, network dynamics, and morphology. Secondly, we will elucidate the main molecular and mechanotransduction mediated role of cortical- Notch signaling in network adaptation to altered flow profiles. The ramifications of altered force applied to vascular islands and a nascent vasculature and how it allows vascular network remodeling will be determined, and ascertain whether inhibition of parts of the cortical-Notch pathway allows the network to revert from being stably quiescent to highly dynamic and proliferative without compromising the overall expression of canonical- Notch expression. More complete understanding of this process is of significant biological and clinical importance as it will allow novel restorative therapies for highly prevalent and deadly vascular diseases such as Peripheral Arterial Disease (PAD) and Ischemic Heart Disease (IHD).