(172g) Spatial-Temporal Distribution of RhoA Controls Vascular Barrier Function

Cardiovascular diseases (CVDs) are one of the major causes of mortality in United States. Changes in vascular integrity are considered the main reasons for CVDs. Although these causes are multifactorial, RhoAGTPases have crucial role in controlling perivascular cells and endothelial cells (ECs) function and overall, the vascular integrity. It is anticipated that the vascular integrity is mediated by spatial-temporal RhoA activation in both cell types. To study this, we developed an in vitro three dimensional (3D) bi-cellular biomimetic model with perfused endothelialized channels lined with perivascular cells and recapitulates the barrier function under disease and normal cases. We introduced into this 3D model different inflammatory factors such as thrombin to identify the behavior of either ECs or perivascular cells under these disease-like conditions. The inflammed bi-cellular microvessels exhibited high vascular permeability due to perivascular detachment and disruption of endothelial integrity. Since these cytokines induce RhoA signaling, we asked whether RhoA would control the vascular barrier and how would affect the perivascular migratory behavior and endothelial barrier function. To probe this, we developed a novel lentiviral construct carrying RhoA(CA), an activating mutant, in which the addition of gibberellin (GA₃-AM) induced coupling of the RhoA construct to the membrane-tethered anchor to rapidly activate it. This tool enables us to capture dynamic behavior of these cells and modulate the degree of RhoA activation. Upon substantial RhoA activation either in perivascular cells or in EC, perivascular cells were detached from the endothelium, indicating that RhoA signaling regulates perivascular behavior. Additionally, RhoA activation in ECs triggered disruption of VE-cadherin mediated junctions and increased vascular permeability. However, we find that lower percentage of RhoA activated endothelial cells decreased vascular permeability and enforced their VE-cadherin mediated junctions. This effect indicates that the spatial distribution of RhoA activity has a critical role in barrier integrity and we quantify it with the use of spatial-temporal correlation functions. We find that the spatial temporal RhoA modulation mediates the homocellular or heterocellular interactions and overall the vascular barrier function