Vascular permeability is an important element of tissue homeostasis and the normal inflammatory response. During inflammation, changes in the endothelium lead to localized leakage of protein-rich fluid and recruitment and activation of circulating leukocytes, accompanied by a breakdown of intercellular junctions and a decrease in barrier function. These events are also characteristic of the endothelial dysfunction seen in many disease states, including arthritis, atherosclerosis, Alzheimer's disease, and tumor angiogenesis. Though many signaling pathways contribute to the regulation of vascular permeability, we still do not understand how these pathways are coordinated in cells and tissues. KRIT1 is a central regulator of multiple signaling pathways involved in the regulation of vascular permeability, and as such, is positioned to act as a rheostat to control the degree of endothelial activation and/or specific responses to inflammatory stimuli. We propose that the regulation of reactive oxygen species (ROS) by KRIT1 is the key linkage between KRIT1's effects on endothelial adherens junction stability and vascular permeability. Moreover, additional studies have revealed that KRIT1 expression in hematopoietic cells is required for the increase in permeability in KRIT1 heterozygous mice, suggesting that KRIT1 plays a novel role in both endothelial- and leukocyte-dependent regulation of permeability. We will test our overall hypothesis in a series of complementary aims. First, we will assess whether ROS are required for the loss of endothelial cell-cell contact integrity and increased vessel permeability in KRIT1 depleted cells and animals by measuring ROS-dependent changes in endothelial barrier function and microvessel permeability in vitro and in vivo. Second, we will determine the mechanism(s) by which KRIT1 limits reactive oxygen species production in endothelial cells, using a series of cell culture experiments designed to evaluate the novel hypothesis that KRIT1 limits ROS production by restricting the interaction of Rap1 with NADPH oxidase. Finally, in Aim 3 we will explore the contribution of hematopoietic KRIT1 to the regulation of vascular permeability. We have established the cell-culture and animal models necessary to examine the role of KRIT1 in this process at the molecular level, and authenticate these findings in the complex environment of the intact vascular network, thus we are uniquely positioned to define the relevant pathways and explore signaling priorities that control the coordination of vascular permeability regulation in vivo.
The normal response to inflammation is marked by regulated changes in blood vessel permeability; however unregulated vessel permeability contributes to many cardiovascular and inflammatory diseases, including atherosclerosis, Alzheimer's disease, and pulmonary edema. Unfortunately, we still do not fully understand how vessel permeability is coordinated in cells and tissues. This proposal will examine the regulation of vascular permeability by KRIT1 in order to understand how KRIT1 coordinates vascular permeability regulation in multiple cell types under inflammatory and non-inflammatory conditions, knowledge necessary in order to develop better and safer treatments for chronic cardiovascular and inflammatory diseases.
|Goitre, Luca; DiStefano, Peter V; Moglia, Andrea et al. (2017) Up-regulation of NADPH oxidase-mediated redox signaling contributes to the loss of barrier function in KRIT1 deficient endothelium. Sci Rep 7:8296|
|DiStefano, Peter V; Smrcka, Alan V; Glading, Angela J (2016) Phospholipase C? Modulates Rap1 Activity and the Endothelial Barrier. PLoS One 11:e0162338|
|Retta, Saverio Francesco; Glading, Angela J (2016) Oxidative stress and inflammation in cerebral cavernous malformation disease pathogenesis: Two sides of the same coin. Int J Biochem Cell Biol 81:254-270|
|Sarelius, Ingrid H; Glading, Angela J (2015) Control of vascular permeability by adhesion molecules. Tissue Barriers 3:e985954|
|DiStefano, Peter V; Kuebel, Julia M; Sarelius, Ingrid H et al. (2014) KRIT1 protein depletion modifies endothelial cell behavior via increased vascular endothelial growth factor (VEGF) signaling. J Biol Chem 289:33054-65|