A central component of inflammatory pathology is breakdown of endothelial integrity and the resulting uncontrolled plasma leakage, leukocyte trafficking and thrombosis. Normal protective inflammatory responses exhibit micron-scale endothelial remodeling dynamics that balance disruptive activities with proactive self-restorative ones, thereby preserving overall integrity. Thus, recruited inflammatory cells breach the endothelial barrier by forming micron-scale para- and trans-cellular discontinuities, which serve as passageways for leukocyte transmigration (i.e., diapedesis). Normally, the endothelium closes these 'micro-wounds'immediately following passage of the leukocyte, thereby largely uncoupling them from plasma leak. In addition, soluble inflammatory mediators cause the endothelium itself to both create and (after minute-scale durations) close similar micro-wounds, thereby inducing controlled, self-limited vascular leak. Thus, the endothelium exhibits dynamic and proactive functions that efficiently 'heal'micro-wounds in order to maintain overall integrity and homeostasis during inflammation. Importantly, in pathologic conditions, such as presence of oxidative stress, homeostasis is lost and leukocyte diapedesis and soluble mediators can cause breakdown of vascular integrity and uncontrolled plasma leak and thrombosis. Formally, pathologic breakdown of integrity could arise from either excessive disruption or inadequate restoration. However, only the former has been studied in detail and neither the normal self-restorative remodeling mechanisms nor basis for their putative perturbation in endothelium are understood. This proposal is aimed at addressing, directly, both of these issues for the first time. Based on our extensive preliminary data we hypothesize that endothelial micro-wounds formed by leukocyte transmigration (as well as those formed by soluble mediators) are closed by novel actin lamellipodial activities that are regulated by intracellular Rac and reactive oxygen species (ROS) signaling pathways and are susceptible to perturbation by oxidative stress. The specific goal of this proposal is to elucidate the detailed mechanisms responsible for closing para- and trans-cellular leukocyte diapedesis passageways. First we will determine the functional role of Rac1 (and associated proteins such as cortactin and IQGAP) in healing diapedesis micro-wounds in cultured endothelium. Then in similar settings we will determine the role for Rac1-associated NADPH oxidase signaling (e.g., p47phox) in micro-wound closure and its sensitivity to perturbation by oxidative stress. Finally, we will establish the physiologic role of these Rac/ROS lamellipodial activities in micro-wound healing via both ex vivo vascular tissue culture models and a combination of in vivo intravital, tissue whole-mount confocal and electron microscopy imaging along with genetic and pharmacologic function perturbation. These studies are will improve our understanding of, and likely reveal new therapeutic strategies for, inflammatory pathology.
The endothelium is the layer of cells that forms the inner surface of the circulatory system and, as such, serves as a critical barrier between the blood and the tissues. Breakdown of this barrier is associated with broad ranging inflammatory diseases and leads to unregulated entry of immune cells and fluid into the tissue, as well as inappropriate blood clotting. Under normal conditions the endothelium displays remarkable self-restorative properties, whereas broad ranging inflammatory diseases are associated with pathologic breakdown in endothelial integrity. The research in this project is designed to uncover for the first time the cellular and molecular mechanisms for endothelial integrity restoration and how this may become abrogated during inflammatory disease.
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