Atherosclerosis, the accumulation of arterial plaques that restrict blood flow, is the leading cause of mortality and morbidity worldwide. Chronic inflammation is the key driver of disease progression, though no widespread treatments exist that target underlying immunological factors. The pro-inflammatory activities of neutrophil are fundamental to disease progression, so therapies that target neutrophil responses to inflammation offer promising treatment alternatives for atherosclerosis. Neutrophils arrive in the early stages of plaque formation via chemotaxis, and their pro-inflammatory functions, including superoxide production and NETosis, cause extensive tissue damage, propagate immune cell activities, and lead to plaque destabilization. These behaviors are coordinated by members of the Rho family of small GTPases, which act as ?molecular switches? to activate distinct signaling cascades at precise times and sub-cellular locations. Organizing decisive responses to complex external cues requires information processing steps within the Rho GTPase network, such as feedback circuits and crosstalk interactions, which shape GTPase activities. However, limitations in the ability to isolate individual processing steps from bulk cellular responses have made it impossible to dissect their unique contributions to final Rho GTPase outputs. Furthermore, a complex network of RhoGEFs and RhoGAPs regulate GTPase signaling activities, though no systematic approaches have been taken to determine the roles of individual proteins in coordinating Rho GTPase dynamics in neutrophils. This study proposes two specific aims to fill critical gaps in our understanding of Rho GTPase signal transduction and enable the development of neutrophil-specific atherosclerosis treatments. First, optogenetic tools for extrinsic Rho GTPase activation will be paired with complementary red-shifted Rho GTPase FRET biosensors to determine crosstalk and feedback signaling dynamics in a neutrophil model cell line. Second, automated live cell imaging assays with arrayed, systematic RhoGEF and RhoGAP knockdown will be used to identify key regulators of Rho GTPase signaling responses to inflammation and determine their roles in mediating neutrophil behaviors. This work will be carried out under the supervision of Dr. Sean Collins (sponsor) and Dr. Karen Zito (Co-sponsor) at the University of California, Davis. Together, Dr. Collins and Dr. Zito have the resources and specialized technical expertise to oversee the successful completion of the research and training goals outlined in this application.
Coronary artery disease from atherosclerosis, the chronic inflammation of arterial walls, is the leading cause for mortality and morbidity worldwide. Neutrophil behaviors such as chemotaxis, superoxide produce, and NETosis are central to disease progression, yet the development of effective targeted therapies has been hindered by our poor understanding of the Rho GTPase signaling pathways that control these activities. The proposed study will promote the development of neutrophil-centric treatments by filling critical gaps in our understanding of Rho GTPase signal transduction, its regulation, and its relation to neutrophil behaviors implicated in atherosclerosis.
