Lisa D. Wilsbacher, Ph, MD - US grants

[unreadable] DESCRIPTION (provided by applicant): Tissue factor (TF) is an integral membrane glycoprotein that initiates the coagulation cascade after exposure to blood components. In classical models, TF is constitutively expressed in epithelia and vascular adventitial cells so as to create a "hemostatic envelope" around but separated from blood. However, endothelial cells and hematopoietic cells can express TF especially after inflammatory stimuli, and it has been postulated that activation of circulating TF is important for clot propagation. We have used a Tie2-Cre transgene to excise a floxed Tf allele to generate mice in which hematopoietic and endothelial cell Tf has been ablated ('Tf hem/endo knockout"). Data using such mice suggest that endothelial and hematopoietic Tf is necessary for disseminated intravascular coagulation in mouse models of endotoxemia. However, the normal role of endothelial and hematopoietic TF in homeostasis and in inflammatory states is unknown. We shallask: Specific Aim 1. Is endothelial and hematopoietic TF important for hemostasis and thrombosis? In Tf hem/endo knockout mice, we will assess ablation of circulating TF by quantitative RT- PCR for Tf mRNA. Hemostasis and thrombosis will be tested in mice using well-validated protocols. Specific Aim 2. Does induction of endothelial and hematopoietic TF by inflammatory stimuli promote hemostasis and thrombosis? We will induce TF mRNA expression via exposure to endotoxin and then examine bleeding times and thrombosis. Enhanced hemostasis and/or thrombosis would support the hypothesis that endothelial and hematopoietic TF may provide a link between inflammation and thrombosis. Specific Aim 3. Might mice lacking factor IX provide a sensitized system in which we might detect normal roles for circulating TF? We will test the hypothesis that these systems are partially redundant by examining hemostasis and thrombosis in mice that lack both circulating TF and factor IX. Our study seeks to define the role of circulating TF, which is increased in important human inflammatory disease states, and its interaction with the intrinsic coagulation pathway. These studies may provide attractive therapeutic targets for treatment of these inflammatory states. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The goal of this Career Development Award is for Dr. Wilsbacher to develop the skills necessary to successfully become an independent physician-scientist at an academic center. Dr. Wilsbacher earned her M.D. and Ph.D. degrees in the Medical Scientist Training Program at Northwestern University, where she investigated genetic and molecular mechanisms that drive circadian rhythms. She trained in Internal Medicine and Cardiology at the University of California, San Francisco. Dr. Wilsbacher's current research objective is to investigate mechanisms of vascular barrier function during normal physiology and disease, and her long-term career goal is to bridge the gap between circadian physiology and endothelial function to help advance understanding of mechanisms governing cardiovascular biology and disease. The training plan includes mentorship by Dr. Shaun Coughlin, a world-class investigator of G protein- coupled receptor (GPCR) signaling in hemostasis, thrombosis, and vascular integrity who has an established record of training young investigators to independence. Additional guidance will be provided from an expert group of scientists who are leaders in GPCR signaling (Dr. Mark von Zastrow), inflammation (Dr. Donald McDonald), and protein chemistry (Dr. James Wells). Outstanding research facilities and equipment are readily available. The training plan also incorporates advanced didactic coursework that focuses on cell signaling, membrane trafficking, protein interactions, and microscopy;attendance at seminar series;participation and presentation in local and national conferences;and mentored guidance with manuscript and grant preparation. Treating or preventing inflammation requires thorough understanding of the mechanisms of endothelial barrier function. The research proposal aims to uncover signaling mechanisms that regulate vascular integrity, particularly through sphingosine-1-phosphate receptor 1 (S1Pr1) and Gi signaling. Aim 1 investigates the role of endothelial Gi and S1Pr1 signaling in barrier function. Specifically, genetic mouse models that express pertussis toxin (which inhibits Gi) in endothelium, express a pertussis-insensitive Gi in endothelium, or conditionally delete S1Pr1 in adult endothelium will be tested for changes in vascular permeability. Aim 2 uses a new GPCR activation detection system to probe when and where S1Pr1 is activated in vivo during inflammation. The new reporter system, which transcriptionally reports on GPCR activation, is an innovative tool that will be widely applicable to other GPCRs in diverse tissues. Aim 3 probes whether caveolae influence S1Pr1-Gi coupling, and whether S1Pr1 localization in caveolae affects 2arrestin-mediated internalization. Dr. Wilsbacher's Career Development Award proposal comprises a promising candidate, outstanding training environment, rigorous training plan, and exciting research proposal that includes an innovative new tool and focuses on a topic with direct relevance to human health and disease. At the completion of this Award, Dr. Wilsbacher should have the skills necessary to succeed as an independent investigator. PUBLIC HEALTH RELEVANCE: A diverse range of acute and chronic human diseases involve abnormal endothelial permeability, including asthma, acute lung injury, inflammatory bowel disease, diabetic retinopathy, rheumatic disease, multiple sclerosis, atherosclerosis, and sepsis. Treating or preventing inflammation and its sequelae requires thorough understanding of the mechanisms of endothelial barrier function. The overall goal of this proposal is to uncover signaling mechanisms regulating vascular integrity that might be utilized in the prevention or treatment of these important human conditions.