Education
1985 B.S.E., University of Tennessee at Chattanooga
1987 M.S., University of Miami, Miami, FL
1991 Ph.D., Vanderbilt University, Nashville, TN
Research Interest
My laboratory focuses on two main areas: (1) acute lung injury, and (2) drug transport in the brain.
(1) Patients with acute respiratory distress syndrome (ARDS) are placed on mechanical ventilators to improve oxygenation, but the ventilator may cause additional injury to the lungs due to either overdistention or airway collapse and reopening. Recent clinical trials have demonstrated a substantial reduction in mortality in ARDS patients when ventilation strategies are used that reduce overdistention (lower tidal volumes) and minimize airway collapse and reopening (positive end expiratory pressure). The lung is a mechanically dynamic organ, and cells in the lung are subjected to shear stress due to fluid flow, tensile and compressive forces due to respiratory motion, and normal forces due to vascular or airway pressure. High tidal volume mechanical ventilation induces mechanical stresses that increase injury to the lung epithelium, stimulate inflammatory responses, and decrease repair mechanisms. We are focusing on the mechanisms by which mechanical forces inhibit wound healing of lung epithelial cells and stimulate inflammation. We are examining cell migration and wound healing, Rho GTPase signaling, cytoskeletal remodeling, stimulation of reactive oxygen species, and regional variations in cellular tension. In addition we are examining lung injury in vivo. My research seeks to identify the levels of mechanical forces and the types of lung injury that cells experience in vivo, to develop in vitro models to evaluate cellular responses, and to identify mechanisms by which mechanical forces are transduced into biological signals.
(2) Tightly regulated cellular barriers limit delivery of pharmacological agents to children with primary tumors in the central nervous system (CNS). The physiological regulation of the blood-brain barrier (BBB) and the blood-cerebrospinal fluid (CSF) barrier (BCB) and the specificity for excluding or allowing drug transport are not well understood. Camptothecin analogs such as topotecan and irinotecan are used to treat children with primary CNS tumors, but the CNS distribution of these drugs is widely different. The discovery of P-glycoprotein (P-gp) and other ATP-binding cassette (ABC) transport proteins has challenged the view that only passive diffusion and physicochemical properties control drug distribution. In collaboration with Dr. Clinton Stewart at St. Jude Children?s Research Hospital we are testing the that the distribution of camptothecin analogs between the blood, brain tissue, and the CSF is largely controlled by drug efflux transporters, including P-pg, multi-drug resistance protein 4 (MRP4), and the breast cancer resistance protein (BCRP or ABCG2). We seek to understand how drug distribution is controlled at the BBB and the BCB. We are developing tissue engineered models of the BBB and BCB to investigate drug transport in vitro. Cells transfected with different ABC transporters are used to determine the specificity of transporters for camptothecin analogs. Mathematical models are being used to evaluate transport parameters from experimental data. We are also examing the mechanisms of camptothecin analog transport in vivo using immunohistochemistry to determine the anatomical distribution of ABC transporters, and microdialysis measurements of brain and CSF drug concentrations in knockout mice deficient in MRP4 and ABCG2. Our long-term goals are to understand the regulation of drug transport in the CNS, to develop in vitro and in vivo models to test camptothecin analogs, and to define and quantify parameters that can be used to compare drugs and ultimately improve therapy for children with primary CNS tumors.
Current Techniques utilized:
- Isolation and culture of airway and alveolar epithelial cells, lung vascular endothelial cells, and brain microvascular endothelial cells.
- microcarrier bead culture.
- application of fluid shear stress to cells - flow chambers, cell columns.
- application of cyclic mechanical strain to cells.
- evaluation of permeability of cell monolayers - cell column (on line), transwell culture.
- transepithelial electrical resistance measurements (including the ECIS system).
- Western analysis to measure cytoskeletal and focal adhesion levels.
- fluorescence microscopy to visualize cell cytoskeleton (f-actin, microtubules), focal adhesions, integrins, junctional assembly.
- Atomic force microscopy to determine stress distribution in wounded cells.
- wound healing assays.
- Spectrophotometry and fluorescence microscopy to measure reactive oxygen species production.
- Pull down assays to measure Rho GTPase activity.
- Adenoviral expression systems to determine the effects of mutations in Rho GTPases.
- Mechanically ventilated rats.
Research Support
Principal Investigator (40% effort): "Biomechanics and Wound Healing in Lung Epithelial Cells," National Institutes of Health (renewal of R01HL064981); 2.2 percentile; 4/01/04-3/31/09.
Principal Investigator (25% effort): "ABC Transporters in CNS Penetration of Camptothecins," National Institutes of Health (R01 GM71321); 6.2 percentile; 7/01/04-6/30/08.
Principal Investigator, sub-contract (10% effort): "Regulation of Airway Epithelial Repair," National Institutes of Health (R01 HL080417), Steven R. White, P.I. (University of Chicago).
Co-Investigator (10% effort): "Stretch and Hyperoxia in Ventilator-Induced Lung Injury," National Institutes of Health (R01 HL081297), Scott Sinclair, P.I.
Mentor: "Role of JAK3-villin interaction in the restitution of intestinal epithelial cells," Chrohn?s and Colitis Foundation of America (#1351), Narendra Kumar, P.I., 7/01/05-6/30/08.
Collaborator: "Mechanotransduction and Eosinophil Function," National Institutes of Health (R01), P.H.S. Sporn, P.I, 4/01/03-3/31/08.