Core C, Pre-Clinical Models of ventilator-induced lung injury (VILI)/acute respiratory distress syndrome (ARDS), is designed to provide PPG investigators with rigorously defined and reproducible murine models of VILI, a two-hit lung injury model induced by exposure to both a ventilator and lipopolysaccharide (LPS) administration (VILI+LPS) that better mimics ARDS in patients, and an E. coli pneumonia model. Core C will comprehensively generate, manage and provide all animal-related experiments, resources, and expertise by accomplishing 6 specific aims.
Specific Aim #1 will provide a complete range of expertise, training, equipment, and data analysis tools to extensively study the pathogenic mechanisms in preclinical models of murine lung injury. We will employ state-of-the-art techniques to a) characterize the role of various intracellular signaling cascades in regulating lung endothelial cell (EC) barrier function, b) determine the effects of specific interventions to provide insight into the efficacy and mechanisms of novel therapeutic strategies; and c) facilitate the translation of basic research to clinical interventions. Toward these goals, Core C will first provide validated quantitative measurements of vascular permeability and inflammation.
Specific Aim #2 will house and care for the genetically-engineered mice and to generate novel transgenic and knockout mice (e.g., inducible endothelium-specific and lung epithelium-specific conditional knockout mice).
Specific Aim #3 will examine selective siRNAs or pharmacological agents for target signaling cascades as potential therapeutic strategies and approaches for preclinical models and ultimately, for ARDS.
Specific Aim #4 will be to provide performance of specific experimental strategies involving VILI, VILI+LPS (?two-hit?), E. coli pneumonia models of ARDS as well as gene-specific rescue interventions.
Specific Aim #5 will evaluate the function of ARDS- associated single nucleotide polymorphisms (SNPs) and sites of functional protein post-translational modification (PTM), utilizing mutated cDNA (high efficiency expression plasmids) targeting the lung endothelium (with ACE antibody-conjugated liposome) in the endothelial conditional knockout mice.
Specific Aim #6 will provide high quality shared data for data storage and well-preserved lung tissue samples to individual projects for more sophisticated molecular and cellular assays. Core C will centralize all mice-related work across all three projects of this program, including generating new strains, breeding and housing of mice, generating preclinical VILI/ARDS and VILI+LPS models, accessing therapeutic effects of siRNAs and chemicals, performing lung inflammation assessment, and providing tissue samples and freshly dissociated murine lung vascular endothelial cells to each project for specific assays (including immunohisto- and immunocytochemistry, western blot analysis, fluorescent microscopy). In addition to its own space and equipment, Core C will have full access to and will utilize resources available at the University of Arizona shared facilities including the Genetically Engineered Mouse Models Core (GEMM).
Acute respiratory distress syndrome (ARDS) is a devastating and life-threatening syndrome that most commonly occurs as a complication of both severe infections by sepsis and excessive mechanical stress from using a ventilator, resulting in ventilator-induced lung injury (VILI). This centralized Core and its experienced leaders have proven to be excellent and will be necessary for all projects in replicating the main focus of this PPG, VILI/ARDS, in preclinical models for the purpose of studying underlying mechanisms and investigating strategies to prevent/treat VILI/ARDS.