Core C — Endothelial Cell Analytic Core

Overall Goal for Core C:

The overall goal of the Cell and Analytical Core is to facilitate completion of the Specific Aims outlined in the scientific Projects 1-3 by providing well-characterized primary endothelial cells from wild type and transgenic mice and human tissue, measurements of endothelial cell (EC) metabolism, barrier function, EndMT, generation of reactive oxygen and nitrogen species and inflammation.

Central Hypothesis

This core will moreover provide support for all projects with standardized measurements of endothelial metabolism, barrier function, levels of ROS/RNS and will distribute reagents for genetic manipulation of endothelial cells. It will also perform analysis of the inflammatory and redox status using cell-based assays for NO bioavailability, measurement of cGMP, multiplex cytokine/chemokine assays, histological staining, chemiluminescence assays and EPR techniques. Endothelial cell glycolytic and oxidative capacity will be assessed by Seahorse assay. For cutting edge quantitative metabolic flux analysis and metabolomics, Dr. Brad Hill (U. Louisville) will provide expertise and guidance on the metabolomics data obtained through the Metabolic Bioanalytical Core at U. of Louisville directed by Dr. Pawel Lorkiewicz. In addition, Dr. Martina Zoccheddu at AUs genomic Core will assist with transcriptomics (bulk and scRNAseq/ATACseq) to assess changes in the expression patterns of metabolic and redox enzymes. These innovative methodologies have been proposed in all 3 Projects.

Project Aims:

Aim 1 – Cell isolation & characterization – service component

To uniformly isolate primary endothelial cells from wild type & transgenic mice as well as from Type 1 diabetes patients. Standardized characterization of isolated EC & to expand & distribute purified, characterized EC in early passages to three research projects.

Aim 2 – Gene delivery & deletion – service component

To construct, purify & deliver recombinant adenovirus, lentivirus & adeno-associated virus. Adenovirus & adeno-associated virus (AAV). To deplete & manipulate endogenous genes in mouse & human EC.

Aim 3 – Analysis – academic component

Metabolic analysis, to measure reactive oxygen (ROS) & nitrogen species (RNS). Detect EC inflammation, to access EndMT-related genes in ECs by transcriptomics, to measure endothelial monolayer permeability.

Core Leaders

Dr. Rudolf Lucas

Core C Lead

Professor of Pharmacology in the VBC at MCG/AU, Dr. Lucas is a leading expert on proinflammatory mechanisms involved in endothelial dysfunction and barrier function, TNF and reactive oxygen species in diseases such as pneumonia, cerebral malaria, glomerulonephritis and obesity/type 2 diabetes as well as developing therapeutic targets. He has expertise in the isolation and culture of endothelial cells from numerous vascular beds.

Together with Co-Directors Drs. David Fulton and Masuko Ushio-Fukai, Dr. Lucas will serve as the Director of Core C.

Dr. David Fulton

Co-Investigator

A Regents Professor and Director of the VBC at MCG/AU and a leading expert in the area of endothelial biology and NO/eNOS and NADPH oxidase in cardiovascular disease for the past twenty years. Dr. Fulton will serve as a Co-I and provide additional studies and mechanistic insight on how glycolysis and PFKFB3 regulate eNOS function and redox balance. Dr. Fulton will also serve as co-director of Core C.