Endothelial Cell Analytic Core

CORE C PROJECT SUMMARY The main goal of the Cell and Analytical Core (Core C) is to facilitate completion of the program investigators’ specific aims. To accomplish this goal, Core C will perform three specific aims, two of which are service components and one of which is an academic component. Aim 1 - Service component 1 - Cell isolation and characterization: a) To uniformly isolate primary mouse endothelial cells from wild type and transgenic mice and from human tissue, using a calibrated system to make cell suspensions from tissue in combination with a negative (CD45-based) and positive selection (CD31-based) endothelial cell purification method. Wild type or transgenic mice will be received from animal Core B or from the three projects. b) To characterize the endothelial character of the isolated EC. c) To expand and distribute purified endothelial cells in early passages to the three research projects. Aim 2 - Service component 2 - Gene Delivery and deletion: a) To construct, purify and deliver recombinant adenovirus, lentivirus and adeno-associated virus (AAV). b) To deplete and manipulate endogenous genes in mouse and human EC, using CRISPR Cas9 and siRNA technology. Aim 3 -Academic component 1- Analysis: a) To measure cell metabolism. O2 consumption rate (OCR) and extracellular acidification rate (ECAR) using Seahorse and performing metabolomics. b) To evaluate oxidative and nitrosative stress. c) To assess inflammation by measuring cytokine/chemokine profiles in cell supernatants using magnetic bead-based Multiplex analysis and adhesion molecule expression relevant for endothelial-leukocyte interactions using an in-house developed cell-based ELISA. This technology will also be applied to measure cytokine/chemokine profile in plasma samples from mice. d) To evaluate endothelial-mesenchymal transition- related genes in transcriptomics, using bulk and single cell RNASeq. e) To assess endothelial barrier function by combining measurements of macromolecular permeability to a macromolecular tracer and electrical impedance of endothelial monolayers. In summary, synergy with the three scientific projects, administrative Core A and core B will be organized as follows: upon receiving wild type or transgenic mice from animal Core B and from the three projects, Core C will isolate, characterize, expand and distribute early passages of primary mouse endothelial cells and of well-characterized in house isolated or purchased human endothelial cells to all four projects. The Core will moreover construct recombinant adenovirus, lentivirus and adeno-associated virus or use CRISPR ON and specific siRNA to manipulate gene expression in human and mouse endothelial cells and will coordinate scRNASeq and proteomics. The Core will moreover organize in a standardized manner cell analysis for all parameters listed above. Finally, Core C will collaborate with administrative Core A and animal Core B to coordinate dissemination of data generated through the collaborations between the cores and projects.