A Spatially Resolved Molecular Atlas of Human Endothelium

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ABSTRACT - ORGAN SPECIFIC PROJECT (Endothelium) The human circulatory system plays a vital role in the proper functioning of every organ in the human body. Because vascular related diseases, such as atherosclerosis, do not occur homogeneously throughout the vascular tree, differences in the endothelium of various organs must exist. There is even evidence of heterogeneity of adjacent endothelial cells. We propose an Organ Specific Project (OSP) that will study normal endothelium throughout the body and across individuals of different ages to characterize these differences. The first step will be to procure normal tissue samples from a wide range of organs. The tissue source will be brainstem dead donors, which allows for collection of sample with minimal ischemic time within an ethical framework allowing for genomic data sharing. From disaggregated samples of these tissues, we will perform single cell combinatorial indexing (sci-) to generate chromatin accessibility (sci-ATAC-seq) and transcriptome (sci-RNA-seq) data from millions of single cells across hundreds of samples. These are established, low-cost, scalable methods that will facilitate the identification of markers that are ubiquitous or heterogeneous in human endothelium. Molecular heterogeneity might correlate with multiple facets of the anatomical distribution of endothelium, including vascular vs. lymphatic, arteries vs. veins, capillaries vs. larger vessels, differences between organs, organ zonation, cell-to-cell differences, etc. Based on heterogeneous markers defined from deep profiling of disaggregated cells, we will employ seqFISH to spatially relate the distinct endothelial subpopulations arising from the single nuclei data to histologic preparations. For example, this will allow us to identify subpopulations with particular markers as arterial, capillary, venous, or lymphatic. Differences in the transcriptome and chromatin accessibility profiles of specific endothelial subpopulations within organs, between organs, and across ages, will be subjected to a unified, integrative analytical framework. The end product of this integration will include multiparameter images in which the complete gene expression and chromatin accessibility profile can be interactively visualized for every cell. The incidental collection of sci-ATAC-seq and sci-RNA-seq data for non-endothelial cell types may be extremely useful to inform and increase the value of the datasets collected by other TMCs and OSPs, as well as to facilitate spatial and molecular integration of the data corresponding to endothelium with organ-specific maps. We anticipate that our spatially resolved map of transcription and chromatin accessibility in normal endothelium in tissues throughout the body will lay the foundation for investigating the molecular basis for vascular diseases.