Abstract 7: In vivo Transcriptional Profiling of Endothelial Cells Identifies Vascular Bed-Specific Changes Upon Lps-Induced Endotoxemia

Abstract

Endothelial cells (ECs) form a critical barrier between blood and parenchymal cells and play an important role in many pathologic conditions, including sepsis. ECs are highly adaptive to their microenvironment and also act as a critical responder to microbial pathogens. Though ECs are thought to display extensive heterogeneity, detailed profiling of the in vivo EC gene expression program has been limited by the challenges of isolating ECs from complex tissues and the phenotypic drift associated with manipulation and expansion of ECs in vitro . We applied an in vivo system in which a conditional hemagglutinin-epitope tag is targeted into the mouse ribosomal protein Rpl22 locus and specifically activated in ECs, allowing immunoisolation of endothelial ribosome-associated mRNA. Both EC-selected and total mRNA from tissue lysates (brain, heart, kidney, liver and lung) were subjected to RNA sequencing followed by differential expression analysis to determine EC-enriched transcripts. These analyses were performed under physiologic conditions as well as in LPS injected mice to study transcriptional changes induced in ECs following endotoxin exposure. LPS-induced endotoxemia resulted in striking changes in the EC transcriptome (~800 per tissue), and included transcripts associated with known sepsis related pathophysiology, including impaired hemostasis, leukocyte recruitment and increased vascular permeability. Gene ontology analysis of transcriptional changes shared between ECs of different tissues identified cellular response to LPS among the highest enriched biologic processes (adjusted p-value 5.2E-5), together with immune (2.0E-14) and inflammatory responses (4.4E-12). Novel transcripts not previously associated with ECs or endotoxemia were also identified, as well as a subset of genes uniquely expressed in distinct vascular beds. In conclusion, our findings demonstrate remarkable heterogeneity of the EC transcriptome across multiple vascular beds in vivo . The EC response to endotoxin challenge is also highly heterogeneous across vascular beds and provides new insight into the endothelial response to infectious challenges, as well as identifying potentially useful biomarkers for the onset of sepsis and response to therapy.