Sustained and intermittent hypoxia combined with TNF additively promote pro-thrombotic gene and protein changes in primary endothelial cells.. Sustained and intermittent hypoxia combined with TNF additively promote pro-thrombotic gene and protein changes in primary endothelial cells.

Venous thromboembolism (VTE) is a common and deadly disease with unclear pathophysiological initiation. Hypoxia and inflammation are believed to contribute to promoting thrombosis, but how and if these potential triggers interact is unclear. We have stimulated primary endothelial cells with sustained and intermittent hypoxia, in the presence or absence of TNF. Effects to the cells were analyzed with RNA sequencing and flow cytometry after 24H and 48H stimulations. RNA sequencing confirmed strong pro-inflammatory effects of TNF, and indicated that hypoxia alone had modest effects, but when combined with TNF there were clear additive effects on the number of differentially expressed genes, as well as modest increases of pro-inflammatory GO terms assessed by GSEA. On protein level, flow cytometry confirmed additive effects of combingin hypoxia and TNF for several adhesion molecules including E-selectin and ICAM-1, as well as on associated molecules including tissue factor. Unsupervised clustering of the flow cytometric data also revealed the expansion of a subset of endothelial cells with strongly pro-thrombotic phenotype. Taken together, our data suggests that hypoxia combines with inflammatory cues in the in vivo situation to promote pro-thrombotic responses by endothelial cells in the venous valve pocket. Overall design: To assess the impact on the transcriptome of hypoxia (sustained and intermittent) alone and combined with TNF, endothelial cells were stimulated for 48 hours in normoxia, sustained hypoxia or intermittent hypoxia in the absence or presence of TNF. RNA seq was performed on RNA isolated from the stimulated endothelial cells from three experiments. **RAW data will be deposited in the Norwegian federated European Genome Archive (EGA)**