PB0839 - Unravelling the molecular dynamics of endothelial inflammation states interplay from a proteomic perspective

Background: Hemostasis and inflammation are tightly intertwined processes in which vascular endothelial cells (ECs) play a central role. Previously we described unique inflammation states induced by TNFα and IFNγ and a synergetic response by combined addition. The design of intervention strategies at the crossroads of thrombo-inflammation requires detailed insight in the mechanistic processes that underly the interplay of these states.

Aims: Understanding the molecular interplay of endothelial inflammatory responses and the inhibition thereof.

Methods: We developed a high-throughput endothelial proteomic screening platform to dissect system wide effects of seven NFKB and JAK/STAT-pathway inhibitors in TNFα, IFNγ and TNFα + IFNγ inflamed ECFCs and the temporal protein dynamics of consecutive addition of both cytokines.

Results: Proteomic screening resulted in >6,000 unique proteins quantified across samples. IFNγ stimulation induced proteins in a single generic induction pattern over time whereas TNFα-induced proteins showed distinct grouping of “immediate” early and “secondary” late upregulated proteins. Interplay between cytokines was most apparent through combined addition, highlighting highly controlled regulation of transcriptional/translational processes underlying inflammation states. The system-wide inhibitor screen enabled further dissection of molecular pathways. Inhibitor Itacitinib (JAK1i) completely inhibited IFNγ responses (e.g. STAT1, HLAs, CXCLs) while TPCA-1 (IKK2/STAT3i) inhibited TNFα-induced responses (e.g. ICAM1, NFKB2) and affected the IFNγ response. Finally, STAT6 inhibitor AS1517499, affected the EC steady-state and all inflammation responses were completely ablated.

Conclusion(s): Our integrated analysis reveals tightly controlled dynamic regulation of inflammatory processes in ECs. Moreover we highlight the feasibility of dissecting inhibition of EC inflammation states from a system-wide perspective, indicating mechanisms beyond classical induction of transcription/translation underlying the regulation of thrombo-inflammation.