Stress‐activated signaling in endothelial cells (609.21)

Endothelial cells (ECs) form the interior layer of blood vessels, creating a selectively permeable barrier. Damage to this monolayer triggers inflammatory responses which may eventually lead to vascular disease, making it relevant to characterize EC reactions to inflammatory stress. Pro‐inflammatory cytokine tumor necrosis factor‐α (TNF‐α) activates several pathways in target cells, which interact to determine cell fate. Binding of TNF‐α to its receptor causes activation of certain caspases, advancing apoptosis. c‐jun N‐terminal kinase (JNK) is activated as well, regulating transcription factor activity and possibly promoting an apoptotic response. Also activated is nuclear factor κ‐light‐chain‐enhancer of activated B‐cells (NFκB), a transcription factor which has pro‐survival effects, partially due to interference with both JNK and caspases. ECs encounter TNF‐α as it travels in the blood, however, while most cells are rarely exposed. Thus, we explored whether EC response to TNF‐α is similar to that of other cell types, using immunofluorescence microscopy and Western blotting to analyze TNF‐α‐induced JNK, NFκB, and caspase‐3 activity in primary bovine aortic ECs (BAOECs). NFκB is usually kept in the cytoplasm by an inhibitor; TNF‐α‐binding leads to inhibitor degradation and allows NFκB to move to the nucleus. Upon TNF‐α treatment (10 ng/mL), this translocation begins by 30 minutes, maximizes from 60 to 120 minutes, and declines thereafter. JNK activation is transient, with p‐JNK levels peaking after 30 minutes before decreasing. Excitation of BAOECs with high TNF‐α levels (50 ng/mL for 20 hours) induces caspase‐3 activation, suggesting significant apoptosis. This reaction profile resembles that of other cell types, though it is possible that there are important differences in pathway cross‐talk and communication. Grant Funding Source : supported in part by NIH grant HL54269