Deactivation and localization of stress-activated protein kinases in subconfluent and wounded vascular endothelial cells

Atherosclerosis is believed to be caused, in part, by chemical and physical damage to the vascular endothelium. Endothelial injury activates the stress-activated protein kinase (SAPK) cascade causing a sequential phosphorylation of proteins that results in de novo gene expression. This work seeks to examine the deactivation and localization of two constituents of the SAPK pathway, p38 and JNK, in subconfluent and wounded vascular endothelial cells. Wounded cells were chosen for study as they are similar to those found in the early stages of atherosclerosis. Our studies utilized cytokines like TNF-α that are found in vivo to mimic pro-atherosclerotic conditions. Based on the constitutive activation and subcellular localization of p-JNK in subconfluent and wounded endothelial cells, we surmise that subconfluent cells are a more accurate model for the study of SAPK activation in wounded vascular endothelial cells than a confluent monolayer. In the study of SAPK activation profiles, we found that unfractionated heparin effectively deactivates active SAPKs through the upregulation of MAP kinase phosphatase-1 (MKP-1), a phosphatase specific for active p38 and JNK. Although heparin itself may be contraindicated for the treatment of atherosclerosis due to hemorrhagic side effects and negation by the presence of human serum in vivo, an understanding of its molecular interactions with the SAPK cascade is useful. This work also compares the effects of low molecular weight and unfractionated heparin on SAPK deactivation and finds that they work in a similar manner, indicating that the clinical superiority of low molecular weight heparin does not lie in the greater deactivation of the SAPK cascade. In addition, we outline the co-localization of active JNK and p38 with cytoskeletal elements in subconfluent vascular endothelial cells. Phospho-p38 was found to localize with focal adhesions, while p-JNK co-localized with actin stress fibers. We examined this subcellular localization through immunofluorescence, treatment with chemical cytoskeletal inhibitors, SDS-PAGE, and Western blotting. These data may support evidence linking the SAPK cascade with mechanotransduction events in the damaged endothelial cell, or may indicate that SAPK constituents are tethered to the cytoskeleton for efficient signal transduction. The effects of heparin on physiological endpoints like IL-8 secretion were also examined. Our results indicate that deactivation of SAPKs in vascular endothelial cells leads to reduced IL-8 secretion and may cause the direct reduction of inflammation in vivo.