| The initial stages of atherosclerosis involve the chronic activation of the endothelium. Endothelium activation results due to stimulation by an array of inflammatory signals such as cytokines like TNF-alpha and reactive oxygen species like hydrogen peroxide, in part generated by circulating immune cells. The endothelial cells themselves, once activated, initiate cytoplasmic signal transduction pathways, such as the classic MAPK and SAPK pathways, that result in the up-regulation of the genes of some of these inflammatory signals such as TNF-alpha and IL-6. The glycosaminoglycan heparin is antiproliferative towards smooth muscle cell growth. Heparin treatment of endothelial cells activated with the cytokine TNF-alpha caused a reduction, at the molecular level, in the activities of the SAPKs JNK and p38. This modulatory effect could be observed at least one step upstream, in particular the MAPKK MEK3 that activates p38 in response to TNF-alpha. The downstream effects of heparin were monitored as well. Heparin was able to attenuate the phosphorylation of the transcription factor c-Jun, a JNK substrate. Heparin also caused a decrease in stress fiber formation induced by TNF-alpha, an effect that is regulated by the p38/MAPKAPK-2/HSP27 pathway. Heparin's attenuation of SAPK activation was reversed by a tyrosine phosphatase inhibitor, sodium orthovanadate, and further experiments revealed that heparin increased the DSP MKP-1 in a concentration- and time-dependent manner. In addition, heparin's effect on the SAPK pathway was found to be sensitive to a cGK inhibitor, signifying a role for the cGMP/cGK pathway in heparin's regulation of SAPK pathways. Heparin receptor antibodies were able to mimic heparin by reducing TNF-alpha-induced SAPK activation as well. Together these indicate that heparin is anti-inflammatory and anti-oxidant towards activated endothelial cells. Finally, the cytoskeletal association of members of the SAPK and MAPK pathways was studied. The results indicated that the phosphorylated forms of the SAM and MAPK associated with cytoskeletal elements, and that their MAPKKs are predominantly found in the nuclei with the exception of pMEK7. The MAPKKK MEKK1 and scaffold protein HP-1, two proteins that regulate JNK activity, were also found associated with stress fibers. |
