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Background: Atherosclerosis is an inflammatory disease in which a perpetuated activation of NFkappaB via the RAGE (receptor for advanced glycation end products)-MAPK signalling pathway may play an important pathogenetic role. As recently S100 proteins have been identified as ligands of RAGE, we sought to determine the effects of the proinflammatory heterodimer of S100A8/S100A9 on the RAGE-NFkappaB mediated induction of proinflammatory gene expression., Methods: Human umbilical vein endothelial cells (HUVEC) were preincubated for 72 h with AGE-albumin or unmodified albumin for control, whereas AGE-albumin induction resulted in an upregulation of RAGE. Following this preactivation, cells were stimulated for 48 h with heterodimeric human recombinant S100A8/S100A9., Results: Heterodimeric S100A8/S100A9 enhanced secretion of IL-6, ICAM-1, VCAM-1 and MCP1 in AGE-albumin pretreated HUVEC in a dose dependent manner. These effects could not be detected after stimulation with the homodimeric proteins S100A8, S100A9, S100A1 and S100B. The effects of heterodimeric S100A8/S100A9 were reduced by inhibition of the MAP-kinase pathways ERK1/2 and p38 by PD 98059 and SB 203580, respectively., Conclusion: The heterodimeric S100A8/S100A9 might therefore play a hitherto unknown role in triggering atherosclerosis in diabetes and renal failure, pathophysiological entities associated with a high AGE burden. Thus, blocking heterodimeric S100A8/S100A9 might represent a novel therapeutic modality in treating atherosclerosis.

Background: The deposition of protein Z was investigated in atherosclerotic vascular lesions of patients with diabetes mellitus or atherosclerotic vascular disease without diabetes in comparison to controls., Patients and Methods: Protein Z antigen was evidenced by immunohistochemistry in arteries of 5 healthy control patients, 11 diabetic patients with arterio-occlusive disease and 7 patients suffering from arterio-occlusive disease without diabetes. For immunohistochemistry, a commercially available antibody was taken as first antibody, and immunopositivity was evaluated independently by two investigators (J.G.; I.K.) as negative (0), positive (+) and strongly positive (++). The results were assessed by the Whitney-Mann-Wilcoxon test., Results: Macrovascular endothelial cells were stained positive for protein Z in all arteries studied. Arteries of controls did not show significant immunopositivity in cells other than macrovascular endothelial cells, while the microvascular endothelial cells of control arteries were largely negative. The proliferating subendothelial space in atherosclerotic vascular lesions showed significant immunopositivity for protein Z. In contrast to control arteries, the microvascular endothelial cells of the proliferating areas stained positive. The staining pattern of the subendothelial space was similar in atherosclerotic vessels independent of the risk factor for atherosclerosis. Plaques were immunopositive for protein Z, too., Conclusions: Protein Z is present in atherosclerotic vascular lesions of diabetic and non-diabetic patients, but not in the subendothelial space and microvascular endothelial cells of healthy controls. Since protein Z-positivity was detected in microvascular endothelium as well as in extra-vascular deposits around plaques, it may play a role in the development of these lesions.

Background: Cellular actions of advanced glycation end-products (AGE) are mediated by a receptor for AGE (RAGE), a novel integral membrane protein. Immunohistochemical studies show only low-level RAGE antigen expression in endothelial cells. Design. It was the purposes of the study to compare expression of RAGE antigen by endothelial cells in non-diabetic uraemic patients (n=8) with non-uraemic controls (n=11). Samples of arterial tissue were obtained at the time of renal transplantation (in uraemic patients) and abdominal surgery (in controls). RAGE antigen was visualized using guinea-pig anti-RAGE IgG and PAP technique., Results: Marked staining for RAGE was noted in endothelial cells, both arterial endothelium and endothelium of vasa vasorum of normoglycaemic uraemic patients, but was not demonstrable in endothelial cells of large arteries and only faintly expressed in vasa vasorum of non-uraemic individuals., Conclusion: Normal endothelial cells do not constitually express RAGE antigen; in contrast it is expressed by arterial and capillary endothelial cells of uraemic patients. The observation is of note in view of the putative role of AGE of causing atherosclerotic and non-atherosclerotic vascular lesions.

This study examines the regulation of the human tissue factor (TF) promotor in vitro and in vivo. Transient transfections were performed in bovine aortic endothelial cells to investigate the role of two fundamentally different AP-1 sites and a closely located NF-kappa B site in the human TF promoter. The NF-kappa B site is functionally active, since overexpression of NF-kappa B(p65) resulted in induction of TF mRNA and activity. Promoter analysis showed that NF-kappa B induction was dependent on the integrity of the region from base pair -188 to -181. Over-expression of Jun/Fos resulted in TF induction of transcription and protein/activity. Functional studies revealed that the proximal AP-1 site, but not the distal, was inducible by Jun/Fos heterodimers. The distal AP-1 site, which has a G-->A switch at position 4, was inductible by Jun homodimers. Electrophoretic mobility shift assays, using extracts of tumor necrosis factor alpha (TNF alpha)-stimulated bovine aortic endothelial cells, demonstrated TNF alpha-inducible binding to the proximal AP-1 site, comprising JunD/Fos heterodimers. At the distal AP-1 site, only minor induction of binding activity, characterized as proteins of the Jun and ATF family, was observed. Consistently, this site only marginally participates in TNF alpha induction. Functional studies with TF promotor plasmids confirmed that deletion of the proximal AP-1 or the NF-kappa B site decreased TNF alpha-mediated TF induction to a higher extend than loss of the distal AP-1 site. However, integrity of both AP-1 sites and the NF-kappa B site was required for optimal TNF alpha stimulation. The relevance of these in vitro data was confirmed in vivo in a mouse tumor model. Expression plasmids for a dominant negative Jun mutant or I-kappa B were packaged in liposomes. When either mutated Jun or I-kappa B were injected intravenously 48 h before TNF alpha, a reduction in TNF alpha-mediated TF expression in the tumor endothelial cells was observed. Simultaneously, fibrin/fibrinogen deposition decreased and free blood flow could be restored. Thus, TNF alpha-induced up-regulation of endothelial cell TF depends on a concerted action of members of the bZIP and NF-kappa B family.