NF-κ B Transcription Factor P50 Critically Regulates Tissue Factor And Play An Important Role In Neointimal Hyperplasia

[Background] Tissue factor (TF)is a 47-kDa transmembrane glycoprotein, consisting of 263 amino acid residues in total, the major part of TF comprises the 219 amino acid extracellular region. In addition, TF contains a 23 amino acid - hydrophobic transmemb- raneregion and a C-terminal intracellular tail of 21 amino acids.It is constitutively expressed in fibroblasts and pericytes surrounding blood vessels but is normally absent from blood cells and vessel wall cells. Under pathophysiological conditions such as sepsis, atherosclerosis, acute myocardial infarction, transplant vasculopathy, or cancer, TF is expressed in monocytes, endothelial cells, smooth muscle cells, and tumor cells. So apart from a key trigger of blood coagulation, TF also play an important role in the process of atherosclerosis, embryonic vessel development, tumor metastasis, proinflammatory, and signal transduction. Thus the efforts to increase our molecular appreciation of the regulation of tissue factor will be of great value in learning to fully exploit this important factor as a therapeutic target.The expression of TF is regulated principally at the transcriptional level. The promoter of human TF contains five binding sites for specificity protein 1 (Sp1), three sites for epidermal growth response-1 (Egr-1), two sites for activator protein-1 (AP-1) and one site for NF-kB. Notably, there has been a non-conservative change of guanine to cytosine in the oligonucleotide sequence of the NF-kB site in human TF promoter, namely the TF-κB site. It is currently believed that Spl sites are mainly responsible for constitutive expression of the basal level of TF, whereas Egr-1, AP-1 and NF-κB sites are chiefly accountable for inducible expression of TF.The family of NF-κB transcription factors has five cellular members, called p105/p50 (NF-κB1), p100/p52 (NF-κB2), p65 (RelA), RelB and c-Rel. Dominant among the five cellular members of NF-κB transcription factors is the p50/p65 heterodimer. The NF-κB signal pathway is involved in many immunological responses. The inactive NF-κB complex (mainly p50/p65 heterodimer) localized in plasma combined with the inhibitor IκB. When activated, IκB is rapidly phosphorylated and degraded to allow the p50/p65 heterodimer shuttle into nucleus. Binding of p50 toκB motif causes transcription of multiple target genes such as cell adhesion molecules (E-selectin, ICAM-1 and VCAM-1), cytokines, immunoreceptors, growth factor receptors and several cell survival genes.The NF-κB transcription factor p65/c-Rel reportedly regulates TF expression in human monocytes and monocytic THP-1 cells in vitro. However, whether other NF-κB transcription factors, such as p50 and p65 (presumably as the p50/p65 heterodimer), modulate the activity and expression of TF in vascular endothelial cells and monocytes/macrophages remains undetermined. Furthermore, the importance of transcriptional regulation of TF by NF-κB transcription factors, including the p65/c-Rel and p50/p65 heterodimers, has not been investigated in vivo.[Objects] In the present study we want to investigate whether the NF-κB transcription factor p50 critically regulates TF expression in stimulated vascular endothelial cells and activated monocytes/macrophages. We further hope to assesse the functional significance of p50 in modulation of TF-mediated thrombosis, using murine models of experimental neointimal hyperplasia. As a part of extending investigation ,we also assesse the p50 inhibitor—Andro derivatives wether can be designed as small molecular weight antagonists of p50 for inhibition of NF-κB activation.[Methods] The present study includes two parts: (1) In. vitro experiments :Human adherent monocytes, murine macrophages of C57, B6, and p50 null mice, HUVECs and murine PVECs were obtined and cultured to be used in vitro experiments. Measurement of TF activity, transfection and luciferase assay showed that suppression p50 or p50 KO decreases TF expression of the stimulated vascular endothelial cells and activated monocytes/macrophages. Electrophoretic mobility shift assay (EMSA), and chromatin immunoprecipitation (ChIP) were utilized to demonstrated the direct interaction of p50/p65 heterodimer with the TF-kB site of human TF promoter. (2) In vivo experiments: Experimental neointimal hyperplasiawas used to assesse the the functional significance of p50 in modulation of TF-mediated thrombosis. H&E staining and EVGstaining were utilized to appreciate the effect of Andro treatment and p50 KO in the neointimal hyperplysia, mainly targeting neointima and intima/media. Futhermore, immunohistochemical and immunofluorescence staining and immunohistoch- emical gray analysis were adopted to detect wether Andro-treated and p50null mice both exhibited blunted TF, E-selectin, and VCAM-1 expression. To correlate our experimental results in mice to clinical findings in patients, we obtained a human sample of thrombotic vasculitis and examined the expression of various NF-kB targeting genes, such as TF, E-selectin and VCAM-1.[Results] (1)Andro ablates TF in stimulated endothelial cells and monocytes /macrophages; (2) p50 is required for TF up-regulation in activated endothelial cells and macrophages;(3)Andro antagonizes the TF-kB site in human TF promoter; (4) p50 binds to the TF-kB site in the promoter of human TF;(5)Inhibition or deletion of p50 suppresses experimental neointimal hyperplasia; (6)Suppression or knockout of p50 decreases TF expression and fibrin deposition; (7) Infiltrated macrophages, aggregated platelets, stimulated endothelial cells and activated smooth muscle cells express TF;(8)Elimination of p50 reduces the expression of NF-κB targeting genes in vivo;(9)Blockade of TF activity attenuates experimental neointimal hyperplasia;(10)NF-κB targeting genes overexpress in human thrombotic vasculitis.[Conclusions] The present study demonstrates that: (1)p50 transcription-ally regulates TF in stimulated human and murine macrophages and vascular endothelial cells in vitro and in in vivo models of experimental neointimal hyperplasia. (2)Andro derivatives can be designed as small molecular weight antagonists of p50 for inhibition of NF-κB activation and therapeutic prevention and treatment of inflammation and thrombosis.